Specific enzymatic assay for D-glucarate in human serum.
A sensitive and specific spectrophotometric assay was developed to determine levels of D-glucarate in human serum. This assay makes use of the Escherichia coli glucarate catabolic enzymes D-glucarate dehydrase, alpha-keto-beta-deoxy-D-glucarate aldolase, and tartronate semialdehyde (TSA) reductase, to convert D-glucarate to equimolar quantities of pyruvate and TSA. In a one-tube reaction that included NADH, lactate dehydrogenase, and the three E. coli enzymes, 1 mumol of D-glucarate was quantitatively converted to 1 mumol each of D-glycerate and L-lactate with concomitant utilization of 2 mumol of NADH. Using this method, D-glucarate in serum was measured, along with quantitative recovery of authentic D-glucarate from duplicate serum samples to which it had been added. Glucarate is a major serum organic acid, approximating blood pyruvate levels previously determined by others.
Department of Microbiology, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois 60153.
H J
HJ
Blumenthal
V L
VL
Lucuta
D C
DC
Blumenthal
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005937:Glucaric Acid / Q000097:blood*; D006801:Humans; D006836:Hydro-Lyases / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D013400:Sugar Acids / Q000097:blood*
D013400:Sugar Acids; D009243:NAD; D000429:Alcohol Oxidoreductases; C064167:2-hydroxy-3-oxopropionate reductase; D000446:Aldehyde-Lyases; C021468:2-keto-3-deoxy-D-glucarate aldolase; D006836:Hydro-Lyases; C020572:glucarate dehydratase; D005937:Glucaric Acid