Combined Gas-liquid Chromatography-mass Spectrometry Research Articles

The metabolism of 16 alpha-ethylprogesterone by rat liver in vitro.

Abstract The metabolism of 16α-ethylprogesterone by rat liver homogenate at different steroid to tissue ratios has been examined. The products, identified by thin-layer chromatography, gas-liquid chromatography and combined gas-liquid chromatography-mass spectrometry were 16α-ethyl-5α-pregnane-3, 20-dione and 16α-ethyl-3α-hydroxy-5α-pregnan-20-one. The yield of these metabolites was 14 and 48% respectively. The results are discussed in relation to the liver in vitro metabolism of progesterone and 16α-hydroxyprogesterone.

The metabolism of dibenzylamine: identification of NN-dibenzylhydroxylamine as the major in vitro metabolic product from rabbit fortified hepatic homogenates

A study of the in vitro metabolism of dibenzylamine with fortified rabbit liver 9000 g supernatant fractions has shown that N-oxidation is the major metabolic process. Of the total amount of dibenzylamine metabolized, 90% is converted to NN-dibenzylhydroxylamine. The primary amine, benzylamine, is formed to the extent of only 6% of total substrate metabolized. The identity of the major metabolite, NN-dibenzylhydroxylamine has been established by thin-layer chromatography, gas-liquid chromatography and combined gas-liquid chromatography-mass spectrometry.

Identification of in vivo liver metabolites of delta 1-tetra-hydrocannabinol, cannabidiol, and cannabinol produced by the guninea-pig.

The metabolites of delta 1-tetrahydroannabinol (delta 1-THC), cannabidiol (CBD), and cannabinol (CBN) produced in vivo by the guinea-pig have been studed by combined gas-liquid chromatography-mass spectrometry, 45 metabolites of delta 1-THC were identified of which 12 have not been reported before. Several other metabolites were detected but not identified. The major metabolic routes involved allylic and aliphatic hydroxylations, oxidations to ketones and acids, oxidative degradation of the side-chain presumably by the beta-oxidation pathway, and formation of glucuronide conjugates. Di- and tri-substituted metabolites were abundant. The metabolism differed considerably from that observed in mouse and rat in that 1''- and 6 beta-hydroxylation and oxidation degradation of the side-chain were major metabolic pathways. 1''-Hydroxy-delta 1-THC was found as a pair of diasteroisomers. Similar metabolic pathways were observed with CBD; twenty metabolites were identified of which two were new. Only 6 metabolites of CBN were identified. These mainly mono-substituted in the same positions as were observed with delta 1-THC and CBD.

Biochemical studies on sphingolipids of Artemia franciscana: novel neutral glycosphingolipids

Neutral glycosphingolipids containing one to six sugars in their oligosaccharide chains have been isolated from cysts of the brine shrimp Artemia franciscana. The structures of these glycolipids were identified by methylation analysis, partial acid hydrolysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and proton nuclear magnetic resonance spectroscopy to be Glcβ1-Cer, Manβ1-4Glcβ1-Cer, Fucα1-3Manβ1-4Glcβ1-Cer, GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GlcNAcα1-2Fucα1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer (CPS), and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer (CHS). Two glycosphingolipids, CPS and CHS, were characterized as novel structures. Because Artemia contains a certain series of glycosphingolipids (-Fucα3Manβ4GlcβCer), which differ from the core sugar sequences reported thus far, we tentatively designated the glycosphingolipids characterized as nonarthro-series ones. Furthermore, CHS exhibited a hybrid structure of arthro-series and nonarthro-series sugar chain. Two novel glycosphingolipids were characterized from the brine shrimp Artemia franciscana; one was composed of arthrotetraose and a branching fucose attached to N-acetylglucosamine residue, and the other was composed of CPS with an additional N-acetylglucosamine residue attached to the branching fucose.

A Novel Sulfatide, GlcCer-I6 Sulfate, from the Ascidian Ciona intestinalis

A novel sulfated glycosphingolipid, SGL-1, was isolated from the ascidian Ciona intestinalis, prepared from chloroform/methanol extracts and fractionated successively on DEAE Sephadex-A25, Florisil and Iatrobeads column chromatographies. Chemical structural analysis was performed using methylation analysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and (1)H-NMR spectroscopy. This chemical structure is presented as GlcCer I(6)-Sulfate. The ceramide moiety was specified by t16:0, t17:0, br,t17:0, t18:0 and br,t18:0 as sphingoids, and 2-hydroxy, saturated fatty acids as represented by docosanoic and tetracosanoic acids.

