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Cholesterol is the precursor of many compounds with functions in the physiology and metabolism of the organism. Methods for the multicomponent analysis of these compounds and their metabolites (metabolomics) are needed to improve our understanding of their roles in different species, organs, cells and metabolic situations and to clarify structure/activity relationships. This review discusses methods based on combinations of ion exchange and reversed-phase separations for sample preparation with derivatization and “charge-tagging” for chromatography-mass spectrometry in qualitative and quantitative characterizations of oxysterol, bile alcohol, bile acid, and steroid hormone metabolomes. Advantages, disadvantages and potential improvements for high-throughput applications are briefly discussed.

The formation of bile acids/bile alcohols is of major importance for the maintenance of cholesterol homeostasis. Besides their functions in lipid absorption, bile acids/bile alcohols are regulatory molecules for a number of metabolic processes. Their effects are structure-dependent, and numerous metabolic conversions result in a complex mixture of biologically active and inactive forms. Advanced methods are required to characterize and quantify individual bile acids in these mixtures. A combination of such analyses with analyses of the proteome will be required for a better understanding of mechanisms of action and nature of endogenous ligands. Mass spectrometry is the basic detection technique for effluents from chromatographic columns. Capillary liquid chromatography-mass spectrometry with electrospray ionization provides the highest sensitivity in metabolome analysis. Classical gas chromatography-mass spectrometry is less sensitive but offers extensive structure-dependent fragmentation increasing the specificity in analyses of isobaric isomers of unconjugated bile acids. Depending on the nature of the bile acid/bile alcohol mixture and the range of concentration of individuals, different sample preparation sequences, from simple extractions to group separations and derivatizations, are applicable. We review the methods currently available for the analysis of bile acids in biological fluids and tissues, with emphasis on the combination of liquid and gas phase chromatography with mass spectrometry.

In humans, the brain accounts for about 20% of the body's free cholesterol, most of which is synthesized de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolized to 24S-hydroxycholesterol, principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover; however, the route by which the remainder is turned over has yet to be elucidated. Here, we describe a novel liquid chromatography (LC) multi-stage fragmentation mass spectrometry (MS(n)) methodology for the identification, with high sensitivity (low pg), of cholesterol metabolites in rat brain. The methodology includes derivatization to enhance ionization, exact mass analysis at high resolution to identify potential metabolites, and LC-MS(n) (n=3) to allow their characterization. 24S-hydroxycholesterol was confirmed as a major oxysterol in rat brain, and other oxysterols identified for the first time in brain included 24,25-, 24,27-, 25,27-, 6,24,- 7alpha,25-, and 7alpha,27-dihydroxycholesterols. In addition, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterized in rat brain.

Farnesoid X receptor knockout (Fxr(-/-)) mice cannot upregulate the bile salt export pump in bile acid loading or cholestatic conditions. To investigate whether Fxr(-/-) mice differ in bile acid detoxification compared with wild-type mice, we performed a comprehensive analysis of bile acids extracted from liver, bile, serum, and urine of naive and common bile duct-ligated wild-type and Fxr(-/-) mice using electrospray and gas chromatography mass spectrometry. In addition, hepatic and renal gene expression levels of Cyp2b10 and Cyp3a11, and protein expression levels of putative renal bile acid-transporting proteins, were investigated. We found significantly enhanced hepatic bile acid hydroxylation in Fxr(-/-) mice, in particular hydroxylations of cholic acid in the 1beta, 2beta, 4beta, 6alpha, 6beta, 22, or 23 position and a significantly enhanced excretion of these metabolites in urine. The gene expression level of Cyp3a11 was increased in the liver of Fxr(-/-) mice, whereas the protein expression levels of multidrug resistance-related protein 4 (Mrp4) were increased in kidneys of both genotypes during common bile duct ligation. In conclusion, Fxr(-/-) mice detoxify accumulating bile acids in the liver by enhanced hydroxylation reactions probably catalyzed by Cyp3a11. The metabolites formed were excreted into urine, most likely with the participation of Mrp4.

Neutral steroids are difficult to analyse using desorption ionisation methods coupled with mass spectrometry (MS). However, steroids with an unhindered ketone group can readily be derivatised with the Girard P (GP) reagent to give GP hydrazones. Steroid GP hydrazones contain a quaternary nitrogen atom and are readily desorbed in the matrix-assisted laser desorption/ionisation (MALDI) process, giving an improvement in sensitivity of two orders of magnitude. Steroids without a ketone group, but with a 3beta-hydroxy-Delta5 function, can be readily converted to 3-oxo-Delta4 steroids and subsequently derivatised to GP hydrazones for MALDI analysis. In addition to giving strong [M]+ ions upon MALDI, steroid GP hydrazones give informative post-source decay (PSD) spectra. By using the accurate mass of the precursor-ion measured by MALDI-MS, in combination with the structural information encoded in its PSD spectrum, steroid structures can readily be determined.

The brain is the almost exclusive site of formation of 24S-hydroxycholesterol in man, and there is a continuous flux of this oxysterol across the blood-brain barrier into the circulation. The hepatic metabolism of 24S-hydroxycholesterol was studied here by three different approaches: incubation of tritium-labeled 24S-hydroxycholesterol with human primary hepatocytes, administration of tritium-labeled 24S-hydroxycholesterol to a human volunteer, and quantitation of free and conjugated 24S-hydroxycholesterol and its neutral metabolites in ileocecal fluid from patients with ileal fistulae. 24S-Hydroxycholesterol as well as 24R-hydroxycholesterol were converted into bile acids by human hepatocytes at a rate of about 40% of that of the normal intermediate in bile acid synthesis, 7α-hydroxycholesterol. There was also a conversion of 24S-hydroxycholesterol into conjugate(s) of 5-cholestene-3β,24S,27-triol at a rate similar to the that of conversion into bile acids. When administered to a human volunteer, labeled 24S-hydroxycholesterol was converted into bile acids at about half the rate of simultaneously administered labeled 7α-hydroxycholesterol. Free, sulfated, and glucuronidated 24S-hydroxycholesterol and 5-cholestene-3β,24,27-triol were identified in ileocecal fluid. The excretion of these steroids was about 3.5 mg/24 h, amounting to more than 50% of the total estimated flux of 24S-hydroxycholesterol from the brain. It is concluded that 24S-hydroxycholesterol is a less efficient precursor to bile acids and that about half of it is conjugated and eliminated in bile as such or as a conjugate of a 27-hydroxylated metabolite. The less efficient metabolism of 24S-hydroxycholesterol may explain the surprisingly high levels of this oxysterol in the circulation and is of interest in relation to the suggested role of 24S-hydroxycholesterol as a regulator of cholesterol homeostasis.

