Incorporation oftrans-8- andcis-8-octadecenoic acid isomers in human plasma and lipoprotein lipids

Mixtures of deuterium-labeledtrans-8-,cis-8- andcis-9-octadecenoic acids (8t–18∶1, 8c–18∶1, 9c–18∶1) were fed as triglycerides (TG) to two adult male subjects. Blood samples were collected sequentially over a 48-hour period. Plasma and lipoprotein lipids were separated by thin layer chromatography and analyzed by gas chromatography-mass spectroscopy. Results indicate (i) absorption of the 8t- and 8c–18∶1 isomers were similar to 9c–18∶1; (ii) the 8t–18∶1 isomer was cleared approximately 30% faster than 9c–18∶1 from plasma TG; (iii) cholesterol ester samples contained 8.4 times less 8t–18∶1 than 9c–18∶1; (iv) incorporation at the 1-acyl phosphatidylcholine (PC) position was higher for 8t–18∶1 and 8c–18∶1 (2.2 and 1.7 times) than for 9c–18∶1; and (v) discrimination at the 2-acyl PC position was 4.6-fold against 8t–18∶1 and 1.3-fold against 8c–18∶1 compared with 9c–18∶1. Discrimination against uptake of the Δ-8 isomers in both neutral and phospholipid classes suggests that both 8t- and 8c–18∶1 may be preferentially oxidized relative to 9c–18∶1. Except for triglycerides, data for each of the lipid classes from total plasma and individual lipoprotein samples were similar. These data indicate that differences for incorporation and turnover of the 8t- and 8c–18∶1 isomers relative to 9c–18∶1 are not substantially influenced by the lipoprotein classes. The maximum isotopic enrichment detected in the chylomicron triglycerides fractions was 60%, which indicates that a substantial amount of endogenous triglycerides was mobilized during absorption of the deuterated fats.     

Conversion of 18∶3 Δ9cis, 12cis, 15trans in rat liver microsomes

Several years ago, it was established that the Δ15 trans isomer of α-linolenic acid is converted in vivo into fatty acids containing 20 and 22 carbons (geometrical isomers of eicosapentaenoic and docosahexaenoic acids). The present study focused on the in vitro Δ6 desaturation, the first step of the biosynthesis of the n-3 long-chain polyunsaturated fatty acids from 18:3n-3. For that purpose, rat liver microsomes were prepared and incubated with radiolabeled 18∶3 Δ9cis, 12cis, 15cis (18∶3 c,c,c) or 18∶3 Δ9cis, 12cis, 15trans (18∶3c,c,t) under desaturation conditions. The data show that 18∶3c,c,t is converted at a lower rate compared with α-linolenic acid. The product of conversion of 18∶3 c,c,t may be 18∶4 Δ6cis, 9cis, 12cis, 15trans resulting from a Δ6 desaturation of the trans substrate. Moreover, the conversion of radiolabeled 18∶3c,c,t was strongly decreased by the presence of 18∶3c,c,c (up to 48%) while the 18∶3c,c,t only slightly decreased the conversion of radiolabeled 18∶3c,c,c. Thus, the desaturation enzyme presented a higher affinity for the native all-cis n-3 substrate.     

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.     

Antioxidant activities of major components of γ-oryzanol from rice bran using a linoleic acid model

The change in hydroperoxides of linoleic acid incubated with constant micro air flow at 37°C was used to evaluate the antioxidant activities of three major components of γ-oryzanol from rice bran (cycloartenyl ferulate, 24-methylene cycloartanyl ferulate, and campesteryl ferulate) compared with α-tocopherol and ferulic acid. The four hydroperoxide isomers of linoleic acid, 9-hydroperoxy-10-trans, 12-cis-octadecadienoic acid [9HPODE(t,c)], 9-hydroperoxy-10-trans, 12-trans-octadecadienoic acid, 13-hydroperoxy-9-cis, 11-trans-octadecadienoic acid [13HPODE(c,t)], and 13-hydroperoxy-9-trans, 11-trans-octadecadienoic acid, were measured using normal-phase high-performance liquid chromatography with an ultraviolet detector. The three components of γ-oryzanol evidenced significant antioxidant activity when they were mixed with linoleic acid in a molar ratio of 1∶100 and 1∶250 but not in a molar ratio of 1∶500 (P<0.05). α-Tocopherol and ferulic acid also demonstrated significant antioxidant activity at all three molar ratios (P<0.05). The highest molar ratio (1∶100) of α-tocopherol, however, caused greater levels of 9HPODE(t,c) and 13HPODE(c,t) than the other two less concentrated treatments.     

