Ion-pair high-performance liquid chromatography of bile salt conjugates: Application to pig bile

The high-performance liquid chromatographic separation and quantitation of conjugated bile salts from pig bile is reported. Synthetic standards and bile samples were chromatographed on a C₁₈ reversed phase column using acetonitrile/water/tetrabutyl ammonium phosphate as an isocratic mobile phase at a flow rate of 1 mL/min. Detection of the ion-pairs was at 214 nm. The method permits efficient separation of all conjugated pig biliary bile salts without prior modification or treatment of the samples. Analysis of 12 pig biles showed that 85% of the bile salts are conjugated to glycine. The three main conjugated bile salts were glyco-3α,6α,7α-trihydroxy-5β-cholanoic acid (GHC), glyco-3α,7α-dihydroxy-5β-cholanoic acid (GCDC), and glyco-3α,6α-dihydroxy-5β-cholanoic acid (GHDC). Glyco-3α-hydroxy-6-oxo-5β-cholanoic acid (G3α6oxo), tauro-3α,7α-dihydroxy-5β-cholanoic acid (TCDC), tauro-3α,6α,7α-trihydroxy-5β-cholanoic acid (THC), and tauro-3α,6α-dihydroxy-5β-cholanoic acid (THDC) were found to contribute each for 4 to 5% ot the total. An excellent correlation was found between the sum of conjugated bile salts quantitated by high-performance liquid chromatography (HPLC) and values obtained by conventional enzymatic assay. Simplicity, efficiency and relative rapidity of the method render it suitable for routine analyses.     

Composition of bile acids in ruminants

The bile acids found in sheep bile, beef bile, beef feces, sheep fetus bile, and beef fetus bile have been analyzed by using conventional techniques. Animals maintained on natural and purified diets were used. The bile acids are a complex mixture of isomeric hydroxy- and keto-5β-cholanoic acids which were substituted at one or several of the carbon atoms 3, 7, and 12. Cholic acid is the predominant bile acid found in these species. Deoxycholic acid was the major product formed from cholic acid when the animals were on a natural diet but the concentration of 3α, 12α-dihydroxy-7-keto-5β-cholanoic acid was elevated in the animals that were maintained on a high concentrated purified diet (without roughage). The fetus bile was found to contain nearly all of the bile acids found in the bile of the mature animal but in different concentrations.     

Bile acids in tissues: Binding of lithocholic acid to protein

Human liver contains two forms of lithocholic acid. One form is readily extractable by 95% ethanol/0.1% ammonia (soluble lithocholate, SL), while the other remains firmly bound to the residue (tissue-bound lithocholate, TBL). TBL could be hydrolytically released using clostridial cholanoylamino acid hydrolase, suggesting a peptide link between lithocholate and protein. With bovine serum albumin (BSA), lithocholic acid showed spontaneous amino group-modifying activity. When small molecular weight lysine (α-t-BOC-1-lysyl-β-naphthylamide) and arginine peptides (α-CBZ-di-arginyl-β-naphthylamide) were used in place of BSA, lithocholate bound specifically to the lysine peptide. The unusual affinity for lysine suggested that this amino acid might be involved as a residue in TBL. Synthesis of lithocholyl lysines and comparison with products of acid hydrolysis of TBL established ε-lithocholyl lysine as the predominant form in which lithocholic acid is found in tissue bound form. Supported in part by the Gomprecht Hepatitis Fund. The systematic nomenclature of bile acids referred to in this report by trivial names are as follows: Cholanic acid, 5β-cholan-24-oic acid; lithocholic acid, 3α-hydroxy-5β-cholan-24-oic acid; 3-epilithocholic acid, 3β-hydroxy-5β-cholan-24-oic acid; glycolithocholic acid, 3α-hydroxy-5β-cholan-24-oyl glycine; 3-ketocholanic acid, 3-keto-5β-cholan-24-oic acid; 12α-hydroxycholanic acid, 12α-hydroxy-5β-cholan-24-oic acid; chenodeoxycholic acid, 3α, 7α-dihydroxy-5β-cholan-24-oic acid; glycochenodeoxycholic acid, 3α, 7α-dihydroxy-5β-cholan-24-oyl glycine; deoxycholic acid, 3α, 12α-dihydroxy-5β-cholan-24-oic acid; glycodeoxycholic acid, 3α, 12α-dihydroxy-5β-cholan-24-oyl glycine; cholic acid, 3α, 7α, 12α-trihydroxy-5β-cholan-24-oic acid, glycocholic acid, 3α, 7α, 12α-trihydroxy-5β-cholan-24-oyl glycine; dehydrocholic acid, 3, 7, 12-triketo-5β-cholan-24-oic acid.     

