Mineralocorticoids (Aldosterone)

Mineralocorticoids bind to the mineralocorticoid receptor (MR) in the distal convoluted tubule and collecting duct of the nephron, the colon, and the salivary glands to promote sodium reabsorption and potassium and hydrogen ion excretion.³⁵² Similar to receptors for other steroid hormones and thyroid hormone, the MR functions as a transcription factor. When mineralocorticoid binds to the cytoplasmic MRs, the mineralocorticoid-MR complex relocates to the nucleus, where it influences cellular DNA regulating gene transcription. The principal mineralocorticoid is aldosterone, but other compounds with mineralocorticoid activity include 11-desoxycorticosterone (DOC), 18-hydroxycorticosterone, corticosterone, and cortisol.

The MR gene encodes a 107 kDa protein and is officially labeled nuclear receptor subfamily 3, group C, member 2 (NR3C2), located on chromosome 4q31.1-31.2. Alternative splicing yields an alpha and beta MR mRNA. Aldosterone stimulates epithelial sodium channel (ENaC) activity via serum and glucocorticoid-induced kinase (SGK) and K-ras, increases expression of mitochondrial ATP-producing genes, and stimulates the basolateral Na⁺/K⁺-ATPase pump. ENaC, a highly selective epithelial sodium channel that is amiloride sensitive (amiloride is a K⁺-sparing diuretic), has three subunits: the alpha subunit (most important of the three subunits: the sodium channel, non–voltage-gated protein 1, alpha coded by SCNN1A, chromosome 12p13), the beta subunit (sodium channel, non–voltage-gated 1, beta SCNN1B, chromosome 16p13-p12), and the gamma subunit (sodium channel, non–voltage-gated 1, gamma SCNN1G, chromosome 16p13-p12).¹⁷⁸ All ENaC subunits are similar and contain a large N-terminal extracellular domain, two transmembrane spanning domains (M1 and M2), and a C-terminal, short intracellular domain.¹⁴⁰ Although the binding of aldosterone to the MR occurs in the cytoplasm with transit of the complex to the nucleus, some free MR is present in the nucleus. The MR binds cortisol and 11-desoxycorticosterone with affinity equal to that of aldosterone. However, the MR is protected from cortisol and 11-desoxycorticosterone by 11 beta-hydroxysteroid dehydrogenase-2 (HSD11B2), where, for example, HSD11B2 catalyzes the conversion of cortisol to cortisone (Figure 54-2). Cortisone does not bind to the MR. Variations in HSD11B2 may be involved in some cases of otherwise “essential” hypertension.⁸⁶ Chronic elevations in angiotensin, which controls aldosterone synthesis and release, produces pathologic changes in the heart and kidney by eliciting myocardial fibrosis and inflammatory changes in the renal vasculature.¹³⁹ The actions of mineralocorticoids are summarized in Table 54-2.

Glucocorticoids (Cortisol)

Glucocorticoids bind to the glucocorticoid receptor (GR) located in a large number of tissues, including lymphocytes, hepatocytes, and bone.³⁴ The GR gene contains 10 exons (exons 1 through 8 plus 9 alpha and 9 beta). Alternative splicing yields GR alpha and GR beta transcripts. Because of the wide distribution of the GR, glucocorticoid effects are diverse, including changes in intermediary metabolism and immunoregulation. Glucocorticoids raise blood glucose concentrations by enhancing the synthesis of gluconeogenic enzymes (e.g., glucose-6-phosphatase and phosphoenol pyruvate carboxykinase), increase liver glycogen content through activation of glycogen synthase, and inhibit glycogen phosphorylase, producing insulin resistance in both muscle and adipose tissue that further raises blood glucose concentrations. Excessive catabolism of skeletal muscle causes myopathy and consequent weakness. Protein catabolism causes thinning of the skin and loss of strength in connective tissues. With excess glucocorticoids, bone loss may result from collagen catabolism and loss of osteoid, which can lead to fractures and compressed vertebrae.

Glucocorticoids cause adipose tissue redistribution centrally to the trunk, neck, and face, increased adipocyte differentiation, and promotion of lipogenesis in these tissues. Insulin resistance raises very low-density lipoprotein (VLDL) and triglyceride concentrations and lowers high-density lipoprotein (HDL) concentrations. The activity of adipose tissue hormone-sensitive lipase (HSL) is decreased because of insulin resistance, allowing triglyceride breakdown to free fatty acids and increased free fatty acid delivery to the liver, which provides substrate for hepatic triglyceride resynthesis and VLDL production and export. Glucocorticoids increase appetite, and a subsequent increase in caloric intake causes weight gain.

