Methodology for the In Vivo Measurement of the Δ9‐Desaturation of Myristic, Palmitic, and Stearic Acids in Lactating Dairy Cattle

Abstract

There is limited methodology available to quantitatively assess the activity of the Delta9-desaturase enzyme in vivo without chemically inhibiting the enzyme or using radioactively labeled substrates. The objective of these experiments was to develop methodology to determine the incorporation and desaturation of 13C-labeled fatty acids into milk lipids. In a preliminary experiment, 3.7 g [1-13C]myristic acid ([1-13C]14:0), 19.5 g [1-13C]palmitic acid ([1-13C]16:0), 20.0 g [1-13C]stearic acid ([1-13C]18:0) were combined and infused into the duodenum of a cow over 24 h. In a following experiment, 5.0 g [1-13C]14:0, 40.0 g [1-13C]16:0, and 50.0 g [1-13C]18:0 were infused into the abomasums of separate cows as a bolus over 20 min or continuously over 24 h. Milk fat was extracted using chloroform:methanol. Fatty acids were methylated, and fatty acid methyl esters (FAME) were converted to dimethyl disulfide derivatives (DMDS). The FAME and DMDS were analyzed by gas chromatography mass spectrometry. In the preliminary experiment, 13C enrichment in 14:0 but not 16:0 or 18:0 was observed. When dosage amounts were increased in the following experiment, peak enrichments from the bolus infusion were observed at 8 h. Enrichments for continuous infusion peaked at 16 h for 14:0 and 18:0, and at 24 h for 16:0. The Delta9-desaturase products of these fatty acids were estimated to be 90% of cis-9 14:1, 50% of cis-9 16:1, and 59% of cis-9 18:1. This study demonstrates that 13C-labeled fatty acids may be utilized in vivo to measure the activity of the Delta9-desaturase enzyme.