Intracerebrally injected monohydroxy and other C24 steroid acids as demyelinating agents in the guinea pig

Sodium salts of lithocholic acid (3α-hydroxy-5β-cholanoic acid), 5β-cholanoic acid, Δ⁵-cholenoic acid and 3-keto-5β-cholanoic acid injected intracerebrally into guinea pigs in doses of 1 mg or higher produced periventricular demyelination. 24-¹⁴C-sodium lithocholate was rapidly released from the brain (only traces remained 2 hr after injection) if injected in quantities ranging from 2 μg to 5 mg. This rapid elimination is believed to account for the relatively high dose of lithocholate required for producing demyelination, and may also account for the limited demyelinating capacity of the other acids injected intracerebrally.     

Effect of chitosan feeding on intestinal bile acid metabolism in rats

The effect of chitosan feeding (for 21 days) on intestinal bile acids was studied in male rats. Serum cholesterol levels in rats fed a commercial diet low in cholesterol were decreased by chitosan supplementation. Chitosan inhibited the transformation of cholesterol to coprostanol without causing a qualitative change in fecal excretion of these neutral sterols. Increased fiber consumption did not increase fecal excretion of bile acids, but caused a marked change in fecal bile acid composition. Litcholic acid increased sigificantly, deoxycholic acid increased to a leasser extent, whereas hyodeoxycholic acid and the 6β-isomer and 5-epimeric 3α-hydroxy-6-keto-cholanoic acid(s) decreased. The pH in the cecum and colon became elevated by chitosan feeding which affected the conversion of primary bile acids to secondary bile acids in the large intestine. In the cecum, chitosan feeding increased the concentration of α-,β-, and ω-muricholic acids, and lithocholic acid. However, the levels of hyodeoxycholic acid and its 6β-isomer, of monohydroxy-monoketo-cholanoic acids, and of 3α, 6ξ, 7ξ-trihydroxy-cholanoic acid decreased. The data suggest that chitosan feeding affects the metabolism of intestinal bile acids in rats.     

Oxidation of 3-oxygenated Δ4- and Δ5-C27 steroids by soybean lipoxygenase and rat liver microsomessteroids by soybean lipoxygenase and rat liver microsomes

The formation of dioxygenated metabolites of cholesterol, epicholesterol (5-cholesten-3α-ol) 4-cholesten-3β-ol, 4-cholesten-3α-ol, 4-cholesten-3-one and 4-stigmasten-3-one was studied after incubations with soybean lipoxygenase and linoleic acid. From cholesterol and epicholesterol were formed the 7α-hydroxy-, 7α-hydroperoxy-, 7β-hydroxy-, 7β-hydroperoxy-, 7-oxo and 5,6-epoxyderivatives as well as 6β-hydroxy-4-cholesten-3-one. All Δ⁴-steroids were hydroxylated in the 6α- and 6β-positions. The ratios between the yields of 6β- and 6α-hydroxylated metabolites varied between 3∶1 and 2∶1. Incubations with 4-cholesten-3α-ol and 4-cholesten-3β-ol also afforded the 4,5-epoxides of these steroids. The ratios between the yields of the 4β,5β- and 4α,5α-epoxides were ca. 4∶1 for 4-cholesten 3β-ol and ca. 3∶2 for 4-cholesten-3α-ol. With iron-supplemented microsomes from rat liver, the compounds formed were qualitatively and quantitatively the same as with soybean lipoxygenase, whereas with 18,000 × g rat liver supernatant fractions the yields of all products formed—except 7α-hydroxycholesterol and 6β-hydroxy-4-cholesten-3-one—were markedly decreased. The results indicate the presence of a rat liver microsomal 6β-hydroxylase which can use 4-cholesten-3-one as a substrate and extend previous findings of similarities between soybean lipoxygenase and a nonspecific lipoxygenase in rat liver microsomes.     

