β-Oxidation of conjugated linoleic acid isomers and linoleic acid in rats

To assess the oxidative metabolism of conjugated linoleic acid (CLA) isomers, rats were force-fed 1.5–2.6 MBq of [1-¹⁴C]-linoleic acid (9c,12c-18∶2),-rumenic acid (9c,11t-18∶2), or-10trans, 12cis-18∶2 (10t, 12c-18∶2), and ¹⁴CO₂ production was monitored for 24 h. The animals were then necropsied and the radioactivity determined in different tissues. Both CLA isomers were oxidized significantly more than linoleic acid. Moreover, less radioactivity was recovered in most tissues after CLA intake than after linoleic acid intake. The substantial oxidation of CLA isomers must be considered when assessing the putative health benefits of CLA supplements.     

Changes in conjugated linolenic acid composition within samples obtained from a single source

Conjugated linoleic acid (CLA; 9c, 11t-18∶2) and CLA isomers have been reported, in animals, to exhibit a variety of health-related benefits. Silver ion high-performance liquid chromatography (Ag-HPLC) was found to provide better resolution of the isomes than gas chromatography. Most commercially available samples of CLA, prepared by base-catalyzed isomerization of linoleic acid (9c, 12c-18∶2), are conposed of mixtures of four major isomers. While these isomers have been characterized, we found significant changes in CLA isomer ratios within samples obtained from the same producer/commercial supplier over a period of 1.5 yr. In the first sample, the four cis/trans isomers (8t, 10c-18∶2, 9c, 11t-18∶2, 10t, 12c-18∶2 and 11c, 13t-18∶2) were present in a ratio of approximately 1∶2∶2∶1, while in the second sample they were present in almost equal proportions. If indeed certain daily levels of CLA intake are required to produce suggested health benefits in humans, changes in concentrations of specific CLA isomers could significantly impact these effects. Care must be taken to analyze the CLA used in human and animal studies.     

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.     

Occurrence of 5c,8c,11c,15t-eicosatetraenoic acid and other unusual polyunsaturated fatty acids in rats fed partially hydrogenated calola oil

Uncommoncis andtrans fatty acids can be desaturated and elongated to produce unusual C₁₈ and C₂₀ polyunsaturated fatty acids in animal tissues. In the present study we examined the formation of such metabolites derived fromcis andtrans isomers of oleic and linoleic acids of partially hydrogenated vegetable oil origin in rats. For two months, aduut male rats were fed a partially hydrogenated canola oil diet containing moderately high levels oftrans fatty acids (9.6 energy%) and an adequate level of linoleic acid (1.46 energy%). Analysis of the phospholipid (PL) fatty acids of liver, heart, serum and brain showed no new C₁₈ polyunsaturated fatty acids, except for those uncommon 18∶2 isomers originating from the diet. However, minor levels (each <0.3% PL fatty acids) of six unusual C₂₀ polyunsaturated fatty acids were detected in the tissues examined, except in brain PL. Identification of their structures indicated that the dietary 9c,13t−18∶2 isomer, which is the majortrans polyunsaturated fatty acid in partially hydrogenated vegetable oils, was desaturated and elongated to 5c,8c,11c,15t−20∶4, possibly by the same pathway that is operative for linoleic acid. Furthermore, dietary 12c−18∶1 was converted to 8c,14c−20∶2 and 5c,8c,14c−20∶3; dietary 9c,12t−18∶2 metabolized to 11c,14t−20∶2 and 5c,8c,11c14t−20∶4, and dietary 9t,12c to 11t,14c−20∶2. These results suggested that of all the possible isomers of oleic and linoleic acids in partially hydrogenated vegetable oils, 12c−18∶1, 9c,13t−18∶2, 9c,12t−18∶2 and 9t,12c−18∶2 are the preferred substrates for desaturation and elongation in rats. However, their conversions to C₂₀ metabolites were not as efficient as that of oleic or linoleic acids.     

Dietary effects of conjugated octadecatrienoic fatty acid (9 cis, 11 trans, 13 trans) levels on blood lipids and nonenzymatic in vitro lipid peroxidation in rats

