Direct quantification of pentosidine in urine and serum by HPLC with column switching

Abstract

Concentrations of pentosidine, an advanced glycation end product, are increased in aging, diabetes mellitus, and uremia. Using HPLC with column switching, we developed a direct method of measuring pentosidine in urine and serum. We inject the sample directly onto a gel-filtration precolumn, select ("heart-cut") the eluate fraction containing pentosidine, and introduce this fraction into a reversed-phased column by use of a switching valve. The recovery rate of the complete method was 97.7-99.9%. The intraassay CV was 5.7%, and the interassay CV was 5.8%. The calibration curve showed significant linearity (r = 0.998, P = 0.0001). We examined urinary concentrations of pentosidine in 12 diabetic patients (mean +/- SD, 8.7 +/- 2.3 micromol/mol of creatinine), 32 patients with chronic renal failure (CRF; 36.1 +/- 39.0), 19 osteoporotic patients (7.9 +/- 5.3), and 29 healthy control subjects (5.2 +/- 2.3). In CRF, urinary pentosidine in the patients undergoing hemodialysis was significantly higher than in CRF patients not being treated by hemodialysis (mean, 58.1 vs 18.2; P <0.001). Also, concentrations of urinary and serum pentosidine were significantly correlated (r = 0.797, P = 0.0011). Because this method does not require pretreatment of samples, it is convenient and useful for measuring urinary and serum pentosidine.