Enzychrom ketone body assay kit

Blood glucose was measured by glutest sensor (Sanwa Kagaku, Aichi, Japan). Serum levels of triglyceride (Triglyceride E‐test, Wako Chemical, Osaka), NEFA (NEFA C‐test Wako Chemical), glycerol (Free Glycerol colorimetric/Fluorometric assay kit, Biovision, USA), β‐hydroxybutyrate (EnzyChrom Ketone Body Assay Kit, BioAssay Systems, CA), and lactate (Lactate colorimetric Assay Kit II, Biovision) were measured according to the manufacturer's protocols.

Weight, height, and samples for metabolomic studies were obtained during the first visit at early pregnancy stages (before 18 weeks of gestation) by trained personnel using standardized techniques. Venous blood samples were collected in the morning after overnight 8 h fasting and serum was kept at 2 to8 °C, centrifuged at 1500 ×g for 15 min, and stored at − 70 °C until their processing at the Instituto Nacional de Medicina Genómica (National Institute of Genomic Medicine, INMEGEN) in Mexico City, Mexico. Serum levels of glucose, triglycerides, and total cholesterol were assessed by an automatic chemistry analyzer (Advia 1800 Siemens, Malvern, PA, USA), while β-Hydroxybutyric acid was determined by EnzyChrom Ketone Body Assay Kit (BioAssay Systems, Hayward, CA) and insulin was determined by ELISA (Human Insulin ELISA, ALPCO, Salem, NH). All methods were standardized with internal controls and an external quality program. HOMA- IR was calculated according to the following formula: HOMA-IR = fasting insulin (µUI/mL) × fasting glucose (mmol/L)/22.5. HOMA-β was calculated according to the following formula: HOMA-β = 20 × fasting insulin (µUI/mL)/(fasting glucose (mmol/L)—3.5) [29 (link), 30 (link)].

Plasma samples were obtained from whole blood collected in EDTA-containing vacutainers post 2000g × 10’ at 4°C and stored in −80°C. Plasma concentrations of cytokines were determined by ELISA following manufacturer’s protocol (Biolegend): IL-1B (Cat: 437004), TNFα (Cat: 430204), IL-6 (Cat: 430504), and IL-10 (Cat: 430601). Plasma concentrations of β-hydroxybutyrate and acetoacetate were determined by EnzyChrom^™ Ketone Body Assay Kit following manufacturer’s protocol (BioAssay Systems, Cat: EKBD-100).

Serum levels of glucose (glutest sensor, Sanwa Kagaku, Aichi), triglyceride (Triglyceride E-test, Wako Chemical, Osaka), non-esterified fatty acid (NEFA C-test Wako Chemical, Osaka) and β-hydroxybutyrate (EnzyChrom Ketone Body Assay Kit, BioAssay Systems, California) and insulin (mouse Insulin ELISA, Mercodia, Sweden) were measured according to the manufacturer’s protocols^{18 (link),19 (link),37 (link)}.

Whole blood was collected from the anesthetized mice via cardiac puncture and centrifuged at 1200× g for 10 min and the serum was aliquoted and stored at −25 °C. The serum was subjected to measurements of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatine phosphokinase (CPK), total bilirubin (T-bil), blood urea nitrogen (BUN), and glucose (GLU) using Spotchem II Stat-1 and Spotchem II Glucose reagents (Arkray, Shiga, Japan) and an automatic biochemical analyzer Spotchem EZ SP-4430 (Arkray). The blood insulin was quantified using Mouse/Rat Insulin ELISA Kit (Morinaga, Tokyo, Japan) according to manufacturer’s instruction. Ketone body was determined using EnzyChrom Ketone Body Assay Kit (BioAssay Systems, CA, USA). Serum glucagon and non-esterified fatty acid (NEFA) were quantified using Glucagon ELISA kit (Mercodia, Uppsala, Sweden) and LabAssay NEFA kit (Wako, 279-75401), respectively, according to the manufacturer’s instruction. Lipoprotein profile of serum was performed by LipoSEARCH service (Immuno-Biological Laboratories, Gunma, Japan) to measure the cholesterol and triglyceride in chylomicron, VLDL, LDL, and HDL.