Sequential enzymes of linoleic acid oxidation in corn germ: lipoxygenase and linoleate hydroperoxide isomerase

Linoleic acid oxidation catalyzed by lipoxygenase (lipoxidase) activity in extracts of defatted corn germ does not terminate in the product, linoleic acid hydroperoxide, unless the lipoxygenase is first partially purified. If purification is not attempted, the hydroperoxide product exists only as a barely detectable intermediate in the synthesis of three products. One of these was identified as 9-hydroxy-10-oxo-cis-12-octadecenoic acid formed from the hydroperoxide by the enzyme, linoleate hydroperoxide isomerase. Another product, 13-hydroxy-10-oxo-trans-11-octadecenoic acid, is believed to be formed by an isomerase also. The third product was the linoleate ester of one of the hydroxy-oxo-fatty acids, 9-(cis-9,cis-12-octadecadienoyl)-10-oxo-cis-12-octadecenoic acid. It is not known if the synthesis of the ester is enzyme-catalyzed. When a mixture of 13-hydroperoxy-cis-9,trans-11-octa-decadienoic acid and 9-hydroperoxy-trans-10,cis-12-octa-decadienoic acid from soybean lipoxygenase oxidation of linoleic acid was used as a substrate, 13-hydroxy-12-oxo-cis-9-octadecenoic acid and 9-hydroxy-12-oxo-trans-10-octadecenoic acid were formed as the major products of catalysis by linoleate hydroperoxide isomerase(s) from corn. Smaller quantities of 9-hydroxy-10-oxo-cis-12-octadecenoic acid and 13-hydroxy-10-oxo-trans-11-octadecenoic acid were also formed.     

Enantioselectivity of the hydrolysis of linoleic acid monoepoxides catalyzed by soybean fatty acid epoxide hydrolase.

Soybean epoxide hydrolase efficiently catalyzes the hydration of the two positional isomers of linoleic acid monoepoxides into their corresponding vic-diols. Kinetic analysis of the progress curves, obtained at low substrate concentrations (i.e. [So] much less than Km), and analysis of the residual substrates by chiral-phase HPLC, indicate that the hydrolase is highly enantioselective, i.e. cis-9R,10S-epoxy-12(Z)-octadecenoic and cis-12R,13S-epoxy-9(Z)-octadecenoic acids are preferentially hydrolyzed (the enantioselectivity ratios are 15 and 28, respectively). Importantly, these two enantiomers are the one formed preponderantly by epoxidation of linoleic acid by peroxygenase, a hydroperoxide-dependent oxidase we have previously described in soybean (Blée, E., and Schuber, F., Biochem. Biophys. Res. Commun. (1990) 173, 1354-1360).     

Metabolism of cis-12-octadecenoic acid and trans-9,trans-12-octadecadienoic acid and their influence on lipogenic enzyme activities in mouse liver

High carbohydrate (65% glucose) diets containing cis-12-octadecenoic acid (12c-18:1) or trans-9,trans-12-octadecadienoic acid (9t,12t-18:2) were fed to weanling mice to investigate the influence of fatty acid structure on six hepatic enzyme activities involved in lipid metabolism. Results with these diets were compared to those with diets containing no fatty acids, saturated fatty acids; cis-9-octadecenoic acid (9c-18:1) and cis-9,cis-12-octadecadienoic acid (9c,12c-18:2). These comparisons show saturated fatty acids, 9c-18:1, 12c-18:1, and 9t,12t-18:2, had little or no influence on the activity levels of fatty acid synthetase, malic enzyme (EC 1.1.1.40)citrate cleavage enzyme (EC 4.1.3.8), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44) and acetyl-CoA carboxylase (EC 6.4.1.2). Neither 12c-18:1 nor 9t,12t-18:2 produced the dramatic enzyme-lowering effect exhibited by the diet containing 9c,12c-18:2 when compared to the diet devoid of fat. Thus, both the 9 and 12 bonds must be present in the same molecule. Also, at least one and probably both bonds must be in the cis configuration to depress liver enzyme activities. Capillary gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) were both used for analysis of the methyl esters derived from the hepatic lipids. The GC and GC-MS data provided (a) direct evidence for incorporation of both isomers into hepatic lipids and (b) indirect evidence that 9t,12t-18:2 lowered liver delta 9-desaturase activity. In addition, since these products were found in the complex liver lipids, there is no doubt that the various enzymes concerned with activation and acylation utilize both of these isomeric fatty acids as substrates.     

