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Lesions at and around the stylomastoid foramen are the commonest abnormality of the facial nerve [VII] and usually result from a viral inflammation of the nerve within the bony canal before exiting through the stylomastoid foramen. Typically the patient has an ipsilateral loss of motor function of the whole side of the face. Not only does this produce an unusual appearance, but it also complicates chewing of food. Lacrimation and taste may not be affected if the lesion remains distal to the greater petrosal and chorda tympani branches that originate deep in the temporal bone.
In the clinic
Trigeminal neuralgia (tic douloureux) is a complex sensory disorder of the sensory root of the trigeminal nerve. Typically the pain is in the region of the mandibular [V3] and maxillary [V2] nerves, and is usually of sudden onset, is excruciating in nature, and may be triggered by touching a sensitive region of skin.
The etiology of trigeminal neuralgia is unknown, although anomalous blood vessels lying adjacent to the sensory route of the maxillary [V2] and mandibular [V3] nerves may be involved.
If symptoms persist and are unresponsive to medical care, surgical exploration of the trigeminal nerve (which is not without risk) may be necessary to remove any aberrant vessels.
In the clinic
The scalp has an extremely rich blood supply from the external carotid arteries, so lacerations of the scalp tend to bleed profusely. Importantly, scalp bleeding is predominantly arterial, because of two reasons. First, in the erect position the venous pressure is extremely low. Second, the vessels do not retract and close when lacerated because the connective tissue in which they are found holds them open.
In the clinic
Fractures of the orbit are not uncommon and may involve the orbital margins with extension into the maxilla, frontal, and zygomatic bones. These fractures are often part of complex facial fractures. Fractures within the orbit frequently occur within the floor and the medial wall; however, superior and lateral wall fractures also occur. Inferior orbital floor fractures are one of the commonest types of injuries. These fractures may drag the inferior oblique muscle and associated tissues into the fracture line. In these instances, patients may have upward gaze failure (upward gaze diplopia) in the affected eye. Medial wall fractures characteristically show air within the orbit in radiographs. This is due to fracture of the ethmoidal labyrinth, permitting direct continuity between the orbit and the ethmoidal paranasal sinuses. Occasionally, patients feel a full sensation within the orbit when blowing the nose.
In the clinic
Horner’s syndrome is caused by any lesion that leads to a loss of sympathetic function in the head. It is characterized by three typical features: pupillary constriction due to paralysis of the dilator pupillae muscle, partial ptosis (drooping of the upper eyelid) due to paralysis of the superior tarsal muscle, and absence of sweating on the ipsilateral side of the face and the neck due to absence of innervation of the sweat glands.
Secondary changes may also include: ipsilateral vasodilation due to loss of the normal sympathetic control of the subcutaneous blood vessels, and enophthalmos (sinking of the eye)—believed to result from paralysis of the orbitalis muscle, although this is an uncommon feature of Horner’s syndrome.
The orbitalis muscle spans the inferior orbital fissure and helps maintain the forward position of orbital contents.
The commonest cause for Horner’s syndrome is a tumor eroding the cervicothoracic ganglion, which is typically an apical lung tumor.
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A surgically induced Horner’s syndrome may be necessary for patients who suffer severe hyperhidrosis (sweating). This often debilitating condition may be so severe that patients are confined to their home for fear of embarrassment. Treatment is relatively straightforward. The patient is anesthetized and a bifurcate endotracheal tube is placed into the left and right main bronchi. A small incision is made in the intercostal space on the appropriate side, and a surgically induced pneumothorax is created. The patient is ventilated through the contralateral lung.
Using an endoscope the apex of the thoracic cavity can be viewed from inside and the cervicothoracic ganglion readily identified. Obliterative techniques include thermocoagulation and surgical excision. After the ganglion has been destroyed, the endoscope is removed, the lung is reinflated, and the small hole is sutured.
In the clinic
Examination of the eye
Examination of the eye includes assessment of the visual capabilities, the extrinsic musculature and its function, and disease processes that may affect the eye in isolation or as part of the systemic process.
Examination of the eye includes tests for visual acuity, astigmatism, visual fields, and color interpretation (to exclude color blindness) in a variety of circumstances. The physician also assesses the retina, the optic nerve and its coverings, the lens, and the cornea.
The extrinsic muscles are supplied by the abducent nerve [VI], the trochlear nerve [IV], and the oculomotor nerve [III].
The extrinsic muscles work synergistically to provide appropriate and conjugate eye movement: lateral rectus—abducent nerve [VI], superior oblique—trochlear nerve [IV], and remainder—oculomotor nerve [III].
The eye may be affected in systemic diseases. Diabetes mellitus typically affects the eye and may cause cataracts, macular disease, and retinal hemorrhage, all impairing vision.
Occasionally unilateral paralysis of the extra-ocular muscles occurs and is due to brainstem injury or direct nerve injury, which may be associated with tumor compression or trauma. The paralysis of a muscle is easily demonstrated when the patient attempts to move the eye in the direction associated with normal action of that muscle. Typically the patient complains of double vision (diplopia).
Loss of innervation of the muscles around the eye
Loss of innervation of the orbicularis oculi by the facial nerve [VII] causes an inability to close the eyelids tightly, allowing the lower eyelid to droop away causing spillage of tears. This loss of tears allows drying of the conjunctiva, which may ulcerate, so allowing secondary infection.
Loss of innervation of the levator palpebrae superioris by oculomotor nerve [III] damage causes an inability of the superior eyelid to elevate, producing a complete ptosis. Usually, oculomotor nerve [III] damage is caused by severe head injury.
Loss of innervation of the superior tarsal muscle by sympathetic fibers causes a constant partial ptosis. Any lesion along the sympathetic trunk can induce this. An apical pulmonary malignancy should always be suspected because the ptosis may be part of Horner’s syndrome (see “In the clinic” on p. 920).
In the clinic
The “H-test”
A simple “formula” for remembering the nerves that innervate the extraocular muscles is “LR6SO4 and all the rest are 3” (lateral rectus [VI], superior oblique [IV], all the rest including levator palpebrae superioris are [III]).
The function of all extrinsic muscles and their nerves [III, IV, VI] that move the eyeball in both orbits can all easily be tested at the same time by having the patient track, without moving his or her head, an object such as the tip of a pen or a finger moved in an “H” pattern—starting from the midline between the two eyes (Fig. 8.98).
In the clinic
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Intraocular pressure will rise if the normal cycle of aqueous humor fluid production and absorption is disturbed so that the amount of fluid increases. This condition is glaucoma and can lead to a variety of visual problems including blindness, which results from compression of the retina and its blood supply.
In the clinic
With increasing age and in certain disease states the lens of the eye becomes opaque. Increasing opacity results in increasing visual impairment. A common operation is excision of the cloudy lens and replacement with a new man-made lens.
In the clinic
Direct visualization of the postremal (vitreous) chamber of the eye is possible in most clinical settings. It is achieved using an ophthalmoscope, which is a small battery-operated light with a tiny lens that allows direct visualization of the postremal (vitreous) chamber and the posterior wall of the eye through the pupil and the lens. It is sometimes necessary to place a drug directly onto the eye to dilate the pupil for better visualization.
The optic nerve, observed as the optic disc, is easily seen. The typical four branches of the central retinal artery and the fovea are also seen.
Using ophthalmoscopy the physician can look for diseases of the optic nerve, vascular abnormalities, and changes within the retina (Fig. 8.109).
In the clinic
High-definition optical coherence tomography (HD-OCT) (Fig. 8.111) is a procedure used to obtain subsurface images of translucent or opaque materials. It is similar to ultrasound, except that it uses light instead of sound to produce high-resolution cross-sectional images. It is especially useful in the diagnosis and management of optic nerve and retinal diseases.
An epiretinal membrane (Fig. 8.112) is a thin sheet of fibrous tissue that develops on the surface of the retina in the area of the macula and can cause visual problems. If the visual problems are significant, surgical removal of the membrane may be necessary.
In the clinic
The eustachian tube links the middle ear and pharynx and balances the pressure between the outer and middle ear. Colds and allergies, particularly in children, can result in swelling of the lining of the eustachian tube, which can then impair normal drainage of fluid from the middle ear. The fluid then builds up behind the tympanic membrane, providing an attractive environment for bacteria and viruses to grow and cause otitis media. Left untreated, otitis media can lead to perforation of the tympanic membrane, hearing loss, meningitis, and brain abscess.
In the clinic
Examination of the ear
The ear comprises three components—the external, middle, and internal ear.
Clinical examination is carried out to assess hearing and balance. Further examination involves use of an otoscope or other imaging techniques.
The external ear is easily examined. The external acoustic meatus and the tympanic membrane require otoscopic examination (Fig. 8.118B). An otoscope is a device through which light can be shone and the image magnified to inspect the external acoustic meatus and the tympanic membrane.
The examination begins by grasping the posterosuperior aspect of the ear and gently retracting it to straighten the external auditory meatus. The normal tympanic membrane is relatively translucent and has a gray–reddish tinge. The handle of the malleus is visible near the center of the membrane. In the 5 o’clock position a cone of light is always demonstrated.
The middle ear is investigated by CT and MRI to visualize the malleus, incus, and stapes. The relationship of these bones to the middle ear cavity is determined and any masses identified.
The inner ear is also assessed by CT and MRI.
In the clinic
Swimmer’s ear, often called otitis externa, is a painful condition resulting from an infection in the external acoustic meatus. It frequently occurs in swimmers.
In the clinic
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Surfer’s ear, which is prevalent among individuals who surf or swim in cold water, results from the development of a “bony lump” in the external acoustic meatus. Growth of the lump eventually constricts the meatus and reduces hearing in the affected ear.
In the clinic
Although perforation of the tympanic membrane (eardrum) has many causes, trauma and infection are the most common.
Ruptures of the tympanic membrane tend to heal spontaneously, but surgical intervention may be necessary if the rupture is large.
Occasionally, it may be necessary to enter the middle ear through the tympanic membrane. Because the chorda tympani runs in the upper one-third of the tympanic membrane, incisions are always below this level. The richer blood supply to the posterior aspect of the tympanic membrane determines the standard surgical approach in the posteroinferior aspect.
Otitis media (infection of the middle ear) is common and can lead to perforation of the tympanic membrane. The infection can usually be treated with antibiotics. If the infection persists, the chronic inflammatory change may damage the ossicular chain and other structures within the middle ear to produce deafness.
In the clinic
Infection within the mastoid antrum and mastoid cells is usually secondary to infection in the middle ear. The mastoid cells provide an excellent culture medium for infection. Infection of the bone (osteomyelitis) may also develop, spreading into the middle cranial fossa.
Drainage of the pus within the mastoid air cells is necessary and there are numerous approaches for doing this. When undertaking this type of surgery, it is extremely important that care is taken not to damage the mastoid wall of the middle ear to prevent injury to the facial nerve [VII]. Any breach of the inner table of the cranial vault may allow bacteria to enter the cranial cavity and meningitis will ensue.
In the clinic
A lingual nerve injury proximal to where the chorda tympani joins it in the infratemporal fossa will produce loss of general sensation from the anterior two-thirds of the tongue, oral mucosa, gingivae, the lower lip, and the chin.
If a lingual nerve lesion is distal to the site where it is joined by the chorda tympani, secretion from the salivary glands below the oral fissure and taste from the anterior two-thirds of the tongue will also be lost.
In the clinic
Anesthesia of the inferior alveolar nerve is widely practiced by most dentists. The inferior alveolar nerve is one of the largest branches of the mandibular nerve [V3], carries the sensory branches from the teeth and mandible, and receives sensory information from the skin over most of the mandible.
The inferior alveolar nerve passes into the mandibular canal, courses through the body of the mandible, and eventually emerges through the mental foramen into the chin.
of the inferior alveolar nerve by local anesthetic. To anesthetize this nerve the needle is placed lateral to the anterior arch of the fauces (palatoglossal arch) in the oral cavity and is advanced along the medial border around the inferior third of the ramus of the mandible so that anesthetic can be deposited in this region.
It is also possible to anesthetize the infra-orbital and buccal nerves, depending on where the anesthesia is needed.
In the clinic
In most instances, access to peripheral veins of the arm and the leg will suffice for administering intravenous drugs and fluids and for obtaining blood for analysis; however, in certain circumstances it is necessary to place larger-bore catheters in the central veins, for example, for dialysis, parenteral nutrition, or the administration of drugs that have a tendency to produce phlebitis.
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“Blind puncture” of the subclavian and jugular veins to obtain central venous access used to be standard practice. However, subclavian vein puncture is not without complications. As the subclavian vein passes inferiorly, posterior to the clavicle, it passes over the apex of the lung. Any misplacement of a needle into or through this structure may puncture the apical pleura, producing a pneumothorax. Inadvertent arterial puncture and vein laceration may also produce a hemopneumothorax.
A puncture of the internal jugular vein (Fig. 8.165) carries fewer risks, but local hematoma and damage to the carotid artery are again important complications.
Current practice is to identify major vessels using ultrasound and to obtain central venous access under direct vision to avoid any significant complication.
In the clinic
The jugular venous pulse is an important clinical sign that enables the physician to assess the venous pressure and waveform and is a reflection of the functioning of the right side of the heart.
In the clinic
The thyroid gland develops from a small region of tissue near the base of the tongue. This tissue descends as the thyroglossal duct from the foramen cecum in the posterior aspect of the tongue to pass adjacent to the anterior aspect of the middle of the hyoid bone. The thyroid tissue continues to migrate inferiorly and eventually comes to rest at the anterior aspect of the trachea in the root of the neck.
Consequently, the migration of thyroid tissue may be arrested anywhere along the embryological descent of the gland. Ectopic thyroid tissue is relatively rare. More frequently seen is the cystic change that arises from the thyroglossal duct. The usual symptom of a thyroglossal duct cyst is a midline mass. Ultrasound easily demonstrates its nature and position, and treatment is by surgical excision. The whole of the duct as well as a small part of the anterior aspect of the hyoid bone must be excised to prevent recurrence.
In the clinic
A thyroidectomy is a common surgical procedure. In most cases it involves excision of part or most of the thyroid gland. This surgical procedure is usually carried out for benign diseases, such as multinodular goiter and thyroid cancer.
Given the location of the thyroid gland, there is a possibility of damaging other structures when carrying out a thyroidectomy, namely the parathyroid glands and the recurrent laryngeal nerve (Fig. 8.181). Assessment of the vocal folds is necessary before and after thyroid surgery because the recurrent laryngeal nerves are closely related to ligaments that bind the gland to the larynx and can be easily traumatized during surgical procedures.
In the clinic
Thyroid gland pathology is extremely complex. In essence, thyroid gland pathology should be assessed from two points of view. First, the thyroid gland may be diffusely or focally enlarged, for which there are numerous causes. Second, the thyroid gland may undersecrete or oversecrete the hormone thyroxine.
One of the commonest disorders of the thyroid gland is a multinodular goiter, which is a diffuse irregular enlargement of the thyroid gland with areas of thyroid hypertrophy and colloid cyst formation. Most patients are euthyroid (i.e., have normal serum thyroxine levels). The typical symptom is a diffuse mass in the neck, which may be managed medically or may need surgical excision if the mass is large enough to affect the patient’s life or cause respiratory problems.
Isolated nodules in the thyroid gland may be a dominant nodule in a multinodular gland or possibly an isolated tumor of the thyroid gland. Isolated tumors may or may not secrete thyroxine depending on their cellular morphology. Treatment is usually by excision.
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Immunological diseases may affect the thyroid gland and may overstimulate it to produce excessive thyroxine. These diseases may be associated with other extrathyroid manifestations, which include exophthalmos, pretibial myxedema, and nail changes. Other causes of diffuse thyroid stimulation include viral thyroiditis. Some diseases may cause atrophy of the thyroid gland, leading to undersecretion of thyroxine (myxedema).
In the clinic
The parathyroid glands develop from the third and fourth pharyngeal pouches and translocate to their more adult locations during development. The position of the glands can be highly variable, sometimes being situated high in the neck or in the thorax. Tumors develop in any of these locations (Fig. 8.182).
In the clinic
Damage to either the right or left recurrent laryngeal nerve may lead initially to a hoarse voice and finally to an inability to speak. Recurrent laryngeal nerve palsy can occur from disruption of the nerves anywhere along their course. Furthermore, interruption of the vagus nerves before the division of the recurrent laryngeal nerves can also produce vocal symptoms.
Lung cancer in the apex of the right lung can affect the right recurrent laryngeal nerve, whereas cancers that infiltrate into the area between the pulmonary artery and aorta, an area known clinically as the “aortopulmonary window,” can affect the left recurrent laryngeal nerve. Thyroid surgery also can traumatize the recurrent laryngeal nerves.
In the clinic
Clinical lymphatic drainage of the head and neck
Enlargement of the neck lymph nodes (cervical lymphadenopathy) is a common manifestation of disease processes that occur in the head and neck. It is also a common manifestation of diffuse diseases of the body, which include lymphoma, sarcoidosis, and certain types of viral infection such as glandular fever and human immunodeficiency virus (HIV) infection.
Evaluation of cervical lymph nodes is extremely important in determining the nature and etiology of the primary disease process that has produced nodal enlargement.
Clinical evaluation includes a general health assessment, particularly relating to symptoms from the head and neck. Examination of the nodes themselves often gives the clinician a clue as to the nature of the pathological process.
Soft, tender, and inflamed lymph nodes suggest an acute inflammatory process, which is most likely to be infective.
Firm multinodular large-volume rubbery nodes often suggest a diagnosis of lymphoma.
Examination should also include careful assessment of other nodal regions, including the supraclavicular fossae, the axillae, the retroperitoneum, and the inguinal regions.
Further examination may include digestive tract endoscopy, chest radiography, and body CT scanning.
Most cervical lymph nodes are easily palpable and suitable for biopsy to establish a tissue diagnosis. Biopsy can be performed using ultrasound for guidance and good samples of lymph nodes may be obtained.
The lymphatic drainage of the neck is somewhat complex, clinically. A relatively simple “level” system of nodal enlargement has been designed that is extremely helpful in evaluating lymph node spread of primary head and neck tumors. Once the number of levels of nodes are determined, and the size of the lymph nodes, the best mode of treatment can be instituted. This may include surgery, radiotherapy, and chemotherapy. The lymph node level also enables a prognosis to be made. The levels are as follows (Fig. 8.199):
Level I—from the midline of the submental triangle up to the level of the submandibular gland.
Level II—from the skull base to the level of the hyoid bone anteriorly from the posterior border of the sternocleidomastoid muscle.
Level III—the inferior aspect of the hyoid bone to the bottom cricoid arch and anterior to the posterior border of the sternocleidomastoid up to the midline.
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Level IV—from the inferior aspect of the cricoid to the top of the manubrium of the sternum and anterior to the posterior border of the sternocleidomastoid muscle.
Level V—posterior to the sternocleidomastoid muscle and anterior to the trapezius muscle above the level of the clavicle.
Level VI—below the hyoid bone and above the jugular (sternal) notch in the midline.
Level VII—below the level of the jugular (sternal) notch.
In the clinic
In emergency situations, when the airway is blocked above the level of the vocal folds, the median cricothyroid ligament can be perforated and a small tube inserted through the incision to establish an airway. Except for small vessels and the occasional presence of a pyramidal lobe of the thyroid gland, normally there are few structures between the median cricothyroid ligament and the skin.
In the clinic
A tracheostomy is a surgical procedure in which a hole is made in the trachea and a tube is inserted to enable ventilation.
A tracheostomy is typically performed when there is obstruction to the larynx as a result of inhalation of a foreign body, severe edema secondary to anaphylactic reaction, or severe head and neck trauma.
The typical situation in which a tracheostomy is performed is in the calm atmosphere of an operating theater. A small transverse incision is placed in the lower third of the neck anteriorly. The strap muscles are deviated laterally and the trachea can be easily visualized. Occasionally it is necessary to divide the isthmus of the thyroid gland. An incision is made in the second and third tracheal rings and a small tracheostomy tube inserted.
After the tracheostomy has been in situ for the required length of time, it is simply removed. The hole through which it was inserted almost inevitably closes without any intervention.
Patients with long-term tracheostomies are unable to vocalize because no air is passing through the vocal cords.
In the clinic
Laryngoscopy is a medical procedure that is used to inspect the larynx. The functions of laryngoscopy include the evaluation of patients with difficulty swallowing, assessment of the vocal cords, and assessment of the larynx for tumors, masses, and weak voice.
The larynx is typically visualized using two methods. Indirect laryngoscopy involves passage of a small rod-mounted mirror (not dissimilar to a dental mirror) into the oropharynx permitting indirect visualization of the larynx. Direct laryngoscopy can be performed using a device with a curved metal tip that holds the tongue and epiglottis forward, allowing direct inspection of the larynx. This procedure can be performed only in the unconscious patient or in a patient in whom the gag reflex is not intact. Other methods of inspection include the passage of fiberoptic endoscopes through either the oral cavity or nasal cavity.
In the clinic
The nasal septum is typically situated in the midline; however, septal deviation to one side or the other is not uncommon, and in many cases is secondary to direct trauma. Extreme septal deviation can produce nasal occlusion. The deviation can be corrected surgically.
In the clinic
Most cancers of the oral cavity, oropharynx, nasopharynx, larynx, sinuses, and salivary glands arise from the epithelial cells that line them, resulting in squamous cell carcinoma. The majority of these are related to cell damage caused by smoking and alcohol use. Certain viruses are also related to cancers in the head and neck, including human papillomavirus (HPV) and Epstein-Barr virus (EBV).
A 50-year-old overweight woman came to the doctor complaining of hoarseness of voice and noisy breathing. She was also concerned at the increase in size of her neck. On examination she had a slow pulse rate (45 beats per minute). She also had an irregular knobby mass in the anterior aspect of the lower neck, which deviated the trachea to the right.
A clinical diagnosis of a multinodular goiter and hypothyroidism was made.
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Enlargement of the thyroid gland is due to increased secretion of thyroid-stimulating hormone, which is usually secondary to diminished output of thyroid hormones. The thyroid undergoes periods of activity and regression, which can lead to the formation of nodules, some of which are solid and some of which are partially cystic (colloid cysts). This nodule formation is compounded by areas of fibrosis within the gland. Other causes of multinodular goiter include iodine deficiency and in certain circumstances, drugs that interfere with the metabolism and production of thyroxine. The typical symptom of a goiter is a painless swelling of the thyroid gland. It may be smooth or nodular, and occasionally it may extend into the superior mediastinum as a retrosternal goiter.
The trachea was deviated.
The enlargement of the thyroid gland due to a multinodular goiter may not be symmetrical. In this case there was significant asymmetrical enlargement of the left lobe of the thyroid deviating the trachea to the right.
The patient had a hoarse voice and noisy breathing.
If the thyroid gland enlargement is significant it can compress the trachea, narrowing it to such an extent that a “crowing sound” is heard during inspiration (stridor).
Other possible causes for hoarseness include paralysis of the vocal cord due to compression of the left recurrent laryngeal nerve from the goiter. Of concern is the possibility of malignant change within the goiter directly invading the recurrent laryngeal nerve. Fortunately, malignant change is rare within the thyroid gland.
When patients have a relatively low production of thyroxine such that the basal metabolic rate is reduced they become more susceptible to infection, including throat and upper respiratory tract infections.
On examination the thyroid gland moved during swallowing.
Characteristically, an enlarged thyroid gland is evident as a neck mass arising on one or both sides of the trachea. The enlarged thyroid gland moves on swallowing because it is attached to the larynx by the pretracheal fascia.
The patient was hypothyroid.
Hypothyroidism refers to the clinical and biochemical state in which the thyroid gland is underactive (hyperthyroidism refers to an overactive thyroid gland). Some patients have thyroid masses and no clinical or biochemical abnormalities—these patients are euthyroid.
The hormone thyroxine controls the basal metabolic rate; therefore, low levels of thyroxine affect the resting pulse rate and may produce other changes, including weight gain, and in some cases depression.
The patient was insistent upon surgery.
After discussion about the risks and complications, a subtotal thyroidectomy was performed. After the procedure the patient complained of tingling in her hands and feet and around her mouth, and carpopedal spasm. These symptoms are typical of tetany and are caused by low serum calcium levels.
The etiology of the low serum calcium level was trauma and bruising of the four parathyroid glands left in situ after the operation. Undoubtedly the trauma of removal of such a large thyroid gland produced a change within the parathyroid gland, which failed to function appropriately. The secretion of parathyroid hormone rapidly decreased over the next 24 hours, resulting in increased excitability of peripheral nerves, manifest by carpopedal spasm and orofacial tingling. Muscle spasms can also be elicited by tapping the facial nerve [VII] as it emerges from the parotid gland to produce twitching of the facial muscles (Chvostek’s sign).
The patient recovered from these symptoms due to a low calcium level over the next 24 hours.
At her return to the clinic the patient was placed on supplementary oral thyroxine, which is necessary after removal of the thyroid gland.
The patient also complained of a hoarse voice.
The etiology of her hoarse voice was damage to the recurrent laryngeal nerve.
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The recurrent laryngeal nerve lies close to the thyroid gland. It may be damaged in difficult surgical procedures, and this may produce unilateral spasm of the ipsilateral vocal cord to produce a hoarse voice.
Since the thyroidectomy and institution of thyroxine treatment, the patient has lost weight and has no further complaints.
A 33-year-old man was playing cricket for his local Sunday team. As the new bowler pitched the ball short, it bounced higher than he anticipated and hit him on the side of his head. He immediately fell to the ground unconscious, but after about 30 seconds he was helped to his feet and felt otherwise well. It was noted he had some bruising around his temple. He decided not to continue playing and went to watch the match from the side. Over the next hour he became extremely sleepy and was eventually unrousable. He was rushed to hospital.
When he was admitted to hospital, the patient’s breathing was shallow and irregular and it was necessary to intubate him. A skull radiograph demonstrated a fracture in the region of the pterion. No other abnormality was demonstrated other than minor soft tissue bruising over the left temporal fossa.
A CT scan was performed.
The CT scan demonstrated a lentiform area of high density within the left cranial fossa.
A diagnosis of extradural hemorrhage was made.
Fractures in the region of the pterion are extremely dangerous. A division of the middle meningeal artery passes deep to this structure and is subject to laceration and disruption, especially in conjunction with a skull injury in this region. In this case the middle meningeal artery was torn and started to bleed, producing a large extradural clot.
The patient’s blood pressure began to increase.
Within the skull there is a fixed volume and clearly what goes in must come out (e.g., blood, cerebrospinal fluid). If there is a space-occupying lesion, such as an extradural hematoma, there is no space into which it can decompress. As the lesion expands, the brain becomes compressed and the intracranial pressure increases. This pressure compresses vessels, so lowering the cerebral perfusion pressure. To combat this the homeostatic mechanisms of the body increase the blood pressure to overcome the increase in intracerebral pressure. Unfortunately, the increase in intracranial pressure is compounded by the cerebral edema that occurs at and after the initial insult.
An urgent surgical procedure was performed.
Burr holes were placed around the region of the hematoma and it was evacuated. The small branch of the middle meningeal artery was ligated and the patient spent a few days in the intensive care unit. Fortunately the patient made an uneventful recovery.
A 35-year-old man was involved in a fight and sustained a punch to the right orbit. He came to the emergency department with double vision.
The double vision was only in one plane.
Examination of the orbits revealed that when the patient was asked to look upward the right eye was unable to move superiorly when adducted. There was some limitation in general eye movement. Assessment of the lateral rectus muscle (abducent nerve [VI]), superior oblique muscle (trochlear nerve [IV]), and the rest of the eye muscles (oculomotor nerve [III]) was otherwise unremarkable.
The patient underwent a CT scan.
A CT scan of the facial bones demonstrated a fracture through the floor of the orbit (Fig. 8.293).
A careful review of this CT scan demonstrated that the inferior oblique muscle had been pulled inferiorly with the fragment of bone in the fracture. This produced a tethering effect, so when the patient was asked to gaze in the upward direction, the left eye was able to do so but the right eye was unable to because of the tethered inferior oblique muscle.
The patient underwent surgical exploration to elevate the small bony fragment and return the inferior oblique to its appropriate position. On follow-up the patient had no complications.
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A 25-year-old man complained of significant swelling in front of his right ear before and around mealtimes. This swelling was associated with considerable pain, which was provoked by the ingestion of lemon sweets. On examination he had tenderness around the right parotid region and a hard nodule was demonstrated in the buccal mucosa adjacent to the right upper molar teeth.
A diagnosis of parotid duct calculus was made.
The formation of stones in the salivary glands is not uncommon, but it is more likely in the submandibular gland than in the parotid gland because the saliva is more mucinous and the duct has a long upward course from the floor of the mouth. Nevertheless, stones do form in the parotid gland and the parotid ducts. Notably, most parotid duct calculi and submandibular duct calculi occur in mouths with excellent dental hygiene and mucosa.
An ultrasound scan was performed.
An initial ultrasound scan demonstrated a stone in the distal end of the right parotid duct with evidence of ductal dilation (eFig. 8.294). Assessment of the gland also demonstrated dilated ducts within the gland and evidence of intraparotid lymphadenopathy.
The patient was treated with antibiotics.
A course of antibiotics was given to remove the bacteria that had produced the inflammation. On return to the doctor some days later the gland was normal in size and there was no evidence of inflammation or infection.
An operation was necessary.
The stone was at the distal end of the parotid duct and it would seem logical and straightforward to make a small incision at the sphincter in the buccal mucosa and deliver the stone, thus permitting the gland to drain normally. Unfortunately, in this patient’s case the gland was significantly destroyed by the chronic obstruction and bacterial infection. Furthermore, smaller calculi were also demonstrated in the gland at ultrasound. On direct questioning it appeared that the patient had had numerous attacks over the previous 4–5 years and it was decided that the parotid gland should be removed surgically.
The patient consented for removal of the parotid gland and a discussion of the possibility for loss of facial function and facial paralysis was had with the patient at this time.
Within the parotid gland the facial nerve [VII] divides into its five terminal branches. At operation the gland is displayed and extremely careful dissection is necessary to peel away the parotid gland from the branches of the facial nerve [VII]. This procedure was made more difficult by the chronic inflammatory change within the gland. After the procedure the patient made a good recovery, though there was some mild paralysis of the whole of the right side of the face. Importantly, taste to the anterior two-thirds of the tongue was preserved. The taste fibers to the anterior two-thirds of the tongue travel in the chorda tympani nerve, which is a branch of the facial nerve [VII]. This nerve leaves the facial nerve [VII] to join the lingual nerve proximal to the parotid gland; therefore, any damage to the facial nerve [VII] within the parotid gland does not affect special sensation (taste).
Over the following week the paralysis improved and was likely due to nerve bruising during the procedure. The patient remained asymptomatic.
A 60-year-old woman was brought to the emergency department with acute right-sided weakness, predominantly in the upper limb, which lasted for 24 hours. She made an uneventful recovery, but was extremely concerned about the nature of her illness and went to see her local doctor.
A diagnosis of a transient ischemic attack (TIA) was made.
A TIA is a neurological deficit resolving within 24 hours. It is a type of stroke.
Neurological deficits may be permanent or transient. Most transient events resolve within 21 days; any failure of resolution beyond 21 days is an established stroke.
An investigation into the cause of the TIA was undertaken.
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Eighty-five percent of all strokes result from cerebral infarction, of which most are due to embolization.
A duplex Doppler scan of the carotid vessels was performed.
The majority of emboli originate from plaques that develop at and around the carotid bifurcation. Emboli consist of platelet aggregates, cholesterol, and atheromatous debris. Emboli may also arise from the heart secondary to cardiac tumors or myocardial infarction.
The lesion in the brain was on the left side.
The motor cortex for the whole of the right side of the body is represented in the left motor strip of the brain, which sits on the precentral gyrus.
The duplex Doppler ultrasound scan demonstrated a significant narrowing (stenosis) of the left internal carotid artery with evidence of plaque formation and abnormal flow in this region. The narrowing was approximately 90%.
Treatment required an operation.
A carotid endarterectomy (removal of the stenosis and the atheromatous plaque) was planned. This procedure is indicated in the presence of an ulcerating plaque with stenosis. The procedure was carried out under general anesthetic and a curvilinear incision was placed in the left side of the neck. The common carotid, external carotid, and internal carotid arteries were displayed. All vessels were clamped and a shunt was placed from the common carotid artery into the internal carotid artery to maintain cerebral blood flow during the procedure. The internal carotid artery was opened and the plaque excised.
After the procedure the patient did extremely well and suffered no further cerebral events. However, a new medical student examined the patient the following day and demonstrated a number of interesting findings. These included altered skin sensation inferior to the left mandible, altered sensation on the left side of the soft palate, a paralyzed left vocal cord, inability to shrug the left shoulder, and a tongue that deviated to the left.
The etiology of these injuries was due to localized nerve trauma.
This constellation of neurological deficits can be accounted for by trauma to the nerves that are close to the carotid bifurcation. The changes in skin sensation can be accounted for by a neurapraxia due to damage to cervical nerves. The alteration in sensation in the soft palate is due to neurapraxia of the glossopharyngeal nerve [IX]. The paralyzed left cord results from neurapraxia of the recurrent laryngeal nerve, while the inability to shrug the shoulder is due to neurapraxia of the accessory nerve [XI]. Deviation of the tongue can be accounted for by damage to the hypoglossal nerve [XII].
Most of these changes are transient and are usually due to traction injuries during the surgical procedure.
A 33-year-old fit and well woman came to the emergency department complaining of double vision and pain behind her right eye. She had no other symptoms. On examination of the right eye the pupil was dilated. There was a mild ptosis. Testing of eye movement revealed that the eye turned down and out and the pupillary reflex was not present.
These findings revealed that the patient had an ipsilateral third nerve palsy (palsy of the oculomotor nerve [III]).
The oculomotor nerve [III] is the main motor nerve to the ocular and extra-ocular muscles. It arises from the midbrain and pierces the dura mater to run in the lateral wall of the cavernous sinus. The oculomotor nerve [III] leaves the cranial cavity and enters the orbit through the superior orbital fissure. Within this fissure it divides into its superior and inferior divisions.
The site of the nerve lesion needs to be assessed.
Third nerve palsy may involve the nucleus of the oculomotor nerve [III], which typically spares the pupil and is painless. The pupillary reflexes are supplied from the autonomic fibers of the Edinger–Westphal nucleus, which pass through the ciliary ganglion.
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The lesion cannot be a primary oculomotor nerve [III] nuclear injury.
As both the pupillary reflexes and vision are affected, the lesion is likely to be along the course of the oculomotor nerve [III]. Medical conditions such as diabetes mellitus and vascular disease may produce an isolated oculomotor nerve [III] injury, but they are not associated with pain.
The lesion was caused by an aneurysm.
One of the commonest causes of a third nerve palsy is pressure on the nerve from a posterior communicating artery aneurysm, which lies parallel to the nerve on the anterior aspect of the brainstem. As the aneurysm abuts the outside of the oculomotor nerve [III], it involves the parasympathetic fibers, which lead to a predominance of the loss of pupillary function over general function.
The aneurysm was imaged with an angiogram.
The patient initially underwent CT and MRI scanning. Currently, the definitive test for assessment of aneurysms arising from the circle of Willis and its branches is a digital subtraction angiogram. The angiogram demonstrated the posterior communicating artery aneurysm. The patient underwent surgery and made an excellent recovery.
A 10-year-old boy was brought to an ENT surgeon (ear, nose, and throat surgeon) with epistaxis (nose bleeding). The bleeding was associated with his nose picking habit. However, the bleeding was profuse and on two occasions required hospital admission and nasal packing.
On inspection an indurated area was noted.
The typical findings are an indurated area in the anterior inferior aspect of the nasal septum (Kiesselbach’s area). This is a very vascular area that has a considerable number of veins, which are often traumatized during nose picking.
The patient underwent treatment.
Typical treatment is cauterization of these prominent veins in Kiesselbach’s area, which is usually performed by a simple local analgesia and the application of silver nitrate.
Unfortunately, the boy was involved in a fight the next day and again developed severe epistaxis, which again was difficult to control.
