Synthesis,isolation, and GC analysis of all the 6,8-to 13,15-<Emphasis Type="Italic">cis/trans</Emphasis> conjugated linoleic acid isomers

Octadecadienoic acids with conjugated double bonds are often referred to as conjugated linoleic acid, or CLA. CLA is of considerable interest because of potentially beneficial effects reported from animal studies. Analysis of CLA is usually carried out by GC elution of FAME. If the presence of low-level isomers is of interest, a complementary technique such as silverion HPLC is also used. These analyses have been hindered by a lack of well-characterized commercially available reference materials. Described here are the synthesis and isolation of selected 6,8-through 13,15-positional CLA isomers, followed by isomerization of these CLA isomers with iodine to produce all the possible cis,cis,cis,trans,trans,cis, and trans,trans combinations. Also present are the GC retention times of the CLA FAME relative to γ-linolenic acid (6c,9c,12c-octadecatrienoic acid) FAME using a 100-m CP Sil-88 capillary column (Varian Inc., Lake Forest, CA). These data include all the CLA isomers that have been identified thus far in foods and dietary supplements and should greatly aid in the future analysis of CLA in these products.     

Quantitative determination of conjugated linoleic acid isomers in beef fat

The amounts of 14 conjugated linoleic acid (CLA) isomers (t12t14, t11t13, t10t12, t9t11, t8t10, t7t9, t6t8; 12,14 c/t, t11c13, c11t13, t10c12, 9,11 c/t, t8c10, t7c9‐18:2) in 20 beef samples were determined by triple‐column silver‐ion high‐performance liquid chromatography (Ag⁺‐HPLC). Quantitation was performed using an external CLA reference standard consisting of cis9,trans11‐18:2,trans9,trans11‐18:2 and cis9,cis11‐18: 2. Linearity was checked as being r > 0.9999 between 0.02 × 10^‐3 to 2 mg/ml. The determination limit (5‐fold signal/noise ratio) of the CLA reference was estimated to be 0.25, 0.50, 1.0 ng/injection for the cis/trans, trans,trans and cis,cis isomers, respectively. As expected, cis9,trans11‐18:2 was the predominant isomer (1.95 ± 0.54 mg/g fat) in beef, followed by trans7,cis9‐18:2 (0.19 ± 0.04 mg/g fat); cis,cis isomers were below the determination limit in most beef samples. Total CLA amounts determined by Ag⁺‐HPLC were compared to total CLAs determined by gas chromatography (GC, 100 m CPSil^TM 88 column). The amounts obtained by GC were generally higher than those determined by Ag⁺ ‐HPLC due to co‐eluting compounds.     

In vitro genotoxicity/antigenotoxicity testing of some conjugated linoleic acid isomers using comet assay

Recently CLA isomers have received considerable attention as potential anti‐cancer agents. The aim of the study was to assess the genotoxicity/antigenotoxicity in vitro of linoleic acid (LA, c,c‐C18:2, Δ‐9), CLA isomer mixtures and homogeneous CLA TAGs (TriCLA) using the comet assay, to evaluate the effects on the extent of DNA injury in human hepatoma (HepG2) cells. The study was carried out both on commercial CLA (CLAc) and on CLA synthesized from grapestone oil (CLAg). The CLA isomer mixtures had different isomer profiles, determined by silver‐ion HPLC (Ag⁺‐HPLC), in particular CLAc was characterized by four main isomers (t8,c10; c9,t11; t10,c12; c11,t13), while CLAg showed two main isomers (c9,t11; t10,c12). As regards antigenotoxicity testing, LA, TriCLAg, and above all TriCLAc were effective antigenotoxic compounds against ethylmethanesulfonate (EMS) induced genotoxicity, while LA and CLAg were almost equally effective against 4‐nitroquinoline N‐oxide (4NQO) induced DNA damage. Both TriCLAc and TriCLAg showed an increased antigenotoxic activity toward EMS and a lower antigenotoxic activity toward 4NQO, with respect to both CLAc and CLAg. The higher capability of CLAg with respect to CLAc in counteracting the genotoxicity of 4NQO could be due to the different CLA isomer composition. Practical applications: CLA isomers have shown many beneficial health effects both on animals and humans. They are widely used in nutritional supplements, as CLA improves body composition by reducing fat storage. In this regard it is very important to know, besides the chemical and analytical aspects, also genotoxic and antigenotoxic effects of different CLA mixtures. To our best knowledge, few results have been reported on CLA antigenotoxic properties by the comet assay, and no data could be retrieved in the literature for TriCLA antigenotoxicity testing. The obtained results are interesting in that they can increase the knowledge on particular fatty acids used in commercial supplements.     

