Liquid chromatography-tandem mass spectrometry (LC-MS/MS) based assay for the simultaneous quantification of 18-hydroxycorticosterone, 18-hydroxycortisol and 18-oxocortisol in human plasma.

18-hydroxycorticosterone (18-OHB), 18-hydroxycortisol (18-OHF) and 18-oxocortisol (18-OXOF) are important biomarkers for the diagnosis of subtypes of primary aldosteronism. The detection of these three analytes by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is free from structurally similar compounds. The aim of this study was to develop and validate a new LC-MS/MS assay for the simultaneous quantification of 18-OHB, 18-OHF and 18-OXOF in plasma and to establish a reference intervals for apparently healthy population. Plasma samples were prepared by solid phase extraction and separated in an ultra-high performance reversed phase column. MS detection was achieved using a triple quadrupole mass spectrometer in both positive and negative ionization modes. The developed assay was then validated against standard guidelines. We collected 691 plasma samples from apparently healthy individuals (M:398, F:293) to establish the reference intervals. The analytes were separated and quantified within 5 min. The newly developed method demonstrated linearity for the detected steroid concentration in range of 5 to 3000 pg/ml for 18-OXOF (r2 = 0.999) and 20 to 3000 pg/ml for 18-OHB (r2 = 0.997) and 18-OHF (r2 = 0.997). The lower limit of quantification (LLOQ) was 2.5 pg/ml, 20 pg/ml and 20 pg/m for 18-OXOF, 18-OHB and 18-OHF respectively. Specificity, precision, accuracy and stability were tested, and met the requirements of the guidelines. 18-OHB was higher in females than in males, but 18-OHF were higher in males than females. The reference intervals of 18-OHB, 18-OHF and 18-OXOF for both genders together were 90.5-1040.6 pg/ml, 224.4-1685.2 pg/ml, 4.0-70.5 pg/ml, respectively. Age was also an important factor influencing the levels of these three hormones. We have developed a sensitive and reliable method for the simultaneous quantification of 18-OHB, 18-OHF, and 18-OXOF. Our work provides a reference interval for the clinical application of these three steroid hormones.

Enabling insights into the maturation of the renin-angiotensin-aldosterone system in children-Development of a low-volume LC-MS assay for the simultaneous determination of aldosterone, its precursor, and main metabolite.

INTRODUCTION: As part of the renin-angiotensin-aldosterone system (RAAS), aldosterone is key to the pathology of cardiovascular and renal diseases, leading to end-organ damage and cardiovascular death. Because of different aetiology and metabolism, pharmacotherapy in adults shows only limited transferability to children. Comprehensive investigations of humoral parameters, their precursors, and metabolites are necessary to establish a more rational and safe therapy in children. The LENA (Labeling of Enalapril from Neonates up to Adolescents) project aims to generate these missing data in neonates up to adolescents and provide insight into the maturing RAAS. METHODS: A HRMS (high-resolution mass spectrometry) assay was developed, utilizing blank serum depleted of the endogenous aldosterone, its precursor, 18-hydroxycorticosterone, and its main metabolite, tetrahydroaldosterone. A TOF-MS (time-of-flight-mass spectrometry) scan run in parallel with the simultaneous determination of all three analytes enriches the acquired data. Validation of aldosterone was conducted according to EMA and FDA bioanalytical guidelines. RESULTS: Using the Sciex TripleTOF 6600, a reliable determination in 50 µL serum was successfully shown. Appropriate calibration ranges from 19.53 pg/mL for aldosterone, 39.06 pg/mL for 18-hydroxycorticosterone, and 78.13 pg/mL for tetrahydroaldosterone to 2500 pg/mL were established to ensure the applicability in diseased paediatric patients. Between-run accuracy and precision for aldosterone ranged between -1.21 and -6.99 % and 2.07 and -10.22 %, respectively, confirming compliance with international guidelines. CONCLUSION: A simultaneous bioanalytical LC-HRMS assay for the determination of the biomarker aldosterone, its precursor, and main metabolite, utilizing 50 µL serum, was successfully established. This assay facilitates insight into the maturing RAAS from neonates up to adolescents.

