Serum level of 19-hydroxyandrostenedione during pregnancy and at delivery determined by gas chromatography/mass spectrometry

19-Hydroxyandrostenedione (19-OHA) is secreted from the adrenal glands in men and women and also from the placenta during pregnancy. It has been found to cause hypertension in animal models. We have synthesized [7,7-2H2]-19-OHA with high deuterium content and, together with [7,7-2H2]A and [9,11-2H2]estrone (E1), have developed a quantitative assay of serum level 19-OHA, A, and E1 using the gas chromatography/mass spectrometry-mass fragmentography method to monitor individual subjects throughout pregnancy. The labeled 19-OHA, used as internal standard, showed only 6.73% of unlabeled compound. Recovery of standard 19-OHA, A, and E1 (5,000 pg each) added to male plasma was 97.4 +/- 2.3%, 96.3 +/- 2.1%, and 100.1 +/- 4.1% (mean +/- SD), respectively; the intraassay coefficient of variation was 2.1%, 3.5%, and 3.8%, respectively. Ten pregnant subjects without complications and 10 pregnant subjects near term with hypertension were selected (with informed consent). The 19-OHA and E1 serum concentrations of maternal venous blood from uncomplicated pregnancies increased significantly as gestation progressed (19-OHA: first trimester, 225 +/- 72; second trimester, 656 +/- 325; third trimester, 1,518 +/- 544 pg/ml), reaching the highest level at delivery (19-OHA: 1,735 +/- 684 pg/ml). Whereas a positive correlation was found between the level of 19-OHA and E1, no apparent change of the A level was observed during pregnancy. Levels of the three steroid hormones in pregnancy complicated by hypertension in the second and third trimester were not found to be significantly different from those of normal pregnancy (19-OHA of hypertensive subjects: second trimester, 762 +/- 349; third trimester, 1,473 +/- 491 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of c-2, 3, 17 and 19-oxygenated androgens

The synthesis of C19-androgens oxygenated at carbons 2, 3, 17 and 19 and of their deuterium or deuterium-tritium labeled analogs is described.     

Aromatase and other inhibitors in breast and prostatic cancer

Estrogens have an important role in the growth of breast and other hormone-sensitive cancers. We have shown that 4-hydroxyandrostenedione (4-OHA) selectively blocks estrogen synthesis by inhibiting aromatase activity in ovarian and peripheral tissues and reduces plasma estrogen levels in rat and non-human primate species. In postmenopausal men and women, estrogens are mainly of peripheral origin. When postmenopausal breast cancer patients were administered either by daily oral or parenteral weekly treatment with 4-OHA, plasma estrogen concentrations were significantly reduced. Complete or partial response to treatment occurred in 34% of 100 patients with advanced breast cancer, while the disease was stabilized in 12%. We recently studied the effects of 4-OHA and other aromatase inhibitors, 10-propargylestr-4-ene-3,17-dione (PED) and imidazo[1,5-]3,4,5,6-tetrahydropyrin-6-yl-(4-benzonitrile) (CGS 16949A) as well as 5-reductase inhibitors, N,N-diethyl-4-methyl-3-oxo-4-aza-5-androstane-17β-carboxyamide (4-MA) and 17β-hydroxy-4-aza-4-methyl-19norandrost-5-en-3-one (L651190) in prostatic tissue from 11 patients with prostatic cancer and six patients with benign prostatic hypertrophy (BPH), and from normal men at autopsy. We attempted to measure aromatase activity in tissue incubation by quantitating ³H₂O released during aromatization of androstenedione or testosterone labeled at the C-1 position. The amount of ³H₂O released from all samples was at least twice that of the heat inactivated tissue samples. The ³H₂O release was significantly inhibited by 4-OHA and 4-MA, but not by the other aromatase inhibitors. However, when HPLC and TLC were used to isolate steroid products, no estrone or estradiol was detected in the incubates. Furthermore, no aromatase mRNA was detected following amplification by PCR. The 4-OHA was found to inhibit 5-reductase in both BPH and cancer tissue, although to a lesser extent than 4-MA. The other aromatase inhibitors were without effect. Although a mechanism involving intraprostatic aromatase is not likely, inhibitors may act to reduce peripherally-formed estrogens. In postmenopausal breast cancer, the results indicate that 4-OHA is of significant benefit.     

