Gas--liquid chromatography--mass spectrophotometry of hydroxylated octadecanols derived from hydroxylated stearic acids

A gas-liquid chromatographic-mass spectrometric method of determining the position of oxygen atoms on polyfunctional fatty acids has been explored. The method consists of reduction of keto, hydroperoxy, epoxy, and carboxylic acid groups with LiAlH(4) to the corresponding alcohols; trimethylsilylation with bis(trimethylsilyl)acetamide; and analysis by means of the combined gas-liquid chromatograph-mass spectrometer. The following compounds were analyzed: 9-mono-, 9,10-di-, 9,10,12-tri-, and 9,10,12,13-tetrahydroxystearic acids and the corresponding derivatives of octadecan-1-ol. The reduction products of 9,10-epoxystearic acid and a mixture of linoleic acid 9- and 13-hydroperoxides were also analyzed. The position of the oxygen function in the original molecule can be deduced rapidly and accurately.     

Microbial Oxidation of Allylic Alcohols

A new method for the oxidation of primary ad secondary allylic alcohols to the corresponding aldehydes/acids and ketones respectively is described utilizing Nocardia corallina B-276. In contrast, Pseudomonas oleovorans TF4-1L oxidized only the primary allylic alcohols but not the secondary allylic alcohols.     

A convenient procedure for the synthesis of ceramides

A procedure for the preparation of ceramides by direct coupling of long-chain bases and fatty acids in the presence of a mixed carbodiimide is described. This method has been used to prepare ceramides containing sphing-4-enine or sphinganine and various saturated and unsaturated fatty acids as well as saturated 2-hydroxy acids. Ceramides containing 4-hydroxy sphinganine and saturated nonhydroxy acids have also been prepared. The yields were 60-75%. The characterization of these compounds by gas-liquid chromatography-mass spectrometry as trimethylsilyl derivatives has been previously reported. Some of the ceramides are further characterized in this report by infrared spectroscopy and one compound, in addition, by elementary analysis. Use of racemic constituents for 2-hydroxy acid ceramide syntheses leads to the formation of diastereoisomers which separate by thin-layer chromatography. These were characterized by gas-liquid chromatography-mass spectrometry as the trimethylsilyl derivatives and by infrared spectroscopy. Their configurations were established by syntheses with optically active constituents.     

Synthesis and stereochemical determination of diunsaturated valproic acid analogs including its major diunsaturated metabolite

Valproic acid, an antiepileptic drug, is transformed into diunsaturated metabolites in humans. Synthesis of the geometric isomers of 2-(1'-propenyl)-2-pentenoic acid and 2-(1'-propenyl)-3-pentenoic acid was attempted using known procedures. The final product, a mixture of isomers, was converted into tert-butyldimethylsilyl or ethyl derivatives. Capillary gas-liquid chromatography-mass spectrometry analysis of the derivatives showed at least three isomeric dienoic acids from synthesized products. Argentation thin-layer chromatography was effective in resolving the isomeric mixture into a single isomer or mixture of two isomers. Thin-layer chromatography and gas-liquid chromatography retention data, photochemical isomerization studies, and nuclear magnetic resonance spectrometry were used to characterize the dienoic acids. By comparison of the retention times of the diunsaturated metabolites with synthesized reference compounds, the structure assigned to the major diunsaturated metabolite is 2-[(E)-1'-propenyl](E)-2-pentenoic acid.     

Isolation of 19,20-dehydroprostaglandins E1 and E2 in human seminal fluid and further studies on 18,19-dehydroprostaglandins E1 and E2

Human seminal fluid was recently found to contain 18,19-dehydroprostaglandins E1 and E2 (E. H. Oliw, H. Sprecher, and M. Hamberg, (1986) J. Biol. Chem. 261, 2675-2683). In the present study, the cis and trans isomers of 18,19-dehydroprostaglandins E1 and E2 were prepared by incubation of microsomes of ram vesicular glands and glutathione with the precursor fatty acids, 8(Z),11(Z),14(Z),18(E/Z)-eicosatetraenoic acids, and 5(Z),8(Z),11(Z),14(Z),18(E/Z)-eicosapentaenoic acids, and used as references to characterize the 18,19-dehydroprostaglandins of human seminal fluid. Based on separation by reversed-phase high-performance liquid chromatography, capillary gas chromatography-mass spectrometry, and ozonolysis of the (-)-menthoxycarbonyl derivatives and on comparison with the authentic compounds, human seminal fluid was found to contain both the cis and trans isomers of 18,19-dehydroprostaglandins E1 and E2. Furthermore, human seminal fluid contained two related compounds, viz. 19,20-dehydroprostaglandins E1 and E2. The structures of these compounds were established by conversion into the corresponding prostaglandin B compounds, by mass spectrometric analysis and by chemical degradation by oxidative ozonolysis, which afforded, inter alia, 2(S)-hydroxy-adipic acid.     

