Enzymatic

Lymphocyte carboxylase enzyme activity was performed by the University of California San Diego Biochemical Genetics Laboratory. Briefly, lymphocytes recovered from blood by density gradient centrifugation (Histopaque-1077, Sigma-Aldrich) were incubated with Na · H¹⁴CO₃ and (a) propionyl-CoA, (b) 3-methylcrotonyl-CoA, and (c) pyruvate plus acetyl-CoA, to produce the nonvolatile products ¹⁴C-methylmalonyl-CoA, ¹⁴C-methylglutaconyl-CoA, and ¹⁴C-citrate through the propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, and pyruvate carboxylase (plus citrate synthase) reactions, respectively. Unreacted Na · H¹⁴CO₃ is removed as ¹⁴CO₂ by acidification with formic acid and drying under heat, and quantification of retained ¹⁴C is used to calculate the activity of each enzyme (Van Hove et al. 2008). As part of the laboratory quality assurance, continuing assay performance validation includes adjustment of normal ranges at semiannual intervals, based on frequency distribution of control results obtained over each interval.