The aromatase inhibitor 4-hydroxyandrostenedione inhibits the formation of 19-norandrostenedione by porcine grakulosa cells in vitro {Poster 32}

Porcine granulosa cells convert androstenedione (A) to 19-norandrostenedione (19-norA) and 19-hydroxyandrostenedione (19-OHA) in the presence of FSH and 10% porcine serum; 19-norA is also formed from independent incubations with 19-OHA. 19-NorA and 19-OHA formation from A, and 19-norA from 19-OHA, is blocked by 4-hydroxyandrostenedione, an irreversible inhibitor of aromatase.     

Age-related changes in 11β-hydroxyandrostenedione concentration in normal and osteoporotic women

The secretion of dehydroepiandrosterone (DHEA) and its sulfate is known to decline gradually with advancing age. Furthermore DHEA is known to be significantly lower in osteoporotic subjects than in normals. Recently 11β-hydroxyandrostenedione (11-OHA) has been proposed as an important indicator of the adrenal source of hormone excess in different hyperandrogenic states. In the present study we measured 11-OHA in 224 normal women aged 20–79 yr and 130 osteoporotic women aged 40–79 yr. RIA of 11-OHA was performed with highly specific antiserum raised in rabbits.

The mean 11-OHA serum concentration was 2.20±0.90 ng/ml in normal women and 1.75±0.58 ng/ml in osteoporotic women. In contrast to DHEA there was no age-related decrease in 11-OHA serum concentrations in normal and osteoporotic women. Osteoporotic subjects showed statistically significantly lower 11-OHA serum concentrations than normal women. Therefore low serum 11-OHA might represent a further risk factor for osteoporosis.     

Metabolism of coenzyme Q10: biliary and urinary excretion study in guinea pigs.

Biliary and urinary metabolites were examined after intravenous administration of 14C-coenzyme Q10 (14C-CoQ) to guinea pigs. Cumulative recovery of administered radioactivity for up to 8 hours by bile drainage was 4.8%. The greater part of radioactivity was detected in conjugate form. After hydrolyzing with beta-glucuronidase, aglycone fragments were subjected to methylation and reductive acetylation. The main metabolite was demonstrated to be Q acid-1 1,4-hydroquinone diacetate methyl ester (M-1) on HPLC. Then, the main metabolite was assumed to be glucuronide of 2,3-dimethoxy-5-methyl-6-(3'-methyl-5'-carboxy-2'-pentenyl)-1, 4-benzohydroquinone [Q acid-I hydroquinone]. The cumulative urinary recovery of the administered radioactivity over 48 hours was 8.3%. The labeled samples were treated similarly to bile. The urinary metabolites of CoQ10 consisted of unconjugated and conjugated forms. Lyophilized urine was treated as a bile sample and analyzed. The two major metabolites were assigned to be M-1 and Q acid-II 1,4-hydroquinone diacetate methyl ester (M-2). Then, the two metabolites were assumed to be composed of Q acid-I and 2,3-dimethoxy-5-methyl-6-(3'-carboxypropyl)-1,4-benzoquinone (Q acid-II) in free and corresponding hydroquinone conjugate forms. To investigate the effect of exogenous labeled CoQ10 on unlabeled CoQ10 (endogenous) metabolites in urine, simultaneous quantitative determination was performed using deuterium labeled CoQ10 (CoQ10-d5). Urine collected over a 72-hour period after intravenous administration of CoQ10-d5 was processed similarly to that described above and two derivatized metabolites (M-1 and M-2) were quantified by gas chromatography-mass fragmentography with the multi-ion detection method. The analytical results showed that the addition of exogenous labeled CoQ10 did not influence the metabolism (or breakdown) of unlabeled (endogenous) CoQ10.     

