E. A. Emken

Biosynthesis of conjugated linoleic acid in humans.

This paper deals with the reanalysis of serum lipids from previous studies in which deuterated fatty acids were administered to a single person. Samples were reanalyzed to determine if the deuterated fatty acids were converted to deuterium-labeled conjugated linoleic acid (CLA, 9c, 11t-18∶2) or other CLA isomers. We found 11-trans-octadecenoate (fed as the triglyceride) was converted (Δ9 desaturase) to CLA, at a CLA enrichment ofca. 30%. The 11-cis-octadecenoate isomer was also converted to 9c, 11c-18∶2, but at <10% the concentration of the 11t-18∶1 isomer. No evidence (within our limits of detection) for conversion of 10-cis-or 10-trans-octadecenoate to the 10,12-CLA isomers (Δ12 desaturase) was found. No evidence for the conversion of 9-cis, 12-cis-octadecadienoate to CLA (via isomerase enzyme) was found. Although these data come from isomerase enzyme) was found. Although these data come from four single human subject studies, data from some 30 similar human studies have convinced us that the existence of a metabolic pathway in one subject may be extrapolated to the normal adult population.

Effect of dietary docosahexaenoic acid on desaturation and uptake in vivo of isotope-labeled oleic, linoleic, and linolenic acids by male subjects.

The effect of dietary docosahexaenoic acid (22∶6n−3, DHA) on the metabolism of oleic, linoleic, and linolenic acids was investigated in male subjects (n=6) confined to a metabolic unit and fed diets containing 6.5 or <0.1 g/d of DHA for 90 d. At the end of the diet period, the subjects were fed a mixture of deuterated triglycerides containing 18∶1n−9[d6], 18∶2n−6[d2], and 18∶3n−3[d4]. Blood samples were drawn at 0, 2, 4, 6, 8, 12, 24, 48, and 72 h. Methyl esters of plasma total lipids, triglycerides, phospholipids, and cholesterol esters were analyzed by gas chromatography-mass spectrometry. Chylomicron triglyceride results show that the deuterated fatty acids were equally well absorbed and diet did not influence absorption. Compared to the low-DHA diet (LO-DHA), clearance of the labeled fatty acids from chylomicron triglycerides was modestly higher for subjects fed the high DHA diet (HI-DHA). DHA supplementation significantly reduced the concentrations of most n-6[d2] and n-3[d4] long-chain fatty acid (LCFA) metabolites in plasma lipids. Accumulation of 20∶5n−3[d4] and 22∶6n−3[d4] was depressed by 76 and 88%, respectively. Accumulations of 20∶3n−6[d2] and 20∶4n−6[d2] were both decreased by 72%. No effect of diet was observed on acyltransferase selectivity or on uptake and clearance of 18∶1n−9[d6], 18∶2n−6[d2], and 18∶3n−3[d4]. The results indicate that accumulation of n−3 LCFA metabolites synthesized from 18∶3n−3 in typical U.S. diets would be reduced from about 120 to 30 mg/d by supplementation with 6.5 g/d of DHA. Accumulation of n−6 LCFA metabolites synthesized from 18∶2n−6 in U.S. diets is estimated to be reduced from about 800 to 180 mg/d. This decrease is two to three times the amount of n−6 LCFA in a typical U.S. diet. These results support the hypothesis that health benefits associated with DHA supplementation are the combined result of reduced accretion of n−6 LCFA metabolites and an increase in n−3 LCFA levels in tissue lipids.

The isolation of omega-3 polyunsaturated fatty acids and methyl esters of fish oils by silver resin chromatography

Multigram quantities of the highly unsaturated ω3 component from samples of fish oil fatty acids and esters were isolated by silver resin chromatography. An XN1010 resin column saturated with silver ions was utilized. Polyunsaturated fatty acid (PUFA) esters from fish oil concentrate (FOC) were fractionated based on the number of double bonds by using solvent programming (acetonitrile in methanol). Larger samples (4–9 g) of FOC acids and esters and menhaden acids and esters were enriched in ω3 polyunsaturates to 82–99% (95–99% total PUFA) by use of a large 100% silver resin column and isocratic elution with 30, 35 or 45% acetonitrile in acetone.

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.

