Jan Fritsche

Conjugated linoleic acid in milk fat: High variation depending on production system

Abstract During one years period bulk milk samples were collected monthly from three different types of farms: 1. conventional farming - indoor feeding with silages the whole year, 2. conventional farming - grazing during summer season, 3. ecological farming - grazing during summer season. Conjugated linoleic acids (CLA), trans vaccenic and other trans isomers of milk fatty acids were analyzed. Variation of CLA in milk fat was substantial (0.26 to 1.14 % of total methyl esters) and was season-dependent. The lowest percentage of CLA (0.34 %) was found in the group, fed only fermented roughage and concentrates (most intensive production farm) the highest (0.80 %) in the ecologically produced milk fat. The concentration of CLA and trans vaccenic acid was positively correlated. There is a growing interest in CLA, considered to be beneficial in prevention of carcinogenesis. Its percentage in milk products can be increased through a suitable dietary regimen.

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.

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.

Analysis, occurrence, and physiological properties of trans fatty acids (TFA) with particular emphasis on conjugated linoleic acid isomers (CLA) - a review

The present review provides an outline of the current knowledge of trans fatty acids (TFA) including their structure and formation, analysis, occurrence in foods, estimation and evaluation of daily intake, contents in human adipose tissues and fluids, and physiological properties. Special emphasis is put on conjugated linoleic acids (CLA), which are related to unique beneficial physiological properties. The effects of CLA on carcinogenesis in in vivo and human cancer cell culture studies, on cancer inhibition via the immune system, and further physiological properties are briefly reported. Ways to affect the CLA contents in foods, e.g. influence of feeding regimens or processing conditions, are also discussed.

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.

The potential anticarcinogenic conjugated linoleic acid, cis-9,trans-11 C18:2, in milk of different species: Cow, goat, ewe, sow, mare, woman

The distribution of the potential anticarcinogenic fatty acid cis-9,trans-11-octadecadienoic acid (rumenic acid) and other trans and cis fatty acids in milk fat of different ruminants and non-ruminants including human milk was determined by gas-liquid chromatography. The CLA isomer cis-9,trans-11 was the predominant found. Its variation in milk fat of the bulk and individual samples was substantial (0.07 – 1.35% of FAME). Because feed composition and rumen microflora influence isomerisation of linoleic acid in the rumen, factors such as farm management and season were taken into consideration. CLA in milk of all ruminants was season-dependent and there exists a close positive correlation to trans vaccenic acid. Ewe milk is rich in CLA (1.1%). Among non-ruminants mare milk was nearly CLA-free (0.09%). Human milk contained significantly more CLA (0.42%, P < 0.01) in comparison wih the analyzed milk of the other monogastrides. There are differences between milk- and non-milk drinkers. The arrangement of the species according to the increasing CLA concentration in milk is: mare, sow, woman, goat, cow, ewe. The higher CLA content of ruminant milk compared with non-ruminant species is inversely correlated to the content of PUFA and partly to MUFA.

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 CPSilTM 88 column). The amounts obtained by GC were generally higher than those determined by Ag+ -HPLC due to co-eluting compounds.

Conjugated linoleic acid (CLA) isomers: formation, analysis, amounts in foods, and dietary intake

The analysis, content, and daily intake of conjugated linoleic acid isomers (CLA) are presented in the following review. Modern analytical techniques such as capillary gas chromatography (GC), silver -ion high performance liquid chromatography (Ag + -HPLC) combined with different detection methods (flame ionisation, mass spectroscopic, ultra violet) are mandatory for the unequivocal determination of geometric and positional CLA isomers. An overview is given on the CLA contents in 139 German foods, e. g. milk/dairy products, meat/meat products, edible oils, margarines, fish, and deep fried products. The dietary intake for men and women is estimated using consumption data.

Enhanced Resolution of Conjugated Linoleic Acid Isomers by Tandem‐Column Silver‐Ion High Performance Liquid Chromatography

A commercial mixture of conjugated linoleic acid (CLA) isomers, reportedly consisting of six components, was recently resolved into 12 peaks attributed to CLA isomers using silver-ion high performance liquid chromatography (Ag+-HPLC). In this study, the coupling of two analytical silver-ion high performance liquid chromatography columns (tandem-column Ag+-HPLC) in series led to the enhanced resolution of CLA isomers. Many CLA isomers were baseline resolved and the pair 18 : 2 8,10 c/t and 18 : 2 7,9 c/t found in cheese products, was resolved for the first time. In this work, a similar commercial CLA mixture was separated into 16 peaks, while CLA isomers from cheese also gave rise to 16 peaks. As expected, the CLA isomers were separated into three geometric groups in the order trans,trans, cis/trans, and cis,cis. Semi-preparative Ag+-HPLC, followed by gas chromatography–mass spectroscopy of the dimethyloxazoline derivatives, was used to confirm the identity of the newly resolved positional CLA isomers. The double bond configuration of CLA isomers was established by gas chromatography–Fourier transform infrared spectroscopy. Two minor t,t CLA isomers found in cheese, presumably 18 : 2 t6t8 and 18 : 2 t13t15, were also separated. The CLA isomeric composition of 16 commercial cheese products was determined.

Lipase-catalyzed fractionation of conjugated linoleic acid isomers

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