Metabolism in humans ofcis-12,rans-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.     

Selective deposition oftrans-8- andcis-9-octadecenoates in egg and tissue lipids of the laying hen

The deposition oftrans-8-octadecenoate-8(9)-³H (8t-18∶1-³H) was compared tocis-9-octadecenoate-10-¹⁴C (9c-18∶1-¹⁴C) in the major egg yolk neutral lipids and phospholipids and in organ lipids from the laying hen.trans-8-Octadecenoate was preferentially incorporated into only the phosphatidylethanolamines (PE), whereas discrimination against 8t-18∶1-³H occurred in the phosphatidylcholines (PC), triglycerides (TG) and cholesteryl esters (CE). The 1-acyl position of both PE and PC contained three times more 8t-18∶1-³H than 9c-18∶1-¹⁴C. Almost total exclusion of the 8t-18∶1-³H from the 2-acyl position of these phospholipids was found. Preferential incorporation of 9c-18∶1-¹⁴C occurred at the combined 1- and 3-acyl positions and at the 2-acyl position of yolk TG. Tissue lipid analyses indicated that there was preferential deposition of 9c-18∶1-¹⁴C into all organs. Individual liver lipid classes displayed the same relative order of discrimination against 8t-18∶1-³H as did egg yolk lipids (CE>TG>PC>PE).     

Absorption and distribution of deuterium-labeledtrans- andcis-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.     

Selective hydrogenation with tris(triphenylphosphine) chlororhodium (I) catalyst: Preparation of octadecenoate isomers

Fractionation of products obtained from partial catalytic hydrogenation of methylcis-9,cis-12-octadecadienoate (9c,12c-18:2) with tris(triphenylphosphine) chlororhodium [RhCl(Ph₃P)₃] provided a facile method for preparation of a nearly equal molar mixture of methylcis-9- andcis-12-octadecenoate (9c-18∶1 and 12c-18∶1). Isolation of products was achieved by silver resin and C18 reverse phase liquid chromatography. Catalytic deuteration of 9c,12c-18∶2 yields a mixture of 9c-18∶1-12,13-d₂ and 12c-18∶1-9,10-d₂ with an isotopic purity of 85%. Final isolated yield of the mixture of 9c- and 12c-18∶1 products was 30%. Isolation of products from partial hydrogenation of conjugated octadecadienoates (9c,11t-18∶2 or 10t,12c-18∶2) provided a convenient method for synthesis of an almost equal molar mixture of methyltrans-10 andtrans-11-octadecenoate (10t-18∶1 and 11t-18∶1). Characterization of the reaction products from hydrogenation of 9c,12c-28∶2 indicates that the 9c- and 12c-18∶1 products are formed by the expected 1,2-hydride addition. The presence of small amounts of 10t- and 11t-18∶1 and conjugated octadecadienoates was evidence for a secondary isomerization-1,4-hydride addition pathway. Isolation and characterization of products from RhCl(Ph₃P)₃-catalyzed hydrogenation of 9c,11t-18∶2 and 10t,12c-18∶2 indicate that both 1,2- and 1,4-hydride addition to the conjugated diene isomers occurs at about equal rates, but only thecis bond is reduced by the 1,2-hydride addition pathway and the 1,4-hydride addition pathway yields only atrans-18∶1. Because of this unusual selectivity for acis bond conjugated with atrans bond, hydrogenation of both 9c,11t-18∶2 and 10t,12c-18∶2 yields the same mixture of t-18∶1 isomers.     

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.     

Competitive deposition oftrans-12- andcis-9-octadecenoates into egg yolk lipids

The deposition oftrans-12-octadecenoate-12(13)-³H (12t-18∶1-³H) was compared tocis-9-octadecenoate-10-¹⁴C (9c-18∶1-¹⁴C) in the major egg yolk neutral lipids and phospholipids.trans-12-Octadecenoate was preferentially incorporated into cholesteryl esters (CE), phosphatidylcholines (PC), and phosphatidylethanolamines (PE) but was discriminated against in triglycerides (TG). Isotopic ratios indicate that 5.9 and 5.6 times more 12t-18∶1-³H than 9c-18∶1-¹⁴C was esterified at the 1-acyl position of PE and PC, respectively. The combined 1- and 3-acyl positions of TG and the 2-acyl position of TG, PE and PC were each preferentially esterified with 9c-18∶1-¹⁴C.     

