Products of docosahexaenoic acid (DHA) metabolism by mixed ruminal microorganisms from sheep

Abstract

The health benefits of long-chain n-3 polyunsaturated fatty acids (PUFA), specifically DHA, have been well recognized. Attempts to enrich ruminant products with DHA have not been successful due to the low transfer efficiency (<4%), the result of rumen biohydrogenation processes that are not well understood. Three sheep served as donors of rumen content that was incubated with 200 ¿g of DHA for up to 6h in anaerobic conditions (3 tubes per incubation time). Total fatty acid methyl esters (FAME) were prepared and analyzed by GC using both a 100 m SP-2560 and a 100 m SLB-IL111 column. DHA was shown to deplete to 18% of starting content, and 60% of the added DHA could be accounted for as FAME peaks eluting after 20:4n-6 compared to non-incubated tubes (0h). To further investigate DHA metabolites, the total FAME mixture was subjected to a GC-on line reduction-GC analysis. The results showed a complex mixture of C22 containing FAMEs from 22:0 to beyond 22:6. To determine the number of double bonds within the different C22 FAMEs, the mixture was analyzed by GC-time of flight-MS. Clusters of FAMEs with increasing number of double bonds from 1 to 6 were identified including FAME peaks eluting after 22:6, 22:5 and 22:4 clusters, which were tentatively identified as their conjugated fatty acids (CFA). No attempt was made to identify all members within each unsaturated C22 cluster. Based on these findings we propose that DHA is metabolized by processes of isomerization leading to CFA followed by reduction, much the same as linoleic and linolenic acids. This process appears to continue systematically until 22:0, and there is no evidence of chain shortening or elongation. The many positional/geometric isomers within each unsaturated C22 FA group suggest that the enzymes are able to attack DHA and subsequent metabolites at multiple sites. This is the first demonstrated evidence of all the DHA metabolites formed by rumen bacteria.