W. K. Rohwedder

Analysis of autoxidized fats by gas chromatography-mass spectrometry: II. Methyl linoleate

The gas chromatography-mass spectrometry (GC-MS) approach developed in the preceding paper was applied for qualitative and quantitative investigations of autoxidation products of methyl linoleate. A GC-MS computer summation method was standardized with synthetic 9- and 13-hydroxyoctadecanoate. Equal amounts of 9- and 13-hydroperoxides were found in all samples of linoleate autoxidized at different temperatures and peroxide levels. The results are consistent with the classical free radical mechanism of autoxidation involving a pentadiene intermediate having equivalent sites for oxygen attack at carbon-9 and carbon-13. Minor oxygenated products of autoxidation indicated by GC-MS include keto dienes, epoxyhydroxy monoenes di- and tri-hydroxy monoenes. These hydroxy compounds are presumed to be present in the form of hydroperoxides. The quantitative GC-MS method was found suitable for the analysis of autoxidized mixtures of oleate and linoleate. By this method, it is possible to determine the origin of the hydroperoxides formed in mixtures of these fatty esters.

Analysis of autoxidized fats by gas chromatography-mass spectrometry: I. Methyl oleate

A structural investigation of autoxidation products of methyl oleate was carried out by gas chromatography-mass spectrometry (GC-MS) of trimethylsilyl (TMS) ether derivatives. GC-MS using computer plots of selected masses afforded structural assignments of GC peaks due to incompletely resolved mixtures. This method provided evidence of epoxy and keto esters which are not completely separated from the main components consisting of the TMS derivatives of the allylic hydroxy esters. Use of an MS-computer system also showed that the hydroxyoctadecanoate TMS ethers were partially separated by GC. The use of synthetic hydroxyoctadecanoates for the first time enabled us to demonstrate the quantitative reliability of a GC-MS computer summation approach to analyze the isomeric composition of oleate hydroperoxides (as the saturated TMS ether derivatives). Consistently higher concentrations were found of the 8- and 11-hydroperoxides than of the 9- and 10-hydroperoxides. Minor products of autoxidation identified by GC-MS include allylic enones, isomeric epoxyoctadecanoates, dihydroxyctadecenoates, and dihydroxyoctadecanoates.

Volatile components from trilinolenin heated in air1

Headspace volatiles derived from trilinolenin heated to 192 C in air were collected, separated and identified using a microroom/GC-MS/ computer system. Data from four analyses indicated more than 80 volatiles were present. Of these volatiles, 38 have been identified, and their combined GC peak areas represent 90% of the total integrated Chromatographic area. Dominant volatiles identified included: ethanal (2%), 2-pentene (3%), ethanol (0.5%), 2-propenal (20%), propanal (7%), ethyl furan (13%), 2-butenal (4%), 2-pentenal (4%), 2,4-heptadienal (21%), 4,5-epoxy-2-heptenal (3%) and 2,4,7-decatrienal (2%). Most of the dominant and some of the minor volatiles are the predicted decomposition products of the four linolenin monohydroperoxides. However, the ratios of the predicted volatiles do not correspond to the monohydroperoxide ratios observed from autoxidized linolenate. Other primary and minor volatiles are present that are not predicted from the classical hydroperoxide decomposition hypothesis, which suggests that trilinolenin and some of the volatile compounds undergo further oxidation.

Analysis of autoxidized fats by gas chromatography-mass spectrometry: III. Methyl linolenate

The gas chromatography-mass spectrometry (GC-MS) method developed in the preceding papers was extended to the analysis of autoxidation products of methyl linolenate. Four isomeric hydroxy allylic trienes with a conjugated diene system were identified after reduction of the linolenate hydroperoxides. All eight geometrictrans,cis- andtrans, trans-conjugated diene isomers of these hydroxy allylic compounds were identified and partially separated by GC of the trimethylsilyl (TMS) ether derivatives. The proportion found of 9- and 16-hydroperoxides was significantly higher (75–81%) than the 12- and 13-hydroperoxides (18–25%). The tendency of the 12- and 13-hydroperoxides to form cyclic peroxides, cyclic peroxidehydroperoxides, and prostaglandin-like endoperoxides was supported by indirect evidence for the presence of 9,10,12- and 13,15,16-trihydroxyoctadecanoate in hydrogenated derivatives of the highly oxygenated products. The quantitative GC-MS method was used to determine the relative contribution of linolenate, linoleate, and oleate in mixtures to the formation of hydroperoxides.

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.

