Separation of phytanic and pristanic acid by high-pressure liquid chromatography: application of the method.

The synthesis of pristanic acid from phytanic acid, and a simple reversed-phase high-pressure liquid chromatographic (HPLC) method for the separation and purification of these acids, is described. A base-line separation of [U-3H]phytanic and [U-3H]pristanic acid is achieved with a graphitized carbon column. The isoprenoid metabolites formed after incubation of cultured fibroblasts with phytanic or pristanic acids are extracted with a Sep-Pak C18 cartridge and separated from the substrates by the same reversed-phase HPLC used for substrate purification. The methods are suitable for studies on the mechanisms for degradation of phytanic acid. Recently, different inborn errors with accumulation of phytanic acid have been defined. The present method will be a useful tool in our efforts to define these metabolic defects and their subcellular localization.

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase.

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca2+ concentration ([Ca2+]i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and alpha-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n-6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n-3 fatty acids (alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n-6 fatty acids (linoleic acid and arachidonic acid), the total n-3 fatty acyl content was reduced in all the phospholipids examined. In n-3 and n-6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n-9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appears to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca2+]i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n-3 and n-6 PUFA but not in n-9 monounsaturated fatty acid modified cells. In Ca2+ free medium, OKT3-induced transient increase in [Ca2+]i representing Ca2+ release from the inositol 1,4,5-trisphosphate-sensitive Ca2+ stores, were similar in control and UFA modified cells. Using Mn2+ entry as an index of plasma membrane Ca2+ permeability, the rate of fura-2 fluorescence quenching as a result of Mn2+ influx stimulated by OKT3 in n-9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n-3 and n-6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca2+ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and monounsaturated fatty acids appears to be important for the maintenance of a functional Ca2+ influx mechanism.

Identification of intermediates in the peroxisomal beta-oxidation of linoleic acid.

Rat liver peroxisomes contain a beta-oxidation system different from that present in the mitochondria. Intermediates in this oxidation have not hitherto been identified by direct methods. Incubation of linoleic acid with isolated peroxisomes (in the absence of detergent) resulted in the accumulation of polar products in addition to the chain-shortened products. Omission of NAD in the incubation mixture considerably increased the accumulation of these products. Two of the products were isolated and characterized by gas chromatography-mass spectrometry. They were identified as 2,3-dehydrolinoleic acid and 3-hydroxylinoleic acid, based on identical chromatographic behaviour and mass spectra compared to synthetic reference compounds. Stereochemical analysis of catalytically hydrogenated 3-hydroxylinoleic acid showed a D/L ratio near to one. The mechanism behind the apparent lack of stereospecificity is discussed in relation to the recently described novel peroxisomal 2-enoyl-CoA hydratase (Smeland, T.E., Li, J., Chu, C.-h., Cuebas, D. and Schultz, H. (1989) Biochem. Biophys. Res. Commun. 160, 988-992 and Hiltunen, J.K., Palosaari, P.M. and Kunau, W.-H. (1989) J. Biol. Chem. 264, 13536-13540). In previous work we have demonstrated that beta-oxidation intermediates accumulate also in the peroxisomal metabolism of C27-bile acid intermediates and prostaglandins. The possibility is discussed that the peroxisomal beta-oxidation system is less tightly coupled than the corresponding system in mitochondria.

Suppression of growth in a leukemic T cell line by n-3 and n-6 polyunsaturated fatty acids.

Proliferation in a leukemic T cell line (Jurkat) was suppressed in a dose dependent manner by n-6 and n-3 polyunsaturated fatty acids (PUFA) added to the culture medium. At high concentrations, PUFA have a cytotoxic effect on Jurkat cells. The inhibitory effect of the PUFA was not due to production of prostaglandins, and lipid peroxidation was only partly responsible. In addition to production of peroxides and aldehydes, lipid peroxidation also reduced the plasmalogen levels in these cells. The antioxidant alpha-tocopherol blocked lipid peroxidation and restored the plasmalogen levels to normal. alpha-Tocopherol did not totally restore cell proliferation although the MDA-like products in these cultures (supplemented with PUFA) were reduced to control level. Cultures supplemented with n-6 PUFA seemed to respond better to alpha-tocopherol than n-3 PUFA. This suggests that n-6 PUFA may exert their growth inhibitory effect predominantly via lipid peroxidation while different mechanisms might be operating for the n-3 PUFA.