Effect of fatty acids on growth of Japanese encephalitis virus cultivated in BHK-21 cells and phospholipid metabolism of the infected cells.

Growth of Japanese encephalitis virus (JEV) in BHK-21 cells was stimulated in the presence of 20 to 40 mug of the sodium salt of oleic acid (cis-9-octadecenoic acid, 9-18:1) per ml supplemented in Waymouth medium. The stimulatory effect of the salt was highest when 9-18:1 was added after adsorption of the virus. Study of the effect of other fatty acids on growth of JEV showed the following results: the longer the chain length of the saturated fatty acid salt, the higher the stimulatory effect on viral growth. In contrast, polyunsaturated fatty acids had an inhibitory effect on viral growth. The effect of isomeric cis-octadecenoic acids on viral growth was variable, depending upon the position of the double bond. The cis-6-octadecenoic acid had the highest inhibitory effect on growth of JEV compared to other isomeric octadecenoic acids. The sodium salt of (1-14C) cis-9-octadecenoic acid (9-18:1, 20 mug/ml) was rapidly incorporated into control and JEV-infected cells. Specific radioactivity in phosphatidylcholine dropped 12 to 24 h after virus inoculation, whereas synthesis of phosphatidylethanolamine increased 12 to 24 h after virus inoculation in infected cells compared to uninfected cells. Results from these studies suggest that phospholipid metabolism of infected cells is markedly changed, which can be associated with altered fatty acid metabolism when using labeled 9-18:1 fatty acid as a marker.     

Solid-liquid phase behavior of binary fatty acid mixtures. 2. Mixtures of oleic acid with lauric acid, myristic acid, and palmitic acid

Solid-liquid phase behavior was investigated for binary fatty acid mixtures composed of oleic acid (OA; cis-9-octadecenoic acid) and saturated fatty acids, lauric acid (LA; dodecanoic acid), myristic acid (MA; tetradecanoic acid), and palmitic acid (PA; hexadecanoic acid), by means of differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). When the mixture was heated immediately after the solidification from the melt, the heat effect due to the gamma-to-alpha transformation of OA varied depending on the composition of the mixture. However, the mixture subjected to an annealing at the temperature slightly below the melting temperature provided the transformation at constant temperature which corresponds to the gamma-to-alpha transformation temperature of pure OA. This suggests that a solid phase formed by cooling of the melt of the mixture is not in an equilibrium state, but it relaxes to a stable solid during the annealing process. The T-X phase diagrams of these mixtures constructed from the DSC measurements demonstrate that the two fatty acid species are completely immiscible in a solid phase regardless of the type of polymorphs of OA, alpha- or gamma-form. According to a thermodynamic analysis of liquidus line basing on the regular solution model for the melt, the non-ideality of mixing tends to increase with the decrease in the acyl chain length of the saturated fatty acid, although the mixing is rather close to ideal.     

Simultaneous UPLC MS/MS analysis of endogenous jasmonic acid,salicylic acid,and their related compounds

Jasmonic acid (JA) and salicylic acid (SA) are plant hormones involved in plant growth and development. Recent studies demonstrated that presence of a complex interplay between JA and SA signaling pathways to response to pathogenesis attack and biotic stresses. To our best knowledge, no method has existed for simultaneous analyses of JA, SA, and their related compounds. Especially, the glucosides are thought to be the storages or the inactivated compounds, but their contribution should be considered for elucidating the amount of the aglycons. It is also valuable for measuring the endogenous amount of phenylalanine, cinnamic acid, and benzoic acid that are the biosynthetic intermediates of SA due to the existence of isochorismate pathway to synthesize SA. We established this method using deuterium labeled compounds as internal standards. This is the first report of simultaneous analysis of endogenous JA, SA, and their related compounds. Measuring the endogenous JA, SA, and their related compounds that had been accumulated in tobacco plants proved the practicality of the newly developed method. It was demonstrated that accumulation of JA, SA and their related compounds were induced in both case of TMV infection and abiotic stresses.     

