A rapid, automated enzymatic fluorometric assay for determination of D-arabinitol in serum.

Abstract

A rapid enzymatic fluorometric assay for measuring D-arabinitol in serum was developed using recombinant D-arabinitol dehydrogenase from Candida albicans (rArDH). rArDH was produced in Escherichia coli and purified by dye-ligand affinity chromatography. rArDH was highly specific for D-arabinitol, cross-reacting only with xylitol (4.9%) among all polyols tested. A Cobas Fara II centrifugal autoanalyzer (Roche) was used to measure NADH fluorometrically when rArDH and NAD were added to serum extracts, and D-arabinitol concentrations were calculated from standard curves derived from pooled human serum containing known amounts of D-arabinitol. The method was precise (mean intra-assay coefficients of variation [CVs], 0.8%, and mean interassay CVs, 1.6%) and rapid (3.5 min per assay) and showed excellent recovery of added D-arabinitol in serum (mean recovery rate, 101%). The mean and median D-arabinitol/creatinine ratios were 2.74 and 2.23 microM/mg/dl, respectively, for the 11 patients with candidemia compared to 1.14 and 1.23 microM/mg/dl, respectively, for 10 healthy controls (P < 0.01). These results confirm earlier studies showing that serum D-arabinitol measurement may help to promptly diagnose invasive candidiasis. The technique shows a significant improvement in terms of accuracy, cost, simplicity, specificity, and speed compared with gas chromatography, mass spectrometry, and earlier enzymatic assays.

• Authors: S F Yeo, Y Zhang, D Schafer, S Campbell, B Wong
• Journal: Journal of clinical microbiology (J Clin Microbiol) Vol. 38 Issue 4 Pg. 1439-43 (Apr 2000) ISSN: 0095-1137 [Print] United States
• PMID: 10747122 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Recombinant Proteins
• Sugar Alcohols
• Sugar Alcohol Dehydrogenases
• D-arabinitol dehydrogenase
• arabitol

Topics

• Candida albicans (enzymology, genetics)
• Candidiasis (diagnosis, microbiology)
• Escherichia coli (enzymology, genetics)
• Fluorometry
• Fungemia (diagnosis, microbiology)
• Humans
• Recombinant Proteins (genetics, metabolism)
• Reproducibility of Results
• Sensitivity and Specificity
• Sugar Alcohol Dehydrogenases (genetics, metabolism)
• Sugar Alcohols (blood)