Determination of glyoxal and methylglyoxal in serum by UHPLC coupled with fluorescence detection.

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 ±â€¯27 and 192 ±â€¯10 nmol/L, respectively. In human serum, GO and MGO levels in non-diabetic subjects (n = 14) were determined to be 154 ±â€¯88 and 98 ±â€¯27 nmol/L, and in serum samples from subjects with diabetes (n = 14) 244 ±â€¯137 and 190 ±â€¯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.

Advanced Glycation End Products and esRAGE Are Associated With Bone Turnover and Incidence of Hip Fracture in Older Men.

Background: Diabetes mellitus is associated with increased fracture risk despite preservation of bone density and reduced bone turnover. Aims: We tested the hypothesis that circulating advanced glycation end products (AGEs) and endogenous secretory receptor for AGEs (esRAGE) differentially modulate bone turnover and predict fracture risk in older men. Participants: A total of 3384 community-dwelling men aged 70 to 89 years. Methods: Collagen type I C-terminal cross-linked telopeptide, N-terminal propeptide of type I collagen (P1NP), and total osteocalcin (TOC) were assayed using immunoassay and undercarboxylated osteocalcin (ucOC) following hydroxyapatite binding. Plasma N-carboxymethyllysine (CML) and esRAGE were assayed using immunoassay. Methylglyoxal and glyoxal were assayed using mass spectrometry. Incident hip fractures were ascertained. Results: Median age was 76.3 years (interquartile range, 74.2 to 79.1 years). Plasma CML was measured in 3011 men, methylglyoxal and glyoxal in 766 men, and esRAGE in 748 men. Plasma CML, methylglyoxal, glyoxal, and esRAGE were similar in men without and with diabetes (all P > 0.05). CML was positively associated with fasting glucose (r = 0.06, P < 0.001), and esRAGE was inversely associated (r = -0.08, P = 0.045). esRAGE was positively associated with bone formation (P1NP, r = 0.17, P < 0.001; ucOC, r = 0.11, P = 0.008; TOC, r = 0.16, P < 0.001). Incident hip fractures occurred in 106 men during follow-up. Men with CML in the third quartile of values had reduced incidence of hip fracture compared with men in the lowest quartile (hazard ratio, 0.49; 95% CI, 0.24 to 0.99; P = 0.045). Conclusions: Glycemia associates positively with CML and reciprocally with esRAGE in older men. Circulating esRAGE modulates bone turnover in older men, whereas CML predicts incidence of hip fracture.