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

A rapid enzymatic fluorometric assay for measuring D-arabinitol in serum wa s developed using recombinant D-arabinitol dehydrogenase from Candida albic ans (rArDH). rArDH was produced in Escherichia coli and purified by dye-lig and affinity chromatography. rArDH was highly specific for D-arabinitol, cr oss-reacting only with xylitol (4.9%) among all polyols tested. A Cobas Far a II centrifugal autoanalyzer (Roche) was used to measure NADH fluorometric ally when rArDH and NAD were added to serum extracts, and D-arabinitol conc entrations were calculated from standard curves derived from pooled human s erum 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 adde d D-arabinitol in serum (mean recovery rate, 101%). The mean and median D-a rabinitol/creatinine ratios mere 2.74 and 2.23 mu M/mg/dl, respectively, fo r the 11 patients with candidemia compared to 1.14 and 1.23 mu M/mg/dl, res pectively, for 10 healthy controls (P < 0.01), These results confirm earlie r studies showing that serum D-arabinitol measurement may help to promptly diagnose invasive candidiasis, The technique shows a significant improvemen t in terms of accuracy, cost, simplicity, specificity, and speed compared w ith gas chromatography, mass spectrometry, and earlier enzymatic assays.