Characterization of the disappearance and formation of biohydrogenation intermediates during incubations of linoleic acid with rumen fluid in vitro

Dietary unsaturated fatty acids are extensively hydrogenated in the rumen, resulting in the formation of numerous intermediates that may exert physiological effects and alter the fat composition of ruminant-derived foods. A batch culture method was used to characterize the hydrogenation of linoleic acid (LeA) by strained rumen fluid in vitro. Incubations (n = 5) were performed in 100-mL flasks maintained at 39 °C containing 400mg of grass hay, 50 mL of buffered rumen fluid, and incremental amounts of LeA (0, 1.0, 2.5, 5.0, or 10.0mg) for 0, 1.5, 3.0, 4.5, 6.0, and 9.0 h. The fatty acid composition of flask contents was determined using complimentary silver-ion thin-layer chromatography, gas chromatography mass-spectrometry, and silver-ion high-performance liquid chromatography. Linoleic acid was extensively (98.1, 97.6, 98.0, and 89.8% for additions of 1.0, 2.5, 5.0, and 10.0mg of LeA, respectively) hydrogenated over time. Complete reduction of LeA to 18:0 was inhibited in direct relation to the amount of added substrate, the extent of which was greatest for the highest amount of LeA addition. Recoveries of 1.0, 2.5, 5.0, and 10.0mg of added LeA as 18:0 averaged 73.6, 65.0, 57.3, and 10.7%, respectively. Incubation of incremental amounts of LeA resulted in a time-dependent accumulation of geometric isomers of 9,11 and 10,12 conjugated linoleic acid, several nonconjugated 18:2 isomers, and a wide range of cis 18:1 and trans 18:1 intermediates. Several unusual intermediates including cis-6,cis-12 18:2; cis-7,cis-12 18:2; and cis-8,cis-12 18:2, were found to accumulate in direct relation to the amount of added LeA, providing the first indications that hydrogenation of LeA by ruminal bacteria may also involve mechanisms other than hydrogen abstraction or isomerization of the cis-12 double bond. Fitting of single-pool, first-order kinetic models to experimental data indicated that the rate of LeA disappearance decreased with increases in substrate availability. Reduction of 18:1 and 18:2 intermediates occurred at much lower rates compared with conjugated linoleic acid and nonconjugated 18:2 isomer formation. In conclusion, the extent of LeA biohydrogenation in vitro was shown to be time- and dose-dependent with evidence that LeA is hydrogenated by ruminal bacteria via several distinct metabolic pathways. The accumulation of several unusual 18:2 isomers indicates that biohydrogenation of LeA also proceeds via mechanisms other than isomerization of the cis-12 double bond.