In vivo distribution and turnover of trans- and cis-10-octadecenoic acid isomers in human plasma lipids

Triacylglycerols containing deuterium-labeled trans-10- and cis-10-octadecenoic acid (10t-18:1, 10c-18:1) plus the triacylglycerol of deuterated cis-9-octadecenoic acid (9c-18:1) were fed as a mixture to two young, adult male subjects. Analysis by mass spectroscopy of the labeled fats in blood samples collected periodically for 48 h allowed the uptake, distribution and turnover of both 10-octadecenoic acid isomers to be directly compared to 9c-18:1. A feature of this study is that actual weight data for the labeled fats were obtained. These data allowed plasma triacylglycerol turnover rates of 3.47-5.13 mg/min per kg to be estimated. Plasma and chylomicron triacylglycerol data also provided evidence that absorption of the deuterated fats mobilized 10-12 g of a triacylglycerol pool present in the intestinal cells. Other results are summarized as follows: the 10t-, 10c- and 9c-18:1 fatty acids were equally well absorbed, both delta 10-18:1 isomers were oxidized more rapidly than 9c-18:1, conversion of the delta 10-18:1 isomers into their corresponding 16:1 isomers was about 3-times faster than for 9c-18:1, the delta 10-18:1 isomers were preferentially incorporated at the 1-acyl and excluded from the 2-acyl position of phosphatidylcholine, esterification of cholesterol with the delta 10-18:1 fatty acids was 2.5-4.3-times slower than for 9c-18:1 and desaturation and elongation rates for the delta 10-18:1 acids were very low.     

Novel multi-component enzyme machinery in lactic acid bacteria catalyzing C=C double bond migration useful for conjugated fatty acid synthesis

Linoleic acid isomerase was identified as a multi-component enzyme system that consists of three enzymes that exist in both the membrane and soluble fractions of Lactobacillus plantarum. One enzyme (CLA-HY) is present in the membrane fraction, while two enzymes (CLA-DH and CLA-DC) exist in the soluble fraction. Three Escherichia coli transformants expressing CLA-HY, CLA-DH, and CLA-DC were constructed. Conjugated linoleic acid (CLA) and 10-hydroxy-12-octadecenoic acid were generated from linoleic acid only when all these three E. coli transformants were used as catalysts simultaneously. CLA-HY catalyzed the hydration reaction, a part of linoleic acid isomerization, to produce 10-hydroxy-12-octadecenoic acid. This multi-component enzyme system required oxidoreduction cofactors such as NADH and FAD. This is the first report to reveal enzymes genes and the elaborate machinery that synthesizes CLA, especially an important isomer of cis-9, trans-11-CLA, in lactic acid bacteria.     

The possible formation of nano-clusters of succinic acid and maleic acid in tetrahydrofuran due to incomplete solvation

In continuation of our work on the conformational analysis of succinic acid (SA) and maleic acid (MA) in different solvents, we present here the experimental dielectric and IR and also the ab initio Hartree–Fock calculations of the two dicarboxylic acids in tetrahydrofuran (THF). The dielectric measurements are carried out at microwave X-band frequency of 9.7 GHz and the calculations are performed at STO-3G and 6-31G(d) basis sets. The dielectric data and the dipole moment determined experimentally are compared with the dipole moment determined from the conformal analysis. It is seen that the dielectric properties of SA/MA in THF are much different from that of SA/MA in 1-4, dioxane (1-4D) that we had reported previously. The IR spectra of SA–THF system is also reported here. The present study indicates the possible formation of nano-clusters of SA/MA in THF due to incomplete solvation by THF.     

Gas chromatography-mass spectrometry of cis-9,10-epoxyoctadecanoic acid (cis-EODA). II. Quantitative determination of cis-EODA in human plasma

Cytochrome P450 dependent epoxidation and non-enzymic lipid peroxidation of oleic acid (cis-9-octadecenoic acid) result in the formation of cis-9,10-epoxyoctadecanoic acid (cis-EODA). This oleic acid oxide has been identified indirectly in blood and urine of humans. Reliable concentrations of circulating cis-EODA have not been reported thus far. In the present article, we report on the first GC-tandem MS method for the accurate quantitative determination in human plasma of authentic cis-EODA as its pentafluorobenzyl (PFB) ester. cis-[9,10-2H2]-EODA (cis-d2-EODA) was synthesized by chemical epoxidation of commercially available cis-[9,10-2H2]-9-octadecenoic acid and used as an internal standard for quantification. Endogenous cis-EODA and externally added cis-[9,10-2H2]-EODA were isolated from acidified plasma samples (1 ml; pH 4.5) by solvent or solid-phase extraction, converted into their PFB esters, isolated by HPLC and quantified by selected reaction monitoring. The parent ions [M-PFB]- at mass-to-charge ratio (m/z) 297 for cis-EODA and m/z 299 for (cis-d2-EODA) were subjected to collisionally-activated dissociation and the corresponding characteristic product ions at m/z 171 and 172 were monitored. In plasma of nine healthy humans (5 females, 4 males), cis-EODA was found to be present at 47.6+/-7.4 nM (mean+/-S.D.). Plasma cis-EODA levels were statistically insignificantly different (P=0.10403, t-test) in females (51.1+/-3.4 nM) and males (43.1+/-2.2 nM). cis-EODA was identified as a considerable contamination in laboratory plastic ware and found to contribute to endogenous cis-EODA by approximately 2 nM. The present GC-tandem MS method should be useful in investigating the physiological role(s) of cis-EODA in humans.     

