Nm Drayer

A persisting secular trend for body measurements in Dutch children. The Oosterwolde II study

To investigate whether the secular trend for growth in Dutch children still exists, the Oosterwolde I study of 1980 was repeated in 1989. A persisting secular trend was visible for height while the z scores of body proportions show no change during the past 10 years, which suggests that there is no change in the timing of puberty.

NEW IDENTIFIED 15-BETA-HYDROXYLATED 21-DEOXY-PREGNANES IN CONGENITAL ADRENAL-HYPERPLASIA DUE TO 21-HYDROXYLASE DEFICIENCY

The identification of 3 new 15 beta-hydroxylated 21-deoxy-pregnanes in the urinary steroid profile of a 4-month-old girl with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is reported here. These steroids were identified by gas chromatography and gas chromatography-mass spectrometry as 3 alpha,15 beta,17-trihydroxy-5 alpha-pregnan-20-one (5 alpha II), 3 alpha,15 beta,17,20 alpha-tetrahydroxy-5 alpha-pregnane, and 3 alpha,15 beta,17,20 alpha-tetrahydroxy-5 beta-pregnane (20 alpha DH-II). Two other compounds in the urine, 3 beta,15 beta,17- trihydroxy-5 alpha-pregnan-20-one and 3 beta,15 beta,17-trihydroxy-5 beta-pregnan-20-one were also characterized. The identification of the former 3 steroids was obtained by comparing their methylene unit values and mass spectral data with the corresponding data of the standard steroids synthesized from 15 beta,17-dihydroxy-4-pregnene-3,20-dione. Seven other synthesized and identified 15 beta-hydroxylated steroids were 3 alpha,15 beta,17-trihydroxy-5 beta-pregnan- 20-one (II), 3 alpha,15 beta,17,20 beta-tetrahydroxy-5 beta-pregnane, 15 beta,17-dihydroxy-5 alpha-pregnane-3,20-dione, 15 beta,17-dihydroxy-5 beta-pregnane-3,20-dione, 3 alpha,15 beta-dihydroxy-5 alpha-androstan-17-one (15 beta OH-An), 3 alpha,15 beta-dihydroxy-5 beta-androstan-17-one (15 beta OH-Et) and 3 alpha,15 beta,17,20 beta- tetrahydroxy-5 alpha-pregnane. Of these the latter two have not been reported previously. This study supports the findings that 15 beta-hydroxylated steroids are common in the neonate and could play an important role in the diagnosis of CAH due to 21OHD, where II and the newly identified steroids from this investigation viz., 5 alpha II and 20 alpha DH-II appear the most important 15 beta-hydroxysteroid markers for this disease.

Measurement of the cortisol production rate in two sisters with 17α-hydroxylase deficiency using [1,2,3,4-13C]cortisol and isotope dilution mass spectrometry

[1,2,3,4-13C]cortisol was i.v. administered to two sisters aged 11 yr (patient I) and 3 yr (patient II) who suffer from 17 alpha-hydroxylase deficiency. This is the first time that the cortisol production rate (CPR) in patients with 17 alpha-hydroxylase deficiency has been measured with a stable labelled tracer using the urinary method. The urine was collected for 3 days. High-performance liquid chromatography (HPLC) of approximately 100 ml urine extracts was carried out to isolate the small amount of cortisol metabolites excreted. The cortisol metabolites were oxidized to 11-oxo-aetiocholanolone. The isotope dilution in the methyl oxime tert-butyldimethylsilyl ether derivatives was measured by selected ion monitoring gas chromatography/mass spectrometry (GC/MS). The CPR calculated from tetrahydrocortisone (THE) and the cortolones was 765 and 536 nmol/day, respectively in patient I. The CPR in patient II was only calculated from THE and was 62 nmol/day. If radioactive labelled cortisol had been used, much larger quantities of urine would have been needed for isolation of sufficient mass of metabolites, even then purification may have been difficult. Steroid profiling of 1 ml urine samples by GC and identification by GC/MS revealed high concentrations of pregnenolone, progesterone, 11 beta-hydroxy progesterone and corticosterone metabolites. Tetrahydrocorticosterone and 5 alpha-tetrahydrocorticosterone were found in urine at elevated excretions of 2.5 and 5.7, 0.9 and 2.0 mumols/24 h, in patients I and II respectively. No cortisol metabolites were detected by routine GC or GC/MS as the low amounts excreted co-eluted with the relatively abundant corticosterone metabolites.

