Ilab 600

The blood samples were drawn into heparinized syringes, and plasma was prepared
rapidly at 4°C and immediately frozen at −80°C before analysis.
The plasma glucose, NEFA, and glycerol concentrations were measured enzymatically
using commercially available kits on an ILAB600 or ILAB650 clinical analyzer
(Instrumentation Laboratory UK, Warrington, UK). Cortisol was measured using a
colorimetric assay (R&D Systems, Abingdon, UK). Insulin and C-peptide were
measured using an enzyme-linked immunosorbent assay (Invitron, Monmouth, UK) in an
accredited reference laboratory (Diabetes Research Unit, Swansea University, Swansea,
UK). Interleukin (IL)-6 was measured using an enzyme-linked immunosorbent assay
(Thermo Fisher Scientific, Loughborough, UK).

Antioxidant capacity assays were carried out on the same extracts used for phenolic profiling. The in vitro antioxidant capacity of each maize sample was evaluated as radical scavenging ability (DPPH assay) and ferric reducing power (FRAP assay), as previously described (Ghisoni et al., 2017 (link)). Briefly, 1.5 mL of maize extract was combined to the same volume of an ethanol solution of DPPH (1.0 mM). The absorbance was recorded at 5-min intervals (until the steady state) to 517 nm using a Perkin Elmer Lambda 12 spectrophotometer (Ontario, Canada). The results were finally expressed as gallic acid equivalents (GAE).
The FRAP antioxidant assay was carried out by means of a clinical analyzer ILAB 600 (Instrumentation Laboratory, Lexington, MA, United States). The FRAP working reagent consist of acetate buffer (300 mM, pH 3.6), TPTZ (10 mM) in 40 mM HCl and FeCl3 (20 mM), in the ratio 10:1:1 (v/v). Each extract (100 μL) was combined to 3 ml of FRAP working reagent, and the absorbance was recorded at λ = 600 nm, after 243 s of incubation at 37°C. The FRAP results were expressed as GAE.

Venous blood samples were collected every 30 min into K3 EDTA (for NEFA, ET-1, insulin and C-peptide) or serum tubes (TG, NOx). To limit in vitro lipolysis, the EDTA samples were placed immediately on ice and processed within 30 min [18] (link). For analysis of C-peptide, 500 KIU of apoprotinin (Fisher Scientific, Loughborough, UK) was added per ml plasma to protect against proteolysis. NEFA and TG were quantified using an ILAB 600 (Instrumentation laboratory, Warrington, UK) with kits by Alpha Laboratories (Eastleigh, UK) and Instrumentation Laboratory respectively. ET-1 was measured by ELISA (R&D systems Europe Ltd, Abingdon, UK) and NOx using a NO quantification kit (Actif Motif, Rixensart, Belgium).
Plasma C-peptide and insulin were quantified using a multiplex assay system (Luminex 100, Invitrogen, Paisley, UK) with a Milliplex Endocrine Panel (Millipore Corp, Watford, UK). NEFA composition analysis was performed by extracting lipids from 800 μl of plasma collected at baseline (the two baseline samples were pooled) and 240 min [14] (link).

Total cholesterol and triglycerides were determined using kits purchased from Instrumentation Laboratory (IL Test) and analyzed at 37 °C by a clinical auto-analyzer (ILAB 600, Instrumentation Laboratory, Lexington, MA). ROMt were measured by commercial kits (Diacron International s.r.l., Grosseto, Italy) by clinical auto-analyzer (ILAB 600). Haptoglobin was determined using the method described by Calamari et al [63 (link)] adapted to the ILAB 600 conditions. Serum amyloid A protein was assayed using an ELISA commercial kit (SAA, by ELISA, Tridelta, Ireland) and a microplate reader (The SynergyTM 2 BioTek Instruments, Inc., Winooski, Vermont, USA).

Blood metabolites were analyzed in lithium heparin samples at 37°C following the procedures previously described by Bionaz et al. [9 (link)] in a clinical auto-analyzer (ILAB 600, Instrumentation Laboratory, Lexington, MA, USA). Glucose, NEFA and BHBA were determined using commercial kits purchased from Instrumentation Laboratory (IL Test), Wako Chemicals GmbH (Neuss, Germany) and Randox Laboratories Ltd. (Crumlin, Co. Antrium, UK) respectively. Adiponectin was measured in serum samples using the ELISA assay developed by Mielenz et al. [10 (link)], while insulin was assayed by a double-antibody radioimmunoassay (RIA) using a primary antiserum to bovine insulin as described by Sosa et al. [11 (link)]. The ratios of NEFA and glucose to insulin were calculated to estimate systemic insulin sensitivity.

Blood samples were obtained from the coccygeal vessel at 0900 h on d −21, −14, −7, 7, 14, 21, and 28 relative to parturition, and immediately after parturition. Blood was taken into evacuated serum separator tubes containing clot activator and into evacuated plasma tubes containing ethylenediaminetetraacetic acid. Tubes for plasma preparation were put on ice immediately after sampling and centrifuged at 3,000×g for 15 min at 4°C. Tubes for serum preparation were allowed to clot for 30 min at room temperature before centrifugation for 15 min at 2,000×g at 4°C. Plasma and serum were aliquoted into 1.5-mL centrifuge tubes and stored at −20°C until analysis. Plasma samples were sent to a clinical laboratory of the 309th Hospital (Beijing, China) for analysis of glucose, NEFA, and plasma urea nitrogen (PUN) using an autoanalyzer (ILAB 600, Instrumentation Laboratory, Lexington, MA, USA) with kits supplied by Instrumentation Laboratory. Plasma concentrations of β-hydroxybutyrate (BHBA) were measured using a BHBA kit (Sigma-Aldrich Chemical Co., Shanghai, China). Radioimmunoassay kits were used to measure serum insulin (Coat-A-Count Insulin, Siemens Medical Solutions USA Inc., Malvern, PA, USA) and plasma glucagon (Double Antibody Glucagon, Siemens Medical Solutions USA Inc., USA).

Concentrations of serum total cholesterol, HDL-cholesterol, triacylglycerol, NEFA, glucose, and CRP were evaluated by iLab 600 (all kits and equipment from Instrumentation Laboratory, Warrington, UK).