Not only is there a rich venous plexus around Kiesselbach’s area, but there is also a significant arterial supply, which is provided from the nasal septal branches of the posterior and anterior ethmoidal arteries and the branches of the greater palatine artery. These are supplemented from the septal branches of the superior labial artery.
In most cases treatment is conservative.
Conservative treatment usually involves packing the nasal cavity until bleeding has stopped and correcting any bleeding abnormality. In patients with bleeding refractory to medical treatment a series of maneuvers have been employed, including ligating the anterior and posterior ethmoidal arteries through a medial incision in the canthus orbit, or by ligating other major arteries supplying the nasal cavity. Unfortunately, many of these procedures fail because of the rich and diverse origin of blood supply to the nasal cavity.
Determination of the specific site of bleeding can be achieved radiologically.
By placing a catheter from the femoral artery through the aorta and into the carotid circulation the sphenopalatine artery can be easily cannulated from the maxillary branch of the external carotid artery. Bleeding can usually be demonstrated and the vessel can be embolized using small particles.
Fortunately in this young boy’s case, bleeding stopped after further medical management and he remained asymptomatic.
A 30-year-old woman came to her doctor with a history of amenorrhea (absence of menses) and galactorrhea (the production of breast milk). She was not pregnant and appeared otherwise fit and well.
Serum prolactin was measured.
Prolactin is a hormone produced by the pituitary gland and necessary for the production of breast milk postpartum. This hormone was markedly elevated.
Further clinical tests demonstrated visual field defects.
The patient went to see an optometrist who performed a visual field assessment and demonstrated a reduction in the lateral aspects of the normal visual fields. This was bilateral and symmetrical—a bilateral temporal hemianopia.
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The visual pathways have now determined the site of the lesion.
Visual information from the temporal fields is projected onto the medial aspect of the retina bilaterally. The visual information from the medial aspects of the retina is carried in fibers that cross the midline through the optic chiasm to the opposite side.
The lesion is in the area of the optic chiasm.
Any disruption of the optic chiasm produces the field defect of bitemporal hemianopia. Tumors of the optic chiasm are unusual, though gliomas do occur. More frequently, compression of the optic chiasm by tumors in the vicinity is the usual cause for bitemporal hemianopia.
A pituitary tumor was diagnosed.
The optic chiasm is anterior and extremely close to the pituitary gland. Given that the patient is producing excessive amounts of prolactin (a pituitary tumor) and there is loss of the function of the chiasm, the most likely clinical explanation is an exophytic pituitary tumor compressing the optic chiasm.
An MRI scan was performed and demonstrated a large tumor (macroadenoma) of the pituitary gland.
Drug treatment was commenced and the tumor shrank (eFig. 8.295). The endocrinological effects of the prolactin secretion also stopped.
Follow-up scans were performed.
Over the ensuing few years the tumor shrank. Unfortunately, the patient again began to secrete prolactin and surgery was performed.
A transsphenoidal approach was undertaken.
With meticulous accuracy a series of very fine instruments was passed through the nasal cavity into the sphenoid bone. The bone was drilled and via this approach the pituitary gland was removed.
Extreme care must be taken because on both sides of the pituitary gland is the cavernous sinus through which the internal carotid artery, oculomotor nerve [III], trochlear nerve [IV], trigeminal nerve [V], and abducent nerve [VI] pass.
1121.e2 1121.e1
Fig. 8.7, cont’d Skull.
Conceptual Overview • Relationship to Other Regions
Fig. 8.16, cont’d
In the clinic—cont’d
In the clinic—cont’d
In the clinic—cont’d
In the clinic—cont’d
In the clinic—cont’d
Table 8.5 Cranial nerves (see Table 8.4 for abbreviations)—cont’d
In the clinic—cont’d
Table 8.7 Muscles of the face—cont’d
In the clinic—cont’d
In the clinic—cont’d
Fig. 8.149, cont’d
In the clinic—cont’d
Fig. 8.235, cont’d
Fig. 8.239, cont’d
Surface Anatomy • Visualizing Structures at the CIII/CIV and CVI Vertebral Levels
Surface Anatomy • How to Locate the Cricothyroid Ligament
Surface Anatomy • Major Features of the Face
Neuroanatomy and neuroscience are fields of science that seek to explain embryonic development, structural organization, and physiological function of the nervous system. Both fields work together to help identify the simple to the most complex questions of human sensory, motor, behavioral, and higher cognitive functions. The focus of this chapter is to introduce the basic structures and functions of the individual and systemic components of the human nervous system.
Part I: Nervous system
Organization of the human nervous system is structurally divided into the central nervous system (CNS) and peripheral nervous system (PNS) (eFig. 9.1). Components of the CNS are the brain and spinal cord, which are enclosed within the cranial cavity and vertebral column of the axial skeleton. Peripheral nervous system structures include cranial nerves, spinal nerves, autonomic nerves, and the enteric nervous system.
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During the third week of development the outermost layer of the embryo—the ectoderm—thickens to form a neural plate (eFig. 9.2A). This plate develops a longitudinally running neural groove, which deepens so that it is flanked on either side by neural folds (eFig. 9.2B). These folds further develop and eventually fuse during a process called neurulation to form a long tubelike structure called the neural tube with an inner lumen called the neural canal (eFig. 9.3). Fusion of the tube starts at the midpoint and extends cranially and caudally so that the tube is fully formed by the fourth week. Continued proliferation of the cells at the cephalic end cause the neural tube to dilate and form the three primary brain vesicles (eFig. 9.4): the prosencephalon (forebrain), mesencephalon (midbrain), and rhombencephalon (hindbrain), which later give rise to the structures of the brain. Caudally, the neural tube lengthens and narrows to form the spinal cord. The neural canal forms the cavities of the ventricular system in the brain and central canal of the spinal cord (eTable 9.1). The peripheral nervous system consists of cranial nerves, spinal nerves, spinal ganglia, the enteric system, and autonomic ganglia. The peripheral nervous system is formed by nerve fibers that extend out of the central nervous system and by neurons and their fibers that develop from migratory neural crest cells (eFig. 9.4A). Like the neural tube, neural crest cells originate from surface ectoderm and initially lie on each side of the developing CNS.
Several terms are used to identify the orientation and location of neural structures. The orientation nomenclature is quite simple in organisms such as fish and reptiles, which have a linear nervous system. For these animals, ventral (Latin for “belly”) is oriented toward the ground, dorsal (Latin for “back”) toward the sky, rostral (Latin for “beak”) toward the snout, and caudal (Latin for “tail) toward the tail (eFig. 9.5). Because humans are bipedal and maintain an erect posture, the nervous system makes an obligatory bend of 80 to 90 degrees at the midbrain–diencephalic junction. Because of this, directional references such as ventral, dorsal, rostral, and caudal have different meanings along different locations of the CNS structures (eFig. 9.6A). An additional set of terms that remain constant in their reference to orientation of nervous system structures are anterior, posterior, superior, and inferior.
When studied through imaging or in histopathology, the nervous system is observed in sections cut from one of three different planes: a coronal plane, which divides the nervous system into anterior and posterior parts; the sagittal plane, which is oriented at a right angle to the coronal plane and divides the nervous system into left and right parts; and a horizontal (also referred to as axial or transverse) plane, which divides the nervous system into superior and inferior parts (eFig. 9.6). Note that a sagittal plane passing through the midline may also be referred to as a midsagittal section, whereas a section taken just lateral to the midline is referred to as a parasagittal section.
Nerve cells (neurons) and glial cells are the primary cellular components of the nervous system. Neurochemical signaling is predominantly carried out through a complex series of physiological connections between adjoining neurons. Glial cells participate in a constellation of functions that are vital for proper brain function. Their historically appreciated contribution to neuronal function has expanded to include recognition of their role in regulating the content of the extracellular space and regulation of neurotransmitters at the synaptic junction.
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Neurons consist of a cell body (or soma), which contains the cell nucleus, short processes called dendrites for receiving input from other neurons, and long processes called axons, which conduct signals away from the cell body (eTable 9.2). Depending on their location, neuronal morphology can be quite variable. The majority of mammalian neurons are multipolar, indicating that there are several dendrites from one end and a single axon that branches extensively at its terminus (eFig. 9.7). Some additional neuronal types are bipolar, unipolar, and pseudounipolar (eFig. 9.8).
To prevent the loss of linear signal propagation, glial cells form a phospholipid-based layer of insulation called the myelin sheath along the length of the axon (eFig. 9.7). The myelin sheath is formed by oligodendrocytes in the CNS and Schwann cells in the PNS. Interspersed between the segments of myelin are exposed segments of the axon called nodes of Ranvier, which have a large population of voltage-gated ion channels. Presence of the ion channels facilitates rapid conduction of the action potential (a transient voltage change in the axonal membrane) from node to node in a process called saltatory conduction (eFig. 9.7).
Functionally, the nervous system is organized into a somatic nervous system and visceral nervous system. The somatic nervous system consists of nerves that carry conscious sensation from peripheral regions back to the CNS and nerves that exit the CNS to innervate voluntary (skeletal) muscles. In contrast, the visceral nervous system consists of nerves that carry sensory information into and motor (autonomic) innervation out of the CNS to regulate homeostatic functions. Further discussion of the somatic and visceral nervous systems will be presented within the context of the subsequent “Spinal Cord” section.
Part II: Brain
Externally, the outer surface of the brain, or cerebral cortex, is composed of six layers of cell bodies referred to as gray matter. Internally, the myelinated axonal processes of these cells extend into the cerebral hemispheres. Because of the whitish appearance of these large bundles of myelinated axons, they are referred to as white matter. In the brain, gray matter is predominantly located on the cortical surface and the white matter runs deep inside the cerebral hemispheres; the opposite is true for the spinal cord, where the white matter is superficial to the gray matter. Topographically, the surface of the cerebral hemispheres has a series of elevations called gyri and infoldings referred to as sulci, both of which significantly increase the surface area of the brain.
Structurally, each cerebral hemisphere is divided into four major anatomical lobes: frontal, parietal, occipital, and temporal (eFig. 9.9A). The frontal lobes are located anteriorly and are separated from the more posterior parietal lobe by the central sulcus (sulcus of Rolando) (eFig. 9.9A). Laterally, the frontal lobe is separated from the temporal lobe by the lateral sulcus (fissure of Sylvius). Although there is no specific demarcation between the parietal and occipital lobe laterally, along the medial aspect of the hemispheres the two lobes are separated by the parieto-occipital sulcus (eFig. 9.9B). Along the midline, the cerebral hemispheres are separated from one another by the longitudinal fissure (interhemispheric fissure, sagittal fissure). Concealing a small area of cortex called the insula laterally are portions of the frontal, parietal, and temporal lobes collectively referred to as the operculum (Latin for “lid”) (eFig. 9.10). The insula represents fusion of the telencephalon and diencephalon and can be seen by gently prying open the lateral sulcus.
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The path to and from the cerebral cortex is achieved through various white matter pathways coursing through the spinal cord, brainstem, and cerebral hemispheres. Beneath the gray matter of the cortical surface is an expansion of white matter referred to as the corona radiata. This white matter pathway condenses to form the internal capsule, a V-shaped structure when viewed in horizontal sections that contains axons traversing to and from various cortical and deep nuclear structures (eFig. 9.11). The internal capsule is divided into three parts based on connections to different parts of the cortex and underlying structures. The most anterior portion of this white matter pathway is the anterior limb, which is bounded medially by the head of the caudate and laterally by the globus pallidus and putamen. The anterior limb transitions into the genu (Latin for “knee”) at the level of the interventricular foramen (of Monro) and completes its course as the posterior limb, situated lateral to the thalamus and medial to the globus pallidus and putamen. In addition to this more vertical stream of axonal connections is the horizontally running corpus callosum. The corpus callosum (eFig. 9.12) is formed by myelinated axons horizontally linking the two cerebral hemispheres to one another, and it is divided into a rostrum, genu, body, and splenium (eFig. 9.12).
The ventricular system is derived from the inner lumen of the developing neural tube. As the brain continues to grow, the caverns and canals of the ventricular system adapt to the shape of the cerebral hemispheres, diencephalon, pons, medulla, and cerebellum, which form the surrounding walls (eFig. 9.13). Inferior and lateral to the corpus callosum are two large, fluid-filled cavities that represent the beginning of the ventricular system. These most rostral cavities are the two C-shaped lateral ventricles, located within the cerebral hemispheres (eFig. 9.14). As the lateral ventricles extend through all of the lobes of the cerebral hemispheres, they are divided into five named parts. In the frontal lobe is the anterior (frontal) horn, which transitions into the body within the frontal and parietal lobes (eFig. 9.15). Projecting into the occipital lobe is the posterior (occipital) horn (eFig. 9.15). A final horn extends inferiorly and anteriorly as the inferior (temporal) horn in the temporal lobe (eFig. 9.15). Near the splenium of the corpus callosum, the body, posterior, and inferior horns come together at the atrium/trigone of the lateral ventricles (eFig. 9.15). Lining most of the ventricles is the choroid plexus (eFig. 9.16), a series of modified ependymal cells responsible for producing 0.5 L of cerebrospinal fluid (CSF) a day in adults.
From the lateral ventricles, CSF flows through the interventricular foramen (of Monro) to the slitlike third ventricle, which is surrounded by the thalamus and hypothalamus (eFig. 9.15). The third ventricle communicates with the fourth ventricle via the cerebral aqueduct (aqueduct of Sylvius), which courses through the midbrain (eFig. 9.15). Surrounded by the pons and medulla anteriorly and the cerebellum posteriorly, the fourth ventricle sends CSF out of the ventricular system and into the subarachnoid space via the lateral foramina of Luschka and midline foramen of Magendie (eFig. 9.15).
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Within the bony encasement of the skull and vertebral column, the CNS is surrounded by three concentric, connective tissue coverings called meninges (from Greek word meninx for “membrane”), which act to support and stabilize the brain and spinal cord. The focus of this section will be on the cranial meninges. The spinal meninges, which have a slightly different configuration, will be discussed in the “Spinal Cord” section.
The outermost covering is the dura (Latin for “hard”) mater (Latin for “mother”), a tough, fibrous sheet composed of two layers. The outer periosteal layer is adherent to the skull, and the inner meningeal layer lies against the underlying arachnoid mater (eFig. 9.17). Although these two layers are closely adherent to one another, they do separate in some regions to form dural venous sinuses, which receive cerebral venous drainage (eFig. 9.18). The anatomy of the venous sinuses will be discussed in the “Cerebral Vasculature” section. Two potential spaces exist as the epidural (extradural) space, superficial to the periosteal layer, and subdural space, deep to the meningeal dural layer. These spaces can become filled with blood during vascular trauma (eTable 9.3). Within the cranial cavity, the meningeal layer of dura mater folds in on itself in several areas to form dural reflections, or septa. These reflections are known as the falx (Latin for “sickle”) cerebri between the cerebral hemispheres, as the tentorium cerebelli between the cerebral hemispheres and cerebellum, and as the falx cerebelli between the cerebellar hemispheres (eFig. 9.17). A smaller reflection, the diaphragm sellae, covers the pituitary fossa and underlying pituitary gland.
Deep to the dura mater is the arachnoid (from the Greek word arachne meaning “spider’s web”) mater. The outer layer of the arachnoid mater is composed of several layers of flattened cells that lie adjacent to the meningeal layer of the dura mater (eFig. 9.19). Strands of connective tissue extend from this outer layer to form arachnoid trabeculae, which connect internally to the pia mater (eFig. 9.19). The pia mater forms a thin, veil-like layer that closely follows the gyri and sulci on the surface of the brain. The pia and arachnoid matter are separated by a subarachnoid space that contains CSF and the major blood vessels supplying the brain.
Vascular supply to the brain is divided into the anterior circulation arising from the internal carotid arteries and posterior circulation from the vertebral arteries (eFig. 9.20). The internal carotid arteries arise from the branching of the common carotid arteries at the level of the fourth cervical vertebra. Bilaterally the arteries course through the neck to enter the middle cranial fossa through the carotid canal. The arteries then make a series of turns to pass through the petrous portion of the temporal bone and cavernous sinus before entering the subarachnoid space just lateral to the optic chiasm. Upon exiting the cavernous sinus, the internal carotid artery gives rise to the ophthalmic artery and then continues superiorly to give off the posterior communicating artery and anterior choroidal arteries before terminating as the anterior and middle cerebral arteries (eFig. 9.21).
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The two anterior cerebral arteries anastomose proximally via the anterior communicating artery, anterior to the optic chiasm (eFig. 9.22). Distal to this connection, the anterior cerebral artery (ACA) courses along the medial aspect of the cerebral hemisphere within the longitudinal fissure and follows the superior border of the corpus callosum to the anterior portion of the parietal lobe. Along its course two large branches arise: the callosomarginal artery, which follows the cingulate sulcus, and the pericallosal artery, which is immediately adjacent to the corpus callosum (eFig. 9.23). Given its course and branches, the ACA perfuses most of the medial aspect of the brain from the frontal lobe to the anterior portion of the parietal lobe.
Branching laterally from the internal carotid artery, the middle cerebral artery (MCA) penetrates the lateral fissure and gives off the lenticulostriate striate arteries (eFig. 9.24) before bifurcating into superior and inferior divisions, which loop extensively along the insula and frontal operculum before emerging on the lateral convexity of the cerebrum. The superior division perfuses the cortex above the lateral fissure, including the lateral frontal lobe and a small portion of the anterior parietal lobe (eFig. 9.21). The inferior division perfuses the cortex below the lateral fissure, including the temporal lobe and the anterolateral portion of the parietal lobe.
The posterior cerebral cortex receives vascular supply from the vertebral-basilar system of arteries. This system begins with the vertebral arteries bilaterally, which arise from the subclavian arteries and ascend through the foramen transversarium of the cervical vertebrae in the neck. After entering the foramen magnum at the level of the pontomedullary junction, the arteries join to form the basilar artery, which courses along the midline of the ventral brainstem (eFig. 9.20). At the level of the midbrain, the basilar artery gives rise to the posterior cerebral artery (PCA), which turns posteriorly and gives rise to branches that perfuse the inferior and medial temporal and occipital lobes. Also at the PCA, a connecting artery, the posterior communicating artery, branches off and connects to the internal carotid artery (eFig. 9.22).
Venous drainage of the cerebral hemispheres follows a system of deep veins, superficial veins, and dural venous sinuses before reaching the internal jugular vein. Before reaching the internal jugular veins, the superficial and deep veins connect to the dural sinuses located between the periosteal and meningeal layers of the dura. None of the vessels in this network have valves present in their lumen.
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Running along the superior edge of the falx cerebri is the superior sagittal sinus. The superior sagittal sinus continues posteriorly to drain into the transverse sinuses bilaterally (eFig. 9.25A). Each transverse sinus turns inferiorly to form the sigmoid sinus, which exits the jugular foramen to become the internal jugular vein. Along the inferior margin of the falx cerebri is the inferior sagittal sinus (eFig. 9.25B). Posteriorly, the inferior sagittal sinus joins the great vein of Galen to form the straight sinus. The point where the straight sinus, superior sagittal sinus, and occipital sinus join is known as the confluence of the sinuses (eFig. 9.25B). The confluence of sinuses is drained by the transverse sinuses. Located on either side of the hypophysial fossa is a plexus of veins referred to as the cavernous sinus (eFig. 9.26). In addition to receiving drainage from the other sinuses, the cavernous sinus also receives the ophthalmic veins. The cavernous sinus is drained by the superior petrosal sinus into the transverse sinus and inferior petrosal sinuses into the internal jugular vein.
Venous drainage from the superficial veins is primarily received by the superior sagittal sinus and cavernous sinus. Although the pattern of superficial veins coursing through the subarachnoid space from the cerebral cortex is quite variable, three veins appear to be fairly constant. Positioned in parallel to the lateral fissure is the superficial middle cerebral vein, which drains into the cavernous sinus from the temporal lobe (eFig. 9.25A). Connecting to the superficial middle cerebral vein perpendicularly is the superior anastomotic vein (of Trolard) (eFig. 9.25A). This vein courses superiorly across the parietal lobe to drain into the superior sagittal sinus. Also connecting to the superficial middle cerebral vein perpendicularly is the inferior anastomotic vein (of Labbé) (eFig. 9.25A). The inferior anastomotic vein passes inferiorly along the temporal lobe to drain into the transverse sinus.
In contrast to the superficial veins, deep veins are more constant in their organization. Most of the deep veins eventually drain into the great cerebral vein (of Galen) before entering the dural venous sinuses (eFig. 9.27). Traveling adjacent to the ACA and MCA are the anterior cerebral vein and deep middle cerebral vein. These deep veins join to form the basal vein (of Rosenthal), which continues around the lateral aspect of the midbrain. Formed at the interventricular foramen by the joining of the septal and thalamostriate veins bilaterally are the internal cerebral veins (eFig. 9.27). Posterior to the midbrain, the internal cerebral veins and basal veins join to form the great cerebral vein (of Galen) (eFig. 9.27). From here the great cerebral vein joins the inferior sagittal sinus to form the straight sinus.
Part III: Thalamus
The thalamus (Greek for “inner chamber”) is a large, egg-shaped mass of gray matter derived from the diencephalon of the developing brain (see eTable 9.1). The most significant role of the thalamus is as a synaptic relay for pathways projecting to the cerebral cortex. However, the thalamus also acts as a gatekeeper to prevent or enhance information transfer, depending on the behavioral state. Sensory, motor, limbic, and modulatory signals from behavioral and arousal circuits all have synaptic relays within the thalamic nuclei.
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Because of its location deep within the brain, the thalamus is neighbored by several structures and also portions of the ventricular system. Anteriorly, the thalamus extends forward to contact the interventricular foramen, which connects the lateral and third ventricles (eFig. 9.28). Together, the thalamic nuclear masses and the ventrally located hypothalamus comprise the lateral walls of the third ventricle. Immediately lateral to the thalamus is the posterior limb of the internal capsule (eFig. 9.29). Related to the dorsal aspect of the thalamus is the body of the lateral ventricle, and extending caudally is the midbrain portion of the brainstem (eFig. 9.28). Across the midline of the third ventricle, the two thalamic masses are interconnected by the interthalamic adhesion.
Based on their relationship to the internal medullary lamina, a Y-shaped band of myelinated axons coursing rostrocaudally through the thalamus, the thalamic nuclei are classified into four groups: (1) anterior, (2) medial, (3) lateral, and (4) intralaminar (eFig. 9.30). In addition to this structural categorization, the nuclei are divided into three major functional classes: (1) relay, (2) intralaminar, and (3) reticular. As mentioned earlier, most of the thalamus is composed of relay nuclei, which have reciprocal excitatory connections with the cortex. Relay nuclei are further subdivided into specific and nonspecific based on their projections to specific areas of the primary sensory and motor cortex or more diffuse cortical projections. The majority of specific relay nuclei reside in the lateral thalamus—in fact, all sensory modalities, with the exception of olfaction, have relays in the lateral thalamus before reaching their primary cortical target. Details of specific and nonspecific nuclei and their cortical connections can be reviewed in eTable 9.4.
Intralaminar nuclei reside within the internal medullary lamina and have numerous reciprocal projections, primarily with the basal ganglia and reticular formation (eFig. 9.30).There are two major functional regions of the intralaminar nuclei: rostral and caudal. The rostral intralaminar nuclei have reciprocal connections with the basal ganglia and also relay input from the ascending reticular activating system. Caudal intralaminar nuclei, which include the large centromedian nucleus, are predominantly involved in basal ganglia circuitry.
The reticular nucleus of the thalamus is a thin, sheet-like structure along the lateral aspect of the thalamus just medial to the posterior limb of the internal capsule (eFig. 9.30). Unlike the rest of the thalamic nuclei, the reticular nucleus does not send projections to the cerebral cortex, but rather receives input from other thalamic nuclei and the cortex, which project back to the thalamus. Functionally, this organization of inputs and outputs, along with the GABAergic neurons of the reticular nucleus, allows it to regulate thalamic activity quite effectively.
Vascular supply to the thalamus arises from penetrating branches from the ACA, anterior choroidal artery branching from the internal carotid, lenticulostriate arteries of the middle cerebral artery and thalamoperforator arteries from the posterior cerebral arteries.
Part IV: Brainstem
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The brainstem is a stalklike structure within the posterior cranial fossa of the skull connecting the forebrain and spinal cord (eFig. 9.31). From rostral to caudal, the brainstem consists of the midbrain, pons, and medulla oblongata. Broadly speaking, the brainstem has three main functions: (1) it is a conduit for tracts ascending and descending through the CNS; (2) it houses cranial nerve nuclei III to XII (note that the CNXI is in the cervical spinal cord); and (3) it is the location for reflex centers related to respiration, cardiovascular function, and regulation of consciousness. Externally, each portion of the brainstem has a distinct appearance and structural features that define its many functional roles.
Approximately 2 cm in length, the midbrain connects the forebrain to the pons caudally and the cerebellum posteriorly (eFig. 9.32A). Along the midline of the anterior surface there is a deep depression, the interpeduncular fossa, which has several perforations on its surface where small vessels perforate through the floor of the fossa. On either side of the interpeduncular fossa are the crus cerebri (eFig. 9.32B). Projecting medially from the crus is cranial nerve III, the oculomotor nerve (eFig. 9.33A). The most prominent features on the posterior surface are the superior and inferior colliculi (eFig. 9.32A). Between the inferior colliculi, cranial nerve IV, the trochlear nerve, emerges, crosses the midline, and wraps around the lateral aspect of the midbrain (eFig. 9.32A). Laterally, the superior brachium and inferior brachium can be seen as they project in an anterolateral direction from the superior and inferior colliculi.
Caudal to the midbrain, the pons is approximately 2.5 cm in length and connects the midbrain to the medulla (eFig. 9.32B). The midline of the anterior surface has a shallow groove, the basilar groove, where the basilar artery resides along its course (eFig. 9.32B). On either side of the basilar groove, the pons has a prominent convex shape as a result of the vast number of fibers bridging through the pons to the cerebellum through the middle cerebellar peduncle. Along the anterolateral surface of the pons, cranial nerve V, the motor and sensory root of the trigeminal nerve, emerges (eFig. 9.33B). At the junction of the pons and medulla, from medial to lateral, cranial nerves VI (abducent), VII (facial), and VIII (vestibulocochlear) emerge (eFig. 9.33C). The posterior surface of the pons faces the cerebellum and forms the floor of the fourth ventricle (eFig. 9.32A). Along the midline of the posterior surface is a prominent bump, the facial colliculus, which represents the relationship between the facial nerve fibers as they wind around the nucleus of the abducent nerve (eFig. 9.33C).
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The medulla is the longest portion of the brainstem, measuring approximately 3 cm in length (eFig. 9.32A). At the rostral connection of the medulla to the pons, it has a broad conical shape that tapers caudally before connecting with the spinal cord at the level of the foramen magnum. Along the midline of the anterior surface is the anterior median fissure, which continues on to the anterior surface of the spinal cord (eFig. 9.33B). Lateral to the fissure are the medullary pyramids composed of motor fibers descending from the cerebral cortex (eFig. 9.33B). Continuing caudally, the pyramids eventually give way to the decussation of the pyramids where the majority of motor fibers decussate to the opposing side. Lateral to the pyramids are the olives, which represent the underlying inferior olivary nuclei. It is at the junction of the pyramid and the olive that the rootlets of cranial nerve XII, the hypoglossal nerve, emerge (eFig. 9.33D). Oriented posterior to the olives are the inferior cerebellar peduncles, which form a connection between the cerebellum and medulla (eFig. 9.33D). At the junction of the olive and inferior cerebellar peduncles from rostral to caudal, rootlets of cranial nerves IX (glossopharyngeal), X (vagus), and XI (spinal accessory) emerge. Like the pons, the posterior surface of the medulla forms the floor of the fourth ventricle (eFig. 9.32A). Coursing down the midline of the posterior medulla is the posterior median sulcus, which continues into the spinal cord. On either side of the sulcus are the gracile and cuneate tubercles, formed by the underlying gracile nucleus and cuneate nuclei (eFig. 9.32A).
Internal structures of the brainstem can be identified by their general location in the tectum (Latin for “roof”), tegmentum (Latin for “covering”), or basis (eFig. 9.34). To identify the structures present in each level of the brainstem, it is best to view serial sections stained for myelin. This way, nuclear groups and myelinated axons can be more easily distinguished from one another.
The tectum is the most obvious landmark of the midbrain, as it consists of the prominent superior colliculi, inferior colliculi, and underlying cerebral aqueduct. Given the short length of the midbrain, stained serial sections typically include either the superior colliculi, which are located rostrally, or the inferior colliculi, which are caudal. In addition to the superior colliculi, within a rostral section of the midbrain prominent nuclei such as the oculomotor nuclei, Edinger–Westphal nuclei, red nuclei, mesencephalic nuclei of cranial nerve V, and substantia nigra can be seen (eFig. 9.35). In caudal sections of the midbrain the inferior colliculus, trochlear nucleus, mesencephalic nuclei of cranial nerve V, and substantia nigra are present (eFig. 9.36).
Sections from the rostral pons are significant for pontine nuclei and multiple transversely oriented pontocerebellar fibers en route to the contralateral cerebellum. Interspersed among these horizontally running axon are longitudinally running corticospinal fibers (eFig. 9.37). Dorsal to this collection of fiber bundles, the medial lemniscus is oriented horizontally, forming a borderlike structure between the basal and tegmental portion of the pons (eFig. 9.37). At the lateral edge of the medial lemniscus, the spinothalamic tract can be seen, because it neighbors the superior cerebellar peduncle (eFig. 9.37). Near the midline, the medial longitudinal fasciculus resides just ventral to the periaqueductal gray matter.
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At mid-pontine levels the superior cerbellar peduncles form the lateral walls of the expanding fourth ventricle (eFig. 9.38). Also at this level, the prominent middle cerebellar peduncles can also be seen, along with the motor nucleus and principal sensory nucleus of cranial nerve V, the trigeminal nerve.
In the caudal aspect of the pons, the abducent nucleus can be appreciated just lateral to the medial longitudinal fasciculus (eFig. 9.39). Also present at this level is the medial vestibular nucleus in addition to the anterior and posterior cochlear nuclei.
From rostral to caudal the internal structures of the medulla will be briefly described from three levels: (1) level of the inferior olivary nucleus, (2) decussation of the internal arcuate fibers, and (3) decussation of the pyramids. Nuclei visible in a transverse section at the level of the inferior olivary nucleus include those associated with cranial nerves VIII (vestibulocochlear), IX (glossopharyngeal), X (vagus), XI (spinal accessory), and XII (hypoglossal) (eFig. 9.40). At this level, the medial lemniscus maintains a vertical position immediately adjacent to the midline. Dorsal to the medial lemniscus are the medial longitudinal fasciculus and hypoglossal nucleus. A large distinguishing structure at this rostral level is the laterally oriented inferior cerebellar peduncle, which connects to the cerebellum posteriorly.
Continuing caudally at the level of the internal arcuate fibers, the dorsal aspect of the medulla is populated by the gracile nucleus medially, followed by the cuneate and spinal trigeminal nucleus laterally. Ventral to the cuneate and gracile nuclei, their neuronal axons can be seen decussating as internal arcuate fibers to form the medial lemniscus near the midline of the medulla. Lateral to the internal arcuate fibers and ventral to the spinal trigeminal tract, the spinocerebellar and anterolateral tracts can be seen along the perimeter of the medulla (eFig. 9.41).
Before transitioning into the spinal cord, the pyramidal decussation can be observed along the midline of the caudal medulla (eFig. 9.36). Dorsal to these decussating fibers, the gracile and cuneate nuclei begin to emerge as their fasciculi continue rostrally. Note that the spinal accessory nucleus (CN XI) is located in the cervical spinal cord and not the medulla.
Vascular supply to the brainstem, and other structures in the posterior cranial fossa, is provided by branches off of the vertebrobasilar system of arteries. As mentioned in the “Cerebral Vasculature” section, the vertebrobasilar system begins with the vertebral arteries bilaterally, which arise from the subclavian arteries and ascend through the foramen transversaria of cervical vertebrae C6 to C2 in the neck. At the pontomedullary junction, the vertebral arteries fuse to form the single basilar artery. The basilar artery continues rostrally and terminates as the paired posterior cerebral arteries at the pontomesencephalic junction.
Before merging and forming the basilar artery, each vertebral artery gives rise to a posterior inferior cerebellar artery (PICA) and posterior spinal artery and contributes to the formation of the anterior spinal artery (eFig. 9.42). The PICA perfuses the lateral aspect of the medulla and the inferior portion of the cerebellum. The medial and anterior portions of the medulla receive vascular supply from the paramedian branches off of the vertebral and anterior spinal arteries.
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At the level of the caudal pons, the anterior inferior cerebellar artery (AICA) branches off of the basilar artery and perfuses the lateral portion of the caudal pons (eFig. 9.43A). Rostral levels of the lateral pons are perfused by circumferential branches of the basilar artery (eFig. 9.43A and B). Medial portions of the pons are perfused by paramedian branches off of the basilar artery as it continues rostrally toward the midbrain.
Just before the midbrain, the superior cerebellar arteries branch off of the basilar artery and supply the superior cerebellar peduncles and caudal aspect of the dorsal midbrain before reaching the superior portion of the cerebellar hemispheres. Paramedian branches from the basilar artery supply the medial aspect of the midbrain (eFig. 9.43C). The final branches emerging from the top of the basilar artery are the PCAs, which perfuse the lateral aspect of the midbrain before reaching the thalamus, medial occipital, and inferior temporal lobes (eFig. 9.43C and D).
Part V: Spinal cord
The spinal cord is continuous with the medulla oblongata near the foramen magnum at the base of the skull. Cylindrical in shape, it occupies the vertebral canal of the vertebral column to the LI and LII vertebral level in an adult (eFig. 9.44). Numerous ascending and descending axonal tracts course through the spinal cord and connect with the brain to convey sensory (afferent) and motor (efferent) information for facilitation of movement, reflexes, sensory input, and feedback mechanisms.
Like the brain, the spinal cord is surrounded by three concentric meninges: the dura mater, arachnoid mater, and pia mater. The spinal dura mater is continuous with the inner meningeal layer of the cranial dura mater and extends inferiorly to the posterior surface of the vertebral body of S2 (eFig. 9.45). It is separated from the bony vertebral canal by the epidural/extradural space (eFig. 9.45). In addition, unlike in the cranial cavity, the underlying arachnoid mater is not tightly adherent to the dura mater, and instead has a theoretical plane or potential space called the subdural space.
Although the arachnoid mater of the spinal cord has a less adherent relationship with the dura mater than in the cranial cavity, the overall structure of the arachnoid mater is the same (eFig. 9.46A). The subarachnoid space created by the loose relationship of the arachnoid and underlying pia mater extends inferiorly to the level of the SII vertebra (eFig. 9.45). Given that the spinal cord terminates near the LI–LII vertebrae, this lower termination point of the subarachnoid space creates a safe and enlarged space for accessing CSF in the clinical setting (eFig. 9.45).
The innermost pia mater is a highly vascular layer that is adherent to the surface of the spinal cord. Midway between the anterior and posterior roots the pia mater forms a flat continuous sheet, the denticulate ligament (eFig. 9.46B). At the posterior and anterior rootlets, sleevelike projections from the denticulate ligament extend out through the arachnoid mater to attach onto the dura mater. These delicate attachments anchor and position the spinal cord within the central area of the subarachnoid space.
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The anterior and posterior surfaces of the spinal cord have several longitudinally running fissures and sulci. Along the midline on the anterior surface of the spinal cord is a deep separation, the anterior median fissure (eFig. 9.47). Posteriorly the spinal cord has a shallower separation, the posterior median sulcus, which is flanked on either side by a posterolateral sulcus (eFig. 9.47). Emerging from the spinal cord are a series of rootlets, which coalesce to form anterior and posterior roots at the corresponding cord segment (eFig. 9.48). These anterior and posterior roots converge to form 31 pairs of spinal nerves, which extend the length of the spinal cord (eFig. 9.44). Along the length of the spinal cord two regions are enlarged to accommodate the numerous neurons innervating the upper and lower extremities. The cervical enlargement extends from C5 to T1 and innervates the upper extremities, whereas the lumbar enlargement extends from L2 to S3 and innervates the lower extremities (eFig. 9.44).