The present study examined the antioxidant activity of conjugated octadecatrienoic fatty acid (9 cis, 11 trans, 13 trans-18∶3), α-eleostearic acid, of karela seed (Momordica charantia), fed to rats for 4 wk. The growth pattern of rats and the effect on plasma cholesterol and high density lipoprotein (HDL) cholesterol and peroxidation of plasma lipid, lipoprotein, eryhrocyte membrane, and liver lipid were measured. Rats were raised on diets containing sunflower oil mixed with three different levels of conjugated trienoic fatty acid (9c,11t,13t-18∶3) 0.5,2, and 10% by weight; the control group was raised with sunflower oil as dietary oil as the source of linoleic acid (9c,12c-18∶2). The growth pattern of the three experimental groups of rats showed no significant difference compared to the control group of rats, but the group with 10% 9c,11t,13t-18∶3 had slightly higher body weight than the control group of rats. Concentrations of total cholesterol, HDL-cholesterol, and non-HDL-cholesterol in plasma were similar in all four groups. Plasma lipid peroxidation was significantly lower in the case of 0.5% 9c,11t,13t-18∶3 group than the control group and the 2 and 10% 9c,11t,13t-18∶3 dietary groups as well. Lipoprotein oxidation susceptibility test with 0.5,2 and 10% 9c,11t,13t-18∶3 dietary groups was significantly less susceptible to lipoprotein peroxidation when compared with sunflower oil dietary group, and the dietary group with 0.5% 9c,11t,13t-18∶3 showed least susceptibility. There was significant lowering in erythrocyte ghost membrane lipid peroxidation in the 0.5,2, and 10% 9c,11t,13t-18∶3 dietary groups compared to the sunflower oil groups. Nonenzymatic liver tissue lipid peroxidation was significantly lower in the group of rats raised on 0.5% 9c,11t,13t-18∶3, but the groups on 2 and 10% 9c,11t,13t-18∶3 acid did not show any significant difference compared with the control group of rats.     

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.     

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.     

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.     

Geometry of conjugated double bonds of CLA isomers in a commercial mixture and in their hepatic 20∶4 metabolites

Rats were fed a fat-free diet for 2 wk. After this period, while maintaining the animals on the same diet, the rats were given intragastrically 180 mg per day of a mixture of conjugated linoleic acids (CLA) as triacylglycerols. Gas chromatography-mass spectrometry (GC-MS) analyses of this mixture, as well as hydrazine reduction and GC-MS and GC-Fourier transform infrared analyses of the resulting monoenes, revealed the presence of two major isomers, the 9c, 11t-and the 10t, 12c-18∶2 accompanied by smaller amounts of the 8t, 10c and the 11c,13t−18∶2 isomers. Minor quantities of cis,cis and trans,trans conjugated isomers also were detected. The total fatty acid methyl esters from the liver lipids were fractionated by reversed-phase high-performance liquid chromatography, and the fraction containing the 20∶4 isomers was further fractionated by silver nitrate thin-layer chromatography. A band containing two 20∶4 conjugated isomers was submitted to hydrazine reduction and the resulting monoenes analyzed by GC-MS as dimethyl-oxazoline derivatives. The two conjugated isomers were tentatively identified as 5c,8c,11c,13t–20∶4 and 5c,8c,12t,14c−20∶4. These could be formed by desaturation and elongation of the 9c,11t-and 10t,12c−18∶2 present in the commerical CLA mixture.     

Fatty Acid Desaturation and Elongation Reactions of <Emphasis Type="Italic">Trichoderma</Emphasis> sp. 1-OH-2-3

The fatty acid desaturation and elongation reactions catalyzed by Trichoderma sp. 1-OH-2-3 were investigated. This strain converted palmitic acid (16:0) mainly to stearic acid (18:0), and further to oleic acid (c9-18:1), linoleic acid (c9,c12-18:2), and α-linolenic acid (c9,c12,c15-18:3) through elongation, and Δ9, Δ12, and Δ15 desaturation reactions, respectively. Palmitoleic acid (c9-16:1) and cis-9,cis-12-hexadecadienoic acid were also produced from 16:0 by the strain. This strain converted n-tridecanoic acid (13:0) to cis-9-heptadecenoic acid and further to cis-9,cis-12-heptadecadienoic acid through elongation, and Δ9 and Δ12 desaturation reactions, respectively. trans-Vaccenic acid (t11-18:1) and trans-12-octadecenoic acid (t12-18:1) were desaturated by the strain through Δ9 desaturation. The products derived from t11-18:1 were identified as the conjugated linoleic acids (CLAs) of cis-9,trans-11-octadecadienoic acid and trans-9,trans-11-octadecadienoic acid. The product derived from t12-18:1 was identified as cis-9,trans-12-octadecadienoic acid. cis-6,cis-9-Octadecadienoic acid was desaturated to cis-6,cis-9,cis-12-octadecatrienoic acid by this strain through Δ12 desaturation. The broad substrate specificity of the elongation, and Δ9 and Δ12 desaturation reactions of the strain is useful for fatty acid biotransformation.     