Progesterone inhibits intracellular transport of lysosomal cholesterol in cultured cells, and thus at least in part mimics the biochemical phenotype of Niemann-Pick type C disease (NPC) in human fibroblasts. The goal of this study was to determine whether metabolism of progesterone to other steroids is affected by the NPC mutation or by P-glycoprotein (a known progesterone target). We found that human fibroblasts metabolize progesterone in three steps: rapid conversion to 5alpha-pregnane-3,20-dione, which is then reduced to 5alpha-pregnane-3beta(alpha)-ol-20-one with subsequent 6alpha-hydroxylation. The pattern and rates of progesterone metabolism were not significantly different in a variety of fibroblasts from normal individuals, NPC patients, and obligate heterozygotes. Inhibition of steroid 5alpha-reductase with finasteride completely blocked metabolism of progesterone but had no effect on inhibition of LDL-stimulated cholesterol esterification (IC50 = 10 microM). Progesterone also partially inhibited 25-hydroxycholesterol-induced cholesterol esterification, with similar dose-dependence in normal and NPC fibroblasts. P-glycoprotein levels varied significantly among the various fibroblasts tested, but no correlation with NPC phenotype or rate of progesterone metabolism was noted, and P-glycoprotein inhibitors did not affect conversion of progesterone to products. These results indicate that metabolism of progesterone in human fibroblasts is largely independent of its ability to interfere with cholesterol traffic and P-glycoprotein function.

Background/Aims and Methods: The etiology of intrahepatic cholestasis of pregnancy (ICP) is unknown. We have performed comprehensive chromatographic and mass spectrometric analyses of progesterone metabolites and bile acids in serum and urine of six patients in order to characterize changes that might be of importance for the development of the disease. Results: Conjugated bile acids were increased in serum and urine of patients with ICP while the levels of unconjugated bile acids were similar in healthy pregnancies and ICP. Unconjugated and conjugated 7α,12α-dihydroxy-3-oxo-4-cholenoic acid was excreted in urine both in healthy pregnancies and in ICP, possibly indicating a rate limitation of 3-oxo-Δ 4 -steroid 5β-reductase in pregnancy. The serum levels and urinary excretion of total sulfated progesterone metabolites were increased in ICP while the glucuronides were unchanged or low. Confirming previous results, the fraction of metabolites with 3α-hydroxy-5α(H) configuration was increased. The urinary excretion of 5α-pregnane-3α,20α-diol 3-sulfate,20- N -acetylglucosaminide was greatly increased in ICP, as was that of 3α-hydroxy-5α-androstane-17β-carboxylic acid, assumed to be a progesterone metabolite. Conclusions: The combined results of this and previous studies are compatible with a primary change in the reductive metabolism of progesterone in ICP, resulting in increased formation of metabolites with a 3α-hydroxy-5α(H) configuration and a larger fraction of sulfates. There also seems to be a selective defect in the biliary secretion of sulfated metabolites, particularly disulfates.

A series of pregnanediols and pregnanetriols doubly conjugated with N-acetylglucosamine and glucuronic or sulfuric acid has been identified in urine from pregnant women. Steroid conjugates were separated by ion-exchange chromatography and the glucuronide and monosulfate fractions were analysed by fast atom bombardment mass spectrometry. After removal of the acid moiety, the neutral steroids were isolated, derivatized, and analysed by gas chromatography-mass spectrometry (GC-MS). The analyses revealed the presence of steroids conjugated with N-acetylhexosamine both in the glucuronide and the monosulfate fractions. Following enzyme hydrolysis, the sugar was identified by GC-MS as N-acetylglucosamine (GlcNAc). The major steroid conjugated with GlcNAc both in the glucuronide and monosulfate fractions was identified as 5alpha-pregnane-3alpha,20alpha-diol. 5beta-Pregnane-3alpha,2Oalpha-diol was also present as a GlcNAc conjugate in both fractions whereas a GlcNAc conjugate of 5alpha-pregnane-3beta,20alpha-diol was only found in the sulfate fraction. 5alpha-Pregnane-3alpha,20alpha,21-triol was a double conjugate with GlcNAc in the sulfate fraction whereas a pregnane-2,3,20-triol was a double conjugate in the glucuronide fraction. The positions of conjugation were determined by collision-induced dissociation of the pseudomolecular anions produced by fast atom bombardment ionization. The sulfate and glucuronic acid moieties were located at C-3 and N-acetylglucosamine at C-20. An alternative localization of GlcNAc at C-21 of 5alpha-pregnane-3alpha,20alpha,21-triol cannot be excluded. Judging from the enzymatic hydrolysis of the conjugates, the sugar was attached in beta-glycosidic linkage. The mean excretion of N-acetylglucosaminides of the pregnanediols and pregnanetriols was 32.2 micromol/g creatinine (range 17.9-49.1 micromol) in five healthy women in the 38th-39th week of pregnancy. The mean excretion of 5beta-pregnane-3alpha,20alpha-diol glucuronide in the same women was 71 micromol/g creatinine, (range 27-127 micromol). This indicates that conjugation with N-acetylglucosamine constitutes a quantitatively important pathway of progesterone metabolism in human pregnancy.

The metabolism of 27-hydroxycholesterol and 25-hydroxycholesterol was studied in cultures of human diploid fibroblasts. Both steroids underwent 7 alpha-hydroxylation with subsequent oxidation to 7 alpha-hydroxy-3-oxo-delta 4 steroids. A minor fraction of the 27-hydroxysteroids was oxidized to acids. Competition experiments indicated that both hydroxycholesterols were hydroxylated by the same enzyme, different from cholesterol 7 alpha-hydroxylase. 7 alpha,25-Dihydroxycholesterol suppressed the activity of HMG-CoA reductase at least as effectively as 25-hydroxycholesterol whereas 7 alpha,25-dihydroxy-4-cholesten-3-one was a less effective suppressor. The results suggest that cholesterol might be converted to 7 alpha-hydroxylated bile acid precursors in extrahepatic tissues in vivo and that the regulation of the activity of HMG-CoA reductase by oxysterols might be modulated by 7 alpha-hydroxylation and subsequent oxidation by 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase.

In order to establish the position of conjugation of bile acids with glucose or N-acetylglucosamine, glucosides of chenodeoxycholic and hyodeoxycholic acids and of 13C-labeled cholic, lithocholic, chenodeoxycholic, hyodeoxycholic, and ursodeoxycholic acids, and N-acetylglucosaminides of ursodeoxycholic, isoursodeoxycholic, 3-dehydro-ursodeoxycholic, and ursodeoxycholylglycine were synthesized in vitro. The conjugates were purified by anion-exchange chromatography and reversed-phase HLPC and were analyzed by gas chromatography-mass spectrometry. The glucosides of chenodeoxycholic and hyodeoxycholic acids were also analyzed after periodate and chronic acid oxidation. All conjugates were analyzed by fast atom bombardment mass spectrometry with collision-induced dissociation. Glucose conjugation was shown to occur at C-3 in all bile acid glucosides studied. In contrast, the selective N-acetylglucosaminidation of 7 beta-hydroxy bile acids was shown to occur at the 7 beta-position.

When analyzed by fast atom bombardment mass spectrometry, taurine-conjugated bile acids give intense [M-H]-pseudomolecular ions that can be subjected to collision-induced fragmentation to give structural information. A method has been developed that permits rapid coupling of taurine to unconjugated, glycine-conjugated, sulfated, and glucuronidated bile acids. The reaction is performed for 2 h at room temperature in aqueous pyridine hydrochloride buffer, with or without dioxane, using 0.1 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as the coupling agent and 0.2 M taurine. The yields are higher than 95%. In contrast to published coupling reactions, the method permits conjugation of bile acids with the labile 7 alpha-hydroxy-3-oxo-4-ene structure.