Determination of serum levels of unesterified lathosterol by isotope dilution-mass spectrometry.

The synthesis of 2H3-labelled lathosterol is described. This compound was used together with 2H7-labelled cholesterol for simultaneous assay of unesterified lathosterol and cholesterol in serum by isotope dilution-mass spectrometry. After addition of a fixed amount of the two internal standards to a fixed amount of serum (in general 25 microliter), the steroids were extracted with chloroform and subjected to Lipidex 5000 chromatography. The fraction containing cholesterol and lathosterol was converted into trimethylsilyl ether and subjected to mass spectrometric analysis with selected monitoring of the ions at m/z 458 (molecular ion of the trimethylsilyl ether derivative of unlabelled cholesterol and lathosterol), m/z 461 (molecular ion of derivative of 2H3-labelled lathosterol) and m/z 465 (molecular ion of derivative of 2H7-labelled cholesterol). Individual standard curves were used for assay of each steroid. Under the conditions employed, the coefficient of variation of the two assays was less than 6%. In different recovery experiments the maximal difference between expected and found values was less than 7%. Using a less accurate method for analysis of lathosterol, we have shown previously that there is a high correlation between the hepatic HMG CoA reductase and the relative concentration of unesterified lathosterol in serum (concentration of lathosterol relative to cholesterol). This was confirmed with the present method and a correlation coefficient of about 0.94 was found between the two parameters. It is concluded that the present method may be suitable for detection of cases with accelerated rate of synthesis of cholesterol.

Simple diagnosis of the Zellweger syndrome by gas-liquid chromatography of dimethylacetals.

The absence of peroxisomes in patients with the cerebrohepatorenal syndrome of Zellweger leads to several biochemical abnormalities, including deficient synthesis of plasmalogens as well as accumulation of very long-chain fatty acids and intermediates in bile acid biosynthesis. Accumulation of very long-chain fatty acids in serum and fibroblasts has hitherto been used most extensively for diagnosis. Due to the relatively small amounts of the very long-chain fatty acids also in the Zellweger patients, and the risk for interfering impurities, such analyses are difficult. Direct assay of plasmalogens is also relatively difficult and time-consuming. In this report, we describe a relatively simple method for diagnosis, based on gas-liquid chromatography of a lipid extract of erythrocytes after methyl transesterification. The alpha, beta-unsaturated ether in the plasmalogen is converted to the dimethylacetal of the corresponding aldehyde, and the relative amount of plasmalogen is thus reflected by the ratio between 18:0 dimethylacetal and methyl stearate as well as by the ratio between 16:0 dimethylacetal and methyl palmitate. The ratio 18:0 dimethylacetal/methyl stearate was found to be 0.28 +/- 0.03 (mean +/- SD) after analyses of erythrocytes from healthy or non-Zellweger infants, but less than 0.02 in erythrocytes from three infants with the Zellweger syndrome. Preliminary work with amniotic fluid suggests that this analysis may be suitable also for prenatal diagnosis of the Zellweger syndrome.

Dietary modulation of retina phospholipids and photoreceptor function with particular reference to phosphatidylinositol.

The effect of dietary partially hydrogenated herring oil (HHO) on the fatty acid composition of the phospholipids of rat retina and on the amplitude of the a-wave of the electroretinogram (ERG) was studied in rats raised for several generations on an essential fatty acid poor diet. The most significant effect of the dietary treatment was a decreased content of arachidonic acid (C20:4 omega 6) and an increased concentration of docosahexaenoic acid (C22:6 omega 2) and 18:1 isomers associated with the phosphatidylinositol (PI). The a-wave of the ERG showed a decreasing amplitude during the experiment and was reduced with about 30% (P less than 0.004) of the initial value in all rats at the end of the experiment. The pronounced change in the arachidonic content in PI and the decreased amplitude of the a-wave of the ERG suggest that arachidonic acid of the PI has an important function in the visual phototransduction process.

Occurrence of short chain aliphatic diols in human blood: identification by gas chromatography-mass spectrometry.