Measurement of 18-hydroxycorticosterone during adrenal vein sampling for primary aldosteronism.

CONTEXT: In primary aldosteronism, elevated serum 18-hydroxycorticosterone (18OHB) suggests aldosterone-producing adenoma (APA) rather than bilateral, idiopathic hyperaldosteronism (IHA), but little is known about the relative production of 18OHB and aldosterone (A) in APAs compared with IHA. OBJECTIVES: We measured 18OHB, A, and cortisol (F) in blood from adrenal vein sampling (AVS) studies. We compared the discriminatory power of gradients in 18OHB/A and 18OHB/F ratios with A/F ratio gradients for distinguishing APA from IHA. DESIGN, SETTING, AND SUBJECTS: We measured 18OHB and A in excess serum from 23 AVS studies performed at our university hospitals. MAIN OUTCOME MEASURES: We calculated the ratios 18OHB/A, 18OHB/F, and A/F for all specimens, and determined the adrenal vein gradients for these ratios. RESULTS: The 18OHB/A ratios were much lower in blood draining APAs (2.17 +/- 0.62) than in blood draining the contralateral adrenals (12.96 +/- 12.76; P < 0.001) but similar to blood draining IHA adrenals (4.69 +/- 4.32; P = 0.02). In contrast, the 18OHB/F ratios were elevated in specimens from APAs (26.03 +/- 11.51) compared with IHA adrenals (9.22 +/- 5.18; P < 0.001) or the contralateral adrenals (6.23 +/- 2.97; P < 0.001). Using 18OHB/F gradient greater than two or 18OHB/A gradient less than 0.5 as criteria for lateralization, interpretations agreed with lateralizations based on A/F gradients in 21 of 23 cases. CONCLUSIONS: High serum 18OHB in APA reflects augmented production of both 18OHB and A, not disproportionate 18OHB secretion relative to A. The 18OHB/A and 18OHB/F gradients are useful adjuncts but not as reliable as A/F gradients for A lateralization during AVS.

Responsiveness of plasma 18-hydroxycorticosterone and aldosterone to angiotensin II or corticotropin in nonazotemic diabetes mellitus.

To assess the function of the final step of the pathway for aldosterone biosynthesis, the responsiveness of plasma 18-hydroxycorticosterone and aldosterone concentrations to angiotensin II infusion was studied in 14 patients with nonazotemic diabetes mellitus as compared with 14 normal controls approximately matched for sex and age. In addition, the responses of both steroids to corticotropin injection were investigated in the diabetic patients. Under basal conditions, plasma aldosterone levels were slightly lower in the patients than in normal controls, while plasma 18-hydroxycorticosterone concentrations were similar in the two study groups. Angiotensin II induced marked and comparable increases in plasma 18-hydroxycorticosterone and aldosterone levels in normal and diabetic subjects. Plasma 18-hydroxycorticosterone and aldosterone levels before and after angiotensin II infusion were significantly interrelated; this correlation was similar in normal subjects (r = 0.61; P less than 0.001) and diabetic patients (r = 0.51; P less than 0.005). Plasma 18-hydroxycorticosterone and aldosterone were significantly increased by corticotropin in the patients. These findings indicate that the terminal step of aldosterone biosynthesis, which involves the production of 18-hydroxycorticosterone and aldosterone, is largely unaltered in patients with nonazotemic diabetes mellitus.

Acquired partial corticosterone methyl oxidase type II defect in diabetes mellitus. Case of hyperreninemic hypoaldosteronism.

The aim of this study was to investigate the pathogenesis of hypoaldosteronism in diabetes. Endogenous elevation of plasma renin activity and exogenous corticotropin were used to study steroidogenesis. Observations were made over 12 yr on the evolution and treatment of hyperkalemia in a diabetic subject. In 1977, potassium, baseline cortisol, aldosterone, and renin activity were normal; renin activity increased normally with posture; and cortisol responded normally to ACTH infusion. Nine yr later, persistent hyperkalemia was documented. Upright renin activity was elevated to 5.26 ng.L-1.s-1, with concomitant elevation of 18-hydroxycorticosterone (18-OHB) and a low-normal aldosterone level. One hour after administration of 0.25 mg i.m. cosyntropin, cortisol increased normally, aldosterone increased from 220 to 360 pM, and 18-OHB increased from 3700 to 4800 pM. During treatment with fludrocortisone, fludrocortisone with furosemide, and furosemide alone, improvement of hyperkalemia was noted. Endogenous hyperreninemia and basal elevations of 18-OHB, accompanied by limited aldosterone responsiveness to renin and ACTH, suggest the presence of a partial corticosterone methyl oxidase type II defect. Evolution of hyperkalemia between 1977 and 1986 suggests this defect was acquired.