When glucocorticoids bind to the cytoplasmic GR, heat shock proteins (HSPs) are released (HSP70 and HSP90).⁶³ Glucocorticoids are powerful anti-inflammatory hormones that inhibit nuclear factor kappaB (NFkappaB) through the induction of IkappaB synthesis.⁹¹ IkappaB binds to NFkappaB in the cytoplasm, impairing the entrance of NFkappaB into the nucleus. Within the nucleus, the GR-cortisol complex binds NFkappaB, preventing its binding to DNA. Finally, within the nucleus, GR-cortisol and NFkappaB compete for cofactors that are available in limited quantities. Although IkappaB synthesis is enhanced, many proinflammatory genes are repressed, such as cyclo-oxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), various interleukins (IL-1, IL-2, and IL-6), tumor necrosis factor-alpha, interferon-gamma, and E-selectin. Adrenocorticotropic hormone (ACTH) also stimulates the release of IL-1, IL-6, and tumor necrosis factor-alpha.²⁷⁵

Glucocorticoids help maintain vascular tone and cardiac output, and stabilize lysosomal membranes.⁴⁴ Glucocorticoids suppress hypersensitivity responses by inhibiting the production of histamine by basophils and mast cells. Modest doses of glucocorticoids may improve one’s mood, yet in pharmacologic concentrations, they may produce psychosis. Pathologically elevated concentrations of glucocorticoids are discussed further under the heading of “Cushing syndrome.” The relative potencies of corticosteroids in terms of glucocorticoid and mineralocorticoid activity are given in Table 54-3. The actions of glucocorticoids are summarized in Table 54-4.

Adrenal Androgens (DHEA and Androstenedione)

The adrenal androgens dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), and androstenedione provide androgenic effects through their peripheral conversion to testosterone, which in turn binds to the androgen receptor (AR) that is described in Chapter 56.¹⁴⁹ Between ages 7 and 8, the urinary excretion of 17-ketosteroids (the breakdown products of adrenal androgens) increases as an early sign that puberty will begin in the coming 3 to 5 years.²³⁹

In males, adrenal androgens, such as DHEA and androstenedione, are normally of negligible importance because testosterone is a much more potent androgen. However, adrenal androgens are important in pubertal and adult women because they produce axillary and pubic hair. Women with Turner syndrome are an excellent example of the effects of adrenal androgens in women. Because of streak gonads (hypoplastic and dysfunctioning gonads mainly composed of fibrous tissue), adolescents with Turner syndrome do not experience gonadarchy (the period during which the gonads begin to secrete sex hormones) because all of their ovarian follicles are atretic before birth. Estrogen deficiency during adolescence is manifest in lack of breast development, primary amenorrhea, and failure of fat redistribution to the hips and buttocks. However, because adrenarchy (the increase in activity of the adrenal glands preceding puberty) is normal in adolescents with Turner syndrome, they will develop axillary and pubic hair despite their lack of estrogenization.

Throughout life, DHEA is sulfated, and circulating concentrations of DHEA-S exceed those of DHEA by 100-fold or more. For example, a typical DHEA reference interval in males is 180 to 1250 ng/dL, whereas the DHEA-S reference interval is 125 to 619 µg/dL.

Aldosterone

Aldosterone production and secretion are controlled through the renin-angiotensin system (Figure 54-3).^216,342 The rate-limiting component in this system is renin release, which is regulated by the juxtaglomerular apparatus. Anatomically, the juxtaglomerular apparatus is composed of (1) the juxtaglomerular cells of the afferent arteriole that immediately leads to the glomerulus, (2) lacis cells (extraglomerular mesangial cells located at the vascular pole of the renal corpuscle), and (3) the macula densa.

The juxtaglomerular cells are modified smooth muscle cells that synthesize and secrete renin. Preprorenin is a 406 amino acid protein. Removal of the 20 amino acid pre-sequence yields prorenin (386 amino acids). Cleavage of the 46 amino acid pro-segment produces the active hormone (340 amino acids, 37 kDa). Both prorenin and renin are released by the juxtaglomerular cells, which function as baroreceptors that detect arterial wall stretch produced by renal perfusion pressure. Therefore, decreased renal perfusion leads to renin release. This is the most important mechanism regulating renin concentrations in the circulation.

