Separation and computerized gas chromatography-mass spectrometry of unconjugated neutral steroids in plasma
Abstract
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.
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Plasma levels of ursodeoxycholic acid in black bears, Ursus americanus: Seasonal changes
2006, Comparative Biochemistry and Physiology - C Toxicology and PharmacologyTo date, no other studies have examined the seasonal changes in circulating levels of various bile acids in the plasma of wild North American black bears, Ursus americanus. Using gas chromatography, bile acid concentrations were measured in plasma samples obtained during either early or late hibernation, and during summer active periods. Thus, specific compositional changes from individual animals were examined through a given year. Total bile acid concentrations in the plasma of these normal animals were found to range between 0.2 and 3.1 μmol/L (0.9 ± 0.2 μmol/L, mean ± SEM). Cholic, ursodeoxycholic and chenodeoxycholic acids were the major bile acid species identified. Ursodeoxycholic acid represented 28.0 ± 2.6% of the total bile acid pool. Deoxycholic and lithocholic acids were found only in small amounts. In addition, total bile acid concentrations were lower in plasma samples obtained during hibernation compared with those obtained during summer active periods (0.6 ± 0.1 and 1.2 ± 0.4 μmol/L, respectively; p < 0.05). However, the relative proportion of ursodeoxycholic acid, was significantly greater in winter than in summer (31.5 ± 3.2% and 22.2 ± 4.5%, p < 0.05). Finally, taurine-conjugated bile acids were the predominant species in bear plasma, accounting for > 67% of the total bile acids. These data demonstrate that ursodeoxycholic acid is a major bile acid in black bear plasma, mostly conjugated with taurine. Further, the finding of seasonal variation in plasma bile acid composition provides evidence to support the possible role that ursodeoxycholic acid may play in cellular protection in hibernating black bears.
The implications of 7-dehydrosterol-7-reductase deficiency (Smith-Lemli-Opitz syndrome) to neurosteroid production
2004, SteroidsSmith–Lemli–Opitz syndrome (SLOS) is an autosomal recessive, multiple malformation/mental retardation syndrome with an estimated incidence among individuals of European ancestry of 1 in 20,000 to 1 in 30,000. It is caused by inactivity of the enzyme 7-dehydrosterol-Δ7-reductase, which catalyses the terminal transformation in cholesterol synthesis. Patients show not only an increased level of 7-dehydrocholesterol in blood and tissues, but also increased 8-dehydrocholesterol because of the presence of an active Δ8-Δ7 isomerase. A major consequence of these biochemical abnormalities is the alteration of normal embryonic and fetal somatic development causing postnatal abnormalities of growth, learning, language and behavior. While deficient cholesterol during early development is the primary cause of central nervous system (CNS) abnormalities and retardation, we questioned whether neurosteroids could also be involved since they can have a profound influence on behavioral characteristics. Disordered neurosteroidogenesis would be expected in SLOS and could be caused by a deficiency in classical neurosteroid synthesis secondary to cholesterol deficiency, or by synthesis from 7- and 8-dehydrocholesterol of novel neurosteroids with Δ7 or Δ8 unsaturation which may have altered activity compared with conventional neurosteroids. In particular we sought analogues of dehydroepiandrosterone sulfate, pregnenolone sulfate and the pregnanolone epimers. We targeted urine from post-pubertal females, as this type of sample would be most likely to yield identifiable amounts of the pregnanolone metabolites of progesterone. Analysis by GC/MS of urinary steroids excreted by post-pubertal females confirmed the presence of neurosteroid-like compounds in SLOS patient’s urine. Even though the new neuroactive steroids identified were unlikely to have been formed in the brain, it is likely that mechanisms for their synthesis are operable in this organ.
Identification of 7(8) and 8(9) unsaturated adrenal steroid metabolites produced by patients with 7-dehydrosterol-Δ<sup>7</sup>-reductase deficiency (Smith-Lemli-Opitz syndrome)
2002, Journal of Steroid Biochemistry and Molecular BiologyPatients with Smith–Lemli–Opitz syndrome have impaired ability to synthesize cholesterol due to attenuated activity of 7-dehydrosterol-Δ7-reductase which catalyses the final step in cholesterol synthesis. Accumulation of 7- and 8-dehydrocholesterol is a result of the disorder and potentially these sterols could be used as precursors of a novel class of Δ7 and Δ8 unsaturated adrenal steroids and their metabolites. In this study, we have analyzed urine from SLOS patients in the anticipation of characterizing such metabolites. Gas chromatography/mass spectrometry (GC/MS) was used in the identification of two major metabolites as 7- and 8-dehydroversions of the well-known steroid pregnanetriol. Other steroids, such as 8-dehydro dehydroepiandrosterone (8-dehydro DHEA) and 7- or 8-dehydroandrostenediol were also identified, and several more steroids are present in urine but remain uncharacterized. As yet, the study provides no evidence for the production of ring-B unsaturated metabolites of complex steroids, such as cortisol. We believe that the following transformations can utilize ring-B dehydroprecursors: StAR transport of cholesterol, p450 side chain cleavage, 17-hydroxylase/17,20-lyase, 3β-hydroxysteroid dehydrogenase, 3α-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase, 20α-hydroxysteroid dehydrogenase and 5β-reductase. We have yet to prove the activity of adrenal 21-hydroxylase, 11β-hydroxylase or 5α-reductase towards 7- or 8-dehydroprecursors.
