Quantitation of 3-Deoxyglucosone Levels in Human Plasma

Abstract

3-Deoxyglucosone (3DG), a reactive dicarbonyl, is an important intermediate in the formation of advanced glycation end products (AGEs). The AGEs are particularly important in diabetes since they have been correlated with the development of diabetic complications. Consequently, measurements of 3DG are likely to provide valuable insights into the role of this metabolite in the etiology of diabetic complications. While several methods of 3DG quantitation in human plasma have been previously published, a significant discrepancy (over 30-fold) exists in the reported values. Knechtet al.(Arch. Biochem. Biophys.294, 130–137, 1992) have reported the levels of plasma 3DG in normoglycemics to be 61 nm, using a GC/MS procedure. In contrast to this, Niwaet al.(Biochem. Biophys. Res. Commun.196, 837–843, 1993) reported 3DG levels to be 1800 nmin normoglycemics, using a totally independent GC/MS method. To resolve this disagreement and fill the need for a robust assay for this dicarbonyl, suitable for absolute quantitation, a GC/MS procedure was devised for its measurement. Plasma samples were deproteinized either by ultrafiltration or by addition of ethanol as described by Niwaet al.(Biochem. Biophys. Res. Commun.196, 837–843, 1993). 3DG in the ultrafiltrate or the supernatant was conjugated with 2,3-diaminonaphthalene to produce a stable adduct which was then converted to a silyl ether and analyzed by GC/MS. The analyte was monitored by selected ion monitoring at anm/zof 295 and 306 and quantitated using an internal standard of [U-13C]3DG. Using this approach, 3DG levels in plasma deproteinized by ultrafiltration were found to be significantly elevated from 58.5 ± 14 (SD) nmin normoglycemics to 98.5 ± 34 (SD) nmin type I diabetics. When deproteinization of the plasma was carried out using ethanol, the levels of 3DG from normoglycemic plasma were similar to those reported by Niwaet al.(1710 ± 750 (SD) nm). These results suggest that 3DG levels measured by ultrafiltration may represent the free circulating 3DG and those obtained by ethanol extraction may represent aform of 3DG bound to a macromolecule (presumbaly protein).