The macula densa consists of specialized cells that line the distal convoluted tubule (DCT). Compared with other tubular cells, these cells are unique in that their nuclei are near the apical (luminal) pole of the cell, whereas the Golgi apparatus is near the basolateral pole of the cell. Acting as a chemoreceptor, the macula densa monitors the sodium concentration in the DCT. If a decline in sodium concentration occurs in the DCT, the macula densa signals the juxtaglomerular cells via prostacyclin to release renin. Anatomically, the DCT passes between the afferent and efferent arterioles of the nephron, which, respectively, supply blood to and drain blood from the glomerular capillaries.

Decreased sodium delivery to the DCT occurs in states of decreased renal perfusion. Decreased sodium delivery to the DCT can result from hyponatremia or a decreased glomerular filtration rate, both of which would elicit renin release. Sympathetic innervation of the juxtaglomerular cells also influences renin secretion via beta₁-adrenoreceptors. For this reason, norepinephrine and dopamine stimulate renin release. Thus, with upright posture and catecholamine release, renin release is enhanced. Potassium also directly stimulates renin release. Overall, renin is physiologically released in response to hypovolemia, reduced cardiac output, systemic vasodilatation, selectively reduced renal perfusion, hyponatremia, and stress (mediated by catecholamines).

Angiotensinogen (≈60 kDa) is an alpha₂-globulin synthesized in hepatocytes.²¹¹ Renin acts as an aspartyl proteolytic enzyme cleaving the 10 N-terminal amino acids of angiotensinogen to form the decapeptide angiotensin I (Table 54-5). Angiotensin I has no endocrine, paracrine, or autocrine effects. Angiotensin-converting enzyme (ACE), a zinc metallopeptidase, removes the two C-terminal residues from angiotensin I to generate the octapeptide angiotensin II.³¹⁵ High concentrations of ACE are expressed in the lung. ACE is pathologically expressed in conditions involving macrophage activation such as sarcoidosis. Further degradation of angiotensin II by aminopeptidase A (a glutamyl aminopeptidase) yields the heptapeptide angiotensin III. The ratio of angiotensin II to angiotensin III is usually 4 to 1. An arginyl aminopeptidase generates angiotensin IV from angiotensin III.

Angiotensin II acts to preserve circulating blood volume and maintain blood pressure through several mechanisms: (1) stimulation of aldosterone synthase (CYP11B2) to produce aldosterone; (2) direct vasoconstriction; (3) increased release of epinephrine and norepinephrine from the adrenal medulla, which will also act as vasoconstrictors; (4) stimulation of sodium reabsorption in the proximal convoluted tubule (PCT); (5) stimulation of thirst; and (6) stimulated release of antidiuretic hormone (ADH). Angiotensin III has equivalent potency in stimulating aldosterone secretion.

The best characterized of the angiotensin receptors are AT1 and AT2 that involve multiple second messenger systems.¹³⁴ Most functions of angiotensin II are mediated via the AT1 receptor. Some actions of the stimulated AT2 receptor oppose those of the AT1 receptor (e.g., AT2 receptor engagement causes vasodilatation).

Cortisol

Cortisol is controlled through a traditional hypothalamic-pituitary-end organ negative feedback system (Figure 54-4). Corticotropin-releasing hormone (CRH) is released by stress, exercise, and hypoglycemia. Examples of physiologic stress include pain, trauma, surgery, and hemorrhage. Examples of psychologic stress include severe anxiety and major depression. Prolonged administration of supraphysiologic doses of glucocorticoids orally or parenterally will suppress the hypothalamic-pituitary-adrenal axis, leading to adrenal atrophy. As a result, abrupt termination of exogenous steroids may induce an acute and possibly life-threatening glucocorticoid insufficiency.

CRH is produced by the paraventricular nucleus of the hypothalamus. Prohormone convertase-1 (PC1) and PC2 liberate a C-terminal, 43 amino acid CRH precursor from the 196 amino acid preprohormone. Peptidylglycine alpha-amidating mono-oxygenase (PAM) removes the two C-terminal residues and adds an amine group, producing the 41 amino acid polypeptide, CRH.