Using a novel combination of analytical chemical and molecular biological techniques, lipophilic components of human plasma separated according to their physico-chemical properties were screened for their ability to activate the rat peroxisome proliferator-activated receptor (rPPAR). Activation of an rPPAR/glucocorticoid receptor chimera stably expressed in CHO cells by fractions in the initial screening guided further subfractionation. Characterization of an active subfraction by gas chromatography alone and in combination with mass spectrometry (GC-MS), indicated the presence of free fatty acids. Individual active components in this mixture were isolated by a final fractionation using high performance liquid chromatography (HPLC). GC-MS analyses of HPLC fractions able to activate the chimeric receptor identified palmitic acid, oleic acid, linoleic acid, and arachidonic acid as endogenous activators of rPPAR. No other activators were identified. This approach is able to specifically extract and identify endogenous activators of PPAR from a complex biological extract and as such may be valuable in the identification of activators of other orphan receptors in the steroid hormone receptor superfamily.

A review is given of methods for analysis of organochlorine compounds in which the lipophilic gel Lipidex is used for extraction and purification. Some of the compounds analysed are DDT, DDE, hexachlorobenzene (HCB), hexachlorocyclohexane (α-, β-, γ-HCH), dieldrin, oxychlordane, trans -nonachlor, pentachlorophenol, polychlorinated biphenyls (PCBs), including the non- ortho planar congeners, polychlorinated dibenzo- p -dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) and metabolites of chlorinated paraffins. Applications to analyses of human milk, cod liver oil, bile, urine, water and indoor air are discussed.

In connection with studies of alternative pathways in bile acid biosynthesis, potential intermediates in a pathway starting with 27-hydroxylation of cholesterol have been prepared in natural and deuterated forms. Established methods were used to prepare 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid. Clemmensen reduction of kryptogenin in unlabeled and deuterated solvents yielded 27-hydroxy-cholesterol and 16-oxo-5-cholestene-3 beta,27-diol, which were separated by adsorption chromatography on Unisil. The labeled 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid derived from it consisted of molecules with seven (50%), six (20%), and eight (20%) deuterium atoms, and unlabeled molecules were not detected. The acetates of 27-hydroxycholesterol and methyl 3 beta-hydroxy-5-cholestenoate were 7 alpha-hydroxylated in a copper-catalyzed reaction with tert-butylperbenzoate, and the products were purified by chromatography on Unisil. The 7 beta-epimers were obtained as side products. Labeled 3 beta,7 alpha-dihydroxy-5-cholenoic acid was prepared in the same way from 3 beta-hydroxy-5-[2,2,4,4,23-2H5]-cholenoic acid. The 3-oxo-delta 4 analogs of the 3 beta-hydroxy-delta 5 compounds were prepared by oxidation with cholesterol oxidase. The labeled products had the same isotopic composition as the starting materials. Gas chromatographic retention indices and mass spectral characteristics of the trimethylsilyl ether derivatives of the neutral steroids and the methylated acids are given for all compounds.

An analytical method is described whereby progesterone is isolated from pregnancy plasma on the basis of the high affinity and specificity of the progesterone receptor for its ligand. Partially purified progesterone receptor ligand-binding domain, expressed as a protein A fusion protein in Escherichia coli, is incubated with a neutral steroid fraction obtained by extraction and ion-exchange chromatography of human late-pregnancy plasma. The incubated sample is passed through two Lipidex 1000 (lipophilic gel) beds. The first, at 4 degrees C, separates the specific ligand-fusion protein complex from nonspecifically bound and unbound compounds, and the second, at 40 degrees C, separates the specific ligand from the protein. Elution of the second bed with methanol yields a fraction containing specific ligand that can be characterized by gas chromatography-mass spectrometry. This methodology may be valuable for identification of endogenous ligands to orphan receptors of the steroid hormone receptor superfamily.

In order to study the glycosidic conjugation of chenodeoxycholic, hyodeoxycholic, and ursodeoxycholic acids in patients with cholestasis after oral administration of pharmacological amounts of the respective bile acids avoiding the application of radioactive tracers we synthesized [24-13C]chenodeoxycholic, [24-13C]hyodeoxycholic, and [24-13C]ursodeoxycholic acids. The reaction intermediates of the bile acid syntheses were characterized by infrared spectroscopy. Purity was confirmed using thin-layer chromatography as well as gas chromatography-mass spectrometry. The 13C atom excess of approximately 90% of the synthesized bile acids was the same as the 13C atom excess of the sodium [13C]cyanide used for the labeling reaction confirming the successful synthesis. After oral administration of 0.5 g of [24-13C]ursodeoxycholic acid to a healthy volunteer, 13C label was detected in the nonamidated and glycine- or taurine conjugated glucosides and the N-acetylglucosaminide of ursodeoxycholic acid in urine. This establishes ursodeoxycholic acid as the first bile acid so far known to undergo both of the recently described glycosidic conjugation reactions in humans.

The plasma concentrations of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid have been compared with that of 7 alpha-hydroxy-4-cholesten-3-one in healthy subjects and in patients with an expected decrease or increase of the bile acid production. In controls and patients with liver disease, the level of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid was positively correlated to that of 3 beta,7 alpha-dihydroxy-5-cholestenoic acid and not to that of 7 alpha-hydroxy-4-cholesten-3-one. In patients with stimulated bile acid formation the levels of the acids were not correlated to each other but there was a significant positive correlation between the levels of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid and 7 alpha-hydroxy-4-cholesten-3-one. These findings indicate that the precursor of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid differs depending on the activity of cholesterol 7 alpha-hydroxylase. Since the activity of this enzyme is reflected by the level of 7 alpha-hydroxy-4-cholesten-3-one in plasma the findings are compatible with a formation of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid from 3 beta,7 alpha-dihydroxy-5-cholestenoic acid when the rate of bile acid formation is normal or reduced and from 7 alpha-hydroxy-4-cholesten-3-one under conditions of increased bile acid synthesis. In support of this interpretation, 7 alpha,26-dihydroxy-4-cholesten-3-one was identified at elevated levels in plasma from patients with ileal resection or treated with cholestyramine. The levels of 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one were also higher than normal in these patients. Based on these findings and previous knowledge, a model is proposed for the biosynthesis of bile acids in man. Under normal conditions, two major pathways, one "neutral" and one "acidic" or "26-oxygenated", lead to the formation of cholic acid and chenodeoxycholic acid, respectively. These pathways are separately regulated. When the activity of cholesterol 7 alpha-hydroxylase is high, the "neutral" pathway is most important whereas the reverse is true when cholesterol 7 alpha-hydroxylase activity is low. In cases with enhanced activity of cholesterol 7 alpha-hydroxylase, the "neutral" pathway is connected to the "acidic" pathway via 7 alpha,26-dihydroxy-4-cholesten-3-one, whereas a flow from the acidic pathway to cholic acid appears to be of minor importance.

