Cytochrome P450

Most steroidogenic enzymes are cytochrome P450 enzymes. “Cytochrome P450” designates a group of oxidative enzymes, all of which have about 500 amino acids and contain a single heme group. “P450” means “pigment 450” because all absorb light at 450 nm in their reduced states. Human beings have 57 genes for P450 enzymes; seven human P450s are targeted to mitochondria and 50 are targeted to the endoplasmic reticulum, especially in the liver, where they metabolize countless toxins, drugs, xenobiotics, and environmental pollutants. Each P450 enzyme, including the steroidogenic P450s, can metabolize multiple substrates, catalyzing a broad array of oxidations.

Five P450s are involved in adrenal steroidogenesis ( Fig. 14.3 ). Mitochondrial P450scc (CYP11A1) is the cholesterol side-chain cleavage enzyme catalyzing the series of reactions formerly termed 20,22 desmolase . The two mitochondrial isozymes of P450c11, P450c11β (CYP11B1) and P450c11AS (CYP11B2), catalyze 11β-hydroxylase, 18-hydroxylase, and 18-methyl oxidase activities. P450c17 (CYP17A1), found in the endoplasmic reticulum, catalyzes both 17α-hydroxylase and 17,20 lyase activities, and P450c21 (CYP21A2) catalyzes the 21-hydroxylation of both glucocorticoids and mineralocorticoids. In the gonads and elsewhere P450aro (CYP19A1) in the endoplasmic reticulum catalyzes aromatization of androgens to estrogens.

Principal pathways of human adrenal steroid hormone synthesis. Other quantitatively and physiologically minor steroids are also produced. The names of the enzymes are shown by each reaction, and the traditional names of the enzymatic activities correspond to the circled numbers. Reaction 1: Mitochondrial cytochrome P450scc mediates 20α-hydroxylation, 22-hydroxylation, and scission of the C20-22 carbon bond. Reaction 2: 3βHSD mediates 3β-hydroxysteroid dehydrogenase and isomerase activities, converting Δ ⁵ steroids to Δ ⁴ steroids. Reaction 3: P450c17 catalyzes the 17α-hydroxylation of pregnenolone to 17OH-pregnenolone and of progesterone to 17OH-progesterone. Reaction 4: The 17,20 lyase activity of P450c17 converts 17OH-pregnenolone to dehydroepiandrosterone (DHEA); only insignificant amounts of 17OH-progesterone are converted to Δ ⁴ androstenedione by human P450c17, although this reaction occurs in other species. Reaction 5: P450c21 catalyzes the 21-hydroxylation of progesterone to deoxycorticosterone (DOC) and of 17OH-progesterone to 11-deoxycortisol. Reaction 6: DOC is converted to corticosterone by the 11-hydroxylase activity of P450c11AS in the zona glomerulosa and by P450c11β in the zona fasciculata. Reaction 7: 11-Deoxycortisol undergoes 11β-hydroxylation by P450c11β to produce cortisol in the zona fasciculata. Reactions 8 and 9: The 18-hydroxylase and 18-methyl oxidase activities of P450c11AS convert corticosterone to 18OH-corticosterone and aldosterone, respectively, in the zona glomerulosa. Reactions 10 and 11 are found principally in the testes and ovaries. Reaction 10: 17βHSD-III converts DHEA to androstenediol and androstenedione to testosterone, whereas 17βHSD-I converts estrone to estradiol. Reaction 11: Testosterone may be converted to estradiol and androstenedione may be converted to estrone by P450aro.

Hydroxysteroid Dehydrogenases

The hydroxysteroid dehydrogenases (HSDs) have molecular masses of about 35 to 45 kDa, do not have heme groups, and require nicotinamide adenine dinucleotide (NAD ⁺ ) or nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) as cofactors. Whereas most steroidogenic reactions mediated by P450 enzymes are catalyzed by a single form of P450, each of the reactions mediated by HSDs can be catalyzed by at least two isozymes. The HSDs include the 3α- and 3β-hydroxysteroid dehydrogenases, two 11β-hydroxysteroid dehydrogenases, and a series of 17β-hydroxysteroid dehydrogenases; the 5α-reductases are unrelated to this family. Based on their structures, the HSDs fall into two groups: the short-chain dehydrogenase/reductase (SDR) family, structurally characterized by a “Rossman fold,” and the aldo-keto reductase (AKR) family, characterized by a triosephosphate isomerase barrel motif. The SDR enzymes include 11β-HSDs 1 and 2, and 17β-HSDs 1, 2, 3, and 4; the AKR enzymes include 17β-HSD5, which is important in extraglandular activation of androgenic precursors, and the 3α-hydroxysteroid dehydrogenases that participate in the so-called backdoor pathway of fetal androgen synthesis (see later). Based on their activities, it is physiologically more useful to classify them as dehydrogenases or reductases. The dehydrogenases use NAD + as their cofactor to oxidize hydroxysteroids to ketosteroids, and the reductases mainly use NADPH to reduce ketosteroids to hydroxysteroids. Although these enzymes are typically bidirectional in vitro, they tend to function in only one direction in intact cells, with the direction determined by the cofactor(s) available.

P450scc

Conversion of cholesterol to pregnenolone in mitochondria is the first, rate-limiting and hormonally regulated step in the synthesis of all steroid hormones. This involves cholesterol 20α-hydroxylation, 22-hydroxylation, and scission of its side chain to yield pregnenolone and isocaproic acid. Because 20-hydroxycholesterol, 22-hydroxycholesterol, and 20,22-hydroxycholesterol could be isolated from adrenals in significant quantities, it was previously thought that three separate enzymes were involved. However, a single protein, termed P450scc (‘scc’ refers to the s ide- c hain c leavage of cholesterol) encoded by the CYP11A1 gene on chromosome 15 catalyzes all the steps between cholesterol and pregnenolone. Deletion of the mouse or rabbit cyp11a1 gene eliminates all steroidogenesis, indicating that all steroidogenesis is initiated by this one enzyme.

Transport of Electrons to P450scc: Ferredoxin Reductase and Ferredoxin

Mitochondrial P450 enzymes (P450scc, P450c11β, P450c11AS, and the vitamin D 1α- and 24-hydroxylases) are terminal oxidases in an electron transport system: NADPH donates electrons to ferredoxin reductase (FDXR, also termed adrenodoxin reductase ), a flavoprotein that is loosely associated with the inner mitochondrial membrane. FDXR transfers the electrons to ferredoxin (FDX, also termed adrenodoxin ), a 14-kDa iron/sulfur protein, which then transfers the electrons to P450scc ( Fig. 14.4 ). FDX forms a 1:1 complex with FDXR, dissociates, then reforms an analogous 1:1 complex with the P450, thus functioning as a diffusible electron shuttle mechanism. FDXR and FDX are widely expressed in human tissues, but FDXR expression is two orders of magnitude higher in steroidogenic tissues. The primary ribonucleic acid (RNA) transcript from the FDXR gene is alternatively spliced yielding two messenger RNA (mRNA) species that encode proteins differing by six amino acids, but only the shorter protein is active in steroidogenesis. FDXR is also essential in the formation of the iron/sulfur centers used by many enzymes. There are two human FDX isozymes encoded by genes on different chromosomes: FDX1 interacts with mitochondrial P450 enzymes; FDX2 is used for synthesis of iron-sulfur clusters, but not for steroidogenesis. FDX1 is ubiquitously expressed but is especially abundant in adrenal cortex. Human mutations in FDX have not been described, but several FDXR mutations that partially impair formation of iron/sulfur centers are associated with neuropathic hearing loss.

Electron transport to mitochondrial forms of cytochrome P450. Adrenodoxin reductase (AdRed), a flavoprotein loosely bound to the inner mitochondrial membrane, accepts electrons (e ⁻ ) from nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), converting it to NADP ⁺ . These electrons are passed to adrenodoxin (Adx), an iron-sulfur protein in solution in the mitochondrial matrix that functions as a freely diffusible electron shuttle mechanism. Electrons from charged adrenodoxin (Adx ⁻ ) are accepted by any available cytochrome P450, such as P450c11, or P450scc shown here. The uncharged adrenodoxin (Adx ⁺ ) may then be again bound to adrenodoxin reductase to receive another pair of electrons. For P450scc, three pairs of electrons must be transported to the P450 to convert cholesterol to pregnenolone. The flow of cholesterol into the mitochondria is facilitated by steroidogenic acute regulatory protein, which is not shown in this diagram.

