The contribution of newly synthesized cholesterol to bile salt synthesis in rats quantified by mass isotopomer distribution analysis.

A new stable isotope procedure has been developed and validated in rats, applying [1-(13)C]acetate infusion to quantify the production of bile salts from de novo synthesized cholesterol making use of the mass isotopomer distribution analysis (MIDA) principle. Ions (m/z) 458-461, 370-373 and 285-288 were monitored by GC/MS (EI-mode) for the methyl trimethylsilylether derivatives of cholate, chenodeoxycholate and beta-muricholate, respectively. Rats with intact exteriorized enterohepatic circulation and rats with chronic bile diversion were infused with [1-(13)C]acetate for up to 14 h. After 10 h of infusion the enterohepatic circulation of the intact group was interrupted to deplete the existing bile salt pool (acute bile diversion). The fractions of biliary cholesterol and individual bile salts derived from newly synthesized cholesterol were determined by MIDA at t=14 h. In rats with acute bile diversion, these fractions were 20, 25, 27 and 23% for biliary cholesterol, cholate, chenodeoxycholate and beta-muricholate, respectively. After bile diversion for 8 days to induce hepatic cholesterol and bile salt synthesis, these fractions increased significantly to 32, 47, 41 and 47%, respectively. Calculated enrichments of the acetyl-CoA precursor pools were similar for all bile salts and biliary cholesterol within the two rat groups. However, chronic enterohepatic interruption decreased the acetyl-CoA pool size almost two-fold. We conclude that MIDA is a validated new stable isotope technique for studying the synthetic pathway from acetyl-CoA to bile salts. This technique provides an important new tool for studying bile salt metabolism in humans using stable isotopes.

Phenylketonuria. The in vivo hydroxylation rate of phenylalanine into tyrosine is decreased.

In phenylketonuria (PKU), the enzyme phenylalanine hydroxylase is deficient, resulting in a decreased conversion of phenylalanine (Phe) into tyrosine (Tyr). The severity of the disease is expressed as the tolerance for Phe at 5 yr of age. In PKU patients it is assumed that the decreased conversion of Phe into Tyr is directly correlated with the tolerance for Phe. We investigated this correlation by an in vivo stable isotope study. The in vivo residual hydroxylation was quantitated using a primed continuous infusion of L-[ring- 2H5]Phe and L-[1-13C]Tyr and the determination of the isotopic enrichments of L-[ring-2H5]Phe, L-[ring-2H4]Tyr, and L-[1-13C]Tyr in plasma. Previous reports by Thompson and coworkers (Thompson, G.N., and D. Halliday. 1990. J. Clin. Invest. 86:317-322; Thompson, G.N., J.H. Walter, J.V. Leonard, and D. Halliday. 1990. Metabolism. 39:799-807; Treacy, E., J.J. Pitt, K. Seller, G.N. Thompson, S. Ramus, and R.G.H. Cotton. 1996. J. Inherited Metab. Dis. 19:595- 602), applying the same technique, showed normal in vivo hydroxylation rates of Phe in almost all PKU patients. Therefore, our study was divided up in two parts. First, the method was re-evaluated. Second, the correlation between the in vivo hydroxylation of Phe and the tolerance for Phe was tested in seven classical PKU patients. Very low (0.13- 0.95 micromol/kg per hour) and normal (4.11 and 6.33 micromol/kg per hour) conversion rates were found in patients and controls, respectively. Performing the infusion study twice in the same patient and wash-out studies of the labels at the end of the experiment in a patient and control showed that the method is applicable in PKU patients and gives consistent data. No significant correlation was observed between the in vivo hydroxylation rates and the tolerances. The results of this study, therefore, showed that within the group of patients with classical PKU, the tolerance does not depend on the in vivo hydroxylation.

Contribution of newly synthesized cholesterol to rat plasma and bile determined by mass isotopomer distribution analysis: bile-salt flux promotes secretion of newly synthesized cholesterol into bile.

