Methyl Ester Trimethylsilyl Ether Derivatives Research Articles

Separation of mono-, di-, and trihydroxy stereoisomers of bile acids by capillary gas-liquid chromatography

Capillary gas-liquid chromatographic separation was studied for the complete set of the 26 theoretically possible isomers of mono-, di-, and trihydroxylated 5 beta-cholanic acids, which differ from one another in the number, position, and configuration of hydroxyl groups at C-3, C-7, and/or C-12 in the nucleus, as well as for some of their related acids. The bile acid samples were chromatographed as their methyl ester-trimethylsilyl (TMSi) ether derivatives and analyzed on three capillary columns coated with nonpolar OV-1, slightly polar OV-17, and polar SP-2340 as liquid phases. The retention times on capillary gas-liquid chromatography (GLC) responded dramatically to the minor structural differences, and almost complete separation of the positional and stereochemical isomers was achieved by the combined use of SP-2340 and OV-17 (or OV-1) capillary columns.

New role for 15-hydroxyeicosatetraenoic acid. Activator of leukotriene biosynthesis in PT-18 mast/basophil cells.

Leukotrienes are vasoactive arachidonic acid metabolites which are released by mast cells during hypersensitivity reactions. The mechanisms for regulating leukotriene biosynthesis are not well understood. A murine mast/basophil cell line (PT-18) was used to investigate this problem. Exogenously supplied [14C]arachidonic acid is not appreciably converted to leukotrienes by untreated PT-18 cells. However, when the cells were preincubated with the lymphocyte product 15-hydroxyeicosatetraenoic acid (15-HETE), addition of [14C]arachidonic acid consistently resulted in a dose-dependent synthesis of large amounts of both [14C]leukotriene B4 and [14C]5-HETE. These metabolites were isolated by high pressure liquid chromatography, converted to the methyl ester trimethylsilyl ether derivatives, and the structures confirmed by gas chromatography-mass spectrometry. These findings indicate that 15-HETE induces a direct activation of a cryptic 5-lipoxygenase in these cells. The closely related 12-HETE was ineffective. The activation phenomenon occurs rapidly and is reversible. Furthermore, the activation appears to be highly cell- and enzyme-specific, since lipoxygenases in three primary cell types including one that contains a 5-lipoxygenase and six other cell lines did not show this specific induction of leukotriene biosynthesis by 15-HETE. This report is the first evidence that 15-HETE, a major arachidonate metabolite in lymphocytes, can act as a signal to activate leukotriene production by susceptible mast cells.

Preparation of biological fluids for simultaneous analysis of prostaglandin cyclo-oxygenase synthesized compounds by gas chromatography with electron capture detection

A method for isolating climax products of the arachidonic acid cascade from biological fluids is described which allows simultaneous measurement of PGs (PGE 2, PGD 2, 6-keto-PGF 1 α , TXB 2, 6-keto-PGE 1, 6, 15-diketo-13,14-dihydro-PGF 1 α ) by electron capture detection of pentafluorobenzyloxime methyl ester trimethylsilyl (TMS) ether derivatives. PGs are adsorbed onto Amberlite XAD-2 from acidified solution and nonadhering substances removed by sequential administration of water, then petroleum ether. PGs are extracted into methanol. Following evaporation and reconstitution in water, the PGs are extracted into ethyl acetate at pH 3 and the ethyl acetate extracts are purified by lipidex column chromatography. Derivatization to pentafluorobenzyloxime methyl ester TMS ethers of PGs in the sample is followed by separation on either glass packed-columns or SCOT capillary columns, and detection by an electron capture detector. PGA 2, added to the unpurified sample, is used as an internal standard for quantification. The methods have performed well on all biological fluids thus far examined. Examples of chromatographs from urine, Krebs-perfused lung effluents, and blood are shown.

Quantitative determination of ursodeoxycholic acid and its deuterated derivative in human bile by gas chromatography-mass fragmentography.

Deuterium-labeled ursodeoxycholic acid (UDCA-d2) was synthesized from cholic acid by heterogeneous catalytic reduction of the 11, 12-unsaturated bile acid with deuterium gas. A new method for the simultaneous determination of UDCA, UDCA-d2, related bile acids and cholesterol in human bile has been developed by gas chromatography-mass fragmentography of the methyl ester trimethylsilyl ether derivatives. The glycine conjugate of 5β-chol-3-en-24-oic acid was prepared from lithocholic acid and used as an internal standard. Calibration plots gave a good linear relationship over the range of 100-500 ng of each compound, and their recoveries (95-99%) relative to the internal standard suggested that the present method is sufficiently accurate for the practical analysis of these bile acids in human bile. The method was applied to investigate the fate of UDCA-d2 orally administered to a patient with complete bile fistula.

Prostaglandin E1 and E2 in bovine semen: Quantification by gas chromatography

Prostaglandins PGE1 and PGE2 extracted from bovine semen, purified via silicic column chromatography were quantified by gas chromatography as their methoxime methyl ester trimethylsilyl ether derivatives. The PGE1 and PGE2 concentrations of 19 bovine semen samples ranged from 395 ± 225 and 487 ± 407 ng/ml, respectively. A constant 1:1 ratio between PGE1 and PGE2 was observed. There was no relationship between PGE and sperm motility, but high sperm counts were apparently associated with decreased PGE levels. The direct precursors of PGE1 and PGE2, i.e. 20:3n6 and 20:4n6, occurred in low concentrations compared to other related unsaturated fatty acids, i.e. 18:2n6 and 22:5n6 of the n-6 family.

