Atherosclerosis 143 (1999) 213 – 215 Technical note Detection of structural alterations in LDL isolated from type 2 diabetic patients: application of the fructosamine assay to evaluate the extent of LDL glycation Silvia M. Sanguinetti, Laura E. Schreier, Alicia Elbert, Vero ´ nica Fasulo, Norma Ferrari, Regina L.W. Wikinski * Laboratory of Lipids and Lipoproteins, Department of Clinical Biochemistry, School of Pharmacy and Biochemistry, Uniersity of Buenos Aires, Buenos Aires, Argentina Received 7 August 1998; accepted 23 September 1998 Keywords: Fructosamine; Type 2 diabetes; Glycated LDL; LDL modifications Modifications in LDL such as glycation contribute to the accelerated development of macrovascular alter- ations in diabetic patients [1], but to date there is no recommended method to determine the extent of plasma LDL glycation. The fructosamine assay origi- nally 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, fruc- tosamine values in LDL from diabetic patients are as yet unknown. Therefore, we re-adapted and evaluated the fructosamine assay for the determination of gly- cated LDL in type 2 diabetic patients. Besides, we characterized LDLs by means of their chemical compo- sition 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 ( S.D.) levels of HbA1c were 8.5 2.5%, serum fructosamine 370 90 mol/l and glycemia in the fast- ing state 213 56 mg/dl. Mean ( S.D.) levels of plasma triglycerides (TG) and total, HDL- and LDL- cholesterol were 173 76, 227 42, 49 13 and 141  36 mg/dl, respectively. Throughout, there was no clini- cal 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 ( S.D.) levels of HbA1c were 4.9 0.5%, serum fructosamine 230 10 mol/l and glycemia in the fast- ing state 86 7 mg/dl. Mean ( S.D.) levels of plasma triglycerides and total, HDL- and LDL- cholesterol were 85 38, 197 44, 59 16 and 118 43 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 fruc- tosamine 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: ssan- guinetti@dbc.ffyb.uba.ar. 0021-9150/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII:S0021-9150(98)00276-7