茉莉酸甲酯与真菌诱导物、水杨酸组合对红豆杉细胞中紫杉醇含量的影响

比较了茉莉酸甲酯与真菌诱导物、水杨酸组合对红豆杉细胞几个抗病相关指标（POD、CAT活力、H2O2含量）及紫杉醇含量的影响,3种信号分子的组合对POD、CAT、H2O2及紫杉醇含量的影响是不一致的,MJ单独添加,MJ与SA联合作用以及MJ与F5联合作用都可使POD活力增加,且12h后H2O2含量均升高,约在48h达到高峰,为对照的2倍左右,但72h后,MJ单独添加和MJ与SA联合作用组中H2O2含量变化不大,F5与MJ联合作用则使H2O2含量持续比对照高。MJ单独添加使CAT酶活在144h后才较对照低,F5、SA的加入都可使CAT酶活下降,SA的作用更显著。说明三者的诱导途径并不完全一样,以SA和MJ联合添加对紫杉醇合成的促进作用最大,含量达到细胞干重的0．04％。     

Synthesis and biological activity of hydroxylated derivatives of linoleic acid and conjugated linoleic acids

Allylic hydroxylated derivatives of the C18 unsaturated fatty acids were prepared from linoleic acid (LA) and conjugated linoleic acids (CLAs). The reaction of LA methyl ester with selenium dioxide (SeO₂) gave mono-hydroxylated derivatives, 13-hydroxy-9Z,11E-octadecadienoic acid, 13-hydroxy-9E,11E-octadecadienoic acid, 9-hydroxy-10E,12Z-octadecadienoic acid and 9-hydroxy-10E,12E-octadecadienoic acid methyl esters. In contrast, the reaction of CLA methyl ester with SeO₂ gave di-hydroxylated derivatives as novel products including, erythro-12,13-dihydroxy-10E-octadecenoic acid, erythro-11,12-dihydroxy-9E-octadecenoic acid, erythro-10,11-dihydroxy-12E-octadecenoic acid and erythro-9,10-dihydroxy-11E-octadecenoic acid methyl esters. These products were purified by normal-phase short column vacuum chromatography followed by high-performance liquid chromatography (HPLC). Their chemical structures were characterized by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR). The allylic hydroxylated derivatives of LA and CLA exhibited moderate in vitro cytotoxicity against a panel of human cancer cell lines including chronic myelogenous leukemia K562, myeloma RPMI8226, hepatocellular carcinoma HepG2 and breast adenocarcinoma MCF-7 cells (IC₅₀ 10-75 μM). The allylic hydroxylated derivatives of LA and CLA also showed toxicity to brine shrimp with LD₅₀ values in the range of 2.30-13.8 μM. However these compounds showed insignificant toxicity to honeybee at doses up to 100 μg/bee.     

Jasmonic acid and salicylic acid activate a common defense system in rice

Jasmonic acid (JA) and salicylic acid (SA) play important roles in plant defense systems. JA and SA signaling pathways interact antagonistically in dicotyledonous plants, but, the status of crosstalk between JA and SA signaling is unknown in monocots. Our rice microarray analysis showed that more than half of the genes upregulated by the SA analog BTH are also upregulated by JA, suggesting that a major portion of the SA-upregulated genes are regulated by JA-dependent signaling in rice. A common defense system that is activated by both JA and SA is thus proposed which plays an important role in pathogen defense responses in rice.     