To quantify the contribution of newly synthesized cholesterol to total plasma and biliary cholesterol under physiological conditions, unrestrained rats were infused intravenously with [1-13C]acetate (0. 6mmol/h per kg) from 12:00 to 24:00 h, and fractional and absolute cholesterol-synthesis rates were determined by mass isotopomer distribution analysis (MIDA). As bile diversion leads to changes in cholesterol metabolism, rats were equipped with permanent catheters in the bile duct and duodenum, allowing sampling of small amounts of bile from an intact enterohepatic circulation. For comparison, rats with chronic bile diversion were also studied. Fractional synthesis of plasma cholesterol was 10.8+/-1.7% (mean+/-S.D.) after 12 h in rats with intact circulation. Fractional synthesis of biliary cholesterol was significantly higher than that of plasma cholesterol, i.e. 16.5+/-2.0% (P<0.05) after 12 h. In contrast, no differences between fractional synthesis of cholesterol in plasma and bile were found in bile-diverted animals (31.8+/-2.1 and 33.1+/-3.3% respectively after 12 h). The calculated absolute rate of cholesterol biosynthesis increased from 53+/-10 to 221+/-19 micromol/day per kg after bile diversion. A comparison of MIDA results with those obtained from balance studies indicated that MIDA does not assess total body synthesis in rats, presumably because of incomplete equilibration of newly synthesized molecules with cholesterol in the plasma compartment. These studies demonstrate that the contribution of newly synthesized cholesterol to biliary cholesterol is higher than to plasma cholesterol under physiological conditions, probably reflecting bile-salt-induced secretion of newly formed cholesterol by the periportal hepatocytes.

Oral administration of deuterium-labelled polyamines to sucking rat pups: luminal uptake, metabolic fate and effects on gastrointestinal maturation.

Non-physiological amounts of oral polyamines have been reported to induce precocious gut maturation in rat pups. The aim of the present study was to investigate organ distribution and metabolic fate of orally administered stable-isotopically labelled polyamines in rat pups. Pups received tetradeuterium-labelled putrescine (Pu-d4; 3 mumol), spermidine (Sd-d4; 5 mumol), spermine (Sp-d4; 3 mumol), or physiological saline twice daily on postnatal days 7-10 or 12-15. They were killed on days 10 and 15. We determined activities of ileal lactase (EC 3.2.1.23), maltase (EC 3.2.1.20), sucrase (EC 3.2.1.48) and diamine oxidase (EC 1.4.3.6) and established villus and crypt lengths. Polyamines and their labelling percentages in organs were determined by GC and mass fragmentography. Treatments did not affect growth rate, but caused lower weights of liver, kidneys and heart. Maltase activity increased, lactase decreased, whereas sucrase and diamine oxidase did not change. Villus and crypt lengths increased. Organ polyamine pools were labelled to different extents. Irrespective of the orally administered polyamine, all organs contained Pu-d4, SD-d4 and Sp-d4. Administered Pu-d4 and Sd-d4 were recovered mainly as Sd-d4, whereas Sp-d4 was recovered as Sp-d4 and Sd-d4. Total polyamines in a caecum, colon and erythrocytes increased, but increases were only to a minor extent with regard to labelled polyamines. Our data confirm precocious gut maturation by exogenous polyamines. Putrescine appears to be limiting factor. The exogenous polyamines were distributed among all investigated organs. They are not only used for the synthesis of higher polyamines, but also retroconverted to their precursors. Changes in erythrocyte polyamine contents suggest precocious stimulation of erythropoiesis.

Optimization of a classical aromatase activity assay and application in normal, adenomatous and malignant breast parenchyma.

The tritium water release assay, originally described for the analysis of aromatase activity in placental tissue, was used to estimate aromatase activity in breast tissue samples. The lower activity in this tissue necessitates longer incubation times and thus optimization of the assay conditions. To prevent oxidative and proteolytic inactivation of aromatase, dithiothreitol and albumin were added to the incubation mixture. Extra NADPH, cofactor in the aromatase reaction, also improved reaction rate in placental incubations, but after approximately 120 min activity rapidly decreased. Inhibitors gradually produced during the incubation could explain this phenomenon. Quantitative gas chromatography-mass spectrometry (GC-MS) analyses of testosterone, oestradiol, oestrone and androstenedione after incubation with non-labelled androstenedione proved that a substantial amount of the substrate is converted into testosterone. Qualitative GC-MS steroid profiling of the incubation mixture demonstrated the presence of hydroxylated oestradiol and hydroxylated testosterone, produced during incubation, which could have caused partial aromatase inhibition. The adjusted assay was used to analyse 84 breast tissue samples, histologically classified as normal, adenoma or carcinoma. Aromatase activity was found in 56% of all samples and ranged from 0.6 to 26 pmol oestrogen/g protein per hour. Aromatase positivity was found in 80% of the normal samples, 56% of the adenoma samples and 48% of the carcinoma samples. Although carcinoma samples were less often aromatase positive than normal tissue samples (chi2 = 4.80; P < 0.050) there was no difference in absolute aromatase activity. Because no less than approximately 50% of the carcinomas contained aromatase activity and because of the non-routine character of the assay we conclude that it is justified to start aromatase inhibition therapy without previous knowledge of the aromatase status.

