Advia 2400 clinical chemistry system

After overnight fasting, blood samples were drawn from peripheral venous blood, collected into serum separating tubes and randomly ordered for testing at 37°C. All analyses were performed at the biochemistry laboratory of Shanghai Pulmonary Hospital by ADVIA 2400 Clinical Chemistry System (Siemens Healthcare) using Siemens reagent 02011954. The method for measurement of GGT enzyme activity was standardized according to the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference measurement procedures (23 (link)). The intra-assay coefficient of variation was estimated at 1.9%. The laboratory technicians were blinded to do and the normal reference values were 0–38 U/L for men and women. Blood samples were obtained within 2 days prior to RHC.

Fasting venous blood samples were taken at 8:00 after a 12 h overnight fast. Glucose was immediately measured by using the glucose/oxidase method (Glucose GOD-PAP; Biolabo, Madrid, Spain). Insulin was measured by using enzyme-linked immunosorbent assay (Diagnostic System Laboratories, Webster, TX, USA). Total cholesterol and triglycerides were determined by enzymatic methods (CHOD-PAP and GPO-PAP, respectively; Roche Diagnostics, Basel, Switzerland). HDL cholesterol was determined after precipitation with phosphotungstic acid. LDL cholesterol was measured by using an Advia 2400 Clinical Chemistry System (Siemens Healthcare Diagnostics, Erlangen, Germany). NEFAs were measured with an ACS-ACOD assay (Wako Chemicals GmbH, Neuss, Germany). Glycated hemoglobin (HbA1c) was measured according to the Standard Operating Procedure of the IFCC Reference, with an automated high-performance liquid chromatographic analyzer (Bio-Rad, Milan, Italy).

The quantitative determination of TBA was performed on the ADVIA 2400 Clinical Chemistry System (Siemens Healthcare, Erlangen, Germany) with the use of the Diazyme Laboratory Total Bile Acids Assay Kit (DZ042A-K; Diazene Lab, Poway, CA, USA) by 2 experienced technicians.

Fasting serum levels of vitamins D2 and D3 were measured as 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, respectively, according to a previously described liquid chromatography-tandem mass spectrometry (LC-MS/MS) method^{21 (link),22 (link)} using deuterated 25-hydroxyvitamin D2 and D3 as internal standards in an API 4000 Tandem Mass Spectrometer (AB Sciex) equipped with a Shimadzu series liquid chromatograph. The fasting level of homocysteine was also measured using an LC-MS/MS method, as described previously.²³ The Analyst, version 1.5, software was used for data collection and quantitative analysis. The hepatic and renal serum profiles were recorded using an Advia 2400 Clinical Chemistry System (Siemens). The serum levels of folic acid and vitamin B12 were measured using an Advia Centaur XP Immunoassay System (Siemens). The accuracy of quality control for low, medium, and high concentrations was between 85% and 115%, and precision was within 15%. Our analysis of the levels of biochemical markers was based on the following reference ranges for healthy subjects ≥65 years of age, which had been established at Shanghai Xuhui District Central Hospital prior to the study period: Creatinine, 40 to 120 μmol/L; folic acid, >5.38 ng/mL; vitamin B12, 0.211 to 0.911 ng/mL; homocysteine, 5.0 to 15 μmol/L; vitamin D2, 2.42 to 22.4 ng/mL; vitamin D3, 10 to 55 ng/mL.

Venous samples were drawn after a minimum fasting period of 12 h. All samples were collected between 06:00 and 07:00 h. The concentrations of serum glucose, total cholesterol, triglyceride (TG), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured by spectrophotometry using an Advia 2400 Clinical Chemistry System (Siemens, USA). Insulin, FT3, FT4, TSH, thyroid peroxidase antibody (TPOAb), and thyroid globulin antibody (TGAb) were determined by a chemiluminescent immunometric assay using a Centaur XP chemiluminescent analyzer (Siemens, USA). The HOMA was used as a measure of insulin sensitivity using the equation = fasting insulin (mU/L) × glucose (mmol/L)/22.5. IR was defined by a HOMA value > 2.7 as suggested by Matthews et al. [16 (link)].