Blood sampling and you may analyses
Samples in the VIP were collected in the morning, and only 12 of 825 study subjects (1.5%) had fasted <4 hours, and 146 (18%) <8 hours. In the MSP, blood samples were collected throughout the day, and 163 of 168 study subjects (97%) had fasted for less than 4 hours. The blood samples used in the present study were collected in EDTA sample tubes, separated into plasma, buffy coat, and erythrocyte fractions, aliquoted, and cryopreserved at ?80°C. Samples are frozen within 1 hour of collection, either at ?80°C or at ?20°C for up to 1 week before transfer to a ?80°C freezer at the Northern Sweden Medical Biobank for long-term storage. All biochemical analyses were performed at Bevital AS, Bergen, Norway (27). Folate and cobalamin concentrations were determined with a microbiologic method, using Lactobacillus casei for folate and Lactobacillus leichmannii for cobalamin, adapted to a microtiter plate format, and carried out by a robotic workstation. Concentrations of total homocysteine were measured with an isotope dilution gas chromatography–mass spectrometry method (28). Concentration of methionine sulfoxide was determined using a liquid chromatography-tandem mass spectrometry method. The coefficients of variation for serum folate and cobalamin are 5%, whereas the coefficients of variations for homocysteine and methionine/methionine sulfoxide are <5% (29). The investigators and laboratory staff were blinded to case and control status.
Analytical analyses
Mann–Whitney and ? 2 tests were used to compare baseline characteristics and study variables in cases and controls. Odds ratios (OR) for disease and 95% confidence intervals (CI) were calculated by conditional logistic regression. In this report, plasma metabolite tertiles were used instead of quintiles, used in our previous report, to preserve power in subgroup analyses. Performance for quintiles are available in Supplementary Table S1 (9). Because cases and controls were matched by cohort, tertile cutoffs were not stratified for cohort. Tests for trend were performed by including tertiles (labeled with sex-specific tertile medians) as continuous variables in regression analyses. Covariates for the multivariate analysis were selected to reproduce the procedure of our previous study (9). Variables included were body mass index (BMI; <25, 25–30, and >30 kg/m ?2 ), smoking (current, not current smoker), intake of alcohol (sex-specific quartiles), and recreational and occupational physical activity (self-reported on a scale from 1 to 5). The selected covariates are all established risk factors for colorectal cancer (30). Stratified analyses were also performed for sex, age at recruitment (divided at 59 years to approximate the median but to avoid dividing the large sampling cluster at 60 years), time from recruitment to diagnosis (divided at the median of 10.8 years), tumor site (right and left colon, and rectum), tumor stage (stage I–II and III–IV), and cohort (VIP and MSP). Missing values in covariates were treated as dummy variables in multivariate analyses. Heterogeneity between results of the subgroup analysis was investigated by a ? 2 -based test. Statistical tests and corresponding P values were two sided and P values <0.05 were considered statistically significant. IBM SPSS Statistics version 21.0 (IBM Corporation) was used for all statistical analysis.
Results
All in all, 331 circumstances and 662 controls had been utilized in the analysis. You to definitely control lacked analysis getting plasma concentrations away from vitamin B12. Standard functions regarding cases and you can control and cyst properties are offered from inside 
the Dining table step one. Average ages during the recruitment are 59.eight both for circumstances and you may controls. By the MSP cohort, female were some overrepresented. Sufferers about MSP were old on employment [average years within employment, 64.six decades (25th–75th percentile, 58.5–67.8 ages) compared with 59.5 years (25th–75th percentile, fifty.0–60.0 age) to the VIP]. Right-sided colon cancer was also more widespread about MSP (forty eight.2% away from tumors, unlike 33.5% from the VIP). Total, no tall variations in standard services were observed between cases and control, no matter if plasma supplement B12 levels was in fact quite high when you look at the controls (P = 0.055). Average go out off blood draw to prognosis out-of instances is 10.8 decades, which have a range of 0.step 3 to help you 19.8 age.