G.P.B. Kraan

Kinetics of intravenously dosed cortisol in four men. Consequences for calculation of the plasma cortisol production rate

The kinetics of cortisol in the serum of 4 healthy men were studied following single i.v. doses of 2 and 0.8 mg of cortisol. The disappearance of cortisol was determined by blood sampling frequently over 2.5 h and analysing the apparently biexponential cortisol decay. The main results, shown as the mean (+/-SD), were: (a) the average distribution volume of cortisol at steady state (Vd,ss), which was 7.1 l/m2 body surface area. The extrapolated distribution volume (Vd,ext) was 8.4 l/m2, being 18% higher than the corresponding Vd,ss. (b) It was confirmed that plasma cortisol disappears biexponentially. Since the rapid phase remains unnoticed if cortisol is measured at an interval of 10 or more minutes, the obscured rapid-phase parameters can be found only if the known ratio of the two rate constants is used. (c) The fraction of cortisol, which during this fast phase irreversibly disappeared according to the two-compartment open model, was 5 to 8% larger than that found using the monocompartment model. (d) The half-life of the slow or beta phase was equal for the 2 and 0.8 mg experiments, namely t1/2(beta) = 66 +/- 18 min. The kinetics of cortisol in the same 4 men were also measured after an i.v. dose of radioactive cortisol (82 +/- 7 kBq 3H/m2). All urine was collected in 15 portions during the next 3 days, followed by measuring the cumulative radioactivity and analysing the triexponential increase of urinary radioactivity [1]. The main results with the urinary model were: (a) the half-life of cortisol elimination from the circulation was 40 +/- 11 min, (b) the maximal radioactivity (69 +/- 7% of the dose) in the first pool (liver) was found at 2 +/- 0.3 h, (c) the half-life of the cortisol metabolites in the body was 6.8 +/- 0.7 h. Forcing the measured cortisol concentrations in plasma to fit a monoexponential function, allowed us to compare the half-life of cortisol decay with that from the urinary model. It was found that these half-lives were similar with values between 30 and 40 min. Finally, the distribution volume has to be measured individually if a 24 h plasma cortisol profile is used for the calculation of the cortisol production rate.

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.

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.

URINARY STEROID PROFILE OF A NEWBORN SUFFERING FROM PSEUDOHYPOALDOSTERONISM

A case is described of a newborn, admitted to hospital because of severe salt loss at the age of 1 month. Subsequent analysis of urinary steroid excretion, by gas chromatography and gas chromatography-mass spectrometry, revealed that the patient suffered from pseudohypoaldosteronism. However, it was difficult to interpret the results unambiguously, since the first urinary analysis appeared to suggest 21-hydroxylase- or 18-hydroxylase deficiency. The final diagnosis was possible only after detecting high urinary levels of aldosterone and tetrahydroaldosterone. It is concluded that neonatal urinary steroid profiles should be interpreted cautiously in order to arrive at the correct diagnosis.

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.