(©W.L. Miller.)

Mitochondrial Cholesterol Uptake: the Steroidogenic Acute Regulatory Protein, StAR

ACTH regulates steroidogenic capacity (chronic regulation) by inducing the transcription of genes for steroidogenic enzymes, but acute regulation, where steroids are released within minutes of a stimulus, is at the level of cholesterol access to P450scc. Treating either steroidogenic cells or intact rats with inhibitors of protein synthesis (e.g., cycloheximide) eliminated the acute steroidogenic response, suggesting that a short-lived protein triggers the response. A long search led to the identification and cloning of the steroidogenic acute regulatory protein, StAR. The central role of StAR in steroidogenesis was proven by finding that mutations of StAR caused congenital lipoid adrenal hyperplasia. Thus StAR is the acute trigger that is required for the rapid flux of cholesterol from the outer to the inner mitochondrial membrane that is needed for the acute response of aldosterone to angiotensin II, of cortisol to ACTH, and of sex steroids to a luteinizing hormone (LH) pulse.

Some adrenal steroidogenesis is independent of StAR; this StAR-independent steroidogenesis is about 14% of the StAR-induced rate. The placenta uses P450scc to initiate steroidogenesis but does not express StAR. The mechanism of StAR-independent steroidogenesis is unclear; it may occur without a triggering protein, or some other protein may exert StAR-like activity to promote cholesterol flux, but without StAR’s rapid kinetics. The mechanism of StAR’s action is unclear, but it is established that StAR acts on the outer mitochondrial membrane (OMM), does not need to enter the mitochondria to be active, and undergoes conformational changes on the OMM that are required for StAR’s activity. Some studies suggest that StAR functions as a component of a molecular machine that consists of StAR, TSPO (the translocator protein formerly known as the peripheral benzodiazepine receptor), and other proteins on the OMM, although studies with TSPO-knockout mice question its role. The precise fashion in which cholesterol is loaded into the OMM and moves from the OMM to P450scc with the assistance of StAR remains unclear, and remains under active investigation.

3 β -Hydroxysteroid Dehydrogenase/ Δ ⁵ − > Δ ⁴ Isomerase

Once pregnenolone is produced from cholesterol, it may undergo 17α-hydroxylation by P450c17 to yield 17-hydroxypregnenolone (17-Preg), or it may be converted to progesterone. Two 42-kDa isozymes of 3β-hydroxysteroid dehydrogenase (3βHSD), encoded by the HSD3B1 and HSD3B2 genes, can catalyze both conversion of the hydroxyl group to a keto group on carbon 3 and the isomerization of the double bond from the B ring (Δ ⁵ steroids) to the A ring (Δ ⁴ steroids). These isozymes share 93.5% amino acid sequence identity and are enzymatically very similar: both can convert pregnenolone to progesterone, 17-Preg to 17α-hydroxyprogesterone (17OHP), DHEA to androstenedione, and androstenediol to testosterone. However, 3βHSD2, the isozyme expressed in the adrenals and gonads, has a high Michaelis-Menten constant (Km) of about 5.5 μM, about 10 times higher than 3βHSD1 expressed in placenta, brain, and “extraglandular” tissues. The low Km of extraglandular 3βHSD1 permits it to act on the low concentrations of some steroids found in the circulation. Ultrastructural data show that 3βHSD can be found in the mitochondria, endoplasmic reticulum, and cytoplasm. It is not clear if this subcellular distribution differs in various types of steroidogenic cells, but this could be a novel point regulating the direction of steroidogenesis.

P450c17

P450c17 (CYP17A1) catalyzes both 17α-hydroxylase and 17,20-lyase activities. The 17α-hydroxylase activity of P450c17 can convert pregnenolone to 17-Preg and progesterone to 17OHP. The 17,20-lyase activity of P450c17 can convert 17-Preg to DHEA, but very little 17OHP is converted to androstenedione because human P450c17 catalyzes this reaction at only around 2% to 3% of the rate for conversion of 17-Preg to DHEA. P450c17 is the key branch point in steroid hormone synthesis: in its absence, as in the adrenal zona glomerulosa, 17α-hydroxylase and 17,20-lyase activities are absent, so that pregnenolone is converted to mineralocorticoids; in the presence of its 17α-hydroxylase activity in the zona fasciculata, only the 17α-hydroxylase activity is present so that pregnenolone is sequentially converted to cortisol; in the zona reticularis where both activities are present, pregnenolone is sequentially converted to sex steroids (see Fig. 14.3 ).

17α-Hydroxylase and 17,20 lyase were once thought to be separate enzymes. The adrenals of prepubertal children synthesize ample cortisol but virtually no DHEA (i.e., have 17α-hydroxylase activity but not 17,20 lyase activity) until adrenarche initiates production of adrenal androgens (i.e., turns on 17,20 lyase activity). Furthermore, patients had been described lacking 17,20 lyase activity but retaining normal 17α-hydroxylase activity. However, both 17α-hydroxylase and 17,20 lyase activities reside in the same active site, and cells transfected with a vector expressing P450c17 acquire both 17α-hydroxylase and 17,20 lyase activities. P450c17 is encoded by the CYP17A1 gene on chromosome 10q24.3, and is structurally related to the CYP21A2 gene for P450c21 (21-hydroxylase). Thus the distinction between 17α-hydroxylase and 17,20 lyase is functional and not genetic or structural. Human P450c17 catalyzes 17α-hydroxylation of pregnenolone and progesterone equally well, but its 17,20 lyase activity uses 17-Preg almost exclusively, and not 17OHP, consistent with the large amounts of DHEA secreted by both the fetal and adult adrenal. The principal factor regulating the 17,20 lyase reaction is electron transport from NADPH via P450 oxidoreductase (POR).

Electron Transport to P450c17: P450 Oxidoreductase and Cytochrome b ₅

POR, a membrane-bound flavoprotein that is unrelated to mitochondrial ferredoxin reductase, receives electrons from NADPH and transfers them to the 50 microsomal forms of cytochrome P450, including P450c17, P450c21, and P450aro. For the 17,20 lyase reaction of P450c17, electron transfer is facilitated by the action of cytochrome b ₅ , acting as an allosteric factor rather than as an alternate electron donor; 17,20 lyase activity is also increased by the serine/threonine phosphorylation of P450c17 by p38α, a cyclic adenosine monophosphate (cAMP)-dependent protein kinase ( Fig. 14.5 ). Thus the availability of electrons determines whether P450c17 performs only 17α-hydroxylation, or also performs 17,20 bond scission: increasing the ratio of POR or cytochrome b ₅ to P450c17 increases the ratio of 17,20 lyase activity to 17α-hydroxylase activity. Thus the regulation of 17,20 lyase activity, and consequently of DHEA production, depends on factors that facilitate the flow of electrons to P450c17: high concentrations of POR, the presence of cytochrome b ₅ , and serine phosphorylation of P450c17.

Electron transport to microsomal forms of cytochrome P450. Nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) interacts with P450 oxidoreductase (POR), bound to the endoplasmic reticulum, and gives up a pair of electrons (e ⁻ ), which are received by the flavin adenine dinucleotide (FAD) moiety. Electron receipt elicits a conformational change, permitting the isoalloxazine rings of the FAD and flavin mononucleotide (FMN) moieties to come close together, so that the electrons pass from the FAD to the FMN. Following another conformational change that returns the protein to its original orientation, the FMN domain of POR interacts with the redox-partner binding site of the P450. Electrons from the FMN domain of POR reach the heme group to mediate catalysis. The interaction of POR and the P450 is coordinated by negatively charged acidic residues on the surface of the FMN domain of POR, and positively charged basic residues in the concave redox-partner binding site of the P450. The active site containing the steroid lies on the iron side of heme ring (Fe) opposite from the redox-partner binding site. In the case of human P450c17, this interaction is facilitated by the allosteric action of cytochrome b ₅ , and by the serine phosphorylation of P450c17.

