Effect of vitamin C on glycosylation of proteins.

Twelve nondiabetic subjects consumed 1 g/day vitamin C for 3 mo. A fasting blood sample was taken at the start of the study and at the end of each month for the measurement of plasma and intraerythrocyte glucose, vitamin C, glycosylated hemoglobin (affinity chromatography and electrophoresis), and glycosylated albumin (affinity chromatography). Although there were no significant changes in fasting glycemia, glycosylated hemoglobin (affinity chromatography) decreased 18%, from 6.18 +/- 0.48% (mean +/- SD) at the start to 5.05 +/- 0.50% (P less than 0.0001) after 3 mo, whereas, HbA1 measured by electrophoresis increased 16%, from 6.17 +/- 0.61 to 7.16 +/- 0.59% (P less than 0.0001) in this period. Glycosylated albumin decreased 33%, from 1.56 +/- 0.24 to 1.04 +/- 1.01% (P less than 0.0001) after 3 mo. This discrepancy between glycosylated hemoglobin measured by electrophoresis and affinity chromatography was due to methodological differences between the two techniques, with affinity chromatography measuring "true" glycosylated hemoglobin. The greater decrease found with glycosylated albumin was probably due to the different distribution of vitamin C between plasma and within the erythrocyte, levels after 1 mo of supplementation being 109 +/- 19 and 59 +/- 9 microM, respectively (P less than 0.001). This indicates that administration of oral vitamin C may inhibit the glycosylation of proteins in vivo by a competitive mechanism.

Investigation of the mechanism underlying the variability of glycated haemoglobin in non-diabetic subjects not related to glycaemia.

The Islington Diabetes Survey identified two groups of non-diabetic individuals, low and high glycators, who remained consistently classified 4.4 +/- 0.2 years after the original study. To investigate the mechanism for this grouping, 12 original subjects, 5 with low and 7 with high levels of glycated haemoglobin relative to their 2 h blood glucose, were studied. Glycated albumin and fructosamine measurements gave comparable classifications, with three individuals being misclassified for each measurement; in addition glycated albumin was positively correlated with mean blood-glucose concentration (r = 0.53; P < 0.05). Fasting plasma glucose concentration was greater than the intra-erythrocyte concentration (P < 0.05), but their ratio was reduced in low compared to high glycators (0.77 +/- 0.12 and 0.94 +/- 0.13, P < 0.0001). No differences between groups were found for plasma insulin, urea or non-esterified fatty acids; plasma or intra-erythrocyte inorganic phosphate or vitamin C; nor plasma, erythrocyte or urinary total amino acids. Erythrocyte 2,3-diphosphoglycerate, a catalyst of glycation, was elevated in high compared to low glycators (5.61 +/- 0.26 and 4.81 +/- 0.24 mmol/l, P < 0.001). Mean centile glycated haemoglobin was positively correlated with intra-erythrocyte pH (r = 0.55; P < 0.05) and negatively with plasma total amino acids (r = -0.57, P < 0.05). These data indicate that the intra-erythrocyte environment of high glycators favours glycation of haemoglobin. This could have important consequences for diabetic patients in terms of monitoring their glycaemic control and in the progression of those complications related to non-enzymic glycation of intracellular proteins.

Biological variation in glycated proteins.

The high degree of individuality in the fructosamine assay has been ascribed to non-specific interferences in the assay. To investigate this, we measured the biological variability of 10 non-diabetic subjects using the fructosamine assay, the new fructosamine plus assay, glycated albumin and glycated total plasma proteins by affinity chromatography. The total variation of the two fructosamine assays was half that of the affinity chromatography assays. This was mainly due to the greater analytical imprecision of the affinity chromatography assays. The resulting high index of heterogeneity for both affinity methods makes it difficult to assess the significance of changes in serial results. The within-subject variation made a small contribution to the total variation for all the assays, and was particularly low for the fructosamine assays. This suggests that any non-specific component makes a constant contribution to the measured fructosamine activity in non-diabetic subjects. The fructosamine assays therefore have significant advantages over the affinity chromatography methods as indices of medium-term glycaemic control.