Effects of metoclopramide on plasma corticosteroid levels in sheep.

This study investigated the role of dopaminergic mechanisms in modulation of corticosteroid secretion in sheep. Administration of the dopamine antagonist metoclopramide (200 micrograms/kg iv) in six mature sheep resulted in rapid and parallel rises in plasma cortisol, corticosterone, 18-hydroxycorticosterone, and aldosterone. Treatment of the sheep with 4 mg dexamethasone im every 6 h for 4 days abolished the response of all four corticosteroids to metoclopramide in the six sheep. These observations suggest that metoclopramide may stimulate corticosteroid secretion in sheep via nonspecific stressor effects.

Dopaminergic modulation of aldosterone secretion in the rhesus monkey: evidence that dopamine affects the late pathway of aldosterone biosynthesis by inhibiting the conversion of corticosterone to 18-hydroxycorticosterone.

This study was designed to investigate the role of dopaminergic mechanisms in the control of aldosterone secretion. Six rhesus monkeys received metoclopramide (1.25 mg, iv), with 5% dextrose (vehicle) or with dopamine (4 micrograms/kg . min) infusions begun 60 min before the administration of the dopamine antagonists. Metoclopramide, in the presence of vehicle, increased plasma aldosterone concentrations from 4.5 +/- 0.5 ng/dl to a maximum of 26 +/- 4.1 ng/dl and PRL concentrations from 8.1 +/- 1.3 ng/ml to a maximum of 118.4 +/- 7.6 ng/ml. Dopamine infusion inhibited the aldosterone and PRL responses to metoclopramide. The administration of metoclopramide resulted in a rise in plasma 18-hydroxycorticosterone from 10.4 +/- 1.5 ng/dl to a maximum concentration of 41 +/- 4.2 ng/dl. The aldosterone and 18-hydroxycorticosterone responses displayed a parallel time course, with significant responses of both occurring 5 min after metoclopramide administration. Plasma concentrations of electrolytes, PRA, plasma cortisol, 11-deoxycorticosterone, corticosterone, and 18-hydroxy-11-deoxycorticosterone were not altered by metoclopramide. The results of this investigation demonstrate that aldosterone and PRL responses to metoclopramide are mediated by their antagonist activities at dopamine receptors. Rather than simply affecting secretion, dopaminergic mechanisms appear to modulate the late pathway of adrenal glomerulosa biosynthesis of aldosterone. A parallel time course of stimulation of 18-hydroxycorticosterone and aldosterone secretion, with no change in other aldosterone precursors, strongly suggests that dopamine modulates the activity of the glomerulosa 18-hydroxylase enzyme.

Mineralocorticoid response to low dose adrenocorticotropin infusion.

