Au480 autoanalyzer

Throughout the study plasma/serum was prepared by centrifuging at 3000 rpm for 10 min at 4 °C (using a GS-6R centrifuge; Beckman, Fullerton, CA, USA). The resulting plasma/serum was stored at −20 °C until analyzed at the completion of the study. All samples from each subject were analyzed within the same analytic run. Samples were analyzed in two batches.
Fasting blood lipids were measured in two consecutive blood samples at baseline (day 1, 2) and end of the 6-week intervention (day 43, 44). Postprandial blood lipids were measured at the end of intervention (day 44) at timepoints −15 min, −5 min and +15 min, +30 min, +45 min, +60 min, +90 min, +120 min, +180 min and +240 min after the mixed meal challenge. Serum TC, LDL-C and HDL-C and TG, and plasma glucose concentrations, were measured using commercial enzymatic kits (Roche Diagnostics, Basel, Switzerland) on a Hitachi 902 auto-analyzer (Roche Diagnostics, Indianapolis, IN). Plasma Lp(a) concentrations were measured using a commercial kit (Randox Pty Ltd, Parramatta, NSW, Australia) on a Beckman AU480 auto-analyzer (Beckman, Fullerton, CA, USA). Serum insulin was measured using a commercial ELISA kit (Mercodia AB, Uppsala, Sweden). All blood measurements were carried out at CSIRO. Non-HDL-C (TC minus HDL-C) and remnant C defined as TC minus (HDL-C and LDL-C) were calculated.

The clinical chemistry analyses of circulating biochemical parameters in blood was performed using a clinical chemistry analyzer (AU480 autoanalyzer, Beckman Coulter, Krefeld, Germany). Fasting plasma lipid and glucose levels at 11–12 weeks of age and a broad set of parameters from fed animals at 19–20 weeks were measured using the respective kits provided by Beckman Coulter, including various enzyme activities as well as plasma concentrations of specific substrates and electrolytes in ad libitum fed mice (50 (link)).

Blood samples were taken from isoflurane-anesthetized mice by puncturing the retro-bulbar sinus with non-heparinized glass capillaries (1.0 mm in diameter; Neolab, Heidelberg, Germany). Blood samples were collected in heparinized tubes (Li-heparin, KABE, Nümbrecht, Germany). Each tube was immediately inverted five times to achieve a homogeneous distribution of the anticoagulant. Li-heparin-coated tubes were stored at room temperature for 1 to 2 hours. Afterwards, cells and plasma were separated by a centrifugation step (10 min, 5000 g, 8 °C). Plasma samples were then transferred into an Eppendorf tube and diluted 1:2 with distilled water. The solution was mixed for a few seconds to prevent clotting and was then centrifuged again (10 min, 5000 g, 8 °C). Lactate and urea measurements were performed using a Beckman-Coulter AU 480 autoanalyzer and reagents from Beckman-Coulter (Brea, CA, USA).

Clinical-chemical parameters in serum were determined using a Cobas 311 system (Hitachi) or an AU480 autoanalyzer (Beckman-Coulter) and adapted reagents from Roche Diagnostics or Beckman-Coulter, respectively. Serum GH concentrations were measured by an ELISA for rat/mouse GH (EZRMGH-45K; Merck) that cross-reacts with porcine GH. To calculate the area under the GH curve, values below the quantification limit of the assay (<0.07 ng/mL) were arbitrarily set to 0.07 ng/mL. IGF1 levels in serum were determined by RIA after dissociation of IGF1 from IGFBPs by acidification and blocking the IGF1 binding sites with an excess of IGF2 [25] (link). IGFBP ligand blot analysis of serum samples was performed as described previously [26] (link) using serial dilutions of recombinant human IGFBP3 (41/38 kDa), IGFBP2 (32 kDa), IGFBP5 (29 kDa) and IGFBP4 (24 kDa) for quantification. Plasma insulin was determined using a species-specific RIA (Merck Millipore) as previously described [27] (link). Blood glucose levels were determined immediately using a Precision Xceed^® glucometer and Precision XtraPlus^® test strips (Abbott) [28] (link). Serum leptin levels were measured using a multi-species leptin radioimmunoassay (Cat. # XL-85K; EMD Millipore Corporation) that has been validated for porcine samples [29] (link).

Participants with a history of CVD or its treatment, pregnant women and those with incomplete data were excluded from study participation. Anthropometric measurements performed were weight, height and WC. Weight (kg) was measured to the nearest 0.1 kg with the respondent wearing light clothes and without shoes using the Omron-B100 scale. Height (m) was measured to the nearest 0.1 m using a stadiometer. Participants were requested to stand vertically on the stadiometer without shoes. Body mass index (BMI) was calculated dividing weight in kilograms by height measured in meters squared. Waist circumference (WC) (cm) was measured to the nearest 0.1 cm at the level of the iliac crest while the participant was at minimal respiration.
Carotid Intima-Media Thickness measurements were done using a LOGIQe ultrasound system (GE Healthcare, CT, USA), according to detailed procedures and methods that were previously published by Ali et al., 2018. For CVD risk factors, Blood pressure was measured using a digital sphygmomanometer (Omron M6, Omron, Kyoto, Japan), detailed procedure [30 (link)]; fasting blood glucose and lipids were determined using the AU480 auto analyzer from Beckman Coulter.

The homeostasis of serum thiol-disulfide was measured using a fully automated method described by Erel and Neselioğlu [18 (link)]. The measurements were carried out using fully automated native thiol, total thiol kits (Rel Assay DC, Gaziantep, Turkey) in a Beckman Chemistry Coulter AU480 autoanalyzer. This method is based on the borohydride-mediated reduction of dynamic disulfide bonds to functional thiol groups. Reducible disulfide bonds are first reduced to functional thiol groups in free form using sodium borohydride (NaBH₄). Excess NaBH₄ is consumed via formaldehyde and is removed from the medium. All thiol groups, the reduced ones and native thiols are reacted with 5,5′-dithiobis-2-nitrobenzoic (DTNB) acid to undertake the measurements. Half of the difference between the total thiol and native thiol yields the amount of dynamic disulfide. After the native thiol, total thiol and disulfide concentrations were detected; disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios were computed. The results were expressed as µmol/L.