R. O. Adlof

Advances in conjugated linoleic acid research

BIOSYNTHESIS AND METABOLIC PROCESSES Conjugated Linoleic Acid Synthesis within the Gut Microbial Ecosystem of Ruminants Detection of Partial b-Oxidation Products of Conjugated Linoleic Acid Isomers and Their Metabolites in Animals and Humans TECHNIQUES OF DETERMINATION OF INDIVIDUAL ISOMERS Syntheses of Conjugated Linoleic Acids Systematic Analysis of trans and Conjugated Linoleic Acids in the Milk and Meat of Ruminants New Developments in Silver Ion and Reverse Phase HPLC of Conjugated Linoleic Acid Structural Characterization of Conjugated Linoleic Acid Methyl Esters with Acetonitrile Chemical Ionization Tandem Mass Spectrometry DIVERSITY OF CLA Growth Inhibition and Apoptotic Cell Death of Cancer Cells Induced by Conjugated Linoleic Acid Modulatory Properties of Conjugated Linoleic Acid on Inflammation and Immune Function: Cellular and Molecular Mechanisms Conjugated Linoleic Acid in Hypertension Antioxidative Activity of Conjugated Linoleic Acid Determined by ESR EFFECTS OF CLA ISOMERS IN HUMANS An Overview of the Effects of Conjugated Linoleic Acid on Body Weight and Body Composition in Humans Conjugated Linoleic Acid in Healthy and Cancerous Human Tissues Lipid-Lowering Actions of trans-10, cis-12 Conjugated Linoleic Acid in Primary Cultures of Human (Pre)Adipocytes Safety Data on Conjugated Linoleic Acid from Animal Studies: An Overview

Improved separation of conjugated fatty acid methyl esters by silver ion-high-performance liquid chromatography

Operating from one to six silver ion-high-performance liquid chromatography (Ag+-HPLC) columns in series progressively improved the resolution of the methyl esters of conjugated linoleic acid (CLA) isomeric mixtures from natural and commercial products. In natural products, the 8 trans, 10 cis-octadecadienoic (18∶2) acid was resolved from the more abundant 7 trans, 9 cis-18∶2, and the 10 trans, 12 cis-18∶2 was separated from the major 9 cis, 11 trans-18∶2 peak. In addition, both 11 trans, 13 cis-18∶2 and 11 cis, 13 trans-18∶2 isomers were found in natural products and were separated; the presence of the latter, 11 cis, 13 trans-18∶2, was established in commercial CLA preparations. Three Ag+-HPLC columns in series appeared to be the best compromise to obtain satisfactory resolution of most CLA isomers found in natural products. A single Ag+-HPLC column in series with one of several normal-phase columns did not improve the resolution of CLA isomers as compared to that of the former alone. The 20∶2 conjugated fatty acid isomers 11 cis, 13 trans-20∶2 and 12 trans, 14 cis-20∶2, which were synthesized by alkali isomerization from 11 cis, 14 cis-20∶2, eluted in the same region of the Ag+-HPLC chromatogram just before the corresponding geometric CLA isomers. Therefore, CLA isomers will require isolation based on chain length prior to Ag+-HPLC separation. The positions of conjugated double bonds in 20∶2 and 18∶2 isomers were established by gas chromatography-electron ionization mass spectrometry as their 4,4-dimethyloxazoline derivatives. The double-bond geometry was determined by gas chromatography-direct deposition-Fourier transform infrared spectroscopy and by the Ag+-HPLC relative elution order.

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.

Preparation, separation, and confirmation of the eight geometrical cis/trans conjugated linoleic acid isomers 8,10-through 11,13–18∶2

Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or I2 catalyst. The resultant mixtures of the eight cis/trans geometric isomers of 8,10-, 9,11-, 10,12-, and 11,13-octadecadienoic (18∶2) acid methyl esters were separated by silver ion-high-performance liquid chromatography (Ag+-HPLC) and gas chromatography (GC). Ag+-HPLC allowed the separation of all positional CLA isomers and geometric cis/trans CLA isomers except 10,12–18∶2. However, one of the 8,10 isomers (8cis, 10trans-18∶2) coeluted with the 9trans,11cis18∶2 isomer. There were differences in the elution order of the pairs of geometric CLA isomers resolved by Ag+-HPLC. For the 8,10 and 9,11 CLA isomers, cis,trans eluted before trans,cis, whereas the opposite elution pattern was observed for the 11,13–18∶2 geometric isomers (trans,cis before cis,trans). All eight cis/trans CLA isomers were separated by GC on long polar capillary columns only when their relative concentrations were about equal. Large differences in the relative concentration of the CLA isomers found in natural products obscured the resolution and identification of a number of minor CLA isomers. In such cases, GC-mass spectrometry of the dimethyloxazoline derivatives was used to identify and confirm coeluting CLA isomers. For the same positional isomer, the cis,trans consistently eluted before the trans,cis CLA isomers by GC. High resolution mass spectrometry (MS) selected ion recording (SIR) of the molecular ions of the 18∶1 18∶2, and 18∶3 fatty acid methyl esters served as an independent and highly sensitive method to confirm CLA methyl ester peak assignments in GC chromatograms obtained from food samples by flame-ionization detection. The high-resolution MS data were used to correct for the nonselectivity of the flame-ionization detector.

