Nefa hr kit

Plasma free fatty acid concentration was measured using an NEFA‐HR kit according to the manufacturer (WAKO Diagnostics GmbH, Germany).
Plasma triglyceride content was determined as the amount of free glycerol produced by hydrolyzation of triglycerides by ethanolic KOH. From each sample, free glycerol was measured using a Free Glycerol Reagent (Sigma Aldrich, Denmark) leading to the production of a quinoneimine dye that was spectrophotometrically detected at 540 nm in a Multiskan (Multiskan FC, Thermo Scientific). A standard curve was generated from a serial dilution of a Glycerol Standard Solution (Sigma Aldrich, Denmark) and used to convert the absorbance of the samples to a free glycerol concentration. The free glycerol concentration was finally converted to triglyceride content according to the manufacturer.

Frozen gastrocnemius muscles were pulverized using a pulverizor (Bio Spec Products Inc.) in liquid nitrogen and weighed. For the Non-Esterified Fatty Acids (NEFA) and Triglyceride (TAG) assay, lipids were extracted using the Bligh & Dyer method [13 (link)]. The organic phase was dried at 50°C and reconstituted in 0.5% Triton X-100 solution. NEFA and TAG were measured using a NEFA-HR Kit (Wako) and a Triglyceride Quantification Kit (BioVision K622-100). For the acylcarnitine assay, 6 volumes of 80% acetonitrile were added to pulverized tissue weight (about 50 mg). Tissue mixtures were sonicated 10 times, and centrifuged at 12,000 rpm 10 min at 4°C. The isolated supernatants were then dried under a stream of nitrogen at 40 °C and resuspended in 100 µl of 50% acetonitrile. The acylcarnitine content was measured by using electrospray ionization tandem mass spectrometry [14 ]. Ceramide content was measured by using high-performance liquid chromatography/mass spectrometry in the Medical University of South Carolina Lipidomics Core as previously described [15 ]. Analytical results were normalized to total protein.

Plasma insulin, glucagon, cortisol, and c-peptide (Millipore, St. Louis, USA) were measured by the Vanderbilt Diabetes Research and Training Center’s (DRTC) hormone assay and analytical services core. Plasma glucose was measured using the glucose oxidase method (ref. ²² ; Beckman Instruments). Glycerol and lactate^{23 (link)} and plasma specific activity^{20 (link)} were measured as described previously and free fatty acids were measured using a commercially available kit (NEFA-HR kit; Wako Chemicals; Osaka, Japan).

CDA-Diagnostic Laboratory (São Paulo, Brazil) performed blood leukocytes, red blood cell counting, hematocrit, hemoglobin using the Sysmex XT 2000 equipment (Kobe, Japan). Plasma creatine phosphokinase (CPK) activity was determined by using Cobas C-311 analyzer (Roche, CA, USA). Plasma concentration of non-esterified free fatty acids (NEFAs) was determined with the NEFA HR Kit (Wako Chemical, Neuss, Germany). This enzymatic and colorimetric assay was performed following the manufacturer’s instructions, as previously reported (30 (link), 40 (link)). Testosterone and cortisol were evaluated using a high-affinity monoclonal antibody that binds specifically against testosterone (41 (link)) or cortisol (42 (link)), following the manufacturer’s instructions (Elecsys, Roche Diagnostics, Penzberg, Germany).

Plasma levels of FFA were evaluated using NEFA-HR kit (Wako Diagnostics, Richmond, VA, USA). Plasma samples (3 µL) were transferred to a 96-well plate and reagents (200 µL) were added following the manufacturer’s instructions. Measurements of absorbance were carried out at 450 nm. Plasma TG concentrations were determined by enzymatic colorimetric assay following the manufacturer’s protocol (Bioclin^®, Belo Horizonte, MG, Brazil).
For determination of TG and FFA levels in the LV, the heart tissue was homogenized in chloroform and methanol solution (2:1). The lower phase was then transferred to a new tube and evaporated in a vacuum centrifuge. Lipid pellets were suspended in 100 µL absolute ethanol. The levels of FFA and TG were determined as described above.