Structure-retention correlation of isomeric bile acids in inclusion high-performance liquid chromatography with methyl β-cyclodextrin

The structure-retention correlation of various C₂₄ bile acid isomers was studied by the addition of methyl β-cyclodextrin (Me-β-CD) to mobile phases in reversed-phase high-performance liquid chromatography (HPLC). The compounds examined include a series of monosubstituted bile acids related to cholanoic acids differing from one another in the position and configuration of an oxygen-containing function (hydroxyl or oxo group) at the position C-3, C-6, C-7, or C-12 and the stereochemistry of the A/B-ring fusion (trans 5α-H and cis 5β-H) in the steroid nucleus. The inclusion HPLC with Me-β-CD was also applied to biologically important 4β- and 6-hydroxylated bile acids substituted by three to four hydroxyl groups in the 5β-steroid nucleus. These bile acid samples were converted into their fluorescence prelabeled 24-pyrenacyl ester derivatives and chromatographed on a Capcell Pak C₁₈ column eluted with methanol-water mixtures in the presence or absence of 5 mM Me-β-CD. The effects of Me-β-CD on the retentions of each compound were correlated quantitatively to the decreasing rate of capacity factors and the relative strength of host-guest inter-actions. On the basis of the retention data, specific and nonspecific hydrogen-bonding interactions between the bile acids and the Me-β-CD were discussed.     

Functionalization of unactivated carbons in 3α,6- and 3α,24-dihydroxy-5β-cholane derivatives by dimethyldioxirane

Direct remote functionalization of unactivated carbons by dimethyldioxirane (DMDO) was examined for 3α,6- and 3α,24-dihydroxy-5β-cholane derivatives. DMDO oxidation of stereoisomeric methyl 3α,6-diacetoxy-5β-cholanoates caused the direct, unexpected 14α- and 17α-hydroxylations, in analogy with that of the 5α-H analogs, regardless of the differences in stereochemical configuration of the A/B-ring junction and of the acetoxyl groups at C-3 and C-6. On the other hand, the ester derivatives of 3α,24-dihydroxy-5β-cholane with DMDO were transformed into the corresponding 5β-, 14α-, and 17α-hydroxy compounds, whereas the ether derivatives yielded the 5β-hydroxy, 3-oxo, and C-24 oxidized products, accompanied by their dehydrated ones.     

Esterified lipids of the freshwater dinoflagellatePeridinium lomnickii

Steryl esters, phytyl esters and triacylglycerols of a naturally occurring freshwater dinoflagellate,Peridinium lomnickii, were identified using capillary gas chromatography-mass spectrometry (GC-MS). Steryl esters differing in degree of unsaturation were separated, prior to analysis, by argentation thin layer chromatography. 5α(H)-Cholestanol was more dominant, relative to 4α-methylstanols, in steryl esters than in the free sterols, but the same sterol moieties occurred in both fractions. Monoenoic fatty acids were enriched in the steryl esters relative to the free fatty acids. Major acyl groups in steryl esters were 16∶0 or 20∶1, with smaller amounts of 14∶0 and 18∶1. In triacylglycerols the acyl moieties were 14∶0, 16∶0, 18∶1, 16∶1 and 12∶0, in order of decreasing abundance. Phytyl esters, previously inferred to occur in a marine dinoflagellate only by analysis of transesterified products, were identified by GC-MS comparison with authentic compounds. Direct analysis of these esterified lipids has not been reported for freshwater phytoplankton. The 4α-methylstanyl esters, 5α(H)-cholestan-3β-yl esters and phytyl esters occurring inP. lomnickii are further features in common with marine dinoflagellates, additional to the 4α-methylsterols reported previously.     