Effects of trans‐10,cis‐12 and cis‐9,trans‐11 CLA on atherosclerosis in apoE/LDLR−/− mice

The objective of our studies was to verify the potential health‐related, anti‐atherogenic potency of CLA isomers, fed to apolipoprotein E and LDL receptor double knockout mice (apoE/LDLR^?/?), representing a reliable model of atherogenesis. Additionally, the effect of CLA isomers on liver steatosis was observed. In a “long experiment” (LONG), 2‐month‐old mice with no atherosclerosis were randomly assigned to three experimental groups and fed for the next 4 months. In a “short experiment” (SHORT), 4‐month‐old mice, with pre‐established atherosclerosis, were randomly assigned to three experimental groups and fed for the next 2 months. The experimental diets were: AIN‐93G (control), AIN‐93G + 0.5% trans‐10,cis‐12 CLA (t10,c12), and AIN‐93G + 0.5% cis9,trans‐11 CLA (c9,t11). In both experiments, c9,t11 CLA increased mice body weight. In mice fed t10,c12 CLA weight of liver was threefold (p<0.05) increased what was linked with hepatic steatosis observed in LONG and SHORT experiment. In LONG experiment, t10,c12 CLA significantly (p<0.05) increased plasma TAGs, whereas no such effect was observed in SHORT one. In mice receiving the CLA isomers the level of PPARα and SREBP‐1 mRNA in liver were significantly decreased. The expression of their target genes like ACO (PPARα) or FAS (SREBP‐1) were not changed. Only c9,t11 increased ACO level in LONG experiment. There were no isomer‐specific effects of CLA isomers on the area of atherosclerotic plaque. In conclusion, our results do not support the notion that CLA isomers supplementation to the diet has anti‐atherosclerotic effects. CLA isomers have no effect on atherosclerosis in apoE/LDLR^?/? mice. Practical applications: CLAs have been shown to occur naturally in food. In the last 10 years, attempts have been made to enrich animal‐derived foods in CLA isomers through animal nutrition strategies. Indeed, these attempts resulted in production of functional food such as CLA‐enriched milk (butter and cheese), ruminant and non‐ruminant meat, as well as eggs. In addition to natural foodstuff, dietary CLA supplements can also contribute to CLA intake in humans. Commercial CLA preparations, fed to laboratory animals, showed several health‐related properties, including anti‐adipogenic, anti‐carcinogenic, anti‐atherogenic, and anti‐inflammatory effects. The underlying mechanisms of action, however, are only poorly understood. The major objective of our studies was to verify the potential health‐related, namely anti‐atherogenic potency of CLA isomers, fed to apoE/LDLR^?/? mice, representing unique and reliable model of atherogenesis. Additionally, effect of CLA isomers on steatosis was observed.     

Relative retention order of all isomers of <Emphasis Type="Italic">cis/trans</Emphasis> conjugated linoleic acid FAME from the 6,8- to 13,15-positions using silver ion HPLC with two elution systems