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.

Measurement of short-chain acyl-CoA dehydrogenase (SCAD) in cultured skin fibroblasts with hexanoyl-CoA as a competitive inhibitor to eliminate the contribution of medium-chain acyl-CoA dehydrogenase.

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency has so far been reported in only very few patients. This is due, in part, to the problems involved in measuring the activity of SCAD unequivocally. The main reason for this difficulty is that butyryl-CoA, the substrate preferably used for SCAD activity measurements, is also dehydrogenated by medium-chain acyl-CoA dehydrogenase (MCAD). Elimination of this contribution can be achieved by means of immune precipitation with a specific MCAD antibody. We now describe a relatively straightforward assay based on the use of gas chromatography/mass spectrometry for detection. The contribution of MCAD to overall butyryl-CoA dehydrogenation was eliminated by adding excess hexanoyl-CoA to the assay medium. The validity of the method developed was checked by SCAD-activity measurements in fibroblasts from an established SCAD-deficient patient.

Synthesis and identification of twelve A-ring reduced 6 alpha- and 6 beta-hydroxylated compounds derived from 11-deoxycortisol, corticosterone and 11-dehydrocorticosterone.

The synthesis and identification of 12 A-ring reduced 6 alpha-(and 6 beta-)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 6 alpha-(and 6 beta-)hydroxy-4-pregnene-3-ones. Selective reduction of the 4,5 double bond yielded 12 6 alpha-(and 6 beta)hydroxy-5 alpha-(and 5 beta)pregnane-3,20-diones. Enzymatic reduction of these compounds with NADH and 3 alpha-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 1H-NMR. 6 beta OH-THS and 6 beta OH-5 alpha THS were identified by 1H-NMR. The structures of the two precursors, i.e. 6 beta OH-5 beta DHS and 6 beta OH-5 alpha DHS were confirmed by 1H-NMR using two-dimensional spectra. 6 alpha OH-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 6 alpha OH-20 beta HHS to the corresponding 20-ketosteroid. The other steroids, e.g. 6 alpha OH-THB and 6 alpha OH-5 alpha THB were identified by using the proved sequence of elution of each of the epimer pairs on the normal phase HPLC column (5 alpha < 5 beta), and by the reversed order of elution of the same epimer pair as the methoxime-trimethylsilyl ethers on the GC column (5 alpha > 5 beta) and by the mass spectra, with the exception of 6 beta OH-THA.

GC-MS determination of ratios of stable-isotope labelled to natural urea using [13C15N2]urea for studying urea kinetics in serum and as a means to validate routine methods for the quantitative assay of urea in dialysate.

A GC-MS determination of urea in serum or spent dialysate is described, using 13C15N2-labelled urea and assaying the area ratio of labelled to natural urea by mass fragmentographic monitoring of fragments m/e 153 and 156, after its eventual conversion into the trimethylsilylether-derivative of 2-hydroxypyrimidine. The procedure can be successfully applied in the follow-up of the disappearance of labelled urea in serum after intravenous injection in man, enabling kinetic parameters of urea to be established, e.g. for purposes of studying the effectiveness of dialysis procedures. Furthermore the method can be used for validation of routine methods for measuring urea in other fluids, in particular dialysate. Examples are given of both applications of the GC-MS method described.

The determination of delta-5-androstenediol and its sulphate in serum and urine by gas chromatography-mass spectrometry.