A new route to steroid ring C aromatization from readily available precursors

3β-Acetoxy-8α,9α-epoxy-5α-cholest-14-ene (1); 3β-acetoxy-14α,15α-epoxy-5α-cholest-8-ene (2); 3β-acetoxy-5α-cholest-8(14)-ene-9α,15α-diol (3); and 3β-acetoxy-5α-cholesta-8(14),9(11)-dien-15α-ol (4) have been aromatized to a 9∶1 mixture of 3β-hydroxy-12-methyl-18-nor-5α,17β(H)-cholesta-8,11,13-triene (5a) and 3β-hydroxy-12-methyl-18-nor-5α,17α(H)-cholesta-8,11,13-triene (5b) in ethanol solution by using hydrochloric acid. The aromatization by action ofp-toluenesulfonic acid gave mainly the epimer with the natural C-17 configuration as the acetate 5c at the appropriatep-toluenesulfonic acid concentration. 3β-Acetoxy-5α-cholesta-7,9(11),14-triene (7a) and 3β-hydroxy-5α-cholesta-8,11,14-triene (8a), 2 intermediary compounds in the aromatization, were isolated and characterized.     

Potential bile acid metabolites. 24. An efficient synthesis of carboxyl-linked glucosides and their chemical properties

A facile and efficient synthesis of the carboxyl-linked glucosides of bile acids is described. Direct esterification of unprotected bile acids with 2,3,4,6-tetra-O-benzyl-d-glucopyranose in pyridine in the presence of 2-chloro-1,3,5-trinitrobenzene as a coupling agent afforded a mixture of the α- and β-anomers (ca. 1∶3) of the 1-O-acyl-d-glucoside benzyl ethers of bile acids, which was separated effectively on a C₁₈ reversedphase chromatography column (isolated yields of α- and β-anomers are 4–9% and 12–19%, respectively). Subsequent hydrogenolysis of the α- and β-acyl glucoside benzyl ethers on a 10% Pd−C catalyst in acetic acid/methanol/EtOAc (1∶2∶2, by vol) at 35°C under atmospheric pressure gave the corresponding free esters in good yields (79–89%). Chemical specificities such as facile hydrolysis and transesterification of the acyl glucosides in various solvents were also discussed.     

The metabolism of lithocholic acid-3α-sulfate by human intestinal microflora

Lithocholic acid-3α-sulfate is metabolized by human intestinal microflora to nonpolar metabolites which have been partially purified by Sephadex LH-20 chromatography. These metabolites were characterized by thin layer and gas liquid chromatography as well as combined gas liquid chromatography-mass spectrometry. The chromatographic properties of one of the metabolites are consistent with those described for a Δ²-or Δ³. The formation of cholenates by the microflora may represent a retoxification of the sulfate ester of lithocholic acid. The following names and abbreviations for chemicals and methods have been used throughout the text: lithocholic acid (LA)=3α-hydroxy-5β-cholan-24-oic acid; isolithocholic acid (ILA)=3β-hydroxy-5β-cholan-24-oic acid; 3-keto=3-keto-5β-cholan-24-oic acid; 5β=5β-cholan-24-oic acid; Δ²-cholenate; = 5β-chol-2-en-24-oate; Δ³-cholenate =5β-chol-3-en-24-oate LASO₄=lithocholic acid-3α-sulfate; methyl lithocholate (MLA)=methyl-3α-hydroxy-5β-cholan-24-oate; methyl isolithocholate (MIL)=methyl-3β-hydroxy-5β-cholan-24-oate; methyl-3-keto (Me-3-keto)=methyl-3-keto-5β-cholan-24-oate; methyl-5β (Me-5β)=methyl-5β-cholan-24-oate; methyl deoxycholate (MEDOXY)=methyl-3α12α-dihydroxy-5β-cholan-24-oate; GLC=gas liquid chromatography; GLC-MS=gas liquid chromatography-mass spectrometry; GFP=glass fiber paper; ITLC-SG=instant thin layer chromatography-silica gel; ITLC-SA=instant thin layer chromatography-silicic acid; BHI=brain heart infusion; MFC=mixed fecal culture.     