The effects of ethynylestradiol on endogenous bile acids, their capacity to conjugate and excrete intra- venously infused cholic acid, the concentrations of biliary cholesterol and lecithin, and the individual molecular species of phosphatidylcholine have been determined in male and female Sprague-Dawley rats. Endogenous biliary bile acids were analyzed by gas-liquid chromatography- mass spectrometry. Eleven bile acids were identified and several minor bile acids, primarily muricholates, could not be completely characterized. After 5 days of treatment with ethynylestradiol (1 mg/kg per day), the percentage of cholic acid decreased and the percentage of GB-hydroxyl- ated bile acids, including several monounsaturated species, increased. Ethynylestradiol caused a decrease in bile acid- independent bile flow. Intravenous infusion of cholic acid at a high concentration caused cholestasis in control ani- mals but, after ethynylestradiol treatment, cholestasis developed during the infusion of a much lower concentra- tion of cholate, indicating a lowered threshhold for bile acid-induced cholestasis. In the treated rats, there was a slight increase in excretion of unconjugated endogenous bile acids, and a striking impairment of conjugation of intravenously administered cholic acid. One of the few sex-related differences observed was an increased con- centration of biliary phospholipids in untreated male rats. Both phospholipid and cholesterol concentrations in the bile were higher in the treated animals. The molar per- centage of cholesterol was always Many investigators have studied the hepatic effects of natural and synthetic estrogens, especially ethynyl- estradiol, a common estrogenic component of contra- ceptive steroids. In the rat, estrogens increase liver size and water content, RNA, DNA, and protein con- tent. They decrease the bile salt-independent frac- tion of bile flow and the maximal transport capacity for bile acids, bilirubin, and bromosulfophthalein (BSP) (1 -6). Ethynylestradiol reduces hepatic cyto- chrome P-450 activity (7) and inhibits microsomal hydroxylation of steroids in vitro (8). It is not known, however, to what extent it affects hydroxylation and conjugation reactions involved in bile acid formation in vivo and whether changes in bile acid metabolism, especially conjugation, are related to the cholestatic effect of ethynylestradiol. In this study, we examined the effect of ethynylestradiol on bile acid metabolism in vivo by analyzing bile acids in bile of both male and female rats given this drug. We also determined the capacity of these rats to conjugate and excrete cholic acid infused intravenously and related the results to the cholestatic effect of ethynylestradiol. Ethynylestradiol, other estrogens, and contracep- tive steroid preparations are also known to alter the composition of biliary lipids in several animal species, including man, so that the bile becomes more saturated with cholesterol (9- 11). This is consistent with the clinically important observation that choles- terol gallstones occur more frequently in women during the childbearing age than in men (12, 13) and that contraceptive steroids cause an increase in

A new bile alcohol was detected in urine from healthy subjects and patients with liver disease. Urine was extracted with Amberlite XAD-2 and a glucuronide fraction was isolated by ion exchange chromatography on Lipidex-DEAP. Following enzymatic hydrolysis, neutral compounds were purified on Lipidex DEAP, and the major bile alcohol was isolated by chromatography on Lipidex 1000. Based on a combination of microchemical reactions and capillary column GC/MS, the new bile alcohol was identified as 27-nor-5β-cholestane-3α,7α,12α,24ξ,25ξ-pentol. GC/MS analysis of the derivatized purified glucuronide fraction indicates that the bile alcohol is excreted conjugated with glucuronic acid.

Three healthy men were given ethanol, 0.3 g/kg, and the excretion of steroids in urine was analyzed by capillary column gas chromatography and mass spectrometry, following group separation of conjugates. The ratio between the excretion rates of 17β-hydroxy- and corresponding 17-oxosteroid monosulphates increased 2–5 times during ethanol metabolism. This change is similar to that previously observed for the concentrations of these steroids in plasma, and is considered to be due to the changed redox state of hepatic pyridine nucleotides. Neither 20α (andβ) -hydroxy-/20-oxo-steroid couples formed from cortisol, nor various 11β-hydroxy-/11-oxosteroid couples in the glucuronide fraction changed towards a more reduced state during ethanol oxidation. The excretion of glucuronides of 11-oxygenated C19 steroids relative to that of cortisol metabolites of the C21 series decreased slightly, indicating an inhibition of side-chain cleavage. The rate of excretion of steroid disulphates was positively correlated to the urine volume.

Molecular species of phosphatidylcholines were analyzed in hepatic and gallbladder bile obtained from six subjects with adenomyoma of the gallbladder (gallstone-free controls) and 27 gallstone patients undergoing cholecystectomy. Seven of the gallstone patients had been treated with cholic acid and seven with chenodeoxycholic acid for at least 8 weeks before operation. The two predominant species were 1-palmitoyl-2-oleoyl- and 1-palmitoyl-2-linoleoyl-sn-glycerophosphocholines which together accounted for 75-80% of the total amount of phosphatidylcholines. Minor species were 1-palmitoyl-2-palmitoleoyl-, 1-stearoyl-2-linoleoyl-, 1-oleoyl-2-linoleoyl-, and 1-palmitoyl-2-arachidonoyl-sn-glycerophosphocholines. Gallstone patients had a higher portion of the 1-palmitoyl-2-oleoyl species and a concomitant lower proportion of the 1-palmitoyl-2-linoleoyl species than gallstone-free subjects. The ratio between the two species was about 0.7 and 0.4, respectively, in the hepatic bile of the two groups of patients. Treatment with bile acids was associated with a normalization of the pattern of phosphatidylcholines.

Pregnenolone (P) and its sulfate ester (PS) have been characterized in the brain of adult male rats. The concentration of P (38.4 +/- 6.9 and 22.1 +/- 2.9 ng/g, mean +/- S.D., in anterior and posterior brain, respectively) exceeded that of PS in brain (15.8 +/- 3.0 and 5.7 +/- 2.1 ng/g in the same fractions) and largely those of P and PS in plasma (1.3 +/- 0.2 and 1.4 +/- 0.3 ng/g, respectively). The level of P in brain was much larger than that of dehydroepiandrosterone sulfate (DS), characterized and measured previously (Corpéchot et al.). Brain P and PS levels did not seem to depend on steroidogenic gland secretion: no meaningful difference occurred in brain 15 days after adrenalectomy plus orchiectomy, compared with sham-operated controls. It is proposed that, as that of DS (ref. 5) P and PS formation or accumulation (or both) in the rat brain depend on in situ mechanisms unrelated to the peripheral endocrine gland system.