Enantioselective epoxidation by an optically active hydroperoxide

Enantioselective epoxidation of prochiral allylic alcohols with optically active 4,6-di-O-acetyl-2,3-dideoxy-α-D -threo-hex-2-enopyranosyl hydroperoxide in the presence of Ti(O-i-Pr)₄ gives chiral epoxy alcohols with moderate enantiomeric excess. © 1993 Wiley-Liss, Inc.     

Analyses of fermentation products of Listeria species by frequency-pulsed electron-capture gas-liquid chromatography

Aerobic fermentation broths of eight Listeria monocytogenes strains, two or more strains of the remaining six Listeria species, and one strain of Jonesia denitrificans were examined by frequency-pulsed electron-capture gas-liquid chromatography for carboxylic acids, alcohols, amines, and hydroxy acids. All species produced acetic, isobutyric, butyric, isovaleric, phenylacetic, lactic, 2-hydroxybutyric, 2-hydroxyvaleric, and 2-hydroxyisocaproic acids. Propionic acid was not formed, and traces of isocaproic acid were observed. Of the alcohol and amine derivatives observed, only acetylmethylcarbinol, butylamine, and putrecine were identified. Recognition of the products of glucose and amino acid metabolism serves to further characterize the members of the genus Listeria both taxonomically and physiologically.     

Stereoselective oxidation of regioisomeric octadecenoic acids by fatty acid dioxygenases

Seven Z-octadecenoic acids having the double bond located in positions 6Z to 13Z were photooxidized. The resulting hydroperoxy-E-octadecenoic acids [HpOME(E)] were resolved by chiral phase-HPLC-MS, and the absolute configurations of the enantiomers were determined by gas chromatographic analysis of diastereoisomeric derivatives. The MS/MS/MS spectra showed characteristic fragments, which were influenced by the distance between the hydroperoxide and carboxyl groups. These fatty acids were then investigated as substrates of cyclooxygenase-1 (COX-1), manganese lipoxygenase (MnLOX), and the (8R)-dioxygenase (8R-DOX) activities of two linoleate diol synthases (LDS) and 10R-DOX. COX-1 and MnLOX abstracted hydrogen at C-11 of (12Z)-18:1 and C-12 of (13Z)-18:1. (11Z)-18:1 was subject to hydrogen abstraction at C-10 by MnLOX and at both allylic positions by COX-1. Both allylic hydrogens of (8Z)-18:1 were also abstracted by 8R-DOX activities of LDS and 10R-DOX, but only the allylic hydrogens close to the carboxyl groups of (11Z)-18:1 and (12Z)-18:1. 8R-DOX also oxidized monoenoic C(14)-C(20) fatty acids with double bonds at the (9Z) position, suggesting that the length of the omega end has little influence on positioning for oxygenation. We conclude that COX-1 and MnLOX can readily abstract allylic hydrogens of octadecenoic fatty acids from C-10 to C-12 and 8R-DOX from C-7 and C-12.     

A 38 kDa allylic alcohol dehydrogenase from the cultured cells of Nicotiana tabacum

An NADP+-dependent alcohol dehydrogenase (allyl-ADH) was isolated from the cultured cells of Nicotiana tabacum. The allyl-ADH was found to be efficient for the dehydrogenation of secondary allylic alcohols rather than saturated secondary alcohols and it was specific for the S-stereoisomer of the alcohols. The enzyme catalyzed the reversible reaction whereby the carbonyl group of enones is reduced to the corresponding allylic alcohol or vice versa. Two possible primary structures of the allyl-ADH were deduced by the sequence analyses of full-length cDNAs (allyl-ADH1 and ally-ADH2), which were cloned by the PCR method. These analyses indicated that the allyl-ADHs are composed of 343 amino acids having the molecular weights 38083 and 37994, respectively, and they showed approximately 70% homology to the NADP+-dependent oxidoreductases belonging to a plant zeta-crystallin family.     