Occurrence of octadecenoic fatty acid isomers from hydrogenated fats in human tissue lipid classes

The level oftrans-18∶1 isomers in several isolated lipid classes of human liver, heart, red blood cells and plasma was determined. Phospholipids contained substantially fewertrans-18∶1 isomers than triglycerides. The double bond distribution of thecis andtrans octadecenoate fraction of triglycerides and phosphatidylcholines from human liver and heart was determined. Whereas the double bond distribution of the triglycerides correlated closely with the pattern found in dietary hydrogenated vegetable oils, the phosphatidylcholine fraction showed evidence of selective incorporation or metabolism of specifictrans positional isomers. In general, isomers with double bonds near the methyl terminus were present at levels higher than expected from their relative abundance in the diet. Refinements in methodology needed to analyze octadecenoate double bond configuration and location in human tissues are presented.

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.

Effect of dietary arachidonic acid on metabolism of deuterated linoleic acid by adult male subjects

The influence of dietary supplementation with 20:4n−6 on uptake and turnover of deuterium-labeled linoleic acid (18:2n−6[d2]) in human plasma lipids and the synthesis of desaturated and elongated n−6 fatty acids from 18:2n−6[d2] were investigated in six adult male subjects. The subjects were fed either a high-arachidonic acid (HIAA) diet containing 1.7 g/d or a low-AA (LOAA) diet containing 0.21 g/d of AA for 50 d. Each subject was then dosed with about 3.5 g of 18:2n−6[d2] as the triglyceride (TG) at 8:00 a.m., 12:00, and 5:00 p.m. The total 18:2n−6[d2] fed to each subject was about 10.4 g and is approximately equal to one-half of the daily intake of 18:2n−6 in a typical U.S. male diet. Nine blood samples were drawn over a 96-h period. Methyl esters of plasma total lipid (TL), TG, phospholipid, and cholesterol ester were analyzed by gas chromatography-mass spectroscopy. Dietary 20:4n−6 supplementation did not affect uptake of 18:2n−6[d2] in plasma lipid classes over the 4-d study period nor the estimated half-life of 24–36 h for 18:2n−6[d2]. The percentages of major deuterium-labeled desaturation and elongation products in plasma TL, as a percentage of total deuterated fatty acids, were 1.35 and 1.34% 18:3n−6[d2]; 0.53 and 0.50% 20:2n−6[d2]; 1.80 and 0.92% 20:3n−6[d2] and 3.13 and 1.51% 20:4n−6[d2] for the LOAA and HIAA diet groups, respectively. Trace amounts (<0.1%) of the TL concentration data for both 20:3n−6[d2] and 20:4n−6[d2] were 48% lower (P<0.05) in samples from the HIAA diet group than in samples from the LOAA diet group. For a normal adult male consuming a typical U.S. diet, the estiamted accumulation in plasma TL of 20:4n−6 synthesized from 20 g/d (68 mmole) of 18:2n−6 is 677 mg/d (2.13 mmole). Dietary supplementation with 1.5 g/d of 20:4n−6 reduced accumulation of 20:4n−6 synthesized from 20 g/d of 18:2n−6 to about 326 mg/d (1.03 mmole).

Comparison of FTIR and capillary gas chromatographic methods for quantitation oftrans unsaturation in fatty acid methyl esters

A computer-assisted method has been developed for estimation of isolatedtrans unsaturation using the peak area of thetrans absorbance band at 966 cm-1from FTIR spectra of fatty acid methyl esters. Peak areas were used to determine thetrans content of weighed standards containing from 0 to 100% methyl elaidate and of hydrogenated soybean oil samples containing up to 36%trans unsaturation. These data for percenttrans by FTIR were compared to corresponding data obtained by capillary gas chromatography and the AOCS Official Method 14-61. Determination of isolatedtrans composition in oils using peak areas gave values with the smallest standard deviation for weighed standards and values within 4% of those obtained by capillary gas chromatography and the AOCS Official Method for hydrogenated samples.

Acetonitrile as eluent in silver resin column chromatography

Acetonitrile (ACN) is shown to be an effective eluent in combina-tion with methanol (MeOH) or acetone for the rapid separation of polyunsaturated fatty acid methyl esters on a fully silver-loaded ion-exchange (XN1010) column. ACN-containing eluents thus provide on one fully siliver-loaded resin column an attractive alternative separation system to a series of partially silvered resin columns (“PARC” columns), which previously have been used for such separations. Solvent programming from 0 to 15% ACN in MeOH allows the separation of methyl oleate, linoleate, linolenate and arachidonate within 3 hr. Preparative (up to 800 mg) isocratic separations of a number of polyunsaturated fatty acid esters from natural sources (methyl linoleate from safflower esters, for example) were readily achieved.