A gas chromatographic-mass spectrometric (GC-MS) method for analysis of unconjugated steroids in a rat testis is described. A combined solvent-solid extraction procedure, utilizing Lipidex 1000 and Sep-Pak C18, gives a 25-fold purified extract. Steroids in this extract are fractionated by straight phase high-performance liquid chromatography (HPLC) on a LiChrosorb DIOL column in n-hexane-2-propanol, 92:8 (v/v). Four fractions are collected and the steroids are converted to tert-butyldimethylsilyl (TBDMS), 3-enol-TBDMS, and mixed TBDMS-trimethylsilyl (TMS) derivatives using TBDMS- and TMS-imidazole with sodium formate as catalyst under conditions suitable for the steroids present in the respective fractions. The derivatives are purified by reversed phase HPLC in 100% methanol and are analyzed by GC-MS, using selected ion monitoring of the major ions of high mass. For quantification, a mixture of known amounts of ten 14C-labelled steroids, [3H]estradiol and [2H3]estradiol are added to the testis homogenate. The mean concentrations (ng/g wet wt) of the twelve steroids determined were: 4-androstene-3, 17-dione, 4.0; testosterone, 127; 17 beta-hydroxy-5 alpha-androstan-3-one, 4.5; 5 alpha-androstane-3 alpha, 17 beta-diol, 5.7; 5 alpha-androstane-3 beta, 17 beta-diol, 1.5; progesterone, 5.5; 17 alpha-hydroxyprogesterone, 14.4; 3 beta-hydroxy-5-androsten-17-one, 0.07; 5-androstene-3 beta, 17 beta-diol, 0.25; 3 beta-hydroxy-5-pregnen-20-one, 10.3; 3 beta, 17 beta-dihydroxy-5-pregnen-20-one, 0.95; and estradiol, 0.025. Variations between animals were large whereas testes from the same animal in most cases had similar steroid concentrations.

A synthesis is reported of 3beta-hydroxy-5alpha-pregnan-20-one sulphate and the disulphate and 3-monosulphate of 5alpha-pregnane-3beta,20alpha-diol, labelled specifically with deuterium in high isotopic purity for metabolic studies in humans. Base-catalyzed equilibration of 3beta-hydroxy-5alpha-25R-spirostan-12-one (hemcogenin, II) with deuterium oxide, followed by removal of the 12-keto group and degradation of the sapogenin side-chain afforded 3beta-hydroxy-5alpha-[11,11-2H2]pregn-16-en-20-one (VII). Further deuterium atoms were introduced at the 3alpha and 20beta positions by reductions with sodium borodeuteride and lithium aluminum deuteride, respectively. These reactions led to 3beta-hydroxy-5alpha-[3alpha,11,11-2H3]pregnan-20-one (X; isotopic purity 87.2%) and 5alpha-[3alpha,11,11,20beta-2H4]pregnane-3beta,20alpha-diol (XIV; isotopic purity 83.9%). The 3-sulphate of the pregnanolone and the 3,20-disulphate of the pregnanediol were prepared directly form the free alcohols, while the 3-monosulphate of the pregnanediol was obtained via 5alpha-[3alpha,11,11,20beta-2H4]pregnane-3beta,20alpha-diol 20-acetate (XVII).

A method for the isolation, separation and analysis of glucosides and N-acetylglucosaminides of non-amidated bile acids and of glycine- and taurine-conjugated bile acid glucosides from normal human urine is described. Total bile acids were extracted from 24-h collections of urine by repetitive use of Sep-Pak C 18 cartridges. After elution with 80% aqueous methanol, a group separation into non-amidated, glycine- and taurine-conjugated bile acids was performed by ion-exchange chromatography on Lipidex-DEAP. The glycosylated compounds were then separated from the corresponding non-glycosylated ones by high-performance liquid chromatography (HPLC) using a reversed-phase system with a linear methanol gradient. The glycosylated compounds isolated by HPLC were analysed by fast atom bombardment mass spectrometry and, after derivatization, by gas chromatography—mass spectrometry. Information about the sugar moieties of the bile acid glycosides was also obtained by treatment with different glycosidases.

A method is described for analysis of metabolic profiles of unconjugated steroids in plasma. After extraction by Amberlite XAD-2 at 64°, the steroids are purified by filtration through a 0.5 g column of sulphoethyl Sephadex LH-20 in methanol, and by chromatography on a 0.5 g column of triethyl-aminohydroxypropyl-hydroxyalkyl Sephadex LH-20. The latter column is used in a reversed-phase system which separates neutral steroids as a group from less polar lipids, and yields phenolic steroids in a separate fraction. The neutral steroids are converted into MO-TMS derivatives which are purified by rapid filtration through a 0.25 g column of Lipidex 5000 in hexane containing pyridine, hexameth-yidisilazane and dimethoxypropane to prevent hydrolysis of TMS ethers. Estrogens are converted into TMS ethers. The recoveries of added [3H]-labelled neutral steroids and estrogens are about 85%. Sample sizes corresponding to 1–2 ml plasma can be injected onto glass capillary columns. The derivatives are analyzed by repetitive scanning GC-MS using a glass capillary column connected via a single-stage adjustable jet separator. Metal surfaces are deactivated with water glass and benzyltri-phenylphosphonium chloride, and a Teflon coupling device is used for glass-glass capillary connections. Characterization and quantitation of steroids is based on computer construction of fragment ion current chromatograms. When the method was applied to the analysis of unconjugated steroids in plasma from pregnant women, 27 steroids in the concentration range 1–300 ng/ml could be identified. The 13 major pregnane derivatives were quantitated. The coefficients of variation were 5–13% when 5 analyses of the same sample were performed.

The intestinal bacterial metabolism of 2-methoxyestrone was studied by incubation in the isolated coecum from rats. Following isolation of estrogens by a combination of ion-exchange and ligand-exchange chromatography, the metabolites were identified by gas chromatography-mass spectrometry. The two main reactions were oxidoreduction at C-17 and extensive demethylation at C-2. Thus, the demethylation of 2-methoxyestrogens known to occur in vivo may be due to the action of microbial enzymes. The study also shows that the intestinal microflora is capable of converting biologically inactive into active steroid hormones.

The effects of ethanol on the concentrations of steroids in testis was studied in adult rats. Testosterone, seven of its potential precursors, three of its metabolites, and estradiol were analyzed by gas chromatography-mass spectrometry of samples from testes removed 2 h after intraperitoneal administration of ethanol, 1.2 g/kg body weight. The same analyses were made on samples from control rats. Ethanol gave a marked increase of all 3 beta-hydroxy-delta 5 steroids analyzed: pregnenolone (60%), 17-hydroxypregnenolone (480%), dehydroepiandrosterone (430%) and 5-androstene-3 beta, 17 beta-diol (60%). This resulted in highly significant increases of the 3 beta-hydroxy-delta 5/3-oxo-delta 4 steroid ratios for all steroid couples analyzed. An analogous increase of the ratio between 5 alpha-androstane-3 beta, 17 beta-diol and dihydrotestosterone was also observed, whereas the ratio between androstenediol and dehydroepiandrosterone was decreased by ethanol. The concentration of estradiol was not affected. The results indicate that moderate doses of ethanol inhibit the conversion of 3 beta-hydroxy-delta 5 to 3-oxo-delta 4 steroids. This may be one mechanism by which ethanol decreases the production of testosterone.

Methods for isolation of steroids by filtration procedures are reviewed. Biological fluids or extracts are filtered through a series of small columns which retain either the steroids or unwanted material. The filters consist of lipophilic-hydrophobic Lipidex gels with and without cation or anion exchanging groups. A filter of Lipidex 1000 extracts steroids of low and medium polarity from aqueous solutions. A nonpolar ion exchanger removes lipids from steroids in aqueous methanol and permits rapid separation of groups of conjugates at the same time. Reagents and polar contaminants are removed from derivatized steroids prior to GC/MS by filtration through Lipidex in a nonpolar solvent. Selective isolation procedures are based on specific group interactions. Phenolic steroids are isolated on a nonpolar anion exchanger, 3-ketosteroids are isolated as oximes on a nonpolar cation exchanger, and ethinyl steroids are isolated on a strong cation exchanger in silver form.