CLA, defined as one or more octadecadienoic acids (18∶2) with conjugated double bonds, has been reported to be active in a number of bological systems. GC and silver ion HPLC (Ag⁺-HPLC) have been the primary techniques for identifying specific CLA isomers in both foods and biological extracts. Recently, GC relative retention times were reported for all c,c, c/t (c,t and t,c), and t,t CLA FAME from the 6,8- to the 13,15-positions in octadecadienoic acid (18∶2). Presented here is the relative retention order of the same CLA FAME using Ag⁺-HPLC with two different elution systems. The first elution system, consisting of 0.1% acetonitrile/0.5% diethyl ether (DE)/hexane, has been used previously to monitor CLA composition in foods. Also presented here is the retention order of CLA FAME using 2% acetic acid/hexane elution solvent, which has advantages of more stable retention volumes and a complementary elution order of CLA FAME isomers. The data are reported using retention volumes (RV) adjusted for toluene, an estimator for dead volume, and relative to c9,t11-18∶2. Measurement of relative RV in the analysis of 88 samples of cow plasma, milk, and rumen fluids using Ag⁺-HPLC is also presented here. The % CV ranged from 1.04 to 1.62 for t,t isomers and from 0 to 0.48 for c/t isomers.     

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.     

High trans,trans Conjugated Linoleic Acid-Rich Triacylglyceride Production by Photo-Irradiation

Recent studies have shown that a 20 % trans,trans conjugated linoleic acid (CLA)‐rich soy oil significantly reduces heart disease and diabetes risk factors in obese rats. Furthermore, trans,trans‐CLA has been reported to have superior anti‐carcinogenic activity than other CLA isomers. Therefore, a more concentrated source of trans,trans‐CLA oil would be highly desirable. The objectives of this study were to (1) determine the yield of trans,trans‐CLA isomers resulting from photo‐irradiation of Tonalin^® (BASF Global, Florham Park, NJ, USA) and identify trans,trans‐CLA positional isomers; and (2) derive a mathematical model of kinetics of trans,trans‐CLA TAG formation from Tonalin^®. Fifty‐five percent trans,trans‐CLA rich oil was obtained in about 140 min when Tonalin^® was photo‐isomerized with 0.35 % iodine, which is almost three times more than is possible with photo‐isomerized soy oil. Photo‐isomerization of Tonalin^® requires about 2 h, compared to 12 h for photo‐isomerization of soy oil. This reaction is a first‐order reversible reaction with the forward rate constant (k_f) = 13.17 × 10^?3min^?1 and backward rate constant (k_b) = 5.334 × 10^?3min^?1. The major isomers identified were trans‐9,trans‐11‐ and trans‐10,trans‐12‐CLA.     

Diet‐dependent occurrence of CLA isomers in rumen and duodenal digesta of slaughtered bulls

This study examined the effects of linolenic acid‐rich vs. linoleic acid‐rich feeding system on the occurrence of individual CLA isomers in the rumen and duodenum digesta of German Holstein and German Simmental bulls using Ag⁺‐HPLC/DAD. The diet affected the biosynthesis of individual CLA isomers in the rumen of the bulls of both breeds. The isomer t‐11,c‐13 CLA was detected as the most abundant isomer in the rumen of linolenic acid‐rich diet‐fed bulls, up to six times higher compared to linoleic acid‐rich diet‐fed bulls. However, the main isomer in muscle lipid, c‐9,t‐11 CLA, was produced to a low extent in the rumen of linolenic acid‐rich diet‐fed bulls compared to higher concentrations of this isomer in the rumen of linoleic acid‐rich diet‐fed bulls. The isomers t‐7,c‐9 CLA and t‐8,c‐10 CLA were not present in the rumen samples of bulls fed both diets; however, abundant t‐7,c‐9 CLA was identified in the duodenum. The CLA isomers t‐12,t‐14 CLA and t‐11,t‐13 CLA were identified as the main t,t CLA isomers in the rumen, and were significantly enhanced in the rumen of linolenic acid‐rich diet‐fed compared to linoleic acid‐rich diet‐fed bulls. In contrast to c‐9,t‐11 CLA, the t,t CLA isomers seem to be biosynthesized predominantly in the rumen, further transported via the duodenum and finally deposited in the tissue lipids mainly in linolenic acid‐rich diet‐fed bulls. This was shown earlier for muscle and subcutaneous fat samples from the same animal experiment.     

