Esther Roitman

Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid

Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.

Androstanediol and 5-androstenediol profiling for detecting exogenously administered dihydrotestosterone, epitestosterone, and dehydroepiandrosterone: Potential use in gas chromatography isotope ratio mass spectrometry

The basis of a potential method for confirming intake of four natural androgens (testosterone, epitestosterone, dihydrotestosterone, and dehydroepiandrosterone is presented. The method relies on isolating from urine a steroid fraction containing androstenediol and androstanediol metabolites of these natural steroids and analyzing their 13C content by gas chromatography, combustion, isotope ratio mass spectrometry. The steroids were recovered from urine by conjugate hydrolysis with a Helix pomatia preparation (sulfatase and beta-glucuronidase), Girard T reagent separation to obtain a nonketonic fraction, and Sephadex LH-20 chromatography for purification. Metabolites appropriate for all of the natural steroids could be separated (as diacetates) by gas chromatography on a DB-17 capillary column viz.: 5 alpha (and beta)-androstane-3 alpha,17 alpha-diol (epitestosterone as precursor); 5 alpha (and beta)-androstane-3 alpha,17 beta-diol (testosterone as precursor); 5-androstene-3 beta,17 beta-diol (dehydroepiandrosterone precursor); and 5 alpha-androstane-3 alpha,17 beta- (and 17 alpha-) diol (dihydrotestosterone precursor). Measurement of the 13C content of the specific analytes after ingestion of the androgen precursors demonstrated a lowering of delta 13C/1000 value compared to normal values. Typically, in the male individual studied, delta 13C/1000 values for all components were -26 to -27 before drug administration and -29 to -30 at 6 h after, the latter values reflecting those obtaining for commercial synthetic steroid compared to in vivo synthesized steroid. While generally the metabolism of the steroids was as expected, this was not the case for 5 alpha-dihydrotestosterone. A major metabolite was 5 alpha-androstane-3 alpha,17 alpha-diol, which had presumably been formed by 17 beta/17 alpha isomerization, a process previously known for unnatural anabolics but not for natural hormones. The isolation, purification, and isotope ratio mass spectrometry techniques described may form the basis of a general method for confirming natural steroid misuse by sports participants.

Quantitation of cortisol and related 3-oxo-4-ene steroids in urine using gas chromatography/mass spectrometry with stable isotope-labeled internal standards

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]6 beta-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), 6 beta-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% beta-glucuronide and 14% as other conjugates. Relatively little (3-8%) of the hydroxylated cortisols were excreted conjugated.

Identification of 7(8) and 8(9) unsaturated adrenal steroid metabolites produced by patients with 7-dehydrosterol-Δ7-reductase deficiency (Smith-Lemli-Opitz syndrome)

Patients with Smith-Lemli-Opitz syndrome have impaired ability to synthesize cholesterol due to attenuated activity of 7-dehydrosterol-delta(7)-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 delta(7) and delta(8) 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, 3beta-hydroxysteroid dehydrogenase, 3alpha-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, 20alpha-hydroxysteroid dehydrogenase and 5beta-reductase. We have yet to prove the activity of adrenal 21-hydroxylase, 11beta-hydroxylase or 5alpha-reductase towards 7- or 8-dehydroprecursors.

Neonatal urinary steroids in Smith-Lemli-Opitz syndrome associated with 7-dehydrocholesterol reductase deficiency

The biosynthetic abnormality in Smith-Lemli-Opitz syndrome (SLOS) is a deficiency of 7-dehydrocholesterol (7DHC) reductase, the enzyme responsible for catalyzing the final step in the Kandutsch-Russell pathway for cholesterol synthesis. Because the disposition of 7DHC and 8-dehydrocholesterol [8DHC; cholesta-5,8(9)-dien-3beta-ol] produced in this syndrome is little understood, we have analyzed urine from three young infants by gas chromatography/mass spectrometry to characterize its steroid metabolites. All steroid metabolites of adrenal origin found in normal infant urine were also found in urine from the patients with SLOS but in reduced amount. Quantitatively, the major steroids in these SLOS patients were identified by mass spectrometry as homologs of normal neonatal steroids possessing an additional double bond. Generally, two forms of each steroid were present in a similar amount. Because of the markedly increased levels of 7DHC and 8DHC in SLOS, these almost certainly represented the 5,7 and 5,8(9) unsaturated forms of each metabolite. The most abundant steroids were tentatively identified as 3beta,16alpha-dihydroxy-5,7-pregnadien-20-one and 3beta,16alpha-dihydroxy-5,8(9)-pregnadien-20-one, although similar 21-hydroxylated steroids and homologs of 16alpha-hydroxy-DHEA were also found. This study shows that all enzymatic steps used by cholesterol in the DHEA synthetic pathway are also functional for 7DHC and 8DHC.