Following hypothalamic secretion, CRH reaches the anterior pituitary gland through the hypothalamic pituitary portal system.²⁴⁴ Corticotrophs represent about 20% of functional anterior pituitary cells and express receptors for CRH that promote synthesis, storage, and release of corticotropin (ACTH; 4.5 kDa).¹¹⁰ ACTH is also released by ADH stimulation but to a lesser degree than CRH. The proinflammatory cytokines IL-1, IL-6, and tumor necrosis factor-alpha also release ACTH. There are two CRH receptors (CRH-R1 and CRH-R2) and three splice variants of CRH-2 (CRH-2alpha, CRH-2beta, and CRH-2gamma). CRH mediates its effects on corticotrophs exclusively through CRH-R1. CRH and its receptors are widely distributed throughout the central nervous system (CNS).

ACTH is released from its 32 kDa precursor protein, 266 amino acid pro-opiomelanocortin (POMC; gene: 8 kb; chromosome 2p23), by proteolysis (Figure 54-5).^133,159 In the corticotrophs, subtilisin-like proprotein convertase (PC1/3) cleaves POMC into two fragments: the 22 kDa pro-ACTH fragment and beta-lipotropin (amino acids 42-134), whose function remains poorly understood. Next, PC1/3 releases ACTH (amino acids 1-39) from pro-ACTH. The resulting N-terminal fragment is further cleaved to pro-gamma-melanocyte-stimulating hormone (MSH) and a joining peptide (JP). ACTH is not further cleaved in corticotrophs.

The action of PC2 in the hypothalamus, skin, and melanotrophs of the intermediate lobe is to release gamma-MSH from pro-opiocortin (N-POC); alpha-melanocyte-stimulating hormone (alpha-MSH; amino acids 1-13) and corticotropin-like intermediate lobe peptide (CLIP; amino acids 18-39) from ACTH; and gamma-lipotropin (amino acids 42-101) and beta-endorphin (amino acids 104-134) from beta-lipotropin (see Figure 54-5). Last, beta-MSH (amino acids 84-101) is derived from gamma-lipotropin via PC2.

Hyperpigmentation that occurs with ACTH excess appears to be a direct consequence of the MSH-like activity of ACTH and not ACTH cleavage into alpha-MSH.³⁴⁹ Whereas (1) a gamma-MSH sequence is contained within the N-terminal fragment, (2) alpha-MSH is contained within ACTH, and (3) beta-MSH is contained within gamma-lipotropin (a fragment of beta-lipotropin), MSH is not released by the human anterior pituitary gland.

ACTH circulates systemically to bind to ACTH receptors located on cells within the adrenal cortex. The ACTH receptor is the G-protein–coupled melanocortin-2 receptor (MC2R; gene location: chromosome 18p11.2).⁷⁷ The second messenger system involves adenyl cyclase and the generation of cyclic AMP. The actions of ACTH are then triggered via protein kinase A and protein kinase C, leading to steroidogenesis, increased size and number of adrenocortical cells, and increased size and functional complexity of cellular organelles. Cortisol is then synthesized and released. Cortisol feeds back centrally at the hypothalamus and to a lesser degree feeds back negatively at the pituitary to suppress CRH and ACTH secretion.¹⁵⁸ Other negative feedback loops include ACTH suppression of hypothalamic CRH and an ultra-short feedback loop whereby ACTH suppresses its own release.

Pulses of CRH cause the release of ACTH, which stimulates cortisol secretion. A wide diurnal variation in the secretion of cortisol is noted, with highest concentrations in the early morning (≈2 hours before awakening) and lowest concentrations near midnight (assuming that the individual is asleep overnight).

Adrenal Androgens

The regulation of adrenal androgen synthesis and secretion remains poorly understood. The existence of a pituitary adrenal androgen–stimulating hormone or a cortical androgen–stimulating hormone remains doubtful despite many years of research that sought its existence.¹¹⁶ The best characterized regulator of androstenedione and DHEA secretion is ACTH. This is not surprising, as CYP17 is regulated by ACTH. A diurnal rhythm in adrenal androgen concentrations parallels cortisol variations. Nevertheless, ACTH regulation of adrenal androgens does not explain the normal prepubertal and pubertal increases in adrenal androgen synthesis that occurs in both boys and girls: ACTH does not increase prior to puberty. Evidence indicates that sympathetic innervation of the zona reticularis may exist and may regulate adrenal androgen secretion. Immune regulation of adrenal androgen secretion has also been proposed.