(©W.L. Miller)

P450c21

After the synthesis of progesterone and 17OHP, these steroids are 21-hydroxylated to yield deoxycorticosterone (DOC) and 11-deoxycortisol, respectively (see Fig. 14.3 ). The nature of the 21-hydroxylating step is of great interest because disordered 21-hydroxylation causes more than 90% of all cases of CAH. The clinical findings in CAH are complex and potentially devastating. Decreased cortisol and aldosterone synthesis often lead to sodium loss, potassium retention, acidosis, and hypotension, which will lead to cardiovascular collapse and death, usually within a month after birth if not treated appropriately. Decreased adrenal steroidogenesis in utero leads to overproduction of adrenal androgens via several pathways (see later), resulting in severe prenatal virilization of female fetuses. Adrenal 21-hydroxylation is catalyzed by P450c21 found in smooth endoplasmic reticulum, using POR to receive electrons from NADPH. Duplicated CYP21A1P and CYP21A2 genes lie on chromosome 6p21, but only the human CYP21A2 gene encodes P450c21. As this gene lies in the middle of the major histocompatibility locus, disorders of adrenal 21-hydroxylation are closely linked to specific human leukocyte antigen (HLA) types. Extraadrenal 21-hydroxylase activity is found in many adult and fetal tissues, especially in the liver, but is catalyzed by other enzymes, notably CYP2C19 and CYP3A4, which are principally involved in drug metabolism. CYP2C19 and CYP3A4 can 21-hydroxylate progesterone but not 17OHP, and hence may contribute to the synthesis of mineralocorticoids but not glucocorticoids.

P450c11 β and P450c11AS

The closely related P450c11β and P450c11AS enzymes catalyze the final steps in the synthesis of glucocorticoids and mineralocorticoids. These two isozymes have 93% amino acid sequence identity and are encoded by tandemly duplicated genes on chromosome 8q21-22. Like P450scc, the two forms of P450c11 are found on the inner mitochondrial membrane, and use ferredoxin and ferredoxin reductase to receive electrons from NADPH. By far the more abundant of the two isozymes is P450c11β (encoded by CYP11B1 ), which is the classic 11β-hydroxylase that converts 11-deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone. The less abundant isozyme, P450c11AS (encoded by CYP11B2 ), is found only in the zona glomerulosa, where it has 11β-hydroxylase, 18-hydroxylase, and 18-methyl oxidase (aldosterone synthase) activities; thus P450c11AS is able to catalyze all the reactions needed to convert DOC to aldosterone. CYP11B1 is induced by ACTH via cAMP and is suppressed by glucocorticoids; CYP11B2 is induced by angiotensin II and K ⁺ . Patients with mutations in CYP11B1 have classic 11β-hydroxylase deficiency but can still produce aldosterone, whereas patients with mutations in CYP11B2 have rare forms of aldosterone deficiency (so-called corticosterone methyl oxidase deficiency ), while retaining the ability to produce cortisol.

17 β -Hydroxysteroid Dehydrogenase

Androstenedione is converted to testosterone, DHEA is converted to androstenediol, and estrone is converted to estradiol by the 17β-hydroxysteroid dehydrogenases (17βHSD; HSD17B), sometimes also termed 17-oxidoreductases or 17-ketosteroid reductases . The terminologies for these enzymes vary, depending on the direction of the reaction being considered. The literature about the 17βHSDs can be confusing because (1) there are several different 17βHSDs; (2) some are preferential oxidases, whereas others are preferential reductases; (3) they differ in their substrate preference and sites of expression; (4) the nomenclature is inconsistent; and (5) some proteins termed 17βHSD actually have very little 17βHSD activity, and are principally involved in other reactions.

Type 1 17βHSD (17βHSD1, encoded by HSD17B1 ), is a 34-kDa reductive, estrogenic SDR enzyme expressed in ovarian granulosa cells (where it produces estradiol) and placenta (where it produces estriol). 17βHSD1 uses NADPH as its cofactor, acts as a dimer, and only accepts steroid substrates with an aromatic A ring, so that its activity is confined to activating estrogens. No genetic deficiency syndrome for 17βHSD1 has been described.

17βHSD2 is a microsomal oxidase that uses NAD ⁺ to inactivate estradiol to estrone and testosterone to Δ ⁴ androstenedione. 17βHSD2 is found in the placenta, liver, small intestine, prostate, secretory endometrium, and ovary. Whereas 17βHSD1 is found in placental syncytiotrophoblast cells, 17βHSD2 is expressed in endothelial cells of placental intravillous vessels, consistent with its apparent role in defending the fetal circulation from transplacental passage of maternal estradiol or testosterone. No deficiency state for 17βHSD2 has been reported.

Microsomal 17βHSD3 is the principal androgenic form of 17βHSD, apparently expressed only in the testis. This enzyme is disordered in the classic syndrome of male pseudohermaphroditism that is often termed 17-ketosteroid reductase deficiency .

An enzyme termed 17βHSD4 was initially identified as an NAD ⁺ -dependent oxidase with activities similar to 17βHSD2, but this peroxisomal protein is primarily an enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase. Deficiency of 17βHSD4 causes a form of Zellweger syndrome, in which bile acid biosynthesis is disturbed but steroidogenesis is not.

17βHSD5, originally cloned as a 3α-hydroxysteroid dehydrogenase, is an AKR enzyme (whereas 17βHSD types 1-4 are SDR enzymes) termed AKR1C3 that catalyzes the reduction of Δ ⁴ androstenedione to testosterone. The 17βHSD activity of 17βHSD5 is quite labile in vitro, and this enzyme catalyzes different activities under different conditions. The adrenal zona reticularis expresses this enzyme at low levels, accounting for the small amount of testosterone produced by the adrenal.

17βHSD6, encoded by the HSD17B6 gene on chromosome 12q13.3, is also known as RoDH for its homology to retinol dehydrogenases. 17βHSD6 is expressed at low but detectable levels in the fetal testes, where it appears to catalyze oxidative 3αHSD activities in the so-called “backdoor pathway” to dihydrotestosterone (DHT) synthesis.

Steroid Sulfotransferase and Sulfatase

Steroid sulfates may be synthesized directly from cholesterol sulfate or may be formed by sulfation of steroids by cytosolic sulfotransferase (SULT) enzymes. SULTs use 3’-phosphoadenine-5’-phosphosulfate (PAPS) as sulfate donor, synthesized from adenosine triphosphate (ATP) and sulfate by two isozymes of PAPS synthase: PAPSS1, which is ubiquitously expressed, and PAPSS2, principally expressed in cartilage, adrenal, and liver. At least 44 distinct isoforms of these enzymes have been identified belonging to five families of SULT genes; many of these genes yield alternately spliced products accounting for the large number of enzymes. The SULT enzymes that sulfonate steroids include SULT1E (estrogens), SULT2A1 (nonaromatic steroids), and SULT2B1 (sterols). SULT2A1 is the principal sulfotransferase expressed in the adrenal, where it sulfates the 3β hydroxyl group of Δ ⁵ steroids (pregnenolone, 17OH-pregnenolone, DHEA, androsterone) but not cholesterol. SULT2B1a will also sulfonate pregnenolone but not cholesterol, whereas cholesterol is the principal substrate for SULT2B1b in the skin, liver, and elsewhere. It is not clear whether most steroid sulfates are simply inactivated forms of steroid or if they serve specific hormonal roles. Knockout of the mouse sult1e1 gene is associated with elevated estrogen levels, increased placental expression of tissue factor, and increased platelet activation, leading to placental thrombi and fetal loss that could be ameliorated by anticoagulant therapy. Mutations ablating the function of human SULT enzymes have not been described, but PAPSS2 mutations deplete adrenal PAPS, increasing production of unconjugated DHEA. African Americans have a high rate of polymorphisms in SULT2A1, apparently influencing plasma ratios of DHEA:DHEAS, which may correlate with risk of prostatic and other cancers.

Steroid sulfates may also be hydrolyzed to the native steroid by steroid sulfatase. Deletions in the steroid sulfatase gene on chromosome Xp22.3 cause X-linked ichthyosis. The fact that males have a single copy of this gene probably accounts for males having higher DHEAS levels than females of the same age. In the fetal adrenal and placenta, diminished or absent sulfatase deficiency reduces the pool of free DHEA available for placental conversion to estrogen, resulting in low concentrations of estriol in the maternal blood and urine. The accumulation of steroid sulfates in the stratum corneum of the skin causes the ichthyosis.