Rumenic acid: A proposed common name for the major conjugated linoleic acid isomer found in natural products

At the last American Oil Chemists’ Society meeting in Chicago, May 10–13, 1998, there was a formal discussion period after a day-long series of presentations on conjugated linoleic acid (CLA) attended by about 100 participants. One of the topics discussed was the possible naming of the major CLA isomer, cis-9, trans-11-octadecadienoic acid—found in milk, other dairy products, and meats of ruminant animals—as rumenic acid. The name rumenic acid has been proposed by Peter W. Parodi and is supported by a number of other scientists. There was extensive debate on this topic. CLA is a mixture of many positional and geometrical isomers of conjugated octadecadienoic acids both in natural products and in commercial preparations. In natural products, the predominant isomer (≥80% of total CLA) is cis-9, trans-11, whereas in commercial preparations the number and proportion of the isomers can vary widely depending on the conditions of preparation. The major arguments presented against naming any CLA isomer were: (i) the term CLA has been in common use for nearly two decades; (ii) cis-9, trans-11 appears also to be formed outside the rumen by desaturation of trans-11-18:1; (iii) the active isomer has yet to be identified; (iv) there appears to be evidence that cis-9, trans-11 isomer may not be the only active CLA isomer, therefore; how shall we name them? On the other hand, the major arguments for naming cis-9, trans-11-octadecadienoic acid rumenic acid were: (i) this is the major naturally occurring conjugated fatty acid in milk, other dairy products, and meats from ruminants; (ii) a major natural component can be named regardless of whether any biological activity has been ascribed to it; (iii) the name would avoid the misconception that it is a CLA having a methylene-interrupted double bond system, and (iv) it is an easy name, associated with the major place of origin, and may thus be less confusing. Additional names were suggested, such as bovinic acid. However, there was agreement that this name did not encompass the broad spectrum of natural products containing cis-9, trans-11-octadecadienoic acid, for example, from sheep and other ruminants. There was no complete consensus, but half the participants said they would use the new name in their future publications. We therefore recommend naming cis-9, trans-11-octadecadienoic acid as rumenic acid.

Quantitative Analysis of Triglycerides Using Atmospheric Pressure Chemical Ionization-Mass Spectrometry

Atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) was used for quantitative analysis of triglycerides (TG) separated by reverse-phase high-performance liquid chromatography. APCI-MS was used for analysis of mono-acid TG standards containing deuterated internal standard, of a synthetic mixture of heterogeneous TG, of randomized and normal soybean oils and of randomized and normal lard samples. Quantitation of the TG by four approaches based on APCI-MS were compared, and these were compared to quantitation obtained using liquid chromatography (LC) with flameionization detection (FID). The APCI-MS methods were based on (i) calibration curves from data for mono-acid TG standards, (ii) response factors obtained from a synthetic mixture of TG, (iii) response factors calculated from comparison of randomized samples to their statistically expected compositions, and (iv) response factors calculated from comparison of fatty acid (FA) compositions calculated from TG compositions to FA compositions obtained by calibrated gas chromatography (GC) with FID. Response factors derived from a synthetic mixture were not widely applicable to samples of disparate composition. The TG compositions obtained using APCI-MS data without application of response factors had average relative errors very similar to those obtained using LC-FID. Numerous TG species were identified using LC/APCI-MS which were undetected using LC-FID. Two quantitation methods, based on response factors calculated from randomized samples or on response factors calculated from FA compositions, both gave similar results for all samples. The TG compositions obtained using response factors calculated from FA compositions showed less average relative error than was obtained from LC-FID data, and were in good agreement with predicted compositions for the synthetic mixture and for randomized soybean oil and lard samples.

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.

A practical guide to the isolation, analysis and identification of conjugated linoleic acid.

Natural and synthetic conjugated linoleic acids (CLA) are reputed to have therapeutic properties that are specific to particular geometrical and positional isomers. Analysis of these has presented unique problems that have brought forward distinctive solutions, especially the use of gas chromatography–mass spectrometry and silver-ion high-performance liquid chromatography. In the analysis of CLA present at low levels in tissue samples, it is sometimes necessary to use concentration methods. In this review, the most useful and practical methods for the isolation and analysis of CLA isomers in tissues and in commercial CLA preparations are described.

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.

Analysis of the monoenoic fatty acid distribution in hydrogenated vegetable oils by silver-ion high-performance liquid chromatography

A silver-ion high-performance liquid chromatography column (hexane/acetonitrile as solvent, ultraviolet detection) was used to analyze the fatty acid distribution (as fatty acid methyl esters) of a representative sample of hydrogenated oil. Fractions containingcis- andtrans-18:1 isomers were readily separated. The positional fatty acid isomers were separated by rechromatographing these fractions. The elution order and percent compositions were compared with results obtained by gas chromatography. Of the Δ8 to Δ14trans-18:1 isomers, only the Δ8 and Δ9 pair could not be separated. The Δ8 and Δ9cis-18:1 pair also could not be separated, and the Δ10 isomer was poorly separated from this pair. Area percents were comparable to results obtained by gas chromatography.