<Emphasis Type="Italic">Trans</Emphasis>-18∶1 isomers in rat milk fat as effective biomarkers for the determination of individual isomeric <Emphasis Type="Italic">trans</Emphasis>-18∶1 acids in the dams' diet

Female rats were fed a diet containing by weight 10% partially hydrogenated sunflower oil, 2% sunflower oil, and 1% rapeseed oil during gestation and lactation. The trans-18∶1 isomer profile of the fat supplement was (in % of total trans 18∶1 acids in the fat supplement): Δ4, 0.5; Δ5, 1.0;Δ6–Δ8, 18∶0; Δ9 (elaidic), 13.5; Δ10, 22.2;Δ11 (vaccenic), 16.0; Δ12, 11.3; Δ13–Δ14, 12.8; Δ15, 2.5; and Δ16, 2.2 (total trans 18∶1 acids in the fat supplement: 40.6%). The cis 18∶1 isomer profile was (in % of total cis-18∶1 isomers):Δ6, Δ8, 2.1; Δ9 (oleics), 70.9; Δ10, 6.1; Δ11, 8.3; Δ12, 4.0; Δ13, 2.8; Δ14, 4.6, and Δ15, 1.2 (total cis-18∶1 acids in the fat supplement: 32.6%). Suckling rats from four litters were sacrificed at day 17 or 18 after birth, and their stomach content (milk) was analyzed. The trans-18∶1 isomer profile of milk was (relative proportions, in % of total): Δ4, 0.3; Δ5, 1.1; Δ6–Δ8, 16.8; Δ9, 15.3; Δ10, 22.0; Δ11, 16.7; Δ12, 11.8; Δ13–14, 11.8; Δ15, 2.5, and Δ16, 1.9 (total trans 18∶1 acids in milk: %). That of cis-18∶1 isomers was (proportions in % relative to total cis-18∶1 isomers): Δ6–Δ8, 4.7; Δ9, 72.5; Δ10, 4.0; Δ11, 8.0; Δ12, 7.1; Δ13, 1.9; Δ14, 1.0, and Δ15, 0.7 (total cis-18∶1 acids in milk: %). These results demonstrate that all isomeric acids, independent of the geometry and the position of the ethylenic bond, are incorporated into milk lipids. With regard to trans-18∶1 isomers, the distribution profile in milk is identical to that in the dams' diet, i.e., there is no discrimination against any positional isomer between their ingestiona nd their deposition into milk lipids. As a consequence, this study indicates that the trans-18∶1 isomer profile of milk reflects that in the dams' diet and supports our earlier hypothesis that the profile of trans-18∶1 isomers in milk can be used to deduce the relative contribution of ruminant fats and partially hydrogenated oils in the diet ot the total intake of trans-18∶1 isomers. On the other hand, the cis-18∶1 isomer profile in milk shows significant differences when compared to that in the dams' diet. Surprisingly, there are no major differences for the cis-Δ9 (oleic) and the cis-Δ11 (asclepic) isomers, which can be synthesized by the mother. However, there seems to be a significant positive selectivity for the group cis-Δ6–Δ8, and for the cis-Δ12 isomer, whereas a negative selectivity occurs for the Δ10 and Δ13 to Δ15 cis isomers. Dr. Robert L. Wolff Robert Wolff passed away at the age of 53 on the 10th of November, 2002. His know-how in the field of lipids was recognized internationally. He had the ability to lead his research projects in both the animal and vegetal worlds. His scientific achievement, more than 100 publications to his name in the field of trans fatty acids, made him highly esteemed by his colleagues. He was Conference Master at Bordeaux 1 University (France) up until 2001, at which time he joined the Nutritional Lipid Unit in I.N.R.A., Dijon (France). His mission there was to develop a research program on plasmalogens and their role in brain and muscle function, for which his analytical and biochemical skills were a guarantee of success. Unfortunately, his state of health did not allow him to complete this project. This publication is his final one.     