Plasma steroids from 11 women in the last trimester of pregnancy were separated into conjugate class by chromatography on Sephadex LH-20. Fractions corresponding to mono- and disulphate conjugates were solvolyzed and the free steroids were separated by lipophilic gel chromatography into groups containing mono-, di- and trihydroxy steroids, respectively. Trimethylsilyl ether derivatives were prepared and repetitive-scanning gas chromatography-mass spectrometry (GC-MS) was used to characterize and quantitate the following 16α-hydroxy steroids in the monosulphate fraction: 3α,16α:-dihydroxy-5α-pregnan-20-one (range 8–80 ng/ml), 3β,16α-dihydroxy-5α-pregnan-20-one (6–55 ng/ml), 5α-pregnane-3α,16α,20α-triol (9–31 ng/ml) and 5α-pregnane-3β,16α,20α-triol (11–77 ng/ml). These compounds were also present as disulphates although at 5–50 times lower concentrations.

A method is described for the analysis of bile acid glucuronides in urine. Following extraction on Amberlite XAD-2 and separation of bile acid conjugates on DEAP-LH-20, the fractions containing bile acid glucuronides are methyl esterified and filtered through DEAP-LH-20, yielding a fraction of purified bile acid glucuronide methyl esters. Individual glucuronides are separated in a reversed phase system on Lipidex 1000 and are analyzed as TMS ethers by GC/MS. Oxidation with periodate, which transforms the glucuronyl moiety into a formate ester, followed by oxidation with chromic acid and conversion of keto groups to O-methyloximes, permits determination of the site of conjugation with glucuronic acid. The daily excretion of bile acid glucuronides in urine of healthy subjects was 0.8–16.2 μmol (12–36% of the total bile acid excretion in urine). Only ether glucuronides were observed, most of which were otherwise unconjugated. A major part of the bile acids were hydroxylated at C-6, and 3α,6α,12α-trihydroxy-5β-cholanoic acid was identified as a quantitatively important new bile acid. The glucuronyl moiety was attached at C-6 of hyodeoxycholic acid and at C-3 of cholic and chenodeoxycholic acids. The results may indicate a possible coupling of glucuronic acid conjugation with 6α-hydroxylation of secondary bile acids.

A method is described for analysis of unconjugated neutral steroids in plasma. It is based on extraction of the sample with Amberlite XAD-2 at elevated temperature, purification of the extract on lipophilic ion exchanging and neutral Sephadex derivatives, and gas chromatography-mass spectrometry-computer analysis of O-methyloxime-trimethylsilyl ether derivatives purified on hydrophobic Sephadex. Examples of the application of the method to analysis of steroids in plasma from pregnant women are given.

The effects of administration of ethinylestradiol (17α-ethinyl-1,3,5(10)-estratriene-3, 17β-diol), 5 or 500 mg · kg −1 per day, and norethisterone (17β-hydroxy-17α-ethinyl-4-estren-3-one), 0.5 or 50 mg · kg −1 per day, for two weeks on the hepatic microsomal metabolism of 5α-[4- 14 C]androstane-3α,17β-diol, 4-[4- 14 C] and rostene-3,17-dione, 4-[4- 14 C] pregnene-3,20-dione and 7α-hydroxy-4-[6β- 3 H] cholesten-3-one and on the fine structure of parenchymal cells were studied in male and female rats. The low dose of ethinylestradiol suppressed most hydroxylase levels and increased 5α-reductase levels in male rats in a manner consistent with a feminizing effect of the drug. Female rats reacted much less to the low dose of ethinylestradiol although 5α-reductase levels were slightly but significantly reduced. The high dose of ethinylestradiol suppressed most enzyme activities both in male and female rats. The low dose of norethisterone stimulated some hydroxylase and oxidoreductase activities in the metabolism of 4-androstene-3,17-dione, 4-pregnene3, 20-dione and 7α-hydroxy-4-cholesten-3-one. In contrast, the high dose reduced these activities in both male and female rats. No alterations in the fine structure of hepatic parenchymal cells were observed following the administration of ethinylestradiol or norethisterone. It is concluded that ethinylestradiol affects hepatic steroid hydroxylation and reduction mainly in male rats and by virtue of its estrogenic, demasculinizing effect, and that it causes a general suppression of the steroid metabolizing enzymes in rats of both sexes when given in the large doses, 500 mg · kg −1 , commonly used in previous studies. Norethisterone, like progesterone, increases the activities of a few hydroxylases but generally suppresses

Rabbit liver mitochondria in the presence of NAD+ were found to catalyze the conversion of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 26-tetrol into 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid. The peroxisomal fraction did not catalyze the reaction. Sonication of the mitochondria or dialysis overnight against a hypotonic buffer increased the rate of oxidation twofold. Most of the enzyme activity was recovered in the supernatant fraction after centrifugation at 100,000xg of sonicated mitochondria. 4-Heptylpyrazole, an inhibitor of cytosolic ethanol dehydrogenase, inhibited the mitochondrial formation of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid by 70%. Disulfiram, an inhibitor of cytosolic acetaldehyde dehydrogenase, did not inhibit the reaction. The role of the mitochondrial dehydrogenase system in bile acid biosynthesis is discussed.

The identification of 3α,6β,11β,17,21-pentahydroxy-5β-pregnan-20-one (6β-hydroxy-THF) from the urine of the baboon ( Papio papio ) is described. Steroids were extracted from urine with Amberlite XAD-2 resin and the polar steroid fraction containing 6β-hydroxy-THF isolated by chromatography on Sephadex LH-20. The reaction products resulting from borohydride reduction, periodate oxidation and chromate oxidation were analysed by gas chromatography and gas chromatography-mass spectrometry.

Conditions are described for conversion of testosterone into the 3-enol, 17-bis-tert.-butyldimethylsilyl ether derivative without formation of side products. The steroid is treated with tert.- butyldimethylsilylimidazole in heptane at 100 degrees C using sodium formate as catalyst. Derivatives are also formed at different rates of 3-keto-5 alpha, 3 alpha/beta-hydroxy-, 6 alpha/beta-hydroxy-, 7 beta-hydroxy-, 16 alpha-hydroxy-, 17 beta-hydroxy(sec.)- and 20 alpha/beta-hydroxysteroids, whereas hydroxyl groups in 1 beta, 7 alpha, 12 alpha/beta, 15 beta and 17 alpha(tert.) positions do not react to a significant extent. These positions are derivatized by subsequent addition of trimethylsilylimidazole , yielding mixed derivatives which are suitable for gas chromatography--mass spectrometry with selected ion monitoring. Conditions are given for conversion of some biologically important androgens, progestins and bile acids into a single form of derivative. The use of the method is illustrated by an analysis of steroids in a rat testis.

