Biochemical measurements

Serum homocysteine, 5-MTHF, leptin and adiponectin levels were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's protocol. The homocysteine ELISA kit was purchased from Cell Biolabs, Inc. (cat no. STA-670; San Diego, CA, USA), the 5-MTHF ELISA kit was purchased from Cusabio Technology LLC (cat no. CSB-E17109h; Houston, TX, USA) while the leptin (cat no. DY398) and adiponectin (cat no. DY1065) ELISA kits were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). To measure homocysteine levels, serum samples were diluted 10-fold. Serum samples were diluted 100-fold to measure the 5-MTHF and leptin levels, and were diluted 8,000-fold to measure adiponectin levels. All dilutions were prepared in phosphate buffer saline containing 0.1% bovine serum albumin (cat no. P3688; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany).

On the other hand, serum BCAAs levels were measured using a commercially available kit purchased from Sigma-Aldrich (Merck KGaA) with leucine used as the standard. The assay used a coupled enzymatic reaction to determine the concentration of all three BCAAs, resulting in a colorimetric product. For all ELISAs performed, the absorbance was measured using an ELx800 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). A wavelength of 450 nm was used for the measurements.