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`JANSSEN EXHIBIT 2086
`
`JANSSEN EXHIBIT 2086
`Wockhardt v. Janssen IPR2016-01582
`
`

`

`Prz'hczp[es and Practice of
`ENDOCRINOLOGY
`
`METABOLISM
`
`THIRD EDITION
`
`I v E
`
`DITOR
`Kenneth L. Becker
`
`ASSOCIATE EDITORS
`
`john P. Bilezikian
`William I. Bremner
`Wellington Hung
`C. Ronald Kahn
`
`D. Lynn Loriaux
`Eric S. Nylén
`Robert W. Rebar
`
`Gary L. Robertson
`Richard H. Snider, Ir.
`Leonard Wartofsky
`
`With 330 Contributors
`
`ALA LIPlDINCOTT WILLIAMS 6 WILKINS
`v A Wolters Kluwer Company
`Philadelphia - Baltimore - New York - London
`Buenos Aires - Hong Kong;
`- Sydney - Tokyo
`
`V'
`
`

`

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`___’__—_____._._——_—h
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`
`.
`.
`s.
`,
`Principles and practice of endocrinology and metabolism / editor, Kenneth [ Becker .
`associate editors, John P. Bilezikian
`[et al.].--3rd ed.
`P‘ ,‘ cm.
`Includes bibliographical references and index.
`ISBN 07817-17505
`1. Endocrinology. 2. Endocrine glai'ids-ADiseases. 3. Metabolism--Disorders. l. Becker,
`Kenneth L.
`{DNLM1 l. Endocrine Diseases. 2. Metabolic Diseases. WK 100 P957 2000]
`RCr’H/‘i .l’h7 2000
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`a particular situation remains the professional responsibilitv of
`of this information in
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`the practitioner.
`The authors, editors, and PUbli>lwr have exerted everv effort to ensure that drug
`selection and dosage set forth in this text are in accordance with current recommenda—
`tions and practice at the time of publication. However, in view of ongoing research
`7
`.
`4
`i
`changes in government regulations, and the constant flow of information relating to
`drug therapy and drug reactions, the reader is urged to check the package insert for each
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`lhis is particularly important when the recommended agent is a new or infrequently
`employed drug.
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`Administrmgh}Ehrlicdlical dc\ lgth lprcscnted in this publication have lood and Drug
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`10987654321
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`

`

`704
`
`PART V: THE ADRENAL GLANDS
`
`There is a close relationship between neural cells of the sympa—
`thetic ganglia and chromaffin cells. Small numbers of ganglion
`cells can be found in the adrenal medulla, and chromaffin cells
`occur in the sympathetic ganglia. Hence, neoplastic disorders
`affecting either cell type can arise throughout this system.
`The diagnosis of adrenal medullary hyperplashia depends on
`an increased volume of adrenal medulla in relation to the cor-
`tex. There is a diffuse nodular proliferation of normal. medul-
`lary elements. This condition is seen mainly in families With
`multiple endocrine neoplasia type 2A and 1s conSidered a pre-
`neoplastic condition (see Chap. 188).
`.
`Neuroblastomas, ganglioneuroblastomas, and ganglioneuro-
`mas arise from neuroblasts. These tumors form a continuum from
`least to most differentiated and from malignant to benign. Neuro-
`blastoma usually is a tumor of infancy and childhood, with a
`median incidence at
`the age of 2 years.
`It originates in the
`paraganglia of the sympathetic nervous system or the adrenal
`medulla, and 60% arise in the abdomen. Grossly, it is a multi-
`nodular tumor with areas of hemorrhage, necrosis, and cystic
`degeneration.“ The cells are arranged in nests and contain small,
`dark-staining nuclei with little cytoplasm. Although the light-
`microscopic features are not necessarily distinct from those of
`other childhood tumors, electron microscopic examination shows
`characteristic cytoplasmic neurosecretory granules. Neuroblasto-
`mas usually secrete catecholamines, their metabolites, or both.
`Characteristically,
`these tumors grow rapidly and metastasme
`early to local lymph nodes, the liver and other abdominal organs,
`and bone. Although spontaneous regression or differentiation into
`a more benign tumor may occur, mortality is high and treatment
`often includes combinations of palliative surgery, radiation, and
`chemotherapy.25 Ganglioneuroblastomas contain. some mature
`ganglion cells and have a better prognosis. Ganglionegirpmas are
`benign tumors arising from mature neuronal elementsrf’l-7
`Pheochromocytomas arise from chromaffin cells. As expected
`from their cellular origin, 95% or more are in the abdomen, with
`most being in the adrenal glands.” Similar to chromaffin cells,
`however, they occasionally can be found anywhere along the sym-
`pathetic chain of ganglia from the base of the skull to the neck of
`the urinary bladder. Ten percent of patients with sporadic pheo-
`chromocytomas and 50% of those with familial pheochromocyto-
`mas have bilateral tumors. The pheochromocytoma that may occur
`in von Hippel-Lindau disease commonly is bilateral.”30 Although
`only 5% to 10% are malignant, it is often impossible to distinguish
`benign from malignant neoplasms histologically. l’heochromocyto-
`mas are highly vascular tumors with local hemorrhage and cystic
`degeneration. lvlicroscopically, they often have a chaotic pattern of
`pleomorphic elongated cells with prominent cytoplasmic granules
`(see Chap. 86). Although the light-microscopic features may not be
`diagnostic, electron microscopic examination demonstrates charac-
`teristic dense ca techolamine secretory granules.