Synthesis of deuterium labeled 17-methyl-testosterone

The synthesis of two forms of selectively deuterated 17-methyl-testosterone is described. 17-Methy1-d₃-testosterone was prepared by the Grignard reaction of dehydroepiandrosterone with deuterium labeled methyl magnesium iodide followed by an Oppenauer oxidation. 17-Methyl-d₃-testosterone-19, 19, 19-d₃ was prepared by treating 3, 3-ethylenedioxy-5, 10-epoxy-5α, 10α-estran-17-one with deuterium labeled methyl magnesium bromide followed by hydrolysis and dehydration of the 5α-hydroxyandrostane derivative.     

Dissociation of 19-hydroxy- 19-oxo-, and aromatizing-activities in human placental microsomes through the use of suicide substrates to aromatase

Suicide substrates of aromatase were used as chemical probes to determine if free 19-hydroxyandrost-4-ene-3,17-dione (19-OHA) and 19-oxoandrost-4-ene-3,17-dione (19-oxoA) are obligatory intermediates in the aromatization of androst-4-ene-3,17-dione (androstenedione) to oestrone by human placental aromatase. A radiometric-HPLC assay was used to monitor 19-hydroxy, 19-oxo-, and aromatized products formed in incubations of [14C]androstenedione and human placental microsomes. When microsomes were preincubated with the suicide substrates 10 beta-mercapto-estr-4-ene-3,17-dione (10 beta-SHnorA), or 17 beta-hydroxy-10 beta-mercaptoestr-4-ene-3-one (10 beta-SHnorT), it was found that 19-hydroxy-, 19-oxo- and aromatase activities were inhibited in parallel. However, when the suicide substrates 4-hydroxyandrost-4-ene-3,17-dione (4-OHA) and 19-mercaptoandrost-4-ene-3,17-dione (19-SHA) were preincubated with placental microsomes, significantly greater inhibition of formation of oestrogens was observed in comparison to the inhibition of formation of 19-hydroxy- and 19-oxo-metabolites. Furthermore, significantly more time-dependent inhibition of 19-oxoA formation was observed in comparison to inhibition of 19-OHA formation with these same inhibitors. These results suggest that 19-hydroxy- and 19-oxo-androstenediones are not free, obligatory intermediates in the aromatization of androstenedione by human placental aromatase, but rather are products of their own autonomous cytochrome P-450-dependent, microsomal enzymatic activities.     

Evidence against an abstraction or direct insertion mechanism for cytochrome P-450 catalysed meta hydroxylations

Warfarin, specifically labeled with deuterium in the 7 position, was incubated with liver microsomes from untreated rats or rats which were pretreated with either phenobarbital of β-napthoflavone. The four phenolic metabolites (6-, 7-, 8- and 4′-hydroxywarfarin) were isolated and quantitated by $\text{GC}\text{MS}$ and the percent deuterium retention calculated. In all induction states the 7-hydroxy metabolite of (7,²H)warfarin retained greater than 77% of the deuterium. These results suggest that hydroxylation at the 7 position (meta hydroxylation) cannot proceed by either a direct insertion or abstraction mechanism.     

Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123.