A cross-section of the spinal cord reveals an inner H-shaped gray matter consisting of neuronal cell bodies and an outer white matter composed of myelinated neuronal axons. The ventral or anterior horns of gray matter contain cell bodies of motor neurons, whereas the dorsal or posterior horns contain cell bodies receiving sensory information (eFig. 9.47). An enlargement of the lateral portion of the gray matter, termed the intermediolateral cell column, can be seen in the T1 to L2 region of the spinal cord (eFig. 9.49). This region enlarges to accommodate the preganglionic cell bodies of the sympathetic nervous system. To further define the diverse cytoarchitecture of the gray matter, it is divided into 10 zones known as Rexed’s laminae (eFig. 9.50). These will be referred to as they relate to later discussions of the ascending and descending tracts within the spinal cord.
The anterior funiculus of the white matter consists of motor axons, whereas the posterior funiculus consists of axons conveying sensory information (eFig. 9.47). The lateral funiculus has a mixture of axons conveying both sensory and motor information.
Sensory information entering the CNS from peripheral sensory receptors is conducted through a series of neurons that synapse with targets in the spinal cord, cerebral cortex, and other brain structures. The sensory modalities carried in these pathways include pain, temperature, tactile, and proprioceptive input. Conscious perception of sensory input is transmitted through neuronal pathways, which reach the primary somatosensory region of the cerebral cortex. In addition to conscious sensory input, there is subconscious sensory input, which is transmitted to other structures such as the cerebellum. For simplicity, in this section we will review the sensory pathways that reach conscious perception and discuss the pathways conveying subconscious sensory input in the section on cerebellum.
Two somatosensory pathways ascend within the spinal cord to reach the cortex: (1) the anterolateral pathways, which convey sensations of pain, temperature, and crude touch; and (2) the posterior column–medial lemniscal pathway, which conveys sensations of discriminative or fine touch, vibration, and conscious proprioception (eFig. 9.51). Both of these pathways transmit information through a series of three neurons. We will review the anterolateral pathway first.
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The anterolateral pathways are composed of three tracts: the spinothalamic, spinoreticular, and spinomesencephalic tract. Separate aspects of pain are conveyed through the spinothalamic tract, so we will follow the course of those neurons first. The first-order neuronal cell body of axons forming the spinothalamic tract is located in a spinal ganglion (eFig. 9.52). Axons then enter the spinal cord through the posterior root to reach the posterior horn. From here, axons have two courses: some synapse immediately on second-order neurons in the posterior horn gray matter (lamina I and V), and others have axonal collaterals that ascend or descend one to two spinal cord segments in the posterolateral tract of Lissauer before synapsing with the second-order neurons in the gray matter (eFig. 9.51). Axons of the second-order neurons then cross obliquely over two to three spinal cord segments within the anterior commissure of the spinal cord to join the anterolateral tract on the contralateral side (eFig. 9.52). These second-order axons ascend through the CNS to reach the third-order neuronal cell bodies in the ventral posterior lateral nucleus of the thalamus (eFig. 9.52). Axons from the third-order neurons then project through the posterior limb of the internal capsule to reach the primary somatosensory cortex (eFig. 9.52).
The spinoreticular and spinomesencephalic tracts have a similar beginning as the spinothalamic tract. The principal difference is the target structure of the second-order axons. Rather than project to the thalamus as the spinothalamic tract does, the spinoreticular tract projects to the reticular formation in the brainstem to convey the emotional and arousal aspects of pain (eFig. 9.52), and the spinomesencephalic tract projects to the periaqueductal gray matter and superior colliculi in the midbrain for the central modulation of pain (eFig. 9.52).
First-order neuronal cells bodies of the posterior column–medial lemniscal pathway are located in a spinal ganglion (eFig. 9.53). Axons then enter the spinal cord through the posterior root to reach either the gracile fasciculus (gracile means “thin”), which carries information from the lower limb and trunk, or the cuneate fasciculus (cuneate means “wedge-shaped”), which carries information from the upper limb and neck. These first-order axons then ascend ipsilaterally to the caudal medulla and synapse with the second-order neuronal cell bodies within the nucleus gracilis and nucleus cuneatus (eFig. 9.53). Axons of these second-order neurons then cross over as the internal arcuate fibers to form the medial lemniscus in the contralateral medulla (eFig. 9.53). These second-order axons ascend through the brainstem to reach the third-order neuronal cell bodies in the ventral posterior lateral nucleus of the thalamus (eFig. 9.53). Axons from the third-order neurons then project through the posterior limb of the internal capsule to reach the primary somatosensory cortex (eFig. 9.53).
Descending tracts through the spinal cord are involved in voluntary movements; postural movements; and coordination of head, neck, and eye movements. These pathways originate from the cerebral cortex and brainstem and are influenced by sensory input and feedback circuitry from the cerebellum and basal ganglia. Structures that influence regulation of motor planning and voluntary control will be discussed in subsequent sections. In this section we will review the tracts of the medial and lateral motor systems. The tracts in each of these systems are composed of an upper motor neuron with cell bodies located in the cerebral cortex or brainstem and a lower motor neuron with cell bodies located in the spinal cord gray matter. We will begin by exploring the tracts of the lateral motor system first.
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Tracts of the lateral motor system include the lateral corticospinal tract and rubrospinal tract. Both are located in the lateral column of the spinal cord white matter and synapse on lower motor neuronal cell bodies in the lateral aspect of the anterior horn gray matter.
Clinically, the most important tract is the lateral corticospinal tract, because it is responsible for controlling movement of the upper and lower extremities. Cell bodies of upper motor neurons forming this tract are located in the primary motor cortex (eFig. 9.54). Axons of these upper motor neurons converge in the corona radiata and descend through the posterior limb of the internal capsule to reach the crus cerebri of the midbrain. These axons continue through the anterior aspect of the pons as small bundles to accommodate the transverse pontocerebellar fibers, which are also present in this location. Once the fibers reach the medulla, they are again grouped together and form a large swelling known as the pyramid (eFig. 9.54). At the caudal medulla, before transitioning into the spinal cord, most of the axons decussate over to the contralateral side to form the lateral corticospinal tract (eFig. 9.54). The remaining axons will stay ipsilateral and form the anterior corticospinal tract, a tract included in the medial motor systems.
After decussating and forming the lateral corticospinal tract, the axons descend through the spinal cord to synapse on the cell bodies of lower motor neurons in the lateral portion of the anterior horn gray matter. Axons of these lower motor neurons then exit the spinal cord through the anterior root (eFig. 9.54).
The other lateral motor system pathway is the rubrospinal tract (eFig. 9.55). Cell bodies of upper motor neurons in this pathway begin in the red nucleus of the midbrain. After leaving the red nucleus, the axons cross the midline as the ventral tegmental decussation and descend as the rubrospinal tract through the brainstem and lateral column of the spinal cord white matter (eFig. 9.55). These axons only descend to cervical regions of the spinal cord, and axons synapse with interneurons in the anterior horn gray matter to facilitate flexor muscle activity and inhibit extensor muscle activity of the upper limb.
Tracts of the medial motor system regulate axial or truncal muscles involved in maintaining posture, balance, automatic gait-related movements, and orientating movements of the head and neck. Unlike the lateral motor system, the tracts in this system primarily project bilaterally on interneurons within the spinal cord. This makes it difficult to test each tract individually in the clinical system. We will briefly review the four tracts of the medial motor system beginning with the anterior corticospinal tract.
The anterior corticospinal tract is formed by the remaining descending upper motor neurons that did not decussate in the caudal medulla to form the lateral corticospinal tract. These upper motor neurons, which remain ipsilateral to form the anterior corticospinal tract, descend through the medial aspect of the anterior spinal cord to the level of the upper thoracic region (eFig. 9.54). These axons project bilaterally to synapse on cell bodies of lower motor neurons in the medial portion of the anterior horn gray matter. Axons of these lower motor neurons then exit the spinal cord through the anterior root.
Tectospinal tract axons arise from cell bodies located in the superior colliculus of the dorsal midbrain (eFig. 9.56). These axons decussate in the dorsal tegmental decussation shortly after leaving the nucleus to form the tectospinal tract along the midline of the brainstem. The tectospinal tract continues through the brainstem near the medial longitudinal fasciculus and into cervical regions of the spinal cord near the anterior median fissure. Within the cervical spinal cord, axons project bilaterally to synapse on cell bodies of interneurons in the anterior horn gray matter. As the superior colliculus receives visual input, it is believed that the tectospinal tract modulates reflex postural movements in response to visual stimuli.
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Vestibulospinal tract axons arise from vestibular nuclei located in the pons and medulla. The medial vestibular nucleus gives rise to the medial vestibulospinal tract, which projects bilaterally to thoracic regions of the spinal cord, and the lateral vestibular nucleus gives rise to the lateral vestibulospinal tract, which descends ipsilaterally through the entire length of the spinal cord to synapse on interneurons in the anterior horn gray matter (eFig. 9.57). Given that the vestibular nuclei receive sensory input from the inner ear and cerebellum, this tract facilitates activity of extensor/antigravity muscles and inhibits activity of flexor muscles to maintain balance and an upright posture. As an example, the change in head position induced during tripping initiates extension of the upper limb and/or lower limb to prevent oneself from falling forward.
Reticulospinal tract axons arise from the reticular formation in the pons and medulla. The axons of the pontine and medullary reticulospinal tracts descend ipsilaterally through the length of the spinal cord in the anterior white matter and synapse with interneurons in the anterior horn gray matter. They are believed to function in regulating voluntary movements in reflex activity and autonomic outflow (eTables 9.5 and 9.6).
Vascular perfusions to the spinal cord are supplied by three longitudinally running vessels and several segmental branches. The longitudinally running vessels are the anterior spinal artery and two posterior spinal arteries. The posterior spinal arteries originate in the cranial cavity as branches of either the vertebral artery or PICA. These arteries descend along the length of the posterior spinal cord on the posterolateral sulcus. The single anterior spinal artery originates within the cranial cavity from the union of two contributing branches from the vertebral arteries. The anterior spinal artery descends along the length of the anterior spinal cord on the anterior median fissure.
Reinforcing vascular supply to these longitudinally running vessels is provided by eight to ten segmental medullary arteries. The largest segmental medullary artery is the artery of Adamkiewicz in the lower thoracic or upper lumbar region. This vessel is typically on the left side and contributes significantly to perfusion of the lower portion of the spinal cord.
Venous drainage of the spinal cord occurs through a series of longitudinally running channels that connect with the anterior and posterior spinal veins on the surface of the cord.
Part VI: Basal nuclei
The basal nuclei are a collection of gray-matter structures named for their location deep within the base of the forebrain. Functionally, the basal nuclei have a significant role in controlling posture and voluntary movement through connections to the thalamus, cortex, and neighboring basal nuclei structures. In addition to their role in posture and movement, the basal nuclei have connections to limbic system pathways, which govern the expression of various behaviors and motivational states. For the purposes of this section, we will focus on reviewing the structures of the basal nuclei and pathways involved in controlling posture and voluntary movement.
A variety of terminology is used to refer to the structures of the basal nuclei individually and based on their collective morphology. To appreciate the three-dimensional shape of the basal nuclei and their relationship to surrounding structures, it is best to view them in horizontal and coronal brain sections taken at different levels of the brain.
The corpus striatum (Latin for “striped body”) includes the caudate nucleus and lentiform nucleus. This collection of structures received their name because of the striated appearance of bands that interconnect the caudate nucleus and putamen of the lentiform nucleus through the anterior limb of the internal capsule (eFig. 9.58).
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The lentiform nucleus (Latin for “lens-shaped”) includes the globus pallidus and putamen, which appear lens-shaped when viewed laterally. Both of these structures are lateral to the internal capsule, which separates them from the thalamus and caudate nucleus medially (eFig. 9.58). Laterally, the putamen is bordered by the external capsule, a thin layer of white matter adjacent to a thin gray-matter layer called the claustrum. Beyond the claustrum is the external capsule, which borders the white matter of the insula (eFig. 9.58).
Medial to the internal capsule is the caudate nucleus. The caudate nucleus is a large C-shaped structure divided into a head, body, and tail, which closely follows the shape of the lateral ventricle (eFig. 9.58). Rostrally, the head of the caudate has a large rounded shape that contributes to the lateral wall of the anterior horn of the lateral ventricle (eFig. 9.58). Also at this level, the head of the caudate is continuous with the putamen (eFig. 9.59). Because of this close relationship the putamen and caudate are referred to collectively as the striatum. At the level of the interventricular foramen, the head of the caudate transitions to the body. The body of the caudate is long and narrows substantially as it transitions from the head to the tail (eFig. 9.59). Along its course the body contributes to the floor of the lateral ventricle. Near the posterior border of the thalamus the body of the caudate transitions into the tail. The tail continues anteriorly within the roof of the inferior horn of the lateral ventricle to terminate in the amygdaloid nucleus (eFig. 9.59).
Input to the basal nuclei is primarily received by the striatum (caudate nucleus and putamen), and output predominantly leaves from the globus pallidus. Many structures send input to the striatum, including all areas of the cerebral cortex, thalamic nuclei, subthalamic nucleus, brainstem, and substantia nigra. To understand how the basal nuclei integrates all of this incoming information to influence motor activity, two simplified neuronal loops are described: the direct pathway and the indirect pathway.
The direct pathway has a series of connections through the basal nuclei, which result in an overall increase in motor activity. This pathway begins with input to the striatum, which sends axonal connections to the globus pallidus (eFig. 9.60A). The globus pallidus then has output connections to the thalamus, which completes the circuit with axonal connections back to the cortex. The indirect pathway has a similar course, with the addition of output connections to the subthalamic nucleus, which results in an overall decrease in motor activity (eFig. 9.60B).
Part VII: Cerebellum
The cerebellum is the largest structure of the hindbrain. It resides within the posterior cranial fossa and is composed of two large hemispheres, which are connected by the vermis in the midline (eFig. 9.61). Functionally, the cerebellum plays a role in maintaining balance and influencing posture and is responsible for coordinating movements by synchronizing contraction and relaxation of voluntary muscles. We will first examine the structural organization of the cerebellum and then review how these structures contribute to the circuitry of the cerebellum.
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Within the posterior cranial fossa, the cerebellum is covered by the tentorium cerebelli of the dura mater (eFig. 9.17) and connects to the posterior surface of the brainstem via the superior, middle, and inferior cerebellar peduncles (eFig. 9.62). Anteriorly, the cerebellum forms the roof of the fourth ventricle (eFig. 9.14). On its surface, the cerebellum has several convoluted folds, or folia, separated by fissures. Two of these fissures serve as landmarks to divide the cerebellum into three lobes. Superiorly, the primary fissure separates the anterior lobe from the posterior lobe (eFig. 9.61). Anteriorly and inferiorly, the posterolateral fissure defines the structures of the flocculonodular lobe, which includes the flocculus from each hemisphere and nodule of the vermis (eFig. 9.63). A third fissure, the horizontal fissure, borders the superior and inferior surfaces of the cerebellum (eFig. 9.64).
Each fold or folia of the cerebellar cortex has a central core of white matter covered by a thin layer of gray matter superficially. In sections parallel to the median plane, the branching pattern of the folia can be appreciated; this is often referred to as the arbor vitae (eFig. 9.28). Deep within the white matter of each hemisphere are four masses of cerebellar nuclei. From lateral to medial they are the dentate, emboliform, globose, and fastigial (eFig. 9.65). Note that the emboliform and globose are collectively referred to as the interposed nuclei. Output from the cerebellum originates from one of these four nuclear complexes before leaving through the superior cerebellar peduncle, predominantly. In general, the output from each cerebellar hemisphere coordinates movement on the ipsilateral side of the body.
Functionally, the cerebellar cortex can be divided into three areas. The vermis in the midline influences movements along the axis of the body, including the neck, trunk, abdomen, and pelvis (eFig. 9.66). Adjacent to the vermis, the intermediate zone controls muscles of the distal upper and lower limbs. The lateral zone participates in motor planning to coordinate sequential movements of the entire body. Input to these functional areas of cerebellar cortex come from the cerebral cortex, spinal cord, and brainstem by passing predominantly through the middle and inferior cerebellar peduncles. Fibers entering the cerebellum proceed as mossy fibers (from various regions) or climbing fibers (from olivary nucleus). Mossy fibers form excitatory synapses with dendrites of the granule cells, in the granule cell layer (eFig. 9.67). From here, the granule cells send axons to the molecular layer, where they bifurcate into parallel fibers that run longitudinally in the folia. The parallel fibers then synapse on Purkinje cells in the outermost Purkinje cell layer (eFig. 9.67). Climbing fibers project to the Purkinje cell layer and form powerful excitatory connections with the Purkinje cells. The Purkinje cells then project to the deep cerebellar nuclei (eFig. 9.67).
As mentioned earlier, the cerebellum receives input from the cerebral cortex, brainstem, and spinal cord. Input from the cerebral cortex to the cerebellum is primarily involved in voluntary muscle control and coordination of movement. Axonal projections from the cerebral cortex destined for the cerebellum descend through the internal capsule and terminate on pontine nuclei (eFig. 9.68). The axons from the pontine nuclei then cross over as transverse fibers to enter the contralateral cerebellum through the middle cerebellar peduncle.
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Input from the spinal cord to the cerebellum conveys information from muscles and joints to influence muscle tone and posture. The primary spinal cord pathways with connections to the cerebellum include the anterior or ventral spinocerebellar and the posterior or dorsal spinocerebellar tracts. These tracts originate from joint and cutaneous mechanoreceptors and ascend through the spinal cord to enter the ipsilateral cerebellum primarily through the inferior cerebellar peduncle (eFig. 9.69).
A final source of cerebellar input arises from the vestibular nuclei and reticular formation in the brainstem. The connections are primarily involved in reflexive maintenance of balance. These nuclei send axonal projections to the ipsilateral cerebellum through the inferior cerebellar peduncle.
Output from the cerebellum originates from one of the four deep cerebellar nuclei. The largest collection of fibers leaving the cerebellum originates from the dentate nucleus. Axons from this nuclear complex project to the contralateral ventral nucleus of the thalamus after decussating in the superior cerebellar peduncle. From here, axons of the thalamic nuclei project to the motor cortex (eFig. 9.70). This pathway influences posture and movement.
The emboliform and globose nuclei, or interposed nuclei, have a similar course as the axons from the dentate, but with the addition of another synaptic target. Axons from the interposed nuclei decussate in the superior cerebellar peduncle to synapse on the contralateral ventral nucleus of the thalamus and the contralateral red nucleus in the midbrain (eFig. 9.70). Axons leaving the red nucleus descend to the inferior olivary nucleus in the medulla. The axonal projections from the interposed nuclei function in monitoring and correcting motor activity of the upper and lower extremities.
Axons from the fastigial nucleus project to the vestibular nuclei, reticular formation, contralateral ventral nucleus of the thalamus, and contralateral tectum. Vestibular axons pass through the inferior cerebellar peduncle to reach the ipsilateral vestibular nucleus and uncinate fasciculus to reach the contralateral vestibular nucleus (eFig. 9.70). Also going through the inferior cerebellar peduncle are axons heading to the reticular formation. Ascending in the superior cerebellar peduncles are axons that will synapse with the contralateral tectum and contralateral ventral nucleus of the thalamus.
The cerebellum is perfused by the vertebrobasilar system of arteries (eFig. 9.42). Before merging and forming the basilar artery, each vertebral artery gives rise to a PICA and posterior spinal artery and contributes to the formation of the anterior spinal artery (eFig. 9.71). The PICA perfuses the inferior portion of the cerebellum. At the level of the caudal pons, the AICA branches off the basilar artery and perfuses the anterior and lateral portion of the cerebellum, as well as the middle and inferior cerebellar peduncles (eFig. 9.71). Just before the midbrain, the superior cerebellar arteries branch off of the basilar artery and supply the superior cerebellar peduncles and superior portion of the cerebellar hemispheres (eFig. 9.71).
Part VIII: Visual System
The visual system is a complex special sensory system that begins in the eyeball and has neuronal connections to the thalamus, brainstem, primary visual cortex, and association cortices. In addition to mediating visual perception, these connections are also involved in higher visual functions, such as determining spatial relationships between objects and structural features of objects. In this section we will explore the primary or geniculate visual pathway from the retina to the primary visual cortex.
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Although the retinal layer of the eyeball is often considered the beginning of visual perception, the anterior structures of the eye play an important role in visual perception too. The anteriormost structure is the cornea and is the first layer through which light, the visual stimulus, enters the eye (eFig. 9.72). This transparent layer overlies the aqueous humor of the anterior chamber and the pupil, a small central aperture that controls how much light is admitted into the eye. After passing through the pupil light is refracted through the lens, which along with the ciliary body, separates the anterior portion of the eye from the posterior portion (eFig. 9.72). The lens is a clear biconvex structure that rounds in shape as the ciliary muscle contracts and relaxes the suspensory ligaments attached to the borders of the lens, a parasympathetically controlled process referred to as accommodation.
After the lens, light passes through the vitreous humor of the posterior chamber and is projected onto the layers of the retina (eFig. 9.73). The retina is composed of a non-neural layer and several layers of neural cells with synaptic connections. To reduce the amount of light reflected in the eye, the choroid layer lining the inner surface of the sclera (eFig. 9.72), along with the pigment epithelium layer (non-neural) of the retina, absorb and refract some of the light stimulus (eFig. 9.73). Interdigitating between the pigment epithelial cells, the photoreceptors transduce the light stimulus into an electrical signal in a process called phototransduction. Images formed on the retina are inverted in both vertical and lateral dimensions (eFig. 9.74). Because of this, the visual field is defined as having four quadrants: left/right and upper/lower.
Photoreceptors include rods, which are very sensitive to light and essential for vision in dimly lit conditions, and cones, which are responsible for color vision and high visual acuity. Although rods and cones are both distributed across the retina, rods outnumber cones by twentyfold and are concentrated in the periphery of the retina. Cones predominate near the macula and are the only photoreceptors present at the fovea (eFig. 9.72). The fovea represents the primary visual axis of the eye and is the location of maximal visual acuity. Despite their functional differences, both the rods and cones have an outer light-sensing segment, an inner segment, and a synaptic terminal. These synaptic terminals contact bipolar cells, the first-order neurons in the visual pathway (eFig. 9.73). Bipolar cells then synapse with ganglion cells, the second-order neurons in the visual pathway. Two other cell types present in the retina are horizontal and amacrine cells. These transversely oriented interneurons moderate the excitation level of bipolar and ganglion cells.
Axons from the ganglion cells coalesce at the optic disc, which is absent of photoreceptors and thus creates a blind spot in the visual field. As they leave the optic disc they form the optic nerve, acquire a myelin sheath provided by oligodendrocytes, and are invested by the cranial meninges (eFig. 9.72). These morphological features derived during embryonic development, define the optic nerve as a component of the CNS. Anterior to the infundibular stalk, the optic nerves converge at the optic chiasm. Within the chiasm, axons from the nasal portion of the retina decussate and enter into the contralateral optic tract (eFig. 9.75). Conversely, axons from the temporal portion of the retina stay ipsilateral to enter the ipsilateral optic tract (eFig. 9.75).
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Gray's Anatomy
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Continuing posteriorly, the optic tracts course around the midbrain to enter the lateral geniculate nucleus of the thalamus (eFig. 9.75). At this level, a small portion of the fibers from the optic tract travel to the pretectal area and superior colliculus to mediate the pupillary light reflex. Axons leaving the lateral geniculate nucleus form the optic radiations, which continue on to the primary visual cortex in the occipital lobe (eFig. 9.76). Fibers traveling in the lower portion of the optic radiations terminate on the lower half of the primary visual cortex, whereas fibers in the upper portion terminate on the upper half of the cortex (eFig. 9.76). A full review of how the visual field is represented throughout the visual pathway can be reviewed in eFig. 9.77.
As mentioned previously, images formed on the retina are inverted in both vertical and lateral dimensions. In addition, the fibers from the nasal portion of the hemiretina decussate at the optic chiasm. Because of this, the optic tracts, thalamus, optic radiations, and primary visual cortex receive information relating only to the contralateral half of the visual field. Knowledge of how the visual field is represented throughout the visual pathway is essential for identifying the location of lesions in patients with visual field deficits. Examples of lesions in the five major structures of the visual pathway (optic nerve, optic chiasm, optic tract, optic radiations, primary visual cortex) and their associated visual field deficits are represented in eFig. 9.78.
Part IX: Auditory and
Cranial nerve VIII, the vestibulocochlear nerve, conveys sensory information from the vestibular and auditory organs of the inner ear to the pontomedullary junction of the brainstem. Although these sensory modalities are conveyed to the brainstem by a common nerve bundle, each of these sensory functions has different central pathways. In this section we will first review the central auditory pathways and then the vestibular pathways.
Sound waves, the auditory stimulus, are directed toward the external acoustic meatus by the pinna of the outer ear and through the ear canal to the tympanic membrane (eFig. 9.79). Vibration of the tympanic membrane, induced by the incoming sound waves, is transmitted to the three ossicle bones (malleus, incus, and stapes) of the middle ear. These bones amplify sound waves so they can be converted to pressure waves at the oval window of the fluid-filled cochlea. To prevent damage from loud or high-decibel sounds, movements of the malleus and stapes are reduced by the tensor tympani and stapedius muscles.
In the cochlea, sound waves are converted into an electrical signal at the organ of Corti. The organ of Corti rests upon the basilar membrane in the scala media, which is filled with endolymph (eFig. 9.80). In addition to contributing to the formation of the scala media with the vestibular membrane, the basilar membrane separates the cochlea into the scala vestibuli and scala tympani, which are filled with perilymph and are continuous with one another at the helicotrema (eFig. 9.80).
As the sound waves move through the perilymph they displace the basilar membrane, which causes deflection of the hair cells in the organ of Corti (eFig. 9.81A). These sensory receptors synapse with sensory neurons, which have cell bodies in spiral ganglion located within the modiolus of the cochlea (eFig. 9.80). Axons leaving the modiolus form the cochlear nerve near the base of the cochlea. The cochlear nerve then passes through the internal acoustic meatus and subarachnoid space to enter the pontomedullary junction at the cerebellopontine angle.
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The first-order axons entering the brainstem from the cochlea terminate ipsilaterally on the dorsal and ventral cochlear nuclei. From here, second-order axons have several synaptic targets. Second-order axons forming the ascending auditory pathway ascend to the pons and project bilaterally on the superior olivary nucleus (eFig. 9.82). These bilateral projections are important for auditory acuity and localizing the origin of a sound. From here, fibers continue to ascend as the lateral lemniscus and terminate in the inferior colliculus of the midbrain. Axons leaving the inferior colliculus then have a synaptic relay in the medial geniculate nucleus of the thalamus before reaching the final synaptic target: the primary auditory cortex (superior temporal gyrus) of the temporal lobe (eFig. 9.82). Throughout this pathway, from the basilar membrane to the primary auditory cortex, auditory stimuli are tonotopically represented. This is analogous to the somatotopic map previously discussed in the section on somatosensory systems pathways.
In addition to the ascending auditory pathway, the superior olivary nucleus receives descending input from the primary auditory cortex as a form of feedback (eFig. 9.82). The superior olivary nucleus then sends descending olivocochlear fibers to the organ of Corti, which has an inhibitory function on the hair cells to prevent damage from harmfully loud sounds. It is also believed that the superior olivary nucleus has connections with the motor nuclei of the trigeminal and facial nerve to mediate reflexive contraction of the tensor tympani and stapedius muscles in response to loud sounds.
The vestibular nerve transmits afferent or sensory information regarding movement and position of the head from the vestibular organs, which include semicircular ducts, utricle, and saccule. Like the cochlear apparatus, each of these vestibular organs is located within a membranous (ductal) portion of the vestibular apparatus, which is surrounded by a bony (canal) portion (eFig. 9.83). The cell bodies for these sensory receptors are located in the vestibular (Scarpa’s) ganglion within the internal acoustic meatus (eFig. 9.83). The vestibular nerve then passes through the internal acoustic meatus and subarachnoid space to enter the pontomedullary junction at the cerebellopontine angle.
The central processes of the vestibular axons predominantly terminate in the four vestibular nuclei (superior, inferior, medial, and lateral), which are located in the rostral medulla and caudal pons (eFig. 9.40). Axons leaving the vestibular nuclei have several ascending and descending synaptic targets. These pathways connect with visual motor, descending motor, and cerebellar pathways to coordinate movement and maintain posture and balance (eFig. 9.84).
Axons ascending after leaving the vestibular nuclei form the medial longitudinal fasciculus to reach the oculomotor, trochlear, and abducent nuclei (eFig. 9.85). These connections coordinate movement of the head and eyes so that visual fixation on an object can be maintained. Other axons ascending from the vestibular nuclei project to the cerebral cortex after a synaptic relay in the ventral posterior nucleus of the thalamus (eFig. 9.84B). This connection assists in consciously orienting oneself in space. In addition to these connections, axons leaving the vestibular nuclei pass through the inferior cerebellar peduncle to reach the cerebellum and modulate equilibrium (eFig. 9.84B).
Axons descending after leaving the vestibular nuclei form the medial and lateral vestibulospinal tracts ipsilaterally (eFig. 9.84B). As mentioned in the previous section on medial motor systems in the spinal cord, the axons in these pathways synapse on interneurons within the anterior horn gray matter to influence spinal motor neuron activity and maintain posture and balance.
Part X: Hypothalamus
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The hypothalamus is a neuroendocrine organ that regulates physiological processes for survival such as consumption of fluid and food, temperature control, the sleep–wake cycle, growth, and reproduction. As a result of this vast array of functions, the nuclei of the hypothalamus make connections with several other neural and endocrine structures in the body (eFig. 9.86). In this section we will explore the nuclei of the hypothalamus and their main connections.
Located in the ventralmost aspect of the diencephalon, the hypothalamus is bounded by the lamina terminalis anteriorly, the hypothalamic sulcus superiorly, and the tegmentum of the midbrain posteriorly (eFig. 9.87). Laterally the hypothalamus is bordered by the substantia innominata rostrally and the posterior limb of the internal capsule caudally. It forms the floor in addition to the lower portion of the lateral walls in the third ventricle. When viewing the external surface of the ventral brain, the area containing the hypothalamus is circumscribed by the optic chiasm, optic tract, crus cerebri, and caudal edge of the mammillary bodies (eFig. 9.88).
Continuous with the hypothalamus inferiorly is the pituitary gland. These two structures are connected by the infundibulum and hypophysial stalk just caudal to the optic chiasm (eFig. 9.87). The intimate relationship of the hypothalamus to the portal circulation of the pituitary gland allows the hypothalamus to be an efficient regulator of hormone synthesis and release. Releasing and inhibiting factors synthesized by the hypothalamus pass through these portal vessels via the tuberoinfundibular tract (eFig. 9.89) to reach the anterior pituitary (adenohypophysis) and control release of hormones produced by the anterior pituitary such as adrenocorticotropic hormone, luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, growth hormone, and prolactin (eFig. 9.90).
A second connection exists between the hypothalamus and pituitary through nerve fibers, which originate in the supraoptic region and paraventricular nuclei of the medial hypothalamus and extend through the hypothalamohypophyseal tract to the posterior lobe of the pituitary for release into the circulatory system (eFig. 9.89).
Internally, the hypothalamus is composed of many small nuclei, which are divided by a parasagittal plane into a medial and lateral zone. Landmarks for this dividing point are the columns of the fornix and the mammillothalamic tract as they reside within this sagittal plane (eFig. 9.91). The hypothalamus is also divided by coronal planes into a preoptic area and periventricular zone. Note that the periventricular zone is not to be confused with the paraventricular nucleus, which is a thin region of cell bodies lying medial to the medial zone.
Within the lateral zone of the hypothalamus is a large bundle of axons forming the medial forebrain bundle (MFB) (eFig. 9.90). Theses fibers interconnect the hypothalamus with the septal nuclei rostrally and nuclear complexes within the brainstem. Axons arising from the large lateral nucleus in this zone enter the MFB and function in promoting feeding behavior. The other and smaller nuclear group in this zone is the tuberal group. Axons from the tuberal nuclei make connections with the anterior pituitary through the tuberoinfundibular tract to regulate release of hormones in the hypophysial portal system and to the cerebellum to regulate motor activity (eFig. 9.89).
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The medial zone is divided into three regions: supraoptic, tuberal, and mammillary. There are four nuclei in the supraoptic region, which play a role in thermoregulation, osmoregulation, and the sleep–wake cycle. The supraoptic and paraventricular nuclei in this region synthesize antidiuretic hormone or vasopressin, which stimulates water uptake and oxytocin for stimulation of uterine contractions and lactation in the mammary glands. Axons from these nuclei are conveyed by the hypothalamohypophyseal tract to the posterior pituitary for release into the circulatory system (eFig. 9.89). A third nuclear group in this region is the suprachiasmatic nucleus. This nucleus receives input directly from the retina to influence circadian rhythms, which contribute to the light–dark cycle. The anterior nucleus is the final group and functions predominantly in regulating body temperature.
The tuberal region in the medial zone contains three nuclei: ventromedial, dorsomedial, and arcuate. The largest and best defined is the ventromedial nucleus, which functions as a satiety center to decrease feeding behavior. Posterior to the ventromedial nucleus is the dorsomedial nucleus, which functions in the behavioral expression of rage or aggressive behavior. Finally, the arcuate nucleus serves as a center for releasing hormones, which are transmitted by the tuberoinfundibular tract and hypophysial portal system to the anterior pituitary (eFig. 9.89).
The mammillary region or mammillary body is the final group of nuclei in the medial zone. Four nuclei comprise this region: medial, intermediate, lateral mammillary, and posterior hypothalamic. The best defined is the medial mammillary nucleus, as it is the primary site for the termination of axons from the postcommissural fornix. This pathway originates from the subiculum of the hippocampal complex and plays a key role in memory. The medial mammillary nuclei also connects to structures of the limbic system.
The periventricular zone resides medial to the medial zone and adjacent to the ependymal cells of the third ventricle. Neurons from this zone predominantly synthesize releasing hormones. Axons from these neuronal cells project through the tuberoinfundibular tract to the hypophysial portal system to influence release of hormones from the anterior pituitary.
Through the review of the hypothalamus thus far, it is apparent that this small, 4-gram structure has a significant role in regulating visceral, endocrine, and behavioral system functions through multiple pathways. It is important to remember the majority of the pathways mentioned represent input–output relationships between the hypothalamus and other structures. For a review of neural and non-neural inputs and outputs, refer to the summary figures (eFig. 9.92A and B).
Part XI: Olfactory and
The sense of olfaction has a role in both pleasurable experiences and survival. The same receptors that allow us to enjoy the food we consume or experience odorants in the environment also help us avoid spoiled food or potentially hazardous situations like a fire. Unlike the other special sensory system pathways, the olfactory sensory pathway is unique in that it does not have a thalamic relay before reaching the primary olfactory cortex. In this section we will review the course of the neurons in the olfactory system and their connection to the limbic system.
Three types of olfactory receptors make up the olfactory epithelium along the lateral and septal walls of the nasal cavity. These cells allow for regeneration (basal stem cells), support (sustentacular cells), and transmission of information (olfactory receptor neurons). Each olfactory receptor neuron has an olfactory vesicle with cilia that contain receptors for odorant molecules and an unmyelinated axon that passes through the cribriform plate to terminate in the olfactory bulb (eFig. 9.93). As the olfactory receptor neurons originate embryologically from the CNS, they are considered part of the CNS and not the PNS.
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After synapsing with the mitral cells in the glomeruli of the olfactory bulb, mitral cell axons converge to form the olfactory tract. The olfactory tract then divides into medial and lateral olfactory striae to reach different synaptic targets (eFig. 9.94). Some of the axons in the medial olfactory striae travel through the diagonal band to reach the septal area, whereas others cross the midline in the anterior commissure and inhibit mitral cell activity in the contralateral olfactory bulb to enhance localization of the olfactory stimulant. Axons in the lateral olfactory stria primarily terminate in the piriform cortex/primary olfactory cortex of the uncus and in the amygdala (eFig. 9.94).