Aldosterone

Within the cytoplasm of steroid-producing cells, cholesterol ester is hydrolyzed to free cholesterol and a free fatty acid via the ACTH-responsive steroidogenesis activator protein, which is functionally a cholesterol esterase. Free cholesterol is then transported across the outer mitochondrial membrane by a sterol transfer protein into the mitochondrial intermembranous space. The 30 kDa steroidogenic acute regulatory protein (StAR) next transports cholesterol across the inner membrane into the mitochondria.205,301 StAR-mediated transport of cholesterol into the mitochondria is a rate-limiting step in steroid hormone synthesis. StAR synthesis is enhanced by rising concentrations of cyclic AMP resulting from ACTH binding to its receptor. In the mitochondria, cholesterol is converted to pregnenolone by the action of CYP11A, which is the cytochrome P450 sidechain cleavage enzyme (P450ssc; gene location: chromosome 15q23-24) that is functionally a 20,22-desmolase that releases isocaproaldehyde. CYP11A therefore initiates the conversion of the C₂₇ steroid cholesterol to the other C₂₁ steroids (the subscript indicates the number of carbons in the compound).

Pregnenolone moves from the mitochondrion to the lumen of the endoplasmic reticulum. In the zona glomerulosa, CYP17 (P450c17) activity is not expressed; therefore pregnenolone is converted to progesterone via the non-P450 enzyme, 3-beta-hydroxysteroid dehydrogenase type 2 (3 beta-HSD; gene location: chromosome 1p13.1). The conversion of pregnenolone to progesterone also requires delta(5)-ketosteroid isomerase, which “moves” the double bond from the 5 position to the 4 position. Indeed, pregnenolone, 17-hydroxypregnenolone, and DHEA are characterized as delta(5) steroids, whereas progesterone, 17-hydroxyprogesterone, and androstenedione are characterized as delta(4) steroids based on the location of their double bond.

CYP21, a P450 21 alpha-hydroxylase (P450c21; gene location: chromosome 6p21.3) also expressed in the zona fasciculata, converts progesterone to 11-desoxycorticosterone (DOC). DOC migrates back into the mitochondrion, where CYP11B2 (P450 aldo; gene location: 8q24.3) catalyzes the conversion of DOC ultimately to aldosterone. Normally, little CYP11B1 (P450c11; gene location: 8q24.3) activity occurs in the zona glomerulosa.

CYP11B2 (aldosterone synthase) encompasses three enzymatic activities: (1) an 11-hydroxylase (DOC → corticosterone), (2) an 18-hydroxylase [corticosterone methyl oxidase I (CMOI); corticosterone → 18-hydroxycorticosterone], and (3) an 18-hydroxydehydrogenase [corticosterone methyl oxidase II (CMOII); 18-hydroxycorticosterone → aldosterone; see Figure 54-6].²³⁴ Aldosterone diffuses out of the mitochondrion into the cytoplasm and across the cell membrane to enter the interstitium and then the circulation. The aldosterone secretion rate per day is 100 to 150 µg, with some estimates varying up to 200 µg. Thus the aldosterone secretion rate is approximately one tenth the secretion rate of cortisol on a weight basis. The half-life of circulating aldosterone is less than 15 minutes.

Whereas CYP11A, CYP17, and CYP11B1 are under ACTH control, CYP11B2 is predominantly controlled by angiotensin II. In this way, control of aldosterone synthesis is mostly independent of the anterior pituitary. Cortisol and adrenal androgens are not formed in the zona glomerulosa because the zona glomerulosa lacks expression of CYP17.

Cortisol

Cortisol production increases within minutes of an increase in circulating ACTH concentrations. In the zona fasciculata, where CYP17 (P450c17; gene location: chromosome 10q24.3) is expressed, CYP17 hydroxylates pregnenolone to 17-hydroxypregnenolone.²⁰⁶ CYP17 also includes P450 17,20-lyase activity, which is important for the formation of adrenal androgens. 3-Beta-HSD and delta(5)-4-isomerase next catalyze the conversion of 17-hydroxypregnenolone to 17-hydroxyprogesterone. The last step outside of the mitochondrion involves CYP21 that 21-hydroxylates 17-hydroxyprogesterone to 11-desoxycortisol. 11-Desoxycortisol travels back to the mitochondrion, where CYP11B1 (P450c11) catalyzes the conversion of 11-desoxycortisol to cortisol via its 11-beta hydroxylase activity. Little CYP11B2 activity is seen in the zona fasciculata. Similar to aldosterone, cortisol diffuses out of the cell to ultimately enter the circulation. The normal cortisol secretion rate is 6 to 14 mg/m² per 24 hours. In adults, this is approximately 10 to 20 mg/d, with some estimates as high as 25 mg/d.

