Cobas b 101

Confirmation of eligibility, informed consent, and randomisation is conducted at the first assessment visit. Comprehensive information is obtained from the woman and her clinical records, including: demographic, socioeconomic, educational and employment data; medical history; obstetric history such as parity, method of conception, gestation and accuracy of estimated date of delivery, previous pregnancy complications (GDM, hypertensive disease, pre-term delivery, caesarean section); family history of diabetes, hypertension and cardiovascular disease; history of smoking, alcohol and other drug use; medications and nutritional supplements; probiotic food ingestion; maternal anthropometrics: weight, height, waist and mid-arm circumference; blood pressure (BP); finger-prick blood lipid testing (Roche cobas b 101 point-of-care system); samples collection : non-fasting blood and urine specimen for biobank; questionnaires including: New Zealand Food Frequency Questionnaire - Short Form [59 (link)], New Zealand Physical Activity Questionnaire [60 ], Edinburgh Postnatal Depression Scale (EPDS) [61 (link)], State-Trait Anxiety Inventory (STAI) [62 (link)], and the 12-item Short-Form Health Survey (SF-12) [63 ]. It takes approximately 30 min to complete these questionnaires.

A finger prick test using a blood glucometer (FreeStyle Freedom Lite) was performed within a 15 min window preceding the MOXO test, which itself lasted a similar time-frame as mentioned below. HbA1c was measured simultaneously using the Cobas B 101 (Roche) point of care system.

In the ILERVAS cohort, a fasting dried blood spot sample was obtained by a fingertip puncture according to standard protocols. Creatinine, uric acid, and total cholesterol levels were assessed with the REFLOTRON^® Plus system (Roche Diagnostics, Germany). It is a validated clinical chemistry system with highly correlated results to well-standardized laboratory methods (21 (link)–23 (link)). The glycosylated hemoglobin test was performed using a point-of-care instrument (Cobas B101^®, Roche Diagnostics, Germany) that meets the generally accepted performance criteria for its measurement (24 (link)). In the NEFRONA cohort, biochemical parameters were obtained from a routine fasting blood test taken no more than 3 months apart from vascular examination. GFR was estimated according to international guidelines using the CKD-EPI equation in both cohorts (25 (link)).

Upon collection, blood samples (EDTA) were inverted and blood lipids (triglycerides, total, HDL, and LDL cholesterol) were analyzed in whole blood at the time of collection using a Cobas b 101 instrument (Roche Diagnostics Ltd., Basel, Switzerland). At Visits 3 and 7, glycated haemoglobin (HbA1c) was also measured from whole blood using the Cobas b 101 instrument prior to centrifugation. The remaining samples were spun at 1800× g for 10 min at 4 °C to obtain plasma samples which were then frozen at −80 °C for later analysis. Glucose concentrations were measured in duplicate from thawed plasma samples using YSI 2900 analyzer (YSI Life Sciences, Yellow Springs, OH, USA; coefficient of variation (CV) < 1.0%). Insulin concentrations were measured using thawed plasma with a commercially available ELISA (Alpco Ltd., Windham, NH, USA), in duplicate with an intra-assay CV of 3.8%. Total area under the curve (AUC) was calculated for venous glucose and insulin concentrations using the trapezoid method using GraphPad Prism (Version 7.01, GraphPad Software Inc., San Diego, CA, USA).

We sampled venous blood before breakfast and after dinner, at baseline (Visit 1), after 5 days of HFD (Visit 2), and after a further 5 days on the HFD, either with daily exercise or no exercise (Visit 3) (Fig. 6). The participants fasted from 22:00 h the night before the blood sampling on all visits, with blood obtained between 07:15 and 07:45. The evening (postprandial) samples were obtained 34 (SD 7) minutes after eating dinner, between 19:20 and 20:00 h. The time since the last bout of exercise at Visit 3 was (by design) different between the EXam and EXpm group: For fasted samples this time was ⁓24 h for EXam and ⁓12 h for EXpm, and for postprandial samples the time was ⁓36 h for EXam and ⁓24 h for EXpm. Circulating concentrations of total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides were analysed in whole blood using Cobas b 101 (Roche Diagnostics Ltd, Switzerland), and have been reported previously^{15 (link)}.