Detection of structural alterations in LDL isolated from type 2 diabetic patients: application of the fructosamine assay to evaluate the extent of LDL glycation

Abstract

Modifications in LDL such as glycation contribute to the accelerated development of macrovascular alterations in diabetic patients [1], but to date there is no recommended method to determine the extent of plasma LDL glycation. The fructosamine assay originally described by Johnson et al. [2] to evaluate medium term glycemic control in diabetic patients, was previously applied to measurements of glycated protein in LDL fractions isolated from in vitro glycated plasma obtained from non-diabetic subjects [3]. However, fructosamine values in LDL from diabetic patients are as yet unknown. Therefore, we re-adapted and evaluated the fructosamine assay for the determination of glycated LDL in type 2 diabetic patients. Besides, we characterized LDLs by means of their chemical composition and estimated the predominance of small dense LDL through the total proteins/cholesterol ratio in the LDL fraction [4]. Twenty-three type 2 diabetic patients of either sex, whose ages ranged from 47 to 82 years, were studied. Mean (9S.D.) levels of HbA1c were 8.592.5%, serum fructosamine 370990 mmol/l and glycemia in the fasting state 213956 mg/dl. Mean (9S.D.) levels of plasma triglycerides (TG) and total, HDLand LDLcholesterol were 173976, 227942, 49913 and 1419 36 mg/dl, respectively. Throughout, there was no clinical or laboratory evidence of impaired liver or kidney function. The control group comprised 19 subjects of either sex, ages ranging from 22 to 87 years, whose mean (9S.D.) levels of HbA1c were 4.990.5%, serum fructosamine 230910 mmol/l and glycemia in the fasting state 8697 mg/dl. Mean (9S.D.) levels of plasma triglycerides and total, HDLand LDLcholesterol were 85938, 197944, 59916 and 118943 mg/dl, respectively. LDL was isolated from fasting plasma supplemented with EDTA 1 g/l, by sequential ultracentrifugation within the 1.019–1.063 g/ml density range. The proposed assay was evaluated as below. An LDL aliquot obtained from controls was used to make up a pool to check the correlation between the degree of in vitro glycation and the measurement of fructosamine in the LDL fraction isolated, as well as to determine assay recovery and reproducibility. For this purpose, an aliquot of the pool was incubated with 100 mmol/l glucose during 1–7 days. Another aliquot was incubated at 37°C with glucose concentrations ranging from 13 to 100 mmol/l during 4 days, a period roughly equal to LDL mean life in plasma. Fructosamine was determined in LDL fractions using Nitro Blue Tetrazolium (NBT) reagent and serum con* Corresponding author. Fax: +54-1-823-7351; e-mail: ssanguinetti@dbc.ffyb.uba.ar.