HPLC METHOD FOR PENTOSIDINE DETERMINATION IN URINE, SERUM, AND TISSUES AS A MARKER OF GLYCATION AND OXIDATION LOADING OF THE ORGANISM

Abstract

ABSTRACT A sensitive reverse phase HPLC method for pentosidine (PEN) determination in urine, serum, and tissues has been elaborated on utilizing a SHIMADZU liquid chromatograph, type CLASS VP, version 5.0. Silica C18 and water–heptafluorobutyric acid (HFBA)-acetonitrile (ACN) as stationary and mobile phase, was used. The fluorescence monitor setting was 335/385 nm as excitation and emission wavelengths, respectively. PEN standard was synthesized utilizing a simple polymer analogous reaction with no requirement for purification of the reaction product. Urine and serum required 250 μL and tissue (cartilage, synovium, granular tissue) required 2–4 mg (dry weight) sample sizes. Twenty five μL of urine and tissue sample volumes were injected into the HPLC column, whereas serum samples required only 10 μL. Reproducibility of the HPLC determination, alone, was slightly above 1% (RSD), and of the whole method (i.e., including sample hydrolysis and purification with cellulose) was 4.44%, respectively. Recovery of the whole method was 77±3.5% with a sensitivity limit of 17.6 femtomols.Urine PEN (U-PEN) concentrations have been determined in a group of patients with osteoarthritis (OA, N=58, age 66.97± 9.89 years, mean±S.D.) and compared with control individuals (N=15, age 30.01±8.67 years, U-PEN 1.65± 0.46 nmol/mmol creatinine). PEN-age dependence between both groups has been eliminated mathematically using a known measured PEN-age relationship. It was found, that U-PEN concentrations in OA were significantly higher as compared with (corrected) healthy controls (7.7±7.8 vs. 2.1±0.5 nmol/mmol creatinine, P=0.00002). Additionally, a small correlation was noted between U-PEN and measured urinary pyridinoline (U-PD) in OA (U-PD=2.3111* U-PEN+55.475, r=0.50). This may be partially related to the immune response of the organism caused by the toxic action of molecular domains containing PEN, secondarily leading to accelerated resorption kinetics and, thus, to a higher pyridinoline level.