Oxidized plant sterols in human serum and lipid infusions as measured by combined gas-liquid chromatography-mass spectrometry

Some oxidized forms of cholesterol (oxysterols) are thought to be atherogenic and cytotoxic. Because plant sterols are structurally related to cholesterol, we examined whether oxidized plant sterols (oxyphytosterols) could be identified in human serum and soy-based lipid emulsions. We first prepared both deuterated and nondeuterated reference compounds. We then analyzed by gas-liquid chromatography-mass spectrometry the oxyphytosterol concentrations in serum from patients with phytosterolemia or cerebrotendinous xanthomatosis, in a pool serum and in two lipid emulsions. 7-Ketositosterol, 7 beta-hydroxysitosterol, 5 alpha, 6 alpha-epoxysitosterol, 3 beta,5 alpha,6 beta-sitostanetriol, and probably also 7 alpha-hydroxysitosterol were present in markedly elevated concentrations in serum from phytosterolemic patients only. Also, campesterol oxidation products such as 7 alpha-hydroxycampesterol and 7 beta-hydroxycampesterol were found. Interestingly, sitosterol was oxidized for approximately 1.4% in phytosterolemic serum, which is rather high compared with the approximate 0.01% oxidatively modified cholesterol normally seen in human serum. The same oxyphytosterols were also found in two lipid emulsions in which the ratio of oxidized sitosterol to sitosterol varied between 0.038 and 0.041. In conclusion, we have shown that oxidized forms of plant sterols are present in serum from phytosterolemic patients and two frequently used soy-based lipid emulsions. Currently, it is unknown whether oxyphytosterols affect health, as has been suggested for oxysterols. However, 7 beta-hydroxycholesterol may be one of the more harmful oxysterols, and both sitosterol and campesterol were oxidized into 7 beta-hydroxysitosterol and 7 beta-hydroxycampesterol. The relevance of these findings therefore deserves further exploration.

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The novel sialic acid 4-O-acetyl-9-O-lactyl-N-acetylneuraminic acid has been identified as a constituent of horse submandibular gland glycoproteins in addition to the already know equine sialic acids, N-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 9-O-acetyl-N-acetylneuraminic acid, 4,9-di-O-acetyl-N-acetylneuraminic acid, N-glycolylneuraminic acid, 4-O-acetyl-N-glycolylneuraminic acidand 9-O-acetyl-N-glycolylneuraminic acid. The structure has been established by combined gas-liquid chromatography-mass spectrometry.

Composition analysis of carboxymethylcellulose by high- pH anion-exchange chromatography with pulsed amperometric detection

A rapid method for the determination of the substituent distribution in carboxymethylcellulose has been developed, involving hydrolysis of carboxymethylcellulose in 1.2 M perchloric acid and mixture of carboxymethylated glucose residues and glucose by high-pH anion-exchange chromatography with pulsed amperometric detection. The peaks in the chromatogran were identified by combined gas-liquid chromatography-mass spectrometry after pertrimethysilylation. Molar response factors for each of the constituent monomers were established by 1H NMR spectroscopy. The degrees of substitution for three carboxymethylcellulose preparations determined by the proposed method and by a standard titration method were found to be in excellent agreement.

Chemical Characterization of Stress-Induced Vascular Coating in Tomato

Indirect evidence suggests that vascular coatings formed by plants in response to stress consist of suberin-like substances containing lipid and phenolic compounds. To provide more direct chemical evidence that coatings are suberin, we used a natural pathogen, Verticillium albo-atrum, or a stress-responsive hormone, abscisic acid, to induce coating in two isolines of tomato (Lycopersicon esculentum L. cultivar Craigella) that are resistant or susceptible to the pathogen. Using treated petioles that had been monitored cytologically, chemical depolymerization followed by combined gas-liquid chromatography-mass spectrometry analysis of alkane-alpha,omega-diol levels confirmed the presence of suberin after induction of coating and showed quantitative differences between the isolines that correlated with cytological measurements of the coating response. Northern analysis of suberization-associated anionic peroxidase mRNA showed corresponding increases, and tissue blot analysis further indicated that induction of the mRNA was localized in the responding vascular bundles, as determined by suberin histochemistry. Taken together, these results provide chemical evidence that the coatings are mainly suberin.