Characterization of trans-monounsaturated alkenyl chains in total plasmalogens (1-O-alk-1′-enyl-2-acyl glycerophospholipids) from sheep heart

In the present study, we investigated the alkenyl chains from sheep heart plasmalogens (1-O-alk-1′-enyl-2-acyl glycerophospholipids) after their conversion into trimethylene dioxyalkanyl (TMDOA) derivatives. Particular attention was given to monounsaturated alkenyl chains (C₁₈ mainly). For this purpose, a combination of silver ion TLC and GLC on highly polar, very long capillary columns was applied to TMDOA derivatives. Approximately 30 different alkenyl chains could be separated, and the main observation was that the component previously reported as a cis-9 18∶1 alkenyl chain in plasmalogens embraces in fact a wide range of trans and cis isomers, in amounts equal to 7.9 and 5.6%, respectively, of total alkenyl chains. Concerning the trans-monoenoate fraction, isomers with their ethylenic bond spanning from Δ6–Δ8 to Δ16 were tentatively identified on the basis of their distribution profile, which was similar to that of trans-18∶1 acids prepared and isolated from sheep adipose tissue. The main trans-monoenoic C₁₈ alkenyl chain in sheep heart plasmalogens would thus have its double bond in position 11, which seems logical, as alkenyl chains are derived from the corresponding alcohols, themselves issued from the corresponding FA, and in this particular case, vaccenic (trans-11 18∶1) acid. cis-Monoenoic C₁₈ alkenyl chains also appear more complex than realized earlier, showing in particular isomers with their ethylenic bond farther than the Δ9 position, in addition to the main isomer derived from oleic acid. Several trans-16∶1 alkenyl chains could be observed (totaling ca. 1%), but cis-16∶1 isomers were present in trace amounts only.     

Distributions of conjugated linoleic acid (CLA) isomers in tissue lipid classes of pigs fed a commercial CLA mixture determined by gas chromatography and silver ion-high-performance liquid chromatography

Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerization of sunflower oil, at 2% of the basal diet, from 61.5 to 106 kg live weight, and were compared to pigs fed the same basal diet with 2% added sunflower oil. The total lipids from liver, heart, inner back fat, and omental fat of pigs fed the CLA diet were analyzed for the incorporation of CLA isomers into all the tissue lipid classes. A total of 10 lipid classes were isolated by three-directional thin-layer chromatography and analyzed by gas chromatography (GC) on long capillary columns and by silver-ion high-performance liquid chromatography (Ag⁺-HPLC); cholesterol was determined spectrophotometrically. Only trace amounts (<0.1%; by GC) of the 9,11–18∶2 cis/trans and trans, trans isomers were observed in pigs fed the control diet. Ten and twelve CLA isomers in the diet and in pig tissue lipids were sepatated by GC and Ag⁺-HPLC, respectively. The relative concentration of all the CLA isomers in the different lipid classes ranged from 1 to 6% of the total fatty acids. The four major cis/trans isomers (18.9% 11 cis, 13 trans-18∶2; 26.3% 10 trans, 12 cis-18∶2; 20.4% 9 cis, 11 trans-18∶2; and 16.1% 8 trans, 10 cis-18∶2) constituted 82% of the total CLA isomers in the dietary CLA mixture, and smaller amounts of the corresponding cis,cis (7.4%) and trans,trans (10.1%) isomers were present. The distribution of CLA isomers in inner back fat and in omental fat of the pigs was similar to that found in the diet. The liver triacylglycerols (TAG), free fatty acids (FFA), and cholesteryl esters showed a similar patterns to that found in the diet. The major liver phospholipids showed a marked increase of 9 cis,11 trans-18∶2, ranging from 36 to 54%, compared to that present in the diet. However, liver diphosphatidylglycerol (DPG) showed a high incorporation of the 11 cis,13 trans-18∶2 isomer (43%). All heart lipid classes, except TAG, showed a high content of 11 cis,13 trans-18∶2, which was in marked contrast to results in the liver. The relative proportion of 11 cis,13 trans-18∶2 ranged from 30% in the FFA to 77% in DPG. The second major isomer in all heart lipids was 9 cis,11 trans-18∶2. In both liver and heart lipids the relative proportions of both 10 trans,12 cis-18∶2 and 8 trans,10 cis-18∶2 were significantly lower compared to that found in the diet. The FFA in liver and heart showed the highest content of trans,trans isomers (31 to 36%) among all the lipid classes. The preferential accumulation of the 11 cis,13 trans-18∶2 into cardiac lipids, and in particular the major phospholipid in the inner mitochondrial membrane, DPG, in both heart and liver, appears unique and may be of concern. The levels of 11 cis,13 trans-18∶2 naturally found in foods have not been established.     