A method for the isolation of 3-ketosteroids based on the positive charge of their oximes is described. The biological extract is filtered through a column of sulphoethyl Sephadex LH-20 (H+). Steroids in the filtrate are converted into oximes and the dried reaction mixture is filtered through a column of diethylaminohydroxypropyl Sephadex LH-20 (OH−) in methanol. After evaporation of the solvent and hydroxylamine, the oximes are taken up by and separated on a column of sulphoethyl Sephadex LH-20 (H+) in methanol. Following elution of other steroids, the oximes of 3-ketosteroids are eluted as a group with methanol-pyridine (20:1, v/v), and are converted into trimethylsilyl ethers. Removal of reagents and further purification of the sample is achieved by rapid filtration through Lipidex 5000 in n-hexane-pyridine-hexamethyldisilazane-dimethoxypropane (97:1:2:10). The derivatives are then analyzed by computerized gas chromatography-mass spectrometry using open-tubular glass capillary columns. Recoveries of picogram amounts of 3H-labelled steroids carried through the entire isolation procedure are 80–90%. The purification achieved in analyses of plasma permits solid injection of the equivalent of 1–2 ml of plasma without overloading of the capillary column. The principle of the isolation procedure might be applicable to other groups of compounds that posses keto or aldehydo groups.

A general method for extraction and purification of ethynyl steroids in urine and plasma prior to GC/MS analysis is described. Twenty ml of urine or 10 ml of plasma is extracted on a column of Amberlite XAD-2 (3 g). The extract is filtered in 72% methanol through SP-Sephadex in H+-form (0.4 g) to remove organic cations. Neutral steroids, less polar lipids, phenolic steroids and conjugated steroids are then separated into groups by passage of the effluent through a column of TEAP-Lipidex 5000 (0.65 g). Fractions containing neutral and phenolic steroids are filtered through SP-Sephadex in Ag+-form (0.15 g). Ethynyl steroids are selectively retained by the ion exchanger and are eluted by displacement with 1-hexyne. Fractions containing conjugated steroids are hydrolyzed, and the liberated steroids are extracted with Amberlite XAD-2. Separation of neutral and phenolic steroids and isolation of ethynyl steroids is then performed as above. Prior to GC/MS, phenolic steroids are converted into TMS ethers and neutral steroids into MO-TMS derivatives, which are purified by rapid filtration through Lipidex 5000 (0.25 g). Recoveries of unconjugated ethynyl steroids added to urine or plasma were 75–90%. A simplified method is also described which can be used when separation of neutral, phenolic and conjugated steroids is not required. Unconjugated steroids of low and medium polarity are extracted from urine or an enzyme hydrolyzate by filtration through Lipidex 1000. Following elution with methanol and filtration through SP-Sephadex in H+-form, ethynyl steroids are isolated on SP-Sephadex in Ag+-form. MO-TMS derivatives are prepared and purified on Lipidex 5000. Recoveries of unconjugated steroids added to urine were 80–90%. GC/MS analysis is performed using an OV-1 or SE-30 open tubular capillary column and single ion detection. A combination of external and internal standards is used. The limit of detection of unconjugated ethynylestradiol in urine was about 5 pg/ml. The precision of quantitation was influenced by a nonspecific carrier effect of compounds present in biological extracts. The separator or ion source appeared to be the main site of the effect, which was minimized when highly purified fractions were analyzed. Examples of analyses of urine from a healthy woman taking 250 μg (−)-norgestrel and 50 μg ethynylestradiol daily are presented.

A new method for the extraction of bile acids from aqueous solutions, urine, plasma, and bile is described. A buffered solution of decyltrimethylammonium bromide is added to the sample to give a 0.03 m concentration of the counter-ion. The mixture is passed through a bed of Lipidex 1000, which is then washed with the buffered solution of counter-ion followed by water. The decyltrimethylammonium salts of bile acids are sorbed by the Lipidex and are eluted with methanol. Recoveries of unconjugated, taurine- and glycine-conjugated, sulfated, and glucuronidated bile acids are close to 100%. Unconjugated bile acids can also be quantitatively extracted from aqueous solutions and urine after acidification with acetic acid.

A method is described for the separation of ketonic from non-ketonic bile acids. Oximes are prepared from the methyl esters and are subjected to chromatography on the lipophilic strong cation exchanger sulphohydroxypropyl Sephadex LH-20 (SP-LH-20) in the H+ form. Three fractions are obtained which contain non-ketonic compounds, compounds having a 7- or 12-oxime but no 3-oxime group, and compounds having a 3-oxime group. These groups can be analysed as trimethylsilyl ethers by gas chromatography and gas chromatography-mass spectrometry by using fused-silica capillary columns with SE-30 as the stationary phase. The method was applied to the analysis of bile acids in faeces. The major 3-oxo acids found were 3-oxo-5 beta- and 12 alpha-hydroxy-3-oxo-5 beta-cholanoic acids. Smaller amounts of 3,12-dioxo-5 beta-cholanoic acid were present and 3-oxo-5 alpha-cholanoic and 3-oxo-4-cholenoic acids were also identified, but could not be quantified. Semiquantitative analyses indicated that bile acids with a 3-oxo group may constitute 1-20% of the corresponding 3 alpha-hydroxy bile acids.

Metabolic profiles of steroids in urine and plasma were analyzed in 14 male and four female alcoholics during withdrawal. The daily excretion of 30 conjugated steroids in urine and the concentration of 13 steroid sulfates in plasma were measured on days 1, 7 and 29 of the period of observation, which started on day 5-7 of abstinence. While the total excretion of cortisol metabolites was normal in most cases, the profiles of metabolites were changed in the alcoholics during the period of observation. The ratio between tetrahydrocortisol and tetrahydrocortisone exceeded the mean normal value by more than one standard deviation in 97% of the samples analyzed. The same was true of the ratio between 20-hydroxy and 20-oxosteroids in 90% of the samples. The differences between alcoholic and healthy subjects were statistically significant (p less than 0.001). The major change in plasma was a significantly increased concentration of 5-androstene-3 beta, 17 beta-diol disulfate on the first day of the study. The concentration decreased to normal values during the first month of withdrawal. The rate of excretion of this steroid in urine was increased in half of the patients and also decreased with time. The rate of excretion and the degree of fatty infiltration in liver biopsies were positively correlated. It is suggested that the ratios between cortisol metabolites in urine might be of value as biochemical markers in alcoholism, and that the absolute or relative concentrations of steroid disulfates in plasma might serve as an indicator of recent alcohol intake.

Work carried out in this laboratory aiming at the development of generally applicable methods for analysis of steroids in biological materials is reviewed. Isolation and separation of groups of steroids is achieved by chromatography in miscible solvent systems on neutral or ion exchanging lipophilic derivatives of Sephadex. Reversed or straight-phase systems can be designed to obtain columns which act as filters to remove unwanted material. The hydrolyzed steroids are converted to O-methyloxime-trimethylsilyl ether derivatives which are purified by liquid-gel chromatography and analyzed by computerized gas chromatography-mass spectrometry. Repetitive magnetic scanning of the entire mass range or repetitive accelerating voltage scanning of a limited mass range is used to monitor individual fragment ion currents. Spectra are recorded on magnetic tape, and peaks in fragment ion current chromatograms are located by the computer which performs a preliminary search for potential molecular ions in spectra taken at the located peaks. Quantitative determinations are based on areas of peaks with correct retention time in chromatograms based on the current of specific fragment ions. Similar methods are used to determine the number and abundance of heavy atoms in steroids formed from precursors labelled with stable isotopes and in metabolites formed from steroids labelled with stable isotopes in specific positions.