Aromatase: P450aro

Estrogens are produced by the aromatization of androgens, including adrenal androgens, by a complex series of reactions catalyzed by aromatase, P450aro. This microsomal cytochrome P450 is encoded by a single gene ( CYP19A1 ) on chromosome 15q21.1 that uses several different promoter sequences, transcriptional start sites, and alternatively chosen first exons to encode identical P450aro in different tissues under different hormonal regulation. Aromatase expression in the extraglandular tissues, especially adipose tissue, can convert adrenal androgens to estrogens. Aromatase in the epiphyses of growing bone converts testosterone to estradiol; the tall stature, delayed epiphyseal maturation and osteopenia of males with aromatase deficiency, and their rapid reversal with estrogen replacement indicate that estrogen, not androgen, is responsible for epiphyseal maturation in males.

Rare patients with aromatase deficiency illustrate that placental aromatase activity and consequent feto-placental estrogen synthesis are not needed for normal fetal development. However, in the absence of placental aromatase activity, fetal adrenal androgens and androgen precursors pass into the maternal circulation, causing maternal virilization. Furthermore, placental aromatase converts maternal androgens to estrogens, protecting the fetus from potential virilization (as in pregnant women with undertreated 21-hydroxylase deficiency [21OHD]). Children with aromatase deficiency grow normally and continue to grow after puberty, because only estrogens, not androgens, mediate epiphyseal fusion. Treating aromatase-deficient patients with estrogens will fuse their epiphyses and arrest growth. These observations underlie the use of aromatase inhibitors for inhibiting accelerated bone maturation.

5 α -Reductases

Testosterone is converted to the more potent androgen, DHT, by 5α-reductase, an enzyme found in testosterone’s target tissues. There are two hydrophobic, 30-kDa isozymes of 5α-reductase that share 50% identity. The type I enzyme, found in the scalp and other peripheral tissues, is encoded by the SRD5A1 gene on chromosome 5; the type II enzyme, the predominant form found in male reproductive tissues, is encoded by the structurally related SRD5A2 gene on chromosome 2p23. The syndrome of 5α-reductase deficiency, a disorder of male sexual differentiation, is caused by a wide variety of mutations in the SRD5A2 gene. In the fetus, the SRD5A1 gene is expressed in the testis, then is expressed briefly in the skin of the newborn, and then remains unexpressed until its activity and protein are again found after puberty. The SRD5A2 gene is expressed in fetal genital skin, in the normal prostate, and in prostatic hyperplasia and adenocarcinoma. Thus the type I enzyme may be responsible for the pubertal virilization seen in patients with classic 5α-reductase deficiency, and the type II enzyme may be involved in male pattern baldness.

11 β -Hydroxysteroid Dehydrogenases

Although certain steroids are typically categorized as glucocorticoids or mineralocorticoids, the “mineralocorticoid” (glucocorticoid type II) receptor has equal affinity for both aldosterone and cortisol. Nevertheless, cortisol does not act as a mineralocorticoid in vivo, even though cortisol concentrations can exceed aldosterone concentrations by 100- to 1000-fold, because mineralocorticoid responsive tissues (such as the kidney) convert cortisol to cortisone, a metabolically inactive steroid. The interconversion of cortisol and cortisone is mediated by two membrane-bound isozymes of 11β-hydroxysteroid dehydrogenase (11βHSD; HSD11B), each of which can catalyze both oxidase and reductase activity, depending on the cofactor available (NADP ₊ or NADPH). The ratio of NADP ₊ to NADPH is regulated by hexose-6-phosphate dehydrogenase (H6PDH).

The type 1 enzyme (HSD11B1; 11βHSD1) is expressed mainly in glucocorticoid-responsive tissues, such as the liver, fat, testis, and lung. HSD11B1 can catalyze both the oxidation of cortisol to cortisone using NADP ⁺ as its cofactor (Km 1–2 μM), or the reduction of cortisone to cortisol using NADPH as its cofactor (Km 0.1–0.3 μM); the reaction catalyzed depends on which cofactor is available, but the enzyme can only function with high (micromolar) concentrations of steroid. HSD11B1 is located on the luminal side of the endoplasmic reticulum, and hence is not in contact with the cytoplasm, permitting HSD11B1 to receive NADPH provided by H6PDH. This links HSD11B1 to the pentose monophosphate shunt, providing a direct paracrine link between local glucocorticoid production and energy storage as fat. Prednisone and cortisone are inactive 11-ketosteroids that must be reduced to their active 11β-hydroxy derivatives by HSD11B1, mainly in the liver. By activating cortisone, HSD11B1 amplifies glucocorticoid action in the tissues in which it is expressed, especially in liver and fat, potentially contributing to obesity, insulin resistance, metabolic syndrome, and possibly nonalcoholic fatty liver disease; consequently, studies of HSD11B1 inhibitors are of substantial pharmaceutic interest.

HSD11B2 (11βHSD2) catalyzes the oxidation of cortisol to cortisone using NADH, and can function with low (nanomolar) steroid concentrations (Km 10–100 nM). HSD11B2 is expressed in mineralocorticoid-responsive tissues, such as the distal nephron and thus serves to “defend” the mineralocorticoid receptor (MR) by inactivating cortisol to cortisone, so that only “true” mineralocorticoids, such as aldosterone or deoxycorticosterone, can exert a mineralocorticoid effect; thus HSD11B2 prevents cortisol from overwhelming renal MRs. Placental HSD11B2 also inactivates maternal cortisol to cortisone (and prednisolone to prednisone), and the placenta has abundant NADP ⁺ favoring the oxidative action of HSD11B1, so that in the placenta both enzymes protect the fetus from high maternal concentrations of cortisol, but not from maternally administered beta-methasone or dexamethasone, which are not substrates for HSD11B enzymes. Thus the HSD11B isozymes determine whether a steroid “crosses the placenta.”

3 α -Hydroxysteroid Dehydrogenases

The 3α-hydroxysteroid dehydrogenases (3αHSDs) are not familiar to most endocrinologists, but are clinically important because of the recent discovery of the “backdoor pathway” of steroidogenesis. This remarkable pathway ( Fig. 14.6 ), first discovered as the mechanism by which the male marsupial fetal testis makes androgens, plays a central role in human male sexual differentiation. In this pathway, 17OHP is converted to DHT without going through DHEA, androstenedione, or testosterone, and hence provides a mechanism for 17OHP to contribute to the virilization of female fetuses with 21OHD. The reductive 3αHSDs are AKR enzymes and the oxidative 3αHSDs are SDR enzymes. There are four reductive 3αHSDs, termed AKR1C4-1. These enzymes are structurally very similar, are encoded by a cluster of very similar genes on chromosome 10p14-15, and catalyze a wide array of steroidal conversions (notably 17-ketosteroid reduction and 20α-reduction) and other reactions. AKR1C3, also known as 17βHSD5, converts androstenedione to testosterone in the adrenal.

Pathways to androgens in congenital adrenal hyperplasia (CAH). In the absence of P450c21 activity, the adrenal can produce androgens by three pathways. First , the pathway from cholesterol to dehydroepiandrosterone (DHEA) remains intact in 21-hydroxylase deficiency, and increased production of DHEA will lead to some DHEA being converted to androstenedione and thence to testosterone. Second , although minimal amounts of 17α-hydroxyprogesterone (17OHP) are converted to androstenedione in the normal adrenal, the huge amounts of 17OHP produced in CAH permit some 17OHP to be converted to androstenedione and then to testosterone. Third , the so-called “backdoor pathway” depends on the 5α and 3α reduction of 17OHP to 17OH-allopregnenolone. This steroid is readily converted to androstanediol, which can then be oxidized to dihydrotestosterone (DHT) by a 3α-hydroxysteroid dehydrogenase (HSD) enzyme, AKR1C2. Mass spectrometric examinations of human urinary steroids indicate this pathway is a major contributor in CAH.

The backdoor pathway is characterized by both reductive and oxidative 3αHSD activities; the reductive activity can apparently be catalyzed by either AKR1C2 or AKR1C4. The oxidative 3αHSDs are SDRs that are similar to the retinol dehydrogenase or cis-retinol/androgen dehydrogenase (RoDH/CRAD) subfamily. The most active member of these is RoDH, also called 17βHSD6, which probably catalyzes the final step in the backdoor pathway. In the brain, 3αHSDs reduce 5α-dihydroprogesterone to allopregnanolone, which is an allosteric activator of the gamma-aminobutyric acid (GABA) _A receptors. Further studies of the role of the backdoor pathway will be central to pediatric endocrinology.

