Fluorimetric determination of the levels of urinary neopterin and serum thiobarbituric acid reactive substances in the nonagenarians

Abstract

Twelve self-sustaining nonagenarians, 10 women and two men, aged 94±3 years, and eight institutionalised nonagenarians, eight women, aged 91±1 year as well as 11 control subjects, seven women and four men, aged 84±5 years entered the study. Urinary neopterin, an indicator of systemic immune activation, and serum thiobarbituric acid reactive substances (TBARS), a marker of lipoperoxidation, were determined initially, and collection of the blood and urine samples was repeated at 3-month interval. Neopterin was measured in the urine specimens by reversed-phase high performance liquid chromatography. A C 18 reversed-phase column 3.3×150 mm, 5 μm-diameter packing Separon SGX was used. Potassium phosphate buffer (15 mmol l −1, pH 6.4) at flow rate of 0.8 ml min −1 was used as mobile phase. After centrifugation (5 min, 1300× g) and diluting 100 μl of urine specimens with 1.0 ml of mobile phase containing 2 g of disodium–EDTA per litre, a 20 μl sample was injected on a column. Neopterin was identified by its native fluorescence (353 nm excitation, 438 nm emission). Creatinine was determined by Jaffé kinetic reaction after dilution of sample 1:50 (v/v). The concentration of neopterin in urine was expressed as neopterin/creatinine ratio (μmol mol −1 creatinine). TBARS were determined spectrofluorometrically using LS-5 spectrofluorimeter (excitation wavelength 528 nm, emission wavelength 558 nm) after extraction with n-butanol treatment with thiobarbituric acid. The significance of differences between nonagenarians and control group was examined by ANOVA–Kruskal–Wallis tests, using statistical software NCSS 6.0.21 (Kaysville, UT, 1996). The decision on significance was based on P=0.05. Urinary neopterin was significantly higher in institutionalised compared to self-sustaining subjects and controls (625±565 vs. 203±63 μmol mol −1 creatinine, and 198±128 μmol mol −1 creatinine, respectively, P=0.006). The serum TBARS were higher in both groups of nonagenarians (3.23±1.16 μmol l −1 and 2.69±0.39 vs. 2.12±0.83 μmol l −1 for the self-sustaining, institutionalised and controls, respectively, P=0.023). We conclude that the fluorimetric determinations of urinary neopterin and serum TBARS can be useful for the monitoring health status in the elderly patients.