Endogenous Synthesis of CLA

As is outlined in the following section, it is now apparent that endogenous synthesis of RA occurs in most, if not all, animal species. The classic studies of Mahfouz et al. (1980) and Pollard et al. (1980) showed independently that positional isomers of trans-octadecenoic acids are desaturated by the enzyme A-9 desaturase in rat liver microsomal systems. All tram monoenes, A-4 to A-13, except A-8, 9, and 10, were substrates, with products being trans-A-x, cis-9 dienes. The rate of A-9 desaturation increased as the trans bond was removed further from the A-9 position, so that trans-A-4 and A-13 monoenes were most rapidly desaturated (Mahfouz et al., 1980). The trans-5, cis-9 18 2 was isomerized rapidly to the cis, cis diene without changing bond positions tram-4, cis-9 18 2 was isomerized similarly, but at a slower rate (Mahfouz et al., 1980). Significant amounts of some of the cis/trans dienes were desaturated further at A-6, to yield cis, tram, cis trienes (Pollard et al., 1980). 

The end-products of rumen microbial metabolism of greatest interest with regard to CLA metabolism in the body are the various CLA isomers and the tram-11 monoenoic acid VA. The latter, being a substrate for A-9-de-saturase, is converted to RA in animal tissues. Hay and Morrison (1970) reported the distribution of monoenoic isomers of milk fat, and the content of various CLA and other 18 2 isomers in the fat of ruminant products has been summarized by Parodi (2003). 

Researchers have used one of two approaches to quantify endogenous synthesis. The first involves inhibiting the A-9-desaturase, thereby inhibiting conversion of VA to RA. Using abomasal infusion of sterculic oil to inhibit A-9-desaturase activity, combined with monoene/saturate pair ratios to correct for the extent of inhibition, Griinari et al. (2000) estimated that a minimum of 64% of RA in their milk fat samples was derived from desaturation of VA. Further experiments using sterculic oil also indicated that the 

The relationship between substrate and product for A-9-desaturase is reflected by the desaturase index, defined as [RA a- (RA + VA)]. Various approaches to calculating desaturase index in milk fat are discussed by Kelsey et al. (2003). In the study by Corl et al. (2001), the desaturase index was 0.23 for the hay and concentrate diet and 0.20 when the diet was supplemented with PH VO. Kay et al. (2004) reported a desaturase index of 0.25 for the pasture diet and 0.22 when the diet was supplemented with sunflower oil. Piperova et al. (2002) observed desaturase indices for highland low-fiber diets of 0.40 and 0.35, respectively. Shingfield et al. (2003) reported desaturase indices of 0.18 and 0.15 with a grass silage diet without or supplemented with fish oil these values are probably lower than others because the analytical methods accounted for minor CLA isomers that typically co-elute with RA or because of inhibition of A-9-desaturase by the long-chain PUFA from the fish oil supplement. The desaturase index, as defined earlier, should approximate the proportion of VA desaturated in the tissues. A summary of endogenous RA synthesis estimates and the proportion of VA desaturated in the tissues is in Table III. 

QUANTITATIVE STUDIES OF ENDOGENOUS CLA SYNTHESIS IN LACTATING COWS 

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Because the presence of CLA in the human diet is reliant on ruminant products, this chapter first addresses the synthesis of CLA in ruminants. The presence of CLA in ruminant milk and meat is related to rumen fermentation and its synthesis by microorganisms through the process of biohydrogenation (BH) of dietary unsaturated fatty acids. Thus, the effect of diet and processes within the rumen is reviewed. The role of endogenous synthesis of CLA in mammalian tissues has been discovered, and this will be discussed also first as it contributes to the occurrence of CLA in ruminant products and second the significance of endogenous synthesis as a source of CLA in humans and other species. 

Figure 3.2. Pathways for ruminal and endogenous synthesis of rumenic acid (cis-9, trans-11 CLA) in the lactating dairy cow. Pathways for biohydrogenation of linoleic and linolenic acids yielding vaccenic acid trans-11 18 1) are shown in the rumen box and endogenous synthesis by A9-desaturase is shown in the mammary gland box. Adapted from Bauman et at. (2003).