The adrenocorticoid responses to low dose ACTH of plasma aldosterone (aldo), corticosterone (B), 11-deoxycorticosterone (DOC), 18-hydroxycorticosterone (18-OHB), 18-hydroxycorticosterone (18-OH-DOC), and cortisol (F) were compared. Alpha ACTH-)1-24) was infused beginning at 0800 h at increasing rates from 12.5-200 mIU/30 min in supine normal subjects under the following conditions: 1) regular Na (120 meq) diet, 2) low Na (10 meq) diet, 3) dexamethasone preadministration (0.5 mg every 6 h for 48 h), and 4) night study (2000 h; 120 meq Na intake). Plasma 18-OH-DOC and B demonstrated quantitatively the greatest responses to ACTH, while DOC and 18-OHB responses were intermediate. Increments in aldo and F were least after ACTH and were maximum at 50 mIU/30 min ACTH, whereas other corticosteroids demonstrated linear responses up to infusion rates of 200 mIU/30 min. All corticosteroids, however, were similar in their threshold responses to ACTH which were at infusion rates of approximately 7-9 mIU/30 min. Na restriction enhanced aldo and 18-OHB responses to ACTH 2- to 3-fold but did not alter the other corticosteroid responses. Dexamethasone pretreatment augmented aldo, 18-OHB, and F responses but did not change the responsitivity of the other corticosteroids to ACTH. Adrenal corticosteroid responses to ACTH were not significantly different between 0800 and 2000 h in subjects on 120-meq Na intake. Thus, corticosteroids show markedly different responses to physiological doses of ACTH, which may have more importance in their regulation than heretofore proposed. Dexamethasone pretreatment enhances aldo, 18-OHB, and F responses to ACTH but does not affect the responses of other corticosteroids. Contrary to reports in experimental animals, corticosteroid responses to ACTH in man do not differ from day to night.

Dopaminergic control of 18-hydroxycorticosterone responses to posture, isometric exercise, and diuretic administration in normal man.

This study investigates dopaminergic mechanisms involved in the control of corticosteroid secretion in man. The responses of plasma 18-hydroxycorticosterone (18-OHB) and aldosterone levels to upright posture and isometric handgrip exercise and furosemide administration as well as PRA and catecholamine responses to posture and exercise were evaluated in six normal subjects with and without bromocriptine (BEC) treatment. To evaluate the role of PRL suppression by BEC in affecting corticosteroid responses, we evaluated the effect of BEC on plasma 18-OHB and aldosterone responses to 20 mg furosemide in two subjects with autoimmune hypoprolactinemia. BEC (2.5 mg, three times a day for 4 days) markedly suppressed basal levels of 18-OHB, but not aldosterone, in the six normal subjects as well as the two subjects with autoimmune hypoprolactinemia. BEC also suppressed the 18-OHB and aldosterone responses to upright posture, isometric exercise, and furosemide administration without altering electrolytes, PRA, or plasma cortisol levels. Additionally, BEC suppressed basal levels of PRL, norepinephrine, and epinephrine as well as norepinephrine and blood pressure responses to upright posture and isometric exercise in the normal subjects. These results offer additional evidence that dopaminergic mechanisms modulate the secretion of 18-OHB and aldosterone, perhaps indirectly via inhibitory effects of dopaminergic pathways on catecholamine secretion.

Suppression of adrenal mineralocorticoid production in prehypertensive young adult men.

We assessed the renin-aldosterone axis in response to treadmill exercise and adrenal steroidogenesis after graded ACTH infusions in two groups, each composed of nine young adult men from Bourbon County, Kentucky, from the upper and lower deciles of the blood pressure distribution. Those from the upper decile with relatively higher blood pressures, termed prehypertensive, had significantly lower plasma aldosterone and 18-hydroxycorticosterone concentrations after graded ACTH than those with relatively lower pressures, and had significantly lower urinary potassium excretions. Renin and aldosterone responses to exercise were also significantly blunted in the prehypertensive subjects. No differences were found in plasma cortisol, deoxycortisol, deoxycorticosterone, corticosterone, or dehydroepiandrosterone sulfate, although dehydroepiandrosterone was higher at one infusion rate in prehypertensives. These findings suggest that the adrenal mineralocorticoid pathway and the renin-aldosterone axis are suppressed in prehypertensive young males perhaps as an appropriate feedback response to their higher blood pressures.

Dopaminergic modulation of 18-hydroxycorticosterone secretion in man.

