Venous whole blood samples were collected after an overnight fasting into Vacutainer® tubes either containing EDTA or lithium/heparin as anticoagulants, to measure biochemical variables and for extracting DNA from peripheral blood mononuclear cells.
After centrifugation at 1500 g for 10 min at room temperature, lithium heparin plasma was separated, stored in aliquots and kept frozen at −70°C until measurement. Fe was determined by the routine method used in the local laboratory (Roche Diagnostics). Ferritin concentration was measured with an automated chemiluminescence method, on Roche Cobas e801 (Roche Diagnostics). DNA was extracted from peripheral blood mononuclear cells by Wizard Genomic DNA Purification Kit (Promega Corporation). Genotyping for one-carbon-related polymorphisms (MTHFR 677C > T, cSHMT 1420C > T, DHFR 19bp ins/del, RFC1 80G > A) was analysed by different methods as previously described(14 (link)).
A plasma Fe concentration > 10·74 μmol/l and ferritin range values 20–200 µg/l were considered as adequate concentrations(15 (link)).
After centrifugation at 1500 g for 10 min at room temperature, lithium heparin plasma was separated, stored in aliquots and kept frozen at −70°C until measurement. Fe was determined by the routine method used in the local laboratory (Roche Diagnostics). Ferritin concentration was measured with an automated chemiluminescence method, on Roche Cobas e801 (Roche Diagnostics). DNA was extracted from peripheral blood mononuclear cells by Wizard Genomic DNA Purification Kit (Promega Corporation). Genotyping for one-carbon-related polymorphisms (MTHFR 677C > T, cSHMT 1420C > T, DHFR 19bp ins/del, RFC1 80G > A) was analysed by different methods as previously described(14 (link)).
A plasma Fe concentration > 10·74 μmol/l and ferritin range values 20–200 µg/l were considered as adequate concentrations(15 (link)).
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