Unusual isomeric polyunsaturated fatty acids in liver phospholipids of rats fed hydrogenated oil.

Linoleic acid (18:2 omega 6) and linolenic acid (18:3 omega 3) are precursors of two series of essential fatty acids (EFA) formed by alternate desaturations and elongations. In EFA deficiency (EFAD), oleic acid (18:1 omega 9) and palmitoleic acid (16:1 omega 7) undergo the same reactions to form polyunsaturated fatty acids (PUFA) of other structures. Partially hydrogenated soybean oil (PHSO) contains isomeric 18:1 acids that can be converted to unusual isomers of 18:2 by liver microsomes. To test whether 18:2, 20:3, and 20:4 of unusual structure occur in phospholipids as a consequence of EFAD or ingestion of PHSO, rats were fed corn oil, an EFA-deficient diet, or PHSO to provide isomeric 18:1 acids. At 2.5 months the phospholipids were isolated from livers and converted to methyl esters, and the 18:2, 20:2, 20:3, and 20:4 fractions were isolated. The 18:2 and 20:2 fractions were ozonized, and, by using a computer solution of simultaneous equations, the structures and proportions of each isomer were calculated. The 20:3 and 20:4 fractions were analyzed by ozonolysis and capillary gas chromatography. When corn oil was fed, the major isomer in each group was 9,12-18:2, 11,14-20:2, 8,11,14-20:3, and 5,8,11,14-20:4. Patterns in EFAD- and PHSO-fed groups were more diverse, with large proportions of unusual isomers. Feeding EFA-deficient diet and PHSO induced measurable amounts of unusual PUFA at each step of the cascade, and these PUFA may compete in metabolism of normal PUFA and are substrates for oxidative formation of autacoids of unknown structures and function.

Composition of mixed octadecadienoates via ozonolysis, chromatography and computer solution of linear equations.

An approach to the analysis of 55 possible nonconjugated positional isomers of octadecadienoic acid is described and tested with mixtures of individual synthetic methyl esters. In the first example, by ozonolysis a seven-component mixture consisting of cis,cis 5,12-, 6,10-, 6,11-, 6,12-, 7,12-, 8,12-, and 9,12-octadecadienoates was converted to aldehydes, aldehyde-esters and dialdehydes. These fragments were separated on a 50 m X 0.2 mm free fatty acid phase (FFAP) vitreous silica capillary column. Equations for an arbitrarily restricted 12 X 15 matrix of linear simultaneous equations and a computer solution of the matrix provided the composition of the initial methyl octadecadienoate mixture. The power and significance of this method became apparent with the observation that only two of the seven isomers in the known mixture were resolved as single peaks by state-of-the-art capillary gas chromatography, but all seven were identified and estimated with acceptable error by the ozonolysis-capillary gas chromatography-computer procedure. In a generalized approach to the analysis of the 55 possible nonconjugated isomers, a computer program selects the appropriate matrix of linear simultaneous equations based on the aldehyde data supplied by the analyst. Twenty of 21 combinations of seven isomeric esters taken five at a time have been analyzed to assess the efficiency of the method. To illustrate applicability at this stage of development, the method has been used to analyze the diene products of the hydrazine reduction of gamma-linolenic acid and the diene products from the biological desaturation of isomeric monoenes.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of deuterium-labeled cis- and trans-12-octadecenoic acids in human plasma and lipoprotein lipids.

Triglycerides containing cis- and trans-12-octadecenoic acid (12c-18:1 and 12t-18:1) and cis-9-octadecenoic acid (9c-18:1) labeled with deuterium were fed to 2 young adult male subjects. These fatty isomers each contained a different number of deuterium lables, which allowed mass spectrometric analysis to distinguish among them after they were fed as a mixture. This approach results in a direct comparison of the absorption and distribution of these 3 monenoic acids into blood plasma and lipoprotein lipids. Plasma lipid data indicated that all phospholipid fractions selectively incorporate 12c-18:1 and 12t-18:1 in preference to 9c-18:1. Discrimination against 12c-18:1 and 12t-18:1 compared to 9c-18:1 was found in the plasma neutral lipids, with a strong discrimination against 12t-18:1 incorporation into the cholesteryl ester fraction. Considerable reduction in the percentage of linoleic and arachidonic acid was observed when 12-18:1 isomers were incorporated in plasma triglyceride, phosphatidylcholine and spingomyelin samples. Chylomicron lipid analyses indicated that all isomers were well absorbed. Variation was observed in the relative distribution of 12c-18:1, 12t-18:1 and 9c-18:1 between the very low density, low density and high density lipoprotein lipid classes. No desaturation of 12c-18:1 to linoleic acid was detected.

Incorporation of deuterium-labeled cis- and trans-9-octadecenoic acids in humans: plasma, erythrocyte, and platelet phospholipids.

The objective of this study was to follow the uptake and distribution of oleic and elaidic acids into human erythrocytes, platelets, and plasma phospholipids. The use of dual and triple labeling methodology permitted a precise comparison of elaidic and oleic acid utilization. Elaidic acid (EI) was selectively concentrated in all the plasma phospholipids except for lysophosphatidylcholine. Three times more elaidic than oleic acid (OI) accumulated in the 1-acyl position of phosphatidylcholine, as determined by hydrolysis with phospholipase A2. Rapid incorporation and removal of elaidate were observed for all samples. These results support the concept that enzymes responsible for acylation of phospholipids are sensitive to double bond configuration and the physical properties of the fatty acid moieties. Labeled fatty acid levels in red cell and platelet phospholipids were much lower than for plasma phospholipids, indicating a relatively slow rate for the in vivo incorporation of fatty acids into blood cell membrane phospholipids. No isotope effect was found when oleic acid labeled with deuterium on the double bond was used.