Hydrolysis of linoleate geometric isomers byGeotrichum candidum lipase

Lipase (EC 3.1.1.3) from the microorganismGeotrichum candidum preferentially hydrolyzescis-9 18∶1 andcis,cis-9,12 18∶2 from triacylglycerols, largely ignoring all other positional isomers ofcis 18∶1 as well astrans-9 18∶1. To obtain additional information about the specificity of the enzyme, two triacylglycerols were prepared and utilized as substrates. The lipase hydrolyzed 85%cis,cis-9,12 18∶2 and 15%trans,trans-9,12 18∶2 from the triacylglycerol, containing ca. 50% of each acid. From the triacylglycerol containing 46.3%cis,trans-9,12 18∶2 and 53.7%trans,cis-9,12 18∶2, 44.8 and 55.2% of the two acids were hydrolyzed. Therefore the enzyme discriminated against thetrans,trans isomer but not between thecis,trans andtrans,cis isomers. Scientific contribution No. 535, Agricultural Experiment Station, University of Connecticut. ARS, USDA.     

Desaturation of isomericcis 18∶1 acids

The desaturation of positionalcis 18∶1 isomers (Δ4 through Δ11) was studied, using essential fatty acid deficient rat liver microsomes. Thecis Δ4, Δ5, Δ6 and Δ7 isomers were not desaturated. Thecis Δ10 and Δ11 isomers were desaturated at a very low rate. The maximum desaturation was obtained for Δ8 and Δ9 isomers. Thecis Δ8 and Δ11 isomers were desaturated by Δ5 desaturase; thecis Δ9 isomer was desaturated by Δ6 desaturase; and thecis Δ10 isomer was desaturated to Δ7,10 and 5,10–18∶2 acids.     

Lipase-catalyzed fractionation of conjugated linoleic acid isomers

The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mixtures of conjugated linoleic acid (CLA) isomers during esterification of mixed CLA free fatty acids and during hydrolysis of mixed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11–18∶2. A commercial CLA methyl ester preparation, containing at least 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-11–18∶2 methyl ester occurred, with negligible hydrolysis of other CLA isomers. The content of cis-9,trans-11–18∶2 in the resulting free fatty acid fraction was between 94 (lipase B reaction) and 77% (GC-4 reaction). The commercial CLA mixture contained only trace amounts of trans-9,cis-11–18∶2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analogous results were obtained with these enzymes in the esterification in organic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a methyl ester fraction that contained >98% cis-9,trans-11–18∶2. Geotrichum candidum lipases B and GC-4 also demonstrated high selectivity in the esterification of CLA with ethanol, generating ethyl ester fractions containing 96 and 80%, respectively, of the cis-9,trans-11 isomer. In a second set of experiments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was subjected to esterification with octanol in an aqueous reaction system using Amano GC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-11 isomer. After adjustment of the reaction conditions, a concentration of 85% trans-10,cis-12–18∶2 could be obtained in the unreacted free fatty acid fraction. These lipase-catalyzed reactions provide a means for the preparative-scale production of high-purity cis-9,trans-11–18∶2, and a corresponding CLA fraction depleted of this isomer.     