Incorporation into lipid classes of products from microsomal desaturation of isomerictrans-octadecenoic acids

The microsomal desaturation of positional isomers oftrans-octadecenoic acids is effected by the Δ9-desaturase and, with concomitant geometric isomerization,cis,trans- andcis,cis-octadecadienoic acids of unusual structure are formed. Incorporation of the substrates and their products into lipids varied from 50.5% for incubations with 14–18∶1 to 81.0% for 6–18∶1. A detailed study of the composition of each of the major lipid classes, i.e., phospholipids, triacylglycerol and cholesteryl esters, as well as the composition of the free fatty acid fraction, revealed a complex picture. Generally, thec,c-18∶2 products were enriched in the phospholipid fraction, whereas thec,t-18∶2 appeared preferentially in cholesteryl esters. The 18∶1 substrates themselves did not show marked preferences for any of the lipid classes. Phospholipase A₂ action on phosphatidylcholine and phosphatidylethanolamine demonstrated enrichment of thec,c- and thec,t-18∶2 products in the 2-position, whereas the 18∶1 substrates were preferentially inserted into the 1-positions. Thec,c- andc,t-18∶2 formed by desaturation oft11–18∶1 varied from this pattern, probably due to their conjugated double bond structures. Linoleic acid,c9,c12–18∶2, formed during desaturation oft12–18∶1, surprisingly showed enrichment in the 1-position of phosphatidylcholine. Incubation experiments witht5- andt6-isomers using liver microsomes from rats fed a corn-oil-supplemented diet showed conversion and incorporation rates similar to the rates obtained with microsomes from EFA-deficient rats. The fatty acid composition of lipid classes and the distributions of products and substrate between the 1- and 2-positions of phosphatidylcholine also agreed with results obtained using microsomes from EFA-deficient rats.     

Identification of conjugated linoleic acid isomers in cheese by gas chromatography, silver ion high performance liquid chromatography and mass spectral reconstructed ion profiles. Comparison of chromatographic elution sequences

Commercial cheese products were analyzed for their composition and content of conjugated linoleic acid (CLA) isomers. The total lipids were extracted from cheese using petroleum ether/diethyl ether and methylated using NaOCH₃. The fatty acid methyl esters (FAME) were separated by gas chromatography (GC), using a 100-m polar capillary column, into nine minor peaks besides that of the major rumenic acid, 9c, 11t-octadecadienoic acid (18∶2), and were attributed to 19 CLA isomers. By using silver ion-high performance liquid chromatography (Ag⁺-HPLC), CLA isomers were resolved into seven trans, trans (5–9%), three cis/trans (10–13%), and five cis, cis (<1%) peaks, totaling 15, in addition to that of the 9c, 11t-18∶2 (78–84%). The FAME of total cheese lipids were fractionated by semipreparative Ag⁺-HPLC and converted to their 4,4-dimethyloxazoline derivatives after hydrolysis to free fatty acids. The geometrical configuration of the CLA isomers was confirmed by GC-direct deposition-Fourier transform infrared, and their double bond positions were established by GC-electron ionization mass spectrometry. Reconstructed mass spectral ion profiles of the m+2 allylic ion and the m+3 ion (where m is the position of the second double bond in the parent conjugated fatty acid) were used to identify the minor CLA isomers in cheese. Cheese contained 7 t,9c-18∶2 and the previously unreported 11t, 13c-18∶2 and 12c, 14t-18∶2, and their trans,trans and cis,cis geometric isomers. Minor amounts of 8,10-, and 10, 12–18∶2 were also found. The predicted elution orders of the different CLA isomers on long polar capillary GC and Ag^*-HPLC columns are also presented.     