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. 

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. 

The second most prevalent CLA isomer in ruminant fat is trans-1, cis-9 CLA, representing 3 16% of total CLA in ruminant fat (Corl et al., 2002 Parodi, 2003 Piperova et al., 2000, 2002 Shingfield et al., 2003 Yurawecz et al., 1998). A number of the studies previously described have determined the source of trans-1, cis-9 CLA in ruminant fat. Corl et al. (2002) showed that the trans-1, cis-9 CLA in milk fat was derived almost exclusively from endogenous synthesis by using both sterculic acid and trans-10, cis-12 CLA to inhibit A-9-desaturase they also found that trans-1, cis-9 CLA concentration in rumen fluid was very low and at the limit of detection. Similarly, Piperova et al. (2002) found that virtually all of the trans-1, cis-9 CLA in milk fat was produced post-ruminally. As mentioned previously, trans-1 18 1, a minor BH intermediate in ruminal contents, also is a substrate for A-9-desaturase (Mahfouz et al., 1980 Pollard et al., 1980). 

The metabolic pathways involved in milk fat synthesis were reviewed recently (6) and are referred to briefly here in the context of CLA synthesis. Fatty acids in milk are derived from two sources, i.e., (i) de novo synthesis from acetate and P-hydrox-ybutyrate in the mammary gland and (ii) dietary fatty acids modified by microbial activity in the rumen followed by uptake from circulating lymph and blood. The composition of the fatty acids from these two sources is very different, e.g., endogenously produced fatty acids are of carbon length C -Cjg, whereas a proportion of Cjg, and all of the Cjg fatty acids arise from blood (7). 

Contribution of Endogenous Synthesis to the c/s-9, trans- 1 CLA Content of Milk Fat in Lactating Dairy Cows... 

Overall, investigations using different diets and experimental approaches have resulted in similar conclusions (Table 10.1). The major pathway for the origin of cis-9, trans-ll CLA in milk fat is endogenous synthesis as shown in Fig. 10.1. This CLA isomer accounts for 75-90% of the total CLA in milk fat, and trans-ll 18 1 derived from rumen output is the precursor for endogenous synthesis. Thus, the relatively constant milk fat ratio observed between trans-ll 18 1 and cis-9, trans-ll CLA reflects the substrate product relationship for A -desatnrase, and the keys to increasing the milk fat content of cis-9, trans-ll CLA are to enhance rumen ontput of trans-l 1 18 1 and increase tissue activity of A -desaturase (Rg. 10.1). 

Yurawecz and co-workers (13) discovered that ruminant fat also contained trans-7, cis-9 CLA it was the second most prevalent CLA isomer, representing 3-16% of total CLA in milk fat. Trans-7, cis-9 CLA had not been detected previously because it coeluted with cis-9, trans- CLA in gas chromatography methods that were in routine use. Thus, concentrations of cis-9, trans-ll CLA reported in the scientific literature typically include trans-7, cis-9 CLA as a component. Milk fat content of trans-7, cis-9 CLA was generally on the order of 10% of cis-9, trans-ll CLA and several-fold greater than any of the remaining CLA isomers (12,13,39,60,61). With their initial discovery, Yurawecz et al. (13) speculated that trans-7, cis-9 CLA might be derived by endogenous synthesis via A -desatnrase. Earlier, Katz and Keeney (62) had shown that trans-7 18 1 is produced in the mmen and Mahfonz et al (30) and Pollard et al. (31) demonstrated that A -desaturase isolated from a rat hver microsomal preparation could convert trans-7 18 1 to trans-7, cis-9 CLA. 

Trans-7, cis-9 CLA is also present in the body fat of ruminants, and, as with milk fat, it is the second most prevalent CLA isomer (13,14). Endogenous synthesis is the likely source, although this has not been investigated directly. Concentrations of trans-7, cis-9 CLA are neghgible in rumen fluid (61), omasal digesta (68), and duodenal samples (39) from cows. In contrast, concentrations of trans-7, cis-9 CLA in beef fat represent 10-20% of values for cis-9, trans-ll CLA and are several-fold... 

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