Dietary fats containing concentrates ofcis ortrans octadecenoates and the patterns of polyunsaturated fatty acids of liver phosphatidylcholine and phosphatidylethanolamine

The effects of the mixedcis- 18∶1 isomers and mixedtrans-18∶1 isomers present in partially hydrogenated soybean oil (PHSO) upon the patterns of polyunsaturated fatty acids (PUFA) in liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were studied in rats fed concentrates ofcis- 18∶1 ortrans- 18∶1 isomers isolated as triacylglycerides from PHSO. Thecis- 18∶1 andtrans- 18∶1 concentrates were fed at levels equal to those present in PHSO fed at 17.9% of the diet. All diets contained the required amounts of both linoleic and linolenic acids. Thetrans- 18∶1 concentrate was found to suppress the levels of 20∶4ω6 and 20∶3ω9, and to increase the levels of 18∶2ω6 and 20∶5ω3 in PC and PE. Thecis- 18∶1 concentrate suppressed 20∶4ω6 in PC, 20∶5ω3 in PC and PE, and 18∶2ω6 was more effective than thetrans concentrate in suppressing 22∶6ω3. Thetrans- 18∶1 concentrate was more effective in suppressing 20∶4ω6. Thetrans-18∶ isomers appear to modify PUFA metabolism by inhibition of PUFA synthesis, whereas thecis- 18∶1isomers appear to compete with 2-position fatty acyl transfer and to inhibit ω3 PUFA acylation.     

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.     

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.     

Effect of dietary conjugated linoleic acid (CLA) on metabolism of isotope-labeled oleic,linoleic, and CLA isomers in women

The purpose of this study was to investigate the effect of dietary CLA on accretion of 9c-18∶1, 9c, 12c-18∶2, 10t, 12c-18∶2, and 9c, 11t-18∶2 and conversion of these FA to their desaturated, elongated, and chain-shortened metabolites. The subjects were six healthy adult women who had consumed normal diets supplemented with 6 g/d of sunflower oil or 3.9 g/d of CLA for 63 d. A mixture of 10t, 12c-18∶2-d ₄, 9c, 11t-18∶2-d ₆, 9c-18∶1-d ₈, and 9c, 12c-18∶2-d ₂, as their ethyl esters, was fed to each subject, and nine blood samples were drawn over a 48-h period. The results show that dietary CLA supplementation had no effect on the metabolism of the deuterium-labeled FA. These metabolic results were consistent with the general lack of a CLA diet effect on a variety of physiological responses previously reported for these women. The ²H-CLA isomers were metabolically different. The relative percent differences between the accumulation of 9c, 11t-18∶2-d ₆ and 10t, 12c-18∶2-d ₄ in plasma lipid classes ranged from 9 to 73%. The largest differences were a fourfold higher incorporation of 10t, 12c-18∶2-d ₄ than 9c, 11t-18∶2-d ₆ in 1-acyl PC and a two- to threefold higher incorporation of 9c, 11t-18∶2-d ₆ than 10t, 12c-18∶2-d ₄ in cholesterol esters. Compared to 9c-18∶1-d ₈ and 9c, 12c-18∶2-d ₂, the 10t, 12c-18∶2-d ₄ and 9c, 11t-18∶2-d ₆ isomers were 20–25% less well absorbed. Relative to 9c-18∶1, incorporation of the CLA isomers into 2-acyl PC and cholesterol ester was 39–84% lower and incorporation of 10t, 12c-18∶2 was 50% higher in 1-acyl PC. This pattern of selective incorporation and discrimination is similar to the pattern generally observed for trans and cis 18∶1 positional isomers. Elongated and desaturated CLA metabolites were detected. The concentration of 6c, 10t, 12c-18∶3-d ₄ in plasma TG was equal to 6.8% of the 10t, 12c-18∶2-d ₄ present, and TG was the only lipid fraction that contained a CLA metabolite present at concentrations sufficient for reliable quantification. In conclusion, no effect of dietary CLA was observed, absorption of CLA was less than that of 9c-18∶1, CLA positional isomers were metabolically different, and conversion of CLA isomers to desaturated and elongated metabolites was low.     

