Fluorimetric determination of the levels of urinary neopterin and serum thiobarbituric acid reactive substances in the nonagenarians (original) (raw)

Twelve self-sustaining nonagenarians, 10 women and two men, aged 949/3 years, and eight institutionalised nonagenarians, eight women, aged 919/1 year as well as 11 control subjects, seven women and four men, aged 849/5 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 mm-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 ml of urine specimens with 1.0 ml of mobile phase containing 2 g of disodium Á/EDTA per litre, a 20 ml 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 (mmol 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 Á/Kruskal Á/ Wallis tests, using statistical software NCSS 6.0.21 (Kaysville, UT, 1996). The decision on significance was based on P0/0.05. Urinary neopterin was significantly higher in institutionalised compared to self-sustaining subjects and controls (6259/565 vs. 2039/63 mmol mol (1 creatinine, and 1989/128 mmol mol (1 creatinine, respectively, P0/0.006). The serum TBARS were higher in both groups of nonagenarians (3.239/1.16 mmol l (1 and 2.699/0.39 vs. 2.129/0.83 mmol l (1 for the self-sustaining, institutionalised and controls, respectively, P0/0.023). We conclude that the