Blood samples were centrifuged for 10 min at 3000 rpm at 4 °C, and plasma was separated from the cells. Glucose was determined immediately using the glucose oxidation method with the Biosen glucose analyzer (EKF Diagnostics, Barleben, Germany). Plasma aliquots were temporarily stored at −20 °C. HbA1c, plasma insulin, and plasma triglyceride levels were measured as described previously (Stenvers et al., 2014 (link)); C-peptide was determined with a 125I radioimmunoassay (Linco Research, Inc., St. Charles, MO); and plasma free fatty acid (FFA) levels were determined with an enzymatic calorimetric method (NEFA-HR(2) test kit, Wako Chemical, Neuss, Germany). In patients with type 2 diabetes, 2 additional measurements were performed: Plasma glucagon was determined with a 125I radioimmunoassay (Millipore, Billerica, MA), and saliva cortisol was determined by online solid phase extraction LC/MS/MS (Waters Corporation, Milford, MA).
Nefa hr 2 test kit
Manufactured by Fujifilm
The NEFA-HR(2) test kit is a laboratory assay used to quantitatively measure the levels of non-esterified fatty acids (NEFAs) in biological samples. It is a colorimetric assay that provides accurate and reliable results for research and clinical applications.
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2 protocols using nefa hr 2 test kit
1
Biomarker Quantification in Type 2 Diabetes
2
Measuring Glucose Metabolism Dynamics
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Blood glucose concentrations were directly measured during the experiment, using a custom glucose meter (Freestyle Freedom Lite, Abbott). Blood samples were immediately chilled on ice in Eppendorf tubes with 5 μL heparin: saline (10×) solution and centrifuged at RT (15 min, 1600 g). Plasma was stored at -20°C until further analysis. Plasma concentrations of insulin and glucagon were measured using radioimmunoassay kits (Millipore and Biochemicals, Costa Mesa, CA, respectively). The amounts of sample, standards, label, antibody and 232:1
precipitating reagent, described in the manufactures' protocol, were divided by four. Previous research from our department has shown that the mean cross-reactivity of Actrapid measured in rat plasma using the insulin radioimmunoassay is 84% (Ackermans, Ann Clin Biochem, 2008) . Plasma [6,6-2 H 2 ] glucose enrichment was measured by gas chromatography-mass spectrometry (GCMS) (Ackermans et al. 2001) (link), EGP and Rd were calculated using Steele equations (Steele 1959 (link)). The FFA concentration was determined with an enzymatic colorimetric method (NEFA-HR(2) test kit, Wako Chemicals GmbH).
precipitating reagent, described in the manufactures' protocol, were divided by four. Previous research from our department has shown that the mean cross-reactivity of Actrapid measured in rat plasma using the insulin radioimmunoassay is 84% (Ackermans, Ann Clin Biochem, 2008) . Plasma [6,6-2 H 2 ] glucose enrichment was measured by gas chromatography-mass spectrometry (GCMS) (Ackermans et al. 2001) (link), EGP and Rd were calculated using Steele equations (Steele 1959 (link)). The FFA concentration was determined with an enzymatic colorimetric method (NEFA-HR(2) test kit, Wako Chemicals GmbH).
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