THE EARLY RECOGNITION OF THE 21-HYDROXYLASE DEFICIENCY VARIETY OF CONGENITAL ADRENAL-HYPERPLASIA

The urinary steroids excreted by three newborn infants with 21-hydroxylase deficiency and by 15 healthy newborns aged two days have been compared after analysis by gas liquid chromatography (GLC). The identity of each steroid was carefully checked by gas chromatography/mass spectroscopy (GC-MS). The enzyme deficiency leads to the elevated excretion of urinary precursor metabolites, mainly 3alpha,17alpha,20alpha-trihydroxy-5beta-pregnan, 3alpha,17alpha,20alpha-trihydroxy-5beta-pregnan-11-one and 3alpha,17alpha-dihydroxy-5beta-pregnan-20-one. In the search for a quick and firm confirmation of suspected 21-hydroxylase deficiency in a newborn baby by means of a GLC-profile of urinary steroids, most attention has up to now been paid to 3alpha,17alpha,20alpha-trihydroxy-5beta-pregnan. However, 3alpha,17alpha-dihydroxy-5beta-pregnan-20-one is a better indicator, as it enables one to confirm the existence of this disease soon after birth directly from the GLC-profile without further analyses by GC-MS.

Improved gas chromatographic—mass fragmentographic assay for tetrahydroaldosterone and aldosterone in urine☆

A newly devised procedure for a simultaneous determination of urinary tetrahydroaldosterone and aldosterone is described. The procedure is based on deconjugation and acetalization, followed by extraction and derivatization of the urinary compounds to their trimethylsilyl ethers and subsequent gas chromatographic-mass fragmentographic detection. To evaluate the assay, aliquots of a urine sample of a healthy individual were analysed in multiplicate; a mean tetrahydroaldosterone concentration of 103 nmol/l and a within-sample, within-day- and day-to-day coefficient of variation of 1.8, 3.2 and 3.4%, respectively, were found. Determination of aldosterone in the same sample yielded a mean concentration of 25.3 nmol/l and the following coefficients of variation: 2.8% (within-sample), 3.8% (within-day) and 4.3% (day-to-day). The urinary excretion of tetrahydroaldosterone and aldosterone in 24-h urine portions was determined in twenty healthy individuals, aged 23-77 years; for tetrahydroaldosterone and aldosterone, an excretion of 94 +/- 66 nmol per 24 h and of 40 +/- 22 nmol per 24 h was found, respectively, in accord with the literature. An example of the usefulness of the described assay is given by establishing the cause of severe salt-wasting in an infant; a highly elevated tetrahydroaldosterone and aldosterone excretion was demonstrated, proving that the child suffered from unresponsiveness to aldosterone (pseudohypoaldosteronism).

Kinetics of cortisol metabolism and excretion. A hypothetic model based on the cumulative urinary radioactivity in eight multiple pituitary deficient patients

A new model is proposed to study the kinetics of [3H]cortisol metabolism by using urinary data only. The model consists of 5 pools, in which changes of the fractions of dose are given by a system of 5 ordinary differential equations. After i.v. administration of [3H]cortisol to 8 multiple pituitary deficient (MPD) patients (group I) the urines from each patient were collected in 9-15 portions during the following 3 days. From the urinary data the rate constants of cortisol metabolism were calculated. A published set of urinary data from patients with a normal cortisol metabolism (group II) was used for comparison. The overall half-life of the label in the circulation was 30 min for both groups; the half-life of the label excretion by both groups was 6 h and the time of maximal activity in the main metabolizing pool was 1.8 h in group I and 1.5 h in group II. The 20% of normal cortisol production rate (CPR) in the 8 MPD patients amounted to 7.2 +/- 1.9 mumol/(m2*d). Therefore, the low CPR but normal rate constants, i.e. a normal metabolic clearance rate of cortisol, in the MPD patients suggest a sensitive adjustment of the cortisol response in the target organs.

Synthesis and identification of twelve A-ring reduced 6α- and 6β-hydroxylated compounds derived from 11-deoxycortisol, corticosterone and 11-dehydrocorticosterone

Abstract The synthesis and identification of 12 A-ring reduced 6α-(and 6β-)hydroxylated compounds derived from 11-deoxycortisol (S), corticosterone (B) and 11-dehydrocorticosterone (A) are reported here. These steroids were prepared in two steps from the corresponding 6 6α-(and 6β-)hydroxy-4-pregnene-3-ones. Selective reduction of the 4,5 double bond yielded 12 6α-(and 6β)hydroxy-5α-(and 5β)pregnane-3,20-diones. Enzymatic reduction of these compounds with NADH and 3α-hydroxysteroid dehydrogenase yielded the corresponding tetrahydro steroids. The steroids were characterized by high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC and GC/MS) and in part by 1 H-NMR. 6βOH-THS and 6βOH-5αTHS were identified by 1 H-NMR. The structures of the two precursors, i.e. 6βOH-5βDHS and 6βOH-5αDHS were confirmed by 1 H-NMR using two-dimensional spectra. 6αOH-THS was identified by comparing its HPLC, GC and MS data with those of the steroid obtained by enzymatic oxidation of the standard reference steroid 6αOH-20βHHS to the corresponding 20-ketosteroid. The other steroids, e.g. 6αOH-THB and 6αOH-5αTHB were identified by using the proved sequence of elution of each of the epimer pairs on the normal phase HPLC column (5 α β ), and by the reversed order of elution of the same epimer pair as the methoxime-trimethylsilyl ethers on the GC column (5α > 5β) and by the mass spectra, with the exception of 6βOH-THA.

