Department of Pharmacology, School of Medicine, Western Sydney University, Locked Bag 1797, Penrith, NSW, 2751, Australia.
Department of Medicine, Peninsula Health & Peninsula Clinical School, Monash University, Victoria, 3199, Australia.
Anal Biochem. 2019 May 15;573:51-66. doi: 10.1016/j.ab.2019.02.014. Epub 2019 Feb 20.
Glyoxal (GO) and methylglyoxal (MGO) are two important biomarkers in diabetes. Analytical methods for determination of GO and MGO in serum samples are either HPLC with UV-Vis (low sensitivity) or MS/MS (expensive) detection. These disadvantages have hampered the introduction of these biomarkers as a routine analyte for diabetes diagnostics into the clinical laboratory. In this study, we introduce a UHPLC method with fluorescence detection for the measurement of GO and MGO in serum samples by pre-column derivatization at neutral pH with 5, 6-diamino-2,4-dihydroxypyrimidine sulfate (DDP) to form lumazines. The method was validated as per FDA guidelines. Using this method, we have determined GO and MGO in a variety of animal serum samples, and for example, determined the GO and MGO concentration in adult bovine serum to be 852 ± 27 and 192 ± 10 nmol/L, respectively. In human serum, GO and MGO levels in non-diabetic subjects (n = 14) were determined to be 154 ± 88 and 98 ± 27 nmol/L, and in serum samples from subjects with diabetes (n = 14) 244 ± 137 and 190 ± 68 nmol/L, respectively. In addition, interference studies showed that physiological serum components did not lead to an artificial increase in the levels of GO and MGO.
乙二醛 (GO) 和甲基乙二醛 (MGO) 是糖尿病的两个重要生物标志物。用于测定血清样品中 GO 和 MGO 的分析方法要么是 HPLC 与 UV-Vis（灵敏度低），要么是 MS/MS（昂贵）检测。这些缺点阻碍了这些生物标志物作为常规分析物引入糖尿病诊断的临床实验室。在这项研究中，我们介绍了一种使用 UHPLC 方法与荧光检测，通过在中性 pH 值下用 5,6-二氨基-2,4-二羟基嘧啶硫酸盐 (DDP) 对血清样品进行柱前衍生化来测定 GO 和 MGO，形成荧光素。该方法是根据 FDA 指南进行验证的。使用该方法，我们已经测定了各种动物血清样品中的 GO 和 MGO，例如，确定成年牛血清中的 GO 和 MGO 浓度分别为 852±27 和 192±10 nmol/L。在非糖尿病患者（n=14）的人血清中，GO 和 MGO 的水平分别为 154±88 和 98±27 nmol/L，而在糖尿病患者（n=14）的血清样本中，GO 和 MGO 的水平分别为 244±137 和 190±68 nmol/L。此外，干扰研究表明，生理血清成分不会导致 GO 和 MGO 水平的人为增加。