Fatty acid and conjugated linoleic acid isomer profiles in human milk fat

The fatty acid composition of 39 mature human milk samples from four Spanish women collected between 2 and 18 weeks during lactation was studied by gas chromatography. The conjugated linoleic acid (CLA) isomer profile was also determined by silver‐ion HPLC (Ag⁺‐HPLC) with three columns in series. The major fatty acid fraction in milk lipids throughout lactation was represented by the monounsaturated fatty acids, with oleic acid being the predominant compound (36–49% of total fatty acids). The saturated fatty acid fraction represented more than 35% of the total fatty acids, and polyunsaturated fatty acids ranged on average between 10 and 13%. Mean values of total CLA varied from 0.12 to 0.15% of total fatty acids. The complex mixture of CLA isomers was separated by Ag⁺‐HPLC. Rumenic acid (RA, cis‐9 trans‐11 C18:2) was the major isomer, representing more than 60% of total CLA. Trans‐9 trans‐11 and 7‐9 (cis‐trans + trans‐cis) C18:2 were the main CLA isomers after RA. Very small amounts of 8‐10 and 10‐12 C18:2 (cis‐trans + trans‐cis) isomers were detected, as were different proportions of cis‐11 trans‐13 and trans‐11 cis‐13 C18:2. Although most of the isomers were present in all samples, their concentrations varied considerably.     

Conjugated linoleic acids: Physiological effects in animal and man with special regard to body composition

Institute of Nutrition, Friedrich Schiller University of Jena, Jena, Germany In the last decade, conjugated linoleic acids (CLA) have been shown to have some beneficial (but also unfavourable) effects: anticarcinogenic properties, immune modulation, reduction of body fat and increase of lean body mass, normalisation of impaired glucose tolerance, promotion of fatty streak formation, and isomer‐specific effects. The research base on CLA has been derived almost exclusively from animal models, while some of the biological properties have been fairly well‐documented, others are still open to question. For about 5 years a lot of commercial CLA mixtures have been offered. These mixtures produced from linoleic acid‐rich oil like sunflower or safflower oil by alkali isomerization contained, besides cis‐9,trans‐11 and trans‐10,cis‐12 CLA isomers (about 20—40% of each), parts of cis,cis and trans,trans isomers as well. The quality of the recent products is significantly improved and they contain only two CLA isomers: cis‐9,trans‐11 and trans‐10,cis‐12. CLA play apparently a key role in regulating body composition. Several studies showed a reduction in body fat mass and a slight increase in lean body mass depending on the species. A possible explanation for the decrease of body fat may be a stimulation of lipolysis and a reduction of lipoprotein lipase activity in adipocytes. In adipose and muscle tissue a CLA‐stimulated increase of carnitine palmitoyltransferase activity resulting in an enhanced fatty acid oxidation was shown. There is evidence that CLA provide protection against cytokine‐induced (Tumour necrosis factor‐α, interleukin‐1) skeletal‐muscle catabolism (anabolic effect). The body composition modulating effects are most impressive in rodents and seem to become smaller in pigs and in humans. Data on humans are insufficient. Further research is essential to characterize the multifunctionality of CLA in humans, in order to identify the specific physiological mechanism of the biologically active isomers and to determine the optimal level of these isomers for beneficial effects.     

Silver-ion high-performance liquid chromatographic separation and identification of conjugated linoleic acid isomers

This is the first report of the application of silverion impregnated high-performance liquid chromatography (Ag⁺-HPLC) to the separation of complex mixtures of conjugated linolenic acid (CLA) isomers present in commercial CLA sources and foods and in biological specimens. This method showed a clear separation of CLA isomers into three groups related to their trans,trans, cis,trans or trans,cis, and cis,cis configuration of the conjugated double-bound system. In addition, this method separated within each geometrical isomeric group. Following Ag⁺-HPLC isolation, gas chromatography (GC)-electron impact mass spectrometry, and GC-direct deposition-Fourier transformed infrared spectroscopy were used to confirm the identity of two major positional isomers in the cis/trans region, i.e., Δ8,10- and Δ11,13-octadecadienoic acid, which had not been chromatographically resolved previously, Furthermore, the potential of this method was demonstrated by showing different Ag⁺-HPLC profiles exhibiting patterns of isomeric distributions for biological specimens from animals fed a diet containing a commerical CLA preparation, as well as for a commerical cheese product.     

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.     

