2.7. Blood Sampling and Biochemical Analyses
Anemia
BLOOD, ANEMIA, LOCAL ANAESTHETICS, EMD
Venous blood samples were collected at baseline and at four and six months of age at least 2 h postprandially. Local anaesthetic topical cream was used prior to sampling. Hemoglobin (Hb) was analyzed within a few hours at the University and Regional Laboratories of Skåne or at Norrland´s University Hospital on a Sysmex XN-10 instrument (Sysmex Corporation, Chuo-ku, Kobe, Japan). Blood was centrifuged at 1300× Serum iron was analyzed on a Cobas 701 instrument (Roche Diagnostics, Basel, Switzerland), serum C-peptide, insulin, and ferritin on a Cobas 601 instrument (Roche Diagnostics), and serum IGF-1 by the IDS-iSYS assay (Immunodiagnostic System Ltd., Boldon, Tyne & Wear, England). All analyses were carried out at the University and Regional Laboratories of Skåne. Anemia was defined as Hb < 90 g/L at baseline and <105 g/L at four and six months of age. Iron depletion was defined as serum ferritin < 40 µg/L at baseline, <20 µg/L at four months, and <12 µg/L at six months [Serum leptin and leptin receptor were analyzed by ELISA (Human Leptin ELISA kit, EMD Millipore; Merck KGaA, Germany and Human Leptin R QuantikineSamples from the subpopulation of 200 infants were analyzed for blood urea nitrogen (BUN) using a urea nitrogen colorimetric detection kit (Life Technologies Corporation, Fredrick, MD, USA) according to the manufacturer´s instructions at the University of California, Davis (UCD). In the same subgroup, serum amino acids were analyzed by ion exchange column chromatography (IEC). Samples were acidified to 2% sulfosalicylic acid (SSA) final concentration and then diluted with 100 nmol/mL AE-Cys Li diluent prior to the 50 ul injection. Free amino acids were separated using ion-exchange chromatography with post-column ninhydrin reaction. The IEC instrument (Hitachi 8900), buffers and column were from Hitachi Tokyo, Japan. Calibration was performed using amino acid standards (Sigma-Aldrich, St. Louis, MO, USA). The included reference standard (AE-Cys) was used to correct for any variances in injection volume due to the autosampler [