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.     

cis-5-olefinic unusual fatty acids in seed lipids of gymnospermae and their distribution in triacylglycerols

Open-tubular gas chromatographic analysis of fatty acids in the lipids from the seeds of 20 species of Gymnospermae showed that they all contained nonmethylene-interrupted polyenoic (NMIP) acids as minor components and palmitic, oleic, linoleic and α-linolenic acids as major components. The NMIP acids have an additional 5,6-ethylenic bond in ordinary plant unsaturated fatty acids and the following C₂ elongation acids:cis-5,cis-9-octadecadienoic acid (5,9–18∶2) (I); 5,9,12–18∶3 (II); 5,9,12,15–18∶4, 5,11–20∶2, 5,11,14–20∶3 (III); and 5,11,14,17–20∶4 (IV). The main NMIP acids found in neutral lipids are I in two species ofTaxus, II in seven species of Pinaceae, III in two species of Podocarpaceae,Torreya nucifera, Cycas revoluta, andGinkgo biloba, and III and IV in each of three species of Taxodiaceae, and Cupressaceae. The polar lipids constitute the minor fraction of seed lipids in general. The content and composition of NMIP acids in these lipids differe considerably from those in neutral lipids. Analysis of the partial cleavage products of triacylglycerols showed that the NMIP acids distribute mainly in the 1,3-position.     

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.     

Influence of dietary arachidonic acid on metabolism in vivo of 8 cis, 11 cis, 14-eicosatrienoic acid in humans

This study investigated the influence of dietary arachidonic acid (20∶4n-6) on Δ5 desaturation and incorporation of deuterium-labeled 8cis, 11cis, 14-eicosatrienoic acid (20∶3n-6) into human plasma lipids. Adult male subjects (n=4) were fed diets containing either 1.7 g/d (H120∶4 diet) or 0.21 g/d (LO20∶4 diet) of arachidonic acid for 50 d and then dosed with a mixture containing ethyl esters of 20∶3n-6[d4] and 18∶1n-9[d2]. A series of blood samples was sequentially drawn over a 72-h period, and methyl esters of plasma total lipid, triacylglycerol, phospholipids, and cholesteryl ester were analyzed by gas chromatography-mass spectrometry. Based on the concentration of 20∶3n-6[d4] in total plasma lipid, the estimated conversion of 20∶3n-6[d4] to 20∶4n-6[d4] was 17.7.±0.79% (HI20∶4 diet) and 2.13±1.44% (LO20∶4 diet). The concentrations of 20∶4n-6[d4] in total plasma lipids from subjects fed the HI20∶4 and LO20∶4 diets were 2.10±0.6 and 0.29±0.2 μmole/mL plasma/mmole of 20∶3n-6[d4] fed/kg of body weight. These data indicate that conversion of 20∶3n-6[d4] to 20∶4n-6[d4] was stimulated 7-8-fold by the HI20∶4 diet. Phospholipid acyltransferase was 2.5-fold more selective for 20∶3n-6[d4] than 18∶1n-9[d2], and lecithin:cholesterol acyltransferase was 2-fold more selective for 18∶1n-9[d2] than 20∶3n-6[d4]. These differences in selectivity were not significantly influenced by diet. Absorption of ethyl 20∶3n-6[d4] was about 33% less than ethyl 18∶1n-9[d2]. The sum of the n-6 retroconversion products from 20∶3n-6[d4] in total plasma lipids was about 2% of the total deuterated fatty acids. Neither absorption nor retroconversion appears to be influenced by diet.     

Structural importance of thecis-5 ethylenic bond in the endogenous desaturation product of dietary elaidic acid,cis-5,trans-9 18∶2 acid,for the acylation of rat mitochondria phosphatidylinositol

When rats were fed elaidic (trans-9 18∶1) acid at a high load in diets that were otherwise marginally or almost completely deficient in linoleic (cis-9,cis-12 18∶2) acid, elaidic acid was desaturated tocis-5,trans-9 18∶2 acid. This polymethylene-interrupted acid was then incorporated into most phospholipids from rat mitochondria, cardiolipin being an exception. Its level of esterification in phospholipids followed the increasing order: phosphatidylethanolamine <phosphatidylcholine < phosphatidylinositol (PI). The content ofci-5,trans-9 18∶2 acid decreased in organs in the order liver > kidney > heart. The levels ofcis-5,trans-9 18∶2 acid increased in mitochondria phospholipids as the level of linoleic acid was lowered in the diet. In liver mitochondria PI, it reached 16% of total fatty acids. After hydrolysis of liver mitochondria PI withNaja naja phospholipase A₂, we observed that elaidic acid was essentially esterified to position 1 at the expense of saturated acids, whereascis-5,trans-9 18∶2 acid was exclusively esterified to position 2, along with 20∶3n−9 and 20∶4n−6 acids. As a consequence, the sums of saturated andtrans-9 18∶1 acids on the one hand, and of 20∶3n−9, 20∶4n−6, andcis-5,trans-9 18⩺2 acids on the other hand, remained fairly constant in liver mitochondria PI (ca. 55 and 30%, respectively). Becausetrans-9 18∶1 andcis-5,trans-9 18∶2 acids differ only by thecis-5 ethylenic bond, which is also present in 20∶3n−9 and 20∶4n−6 acids, this distribution pattern indicates that thecis-5 double bond, rather than any other ethylenic bond, may be of major structural importance for channeling fatty acids to position 2 of PI.     