OBJECTIVES: We developed an assay for delta-5-androstenediol (adiol) and delta-5-androstenediol-3-sulphate (adiol-3S) in serum and adiol and total adiol sulphate (adiol-S) in urine. DESIGN: An analytical procedure using HPLC and gas chromatography-mass spectrometry was devised and tested for its reliability. MEASUREMENTS: After addition of deuterated androstenediol as internal standard, serum and urine samples were extracted. Steroid sulphates were hydrolysed. The extracts and hydrolysates were purified on HPLC, adiol was derivatized using heptafluorobutyric anhydride and finally quantified by gas chromatography-mass spectrometry. RESULTS: The assay is accurate and reproducible. The coefficient of variation (CV) for the determination of adiol in serum samples is 4% (intra-assay) and 9% (interassay) and for urine samples 3 and 8% respectively. The intra-assay CV for the adiol-3S analyses is 5% for serum and 2% for urine samples while the interassay CV values for adiol-3S are 10% for serum and 7% for urine samples. The recovery of adiol and adiol-3S from serum and urine samples is 97%. CONCLUSIONS: The developed assay meets the analytical demands needed for clinical applications.

Delta-5-androstenediol and its sulphate in serum and urine of normal adults and patients with endocrine diseases.

OBJECTIVES: We evaluated the role of delta-5-androstenediol (adiol) and its sulphates in health and endocrine diseases. DESIGN: Serum and urine samples from healthy adult men and pre and post-menopausal women were analysed by gas chromatography-mass spectrometry to establish reference values. In patients who were either evaluated or treated for endocrine diseases, sequential serum samples were collected and analysed. PATIENTS: Reference values were obtained from 24 healthy male, 23 premenopausal and 30 post-menopausal female volunteers. Adiol and delta-5-androstenediol-3-sulphate (adiol-3S) concentrations were determined in combination with other relevant steroids in patients with either pituitary (n = 5), adrenal (n = 2) or gonadal dysfunction (n = 1), or testicular carcinoma (n = 19). MEASUREMENTS: After addition of deuterated adiol as internal standard, serum and urine samples were extracted. Steroid sulphates were hydrolysed. The extracts and hydrolysates were purified on HPLC, adiol was derivatized and finally quantified by gas chromatography-mass spectrometry. RESULTS: The calculated reference ranges for adiol and adiol-3S concentrations in serum are respectively: in men 1.78-7.24 and 123-579 nmol/l, in premenopausal women 0.65-6.93 and 21.2-298 nmol/l and in post-menopausal women 0.29-2.90 and 6.1-184 nmol/l. Urinary values varied considerably. In the population with endocrine abnormalities serum adiol and adiol-3S concentrations were compared with other relevant steroids. CONCLUSIONS: The wide concentration range of adiol and adiol-3S in urine makes analysis of these steroids in urine of little clinical value. Serum concentrations of adiol and adiol-3S are higher in men than in women. Premenopausal values are higher than post-menopausal. Adiol and adiol-3S in serum are significantly correlated in pre and post-menopausal women, r = 0.51 and r = 0.69 respectively, but not in men. In endocrine patients the serum concentrations of adiol show an ACTH or LH dependency in women; adiol correlates with cortisol, dehydroepiandrosterone or androstenedione and, in males, additionally with testosterone. However, in several situations adiol correlates with none of these steroids. Although adiol secretion can be stimulated by ACTH and LH, the level of serum adiol is also determined by other factors. Finally, in adrenal carcinoma serum adiol and adiol-3S may be used as tumour markers.

Synthesis and characterization of the 6 alpha- and 6 beta-hydroxylated derivatives of corticosterone, 11-dehydrocorticosterone, and 11-deoxycortisol.

This report describes the synthesis of 6 alpha, 17,21- and 6 beta, 17,21-trihydroxypregn-4-ene-3,20-dione, 6 alpha, 7,21- and 6 beta, 11 beta, 21-trihydroxypregn-4-ene-3,20-dione, and--for the first time--that of 6 alpha, 21- and 6 beta, 21-dihydroxypregn-4-ene-3,11,20-trione. The former four compounds were prepared by 6-hydroxylation of 17,21-trihydroxypregn-4-ene-3,20-dione and 11 beta, 21-dihydroxypregn-4-ene-3,20-dione, respectively. This was achieved by autoxidation or by oxidation with 3-chloroperbenzoic acid, of the 3-methoxy-pregna-3,5-dienes of the latter two steroids. The yield of the 6 beta-hydroxylated steroids, but not of their corresponding 6 alpha-epimers, was higher using autoxidation than the peracid. The two 6-hydroxylated pregnenetriones were prepared from 6 alpha, 21-diacetoxy-11 beta-hydroxypregn-4-ene-3,20-dione and 6 beta, 21-diacetoxy-11 beta-hydroxypregn-4-ene-3,20-dione, respectively, by oxidation with pyridinium chlorochromate. The above-mentioned six steroids were identified and characterized by nuclear magnetic resonance, infrared, ultraviolet, high performance liquid chromatography, gas chromatography, and mass spectrometry.

