Hormonal circadian rhythms in patients with congenital adrenal hyperplasia: identifying optimal monitoring times and novel disease biomarkers.

OBJECTIVES: The treatment goal in congenital adrenal hyperplasia (CAH) is to replace glucocorticoids while avoiding androgen excess and iatrogenic Cushing's syndrome. However, there is no consensus on how to monitor disease control. Our main objectives were to evaluate hormonal circadian rhythms and use these profiles to identify optimal monitoring times and novel disease biomarkers in CAH adults on intermediate- and long-acting glucocorticoids. DESIGN: This was an observational, cross-sectional study at the National Institutes of Health Clinical Center in 16 patients with classic CAH. METHODS: Twenty-four-hour serum sampling for ACTH, 17-hydroxyprogesterone (17OHP), androstenedione (A4), androsterone, DHEA, testosterone, progesterone and 24-h urinary pdiol and 5ß-pdiol was carried out. Bayesian spectral analysis and cosinor analysis were performed to detect circadian rhythmicity. The number of hours to minimal (TminAC) and maximal (TmaxAC) adrenocortical hormone levels after dose administration was calculated. RESULTS: A significant rhythm was confirmed for ACTH (r(2), 0.95; P<0.001), 17OHP (r(2), 0.70; P=0.003), androstenedione (r(2), 0.47; P=0.043), androsterone (r(2), 0.80; P<0.001), testosterone (r(2), 0.47; P=0.042) and progesterone (r(2), 0.64; P=0.006). The mean (s.d.) TminAC and TmaxAC for 17OHP and A4 were: morning prednisone (4.3 (2.3) and 9.7 (3.5) h), evening prednisone (4.5 (2.0) and 10.3 (2.4) h), and daily dexamethasone (9.2 (3.5) and 16.4 (7.2) h). AUC0-24 h progesterone, androsterone and 24-h urine pdiol were significantly related to 17OHP. CONCLUSION: In CAH patients, adrenal androgens exhibit circadian rhythms influenced by glucocorticoid replacement. Measurement of adrenocortical hormones and interpretation of results should take into account the type of glucocorticoid and time of dose administration. Progesterone and backdoor metabolites may provide alternative disease biomarkers.

A new marker for early diagnosis of 21-hydroxylase deficiency: 3beta,16alpha,17alpha-trihydroxy-5alpha-pregnane-7,20-dione.

In neonates with 21-hydroxylase deficiency the specific marker 11-oxo-pregnanetriol is at low levels in the first days of life and this drives the search for alternatives. We describe the structural characterisation of a new early marker, 3beta,16alpha,17alpha-trihydroxy-5alpha-pregnane-7,20-dione. Urine samples from 87 untreated and 11 recently treated newborns with 21-hydroxylase deficiency (42 males and 56 females) between birth and 40 days of age and control samples from 7 healthy neonates (4 males, 3 females) were compared. Steroids were analyzed as methyloxime-trimethylsilyl ether derivatives by GC-MS and GC-MS/MS, after extraction and enzymatic conjugate hydrolysis. Microchemical methods and deuterated derivatives were used. The new steroid was identified by comparison with 3beta,16alpha,17alpha-trihydroxy-preg-5-en-20-one and 3beta-hydroxy-5alpha-pregnane-7,20-dione standards. It was present for the first 4 weeks after birth (with a maximum around day 4) and showed a marked inter-individual variability. No effect of treatment was evident and levels were much higher than for 11-oxo-pregnanetriol in the first days of life. Only traces were found in controls. The likely involvement of oxysterol 7alpha-hydroxylase (CYP7B1) from the 'acidic' pathway of bile acid synthesis and 11beta-hydroxysteroid dehydrogenase-1 in the generation of the 7-oxo group is discussed. We conclude that this steroid is a useful early marker of 21-hydroxylase deficiency.

Urine steroid hormone profile analysis in cytochrome P450 oxidoreductase deficiency: implication for the backdoor pathway to dihydrotestosterone.

CONTEXT: Although the "backdoor" pathway to dihydrotestosterone has been postulated in the fetal-to-early-infantile period of patients with cytochrome P450 oxidoreductase deficiency (PORD), clinical data in support of this pathway remain limited. OBJECTIVE: The objective of this study was to obtain clinical evidence for the presence of the backdoor pathway in PORD. SETTING: This was a collaboration study between laboratories and hospitals. SUBJECTS: Twenty-two Japanese patients with molecularly confirmed PORD and 1763 control subjects participated in this study. INTERVENTION: Urine steroid profile analysis was performed by gas chromatography/mass spectrometry. In five patients and 776 control subjects, urine samples were obtained before 12 months of age. MAIN OUTCOME MEASURE: The main outcome measure was identification of a urine steroid(s) indicating the backdoor pathway. RESULTS: In the PORD patients, pregnanediol, pregnanetriolone, and pregnanetriol were obviously elevated, and the urine steroid ratios reflecting CYP17A1 and CYP21A2 activities were decreased throughout the examined ages. Furthermore, etiocholanolone and 11-hydroxyandrosterone, which should originate almost exclusively from androstenedione in the conventional "frontdoor" pathway, were grossly normal or somewhat decreased since early infancy, whereas androsterone, which can be derived not only from androstenedione and dihydrotestosterone in the conventional frontdoor pathway but also from 5alpha-pregnane-3alpha,17alpha-diol-20-one in the backdoor pathway, was increased during early infancy and remained grossly normal thereafter. Thus, the androsterone to etiocholanolone ratio was increased during early infancy and remained grossly normal thereafter. 5alpha-Pregnane-3alpha,17alpha-diol-20-one was elevated throughout the examined ages. CONCLUSIONS: The increased androsterone excretion during early infancy, as compared with the etiocholanolone and 11-hydroxyandrosterone excretions in the same period, suggests the presence of the backdoor pathway in PORD.