The medial forebrain bundle, traveling through the lateral hypothalamus, connects the olfactory cortex with both the hypothalamus and the brainstem to regulate autonomic responses such as arousal through the reticular formation, salivation, and gastric contraction.
The limbic system is composed of several cortical and subcortical structures that participate in an intricate network of connections to regulate complicated behaviors such as memory, emotions, homeostatic functions, and motivational state. In this section we will review the major structures and pathways that form the limbic system.
Grossly, the limbic lobe includes a ring-shaped area of cortical structures that border the brainstem. These cortical areas include the cingulate gyrus, parahippocampal gyrus, and subcallosal area (eFig. 9.12). Laterally, the insular cortex also participates in limbic system function (eFig. 9.10). Nuclear structures of the limbic system include the amygdala, hippocampal formation, anterior and mediodorsal thalamic nuclei, septal nuclei in the forebrain, and nucleus accumbens (eFig. 9.95).
The amygdaloid nucleus is an almond-shaped structure located anterior to the inferior horn of the lateral ventricle and tail of the caudate within the temporal lobe (eFig. 9.96). Structurally, the amygdala consists of three nuclear regions: a large basolateral group and a smaller corticomedial group, which includes the central nucleus. Functionally, the amygdala is primarily associated with the emotion of fear, but it also has an important role in autonomic and neuroendocrine pathways. Connections of the amygdala are predominantly bidirectional and follow three different pathways: the uncinate fasciculus, stria terminalis, and ventral amygdalofugal pathway (eFig. 9.97). Connections to cortical areas pass through the uncinate fasciculus, which progresses anterior to the amygdala. Projections to the septal area and hypothalamus follow the stria terminalis (eFig. 9.98A–D). Fibers forming the ventral amygdalofugal pathway project to thalamic nuclei and several brainstem and forebrain structures (eFig. 9.98A–D).
The nucleus accumbens resides with the ventral forebrain adjacent to where the putamen and head of the caudate become continuous with one another (eFig. 9.99). Afferent axons to the nucleus accumbens come from the amygdala through the amygdalofugal pathway, hippocampal formation by way of the fornix, basal forebrain area from the stria terminalis, and ventral tegmentum through the medial forebrain bundle (eFig. 9.98A–D). Efferent axons leaving the nucleus accumbens project directly to the hypothalamus and globus pallidus and reach nuclei in the brainstem through the medial forebrain bundle. Its connections to the globus pallidus represent an important connection of the limbic system to the motor system. The overall function of the nucleus accumbens is recognized as a gratification center and has been shown to play a role in behaviors related to addiction.
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The septal region is located rostral to the anterior commissure along the medial aspect of the cerebral hemispheres (eFig. 9.99). This region appears to play a role in pleasurable behaviors. Conversely, lesion studies indicate that damage to this area evokes behaviors of extreme displeasure or rage. Afferent axons to the septal area arise from the amygdala, hippocampus, olfactory tract, and monoaminergic nuclei in the brainstem (eFigs. 9.100 and 9.101). The septal area also connects to a collection of cholinergic neurons along the wall and roof of the third ventricle known as the habenular nuclei. Axons from the habenular nuclei project to the interpeduncular nucleus of the reticular formation, which is believed to play a role in the sleep–wake cycle (eFig. 9.100).
The hippocampal formation is located in the medial ventral temporal lobe (eFig. 9.102). It consists of the hippocampus, dentate gyrus, and subiculum (eFig. 9.103A and B). The hippocampal formation plays a role in memory processes such as episodic memory, short-term memory, working memory, and consolidation of memories. Input to the hippocampal formation is primarily received by the entorhinal cortex from association cortices. Because of this, it is believed that the “storage” of memories is in the association and primary cortices, not in the medial temporal lobe. Neurons from the entorhinal cortex project to the hippocampal formation by two pathways: the perforant pathway and alvear pathway. The perforant courses directly through the hippocampal sulcus to reach the dentate gyrus (eFig. 9.104B). As the hippocampus resembles the appearance of a ram’s horn, early on it was named cornu ammonis (horn of Ammon). Based on its cytoarchitecture, it was subdivided into four regions termed the cornu ammonis 1 to 4 (eFig. 9.104A). From the dentate gyrus, axons project to CA3 of the hippocampus. Axons from the hippocampus leave via the fornix or as Shaffer collaterals to reach CA1. Axons from CA1 may enter the fornix or project to the subiculum. Finally, axons from the subiculum enter the fornix or go back to the entorhinal cortex.
A second afferent pathway from the entorhinal cortex to the hippocampal formation is through the alvear pathway. Axons in the alvear pathway project directly on to CA1 and CA3 of the hippocampus (eFig. 9.104B). Similar to the perforant pathway, axons leaving the alvear pathway primarily originate from CA1 and CA3, which then project to the subiculum.
Efferent axons leaving the hippocampal formation primarily exit from the subiculum and form the fornix (Latin for “arch”), a white matter structure that arches over the ventricular system (eFig. 9.95). The fornix begins with axons exiting the hippocampus to form the alveus along the ventricular surface of the hippocampus. As the axons come together medially, they form a bundle referred to as the fimbria of the fornix. The fornix then emerges from the hippocampal formation and curves under the corpus callosum before coursing medially to run adjacent to the midline (eFig. 9.105). At the anterior commissure, the fornix divides into a precommissural fornix and postcommissural fornix to reach the nucleus accumbens, septal nuclei, medial frontal cortex, mammillary nucleus, ventromedial nucleus of the hypothalamus, and anterior nucleus of the dorsal thalamus (eFig. 9.95).
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Through this section we have described a collection of anatomical structures and defined their connections with other areas of the brain and brainstem without exploring how these individual structures are interconnected with one another. In the 1930s James Papez, an American neurologist, described a circuit that links these structures and cortical areas together in a way that was thought to be involved in the experience and expression of emotion. This is referred to as the Papez circuit (eFig. 9.106). The circuit begins with fibers from the subiculum, which then enter the fornix to reach the mammillary nuclei. These axons then project through the mammillothalamic tract to the anterior nucleus of the thalamus. Next, the axons from the anterior nucleus of the thalamus project through the internal capsule to the cingulate gyrus. Lastly, the cingulum fibers from the cingulate cortex project to the parahippocampal gyrus and then the entorhinal cortex and hippocampal formation (eFig. 9.106). Papez’s description of this circuit is useful for reviewing the major limbic system pathways; however, the role of some of the structures in the pathway has been shown to play little or no role in the expression of emotion. In addition, many of the structures that do play a role in the expression of emotion also have a role in other functions.
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For additional ancillary materials related to this chapter, please visit thePoint.
I. OVERVIEW
Proteins are the most abundant and functionally diverse molecules in living systems. Virtually every life process depends on this class of macromolecules. For example, enzymes and polypeptide hormones direct and regulate metabolism in the body, whereas contractile proteins in muscle permit movement. In bone, the protein collagen forms a framework for the deposition of calcium phosphate crystals, acting like the steel cables in reinforced concrete. In the bloodstream, proteins, such as hemoglobin and albumin, transport molecules essential to life, whereas immunoglobulins fight infectious bacteria and viruses. In short, proteins display an incredible diversity of functions, yet all share the common structural feature of being linear polymers of amino acids. This chapter describes the properties of amino acids. Chapter 2 explores how these simple building blocks are joined to form proteins that have unique three-dimensional structures, making them capable of performing specific biologic functions.
II. STRUCTURE
Although >300 different amino acids have been described in nature, only 20 are commonly found as constituents of mammalian proteins. [Note: These standard amino acids are the only amino acids that are encoded by DNA, the genetic material in the cell (see p. 411). Nonstandard amino acids are produced by chemical modification of standard amino acids (see p. 45).] Each amino acid has a carboxyl group, a primary amino group (except for proline, which has a secondary amino group), and a distinctive side chain (R group) bonded to the α carbon atom. At physiologic pH (~7.4), the carboxyl group is dissociated, forming the negatively charged carboxylate ion (−COO−), and the amino group is protonated (−NH3+) (Fig. 1.1A). In proteins, almost all of these carboxyl and amino groups are combined through peptide linkage and, in general, are not available for chemical reaction except for hydrogen bond formation (Fig. 1.1B). Thus, it is the nature of the side chains that ultimately dictates the role an amino acid plays in a protein. Therefore, it is useful to classify the amino acids according to the properties of their side chains, that is, whether they are nonpolar (have an even distribution of electrons) or polar (have an uneven distribution of electrons, such as acids and bases) as shown in Figures 1.2 and 1.3.
and polarity of their side chains at acidic pH (continued from Fig. 1.2). [Note: At physiologic pH (7.35 to 7.45), the α-carboxyl groups, the acidic side chains, and the side chain of free histidine are deprotonated.]
A. Amino acids with nonpolar side chains
Each of these amino acids has a nonpolar side chain that does not gain or lose protons or participate in hydrogen or ionic bonds (see Fig. 1.2). The side chains of these amino acids can be thought of as “oily” or lipid-like, a property that promotes hydrophobic interactions (see Fig. 2.10, p. 19).
1. Location in proteins: In proteins found in aqueous solutions (a polar environment), the side chains of the nonpolar amino acids tend to cluster together in the interior of the protein (Fig. 1.4). This phenomenon, known as the hydrophobic effect, is the result of the hydrophobicity of the nonpolar R groups, which act much like droplets of oil that coalesce in an aqueous environment. By filling up the interior of the folded protein, these nonpolar R groups help give the protein its three-dimensional shape. However, for proteins that are located in a hydrophobic environment, such as a membrane, the nonpolar R groups are found on the outside surface of the protein, interacting with the lipid environment (see Fig. 1.4). The importance of these hydrophobic interactions in stabilizing protein structure is discussed on p. 19.
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Sickle cell anemia, a disease of red blood cells that causes them to become sickle shaped rather than disc shaped, results from the replacement of polar glutamate with nonpolar valine at the sixth position in the β subunit of hemoglobin A (see p. 36).
2. Proline: Proline differs from other amino acids in that its side chain and α-amino nitrogen form a rigid, five-membered ring structure (Fig. 1.5).
Proline, then, has a secondary (rather than a primary) amino group. It is frequently referred to as an “imino acid.” The unique geometry of proline contributes to the formation of the fibrous structure of collagen (see p. 45), but it interrupts the α-helices found in globular proteins (see p. 16).
B. Amino acids with uncharged polar side chains
These amino acids have zero net charge at physiologic pH, although the side chains of cysteine and tyrosine can lose a proton at an alkaline pH (see Fig. 1.3). Serine, threonine, and tyrosine each contain a polar hydroxyl group that can participate in hydrogen bond formation (Fig. 1.6). The side chains of asparagine and glutamine each contain a carbonyl group and an amide group, both of which can also participate in hydrogen bonds.
1. Disulfide bond: The side chain of cysteine contains a sulfhydryl (thiol) group (−SH), which is an important component of the active site of many enzymes. In proteins, the –SH groups of two cysteines can be oxidized to form a covalent cross-link called a disulfide bond (−S–S–). Two disulfide-linked cysteines are referred to as cystine. (See p. 19 for a further discussion of disulfide bond formation.)
Many extracellular proteins are stabilized by disulfide bonds. Albumin, a blood protein that functions as a transporter for a variety of molecules, is an example.
2. Side chains as attachment sites for other compounds: The polar hydroxyl group of serine, threonine, and (rarely) tyrosine can serve as a site of attachment for structures such as a phosphate group. In addition, the amide group of asparagine, as well as the hydroxyl group of serine or threonine, can serve as a site of attachment for oligosaccharide chains in glycoproteins (see p. 165).
C. Amino acids with acidic side chains
The amino acids aspartic acid and glutamic acid are proton donors. At physiologic pH, the side chains of these amino acids are fully ionized, containing a negatively charged carboxylate group (−COO−). The fully ionized forms are called aspartate and glutamate.
D. Amino acids with basic side chains
The side chains of the basic amino acids accept protons (see Fig. 1.3). At physiologic pH, the R groups of lysine and arginine are fully ionized and positively charged. In contrast, the free amino acid histidine is weakly basic and largely uncharged at physiologic pH. However, when histidine is incorporated into a protein, its R group can be either positively charged (protonated) or neutral, depending on the ionic environment provided by the protein. This important property of histidine contributes to the buffering role it plays in the functioning of such proteins as hemoglobin (see p. 30). [Note: Histidine is the only amino acid with a side chain that can ionize within the physiologic pH range.]
E. Abbreviations and symbols for commonly occurring amino acids letter symbol (Fig. 1.7). The one-letter codes are determined by the following rules.
1.
Unique first letter: If only one amino acid begins with a given letter, then that letter is used as its symbol. For example, V = valine.
2.
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Most commonly occurring amino acids have priority: If more than one amino acid begins with a particular letter, the most common of these amino acids receives this letter as its symbol. For example, glycine is more common than glutamate, so G = glycine.
3.
Similar sounding names: Some one-letter symbols sound like the amino acid they represent. For example, F = phenylalanine, or W = tryptophan (“twyptophan” as Elmer Fudd would say).
4.
Letter close to initial letter: For the remaining amino acids, a one-letter symbol is assigned that is as close in the alphabet as possible to the initial letter of the amino acid, for example, K = lysine. Furthermore, B is assigned to Asx, signifying either aspartic acid or asparagine; Z is assigned to Glx, signifying either glutamic acid or glutamine; and X is assigned to an unidentified amino acid.
F. Amino acid isomers
Because the α-carbon of an amino acid is attached to four different chemical groups, it is an asymmetric (chiral) atom. Glycine is the exception because its α-carbon has two hydrogen substituents. Amino acids with a chiral αcarbon exist in two different isomeric forms, designated D and L, which are enantiomers, or mirror images (Fig. 1.8). [Note: Enantiomers are optically active. If an isomer, either D or L, causes the plane of polarized light to rotate clockwise, it is designated the (+) form.] All amino acids found in mammalian proteins are of the L configuration. However, D-amino acids are found in some antibiotics and in bacterial cell walls (see p. 252). [Note: Racemases enzymatically interconvert the D-and L-isomers of free amino acids.]
III. ACIDIC AND BASIC PROPERTIES
Amino acids in aqueous solution contain weakly acidic α-carboxyl groups and weakly basic α-amino groups. In addition, each of the acidic and basic amino acids contains an ionizable group in its side chain. Thus, both free amino acids and some amino acids combined in peptide linkages can act as buffers. Acids may be defined as proton donors and bases as proton acceptors. Acids (or bases) described as weak ionize to only a limited extent. The concentration of protons ([H+]) in aqueous solution is expressed as pH, where pH = log 1/[H+] or –log [H+]. The quantitative relationship between the pH of the solution and concentration of a weak acid (HA) and its conjugate base (A−) is described by the Henderson-Hasselbalch equation.
A. Equation derivation
Consider the release of a proton by a weak acid represented by HA: The salt or conjugate base, A−, is the ionized form of a weak acid. By definition, the dissociation constant of the acid, Ka, is: [Note: The larger the Ka, the stronger the acid, because most of the HA has dissociated into H+ and A−. Conversely, the smaller the Ka, the less acid has dissociated and, therefore, the weaker the acid.] By solving for the [H+] in the above equation, taking the logarithm of both sides of the equation, multiplying both sides of the equation by −1, and substituting pH = −log [H+] and pKa = −log Ka, we obtain the Henderson-Hasselbalch equation:
B. Buffers
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A buffer is a solution that resists change in pH following the addition of an acid or base. A buffer can be created by mixing a weak acid (HA) with its conjugate base (A−). If an acid such as HCl is added to a buffer, A− can neutralize it, being converted to HA in the process. If a base is added, HA can likewise neutralize it, being converted to A− in the process. Maximum buffering capacity occurs at a pH equal to the pKa, but a conjugate acid-base pair can still serve as an effective buffer when the pH of a solution is within approximately ±1 pH unit of the pKa. If the amounts of HA and A− are equal, the pH is equal to the pKa. As shown in Figure 1.9, a solution containing acetic acid (HA = CH3 – COOH) and acetate (A− = CH3 – COO−) with a pKa of 4.8 resists a change in pH from pH 3.8 to 5.8, with maximum buffering at pH 4.8. At pH values less than the pKa, the protonated acid form (CH3 – COOH) is the predominant species in solution.
At pH values greater than the pKa, the deprotonated base form (CH3 – COO−) is the predominant species.
C. Amino acid titration
The titration curve of an amino acid can be analyzed in the same way as described for acetic acid.
1.
Carboxyl group dissociation: Consider alanine, for example, which contains an ionizable α-carboxyl and α-amino group. [Note: Its –CH3 R group is nonionizable.] At a low (acidic) pH, both of these groups are protonated (Fig. 1.10). As the pH of the solution is raised, the −COOH group of form I can dissociate by donating a H+ to the medium. The release of a H+ results in the formation of the carboxylate group, −COO−. This structure is shown as form II, which is the dipolar form of the molecule (see Fig. 1.10). This form, also called a zwitterion (from the German word for “hybrid”), is the isoelectric form of alanine, that is, it has an overall (net) charge of zero.
2.
Application of the Henderson-Hasselbalch equation: The dissociation constant of the carboxyl group of an amino acid is called K1, rather than
Ka, because the molecule contains a second titratable group. The Henderson-Hasselbalch equation can be used to analyze the dissociation of the carboxyl group of alanine in the same way as described for acetic acid: where I is the fully protonated form of alanine and II is the isoelectric form of alanine (see Fig. 1.10). This equation can be rearranged and converted to its logarithmic form to yield: 3.
Amino group dissociation: The second titratable group of alanine is the amino (−NH3+) group shown in Figure 1.10. Because this is a much weaker acid than the –COOH group, it has a much smaller dissociation constant, K2. [Note: Its pKa is, therefore, larger.] Release of a H+ from the protonated amino group of form II results in the fully deprotonated form of alanine, form III (see Fig. 1.10).
4.
Alanine pKs: The sequential dissociation of H+ from the carboxyl and amino groups of alanine is summarized in Figure 1.10. Each titratable group has a pKa that is numerically equal to the pH at which exactly one half of the H+ have been removed from that group. The pKa for the most acidic group (−COOH) is pK1, whereas the pKa for the next most acidic group (−NH3+) is pK2. [Note: The pKa of the α-carboxyl group of amino acids is ~2, whereas that of the α-amino group is ~9.] 5.
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Alanine titration curve: By applying the Henderson-Hasselbalch equation to each dissociable acidic group, it is possible to calculate the complete titration curve of a weak acid. Figure 1.11 shows the change in pH that occurs during the addition of base to the fully protonated form of alanine (I) to produce the completely deprotonated form (III). Note the following: a.
Buffer pairs: The –COOH/–COO− pair can serve as a buffer in the pH region around pK1, and the –NH3+/–NH2 pair can buffer in the region around pK2.
b.
When pH = pK: When the pH is equal to pK1 (2.3), equal amounts of forms I and II of alanine exist in solution. When the pH is equal to pK2 (9.1), equal amounts of forms II and III are present in solution.
c.
Isoelectric point: At neutral pH, alanine exists predominantly as the dipolar form II in which the amino and carboxyl groups are ionized, but the net charge is zero. The isoelectric point (pI) is the pH at which an amino acid is electrically neutral, that is, in which the sum of the positive charges equals the sum of the negative charges. For an amino acid, such as alanine, that has only two dissociable hydrogens (one from the α-carboxyl and one from the α-amino group), the pI is the average of pK1 and pK2 (pI = [2.3 + 9.1]/2 = 5.7) as shown in Figure 1.11. The pI is, thus, midway between pK1 (2.3) and pK2 (9.1). pI corresponds to the pH at which the form II (with a net charge of zero) predominates and at which there are also equal amounts of forms I (net charge of +1) and III (net charge of −1).
Separation of plasma proteins by charge typically is done at a pH above the pI of the major proteins. Therefore, the charge on the proteins is negative. In an electric field, the proteins will move toward the positive electrode at a rate determined by their net negative charge. Variations in the mobility pattern are suggestive of certain diseases.
6. Net charge at neutral pH: At physiologic pH, amino acids have a negatively charged group (−COO−) and a positively charged group (−NH3+), both attached to the α-carbon. [Note: Glutamate, aspartate, histidine, arginine, and lysine have additional potentially charged groups in their side chains.] Substances such as amino acids that can act either as an acid or a base are defined as amphoteric and are referred to as ampholytes (amphoteric electrolytes).
D. Other applications of the Henderson-Hasselbalch equation
The Henderson-Hasselbalch equation can be used to calculate how the pH of a physiologic solution responds to changes in the concentration of a weak acid and/or its corresponding salt form. For example, in the bicarbonate buffer system, the Henderson-Hasselbalch equation predicts how shifts in the bicarbonate ion concentration, [HCO3−], and the carbon dioxide concentration [CO2] influence pH (Fig. 1.12A). The equation is also useful for calculating the abundance of ionic forms of acidic and basic drugs. For example, most drugs are either weak acids or weak bases (Fig. 1.12B). Acidic drugs (HA) release a H+, causing a charged anion (A−) to form.
Weak bases (BH+) can also release a H+. However, the protonated form of basic drugs is usually charged, and the loss of a proton produces the uncharged base (B).
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A drug passes through membranes more readily if it is uncharged. Thus, for a weak acid, such as aspirin, the uncharged HA can permeate through membranes, but A− cannot. Likewise, for a weak base, such as morphine, the uncharged B form permeates through the cell membrane, but BH+ does not. Therefore, the effective concentration of the permeable form of each drug at its absorption site is determined by the relative concentrations of the charged (impermeant) and uncharged (permeant) forms. The ratio between the two forms is determined by the pH at the site of absorption and by the strength of the weak acid or base, which is represented by the pKa of the ionizable group. The Henderson-Hasselbalch equation is useful in determining how much drug is found on either side of a membrane that separates two compartments that differ in pH, for example, the stomach (pH 1.0–1.5) and blood plasma (pH 7.4).
IV. CONCEPT MAPS
Students sometimes view biochemistry as a list of facts or equations to be memorized, rather than a body of concepts to be understood. Details provided to enrich understanding of these concepts inadvertently turn into distractions. What seems to be missing is a road map—a guide that provides the student with an understanding of how various topics fit together to “tell a story.” Therefore, in this text, a series of biochemical concept maps have been created to graphically illustrate relationships between ideas presented in a chapter and to show how the information can be grouped or organized. A concept map is, thus, a tool for visualizing the connections between concepts. Material is represented in a hierarchic fashion, with the most inclusive, most general concepts at the top of the map, and the more specific, less general concepts arranged beneath. The concept maps ideally function as templates or guides for organizing information, so the student can readily find the best ways to integrate new information into knowledge they already possess. Concept map construction is described below.
A. Concept boxes and links
Educators define concepts as “perceived regularities in events or objects.” In the biochemical maps, concepts include abstractions (for example, free energy), processes (for example, oxidative phosphorylation), and compounds (for example, glucose 6-phosphate). These broadly defined concepts are prioritized with the central idea positioned at the top of the page. The concepts that follow from this central idea are then drawn in boxes (Fig. 1.13A). The size of the type indicates the relative importance of each idea. Lines are drawn between concept boxes to show which are related. The label on the line defines the relationship between two concepts, so that it reads as a valid statement (that is, the connection creates meaning). The lines with arrowheads indicate in which direction the connection should be read (Fig. 1.14).
B. Cross-links
Unlike linear flow charts or outlines, concept maps may contain cross-links that allow the reader to visualize complex relationships between ideas represented in different parts of the map (Fig. 1.13B) or between the map and other chapters in this book (Fig. 1.13C). Cross-links can, thus, identify concepts that are central to more than one topic in biochemistry, empowering students to be effective in clinical situations and on the United States Medical Licensure Examination (USMLE) or other examinations that require integration of material. Students learn to visually perceive nonlinear relationships between facts, in contrast to cross-referencing within linear text.
V. CHAPTER SUMMARY
Each amino acid has an α-carboxyl group and a primary α-amino group (except for proline, which has a secondary amino group). At physiologic pH, the α-carboxyl group is dissociated, forming the negatively charged carboxylate ion (−COO−), and the α-amino group is protonated (−NH3+).
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Each amino acid also contains one of 20 distinctive side chains attached to the α-carbon atom. The chemical nature of this R group determines the function of an amino acid in a protein and provides the basis for classification of the amino acids as nonpolar, uncharged polar, acidic (polar negative), or basic (polar positive). All free amino acids, plus charged amino acids in peptide chains, can serve as buffers. The quantitative relationship between the pH of a solution and the concentration of a weak acid (HA) and its conjugate base (A−) is described by the Henderson-Hasselbalch equation. Buffering occurs within ±1 pH unit of the pKa and is maximal when pH = pKa, at which [A−] = [HA]. Because the α-carbon of each amino acid (except glycine) is attached to four different chemical groups, it is asymmetric (chiral), and amino acids exist in D-and L-isomeric forms that are optically active mirror images (enantiomers). The L-form of amino acids is found in proteins synthesized by the human body.
Choose the ONE best answer.
.1. Which one of the following statements concerning the titration curve for a nonpolar amino acid is correct? The letters A through D designate certain regions on the curve below.
A. Point A represents the region where the amino acid is deprotonated.
B. Point B represents a region of minimal buffering.
C. Point C represents the region where the net charge on the amino acid is zero.
D. Point D represents the pK of the amino acid’s carboxyl group.
E. The amino acid could be lysine.
Correct answer = C. Point C represents the isoelectric point, or pI, and as such is midway between pK1 and pK2 for a nonpolar amino acid. The amino acid is fully protonated at Point A. Point B represents a region of maximum buffering, as does Point D. Lysine is a basic amino acid, and free lysine has an ionizable side chain in addition to the ionizable α-amino and α-carboxyl groups.
.2. Which one of the following statements concerning the peptide shown below is correct?Val-Cys-Glu-Ser-Asp-Arg-Cys
A. The peptide contains asparagine.
B. The peptide contains a side chain with a secondary amino group.
C. The peptide contains a side chain that can be phosphorylated.
D. The peptide cannot form an internal disulfide bond.
E. The peptide would move to the cathode (negative electrode) during electrophoresis at pH 5.
Correct answer = C. The hydroxyl group of serine can accept a phosphate group. Asp is aspartate. Proline contains a secondary amino group. The two cysteine residues can, under oxidizing conditions, form a disulfide (covalent) bond. The net charge on the peptide at pH 5 is negative, and it would move to the anode.
.3. A 2-year-old child presents with metabolic acidosis after ingesting an unknown number of flavored aspirin tablets. At presentation, her blood pH was 7.0. Given that the pKa of aspirin (salicylic acid) is 3, calculate the ratio of its ionized to unionized forms at pH 7.0.
Correct answer = 10,000 to 1. pH = pKa + log [A−]/[HA]. Therefore, 7 = 3 + × and × = 4. The ratio of A− (ionized) to HA (unionized), then, is 10,000 to 1 because the log of 10,000 is 4.
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I. OVERVIEW
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The 20 amino acids commonly found in proteins are joined together by peptide bonds. The linear sequence of the linked amino acids contains the information necessary to generate a protein molecule with a unique three-dimensional shape that determines function. The complexity of protein structure is best analyzed by considering the molecule in terms of four organizational levels: primary, secondary, tertiary, and quaternary (Fig. 2.1). An examination of these hierarchies of increasing complexity has revealed that certain structural elements are repeated in a wide variety of proteins, suggesting that there are general rules regarding the ways in which proteins achieve their native, functional form. These repeated structural elements range from simple combinations of α-helices and β-sheets forming small motifs to the complex folding of polypeptide domains of multifunctional proteins (see p. 19).
II. PRIMARY STRUCTURE
The sequence of amino acids in a protein is called the primary structure of the protein. Understanding the primary structure of proteins is important because many genetic diseases result in proteins with abnormal amino acid sequences, which cause improper folding and loss or impairment of normal function. If the primary structures of the normal and the mutated proteins are known, this information may be used to diagnose or study the disease.
A. Peptide bond
In proteins, amino acids are joined covalently by peptide bonds, which are amide linkages between the α-carboxyl group of one amino acid and the αamino group of another. For example, valine and alanine can form the dipeptide valylalanine through the formation of a peptide bond (Fig. 2.2). Peptide bonds are resistant to conditions that denature proteins, such as heating and high concentrations of urea (see p. 20). Prolonged exposure to a strong acid or base at elevated temperatures is required to break these bonds nonenzymically (see p. 14).
1.
Naming the peptide: By convention, the free amino end (N-terminal) of the peptide chain is written to the left and the free carboxyl end (C-terminal) to the right. Therefore, all amino acid sequences are read from the N-to the C-terminal end. For example, in Figure 2.2A, the order of the amino acids in the dipeptide is valine, alanine. Linkage of ≥50 amino acids through peptide bonds results in an unbranched chain called a polypeptide, or protein. Each component amino acid is called a residue because it is the portion of the amino acid remaining after the atoms of water are lost in the formation of the peptide bond. When a peptide is named, all amino acid residues have their suffixes (-ine, -an, -ic, or -ate) changed to -yl, with the exception of the C-terminal amino acid. For example, a tripeptide composed of an N-terminal valine, a glycine, and a C-terminal leucine is called valylglycylleucine.
2.
Peptide bond characteristics: The peptide bond has a partial double-bond character, that is, it is shorter than a single bond and is rigid and planar (Fig. 2.2B). This prevents free rotation around the bond between the carbonyl carbon and the nitrogen of the peptide bond. However, the bonds between the α-carbons and the α-amino or α-carboxyl groups can be freely rotated (although they are limited by the size and character of the R groups). This allows the polypeptide chain to assume a variety of possible conformations. The peptide bond is almost always in the trans configuration (instead of the cis; see Fig. 2.2B), in large part because of steric interference of the R groups (side chains) when in the cis position.
3.
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Peptide bond polarity: Like all amide linkages, the −C = O and −NH groups of the peptide bond are uncharged, and neither accept nor release protons over the pH range of 2–12. Thus, the charged groups present in polypeptides consist solely of the N-terminal (α-amino) group, the C-terminal (α-carboxyl) group, and any ionized groups present in the side chains of the constituent amino acids. The −C = O and −NH groups of the peptide bond are polar, however, and are involved in hydrogen bonds (for example, in α-helices and β-sheets), as described on pp. 16–17.
B. Determining the amino acid composition of a polypeptide
The first step in determining the primary structure of a polypeptide is to identify and quantitate its constituent amino acids. A purified sample of the polypeptide to be analyzed is first hydrolyzed by strong acid at 110°C for 24 hours. This treatment cleaves the peptide bonds and releases the individual amino acids, which can be separated by cation-exchange chromatography. In this technique, a mixture of amino acids is applied to a column that contains a resin to which a negatively charged group is tightly attached. [Note: If the attached group is positively charged, the column becomes an anion-exchange column.] The amino acids bind to the column with different affinities, depending on their charges, hydrophobicity, and other characteristics. Each amino acid is sequentially released from the chromatography column by eluting with solutions of increasing ionic strength and pH (Fig. 2.3). The separated amino acids contained in the eluate from the column are quantitated by heating them with ninhydrin (a reagent that forms a purple compound with most amino acids, ammonia, and amines). The amount of each amino acid is determined spectrophotometrically by measuring the amount of light absorbed by the ninhydrin derivative. The analysis described above is performed using an amino acid analyzer, an automated machine whose components are depicted in Figure 2.3.
C. Sequencing the peptide from its N-terminal end
Sequencing is a stepwise process of identifying the specific amino acid at each position in the peptide chain, beginning at the N-terminal end. Phenylisothiocyanate, known as Edman reagent, is used to label the amino-terminal residue under mildly alkaline conditions (Fig. 2.4). The resulting phenylthiohydantoin (PTH) derivative introduces an instability in the N-terminal peptide bond such that it can be hydrolyzed without cleaving the other peptide bonds. The identity of the amino acid derivative can then be determined. Edman reagent can be applied repeatedly to the shortened peptide obtained in each previous cycle. Automated sequencers are now used.
D. Cleaving the polypeptide into smaller fragments
Many polypeptides have a primary structure composed of >100 amino acids. Such molecules cannot be sequenced directly from end to end. However, these large molecules can be cleaved at specific sites and the resulting fragments sequenced. By using more than one cleaving agent (enzymes and/or chemicals) on separate samples of the purified polypeptide, overlapping fragments can be generated that permit the proper ordering of the sequenced fragments, thereby providing a complete amino acid sequence of the large polypeptide (Fig. 2.5). Enzymes that hydrolyze peptide bonds are termed peptidases (proteases). [Note: Exopeptidases cut at the ends of proteins and are divided into aminopeptidases and carboxypeptidases. Carboxypeptidases are used in determining the C-terminal amino acid. Endopeptidases cleave within a protein.]
E. Determining a protein’s primary structure by DNA sequencing
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The sequence of nucleotides in a protein-coding region of the DNA specifies the amino acid sequence of a polypeptide. Therefore, if the nucleotide sequence can be determined, knowledge of the genetic code (see p. 447) allows the sequence of nucleotides to be translated into the corresponding amino acid sequence of that polypeptide. This indirect process, although routinely used to obtain the amino acid sequences of proteins, has the limitations of not being able to predict the positions of disulfide bonds in the folded chain and of not identifying any amino acids that are modified after their incorporation into the polypeptide (posttranslational modification; see p. 459). Therefore, direct protein sequencing is an extremely important tool for determining the true character of the primary sequence of many polypeptides.
III. SECONDARY STRUCTURE
The polypeptide backbone does not assume a random three-dimensional structure but, instead, generally forms regular arrangements of amino acids that are located near each other in the linear sequence. These arrangements are termed the secondary structure of the polypeptide. The α-helix, β-sheet, and βbend (or, β-turn) are examples of secondary structures commonly encountered in proteins. Each is stabilized by hydrogen bonds between atoms of the peptide backbone. [Note: The collagen α-chain helix, another example of secondary structure, is discussed on p. 45.]
A. α-Helix
Several different polypeptide helices are found in nature, but the α-helix is the most common. It is a rigid, right-handed spiral structure, consisting of a tightly packed, coiled polypeptide backbone core, with the side chains of the component L-amino acids extending outward from the central axis to avoid interfering sterically with each other (Fig. 2.6). A very diverse group of proteins contains α-helices. For example, the keratins are a family of closely related, rigid, fibrous proteins whose structure is nearly entirely αhelical. They are a major component of tissues such as hair and skin. In contrast to keratin, myoglobin, whose structure is also highly α-helical, is a globular, flexible molecule (see p. 26) found in muscles.
1.
Hydrogen bonds: An α-helix is stabilized by extensive hydrogen bonding between the peptide bond carbonyl oxygens and amide hydrogens that are part of the polypeptide backbone (see Fig. 2.6). The hydrogen bonds extend up and are parallel to the spiral from the carbonyl oxygen of one peptide bond to the –NH group of a peptide linkage four residues ahead in the polypeptide. This insures that all but the first and last peptide bond components are linked to each other through intrachain hydrogen bonds. Hydrogen bonds are individually weak, but they collectively serve to stabilize the helix.
2.
Amino acids per turn: Each turn of an α-helix contains 3.6 amino acids. Thus, amino acids spaced three or four residues apart in the primary sequence are spatially close together when folded in the α-helix.
3.
Amino acids that disrupt an α-helix: The R group of an amino acid determines its propensity to be in an α-helix. Proline disrupts an α-helix because its rigid secondary amino group is not geometrically compatible with the right-handed spiral of the α-helix. Instead, it inserts a kink in the chain, which interferes with the smooth, helical structure. Glycine is also a “helix breaker” because its R group (a hydrogen) confers high flexibility. Additionally, amino acids with charged or bulky R groups (such as glutamate and tryptophan, respectively) and those with a branch at the β-carbon, the first carbon in the R group (for example, valine), have low α-helix propensity.
