Related Experiment Videos
Specific enzymatic assay for D-glucarate in human serum
H J Blumenthal1, V L Lucuta, D C Blumenthal
1Department of Microbiology, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois 60153.
Analytical Biochemistry
|March 1, 1990
Summary
A new spectrophotometric assay accurately measures D-glucarate in human serum. This method uses specific enzymes to quantify D-glucarate, a significant serum organic acid.
Area of Science:
- Biochemistry
- Analytical Chemistry
Background:
- D-glucarate is a major organic acid found in human serum.
- Accurate quantification of serum D-glucarate is essential for metabolic studies.
Purpose of the Study:
- To develop a sensitive and specific spectrophotometric assay for determining D-glucarate levels in human serum.
Main Methods:
- Utilized Escherichia coli glucarate catabolic enzymes: D-glucarate dehydrase, alpha-keto-beta-deoxy-D-glucarate aldolase, and tartronate semialdehyde (TSA) reductase.
- Developed a one-tube reaction converting D-glucarate to pyruvate and TSA, followed by NADH utilization measurement via lactate dehydrogenase.
Main Results:
- The assay quantitatively converts D-glucarate to equimolar pyruvate and TSA.
- Demonstrated quantitative conversion of 1 µmol D-glucarate to 1 µmol D-glycerate and L-lactate with 2 µmol NADH consumption.
- Achieved accurate measurement of D-glucarate in human serum samples with validated recovery.
Conclusions:
- Established a reliable spectrophotometric method for serum D-glucarate determination.
- The assay's sensitivity and specificity enable accurate quantification of this major serum organic acid.