Gas chromatography-mass spectrometry of cis-9,10-epoxyoctadecanoic acid (cis-EODA). I. Direct evidence for cis-EODA formation from oleic acid oxidation by liver microsomes and isolated hepatocytes

Oleic acid, cis-9-octadecenoic acid, is the major fatty acid in mammals. Its oxide, cis-9,10-epoxyoctadecanoic acid (cis-EODA), has been identified in blood and urine of humans, its origin is, however, still unknown. Lipid peroxidation and enzyme-catalyzed epoxidation of oleic acid are two possible sources. In the present article, we investigated by HPLC and GC-MS whether cis-EODA is formed enzymatically from oleic acid by the cytochrome P450 (CYP) system. Oleic acid, cis-EODA and its hydratation product threo-9,10-dihydroxyoctadecanoic acid (threo-DiHODA) were quantitated by HPLC as their p-bromophenacyl esters. For structure elucidation by GC-MS, the pentafluorobenzyl (PFB) esters of these compounds were isolated by HPLC and converted to their trimethylsilyl ether derivatives. Liver microsomes of rats, rabbits and humans oxidized oleic acid into cis-EODA. This is the first direct evidence for the enzymatic formation of cis-EODA from oleic acid. The epoxidation of oleic acid was found to depend on CYP, NADPH+H(+), and O(2). cis-EODA was measurable in incubates of liver microsomes for up to 30 min of incubation. Maximum cis-EODA concentrations were reached after 5-7 min of incubation and found to depend upon oleic acid concentration. Isolated rat hepatocytes hydratated cis-EODA into threo-DiHODA which was further converted to unknown metabolites. However, from incubation of oleic acid with these cells we could not detect threo-DiHODA or cis-EODA. Our study suggests that circulating and excretory cis-EODA may originate, at least in part, from CYP-catalyzed epoxidation of oleic acid. GC-MS of intact cis-EODA as its PFB ester in the negative-ion chemical ionization mode should be useful in investigating the physiological role of cis-EODA in man.     

Conversion of linoleic acid into 10-Hydroxy-12(Z)-octadecenoic acid by whole cells of Stenotrophomonas nitritireducens

The conversion of linoleic acid into 10-hydroxy-12(Z)-octadecenoic acid by whole cells of Stenotrophomonas nitritireducens as an isolated bacterium was optimized, and the optimal temperature, pH, and cell and substrate concentrations were 30 degrees C, 7.5, and 20 and 20 g/L, respectively. Under these conditions, whole cells in a bioreactor produced 15 g/L 10-hydroxy-12(Z)-octadecenoic acid in 2 h of reaction time without detectable byproducts. Using 2 g/L linoleic acid, the cells produced 1.92 g/L 10-hydroxy-12(Z)-octadecenoic acid. These are the highest concentration and yield of 10-hydroxy-12(Z)-octadecenoic acid ever reported.     

Linking pattern recognition and salicylic acid responses in Arabidopsis through ACCELERATED CELL DEATH6 and receptors

The Arabidopsis membrane protein ACCELERATED CELL DEATH 6 (ACD6) and the defense signal salicylic acid (SA) are part of a positive feedback loop that regulates the levels of at least 2 pathogen-associated molecular patterns (PAMP) receptors, including FLAGELLIN SENSING 2 (FLS2) and CHITIN ELICITOR RECEPTOR (LYSM domain receptor-like kinase 1, CERK1). ACD6- and SA-mediated regulation of these receptors results in potentiation of responses to FLS2 and CERK1 ligands (e.g. flg22 and chitin, respectively). ACD6, FLS2 and CERK1 are also important for callose induction in response to an SA agonist even in the absence of PAMPs. Here, we report that another receptor, EF-Tu RECEPTOR (EFR) is also part of the ACD6/SA signaling network, similar to FLS2 and CERK1.     

Conjugated linolenic acid (CLnA), conjugated linoleic acid (CLA) and other biohydrogenation intermediates in plasma and milk fat of cows fed raw or extruded linseed