B. β-Sheet
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The β-sheet is another form of secondary structure in which all of the peptide bond components are involved in hydrogen bonding (Fig. 2.7A). Because the surfaces of β-sheets appear “pleated,” they are often called βpleated sheets. [Note: Pleating results from successive α-carbons being slightly above or below the plane of the sheet.] Illustrations of protein structure often show β-strands as broad arrows (Fig. 2.7B).
1.
Formation: A β-sheet is formed by two or more peptide chains (βstrands) aligned laterally and stabilized by hydrogen bonds between the carboxyl and amino groups of amino acids that either are far apart in a single polypeptide (intrachain bonds) or are in different polypeptide chains (interchain bonds). The adjacent β-strands are arranged either antiparallel to each other (with the N-termini alternating as shown in Fig. 2.7B) or parallel to each other (with the N-termini together as shown in Fig. 2.7C). On each β-strand, the R groups of adjacent amino acids extend in opposite directions, above and below the plane of the β-sheet. [Note: β-sheets are not flat and have a right-handed curl (twist) when viewed along the polypeptide backbone.] 2.
Comparing α-helices and β-sheets: In β-sheets, the β-strands are almost fully extended and the hydrogen bonds between the strands are perpendicular to the polypeptide backbone (see Fig. 2.7A). In contrast, in α-helices, the polypeptide is coiled and the hydrogen bonds are parallel to the backbone (see Fig. 2.6).
The orientation of the R groups of the amino acid residues in both the αhelix and the β-sheet can result in formation of polar and nonpolar sides in these secondary structures, thereby making them amphipathic.
C. β-Bends (reverse turns, β-turns) β-Bends reverse the direction of a polypeptide chain, helping it form a compact, globular shape. They are usually found on the surface of protein molecules and often include charged residues. [Note: β-Bends were given this name because they often connect successive strands of antiparallel βsheets.] β-Bends are generally composed of four amino acids, one of which may be proline, the amino acid that causes a kink in the polypeptide chain. Glycine, the amino acid with the smallest R group, is also frequently found in β-bends. β-Bends are stabilized by the formation of hydrogen bonds between the first and last residues in the bend.
D. Nonrepetitive secondary structure
Approximately one half of an average globular protein is organized into repetitive structures, such as the α-helix and β-sheet. The remainder of the polypeptide chain is described as having a loop or coil conformation. These nonrepetitive secondary structures are not random but rather simply have a less regular structure than those described above. [Note: The term “random coil” refers to the disordered structure obtained when proteins are denatured (see p. 20).]
E. Supersecondary structures (motifs)
Globular proteins are constructed by combining secondary structural elements (that is, α-helices, β-sheets, and coils), producing specific geometric patterns, or motifs. These form primarily the core (interior) region of the molecule. They are connected by loop regions (for example, β-bends) at the surface of the protein. Supersecondary structures are usually produced by the close packing of side chains from adjacent secondary structural elements. For example, α-helices and β-sheets that are adjacent in the amino acid sequence are also usually (but not always) adjacent in the final, folded protein. Some of the more common motifs are illustrated in Figure 2.8.
Motifs may be associated with particular functions. Proteins that bind to DNA contain a limited number of motifs. The helix–loop–helix motif is an example found in a number of proteins that function as transcription factors (see p. 438).
IV. TERTIARY STRUCTURE
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The primary structure of a polypeptide chain determines its tertiary structure. “Tertiary” refers both to the folding of domains (the basic units of structure and function; see A. below) and to the final arrangement of domains in the polypeptide. The tertiary structure of globular proteins in aqueous solution is compact, with a high density (close packing) of the atoms in the core of the molecule. Hydrophobic side chains are buried in the interior, whereas hydrophilic groups are generally found on the surface of the molecule.
A. Domains
Domains are the fundamental functional and three-dimensional structural units of polypeptides. Polypeptide chains that are >200 amino acids in length generally consist of two or more domains. The core of a domain is built from combinations of supersecondary structural elements (motifs). Folding of the peptide chain within a domain usually occurs independently of folding in other domains. Therefore, each domain has the characteristics of a small, compact globular protein that is structurally independent of the other domains in the polypeptide chain.
B. Stabilizing interactions
The unique three-dimensional structure of each polypeptide is determined by its amino acid sequence. Interactions between the amino acid side chains guide the folding of the polypeptide to form a compact structure. The following four types of interactions cooperate in stabilizing the tertiary structures of globular proteins.
1. Disulfide bonds: A disulfide bond (–S–S–) is a covalent linkage formed from the sulfhydryl group (−SH) of each of two cysteine residues to produce a cystine residue (Fig. 2.9). The two cysteines may be separated from each other by many amino acids in the primary sequence of a polypeptide or may even be located on two different polypeptides. The folding of the polypeptide(s) brings the cysteine residues into proximity and permits covalent bonding of their side chains. A disulfide bond contributes to the stability of the three-dimensional shape of the protein molecule and prevents it from becoming denatured in the extracellular environment. For example, many disulfide bonds are found in proteins such as immunoglobulins that are secreted by cells. [Note: Protein disulfide isomerase breaks and reforms disulfide bonds during folding.] 2.
Hydrophobic interactions: Amino acids with nonpolar side chains tend to be located in the interior of the polypeptide molecule, where they associate with other hydrophobic amino acids (Fig. 2.10). In contrast, amino acids with polar or charged side chains tend to be located on the surface of the molecule in contact with the polar solvent. [Note: Recall that proteins located in nonpolar (lipid) environments, such as a membrane, exhibit the reverse arrangement (see Fig. 1.4, p. 4).] In each case, a segregation of R groups occurs that is energetically most favorable.
3.
Hydrogen bonds: Amino acid side chains containing oxygen-or nitrogen-bound hydrogen, such as in the alcohol groups of serine and threonine, can form hydrogen bonds with electron-rich atoms, such as the oxygen of a carboxyl group or carbonyl group of a peptide bond (Fig. 2.11; see also Fig. 1.6, p. 4). Formation of hydrogen bonds between polar groups on the surface of proteins and the aqueous solvent enhances the solubility of the protein.
4. Ionic interactions: Negatively charged groups, such as the carboxylate group (−COO−) in the side chain of aspartate or glutamate, can interact with positively charged groups such as the amino group (−NH3+) in the side chain of lysine (see Fig. 2.11).
C. Protein folding
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Interactions between the side chains of amino acids determine how a linear polypeptide chain folds into the intricate three-dimensional shape of the functional protein. Protein folding, which occurs within the cell in seconds to minutes, involves nonrandom, ordered pathways. As a peptide folds, secondary structures form, driven by the hydrophobic effect (that is, hydrophobic groups come together as water is released). These small structures combine to form larger structures. Additional events stabilize secondary structure and initiate formation of tertiary structure. In the last stage, the peptide achieves its fully folded, native (functional) form characterized by a low-energy state (Fig. 2.12). [Note: Some biologically active proteins or segments thereof lack a stable tertiary structure. They are referred to as intrinsically disordered proteins.]
D. Protein denaturation
Denaturation results in the unfolding and disorganization of a protein’s secondary and tertiary structures without the hydrolysis of peptide bonds. Denaturing agents include heat, urea, organic solvents, strong acids or bases, detergents, and ions of heavy metals such as lead. Denaturation may, under ideal conditions, be reversible, such that the protein refolds into its original native structure when the denaturing agent is removed. However, most proteins remain permanently disordered once denatured. Denatured proteins are often insoluble and precipitate from solution.
E. Chaperones in protein folding
The information needed for correct protein folding is contained in the primary structure of the polypeptide. However, most denatured proteins do not resume their native conformations even under favorable environmental conditions. This is because, for many proteins, folding is a facilitated process that requires a specialized group of proteins, referred to as molecular chaperones, and ATP hydrolysis. The chaperones, also known as heat shock proteins (HSP), interact with a polypeptide at various stages during the folding process. Some chaperones bind hydrophobic regions of an extended polypeptide and are important in keeping the protein unfolded until its synthesis is completed (for example, Hsp70). Others form cage-like macromolecular structures composed of two stacked rings. The partially folded protein enters the cage, binds the central cavity through hydrophobic interactions, folds, and is released (for example, mitochondrial Hsp60). [Note: Cage-like chaperones are sometimes referred to as chaperonins.] Chaperones, then, facilitate correct protein folding by binding to and stabilizing exposed, aggregation-prone hydrophobic regions in nascent (and denatured) polypeptides, preventing premature folding.
V. QUATERNARY STRUCTURE
Many proteins consist of a single polypeptide chain and are defined as monomeric proteins. However, others may consist of two or more polypeptide chains that may be structurally identical or totally unrelated. The arrangement of these polypeptide subunits is called the quaternary structure of the protein. Subunits are held together primarily by noncovalent interactions (for example, hydrogen bonds, ionic bonds, and hydrophobic interactions). Subunits either may function independently of each other or may work cooperatively, as in hemoglobin, in which the binding of oxygen to one subunit of the tetramer increases the affinity of the other subunits for oxygen (see p. 29).
Isoforms are proteins that perform the same function but have different primary structures. They can arise from different genes or from tissue-specific processing of the product of a single gene. If the proteins function as enzymes, they are referred to as isozymes (see p. 65).
VI. PROTEIN MISFOLDING
Protein folding is a complex process that can sometimes result in improperly folded molecules. These misfolded proteins are usually tagged and degraded within the cell (see p. 247). However, this quality control system is not perfect, and intracellular or extracellular aggregates of misfolded proteins can accumulate, particularly as individuals age. Deposits of misfolded proteins are associated with a number of diseases.
A. Amyloid diseases
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Misfolding of proteins may occur spontaneously or be caused by a mutation in a particular gene, which then produces an altered protein. In addition, some apparently normal proteins can, after abnormal proteolytic cleavage, take on a unique conformation that leads to the spontaneous formation of long, fibrillar protein assemblies consisting of β-pleated sheets. Accumulation of these insoluble fibrous protein aggregates, called amyloids, has been implicated in neurodegenerative disorders such as Parkinson disease and Alzheimer disease (AD). The dominant component of the amyloid plaque that accumulates in AD is amyloid β (Aβ), an extracellular peptide containing 40–42 amino acid residues. X-ray crystallography and infrared spectroscopy demonstrate a characteristic βpleated sheet secondary structure in nonbranching fibrils. This peptide, when aggregated in a β-pleated sheet conformation, is neurotoxic and is the central pathogenic event leading to the cognitive impairment characteristic of the disease. The Aβ that is deposited in the brain in AD is derived by enzymic cleavages (by secretases) from the larger amyloid precursor protein, a single transmembrane protein expressed on the cell surface in the brain and other tissues (Fig. 2.13). The Aβ peptides aggregate, generating the amyloid that is found in the brain parenchyma and around blood vessels. Most cases of AD are not genetically based, although at least 5% of cases are familial. A second biologic factor involved in the development of AD is the accumulation of neurofibrillary tangles inside neurons. A key component of these tangled fibers is an abnormal form (hyperphosphorylated and insoluble) of the tau (τ) protein, which, in its healthy version, helps in the assembly of the microtubular structure. The defective τ appears to block the actions of its normal counterpart. [Note: In Parkinson disease, amyloid is formed from α-synuclein protein.]
B. Prion (proteinaceous infectious particle) diseases
The prion protein (PrP) is the causative agent of transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle (popularly called “mad cow” disease). After an extensive series of purification procedures, scientists were surprised to find that the infectivity of the agent causing scrapie in sheep was associated with a single protein species that was not complexed with detectable nucleic acid. This infectious protein is designated PrPSc (Sc = scrapie). It is highly resistant to proteolytic degradation and tends to form insoluble aggregates of fibrils, similar to the amyloid found in some other diseases of the brain. A noninfectious form of PrPC (C = cellular), encoded by the same gene as the infectious agent, is present in normal mammalian brains on the surface of neurons and glial cells. Thus, PrPC is a host protein. No primary structure differences or alternate posttranslational modifications have been found between the normal and the infectious forms of the protein. The key to becoming infectious apparently lies in changes in the three-dimensional conformation of PrPC. Research has demonstrated that a number of αhelices present in noninfectious PrPC are replaced by β-sheets in the infectious form (Fig. 2.14). This conformational difference is presumably what confers relative resistance to proteolytic degradation of infectious prions and permits them to be distinguished from the normal PrPC in infected tissue. The infective agent is, thus, an altered version of a normal protein, which acts as a template for converting the normal protein to the pathogenic conformation. The TSE are invariably fatal, and no treatment is currently available that can alter this outcome.
VII. CHAPTER SUMMARY
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Central to understanding protein structure is the concept of the native conformation (Fig. 2.15), which is the functional, fully folded protein structure (for example, an active enzyme or structural protein). The unique three-dimensional structure of the native conformation is determined by its primary structure, that is, its amino acid sequence. Interactions between the amino acid side chains guide the folding of the polypeptide chain to form secondary, tertiary, and (sometimes) quaternary structures, which cooperate in stabilizing the native conformation of the protein. In addition, a specialized group of proteins named chaperones is required for the proper folding of many species of proteins. Protein denaturation results in the unfolding and disorganization of the protein’s structure, which are not accompanied by hydrolysis of peptide bonds. Denaturation may be reversible or, more commonly, irreversible. Disease can occur when an apparently normal protein assumes a conformation that is cytotoxic, as in the case of Alzheimer disease (AD) and the transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease. In AD, normal proteins, after abnormal chemical processing, take on a unique conformational state that leads to the formation of neurotoxic amyloid β peptide (Aβ) assemblies consisting of β-pleated sheets. In TSE, the infective agent is an altered version of a normal prion protein that acts as a template for converting normal protein to the pathogenic conformation.
Choose the ONE best answer.
.1. Which one of the following statements concerning protein structure is correct?
A. Proteins consisting of one polypeptide have quaternary structure that is stabilized by covalent bonds.
B. The peptide bonds that link amino acids in a protein most commonly occur in the cis configuration.
C. The formation of a disulfide bond in a protein requires the participating cysteine residues to be adjacent in the primary structure.
D. The denaturation of proteins leads to irreversible loss of secondary structural elements such as the α-helix.
E. The primary driving force for protein folding is the hydrophobic effect.
Correct answer = E. The hydrophobic effect, or the tendency of nonpolar entities to associate in a polar environment, is the primary driving force of protein folding. Quaternary structure requires more than one polypeptide, and, when present, it is stabilized primarily by noncovalent bonds. The peptide bond is almost always trans. The two cysteine residues participating in disulfide bond formation may be a great distance apart in the amino acid sequence of a polypeptide (or on two separate polypeptides) but are brought into close proximity by the three-dimensional folding of the polypeptide. Denaturation may be reversible or irreversible.
.2. A particular point mutation results in disruption of the α-helical structure in a segment of the mutant protein. The most likely change in the primary structure of the mutant protein is:
A. glutamate to aspartate.
B. lysine to arginine.
C. methionine to proline.
D. valine to alanine.
Correct answer = C. Proline, because of its secondary amino group, is incompatible with an α-helix. Glutamate, aspartate, lysine, and arginine are charged amino acids, and valine is a branched amino acid. Charged and branched (bulky) amino acids may disrupt an α-helix. [Note: The flexibility of glycine’s R group can also disrupt an α-helix.] .3. In comparing the α-helix to the β-sheet, which statement is correct only for the β-sheet?
A. Extensive hydrogen bonds between the carbonyl oxygen (C=O) and the amide hydrogen (N−H) of the peptide bond are formed.
B. It may be found in typical globular proteins.
C. It is stabilized by interchain hydrogen bonds.
D. It is an example of secondary structure.
E. It may be found in supersecondary structures.
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Correct answer = C. The β-sheet is stabilized by interchain hydrogen bonds formed between separate polypeptide chains and by intrachain hydrogen bonds formed between regions of a single polypeptide. The α-helix, however, is stabilized only by intrachain hydrogen bonds. Statements A, B, D, and E are true for both of these secondary structural elements.
.4. An 80-year-old man presented with impairment of intellectual function and alterations in behavior. His family reported progressive disorientation and memory loss over the last 6 months. There is no family history of dementia. The patient was tentatively diagnosed with Alzheimer disease (AD). Which one of the following best describes AD?
A. It is associated with β-amyloid, an abnormal protein with an altered amino acid sequence.
B. It results from accumulation of denatured proteins that have random conformations.
C. It is associated with the accumulation of amyloid precursor protein.
D. It is associated with the deposition of neurotoxic amyloid β peptide aggregates.
E. It is an environmentally produced disease not influenced by the genetics of the individual.
F. It is caused by the infectious β-sheet form of a host-cell protein.
Correct answer = D. Alzheimer disease (AD) is associated with long, fibrillar protein assemblies consisting of β-pleated sheets found in the brain and elsewhere. The disease is associated with abnormal processing of a normal protein. The accumulated altered protein occurs in a β-pleated sheet conformation that is neurotoxic. The amyloid β that is deposited in the brain in AD is derived by proteolytic cleavages from the larger amyloid precursor protein, a single transmembrane protein expressed on the cell surface in the brain and other tissues. Most cases of AD are sporadic, although at least 5% of cases are familial. Prion diseases, such as Creutzfeldt-Jakob, are caused by the infectious β-sheet form (PrPSc) of a host-cell protein (PrPC).
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I. OVERVIEW
The previous chapter described the types of secondary and tertiary structures that are the bricks and mortar of protein architecture. By arranging these fundamental structural elements in different combinations, widely diverse proteins can be constructed that are capable of various specialized functions. This chapter examines the relationship between structure and function for the clinically important globular hemeproteins. Fibrous structural proteins are discussed in Chapter 4.
II. GLOBULAR HEMEPROTEINS
Hemeproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic group. (See p. 54 for a discussion of prosthetic groups.) The role of the heme group is dictated by the environment created by the three-dimensional structure of the protein. For example, the heme group of a cytochrome functions as an electron carrier that is alternately oxidized and reduced (see p. 75). In contrast, the heme group of the enzyme catalase is part of the active site of the enzyme that catalyzes the breakdown of hydrogen peroxide (see p. 148). In hemoglobin and myoglobin, the two most abundant hemeproteins in humans, the heme group serves to reversibly bind oxygen (O2).
A. Heme structure
Heme is a complex of protoporphyrin IX and ferrous iron (Fe2+), as shown in Figure 3.1. The iron is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring. The heme Fe2+ can form two additional bonds, one on each side of the planar porphyrin ring. In myoglobin and hemoglobin, one of these positions is coordinated to the side chain of a histidine residue of the globin molecule, whereas the other position is available to bind O2 (Fig. 3.2). (See pp. 278 and 282, respectively, for a discussion of heme synthesis and degradation.)
B. Myoglobin structure and function
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Myoglobin, a hemeprotein present in heart and skeletal muscle, functions both as an oxygen reservoir and as an oxygen carrier that increases the rate of oxygen transport within the muscle cell. [Note: Surprisingly, mouse myoglobin double knockouts (see p. 502) have an apparently normal phenotype.] Myoglobin consists of a single polypeptide chain that is structurally similar to the individual polypeptide chains of the tetrameric hemoglobin molecule. This homology makes myoglobin a useful model for interpreting some of the more complex properties of hemoglobin.
1.
α-Helical content: Myoglobin is a compact molecule, with ~80% of its polypeptide chain folded into eight stretches of α-helix. These α-helical regions, labeled A to H in Figure 3.2A, are terminated either by the presence of proline, whose five-membered ring cannot be accommodated in an α-helix (see p. 16) or by β-bends and loops stabilized by hydrogen bonds and ionic bonds (see p. 19). [Note: Ionic bonds are also termed electrostatic interactions or salt bridges.] 2.
Location of polar and nonpolar amino acid residues: The interior of the globular myoglobin molecule is composed almost entirely of nonpolar amino acids. They are packed closely together, forming a structure stabilized by hydrophobic interactions between these clustered residues (see p. 19). In contrast, polar amino acids are located almost exclusively on the surface, where they can form hydrogen bonds, both with each other and with water.
3.
Binding of the heme group: The heme group of the myoglobin molecule sits in a crevice, which is lined with nonpolar amino acids. Notable exceptions are two histidine residues (see Fig. 3.2B). One, the proximal histidine (F8), binds directly to the Fe2+ of heme. The second, or distal histidine (E7), does not directly interact with the heme group but helps stabilize the binding of O2 to Fe2+ . Thus, the protein, or globin, portion of myoglobin creates a special microenvironment for the heme that permits the reversible binding of one oxygen molecule (oxygenation). The simultaneous loss of electrons by Fe2+ (oxidation to the ferric [Fe3+] form) occurs only rarely.
C. Hemoglobin structure and function
Hemoglobin is found exclusively in red blood cells (RBC), where its main function is to transport O2 from the lungs to the capillaries of the tissues. Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide chains (two α chains and two β chains) held together by noncovalent interactions (Fig. 3.3). Each chain (subunit) has stretches of αhelical structure and a hydrophobic heme-binding pocket similar to that described for myoglobin. However, the tetrameric hemoglobin molecule is structurally and functionally more complex than myoglobin. For example, hemoglobin can transport protons (H+) and carbon dioxide (CO2) from the tissues to the lungs and can carry four molecules of O2 from the lungs to the cells of the body. Furthermore, the oxygen-binding properties of hemoglobin are regulated by interaction with allosteric effectors (see p. 29).
Obtaining O2 from the atmosphere solely by diffusion greatly limits the size of organisms. Circulatory systems overcome this, but transport molecules such as hemoglobin are also required because O2 is only slightly soluble in aqueous solutions such as blood.
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1. Quaternary structure: The hemoglobin tetramer can be envisioned as composed of two identical dimers, (αβ)1 and (αβ)2. The two polypeptide chains within each dimer are held tightly together primarily by hydrophobic interactions (Fig. 3.4). [Note: In this instance, hydrophobic amino acid residues are localized not only in the interior of the molecule but also in a region on the surface of each subunit. Multiple interchain hydrophobic interactions form strong associations between α-subunits and β-subunits in the dimers.] In contrast, the two dimers are held together primarily by polar bonds. The weaker interactions between the dimers allow them to move with respect to one other. This movement results in the two dimers occupying different relative positions in deoxyhemoglobin as compared with oxyhemoglobin (see Fig. 3.4).
a.
T form: The deoxy form of hemoglobin is called the “T,” or taut (tense) form. In the T form, the two αβ dimers interact through a network of ionic bonds and hydrogen bonds that constrain the movement of the polypeptide chains. The T conformation is the low-oxygen-affinity form of hemoglobin.
b.
R form: The binding of O2 to hemoglobin causes the rupture of some of the polar bonds between the two αβ dimers, allowing movement. Specifically, the binding of O2 to the heme Fe2+ pulls the iron into the plane of the heme (Fig. 3.5). Because the iron is also linked to the proximal histidine (F8), the resulting movement of the globin chains alters the interface between the αβ dimers. This leads to a structure called the “R,” or relaxed form (see Fig. 3.4). The R conformation is the high-oxygen-affinity form of hemoglobin.
oxygen (O2) is not bound. B. Into the plane of the heme upon O2 binding.
D. Oxygen binding to myoglobin and hemoglobin
Myoglobin can bind only one molecule of O2, because it contains only one heme group. In contrast, hemoglobin can bind four molecules of O2, one at each of its four heme groups. The degree of saturation (Y) of these oxygen-binding sites on all myoglobin or hemoglobin molecules can vary between zero (all sites are empty) and 100% (all sites are full), as shown in Figure 3.6. [Note: Pulse oximetry is a noninvasive, indirect method of measuring the oxygen saturation of arterial blood based on differences in light absorption by oxyhemoglobin and deoxyhemoglobin.] 1. Oxygen-dissociation curve: A plot of Y measured at different partial pressures of oxygen (pO2) is called the oxygen-dissociation curve. [Note: pO2 may also be represented as PO2.] The curves for myoglobin and hemoglobin show important differences (see Fig. 3.6). This graph illustrates that myoglobin has a higher oxygen affinity at all pO2 values than does hemoglobin. The partial pressure of oxygen needed to achieve half saturation of the binding sites (P50) is ~1 mm Hg for myoglobin and 26 mm Hg for hemoglobin. The higher the oxygen affinity (that is, the more tightly O2 binds), the lower the P50.
a. Myoglobin: The oxygen-dissociation curve for myoglobin has a hyperbolic shape (see Fig. 3.6). This reflects the fact that myoglobin reversibly binds a single molecule of O2. Thus, oxygenated (MbO2) and deoxygenated (Mb) myoglobin exist in a simple equilibrium:
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The equilibrium is shifted to the right or to the left as O2 is added to or removed from the system. [Note: Myoglobin is designed to bind O2 released by hemoglobin at the low pO2 found in muscle. Myoglobin, in turn, releases O2 within the muscle cell in response to oxygen demand.] b. Hemoglobin: The oxygen-dissociation curve for hemoglobin is sigmoidal in shape (see Fig. 3.6), indicating that the subunits cooperate in binding O2. Cooperative binding of O2 by the four subunits of hemoglobin means that the binding of an oxygen molecule at one subunit increases the oxygen affinity of the remaining subunits in the same hemoglobin tetramer (Fig. 3.7). Although it is more difficult for the first oxygen molecule to bind to hemoglobin, the subsequent binding of oxygen molecules occurs with high affinity, as shown by the steep upward curve in the region near 20–30 mm Hg (see Fig. 3.6).
E. Allosteric effectors
The ability of hemoglobin to reversibly bind O2 is affected by the pO2, the pH of the environment, the partial pressure of carbon dioxide (pCO2), and the availability of 2,3-bisphosphoglycerate (2,3-BPG). These are collectively called allosteric (“other site”) effectors, because their interaction at one site on the tetrameric hemoglobin molecule causes structural changes that affect the binding of O2 to the heme iron at other sites on the molecule. [Note: The binding of O2 to monomeric myoglobin is not influenced by allosteric effectors.] 1. Oxygen: The sigmoidal oxygen-dissociation curve reflects specific structural changes that are initiated at one subunit and transmitted to other subunits in the hemoglobin tetramer. The net effect of this cooperativity is that the affinity of hemoglobin for the last oxygen molecule bound is ~300 times greater than its affinity for the first oxygen molecule bound. Oxygen, then, is an allosteric effector of hemoglobin. It stabilizes the R form.
a. Loading and unloading oxygen: The cooperative binding of O2 allows hemoglobin to deliver more O2 to the tissues in response to relatively small changes in the pO2. This can be seen in Figure 3.6, which indicates pO2 in the alveoli of the lung and the capillaries of the tissues. For example, in the lung, oxygen concentration is high, and hemoglobin becomes virtually saturated (or “loaded”) with O2. In contrast, in the peripheral tissues, oxyhemoglobin releases (or “unloads”) much of its O2 for use in the oxidative metabolism of the tissues (Fig. 3.8).
b. Significance of the sigmoidal oxygen-dissociation curve: The steep slope of the oxygen-dissociation curve over the range of oxygen concentrations that occur between the lungs and the tissues permits hemoglobin to carry and deliver O2 efficiently from sites of high to sites of low pO2. A molecule with a hyperbolic oxygen-dissociation curve, such as myoglobin, could not achieve the same degree of O2 release within this range of pO2. Instead, it would have maximum affinity for O2 throughout this oxygen pressure range and, therefore, would deliver no O2 to the tissues.
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2. Bohr effect: The release of O2 from hemoglobin is enhanced when the pH is lowered (proton concentration [H+] is increased) or when the hemoglobin is in the presence of an increased pCO2. Both result in decreased oxygen affinity of hemoglobin and, therefore, a shift to the right in the oxygen-dissociation curve (Fig. 3.9). Both, then, stabilize the T (deoxy) form. This change in oxygen binding is called the Bohr effect. Conversely, raising the pH or lowering the concentration of CO2 results in a greater oxygen affinity, a shift to the left in the oxygen-dissociation curve, and stabilization of the R (oxy) form.
a. Source of the protons that lower pH: The concentration of both H+ and
CO2 in the capillaries of metabolically active tissues is higher than that observed in alveolar capillaries of the lungs, where CO2 is released into the expired air. In the tissues, CO2 is converted by zinc-containing carbonic anhydrase to carbonic acid: which spontaneously loses a H+, becoming bicarbonate (the major blood buffer):
The H+ produced by this pair of reactions contributes to the lowering of pH. This differential pH gradient (that is, lungs having a higher pH and tissues a lower pH) favors the unloading of O2 in the peripheral tissues and the loading of O2 in the lung. Thus, the oxygen affinity of the hemoglobin molecule responds to small shifts in pH between the lungs and oxygen-consuming tissues, making hemoglobin a more efficient transporter of O2.
b. Mechanism of the Bohr effect: The Bohr effect reflects the fact that the deoxy form of hemoglobin has a greater affinity for H+ than does oxyhemoglobin. This is caused by ionizable groups such as specific histidine side chains that have a higher pKa (see p. 6) in deoxyhemoglobin than in oxyhemoglobin. Therefore, an increase in the concentration of H+ (resulting in a decrease in pH) causes these groups to become protonated (charged) and able to form ionic bonds (salt bridges). These bonds preferentially stabilize the deoxy form of hemoglobin, producing a decrease in oxygen affinity. [Note: Hemoglobin, then, is an important blood buffer.]
The Bohr effect can be represented schematically as: where an increase in H+ (or a lower pO2) shifts the equilibrium to the right (favoring deoxyhemoglobin), whereas an increase in pO2 (or a decrease in H+) shifts the equilibrium to the left.
3. 2,3-BPG effect on oxygen affinity: 2,3-BPG is an important regulator of the binding of O2 to hemoglobin. It is the most abundant organic phosphate in the RBC, where its concentration is approximately that of hemoglobin. 2,3-BPG is synthesized from an intermediate of the glycolytic pathway (Fig. 3.10; see p. 101 for a discussion of 2,3-BPG synthesis in glycolysis).
a.
2,3-BPG binding to deoxyhemoglobin: 2,3-BPG decreases the oxygen affinity of hemoglobin by binding to deoxyhemoglobin but not to oxyhemoglobin. This preferential binding stabilizes the T conformation of deoxyhemoglobin. The effect of binding 2,3-BPG can be represented schematically as: b.
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2,3-BPG binding site: One molecule of 2,3-BPG binds to a pocket, formed by the two β-globin chains, in the center of the deoxyhemoglobin tetramer (Fig. 3.11). This pocket contains several positively charged amino acids that form ionic bonds with the negatively charged phosphate groups of 2,3-BPG. [Note: Replacement of one of these amino acids can result in hemoglobin variants with abnormally high oxygen affinity that may be compensated for by increased RBC production (erythrocytosis).] Oxygenation of hemoglobin narrows the pocket and causes 2,3-BPG to be released.
c.
Oxygen-dissociation curve shift: Hemoglobin from which 2,3-BPG has been removed has high oxygen affinity. However, as seen in the RBC, the presence of 2,3-BPG significantly reduces the oxygen affinity of hemoglobin, shifting the oxygen-dissociation curve to the right (Fig. 3.12). This reduced affinity enables hemoglobin to release O2 efficiently at the partial pressures found in the tissues.
d. 2,3-BPG levels in chronic hypoxia or anemia: The concentration of 2,3-BPG in the RBC increases in response to chronic hypoxia, such as that observed in chronic obstructive pulmonary disease (COPD) like emphysema, or at high altitudes, where circulating hemoglobin may have difficulty receiving sufficient O2. Intracellular levels of 2,3-BPG are also elevated in chronic anemia, in which fewer than normal RBC are available to supply the body’s oxygen needs. Elevated 2,3-BPG levels lower the oxygen affinity of hemoglobin, permitting greater unloading of O2 in the capillaries of tissues (see Fig. 3.12).
e. 2,3-BPG in transfused blood: 2,3-BPG is essential for the normal oxygen transport function of hemoglobin. However, storing blood in the currently available media results in the gradual depletion of 2,3BPG. Consequently, stored blood displays an abnormally high oxygen affinity and fails to unload its bound O2 properly in the tissues. Thus, hemoglobin deficient in 2,3-BPG acts as an oxygen “trap” rather than as an oxygen delivery system. Transfused RBC are able to restore their depleted supplies of 2,3-BPG in 6–24 hours. However, severely ill patients may be compromised if transfused with large quantities of such 2,3-BPG–depleted blood. Stored blood, therefore, is treated with a “rejuvenation” solution that rapidly restores 2,3-BPG. [Note: Rejuvenation also restores ATP lost during storage.] 4. CO2 binding: Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion (see Fig. 1.12 on p. 9). However, some CO2 is carried as carbamate bound to the terminal amino groups of hemoglobin (forming carbaminohemoglobin as shown in Fig. 3.8), which can be represented schematically as follows:
The binding of CO2 stabilizes the T, or deoxy, form of hemoglobin, resulting in a decrease in its oxygen affinity (see p. 28) and a right shift in the oxygen-dissociation curve. In the lungs, CO2 dissociates from the hemoglobin and is released in the breath.
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5. CO binding: Carbon monoxide (CO) binds tightly (but reversibly) to the hemoglobin iron, forming carboxyhemoglobin. When CO binds to one or more of the four heme sites, hemoglobin shifts to the R conformation, causing the remaining heme sites to bind O2 with high affinity. This shifts the oxygen-dissociation curve to the left and changes the normal sigmoidal shape toward a hyperbola. As a result, the affected hemoglobin is unable to release O2 to the tissues (Fig. 3.13). [Note: The affinity of hemoglobin for CO is 220 times greater than for O2. Consequently, even minute concentrations of CO in the environment can produce toxic concentrations of carboxyhemoglobin in the blood. For example, increased levels of CO are found in the blood of tobacco smokers. CO toxicity appears to result from a combination of tissue hypoxia and direct CO-mediated damage at the cellular level.] CO poisoning is treated with 100% O2 at high pressure (hyperbaric oxygen therapy), which facilitates the dissociation of CO from the hemoglobin. [Note: CO inhibits Complex IV of the electron transport chain (see p. 76).] In addition to O2, CO2, and CO, nitric oxide gas (NO) also is carried by hemoglobin. NO is a potent vasodilator (see p. 151). It can be taken up (salvaged) or released from RBC, thereby modulating NO availability and influencing vessel diameter.
F. Minor hemoglobins
It is important to remember that human hemoglobin A (HbA) is just one member of a functionally and structurally related family of proteins, the hemoglobins (Fig. 3.14). Each of these oxygen-carrying proteins is a tetramer, composed of two α-globin (or α-like) polypeptides and two βglobin (or β-like) polypeptides. Certain hemoglobins, such as HbF, are normally synthesized only during fetal development, whereas others, such as HbA2, are synthesized in the adult, although at low levels compared with HbA. HbA can also become modified by the covalent addition of a hexose (see 3. below).
1. Fetal hemoglobin: HbF is a tetramer consisting of two α chains identical to those found in HbA, plus two γ chains (α2γ2; see Fig. 3.14). The γ chains are members of the β-globin gene family (see p. 34).
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a. HbF synthesis during development: In the first month after conception, embryonic hemoglobins such as Hb Gower 1, composed of two α-like zeta (ζ) chains and two β-like epsilon (ε) chains (ζ2ε2), are synthesized by the embryonic yolk sac. In the fifth week of gestation, the site of globin synthesis shifts, first to the liver and then to the marrow, and the primary product is HbF. HbF is the major hemoglobin found in the fetus and newborn, accounting for ~60% of the total hemoglobin in the RBC during the last months of fetal life (Fig. 3.15). HbA synthesis starts in the bone marrow at about the eighth month of pregnancy and gradually replaces HbF. Figure 3.15 shows the relative production of each type of hemoglobin chain during fetal and postnatal life. [Note: HbF represents <2% of the hemoglobin in most adults and is concentrated in RBC known as F cells.] b. 2,3-BPG binding to HbF: Under physiologic conditions, HbF has a higher oxygen affinity than does HbA as a result of HbF only weakly binding 2,3-BPG. [Note: The γ-globin chains of HbF lack some of the positively charged amino acids that are responsible for binding 2,3BPG in the β-globin chains.] Because 2,3-BPG serves to reduce the oxygen affinity of hemoglobin, the weaker interaction between 2,3BPG and HbF results in a higher oxygen affinity for HbF relative to HbA. In contrast, if both HbA and HbF are stripped of their 2,3-BPG, they then have a similar oxygen affinity. The higher oxygen affinity of HbF facilitates the transfer of O2 from the maternal circulation across the placenta to the RBC of the fetus.