Improved method for simultaneous determination of alcohols, volatile fatty acids, lactic acid or 2,3-butanediol in biological samples

A rapid and sample procedure was developed to determine, by gas-liquid chromatography, the concentrations of C₂---C₄ alcohols, C₂---C₆ volatile fatty acids (VFA) and lactic acid or 2,3-butanediol in fermentation liquids. both lactic acid and 2,3-butanediol are oxidized to acetaldehyde by periodic acid and acetaldehyde was eluted before ethanol. A complete separation of the alcohols and acids was performed in <15 min on a column packed with 80/100 Chromosorb WAW, having GP 10% SP-1200/1% H₃PO₄ as the liquid phase. The method was suitable for the analysis of rumen fluid and fermentation products from microbial cultures. The detection limits for all compounds were <0.13 nmol · injection⁻¹.     

Gas chromatographic enantiomer separation of C-3 and C-4 synthons: prediction of absolute configuration from elution order and enzymatic resolution

Enantiomers of C-3 secondary alcohols, 3-hydroxybutanoates, and cyclic 1,3-dithioacetals were separated by chiral GLC using CP-Chirasil-Dex CB and Chiraldex G-TA columns. The former was most successful for analysis of n-alkyl esters of secondary alcohols and the separation depended on the chain length of the acyl group and the electronic and steric properties of the other substituents. The Chiraldex G-TA column was efficient for analysis of secondary alcohols, derivatized as their trifluoroacetates. The elution order of the secondary alcohols and the corresponding acetates was always the same with respect to the size of groups connected to the stereocenter. However, when the secondary alcohols were analyzed as their trimethylsilyl derivatives, the elution order was reversed. Elution order on chiral columns and enantiomeric ratios, E-values, obtained in kinetic resolutions catalyzed by lipase B from Candida antarctica (CALB) were evaluated as a method for prediction of absolute configuration. The usefulness of the method was demonstrated using 22 pairs of enantiomers. Copyright 1999 Wiley-Liss, Inc.     

Improved identification of monomethyl paraffin chain branching (close to the methyl end) of long-chain compounds by gas chromatography and mass spectrometry

An improved method for the identification of monomethyl substituted paraffin chains (n?2, n?3 and n?4) by gas chromatography and mass spectrometry is presented. Fatty acids and sphingolipid long-chain bases were converted to their corresponding alcohols, which were analyzed as methyl ethers. These compounds were better separated on packed columns (XE-60) than other derivatives, and the low temperature of analysis minimized the bleeding of stationary phase into the mass spectrometer. Mass spectra of methyl ethers allowed a more conclusive identification of branches than of other derivatives. The method may be generally applied to fatty acids, aldehydes and alcohols.     

Regioselective oxygenation of fatty acids, fatty alcohols and other aliphatic compounds by a basidiomycete heme-thiolate peroxidase

Reaction of fatty acids, fatty alcohols, alkanes, sterols, sterol esters and triglycerides with the so-called aromatic peroxygenase from Agrocybe aegerita was investigated using GC-MS. Regioselective hydroxylation of C(12)-C(20) saturated/unsaturated fatty acids was observed at the ω-1 and ω-2 positions (except myristoleic acid only forming the ω-2 derivative). Minor hydroxylation at ω and ω-3 to ω-5 positions was also observed. Further oxidized products were detected, including keto, dihydroxylated, keto-hydroxy and dicarboxylic fatty acids. Fatty alcohols also yielded hydroxy or keto derivatives of the corresponding fatty acid. Finally, alkanes gave, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule; and sterols showed side-chain hydroxylation. No derivatives were found for fatty acids esterified with sterols or forming triglycerides, but methyl esters were ω-1 or ω-2 hydroxylated. Reactions using H(2)(18)O(2) established that peroxide is the source of the oxygen introduced in aliphatic hydroxylations. These studies also indicated that oxidation of alcohols to carbonyl and carboxyl groups is produced by successive hydroxylations combined with one dehydration step. We conclude that the A. aegerita peroxygenase not only oxidizes aromatic compounds but also catalyzes the stepwise oxidation of aliphatic compounds by hydrogen peroxide, with different hydroxylated intermediates.     

Determination of plasma bile acids by capillary gas-liquid chromatography-electron capture negative chemical ionization mass fragmentography

Combined capillary gas-liquid chromatography-electron capture negative chemical ionization mass spectrometry of pentafluorobenzyl ester-TMSi ether derivatives of bile acids and isotope dilution using deuterated internal standards are introduced as a sensitive and selective analysis technique for plasma bile acids. As a result of the high ionization efficiency of pentafluorobenzyl derivatives under electron capturing conditions and minimal fragmentation, the detection limit of this technique is low: 1 pg for each bile acid. The high sensitivity enabled the detection and quantitation of atypical bile acids in 200-microliters aliquots of plasma from fasting healthy adults as exemplified by trihydroxycoprostanic acid (0.002 +/- 0.001 mumol/l) and dihydroxycoprostanic acid (0.013 +/- 0.002 mumol/l).     