In vivo metabolism of labeled oleic and linoleic acids by the laying hen.

Radioactive oleic and linoleic acids, labeled with 3H in the chain and 14C in the carbonyl group, were administered to white leghorn laying hens. Mixtures fed in separate experiments included: (1) 3H- and 14C-labeled oleic acid, (2) 3H- and 14C-labeled linoleic acid and (3) [3H]oleic aicd and [14C] linoleic acid. The 3H/14C ratios of both the neutral lipid and phospholipid fractions from the egg yolk and of the isolated acids from these lipid fractions were compared to that in the administered mixture. Agreement in the 3H/14C ratios for the neutral lipid fraction from each of the feeding experiments indicated that neither the 3H- and 14C labeled acids nor the oleic or linoleic acids were distinguishable during synthesis of the neutral lipid. Analysis of the phospholipid fractions showed that when dual-labeled mixtures of oleic acid were administered, 3H/14C ratios were elevated and, therefore, there was selective elimination of the 14C label. When dual-labeled mixtures of linoleic acid were administered, the 3H/14C ratios were in agreement; and when the two acids were administered simultaneously as a dual-labeled mixture, there was selective incorporation of linoleic acid. These findings indicate separate metabolic pathways for synthesis of neutral lipid and phospholipid in egg yolk as expected, as well as preferential use of the essential fatty acid in the phospholipid by the hen.

Dual-labeled technique for human lipid metabolism studies using deuterated fatty acid isomers.

Two deuterated fatty acids, elaidated2 and oleate-d4, were fed simultaneously to a human subject as a mixture of trielaidin-d6 and triolein-d12. Periodically, blood samples were drawn, and red blood cells were separated from the plasma. Red blood cells and plasma lipids were fractionated and analyzed by combined gas chromatography--multiple ion mass spectroscopy. Dual deuterium-labeling allows rate and extent of fatty acid incorporation to be followed in various plasma and red cell neutral and phospholipid fractions. Maximum amount of deuterated fat varied from 4% in cholescurred in either 6-, 8-, or 12-hr samples; generally, less than 1% labeled fatty acids could be detected in 72-hr samples. Because the method is based on dual-labeling, differences in the relative incorporation of both fatty acid isomers can be compared directly. Differences in rates of incorporation, rates of removal, and extent of incorporation of labeled fatty acids into blood plasma can also be determined reliably. Our experimental labeling of fats with deuterium permits for the first time the metabolism of two fatty acid isomers to be compared simultaneously in human subjects. This new method should be applicable to a variety of other lipid metabolic studies.

Determination of petroselinic acid by microreactor chromatography.

The microreactor-ozonolysis technique was applied to the quantitative determination of the relative amounts of petroselinic and oleic acids in seven Umbelliferae seed oils. The operation was easy and rapid. Results were excellent when the method was tested on ester mixtures of known composition. When used on esters prepared from Umbelliferae seed oils, the method gave results comparable with those found by another procedure, also described, which combined thin-layer chromatography with either gas-liquid chromatography or ultraviolet spectrophotometry.

Microreactor for methanolysis of triglycerides before gas-liquid chromatography.

A rapid, accurate microtechnique has been developed for gas chromatographic determination of the fatty acid composition of small (2-3 micro l) samples of vegetable oils. This microtechnique combines transesterification and sample injection into a single operation. The fatty acid compositions of soybean, linseed, and safflower oils thus determined are compared with those obtained by the usual two-step procedure.

Metabolic conversions and the positional distributions in liver lecithin of some unnatural dienoic acids in the rat.

The metabolism of 9,15-octadecadienoate, 12,15-octadecadienoate and 7,13-eicosadienoate was investigated in the fatdeficient rat. The liver lecithins of those animals receiving 9,15- and 12,15-octadecadienoate, as well as the animal remaining on the fat-deficient control, were isolated. The fatty acid distributional pattern was determined by the use of lecithinase A. All three of the experimental dienoic acids were incorporated into tissue lipids. None of the acids, however, was converted to longer chain polyunsaturated fatty acids. The 9,15-octadecadienoate was esterified almost exclusively to the beta-position of lecithin, whereas 12,15-octadecadienoate was about equally distributed between the alpha- and beta-positions.

Mechanism of lipoxidase reaction. I. Specificity of hydroperoxidation of linoleic acid.

Linoleate hydroperoxides from autoxidation of methyl linoleate and from lipoxidase oxidation of linoleic acid are compared. Data indicate an equal amount of methyl 9- and 13-hydroperoxyoctadecadienoate produced by autoxidation of methyl linoleate, and the exclusive formation of 13-hydroperoxyoctadeca-9,11-dienoic acid from the incubation of lipoxidase with linoleic acid. As a result of these findings, a specific mechanism for the reaction of lipoxidase with linoleic acid is postulated.

Mechanism of lipoxidase reaction. II. Origin of the oxygen incorporated into linoleate hydroperoxide.

Two different series of experiments were performed to establish the origin of the oxygen molecule incorporated into hydroperoxide during the incubation of lipoxidase with linoleic acid. These showed, as previously assumed but never demonstrated, that the oxygen introduced into the hydroperoxide molecules comes from the gaseous phase and not from the aqueous phase. Furthermore, soybean lipoxidase does not catalyze the exchange between gaseous oxygen and water oxygen. Possibly, lipoxidase may be involved in the biosynthesis of hydroxytrans,cis conjugated octadecadienoates present in various seeds.