Adrenal Androgens

Debate continues as to the anatomic location of adrenal androgen synthesis.²⁵⁸ Traditionally, it has been taught that adrenal androgens are synthesized exclusively in the zona reticularis. However, the enzymatic activity of the 17,20-lyase (17,20-desmolase) that converts 17-hydroxypregnenolone to DHEA [a delta(5) steroid] and 17-hydroxyprogesterone to androstenedione [a delta(4) steroid] is included in the CYP17 protein, whose 17-hydroxylase activity was described previously. Therefore, adrenal androgens theoretically could be synthesized within both the zona fasciculata and the zona reticularis. However, a predominance of adrenal androgen synthesis in the zona reticularis may result from high cytochrome 5b expression, which increases the 17,20-lyase activity of CYP17. The 17,20-lyase activity of CYP17 converts C₂₁ steroids into C₁₉ steroids. Likewise, there is nothing to prevent cortisol synthesis in the zona reticularis. The conversion of DHEA to DHEA-S is catalyzed by DHEA sulfotransferase (SULT2A1; gene location: chromosome 19q13.3). Based on the model presented so far of enzymes partitioned into the mitochondrion or the endoplasmic reticulum, adrenal androgen synthesis is completed within the endoplasmic reticulum because this synthesis does not require CYP21, CYP11B1, or CYP11B2, which are located in the mitochondrion.

A small amount of testosterone is produced by the adrenal cortex. DHEA is converted to androstenediol by 17-ketosteroid reductase (17 beta-hydroxysteroid dehydrogenase, 17 beta-OHSD); this is followed by conversion of androstenediol to testosterone via the actions of 3-beta HSD and delta(4)-5-isomerase. The peripheral conversion of adrenal androgens to testosterone involves the conversion of DHEA to androstenedione and the conversion of androstenedione to testosterone via 17-ketosteroid reductase. Finally, testosterone is activated to dihydrotestosterone by 5-alpha reductase in target tissues (penis, scrotum, and beard). Peripheral aromatization of androstenedione yields estrone, whereas peripheral aromatization of testosterone yields estradiol.

Adrenal androgens are the major product of the adrenal cortex, whose total synthesis exceeds 20 to 25 mg/d. The adult adrenal secretes approximately 6 to 8 mg/d of DHEA, 8 to 16 mg/d of DHEA-S, 1.5 mg/d of androstenedione, and 0.05 mg/d of testosterone. Only small amounts of the estrogens estradiol and estrone and insignificant amounts of progesterone and other precursor steroids are produced.

Adrenal Steroid Synthesis in the Fetus

The fetal adrenal lacks adult concentrations of 3-beta-HSD expression with resulting elevated production of DHEA, DHEA-S, and 16-hydroxy DHEA-S (from hydroxylation of DHEA-S in the fetal liver). In utero, pregnenolone and 17-hydroxypregnenolone from the fetal adrenal enter the fetal circulation to be converted in the placenta, respectively, into progesterone and 17-hydroxyprogesterone via placental 3-beta-HSD. Progesterone and 17-hydroxyprogesterone then return to the adrenal (via the circulation), where they serve as substrates for aldosterone and cortisol, respectively.²²⁵

Summary of ACTH Activity

The biochemical actions of ACTH on the adrenal cortex is summarized as follows: (1) immediate ACTH effects via steroidogenesis activator protein and StAR; (2) increased cholesterol esterase activity; (3) decreased cholesterol ester synthesis; (4) increased cholesterol transport into the mitochondrion; (5) increased CYP11A binding of cholesterol; and (6) increased synthesis of pregnenolone. Through the action of transcription factors (SF-1), expression of CYP11A, CYP17, CYP11B1 (but not CYP11B2), and the LDL receptor is increased. CYP11B2 is controlled by angiotensin II, separating control of the zona glomerulosa from the zona fasciculata (CYP11B2 is controlled by ACTH). In addition, ACTH and other factors yet not discovered control adrenal androgen synthesis.