Steric analysis of 3-, ω4-, ω3- and ω2-hydroxy acids and various alkanols by gas-liquid chromatography

D-Phenylpropionate (PP) derivatives of racemic hydroxy acid methyl esters and alkanols were prepared and the gas-liquid chromatographic separation of diastereoisomers was investigated using QF-1 as stationary phase. Good separations were obtained for the diasteroisomeric PP-derivatives of methyl 3-, 15-, 16-, and 17-hydroxyoctadecanoates. Less separation was observed for methyl 2- and 14-hydroxyoctadecanoates as PP-derivatives and there was no visible separation for PP-derivatives of 4-, 7-, or 13-hydroxyoctadecanoic acid methyl esters. The use of optically active 15-, 16- and 17-hydroxyoctadecanoic acids showed, that in these cases, the diastereoisomers containing L-hydroxy acids had shorter retention times than the ones containing D-hydroxy acids. On the other hand, the D-phenylpropionate derivative of methyl 3D-hydroxydecanoate had shorter retention time than the derivative of its L-enantiomer. PP-derivatives of 3-hexanol, 3-heptanol, 3-octanol, 2-octanol and 2-eicosanol could be resolved by gas-liquid chromatography.     

Capillary gas-liquid chromatography of glycine-conjugated bile acids without prior hydrolysis

A method is described for the gas-liquid chromatographic (GLC) analysis of intact glycine conjugates of the major bile acids present in human plasma. It is, therefore, now possible to analyze glycine-conjugated and unconjugated bile acids together on a single GLC column without the necessity for a hydrolytic step. A large number of derivatives of bile acid glycine conjugates were examined, but only acetate- and silyl ether-derivatives of carboxylic acid methyl esters were found initially to be suitable. It was not possible to make acetates consistently, and trimethylsilyl ethers did not allow resolution of the glycine conjugates of cholic and chenodeoxycholic acids. Dimethylethylsilyl ether methyl ester derivatives were subsequently found to give the best results. Chromatographic conditions for successful analysis of these derivatives were examined and it was found to be necessary to use wall-coated capillary columns of thin film thickness (0.12 micron) and very high carrier gas flow rates (ca. 20 ml/min hydrogen). Using acetonitrile and Bond Elut extraction, fractionation on Sep-Pak SIL cartridges, and derivatization as dimethylethylsilyl ether methyl esters, the capillary gas-liquid chromatography of intact glycine-conjugated bile acids from human plasma was demonstrated for the first time.     

Novel biological transformations of 15-Ls-hydroperoxy-5,8,11,13-eicosatetraenoic acid

[1-14C]Arachidonic acid was incubated with homogenates of the fungus, Saprolegnia parasitica. The products consisted of comparable amounts of two epoxy alcohols, 15-Ls-hydroxy-11,12-epoxy-5cis,8cis,13trans- eicosatrienoic acid and 15-hydroxy-13,14-epoxy-5cis,8cis,11cis-eicosatrienoic acid. Results of incubations carried out in the presence of nordihydroguaiaretic acid, 5,8,11,14-eicosatetraynoic acid, p-hydroxymercuribenzoate as well as glutathione peroxidase plus reduced glutathione demonstrated that transformation of arachidonic acid into epoxy alcohols occurred with the formation of 15-Ls-hydroperoxy-5cis,8cis,11cis,13trans- eicosatetraenoic acid (15-HPETE) as an intermediate. The pathway involved a lipoxygenase catalyzing the oxygenation of arachidonic acid at the 15L position to produce 15-HPETE, and a hydroperoxide isomerase activity which catalyzed conversion of 15-HPETE into the two epoxy alcohols. Studies with 15-[18O2]HPETE demonstrated that both oxygens of 15-HPETE were retained in the epoxy alcohols. Furthermore, experiments with mixtures of 15-[18O2]-and 15-[16O2]HPETE showed that conversion of 15-HPETE into epoxy alcohols occurred by an intramolecular transfer of hydroperoxide oxygen.     

Metabolism of 18:2(n - 6), 18:3(n - 3), 20:4(n - 6) and 20:5(n - 3) by the fungus Gaeumannomyces graminis: identification of metabolites formed by 8-hydroxylation and by w2 and w3 oxygenation.