`
`ADRENAL MEDULLARY HYPOFUNCTION
`
`The adrenal medulla is affected by the same systemic diseases
`(tubercular and fungal infections, sarcoidosis, amyloidosis) as
`is the cortex and also is often the initial site of hemorrhage. Iso-
`lated adrenal medullary hypofunction is uncommon and usu-
`ally occurs in the setting of diffuse autonomic insufficiency.
`Sparse amounts of medullary tissue may be found in some eld-
`erly persons, but no specific abnormality has been described.
`
`REFERENCES
`
`1. Neville AM, O’Hare MJ. Histopatliologv of the adrenal cortex. J Clin Endo-
`crinol Metab 1985; 14:791.
`‘
`'
`2. Neville AM, O’Hare M]. The human adrenal cortex. Pathology and biol-
`ogy~an integrated approach. New York: Springer-Verlag, 1982.
`3. Symington '1’. The adrenal cortex. In: Bloodworth JMB Jr, ed. Endocrine
`pathology. General and surgical. Baltimore: Williams 8»: Wilkins, 19822419.
`4. Could VE, Sommers SC. Adrenal medulla and paraganglia. In: Bloodworth
`
`pi
`
`JMB Jr, ed. Endocrine pathology. General and surgical. Baltimore: Williams
`& Wilkins, 19822473.
`Francis IR, Cross MD, Shapiro B, et a1. Integrated imaging of adrenal dis
`ease. Radiology 1992; 184:1.
`6. Breslow M]. Regulation of adrenal medullary and cortical blood flow. Am]
`l’liysiol 1992; 262:1'11317.
`7. Hornsby 1’]. Regulation of adrenocortical function by control of growth
`and structure. In: Anderson DC, Winter JSD, eds. Adrenal cortex: BlMR
`clinical endocrinology Boston: Butterworth, 198.51
`8. Hyatt 1’]. Functional significance of the adrenal zones. 1n: D'Agata R,
`Chrousos Gl’, eds. Recent advances in adrenal regulation and function.
`New York: Raven 1’ress, 1987235.
`In:
`9. Winter JSD. Functional changes in the adrenal gland during life.
`D’Agata R, Chrousos CI’, eds. Recent advances in adrenal regulation and
`function. New York: Raven Press, 1987151.
`10. Copeland I’M. The incidentally discovered adrenal mass. Ann Intern Med
`1983;961:940.
`11. Doppman ]L, Miller DL, Dryer A], et a1. Macronodular adrenal hyperplasia
`in Cushing’s disease. Radiology 1988; 166:347.
`12. Carney JA, Young WF Jr. Primary pigmented nodular adrenocortical dis-
`ease and its associated conditions. The Endocrinologist 1992; 2:6.
`13. Ruder HJ, Loriaux DL, Lipsett MB. Severe osteopenia in young adults asso—
`ciated with Cushing’s syndrome due to micronodular adrenal disease. J
`Clin Endocrinol Metab 1974; 3911138.
`14. Sakai Y, Yanase '1', Hara '1', et a1. Mechanism of abnormal production of
`adrenal androgens in patients with adrenocortical adenomas and carcino-
`mas. J Clin Endocrinol Metab 1994; 78:36.
`15. Del Gaudio A, Solidoro G. Myelolipoma of the adrenal gland: report of two
`cases with a review of the literature. Surgery 19861901293.
`16. Kelley RI, Datta NS, Dobyns WB, et a1. Neonatal adrenoleukodystrophy: new
`cases, biochemical studies, and differentiation from Zellweger and related
`peroxisomal polydystrophy syndromes. Am] Med Genet 1986; 23:869.
`17. Schwartz RE, Stayer SA, I’asquariello CA, et al. Anesthesia for the patient
`with neonatal adrenoleukodystrophy. Can J Anaesth 1994; 41:56.
`18. Xarli VI’, Steele AA, Davis 1’J, et a1. Adrenal hemorrhage in the adult. Med«
`icine (Baltimore) 1978; 57:211.
`19. Rao Rll, Vagnucci AH, Amico JA. Bilateral massive adrenal hemorrhage:
`early recognition and treatment. Ann Intern Med 1989; 110:227.