Specificity ofGeotrichum candidum lipase with respect to double bond position in triglycerides containingcis-octadecenoic acids

Fifteen approximately random, mixed, triacylglycerols, each of which contained 12∶0, 14∶0, 9, 12–18∶2, 16∶0 and only one positional isomer ofcis 18∶1 (Δ2 through Δ16), were synthesized. These mixtures were used as substrates for the lipase from the microorganismGeotrichum candidum to define the specificity of the enzyme for unsaturated fatty acids. Comparatively small quantities of the 18∶1 isomers, other than 9–18∶1, were hydrolyzed. Relatively large amounts of 18∶2 were released from all substrates. There was no preference between 9–18∶1 and 18∶2. The positional isomers other than 9–18∶1 accumulated in the di- and mono-acylglycerols. Scientific Contribution No. 497, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, Conn. 06268.     

Quantitative analyses of methyl esters of fatty acid geometrical isomers,and of triglycerides differing in unsaturation,by the Iatroscan TLC/FID technique using AgNO3 impregnated rods

The relative FID responses for Iatroscan analyses ofcis andtrans isomers of methyl esters of 18∶1†6, 18∶1†9 and 18∶1†11 on Chromarods-S impregnated with AgNO₃ were studied at load levels ranging from 0.5 to 20 μg, using methyl stearate as internal standard. The FID response correction factors were greater for thecis than for thetrans isomers. The correction factors were relatively constant in the 10–20 μg interval, but increased in the range 0.5–5 μg. Separation of tristearin, triolein, trilinolein and trilinolenin also was obtained on Chromarods-S impregnated with AgNO₃ using a mixture of benzene: chloroform: acetic acid (90∶8∶2) as the solvent system. The relative FID responses for the triolein, trilinolein and trilinolenin were determined at load levels ranging from 0.5 to 14.3 μg using tristearin as an internal standard. The FID response correction factors of these three triglycerides differed significantly for load levels of 1.0, 2.5 and 5.0 μg. However, the factors could be considered as being equal in the range 10 to 14.3 μg. Correction factors were not affected by repeated re-use of the same set of Chromarods. Several hundred separations and scans appeared feasible.     

Comparative studies on individual isomeric 18:1 acids in cow, goat, and ewe milk fats by low-temperature high-resolution capillary gas-liquid chromatography

The trans- as well as the cis-18∶1 isomer profiles were established in cow, goat, and ewe cheese fats, with the assumption that these are representative of the corresponding milks. Argentation thin-layer chromatography was combined with low-temperature high-resolution gas-liquid chromatography on 100-m highly polar capillary columns, thus adding precision to earlier data for these species. Despite differences in the absolute content of trans-18∶1 isomers between species, the relative profiles were essentially similar. Except for the minor trans Δ6–Δ8 group, all trans-18∶1 isomers with their ethylenic bonds between positions Δ4 and Δ16 (including the resolved critical pair Δ13/Δ14) were separated and quantitated individually. As expected, vaccenic (trans Δ9−18∶1) acid was the main isomer, accounting for as much as 37 to 50% of the total fraction. It was observed that the goat trans-18∶1 isomer profile was usually rather close to that of cows in winter (barn feeding), whereas that of the ewe shows a seasonal dependence. The trans-18∶1 profile of ewe milk fats from this study resembles that of cows in the transition period between winter and summer (pasture) feeding. Regarding the cis-18∶1 acid fraction, two isomers (oleic and cis-vaccenic acids) accounted for ca. 97% of that fraction for the three species, with the cis-Δ12 isomer ranked third. The analytical procedure employed here appears a convenient alternative to oxidative-based procedures (generally ozonolysis), taking less time and alleviating some draw-backs of the latter procedure.     

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.