Aldosterone²⁰

Aldosterone is reduced to tetrahydroaldosterone. Glucuronidation produces tetrahydroaldosterone 3-glucuronide, which can be excreted by the kidney. Aldosterone is 3 alpha- and 5 alpha-reduced, or 3 alpha- and 5 beta-reduced (Figure 54-7). Only ≈0.5% of aldosterone is excreted unchanged in the urine. Aldosterone is glucuronidated at the carbon 18 position. Cirrhosis, severe congestive heart failure, and ascites impair hepatic aldosterone clearance.

Cortisol³¹¹

Cortisone is formed from cortisol via 11 beta-hydroxysteroid dehydrogenase type 2 (see Figure 54-7). In this reaction, the 11-hydroxyl group is converted to an 11-oxo group by removal of two hydrogens. Cortisone lacks glucocorticoid activity. [Note: When cortisone is used as a drug, it can be activated back to cortisol in the liver via 11-beta hydroxysteroid dehydrogenase type 1, an nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxo-reductase.] Reduction of the double bonds at carbons 4-5 via delta(4)-5-beta reductase or delta(4)-5-alpha reductase yields dihydrocortisol and dihydrocortisone (DHE). Metabolism with reduction of the ketone groups at carbon 3 results in tetrahydrocortisol (THF) and tetrahydrocortisone (THE), which account for the major portion of cortisol clearance (≈50%). The only difference in the outcome of delta(4)-5-beta reductase versus delta(4)-5-alpha reductase activity is the alpha or beta orientation of the hydrogen (5 beta-THF or 5-alpha-THF). Normally, the beta metabolite predominates (5-beta THF:5-alpha-THF ratio = 2 : 1).

Further metabolism of THF and THE via 20-alpha-hydroxysteroid dehydrogenase or 20-beta-hydroxysteroid dehydrogenase produces alpha and beta cortol and cortolone (the cortoic acids), which account for ≈30% of cortisol excretion. Opening the carbon 17-20 bond creates a ketone and gives rise to 11 beta-hydroxyetiocholanolone and 11-ketoetiocholanolone, representing ≈10% of cortisol excretion. Only about 1% of cortisol is normally excreted as free cortisol or cortisone. Minor cortisol metabolites include (1) 20 alpha-hydroxycortisol and 20 beta-hydroxycortisol, which result from reduction of the carbon 20 ketone, and (2) 6 beta-hydroxycortisol, which results from hydroxylation of carbon 6. Oxidation of carbon 17 in THF and THE yields oxo or hydroxy metabolites (Figure 54-8). Minor cortisone metabolites are shown in Figure 54-8.

Of the many cortisol metabolites, more than 95% are conjugated at carbon 3 to glucuronic acid, or sulfated at the C-21 position. Proportionately, the glucuronide metabolites predominate over sulfated metabolites.

Adrenal Androgens¹⁰⁰

Regarding the metabolism of the adrenal androgens, DHEA is sulfated to DHEA-S (Figure 54-9). These compounds are 7 alpha-hydroxylated or 16-alpha-hydroxylated. Alternatively, 17-ketosteroid reductase reduces the ketone at carbon 17 to a hydroxyl group, yielding androstenediol from DHEA and androstenediol sulfate from DHEA-S. Androstenedione is converted to androsterone via 3-alpha- and 5-alpha-reduction, whereas 3-alpha- and 5-beta-reduction yields etiocholanolone (see Figure 54-9). Similar to metabolites of cortisol, metabolites of the adrenal androgens are glucuronidated or sulfated for urinary excretion.

Urinary Metabolites

Whereas measurement of biliary excretion of adrenal steroids is not clinically useful, measurement of the urinary excretion of these compounds is common in the laboratory assessment of adrenal disease. Immunoassays for the major circulating steroid hormones are widely available.

Biochemically17-hydroxyprogesterone is reduced to pregnanetriol, which is measured in urine. The reduction converts keto groups at the 3-position and the 20-position to hydroxyl groups, giving the molecule three hydroxyl groups (hence the term triol). Hydroxylation helps solubilize the compound for renal excretion. Before the development of immunoassays for 17-hydroxyprogesterone, a 24 hour urine was collected for measurement of pregnanetriol excretion in cases of congenital adrenal hyperplasia (CAH) due to CYP21 or CYP11B1 deficiency. If these forms of CAH were managed appropriately, ideally the pregnanetriol excretion would return to within it’s reference interval.

