Development of a new method for d-xylose detection and quantification in urine, based on the use of recombinant xylose dehydrogenase from Caulobacter crescentus

Analytical Validation of a New Enzymatic and Automatable Method for d-Xylose Measurement in Human Urine Samples

“…The mean signals obtained were 0.16 ± 0.09 mg/dL, 0.10 ± 0.09 mg/dL, and 0.07 ± 0.05 mg/dL in Roche Cobas c502, Werfen ILab 600, and Siemens Dimension Vista 1500 analyzers, respectively, being lower than 20% of the LLoQ values and demonstrating the absence of interfering components in basal urine samples [26, 27]. Furthermore, selectivity of the enzymatic method in the presence of different monosaccharides was previously tested [20], showing that the assay displays high specificity for xylose over the other assayed sugars.…”

“…Stability of the freeze-dried and rehydrated XylB enzyme has been previously reported [20]. In this work, we evaluated the on-board stability of the reagents of the enzymatic xylose quantification kit.…”

“…XylB was expressed and purified from Escherichia coli overproducer strain BL21(DE3)/pET-xylB-wt [20]. Two standard d -xylose solutions (FAR Diagnostics) were used as controls: control C1 (0.84 mg/dL) and control C2 (5.443 mg/dL).…”

“…To overcome these difficulties, we recently developed a new enzymatic quantification method for xylose measurement in urine human samples, based on the activity of the enzyme xylose dehydrogenase from Caulobacter crescentus (XylB) (EC 1.1.1.175) [20]. In a preliminary manual study, the linearity of the procedure was demonstrated to cover an adequate range of xylose concentrations, using both urine and buffer as sample matrix.…”

“…In a preliminary manual study, the linearity of the procedure was demonstrated to cover an adequate range of xylose concentrations, using both urine and buffer as sample matrix. Additionally, the technique was proved to be efficient and sensitive, representing a potentially good alternative to the phloroglucinol colorimetric reaction [20]. Taking these results into consideration, in this work we have optimized and validated the protocol in order to be implemented in three automated analyzers used in clinical laboratories.…”