A novel method for solvent fractionation of two CLA isomers

Conjugated linoleic acid (CLA) is commercially available as a mixture consisting of almost equal amounts of the cis-9,trans-11-CLA (c9, t11) and trans-10, cis-12-CLA (t10, c12) isomers. Separation of the two isomers is highly significant since each exhibits different biochemical properties. Highly efficient separation could be accomplished by crystallization in acetone (solvent) of the two CLA isomers (solutes) in the presence of medium-chain fatty-acid (MCFA) additives. The relative concentration ratios of the two CLA isomers in the solvent-crystallized materials varied depending on which MCFA were added. Addition of lauric and decanoic acids resulted in the crystals predominantly containing t10,c12, whereas octanoic acid yielded those predominantly containing c9,t11. We have confirmed that onetime solvent crystallization using decanoic acid and octanoic acid additives increased the t10,c12 and c9,t11 concentrations, and that repeated solvent crystallization resulted in the ratio of c9,t11 to t10,c12 of at least 4∶96 or 98∶2.     

Effects of conjugated linoleic acid isomers on the hepatic microsomal desaturation activities in vitro

The influence of individual conjugated linoleic acid (CLA) isomers on the Δ6 desaturation of linoleic and α-linolenic acids and on the Δ9 desaturation of stearic acid was investigated in vitro, using rat liver microsomes. The Δ6 desaturation of 18∶2n−6 was decreased from 23 to 38% when the ratio of 9cis,11trans-18∶2 to 18∶2n−6 increased from 0.5 to 2. The compound 10trans,12cis-18∶2 exhibited a similar effect only at the highest concentration. The Δ6 desaturation of α-linolenic acid was slightly affected by the presence of CLA isomers. The sole isomer to induce an inhibitory effect on the Δ9 desaturation of stearic acid was 10trans,12cis-18∶2.     

Placental transport oftrans fatty acids in the rat

Placental transport of 9-trans [1-¹⁴C] octadecenoic (elaidic) and 9-trans,12-trans [1-¹⁴C] octadecadienoic (linoelaidic) acids was demonstrated in rats. On the 18th day of gestation, a¹⁴C-labeled albumin complex of elaidic or linoelaidic acid was injected into the jugular vein of pregnant rats. For comparison, 9-cis [1-¹⁴C] octadecenoic (oleic) or 9-cis,12-cis [1-¹⁴C] octadecadienoic (linoleic) acid also was injected into the maternal circulation of rats. All animals were sacrificed 1 hr following injection. Lipid composition and distribution of label were determined in maternal plasma, placental and fetal tissues. Differences in specific activities of plasma, placental and fetal total lipids indicated a decreasing concentration gradient for bothcis andtrans isomers of octadecenoic and octadecadienoic acids. Distribution of radioactivity in various lipid components was determined by thin layer chromatography. Irrespective of the label, the highest percentage of total radioactivity was carried by triglycerides (TG) in maternal plasma (∼60–80%), and was incorporated mainly in phospholipids (PL) of fetal tissues (∼50–60%). A nearly equal distribution of the label was found between PL and TG of placental lipids (∼40%). Radioactivity of fatty acid methyl esters (FAME) determined by radiogas liquid chromatography indicated that after injection of linoelaidate, radioactivity of maternal plasma, placental and fetal tissue FAME was associated only witht,t-18∶2. Following injection of elaidate, all the radioactivity in placental FAME was associated witht-18∶1; however, in fetal tissues, the label was distributed between 16∶0 andt-18∶1. These findings suggest that, in contrast to linoelaidic acid, rat fetal tissues can metabolize elaidic acid via β oxidation to form acetyl CoA and palmitic acid.     

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.     

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.     

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.     

Inhibitory effect of conjugated linoleic acid on linoleic acid elongation in transformed yeast with human elongase

Conjugated linoleic acid (CLA; 18∶2) refers to a group of positional and geometric isomers derived from linoleic acid (LA; Δ9, 12–18∶2). Using a growing baker's yeast (Saccharomyces cerevisiae) transformed with human elongase gene, we examined the inhibitory effect of CLA at various concentrations (10, 25, 50, and 100 μM) on elongation of LA (25 μM) to eicosadienoic acid (EDA; Δ11,14–20∶2). Among four available individual CLA isomers, only c9,t11- and t10,c12-isomers inhibited elongation of LA to EDA. The extent of inhibition (ranging from 20 to 60%) was related to the concentration of CLA added to the medium. In the meantime, only these two isomers, when added at 50 μM to the media, were elongated to conjugated EDA (c11,t13- and t12,c14–20∶2) by the same recombinant elongase at the rate of 28 and 24%, respectively. The inhibitory effect of CLA on LA elongation is possibly due to competition between CLA isomers and LA for the recombinant elongase. Thus, results from this study and a previous study suggest that the biological effect of CLA is exerted through its inhibitory effect on Δ6-desaturation as well as elongation of LA which results in a decrease in long-chain n−6 fatty acids and consequently the eicosanoid synthesis.     

