The plasma of mice was centrifuged at 28,000 rpm for 30 minutes at 4 °C to remove chylomicrons. Amylase activity was measured by 5-ethylidene-G7PNP as a substrate with a commercial kit (Beijing Zhongsheng Beikong Biochemistry Company, PR China), and lipase activity was also measured with a commercial kit (Nanjing Jiancheng Biochemistry Company, PR China), as described in the manual from the manufacturer. Serum alanine-aminotransferase (ALT), total bilirubin (TBIL), creatinine (Cr) and urea nitrogen (BUN) were measured with dry chemistry method in General Surgery Biochemistry Laboratory of Jinling Hospital (AU680 automatic biochemical analysis system, Beckman Coulter Inc., USA). Serum ApoCIII levels (Cloud-Clone Crop., Wuhan, PR China) and free fatty acids(Wako Pure Chemical Industries, Ltd., Osaka, Japan) were determined according to the manual from the manufacturer.
Free fatty acid
Manufactured by Fujifilm
Sourced in United States, Japan, Germany, Israel
Free fatty acids are lipids that are not bound to other molecules. They are a component of fats and oils and can be used in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Total cholesterol, by Fujifilm (2 mentions)
Cholesterol, by Thermo Fisher Scientific (2 mentions)
Insulin elisa kit, by Merck Group (2 mentions)
Cholesterol triglyceride, by Abbott (2 mentions)
Leptin adiponectin, by Merck Group (2 mentions)
Edta coated microvette tubes, by Sarstedt (1 mentions)
Cholesterol, by Fujifilm (1 mentions)
Insulin, by ALPCO (1 mentions)
Cobas integra 400, by Roche (1 mentions)
Oasis max, by Waters Corporation (1 mentions)
Aeris widepore column, by Phenomenex (1 mentions)
μ elution plate, by Waters Corporation (1 mentions)
Glucagon, by R&D Systems (1 mentions)
Contour glucometer, by Bayer (1 mentions)
Total cholesterol, by Roche (1 mentions)
Hdl cholesterol, by Roche (1 mentions)
Plasma glucose, by Horiba (1 mentions)
Cobas pentra c400 analyzer, by Horiba (1 mentions)
1.5 ml tube, by Eppendorf (1 mentions)
Cobas mira, by Roche (1 mentions)
29 protocols using free fatty acid
1
Characterization of Metabolic Markers in Mice
2
Comprehensive Serum Metabolic Profiling
3
Plasma Glucose and Lipid Analysis
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Plasma glucose and insulin were analysed by enzymic assay (Sigma‐Aldrich, St. Louis, MO, USA and Crystal Chem, Downers Grove, IL, USA). Blood and liver TG levels were also measured by enzymic assay (Sigma‐Aldrich). Commercial kits were employed for the measurement of free fatty acids (Wako Pure Chemical Industries, Osaka, Japan) and total cholesterol (Cayman Chemical Com., Ann Arbor, MI, USA). Liver metabolic parameters were normalized to respective liver weights.
4
Blood Lipid Profiling and Hepatic Lipid Analysis
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Blood samples were collected 2 h after the last injection. Blood was obtained from tail veins into EDTA-coated microvette tubes (Sarstedt), centrifuged at 5,000 g, and plasma was stored at −80°C. Commercially available kits were used to measure plasma levels of cholesterol (Wako Chemicals, Neuss, Germany), free fatty acids, NEFA (Wako Chemicals, Neuss, Germany), TG (Wako Chemicals, Neuss, Germany) and insulin (ALPCO Diagnostics, Salem, NH) as per the manufacturer's instructions. For lipoprotein separation, samples from eight animals per group were pooled (0.20 ml) and subjected to fast-performance liquid chromatography (FPLC) gel filtration on two Superose 6 columns connected in series as described previously (Hofmann et al, 2008 (link)). Hepatic lipids were extracted as described previously (Folch et al, 1957 (link)), and cholesterol content was quantified using a commercially available kit (Thermo Scientific, Middletown, VA).