Patients attending a general hospital for various reasons were screened for raised serum gamma glutamyltransferase (gamma-GT) and positive blood alcohol concentration (BAC). The results served as objective biochemical tests of heavy drinking. Among 419 individuals, 50 (11.9%) met these requirements and blood samples were used to determine the presence of low molecular mass aliphatic diols. 1,2-Propanediol, 2,3-butanediol and 2,2-dimethyl-1,3-propanediol were determined by gas liquid chromatography-mass spectrometry (GC-MS). In patients with blood ethanol concentration less than 2 mmol/l, 1,2-propanediol and 2,2-dimethyl-1, 3-propanediol mainly occurred together at median concentrations of about 200 mumol/l. When blood ethanol was 2-42 mmol/1,2,3-butanediol was also present covering a wide concentration range: three patients had concentrations between 6 and 10 mmol/l. There was no apparent correlation between the concentration of 2,3-butanediol and the concentration of blood ethanol. The diols were below the limits of detection in blood from nonintoxicated control individuals and hospital in-patients.

Influence of dietary partially hydrogenated vegetable and marine oils on membrane composition and function of liver microsomes and platelets in the rat.

The aim of the present study was to investigate the influence of partially hydrogenated vegetable and marine oils on membrane composition and function of liver microsomes and platelets with particular reference to the metabolism of linoleic acid and the production of arachidonic acid metabolites. Four groups of male weanling rats were fed linoleic acid supplemented diets containing 20% (w/w) of partially hydrogenated low erucic acid rapeseed oil (HLRSO), partially hydrogenated herring oil (HHO), olive oil (OO) and trierucin + triolein (TE) for 10 weeks. An additional two groups were fed partially hydrogenated low erucic acid rapeseed oil and partially hydrogenated herring oil without linoleic acid supplementation (HLRSO- and HHO-, respectively). Substantial amounts of trans fatty acids were incorporated into liver microsomes (12.6% in group HLRSO) and platelets (7.0% in group HLRSO-). This incorporation was not dependent on the dietary linoleic acid level. Hepatic microsomal delta5 -desaturase activity was significantly increased after HLRSO feeding compared to 00 feeding. Delta6 -Desaturase activity did not vary in the linoleic acid supplemented groups. Both delta5 -and delta6 -desaturase activities were significantly increased in groups without linoleic acid supplementation. Docosenoic acid was incorporated into platelet phospholipids in contrast to liver microsomes. In the platelet, docosenoic acid seemed to have a special preference for phosphatidylserine. Very small amounts were incorporated into platelet phosphatidylinositol. Feeding diets HLRSO, HHO and 00 did not influence rat platelet cyclooxygenase or 12-lipoxygenase activity. Platelets from rats fed TE, however, produced significantly less 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) than platelets from rats fed OO. Feeding of HLRSO- and HHO- resulted in a significantly diminished production of the arachidonic acid metabolites 12-HETE, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 6-keto-prostaglandin F1alpha in stimulated platelets and aorta. Thus, high dietary levels of trans isomers of monoenoic acids do not interfere with platelet cyclooxygenase or lipoxygenase activity provided sufficient amounts of linoleic acid are available.

High performance liquid chromatography and glass capillary gas chromatography of geometric and positional isomers of long chain monounsaturated fatty acids.

Positional and geometrical isomers of monounsaturated long chain fatty acids were analyzed by the combination of high performance liquid chromatography (HPLC) and glass capillary gas chromatography (GC). A preparative group separation ofcis andtrans isomers of the monounsaturated fatty acid methyl esters was achieved according to chain length by reversed-phase HPLC, and using a highly sensitive interference refractive index detector. After collection of the different fractions containingcis andtrans forms of the monounsaturated fatty acid methyl esters, the fractions were analyzed for their content of positional isomers using glass capillary GC with Silar-5 CP as stationary phase. The preparative step in the HPLC was also used analytically for the determination of the ratio between thecis andtrans monounsaturated fatty acids. A comparison was made between the results obtained with the HPLC technique and the results of a GLC technique with a packed OV-275 column. There was a good correlation between the 2 techniques with a tendency to highertrans values with the HPLC technique (4%). It was shown with reference substances that 18∶1ω6-cis to ω11-cis and 18∶1ω5-trans to ω12-trans, the most common monounsaturated fatty acid isomers in partially hydrogenated vegetable oils, could be almost quantitatively recovered in the HPLC step. Most of the individual positional isomers of monounsaturated fatty acids of varying chain length could be separated and determined in the glass capillary GC step with the exception of those isomers containing the double bond in a relatively high ω-position. The relative standard deviation of the technique as determined with reference substances was better than 4%. The described technique was applied to the analysis of the isomeric monounsaturated fatty acid content in partially hydrogenated vegetable and marine oils, and about 5 samples a day could be executed.