SYMPOSIA AND ORAL PRESENTATIONS Graduate Student Paper Competition: ADSA Southern Section

There are no reported pathways of docosahexanoic (DHA) biohydrogenation; however, DHA is metabolized by ruminal microorganisms. In this study a 0.5mg pulse dose of uniformly 13C labeled DHA in ethanol was injected at 0h as a metabolic tracer. This study did not examine every fatty acid metabolite, but rather identified several saturated and trans-18:1 fatty acid metabolites and set a foundation for future work. Ruminal microorganisms collected from a lactating Holstein cow were incubated in 10 mL batch cultures for 0, 6, 24 and 48 h. Treatment groups were 0.5% 13C DHA and 0.5% DHA. Duplicate cultures were methylated and then separated on a 100-m CP-Sil 88 column. Abundances of the quasimolecular (M) and M+n ions were determined by mass spectroscopy in chemical ionization mode. Enrichment was calculated as {M+n/[M+(M+n)]} × 100 in labeled minus unlabeled cultures and tested for their difference from zero by t-test (P < 0.01). DHA decreased from 0.207mg to 0.041mg between 0 and 48h and had an enrichment of 98.1%. Oleic, linoleic, and linolenic acids decreased 0.431, 0.962 and 0.081mg in the same time period. Stearic acid increased 1.37mg and trans-18:1 monenes increased 0.415 mg. Palmitic, eicosanoic, and docosanoic acids increased 0.196, 0.008 and 0.005 mg respectively. Palmitic, stearic, all trans-18:1, eicosanoic, and docosanoic acids were examined for enrichment. Docosanoic acid was highly enriched at 24h and 48h to 20.2% and 16.3% (SE = 0.466; P < 0.01). Neither eicosanoic acid, nor any trans-18:1 monenes were enriched at any time (P < 0.05). Low levels of enrichment (<0.1%; P < 0.01) were found in palmitic and stearic acids. This study clearly identifies docosanoic acid as an intermediate of DHA biohydrogenation. It also identifies that, in this system, eicosanoic acid and trans-18:1 fatty acids were not intermediates. Stearic acid had minor enrichments, but DHA only made up 3.13% of total fat or 10.3% of metabolized unsaturated fat.