5
Quantification of Plasma Peptides and Metabolites
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Quantification of peptides in plasma was performed using liquid chromatography-tandem mass spectrometry. Samples were prepared by solid phase extraction using an OASIS MAX (BI-3434) or WCX (stapled secretin) μElution plate (Waters, Milford, MA, USA). Separation was performed on an Aeris WIDEPORE column (Phenomenex, Torrance, CA, USA) and samples were analyzed on a 6500+ Triple Quad System (ABSciex, Darmstadt, Germany) operated in positive ionization mode.
Liver triglycerides, plasma amylase and lipase were measured using an automated analyzer Cobas Integra 400 (Roche, Rotkreuz, Switzerland). Plasma glucagon, insulin und active GLP-1 were measured using an enzyme-linked immunosorbent and colorimetric assay kits (MSD, Metabolic Panel, Rockville, Maryland, USA). Plasma somatostatin was measured using an enzyme-linked immunosorbent and colorimetric assay kit (Phoenix Pharmaceuticals, Inc., Karlsruhe, Germany). Commercially available enzyme-linked immunosorbent and colorimetric assay kits were used to measure free fatty acids and gycerol (Wako Pure Chemicals, Osaka, Japan) in EDTA plasma.
Liver triglycerides, plasma amylase and lipase were measured using an automated analyzer Cobas Integra 400 (Roche, Rotkreuz, Switzerland). Plasma glucagon, insulin und active GLP-1 were measured using an enzyme-linked immunosorbent and colorimetric assay kits (MSD, Metabolic Panel, Rockville, Maryland, USA). Plasma somatostatin was measured using an enzyme-linked immunosorbent and colorimetric assay kit (Phoenix Pharmaceuticals, Inc., Karlsruhe, Germany). Commercially available enzyme-linked immunosorbent and colorimetric assay kits were used to measure free fatty acids and gycerol (Wako Pure Chemicals, Osaka, Japan) in EDTA plasma.
6
Blood Metabolite Measurement Protocol
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Fasting glucose was measured in blood obtained by retro‐orbital collection by directing the collection tube gently in a ventro‐lateral direction while rotating the tube. For multiple collection times, alternate right and left eyes were used. Kits were used to measure the concentrations of several metabolites in serum: insulin, IGF1 (IBL International, Hamburg, Germany), and free fatty acids (WAKO, Neuss, Germany).
7
Metabolic Markers ELISA Analysis
8
Blood Metabolite Measurement Protocol
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Blood for measurement of lactate, pyruvate and ketones was treated immediately after withdrawal with an equal volume of 1 M perchloric acid and subsequently stored at −20 °C. The remainder of the blood was used for plasma preparation. Plasma was stored at −20 °C. Blood glucose was determined using a Contour glucometer (Bayer, Mijdrecht, The Netherlands). Blood lactate, pyruvate, β-hydroxybutyrate and plasma amino acids and acylcarnitines were measured using tandem mass spectrometry [[11] (link), [12] (link), [13] (link), [14] (link)]. Glycerol (Instruchemie, 2913), free fatty acids (Wako, 434–91,795), triglycerides (Human Gesellschaft, 10,724) and creatine kinase were measured in plasma using standard enzymatic assays as described [9 (link),15 (link)].
9
Fasting Blood and Adiponectin Analysis
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Mice were denied access to food for 16 h before the measurements. 30-μl blood samples were collected from the tail vein in accordance with the guidelines for animal experiments of the University of Tokyo. Fasting blood glucose was measured with an automatic glucometer (Glutest Ace. Sanwa Chemical Co., Japan). Plasma levels of insulin (Morinaga Co., Ltd., Japan), triglyceride, total cholesterol, free fatty acids and high-density lipoprotein (Wako Pure Chemical Industries, Ltd., Japan) were assayed by enzymatic methods. Plasma total adiponectin levels (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) and plasma HMW adiponectin levels (ALPCO Diagnostics., USA) were measured with ELISA kits.
10
Quantifying Lipids in Liver and Serum
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Lipids were isolated by homogenizing liver tissue in 7:11:0.1 chloroform/isopropanol/NP-40 followed by evaporation at 60°C. Commercially available kits and reagents were used to quantify the total cholesterol (BioVision Incorporated, Milpitas, CA), triglycerides (Sekisui Diagnostics, Lexington, MA), and free fatty acids (Wako Chemicals, Richmond, VA) in liver and serum.
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