The present study was aimed at developing a cell-free preparation of Gaeumannomyces graminis to biosynthesize w2-hydroxy, w3-hydroxy and related metabolites of essential fatty acids. 14C-labelled linoleic acid (18:2(n - 6)), linolenic acid (18:3(n - 3)), arachidonic acid (20:4(n - 6)) and eicosapentaenoic acid (20:5(n - 3)) were incubated with the cytosolic and microsomal fractions and NADPH. Significant metabolism was only found in the cytosol. The main products were purified by high-performance liquid chromatography and identified by gas chromatography-mass spectrometry (GC-MS). 18:2(n - 6) was metabolized mainly to 8-hydroxy-9,12-octadecadienoic acid (8-HODE), while the w2 and the w3 alcohols were formed in relatively small amounts. The absolute configuration of the 8-hydroxyl was found to be R by ozonolysis of the diastereoisomeric (-)-menthoxycarbonyl derivative of 8-HODE and GC-MS analysis. In analogy, 18:3(n - 3) was converted to 8-hydroxy-9,12,15-octadecatrienoic acid and to smaller amounts of the 15,16-diol (15,16-DiHODE). In contrast, 8-hydroxy metabolites of 20:4(n - 6) or 20:5(n - 3) could not be detected. 20:4(n - 6) was efficiently converted to 18(R)-hydroxyeicosatetraenoic acid (18(R)-HETE) and 19(R)-HETE and to traces of 17-HETE, while 20:5(n - 3) was mainly metabolized to the 17,18-diol (17,18-DiHETE) and to smaller amounts of the w2 alcohol. In conclusion, the cytosol of G. graminis can be used for stereoselective biosynthesis of some hydroxy metabolites of essential fatty acids.     

Bile salts of the coelacanth, Latimeria chalumnae

Bile salts of the coelacanth, Latimeria chalumnae, Smith, have been analyzed and shown to have three bile alcohols, latimerol, 5 alpha-cyprinol, and 5 alpha-cholestane-3 beta, 7 alpha,-12 alpha,25,26-pentol, two C24 bile acids, chenodeoxycholic acid and cholic acid, one C26 bile acid, probably 3 beta, 7 alpha, 12 alpha-trihydroxy-27-nor-5 alpha-cholestan-26-oic acid, and two C27 bile acids, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-26-oic acid and 3 beta,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-26-oic acid as determined by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry.     

Studies of the cryptic allylic pyrophosphate isomerase activity of trichodiene synthase using the anomalous substrate 6,7-dihydrofarnesyl pyrophosphate

Two enantiomeric analogues of farnesyl pyrophosphate (1) were tested as inhibitors and anomalous substrates of trichodiene synthase, which catalyzes the cyclization of trans,trans-farnesyl pyrophosphate (1) to the sesquiterpene hydrocarbon trichodiene (2). The reaction has been shown to involve preliminary isomerization of 1 to the tertiary allylic isomer nerolidyl pyrophosphate (3) which is cyclized without detectable release of the intermediate from the active site of the cyclase. Both (7S)-trans-6,7-dihydrofarnesyl pyrophosphate (7a) and (7R)-trans-6,7-dihydrofarnesyl pyrophosphate (7b), prepared from (3R)- and (3S)- citronellol (9a and 9b), respectively, proved to be modest competitive inhibitors of trichodiene synthase. The values of Ki(7a), 395 nM, and Ki(7b), 220 nM, were 10-15 times the observed Km for 1 and half the Ki of inorganic pyrophosphate alone. Incubation of either 7a or 7b with trichodiene synthase resulted in formation of a mixture of products which by radio/gas-liquid chromatographic and GC/selected ion mass spectrometric analysis was shown to be composed of 80-85% isomeric trienes 19-21 and 15-20% allylic alcohols 12 and 18. Examination of the water-soluble products resulting from incubation of 7a also revealed the generation of 24% of the isomeric cis-6,7-dihydrofarnesyl pyrophosphate (26). The combined rate of formation of anomalous alcoholic and olefinic products was 10% the Vmax determined for the conversion of 1 to 2. The results can be explained by initial enzyme-catalyzed isomerization of dihydrofarnesyl pyrophosphate (7) to the corresponding tertiary allylic isomer dihydronerolidyl pyrophosphate (8). Since the latter intermediate is unable to cyclize due to the absence of the 6,7-double bond, ionization of 8 and quenching of the resulting ion pair by deprotonation, capture of water, or collapse to the isomeric primary pyrophosphate esters will generate the observed spectrum of anomalous products.