Serum and hepatic concentrations of triglycerides, total cholesterol, and free fatty acids (FFA) were measured by colorimetric assays using Triglyceride E‐Test Wako, Cholesterol E‐Test Wako, and NEFA C‐Test Wako (FUJIFILM Wako Pure Chemical Corp.), respectively. Serum and hepatic concentrations of sterols and oxysterols were measured as described previously.(17 ) Fecal sterol concentrations were quantified using our previously described LC/MS‐MS method(18 ) with minor modifications. Briefly, 5 mg of wet feces were incubated in 250 µL of 1N ethanolic KOH at 60°C for 1 hour. After the addition of 1 µg of [2H7]cholesterol (internal standard) to a 10 µL aliquot of the hydrolysate, sterols were extracted with n‐hexane, derivatized to picolinyl esters, and analyzed by LC/MS‐MS. The fecal fatty acid concentration was determined as follows: Fatty acids were extracted from a 5‐µL aliquot of this hydrolysate by the Bligh‐Dyer method(19 ) and quantified using NEFA C‐Test Wako.
Nefa c test wako
Manufactured by Fujifilm
Sourced in Japan
The NEFA C-Test Wako is a laboratory equipment product designed for the quantitative determination of non-esterified fatty acids (NEFAs) in biological samples. It provides a reliable and accurate method for measuring NEFA levels, which is essential for various clinical and research applications.
Automatically generated - may contain errors
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40 protocols using nefa c test wako
1
Quantification of Lipid Metabolites
2
Comprehensive Metabolic Profiling in Rodents
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Blood glucose was measured using a glucometer (Glutest PRO R; Sanwa Kagaku Kenkyusho Co., Ltd., Aichi, Japan). Serum non-esterified fatty acid (NEFA), triglyceride (TG), and 3-hydroxybutyrate levels were determined with NEFA C-Test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan), TG E-Test Wako (Wako Pure Chemical Industries, Ltd.), and beta Hydroxybutyrate Assay Kit (Abcam plc, Cambridge, UK), respectively. Serum alanine aminotransferase (ALT) levels were measured using Fuji Dry-chem 7000V (Fujifilm corporation, Tokyo, Japan). Urine glucose levels were analyzed with enzymatic assays in a laboratory of Oriental Yeast Co., Ltd. (Tokyo, Japan). Serum insulin and plasma glucagon levels were measured with an enzyme-linked immunosorbent assay kit (Morinaga Institute of Biological Science, Inc., Kanagawa, Japan) and Mercodia Glucagon ELISA (Mercodia AB, Uppsala, Sweden), respectively. Total lipids were extracted from the liver with chloroform and methanol (2:1 v/v), and liver TG content was assayed with TG E-Test Wako.
3
Plasma Biomarker Quantification Protocol
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Blood collected in EDTA-coated tubes was immediately separated by centrifugation for 10 min at 5,800 × g. Plasma was collected and stored at −80 °C. Plasma albumin, glucose, insulin, alanine, β-hydroxybutyrate, free fatty acid (FFA), glucocorticoid, and IGF-1 values were measured using A/G B-Test Wako (Wako Pure Chemical Industries, Osaka, Japan), Glucose CII-test Wako (Wako), Mouse Insulin ELISA kits (Morinaga Institute of Biological Science, Kanagawa, Japan), Alanine Colorimetric/Fluorometric Assay Kits (BioVision, Milpitas, CA, USA), β-Hydroxybutyrate (β-HB) Colorimetric Assay Kits (BioVision), NEFA C-test Wako (Wako), AssayMax corticosterone ELISA kits (AssayPro, St. Charles, MO, USA) and Mouse/Rat IGF-1 Quantikine ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) respectively.
4
Plasma and Serum Biomarker Measurements
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The collected blood samples were centrifuged at 3000×g for 5 min. Plasma and serum were stored at – 80 °C until the time of analysis. The concentrations of plasma glucose and free fatty acid (FFA) were measured using Glucose C2-test Wako and NEFA C-test Wako (FUJIFILM Wako Pure Chemical, Osaka, Japan), respectively. Serum triglyceride, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol were determined by Kyoto Biken Laboratories, Inc. (Kyoto, Japan). Plasma FGF21 concentration was determined using a commercially available enzyme-linked immunosorbent assay kit (DF2100; R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions.