`20. Caron 1’, Chalianier MH, Cainbus ]1’, et a1. Definitive adrenal insufficiency
`due to bilateral adrenal hemorrhage and primary antiphospholipid syn-
`d roine. J Clin Endocrinol Metab 1998;831:1437.
`.
`21. Stolai'czykk, Ruio SI, Sinolyar D, et a1. Twenty-torir-hour urinary free cortisol in
`patients with acquired immunodeficiency syndrome. Metabolism 1998; 47:690.
`22. Verges I}, Chdvanct 1’, Degres J, et a1. Adrenal function in HIV infected
`patients. Acta Endocrinol (Copenh) 1989; 1213633-
`23. Bernstein SR, Gonzalez—Hernandez JA, Erhart-Bornstein M, et al. Intimate
`contact of chromaffin and cortical cells within 1116 Iiuinan adrenal gland
`forms the basis for important intraadrenal interactions. J Clin Endocrinol
`Metab 1994; 78:225.
`,
`24. Askin PB, I’erlman E]. Neuroblastoma and peripheral neuroectmtermal
`tumors. Am J Clin l’athol 1998; 109(4 Suppl 11153.3:
`.
`25. Katzenstein HM, Cohn SL. Advances in the diagnosis and treatment of
`neuroblastoma. Curr Opiii Oncol 1998; 10743:
`,.
`.
`26. Schulman H, Laufer L, Barki Y, 01 81 GJT‘Sllmwurmn“: an meidentaloma
`of childhood. Eur Radiol 1998; 8:582.
`.
`)
`27. Fujiwara '1', Kawamura M, Sasou S, Hiramori K. [\L‘Slllts of surgery for a
`.
`,
`-
`(2"
`compound tumor consisting of pheochromocytoma and ganglioneurohlas—
`toma in an adult; 5—year follow-up. Intern l\/1Ld 2000, 3).38.
`28‘ Melddms Ll, Wolf BC, Balogh K, Federman M. Adrenal pheochroinoC),_
`of 60 cases. IIum I’athol 1985; 16:580.
`toma: a clinicopathologic review
`‘
`29, Chew SL, Dacie J15, Reznik RH, et aI. Bilateral pheochromocytomas in von
`HippeI-I.indau disease. Q] Med 1994; 87:49
`,
`.
`.
`30. Couch V, Lindor 11M, Karnes 1’5. MiC11915 VV' V0” llIPPL’l‘lsmddu ‘1'59‘150:
`Mayo Clin I’roc 2000; 75:265.
`
`
`
`CHAPTER 72
`
`
`SYNTHESIS AND
`METABOLISM OF
`CORTICOSTEROIDS
`
`PERRIN C. WHITE
`
`ogically complex organs that
`The adrenal glands are endOcrinol
`‘
`tissues derived from
`are composed of two distinct endocrlne
`). The adrenal cor—
`different embryologic sources1 (see Chap: 71
`land and is the
`tex (OUter 1aYer) constitutes 80% to 90% 01c the t;
`
`

`

`
`
`Cyclopenianoperhydrophenanthrene
`
`CH3
`
`Ch. 72: Synthesis and Metabolism of Corticosteroids
`
`705
`
`
`
`or
`AzB—trans
`
`9
`10 B 87
`6
`
` (3
`
`AzB—cis
`
`9
`
`10 B 87
`6
`
`FIGURE 72-2. ClS‘il‘rlllS orientation of the steroid nucleus and location
`of the (x- and B—SLibstituents.
`
`BIOSYNTHESIS
`
`IMPORTATION INTO MITOCHONDRIA
`
`The rate-limiting step in steroid biosynthesis is importation of
`cholesterol from cellular stores to the matrix side of the mito-
`chondria inner membrane where the cholesterol side-chain
`
`cleavage system (CYPl'lA, adrenodoxin, adrenodoxin reduc—
`tase) is located. This is controlled by the steroidogeiii'c acute regu-
`latory protein (StAR),3'2“ the synthesis of which is increased
`within minutes by trophic stimuli such as adrenocorticotropic
`hormone (ACTH) or, in the zona glomerulosa, by increased
`intracellular calcium. StAR is synthesized as a 37-kDa phospho-
`protein that contains a mitochondrial importation signal pep-
`tide. However, importation into mitochondria is not necessary
`for StAR to stimulate steroidogenesis; to the contrary, the likeli—
`
`TABLE 72-1.