2.
Hemoglobin A2: HbA2 is a minor component of normal adult hemoglobin, first appearing shortly before birth and, ultimately, constituting ~2% of the total hemoglobin. It is composed of two α-globin chains and two δ-globin chains (α2δ2; see Fig. 3.14).
3.
Hemoglobin A1c: Under physiologic conditions, HbA is slowly glycated (nonenzymically condensed with a hexose), the extent of glycation being dependent on the plasma concentration of the hexose. The most abundant form of glycated hemoglobin is HbA1c. It has glucose residues attached predominantly to the amino groups of the N-terminal valines of the βglobin chains (Fig. 3.16). Increased amounts of HbA1c are found in RBC of patients with diabetes mellitus, because their HbA has contact with higher glucose concentrations during the 120-day lifetime of these cells. (See p. 340 for a discussion of the use of HbA1c levels in assessing average blood glucose levels in patients with diabetes.)
III. GLOBIN GENE ORGANIZATION
To understand diseases resulting from genetic alterations in the structure or synthesis of hemoglobin, it is necessary to grasp how the hemoglobin genes, which direct the synthesis of the different globin chains, are structurally organized into gene families and also how they are expressed.
A. α-Gene family
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The genes coding for the α-globin and β-globin subunits of the hemoglobin chains occur in two separate gene clusters (or families) located on two different chromosomes (Fig. 3.17). The α-gene cluster on chromosome 16 contains two genes for the α-globin chains. It also contains the ζ gene that is expressed early in development as an α-globin-like component of embryonic hemoglobin. [Note: Globin gene families also contain globinlike genes that are not expressed, that is, their genetic information is not used to produce globin chains. These are called pseudogenes.]
B. β-Gene family
A single gene for the β-globin chain is located on chromosome 11 (see Fig. 3.17). There are an additional four β-globin-like genes: the ε gene (which, like the ζ gene, is expressed early in embryonic development), two γ genes (Gγ and Aγ that are expressed in HbF), and the δ gene that codes for the globin chain found in the minor adult hemoglobin HbA2.
C. Steps in globin chain synthesis
Expression of a globin gene begins in the nucleus of RBC precursors, where the DNA sequence encoding the gene is transcribed. The ribonucleic acid (RNA) produced by transcription is actually a precursor of the messenger RNA (mRNA) that is used as a template for the synthesis of a globin chain. Before it can serve this function, two noncoding stretches of RNA (introns) must be removed from the mRNA precursor sequence and the remaining three fragments (exons) joined in a linear manner. The resulting mature mRNA enters the cytosol, where its genetic information is translated, producing a globin chain. (A summary of this process is shown in Figure 3.18. A more detailed description of gene expression is presented in Unit VII, Chapters 30–32.)
IV. HEMOGLOBINOPATHIES
Hemoglobinopathies are defined as a group of genetic disorders caused by production of a structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin, or, rarely, both. Sickle cell anemia (HbS), hemoglobin C disease (HbC), hemoglobin SC disease (HbS + HbC = HbSC), and the thalassemias are representative hemoglobinopathies that can have severe clinical consequences. The first three conditions result from production of hemoglobin with an altered amino acid sequence (qualitative hemoglobinopathy), whereas the thalassemias are caused by decreased production of normal hemoglobin (quantitative hemoglobinopathy).
A. Sickle cell anemia (hemoglobin S disease)
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Sickle cell anemia, the most common of the RBC sickling diseases, is a genetic disorder caused by a single nucleotide substitution (a point mutation, see p. 449) in the gene for β-globin. It is the most common inherited blood disorder in the United States, affecting 50,000 Americans. It occurs primarily in the African American population, affecting 1 in 500 newborn African American infants. Sickle cell anemia is an autosomalrecessive disorder. It occurs in individuals who have inherited two mutant genes (one from each parent) that code for synthesis of the β chains of the globin molecules. [Note: The mutant β-globin chain is designated βS, and the resulting hemoglobin, α2βS2, is referred to as HbS.] An infant does not begin showing symptoms of the disease until sufficient HbF has been replaced by HbS so that sickling can occur (see p. 36). Sickle cell anemia is characterized by lifelong episodes of pain (“crises”), chronic hemolytic anemia with associated hyperbilirubinemia (see p. 284), and increased susceptibility to infections, usually beginning in infancy. [Note: The lifetime of a RBC in sickle cell anemia is <20 days, compared with 120 days for normal RBC, hence, the anemia.] Other symptoms include acute chest syndrome, stroke, splenic and renal dysfunction, and bone changes due to marrow hyperplasia. Life expectancy is reduced. Heterozygotes, representing 1 in 12 African Americans, have one normal and one sickle cell gene. The blood cells of such heterozygotes contain both HbS and HbA, and these individuals have sickle cell trait. They usually do not show clinical symptoms (but may under conditions of extreme physical exertion with dehydration) and can have a normal life span.
1.
Amino acid substitution in HbS β chains:: A molecule of HbS contains two normal α-globin chains and two mutant β-globin chains (βS), in which glutamate at position six has been replaced with valine (Fig. 3.19). Therefore, during electrophoresis at alkaline pH, HbS migrates more slowly toward the anode (positive electrode) than does HbA (Fig. 3.20). This altered mobility of HbS is a result of the absence of the negatively charged glutamate residues in the two β chains, thereby rendering HbS less negative than HbA. [Note: Electrophoresis of hemoglobin obtained from lysed RBC is routinely used in the diagnosis of sickle cell trait and sickle cell anemia (or, sickle cell disease). DNA analysis also is used (see p. 493).] 2.
Sickling and tissue anoxia: The replacement of the charged glutamate with the nonpolar valine forms a protrusion on the β chain that fits into a complementary site on the β chain of another hemoglobin molecule in the cell (Fig. 3.21). At low oxygen tension, deoxyhemoglobin S polymerizes inside the RBC, forming a network of insoluble fibrous polymers that stiffen and distort the cell, producing rigid, misshapen RBC. Such sickled cells frequently block the flow of blood in the narrow capillaries. This interruption in the supply of O2 leads to localized anoxia (oxygen deprivation) in the tissue, causing pain and eventually ischemic death (infarction) of cells in the vicinity of the blockage. The anoxia also leads to an increase in deoxygenated HbS. [Note: The mean diameter of RBC is 7.5 µm, whereas that of the microvasculature is 3–4 µm. Compared to normal RBC, sickled cells have a decreased ability to deform and an increased tendency to adhere to vessel walls. This makes moving through small vessels difficult, thereby causing microvascular occlusion.] 3.
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Variables that increase sickling: The extent of sickling and, therefore, the severity of disease are enhanced by any variable that increases the proportion of HbS in the deoxy state (that is, reduces the oxygen affinity of HbS). These variables include decreased pO2, increased pCO2, decreased pH, dehydration, and an increased concentration of 2,3-BPG in RBC.
4.
Treatment: Therapy involves adequate hydration, analgesics, aggressive antibiotic therapy if infection is present, and transfusions in patients at high risk for fatal occlusion of blood vessels. Intermittent transfusions with packed RBC reduce the risk of stroke, but the benefits must be weighed against the complications of transfusion, which include iron overload that can result in hemosiderosis (see p. 404), bloodborne infections, and immunologic complications. Hydroxyurea (hydroxycarbamide), an antitumor drug, is therapeutically useful because it increases circulating levels of HbF, which decreases RBC sickling. This leads to decreased frequency of painful crises and reduces mortality. Stem cell transplantation is possible. [Note: The morbidity and mortality associated with sickle cell anemia has led to its inclusion in newborn screening panels to allow prophylactic antibiotic therapy to begin soon after the birth of an affected child.] 5.
Possible selective advantage of the heterozygous state: The high frequency of the βS mutation among black Africans, despite its damaging effects in the homozygous state, suggests that a selective advantage exists for heterozygous individuals. For example, heterozygotes for the sickle cell gene are less susceptible to the severe malaria caused by the parasite Plasmodium falciparum. This organism spends an obligatory part of its life cycle in the RBC. One theory is that because these cells in individuals heterozygous for HbS, like those in homozygotes, have a shorter life span than normal, the parasite cannot complete the intracellular stage of its development. This may provide a selective advantage to heterozygotes living in regions where malaria is a major cause of death. For example, in Africa, the geographic distribution of sickle cell anemia is similar to that of malaria.
B. Hemoglobin C disease
Like HbS, HbC is a hemoglobin variant that has a single amino acid substitution in the sixth position of the β-globin chain (see Fig. 3.19). In HbC, however, a lysine is substituted for the glutamate (as compared with a valine substitution in HbS). [Note: This substitution causes HbC to move more slowly toward the anode than HbA or HbS does (see Fig. 3.20).] Rare patients homozygous for HbC generally have a relatively mild, chronic hemolytic anemia. They do not suffer from infarctive crises, and no specific therapy is required.
C. Hemoglobin SC disease
HbSC disease is another of the RBC sickling diseases. In this disease, some β-globin chains have the sickle cell mutation, whereas other β-globin chains carry the mutation found in HbC disease. [Note: Patients with HbSC disease are doubly heterozygous. They are called compound heterozygotes because both of their β-globin genes are abnormal, although different from each other.] Hemoglobin levels tend to be higher in HbSC disease than in sickle cell anemia and may even be at the low end of the normal range. The clinical course of adults with HbSC anemia differs from that of sickle cell anemia in that symptoms such as painful crises are less frequent and less severe. However, there is significant clinical variability.
D. Methemoglobinemias
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Oxidation of the heme iron in hemoglobin from Fe2+ to Fe3+ produces methemoglobin, which cannot bind O2. This oxidation may be acquired and caused by the action of certain drugs, such as nitrates, or endogenous products such as reactive oxygen species (see p. 148). The oxidation may also result from congenital defects, for example, a deficiency of NADH-cytochrome b5 reductase (also called NADH-methemoglobin reductase), the enzyme responsible for the conversion of methemoglobin (Fe3+) to hemoglobin (Fe2+), leads to the accumulation of methemoglobin (Fig. 3.22). [Note: The RBC of newborns have approximately half the capacity of those of adults to reduce methemoglobin.] Additionally, rare mutations in the α-or β-globin chain can cause the production of HbM, an abnormal hemoglobin that is resistant to the reductase. The methemoglobinemias are characterized by “chocolate cyanosis” (a blue coloration of the skin and mucous membranes and brown-colored blood) as a result of the dark-colored methemoglobin. Symptoms are related to the degree of tissue hypoxia and include anxiety, headache, and dyspnea. In rare cases, coma and death can occur. Treatment is with methylene blue, which is oxidized as Fe3+ is reduced.
E. Thalassemias
The thalassemias are hereditary hemolytic diseases in which an imbalance occurs in the synthesis of globin chains. As a group, they are the most common single-gene disorders in humans. Normally, synthesis of the αand β-globin chains is coordinated, so that each α-globin chain has a βglobin chain partner. This leads to the formation of α2β2 (HbA). In the thalassemias, the synthesis of either the α-or the β-globin chain is defective, and hemoglobin concentration is reduced. A thalassemia can be caused by a variety of mutations, including entire gene deletions, or substitutions or deletions of one of many nucleotides in the DNA. [Note: Each thalassemia can be classified as either a disorder in which no globin chains are produced (α0-or β0-thalassemia), or one in which some chains are synthesized but at a reduced level (α+-or β+-thalassemia).] 1.
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β-Thalassemias: In these disorders, synthesis of β-globin chains is decreased or absent, typically as a result of point mutations that affect the production of functional mRNA. However, α-globin chain synthesis is normal. Excess α-globin chains cannot form stable tetramers and so precipitate, causing the premature death of cells initially destined to become mature RBC. Increase in α2δ2 (HbA2) and α2γ2 (HbF) also occurs. There are only two copies of the β-globin gene in each cell (one on each chromosome 11). Therefore, individuals with β-globin gene defects have either β-thalassemia trait (β-thalassemia minor) if they have only one defective β-globin gene or β-thalassemia major (Cooley anemia) if both genes are defective (Fig. 3.23). Because the β-globin gene is not expressed until late in prenatal development, the physical manifestations of β-thalassemias appear only several months after birth. Those individuals with β-thalassemia minor make some β chains and usually do not require specific treatment. However, those infants born with β-thalassemia major are seemingly healthy at birth but become severely anemic, usually during the first or second year of life, due to ineffective erythropoiesis. Skeletal changes as a result of extramedullary hematopoiesis also are seen. These patients require regular transfusions of blood. [Note: Although this treatment is lifesaving, the cumulative effect of the transfusions is iron overload. Use of iron chelation therapy has improved morbidity and mortality.] The only curative option available is hematopoietic stem cell transplantation.
2.
α-Thalassemias: In these disorders, synthesis of α-globin chains is decreased or absent, typically as a result of deletional mutations. Because each individual’s genome contains four copies of the α-globin gene (two on each chromosome 16), there are several levels of α-globin chain deficiencies (Fig. 3.24). If one of the four genes is defective, the individual is termed a “silent” carrier of α-thalassemia, because no physical manifestations of the disease occur. If two α-globin genes are defective, the individual is designated as having α-thalassemia trait. If three α-globin genes are defective, the individual has hemoglobin H (β4) disease, a hemolytic anemia of variable severity. If all four α-globin genes are defective, hemoglobin Bart (γ4) disease with hydrops fetalis and fetal death results, because α-globin chains are required for the synthesis of HbF. [Note: Heterozygote advantage against malaria is seen in both α-and β-thalassemias.]
V. CHAPTER SUMMARY
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Hemoglobin A (HbA), the major hemoglobin in adults, is composed of four polypeptide chains (two α chains and two β chains, α2β2) held together by noncovalent interactions (Fig. 3.25). The subunits occupy different relative positions in deoxyhemoglobin compared with oxyhemoglobin. The deoxy form of Hb is called the “T,” or taut (tense), conformation. It has a constrained structure that limits the movement of the polypeptide chains. The T form is the low-oxygen-affinity form of Hb. The binding of oxygen (O2) to the heme iron causes rupture of some of the ionic and hydrogen bonds and movement of the dimers. This leads to a structure called the “R,” or relaxed, conformation. The R form is the high-oxygen-affinity form of Hb. The oxygen-dissociation curve for Hb is sigmoidal in shape (in contrast to that of myoglobin, which is hyperbolic), indicating that the subunits cooperate in binding O2. The binding of an oxygen molecule at one heme group increases the oxygen affinity of the remaining heme groups in the same Hb molecule (cooperativity). Hb’s ability to bind O2 reversibly is affected by the partial pressure of oxygen (pO2), the pH of the environment, the partial pressure of carbon dioxide (pCO2), and the availability of 2,3-bisphosphoglycerate (2,3-BPG). For example, the release of O2 from Hb is enhanced when the pH is lowered or the pCO2 is increased (the Bohr effect), such as in exercising muscle, and the oxygen-dissociation curve of Hb is shifted to the right. To cope long-term with the effects of chronic hypoxia or anemia, the concentration of 2,3-BPG in red blood cells increases. 2,3-BPG binds to the Hb and decreases its oxygen affinity. It therefore also shifts the oxygen-dissociation curve to the right. Fetal hemoglobin (HbF) binds 2,3-BPG less tightly than does HbA and has a higher oxygen affinity. Carbon monoxide (CO) binds tightly (but reversibly) to the Hb iron, forming carboxyhemoglobin. Hemoglobinopathies are disorders primarily caused either by production of a structurally abnormal Hb molecule as in sickle cell anemia or synthesis of insufficient quantities of normal Hb subunits as in the thalassemias (Fig. 3.26).
Choose the ONE best answer.
.1. Which one of the following statements concerning the hemoglobins is correct?
A. HbA is the most abundant hemoglobin in normal adults.
B. Fetal blood has a lower affinity for oxygen than does adult blood because HbF has an increased affinity for 2,3-bisphosphoglycerate.
C. The globin chain composition of HbF is α2δ2.
D. HbA1c differs from HbA by a single, genetically determined amino acid substitution.
E. HbA2 appears early in fetal life.
Correct answer = A. HbA accounts for over 90% of the hemoglobin in a normal adult. If HbA1c is included, the percentage rises to ~97%. Because 2,3bisphosphoglycerate (2,3-BPG) reduces the affinity of hemoglobin for oxygen, the weaker interaction between 2,3-BPG and HbF results in a higher oxygen affinity for HbF relative to HbA. HbF consists of α2γ2. HbA1c is a glycated form of HbA, formed nonenzymically in red blood cells. HbA2 is a minor component of normal adult hemoglobin, first appearing shortly before birth and rising to adult levels (~2% of the total hemoglobin) by age 6 months.
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.2. Which one of the following statements concerning the ability of acidosis to precipitate a crisis in sickle cell anemia is correct?
A. Acidosis decreases the solubility of HbS.
B. Acidosis increases the oxygen affinity of hemoglobin.
C. Acidosis favors the conversion of hemoglobin from the taut to the relaxed conformation.
D. Acidosis shifts the oxygen-dissociation curve to the left.
E. Acidosis decreases the ability of 2,3-bisphosphoglycerate to bind to hemoglobin.
Correct answer = A. HbS is significantly less soluble in the deoxygenated form, compared with oxyhemoglobin S. Decreased pH (acidosis) causes the oxygen-dissociation curve to shift to the right, indicating decreased oxygen affinity (increased delivery). This favors the formation of the deoxy, or taut, form of hemoglobin and can precipitate a sickle cell crisis. The binding of 2,3bisphosphoglycerate is increased, because it binds only to the deoxy form of hemoglobin.
.3. Which one of the following statements concerning the binding of oxygen by hemoglobin is correct?
A. The Bohr effect results in a lower oxygen affinity at higher pH values.
B. Carbon dioxide increases the oxygen affinity of hemoglobin by binding to the C-terminal groups of the polypeptide chains.
C. The oxygen affinity of hemoglobin increases as the percentage saturation increases.
D. The hemoglobin tetramer binds four molecules of 2,3bisphosphoglycerate.
E. Oxyhemoglobin and deoxyhemoglobin have the same affinity for protons.
Correct answer = C. The binding of oxygen at one heme group increases the oxygen affinity of the remaining heme groups in the same molecule. A rise in pH results in increased oxygen affinity. Carbon dioxide decreases oxygen affinity because it lowers the pH. Moreover, binding of carbon dioxide to the N-termini stabilizes the taut, deoxy form. Hemoglobin binds one molecule of 2,3-bisphosphoglycerate. Deoxyhemoglobin has a greater affinity for protons than does oxyhemoglobin.
.4. β-Lysine 82 in HbA is important for the binding of 2,3-bisphosphoglycerate. In Hb Helsinki, this amino acid has been replaced by methionine. Which of the following should be true concerning Hb Helsinki?
A. It should be stabilized in the taut, rather than the relaxed, form.
B. It should have increased oxygen affinity and, consequently, decreased oxygen delivery to tissues.
C. Its oxygen-dissociation curve should be shifted to the right relative to HbA.
D. It results in anemia.
Correct answer = B. Substitution of lysine by methionine decreases the ability of negatively charged phosphate groups in 2,3-bisphosphoglycerate (2,3-BPG) to bind the β subunits of hemoglobin. Because 2,3-BPG decreases the oxygen affinity of hemoglobin, a reduction in 2,3-BPG should result in increased oxygen affinity and decreased oxygen (O2) delivery to tissues. The relaxed form is the high-oxygen-affinity form of hemoglobin. Increased oxygen affinity (decreased delivery) results in a left shift in the oxygen-dissociation curve. Decreased delivery of O2 is compensated for by increased RBC production.
.5. A 67-year-old man presented to the emergency department with a 1-week history of angina and shortness of breath. He complained that his face and extremities had taken on a blue color. His medical history included chronic stable angina treated with isosorbide dinitrate and nitroglycerin. Blood obtained for analysis was brown. Which one of the following is the most likely diagnosis?
A. Carboxyhemoglobinemia
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B. Hemoglobin SC disease
C. Methemoglobinemia
D. Sickle cell anemia
E. β-Thalassemia
Correct answer = C. Oxidation of the ferrous (Fe2+) iron to the ferric (Fe3+) state in the heme prosthetic group of hemoglobin forms methemoglobin. This may be caused by the action of certain drugs such as nitrates. The methemoglobinemias are characterized by chocolate cyanosis (a blue coloration of the skin and mucous membranes and chocolate-colored blood) as a result of the dark-colored methemoglobin. Symptoms are related to tissue hypoxia and include anxiety, headache, and dyspnea. In rare cases, coma and death can occur. [Note: Benzocaine, an aromatic amine used as a topical anesthetic, is a cause of acquired methemoglobinemia.] .6. Why is hemoglobin C disease a nonsickling disease?
In HbC, the polar glutamate is replaced by polar lysine rather than by nonpolar valine as in HbS.
.7. What would be true about the extent of red blood cell sickling in individuals with HbS and hereditary persistence of HbF?
It would be decreased because HbF reduces HbS concentration. It also inhibits polymerization of deoxy HbS.
For additional ancillary materials related to this chapter, please visit thePoint.
I. OVERVIEW
Collagen and elastin are examples of common, well-characterized fibrous proteins of the extracellular matrix (ECM) that serve structural functions in the body. For example, collagen and elastin are found as components of skin, connective tissue, blood vessel walls, and the sclera and cornea of the eye. Each fibrous protein exhibits special mechanical properties, resulting from its unique structure, which is obtained by combining specific amino acids into regular, secondary structural elements. This is in contrast to globular proteins (discussed in Chapter 3), whose shapes are the result of complex interactions between secondary, tertiary, and, sometimes, quaternary structural elements.
II. COLLAGEN
Collagen is the most abundant protein in the human body. A typical collagen molecule is a long, rigid structure in which three polypeptides (referred to as α chains) are wound around one another in a rope-like triple helix (Fig. 4.1). Although these molecules are found throughout the body, their types and organization are dictated by the structural role collagen plays in a particular organ. In some tissues, collagen may be dispersed as a gel that gives support to the structure, as in the ECM or the vitreous humor of the eye. In other tissues, collagen may be bundled in tight, parallel fibers that provide great strength, as in tendons. In the cornea of the eye, collagen is stacked so as to transmit light with a minimum of scattering. Collagen of bone occurs as fibers arranged at an angle to each other so as to resist mechanical shear from any direction.
A. Types
The collagen superfamily of proteins includes >25 collagen types as well as additional proteins that have collagen-like domains. The three polypeptide α chains are held together by interchain hydrogen bonds. Variations in the amino acid sequence of the α chains result in structural components that are about the same size (~1,000 amino acids long) but with slightly different properties. These α chains are combined to form the various types of collagen found in the tissues. For example, the most common collagen, type I, contains two chains called α1 and one chain called α2 (α12α2), whereas type II collagen contains three α1 chains (α13). The collagens can be organized into three groups, based on their location and functions in the body (Fig. 4.2).
1.
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Fibril-forming collagens: Types I, II, and III are the fibrillar collagens and have the rope-like structure described above for a typical collagen molecule. In the electron microscope, these linear polymers of fibrils have characteristic banding patterns, reflecting the regular staggered packing of the individual collagen molecules in the fibril (Fig. 4.3). Type I collagen fibers (composed of collagen fibrils) are found in supporting elements of high tensile strength (for example, tendons and corneas), whereas fibers formed from type II collagen molecules are restricted to cartilaginous structures. The fibers derived from type III collagen are prevalent in more distensible tissues such as blood vessels.
2.
Network-forming collagens: Types IV and VIII form a three-dimensional mesh, rather than distinct fibrils (Fig. 4.4). For example, type IV molecules assemble into a sheet or meshwork that constitutes a major part of basement membranes.
Basement membranes are thin, sheet-like structures that provide mechanical support for adjacent cells and function as a semipermeable filtration barrier to macromolecules in organs such as the kidney and the lung.
3. Fibril-associated collagens: Types IX and XII bind to the surface of collagen fibrils, linking these fibrils to one another and to other components in the ECM (see Fig. 4.2).
B. Structure
Unlike most globular proteins that are folded into compact structures, collagen, a fibrous protein, has an elongated, triple-helical structure that is stabilized by interchain hydrogen bonds.
1.
Amino acid sequence: Collagen is rich in proline and glycine, both of which are important in the formation of the triple-stranded helix. Proline facilitates the formation of the helical conformation of each α chain because its ring structure causes “kinks” in the peptide chain. [Note: The presence of proline dictates that the helical conformation of the α chain cannot be an α helix (see p. 16).] Glycine, the smallest amino acid, is found in every third position of the polypeptide chain. It fits into the restricted spaces where the three chains of the helix come together. The glycine residues are part of a repeating sequence, −Gly–X–Y–, where X is frequently proline, and Y is often hydroxyproline (but can be hydroxylysine, Fig. 4.5). Thus, most of the α chain can be regarded as a polytripeptide whose sequence can be represented as (−Gly–Pro– Hyp–)333.
2.
Hydroxyproline and hydroxylysine: Collagen contains hydroxyproline and hydroxylysine, which are nonstandard amino acids (see p. 1) not present in most other proteins. They result from the hydroxylation of some of the proline and lysine residues after their incorporation into polypeptide chains (Fig. 4.6). Therefore, the hydroxylation is a posttranslational modification (see p. 460). [Note: Generation of hydroxyproline maximizes formation of interchain hydrogen bonds that stabilize the triple-helical structure.] 3. Glycosylation: The hydroxyl group of the hydroxylysine residues of collagen may be enzymatically glycosylated. Most commonly, glucose and galactose are sequentially attached to the polypeptide chain prior to triple-helix formation (Fig. 4.7).
C. Biosynthesis
The polypeptide precursors of the collagen molecule are synthesized in fibroblasts (or in the related osteoblasts of bone and chondroblasts of cartilage). They are enzymically modified and form the triple helix, which gets secreted into the ECM. After additional enzymic modification, the mature extracellular collagen fibrils aggregate and become cross-linked to form collagen fibers.
1.
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Pro-α chain formation: Collagen is one of many proteins that normally function outside of cells. Like most proteins produced for export, the newly synthesized polypeptide precursors of α chains (prepro-α chains) contain a special amino acid sequence at their amino (N)-terminal ends. This sequence acts as a signal that, in the absence of additional signals, targets the polypeptide being synthesized for secretion from the cell. The signal sequence facilitates the binding of ribosomes to the rough endoplasmic reticulum (RER) and directs the passage of the prepro-α chain into the lumen of the RER. The signal sequence is rapidly cleaved in the lumen to yield a precursor of collagen called a pro-α chain (see Fig. 4.7).
2.
Hydroxylation: The pro-α chains are processed by a number of enzymic steps within the lumen of the RER while the polypeptides are still being synthesized (see Fig. 4.7). Proline and lysine residues found in the Y-position of the –Gly–X–Y– sequence can be hydroxylated to form hydroxyproline and hydroxylysine residues. These hydroxylation reactions require molecular oxygen, ferrous iron (Fe2+), and the reducing agent vitamin C (ascorbic acid, see p. 381), without which the hydroxylating enzymes, prolyl hydroxylase and lysyl hydroxylase, are unable to function (see Fig. 4.6). In the case of ascorbic acid deficiency (and, therefore, a lack of proline and lysine hydroxylation), interchain H-bond formation is impaired, as is formation of a stable triple helix. Additionally, collagen fibrils cannot be cross-linked (see 7. below), greatly decreasing the tensile strength of the assembled fiber. The resulting deficiency disease is known as scurvy. Patients with scurvy often show ecchymoses (bruise-like discolorations) on the limbs as a result of subcutaneous extravasation (leakage) of blood due to capillary fragility (Fig. 4.8).
3.
Glycosylation: Some hydroxylysine residues are modified by glycosylation with glucose or glucosyl-galactose (see Fig. 4.7).
4.
Assembly and secretion: After hydroxylation and glycosylation, three pro-α chains form procollagen, a precursor of collagen that has a central region of triple helix flanked by the nonhelical N-and carboxyl (C)terminal extensions called propeptides (see Fig. 4.7). The formation of procollagen begins with formation of interchain disulfide bonds between the C-terminal extensions of the pro-α chains. This brings the three α chains into an alignment favorable for triple helix formation. The procollagen molecules move through the Golgi apparatus, where they are packaged in secretory vesicles. The vesicles fuse with the cell membrane, causing the release of procollagen molecules into the extracellular space.
5.
Extracellular cleavage of procollagen molecules: After their release, the triple-helical procollagen molecules are cleaved by N-and Cprocollagen peptidases, which remove the terminal propeptides, producing tropocollagen molecules.
6.
Collagen fibril formation: Tropocollagen molecules spontaneously associate to form collagen fibrils. They form an ordered, parallel array, with adjacent collagen molecules arranged in a staggered pattern, each overlapping its neighbor by a length approximately three quarters of a molecule (see Fig. 4.7).
7.
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Cross-link formation: The fibrillar array of collagen molecules serves as a substrate for lysyl oxidase. This copper-containing extracellular enzyme oxidatively deaminates some of the lysine and hydroxylysine residues in collagen. The reactive aldehydes that result (allysine and hydroxyallysine) can spontaneously condense with lysine or hydroxylysine residues in neighboring collagen molecules to form covalent cross-links and, thus, mature collagen fibers (Fig. 4.9). [Note: Cross-links can form between two allysine residues.] peroxide.
Lysyl oxidase is one of several copper-containing enzymes. Others include ceruloplasmin (see p. 404), cytochrome c oxidase (see p. 76), dopamine hydroxylase (see p. 286), superoxide dismutase (see p. 148), and tyrosinase (see p. 273). Disruption in copper homeostasis causes copper deficiency (X-linked Menkes syndrome) or overload (Wilson disease) (see p. 402).
D. Degradation
Normal collagens are highly stable molecules, having half-lives as long as several years. However, connective tissue is dynamic and is constantly being remodeled, often in response to growth or injury of the tissue. Breakdown of collagen fibers is dependent on the proteolytic action of collagenases, which are part of a large family of matrix metalloproteinases. For type I collagen, the cleavage site is specific, generating three-quarter and one-quarter length fragments. These fragments are further degraded by other matrix proteinases.
E. Collagenopathies
Defects in any one of the many steps in collagen fiber synthesis can result in a genetic disease involving an inability of collagen to form fibers properly and, therefore, an inability to provide tissues with the needed tensile strength normally provided by collagen. More than 1,000 mutations have been identified in 23 genes coding for 13 of the collagen types. The following are examples of diseases (collagenopathies) that are the result of defective collagen synthesis.
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1. Ehlers-Danlos syndrome: Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders that result from heritable defects in the metabolism of fibrillar collagen molecules. EDS can be caused by a deficiency of collagen-processing enzymes (for example, lysyl hydroxylase or N-procollagen peptidase) or from mutations in the amino acid sequences of collagen types I, III, and V. The classic form of EDS, caused by defects in type V collagen, is characterized by skin extensibility and fragility and joint hypermobility (Fig. 4.10). The vascular form, due to defects in type III collagen, is the most serious form of EDS because it is associated with potentially lethal arterial rupture. [Note: The classic and vascular forms show autosomaldominant inheritance.] Collagen that contains mutant chains may have altered structure, secretion, or distribution, and it frequently is degraded. [Note: Incorporation of just one mutant chain may result in degradation of the triple helix. This is known as a dominant-negative effect.] 2. Osteogenesis imperfecta: This syndrome, known as “brittle bone disease,” is a genetic disorder of bone fragility characterized by bones that fracture easily, with minor or no trauma (Fig. 4.11). Over 80% of cases of osteogenesis imperfecta (OI) are caused by dominant mutations to the genes that encode the α1 or α2 chains in type I collagen. The most common mutations cause the replacement of glycine (in –Gly–X–Y–) by amino acids with bulky side chains. The resultant structurally abnormal α chains prevent the formation of the required triple-helical conformation. Phenotypic severity ranges from mild to lethal. Type I OI, the most common form, is characterized by mild bone fragility, hearing loss, and blue sclerae. Type II, the most severe form, is typically lethal in the perinatal period as a result of pulmonary complications. In utero fractures are seen (see Fig. 4.11). Type III is also a severe form and is characterized by multiple fractures at birth, short stature, spinal curvature leading to a humped-back (kyphotic) appearance, and blue sclerae. Dentinogenesis imperfecta, a disorder of tooth development, may be seen in OI.
III. ELASTIN
In contrast to collagen, which forms fibers that are tough and have high tensile strength, elastin is a connective tissue fibrous protein with rubber-like properties. Elastic fibers composed of elastin and glycoprotein microfibrils are found in the lungs, the walls of large arteries, and elastic ligaments. They can be stretched to several times their normal length but recoil to their original shape when the stretching force is relaxed.
A. Structure
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Elastin is an insoluble protein polymer generated from a precursor, tropoelastin, which is a soluble polypeptide composed of ~700 amino acids that are primarily small and nonpolar (for example, glycine, alanine, and valine). Elastin is also rich in proline and lysine but contains scant hydroxyproline and hydroxylysine. Tropoelastin is secreted by the cell into the ECM. There, it interacts with specific glycoprotein microfibrils, such as fibrillin, which function as a scaffold onto which tropoelastin is deposited. Some of the lysyl side chains of the tropoelastin polypeptides are oxidatively deaminated by lysyl oxidase, forming allysine residues. Three of the allysyl side chains plus one unaltered lysyl side chain from the same or neighboring polypeptides form a desmosine cross-link (Fig. 4.12). This produces elastin, an extensively interconnected, rubbery network that can stretch and bend in any direction when stressed, giving connective tissue elasticity (Fig. 4.13). Mutations in the fibrillin-1 protein are responsible for Marfan syndrome, a connective tissue disorder characterized by impaired structural integrity in the skeleton, the eye, and the cardiovascular system. With this disease, abnormal fibrillin protein is incorporated into microfibrils along with normal fibrillin, inhibiting the formation of functional microfibrils. [Note: Patients with Marfan syndrome, OI, or EDS may have blue sclerae due to tissue thinning that allows underlying pigment to show through.]
B. α1-Antitrypsin in elastin degradation
Blood and other body fluids contain a protein, α1-antitrypsin (AAT), which inhibits a number of proteolytic enzymes (called peptidases, proteases, or proteinases) that hydrolyze and destroy proteins. [Note: The inhibitor was originally named AAT because it inhibits the activity of trypsin, a proteolytic enzyme synthesized as trypsinogen by the pancreas (see p. 248).] AAT has the important physiologic role of inhibiting neutrophil elastase, a powerful protease that is released into the extracellular space and degrades elastin of alveolar walls as well as other structural proteins in a variety of tissues (Fig. 4.14). Most of the AAT found in plasma is synthesized and secreted by the liver. Extrahepatic synthesis also occurs.
1.
α1-Antitrypsin in the lungs: In the normal lung, the alveoli are chronically exposed to low levels of neutrophil elastase released from activated and degenerating neutrophils. The proteolytic activity of elastase can destroy the elastin in alveolar walls if unopposed by the action of AAT, the most important inhibitor of neutrophil elastase (see Fig. 4.14). Because lung tissue cannot regenerate, the destruction of the connective tissue of alveolar walls caused by an imbalance between the protease and its inhibitor results in pulmonary disease.
2.
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α1-Antitrypsin deficiency and emphysema: In the United States, ~2%– 5% of patients with emphysema are predisposed to the disease by inherited defects in AAT. A number of different mutations in the gene for AAT are known to cause a deficiency of the protein, but one single purine base mutation (GAG to AAG, resulting in the substitution of lysine for glutamic acid at position 342 of the protein) is clinically the most widespread and severe. [Note: The mutated protein is termed the Z variant.] The mutation causes the normally monomeric AAT to misfold, polymerize, and aggregate within the RER of hepatocytes, resulting in decreased secretion of AAT by the liver. AAT deficiency is, therefore, a misfolded protein disease. Consequently, blood levels of AAT are reduced, decreasing the amount that gets to the lung. The polymer that accumulates in the liver may result in cirrhosis (scarring of the liver). In the United States, the AAT mutation is most common in Caucasians of Northern European ancestry. An individual must inherit two abnormal AAT alleles to be at risk for the development of emphysema. In a heterozygote, with one normal and one defective gene, the levels of AAT are sufficient to protect the alveoli from damage. [Note: Methionine 358 in AAT is required for the binding of the inhibitor to its target proteases. Smoking causes the oxidation and subsequent inactivation of the methionine, thereby rendering the inhibitor powerless to neutralize elastase. Smokers with AAT deficiency, therefore, have a considerably elevated rate of lung destruction and a poorer survival rate than nonsmokers with the deficiency.] The deficiency of elastase inhibitor can be treated by weekly augmentation therapy, that is, intravenous administration of AAT. The AAT diffuses from the blood into the lung, where it reaches therapeutic levels in the fluid surrounding the lung epithelial cells.