Identification of short side chain bile acids in urine of patients with cerebrotendinous xanthomatosis

Urine from patients with cerebrotendinous xanthomatosis (CTX) was found to contain a number of minor bile acids along with three major bile acids, 7-epicholic acid, norcholic acid, and cholic acid. The following minor bile acids were identified by combined gas-liquid chromatography-mass spectrometry: 7-ketobisnordeoxycholic acid; 12-ketobisnorchenodeoxycholic acid; 7-ketonordeoxycholic acid; 12-ketochenodeoxycholic acid; 7-ketodeoxycholic acid; 12-ketochendeoxycholic acid; bisnorcholic acid; allonorcholic acid; allocholic acid; 1 beta-hydroxybisnorcholic acid; 1 beta-hydroxynorcholic acid; 1 beta-hydroxycholic acid; 2 beta-hydroxybisnorcholic acid; 2 beta-hydroxy-norcholic acid; 2 beta-hydroxycholic acid. The presence of C22 and C23 bile acids in urine of the CTX patients suggests that bile alcohols having a hydroxyl group at C22 or C23 in the side chain may be further degraded to these bile acids.

Identification of 3α,7α,12α-trihydroxy-5β-cholest-24-enoic acid as an intermediate in the peroxisomal conversion of 3α,7α,12α-trihydroxy-5β-cholestanoic acid to cholic acid

In this study, we have identified a Δ 24-unsaturated intermediate involved in the conversion of 3α,7α,12α-trihydroxy-5β- cholestanoic acid to cholic acid by the peroxisomal fraction of rat liver. An accumulation of this intermediate was observed when NAD + was omitted from the reaction mixture. The intermediate was isolated by reversed-phase high-pressure liquid chromatography and identified by combined gas-liquid chromatography-mass spectrometry. The peroxisomal fraction was able to catalyze the conversion of the Δ 24-unsaturated intermediate to cholic acid in the presence of CoA, ATP, Mg 2+ and NAD +. The identification of 3α,7α,12α-trihydroxy-5β-cholest-24-enoic acid in cholic acid formation supports the proposed reaction mechanism in which the side-chain cleavage of C 27-steroids is similar to that of peroxisomal β-oxidation of fatty acids. This involves an FAD-dependent oxidase acting on 3α,7α,12α-trihydroxy-5β-cholestanoyl-CoA.

Reaffirmation of the validity of enzymatic cleavage of lithocholic acid from N-epsilon-lithocholyl-L-lysine and N-alpha-CBZ-N-epsilon-lithocholyl-L-lysine.

N-epsilon-lithocholyl-L-lysine or N-alpha-CBZ-N-epsilon-lithocholyl-L-lysine when incubated overnight at 37 degrees C with 3 K units of clostridial cholanoylaminoacid hydrolase (from Clostridium perfringens ATCC 19574) in the presence of disodium EDTA (0.1 M), beta-mercaptoethanol (0.1 M), and sodium acetate buffer, pH 5.6, released free lithocholic acid. The latter material was isolated by thin-layer chromatography and identified by combined gas-liquid chromatography-mass spectrometry in the full scan and selected-ion mode. In order to maintain its activity, the enzyme was always stored in 1.0-ml aliquots at temperatures below -20 degrees C and each aliquot when thawed was used immediately; any left over enzyme was never reused. Contrary to the observations of Yanagisawa et al. (J. Lipid Res. 1984. 25: 1263-1271) the results of this study reaffirm the validity of the original observations on the enzymatic cleavage of lithocholic acid from tissue-bound form.

