Branched-chain Amino Acid Nitrogen Transfer to Alanine In Vivo in Dogs

Abstract

To investigate the contribution of branched-chain amino acids as a nitrogen source for alanine in vivo, dogs were infused with l-[¹⁵N]leucine, l-[U-¹⁴C]leucine, l-[2,3,3,3-²H₄]alanine, and d-[6,6-²H₂]-glucose. ¹⁴C and ¹⁵N isotopic equilibrium in plasma leucine, and deuterium enrichment in arterial and femoral plasma glucose and alanine were achieved within 3 h of initiation of the respective isotope infusion in all animals. The average flux of leucine determined by [¹⁵N]leucine was 5.4 μmol·kg⁻¹·min⁻¹, whereas using [¹⁴C]leucine it was 3.7 μmol·kg⁻¹·min⁻¹. Turnover rates for alanine and glucose were 11.0 and 17.2 μmol·kg⁻¹·min⁻¹, respectively. [¹⁵N]alanine was detected as early as 30 min, but nitrogen isotopic equilibrium in alanine was not achieved until 6 h. The absolute rate of leucine nitrogen transfer to alanine was 1.92 μmol·kg⁻¹·min⁻¹, which represented 41-73% (mean 53%) of leucine's nitrogen and 15-20% (mean 18%) of alanine's nitrogen. Fractional extraction of alanine and leucine by the dog hindlimb was 35 and 24%, respectively. Average net alanine balance was −6.7 μmol·leg⁻¹·min⁻¹, reflecting a release rate (17.4 μmol·kg⁻¹·min⁻¹) that exceeded the rate of uptake (10.8 μmol·leg⁻¹·min⁻¹). Of the leucine taken up by the hindlimb, 34% transferred its nitrogen to alanine and 8% was oxidized to CO₂. Since the latter value reflects transamination as well as irreversible catabolism, the nitrogen derived from the oxidation of leucine by the hindlimb could account for only 25% of the observed ¹⁵N incorporation into alanine. The significantly faster flux of leucine nitrogen when compared with leucine carbon suggests significant recycling of the leucine α-ketoacid. These studies demonstrate that leucine is a major donor of nitrogen to circulating alanine in vivo.