Materials
LDL Cholesterol kit (cat. Number 61532) was provided by bioMerieux (Marcy l'Etoile France). Hydragel Lipo+Lp(a) kit for agarose electrophoresis was obtained from Sebia. Thiobarbituric acid was obtained from Merck. 1,1',3,3'- tetra-methoxy-propane or malondialdehyde (MDA) was used as standard and purchased from SIGMA Co. St. Louis, MO, USA. All chemicals were of analytical grade and used without further purification.
Sample collection
Twelve type 2 diabetic patients of both sexes (37 - 65 years old) were studied. The degree of metabolic control was assessed by the measurement of fasting plasma glucose (mean 7.2 ± 1.1 mmol/l), fasting plasma HbA1c (mean 6.8 ± 1.3 %; NV 4.8-6.0 %) and they were normolipidemic. A series of 30 control non-diabetic subjects of both sexes (age range, 35-60 year old) was processed in parallel. All controls were normolipidemic according to the Alfedian criteria [18], and none of the subjects were taking any drug known to influence lipid or lipoprotein metabolism. Blood samples were obtained on heparin (5 U/ml) by venipuncture from subjects with 12 hours fasting. Plasma was separated rapidly and processed immediately. Alternatively, the samples were stored at 4 ºC for 24 hours or at -20º C for not more than 2 days.
Method of LDL isolation
LDL was selectively precipitated from 100 μl of plasma by addition of bioMerieux precipitating reagent of LDL-Cholesterol kit and vortex-mixed [14]. The mixture was incubated for 30 min at 2-8 ºC, and centrifuged for 5 min. The supernatant was discarded and the precipitate was washed with precipitating reagent. The washed precipitate was redissolved in different volumes of solubilizing solution (0.01% Triton X100 in 50 g/l NaCl) at 37 ºC, and vortex-mixed (resuspended LDL sample) [15]. Bradford's method [19] was used to determine the total protein content of the resuspended LDL sample, using bovine serum albumin as a standard. For selected experiments, the LDL fraction was obtained by density gradient ultracentrifugation as has been previously described [13].
Characterization of LDL isolated by selective precipitation
Representative samples were subjected to ultracentrifugation and selective precipitation (0, 1 or 2 washes) in order to isolate the LDL fraction. Subsequently, LDL fractions obtained by both methods, as well as the whole plasma, were electrophoresed in agarose according to the manufacturer's instructions. Briefly, electrophoresis was performed at a constant voltage of 130 V and initial intensity of 25 mA, for 80 minutes. The gel was dried and bands were revealed with either Sudan Black or Coomasie brillant blue.
LDL-cholesterol determination
LDL was obtained by selective precipitation of representative samples, and the resulting precipitates were washed once, twice, or not at all with the precipitating reagent, prior to resuspending with the solubilizing solution. Cholesterol content of the resulting resuspended LDL samples was determined by a commercial enzymatic kit (Colestat, Wiener Laboratories Argentina).
Basal and induced LDL oxidation
Basal LDL oxidation was determined by incubating an aliquot of 100 μl resuspended LDL sample, containing 50-90 μg protein, with 30 μl of 1 mM EDTA and 45 μl of distilled water. The corresponding blank was determined substituting the resuspended LDL sample by solubilizing solution.
In other experiments, resuspended LDL sample was mixed with 50 μl of 100 μM Cu2+ (freshly prepared in phosphate buffer saline solution, PBS, pH 7.4) and 25 μl of H2O2 solution (300 ml/l H2O2 in PBS, stock solution corresponds to 10 volume commercial H2O2). Blank was performed with solubilizing solution instead of resuspended LDL sample. In all cases, sample and blank were incubated at 37 ºC for different periods of time with occasional stirring. At the end of the incubation period, the lipid peroxidation was stopped by cooling and addition of 30 μl of 1 mM EDTA.
TBARS determination
Lipid peroxidation of LDL was assessed by TBARS formation [20]. Briefly, samples were incubated with 0.5 ml of 20% acetic acid, pH 3.5 and 0.5 ml of 0.78% aqueous solution of thiobarbituric acid. After heating at 95 °C for 45 minutes, the samples were centrifuged at 4000 r.p.m. for 5 minutes. The red pigment in the supernatant fractions was estimated by absorbance at 532 nm. A calibration curve was prepared with an MDA standard. Results were expressed as nmol MDA /mg LDL protein. All samples gave results which were within the linear portion of the MDA standard curve. A recovery assay was also performed by adding a defined amount of MDA before incubating with the oxidant mixture.
Statistical analysis
Results were expressed as mean ± SD and mean ± SEM. Statistical analysis was performed by Student's t test; a p value < 0.05 was considered statistically significant. Linear regression analysis was used for testing correlations between variables.