Isotope dilution ammonia chemical ionization mass fragmentographic analysis of urinary 3-O-methylated catecholamine metabolites. Rapid sample clean-up by derivatization and extraction of lyophilized samples.

We developed a method for simultaneous quantification of the urinary 3-O-methylated catecholamine metabolites 3-methoxytyramine, normetanephrine and metanephrine by stable isotope-dilution ammonia chemical ionization mass fragmentography. Prepurification of lyophilized samples was done by simultaneous deconjugation and pentafluoropropionylation, followed by extraction and rederivatization. Compared with our previously described method, based on acid hydrolysis, alkaline extraction, derivatization and electron-impact mass fragmentography, the present method was found to be less laborious, more sensitive and presumably more accurate. New urinary excretion values were established for apparently healthy adults. The present prepurification method may prove applicable for profiling of a variety of naturally occurring mono-, di- and polyamines in biological samples.

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.

A new, simple assay for long-chain acyl-CoA dehydrogenase in cultured skin fibroblasts using stable isotopes and GC-MS.

In this paper, we present a new method for measurement of long-chain acyl-CoA dehydrogenase (LCAD) activities in cultured skin fibroblasts. The method is based upon gas chromatographic/mass spectrometric determination of 3-OH-hexadecanoic acid formed during incubation of fibroblasts in a medium containing palmitoyl-CoA and crotonase, to convert the enoyl-CoA ester produced into the 3-hydroxyacyl-CoA ester. The validity of the method is demonstrated by the finding of a full deficiency of LCAD in fibroblasts from three patients with an established deficiency of LCAD.

Measurement of the cortisol production rate in two sisters with 17 alpha-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.

Use of determinations of 7-lathosterol (5 alpha-cholest-7-en-3 beta-ol) and other cholesterol precursors in serum in the study and treatment of disturbances of sterol metabolism, particularly cerebrotendinous xanthomatosis.

The sterol composition of sera from patients with cerebrotendinous xanthomatosis (CTX) was investigated by gas chromatographic analysis of saponified extracts, using a polar (CP Wax 52CB) and an apolar (CP Sil 5CB) capillary column. Apart from already known sterols, the presence of increased amounts of 8-lathosterol (5 alpha-cholest-8(9)-en-3 beta-ol) and significant amounts of 8-dehydrocholesterol (cholesta-5,8-dien-3 beta-ol) were noticed. The latter compound has not been detected previously in human serum and possibly represents a hitherto unknown cholesterol precursor. The apparently elevated levels of delta 8-sterols in CTX serum suggests partial inhibition of migration of the 8,9 double bond to the 7,8 position in this condition. The concentration of 7-lathosterol, an indicator of cholesterol production rate, is also highly elevated in CTX serum and quickly returns to normal values after oral bile acid therapy. Determinations of serum lathosterol are not only useful in the follow-up of therapy of CTX patients, but also in the follow-up of hypercholesterolemic patients treated with either HMG-CoA reductase inhibitors or bile acid sequestrants.

Determination of megestrol acetate and cyproterone acetate in serum of patients with advanced breast cancer by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method with ultraviolet detection of megestrol acetate and cyproterone acetate in human sera is described. The proposed assay is linear up to 1400 ng/ml (r = 0.999) and has a detection limit of 5 ng/ml. Recoveries of both compounds in spiked sera were ca. 95%; inter-assay coefficients of variation were 4.0 and 3.1% and intra-assay values were 1.3 and 1.4%, respectively. For validation of the method we also developed a gas chromatographic-mass spectrometric method for both steroids. The results obtained by the two methods showed good correlation: for megestrol acetate r = 0.98, n = 31, p less than 0.0001, and for cyproterone acetate r = 0.94, n = 0, p less than 0.0001. Large inter-individual differences in the serum concentrations of both substances were found in groups of patients with metastatic breast cancer receiving the same oral load of either steroid.