Elevated levels of bile acids in colostrum of patients with cholestasis of pregnancy are decreased following ursodeoxycholic acid therapy
1998, Journal of HepatologyBackground/Aims: Intrahepatic cholestasis of pregnancy is characterised by increased levels of serum bile acids. Ursodeoxycholic acid therapy corrects the serum bile acid profile. The aims of this study were: (i) to investigate bile acid excretion into colostrum of women with intrahepatic cholestasis of pregnancy; (ii) to compare concentrations of bile acids in serum and colostrum of non-treated and ursodeoxycholic acid-treated patients; and (iii) to clarify wether ursodeoxycholic acid is eliminated into colostrum following treatment.
Methods: Bile acids were assessed by gas chromatography and high-performance liquid chromatography in serum collected at delivery, and in colostrum obtained at 2±1 days after labour, from patients with intrahepatic cholestasis of pregnancy, non-treated (n=9) and treated (n=7) with ursodeoxycholic acid (14 mg/kg bw per day, for 14±7 days) until parturition.
Results: The concentration of total bile acids in colostrum from patients with intrahepatic cholestasis of pregnancy was higher than in normals (23.3±14.8 μmol/l vs 0.7±μmol/l, p<0.01) and cholic acid was a major species (19.0±13.1 μmol/l), reflecting the elevated concentrationsf in maternal serum (48.9±21.0 μmol/l, total bile acids; 33.9±16.7 μmol/l, cholic acid. Following ursodeoxycholic acid administration, total bile acids and cholic acid levels in colostrum diminished to 5.7±2.5 μmol/l and 3.6±3.6±1.5 μmol/l, respectively; the proportion of cholic acid decreased (60.6±8.0% vs. 76.8|+-5.0%, p0.05). The ursodeoxychlic acid concentration in colostrum was maintained following treatment; its increased percentage (9.4±3.2% vs. 1.0±0.2%, p<0.01) was still lower than in maternal serum (20.8±3.6%, p<0.05). Only a small proportion (<1%) of lithocholic acid was found in colostrum following therapy.
Conclusions: Bile acid concentrations are elevated and cholic acid is the major species accumulating in colostrum, reflecting serum bile acid profiles in intrahepatic cholestasis of pregnancy. Ursodeoxycholic acid therapy decreases endogenous bile acid levels in colostrum.
Method for combined analysis of profiles of conjugated progesterone metabolites and bile acids in serum and urine of pregnant women
1997, Journal of Chromatography B: Biomedical ApplicationsA method for analysis of profiles of conjugated progesterone metabolites and bile acids in 10 ml of urine and 1–4 ml of serum from pregnant women is described. Total bile acids and neutral steroids from serum and urine were extracted with octadecylsilane-bonded silica. Groups of conjugates were separated on the lipophilic ion-exchanger triethylaminohydroxypropyl Sephadex LH-20 (TEAP-LH-20). Fractions were divided for steroid or bile acid analyses. Sequences of hydrolysis/ solvolysis and separations on TEAP-LH-20 permitted separate analyses of steroid glucuronides, monosulfates and disulfates and bile acid aminoacyl amidates, sulfates, glucuronides and sulfate-glucuronides. Radiolabelled compounds were added at different steps to monitor recoveries and completeness of separation, and hydrolysis/solvolysis of conjugates was monitored by fast-atom bombardment mass spectrometry. The extraction and solvolysis of steroid disulfates in urine were studied in detail, and extraction recoveries were found to be pH-dependent. Following methylation of bile acids, all compounds were analysed by capillary gas chromatography and gas chromatography—mass spectrometry of their trimethylsilyl ether derivatives. Semiquantification of individual compounds in each profile by gas—liquid chromatography had a coefficient of variation of less than 30%. The total analysis required 3 days for serum and 4 days for urine.
A method for the profiling of several important 3-oxo-4-ene urinary steroids is reported. The methodology is combined gas chromatography/mass spectrometry (GC/MS) utilizing stable isotope-labeled internal standards. The following standards were obtained or easily synthesized: [9,11,12,12-2H4]cortisol, [1,2-2H2] and [9,12,12-2H2]cortisone, [1,2-2H2]6β-hydroxycortisol, and [1,2-2H2]18-hydroxycortisol. We found the following excretions of free steroids for normal adult males and females: cortisol (males mean ± SD, 35 ± 13; females mean ± SD, 23 ± 13), cortisone (males mean ± SD, 58 ± 23; females mean ± SD, 50 ± 22), 6β-hydroxycortisol (males mean ± SD, 164 ± 59; females mean ± SD, 108 ± 55), and 18-hydroxycortisol (males mean ± SD, 148 ± 55; females mean ± SD, 71 ± 30). For 18-hydroxycortisol in particular, the excretions were much higher for males than for females. We found that the larger part of urinary cortisol and cortisone is not free but is released from conjugation by enzymes present in snail digestive juice. Using a pooled urine sample from an equal number of male and female subjects, we found that for cortisol 29% was excreted free, 28% as glucuronide and 43% as other conjugates (probably sulfates). For cortisone 41% was free, 45% β-glucuronide and 14% as other conjugates. Relatively little (3–8%) of the hydroxylated cortisols were excreted conjugated.