A new conjugated linoleic acid isomer, 7 trans, 9 cis-octadecadienoic acid, in cow milk, cheese, beef and human milk and adipose tissue

The identity of a previously unrecognized conjugated linoleic acid (CLA) isomer, 7 trans, 9 cis-octadecadienoic acid (18∶2) was confirmed in milk, cheese, beef, human milk, and human adipose tissue. The 7 trans, 9 cis-18∶2 isomer was resolved chromatographically as the methyl ester by silver ion-high-performance liquid chromatography (Ag⁺-HPLC); it eluted after the major 9 cis, 11 trans-18∶2 isomer (rumenic acid) in the natural products analyzed. In the biological matrices in-vestigated by Ag⁺-HPLC, the 7 trans, 9 cis-18∶2 peak was generally due to the most abundant minor CLA isomer, ranging in concentration from 3 to 16% of total CLA. By gas chromatography (GC) with long polar capillary columns, the methyl ester of 7 trans, 9 cis-18∶2 was shown to elute near the leading edge of the major 9 cis, 11 trans-18∶2 peak, while the 4,4-dimethyloxazoline (DMOX) derivative permitted partial resolution of these two CLA isomers. The DMOX derivative of this new CLA isomer was analyzed by gas chromatography-electron ionization mass spectrometry (GC-EIMS). The double bond positions were at Δ7 and Δ9 as indicated by the characteristic mass spectral fragment ions at m/z 168, 180, 194, and 206, and their allylic cleavages at m/z 154 and 234. The cis/trans double-bond configuration was established by GC-direct deposition-Fourier transform infrared as evidenced from the doublet at 988 and 949 cm⁻¹ and absorptions at 3020 and 3002 cm⁻¹. The 7 trans, 9 cis-18∶2 configuration was established by GC-EIMS for the DMOX derivative of the natural products examined, and by comparison to a similar product obtained from treatment of a mixture of methyl 8-hydroxy-and 11-hydroxyoctadec-9 cis enoates with BF₃, in methanol. Contribution number S010 from the Food Research Center, Guelph, Ontario, Canada.     

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.     

The 9-hexadecenoic and 11-octadecenoic acid content of natural fats and oils

The monoenoic methyl esters from numerous fats and oils which contained appreciablecis-9-hexadecenoic acid (cis-9-16∶1) were isolated by liquid-solid chromatography on silver nitrate-silica gel. Analysis of the monoenes by packed and capillary column gas-liquid chromatography showed that significant amounts ofcis-11-octadecenoic acid (cis-11-18∶1) were present in all samples. The amount ofcis-11-18∶1 found in the monoenoic methyl esters increased proportionally to logarithmic increases in thecis-9-16∶1 level. Most analyses reported in the literature also show this proportionality. This mathematical relationship suggests that chain elongation ofcis-9-16∶1 tocis-11-18∶1 is a biosynthetic pathway operative in a wide variety of species.     

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.     

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.     

