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.     

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.     

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.     

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.     

Fractionation and enrichment of CLA isomers by selective esterification with Candida rugosa lipase

A commercial product of CLA contains almost equal amounts of cis-9,trans-11 (c9,t11)-CLA and trans-10,cis-12 (t10,c12)-CLA. We attempted to enrich the two isomers by a two-step selective esterification using Candida rugosa lipase that acted on c9,t11-CLA more strongly than on t10,c12-CLA. An FFA mixture containing CLA isomers was esterified with an equimolar amount of lauryl alcohol in a mixture of 20% water and the lipase. When the esterification of total FA reached 50%, two isomers were fractionated in a good yield: t10,c12-CLA was enriched in FFA, and c9,t11-CLA was recovered in lauryl esters. The FFA were esterified again to enrich t10,c12-CLA. At 27.3% esterification of total FA, the t10,c12-CLA content in FFA increased to 64.8 wt% with 89.3% recovery: The ratio of the content of t10,c12-CLA to that of two isomers was 95.9%. Lauryl esters obtained by the single esterification were employed for enrichment of c9,t11-CLA. After the esters were hydrolyzed, the resulting FFA were esterified again with lauryl alcohol. At 62.0% esterification of total FA, the c9,t11-CLA content in lauryl esters increased to 73.3 wt% with 79.4% recovery: The ratio of the content of c9,t11-CLA to that of two isomers was 95.6%. In a 600-g-scale purification, molecular distillation was effective in separating the reaction mixture into lauryl alcohol, FFA, and lauryl ester fractions.     

<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.     

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.     

Enrichment of CLA isomers by selective esterification with l-menthol using Candida rugosa lipase

Commercially available preparations of CLA are composed of almost equal amounts of 9-cis,11-trans (9c,11t)-CLA and 10-trans,12-cis (10t,12c)-CLA. Each isomer was fractionated and enriched, for availability as a food supplement, by a process comprising selective esterification with l-menthol by Candida rugosa lipase, distillation, and n-hexane extraction. The first selective esterification of CLA isomers was conducted with an equimolar amount of l-menthol of 30°C. The oil phase of the reaction mixture was fractionated into an l-menthyl ester fraction (9c,11t-CLA rich) and an FFA fraction (10t,12c-CLA rich) by distillation. The FFA fraction was esterified again with an equimolar amount of l-menthol to enrich 10t,12c-CLA. The 10t,12c-CLA preparation was obtained as the resulting FFA fraction by distillation. 10t,12c-CLA was enriched to 91% with 40% recovery. To enrich 9c,11t-CLA, the l-menthyl ester fraction in the first esterification was chemically hydrolyzed, and the resulting FFA were esterified again with an equimolar amount of l-menthol. The 9c, 11t-CLA preparation was obtained by chemical hydrolysis of the resulting l-methyl ester fraction, followed by n-hexane extraction. 9c,11t-CLA was enriched to 94% with 42% recovery. This effective process for purification of CLA isomers using l-methol is applicable to the production of food supplements.     

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.     

Variations in isomer distribution in commercially available conjugated linoleic acid

Conjugated linoleic acid (CLA) has been reported to have anticarcinogenic and antiatherogenic properties, to repartition body fat, to build bone mass, to normalize glucose tolerance, and to reduce hyperglycemia and diabetes. CLA products are now commercially available, and there is considerable interest in studying CLA because of this range of reported beneficial effects. However, little is known about the composition of these preparations. Representative commercial CLA products in capsule or liquid (aqueous or oily) form were analyzed for their CLA content and isomer composition using gas chromatography (GC), silver ion-high performance liquid chromatography (Ag⁺-HPLC) and spectroscopic techniques. The content of CLA in the preparations varied widely. Based on the GC-internal standard technique, total CLA varied from 20 to 89% by total weight and 28 to 94% of total fat. One product contained no CLA. The isomer distributions were generally of two types: those with two major CLA positional isomers, and those with four major CLA positional isomers. All the CLA preparations in capsule form contained the four isomer mixture, while the liquid preparations contained from two to four CLA positional isomers.     

Determination of <Emphasis Type="Italic">c</Emphasis>9,<Emphasis Type="Italic">t</Emphasis>11-CLA in major human plasma lipid classes using a combination of methylating methodologies