Metabolism and cholestatic effect of 3α-hydroxy-7ζ-methyl-5β-cholanoic acid

3α-Hydroxy-7ζ-methyl-5β-cholanoic acid (7ζ-methyl-LA) was infused intravenously into bile fistula hamsters to investigate its metabolism and effect on the bile flow as compared with lithocholic acid. Following infusion of the labeled bile acids, bile was collected quantitatively to allow measurement of bile flow and bile acid composition. More than 80% of radioactivity was recovered in bile within 4 hr. 7ζ-Methyl-LA and lithocholic acid in bile were present as the taurine and glycine conjugates; no free bile acids were detected. 7ζ-Methyl-LA was neither hydroxylated nor metabolized to any measurable extent, though lithocholic acid was 7α-hydroxylated to chenodeoxycholic acid (30–45%). At the infusion rate at which lithocholic acid induced a severe cholestasis (264 nmol/min), 7ζ-methyl-LA did not decrease the bile flow. In fact, the infusion of 7ζ-methyl-LA produced a mild choleresis under conditions where endogenous bile acid excretion was not changed appreciably compared to control infusions with albumin. It is concluded that 7ζ-methyl-LA is not metabolized in the hamster but is conjugated with taurine and glycine, and that the introduction of a methyl group at the 7-position of lithocholic acid appears to alleviate the cholestatic effect of lithocholic acid in the hamster.     

Occurrence of geometrical isomers of eicosapentaenoic and docosahexaenoic acids in liver lipids of rats fed heated linseed oil

Monotrans geometrical isomers of 20∶5 n−3 and 22∶6 n−3 were detected in liver lipid of rats fed heated linseed oil. The isomers were identified as being 20∶5 δ5c, 8c, 11c, 14c, 17t and 22∶6 δ4c, 7c, 10c, 13c, 16c, 19t. These fatty acids were isolated as methyl esters by preparative high-performance liquid chromatography (HPLC) on reversed phase columns followed by silver nitrate thin layer chromatography (AgNO₃-TLC). The structures were identified using partial hydrazine reduction, AgNO₃-TLC of the resulting monoenes, oxidative ozonolysis of each monoene band, and gas-liquid chromatography (GLC) of the resulting dimethyl esters and monomethyl esters. Fourier-transform-infrared spectrometry confirmed thetrans geometry in isolated 20∶5 and 22∶6 isomers. The isomers of eicosapentaenoic and docosahexaenoic acids in liver lipids probably resulted from desaturation and elongation of 18∶3 δ9c, 12c, 15t, a geometrical isomer of linolenic acid present in the heated dietary oil.     

Rapid hydrolysis of bile acid conjugates using microwaves: Retention of absolute stereochemistry in the hydrolysis of (25R) 3α,7α,12α-trihydroxy-5β-cholestan-26-oyltaurine

In recent years, defects of bile acid synthesis caused by disorders of peroxisome biogenesis have led to increased interest in C₂₇ bile acids. In humans, while the majority of bile acids are C₂₄ carboxylic acids, the presence of increased concentrations of C₂₇ bile acids and their metabolites in hereditary diseases associated with peroxisomal dysfunction can serve as a useful marker for the intensity of the metabolic disorder. Our present studies describe an efficient method for the rapid hydrolysis of C₂₇ and C₂₄ bile acid conjugates using a commercial microwave oven. The advantages of this method include freedom from racemization, minimal activation, mild reaction conditions, and the highly stereocontrolled nature of the reaction, thus allowing for free bile acid recovery in high yield. For example, when (25R) 3α,7α,12α-trihydroxy-5β-cholestan-26-oyl taurine, a major compound present in the bile of Alligator mississippiensis, was deconjugated with 4% NaOH/diethylene glycol or 1 M LiOH/propylene glycol in the microwave oven for 4–6 min, 3α,7α,12α-trihydroxy-5β-cholestan-26-oic acid (THCA) was obtained in 81% yield with retention of configuration at C-25. It is suggested that present studies will be helpful in delineating the absolute stereochemistry of 3α,7α,12α-trihydroxy-5β-cholestanoyl-CoA oxidase, the peroxisomal enzyme that catalyzes the first step in the oxidation of THCA.     