Conifer seeds: Oil content and fatty acid composition

The seed oils from twenty-five Conifer species (from four families—Pinaceae, Cupressaceae, Taxodiaceae, and Taxaceae) have been analyzed, and their fatty acid compositions were established by capillary gas-liquid chromatography on two columns with different polarities. The oil content of the seeds varied from less than 1% up to 50%. Conifer seed oils were characterized by the presence of several Δ5-unsaturated polymethylene-interrupted polyunsaturated fatty acids (Δ5-acids) with either 18 (cis-5,cis-9, 18∶2,cis-5,cis-9,cis-12 18∶3, andcis-5,cis-9,cis-12,cis-15 18∶4 acids) or 20 carbon atoms (cis-5,cis-11 20∶2,cis-5,cis-11,cis-14, 20∶3, andcis-5,cis-11,cis-14,cis-17 20∶4 acids). Pinaceae seed oils contained 17–31% of Δ5-acids, mainly with 18 carbon atoms. The 20-carbon acids present were structurally derived from 20∶1n-9 and 20∶2n-6 acids. Pinaceae seed oils were practically devoid of 18∶3n-3 acid and did not contain either Δ5-18∶4 or Δ5-20∶4 acids. Several Pinaceae seeds had a Δ5-acid content higher than 50 mg/g of seed. The only Taxaceae seed oil studied (Taxus baccata) had a fatty acid composition related to those of Pinaceae seed oils. Cupressaceae seed oils differed from Pinaceae seed oils by the absence of Δ5-acids with 18 carbon atoms and high concentrations in 18∶3n-3 acid and in Δ5-acids with 20 carbon atoms (Δ5-20∶3 and Δ5-20∶4 acids). Δ5-18∶4 Acid was present in minute amounts. The highest level of Δ5-20∶4 acid was found inJuniperus communis seed oil, but the best source of Δ5-acids among Cupressaceae wasThuja occidentalis. Taxodiaceae seed oils had more heterogeneous fatty acid compositions, but the distribution of Δ5-acids resembled that found in Cupressaceae seed oils. Except forSciadopytis verticillata, other Taxodiaceae species are not interesting sources of Δ5-acids. The distribution profile of Δ5-acids among different Conifer families appeared to be linked to the occurrence of 18∶3n-3 acid in the seed oils.     

Trans- and cis-octadecenoic acid isomers in the hump and milk lipids from Camelus dromedarius

The distribution profiles of individual trans- as well as cis-18∶1 isomers from the fat prepared from the hump adipose tissue and the milk from Camelus dromedarius (the single-humped Arabian species) are described. Gas-liquid chromatography on two capillary columns with different polarities and lengths were used for this purpose in combination with argentation thin-layer chromatography. A comparison of the profiles established is made with that of true ruminant fats. In the fats from the dromedarius as well as from true ruminants, the trans-18∶1 isomers have their ethylenic bonds in all positions between Δ4 and Δ16. The prominent trans isomer is the 11–18∶1 (vaccenic) acid in all species, and the complete distribution profiles are quite similar. Concerning the cis isomers, the prominent isomer is oleic acid, followed by cis-vaccenic acid, as in true ruminant fats. Other cis isomers encompass the Δ6–8 and the Δ12 to Δ15 isomers. Camelidae (suborder Tylopoda) and Bovidae (suborder Ruminantia) have evolved independently since the Eocene, that is for approximately 50 million years. Despite this considerable period, and the profound differences in anatomy, morphology, physiology, ecological and dietary habits between the extant species of these suborders, the rumen microflora has continued to synthesize the same trans- and cis-octadecenoic acid isomers, in comparable proportions, at least as deduced from their composition profiles. We conclude that the trans-18∶1 acid profile is not intrinsically species-dependent, but it can be affected by the nature and the proportions of dietary unsaturated fatty acids that themselves depend on the feed, and that may be species-specific.     

<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.     

Incorporation ofcis-octadecenoic acids into the rat liver mitochondrial membrane phospholipids and adipose tissue triglycerides

The incorporation of the dietarycis 18∶1 (n−12) andcis 18∶1 (n−10) into liver mitochondrial membrane phospholipids and adipose tissue trigly cerides was studied in 4 groups of rats fed diets containing 10 weight percent (wt%) of fat with the following contents of octadecenoic acids: 50%cis 18∶1(n−12) +9%cis 18∶1 (n−9); 25%cis 18∶1 (n−12)+32%cis 18∶1 (n−9); 50%cis 18∶1 (n−10)+10%cis 18∶1 (n−9); or 54%cis 18∶1 (n−9). Dietary linoleic acid was 3 wt% in all 4 groups. In the mitochondrial membranes, the isomeric octadecenoic acids were primarily incorporated into the 1-position of phosphatidylcholines and phosphatidylethanolamines at the expense of saturated fatty acids. The maximal incorporations observed in the 1-position of phosphatidylethanolamines were 4.8% 18∶1 (n−12) and 8.9% 18∶1 (n−10). No effects on the contents of polyunsaturated fatty acids in the phospholipids were seen. In the adipose tissue, the isomeric octadecenoic acids were incorporated at a level of 13%cis 18∶1 (n−12) or 23%cis 18∶1 (n−10), paralleled by a reduction in the content of oleic acid. Presented in part at the 9th Scandinavian Symposium on Lipids, Visby, Sweden, June 1977.