Treatment of rhamnogalacturonan I with lithium in ethylenediamine

Rhamnogalacturonan I is a pectic polysaccharide that is solubilized from the walls of suspension-cultured sycamore cells ( Acer pseudoplatanus) by the action of a highly purified endo-1,4-α-polygalacturonanase. Rhamnogalacturonan I has a linear backbone consisting of the diglycosyl repeating unit, →4)-α- d-Gal pA-(1→2)-α- l-Rha p-(1→. Approximately half of the α- l-rhamnosyl residues of the backbone are branched at O-4. Selective cleavage at the galactosyluronic acid residues of the backbone by treatment of rhamnogalacturonan I wit lithium in ethylenediamine resulted in the release of the neutral glycosyl-residue sidechains that had been attached to the backbone. Various analytical techniques, including combined liquid chromatography-mass spectrometry, combined gas-liquid chromatography-mass spectrometry, and 1H-nuclear magnetic resonance spectroscopy, were used to determine the structure of the side chains. The majority of the sidechains were isolated as oligoglycosylalditols, with rhamnitol at the “reducing” end. Terminal 2-, 4-, or 6-linked galactosyl residues were found attached to O-4 of the rhamnitol residues The 2-, 4-, and 6-linked galactosyl residues had terminal or 2-linked arabinosyl, or additional galactosyl, residues attached to them. Based on the results of fast-atom-bombardment mass spectrometry, the side chains were found to range in size from one to fourteen glycosyl residues. The side-chain structures suggest that there are four or more distinct families of side chains attached to the backbone of rhamnogalacturonan I.

Serum salbutamol concentrations during oral and intravenous treatment in pregnant women.

The serum concentration of salbutamol was determined in 29 pregnant women during oral treatment (4 mg five times per day) and in seven during intravenous infusion (6-30 micrograms/min) because of premature labour. The concentration of salbutamol was determined by combined gas-liquid chromatography mass spectrometry. The mean concentration of serum salbutamol was twice as high during intravenous treatment (24; SD 9 ng/ml), than during oral treatment (12; SD 3 mg/ml). During oral treatment, the salbutamol levels were not correlated to maternal height, weight or the incidence or severity of side-effects. The serum concentrations of salbutamol in patients with twin pregnancies did not differ from those with singleton pregnancies. After stopping intravenous treatment, serum salbutamol levels remained high for several hours and oral treatment can be started 4-6 h after stopping intravenous infusion.

The molecular basis of the density increase of phenylalanine ammonia-lyase, extracted from the cotyledons of mustard (Sinapis alba) incubated on deuterium oxide

Abstract Previous studies of deuterium labelling of proteins have measured shifts in buoyant density and inferred the incorporation of deuterium into the free amino acids. This paper reports the use of combined gas-liquid chromatography-mass spectrometry to measure the incorporation of deuterium into the free amino acids of Sinapis alba cotyledons, from which the expected density shift of phenylalanine ammonia-lyase (EC 4.3.1.5) has been calculated and compared with that measured by isopycnic density-gradient centrifugation. The quantitative results show that most of the amino acids entering phenylalanine ammonia-lyase are only deuterated to a small extent and pass from storage protein to phenylalamine ammonia-lyase virtually without engaging in enzyme-catalysed exchange reactions. Most of the deuterium incorporated into phenylalanine ammonia-lyase is transferred with the amino acids alanine (approx. 32%), glutamate (approx. 21%), and glutamine (approx. 16%). Possible reasons for the discrepancy between the calculated and observed shift in buoyant density of phenylalanine ammonia-lyase are discussed.

Mevalonic aciduria: an inborn error of cholesterol biosynthesis?

The application of combined gas-liquid chromatography mass spectrometry in the analysis of physiological fluids from patients in whom a metabolic disorder is suspected has revealed many new inborn errors of metabolism, especially organic acidurias. Inborn errors have been identified in the conversion of cholesterol into steroid hormones and bile acids (for review see ref. 1). To our knowledge no inborn error of cholesterol biosynthesis has been reported. In view of the important function of cholesterol with respect to synthesis of biolo~cally active steroid compounds and membrane function we wish to report a patient with a persistent urinary excretion of mevalonic acid, a precursor in the synthesis of sterols.

Identification of bile alcohols in urine from healthy humans.

Bile alcohols present in urine from healthy adults were studied. Urine was extracted with octadecylsilane-bonded silica gel, and a neutral fraction and a glucuronide fraction were isolated by ion exchange chromatography on piperidinohydroxypropyl Sephadex LH-20. Following hydrolysis of the glucuronide fraction with beta-glucuronidase and purification by silica gel column chromatography, the bile alcohols were analyzed by a combined gas-liquid chromatography-mass spectrometry. By direct comparison with reference standards, the structures of four major bile alcohols were elucidated as follows: 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25- pentol ; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24, 25- pentol ; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26- pentol ; and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26,27- pentol . The daily excretion of these four bile alcohols in urine was 0.5-1.0 mumoles. After purification by silica gel column chromatography, the neutral fraction was analyzed by a combined gas-liquid chromatography-mass spectrometry. The major bile alcohol excreted in urine as the unconjugated form was 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25- pentol . The daily excretion of the unconjugated C26 pentol (0.02-0.05 mumoles) was far lower than that of the conjugated C26 pentol (0.34-0.86 mumoles).

