Dietary supplementation of branched-chain amino acids increases muscle net amino acid fluxes through elevating their substrate availability and intramuscular catabolism in young pigs.

Branched-chain amino acids (BCAA) have been clearly demonstrated to have anabolic effects on muscle protein synthesis. However, little is known about their roles in the regulation of net AA fluxes across skeletal muscle in vivo. This study was aimed to investigate the effect and related mechanisms of dietary supplementation of BCAA on muscle net amino acid (AA) fluxes using the hindlimb flux model. In all fourteen 4-week-old barrows were fed reduced-protein diets with or without supplemental BCAA for 28 d. Pigs were implanted with carotid arterial, femoral arterial and venous catheters, and fed once hourly with intraarterial infusion of p-amino hippurate. Arterial and venous plasma and muscle samples were obtained for the measurement of AA, branched-chain α-keto acids (BCKA) and 3-methylhistidine (3-MH). Metabolomes of venous plasma were determined by HPLC-quadrupole time-of-flight-MS. BCAA-supplemented group showed elevated muscle net fluxes of total essential AA, non-essential AA and AA. As for individual AA, muscle net fluxes of each BCAA and their metabolites (alanine, glutamate and glutamine), along with those of histidine, methionine and several functional non-essential AA (glycine, proline and serine), were increased by BCAA supplementation. The elevated muscle net AA fluxes were associated with the increase in arterial and intramuscular concentrations of BCAA and venous metabolites including BCKA and free fatty acids, and were also related to the decrease in the intramuscular concentration of 3-MH. Correlation analysis indicated that muscle net AA fluxes are highly and positively correlated with arterial BCAA concentrations and muscle net BCKA production. In conclusion, supplementing BCAA to reduced-protein diet increases the arterial concentrations and intramuscular catabolism of BCAA, both of which would contribute to an increase of muscle net AA fluxes in young pigs.

HPLC-MS analysis of iodotyrosines produced by sample hydrolysis: A simple method for monitoring iodinated casein in feed premixes.

A new and simple HPLC-MS method was developed for monitoring iodinated casein in feed premixes. In this method, feed premixes were hydrolyzed, and the iodotyrosines thus released were analyzed. Sample pretreatment included precipitation of transition metals ions with Na(2)S, hydrolysis with sodium hydroxide, and cleaning up with an Oasis SAX cartridge. Gradient elution was carried out on a C(18) column with water (containing 0.1% formic acid) and acetonitrile. Ion detection was performed using ESI positive SIM at m/z 262, 308, 388, and 434. Iodinated casein levels were monitored by qualitative analysis of the iodotyrosines released upon sample hydrolysis and by quantifying the 3,5-diiodotyrosine released. The validation data demonstrated that the method was selective and sensitive (