The urinary metabolites of 11-desoxycortisol and cortisol are classified as 17-hydroxycorticosteroids (17-OHCS). Analytically, 17-OHCS are photometrically determined by the reaction of 17,21-dihydroxy-20-oxosteroids with a phenylhydrazine-ethanol-sulfuric acid reagent, producing yellow phenylhydrazones that are termed Porter-Silber chromogens. Measurements of 17-OHCS have been used to differentiate CYP21 deficiency from CYP11B1 deficiency. In CYP21 deficiency, 17-OHCS would not be elevated because both 11-desoxycortisol and cortisol are low. However, because CYP11B1 deficiency increases 11-desoxycortisol (the metabolic block is between 11-desoxycortisol and cortisol), 17-OHCS would be elevated in untreated or undertreated CYP11B1 deficiency. Also, CYP11B1 deficiency and CYP21 deficiency are differentiated by the greatly elevated plasma 11-desoxycortisol concentrations in the former.

The urinary metabolites of 17-hydroxyprogesterone, 11-desoxycortisol, and cortisol are 17-ketogenic steroids (17-KGS) because oxidation of these compounds yields a keto group at the 17 position. Ketogenic steroids have been measured using the Zimmermann reaction, in which an alkaline solution of meta-dinitrobenzene reacts with methylene groups at carbon-16 of the 17-ketosteroids. In CYP17 deficiency, 17-KGS would not be elevated. However, in both CYP21 and CYP11B1 deficiencies, when untreated or undertreated, 17-KGS are elevated.

DHEA and androstenedione metabolites are measured in the same way as 17-ketosteroids because both have keto groups in the C-17 position. Because testosterone has a hydroxyl group at the C-17 position, it is not a 17-ketosteroid. DHEA and androstenedione are elevated in untreated and undertreated CYP21 and CYP11B1 deficiencies. Figure 54-10 summarizes these urine steroid measurements.

Factors Affecting Adrenal Steroid Metabolism

Cortisol clearance (or metabolism) affects cortisol concentrations. If the clearance of cortisol is reduced, plasma cortisol concentrations can increase, whereas enhanced clearance of cortisol decreases its concentration. Rifampin-induced Addisonian crisis from increased cortisol metabolism has been reported. However, in most cases, cortisol, free cortisol, and ACTH are normal in these conditions, presumably because alterations in the free cortisol concentration will be sensed by the hypothalamus, which will respond by secreting CRH to ultimately return the free cortisol concentration to within it’s reference interval. Table 54-7 lists a number of conditions that affect cortisol concentrations.

ACTH Stimulation (Cosyntropin) Test

ACTH stimulation tests, sometimes referred to as the cosyntropin tests, are designed to document the functional capacity of the adrenal glands to synthesize cortisol (Boxes 54-1 and 54-2). Cosyntropin (Cortrosyn) is a synthetic polypeptide that is the N-terminal 24 amino acid sequence of ACTH, which contains the biologically active domain. Another name for cosyntropin is tetracosactrin (Synacthen). Cosyntropin, a potent stimulator of cortisol secretion, has a very brief half-life and minimal antigenicity. The protocols for 1 hour and multiple-day ACTH stimulation tests are shown in Box 54-1.

BOX 54-1   Protocol for the 1 Hour (Rapid) Cosyntropin Stimulation Test

Rationale

Administration of ACTH to normal subjects results in a rapid rise in the serum cortisol concentration. Patients with adrenal destruction (e.g., Addison disease) show no change or an inadequate change in serum cortisol concentration after ACTH injection. Patients with atrophy of the adrenal cortex caused by exogenous glucocorticoid treatment or dysfunction of the pituitary gland or hypothalamus may show a slight rise in serum cortisol concentration, but not one of normal magnitude.

Procedure

A baseline blood sample is drawn for determination of serum cortisol concentration; then 250 µg of cosyntropin is given intramuscularly or intravenously. Additional samples for serum cortisol determination are drawn 30 and 60 minutes after injection.

Interpretation

A peak serum cortisol concentration of 18 to 20 µg/dL or greater is a normal response. The expected change in cortisol concentration is 7 to 10 µg/dL (the delta cortisol). The peak cortisol value is more important than the incremental change. The incremental change may not be seen in patients who are tested at times of stress, when their adrenal output of cortisol is already maximally stimulated by endogenous ACTH.

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