The effect of conjugated linoleic acid isomers on fatty acid profiles of liver and adipose tissues and their conversion to isomers of 16:2 and 18:3 conjugated fatty acids in rats

Conjugated linoleic acid (CLA) is a collective term that describes different isomers of linoleic acid with conjugated double bonds. Although the main dietary isomer is 9cis,11trans-18∶2, which is present in dairy products and ruminant fat, the biological effects of CLA generally have been studied using mixtures in which the 9cis,11trans- and the 10trans,12cis-18∶2 were present at similar levels. In the present work, we have studied the impact of each isomer (9cis,11trans- and 10trans,12cis-18∶2) given separately in the diet of rats for 6 wk. The 10trans,12cis-18∶2 decreased the triacylglycerol content of the liver (−32%) and increased the 18∶0 content at the expense of 18∶1n−9, suggesting an alteration of the Δ9 desaturase activity, as was already demonstrated in vitro. This was not observed when the 9cis,11trans-18∶2 was given in the diet. Moreover, the 10trans,12cis-18∶2 induced an increase in the C₂₂ polyunsaturated fatty acids in the liver lipids. The 10trans,12cis-18∶2 was mainly metabolized into conjugated 16∶2 and 18∶3, which have been identified. The 9cis,11trans isomer was preferentially metabolized into a conjugated 20∶3 isomer. Thus, the 9cis,11trans- and the 10trans,12cis-CLA isomers are metabolized differently and have distinct effects on the metabolism of polyunsaturated fatty acids in rat liver while altering liver triglyceride levels differentially.     

Positional specificity of γ-ketol formation from linoleic acid hydroperoxides by a corn germ enzyme

We have shown unequivocally that the positional specificity of γ-ketol formation by a corn germ enzyme was different from that observed previously by others with an alfalfa seedling enzyme. When the pure positional isomers of linoleic acid hydroperoxide served as substrates, the corn germ enzyme formed one of two γ-ketols: 12-oxo-9-hydroxy-trans-10-octadecenoic acid from 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid (99+% pure) and 10-oxo-13-hydroxy-trans-11-octadecenoic acid from 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid (96% pure). Also isolated from these reactions was one of two α-ketols commonly found as a result of catalysis by linoleic acid hydroperoxide isomerase: 12-oxo-13-hydroxy-cis-9-octadecenoic acid from the 13-hydroperoxide and 10-oxo-9-hydroxy-cis-12-octadecenoic acid from the 9-hydroperoxide. Evidence is offered that γ-ketol formation is catalyzed by linoleic acid hydroperoxide isomerase, the same enzyme responsible for α-ketol production. Presented at the AOCS Spring Meeting, Dallas, Texas, April, 1975.     

In vitro desaturation and elongation of rumenic acid by rat liver microsomes

Various nutritional studies on CLA, a mixture of isomers of linoleic acid, have reported the occurrence of conjugated long-chain PUFA after feeding experimental animals with rumenic acid, 9c,11t–18∶2, the major CLA isomer, probably as a result of successive desaturation and chain elongation. In the present work, in vitro studies were carried out to obtain information on the conversion of rumenic acid. Experiments were first focused on the in vitro Δ6-desaturation of rumenic acid, the regulatory step in the biosynthesis of long-chain n−6 PUFA. The conversion of rumenic acid was compared to that of linoleic acid (9c,12c–18∶2). Isolated rat liver microsomes were incubated with radiolabeled 9c,12c–18∶2 and 9c,11t–18∶2 under desaturation conditions. The data indicated that [1-¹⁴C]9c,11t–18∶2 was a poorer substrate for Δ6-desaturase than [1-¹⁴C]-9c,12c–18∶2. Next, in vitro elongation of 6c,9c,11t–18∶3 and 6c,9c,12c–18∶3 (γ-linolenic acid) was investigated in rat liver microsomes. Under elongation conditions, [1-¹⁴C]6c,9c,11t–18∶3 was 1.5-fold better converted into [3-¹⁴C]8c,11c,13t–20∶3 than [1-¹⁴C]6c,9c,12c–18∶3 into [3-¹⁴C]8c,11c,14c–20∶3. Finally, in vitro Δ5-desaturation of 8c,11c,13t–20∶3 compared to 8c,11c,14c–20∶3 was investigated. The conversion level of [1-¹⁴C]8c,11c,13t–20∶3 into [1-¹⁴C]5c,8c,11c,13t–20∶4 was 10 times lower than that of [1-¹⁴C]8c,11c,14c–20∶3 into [1-¹⁴C]5c,8c,11c,14c–20∶4 at low substrate concentrations and 4 times lower at the saturating substrate level, suggesting that conjugated 20∶3 is a poor substrate for the Δ5-desaturase.