IV. CHAPTER SUMMARY
Collagen and elastin are structural fibrous proteins of the extracellular matrix (Fig. 4.15). Collagen contains an abundance of proline, lysine, and glycine, the latter occurring at every third position in the primary structure. It also contains hydroxyproline, hydroxylysine, and glycosylated hydroxylysine, each formed by posttranslational modification. Fibrillar collagen has a long, rigid structure, in which three collagen polypeptide α chains are wound around one another in a rope-like triple helix stabilized by interchain hydrogen bonds. Diseases of fibrillar collagen synthesis affect bones, joints, skin, and blood vessels. Elastin is a connective tissue protein with rubber-like properties in tissues such as the lung. α1-Antitrypsin (AAT), produced primarily by the liver, inhibits elastase-catalyzed degradation of elastin in the alveolar walls. A deficiency of AAT increases elastin degradation and can cause emphysema and, in some cases, cirrhosis of the liver.
Choose the ONE best answer.
.1. A 30-year-old woman of Northern European ancestry presents with progressive dyspnea (shortness of breath). She denies the use of cigarettes. Family history reveals that her sister also has problems with her lungs. Which one of the following etiologies most likely explains this patient’s pulmonary symptoms?
A. Deficiency in dietary vitamin C
B. Deficiency of α1-antitrypsin
C. Deficiency of prolyl hydroxylase
D. Decreased elastase activity
E. Increased collagenase activity
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Correct answer = B. α1-Antitrypsin (AAT) deficiency is a genetic disorder that can cause pulmonary damage and emphysema even in the absence of cigarette use. A deficiency of AAT permits increased elastase activity to destroy elastin in the alveolar walls. AAT deficiency should be suspected when chronic obstructive pulmonary disease develops in a patient younger than age 45 years who does not have a history of chronic bronchitis or tobacco use or when multiple family members develop obstructive lung disease at an early age. Choices A, C, and E refer to collagen, not elastin.
.3. A 7-month-old child “fell over” while crawling and now presents with a swollen leg. Imaging reveals a fracture of a bowed femur, secondary to minor trauma, and thin bones (see x-ray at right). Blue sclerae are also noted. At age 1 month, the infant had multiple fractures in various states of healing (right clavicle, right humerus, and right radius). A careful family history has ruled out nonaccidental trauma (child abuse) as a cause of the bone fractures. Which pairing of a defective (or deficient) molecule and the resulting pathology best fits this clinical description?
A. Elastin and emphysema
B. Fibrillin and Marfan disease
C. Type I collagen and osteogenesis imperfecta
D. Type V collagen and Ehlers-Danlos syndrome
E. Vitamin C and scurvy
Correct answer = C. The child most likely has osteogenesis imperfecta. Most cases arise from a defect in the genes encoding type I collagen. Bones in affected patients are thin, osteoporotic, often bowed, and extremely prone to fracture. Pulmonary problems are not seen in this child. Individuals with Marfan syndrome have impaired structural integrity of the skeleton, eyes, and cardiovascular system. Defects in type V collagen cause the classic form of Ehlers-Danlos syndrome characterized by skin extensibility and fragility and joint hypermobility. Scurvy caused by vitamin C deficiency is characterized by capillary fragility.
.2. What is the differential basis of the liver and lung pathology seen in α1antitrypsin deficiency?
With α1-antitrypsin (AAT) deficiency, the cirrhosis that can result is due to polymerization and retention of AAT in the liver, its site of synthesis. The alveolar damage is due to the retention-based deficiency of AAT (a serine protease inhibitor) in the lung such that elastase (a serine protease) is unopposed.
.4. How and why is proline hydroxylated in collagen?
Proline is hydroxlyated by prolyl hydroxylase, an enzyme of the endoplasmic reticulum that requires oxygen, ferrous iron, and vitamin C. Hydroxylation increases interchain hydrogen bond formation, strengthening the triple helix of collagen. Vitamin C deficiency impairs hydroxylation.
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I. OVERVIEW
Virtually all reactions in the body are mediated by enzymes, which are protein catalysts that increase the rate of reactions without being changed in the overall process. Among the many biologic reactions that are energetically possible, enzymes selectively channel reactants (called substrates) into useful pathways. Thus, enzymes direct all metabolic events. This chapter examines the nature of these catalytic molecules and their mechanisms of action.
II. NOMENCLATURE
Each enzyme is assigned two names. The first is its short, recommended name, convenient for everyday use. The second is the more complete systematic name, which is used when an enzyme must be identified without ambiguity.
A. Recommended name
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Most commonly used enzyme names have the suffix “-ase” attached to the substrate of the reaction (for example, glucosidase and urease) or to a description of the action performed (for example, lactate dehydrogenase and adenylyl cyclase). [Note: Some enzymes retain their original trivial names, which give no hint of the associated enzymic reaction, for example, trypsin and pepsin.]
B. Systematic name
In the systematic naming system, enzymes are divided into six major classes (Fig. 5.1), each with numerous subgroups. For a given enzyme, the suffix ase is attached to a fairly complete description of the chemical reaction catalyzed, including the names of all the substrates, for example, lactate:nicotinamide adenine dinucleotide (NAD+) oxidoreductase. [Note: Each enzyme is also assigned a classification number. Lactate:NAD+ oxidoreductase is 1.1.1.27.] The systematic names are unambiguous and informative but are frequently too cumbersome to be of general use.
inorganic phosphate.
Potentially confusing enzyme nomenclature includes synthetase (requires ATP), synthase (no ATP required), phosphatase (uses water to remove a phosphate group), phosphorylase (uses inorganic phosphate to break a bond and generate a phosphorylated product), dehydrogenase (NAD+ or flavin adenine dinucleotide [FAD] is an electron acceptor in a redox reaction), oxidase (oxygen is the acceptor, and oxygen atoms are not incorporated into substrate), and oxygenase (one or both oxygen atoms are incorporated).
III. PROPERTIES
Enzymes are protein catalysts that increase the velocity of a chemical reaction and are not consumed during the reaction. [Note: Some ribonucleic acids (RNA) can catalyze reactions that affect phosphodiester and peptide bonds. RNAs with catalytic activity are called ribozymes (see p. 434) and are much less common than protein catalysts.]
A. Active site
Enzyme molecules contain a special pocket or cleft called the active site. The active site, formed by folding of the protein, contains amino acid side chains that participate in substrate binding and catalysis (Fig. 5.2). The substrate binds the enzyme noncovalently, forming an enzyme–substrate (ES) complex. Binding is thought to cause a conformational change in the enzyme (induced fit model) that allows catalysis. ES is converted to an enzyme–product (EP) complex that subsequently dissociates to enzyme and product.
B. Efficiency
Enzyme-catalyzed reactions are highly efficient, proceeding from 103 to 108 times faster than uncatalyzed reactions. The number of substrate molecules converted to product per enzyme molecule per second is called the turnover number, or kcat, and typically is 102–104 s−1 . [Note: kcat is the rate constant for the conversion of ES to E + P (see p. 58).]
C. Specificity
Enzymes are highly specific, interacting with one or a few substrates and catalyzing only one type of chemical reaction. The set of enzymes made in a cell determines which reactions occur in that cell.
D. Holoenzymes, apoenzymes, cofactors, and coenzymes
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Some enzymes require nonproteins for enzymic activity. The term holoenzyme refers to the active enzyme with its nonprotein component, whereas the enzyme without its nonprotein moiety is termed an apoenzyme and is inactive. If the nonprotein moiety is a metal ion, such as zinc (Zn2+) or iron (Fe2+), it is called a cofactor (see Chapter 29). If it is a small organic molecule, it is termed a coenzyme. Coenzymes that only transiently associate with the enzyme are called cosubstrates. Cosubstrates dissociate from the enzyme in an altered state (NAD+ is an example; see p. 101). If the coenzyme is permanently associated with the enzyme and returned to its original form, it is called a prosthetic group (FAD is an example; see p. 110). Coenzymes commonly are derived from vitamins. For example, NAD+ contains niacin, and FAD contains riboflavin (see Chapter 28).
E. Regulation
Enzyme activity can be regulated, that is, increased or decreased, so that the rate of product formation responds to cellular need.
F. Location within the cell
Many enzymes are localized in specific organelles within the cell (Fig. 5.3). Such compartmentalization serves to isolate the reaction substrate or product from other competing reactions. This provides a favorable environment for the reaction and organizes the thousands of enzymes present in the cell into purposeful pathways.
IV. MECHANISM OF ENZYME ACTION
The mechanism of enzyme action can be viewed from two different perspectives. The first treats catalysis in terms of energy changes that occur during the reaction. That is, enzymes provide an alternate, energetically favorable reaction pathway different from the uncatalyzed reaction. The second perspective describes how the active site chemically facilitates catalysis.
A. Energy changes occurring during the reaction
Virtually all chemical reactions have an energy barrier separating the reactants and the products. This barrier, called the activation energy (Ea), is the energy difference between that of the reactants and a high-energy intermediate, the transition state (T*), which is formed during the conversion of reactant to product. Figure 5.4 shows the changes in energy during the conversion of a molecule of reactant A to product B as it proceeds through the transition state.
1.
Activation energy: The peak of energy in Figure 5.4 is the difference in free energy between the reactant and T*, in which the high-energy, short-lived intermediate is formed during the conversion of reactant to product. Because of the high Ea, the rates of uncatalyzed chemical reactions are often slow.
2.
Rate of reaction: For molecules to react, they must contain sufficient energy to overcome the energy barrier of the transition state. In the absence of an enzyme, only a small proportion of a population of molecules may possess enough energy to achieve the transition state between reactant and product. The rate of reaction is determined by the number of such energized molecules. In general, the lower the Ea, the more molecules have sufficient energy to pass through the transition state and, therefore, the faster the rate of the reaction.
3.
Alternate reaction pathway: An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower Ea (see Fig. 5.4). The enzyme does not change the free energies of the reactants (substrates) or products and, therefore, does not change the equilibrium of the reaction (see p. 70). It does, however, accelerate the rate by which equilibrium is reached.
B. Active site chemistry
The active site is not a passive receptacle for binding the substrate but, rather, is a complex molecular machine employing a diversity of chemical mechanisms to facilitate the conversion of substrate to product. A number of factors are responsible for the catalytic efficiency of enzymes, including the following examples.
1.
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Transition-state stabilization: The active site often acts as a flexible molecular template that binds the substrate and initiates its conversion to the transition state, a structure in which the bonds are not like those in the substrate or the product (see T* at the top of the curve in Fig. 5.4). By stabilizing the transition state, the enzyme greatly increases the concentration of the reactive intermediate that can be converted to product and, thus, accelerates the reaction. [Note: The transition state cannot be isolated.] 2.
Catalysis: The active site can provide catalytic groups that enhance the probability that the transition state is formed. In some enzymes, these groups can participate in general acid–base catalysis in which amino acid residues provide or accept protons. In other enzymes, catalysis may involve the transient formation of a covalent ES complex. [Note: The mechanism of action of chymotrypsin, an enzyme of protein digestion in the intestine, includes general base, general acid, and covalent catalysis. A histidine at the active site of the enzyme gains (general base) and loses (general acid) protons, mediated by the pK of histidine in proteins being close to physiologic pH. Serine at the active site forms a transient covalent bond with the substrate.] 3.
Transition-state visualization: The enzyme-catalyzed conversion of substrate to product can be visualized as being similar to removing a sweater from an uncooperative infant (Fig. 5.5). The process has a high Ea because the only reasonable strategy for removing the garment (short of ripping it off) requires that the random flailing of the baby results in both arms being fully extended over the head, an unlikely posture. However, we can envision a parent acting as an enzyme, first coming in contact with the baby (forming ES) and then guiding the baby’s arms into an extended, vertical position, analogous to the transition state. This posture (conformation) of the baby facilitates the removal of the sweater, forming the disrobed baby, which here represents product. [Note: The substrate bound to the enzyme (ES) is at a slightly lower energy than unbound substrate (S) and explains the small dip in the curve at ES.]
V. FACTORS AFFECTING REACTION VELOCITY
Enzymes can be isolated from cells and their properties studied in a test tube (that is, in vitro). Different enzymes show different responses to changes in substrate concentration, temperature, and pH. This section describes factors that influence the reaction velocity of enzymes. Enzymic responses to these factors give us valuable clues as to how enzymes function in living cells (that is, in vivo).
A. Substrate concentration 1.
Maximal velocity: The rate or velocity of a reaction (v) is the number of substrate molecules converted to product per unit time. Velocity is usually expressed as µmol of product formed per minute. The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached (Fig. 5.6). The leveling off of the reaction rate at high substrate concentrations reflects the saturation with substrate of all available binding sites on the enzyme molecules present.
2.
Shape of the enzyme kinetics curve: Most enzymes show Michaelis-Menten kinetics (see p. 58), in which the plot of initial reaction velocity (vo) against substrate concentration is hyperbolic (similar in shape to that of the oxygen-dissociation curve of myoglobin; see p. 29). In contrast, allosteric enzymes do not follow Michaelis-Menten kinetics and show a sigmoidal curve (see Fig. 5.6) that is similar in shape to the oxygen-dissociation curve of hemoglobin (see p. 29).
B. Temperature 1.
Velocity increase with temperature: The reaction velocity increases with temperature until a peak velocity is reached (Fig. 5.7). This increase is the result of the increased number of substrate molecules having sufficient energy to pass over the energy barrier and form the products of the reaction.
2.
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Velocity decrease with higher temperature: Further elevation of the temperature causes a decrease in reaction velocity as a result of temperature-induced denaturation of the enzyme (see Fig. 5.7).
The optimum temperature for most human enzymes is between 35°C and 40°C. Human enzymes start to denature (see p. 20) at temperatures above 40°C, but thermophilic bacteria found in hot springs have optimum temperatures of 70°C.
C. pH 1.
pH effect on active site ionization: The concentration of protons ([H+]) affects reaction velocity in several ways. First, the catalytic process usually requires that the enzyme and substrate have specific chemical groups in either an ionized or unionized state in order to interact. For example, catalytic activity may require that an amino group of the enzyme be in the protonated form (−NH3+). Because this group is deprotonated at alkaline pH, the rate of the reaction declines.
2.
pH effect on enzyme denaturation: Extremes of pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of the amino acid side chains.
3.
Variable pH optimum: The pH at which maximal enzyme activity is achieved is different for different enzymes and often reflects the [H+] at which the enzyme functions in the body. For example, pepsin, a digestive enzyme in the stomach, is maximally active at pH 2, whereas other enzymes, designed to work at neutral pH, are denatured by such an acidic environment (Fig. 5.8).
VI. MICHAELIS-MENTEN KINETICS
Leonor Michaelis and Maude Menten proposed a simple model that accounts for most of the features of many enzyme-catalyzed reactions. In this model, the enzyme reversibly combines with its substrate to form an ES complex that subsequently yields product, regenerating the free enzyme. The reaction model, involving one substrate molecule, is represented below: where S is the substrate. E is the enzyme. ES is the enzyme–substrate complex. P is the product. k1, k−1, and k2 (or, kcat) are rate constants.
A. Michaelis-Menten equation
The Michaelis-Menten equation describes how reaction velocity varies with substrate concentration:
The following assumptions are made in deriving the Michaelis-Menten rate equation.
1.
Enzyme and substrate relative concentrations: The substrate concentration ([S]) is much greater than the concentration of enzyme so that the percentage of total substrate bound by the enzyme at any one time is small.
2.
Steady-state assumption: The concentration of the ES complex does not change with time (the steady-state assumption), that is, the rate of formation of ES is equal to that of the breakdown of ES (to E + S and to E + P). In general, an intermediate in a series of reactions is said to be in steady state when its rate of synthesis is equal to its rate of degradation.
3.
Initial velocity: Initial reaction velocities (vo) are used in the analysis of enzyme reactions. This means that the rate of the reaction is measured as soon as enzyme and substrate are mixed. At that time, the concentration of product is very small, and therefore, the rate of the back reaction from product to substrate can be ignored.
B. Important conclusions 1. Km characteristics: Km, the Michaelis constant, is characteristic of an enzyme and its particular substrate and reflects the affinity of the enzyme for that substrate. Km is numerically equal to the substrate concentration at which the reaction velocity is equal to one half Vmax. Km does not vary with enzyme concentration.
a. Small Km: A numerically small (low) Km reflects a high affinity of the enzyme for substrate, because a low concentration of substrate is needed to half-saturate the enzyme—that is, to reach a velocity that is one half Vmax (Fig. 5.9).
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b. Large Km: A numerically large (high) Km reflects a low affinity of enzyme for substrate because a high concentration of substrate is needed to half-saturate the enzyme.
2.
Velocity relationship to enzyme concentration: The rate of the reaction is directly proportional to the enzyme concentration because [S] is not limiting. For example, if the enzyme concentration is halved, the initial rates of the reaction (vo) and that of Vmax are reduced to half that of the original.
3.
Reaction order: When [S] is much less (<<) than Km, the velocity of the reaction is approximately proportional to the substrate concentration (Fig. 5.10). The rate of reaction is then said to be first order with respect to substrate. When [S] is much greater (>>) than Km, the velocity is constant and equal to Vmax. The rate of reaction is then independent of substrate concentration (the enzyme is saturated with substrate) and is said to be zero order with respect to substrate concentration (see Fig. 5.10).
D. Lineweaver-Burk plot
When vo is plotted against [S], it is not always possible to determine when Vmax has been achieved because of the gradual upward slope of the hyperbolic curve at high substrate concentrations. However, if 1/vo is plotted versus 1/[S], a straight line is obtained (Fig. 5.11). This plot, the
Lineweaver-Burk plot (also called a double-reciprocal plot) can be used to calculate Km and Vmax as well as to determine the mechanism of action of enzyme inhibitors.
The equation describing the Lineweaver-Burk plot is: where the intercept on the x axis is equal to − 1/Km, and the intercept on the y axis is equal to 1/Vmax. [Note: The slope = Km/Vmax.]
VII. ENZYME INHIBITION
Any substance that can decrease the velocity of an enzyme-catalyzed reaction is called an inhibitor. Inhibitors can be reversible or irreversible. Irreversible inhibitors bind to enzymes through covalent bonds. Lead, for example, forms covalent bonds with the sulfhydryl side chain of cysteine in proteins. Ferrochelatase, an enzyme involved in heme synthesis (see p. 279), is irreversibly inhibited by lead. [Note: An important group of irreversible inhibitors are the mechanism-based inhibitors that are converted by the enzyme itself to a form that covalently links to the enzyme, thereby inhibiting it. They also are referred to as “suicide” inhibitors.] Reversible inhibitors bind to enzymes through noncovalent bonds and, thus, dilution of the enzyme–inhibitor complex results in dissociation of the reversibly bound inhibitor and recovery of enzyme activity. The two most commonly encountered types of reversible inhibition are competitive and noncompetitive.
A. Competitive inhibition
This type of inhibition occurs when the inhibitor binds reversibly to the same site that the substrate would normally occupy and, therefore, competes with the substrate for that site.
1.
Effect on Vmax: The effect of a competitive inhibitor is reversed by increasing the concentration of substrate. At a sufficiently high [S], the 2.
Effect on Km: A competitive inhibitor increases the apparent Km for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve one half Vmax.
3.
Effect on the Lineweaver-Burk plot: Competitive inhibition shows a characteristic Lineweaver-Burk plot in which the plots of the inhibited and uninhibited reactions intersect on the y axis at 1/Vmax (Vmax is reaction velocity reaches the Vmax observed in the absence of inhibitor, that is, Vmax is unchanged (Fig. 5.12).
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unchanged). The inhibited and uninhibited reactions show different x-axis intercepts, indicating that the apparent Km is increased in the presence of the competitive inhibitor because − 1/Km moves closer to zero from a negative value (see Fig. 5.12). [Note: An important group of competitive inhibitors are the transition state analogs, stable molecules that approximate the structure of the transition state, and, therefore, bind the enzyme more tightly than does the substrate.] 4. Statin drugs as examples of competitive inhibitors: This group of antihyperlipidemic agents competitively inhibits the rate-limiting (slowest) step in cholesterol biosynthesis. This reaction is catalyzed by hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase; see p. 221). Statins, such as atorvastatin (Lipitor) and pravastatin (Pravachol), are structural analogs of the natural substrate for this enzyme and compete effectively to inhibit HMG CoA reductase. By doing so, they inhibit de novo cholesterol synthesis, thereby lowering plasma cholesterol levels (Fig. 5.13).
B. Noncompetitive inhibition
This type of inhibition is recognized by its characteristic effect on Vmax (Fig. 5.14). Noncompetitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme. The noncompetitive inhibitor can bind either free enzyme or the enzyme–substrate complex, thereby preventing the reaction from occurring (Fig. 5.15).
1.
Effect on Vmax: Noncompetitive inhibition cannot be overcome by increasing the concentration of substrate. Therefore, noncompetitive inhibitors decrease the apparent Vmax of the reaction.
2.
Effect on Km: Noncompetitive inhibitors do not interfere with the binding of substrate to enzyme. Therefore, the enzyme shows the same Km in the presence or absence of the noncompetitive inhibitor, that is, Km is unchanged.
3.
Effect on Lineweaver-Burk plot: Noncompetitive inhibition is readily differentiated from competitive inhibition by plotting 1/vo versus 1/[S] and noting that the apparent Vmax decreases in the presence of a noncompetitive inhibitor, whereas Km is unchanged (see Fig. 5.14). [Note: Oxypurinol, a metabolite of the prodrug allopurinol, is a noncompetitive inhibitor of xanthine oxidase, an enzyme of purine degradation (see p. 301).]
C. Enzyme inhibitors as drugs
At least half of the ten most commonly prescribed drugs in the United States act as enzyme inhibitors. For example, the widely prescribed βlactam antibiotics, such as penicillin and amoxicillin, act by inhibiting enzymes involved in bacterial cell wall synthesis. Drugs may also act by inhibiting extracellular reactions. This is illustrated by angiotensinconverting enzyme (ACE) inhibitors. They lower blood pressure by blocking plasma ACE that cleaves angiotensin I to form the potent vasoconstrictor, angiotensin II. These drugs, which include captopril, enalapril, and lisinopril, cause vasodilation and, therefore, a reduction in blood pressure. Aspirin, a nonprescription drug, irreversibly inhibits prostaglandin and thromboxane synthesis by inhibiting cyclooxygenase (see p. 214).
VIII. ENZYME REGULATION
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The regulation of the reaction velocity of enzymes is essential if an organism is to coordinate its numerous metabolic processes. The rates of most enzymes are responsive to changes in substrate concentration, because the intracellular level of many substrates is in the range of the Km. Thus, an increase in substrate concentration prompts an increase in reaction rate, which tends to return the concentration of substrate toward normal. In addition, some enzymes with specialized regulatory functions respond to allosteric effectors and/or covalent modification or they show altered rates of enzyme synthesis (or degradation) when physiologic conditions are changed.
A. Allosteric enzymes
Allosteric enzymes are regulated by molecules called effectors that bind noncovalently at a site other than the active site. These enzymes are almost always composed of multiple subunits, and the regulatory (allosteric) site that binds the effector is distinct from the substrate-binding site and may be located on a subunit that is not itself catalytic. Effectors that inhibit enzyme activity are termed negative effectors, whereas those that increase enzyme activity are called positive effectors. Positive and negative effectors can affect the affinity of the enzyme for its substrate (K0.5), modify the maximal catalytic activity of the enzyme (Vmax), or both (Fig. 5.16). [Note: Allosteric enzymes frequently catalyze the committed step, often the rate-limiting step, early in a pathway.] 1.
Homotropic effectors: When the substrate itself serves as an effector, the effect is said to be homotropic. Most often, an allosteric substrate functions as a positive effector. In such a case, the presence of a substrate molecule at one site on the enzyme enhances the catalytic properties of the other substrate-binding sites. That is, their binding sites exhibit cooperativity. These enzymes show a sigmoidal curve when vo is plotted against substrate concentration, as shown in Figure 5.16. This contrasts with the hyperbolic curve characteristic of enzymes following Michaelis-Menten kinetics, as previously discussed. [Note: The concept of cooperativity of substrate binding is analogous to the binding of oxygen to hemoglobin (see p. 29).] 2.
Heterotropic effectors: The effector may be different from the substrate, in which case the effect is said to be heterotropic. For example, consider the feedback inhibition shown in Figure 5.17. The enzyme that converts D to E has an allosteric site that binds the end product, G. If the concentration of G increases (for example, because it is not used as rapidly as it is synthesized), the first irreversible step unique to the pathway is typically inhibited. Feedback inhibition provides the cell with appropriate amounts of a product it needs by regulating the flow of substrate molecules through the pathway that synthesizes that product. Heterotropic effectors are commonly encountered. For example, the glycolytic enzyme phosphofructokinase-1 is allosterically inhibited by citrate, which is not a substrate for the enzyme (see p. 99).
Figure5.17Feedbackinhibitionofametabolicpathway.
B. Covalent modification
Many enzymes are regulated by covalent modification, most often by the addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme. Protein phosphorylation is recognized as one of the primary ways in which cellular processes are regulated.
1.
Phosphorylation and dephosphorylation: Phosphorylation reactions are catalyzed by a family of enzymes called protein kinases that use ATP as the phosphate donor. Phosphate groups are cleaved from phosphorylated enzymes by the action of phosphoprotein phosphatases (Fig. 5.18).
2.
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Enzyme response to phosphorylation: Depending on the specific enzyme, the phosphorylated form may be more or less active than the unphosphorylated enzyme. For example, hormone-mediated phosphorylation of glycogen phosphorylase (an enzyme that degrades glycogen) increases activity, whereas phosphorylation of glycogen synthase (an enzyme that synthesizes glycogen) decreases activity (see p. 132).
C. Enzyme synthesis
The regulatory mechanisms described above modify the activity of existing enzyme molecules. However, cells can also regulate the amount of enzyme present by altering the rate of enzyme degradation or, more typically, the rate of enzyme synthesis. The increase (induction) or decrease (repression) of enzyme synthesis leads to an alteration in the total population of active sites. Enzymes subject to regulation of synthesis are often those that are needed at only one stage of development or under selected physiologic conditions. For example, elevated levels of insulin as a result of high blood glucose levels cause an increase in the synthesis of key enzymes involved in glucose metabolism (see p. 105). In contrast, enzymes that are in constant use are usually not regulated by altering the rate of enzyme synthesis. Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days), compared with allosterically or covalently regulated changes in enzyme activity, which occur in seconds to minutes. Figure 5.19 summarizes the common ways that enzyme activity is regulated.
IX. Enzymes in Clinical Diagnosis
Plasma enzymes can be classified into two major groups. First, a relatively small group of enzymes are actively secreted into the blood by certain cell types. For example, the liver secretes zymogens (inactive precursors) of the enzymes involved in blood coagulation. Second, a large number of enzyme species are released from cells during normal cell turnover. These enzymes almost always function intracellularly and have no physiologic use in the plasma. In healthy individuals, the levels of these enzymes are fairly constant and represent a steady state in which the rate of release from damaged cells into the plasma is balanced by an equal rate of removal from the plasma. Increased plasma levels of these enzymes may indicate tissue damage (Fig. 5.20).
(B) cells.
Plasma is the fluid, noncellular part of blood. Laboratory assays of enzyme activity most often use serum, which is obtained by centrifugation of whole blood after it has been allowed to coagulate. Plasma is a physiologic fluid, whereas serum is prepared in the laboratory.
A. Plasma enzyme levels in disease states
Many diseases that cause tissue damage result in an increased release of intracellular enzymes into the plasma. The activities of many of these enzymes are routinely determined for diagnostic purposes in diseases of the heart, liver, skeletal muscle, and other tissues. The level of specific enzyme activity in the plasma frequently correlates with the extent of tissue damage. Therefore, determining the degree of elevation of a particular enzyme activity in the plasma is often useful in evaluating the prognosis for the patient.
B. Plasma enzymes as diagnostic tools
Some enzymes show relatively high activity in only one or a few tissues. Therefore, the presence of increased levels of these enzymes in plasma reflects damage to the corresponding tissue. For example, the enzyme alanine aminotransferase (ALT; see p. 251) is abundant in the liver. The appearance of elevated levels of ALT in plasma signals possible damage to hepatic tissue. [Note: Measurement of ALT is part of the liver function test panel.] Increases in plasma levels of enzymes with a wide tissue distribution provide a less specific indication of the site of cellular injury and limits their diagnostic value.
C. Isoenzymes and heart disease
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Isoenzymes (also called isozymes) are enzymes that catalyze the same reaction. However, they do not necessarily have the same physical properties because of genetically determined differences in amino acid sequence. For this reason, isoenzymes may contain different numbers of charged amino acids, which allows electrophoresis (the movement of charged particles in an electric field) to separate them (Fig. 5.21). Different organs commonly contain characteristic proportions of different isoenzymes. The pattern of isoenzymes found in the plasma may, therefore, serve as a means of identifying the site of tissue damage. For example, the plasma levels of creatine kinase (CK) are commonly determined in the diagnosis of myocardial infarction (MI). They are particularly useful when the electrocardiogram (ECG) is difficult to interpret such as when there have been previous episodes of heart disease.
1.
Isoenzyme quaternary structure: Many isoenzymes contain different subunits in various combinations. For example, CK occurs as three isoenzymes. Each isoenzyme is a dimer composed of two polypeptides (called B and M subunits) associated in one of three combinations: CK1 = BB, CK2 = MB, and CK3 = MM. Each CK isoenzyme shows a characteristic electrophoretic mobility (see Fig. 5.21). [Note: Virtually all CK in the brain is the BB isoform, whereas it is MM in skeletal muscle. In cardiac muscle, about one third is MB with the rest as MM.] 2.
Diagnosis of myocardial infarction: Measurement of blood levels of proteins with cardiac specificity (biomarkers) is used in the diagnosis of MI. Myocardial muscle is the only tissue that contains >5% of the total CK activity as the CK2 (MB) isoenzyme. Appearance of this hybrid isoenzyme in plasma is virtually specific for infarction of the myocardium. Following an acute MI, CK2 appears in plasma within 4–8 hours following onset of chest pain, reaches a peak of activity at ~24 hours, and returns to baseline after 48–72 hours (Fig. 5.22). Troponins T (TnT) and I (TnI) are regulatory proteins involved in muscle contractility. Cardiac-specific isoforms (cTn) are released into the plasma in response to cardiac damage. They are highly sensitive and specific for damage to cardiac tissue. cTn appear in plasma within 4–6 hours after an MI, peak in 24–36 hours, and remain elevated for 3–10 days. Elevated cTn, in combination with the clinical presentation and characteristic changes in the ECG, are currently considered the “gold standard” in the diagnosis of an MI.
X. CHAPTER SUMMARY
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Enzymes are protein catalysts that increase the velocity of a chemical reaction by lowering the energy of the transition state (Fig. 5.23). They are not consumed during the reaction. Enzyme molecules contain a special cleft called the active site, which contains amino acid side chains that participate in substrate binding and catalysis. The active site binds the substrate, forming an enzyme–substrate (ES) complex. Binding is thought to cause a conformational change in the enzyme (induced fit) that allows catalysis. ES is converted to enzyme and product. An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower activation energy (Ea). Because the enzyme does not change the free energies of the reactants or products, it does not change the equilibrium of the reaction. Most enzymes show Michaelis-Menten kinetics, and a plot of the initial reaction velocity (vo) against substrate concentration ([S]) has a hyperbolic shape similar to the oxygen-dissociation curve of myoglobin. A Lineweaver-Burk plot of 1/v and 1/[S] allows determination of Vmax (maximal velocity) and Km (Michaelis constant, which reflects affinity for substrate). Any substance that can decrease the velocity of an enzyme-catalyzed reaction is called an inhibitor. The two most common types of reversible inhibition are competitive (which increases the apparent Km) and noncompetitive (which decreases the apparent Vmax). In contrast, the multisubunit allosteric enzymes show a sigmoidal curve similar in shape to the oxygen-dissociation curve of hemoglobin. They typically catalyze the committed step of a pathway. Allosteric enzymes are regulated by molecules called effectors that bind noncovalently at a site other than the active site. Effectors can be either positive (increase enzyme activity) or negative (decrease enzyme activity). An allosteric effector can alter the affinity of the enzyme for its substrate (K0.5), the maximal catalytic activity of the enzyme (Vmax), or both. Enzymes can also be regulated by covalent modification and by changes in the rate of synthesis or degradation.
Enzymes have diagnostic and therapeutic value in medicine.
Choose the ONE best answer.
.1. In cases of ethylene glycol poisoning and its characteristic metabolic acidosis, treatment involves correction of the acidosis, removal of any remaining ethylene glycol, and administration of an inhibitor of alcohol dehydrogenase (ADH), the enzyme that oxidizes ethylene glycol to the organic acids that cause the acidosis. Ethanol (grain alcohol) frequently is the inhibitor given to treat ethylene glycol poisoning. Results of experiments using ADH with and without ethanol are shown to the right. Based on these data, what type of inhibition is caused by the ethanol?
A. Competitive
B. Feedback
C. Irreversible
D. Noncompetitive
Correct answer = A. A competitive inhibitor increases the apparent Km for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve one half Vmax. The effect of a competitive inhibitor is reversed by increasing substrate concentration ([S]). At a sufficiently high [S], the reaction velocity reaches the Vmax observed in the absence of inhibitor.
.2. Alcohol dehydrogenase (ADH) requires oxidized nicotinamide adenine dinucleotide (NAD+) for catalytic activity. In the reaction catalyzed by ADH, an alcohol is oxidized to an aldehyde as NAD+ is reduced to NADH and dissociates from the enzyme. The NAD+ is functioning as a/an:
A. apoenzyme.
B. coenzyme–cosubstrate.
C. coenzyme–prosthetic group.
D. cofactor.
E. heterotropic effector.
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Correct answer = B. A Coenzymes–cosubstrates are small organic molecules that associate transiently with an enzyme and leave the enzyme in a changed form. Coenzyme–prosthetic groups are small organic molecules that associate permanently with an enzyme and are returned to their original form on the enzyme. Cofactors are metal ions. Heterotropic effectors are not substrates.
For Questions 5.3 and 5.4, use the graph below that shows the changes in free energy when a reactant is converted to a product in the presence and absence of an enzyme. Select the letter that best represents: .3. the activation energy of the catalyzed forward reaction. .4. the free energy of the reaction.
Correct answers = B; D. Enzymes (protein catalysts) provide an alternate reaction pathway with a lower activation energy. However, they do not change the free energy of the reactant or product. A is the activation energy of the uncatalyzed reaction. C is the activation energy of the catalyzed reverse reaction.
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I. OVERVIEW
Bioenergetics describes the transfer and utilization of energy in biologic systems. It concerns the initial and final energy states of the reaction components, not the reaction mechanism or how much time it takes for the chemical change to occur. Bioenergetics makes use of a few basic ideas from the field of thermodynamics, particularly the concept of free energy. Because changes in free energy provide a measure of the energetic feasibility of a chemical reaction, they allow prediction of whether a reaction or process can take place. In short, bioenergetics predicts if a process is possible, whereas kinetics measures the reaction rate (see p. 54).
II. FREE ENERGY
The direction and extent to which a chemical reaction proceeds are determined by the degree to which two factors change during the reaction. These are enthalpy (∆H, a measure of the change [∆] in heat content of the reactants and products) and entropy (∆S, a measure of the change in randomness or disorder of the reactants and products), as shown in Figure 6.1. Neither of these thermodynamic quantities by itself is sufficient to determine whether a chemical reaction will proceed spontaneously in the direction it is written. However, when combined mathematically (see Fig. 6.1), enthalpy and entropy can be used to define a third quantity, free energy (G), which predicts the direction in which a reaction will spontaneously proceed.
