Identification of C18 Intermediates Formed During Stearidonic Acid Biohydrogenation by Rumen Microorganisms In Vitro

Abstract

In vitro batch incubations were used to study the rumen biohydrogenation of unsaturated fatty acids. An earlier study using increasing supplementation levels of stearidonic acid (18:4n-3), revealed that the rumen microbial population extensively biohydrogenates 18:4n-3 after 72 h of in vitro incubation, though several intermediates formed were not completely characterized. Therefore, in the present study, samples were reanalyzed in order to identify the 18:2, 18:3 and 18:4 biohydrogenation intermediates of 18:4n-3. Gas-liquid chromatography coupled to mass spectrometry was used to characterize these intermediates. The acetonitrile chemical ionization mass spectrometry of the fatty acid methyl esters derivatives enabled the discrimination of fatty acids as non-conjugated or conjugated biohydrogenation intermediates. In addition, the acetonitrile covalent adduct chemical ionization tandem mass spectrometry yielded prominent ions indicative of the double bond position of the major 18:3 isomers, i.e. Δ5,11,15 18:3. Furthermore, the 4,4-dimethyloxazoline derivatives prepared from the fatty acid methyl esters enabled the structure of novel 18:2, 18:3 and 18:4 biohydrogenation intermediates to be elucidated. The intermediates accumulated in the fermentation media after 72 h of incubation of 18:4n-3 suggest that similar to the biohydrogenation pathways of linoleic (18:2n-6) and α-linolenic (18:3n-3) acids, the pathway of the 18:4n-3 also proceeds with the formation of conjugated fatty acids followed by hydrogenation, although no conjugated dienes were found. The formation of the novel biohydrogenation intermediates of 18:4n-3 seems to follow an uncommon isomerization pattern with distinct double bond migrations.

Characterization of the rumen lipidome and microbiome of steers fed a diet supplemented with flax and echium oil

“…Irrespective, some 18:4 n -3 remained unbiohydrogenated resulting in its higher concentrations within the rumen of GSE-fed steers compared with GS- and GSF-fed steers. The biohydrogenation of 18:4 n -3 by the rumen microbiota has previously been shown in vitro (Alves et al ., 2012 ; Maia et al ., 2012 ), with similar effects on the lipidome as seen within our animal trial in terms of its rapid biohydrogenation. Alves and colleagues ( 2012 ), nonetheless, showed that 18:4 n -3 biohydrogenation seems to follow an isomerization pattern with the migration of distinct double bonds shown to triene intermediates.…”

“…The biohydrogenation of 18:4 n -3 by the rumen microbiota has previously been shown in vitro (Alves et al ., 2012 ; Maia et al ., 2012 ), with similar effects on the lipidome as seen within our animal trial in terms of its rapid biohydrogenation. Alves and colleagues ( 2012 ), nonetheless, showed that 18:4 n -3 biohydrogenation seems to follow an isomerization pattern with the migration of distinct double bonds shown to triene intermediates. In our study, we did not see these unique 18:3 intermediates likely due to our detection method, as Alves and colleagues ( 2012 ) used gas liquid chromatography-mass spectrophotometry to find these intermediates.…”

“…Alves and colleagues (), nonetheless, showed that 18:4 n ‐3 biohydrogenation seems to follow an isomerization pattern with the migration of distinct double bonds shown to triene intermediates. In our study, we did not see these unique 18:3 intermediates likely due to our detection method, as Alves and colleagues () used gas liquid chromatography‐mass spectrophotometry to find these intermediates. When comparing results for rumen fatty acid concentration from steers‐fed GSF as compared with those fed GSE, a higher accumulation of the biohydrogenation intermediates 18:1 trans ‐11 and 18:2 cis ‐9, trans ‐11, was seen in the rumen of steers fed GSE as compared with GSF diets; nonetheless, no significant differences in resultant 18:0 were seen.…”

“…(plant rich in 18:4 n -3), has been suggested as a potentially beneficial dietary supplement. However, two recent publications suggest that 18:4 n -3 is largely biohydrogenated in the presence of rumen microbes in vitro (Alves et al ., 2012 ; Maia et al ., 2012 ), suggesting that little 18:4 n -3 reaches the muscle or liver for enhancement of chain elongation in vivo .…”

Sources and Bioactive Properties of Conjugated Dietary Fatty Acids

“…Indeed, some strains of Lactobacillus, Bifidobacterium and Propionibacterium have been identified that possess this ability [33,105]. Furthermore, in vitro ruminal studies assessing the biohydrogenation of stearidonic acid have detected a number of CSA isomers including 5, 7, 11, 15-CSA and at least three 5, 8, 10, 15-CSA isomers suggesting substantial potential for CSA production among the ruminal microbiota [49] (Table 1).…”