Hydroperoxide formation during autoxidation of conjugated linoleic acid methyl ester

The aim of this study was to investigate whether hydroperoxides are formed in the autoxidation of conjugated linoleic acid (CLA) methyl ester both in the presence and absence of α‐tocopherol. The existence of hydroperoxide protons was confirmed by D₂O exchange and by chemoselective reduction of the hydroperoxide groups into hydroxyl groups using NaBH₄. These experiments were followed by nuclear magnetic resonance (NMR) spectroscopy. The ¹³C and ¹HNMR spectra of a mixture of 9‐hydroper‐oxy‐10‐trans,12‐cis‐octadecadienoic acid methyl ester (9‐OOH) and 13‐hydroperoxy‐9‐cis, 11‐trans‐octadecadienoic acid methyl ester (13‐OOH), which are formed during the autoxidation of methyl linoleate, were studied in detail to allow the comparison between the two linoleate hydroperoxides and the CLA methyl ester hydroperoxides. The ¹³CNMR spectra of samples enriched with one of the two linoleate hydroperoxide isomers were assigned using 2D NMR techniques, namely Correlated Spectroscopy (COSY), gradient Heteronuclear Multiple Bond Correlation (gHMBC), and gradient Heteronuclear Single Quantum Correlation (gHSQC). The ¹³C and ¹H NMR experiments performed in this study show that hydroperoxides are formed during the autoxidation of CLA methyl ester both in the presence and absence of α‐tocopherol and that the major isomers of CLA methyl ester hydroperoxides have a conjugated monohydroperoxydiene structure similar to that in linoleate hydroperoxides.     

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.     

Protective action of CLA against oxidative inactivation of paraoxonase 1, an antioxidant enzyme

The effect of CLA on paraoxonase 1 (PON1), one of the antioxidant proteins associated with HDL, was investigated for its protective action against oxidative inactivation as well as its stabilization activity. When cis-9 (c9),trans-11 (t11)-CLA and t10,c12-CLA were examined for their protective activity against ascorbate/Cu²⁻-induced inactivation of PON1 in the presence of Ca²⁺, two CLA isomers exhibited a remarkable protection (E _max, 71–74%) in a concentration-dependent manner (50% effective concentration, 3–4 μM), characterized by a saturation pattern. Such a protective action was also reproduced with oleic acid, but not linoleic acid. Rather, linoleic acid antagonized the protective action of CLA isomers in a noncompetitive fashion. Additionally, the two CLA isomers also protected PON1 from oxidative inactivation by H₂O₂ or cumene hydroperoxide. The concentration-dependent protective action of CLA against various oxidative inactivation systems suggests that the protective action of CLA isomers may be mediated through their selective binding to a specific binding site in a PON1 molecule. Separately, the inactivation of PON1 by p-hydroxymercuribenzoate (PHMB), a modifier of the cysteine residue, was also prevented by CLA isomers, suggesting the possible existence of the cysteine residue in the binding site of CLA. The c9,t11-CLA isomer seems to be somewhat more effective than t10,c12-CLA in protecting against the inactivation of PON1 by either peroxides or PHMB, in contrast to the similar efficacy of these two CLA isomers in preventing ascorbate/Cu²⁺-induced inactivation of PON1. Separately, CLA isomers successfully stabilized PON1, but not linoleic acid. These data suggest that the two CLA isomers may play a beneficial role in protecting PON1 from oxidative inactivation as well as in its stabilization.     

Determination of CLA <Emphasis Type="Italic">trans</Emphasis>,<Emphasis Type="Italic">trans</Emphasis> Positional Isomerism in CLA-Rich Soy Oil by GC–MS and Silver Ion HPLC