To detect intracellular oxidant formation during reoxygenation of anoxic endothelium, the oxidant-sensing fluorescent probes, 2',7'-dichlorodihydrofluorescein diacetate, dihydrorhodamine 123, or 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate were added to human umbilical vein endothelial cells during reoxygenation. None of these fluorescent probes were able to differentiate the controls from the reoxygenated cells in the confocal microscope. However, dihydrofluorescein diacetate demonstrated fluorescence of linear structures, consistent with mitochondria, in reoxygenated endothelium. This work tests the hypothesis that dihydrofluorescein diacetate is a better fluorescent probe for detecting intracellular oxidants because it is more reactive toward specific oxidizing species. To investigate this, dihydrofluorescein diacetate was exposed to various oxidizing species (hydrogen peroxide, superoxide [KO2], peroxynitrite, nitric oxide, horseradish peroxidase, ferric iron, xanthine oxidase, cytochrome c, and lipoxygenase) and compared with the three other popular probes. Though oxidized dihydrofluorescein has higher molar fluorescence, comparison of the reactions of dihydrofluorescein with these other three probes in a cell-free system indicates that dihydrofluorescein is sometimes less fluorescent than the other probes. In addition, we find that the reactivity of all of the probes is very complex. Based on the results reported here, it is no longer appropriate to think of these probes as detecting a specific oxidizing species in cells, such as H2O2, but rather as detectors of a broad range of oxidizing reactions that may be increased during intracellular oxidant stress. Cell-loading studies indicate that dihydrofluorescein achieves higher intracellular concentrations than the second brightest intracellular probe, 2',7'-dichlorodihydrofluorescein. This fact and its higher molar fluorescence may account for the superior brightness of dihydrofluorescein diacetate. Dihydrofluorescein diacetate may be a superior fluorescent probe for many cell-based studies.     

The influence of 4-hydroxy-4-androstene-3,17-dione on androgen metabolism and action in cultured human foreskin fibroblasts

4-hydroxy-4-androstene-3,17-dione (4-OHA) has been shown to be a potent inhibitor of aromatase activity. It is effective in the control of estrogen-dependent processes in female subjects and may potentially be useful in the treatment of estrogen-dependent processes in men. Human foreskin fibroblasts grown in cell culture provide a model to investigate the effects of 4-OHA on extraglandular aromatase activity as well as the ability of the compound to influence androgen receptor binding and the 5 alpha-reduction of testosterone (T). Initial experiments were carried out to determine the potency of 4-OHA in genital skin fibroblasts by incubating cells with 4-OHA over a range of concentrations. When aromatase activity was determined at a substrate concentration close to the apparent Km of the enzyme, a 44% inhibition of enzyme activity occurred at a mean concentration of 5 nM 4-OHA. Enzyme kinetic studies analyzed by Eadie-Hofstee plots demonstrated competitive inhibition by 4-OHA with a mean apparent Ki of 2.7 nM. When 5 alpha-reductase activity was determined in the presence of 200 nM [3H]T, in the absence or presence of 4-OHA, a 50% inhibition of enzyme activity occurred at an inhibitor concentration of 3 microM. In androgen receptor binding studies, 4-OHA possessed 1% of the affinity of dihydrotestosterone (DHT) for [3H]DHT binding sites. In summary: 4-OHA is a potent and specific inhibitor of aromatase activity in human genital skin fibroblasts, the affinity of the enzyme for 4-OHA being greater than its affinity for the substrate, androstenedione. The influence of 4-OHA on 5 alpha-reductase activity and androgen receptor binding is minimal.     

Synthesis of [19- 2H3]-analogs of dehydroepiandrosterone and pregnenolone and their sulfates

Deuterated analogs of pregnenolone and pregnenolone sulfate with three atoms of deuterium in position 19 were prepared. The synthetic approach was developed on derivatives of dehydroepiandrosterone, where initial intermediates were well characterized, and then applied to the pregnenolone series. Starting 19-hydroxy compounds were transformed into 3alpha,5-cycloderivatives to simplify the Jones oxidation into the corresponding 19-oic acids. After oxidation, rearrangement to 3-hydroxy-5-enes, and suitable protection, two deuterium atoms were introduced by lithium aluminum deuteride reduction. Mesylate exchange by iodide in the presence of zinc and deuterium oxide added third deuterium atom. Deprotection gave title analogs with about 93-95% content of d3-derivative, the rest was mainly not fully deuterated d2-analogue as followed from the mass spectra analysis. Thus, 3beta-hydroxy[19-2H3]androst-5-en-17-one was prepared in 14 steps from 19-hydroxy-17-oxoandrost-5-en-3beta-yl acetate in 8.9% yield, the analogous sequence in the pregnenolone series gave 3beta-hydroxy[19-2H3]pregn-5-en-20-one in 7.3% yield. Corresponding sulfates were prepared via pyridinium salts in 53 and 57% yields, respectively. Fully assigned NMR data of selected pregnenolone derivatives were given.     

