Fluorometry of citrate in serum, with use of citrate (pro-3S)-lyase.
We describe a procedure for enzymatic assay of citrate in human serum. The citrate is degraded to acetate and oxaloacetate with citrate oxaloacetate-lyase (pro-3S-CH2-COO- yields acetate) (EC 4.1.3.6). Some oxaloacetate loses CO2 to form pyruvate. Addition of malate and lactate dehydrogenases (EC 1.1.1.37 and 1.1.1.27) permits determination of the oxaloacetate and pyruvate generated, and thus of the citrate concentration. The decrease in NADH concentration is measured fluorometrically. Results obtained for 30 consecutive human sera by this procedure were compared to the procedure in which the citrate is converted to pentabromoacetone. There was no statistically significant difference in values obtained by the two procedures. The range of values (mean plus or minus 2 SD) found for sera from 25 blood donors by this procedure was 12.8-27.2 mg/liter (mean, 19.0 mg/liter). Serum citrate as measured by both procedures during a glucose tolerance test was decreased from initial values under the influence of administered glucose (and endogenous insulin). Insulin concentrations were also measured during these glucose-tolerance tests. Citrate concentrations remain subnormal after the glucose and insulin concentrations return to their initial values. This accords with published reports.
D000096:Acetone / Q000032:analysis; D001966:Bromine; D002951:Citrates / Q000097:blood*; D005069:Evaluation Studies as Topic; D005951:Glucose Tolerance Test; D006801:Humans; D007328:Insulin / Q000097:blood / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase; D009243:NAD / Q000032:analysis; D010071:Oxaloacetates / Q000032:analysis; D007652:Oxo-Acid-Lyases; D011773:Pyruvates / Q000032:analysis; D013050:Spectrometry, Fluorescence / Q000379:methods
D002951:Citrates; D007328:Insulin; D010071:Oxaloacetates; D011773:Pyruvates; D009243:NAD; D000096:Acetone; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase; D007652:Oxo-Acid-Lyases; D001966:Bromine