Hypothalamus: Corticotropin-Releasing Factor and Arginine Vasopressin

The principal steroidal product of the human adrenal is cortisol, which is mainly secreted in response to ACTH (corticotropin) produced in the pituitary; secretion of ACTH is stimulated primarily by corticotropin-releasing factor (CRF) from the hypothalamus. The history of the discovery of the components of the HPA axis and their interrelationships has been reviewed recently. Hypothalamic CRF is a 41-amino acid peptide synthesized mainly by neurons in the paraventricular nucleus. These same hypothalamic neurons also produce the decapeptide arginine vasopressin (AVP, also known as antidiuretic hormone or ADH). Both CRF and AVP are proteolytically derived from larger precursors, with the AVP precursor containing the sequence for neurophysin, which is the AVP-binding protein. CRF and AVP travel through axons to the median eminence, which releases them into the pituitary portal circulation, although most AVP axons terminate in the posterior pituitary. AVP is cosecreted with CRF in response to stress, and both CRF and AVP stimulate the synthesis and release of ACTH, but they appear to do so by different mechanisms. CRF binds to a G protein–coupled receptor on the membranes of pituitary corticotropes and activates adenylyl cyclase, increasing cAMP, which activates the protein kinase A (PKA) signaling pathway. PKA triggers ACTH secretion by concerted regulation of cellular potassium and calcium fluxes, and enhances proopiomelanocortin ( POMC ) gene transcription. AVP binds to its G protein–coupled receptor and activates phospholipase C, which leads to the release of intracellular Ca ⁺⁺ and to the activation of protein kinase C (PKC). AVP seems to amplify the effects of CRF on ACTH secretion without affecting synthesis. However, CRF is the more important physiologic stimulator of ACTH release, although maximal doses of AVP can elicit a maximal ACTH response. When given together, CRF and AVP act synergistically, as would be expected from their independent mechanisms of action.

Pituitary: Adrenocorticotropic Hormone and Proopiomelanocortin

Pituitary ACTH is a 39-amino acid peptide derived from POMC, a 241-amino acid protein. POMC undergoes a series of proteolytic cleavages, yielding several biologically active peptides ( Fig. 14.8 ). The N-terminal glycopeptide (POMC 1-75) can stimulate steroidogenesis and may function as an adrenal mitogen. POMC 112-150 is ACTH 1-39, POMC 112-126 and POMC 191-207 constitute α- and β-MSH (melanocyte stimulating hormone), respectively, and POMC 210-241 is β-endorphin. POMC is also produced in small amounts by the brain, testis, liver, kidney, and placenta, but this extrapituitary POMC does not contribute significantly to circulating ACTH. Malignant tumors will commonly produce “ectopic ACTH” in adults and rarely in children; this ACTH derives from ectopic biosynthesis of the same POMC precursor. Only the first 20 to 24 amino acids of ACTH are needed for its full biologic activity, and synthetic ACTH 1-24 is widely used in diagnostic tests of adrenal function. However, these shorter forms of ACTH have a shorter half-life than does native ACTH 1-39. POMC gene transcription is stimulated by CRF and inhibited by glucocorticoids.

Structure of human preproopiomelanocortin (POMC). The numbers refer to amino acid positions, with No. 1 assigned to the first amino acid of POMC after the 26-amino acid signal peptide. The α-, β-, and γ- MSH regions, which characterize the three “constant” regions, are indicated by diagonal lines; the “variable” regions are solid. The amino acid numbers shown refer to the N-terminal amino acid of each cleavage site; because these amino acids are removed, the numbers do not correspond exactly with the amino acid numbers of the peptides as used in the text. ACTH, Adrenocorticotropic hormone; CLIP, corticotropin-like intermediate lobe peptide; LPH, lipotropic hormone; MSH, melanocyte-stimulating hormone.

Actions of Adrenocorticotropic Hormone

ACTH stimulates the G protein–coupled melanocortin 2 receptor (MCR2), which is located almost exclusively in the adrenal cortex. Activation of MCR2 triggers the production of cAMP, activating PKA that catalyzes the phosphorylation of many proteins involved in steroidogenesis, thereby modifying their activity. ACTH elicits both acute and long-term effects. ACTH stimulates the biosynthesis of LDL receptors and the uptake of LDL, which provides most of the cholesterol used for steroidogenesis, and stimulates transcription of the gene for HMG-CoA reductase, the rate-limiting step in cholesterol biosynthesis, but adrenal biosynthesis of cholesterol is quantitatively much less important than the uptake of LDL cholesterol. Cholesterol is stored in steroidogenic tissues as cholesterol esters in lipid droplets. ACTH stimulates the activity of cholesterol esterase, while inhibiting cholesterol ester synthetase, thus increasing the intracellular pool of free cholesterol, the substrate for P450scc. Finally, ACTH facilitates transport of cholesterol into mitochondria, by stimulating the synthesis and phosphorylation of StAR, thus increasing the flow of free cholesterol into the mitochondria. All of these actions are mediated by cAMP and occur within minutes, constituting the “acute” effect of ACTH on steroidogenesis. The adrenal contains relatively modest amounts of steroid hormones; thus release of preformed cortisol does not contribute significantly to the acute response to ACTH; acute responses occur by the rapid provision of large supplies of cholesterol to mitochondrial P450scc.

The long-term “chronic” effects of ACTH are mediated directly at the level of the steroidogenic enzymes. ACTH via cAMP stimulates the accumulation of the steroidogenic enzymes and their mRNAs by stimulating the transcription of their genes. ACTH also increases adrenal blood flow, increasing the influx of oxygen and metabolic fuel and the delivery of newly secreted hormones to the circulation. Thus ACTH increases both the uptake of the cholesterol substrate and its conversion to steroidal products. The stimulation of this steroidogenesis occurs at each step in the pathway, and not only at the rate-limiting step, P450scc.

The role of ACTH and other peptides derived from POMC, in stimulating growth of the adult adrenal, is supported by the observations that lack of pituitary POMC causes severe adrenal hypoplasia, and chronic ACTH excess causes adrenal hyperplasia. In the fetal adrenal, ACTH stimulates the local production of IGF-2, FGF2, and EGF. These, and possibly other factors work together to mediate ACTH-induced growth of the fetal adrenal.

Diurnal Rhythms of Adrenocorticotropic Hormone and Cortisol

Plasma concentrations of ACTH and cortisol tend to be high in the morning and low in the evening. Peak ACTH levels are usually seen at 4 to 6 AM and peak cortisol levels follow at about 8 AM. Both ACTH and cortisol are released episodically in pulses every 30 to 120 minutes throughout the day, but the frequency and amplitude of these is much greater in the morning. The basis of this diurnal rhythm is complex and poorly understood. The hypothalamic content of CRF itself shows a diurnal rhythm with peak content at about 4 AM. At least four factors appear to play a role in the rhythm of ACTH and cortisol: intrinsic rhythmicity of synthesis and secretion of CRF by the hypothalamus; light/dark cycles; feeding cycles; and inherent rhythmicity in the adrenal, possibly mediated by adrenal innervation. These factors are clearly interdependent and related. Dietary rhythms may play as large a role as light/dark cycles. Animal experiments show that altering the time of feeding can overcome the ACTH/cortisol periodicity established by a light/dark cycle. In normal human subjects, cortisol is released before lunch and supper, but not at these times in persons eating continuously during the day. Thus glucocorticoids, which increase blood sugar, appear to be released at times of fasting and are inhibited by feeding.

As all parents know, infants do not have a diurnal rhythm of sleep or feeding. Infants acquire such behavioral rhythms in response to their environment long before they acquire a rhythm of ACTH and cortisol. The diurnal rhythms of ACTH and cortisol begin to be established at 6 to 12 months and often are not well established until after 3 years of age. Once the rhythm is well established in the older child or adult, it is changed only with difficulty. When people move to different parts of the world, their ACTH/cortisol rhythms generally take 15 to 20 days to adjust appropriately. Reentrainment of the circadian clock following jet lag requires approximately 1 to 2 hours/day, more for eastward and less for westward travel.