Metabolic dissimilarity between (9,12,12-2H) cortisol and natural cortisol in vivo. Can deuterated cortisol be used for the measurement of the urinary cortisol production rate?

The metabolism of deuterated cortisol (9,12,12,-2H)cortisol, 2H3-F) was compared to that of radioactive cortisol (3H2-F) and natural cortisol, when these three compounds were administered simultaneously to an adrenalectomized piglet. The relative isotope dilution of tritium was determined from the specific activities of the main urinary neutral cortisol metabolites, tetrahydrocortisone (THE) and tetrahydrocortisol (THF), normalized to that of the cortisol mixture administered. To obtain a comparison of the isotope dilution of deuterium in the metabolites THE and THF to that in the cortisol mixture, the three steroids were converted to the common oxidation product 11-oxo-aetiocholanolone, and derivatized to the methoxime-tert-butyl-dimethylsilyl ether. The relative 2H-isotope dilution then was measured by gas chromatography/mass spectrometry. It was found that the specific activity of THE in the cumulative urine collections was similar to that of the cortisol mixture administered; the two-day value was, however, less. The specific activity of THF was slightly but significantly smaller than 1 (approximately 0.9) at all times. The relative 2H-isotope dilution in THE was slightly but significantly larger than one (approximately 1.1) at all times, whereas that in the THF was larger than 1.0 at 9 and 32 h or equal to 1.0 at 20 and 47 h of urine collection. When comparing the metabolism of the two tracer cortisol species the quotient of the 3H- and the 2H-isotope dilutions in THE and THF was smaller than 1.0. It can be concluded that (2H3)cortisol may be used for the determination of the cortisol production rate.

Are (13C)cortisol and (3H)cortisol metabolized identically to natural cortisol in adrenalectomized piglets?

Adrenalectomized piglets were intravenously administered a mixture of (13C4)cortisol and (3H)cortisol and natural cortisol to determine if the two tracers are metabolized identically to natural cortisol. Urine was collected after 0.5, 1.0, 1.5 and 2.0 days and the isotope dilution was measured in the four major urinary cortisol metabolites, namely tetrahydrocortisone (THE), tetrahydrocortisol (THF), alpha- and beta-cortolone in the cumulative urines. In contrast to other studies, because of the sensitivity of the method used to measure the 13C4 enrichment, non-cumulative urine collections were also analysed. Quantification of the 13C4 isotope enrichment was carried out by gas chromatography/mass spectrometry with selected ion monitoring. The specific activities of the metabolites from the cumulative urine collections were determined by high-performance liquid chromatography and scintillation counting. Small secondary isotope effects seemed to occur during the metabolism of (13C4)cortisol, as a decrease in isotope enrichment in all four metabolites was measured. These effects were easily observed with alpha- and beta-cortolone isolated from the cumulative urine collections; the enrichment decreased by 19% and 14%, respectively. The lowering in isotope dilution in THE observed in the 2.0 day cumulative urine collection in piglets 1 and 2 were 4% and 3%, respectively. A lowering in isotope dilution in THF in the 2.0 day cumulative urine collection could be observed in piglet 2, namely 7%, but no change in isotope dilution could be seen in piglet 1. These secondary isotope effects could only be observed in the 2 days cumulative urine, and not in the cumulative urines collected over shorter times. The non-cumulative urines collected at half-day periods showed a significant decrease in isotope dilution in THE and THF isolated from the urine collected after 1 day. No statistically significant isotope effects were observed with the metabolism of (3H)cortisol, except at 0.5 day when the specific activity in the cortolones was lower and that in THF was higher. However, at 0.5 day with THE and 1.0 day with THF and the cortolones the specific activities remained approximately 6% higher than that administered in the cortisol. Secondary isotope effects with tritiated cortisol may have occurred but because of the relatively large imprecision of the measurement (SD = 3-4% with THE and THF and the cortolones (SD approximately 8%) compared to the measurements of the 13C4 enrichment (SD approximately 2%) these effects could not statistically be proven.(ABSTRACT TRUNCATED AT 400 WORDS)