A simple, rapid selected ion monitoring method for the determination of prostaglandins E2 and F2 alpha in human urine

A new procedure for the simultaneous measurement of prostaglandins E2 and F2 alpha in human urine is described. Quantification was achieved by gas chromatography mass spectrometry with selected ion monitoring and deuterium labeled internal standards. The levels of measured prostaglandins ranged from 25 pg ml-1 of urine (PGE2) to 2500 pg ml-1 (PGF2 alhpa). The interassay coefficient of variation, determined by replicate analysis of four identical samples, was 1.9% for PGE2 and 0.8% for PGE2 alpha. The procedure takes a fraction of the time needed with published methods and can be conducted with as little as one-tenth of the daily urinary output.     

Quantitation of gluconeogenesis by (2)H nuclear magnetic resonance analysis of plasma glucose following ingestion of (2)H(2)O

We present a simple (2)H NMR assay of the fractional contribution of gluconeogenesis to hepatic glucose output following ingestion of (2)H(2)O. The assay is based on the measurement of relative deuterium enrichment in hydrogens 2 and 3 of plasma glucose. Plasma glucose was enzymatically converted to gluconate, which displays fully resolved deuterium 2 and 3 resonances in its (2)H NMR spectrum at 14.1 T. The signal intensity of deuterium 3 relative to deuterium 2 in the gluconate derivative as quantitated by (2)H NMR was shown to provide a precise and accurate measurement of glucose enrichment in hydrogen 3 relative to hydrogen 2. This measurement was used to estimate the fractional contribution of gluconeogenesis to hepatic glucose output for two groups of rats; one group was fasted for 7 h and the other was fasted for 29 h. Rats were administered (2)H(2)O to enrich total body water to 5% over the last 4-5 h of each fasting period. For the 7-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.32 +/- 0.09 (n = 7). This indicates that gluconeogenesis contributed 32 +/- 9% of total hepatic glucose output with glycogenolysis contributing the remainder. For the 29-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.81 +/- 0.10 (n = 6), indicating that gluconeogenesis supplied the bulk of hepatic glucose output (81 +/- 10%).     

Biochemical aromatization of 2-methyleneandrostenedione: stereochemistry of hydrogen removal at the C-1 position

To explore a stereochemistry of hydrogen removal at C-1 of the powerful aromatase inhibitor 2-methyleneandrostenedione (1), of which the A-ring conformation is markedly different from that of the natural substrate androstenedione (AD), in the course of the aromatase-catalyzed A-ring aromatization producing 2-methylestrone (2), we synthesized [1-²H]labeled steroid 1 and its [1β-²H]stereoisomer, and the metabolic fate of the C-1 deuterium in aromatization was analyzed by gas chromatography–mass spectrometry (GC–MS) in each. Parallel experiments with the natural substrates [1-²H] and [1β-²H]ADs were also carried out. The GC–MS analysis indicated that 2-methyl estrogen 2 produced from [1-²H]labeled substrate 1 retained completely the 1-deuterium (1β-H elimination), while product 2 obtained from [1β-²H]isomer 1 lost completely the 1β-deuterium. Stereospecific 1β-hydrogen elimination was also observed in the parallel experiments with the labeled ADs as established previously. The results indicate that biochemical aromatization of the 2-methylene steroid 1 proceeds through the 1β-hydrogen removal concomitant with cleavage of the C₁₀–C₁₉ bond, yielding 1(10),4-dienone 9, in a similar manner to that involved in AD aromatization. This would give additional evidence for the stereomechanisms for the last step of aromatization of AD, requiring the stereospecific 1β-hydrogen abstraction and cleavage of the C₁₀–C₁₉ bond, and for the enolization of a carbonyl group at C-3 in the A-ring aromatization.     

