Enzymic fluorometric continuous-flow assays for blood glucose, lactate, pyruvate, alanine, glycerol, and 3-hydroxybutyrate.

We describe enzymic fluorometric methods of automated analysis for glucose, lactate, pyruvate, 3-hydroxybutyrate, glycerol, and alanine in perchloric acid extracts of blood. Unmodified Technicon AutoAnalyzer II apparatus is used. The usual concentrations of all these metabolites can be measured in as little as 0.1 ml of blood from a fasting subject. Within-batch and between-batch coefficients of variation ranged from 0.4 to 4.4% for all metabolites except 3-hydroxybutyrate, for which CV's were higher for low concentrations. Analytical recovery of added metabolites ranged from 92 to 98%. Glucose, lactate, alanine, and 3-hydroxybutyrate are stable in perchloric acid extracts for at least 13 days at room temperature, and a year at -20 degrees C; pyruvate shows a 6--8% loss after 3 days and 52% by one year at -20 degrees C; glycerol concentrations were stable at -20 degrees C for at least 13 days. Blank fluorescence is found in perchloric acid extracts of blood, necessitating blank runs for pyruvate, 3-hydroxybutyrate, glycerol, and alanine. The systems are simple to use, relatively inexpensive to operate, and are recommended for any laboratory with high throughput of samples.

Identification of Lithocholic Acid and Measurement of Other Bile Acids in Serum of Healthy Humans

Combined gas—liquid chromatography mass-spectroscopy was used to identify lithocholic acid and confirm the presence of other bile acids in serum of a healthy fasting subject. GLC was used to measure deoxycholic (DCA), chenodeoxycholic (CDCA), and cholic (CA) acids in sera. Before analysis, serum bile acids were purified by (a) enzymatic hydrolysis of conjugates, (b) anion-exchange chromatography, (c) alumina adsorption chromatography, and (d) GLC of methyl trifluoroacetate derivatives on QF-1. Recovery of bile acids (determined by adding [14C]cholic acid to each sample), after correction for loss during purification, was 63-83%. Fasting values for 28 healthy subjects were: 1.4-46.5 (av 7.1), 1.4-49.6 (12.8), and 1.4-46.0 (16.0) µg/100 ml for DCA, CDCA, and CA, respectively. Traces of lithocholic acid were found in 20% of the cases studied. The smaller ranges we found for serum bile acid concentrations in a healthy fasting population are attributed to the careful health-screening of subjects and improved techniques.

Detection of Phenylketonuric Heterozygotes

Estimation of plasma phenylalanine and tyrosine by an abridged (30 min) column chromatography procedure was assessed. Plasma phenylalanine and tyrosine concentrations, determined in 112 phenylketonuric obligate heterozygotes and in 88 normal controls, did not differ significantly from other reported series in which the data were obtained by column chromatography. All these series were combined and analyzed statistically. Phenylketonuric heterozygotes can be detected with a high degree of discrimination by the ratio of phenylalanine to tyrosine in the plasma of the fasting subject, in combination with the plasma phenylalanine concentration.