Abstract
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, α-keto-β-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 μmol of d-glucarate was quantitatively converted to 1 μmol each of d-glycerate and l-lactate with concomitant utilization of 2 μmol 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.
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