`Adrenal Steroidogenesis: Nomenclature
`Nomenclature of the Major Naturally Occurring Steroid Hormones
`
`Common Name
`Biochemical Name
`Latter
`——__—____‘
`Aldosterone
`
`l’regn-Jt-en-ll (3,21-diol-18—al-3,20-
`dione
`
`Corticosterone
`
`Cortisol (hydrocortisone)
`
`Cortisone
`
`Dehydroepiandrosterone
`(DHA, DHEA)
`
`l’i‘egii-4-eii-l‘l (3,21-diol«3,20«dione
`Pregnel-en-l1(3,'l7(1,Zl-triols3,20-
`dione
`
`I’regn-«lven—1701,21-diol—3,11,20-trione
`Androst—S-en—CtB-ol-l7—one
`
`B
`F
`
`E
`
`0
`
`0
`
`OH
`
`021 Pregnane
`(Progesterone)
`
`C19 Androstane
`(Testosterone)
`
`Cia Estrane
`(Estradiol)
`
`FIGURE 72-1. The steroid nucleus. The four rings are labeled A, B, C, D,
`and the carbons are numbered as shown. Examples are shown of ster-
`oids from each of the three structural categories of steroid hormones:
`the Zl-carbon pregnane derivatives, the 19-carbon androstane deriva-
`tives, and the l8—carbon estrane derivatives. '1 he names of the indiv1d—
`ual steroids shown are indicated in parentheses.
`
`source of the steroid hormones, whereas the adrenal medulla is
`the source of catecholamines. Although the adrenal cortex and
`medulla are in close proximity, they function independently.
`This chapter describes the biosynthesrs, metabolism, mecha-
`nisms of action, and regulation of the ster01d products of the
`adrenal cortex.
`Three major groUPS of hormones are produced by the adre—
`nal cortex: mi,I‘vllnlocorticoids, gliicocorticmds, and sex sterords.
`Mineralocorticoids are produced primarily by the zona glomer-
`ulosa; glucocorticoids are produced by the zona fasciculata;
`and sex steroids originate primarily from the zona reticularis
`The hormonal products of the adrenal cortex share cholesterol
`as a common precursor. Cholesterol is also the precursor for. the
`gonadal steroids, vitamin D and derivatives, and the bile ac1ds.
`
`STRUCTURE AND NOMENCLATURE
`
`Steroids have a common structure with '17-carbon atoms
`arranged in three six-membered rings and. a fourth five-
`membered ring labeled A, B, C, and D, respectively (Fig. 72-1),
`Each of the 17 carbons is numbered in a standard way. Two
`additional carbons, numbered 18 and 19, may be attached at
`carbons '13 and ,10, respectively. Carbon atoms 20 and 21 may
`be attached at the 17 position. These various additions yield
`three steroid families: the C18 (’Sfl‘llllL’S With an aromatic ring
`(estrogens); the C10 amlrostaiivs (androgens); and the (:21 Frog
`iiaiics (corticoids and progestins) (see Fig. 72-1). The ‘steiold
`nucleus lies in a plane that can be modified by the addition of
`substituents either above or below (Fig. 72-2). The (x-subsm“-
`wits occur bylaw the plane (indicated by dotted lines in Fig. 72-?)
`and the B—sabstitiwiits lie above the plane (indicated by sohd
`lines). The A and B rings may be attached so that the substitu-
`ents at positions 5 and 10 are in either the Cis or trans orienta-
`'
`..:~, 2—2.
`’
`“01X igititltitpljicfty o)f trivial and systematic or pioclieiiczizcalggames
`exist for each steroid (Table 72—1). between 1)30 an 19p1i two
`groups under the direction of Reichstein and of Kent a
`iso-
`naturally occurring stermdsz Each group
`lated most of the
`labeled steroids alphabetically in the order in which they were
`discovered, with the result
`that
`the same compound 'Was
`sometimes given two different alphabetical designation/s.
`Thus, Kendall’s compound A is not the same as lteichstein s
`compound A.
`
`Deoxycorticosterone (DOC)
`Deoxycortisol
`Estradiol
`
`Progesterone
`Testosterone
`
`l’regn-ten-Z1-01-3,20-dione
`Pregn-l-errl70.,2l-diol-3,20-dione
`Esti‘a-1,3,4( 10)-trien-3,17B—diol
`l’regn—4-en—3,20—dione
`And rost—l—en- l 7B-ol-3-one
`H
`
`S
`
`

`

`706
`
`PART V: THE ADRENAL GLANDS
`
`TABLE 72-2.
`
`Characteristics of Enzymes Involved in Adrenal Steroid Biosynthesis
`———h———____—____—__
`Number
`Number
`'
`Exons
`Amino Acids”
`Subcellular Location
`
`Chromosome
`
`Gene Size (kb)
`
`Gene
`
`Alias
`
`Enzyme
`
`M C
`
`YI’llA
`CYP17
`HSDBBZ
`
`P450scc
`P450cl7
`3B-HSD
`
`Cholesterol desmolase
`17(1—Hydroxylase/17,20-lyase
`30<l~lydroxysteroid dehydroge-
`nase/Al ~Al-isomerase
`
`15q23-24
`qu24.3
`lpll-13
`
`20
`6.7
`8
`
`9
`8
`4
`
`521/482
`508
`371
`
`Mitochondria
`ER
`ER
`
`P450c21
`CYPZI
`P450c ll
`CYI’llBl
`P450aldo
`CYPllBZ
`P450arom
`CYI’19
`11-HSD2
`HSDHBZ
`ER, endoplasmic reticulum
`*h/iitochondrial enzymes are synthesized as prohormones that are cleaved in mitochondria to the mature proteins; both sizes are given.