Thirty lactating dairy cows were used in a 3 × 3 Latin-square design to investigate the effects of a raw or extruded blend of linseed and wheat bran (70:30) on plasma and milk fatty-acids (FA). Linseed diets, containing 16.6% linseed blend on a dry-matter basis, decreased milk yield and protein percentage. They decreased the proportions of FA with less than 18 carbons in plasma and milk and resulted in cis-9, cis-12, cis-15 18:3 proportions that were more than three and four times higher in plasma and milk, respectively, whereas cis-9, cis-12 18:2 proportions were decreased by 10-15%. The cis-9, trans-11, cis-15 18:3 isomer of conjugated linolenic acid was not detected in the milk of control cows, but was over 0.15% of total FA in the milk fat of linseed-supplemented cows. Similarly, linseed increased plasma and milk proportions of all biohydrogenation (BH) intermediates in plasma and milk, including the main isomer of conjugated linoleic acid cis-9, trans-11 18:2, except trans-4 18:1 and cis-11, trans-15 18:2 in plasma lipids. In milk fat, compared with raw linseed, extruded linseed further reduced 6:0-16:0 even-chain FA, did not significantly affect the proportions of 18:0, cis-9 18:1 and cis-9, cis-12 18:2, tended to increase cis-9, cis-12, cis-15 18:3, and resulted in an additional increase in the proportions of most BH intermediates. It was concluded that linseed addition can improve the proportion of conjugated linoleic and linolenic acids, and that extrusion further increases the proportions of intermediates of ruminal BH in milk fat.     

An ab initio and DFT study of structure and vibrational spectra of γ form of Oleic acid: Comparison to experimental data

Oleic acid (cis-9-octadecenoic acid) is the most abundant cis-unsaturated fatty acid in nature; it is distributed in almost all organisms. In this work, we present a detailed vibrational spectroscopy investigation of Oleic acid by using infrared and Raman spectroscopies. These data are supported by quantum mechanical calculations, which allow us to characterize completely the vibrational spectra of this compound. The equilibrium geometry, harmonic vibrational frequencies, infrared intensities and activities of Raman scattering were calculated by ab initio Hartree-Fock (HF) and density functional theory (DFT) employing B3LYP with complete relaxation in the potential energy surface using 6-311G(d, p) basis set. After a proper scaling the calculated wavenumbers show a very good agreement with the observed values. A complete vibrational assignment is provided for the observed Raman and infrared spectra of Oleic acid. In this work, we also investigate the deviation of vibrational wavenumbers computed with two quantum chemical methods (HF and B3LYP).     

Isolation and characterization of a lipoxygenase from Pseudomonas 42A2 responsible for the biotransformation of oleic acid into (S)-(E)-10-hydroxy-8-octadecenoic acid

The isolation of a new lipoxygenase-like (LOX-like) enzyme from Pseudomonas 42A2 and its characterization is described. The enzyme, located in the periplasm of the cell, which contained 0.55 mol of Fe2+ per mol of protein, is monomeric and has a molecular mass of 45 kDa. In the presence of oxygen, the enzyme converts oleic acid into (E)-10-hydroperoxy-8-octadecenoic acid (HPOD), which decomposes to the corresponding (E)-10-hydroxy-8-octadecenoic acid (HOD). The absolute configuration of this acid was determined as S on the basis of exciton-coupled CD data, and specific rotation and NMR analysis of the corresponding p -bromobenzoate derivative. The reaction in vivo leads to the dihydroxy derivative (E)-7,10-dihydroxy-8-octadecenoic acid (DHOD), so that the three hydroxy-fatty acids can be isolated from the culture medium. The activity of the enzyme was optimal between 25 and 30 degrees C and 44% of its activity still remained at 55 degrees C. Its optimal pH is 8.5-9; and the presence of magnesium ions increased LOX activity by 1.5. The activity of the LOX is highest in unsaturated fatty acids containing double bonds in position 9 (oleic, linoleic and linolenic acids), linoleic acid being preferred (100% activity) over linolenic (60.4%) and oleic acids (46%). However, kinetic studies showed that the affinity of the enzyme is similar for the three substrates.     

Measurement of salicylic acid in human serum using stable isotope dilution and gas chromatography-mass spectrometry

A simple, highly selective, and sensitive method using stable isotope dilution and gas chromatography-mass spectrometry has been developed to quantify salicylic acid (SA) at concentrations naturally occurring in biological fluids, such as in the serum of subjects not taking aspirin. After extraction of liquid-liquid with diethyl ether and ethyl acetate and preparation of the tert-butyldimethylsilyl derivative, SA content was detected using deuterated SA as internal standard. The mean recovery of SA from serum was 85 +/- 6%. Intra- and interday precision and % relative error were <15% in all cases.With a detection limit of 0.6 ng and a quantification limit of 2 ng, the method is therefore also adequate for population studies because of the small amount of blood necessary to perform the analyses.