5
Serum Analysis of Metabolic Markers
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After completing the measurement of blood flow, a blood sample (∼500 µL) was collected from the abdominal vein, transferred to a micro-blood collection tube, and serum was collected after centrifugation (1,609 × g, for 10 minutes at room temperature) using a Model 6000 centrifuge (KUBOTA Manufacturing Corporation, Osaka, Japan). All measurements were performed in duplicate. Insulin was measured using a Mouse Insulin ELISA Kit (TMB) (AKRIN-011T; Shibayagi Co., Ltd., Gunma, Japan) according to the manufacturer's instructions. Glucose, total cholesterol, triglycerides, and free fatty acids were measured using the Glucose C2-Test Wako (mutarotase-GOD method), the Cholesterol E-Test Wako (cholesterol oxidase DAOS method), the triglyceride E-Test Wako (glycerol-3-phosphate oxidase DAOS method), and the NEFA C-Test Wako (acyl-CoA synthetase and acyl-CoA oxidase method; WAKO Pure Chemical Industries, Ltd., Osaka, Japan), respectively. All measurements were performed according to the instructions included in each kit.
6
Lipid Profiling in HFD-Fed Mice
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Liver and fecal samples were obtained from mice fed a HFD from 5 to 16 weeks of age. Total lipid extraction was performed by the Folch extraction procedure (Folch et al., 1957 (link)). The content of triglyceride, total cholesterol and free fatty acid were measured by using Triglyceride E-test Wako, Cholesterol E-test Wako and NEFA C-test Wako (Wako Pure Chemical Industries, Osaka, Japan), respectively.
7
Lipid Analysis in Liver and Plasma
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Total lipids in the liver were extracted using the Folch method. Triglyceride (TG), free fatty acid (FFA), and total cholesterol (TC) levels in total lipids in the liver and plasma were measured using the Triglyceride E Test Wako, NEFA C Test Wako, and Cholesterol C Test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan), according to the manufacturer’s protocol.
8
Mouse metabolic parameters assessment
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Blood was obtained from the retro-orbital sinus under ad libitum feeding unless otherwise mentioned. Plasma glucose concentration was measured using a glucose assay kit (Wako Pure Chemical Industries). Plasma insulin concentration was measured using an insulin-ELISA kit (Morinaga Institute of Biological Science). Plasma TG, NEFA, and total cholesterol concentrations were measured using enzymatic kits (Triglyceride E-test Wako, NEFA C-test Wako, and Cholesterol E-test Wako, respectively; Wako Pure Chemical Industries). To measure liver TG content, we sampled livers from 20-week-old mice and immediately froze them in liquid nitrogen. Lipids were extracted with isopropyl alcohol heptane (1:1 volume for volume). After evaporation of the solvent, we resuspended lipids in 99.5% volume for volume ethanol, and TG content was measured by an enzymatic kit (Triglyceride E-test Wako).
9
Measuring Anti-EL Antibody Inhibition
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Example 6
Measurement of Inhibitory Activity of Anti-EL Antibodies Against EL
The inhibitory activity of anti-EL antibodies (55A1, 7D4, 14A1, 2D5, 53A11, 13B3, 23H8, 16B3, 16E7, 14E1 and 19E7) against baboon EL, cynomolgus monkey EL, human (1-157) mouse chimeric EL, human (1-53) mouse chimeric EL, human (61-111) mouse chimeric EL, rabbit EL, mouse EL, human (1-305) mouse chimeric EL and human (202-500) mouse chimeric EL were measured as follows. Anti-EL antibody solution, assay buffer (20 mM Tris/HCl (pH 7.5), 0.5% BSA, 4 mM CaCl2, 150 mM NaCl) and 2 mg/mL human HDL (Athens Research & Technology) were mixed in a microtiter plate, followed by adding EL heparin extract. After incubation at 37° C. for 90 min, non-esterified Fatty Acid (NEFA) released from HDL was determined using a commercially available kit (NEFA C test-Wako, Wako). The NEFA amount was used as enzyme activity index. Enzyme activity in the case of adding no anti-EL antibody was determined as control value and specific activity was calculated against the control value at each concentration of the anti-EL antibody (
10
Regulation of Fatty Acid Metabolism in Myotubes
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Differentiated L6 myotube cells were incubated with the medium containing 0.6 μmol palmitic acid (300 μmol/L) for 24 and 48 hrs in the presence or absence of 0.5 mM acetic acid or 0.5 mM AICAR. After the incubation, mediums and cells were collected separately and the concentration of NEFA in the mediums and the concentration of TG in the cells were determined by using commercial assay kits (NEFA C-Test Wako and Triglyceride E-Test Wako, respectively; Wako Pure Chemical Industries Ltd., Osaka, Japan). The amount of fatty acid uptake was calculated by using the concentration of fatty acid remaining in the medium, which averaged 0.263 μmol and 0.075 μmol in 24 hr and 48 hr of the treatment of acetic acid, respectively, and 0.183 μmol and 0.149 μmol in 24 hr and 48 hr of the treatment of AICAR, respectively. The amount of fatty acid remaining in the control medium averaged 0.316 μmol and 0.159 μmol in 24 hr and 48 hr, respectively.
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