Biosynthesis of cholic acid in rat liver: Formation of cholic acid from 3α, 7α, 12α-trihydroxy- and 3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoic acids

Conversion of 3α,7α,12α-trihydroxy-5β-cholestanoic acid into 3α,7α,12α24-tetrahydroxy-5β-cholestanoic and cholic acids was catalyzed either by the mitochondrial fraction fortified with coenzyme A, ATP, MgCl₂ and NAD or by the combination of microsomal fraction and 100,000 x g supernatant fluid fortified with coenzyme A, ATP and NAD. 24-Hydroxylation and formation of cholic acid occurred at similar rates with the 25R- and the 25S-forms of 3α,7α,12α-trihydroxy-5β-cholestanoic acid. The 25R- and 25S-forms of 3α,7α,12α-trihydroxy-and 3α,7α,12α,24-tetrahydroxy-5β-cholestanoic acids were administered to bile fistula rats. Labeled cholic acid was isolated from the bile. The initial specific radioactivity of cholic acid was higher and the disappearance of radioactivity more rapid after administration of 3α,7α,12α-trihydroxy-5β-cholestanoic acid than of 3α,7α,12α,24-tetrahydroxy-5β-cholestanoic acid. The findings are discussed in relation to the assumed pathway for side chain cleavage in cholic acid biosynthesis.     

Effects of bile acid oxazolines on gallstone formation in prairie dogs

The effects of 2 bile acid analogs, chenodeoxy-oxazoline [2-(3α,7α-dihydroxy-24-nor-5β-cholanyl)-4,4-dimethyl-2-oxazoline] and ursodeoxy-oxazoline [2-(3α, 7β-dihydroxy-24-nor-5β-cholanyl)-4,4-dimethyl-2-oxazoline] were examined in the prairie dog model of cholesterol cholelithiasis. Gallstones and biliary cholesterol crystals were induced in 5 out of 6 male prairie dogs fed a semisynthetic diet containing 0.4% cholesterol for 8 weeks. Six animals maintained on a low cholesterol control diet (0.08% cholesterol) exhibited neither gallstones nor biliary cholesterol crystals. The addition of 0.06% chenodeoxy-oxazoline to the lithogenic diet did not prevent induced cholelithiasis or the appearance of cholesterol crystals in bile. In contrast, 0.06% dietary ursodeoxy-oxazoline prevented gallstones in 5 out of 6 prairie dogs (but cholesterol crystals were present in the bile of 4 of these animals). Histologically, most of the livers from the prairie dogs fed the cholesterol-supplemented semisynthetic diet showed bile duct proliferation, inflammatory infiltration and fibrosis along the portal tracts. These pathologic changes were generally not ameliorated by adding chenodeoxy-oxazoline or chenodeoxy-oxazoline plus chenodeoxycholic acid to the diet. Portal tract pathology was markedly reduced in most animals by adding ursodeoxy-oxazoline to the cholesterol-supplemented diet. The pathologic changes overall could best be correlated with the presence of gallstones, but not with the incidence of biliary cholesterol crystals.     

Differential effects of ursodeoxycholic acid and ursocholic acid on the formation of biliary cholesterol crystals in mice