A ~20% CLA-rich soy oil with low saturated fat and no cholesterol was produced by photoisomerizing soy oil linoleic acid. The oil is predominately trans,trans CLA, with the oil containing 17% trans,trans CLA. Recent studies have shown that trans,trans CLA-rich soy oil significantly reduces heart disease and diabetes risk factors in obese rats. However, the positional isomerism of these geometrical isomers has not been identified. The objectives of the study were to determine trans,trans CLA positional isomerism of CLA fatty acids in CLA-rich soy oil and determine the resolution of trans,trans CLA positional isomers by silver ion chromatography. GC–MS studies of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatives of CLA-rich oil showed that 9,11 CLA and 10,12 CLA were the major positional isomers. These were hypothesized to be the trans,trans CLA isomers, which was confirmed by silver ion chromatography and subsequent GC–FID fatty acid and ATR-FTIR geometrical isomer analysis of the collected fractions. The identification of 9,11 trans,trans CLA and 10,12 trans,trans CLA as the major CLA isomers in CLA-rich oil then allowed the deduction of the synthetic mechanism of the photoisomerism of soy oil linoleic acid to trans,trans CLA.     

<Emphasis Type="Italic">9-trans,11-trans</Emphasis>-CLA: Antiproliferative and proapoptotic effects on bovine endothelial cells

Endothelial cell function can be influenced by nutrition, especially dietary FA and antioxidants. One class of dietary FA that is found in meat and dairy products derived from ruminant animals is conjugated linoleic acids (CLA). We have examined the effects of several CLA isomers on endothelial cell proliferation. 9t,11t-CLA was the only isomer that inhibited bovine arotic endothelial cell (BAEC) [³H]methylthymidine incorporation (I₅₀=35 μM), and this antiproliferative effect was time-dependent. A small decrease (20%) in cell number was observed only at the highest concentration (60 μM) tested. The 9c,11t-, 9c,11c-, 10t 12c-, and 11c,13t-CLA isomers did not exhibit any antiproliferative effects over a 5–60 μM concentration range. α-Tocopherol and BHT decreased BAEC proliferation, but pretreatment of cells with either of these antioxidants substantially attenuated the antiproliferative effect of 9t,11t-CLA. No difference in lipid peroxidation, as measured by the thiobarbituric acid assay for malondialdehyde, was observed on treatment of endothelial cells with either 9t,11t- or 9c,11t-CLA. However, a 43% increase in caspase-3 activity was observed after incubating BAEC with 9t,11t-CLA, suggesting that the antiproliferative effect of this isomer is partially due to an apoptotic pathway. In contrast to the above results with normal endothelial cells, these five CLA isomers all inhibited proliferation of the human leukemic cell line THP-1, with the 9t,11t isomer again being the most (I₅₀=60 μM) effective. These results confirm that different CLA isomers have different inhibitory potencies on the proliferation of normal and leukemic cells.     

Photochemical production of conjugated linoleic acid from soybean oil

Conjugated linoleic acid (CLA), an anticarcinogenic compound with numerous other health benefits, is present mainly in dairy and beef lipids. The main CLA isomer present in dairy and beef lipids is cis 9, trans 11 CLA at a 0.5% concentration. The typical minimum human dietary intake of CLA is 10 times less than the 3 g/d suggested requirement that has been extrapolated from animal and cell-line studies. The objectives of this study were to produce CLA isomers from soybean oil by photoisomerization of soybean oil linoleic acid and to study the oxidation status of the oil. Refined, bleached, and deodorized soybean oil with added iodine concentrations of 0, 0.1, 0.25, and 0.5% was exposed to a 100-W mercury lamp for 0 to 120 h. An SP-2560 fused-silica capillary GC column with FID was used to analyze the esterified CLA isomers in the photoisomerized oil. The CLA content of the individual isomers was optimized by response surface methodology. Attenuated total reflectance (ATR)-FTIR spectra in the 3400 to 3600 cm⁻¹ range and ¹H NMR spectra in the 8 to 12 ppm range of the photoisomerized soybean oil were obtained to follow hydroperoxide formation. The largest amount of cis 9, trans 11 CLA isomer in soybean oil was 0.6%, obtained with 0.25% iodine and 84 h of photoisomerization. Lipid hydroperoxide peaks in the ATR-FTIR spectra and aldehyde peaks in the ¹H NMR spectra were not observed in the photoisomerized soybean oil, and the spectra were similar to that of fresh soybean oil. This study shows that CLA isomers can be produced simply and inexpensively from soybean oil by photoisomerization.     

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.