Volatile components from tristearin heated in air

Tristearin was heated to 192 C in air, and its volatile oxidation products were collected directly on a cooled (−60 C) gas liquid chromatography column. Subsequently, the volatile products were separated by temperature programing up to 250 C and identified by mass spectrometry. Methyl ketones and aldehydes were the major degradation products along with minor amounts of monobasic acids, n-hydrocarbons, primary alcohols, and γ-lactones. Qualitative results indicated all the fatty acid methylene carbon atoms are susceptible to oxidation. Quantities of aldehydes and ketones were found to be in excess of their taste threshold concentrations, suggesting thermally oxidized saturated fatty acids may be precursors of some odors and flavors associated with heated lipids.

Distribution of deuterium-labeledcis-andtrans-12-octadecenoic acids in human plasma and lipoprotein lipids

Triglycerides containingcis- andtrans-12-octadecenoic acid (12c-18∶1 and 12t-18∶1) andcis-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 labels, 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 monoenoic 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 sphingomyelin 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.

Metabolism in humans of cis-12,trans-15-octadecadienoic acid relative to palmitic, stearic, oleic and linoleic acids

Mixtures of triglycerides containing deuterium-labeled hexadecanoic acid (16∶0), octadecanoic acid (18∶0),cis-9-octadecenoic acid (9c–18∶1),cis-9,cis-12-octadecadienoic acid (9c, 12c–18∶2) andcis-12,trans-15-octadecadienoic acid (12c,15t–18∶2) were fed to two young-adult males. Plasma lipid classes were isolated from samples collected periodically over 48 hr. Incorporation and turnover of the deuterium-labeled fats in plasma lipids were followed by gas chromatography-mass spectrometry (GC-MS) analysis of the methyl ester derivatives. Absorption of the deuterated fats was followed by GC-MS analysis of chylomicron triglycerides isolated by ultracentrifugation.Results were the following: (i) endogenous fat contributed about 40% of the total fat incorporated into chylomicron triglycerides; (ii) elongation, desaturation and chain-shortened products from the deuterated fats were not detected; (iii) the polyunsaturated isomer 12c,15t–18∶2 was metabolically more similar to saturated and 9c–18∶1 fatty acids than to 9c,12c–18∶2 (iv) relative incorporation of 9c,12c–18∶2 into phospholipids did not increase proportionally with an increase of 9c,12c–18∶2 in the mixture of deuterated fats fed; (v) absorption of 16∶0, 18∶0, 9c–18∶1, 9c,12c–18∶2 and 12c,15t–18∶2 were similar; and (vi) data for the 1- and 2-acyl positions of phosphatidylcholine and for cholesteryl ester fractions reflected the known high specificity of phosphatidylcholine acyltransferase and lecithin:cholesteryl acyltransferase for 9c,12c–18∶2.These results illustrate that incorporation of dietary fatty acids into human plasma lipid classes is selectively controlled and that incorporation of dietary 9c,12c–18∶2 is limited. These results suggest that nutritional benefits of diets high in 9c,12c–18∶2 may be of little value to normal subjects and that the 12c,15t–18∶2 isomer in hydrogenated fat is not a nutritional liability at the present dietary level.

Absorption and distribution of deuterium-labeled trans- and cis-11-octadecenoic acid in human plasma and lipoprotein lipids.

Triglycerides of deuterium-labeledtrans-11-,trans-11-cis-11- andcis-9-octadecenoic acid (11t-18∶1-2H, 11c-18∶1-2H) were simultaneously fed to two young adult male subjects. Plasma lipids from blood samples collected periodically for 48 hr were analyzed by gas chromatography-mass spectroscopy. The results indicate (i) the Δ11-18∶1-2H acids and 9c-18∶1-2H were equally well absorbed; (ii) relative turnover rates were higher for the Δ11-18-1-2H acids in plasma triglycerides; (iii) incorporation of the Δ11-18∶1-2H acids into plasma phosphatidylcholine was similar to 9c-18∶1-2H, but distribution at the 1-and 2-acyl positions was substantially different; (iv) esterification of cholesterol with 11t-18∶1 was extremely low; (v) chain shortening of the Δ11-18∶1-2H acids was 2–3 times greater than for 9c-18∶1-2H; (vi) no evidence for desaturation or elongation of the 18∶1-2H acids was detected; and (vii) a 40% isotopic dilution of the 18∶1-2H acids in the chylomicron triglyceride fraction indicated the presence of a substantial intestinal triglyceride pool. Based on our present knowledge, these metabolic results for Δ11-18∶1 acids present in hydrogenated oils and animal fats indicate that the Δ11 isomers are no more likely than 9c-18∶1 to contribute to dietary fat-related health problems.

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

Two deuterated fatty acids, elaidate-d2 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 cholesterol ester to 64% in phosphatidyl ethanolamine. The highest levels of deuterated fat occurred in either 6-, 8-, or 12-hr samples; generally, <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.