NOVEL ANALYTICAL APPROACH TO MONITORING ADVANCED GLYCOSYLATION END-PRODUCTS IN HUMAN SERUM WITH ONLINE SPECTROPHOTOMETRIC AND SPECTROFLUOROMETRIC DETECTION IN A FLOW SYSTEM

We proposed a simple analytical procedure for measurement of serum adv anced glycosylation end products (AGEs) based on simultaneous detectio n of low-molecular-mass peptides and AGEs with a flow system and two d etectors connected on-line: spectrophotometric for peptides (lambda = 280 nm) and spectrofluorometric for AGEs (lambda(ex) = 247 nm, lambda( em) = 440 nm). Sample pretreatment was carried out in microcentrifuge tubes: Serum (20 mu L) was deproteinized with trichloroacetic acid (48 0 mu L, 0.15 mol/L) and lipids were extracted with chloroform (100 mu L). Twenty microliters of the filtered aqueous layer was injected to t he flow system and the relation between fluorescence and absorption si gnals was measured. A peptide-derived AGE calibrator was used for cali bration. Within-day and between-day CVs were 6.7% and 9.1%, respective ly, at an AGE concentration corresponding approximately to that in hea lthy individuals. Mean results (+/-SD) in 10 healthy individuals were 10.1% +/- 1.0%, in 21 patients with diabetes without complications 18. 0% +/- 6.2%, in 25 patients with complications 24.1% +/- 15.4%, and in 12 diabetic patients in end-stage renal disease 92% +/- 30%. Comparis on with an ELISA procedure (x, in arbitrary units/L) yields a regressi on equation y = 0.713x + 1.24 (S-y/k = 6777, r = 0.8477, n = 41).