Amino Acid Analysis in Dairy Cow Plasma by Chloroformate Derivatization and Gas Chromatography
The objective of the experiment was to evaluate gas chromatography (GC) after chloroformate derivatization of AA to quantify dietary effects on plasma AA concentrations in dairy cows. Plasma was obtained from 72 cows participating in a previous trial (Souza et al., J. Dairy Sci. 95(Suppl. 2):353, 2012, abstract) where positive performance effects to Met supplementation were reported. Starting at calving, cows were fed isoenergetic diets formulated to deliver equal metabolizable protein (2875 g MP/d). The control diet (CTR) provided 1.89% Met (% of MP), while the treatment diet (MET) was supplemented with sufficient Smartamine M® to increase Met to 2.43% of MP. Results indicated increased milk concentrations of protein (2.92 vs. 2.75%, P<0.01) and SNF (8.73 vs. 8.54%, P<0.01) in cows fed MET. Plasma was harvested from blood samples collected at 50 DIM, and kept frozen at -18 °C until analysis. Samples were separated by treatment group (supplemented = 37, control = 35) and combined randomly within parity, into 7 plasma composites of 4 to 6 cows on each diet. Composites were prepared for GC analysis by using a commercial kit (EZ:faast™ GC-FID Physiological, Phenomenex®). This method involves amino acid collection using solid phase extraction (SPE), derivatization with chloroformate, GC separation in a mid-polar capillary column and flame ionization detection. Quantification was based on area under the curve, using the internal standard ratio method (norvaline). Consistent with performance data, analysis of variance indicated higher plasma free Met levels in MET cows vs. CTR cows (22.9 vs. 16.8 μM, P<0.001). Although the kit manual indicated deproteinization was not required, untreated plasma was difficult to work with because variable plasma concentrations of proteins and phospholipids clogged the SPE resin. Mixing plasma at a 1:1 volume ratio with 5% trichloroacetic acid, followed by centrifugation (12,000 x g), allowed satisfactory SPE of all composites. Ion exchange chromatography coupled with ninhydrin quantification is currently the “gold standard” for AA analysis. The drawbacks of that methodology are high cost and long runtimes. The modifications to the GC-chloroformate method here presented allow rapid (15 minute) determination of differences in free amino acids in cow plasma at a reasonable cost.
rumen-protected, methionine, chromatography