Patterns of urinary and fecal steroid excretion during the ovarian cycle and pregnancy in the African elephant (Loxodonta africana).

The aims of the present study were to (i) determine the relative abundance of the 5alpha-reduced progestins 5alpha-pregnane-3-ol-20-one (5alpha-P-3OH) and 5alpha-dihydroprogesterone (5alpha-DHP) and progesterone (P4) in African elephant feces and to establish improved fecal progestin assays for monitoring ovarian function; and (ii) describe longitudinal profiles of urinary and fecal progestin and estrogen metabolites during pregnancy. Matched urine and fecal samples were collected weekly from six adult females throughout 18 nonfertile cycles and two complete pregnancies (89 and 93 weeks duration). Fecal samples were lyophilized and extracted with 80% methanol in water and immunoreactive 5alpha-P-3OH, 5alpha-DHP, and P4 and (for pregnant females only) estrone (E1) and estradiol (E2) determined by enzyme immunoassay. Urine samples were hydrolyzed, ether-extracted, and assayed for 5alpha-P-3OH, E1, and E2. HPLC cochromatography of fecal extracts with various radioactive progestin tracers confirmed the presence of large amounts of both 5-reduced progestins (5alpha-P-3OH > 5alpha-DHP) but not of P4. 5-Reduced progestins (but not P4) were excreted in a cyclic pattern and levels were significantly correlated with urinary 5alpha-P-3OH. Fecal 5alpha-P-3OH showed the more pronounced and consistent luteal-phase elevation and a better correspondence to urine with respect to timing of the luteal-phase rise. Fecal and urinary 5-reduced progestins increased gradually during early pregnancy to maximum values around week 40-45. Levels gradually declined during the second half of pregnancy, reaching baseline values 2 days before parturition. Urinary estrogens did not show any cyclic pattern during the preconception period and levels remained low during the first 30 weeks of gestation. Thereafter, there was a rapid 10- to 20-fold increase to maximum values at mid-pregnancy, followed by a gradual decline to birth. There was no mid-pregnancy elevation in fecal estrogens, but there was a modest increase in E1 during the second half of gestation.

Metabolism of 5 alpha-dihydroprogesterone in women and men: 3 beta- and 3 alpha-,6 alpha-dihydroxy-5 alpha-pregnan-20-ones are major urinary metabolites.

The metabolism of 5 alpha-dihydroprogesterone (5 alpha-DHP) in women and men was evaluated by defining the pattern and identity of selected metabolites excreted in urine after the iv infusion of radiolabeled 5 alpha-DHP. Virtually all of the radioactivity in urine (approximately 37% of the administered dose) was excreted within 72 h. Quantitatively, the 2 major urinary metabolites of 5 alpha-DHP in each of 13 studies conducted in 7 women and 2 men were 3 beta,6 alpha-dihydroxy-5 alpha-pregnan-20-one and 5 alpha-pregnane-3 alpha,20 alpha-diol, which could be extracted after beta-glucuronidase, but not solvolysis, treatment of the urine. Radiolabeled 3 alpha,6 alpha dihydroxy-5 alpha-pregnan-20-one (glucuronoside), in lesser amounts, also was identified in the urine of each subject. The 3 alpha/beta, 6 alpha-dihydroxy-5 alpha-pregnan-20-ones arise through specific extrahepatic pathways of progesterone/5 alpha-DHP metabolism. These metabolites are not the products of the enzyme reaction catalyzed by the cytochrome P450 steroid 6 alpha-hydroxylase of human liver (and other tissues), which affects the 6 alpha-hydroxylation of C19- and C21-delta 4-3-ketosteroids (e.g., progesterone, testosterone, and cortisol), but does not act upon 5 alpha-reduced steroids. Moreover, the steroid 5 alpha-reductases do not act upon 6 alpha-hydroxy-delta 4-3-ketosteroids. In addition, the 6 alpha-hydroxylation of 5 alpha-reduced-3 alpha/beta-hydroxysteroids is not demonstrable in adult liver tissue. Rather, the formation of 6 alpha-hydroxylated-5 alpha-pregnane-3 alpha/beta-ol-20-ones is indicative of an extrahepatic pathway of progesterone metabolism, viz. progesterone-->5 alpha-DHP-->5 alpha-pregnan-3 beta/alpha-ol-20-one(s)-->3 beta/alpha,6 alpha-dihydroxy-5 alpha-pregnan-20-one(s), in which 5 alpha-pregnan-3 alpha/beta-ol-20-ones are metabolized by an enzyme(s) that catalyzes the 6 alpha-hydroxylation of saturated substrates. There are important differences among mammalian species in the enzymes that catalyze the C-6-hydroxylation of 5 alpha-reduced C19- and C(21)-3 beta/alpha-hydroxysteroids, but in all species studied, these enzymatic reactions are the final steps in the extrahepatic inactivation of 5 alpha-reduced bioactive metabolites of progesterone (or testosterone).