Isomeric CLA exhibit several significant biological activities in animals and humans and are easily isomerized to their corresponding t,t-CLA isomers during methylation with various acid-catalyzed reagents. To minimize such isomerization and provide a valid quantification of human plasma CLA content, several methylation methods were tested. Plasma neutral lipid, nonesterified FA (NEFA), and polar lipid classes were separated into the following fractions: (i) cholesteryl ester (CE, 1.2 mg/12 mL, 37.5% lipids), (ii) TAG (0.8 mg/12 mL, 25% lipids), (iii) NFFA (0.2 mg/12 mL, 6.2% lipids), (iv) MAG/DAG/cholesterol (0.3 mg/12 mL, 9.4% lipids), and (v) phospholipid (PL, 0.5 mg/20 mL, 15.6% lipids). Data showed that c9,t11-CLA found in TAG, MAG/DAG/cholesterol, and PL fractions were converted to methyl esters with sodium methoxide within 2 h at 55°C. However, the c9,t11-CLA in the CE fraction could not be completely converted to methyl esters by sodium methoxide/acetylchloride in methanol or methanolic KOH; instead, CE was treated with sodium methoxide and methyl acetate in diethyl ether for 1 h. NEFA were converted to methyl esters with trimethylsilyldiazomethane (TMSDAM). All reaction mixtures were monitored by TLC prior to GLC analysis. The highest enrichment of c9,t11-18∶2 (% FA) was in TAG (0.31%), followed by CE (0.14%) and PL (0.13%). The above methylation methods were then applied to a small subset (n=10) of nonfasting plasma lipid fractions to confirm the applicability of these data. Results from this subset of samples also indicated that the greatest enrichment of c9,t11-CLA was present in the TAG fraction (0.39%), followed by CE (0.27%) and PL (0.22%). These data indicate that different plasma fractions have different c9,t11-CLA contents.     

cis-9,trans-11 and trans-10,cis-12 CLA affect lipid metabolism differently in primary white and brown adipocytes of Djungarian hamsters

We explored whether CLA isomers and other C₁₈ FA affect (i) lipid content and FA concentrations in total adipocyte lipids, (ii) FA synthesis from glucose in TAG and phospholipids of primary brown (BAT) and white adipocytes (WAT), and (iii) mRNA expression of uncoupling protein 1 (UCP1) in primary brown adipocytes of Djungarian hamsters (Phodopus sungorus). c9,t11-CLA, oleic, linoleic, and α-linolenic acid increased whereas t10,c12-CLA decreased lipid accumulation in both adipocyte types. t10,c12-CLA treatment affected FA composition mainly in BAT cells. CLA incorporation into lipids, in particular c9,t11-CLA, was higher in BAT. In both cell types, t10,c12-CLA treatment reduced the incorporation of glucose ¹³C carbon into FA of TAG and phospholipids, whereas c9,t11-CLA, linoleic, and α-linolenic acid either did not influence or dose-dependently increased glucose carbon incorporation into FA. UCP1 mRNA expression was inhibited by t10,c12-CLA but increased by c9,t11-CLA, linoleic, and α-linolenic acid. It is concluded that c9,t11-CLA and t10,c12-CLA have distinctly different effects on lipid metabolism in primary adipocytes. The effects of c9,t11-CLA are similar to those of other unsaturated C₁₈ FA. The opposite effects of c9,t11-CLA and t10,c12-CLA are evident in both WAT and BAT cultures; however, brown adipocytes seem to be more susceptible to CLA treatment.     

Synthesis and isolation of <Emphasis Type="Italic">trans</Emphasis>-7, <Emphasis Type="Italic">cis</Emphasis>-9 octadecadienoic acid and other CLA isomers by base conjugation of partially hydrogenated γ-linolenic acid

CLA is of considerable interest because of reported potentially beneficial effects in animal studies. CLA, while not yet unambiguously defined, is a mixture of octadecadienoic acids with conjugated double bonds. The major isomer in natural products is generally considered to be cis-9,trans-11-octadecadienoic acid (c9, t11), which represents >75% of the total CLA in most cases. Other isomers are drawing increased attention. The t7,c9 isomer, which is often the second-most prevalent CLA in natural products, has been reported to represent as much as 40% of total CLA in milk from cows fed a high-fat diet. The need for a reference material became apparent in a recent study directed specifically at measuring t7,c9-CLA in milk, plasma, and rumen. A suitable standard mixture was produced by stirring 0.5 g of γ-linolenic acid (all cis-6,9, 12-C_18∶3) with 100 mL of 10% hydrazine hydrate in methanol for 2.5 h at 45°C. The solution was diluted with H₂O and acidified with HCl. The resulting partially hydrogenated FA were extracted with ether/petroleum ether, dried with Na₂SO₄, and conjugated by adding of 6.6% KOH in ethlylene glycol and heating for 1.5 h at 150–160°C. Approximately 20 mg each of cis-6, trans-8; trans-7, cis-9; cis-9, trans-11; and rans-10, cis-12 were obtained along with other FA. Methyl esters (FAME) of these four cis/trans isomers were resolved by Ag⁺HPLC (UV 233) and partially resolved by GC/(MS or FID) (CP-Sil 88). Treatment of these FAME with I₂ yielded all possible cis/trans (geometric) isomers for the four positions 6,8; 7,9; 9,11; and 10,12.     