Incorporation of deuterium into different positions of individual molecular species of biliary phosphatidylcholines was determined in bile fistula rats given [1,1-2H2]ethanol at a constant rate for 24 h. The distribution of deuterium between the different positions of the glycerol moiety was the same for all major molecular species. Based on this distribution and on the isotopic composition of the glycerol moiety, the deuterium excess in phosphatidylcholine molecules synthesized de novo could be calculated. The values obtained were the same for all molecular species analyzed and remained constant throughout the experiment. The deuterium excess was 35–55 atom % at C-2, 18–20 atom % at C-3 and 8–10 atom % at C-1. Administration of glucose and fructose decreased the deuterium excess at C-3 to 13–15 and 2–4 atom %, respectively, but did not change it at C-1 or C-2. It was concluded that all molecular species shared the same precursor pools of dihydroxyacetone phosphate and pyridine nucleotide, and that these pools were closely coupled to ethanol oxidation. The deuterium labelling at C-3 compared with that of potential precursors, as well as the effects of glucose and fructose on the labelling at C-3, indicated that at least 65–70% of the glycerol moieties of the phosphatidylcholines were formed from malate via a gluconeogenetic pathway. About 20–30% of the palmitoyl residues were synthesized de novo. This value increased to 31–64% when glucose was administered and to 79–100% when fructose was given. This change of origin was accompanied by a marked increase of the 1-palmitoyl-2-palmitoleoyl, 1-palmitoyl-2-oleoyl, and 1-palmitoleoyl-2-oleoyl species, and a decrease of species having linoleoyl or arachidonoyl residues at C-2. The deuterium excess of the NADPH pool used in palmitic acid synthesis was calculated to be 5–10 atom %. The rates of deuterium incorporation indicated the presence of at least two pools of phosphatidylcholines with widely different turnover. The apparent half-life times of the glycerol moiety of molecular species in the pool having a rapid turnover varied between about 1 h (1,2-dipalmitoyl species) and about 8 h (1-stearoyl-2-linoleoyl species). The apparent half-life times of 1-palmitoyl residues in different molecular species were about 4 and 10 h in rats given glucose and fructose, respectively. The values were the same for all species studied, indicating that labelled palmitoyl residues in different molecular species were derived from the same palmitic acid pool.

A method using the lipophilic gel Lipidex® 5000 for sorption of pentachlorophenol (PCP) from water and urine is described. The procedure for water gives a simultaneous clean-up from lipophilic contaminants and also offers the possibility for determination of less polar compounds, such as 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (p,p′-DDT) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), collected in a separate fraction. The extracted PCP is derivatized to pentachlorophenyl acetate, which is determined by electron-capture gas chromatography. The average recovery of 0.01–0.15 ng PCP per ml water was 96% and of labelled p,p′-DDT (ca. 5 ng/ml) and TCDD (ca. 0.001 ng/ml) 95 and 92%, respectively. A similar method for enrichment of PCP was applied to acidified urine and to urine hydrolysed with hydrochloric acid or with digestive juice from Helix pomatia. Recoveries of 0.1–2.5 ng PCP per ml non-hydrolysed urine were on an average 92% and of 2.5–10 ng PCP per ml hydrolysed urine 96%. The analyses indicate that PCP in urine from non-occupationally exposed persons is originally conjugated and to some extent liberated when stored. The contamination of organic solvents and laboratory environments with PCP is discussed.

The concentrations and turnover of estradiol isolated from cytosolic and nuclear fractions of uteri from ovariectomized rats given estradiol, either in single injections or in continuous infusion, were analyzed by gas chromatography-mass spectrometry. The analytical method was validated for different organs and lower limits of analysis were established. After infusion of 20 ng x h-1 for 18-22 h, mean estradiol levels were 2.0-2.4 fmol x mg-1 uterine wet weight in the nuclear fraction, and 1.2-1.5 fmol x mg-1 in the cytosolic fraction. The concentrations were about five times higher after a single injection of one microgram estradiol but the distribution between nuclear and cytosolic fractions was almost the same. The concentrations of estradiol in nuclei from liver and spleen were 50-200 times lower than those in uterus. Taken together with previous knowledge, the results indicate that the distributions of estradiol and its receptor are not the same and that hormone response cannot be predicted from the concentration of receptors alone. The exchange of estradiol molecules in the uterus was followed after a change of the infusion from unlabelled to [11,12,12-2H3]-labelled estradiol, or vice versa. The uterine uptake of estradiol was calculated to be about 0.7 fmol x h-1 x mg-1 uterine wet weight. The half-life time was calculated to be at least 4 h for estradiol molecules isolated from the nuclear fraction and 3 h (significantly shorter) for those isolated from the cytosolic fraction. The results indicate an uptake of 40-90% of all estradiol passing through the uterus in proestrus with only about 10% of available receptors becoming occupied. When the infusion was changed from estradiol to ethynylestradiol, estradiol disappeared from the uterus at the same rate as in the experiments above. Ethynylestradiol was taken up at a rate of about 0.3-0.4 fmol x h-1 x mg-1 tissue. The percentage of total steroid found in the nuclear fraction was higher for ethynylestradiol, about 70%, than for estradiol, about 60%, indicative of a more stable association of receptor to nuclear binding sites when ethynylestradiol is the ligand.

Cholesterol was shown to be the precursor of 27-nor-5β-cholestane-3α,7α,12α,24,25-pentol which is the major bile alcohol in human urine. 4-[14C]-Labelled cholesterol and β-sitosterol were administered to patients with primary biliary cirrhosis. Urine was extracted with Amberlite XAD-2 and sterol glucuronides and bile acid conjugates were isolated by ion exchange chromatography on Lipidex-DEAP. Following hydrolysis and further purification on Lipidex-DEAP, the C26 bile alcohol and methyl esters of cholic and chenodeoxycholic acids were isolated by HPLC. The specific radioactivity of the C26-pentol was the same as that of cholic acid after administration of [14C]-cholesterol. In contrast, little if any radioactivity could be detected in the C26-pentol after administration of labelled β-sitosterol.

Liver alcohol dehydrogenase (EC 1.1.1.1) is believed to catalyze the oxidation of 26-hydroxylated intermediates in the biosynthesis of bile acids from cholesterol. We have therefore analyzed the composition and size of the bile acid pool in deer-mice genetically lacking alcohol dehydrogenase. Cholic acid was found to be the major primary bile acid accompanied by small amounts of chenodeoxycholic acid. Variable amounts of secondary bile acids were also present, mainly deoxycholic acid and 3 alpha, 12 alpha-dihydroxy-7-oxo-5 beta-cholanoic acid. The same bile acids were found in animals with normal levels of alcohol dehydrogenase. The pool of bile acids in the gallbladder, small intestine and large intestine varied between 4.2 and 8.4 mumol in four animals lacking alcohol dehydrogenase and between 6.0 and 8.4 mumol in four control animals. Ethanol did not influence pool size or composition of bile acids in the animal studied. It is concluded that alcohol dehydrogenase is not obligatory for normal bile acid biosynthesis.