Nonfasting blood samples were obtained from each participant during the first visit between weeks 10–13th of pregnancy and analysed in a central laboratory (Queen’s University, Belfast). All participants were instructed by the interviewers not to consume fruits, vegetables or juice for breakfast on the day of the blood test. A thorough protocol was designed to collect, transport and measure the blood samples for vitamin C, E, B12, folate and carotenoids. Blood samples were separated by centrifugation and stored at -80°C. The blood samples for vitamin C determination were collected at clinical examination under subdued light, wrapped in tin foil, stabilized with meta-phosphoric acid and placed in insulated dry containers at 4°C to exclude light and, therefore, avoid vitamin C degradation. Blood samples packed in dry ice were shipped to the central laboratory by dedicated couriers. Plasma cholesterol was measured to adjust carotenoid concentrations. Folate and vitamin B12 concentrations in serum were measured using a commercially available radioassay (SimulTRAC-SNB ICN Pharmaceuticals, California, USA). Serum carotenoids were measured by HPLC with diode array detection as described by Craft [21 (link)]. Lutein+zeaxanthin plasma concentrations were combined as information for these nutrients is combined in the main food composition tables. Serum concentrations of α-tocopherol were measured by high-performance liquid chromatography (HPLC) with UV detection at 292 nm [21 (link)]. Plasma vitamin C was measured using an ascorbate oxidase-based assay as described by Vuillemier & Keck [22 ]. The inter-assay CV were <10.0% and intra-assay CV<5.0% for all species. The assays were standardized against the appropriate National Institute of Standards and Technology standard reference materials.
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