Prostaglandin E 1 and E 2 in bovine semen: Quantification by gas chromatography

Prostaglandins PGE 1 and PGE 2 extracted from bovine semen, purified via silicic column chromatography were quantified by gas chromatography as their methoxime methyl ester trimethylsilyl ether derivatives. The PGE 1 and PGE 2 concentrations of 19 bovine semen samples ranged from 395 ± 225 and 487 ± 407 ng/ml, respectively. A constant 1:1 ratio between PGE 1 and PGE 2 was observed. There was no relationship between PGE and sperm motility, but high sperm counts were generally associated with decreased PGE levels. The direct precursors of PGE 1 and PGE 2, i.e. 20:3n6 and 20:4n6, occurred in low concentrations compared to other related unsaturated fatty acids, i.e. 18:2n6 and 22:5n6 of the n-6 family.

Simultaneous mass spectrometric measurement of prostaglandins E1 (PGE1) and PGE2 with a deuterated internal standard.

The use of (3,3,4,4-2H4)PGE2 as internal standard for the quantitative measurement of PGE1 by gas chromatography-mass spectrometry is reported. A method for the simultaneous analysis of PGE1 and PGE2 is also described. The prostaglandins were analyzed by selected ion monitoring as the methyl ester-trimethylsilyl ether (ME-TMS) derivatives of PGB1 and PGB2, respectively. In all cases, a linear response over the range 1 to 70 ng (2.8 to 198 pmoles) was demonstrated. The use of this procedure is exemplified by its application to the PG analysis of sheep seminal vesicles.

Analysis of bile acids in serum and bile by capillary gas-liquid chromatography.

Various liquid phases for glass capillary columns have been evaluated for gas-liquid chromatographic analysis of methyl ester trimethylsilylether derivatives of bile acids from serum and bile. Bile acid analysis is rapid and exhibits high separation efficiency with a 20 X 0.3 mm glass capillary column whose internal surface is covered with a crystal layer of barium carbonate and coated with polyethyleneglycol 20000 as liquid phase according to Grob et al.

Glucuronides of unconjugated 6-hydroxylated bile acids in urine of a patient with malabsorption

The excretion of bile acids in urine from a patient with chronic malabsorption was investigated. Bile acids were separated according to mode of conjugation using a lipophilic anion exchanger, diethylaminohydroxypropyl Sephadex LH-20. Following hydrolysis, individual bile acids were analyzed by computerized GC/MS. In addition, bile acid glucuronides were isolated and their methyl ester trimethylsilyl ether derivatives were directly analyzed by GC/MS. The patient had a normal or slightly increased excretion of bile acids in urine. Bile acids carrying a hydroxyl group at C-6 constituted about 40% of the total. Tetrahydroxylated bile acids were present which have not been found in healthy subjects. Glucuronides of otherwise unconjugated bile acids accounted for 20% of the total. About 90% of these conjugates were 6-hydroxylated, hyodeoxycholic acid being the major bile acid. It is suggested that a specific abnormality of bile acid metabolism is related to the disease in this patient.

Analysis of metabolic profiles of bile acids in urine using a lipophilic anion exchanger and computerized gas-liquid chromatorgaphy-mass spectrometry

A method is described for quantitative analysis of bile acids in urine. Urine is acidified and bile acids are extracted on an Amberlite XAD-2 column. Bile salts are converted to acids on an Amberlyst A-15 column and are separated into groups of unconjugated, glycine, taurine, monosulfated, and polysulfated conjugates using the lipophilic anion exchanger diethylaminohydroxypropyl Sephadex LH-20 (DEAP-LH-20). After solvolysis and hydrolysis, the deconjugated bile acids are purified on DEAP-LH-20, and are converted to methyl ester trimethylsilyl ether derivatives. Identification and quantitation of the individual bile acids is accomplished by computerized gas-liquid chromatography-mass spectrometry. The daily excretion of bile acids in urine from healthy subjects was 6.4-11 micro moles. The mixture of bile acids was quite complex and differed from that in bile. About 30 bile acids were identified or partially characterized. Three of these were monosubstituted: lithocholic, allolithocholic, and 3beta-hydroxy-5-cholenoic acids. Fourteen disubstituted bile acids included epimers of deoxycholic, allodeoxycholic, chenodeoxycholic, allochenodeoxycholic, and hyodeoxycholic acids. 3alpha-Hydroxy-12-keto-5beta-cholanoic acid was the major ketonic bile acid and 3beta,12alpha-dihydroxy-5-cholenoic acid was the major unsaturated bile acid in this group. Nine trihydroxy bile acids included cholic and allocholic acids, epimers of these compounds, hyocholic acid, and a 1-hydroxylated bile acid tentatively characterized as 1,3,12-trihydroxycholanoic acid. Cholestatic subjects excreted tetrahydroxycholanoates carrying hydroxyl groups in positions 1, 3, 6, 7, 12, or 23. All monohydroxy and the predominant part of dihydroxy bile acids were present in the monosulfate fraction. Exceptions were 3alpha,12beta-dihydroxy- and 3alpha-hydroxy-12-keto-5beta-cholanoic acids, which were found mainly in the glycine conjugate fraction. Most of the trihydroxy bile acids were nonsulfated, and cholic and norcholic acids were the major unconjugated bile acids. The tetrahydroxy bile acids and hyocholic acid were present mainly in the taurine conjugate fraction, while 1,3,12-trihydroxycholanoic acid was predominantly found in the glycine conjugate fraction. Sulfation of trihydroxy bile acids was increased in patients with marked cholestasis. All bile acids in the monosulfate fraction were conjugated and carried the sulfate ester group at C-3. Significant amounts of di- and trisulfates were not found. The results indicate selective mechanisms for sulfation, hydroxylation, and renal elimination of bile acid conjugates. Analysis of metabolic profiles of bile acids in urine may be a useful method in studies of the function of organs involved in bile acid metabolism.