This study was designed to investigate mechanisms of dopaminergic control of corticosteroid secretion and to determine on which step in the aldosterone biosynthetic pathway dopamine exerts its effects. Plasma concentrations of electrolytes, PRA, plasma cortisol, 11-deoxycorticosterone, corticosterone (18-OHB), and 18-hydroxy-11-deoxycorticosterone were not altered by the iv administration of 10 mg metoclopramide in six healthy male volunteers. Metoclopramide increased plasma aldosterone from 6.3 +/- 0.9 ng/dl to a maximum of 23.0 +/- 3.4 ng/dl, plasma 18-OHB from 11.4 +/- 1.1 ng/dl to a maximum level of 42.8 +/- 4.4 ng/dl, and PRL from 9.9 +/- 1.4 ng/ml to a maximum of 71.0 +/- 5.5 ng/ml. The aldosterone and 18-OHB responses displayed a parallel time course, with significant responses of both occurring with 5 min after metoclopramide administration. Dopamine infusions (3 micrograms/kg . min) begun 60 min before the administration of metoclopramide markedly decreased the 18-OHB as well as the aldosterone and PRL responses to the dopamine antagonist. A parallel time course of stimulation of 18-OHB and aldosterone secretion with no change in other aldosterone precursors suggests that dopamine may modulate the activity of the glomerulosa 18-hydroxylase enzyme. Thus, rather than simply affecting aldosterone secretion, dopaminergic mechanisms appear to modulate the biosynthesis of aldosterone.

Distribution of 18-hydroxycorticosterone between red blood cells and plasma.

Plasma 18-hydroxycorticosterone (18-OH-B) to aldosterone (aldo) concentration ratios reflect adrenal corticosterone methyloxidase type II activity. This ratio is determined not only by the relative secretion rates of the two steroids but also by differences in binding, distribution, and metabolism. Plasma cortisol alters the distribution of aldo between red blood cells (RBC) and plasma. We postulated that the distribution of 18-OH-B, like that of aldo, is determined by the availability of high affinity binding sites on plasma transcortin. Double equilibrium dialyses demonstrated that 18-OH-B, aldo, and cortisol compete for binding sites on transcortin. Increasing amounts of each of the three unlabeled steroids produced progressive decrements in the binding of all three labeled steroids to transcortin. The affinity of 18-OH-B (2 X 10(6) M-1) for transcortin was intermediate between those of cortisol (3 X 10(7) M-1) and aldo (0.9 X 10(6) M-1). Heat treatment of plasma decreased the binding of 18-OH-B and cortisol to transcortin by 82% and 75%, respectively. Gel filtration of plasma revealed that protein-bound [3H]18-OH-B and [14C]cortisol eluted in the same fractions. The addition of increasing quantities of unlabeled cortisol to whole blood in vitro produced similar increments in RBC to plasma concentration ratios of [3H]18-OH-B and [14C]aldo. The ratio of the percentage of circulating 18-OH-B in plasma to the percentage of circulating aldo in plasma was constant in blood containing low and high cortisol concentrations. Therefore, changes in plasma cortisol have similar effects on the distribution of 18-OH-B and aldo between RBC and plasma.

Dose-response relationships between plasma adrenocorticotropin (ACTH), cortisol, aldosterone, and 18-hydroxycorticosterone after injection of ACTH-(1-39) or human corticotropin-releasing hormone in man.

The effects of sc injections (at 1500 h) of increasing amounts of synthetic human ACTH-(1-39) (1.25-30 micrograms) on plasma ACTH, cortisol, aldosterone, and 18-hydroxycorticosterone were compared with those of iv injections of 30 and 100 micrograms synthetic human CRH in nine normal men. Five micrograms of ACTH, sc, was the lowest dose that significantly increased plasma levels of the three steroids. CRH (30 micrograms, iv) increased plasma cortisol and 18-hydroxycorticosterone, but not aldosterone, while 100 micrograms CRH also raised aldosterone secretion. The dose-response curve (peak plasma ACTH level vs. maximum increment of plasma cortisol within the first hour) was initially very steep. Plasma ACTH levels between 50 and 60 ng/L (11-13 pmol/L) stimulated cortisol to almost 80% of the maximal increment obtained with plasma ACTH levels above 300 ng/L (greater than 66 pmol/L). This dose-response relationship is similar to that found in clinical tests of the pituitary-adrenal axis (insulin test, metyrapone test). The effects of plasma ACTH released by CRH on cortisol secretion were not significantly different from those of injected ACTH. Our results argue against the hypothesis that the effect of CRH on steroid secretion is mediated or modulated by POMC-derived peptides other than ACTH.