Physical stress (such as major surgery, severe trauma, blood loss, high fever, or serious illness) can increase the secretion of both ACTH and cortisol, but minor surgery and minor illnesses (such as upper respiratory infections) have little effect on ACTH and cortisol secretion. Infection, fever, and pyrogens can stimulate the release of cytokines, such as interleukin (IL)-1 and IL-6, which stimulate secretion of corticotropin-releasing hormone (CRH), and also stimulate IL-2 and tumor necrosis factor (TNF), which stimulate release of ACTH, providing further stimulus to cortisol secretion during inflammation ; furthermore, IL-6 can directly stimulate adrenal synthesis and release of cortisol. Conversely, glucocorticoids inhibit cytokine production in the immune system, providing a negative feedback loop. Most psychoactive drugs, such as anticonvulsants, neurotransmitters, and antidepressants, do not affect the diurnal rhythm of ACTH and cortisol, although cyproheptadine (a serotonin antagonist) can suppress ACTH release.

Adrenal: Glucocorticoid Feedback

The HPA axis is a classic example of an endocrine feedback system. ACTH increases production of cortisol, and cortisol decreases production of ACTH. Cortisol and other glucocorticoids exert feedback inhibition of both CRF and ACTH (and AVP), principally through the GR. Like the acute and chronic phases of the action of ACTH on the adrenal, there are acute and chronic phases of the feedback inhibition of ACTH (and presumably CRF). The acute phase, which occurs within minutes, inhibits release of ACTH (and CRF) from secretory granules. With prolonged exposure, glucocorticoids inhibit ACTH synthesis by directly inhibiting the transcription of the gene for POMC (and AVP), which can result in secondary adrenal insufficiency. Some evidence also suggests that glucocorticoids can directly inhibit steroidogenesis at the level of the adrenal fasciculata cell itself, but this appears to be a physiologically minor component of the regulation of cortisol secretion.

Major Enzymes That Metabolize Steroids

The liver is the primary site of glucocorticoid metabolism. Cortisol is reduced, oxidized, or hydroxylated and then conjugated with sulfate or glucuronic acid, rendering it water soluble, and is excreted in the urine. 3α-Hydroxysteroid dehydrogenase (e.g., AKR1C2 and AKR1C4) is the major enzyme group that reduces cortisol at the 3-keto group, and 5α- or 5β-reductases (SRD5A1 and AKR1D1, respectively) reduce the 4-5 double bond in the A ring. The tetrahydrocortisols and tetrahydrocortisones that result from these reactions can be reduced by 20-hydroxysteroid dehydrogenases, producing cortols and cortolones. Depending on cofactor availability, 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) can have either dehydrogenase (inactivation) or reductase (activation) activities, but in the liver, this enzyme usually functions as a reductase to activate cortisone to cortisol. Cortisol, and the metabolites generated in the reactions described earlier, are oxidized. This process removes the C20-C21 side chain, resulting in C19 steroids with a 17-ketone group, but the enzyme that drives this reaction has not yet been identified. CYP3A4 can 6β-hydroxylate cortisol; when cortisol levels are normal, the rate of this reaction is very low. However, when cortisol levels are elevated, the rate of the reaction increases, which can make the measurement of urinary 6β-hydroxycortisol a useful adjunct test for excess glucocorticoids. Finally, uridine diphosphoglucuronosyl transferases can conjugate glucuronic acid or sulfate to C19 and C21 metabolites, increasing their water solubility and facilitating their urinary excretion.

Similarly to cortisol, aldosterone is reduced and conjugated in the liver. 4,3-Ketosteroid-reductase (which also has 5β-hydroxysteroid reductase activity) and a 3α-hydroxysteroid dehydrogenase are the major enzymes that result in the conversion of aldosterone to 3α, 5β-tetrahydroaldosterone. Tetrahydroaldosterones are conjugated to glucuronic acid at the 3-keto position (which increases water solubility) and is the major metabolite of aldosterone that is excreted in the urine.

DHEA is the adrenal steroid produced in greatest quantity. DHEA is converted to androstenedione and 5α-reduced to androsterone, which is then converted to etiocholanolone by 5β-reductase. Finally, 17β-hydroxysteroid dehydrogenases reduce etiocholanolones into -diol derivatives, which can be conjugated and excreted in the urine. However, fecal excretion of DHEA and its metabolites is higher than other steroids. The sulfate ester of DHEA (DHEAS), on the other hand, can be directly excreted in the urine. Although these pathways can seem somewhat esoteric, as illustrated later, these pathways can be perturbed by common therapies and conditions, hence they need to be considered in clinical diagnostic and treatment plans.

Examples of Drugs That Alter Steroid Metabolism

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  Thyroid Hormone accelerates cortisol metabolism by inducing hepatic 5α- and 5β-reductase activity and inhibiting CYP3A enzymes. The converse process occurs in patients with hypothyroidism. In both situations, serum cortisol levels are usually normal because the HPA axis remains intact. However, in cases of hypopituitarism, it is prudent to replace cortisol before initiating thyroid hormone replacement, to avoid an acute adrenal crisis because of accelerated cortisol clearance.

  
  
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  Spironolactone increases serum aldosterone levels, in part by inhibition of 18-glucuronidation of aldosterone.

  
  
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  Troglitazone induces CYP3A4 activity, disrupting cortisol metabolism. Although neither rosiglitazone nor pioglitazone appear to affect CYP3A4, thiazolidinedione drugs generally inhibit steroidogenic P450c17 and HSD3B2.

  
  
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  Drugs That Alter P450 Enzymes . The results of a randomized control trial supported reports that consuming Hypericum perforatum (St. John’s Wort, a common herbal remedy) decreased efficacy of estradiol-based oral contraception. St. John’s Wort induces CYP3A4, CYP2C19, and CYP2C9, accelerating the metabolism of the oral contraceptives, decreasing their effectiveness. In contrast, the antifungal drug ketoconazole acts principally by inhibiting CYP51 (lanosterol demethylase), but it also inhibits several steroidogenic P450 enzymes and has been used as adjunctive therapy in prostate cancer and Cushing syndrome.

Examples of Disorders That Alter Steroid Metabolism

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  Stress: Surprisingly, elevations in ACTH levels are usually a transient processes, even when stress is prolonged. The persistently elevated cortisol levels during chronic stress are caused by stimulating cytokines and diminished clearance of cortisol through enzymatic inactivation by 5α- and 5β-reductases and by HSD11B2.

  
  
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  Cushing Syndrome: It is thought that when glucocorticoid levels remain persistently elevated, the oxidation and reduction pathways that metabolize them become saturated and CYP3A4 is induced. This results in increased levels of 6β-hydroxylation (and concomitantly lower levels of cortols, cortolones, tetrahydrocortisone, and 5α- tetrahydrocortisol) excreted, which can be confirmed by analysis of the urine. Elevations in the levels of glucocorticoids also stimulate hepatic HSD11B2 activity.

  
  
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  Obesity: Obese individuals have elevated levels of cortisol metabolites in their urine, even when normalized for body surface area. It is important to distinguish this process from Cushing syndrome. In obese patients, serum cortisol levels are normal as the higher excretion rate is matched by higher production rates.

  
  
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  Insulin Resistance: Individuals with insulin resistance, including those with hyperinsulinemia, have increased levels of hepatic SRD5A1 activity, increasing urinary excretion of 5α-reduced metabolites of cortisol.

  
  
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  Renal Disease: Although metabolism of cortisol is usually normal in patients with renal disease, clearance of glucuronides can be reduced, causing accumulation of the inactive conjugated compounds in the circulation. This process could potentially be used to monitor disease but is not thought to contribute to pathology.

  
  
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  Heart Failure: Patients with severe congestive heart failure and impaired perfusion of the liver also have impaired aldosterone metabolism, resulting in lower levels of urinary tetrahydroaldosterone glucuronide.

Measurement of Steroids

Plasma Renin

Renin may be assayed by its enzymatic activity or by direct measurements of its concentration. Plasma renin activity (PRA) is simply an assay of the amount of angiotensin I generated per milliliter of serum, per hour at 37° C. In normal serum, the concentration of both renin and angiotensinogen (the renin substrate) are limiting. Another test, which is technically more challenging, directly measures plasma renin content (PRC) by immunoassay.

PRA is sensitive to dietary sodium intake, posture, diuretic therapy, activity, and sex steroids. Because PRA values can vary widely, it is best to measure PRA twice, once in the morning, after overnight supine posture, and then again after maintenance of upright posture for 4 hours. A simultaneous 24-hour urine for total sodium excretion is generally needed to interpret PRA results. Decreased dietary and urinary sodium, decreased intravascular volume, diuretics, and estrogens will increase PRA. Sodium loading, hyperaldosteronemia, and increased intravascular volume decrease PRA.

