Quantifying Serum Vitamin and Homocysteine Levels

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.