Gas chromatographic–mass spectrometric determination of erythrocyte 3-deoxyglucosone in diabetic patients
Introduction
The Maillard reaction is nonenzymatic glycation between reducing sugars and protein amino groups. In the early stage, glucose binding to protein forms Schiff base adducts which are then converted to relatively stable Amadori products. It further undergoes a series of reaction to form advanced glycation end products (AGEs). AGEs show a number of biological activities, and are involved in the pathogenesis of diseases such as aging, diabetes mellitus, uremia and Alzheimer’s disease [1], [2], [3], [4].
Hyperglycemia has been thought to be responsible for increased AGEs in diabetes. Some patients with insufficient glycemic control suffer from severe complications. AGEs are supposed to be produced more rapidly and directly from some highly reactive dicarbonyl compounds such as 3-deoxyglucosone (3-deoxy-d-erythro-hexos-2-ulose: 3-DG). 3-DG not only stimulates the formation of AGEs, but also shows some biological activities [4], [5], [6], [7], [8]. 3-DG is generated from degradation and rearrangement of the Amadori compound in the intermediate stage of the Maillard reaction [9]. 3-DG is also generated from fructose and fructose-3-phosphate via the polyol pathway. We consider that 3-DG is a clinically more relevant precursor for AGEs responsible for the occurrence of diabetic and uremic complications [3], [4], [5].
We studied the effect of an aldose reductase inhibitor (ARI) on erythrocyte 3-DG in diabetic hemodialysis (HD) patients by using gas chromatography–mass spectrometry (GC–MS) with a stable isotope-labeled 3-DG ([13C6]-3-DG) [10], [11]. We demonstrated that the polyol pathway is at least partly responsible for intracellular production of 3-DG, because the administration of ARI which suppresses the polyol pathway, successfully decreased erythrocyte 3-DG. In the diabetic HD patients, however, 3-DG before ARI treatment did not correlate with HbA1c. Intracellular accumulation of 3-DG depends on the balance between the speed of generation and the capacity of detoxifying or excreting system. In uremic patients, the capacity of detoxifying or excreting system may be decreased. Thus, it is only natural that 3-DG level did not correlate with glycemic status in diabetic HD patients.
To determine whether hyperglycemia is responsible for production of erythrocyte 3-DG, we measured erythrocyte 3-DG in diabetic patients without renal disease using GC–MS, and analyzed the correlation with a glycemic parameter, hemoglobin A1c (HbA1c). HbA1c is glycated hemoglobin, produced by nonenzymatic conjugation of glucose with N-terminal valine in β-chain of HbA.
Section snippets
Patients
Blood samples were obtained from thirteen diabetic patients (10.1±3.6 years old, mean±SD; five males, eight females), and eleven healthy subjects (20.7±3.3 years old; six males, five females). Eleven out of the patients had suffered from insulin-dependent diabetes mellitus (IDDM), and two from non-insulin-dependent diabetes (NIDDM). The onset age of diabetes was 7.0±3.9 years (mean±SD). They had been treated for 3.0±3.6 years (mean±SD), and were free from proteinuria or renal disease. The
Results
Fig. 1 shows total ion chromatogram (TIC) and SIM chromatograms of 3-DG and [13C6]-3-DG in a standard sample (upper figure) and an erythrocyte sample from a diabetic patient (lower figure). Two peaks, which consist of 3-DG isomers, are recognized between 12 and 13 min in the chromatograms. The major peak is detected at a retention time of about 12.1 min, and the minor peak at about 12.5 min. The base ions at m/z 437, (M+H)+, for 3-DG, and at m/z 443, (M+H)+, for [13C6]-3-DG were monitored for
Discussion
3-DG rapidly reacts with arginine or lysine residues of proteins to form AGEs such as imidazolone or pyrraline, which are involved in the pathogenesis of aging, diabetes and uremia [3], [4], [5]. 3-DG also shows some biological activities, including induction of heparin-binding epidermal growth factor-like growth factor in rat aortic smooth muscle cells [6], promotion of apoptotic cell death in macrophage-derived cell lines [7], suppression of cell-cycle progression during the S phase of rat
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