CLA isomers

In order to assign the chemical shifts of the carbon atoms of the conjugated diene system of each CLA isomer, it was necessary to conduct INADEQUATE, HMBC (heteronuclear multiple bond correlation) and two-dimensional 1H-13C correlation spectroscopy (COSY) techniques on the carbon signals of the diene system of the ,Z-isomers. The results of these experiments for the CLA isomers are summarized in Table 13. 

The first step in the biohydrogenation of linoleic acid to stearic acid by the mmen microorganism, Butyrivibrio fibrisolvens, is the formation of the cis-9, trans-11 CLA isomer (Figure 1). This reaction is catalyzed by a membrane-bound enzyme, linoleate isomerase, which acts only on fatty acids possessing cis double bonds in positions and and a free carboxyl group (72). 

Significant amounts of CLA are found in muscle tissue from ruminant animals (Table 1). The CLA content for beef ranged from 2.9 to 4.3 mg CLA/g fat. Among ruminants lamb was the highest (5.6 mg CLA/g fat) and veal was the lowest (2.7 mg CLA/g fat). The cis-9, trans-11 isomer accounted for more than 79% of the total CLA isomers in meats. 

Figure 1. The chemical structures of linoleic acid (cis-9, cis-12-octadecadienoic acid), and the cis-9, trans-11 CLA isomer (cis-9,trans-l 1-octadecadienoic acid). (Reproduced with permission from the Food Research Institute Annual Report 1990. Copyright Food Research Institute 1991.)...

Values are means for the number of samples indicated. All standard error values are less than 3%. Data were expressed as % of total CLA isomers. 

CLA Formation in Nonruminant Animals. CLA has been detected in the serum, bile and duodenal juice of humans (7 ). It has been confirmed that both the cis-9, trans-11 and trans-9, trans-11 CLA isomers are present in human depot fats (79). 

CLA has also been identified in the tissue lipids of other nonruminant animals. The CLA concentration in nonruminant animals (chicken and pig) is considerably lower than ruminant animals (Table 1). The exception among nonruminants is turkey (2.51 mg CLA/g fat), which is about five fold higher than chicken or pork. More than 76% of the CLA isomer found in nonruminant tissues is cis-9, trans-11 isomer. 

The accumulation of different CLA isomers in various tissues was also reported. When diets containing 1% of either cis, trans or trans, trans CLA isomers were fed to rats, conjugated dienes did not accumulate in testis or brain lipid. By contrast, CLA was incorporated into adipose tissue (26). Substantial deposition of conjugated dienes occurred in heart lipid when animals were fed cis, trans isomers, but no increase was observed when animals were fed trans, trans CLA isomers. 

Why cis-9, trans-11 alone is found in phospholipids is unclear. However, the cis-9, trans-11 isomer exhibits a configuration that is most similar to linoleic acid (Figure 1). We have proposed that the cis-9, trans-11 isomer may be the biologically active anticarcinogenic CLA isomer. 

Table 9.2 Concentration of conjugated linoleic acid (CLA) isomers in selected foods (modified from Ha, Grimm and Pariza, 1989)...

BASIC PROTOCOL I PREPARATION OF FATTY ACID METHYL ESTERS FROM LIPID SAMPLES CATALYZED WITH BORON TRIFLUORIDE IN METHANOL In this method, lipid samples are first saponified with an excess of NaOH in methanol. Liberated fatty acids are then methylated in the presence of BF3 in methanol. The resulting fatty acid methyl esters (FAMEs) are extracted with an organic solvent (isooctane or hexane), and then sealed in GC sample vials for analysis. Because of the acidic condition and high temperature (100°C) used in the process, isomerization will occur to those fatty acids containing conjugated dienes, such as in dairy and ruminant meat products, that contain conjugated linoleic acids (CLA). If CLA isomers are of interest in the analysis, Basic Protocol 2 or the Alternate Protocol should be used instead. Based on experience, this method underestimates the amount of the naturally occurring cis-9, trans-11 CLA isomer by -10%. The formulas for the chemical reactions involved in this protocol are outlined in Equation D1.2.1 Saponification RCOO-R + NaOH, RCOO-Na + R -OH v 100°C DC Esterification RCOO-Na + CH,OH r 3 v RCOO-CH, + NaOH ioo°c ... 

Another important dietary source of trans fat is conjugated linoleic acid, a class of compounds collectively known as CLA. Many CLA isomers contain conjugated cis/trans and trans/trans double bonds. Interest in CLA research has increased significantly in the past few years because several cis/trans CLA isomers have been reported to exhibit different beneficial physiological effects in animal studies (Yurawecz et al., 1999). The reader is referred to a collection of analytical papers published in a dossier (Mossoba, 2001, and references therein) that details several chromatographic and spectroscopic techniques and procedures that have been successfully applied to CLA analysis. 