共聚焦显微技术研究ABA诱导蚕豆气孔保卫细胞H2O2的产生（简报）

The methods of confocal laser scanning microscopy (CLSM) and microinjection were used to study ABA-induced H2O2 in guard cells (Vicia faba), which were labeled with H2O2 specific probe-2, 7-dichlorofluorescin diacetate(H2DCFDA). The results indicated 100 U/mL catalase (CAT) could inhibit partly stomatal closure induced by ABA. 10(-3) mmol/L ABA could significantly induce H2O2 production in chloroplast in guard cells of Vicia faba following microinjection, and 100 U/mL CAT could partly abolish the effects following simultaneous microinjection of ABA and CAT. These suggest that H2O2 is possibly involved in ABA signaling leading to stomatal closure.     

共聚焦显微技术研究ABA诱导蚕豆气孔保卫细胞H_2O_2的产生(简报)

逆境下,植物细胞内ABA含量急剧增加,同时植物也可通过一些酶代谢反应积累活性氧,如H_2O_2,O_2~-。ABA作为逆境信号对气孔运动的显著调节作用已被诸多实验所证实,但关于其对气孔运动调节的细节还知之甚少。H_2O_2作为氧化信号分子在植物抗病信号转导中已得到广泛研究,但H_2O_2是否介导保卫细胞的气孔运动还缺乏直接的证据。我们已初步发现H_2O_2可参与外源ABA诱     

The twin ion technique for detection of metabolites by gas chromatography-mass spectrometry: intermediates in estrogen biosynthesis

[4-¹⁴C + 7-D_0.44]Androstenedione and [4-¹⁴C + 7β-D_0.42]testosterone were prepared. When they were examined by mass spectrometry, the above proportion of deuterium and protium forms resulted in mass spectra in which the molecular ion (M⁺) and (M⁺ + 1) were of equal intensity. Fragment ions that contained deuterium were also twins. When doubly-labeled androstenedione and testosterone were used as substrates for the aromatizing enzymes of human placenta, the mass spectra of metabolites were characteristically labeled and thus readily distinguished from unlabeled material. Metabolites were quantitated by counting ¹⁴C. 17β,19-Dihydroxyandrost-4-en-3-one, 19-hydroxyandrost-4-ene-3,17-dione, 17β-hydroxy-3-oxoandrost-4-en-19-al, 3,17-dioxoandrost-4-en-19-al, estradiol-17β, and estrone were isolated, identified by their mass spectra, and quantitated following incubation of doubly-labeled androstenedione and testosterone with human placental microsomes.     

Variation of transition-state structure as a function of the nucleotide in reactions catalyzed by dehydrogenases. 2. Formate dehydrogenase