The preventive effect of 3α,7β,12α-trihydroxy-5β-cholanoic acid (ursocholic acid) and ursodeoxycholic acid on the formation of biliary cholesterol crystals was studied in mice. Cholesterol crystals developed with 80% incidence after feeding for five weeks a lithogenic diet containing 0.5% cholesterol and 0.25% sodium cholate. When 0.25% ursocholic acid or ursodeoxycholic acid was added to the lithogenic diet, the incidence as well as the grade (severity) of the gallstones were reduced. Plasma and liver cholesterol levels were decreased by ursodeoxycholic acid but not by ursocholic acid. Gallbladder cholesterol and phospholipid levels were decreased by both bile acids. The biliary bile acid level was decreased by ursocholic acid but not by ursodeoxycholic acid. After feeding ursocholic acid, its level in the bile was about 25% and the levels of cholic acid and β-muricholic acid decreased. Fecal sterol excretion was not changed by ursocholic acid, but was increased by ursodeoxycholic acid. After feeding ursocholic acid, fecal excretion of deoxycholic acid, cholic acid, and ursocholic acid increased. No differences were found between mice, with or without gallstones, in plasma and liver cholesterol levels, biliary phospholipid and bile acid levels, fecal sterol and bile acid levels, and biliary and fecal bile acid composition. The results suggest that the lower incidence of crystal formation after treatment with ursocholic acid is probably by a different mechanism than with ursodeoxycholic acid. In the mouse model, ursodeoxycholic acid exerts its effect at least partially, by decreasing cholesterol absorption. Ursocholic acid is well absorbed and excreted into bile and transformed into deoxycholic acid by the intestinal microflora in mice.     

A1H NMR anomaly for steroidal alcohols? Reactions of a steroidal γ-ketoacid

The chemical and spectroscopic properties of several reaction products of 17β-hydroxy-1,3-seco-2-nor-5α-estran-1-oic acid and 17β-hydroxy-1,3-seco-2-nor-4-oxo-5α-estran-1-oic acid were examined and found to be atypical. For instance, the methyl ester of the first acid was resistant to basic hydrolysis conditions but partly hydrolyzed with 100% H₂SO₄. Reduction of the ester by LiA1H₄ gave 1,3-seco-2-nor-5α-estrane-1,17β-diol from which diacetate, ditosylate and 17-monotosylate derivatives were prepared. The C-1 methylene protons of each appeared as a singlet in 60, 100 and/or 270 MHz NMR spectra. The methyl ester and the diacetate of the diol were synthesized by alternate methods to verify the assigned structures. A 470-MHz spectrum eventually resolved the C-1 methylene protons of the monotysylate into the AB portion of an ABX pattern, further confirming the assigned structures. Also, 2,3-seco-1-oxo-5α-estran-17β-ol 17-nitrate was synthesized.     

Identification of core aldehydes amongin vitro peroxidation products of cholesteryl esters

Synthetic cholesteryl 5-oxovalerate and 9-oxononanoate were used as reference standards for the isolation and identification of cholesteryl ester core aldehydes fromtert-butyl hydroperoxide/Fe⁺⁺ oxidation of synthetic and natural cholesteryl esters. The core aldehydes were recovered from the peroxidation products by thin-layer chromatography as the free aldehydes or the 2,4-dinitrophenylhydrazones and were identified, respectively, by gas-liquid chromatography (GLC) and by GLC combined with mass spectrometry (GC/MS) or by reverse-phase high-performance liquid chromatography (HPLC) and by HPLC with MS (LC/MS). The core aldehydes produced by peroxidation of cholesteryl linoleate were identified as mainly 9-oxononanoates of cholesterol and oxycholesterols, with smaller amounts of the 8-oxooctenoates, 10-oxodecenoates, 11-oxoundecenoates and 12-oxododecenoates. Peroxidation of cholesteryl arachidonate yielded 5-oxovalerates of cholesterol and the oxycholesterols as the main products with smaller amounts of the 4-oxobutyrates, 6-oxohexenoates, 7-oxoheptenoates, 8-oxooctenoates, 9-oxononenoates, 9-oxononadienoates and 10-oxodecadienotes. The oxycholesterols resulting from the peroxidation of the steroid ring were identified as mainly 7-keto-, 7α-hydroxy- and 7β-hydroxy-cholesterols and 5α, 6α-and 5β,6β-epoxy-cholestanols. Cholesteryl palmitate and oleate did not yield core aldehydes in the present peroxidation system. In these esters, the sterol and linoleic acid moieties appeared to be oxygenated at about the same rate, while the arachidonic acid moiety reacted more rapidly than did the sterol moiety.