`w
`
`3.1
`7
`7
`70
`7
`
`10
`9
`9
`9
`5
`
`494
`503/479
`503/ 479
`503
`405
`
`BR
`Mitochondria
`Mitochondria
`ER
`ER
`
`Zl-tlydroxylase
`1 lB—Hydroxylase
`Aldosterone syntliase
`Aromatase
`1lli—Hydroxysteroid dehydrogenase
`
`6p21.3
`8(122
`8q22
`15q21.1
`l6q22
`
`alyze oxidative conversions of an extremely Wide variety of
`mostl
`1i 0 hilic substrates.
`‘
`Th: rgdficing equivalents are not accepted directly from
`NADPH but instead from accessory electron transport proteins.
`These accessory proteins are necessary because NAPIH
`donates electrons in pairs, whereas P4505 can only accept single
`electrons.8 Microsomal P4505 use a single accessory protein,
`NADPH-dependent cytochrome P450 reductase. Mitochondrial
`P4505 require two proteins; NADPH-dependent adrenodoxm
`reductase donates electrons to adrenodoxrn, which in turn
`transfers them to the P450.9 Adrenodoxin (or ferredoxrn) reduc-
`tase, like cytochrome P450 reductase, is a flavoprotein. Adreno-
`doxin (or ferredoxin) contains nonheme iron‘complexed With
`sulfur. One gene encodes each accessory prOtem In mammals.
`The heart of the P450 catalytic Site 15 a heme group that
`interacts with a highly conserved peptide ofthe 5450 A can};
`pletely conserved cysteine near the C terminus
`is the it
`
`importation rapidly
`hood now appears that mitochondrial
`inactivates StAR.3 The mechanism by which StAR mediates
`cholesterol transport across the mitochondrial membrane is not
`yet known.
`StAR clearly is not the only protein that mediates cholesterol
`transfer across the mitochondrial membrane. Another protein
`that appears necessary (but not sufficient, at
`least
`in the
`adrenals and gonads) for this process is the peripheral benzodinz-
`epine receptor, an 18-kDa protein in the mitochondrial outer
`membrane that is complexed with the mitochondrial voltage-
`dependent anion carrier in contact sites between the outer and
`inner mitochondrial membranes} This protein does not appear
`to be directly regulated by typical trophic stimuli but is stimu-
`lated by endozepincs, peptide hormones also called diazepmn—
`binding inhibitors. Endozepines may be regulated by ACTH to
`some extent, but not with a rapid time course. Thus far,
`whether or not a direct physical
`interaction exists between
`StAR and the peripheral benzodiazepine receptor is not clear.
`
`ENZYMES
`
`The enzymes required for adrenal steroid biosynthesis com-
`prise two classes: cytochrome P450 enzymes and short chain dehy-
`drogennses (Table 72—2); These enzymes are located in either the
`lipophilic membranes of the smooth endoplasmic reticulum or
`the mitochondrial inner membrane. As the successive biosyn-
`thetic reactions involved in steroidogenesis occur, the adrenal
`steroids shuttle between the mitochondria and the smooth
`endoplasmic reticulum (Fig. 72-3).
`
`CYTOCH ROME P450 ENZYMES
`
`Cytochrome P450 (CYP) enzymes are responsible for most of the
`enzymatic conversions from cholesterol to biologically active
`steroid hormones.“ Five P450 enzymes are involved in cortisol
`and aldosterone synthesis (see Table 72-2). Three—CYPllA
`(cholestemI desmolase, P450scc;
`the “sec” stands for ”side
`Chain cleavage”), CYPllBl
`('llli-hydroxylase, P450c11), and
`CYl’llBZ (aldosterone synthase, P450aldo)—have been local-
`ized in mitochondria;
`taro—~CYP17 (‘17(r-l'iydroxylase/17,20-
`lyase, P450c17) and CYP21 (2'1~hydroxylase, P450c21)—are
`located in the endoplasmic reticulum (see Fig. 72-3).
`P450 enzymes are membrane-bound hemoproteins with
`molecular masses of ~50 kDa. Their name arises from their
`property of absorbing light at a peak wavelength of 450 nm.
`These enzymes are located in the membranes of the smooth
`endoplasmic reticulum and the mitochondrial inner membrane
`and are encoded by a large superfamily of genes.7
`P4503 are mixedfnncthm oxidnses. They use molecular oxygen
`and reducing equivalents (i.e., electrons) provided by reduced
`nicotinamide—adenine dinucleotide phosphate (NADI’H) to cat-
`
`
`
`
`
`
`Mitochondrion
`
`Pregnenolone
`CYP1lB1
`
`
`
`
`EndoplasmicReticulum
`
`Plasma Membrane
`
`Cholesterol
`
`CYP11A
`
`CYP17
`
`1 1—Deoxycortisol
`
`17OH Pregnenolone
`
`CYP21
`
`17OH progesterone
`
`Smooth
`
`SBHSD
`A5—A4 Isomerase
`
`
`
`
`
`E‘_
`
`FIGURE 72-3. Biosynthesis of cortisol fr
`names of the steroidogcnic enzymes are
`72-4.
