Nefa c

Non-esterified fatty acid (NEFA, mmol/L) concentrations were analyzed in duplicate, using an in vitro enzymatic colorimetric method (NEFA-C^®, Wako Chemicals GmbH, Neuss, Germany). NEFA in samples was converted to Acyl-CoA by the action of Acyl-CoA synthetase, under the coexistence with coenzyme A. Obtained Acyl-CoA was oxidized and yielded hydrogen peroxide by the action of Acyl-CoA oxidase. In the presence of peroxidase, the hydrogen peroxide formed yields a blue purple pigment. NEFA concentration was obtained by measuring absorbance of the blue purple colour.
Duplicate aliquots of plasma for the sample tube were assayed. The leptin concentrations were measured by RIA antibody using the multi-species leptin kit (XL-85K, Linco Research Inc.^®, St. Charles, MO, USA). The detection limit was 1.0 to 50.0 ng/mL Human Equivalents (HE). The 17β-estradiol and progesterone concentrations were assayed using a commercial 125I RIA kit (07-238102 and 07-270102, respectively; ICN Pharmaceuticals Inc.^®, Diagnostic Division, Costa Mesa, CA, USA). The detection range was 10 to 3000 pg/mL and 0.15 to 80.00 ng/mL, respectively. Intra and inter-assay coefficients of variations were <5% for all hormones.

Plasma insulin was measured with an ELISA kit (Millipore). NEFA, TG, and cholesterol concentration in serum were measured with NEFA-C and Triglyceride E tests (Wako), respectively. Serum adiponectin and leptin levels were measured with ELISA kits from R&D Systems.

Blood samples collected retro-orbitally were left to clot for 30 min at room temperature in non-coated polypropylene tubes. Samples were then centrifuged at 10,000× g for 5 min at 4 °C, and the resulting serum supernatant was aliquoted and stored at −80 °C. Hormone and lipid concentrations were assessed in these aliquots using leptin (Abcam, Cambridge, UK), glucagon, and ultra-sensitive mouse insulin (Crystal Chem, Elk Grove Village, IL, USA) ELISA kits, and NEFA-C (Wako Pure Chemical Industries, Osaka, Japan) and TAG (Abcam) colorimetric assay kits.

We enrolled 24 male T1D patients with an HbA1c > 7.0% (53 mmol/L) and 24 age-matched male healthy controls. Participants were all between 20 and 70 years old. For T1D patients, the minimal duration of diabetes was 1 year. Patients using medication other than insulin were excluded. HbA1c was measured by standard laboratory methods. Plasma insulin was measured by radioimmunoassay [9 (link)]. Plasma cholesterol, triglyceride (TG), glucose (Liquicolor; Human GmbH, Wiesbaden, Germany) and free fatty acids (NEFA C; WAKO Chemicals, GmbH, Neuss, Germany) were measured enzymatically following the manufacturers’ protocols. Blood was drawn from a cubital vein and collected into sterile EDTA tubes for isolation of peripheral blood mononuclear cells (PBMCs) or from serum tubes (BD Biosciences, Franklin Lakes, NJ, USA). The study was approved by the institutional review board and written informed consent was obtained from all subjects. Using similar criteria, an additional six T1D and six controls were recruited for a follow-up experiment.