The minor bile acids of the toad, Bufo vulgaris formosus.

Bile from the toad, Bufo vulgaris formosus, was found to contain a number of minor bile acids along with two major bile acids, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-22-ene-24-carboxylic acid and 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-23-en-26-oic acid. The following minor bile acids were identified by combined gas-liquid chromatography-mass spectrometry: cholic acid, allocholic acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 alpha-cholestan-26-oic acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 alpha-cholest-22-ene-24-carboxylic acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 alpha-cholest-23-en-26-oic acid, varanic acid, and 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-24-methyl-5 beta-cholestan-26-oic acid. The fact that the toad bile contains not only cholic acid but also 3 alpha, 7 alpha, 12 alpha-trihydroxy- and 3 alpha, 7 alpha, 12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acids, which have been recognized as biosynthetic intermediates of cholic acid in mammals, suggests that the toad is capable of synthesizing cholic acid by the same pathway as that for the biosynthesis of the C24 bile acid in mammals.

Sterols of the spongeTethya amamensis: Occurrence of (24E)-24-ethylidenecholesta-5,7-dienol, (24E)-24-propylidenecholesta-5,7-dienol, and (24Z)-24-propylidenecholesta-5,7-dienol.

The spongeTethya amamensis, collected from Kagoshima Bay, Japan, contained at least 24 sterols, including Δ(5)-sterols (82.2% of total sterols) and Δ(5, 7)-sterols (17.8%). The predominant sterols were cholesterol (29.0%), cholesta-5,22-dienol (13.8%), 24-methylcholesta-5,22-dienol (10.9%), 24-methylenecholesterol (8.3%), 24-methylcholesta-5,7,22-trienol (6.8%), 24-ethylcholest-5-enol (6.1%), and isofucosterol *4.1%). Combined gas liquid chromatography-mass spectrometry suggested the presence of 3 uncommon sterols, (24E)-24-ethylidenecholesta-5,7-dienol, (24E)-24-propylidenecholesta-5,7-dienol, and (24Z)-24-propylidenecholesta-5,7-dienol as minor components. The sterols ofT. amamensis also contained small amounts of 24-norcholesta-5,7,22-trienol and (24Z)-24-ethylidenecholesta-5,7-dienol.

26-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol by isolated nonparenchymal cells and hepatocytes from rat liver

The ability of isolated nonparenchymal and parenchymal rat liver cells to metabolize 5beta-cholestane-3alpha,7alpha,12alpha-triol and other bile acid intermediates has been investigated. Incubation of nonparenchymal cells with 5beta-cholestane-3alpha,7alpha,12alpha-triol resulted in the formation of one more polar product identified as 5beta-cholestane-3alpha,7alpha,12alpha,26-tetrol. The formation was linear with time up to 2 hr and with the number of cells, and showed saturation kinetics with respect to substrate concentration. The maximum rate of conversion was 90 pmol/10(6) cells per hr. Incubation of hepatocytes with the triol resulted in the formation of several more polar products. In addition to 5beta-cholestane-3alpha,7alpha,12alpha,26-tetrol, products with retention time on high pressure liquid chromatography identical with 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-26-oic acid and cholic acid were observed. The identity of these products was verified by combined gas-liquid chromatography-mass spectrometry. The rate of conversion was linear with time for about 10 min and saturation with respect to substrate concentration was not attained. At identical substrate concentrations, the total rate of conversion (12.5 nmol/10(6) cells per hr) was at least two orders of magnitude higher than with the nonparenchymal cells. Similar differences in rates of conversion were observed with other C(27)-bile acid intermediates. It is concluded that the nonparenchymal cells do not play any significant role in the conversion of bile acid intermediates, either under physiological conditions or under experimental conditions where such steroids have been administered intravenously.-Dueland, S., J. I. Pedersen, C. A. Drevon, and I. Björkhem. 26-Hydroxylation of 5beta-cholestane-3alpha,7alpha,12alpha-triol by isolated nonparenchymal cells and hepatocytes from rat liver.