Kinetics and metabolism of 11-deoxycortisol in a patient with congenital adrenal hyperplasia due to 11β-hydroxylase deficiency

The kinetic features of 11-deoxycortisol (S) were studied in a 11 beta-hydroxylase deficient boy. After i.v. administration of 35 kBq [3H]S (11 pmol) together with 44 nmol [13C]cortisol all his urine was collected during the next 3 days. A recently reported kinetic model, by which the fate of radioactive cortisol (F) in the body can be described by analysis of only the urinary radioactivity, has been used to calculate the rate constants of S metabolism. The overall half-life of S in the circulation was 4.7 min, which is very close to a reported half-live of the rapid phase: 4.1 min determined from the plasma radioactivity. The time of maximal accumulation of S in the first metabolic pool--26 min is about one quarter of that found for F--109 +/- 20 min (n = 8). The half-live of the S metabolites in the body was 7.0 h, equal to that of F: 6.1 +/- 0.9 h (n = 8). Obviously S is taken up into the metabolic organs 4 times faster than F, but it is not metabolized faster. The production rates of S and F were 127 and 2.1 mumol/(m2*d), respectively, pointing to a severely deficient synthesis of F. However, from the urinary excretion of 3 alpha,21-dihydroxy-5 beta-pregnan-20-one in relation to 3 alpha,11 beta,21-trihydroxy-5 beta-pregnan-20-one it cannot be concluded that the synthesis of corticosterone was strongly impaired.

USE OF 13C-CORTISOL TO DETERMINE THE CORTISOL PRODUCTION-RATE (CPR) IN CONGENITAL ADRENAL-HYPERPLASIA DUE TO 17-ALPHA-HYDROXYLASE DEFICIENCY

1, 2, 3, 4-13C cortisol(13C-F) was used to measure the CPR in two sisters(karyotype 46, XX)of 11(I) and 3(II) yrs of age with 17α-hydroxylase deficiency. The disease was diagnosed on hyper tension, low plasma cone, of 17β-estradiol, cortisol, PRA and aldosterone and high conc, of progesterone and in patient I of ACTH, FSH and LH. Both children excreted in the urine high amounts of tetra-and hexahydro-metabolites of corticosterone and deoxycorticosterone and (very) low amounts of metabolites of cortisol (F), androstenedione and testosterone.After the i.v. dosage of 60 and 2.4 μg to patient I and II, urine was collected for 3 days. After extraction, hydrolysis and HPLC Chromatography the metabolites of F were oxidised to 11-oxoetiocholanolone (11-OET) and -androsterone. The 13C-enrichments in the methoxime-tert.butyldimethylsily ethers were measured by gas Chromatography mass fragmentography at the leading ions m/z 348 (13C4) and 344 (12C). Calibration standards were prepared from mixtures of 13C-F and F, which were oxidised to cortisone (E), reduced to tetrahydrocortisone (THE) by Cl.paraputrificum and oxidised to 11-OET. The CPR (mg/day) was for I:0.28±0.003 (THE) and 0.19±0.007 (α+βcortolone) and for II:0.022±0.003 (THE). Owing to the very low amounts of cortisol metabolites the CPR determinations would not have been possible if tritiated cortisol was used. Financial support was obtained from the Netherlands Organisation for the Advancement of Pure Research (ZWO).

HYPERCALCIURIA OF RENAL ORIGIN IN DIABETIC CHILDREN - INCIDENCE AND RESPONSE TO INDOMETHACIN

From 47 diabetic children 19 had a urinary calcium excretion 2 S.D. above the mean of 58 healthy children. In 13 of these 19 children the hypercalciuria persisted on follow-up and was independent of the glucose excretion. The hypercalciuric group compared to the normocalciuric diabetic group had a significantly higher pH and calcium/creatinine ratio in fasting urine samples, whereas the blood pH, bicarbonate, glucose and the serum calcium, ionised calcium, phosphate, alkaline phosphatase, iPTH, hCT, hGH levels were not different. In the hypercalciuric group an oral calcium load increased the TmP/GFR but not the urinary calcium/creatinine ratio and decreased the urinary cAMP excretion. Indomethacin lowered the calcium/creatinine ratio in fasting urine samples of the hypercalciuric, but not of the normocalciuric group without changing the sodium excretion.It is suggested that prostaglandins contribute to a defective renal tubular function, causing hypercalciuria.