`
`om cholesterol. The functional
`listed in Table 72—2 and Figure
`
`.J
`
`

`

`Ch. 72: Synthesis and Metabolism of Corticosteroids
`
`707
`
`GHQ
`
`"OH
`
`
`
`“OH
`
`HO
`
`
`
`CORTISOL
`GLUCOCORTICOIDS
`
`18 -HYDROXY-
`
`CORTICOSTERONE
`CORTlCOSTERONE
`
`o
`
`
`MINERALOCORTICOIDS
`
`ALDOSTERONE
`
`
`
`
`
`Enzymatic Steps:
`1 C P
`'20 22 H
`~ Y “A‘
`'
`‘ Yd’OXY'ase
`20, 22-Desmolase
`
`2, 35-Hydroxysteroid Dehydrogenase
`A5-A‘ isomerase
`
`3. CYP17: 17 a-Hydroxylase (3a)
`17. 20-Lyase (3b)
`
`4. CYP21: 21-Hydroxylase
`
`5. CYP1 181: 11 fi-Hydroxylase
`
`6, CYP1182: 11 B—Hydroxylase (6a)
`18-Hydroxyiase (6b)
`18-Oxidase (6c)
`
`7. 17-Ketosteroid Reductase
`8. CYP19: Aromatase
`
`9. 3 d-Hydroxysteroid Sulfotransterase
`
`HO
`
`HO
`
`CHOLESTEROL
`1
`
`2
`
`PREGNENOLONE
`
`O
`PROGESTERONE
`3
`
`l
`O
`0
`(:66 0%
`331
`o
`a
`o
`
`”OH 2
`—-—->
`
`”OH 4
`—‘>
`
`1 1»DEOXY -
`
` CORTICOSTERONE
`
`HO
`
`17-HYDROXY.
`PREGNENOLONE
`
`O
`17-HYDROXY-
`PROGESTERONE
`
`O
`
`11<DEOXY-
`coansor
`
`
`
`
`
`7i!'2
`
`205%) Boéjfi
`
`ESTRONE
`
`o
`
`OH
`
`
`
`HO
`
`TL—V
`
`HO
`
`ESTRADIOL
`_—E‘J
`ESTROGENS
`
`so4
`
`
`
`DEHYDROEPI-
`ANDROSTERONE
`SULFATE
`
`HO
`
`HO
`
`DEHYDROEPI-
`ANDROSTERONE
`7 lA OH
`
`A5-ANDROSTENE—
`DIOL
`ANDROGENS
`
`A4—ANDROSTENEDIONE
`7 A
`E
`
`.__—>
`
`0
`
`TESTOSTERONE
`
`OH
`
`FIGURE 724- The sterOidogeniC pathway and enzymatic steps.
`
`ligand of the heme iron atom. The other ax}alb11§:1rédt11:eellg‘:r
`water or molecular oxygen- When oxygelnt:
`:is of, the hem:
`axis of the oxygen molecule is parallel Wit 1 ‘ Eta
`dels of cat11-
`iron According to one of the sever,al Propose mo
`‘
`‘ .
`‘
`'
`,
`'
`15 binding of substrate to ox1-
`y31s,“ the flrSt step Of the reaction
`‘ donated from the
`diZEd (ferric, Fe”) enzyme- One electrO? 15
`, me so that the
`accessory electron transport protei? t? ttie Tlifsycom lex binds
`iron is in the reduced (ferrouS/ Feb ) Sta e‘nd electrofx from the
`molecular oxygen and then accepts 3159“) en molecule with .1
`accessory Protein, leaving the boun toggicce ts two rotoncs
`negative charge. The distal oxygen a OX 'bl via) abounEl water
`from a conserved threonine resrdue, POE:
`ieletcsed "is 1 water
`molecule 13 The distal oxygen atom IS 3+ er:
`t
`(Tl
`; inf "
`r
`.
`1 wing the iron in the Fe
`s a e.
`1e
`e
`(ining
`mOIECLlle/ 9‘ .
`‘ hi vhly reactiVe (the iron-oxygen complex is a
`0:Y8ciifl::$£1t;) find attacks the substrate, resulting in an
`erry
`‘
`‘
`hydroxylatiOHI-Desmo|ase (CYP11A, P450500)_.
`' The first enzy-
`(.‘zho‘ltesterlofl111 Steroid hormone biosynthems is the converswn
`{Eaéifoie‘it’e‘fiof to pregnenolone by CW“? (21:55:13Zéigieitiiéfi
`' 7
`-
`This enzYme catalyzes 200" an
`y
`y c
`-
`Fig. 72 3).