Changes in the milk and cheese fat composition of ewes fed commercial supplements containing linseed with special reference to the CLA content and isomer composition

A study was carried out to increase the CLA contents in ewes’ milk fat under field conditions by dietary means and to investigate the extent of the changes and consequences for milk processing and cheese quality. During a 3-mon period, ewes’ bulk milk samples were collected every week from two different herds. For the first 4 wk the ewes were fed a conventional diet. Then the following 6 wk a supplement enriched in α-linolenate (whole linseed) was incorporated into the ovine diet. Finally, in the last 3 wk the feeding was the same as in the first 4 wk. The FA profile in milk fat was monitored by GC, and the distribution of CLA isomers was thoroughly tested by combining GC-MS of 4,4-dimethyloxazoline derivatives (DMOX) with silver ion-HPLC (Ag⁺-HPLC) of FAME. Reconstructed mass spectral profiles of CLA characteristic ions from DMOX were used to identify positional isomers, and Ag⁺-HPLC was used to quantify them. An increase in total CLA in milk fat was observed, and total CLA remained elevated during the weeks of enriched α-linolenate feeding. In our experimental conditions there was a linear relationship between trans-vaccenic acid (trans-11-octadecenoic acid; trans-11 18∶1) and 9-cis, 11-trans CLA in ewes’ milk fat. Concerning the CLA isomer profile, increases in the 11,13- and 12,14–18∶2 positional isomers were considerable when linseed was included in the diet. Organoleptic characteristics of cheeses made with CLA-enriched milk did not substantially differ from those made with nonsupplemented ewes’ milk. CLA total content and isomer profile did not change during ripening.     

Modulation of body composition and immune cell functions by conjugated linoleic acid in humans and animal models: Benefits vs. risks

We have reviewed the published literature regarding the effects of CLA on body composition and immune cell functions in humans and in animal models. Results from studies in mice, hamsters, rats, and pigs generally support the notion that CLA reduced depot fat in the normal or lean strains. However, in obese rats, it increased body fat or decreased it less than in the corresponding lean controls. These studies also indicate that t10,c12-CLA was the isomer that reduced adipose fat; however, it also increased the fat content of several other tissues and increased circulating insulin and the saturated FA content of adipose tissue and muscle. Four of the eight published human studies found small but significant reductions in body fat with CLA supplementation; however, the reductions were smaller than the prediction errors for the methods used. The other four human studies found no change in body fat with CLA supplementation. These studies also report that CLA supplementation increased the risk factors for diabetes and cardiovascular disease including increased blood glucose, insulin, insulin resistance, VLDL, C-reactive protein, lipid peroxidation, and decreased HDL. Most studies regarding the effects of CLA on immune cell functions have been conducted with a mixture of isomers, and the results have been variable. One study conducted in mice with the purified c9,t11-CLA and t10,c12-CLA isomers indicated that the two isomers have similar effects on immune cell functions. Some of the reasons for the discrepancies between the effects of CLA in published reports are discussed. Although significant benefit to humans from CLA supplementation is questionable, it may create several health risks in both humans and animals. On the basis of the published data, CLA supplementation of adult human diets to improve body composition or enhance immune functions cannot be recommended at this time.     

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.     

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.     

Selective Enrichment of Conjugated Linoleic Acid Isomers in Their Mixtures Using Combined Chemical and Enzymatic Methods

The aim of this study was to selectively enrich t10,c12-conjugated linoleic acid (t10,c12-CLA) and c9,t11-CLA in commercial CLA mixtures using a combination of urea crystallization and lipase-catalyzed esterification. The objective of the urea fractionation is to remove saturated and monounsaturated fatty acids (FA) from the CLA mixtures. CLA-enriched free FA (FFA) mixtures containing 53.8 wt% t10,c12-CLA and 39.1 wt% c9,t11-CLA were produced from the CLA mixtures containing ~34 wt% each of the two CLA isomers by a urea crystallization using methanol and the urea-to-FA weight ratio of 2.5:1. The CLA-enriched FFA mixtures were partially esterified with dodecan-1-ol in a recirculating packed-bed reactor using an immobilized lipase from Candida rugosa to further enrich the t10,c12-CLA and c9,t11-CLA in an FFA fraction and an FA dodecyl ester fraction, respectively, under the optimal conditions, i.e., temperature, 20 °C; FA-to-dodecan-1-ol molar ratio, 1:1; water content, 2 wt% of total substrates; residence time, 5 min; and reaction time, 24 h (for t10,c12-CLA enrichment) and 12 h (for c9,t11-CLA enrichment). After the reaction, an FFA fraction with 72.6 wt% t10,c12-CLA was obtained. Another FFA fraction with 62.0 wt% c9,t11-CLA was recovered after the saponification of the FA dodecyl ester fraction. The yields of t10,c12-CLA and c9,t11-CLA in the FFA fractions were 43.6 and 21.5 wt%, respectively, based on their initial weights in the CLA mixtures.     

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.