Plasma alloisoleucine: analytical method and clearance in ketoacid-supplemented normals

Chromatographic separation of alloisoleucine (ALE) was achieved using a Beckman 6300 Amino Acid Analyzer equipped with a 20 cm lithium High Performance column and Li-A buffer. Twenty microliter samples were injected and the initial column temperature was 57 degrees C. After 1.0 minute, the temperature was increased at a rate of 0.75 degrees C/min to 70 degrees C. Flow rate was maintained at 22 ml/hr. ALE eluted at 46 minutes. Accuracy of the method was 97.2% with spike recovery studies. Repeated analyses (N = 9) of a plasma sample containing ALE gave a SD of 0.04 and 1.37% RSD. Linearity using standard solutions of 1.25 to 100 mumols/100 ml was confirmed by a correlation coefficient of r = 0.999. Limit of quantitation was estimated at 0.2 mumols/100 ml. This method was used to determine the half-life of ALE in plasma of healthy humans fed ketoanalogue-supplemented diets on the first day and after 28 days of consumption. Plasma samples were taken at 0, 0.5, 1.0, 1.5, 2, 3, 4, 6, 8, 10, 12 and 24 hours post-dose of either 6.2 +/- 0.25 g or 9.3 +/- 0.55 g (meal dose) of a ketoacid mixture (KAM) containing 10.9% (R,S) alpha-keto-beta-methylvalerate.(ABSTRACT TRUNCATED AT 250 WORDS)