Floridi A, Trizza V, Paolotti P, Lucarelli C
Dipartimento di Biologia Cellulare e Molecolare, Università di Perugia, Italy.
J Chromatogr A. 1999 Jun 18;846(1-2):65-71. doi: 10.1016/s0021-9673(99)00442-2.
We propose a newly integrated procedure for the analysis of furosine (early glycation product) and pentosidine (glycoxidation end-product) in plasma proteins and the simultaneous assessment of advanced glycation end-product (AGE) peptides and free pentosidine in plasma. In order to determine furosine and protein-linked pentosidine, plasma proteins were hydrolyzed in 8 M HCl and each analyte was purified by solid-phase extraction. Furosine was determined by ion-pair RP-HPLC methodology with isocratic elution and spectrophotometric detection at 280 nm and pentosidine by ion-pair RP-HPLC by using gradient elution and fluorimetric detection at 335/385 nm. To assess free pentosidine concentration and simultaneously evaluate the AGE peptides, an aliquot of plasma sample was diluted and ultrafiltered by using Centricon 10 M(r) < or = 10,000) ultrafiltration membranes. Free pentosidine and AGE peptides were analysed by ion-pair RP-HPLC, by using gradient elution and fluorimetric detection at 385 nm upon excitation at 335 nm. The HPLC methodology has been successfully used for the determination of glycation and glycoxidation protein status in uremic patients.
我们提出了一种新的综合方法，用于分析血浆蛋白中的呋喃素（早期糖基化产物）和戊糖苷（糖氧化终产物），并同时评估血浆中的晚期糖基化终产物（AGE）肽和游离戊糖苷。为了测定呋喃素和与蛋白质结合的戊糖苷，将血浆蛋白在8M盐酸中水解，然后通过固相萃取对每种分析物进行纯化。呋喃素通过离子对反相高效液相色谱法（RP-HPLC），采用等度洗脱并在280nm处进行分光光度检测来测定；戊糖苷则通过离子对反相高效液相色谱法，采用梯度洗脱并在335/385nm处进行荧光检测来测定。为了评估游离戊糖苷浓度并同时评估AGE肽，取一份血浆样品等分试样进行稀释，并使用Centricon 10 M（r）（≤10,000）超滤膜进行超滤。游离戊糖苷和AGE肽通过离子对反相高效液相色谱法进行分析，采用梯度洗脱并在335nm激发下于385nm处进行荧光检测。该高效液相色谱方法已成功用于测定尿毒症患者的糖基化和糖氧化蛋白状态。
