In a retrospective mono-centered approach, we analyzed the routine laboratory database (2003-2006) of the University Hospital Bonn, Germany using data that were made anonymous. We selected all in- and outpatients in whom vitamin B12 and folate as well as lipoproteins had been determined (table). Serum triglycerides (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL) and low density lipoprotein cholesterol (LDL-C) were measured on the Dimension RxL clinical chemistry system (Dade Behring, Schwalbach, Germany) with enzymatic methods using the manufacturer's reagents and instructions.
Hcys was determined by means of fully automated particle-enhanced immunonephelometry with a BN II System (Dade Behring). Bound homocysteine in the heparinized plasma sample is reduced to free homocysteine by the action of dithiothreitol, and then converted enzmatically to S-adenosyl-homocysteine (SAH) in the next step. Conjugated S-adenosyl-cysteine (SAC), added at the onset of the reaction, competes with SAH in the sample for bonding by anti-SAH antibodies bound to polystyrene particles. The result is evaluated by comparison with a standard of known concentration.
Serum concentrations of vitamin B12 and folate were measured by means of a competitive chemiluminescent immunoassay with an Accessâ„¢ Immunoassy System (Beckman Coulter, Krefeld, Germany) according to the manufacturer's instructions.
Due to anonymous data extraction, information on diagnosis and diet were not available.
Because some parameters were not normally distributed, log transformation was performed for statistical analysis. Multiple linear regression analysis with α = 0.01 (multiple testing of four different lipoproteins) was used to simultaneously analyze independent associations of age, gender, folate and vitamin B12 on the lipoprotein levels as dependent variables. We used stepping mode criteria with a probability of 0.05 for entry and of 0.10 for removal from the equation. Due to the low number of patients for whom homocysteine was known, homocysteine was not included as covariable in the multivariate model. Instead, bivariate Pearson's analysis was calculated. The lipoproteins values of folate and vitamin B12 quartiles were compared by ANOVA. Since this analysis was done following the results of the regression analysis, we did not correct for multiple testing (α = 0.05).