`'dqtive cleavage of the €20-22 carbon bond of cho
`followed bgeigfsé the C2747 side chain.13 These reactions require
`aciofitgl) 01?three oxygen molecules and SIX elegfiglfiklfigcoeg:
`mitochondrial cytochrome P450 EllzlyrgeSchcessor proteins
`these electrons from NADPH throug 1'
`1{21‘ h'
`Y 'd t'
`t
`'
`-ductase and adrenocloxm. Tie t 1638 0X1 a ions
`adrenocrlgzlfi reperformed in succession Without
`release of
`ilg’cdrggylated/ intermediates from the frag/$113113 11
`1 d
`CYI’llA is structurally related to tie?
`( B' 1)? “’XY'
`lase) isozymes (see later). It is synthesrzed as a precursor pro-
`tein. As is the case with other mitochondrial proteins encoded
`TL:- _.-.-._:-|
`
`by nuclear genes, an amino—terminal peptide is required for
`transport to the mitochondrial inner membrane. This Peptide
`of 39 residues is removed in the mitochondria to yield the
`mature protein.
`17(x-Hydroxylase/17,20-Lyase (CYP17, P450017).
`CYP 17
`catalyzes conversion of pregnenolone to 17
`-hydroxypreg-
`nenolone. In rats, this enzyme also converts progesterone to 17-
`hydroxyprogesterone, but progesterone is not a good substrate
`for the human enzyme. CYP17 also catalyzes an oxidative
`cleavage of
`the 17,20 carbon-carbon bond, converting 17_
`hydroxypregnenolone and 17-hydroxyprogesterone to dehydro~
`epiandrosterone (DHEA) and androstenedione, respectively};
`A pair of electrons and a molecule of oxygen are required for
`each hydroxylation or lyase reaction. CYP17 is a microsomal
`cytochrome P450 and accepts these electrons from NADI>H_
`dependent cytochrome P450 reductase.
`In the human adrenal cortex, 170t-hydroxylase activity is
`required for the synthesis of cortisol. The human enzyme pref-
`erentially uses pregnenolone rather than progesterone, and
`3B-hydroxysteroid dehydrogenase then converts 17-hydr0xy_
`pregnenolone to 17—hydroxyprogesterone. Because most
`'
`rOdents
`do not express CYI’17 in the adrenal cortex, they secrete c
`.
`.
`.
`.
`om-
`costerone as their primary glucocorticmd. Both 170L-hyd1-0Xy_
`lase and 17,20-1yase activities are required for androgen and
`estrogen biosynthesis.
`Because the same enzyme catalyzes production of cortis
`01 in
`the adrenal cortex and androgens in the gonads, the relatiVe
`level of 17,20-lyase activity must be independently regulated b
`a mechanism other than gene expression, or else excessiv:
`amounts of androgens would be synthesized in the adrenal Cor-
`tex. Cytochrome b5 levels are likely to be one such mechanism
`because interactions with cytochrome b5 increase 17'20‘1yasé
`.,_\r-- -_—:_,J
`
`

`

`708
`
`PART V: THE ADRENAL GLANDS
`
`activity, and mutations in cytochrome b5 interfere with andro-
`gen biosynthesis. Phosphorylation of specific serines and thre-
`onines in CYI’17 may also be important.15
`21-Hydroxylase (CYP21, P450021).
`The enzyme 21~hydroxy-
`lase resides in the endoplasmic reticulum and is responsible for
`hydroxylating 17-hydroxyprogesterone to Il-deoxycortisol and
`progesterone to 11-deoxycorticosterone. The preferred substrate
`for the human enzyme is 17-hydroxyprogesterone. The structural
`gene encoding human CYPZI (CYP21, CYPZIAZ, or CYPZIB) and
`a pseudogene (CYPZIP, CYI’21A1I’, or CYI’21A) are located in the
`HLA major histocompatibility complex on chromosome 6p21.3
`~30 kilobases (kb) apart, adjacent to and alternating with the C48
`and Gift genes encoding the fourth component of serum comple-
`ment. Both the CYP2'I and C4 genes are transcribed in the same
`direction. CYPZI and CYI’ZII’ each contain 10 exons spaced over
`3.1 kb. Their nucleotide sequences are 98% identical in exons and
`~96% identical in introns. This tandem duplication is genetically
`unstable and frequent recombinations occur between CYPZI and
`CYI’ZII’, causing congenital adrenal hyperplasia due to 21-
`hydroxylase deficiencyl'3 (see Chap. 77),
`The two microsomal enzymes, CYPZI and CYI’I7, are 36%
`identical in amino-acid sequence and their genes have a similar
`intron-exon organization, a finding which suggests that both
`genes evolved from a common ancestor.17
`11B-Hydroxylase (CYP11 B1, P450011) and Aldosterone
`Synthase (CYP11BZ, P450aldo). Humans have distinct IIB-
`hydroxylase isozymes, CYI’lIBI and CYI’IIBZ, that are l'eSpOnSi-
`ble for cortisol and aldosterone biosynthesis, respectively. In vitro,
`both isozymes can convert IlB-hydroxylate ll-deoxycorticoster-
`one to corticosterone and 11-deoxycortisol to cortisol. CYPIIBZ
`also has I8-hydroxylase and 18-oxidase activities, so that it can
`convert deoxycorticosterone to aldosterone.
