Fa115

Insulin, glycaemia, C-peptide and lipids were measured by standard methods (Modular P800, Roche, Milan). LDL-C levels were measured using the Friedewald formula [total cholesterol -(HDL + (TG/5)]. AIP was calculated as TG/HDL-C log. HbA1c levels were determined by HPLC with an ion-exchange resin (Bio-Rad Laboratories, Milan, Italy).
ACTH and UFC levels were detected by electrochemiluminescence immunoassay (ECLIA, Elecsys, Roche, Milan) following the manufacturer's instructions. Normal values for hormonal markers were defined as follows: ACTH 2.2-14 pmol/L and UFC 59-378 nmol/24 h.
Human leptin (ng/ml), Ob-R (ng/ml), adiponectin (µg/ ml), resistin (ng/ml), visfatin (ng/ml) and AFABP (ng/ml) were assayed using an ELISA sandwich enzyme immunoassay (BioVendor, Heidelberg, Germany). NEFAs (mmol/L) were assayed by the colorimetric method (FA115, Randox Laboratories, County Antrim, UK).
The conversion factors for the International System (SI) were as follows: glucose mg/dl vs. mmol/l: 0.0555; insulin mUI/ml vs. pmol/l: 6.945; total cholesterol and HDL-C mg/dl vs. mmol/l: 0.0259; triglycerides mg/dl vs. mmol/l: 0.0113; HbA1c % vs mmol/mol: 10.93% -23.5;cortisol µg/dl vs. nmol/l: 27.59, UFC µg/24 h vs. nmol/24 h: 2.76; ACTH pg/ml vs. pmol/l: 0.22.

Plasma concentrations of nonesterified fatty acids (NEFA) and BHB were measured by colorimetric and enzymatic methods, respectively (kit no. FA115 and RB1007; Randox Laboratories Ltd., Crumlin, UK). The interassay CV for NEFA and BHB were 8.5 and 8.3%, respectively. An autoanalyzer (Technicon Instruments Corp., Chauncey, NY) was used to measure concentrations of plasma glucose (Bran and Luebbe industrial method 339-19; Gochman and Schmitz, 1972) . The intra-and interassay CV were 2.2 and 12.7%, respectively.

Blood samples were collected via jugular venipuncture at 24 ± 1 h of age (after colostrum feeding) and at d 7, 14, 21, 28, 35, 42, 49, and 56 between 0900 and 1000 h. Blood was obtained using serum and sodium heparin vacutainer blood collection tubes (BD catalog #366430 and 366480, Franklin Lakes, NJ). After collection, sodium heparin tubes were placed on ice and serum tubes clotted for 20 min at room temperature. Within 1 h, all tubes were centrifuged at 3,000 × g for 20 min to harvest plasma and serum fractions. Blood hematocrit was analyzed from whole blood, and total protein was quantified from fresh plasma using a digital Brix Refractometer (MA871, Milwaukee Instruments, Rocky Mount, NC). For hematocrit, capillary tubes (Fisher Scientific #22-362-566, Pittsburgh, PA) filled with whole blood were centrifuged at 2,240 × g at 25°C for 3 min in a microhematocrit centrifuge and read using a circular hematocrit reader. Remaining serum and plasma samples were aliquoted and stored at -20°C until further analysis. Plasma concentrations of fatty acids, BHB, and glucose were determined by colorimetric, enzymatic, and GOD-PAP methods, respectively (kit #FA115, RB1007, and GL3981, Randox Laboratories Ltd., Crumlin, UK) by Randox Rx Daytona. The interassay coefficients of variation for fatty acid, BHB, and glucose analysis were 11.4, 5.7, and 8.7%, respectively.