Steroid sulphatase deficiency is the major cause of extremely low oestriol production at mid-pregnancy: a urinary steroid assay for the discrimination of steroid sulphatase deficiency from other causes

A method for determining whether a pregnant woman with an extremely low serum oestriol (ELSE) measurement of mid‐trimester is carrying a fetus with steroid sulphatase deficiency or another more serious disorder is described. We undertook GC/MS analysis of steroids in random maternal urine samples and quantified oestriol, oestriol precursors (dehydroepiandrosterone (DHEA), 5‐androstene‐3β,17β‐diol, 16α‐hydroxy‐dehydroepiandrosterone and 5‐androstene‐3β,16α,17β‐triol), pregnanediol, and five other steroids largely unaffected by pregnancy (androsterone, etiocholanolone, tetrahydrocortisol, 5α‐tetrahydrocortisol and tetrahydrocortisone). Thirty‐two samples collected from seven normal pregnant women between the 7th and 27th week of pregnancy and 22 from individuals with ELSE were analysed. Diagnostic ratios of excreted products were developed. These included ratios of oestriol and oestriol precursors to the cumulative value for the five non‐pregnancy‐related steroids and ratios of oestriol and oestriol precursors to pregnanediol and to each other. Our data demonstrated high 3β‐hydroxy‐5‐ene steroid excretion in all ELSE patients together with low urinary oestriol excretion, a situation only consistent with deficiency of steroid sulphatase. The normal individuals had high oestriol and low excretion of oestriol precursors. No patient in our series showed the low oestriol levels and low oestriol precursor values that would indicate a fetal adrenal abnormality as the underlying defect.

Midgestational maternal urine steroid markers of fetal Smith-Lemli- Opitz (SLO) syndrome (7-dehydrocholesterol 7-reductase deficiency)

Smith-Lemli-Opitz syndrome (SLOS) is a malformation syndrome associated with 7-dehydrocholesterol (7DHC) 7-reductase deficiency. Although SLOS can be detected in an affected fetus before midpregnancy by measurement of 7DHC levels in amniotic fluid or chorionic villus cells, a noninvasive, more routine method is needed. Accordingly, this study was instigated to search for specific steroids in maternal urine in an affected pregnancy that reflect the 7-reductase deficiency of the fetus, ie, steroids retaining 7,8-unsaturation. Steroids were characterized by gas chromatography/mass spectrometry after urinary extraction, conjugate separation, and derivatization. Most steroids in maternal urine from a patient carrying a SLOS fetus were identified as progesterone metabolites, and these were entirely conventional, showing no evidence of additional unsaturation. Unsaturated homologues of the cortisol metabolites were also not detected. However, unsaturated homologues of pregnane-3,16,20-triols and pregnane-3,17,20-triol were found. Most likely, these are 7,8-unsaturated homologues, but 8,9-unsaturation is also possible because of the known activity of delta7-delta8-isomerase on 7DHC, which results in 8DHC being a prominent sterol in SLOS. Among these novel human steroids, the following were provisionally characterized: 5beta-pregn-7(or 8)-ene-3alpha,17alpha,20alpha-triol, 5beta-pregn-7(or 8)-ene-3alpha,16alpha,20alpha-triol, and 5alpha-pregn-7(or 8)-ene-3,16alpha,20alpha-triol. Confirmation of the position of unsaturation will require steroid synthesis. These novel steroids are not present in normal pregnancy urine and, therefore, are valuable for prenatal diagnosis of SLOS. In addition, separate studies have shown that 5beta-pregn-7(or 8)-ene-3alpha,17alpha,20alpha-triol is present in urine of children and adults with SLOS, and so is a useful analyte for confirmation of the disorder throughout life.

Estrogen biosynthesis: 2β-hydroxy-19-oxoandrost-4-ene-3,17-dione revisited

Current evidence is consistent with the view that biosynthesis of estrogens by human microsomal placental aromatase proceeds through several different pathways. One of the proposed mechanisms of aromatization of androgens involves the intermediacy of 2β-hydroxy-19-oxoandrost-4-ene-3.17-dione 1a. It is shown that incubation of 2β[18OH]-1b with placental aromatase gave HC18OOH which differs from previous observations. The incorporation of isotopic oxygen in the formic acid is consistent with the view that one of the alternative routes of estrogen elaboration may involve a 2β,19-dioxygenated androgen.

Biosynthesis of estrogens. Estr-5(10)-ene-3,17-dione: isolation, metabolism and mechanistic implications

The 16- 2H 2 title compound 5b constituted a significant amount of the non-aromatic metabolites recovered from incubations of 3,17-dioxo-[16,16,19- 2H 3]androst-4-en-19-al 1 with placental aromatase. For the evaluation of the role of compound 5b in the elaboration of estrogens, its transformations at pH 6.5 and 7.2 in the presence and absence of microsomal placental aromatase were investigated. In the presence of the aromatase at pH 6.5, estrogens (6.8%), products of isomerization of the double bond [Δ 5(10)→Δ 4] and products of reduction of the carbonyl groups were formed. When the incubation was carried out at pH 7.2, products similar to those obtained above were isolated but in different yields. Noticeably more estrogens (22.7%) and less of the reduced products were formed. Additionally, at pH 7.2, 10β-hydroxy-[16,16- 2H 2]estr-4-ene-3,17-dione 4a was obtained. In the absence of the aromatase, which was replaced with bovine albumin at both pH 6.5 and 7.2, [16,16- 2H 2]estr-4-ene-3,17-dione 3a and its 10β-hydroxy derivative 4a were formed in large amounts and were the only products detected. The ramifications of our observations in the context of estrogen biosynthesis are discussed.

New synthesis of 2β-hydroxy-19-oxoandrost-4-ene-3,17-dione and its 2β-18O analog

Abstract Treatment of 19-[oxygenated]-androst-4-ene-3,17-dione with Mn(AcO) 3 and ClCH 2 COOH in benzene gave epimeric mixtures of the corresponding 2ξ-chloroacetates and 2ξ-acetates. The products were processed to give the title compound. For the synthesis of the 2- 18 O analog, ClCH 2 C 18 OOH was used, which was prepared from ClCH 2 COCl.