Clinical and biochemical variability of congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency. A study of 25 patients.

Twenty five patients (10 males and 15 females) aged 0-23 yr with congenital adrenal hyperplasis due to 11 beta-hydroxylase deficiency were studied. They were divided into 13 classic (group A), and 12 mild (group B) patients. The patients of group A were diagnosed at a younger age and had more severe clinical symptoms (ambiguous genitalia in girls, pseudoprecocious puberty in boys). Two had neonatal salt wasting before treatment, and one gynecomastia. Seven had moderate to severe hypertension. Their mean 3 alpha,17,21-trihydroxy-5 beta-pregnan-20-one (THS) and 3 alpha, 21-dihydroxy-5 beta-pregnane-11,20-dione (THDOC) excretion was 14.2 +/- 4.1 and 7.2 +/- 4.2 mg/m2 . day, respectively. The patients of group B had mostly late onset of symptoms (hirsutism, amenorrhea in girls, pseudoprecocious puberty in boys, tall stature, and advanced bone age in both sexes). One boy had bilateral cryptorchidism. Four had moderate hypertension. In seven patients, THS (5.3 +/- 2.3 mg/m2 . day) and THDOC (3.9 +/- 0.5 mg/m2 . day) responded to ACTH. In five, only THS (4.3 +/- 1.1 mg/m2 . day) responded, but THDOC remained undetectable. It is concluded that the clinical and biochemical expression of 11 beta-hydroxylase deficiency is variable, that hypertension in not directly related to deoxycorticosterone, and that, regardless of the intensity of the defect, there are patients in whom the 11 beta-hydroxylation of 17 alpha-hydroxylated steroids only is impaired, and others in whom both the conversion of 17,20-dihydroxy-4-pregnene-3,20-dione and deoxycorticosterone are reduced.

[Determination of alphaxalone in serum and urine by gas-liquid chromatography. Application in anaesthesiological pharmacology (author's transl)]. / Dosage de l'alphaxalone dans le sérum et les urines par chromatographie gaz-liquide. Application en pharmacologie anesthésiologique.

The authors propose a simple and reliable method for determination of alphaxalone [3 alpha-hydroxy(5 alpha)pregnane 11,20 dione] in serum and urine, during long-duration anaesthesia using Althesin, mixture of alphaxalone and alphadolone acetate [21 acetoxy, 3 alpha-hydroxy(5 alpha)pregnane 11,20 dione]. If assays of alphadolone appear difficult and without any precision, because of numerous interferences, the determination of alphaxalone in serum and urine seems interesting for pharmacokinetic and metabolic investigation. We confirm the quick rate of disappearance of alphaxalone from blood, associated with a very active metabolism in the liver. The results show that it exists a relation between the concentration of alphaxalone in blood and the value of anaesthesia judged on clinical and electrical criteria. We cannot however conclude that Althesin presents a cumulative effect, because we used important amounts of anaesthetic agent.

Effect of exogenous estrogens on LH and FSH secretion in women with hypothalamic amenorrhea.

The effect of estradiol on FSH and LH secretion in 5 women with hypothalamic amenorrhea are presented. The functional capacity of the pituitary to secrete FSH and LH had been proved by testing with synthetic luteinizing hormone-releasing hormone (LH-RH) 100 mug i.m. The thyroid and adrenal functions were normal. In 3 patients estrogens were low-normal and in the other 2 extreme hypoestrogenism was found. Twelve days after the appearance of withdrawal bleeding each patient was given 10 mg estradiol benzoat (EB) i.m. In 3 patients a significant increase in the LH and a slightly smaller increase in the FSH concentration were noted, with an LH peak 60 to 84 hours after injection of EB. The first of these 3 patients became pregnant, in the second the biphasic BBT curve, urine pregnanediol excretion above 2.5 mg/24 hr and the histological finding of secretory endometrium indicated that ovulation had been successfully induced, while in the third patient, despite a satisfactory increase in the LH and FSH concentration, ovulation could not be induced. In the remaining 2 patients no increase in the LH and FSH concentrations was noted even as late as 96 hours after the injection of EB. In these women bleeding began on the 18th and 22nd day of the cycle, respectively, when histological examination revealed proliferative endometrium. The first 3 cases are classified as hypothalamic amenorrhea with dysfunction of the cyclic centre and the other 2 as hypothalamic amenorrhea with dysnfunction of the tonic centre. The effect of exogenous estrogens in patients with hypothalamic amenorrhea depends both on the functional capacity of the hypophysis and the capacity and condition of the tonic and cyclic centres in the hypothalamus.