Klaus Eulitz

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.

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.

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.

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.

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.

Gas chromatography-high resolution selected-ion mass spectrometric identification of trace 21:0 and 20:2 fatty acids eluting with conjugated linoleic acid isomers.

High-resolution selected-ion recording (SIR) of the exact molecular ion mass was used to confirm unambiguously the presence of conjugated linoleic acid (CLA) derivatives in biological matrices and standard mixtures and to differentiate non-CLA derivatives from CLA derivatives in the CLA region of the gas chromatogram. The success of this method was based on the selectivity of the SIR technique and its sensitivity, which was comparable to that of flame-ionization detection. A minor fatty acid methyl ester (FAME) was identified as methyl heneicosanoate (21∶0), and six isomers of 20∶2 FAME were found to elute in the CLA region. Isomerization of a standard CLA mixture resulted in a non-CLA flame-ionization response eluting in the CLA region of the gas chromatogram. It is therefore recommended that the identification of minor CLA isomers in natural products of biological matrices should include their direct confirmation by mass spectrometry

Impact of novel methodologies on the analysis of conjugated linoleic acid (CLA). Implications of CLA feeding studies

Interest in conjugated linoleic acid (CLA) has increased in the past decade as a result of reports of several health benefits related to its consumption. Naturally occurring CLA isomers are found in milk, dairy, and meat products from ruminants. Detailed isomeric composition of CLA in different chemical and biological matrices had been hindered by the lack of adequate analytical techniques. New methodologies were developed and used to determine the distribution of major and minor geometric and positional CLA isomers in cheese, beef, cow milk, human adipose, and human milk. Base-catalyzed methylation was used. A novel silver ion high-performance liquid chromatographic procedure was developed, which successfully resolved up to 16 isomers. The double bond configuration and position for CLA isomers were confirmed by gas chromatography (GC)-direct deposition-Fourier transform infrared spectroscopy and GC-electron ionization mass spectrometry, respectively: The incorporation of CLA isomers in tissues of animals fed CLA diets was also determined. Currently available analytical data suggest the need to re-evaluate prior CLA studies and their nutritional and biological implications. Einflus neuer Methoden auf die Analyse von konjugierter Linolsaure (CLA). Die Bedeutung von CLA-Futterungsstudien.In den letzten zehn Jahren ist das Interesse an konjugierter Linolsaure aufgrund von Berichten uber verschiedene gesundheitsfordernde Aspekte, die man ihrem Verzehr zuschrieb, gewachsen. Naturlich vorkommende CLA werden in Milch, Milchprodukten und in Fleischprodukten von Wiederkauern gefunden. Eine detaillierte Zusammensetzung der Isomeren in verschiedenen chemischen und biologischen Matrizes war nicht zuganglich aufgrund fehlender adaquater analytischer Techniken. Neue Methoden wurden entwickelt und eingesetzt, um die Verteilung von geometrischen und Positionsisomeren dieser Major- und Minor-fettsauren in Kase, Rindfleisch, Kuhmilch, menschlichem Fettgewebe und Milch zu bestimmen. Eine neue Silberionen-Hochleistungsflussigkeitschromatographie-Methode wurde entwickelt, die bis zu 16 Isomere erfolgreich auftrennte. Die Doppelbindungskonfiguration und -position wurde durch gaschromatographie-Fourier-transformierte Infrarotspektroskopie und GC-Elektronenionisierungs-Massenspektrometrie bestatigt: Der Einbau von CLA-Isomeren in Gewebe von Tieren, die mit einer CLA-Diat gefuttert wurden, wurde ebenso bestimmt. Aktuelle analytische Daten zeigen die Notwendigkeit, bisherige CLA-Studien und ihre ernahrungsspezifischen und biologischen Auswirkungen erneut zu evaluieren.

Effects of conjugated linoleic acid on oxygen diffusion-concentration product and depletion in membranes by using electron spin resonance spin-label oximetry.

The effect of conjugated linoleic acid (CLA) on the relation between structure and function of membranes is described in this paper. Electron spin resonance (ESR) spin-label oximetry was used in the present study to evaluate if oxygen transport and oxygen depletion were affected by incorporation of CLA instead of linoleic acid into membrane phospholipids. Specifically, 1-stearoyl-2(9 cis, 11 trans-octadecadienoyl)-phosphorylcholine (SCLAPC) was incorporated into soy plant phosphatidylcholine (soy PC) or egg yolk PC (EYPC) bilayers. The use of spin labels attached to different carbons along the fatty acid chain makes it possible to carry out structural and oximetric determinations with the same test sample. For example, the incorporation of 5 mol% SCLAPC increased the oxygen diffusion-concentration product in soy PC or EYPC liposomes at 37°C, slightly decreased the ordering of the hydrocarbon chains at the C10 and C12 positions (in the region of the conjugated double bonds), and increased the rate of oxygen depletion from the aqueous medium. Similar results were not obtained by incorporating 5 mol% of 1-stearoyl-2-linoleoyl-PC (SLPC). In our model system, free-radical generation was initiated by extended incubation of the liposomes, by induction by 2,2′-azobis(2-amidinopropane)hydrochloride, or by ultraviolet irradiation of H2O2. The rate of consumption of molecular oxygen was studied by monitoring the oxygen concentration in the aqueous phases of the liposomes. The effect of 5 mol% SCLAPC in soy PC was significantly larger than 5 mol% SLPC in soy PC; the response patterns with soy PC and EYPC were similar. Furthermore, 5 mol% SCLAPC in 1-palmitoyl-2-linoleoyl-PC showed similar oxygen consumption to that observed with 5 mol% SCLAPC in EYPC. On the other hand, 5 mol% SCLAPC in synthetic PC membranes containing saturated or monounsaturated fatty acids showed low oxygen depletion rates. The perturbation of membrane structure and the increase of the relative oxygen diffusion-concentration products provided a potential mechanism by which CLA incorporated into membrane lipids could affect oxidative stress.