Impaired mineralocorticoid hormone responses to adrenocorticotropin stimulation: additional characterization of heterozygosity for the 21-hydroxylase deficiency type of congenital adrenal hyperplasia.

In 12 obligate heterozygotes for the simple virilizing form of congenital adrenal hyperplasia (21-hydroxylase deficiency), basal and ACTH-stimulated levels of aldosterone, corticosterone, deoxycorticosterone, 18-hydroxycorticosterone, and 18-hydroxydeoxycorticosterone were examined. The responses to ACTH were significantly impaired (P less than 0.025 less than 0.001) compared with those of normal subjects. In addition to the often exaggerated stimulation by ACTH of the immediate precursor to 21-hydroxylation, 17 alpha-hydroxyprogesterone, the heterozygotes can now be characterized further by the impaired ACTH responses of mineralocorticoids distal to the block in the zona fasciculata; the ACTH-stimulated 17 alpha-hydroxyprogesterone/18-hydroxydeoxycorticosterone ratio was greater than normal in 94% of the heterozygotes. A limitation of 21-hydroxylation may also exist in the zona glomerulosa.

Adrenal steroid responses to ACTH in glucocorticoid-suppressible aldosteronism.

To investigate adrenal responses to adrenocorticotrophin (ACTH), we infused graded doses of ACTH (1.25 to 20.0 mIU/30 minutes) in normal subjects, patients with low-renin essential hypertension (LREH), primary aldosteronism (PA), and glucocorticoid-suppressible hyperaldosteronism (GSH). Plasma aldosterone, cortisol, corticosterone, and 18-hydroxycorticosterone were measured. The results revealed a greater increase in the plasma aldosterone and 18-hydroxycorticosterone levels evoked by ACTH in the GSH group than in any other group, which suggested enhanced responsiveness of the aldosterone-producing cells to ACTH and a probable adrenal abnormality.

Hereditary defect in biosynthesis of aldosterone: aldosterone synthase deficiency 1964-1997.

We studied two of the three patients with a hereditary defect in the biosynthesis of aldosterone originally described by Visser and Cost in 1964. All three presented as newborns with salt-losing syndrome and failure to thrive. The original biochemical studies showed a defect in the 18-hydroxylation of corticosterone. According to the nomenclature proposed by Ulick, this defect would be termed corticosterone methyl oxidase deficiency type I. We measured plasma steroids in the untreated adult patients and performed molecular genetic studies. Aldosterone and 18-OH-corticosterone were decreased, whereas corticosterone and 11-deoxycorticosterone were elevated, thus confirming the diagnosis of corticosterone methyl oxidase deficiency type I. Cortisol and its precursors were in the normal range. Genetic defects in the gene CYP11B2 encoding aldosterone synthase (P450c11Aldo) have been described in a few cases. We identified a homozygous single base exchange (G to T) in codon 255 (GAG) causing a premature stop codon E255X (TAG). This mutation destroys a Aoc II restriction site. Digestion of a PCR fragment containing exon 4 of CYP11B2 (261 bp) with this restriction enzyme revealed in the two patients homozygous for the E255X mutation only a 261-bp fragment, whereas the heterozygous parents had three fragments (261 bp from the mutant allele and 194 and 67 bp from the wild-type allele). The mutant enzyme had lost the five terminal exons containing the heme binding site, and thus there was a loss of function enzyme. We conclude that the biochemical phenotype of these prismatic cases of congenital hypoaldosteronism can be explained by the patients genotype.

The significance of elevated levels of plasma 18-hydroxycorticosterone in patients with primary aldosteronism.

Plasma 18-hydroxycorticosterone (180HB) concentration was measured in 23 patients with primary aldosteronism. After overnight recumbency, the levels were markedly elevated and were 6 times higher in patients with aldosterone-producing adenomas than in patients with hyperplasia. A value of 100 ng/dl or greater at 0800 h after overnight recumbency distinguished an adenoma from hyperplasia. There was no overlap of values, as was observed with plasma aldosterone concentration (PAC) at 0800 h and after 4 h of upright posture at 1200 h. There was a significant negative correlation between the ratio of 180HB to PAC and the potassium concentration in patients with primary aldosteronism due to hyperplasia. Potassium repletion with potassium chloride and/or spironolactone in patients with aldosterone-producing adenomas decreased the 180HB:PAC ratio by decreasing 180HB and increasing PAC. 180HB is an effective discriminator of an adenoma and may be a useful marker of the events in late aldosterone synthesis.

