Ion exchange high performance liquid chromatography

At the initial visit, fasting glucose, glycated hemoglobin (HbA1c), fasting total serum cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, aspartate aminotransaminase, alanine aminotransferase (ALT), and creatinine were measured. Serum creatinine was assayed by calorimetry using Beckman Coulter AU analyzer (Beckman Coulter Inc., Brea, CA, USA) following the Jaffe method and it was IDMS standardized [14 (link)]. HbA1c was measured using ion-exchange high-performance liquid chromatography (Bio-Rad, Hercules, CA, USA) (4.0–6.0%). Albuminuria was measured using a radioimmunoassay (Immunotech, Marseille, France) using spot urine or time-collected urine. Microalbuminuria was defined as an albumin excretion rate of 20–200 μg/min, an albumin/creatinine ratio in spot urine of 30–300 mg/g, or a 24-h urine protein of 30–300 mg/day. Overt albuminuria was defined as an albumin excretion rate > 200 μg/min, an albumin/creatinine ratio in spot urine of > 300 mg/g, or a 24-h urine protein of > 300 mg/day. All laboratory tests were measured in one laboratory.

We estimated clinical and laboratory data using a previously described method^{26 (link)}. Briefly, physical examinations, including heights, weights, WC, and BP were measured in accordance with standard protocol. All parameters described in this study were measured in the central, certified laboratory at AMC. Blood sample data, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), and triglycerides (TG), were measured by Toshiba 200FR Neo analyzer (Toshiba Medical System Co., Ltd., Tokyo, Japan) using the enzymatic colorimetric method. The fasting plasma glucose (FPG) and HbA1c levels were measured by Toshiba 200 FR auto-analyzer (Tosiba) and ion-exchange high-performance liquid chromatography (Bio-Rad Laboratories, Inc., Hercules, CA), respectively, according to the manufacturer’s protocol.

Height and body mass were measured with the participants wearing light clothing and no shoes. BMI was calculated as body mass in kilograms divided by the square of the height in meters. WC (in cm) was measured mid-way between the costal margin and the iliac crest at the end of normal expiration. BP was measured on the right arm after resting for 5 min using an automatic manometer with an appropriate cuff size. After overnight fasting, early-morning blood samples were drawn from the antecubital vein into vacuum tubes and subsequently analysed by the central, certified laboratory at AMC. Measurements included concentrations of fasting glucose, insulin, hsCRP, several lipid parameters and liver enzymes.
Fasting total cholesterol, HDL-C, LDL-C, TG, uric acid, AST and ALT levels were measured using enzymatic colorimetric methods on a Toshiba 200FR Neo analyser (Toshiba Medical System Co., Ltd.). GGT was measured using the L-g-glutamyl-p-nitroanilide method (Toshiba Medical System Co., Ltd.). FPG and hsCRP were measured using the enzymatic colorimetric method on the Toshiba 200 FR auto-analyser and the immunoturbidimetric method (Toshiba Medical System Co., Ltd.), respectively. Ion-exchange high-performance liquid chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to measure the HbA1c levels. All enzyme activities were measured at 37 °C.