A rapid, automated enzymatic fluorometric assay for determination of D-arabinitol in serum.
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.
Yeo
S F
SF
Departments of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA.
Zhang
Y
Y
Schafer
D
D
Campbell
S
S
Wong
B
B
D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D002177:Candidiasis / Q000175:diagnosis* / Q000382:microbiology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005470:Fluorometry; D016469:Fungemia / Q000175:diagnosis / Q000382:microbiology; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D013402:Sugar Alcohols / Q000097:blood*
D011994:Recombinant Proteins; D013402:Sugar Alcohols; D013401:Sugar Alcohol Dehydrogenases; C022751:D-arabinitol dehydrogenase; C014999:arabitol