The term conjugated linoleic add (CLA) refers to a mixture of positional and geometric isomers of linoleic add with a conjugated double bond system milk fat can contain over 20 different isomers of CLA. CLA isomers are produced as transient intermediates in the rumen biohydrogenation of unsaturated fatty acids consumed in the diet. However, cis-9, trans-11 CLA, known as rumenic acid (RA), is the predominant isomer (up to 90% of total) because it is produced mainly by endogenous synthesis from vaccenic acid (VA). VA is typically the major biohydrogenation intermediate produced in the rumen and it is converted to RA by A9-desaturase in the mammary gland and other tissues. 

Trans-10, cis-12 CLA is another CLA isomer in milk fat which can affect lipid metabolism. It is generally present at low concentrations in milk fat (typically <0.2% of CLA) under some dietary conditions, a portion of the rumen biohydrogenation shifts to produce more of this isomer, although it is still only a minor portion of total CLA. These dietary conditions are associated with milk fat depression and as little as 2 g/d of tram-10, cis-12 leaving the rumen will reduce milk fat synthesis by 20%. Because of the potency and specificity of this CLA isomer, it is being developed as a dairy management tool to allow for a controlled reduction in milk fat output. 

CLA isomers in milk fat and how they relate to both animal agriculture and human health are rapidly expanding fields. Milk and dairy products offer exciting opportunities in the area of functional foods, and the functional properties of YA and RA in milk further serve to illustrate the value of dairy products in the human diet. 

CLA refers to a mixture of positional and geometric isomers of linoleic acid (cis-9, cis-12 octadecadienoic acid) with a conjugated double bond system. The structure of two CLA isomers is contrasted with linoleic and vaccenic acids in Figure 3.1. The presence of CLA isomers in ruminant fat is related to the biohydrogenation of polyunsaturated fatty acids (PUFAs) in the rumen. Ruminant fats are relatively more saturated than most plant oils and this is also a consequence of biohydrogenation of dietary PUFAs by rumen bacteria. Increases in saturated fatty acids are considered undesirable, but consumption of CLA has been shown to be associated with many health benefits, and food products derived from ruminants are the major dietary source of CLA for humans. The interest in health benefits of CLA has its genesis in the research by Pariza and associates who first demonstrated that... 

CLA isomers are functional food components when their search for mutagens in cooked meat instead identified CLA as an antimutagen (see Pariza, 1999). As a result of this discovery, research on CLA has increased exponentially over the last decade and a number of potential health benefits of CLA have been reported. The anticarcinogenic activity of CLA has been established clearly, but biomedical studies with animal models have identified an impressive range of additional positive health effects for CLA as summarized in Chapter 17. Particularly noteworthy is the fact that CLA is a potent anticarcinogen when supplied as a natural food component in the form of CLA-enriched butter as discussed later in this review. 

Analysis of the CLA content and profile of animal tissues or biological fluids containing a mixture of lipid classes is more difficult. In order for all of the fatty acids to be methylated, a two-stage methylation procedure is recommended. Kramer et al. (1997) evaluated many different combinations of acid/base catalysts and concluded that the best compromise was the use of sodium methoxide followed by a mild acidic methylation, which resulted in the methylation of the majority of the fatty acids with minimal isomerization of the CLA isomers. However, mild boron triflouride or 1% methanolic sulphuric acid with a minimal temperature and reaction time are often used with good success. 

Additional analytical methods are appropriate when a more complete characterization of the CLA isomers in biological samples is required. Most often, a combination of GC and silver ion-HPLC is used and permits excellent separation and identification of positional and geometrical isomers of CLA (see Adlof, 2003, and Kramer et al., 2004, for detailed reviews of this approach). In addition, the use of gas chromatography-mass spectrometry (GC-MS) has become increasingly popular and represents a very powerful technique for identification of the position of double bonds in fatty acids (see Dobson, 2003), and the orientation of those bonds in CLA isomers (Michaud et al., 2003). 

In summary, the analysis of CLA can be simple or extensive. The particular objectives and the anticipated use of the analytical data will determine the extent to which individual CLA isomers need to be separated, identified and quantified (Christie, 2003). Methodology for the analysis of CLA and related fatty acids continues to evolve and it is recommended that the reader consult recent reviews and publications in this area before undertaking such analysis for the first time. We recommend Christie (2003) and Kramer et al. (2004) as excellent practical guides on the analysis of CLA. 

Overall, investigators using different diets and experimental approaches have found similar results the major source of RA in milk fat is endogenous synthesis (Figure 3.2). Thus, endogenous synthesis is the basis for cis-9, trans-11 being the predominant CLA isomer in milk fat and the relatively constant ratio between VA and RA observed in milk fat reflects the substrate product relationship for A9-desaturase. 

A broad overview of the biological effects of CLA is presented elsewhere in this volume (Chapter 17), so the emphasis in the following section will be two-fold. Firstly, the biology of trans-10, cis-12 CLA in the dairy cow will be summarized because under certain dietary conditions, production of this isomer in the rumen can profoundly affect milk fat synthesis. Secondly, the biological effects of RA when supplied as a natural component of the diet will be reviewed because this CLA isomer represents a functional component of milk fat that has potential health benefits. Although other CLA isomers are present in milk fat, they are present at concentrations much too low to have a significant effect. 

---