Plasma beta-endorphin levels in primary aldosteronism.

Excessive production of an as yet unidentified aldosterone-stimulating factor may cause idiopathic hyperaldosteronism (IHA). This putative factor may be related to proopiomelanocortin-derived peptides, some of which have aldosterone-stimulating properties. The present study evaluated plasma beta-endorphin, ACTH, cortisol, and aldosterone levels in patients with IHA (n = 10), aldosterone-producing adenomas (n = 4), essential hypertension (n = 11), and normal subjects (n = 10). Plasma and urinary hormone measurements were obtained at timed intervals during an isocaloric, fixed electrolyte intake (Na+, 128 meq/day; K+, 80 meq/day) in a metabolic unit. Plasma for beta-endorphin assay was preincubated with sepharose-bound anti-beta-lipotropin to remove beta-lipotropin that cross-reacted with the beta-endorphin RIA. Mean +/- SE plasma beta-endorphin levels at 0800 h were elevated in IHA patients (47 +/- 13 fmol/ml) compared to those in aldosterone-producing adenoma (25 +/- 9), essential hypertension (16 +/- 1), and normal control (20 +/- 2; P less than 0.05) subjects. Plasma ACTH, plasma cortisol, and urinary cortisol levels were not different in these four groups. These data support the hypothesis that excess production of either beta-endorphin or related proopiomelanocortin-derived peptides may function as aldosterone secretogogue(s) in IHA.

Effects of continued adrenocorticotropin stimulation on the mineralocorticoid hormones in classical and nonclassical simple virilizing types of 21-hydroxylase deficiency.

Superphysiological doses of ACTH were administered for 3 consecutive days to nine patients with CAH, five with the classical simple virilizing (CSV) type and four with nonclassical simple virilizing type (NCSV; late onset), receiving a sodium-restricted diet. In the CSV patients, cortisol levels were lower [4.9 +/- 2.2 (+/- SEM) micrograms/dl] than in the NCSV patients (10.9 +/- 3.8; P less than 0.005) and normal subjects (10.7 +/- 4.0; P less than 0.05). ACTH produced a subnormal increase to only 14.5 micrograms/dl by day 3. In the NCSV patients, cortisol rose slowly during the first 24 h, but reached normal response levels by 48 h (42.5 +/- 11.5 micrograms/dl). In all patients, basal plasma corticosterone and 18-hydroxydeoxycorticosterone (18-OHDOC) levels were normal, but deoxycorticosterone (DOC) was elevated at 25.3 +/- 5.0 ng/dl (P less than 0.05). ACTH failed to increase plasma levels of DOC, corticosterone, and 18-OHDOC. Aldosterone and 18-hydroxycortisol were elevated in both groups [29.1 +/- 5.8 (P less than 0.02) and 83.8 +/- 15.3 (P less than 0.01) ng/dl, respectively] and increased briskly after the first 24 h of ACTH. However, neither steroid returned to normal levels in the CSV group, but both did in the NCSV group. Paired values of stimulated cortisol and aldosterone in normal subjects and CSV and NCSV patients (n = 76) were significantly negatively correlated (r = -0.63; P less than 0.001), suggesting that cortisol inhibits aldosterone biosynthesis. Prolonged ACTH administration after initial increases returned aldosterone and 18-hydroxycortisol levels from the zona glomerulosa to baseline values in the NCSV type, but not in the CSV type. The capacity to increase cortisol levels, which occurred only in NCSV patients, is linked to the reduction of aldosterone in the zona glomerulosa. In contrast, in both types of 21-hydroxylase deficiency, sustained impairment of both the 17-hydroxy pathway (cortisol) and the 17-deoxy pathway of the zona fasciculata (DOC, corticosterone, and 18-OHDOC) was demonstrated.