Advia chemistry

A total of 15 ml of fasting blood was withdrawn from each student by a phlebotomist. The students were asked to fast for at least 10 h prior to blood taking. All blood samples were sent to the hospital laboratory before storing it temporarily at 4°C in a cool box upon blood withdrawal. The blood samples were processed at the field laboratories in each state. The samples were spun and stored as serum and divided into several aliquots of 0.5 ml of serum for individual tests. In a plain test tube, 3 ml of blood was collected for the measurement of fasting lipids (Advia Chemistry, Siemens, Germany—triglyceride, total cholesterol, high density lipoprotein cholesterol and low-density lipoprotein cholesterol).

Total protein was determined using Siemens ADVIA 1800 automated chemistry analyzer according to the product manual (Siemens Healthineers, Germany). In brief, a volume of 30 μL plasma was diluted with 120 μL saline solution (physiological 0.9%) and 17.5 μL of the diluted sample was transferred to a test tube for further extraction with two reagents. Reagent 1 (68.5 μL, TP R1, Lot: 129TP) contained sodium hydroxide (400 mmol/L) and Na-K-tartrate (92 mmol/L), while reagent 2 (68.5 μL, TP R2, Lot: 130TP) contained the two prior ingredients as well as potassium iodide (60 mmol/L) and cupric sulphate (24 mmol/L) (Advia Chemistry, Siemens Healthineers, Germany). The cupric ions in reagent 2 interact with the protein peptide bonds and form a purple complex. The intensity of the purple colour is then used to measure the amount of total protein in the sample by spectrophotometry at 545 nm [43 (link)].

A 12-h fasting blood sample was drawn in the morning soon after arrival at the research center, following standardized procedures. A standardized 75 g oral glucose tolerance test (OGTT) was performed in all participants without known diabetes, utilizing an anhydrous glucose solution with plasma glucose levels measured after 2 h. Plasma glucose was measured by the hexokinase method (ADVIA Chemistry; Siemens, Deerfield, Illinois). HbA1C was measured by high-pressure liquid chromatography (Bio-Rad Laboratories, Hercules, California) using a method certified by the National Glycohemoglobin Standardization Program [17 (link)]. Meanwhile, diabetes status was defined in a comprehensive fashion as follows: a previously diagnosed diabetes was classified when answering “yes” to either “Have you been previously told by a physician that you had/have diabetes (sugar in the blood)?” or “Have you used medication for diabetes in the past 2 weeks?” Previously undiagnosed diabetes was classified based on laboratory values when reaching the threshold for fasting plasma glucose (FPG; ≥7.0 mmol/L), 2-h plasma glucose (2h-PG ≥11.1 mmol/L), or HbA1c (≥6.5%; ≥47.5 mmol/mol) [15 ]. Pre-diabetes was defined through fasting plasma glucose (FPG; ≥5.6 mmol/L to 6.9 mmol/L); or 2-h plasma glucose (2-h PG; ≥7.8 mmol/L to 11.0 mmol/L) or HbA1c (5.7–6.4%) [18 ].

In HNR, all laboratory data had been analyzed centrally in the laboratory of the university hospital of Essen. Serum creatinine (according to Jaffé) was determined on a Siemens Healthcare Diagnostics ADVIA Chemistry. Serum creatinine was not standardized to isotope dilution mass spectrometry. Hypertension was defined as either a blood pressure of at least 140 mmHg systolic or at least 90 mmHg diastolic or taking antihypertensive medication. Blood pressure cut-offs were selected according to the cut-offs used to define hypertension in the validated risk models. Diabetes was defined according to the respective definitions used in the risk models: either self-reported prevalent diabetes [27 (link), 28 (link)] or using a combination of known diabetes or taking antidiabetic drugs [29 (link), 30 (link)]. Albuminuria was defined as albumin/creatinine ratio (ACR) ≥ 30 mg/dl. In all models except the Kwon model, anemia was coded if hemoglobin levels were < 12 g/dl. In the Kwon model, the threshold for hemoglobin was < 12 g/dl for women and < 13 g/dl for men. Peripheral vascular artery disease was defined according to clinical information.

Weight was measured using a digital scale with 200 kg capacity, height using a fixed stadiometer and BMI was calculated as weight in kilograms divided by height in meters squared. Blood pressure (BP) was measured with a standard oscillometric device (Omron HEM 705CPINT, Omron Health Care, Lake Forest, IL, USA). Blood samples were taken after an overnight fasting. Plasma glucose was measured by the hexokinase method (ADVIA Chemistry; Siemens, Deerfield, IL, USA). Measurements of total cholesterol, triglyceride, and high-density lipoprotein (HDL-c) were assessed by enzymatic methods. Low-density lipoprotein cholesterol (LDL-c) was calculated by the Friedewald equation.

Plasma glucose was determined by the hexokinase method, and HDL-cholesterol by homogeneous colorimetric, without precipitation (ADVIA Chemistry; Siemens, Deerfield, Illinois, USA). LDL-cholesterol was calculated using the Friedewald equation. When triglyceride concentration was greater than 400 mg/dL, the LDL-cholesterol level was directly measured [31 (link)]. Immunochemistry was used to measure high-sensitivity C-reactive protein (Dade Behring, Siemens, Marburg, Germany), and enzyme-linked immunoenzymatic assay for insulin (Siemens, Tarrytown, USA) and adiponectin determinations (Enzo Life Sciences, Farmingdale, NY, USA). Intra-assay and inter-assay coefficients of variation ranged from 1.8 to 7.2%, and from 0.9 to 14.4%, respectively [21 (link)].