III. FREE ENERGY CHANGE
The change in free energy is represented in two ways, ∆G and ∆G0. The first, ∆G (without the superscript “0”), represents the change in free energy and, thus, the direction of a reaction at any specified concentration of products and reactants. ∆G, then, is a variable. This contrasts with the standard free energy change, ∆G0 (with the superscript “0”), which is the energy change when reactants and products are at a concentration of 1 mol/l. [Note: The concentration of protons (H+) is assumed to be 10−7 mol/l (that is, pH = 7). This may be shown by a prime sign (ʹ ), for example, ∆G0ʹ.] Although ∆G0, a constant, represents energy changes at these nonphysiologic concentrations of reactants and products, it is nonetheless useful in comparing the energy changes of different reactions. Furthermore, ∆G0 can readily be determined from measurement of the equilibrium constant (see p. 71). [Note: This section outlines the uses of ∆G, and ∆G0 is described in D. below.]
A. ∆G and reaction direction
The sign of ∆G can be used to predict the direction of a reaction at constant temperature and pressure. Consider the reaction: 1. Negative ∆G: If ∆G is negative, then there is a net loss of energy, and the reaction goes spontaneously as written (that is, A is converted into B) as shown in Figure 6.2A. The reaction is said to be exergonic.
2.
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Positive ∆G: If ∆G is positive, then there is a net gain of energy, and the reaction does not go spontaneously from B to A (Fig. 6.2B). Energy must be added to the system to make the reaction go from B to A. The reaction is said to be endergonic.
3.
Zero ∆G: If ∆G = 0, then the reaction is in equilibrium. [Note: When a reaction is proceeding spontaneously (that is, ∆G is negative), the reaction continues until ∆G reaches zero and equilibrium is established.]
B. ∆G of the forward and back reactions
The free energy of the forward reaction (A → B) is equal in magnitude but opposite in sign to that of the back reaction (B → A). For example, if ∆G of the forward reaction is −5 kcal/mol, then that of the back reaction is +5 kcal/mol. [Note: ∆G can also be expressed in kilojoules per mole or kJ/mol (1 kcal = 4.2 kJ).]
C. ∆G and reactant and product concentrations
The ∆G of the reaction A → B depends on the concentration of the reactant and product. At constant temperature and pressure, the following relationship can be derived: where ∆G0 is the standard free energy change (see D. below) R is the gas constant (1.987 cal/mol K) T is the absolute temperature (K) [A] and [B] are the actual concentrations of the reactant and product ln represents the natural logarithm.
A reaction with a positive ∆G0 can proceed in the forward direction if the ratio of products to reactants ([B]/[A]) is sufficiently small (that is, the ratio of reactants to products is large) to make ∆G negative. For example, consider the reaction:
D. Standard free energy change
The standard free energy change, ∆G0, is so called because it is equal to the free energy change, ∆G, under standard conditions (that is, when reactants and products are at 1 mol/l concentrations; Fig. 6.3B). Under these conditions, the natural logarithm of the ratio of products to reactants is zero (ln1 = 0), and, therefore, the equation shown at the bottom of the previous page becomes: 1.
∆G0 and reaction direction: Under standard conditions, ∆G0 can be used to predict the direction a reaction proceeds because, under these conditions, ∆G0 is equal to ∆G. However, ∆G0 cannot predict the direction of a reaction under physiologic conditions because it is composed solely of constants (R, T, and Keq [see 2. below]) and is not, therefore, altered by changes in product or substrate concentrations.
2.
Relationship between ∆G0 and Keq: In a reaction A ⇄ B, a point of equilibrium is reached at which no further net chemical change takes place (that is, when A is being converted to B as fast as B is being converted to A). In this state, the ratio of [B] to [A] is constant, regardless of the actual concentrations of the two compounds: where Keq is the equilibrium constant, and [A]eq and [B]eq are the concentrations of A and B at equilibrium. If the reaction A ⇄ B is allowed to go to equilibrium at constant temperature and pressure, then, at equilibrium, the overall ∆G is zero (Fig. 6.3C). Therefore, where the actual concentrations of A and B are equal to the equilibrium concentrations of reactant and product ([A]eq and [B]eq), and their ratio is equal to the Keq. Thus,
This equation allows some simple predictions: 3.
∆G0s of two consecutive reactions: The ∆G0s are additive in any sequence of consecutive reactions, as are the ∆Gs. For example: 4.
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∆Gs of a pathway: The additive property of ∆G is very important in biochemical pathways through which substrates (reactants) must pass in a particular direction (for example, A → B → C → D → …). As long as the sum of the ∆Gs of the individual reactions is negative, the pathway can proceed as written, even if some of the individual reactions of the pathway have a positive ∆G. However, the actual rates of the reactions depend on the lowering of activation energies (Ea) by the enzymes that catalyze the reactions (see p. 55).
IV. ATP: AN ENERGY CARRIER
Reactions or processes that have a large positive ∆G, such as moving ions against a concentration gradient across a cell membrane, are made possible by coupling the endergonic movement of ions with a second, spontaneous process with a large negative ∆G such as the exergonic hydrolysis of ATP (see p. 87). [Note: In the absence of enzymes, ATP is a stable molecule because its hydrolysis has a high Ea.] Figure 6.4 shows a mechanical model of energy coupling. The simplest example of energy coupling in biologic reactions occurs when the energy-requiring and the energy-yielding reactions share a common intermediate.
A. Common intermediates
Two chemical reactions have a common intermediate when they occur sequentially in that the product of the first reaction is a substrate for the second. For example, given the reactions
D is the common intermediate and can serve as a carrier of chemical energy between the two reactions. [Note: The intermediate may be linked to an enzyme.] Many coupled reactions use ATP to generate a common intermediate. These reactions may involve the transfer of a phosphate group from ATP to another molecule. Other reactions involve the transfer of phosphate from an energy-rich intermediate to adenosine diphosphate (ADP), forming ATP.
B. Energy carried by ATP
ATP consists of a molecule of adenosine (adenine + ribose) to which three phosphate groups are attached (Fig. 6.5). Removal of one phosphate produces ADP, and removal of two phosphates produces adenosine monophosphate (AMP). For ATP, the ∆G0 of hydrolysis is approximately – 7.3 kcal/mol for each of the two terminal phosphate groups. Because of this large negative ∆G0 of hydrolysis, ATP is called a high-energy phosphate compound. [Note: Adenine nucleotides are interconverted (2 ADP ⇄ ATP + AMP) by adenylate kinase.]
V. ELECTRON TRANSPORT CHAIN
Energy-rich molecules, such as glucose, are metabolized by a series of oxidation reactions ultimately yielding carbon dioxide and water (H2O), as shown in Figure 6.6. The metabolic intermediates of these reactions donate electrons to specific coenzymes, nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD), to form the energy-rich reduced forms, NADH and FADH2. These reduced coenzymes can, in turn, each donate a pair of electrons to a specialized set of electron carriers, collectively called the electron transport chain (ETC), described in this section. As electrons are passed down the ETC, they lose much of their free energy. This energy is used to move H+ across the inner mitochondrial membrane, creating a H+ gradient that drives the production of ATP from ADP and inorganic phosphate (Pi), described on p. 77. The coupling of electron transport with ATP synthesis is called oxidative phosphorylation, sometimes denoted as OXPHOS. It proceeds continuously in all tissues that contain mitochondria. [Note: The free energy not trapped as ATP is used to drive ancillary reactions such as transport of calcium ions into mitochondria and to generate heat.]
A. Mitochondrial electron transport chain
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The ETC (except for cytochrome c, see p. 75) is located in the inner mitochondrial membrane and is the final common pathway by which electrons derived from different fuels of the body flow to oxygen (O2), reducing it to H2O (see Fig. 6.6).
1. Mitochondrial membranes: The mitochondrion contains an outer and an inner membrane separated by the intermembrane space. Although the outer membrane contains special channels (formed by the protein porin), making it freely permeable to most ions and small molecules, the inner membrane is a specialized structure that is impermeable to most small ions, including H+, and small molecules such as ATP, ADP, pyruvate, and other metabolites important to mitochondrial function (Fig. 6.7). Specialized carriers or transport systems are required to move ions or molecules across this membrane. The inner mitochondrial membrane is unusually rich in proteins, over half of which are directly involved in oxidative phosphorylation. It also contains convolutions, called cristae, which greatly increase its surface area.
2. Mitochondrial matrix: The gel-like solution of the matrix (interior) of mitochondria is also rich in proteins. These include the enzymes responsible for the oxidation of pyruvate, amino acids, and fatty acids (by β-oxidation) as well as those of the tricarboxylic acid (TCA) cycle. The synthesis of glucose, urea, and heme occurs partially in the matrix of mitochondria. In addition, the matrix contains NAD+ and FAD (the oxidized forms of the two coenzymes that are required as electron acceptors), and ADP and Pi, which are used to produce ATP. [Note: The matrix also contains mitochondrial deoxyribonucleic acid (mtDNA), ribonucleic acid (mtRNA), and ribosomes.]
B. Organization
The inner mitochondrial membrane contains four separate protein complexes, called Complexes I, II, III, and IV that each contain part of the ETC (Fig. 6.8). These complexes accept or donate electrons to the relatively mobile electron carrier coenzyme Q (CoQ) and cytochrome c. Each carrier in the ETC can receive electrons from an electron donor and can subsequently donate electrons to the next acceptor in the chain. The electrons ultimately combine with O2 and H+ to form H2O. This requirement for O2 makes the electron transport process the respiratory chain, which accounts for the greatest portion of the body’s use of O2.
C. Reactions
With the exception of CoQ, which is a lipid-soluble quinone, all members of the ETC are proteins. These may function as enzymes as is the case with the flavin-containing dehydrogenases, may contain iron as part of an iron-sulfur (Fe-S) center, may contain iron as part of the porphyrin prosthetic group of heme as in the cytochromes, or may contain copper (Cu) as does the cytochrome a + a3 complex.
1.
NADH formation: NAD+ is reduced to NADH by dehydrogenases that remove two hydrogen atoms from their substrate. [Note: For examples of these reactions, see the discussion of the dehydrogenases of the TCA cycle, p. 112.] Both electrons but only one H+ (that is, a hydride ion [:H−]) are transferred to the NAD+, forming NADH plus a free H+.
2.
NADH dehydrogenase: The free H+ plus the hydride ion carried by NADH are transferred to NADH dehydrogenase, a protein complex (Complex I) embedded in the inner mitochondrial membrane. Complex I has a tightly bound molecule of flavin mononucleotide (FMN), a coenzyme structurally related to FAD (see Fig. 28.15, p. 384) that accepts the two hydrogen atoms (2 electrons + 2 H+), becoming FMNH2.
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NADH dehydrogenase also contains peptide subunits with Fe-S centers (Fig. 6.9). At Complex I, electrons move from NADH to FMN to the iron of the Fe-S centers and then to CoQ. As electrons flow, they lose energy. This energy is used to pump four H+ across the inner mitochondrial membrane, from the matrix to the intermembrane space.
3.
Succinate dehydrogenase: At Complex II, electrons from the succinate dehydrogenase–catalyzed oxidation of succinate to fumarate move from the coenzyme, FADH2, to an Fe-S protein, and then to CoQ. [Note:
Because no energy is lost in this process, no H+ are pumped at Complex II.] 4.
Coenzyme Q: CoQ is a quinone derivative with a long, hydrophobic isoprenoid tail. It is made from an intermediate of cholesterol synthesis (see p. 221). [Note: It is also called ubiquinone because it is ubiquitous in biologic systems.] CoQ is a mobile electron carrier and can accept electrons from NADH dehydrogenase (Complex I), from succinate dehydrogenase (Complex II) and from other mitochondrial dehydrogenases, such as glycerol 3-phosphate dehydrogenase (see p.
80) and acyl CoA dehydrogenases (see p. 192). CoQ transfers electrons to Complex III (cytochrome bc1). Thus, a function of CoQ is to link the flavoprotein dehydrogenases to the cytochromes.
5.
Cytochromes: The remaining members of the ETC are cytochrome proteins. Each contains a heme group (a porphyrin ring plus iron). Unlike the heme groups of hemoglobin, the cytochrome iron is reversibly converted from its ferric (Fe3+) to its ferrous (Fe2+) form as a normal part of its function as an acceptor and donor of electrons. Electrons are passed along the chain from cytochrome bc1 (Complex III), to cytochrome c, and then to cytochromes a + a3 ([Complex IV] see Fig. 6.8). As electrons flow, four H+ are pumped across the inner mitochondrial membrane at Complex III and two at Complex IV. [Note: Cytochrome c is located in the intermembrane space, loosely associated with the outer face of the inner membrane. As seen with CoQ, cytochrome c is a mobile electron carrier.] 6.
Cytochrome a + a3: Because this cytochrome complex (Complex IV) is the only electron carrier in which the heme iron has an available coordination site that can react directly with O2, it also is called cytochrome c oxidase. At Complex IV, the transported electrons, O2, and free H+ are brought together, and O2 is reduced to H2O (see Fig. 6.8). [Note: Four electrons are required to reduce one molecule of O2 to two molecules of H2O.] Cytochrome c oxidase contains Cu atoms that are required for this complicated reaction to occur. Electrons move from CuA to cytochrome a to cytochrome a3 (in association with CuB) to O2.
7.
Site-specific inhibitors: Inhibitors of specific sites in the ETC have been identified and are illustrated in Figure 6.10. These respiratory inhibitors prevent the passage of electrons by binding to a component of the chain, blocking the oxidation-reduction reaction. Therefore, all electron carriers before the block are fully reduced, whereas those located after the block are oxidized. [Note: Inhibition of the ETC inhibits ATP synthesis because these processes are tightly coupled (see p. 78).]
NaN3 = sodium azide.
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Leakage of electrons from the ETC produces reactive oxygen species (ROS), such as superoxide (O2−·), hydrogen peroxide (H2O2), and hydroxyl radicals (OH·). ROS damage DNA and proteins and cause lipid peroxidation. Enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase are cellular defenses against ROS (see p. 148).
D. Free energy release during electron transport
The free energy released as electrons are transferred along the ETC from an electron donor (reducing agent or reductant) to an electron acceptor (oxidizing agent or oxidant) is used to pump H+ at Complexes I, III, and IV. [Note: The electrons can be transferred as hydride ions to NAD+; as hydrogen atoms to FMN, CoQ, and FAD; or as electrons to cytochromes.] 1.
Redox pairs: Oxidation (loss of electrons) of one substance is always accompanied by reduction (gain of electrons) of a second. For example, Figure 6.11 shows the oxidation of NADH to NAD+ by NADH dehydrogenase at Complex I, accompanied by the reduction of FMN, the prosthetic group, to FMNH2. Such redox reactions can be written as the sum of two separate half reactions, one an oxidation and the other a reduction (see Fig. 6.11). NAD+ and NADH form a redox pair, as do FMN and FMNH2. Redox pairs differ in their tendency to lose electrons. This tendency is a characteristic of a particular redox pair and can be quantitatively specified by a constant, E0 (the standard reduction potential), with units in volts.
2.
Standard reduction potential: The E0 of various redox pairs can be ordered from the most negative E0 to the most positive. The more negative the E0 of a redox pair, the greater the tendency of the reductant member of that pair to lose electrons. The more positive the E0, the greater the tendency of the oxidant member of that pair to accept electrons. Therefore, electrons flow from the pair with the more negative
E0 to that with the more positive E0. The E0 values for some members of the ETC are shown in Figure 6.12. [Note: The components of the chain are arranged in order of increasingly positive E0 values.] 3. Relationship of ∆G0 to ∆E0: The ∆G0 is related directly to the magnitude of the change in E0: where n = number of electrons transferred (1 for a cytochrome, 2 for NADH, FADH2, and CoQ)
F = Faraday constant (23.1 kcal/volt mol) ∆E0 = E0 of the electron-accepting pair minus the E0 of the electron-donating pair ∆G0 = change in the standard free energy 4. ∆G0 of ATP: The ∆G0 for the phosphorylation of ADP to ATP is +7.3 kcal/mol. The transport of a pair of electrons from NADH to O2 through the ETC releases 52.6 kcal. Therefore, more than sufficient energy is available to produce three ATP from three ADP and three Pi (3 × 7.3 = 21.9 kcal/mol), sometimes expressed as a P/O ratio (ATP made per O atom reduced) of 3:1. The remaining calories are used for ancillary reactions or released as heat. [Note: The P:O for FADH2 is 2:1 because
Complex I is bypassed.]
VI. PHOSPHORYLATION OF ADP TO ATP
The transfer of electrons down the ETC is energetically favored because NADH is a strong electron donor and O2 is an avid electron acceptor. However, the flow of electrons does not directly result in ATP synthesis.
A. Chemiosmotic hypothesis
The chemiosmotic hypothesis (also known as the Mitchell hypothesis) explains how the free energy generated by the transport of electrons by the ETC is used to produce ATP from ADP + Pi.
1.
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Proton pump: Electron transport is coupled to ADP phosphorylation by the pumping of H+ across the inner mitochondrial membrane, from the matrix to the intermembrane space, at Complexes I, III, and IV. For each pair of electrons transferred from NADH to O2, 10 H+ are pumped. This creates an electrical gradient (with more positive charges on the cytosolic side of the membrane than on the matrix side) and a pH (chemical) gradient (the cytosolic side of the membrane is at a lower pH than the matrix side), as shown in Figure 6.13. The energy (proton-motive force) generated by these gradients is sufficient to drive ATP synthesis. Thus, the H+ gradient serves as the common intermediate that couples oxidation to phosphorylation.
2.
ATP synthase: The multisubunit enzyme ATP synthase ([Complex V] Fig. 6.14) synthesizes ATP using the energy of the H+ gradient. It contains a membrane domain (Fo) that spans the inner mitochondrial membrane and an extramembranous domain (F1) that appears as a sphere that protrudes into the mitochondrial matrix (see Fig. 6.13). The chemiosmotic hypothesis proposes that after H+ have been pumped to the cytosolic side of the inner mitochondrial membrane, they reenter the matrix by passing through a H+ channel in the Fo domain, driving rotation of the c ring of Fo and, at the same time, dissipating the pH and electrical gradients. Rotation in Fo causes conformational changes in the three β subunits of F1 that allow them to bind ADP + Pi, phosphorylate ADP to ATP, and release ATP. One complete rotation of the c ring produces three ATP. [Note: ATP synthase is also called F1/Fo-ATPase because the enzyme can also catalyze the hydrolysis of ATP to ADP and Pi.] contains eight subunits. One complete turn of the ring is driven by eight H+ (protons) moving through the Fo domain. The resulting conformational changes in the three β subunits of the F1 domain allow phosphorylation of three adenosine diphosphates (ADP) to three ATP.] Pi = inorganic phosphate.
a. Coupling in oxidative phosphorylation: In normal mitochondria, ATP synthesis is coupled to electron transport through the H+ gradient. Increasing (or decreasing) one process has the same effect on the other. For example, hydrolysis of ATP to ADP and Pi in energy-requiring reactions increases the availability of substrates for ATP synthase and, thus, increases H+ flow through the enzyme. Electron transport and H+ pumping by the ETC increase to maintain the H+ gradient and allow ATP synthesis.
b. Oligomycin: This drug binds to the Fo (hence the letter “o”) domain of ATP synthase, closing the H+ channel and preventing reentry of H+ into the matrix, thereby inhibiting phosphorylation of ADP to ATP. Because the pH and electrical gradients cannot be dissipated in the presence of this phosphorylation inhibitor, electron transport stops because of the difficulty of pumping any more H+ against the steep gradient. This dependency of cellular respiration on the ability to phosphorylate ADP to ATP is known as respiratory control and is the consequence of the tight coupling of these processes.
c.
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Uncoupling proteins: Uncoupling proteins (UCP) occur in the inner mitochondrial membrane of mammals, including humans. These proteins form channels that allow H+ to reenter the mitochondrial matrix without energy being captured as ATP (Fig. 6.15). The energy is released as heat, and the process is called nonshivering thermogenesis. UCP1, also called thermogenin, is responsible for heat production in the mitochondria-rich brown adipocytes of mammals. [Note: Cold causes catecholamine-dependent activation of UCP1 expression.] In brown fat, unlike the more abundant white fat, ~90% of its respiratory energy is used for thermogenesis in infants in response to cold. Thus, brown fat is involved in energy expenditure, whereas white fat is involved in energy storage. [Note: Brown fat depots have recently been shown to be present in adults.] d.
Synthetic uncouplers: Electron transport and phosphorylation of ADP can also be uncoupled by compounds that shuttle H+ across the inner mitochondrial membrane, dissipating the gradient. The classic example is 2,4-dinitrophenol, a lipophilic H+ carrier (ionophore) that readily diffuses through the mitochondrial membrane (Fig. 6.16). This uncoupler causes electron transport to proceed at a rapid rate without establishing a H+ gradient, much as do the UCP. Again, energy is released as heat rather than being used to synthesize ATP. [Note: In high doses, aspirin and other salicylates uncouple oxidative phosphorylation. This explains the fever that accompanies toxic overdoses of these drugs.]
B. Membrane transport systems
The inner mitochondrial membrane is impermeable to most charged or hydrophilic substances. However, it contains numerous transport proteins that permit passage of certain molecules from the cytosol to the mitochondrial matrix.
1.
ATP and ADP transport: The inner membrane requires specialized carriers to transport ADP and Pi from the cytosol (where ATP is hydrolyzed to ADP in many energy-requiring reactions) into mitochondria, where ATP can be resynthesized. An adenine nucleotide antiporter imports one ADP from the cytosol into the matrix, while exporting one ATP from the matrix into the cytosol (see Fig. 6.13). A symporter cotransports Pi and H+ from the cytosol into the matrix.
2.
Reducing equivalent transport: The inner mitochondrial membrane lacks an NADH transporter, and NADH produced in the cytosol (for example, in glycolysis; see p. 101) cannot directly enter the mitochondrial matrix. However, reducing equivalents of NADH are transported from the cytosol into the matrix using substrate shuttles. In the glycerol 3phosphate shuttle (Fig. 6.17A), two electrons are transferred from NADH to dihydroxyacetone phosphate by cytosolic glycerol 3-phosphate dehydrogenase. The glycerol 3-phosphate produced is oxidized by the mitochondrial isozyme as FAD is reduced to FADH2. CoQ of the ETC oxidizes the FADH2. Therefore, the glycerol 3-phosphate shuttle results in the synthesis of two ATP for each cytosolic NADH oxidized. This contrasts with the malate-aspartate shuttle (Fig. 6.17B), which produces NADH (rather than FADH2) in the mitochondrial matrix, thereby yielding three ATP for each cytosolic NADH oxidized by malate dehydrogenase as oxaloacetate is reduced to malate. A transport protein moves malate into the mitochondrial matrix.
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inner mitochondrial membrane. A. Glycerol 3-phosphate shuttle. B. Malate aspartate shuttle. DHAP = dihydroxyacetone phosphate; NAD(H) = dinucleotide; CoQ = coenzyme Q.
C. Inherited defects in oxidative phosphorylation
Thirteen of the ~90 polypeptides required for oxidative phosphorylation are encoded by mtDNA and synthesized in mitochondria, whereas the remaining proteins are encoded by nuclear DNA, synthesized in the cytosol, and then transported into mitochondria. Defects in oxidative phosphorylation are more likely a result of alterations in mtDNA, which has a mutation rate about 10 times greater than that of nuclear DNA. Tissues with the greatest ATP requirement (for example, the central nervous system, skeletal and heart muscle, and the liver) are most affected by defects in oxidative phosphorylation. Mutations in mtDNA are responsible for several diseases, including some cases of mitochondrial myopathies, and Leber hereditary optic neuropathy, a disease in which bilateral loss of central vision occurs as a result of neuroretinal degeneration, including damage to the optic nerve. [Note: mtDNA is maternally inherited because mitochondria from the sperm cell do not enter the fertilized egg.]
D. Mitochondria and apoptosis
The process of apoptosis (programmed cell death) may be initiated through the intrinsic (mitochondrial-mediated) pathway by the formation of pores in the outer mitochondrial membrane. These pores allow cytochrome c to leave the intermembrane space and enter the cytosol. There, cytochrome c, in association with proapoptotic factors, activates a family of proteolytic enzymes (the caspases), causing cleavage of key proteins and resulting in the morphologic and biochemical changes characteristic of apoptosis.
VII. CHAPTER SUMMARY
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The change in free energy (∆G) occurring during a reaction predicts the direction in which that reaction will spontaneously proceed. If ∆G is negative (that is, the product has a lower free energy than the substrate), then the reaction is spontaneous as written. If ∆G is positive, then the reaction is not spontaneous. If ∆G = 0, then the reaction is in equilibrium. The ∆G of the forward reaction is equal in magnitude but opposite in sign to that of the back reaction. The ∆G are additive in any sequence of consecutive reactions, as are the standard free energy changes (∆G0). Therefore, reactions or processes that have a large, positive ∆G are made possible by coupling with those that have a large, negative ∆G such as ATP hydrolysis. The reduced coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) each donate a pair of electrons to a specialized set of electron carriers, consisting of flavin mononucleotide (FMN), iron-sulfur centers, coenzyme Q, and a series of heme-containing cytochromes, collectively called the electron transport chain. This pathway is present in the inner mitochondrial membrane (impermeable to most substances) and is the final common pathway by which electrons derived from different fuels of the body flow to oxygen (O2), which has a large, positive reduction potential (E0), reducing it to water. The terminal cytochrome, cytochrome c oxidase, is the only cytochrome able to bind O2. Electron transport results in the pumping of protons (H+) across the inner mitochondrial membrane from the matrix to the intermembrane space, 10 H+ per NADH oxidized. This process creates electrical and pH gradients across the inner mitochondrial membrane. After H+ have been transferred to the cytosolic side of the membrane, they reenter the matrix by passing through the Fo H+ channel in ATP synthase (Complex V), dissipating the pH and electrical gradients and causing conformational changes in the F1 β subunits of the synthase that result in the synthesis of ATP from ADP + inorganic phosphate. Electron transport and phosphorylation are tightly coupled in oxidative phosphorylation ([OXPHOS] Fig. 6.18). Inhibition of one process inhibits the other. These processes can be uncoupled by uncoupling protein-1 of the inner mitochondrial membrane of brown adipocytes and by synthetic compounds such as 2,4-dinitrophenol and aspirin, all of which dissipate the H+ gradient. In uncoupled mitochondria, the energy produced by electron transport is released as heat rather than being used to synthesize ATP. Mutations in mitochondrial DNA, which is maternally inherited, are responsible for some cases of mitochondrial diseases such as Leber hereditary optic neuropathy. The release of cytochrome c into the cytoplasm and subsequent activation of proteolytic caspases results in apoptotic cell death.
Choose the ONE best answer.
.1. 2,4-Dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, was used as a weight-loss agent in the 1930s. Reports of fatal overdoses led to its discontinuation in 1939. Which of the following would most likely be true concerning individuals taking 2,4-DNP?
A. ATP levels in the mitochondria are greater than normal.
B. Body temperature is elevated as a result of hypermetabolism.
C. Cyanide has no effect on electron flow.
D. The proton gradient across the inner mitochondrial membrane is greater than normal.
E. The rate of electron transport is abnormally low.
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Correct answer = B. When phosphorylation is uncoupled from electron flow, a decrease in the proton gradient across the inner mitochondrial membrane and, therefore, impaired ATP synthesis are expected. In an attempt to compensate for this defect in energy capture, metabolism and electron flow to oxygen are increased. This hypermetabolism will be accompanied by elevated body temperature because the energy in fuels is largely wasted, appearing as heat. The electron transport chain will still be inhibited by cyanide.
.2. Which of the following has the strongest tendency to gain electrons?
A. Coenzyme Q
B. Cytochrome c
C. Flavin adenine dinucleotide
D. Nicotinamide adenine dinucleotide
E. Oxygen
Correct answer = E. Oxygen is the terminal acceptor of electrons in the electron transport chain (ETC). Electrons flow down the ETC to oxygen because it has the highest (most positive) reduction potential (E0). The other choices precede oxygen in the ETC and have lower E0 values.
.3. Explain why and how the malate-aspartate shuttle moves nicotinamide adenine dinucleotide reducing equivalents from the cytosol to the mitochondrial matrix.
There is no transporter for nicotinamide adenine dinucleotide (NADH) in the inner mitochondrial membrane. However, cytoplasmic NADH can be oxidized to NAD+ by malate dehydrogenase as oxaloacetate (OAA) is reduced to malate. The malate is transported across the inner membrane to the matrix where the mitochondrial isozyme of malate dehydrogenase oxidizes it to OAA as mitochondrial NAD+ is reduced to NADH. This NADH can be oxidized by Complex I of the electron transport chain, generating three ATP through the coupled processes of oxidative phosphorylation.
.4. Carbon monoxide (CO) binds to and inhibits Complex IV of the electron transport chain. What effect, if any, should this respiratory inhibitor have on phosphorylation of adenosine diphosphate (ADP) to ATP?
Inhibition of electron transport by respiratory inhibitors such as CO results in an inability to maintain the proton (H+) gradient. Therefore, phosphorylation of ADP to ATP is inhibited, as are ancillary reactions such as calcium uptake by mitochondria, because they also require the H+ gradient.
Introduction to Carbohydrates 7
For additional ancillary materials related to this chapter, please visit thePoint.
I. OVERVIEW
Carbohydrates (saccharides) are the most abundant organic molecules in nature. They have a wide range of functions, including providing a significant fraction of the dietary calories for most organisms, acting as a storage form of energy in the body, and serving as cell membrane components that mediate some forms of intercellular communication. Carbohydrates also serve as a structural component of many organisms, including the cell walls of bacteria, the exoskeleton of insects, and the fibrous cellulose of plants. [Note: The full set of carbohydrates produced by an organism is its glycome.] The empiric formula for many of the simpler carbohydrates is (CH2O)n, where n ≥3, hence the name “hydrate of carbon.”
II. CLASSIFICATION AND STRUCTURE
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Monosaccharides (simple sugars) can be classified according to the number of carbon atoms they contain. Examples of some monosaccharides commonly found in humans are listed in Figure 7.1. They can also be classified by the type of carbonyl group they contain. Carbohydrates with an aldehyde as their carbonyl group are called aldoses, whereas those with a keto as their carbonyl group are called ketoses (Fig. 7.2). For example, glyceraldehyde is an aldose, whereas dihydroxyacetone is a ketose. Carbohydrates that have a free carbonyl group have the suffix -ose. [Note: Ketoses have an additional “ul” in their suffix such as xylulose. There are exceptions, such as fructose, to this rule.] Monosaccharides can be linked by glycosidic bonds to create larger structures (Fig. 7.3). Disaccharides contain two monosaccharide units, oligosaccharides contain three to ten monosaccharide units, and polysaccharides contain more than ten monosaccharide units and can be hundreds of sugar units in length.
A. Isomers and epimers
Compounds that have the same chemical formula but have different structures are called isomers. For example, fructose, glucose, mannose, and galactose are all isomers of each other, having the same chemical formula, C6H12O6. Carbohydrate isomers that differ in configuration around only one specific carbon atom (with the exception of the carbonyl carbon, see C.
1. below) are defined as epimers of each other. For example, glucose and galactose are C-4 epimers because their structures differ only in the position of the –OH (hydroxyl) group at carbon 4. [Note: The carbons in sugars are numbered beginning at the end that contains the carbonyl carbon (that is, the aldehyde or keto group), as shown in Fig. 7.4.] Glucose and mannose are C-2 epimers. However, because galactose and mannose differ in the position of –OH groups at two carbons (carbons 2 and 4), they are isomers rather than epimers (see Fig. 7.4).
B. Enantiomers
A special type of isomerism is found in the pairs of structures that are mirror images of each other. These mirror images are called enantiomers, and the two members of the pair are designated as a D-and an L-sugar (Fig. 7.5). The vast majority of the sugars in humans are D-isomers. In the D-isomeric form, the –OH group on the asymmetric carbon (a carbon linked to four different atoms or groups) farthest from the carbonyl carbon is on the right, whereas in the L-isomer, it is on the left. Most enzymes are specific for either the D or the L form, but enzymes known as isomerases are able to interconvert D-and L-isomers.
comparison to a triose, glyceraldehyde. [Note: The asymmetric carbons are shown in green.]
C. Monosaccharide cyclization
Less than 1% of each of the monosaccharides with five or more carbons exists in the open-chain (acyclic) form in solution. Rather, they are predominantly found in a ring (cyclic) form, in which the aldehyde (or keto) group has reacted with a hydroxyl group on the same sugar, making the carbonyl carbon (carbon 1 for an aldose, carbon 2 for a ketose) asymmetric. This asymmetric carbon is referred to as the anomeric carbon.
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1. Anomers: Creation of an anomeric carbon (the former carbonyl carbon) generates a new pair of isomers, the α and β configurations of the sugar (for example, α-D-glucopyranose and β-D-glucopyranose), as shown in Figure 7.6, that are anomers of each other. [Note: In the α configuration, the –OH group on the anomeric carbon projects to the same side as the ring in a modified Fischer projection formula (see Fig. 7.6A) and is trans to the CH2OH group in a Haworth projection formula (see Fig. 7.6B).
The α and β forms are not mirror images, and they are referred to as diastereomers.] Enzymes are able to distinguish between these two structures and use one or the other preferentially. For example, glycogen is synthesized from α-D-glucopyranose, whereas cellulose is synthesized from β-D-glucopyranose. The cyclic α and β anomers of a sugar in solution spontaneously (but slowly) form an equilibrium mixture, a process known as mutarotation (see Fig. 7.6). [Note: For glucose, the α form makes up 36% of the mixture.] six-membered ring (5 C + 1 O) is termed a pyranose, whereas one with a five-membered ring (4 C + 1 O) is a furanose. Virtually all glucose in solution is in the pyranose form.] 2. Reducing sugars: If the hydroxyl group on the anomeric carbon of a cyclized sugar is not linked to another compound by a glycosidic bond (see E. below), the ring can open. The sugar can act as a reducing agent and is termed a reducing sugar. Such sugars can react with chromogenic agents (for example, the Benedict reagent) causing the reagent to be reduced and colored as the aldehyde group of the acyclic sugar is oxidized to a carboxyl group. All monosaccharides, but not all disaccharides, are reducing sugars. [Note: Fructose, a ketose, is a reducing sugar because it can be isomerized to an aldose.]
A colorimetric test can detect a reducing sugar in urine. A positive result is indicative of an underlying pathology (because sugars are not normally present in urine) and can be followed up by more specific tests to identify the reducing sugar.
D. Monosaccharide joining
Monosaccharides can be joined to form disaccharides, oligosaccharides, and polysaccharides. Important disaccharides include lactose (galactose + glucose), sucrose (glucose + fructose), and maltose (glucose + glucose). Important polysaccharides include branched glycogen (from animal sources) and starch (plant sources) and unbranched cellulose (plant sources). Each is a polymer of glucose.
E. Glycosidic bonds
The bonds that link sugars are called glycosidic bonds. They are formed by enzymes known as glycosyltransferases that use nucleotide sugars (activated sugars) such as uridine diphosphate glucose as substrates. Glycosidic bonds between sugars are named according to the numbers of the connected carbons and with regard to the position of the anomeric hydroxyl group of the first sugar involved in the bond. If this anomeric hydroxyl is in the α configuration, then the linkage is an α-bond. If it is in the β configuration, then the linkage is a β-bond. Lactose, for example, is synthesized by forming a glycosidic bond between carbon 1 of β-galactose and carbon 4 of glucose. Therefore, the linkage is a β(1→4) glycosidic bond (see Fig. 7.3). [Note: Because the anomeric end of the glucose residue is not involved in the glycosidic linkage, it (and, therefore, lactose) remains a reducing sugar.]
F. Carbohydrate linkage to noncarbohydrates
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