Renin measurements are used in the evaluation of hypertension and in the management of CAH. Assessment of the renin-angiotensin system is essential in children with simple virilizing (SV) CAH who lack clinical evidence of urinary salt loss (hyponatremia, hyperkalemia, acidosis, hypotension, shock), but who may nevertheless have increased PRA, especially when dietary sodium is restricted. This is a clinical sign that this form of 21OHD is simply a milder form of the more common salt-wasting form. Treatment of SV CAH with sufficient mineralocorticoid, to suppress PRA into the normal range, will reduce the child’s requirement for glucocorticoids, thus maximizing final adult height and reducing exposure to excess glucocorticoids. Children with CAH need to have their mineralocorticoid replacement therapy monitored routinely by measuring PRA. Measurement of angiotensin II is also possible in some research laboratories, but most antibodies to angiotensin II strongly cross-react with angiotensin I. Thus PRA remains the usual way of evaluating the renin-angiotensin-aldosterone system.

Plasma Adrenocorticotropic Hormone and other Proopiomelanocortin Peptides

Accurate immunoassay of plasma ACTH is available in most centers, but its measurement remains more difficult and variable than the assays for most other pituitary hormones. Handling of the samples must be done with care; samples must be drawn into a plastic syringe containing heparin or ethylenediamine tetraacetic acid (EDTA) and quickly transported in plastic tubes on ice, as ACTH adheres to glass and is quickly inactivated. Thus elevated plasma ACTH concentrations can be highly informative, but most assays cannot detect low or low-normal values, and such values can be spurious if the samples are handled badly. In adults and older children who have well-established diurnal rhythms of ACTH, normal 8 AM values rarely exceed 50 pg/mL, whereas 8 PM values are usually undetectable. Patients with Cushing disease often have normal morning values, but the diagnosis can be suggested by consistently elevated afternoon and evening values; patients with the ectopic ACTH syndrome can have values up to 1000 pg/mL.

Secretory Rates

The secretory rates of cortisol and aldosterone (or other steroids) can be measured by administering a small dose of tritiated cortisol or aldosterone and measuring the specific activity of one or more known metabolites in a 24-hour urine collection. This procedure permitted measurement of certain steroids, such as aldosterone, before specific immunoassays were available. These procedures have also provided much information about the normal rate of production of various steroids. Based on this procedure, most authorities have agreed that children and adults secrete about 6 to 9 mg of cortisol per square meter of body surface area per day.

Dexamethasone Suppression Test

Administration of dexamethasone, a potent synthetic glucocorticoid, will suppress secretion of pituitary ACTH and adrenal cortisol; the dexamethasone suppression test is useful for distinguishing whether glucocorticoid excess is caused primarily by pituitary disease or adrenal disease. As dexamethasone also suppresses adrenal androgen secretion, this test is useful for distinguishing between adrenal and gonadal sources of sex steroids. A complete formal dexamethasone suppression test requires the measurement of basal values and those obtained in response to both low- and high-dose dexamethasone. This is described in the section on the evaluation of Cushing syndrome. Variations of this test are commonly used, notably the single dose 1.0-mg in adults or 0.3 mg/m ² in children. This is a useful outpatient screening procedure for distinguishing Cushing syndrome from exogenous obesity. It can be useful for the same purpose in adolescents and older children, but is otherwise of limited utility in pediatrics. An overnight high-dose dexamethasone suppression test is probably more reliable than the standard 2-day, high-dose test in differentiating adults with Cushing disease from those with the ectopic ACTH syndrome. However, the utility of this test in pediatric patients has not been established.

Stimulation Tests

Direct stimulation of the adrenal with ACTH is a rapid, safe, easy way to evaluate adrenocortical function. The original ACTH test consisted of a 4- to 6-hour infusion of 0.5 units/kg of ACTH(1-39). This will maximally stimulate adrenal cortisol secretion, and thus effectively distinguishes primary adrenal insufficiency (Addison disease), in which the adrenal is incapable of responding, from secondary adrenal insufficiency caused by hypopituitarism. In secondary adrenal insufficiency, some steroidogenic capacity is present, therefore some cortisol is produced in response to the ACTH; thus cortisol secretion is less than normal but greater than the negligible values seen in primary adrenal insufficiency. The 4- to 6-hour intravenous ACTH test has been replaced by the 60-minute test, wherein a single bolus of ACTH(1-24) is administered intravenously and cortisol and possibly other steroids are measured at 0 and 60 minutes. Normal responses to a 60-minute test are shown in Table 14.4 . Synthetic ACTH(1-24) (cosyntropin) is preferred, as it has a more rapid action and shorter half-life than ACTH(1-39). The usual dose is 15 μg/kg in children up to 2 years of age, and 0.25 mg for children older than 2 years and adults. All of these doses are pharmacologic. A very-low-dose (1 μg) test may be useful in assessing adrenal recovery from glucocorticoid suppression. Newer data show that maximal steroidal responses can be achieved after only 30 minutes, but the best available standards are for a 60-minute test. One of the widest uses of intravenous ACTH tests in pediatrics is in diagnosing CAH. Stimulating the adrenal with ACTH increases steroidogenesis, resulting in accumulation of steroids proximal to the disordered enzyme. For example, inspection of Fig. 14.3 shows that impaired activity of P450c21 (21-hydroxylase) should lead to the accumulation of progesterone and 17OHP. However, progesterone does not accumulate in appreciable quantities, because it is converted to 17OHP. In routine practice, measuring the response of 17OHP to a 60-minute challenge, with intravenous ACTH, is the single most powerful and reliable means of diagnosing 21OHD; genetic testing can provide a useful confirmation. Comparing the patient’s basal and ACTH-stimulated values of 17OHP against those from large numbers of well-studied patients usually permits the discrimination of normal persons, heterozygotes, patients with nonclassic CAH, and patients with classic CAH, although there inevitably is some overlap between groups ( Fig. 14.12 ). Measurement of testosterone or Δ ⁴ androstenedione in response to ACTH can distinguish normal persons from patients with classic CAH, but heterozygotes and patients with cryptic CAH have values overlapping both normal and classic CAH.

17α-Hydroxyprogesterone values (in ng/100 mL) before and after stimulation with adrenocorticotropic hormone in normals, patients with congenital adrenal hyperplasia, and heterozygotes.

Longer ACTH tests of up to 3 days have also been used to evaluate adrenal function. It is important to remember that ACTH has both acute and chronic effects. Thus short tests measure only the acute effects of ACTH, that is, the maximal stimulation of preexisting steroidogenic machinery. In contrast, a 3-day test will examine the more chronic effects of ACTH to stimulate increased capacity for steroidogenesis by increasing the synthesis of steroidogenic machinery. Few situations exist where a 3-day intramuscular ACTH test is indicated, although it is occasionally useful in diagnosing the rare syndromes of hereditary unresponsiveness to ACTH.

Insulin-induced hypoglycemia is an effective but potentially risky test, and hence is rarely used. Insulin (0.1 U/kg/IV) is administered and blood is obtained at 0, 30, 45, and 60 minutes. The insulin-induced hypoglycemia will stimulate the release of “counterregulatory” hormones that have actions to increase plasma glucose concentrations: ACTH and cortisol, growth hormone, epinephrine, and glucagon. Because of the inherent risk of convulsions as a result of hypoglycemia, an experienced physician must be in attendance (not merely “available”) throughout the entire course of the test. Blood glucose must fall to half of the initial value or to 45 mg/dL to achieve an adequate test, and it is wise to terminate the test after this level is reached. Most patients will experience hunger, irritability, diaphoresis, and tachycardia; when these are followed by drowsiness or sleep, blood sugar levels are likely below acceptable limits. If this occurs, a blood sample should be obtained and 2 mL/kg of 20% to 25% glucose given intravenously, to a maximum of 100 mL.

Metyrapone Test

Metyrapone blocks the action of P450c11β and, to a much lesser extent, P450scc. It is thus a chemical means of inducing a transient deficiency of 11-hydroxylase activity, which results in decreased cortisol secretion and subsequently increased ACTH secretion. Metyrapone testing is done to assess the capacity of the pituitary to produce ACTH in response to a physiologic stimulus. This test is useful in evaluating the hypothalamic-pituitary axis in the presence of central nervous system lesions, after neurosurgery, or long-term suppression by glucocorticoid therapy. Patients with a previous history of hypothalamic, pituitary, or adrenal disease, or those who have been withdrawn from glucocorticoid therapy should be reevaluated with a metyrapone test. A normal response indicates recovery of the HPA axis and predicts that the patient will respond normally to the stress of surgery.