Since hydride transfer is completely rate limiting for yeast formate dehydrogenase [Blanchard, J.S., & Cleland, W. W. (1980) Biochemistry 19, 3543], the intrinsic isotope effects on this reaction are fully expressed. Primary deuterium, 13C, and 18O isotope effects in formate and the alpha-secondary deuterium isotope effect at C-4 of the nucleotide have been measured for nucleotide substrates with redox potentials varying from -0.320 (NAD) to -0.258 V (acetylpyridine-NAD). As the redox potential gets more positive, the primary deuterium isotope effect increases from 2.2 to 3.1, the primary 13C isotope effect decreases from 1.042 to 1.036, the alpha-secondary deuterium isotope effect drops from 1.23 to 1.06, and Vmax decreases. The 18O isotope effects increase from 1.005 to 1.008 per single 18O substitution in formate (these values are dominated by the normal isotope effect on the dehydration of formate during binding; pyridinealdehyde-NAD gives an inverse value, possibly because it is not fully dehydrated during binding). These isotope effects suggest a progression toward earlier transition states as the redox potential of the nucleotide becomes more positive, with NAD having a late and acetyl-pyridine-NAD a nearly symmetrical transition state. By contrast, the I2 oxidation of formate in dimethyl sulfoxide has a very early transition state (13k = 1.0154; Dk = 2.2; 18k = 0.9938), which becomes later as the proportion of water in the solvent increases (13k = 1.0265 in 40% dimethyl sulfoxide and 1.0362 in water). alpha-secondary deuterium isotope effects with formate dehydrogenase are decreased halfway to the equilibrium isotope effect when deuterated formate is the substrate, showing that the bending motion of the secondary hydrogen is coupled to hydride transfer in the transition state and that tunneling of the two hydrogens is involved. The 15N isotope effect of 1.07 for NAD labeled at N-1 of the nicotinamide ring suggests that N-1 becomes pyramidal during the reaction. 18O fractionation factors for formate ion relative to aqueous solution are 1.0016 in sodium formate crystal, 1.0042 bound to Dowex-1, and 1.0040 as an ion pair (probably hydrated) in CHCl3. The CO2 analogue azide binds about 10(4) times better than the formate analogue nitrate to enzyme-nucleotide complexes (even though the Ki values for both and the affinity for formate vary by 2 orders of magnitude among the various nucleotides), but the ratio is not sensitive to the redox potential of the nucleotide. Thus, not the nature of the transition state but rather the shape of the initial binding pocket for formate is determining the relative affinity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sterol synthesis. Preparation and characterization of fluorinated and deuterated analogs of oxygenated derivatives of cholesterol.

Oxygenated sterols, including both autoxidation products and sterol metabolites, have many important biological activities. Identification and quantitation of oxysterols by chromatographic and spectroscopic methods is greatly facilitated by the availability of authentic standards, and deuterated and fluorinated analogs are valuable as internal standards for quantitation. We describe the preparation, purification and characterization of 43 oxygenated sterols, including the 4 beta-hydroxy, 7 alpha-hydroxy, 7 beta-hydroxy, 7-keto, and 19-hydroxy derivatives of cholesterol and their analogs with 25,26,26,26,27,27,27-heptafluoro (F7) and 26,26,26,27,27,27-hexadeuterio (d6) substitution. The 7 alpha-hydroxy, 7 beta-hydroxy, and 7-keto derivatives of (25R)-cholest-5-ene-3 beta, 26-diol (1d) and their 16,16-dideuterio analogs were also prepared. These d2-26-hydroxysterols and [16,16-2H2]-(25R)-cholest-5-ene-3 beta, 26-diol (1e) were synthesized from [16,16-2H2]-(25R)-cholest-5-ene-3 beta, 26-diol diacetate (2e), which can be prepared from diosgenin. The highly specific deuterium incorporation at C-16 in 1e and 2e should be useful in mass spectral analysis of 26-hydroxycholesterol samples by isotope dilution methods. The delta 5-3 beta, 7 alpha, 26- and delta 5-3 beta, 7 beta, 26-triols were regioselectively oxidized/isomerized to the corresponding delta 4-3-ketosteroids with cholesterol oxidase. Also described are 5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol, its 5 beta,6 beta-isomer, cholestane-3 beta, 5 alpha,6 beta-triol, their F7 and d6 derivatives, and d3-25-hydroxycholesterol, which was prepared from 3 beta-acetoxy-27-norcholest-5-en-25-one (30). The 43 oxysterols and most synthetic intermediates were isolated in high purity and characterized by chromatographic and spectroscopic methods, including mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Detailed mass spectral assignments are presented, and 1H NMR stereochemical assignments are derived for the C-19 protons of 19-hydroxysterols and for the side-chain protons of 30.