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Clinical Chemistry 43: 1563-1569, 1997;
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(Clinical Chemistry. 1997;43:1563-1569.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Novel analytical approach to monitoring advanced glycosylation end products in human serum with on-line spectrophotometric and spectrofluorometric detection in a flow system

Kazimierz Wróbel1,a, Katarzyna Wróbel1, Ma. Eugenia Garay-Sevilla2, Laura Eugenia Nava2 and Juan Manuel Malacara2

1 Instituto de Investigaciones Científicas, Universidad de Guanajuato, 36000 Guanajuato, Mexico.

2 Instituto de Investigaciones Médicas, Universidad de Guanajuato, 37320 Leon, Mexico.
a Author for correspondence. Fax 473–26252; e-mail katarzyn{at}quijote.ugto.mx

We proposed a simple analytical procedure for measurement of serum advanced glycosylation end products (AGEs) based on simultaneous detection of low-molecular-mass peptides and AGEs with a flow system and two detectors 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 µL) was deproteinized with trichloroacetic acid (480 µL, 0.15 mol/L) and lipids were extracted with chloroform (100 µL). Twenty microliters of the filtered aqueous layer was injected to the flow system and the relation between fluorescence and absorption signals was measured. A peptide-derived AGE calibrator was used for calibration. Within-day and between-day CVs were 6.7% and 9.1%, respectively, at an AGE concentration corresponding approximately to that in healthy 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%. Comparison with an ELISA procedure (x, in arbitrary units/L) yields a regression equation y = 0.713x + 1.24 (Sy||x = 6777, r = 0.8477, n = 41).




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