Metyrapone is generally given orally as 300 mg/m ² every 4 hours for a total of six doses (24 hours). Unlike many other drugs, it is appropriate to continue to increase the dose in older or overweight patients, but the total dose should not exceed 3.0 g. Blood should be obtained for cortisol, 11-deoxycortisol, and ACTH, before and after the test. In a normal response to metyrapone, cortisol decreases, ACTH increases, and 11-deoxycortisol (the substrate for P450c11β) increases greatly, to about 5 μg/dL. Adults and older children can be tested with the administration of a single oral dose of 30 mg/kg at midnight, given with food to reduce the gastrointestinal irritation. Blood samples are drawn at 8 AM the mornings before and after administering the drug.

Wolman Disease and Cholesteryl Ester Storage Disease

Most cellular cholesterol derives from the uptake of circulating lipoproteins that contain cholesterol esters; these esters are cleaved to free cholesterol by lysosomal acid lipase (cholesterol esterase), encoded by the LIPA gene on chromosome 10q23.31. Mutations of LIPA cause Wolman disease (WD, primary xanthomatosis) and its milder variant, cholesteryl ester storage disease (CESD). In WD, cholesteryl esters and triglycerides accumulate in the liver, spleen, lymph nodes, and other tissues. In the adrenal, there is insufficient free cholesterol available for steroidogenesis, causing adrenal insufficiency. The disease is less severe than lipoid CAH with respect to steroidogenesis, and patients may survive for several months after birth. However, the disease affects all cells, not just steroidogenic cells, as all cells must store and use cholesterol; hence, the disorder is relentless and fatal. Vomiting, steatorrhea, failure to thrive, hepatosplenomegaly, jaundice, and anemia, are the usual presenting findings sometimes beginning in the first week of life, leading to developmental delay, and malabsorptive malnutrition. Characteristic bilateral subcapsular adrenal calcification that outlines the adrenals may be seen on pre- or postnatal ultrasound scan. The diagnosis is established by bone marrow aspiration, yielding foam cells containing large lysosomal vacuoles, engorged with cholesterol esters, and is confirmed by finding absent cholesterol esterase activity in fibroblasts, leukocytes, bone marrow cells, or cultured amniocytes (for prenatal diagnosis). Treatment by bone marrow transplantation appears to ameliorate the course of the disease in about half of cases, but the mechanism of this effect is unclear ; enzyme replacement therapy may ameliorate disease progression. Supportive treatments include corticosteroid replacement, vitamin, and mineral supplementation. One survey found WD in around 3% of pediatric adrenal insufficiency.

CESD is a milder, later-onset form of WD caused by LIPA mutations that retain partial activity. CESD typically presents in later childhood and adolescence with hypercholesterolemia, atherosclerosis, and hepatosplenomegaly (hepatic infiltration with macrophages containing cholesteryl esters); hepatic fibrosis may lead to esophageal varices. There may be early onset with severe cirrhosis or later onset with more slowly progressive hepatic disease. Some patients reach adulthood.

Niemann-Pick Type C Disease

The endosomal processing of cholesterol esters requires the action of NPC1 and NPC2, two proteins named for NPC disease. NPC is a rare neurological disorder of cholesterol trafficking characterized by accumulated cholesterol and glycosphingolipids in endosomes, leading to progressive neurodegeneration, robust glial infiltration, and death. NPC typically presents at 2 to 4 years, with mild intellectual impairment, speech loss, supranuclear vertical gaze paresis, ataxia, dystonia, dementia, seizures, and extrapyramidal deficits. An infantile, rapidly progressive form presents with severe hepatic dysfunction, psychomotor delay, supranuclear vertical gaze paresis, ataxia, spasticity, and dementia; the hepatosplenomegaly and childhood cirrhosis can be lethal. However, despite the seemingly central role of the NPC proteins in intracellular cholesterol trafficking, adrenal insufficiency is not reported in NPC. Thus although there is good evidence for disordered steroidogenesis in the NPC brain, the adrenal appears to escape, suggesting it may use other pathways for intracellular cholesterol trafficking.

Pathophysiology

In severe 21OHD, there is an inability to convert progesterone to DOC, resulting in aldosterone deficiency that causes severe hyponatremia, hyperkalemia, and acidosis. The associated hypotension, shock, and cardiovascular collapse may result in death in an untreated newborn. As the control of fluids and electrolytes in the fetus are maintained by the placenta and the mother’s kidneys, this salt-losing crisis develops only after birth, usually during the second week of life. The inability to convert 17OHP to 11-deoxycortisol results in cortisol deficiency, which impairs postnatal carbohydrate metabolism and exacerbates cardiovascular collapse, because a permissive action of cortisol is required for full pressor action of catecholamines. High concentrations of cortisol in the adrenocortical capillary effluent that bathes the medulla are needed for the transcription of medullary phenylethanolamine N-methyltransferase, which converts norepinephrine to epinephrine, hence children with 21OHD have low epinephrine concentrations, which may exacerbate the hypoglycemia associated with cortisol deficiency.

Although a role for cortisol in fetal physiology is not well established, cortisol deficiency is manifested prenatally. Low fetal cortisol stimulates ACTH secretion, which stimulates adrenal hyperplasia and transcription of the genes for all the steroidogenic enzymes, especially for P450scc, the rate-limiting enzyme in steroidogenesis. This increased transcription increases enzyme production and activity, with consequent accumulation of non-21-hydroxylated steroids. There are four pathways by which these steroids are diverted to androgens (see Fig. 14.6 ). First, 17-Preg may be converted to DHEA by the 17,20 lyase activity of P450c17; if not inactivated by sulfation to DHEAS, DHEA may be converted to androstenedione by 3βHSD2. Androstenedione may then be converted to testosterone in the fetal adrenal or in target tissues by 17βHSD5 (see Fig. 14.3 ). Second, although 17OHP is not normally an effective substrate for the 17,20 lyase activity of P450c17, the very high levels of 17OHP characteristic of 21OHD will “force” some conversion to androstenedione by mass action. Third, as described in Section III (Adrenal Androgen Secretion and the Regulation of Adrenarche), androstenedione may be converted to 11-keto-testosterone, the principal adrenal androgen, in the zona reticularis. Fourth, in the alternative or backdoor pathway to DHT, 17OHP is 5α- and 3α-reduced to 17OH-allopregnenolone, which is readily converted by P450c17 to androsterone and thence by 17βHSD to androstanediol and an oxidative 3αHSD to DHT, so that this most potent androgen is produced without DHEA, androstenedione, or testosterone as intermediates. Recent work has established that this pathway exists in the human fetus and that it contributes substantially to the production of androgens in children with 21OHD. Also as described earlier (see Fetal Adrenal Steroidogenesis), the fetal adrenal transiently expresses 3βHSD2 at about 7 to 12 weeks, permitting cortisol synthesis, which in turn suppresses ACTH and hence suppressing fetal adrenal production of DHEA and other C-19 steroids. Thus fetal adrenal androgen synthesis is normally suppressed during the time when the external genitalia may become virilized. In 21OHD, this transient production of cortisol is not possible, further driving the androgenization of female fetuses.

Early in gestation, the fetal testes produce testosterone by the conventional androgenic pathway and DHT by the backdoor pathway, both of which are needed to differentiate the pluripotent embryonic precursor structures into male external genitalia. In the male fetus with 21OHD, the additional androgens produced in the adrenals have no phenotypic effect. In contrast, the fetal ovaries normally produce minimal sex steroids or other factors needed for differentiation of the female external genitalia. The androgens inappropriately produced by the adrenals of the 21OHD female fetus cause varying degrees of external genital virilization, which can range from mild clitoromegaly, with or without posterior fusion of the labioscrotal folds, to complete labioscrotal fusion that includes a urethra traversing the enlarged clitoris ( Fig. 14.14 ). These infants have normal ovaries, fallopian tubes, and a uterus, but have “ambiguous” external genitalia or may be sufficiently virilized so that they appear to be male, resulting in errors of sex assignment at birth. 21OHD is the most common form of 46,XX DSD.

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