`In contrast,
`the
`CYI’I'IB’I
`isozyme does not synthesize detectable amounts of
`aldosterone from corticosterone or l8—hyclroxycorticosterone.18
`These isozymes are mitochondrial cytochrome P450 enzymes
`and are synthesized with a signal peptide that is cleaved in mito-
`chondria. The sequences of
`the proteins are 93% identical.
`CYI’IIBI and CYP‘IIBZ are encoded by two genes on chromo-
`some 8q21-q22. CYI’IIBZ is located to the left of CYI’IIBI if the
`genes are pictured as being transcribed from left to right; the genes
`are located approximately 40 kb apart. The location of introns in
`each gene is identical to that seen in the CYI’IIA gene encoding
`cholesterol desmolase, and the predicted protein sequences of the
`CYP'IIB isozymes are each ~36% identical to that of CYPI‘IA.
`CYI’IIBI
`is expressed at high levels in normal adrenal
`glands. CYP'IlBZ is normally expressed at low levels, but its
`expression is dramatically increased in aldosterone-secreting
`tumors. Transcription of CYI’IIBI
`is regulated mainly by
`ACTH, whereas CYI’IIBZ is regulated by angiotensin II and
`potassium levels (see later). Recombinations between these two
`genes can lead to abnormal regulation of CYI’IIBZ and cause
`hypertension,
`a condition termed glucocorti’coid suppressiblc
`llyiiern/riostrrmiism (see Chap. 80).
`
`SHORT CHAIN DEHYDROGENASES
`Most short chain dehydrogenases catalyze reversible reactions.”
`In the dehydrogenase direction, a hydride (i.e., a proton plus
`two electrons) is removed from the substrate and transferred to
`an electron acceptor that, depending on the enzyme, is oxidized
`nicotinamide-adenine dinucleotide (NAD+) or oxidized nico-
`tinamide-adenine dinucleotide phosphate (NADI’+).
`If,
`for
`example,
`the reaction involves a hydroxylated substrate,
`the
`reaction converts the hydroxyl
`to a keto group; NADH or
`NADI’H and a proton are also produced. Oxoreductase reac-
`tions reverse this process. Unlike oxidations mediated by cyto-
`chrome P450 enzymes, no acceSsory protein is required for these
`reactions.
`
`Most short chain dehydrogenases contain 250 to 300 amino-
`acid residues. Some enzymes are found in cytosol, whereas oth-
`ers are located mainly in the endoplasmic reticulum. These
`
`enzymes have a conserved cofactor-binding domain near the N
`terminus and all have tyrosine and lysine residues near the C
`terminus that are crucial for catalysis:0 The lysine residue low-
`ers the apparent pKJ of the phenolic hydroxyl of'tyrosine from
`10.0,
`its value in solution,
`into the physmlogic range.
`Ihe
`deprotonated hydroxyl is then able to remove a proton f‘rfom tfhe
`hydroxyl group of the substrate, which faCilitates trans er 0 a
`hydride from the substrate to the ox1d1zed co'factor.
`The most important enzyme of this type in the adrenal colr-
`tex is 3B-hydroxysteroid dehydrogenase. Ihis enzyme is on y
`~15% identical
`to most other short chain deliydrogenases,
`although it retains the essential structural features of this class
`of enzymes. Other enzymes of this type that are important in
`steroid metabolism include 11p-hydroxysteroid dehydroge-
`nase which interconverts cortisol and cortisone, and l7-keto-
`steroid reductase, which converts andrqstenedione and estrone
`v
`.
`i d stradiol, res ective y.
`warmisssizma
`version of A5-3B-hydroxyster0ids (pregnenolone, 17- iy )ioxy-
`pregnenolone, DHEA)
`to A.i_3-ket()ster()ids (pirogcsitgrroigie,
`17-hydroxyprogesterone, androstenedione)‘ is tiliiec ‘18th 1 y _ I3-
`hydroxysteroid dehydrogenase (3l3‘PI§D) 1?
`‘8 U“ 0? tasmIic
`reticulum. This enzyme uses NAD+ 35 an e‘eFtFE’“ “C.“‘P onfl n
`addition to 3fi-11ydroxysteroid dehydrogenasel activ1ty,f
`‘16
`enzyme mediates a A5_A»1,ene isomerase reaction t‘iat trans ers a
`double bond from the B ring to the A ring of the steroid so‘ t