Erythrocytes were separated from the plasma by centrifugation (3000 rpm, 1500 ×g, for 10 min) and washed with an equal volume of saline. These erythrocytes resuspended with saline were stored in a freshly 0.01% butylated hydroxyl toluene- (BHT-) treated Eppendorf vials at −80°C. The fatty acids composition was determined using the method by Lepage and Roy [19 (link)]; erythrocyte's membranes were extracted from aliquots of 200 μL of erythrocyte suspensions and the fatty acids converted to methyl esters by reaction with acetyl chloride for 60 min at 100°C. Methyl ester fatty acids (FAME) were separated and analyzed by gas chromatography performed on a Shimadzu GCMS-QP2010 Plus gas chromatograph/mass spectrometer (Shimadzu, Kyoto, Japan) and peaks were identified through mass spectra and by comparing with respect to a reference FAME mixture (GLC-744 Nu-Chek Prep. Inc., Elysian MN, USA) the elution pattern and relative retention times of FAME. The O3Ix was calculated as erythrocyte (EPA + DHA)/(total fatty acids) × 100% (percentage molar of total fatty acids) [11 (link)].
Glc 744
Manufactured by Nu-Chek Prep
Sourced in United States
The GLC-744 is a gas chromatography (GC) instrument designed for analytical applications. It is capable of separating and analyzing complex mixtures of chemical compounds. The GLC-744 features a high-performance oven and a sensitive detector, enabling precise and reliable analysis of various samples.
Automatically generated - may contain errors
Lab products found in correlation
Gcms qp2010 plus gas chromatograph mass spectrometer, by Shimadzu (2 mentions)
Glc 68a, by Nu-Chek Prep (2 mentions)
Agilent 7820a gc, by Agilent Technologies (2 mentions)
Cis trans 18 2n 6, by Merck Group (2 mentions)
Glc 532, by Nu-Chek Prep (2 mentions)
Contour blood glucose monitoring system, by Bayer (1 mentions)
6890 series gc system, by Hewlett-Packard (1 mentions)
Synergy neo2, by Agilent Technologies (1 mentions)
Hr series nefa hr kit, by Fujifilm (1 mentions)
Enzymatic assay kit, by Fujifilm (1 mentions)
Elisa assay, by Crystal Chem (1 mentions)
Fame 37, by Merck Group (1 mentions)
Sp 2560, by Merck Group (1 mentions)
6 protocols using glc 744
1
Erythrocyte Fatty Acid Profiling
2
Fatty Acid Profiling in Erythrocyte Membranes
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Analysis of the fatty acid profile in the erythrocyte membrane was analyzed as follows: 10-mL blood samples were taken from the antecubital vein 10 min before the physical tests in tubes with tripotassium EDTA (ethylenediamine tetraacetic acid) washed with isotonic saline and purged with nitrogen gas and centrifuged at 2000 rpm for 30 min at 4 °C. The supernatant was collected and frozen at −80 °C and the cell phase (erythrocytes) was frozen at −80 °C for fatty acid analysis. Erythrocyte lipids were determined as methyl esters after methylation reaction, using the method of Lepage and Roy [31 (link)]. GC analysis was performed on a Shimadzu GCMS-QP2010 Plus gas chromatograph/mass spectrometer (Shimadzu, Kyoto, Japan). Fatty acid methyl esters (FAMEs) were identified through mass spectrometry and through comparison of the elution pattern and relative retention times of FAMEs with the reference FAME mixture (GLC-744 Nu-Chek Prep. Inc., Elysian, MN, USA). The results were expressed in relative amounts (percentage molar of total fatty acids) [32 (link)]. The fatty acid methyl esters are analyzed by capillary gas chromatography with an ionized flame detector [33 (link)].
3
Plasma and Tissue Lipid Analysis
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Blood was collected from tail veins in citrate-EDTA tubes, and plasma was isolated by centrifugation at 2000g for 10 minutes at 4°C. Plasma levels of TGs and cholesterol were measured using Infinity series kits (Thermo Fisher Scientific). NEFA levels were measured in duplicate or triplicate using the HR Series NEFA-HR Kit (Fujifilm Wako Diagnostics). Results were obtained using the Synergy Neo2 plate reader (BioTek Instruments). Plasma glucose levels were measured using Contour Blood Glucose Monitoring System (Bayer).
For fatty acid analysis of tissues, lipids were extracted from the blood (51 (link)) of the fasting and refed animals, derivatized to form methyl esters, and separated by gas/liquid chromatography using a Hewlett Packard 6890 Series GC system. The identity of the fatty acid methyl ester was determined by comparing the retention times with fatty acid standards (GLC-744; NU-Chek Prep). The abundance of each fatty acid was determined from the peak intensity and the internal standard. Fatty acid profiles were generated using a modified GC-MS method, as previously described (52 (link)).
For fatty acid analysis of tissues, lipids were extracted from the blood (51 (link)) of the fasting and refed animals, derivatized to form methyl esters, and separated by gas/liquid chromatography using a Hewlett Packard 6890 Series GC system. The identity of the fatty acid methyl ester was determined by comparing the retention times with fatty acid standards (GLC-744; NU-Chek Prep). The abundance of each fatty acid was determined from the peak intensity and the internal standard. Fatty acid profiles were generated using a modified GC-MS method, as previously described (52 (link)).
4
Liver Lipid Profiling and Serum Biomarkers
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Lipids were extracted from livers (∼100 mg) using the method of Folch et al.20 (link) The levels of TG, cholesterol, cholesteryl ester, free cholesterol, and phosphatidylcholine were measured using enzymatic assays (Infinity, Thermo Electron, Madison, WI, and Wako, Richmond, VA) and normalized to sample weight. Lipids were also separated into cholesterol esters, TGs, and phospholipids using thin-layer chromatography as previously described.21 (link) Lipids in each fraction were hydrolyzed, derivatized with trimethylsilane, and separated by gas-liquid chromatography (GLC) using a Hewlett Packard 6890 Series gas chromatograph (Agilent, Santa Clara, CA). The fatty acids were identified by retention times established using purified fatty acid standards (GLC-744, NU-Chek Prep, Elysian, MN). The percentage of each fatty acid in each fraction was calculated using pentadecanoin (C15:0) as an internal standard.
Serum levels of liver enzymes, TG, cholesterol, and glucose were measured using the Vitros 250 system (GMI, Ramsey, MN). Enzymatic assay kits were used to determine serum levels of NEFA (Wako). Serum levels of insulin were measured using an enzyme-linked immunosorbent assay (ELISA) assay (Crystal Chem, Downers Grove, IL).
Serum levels of liver enzymes, TG, cholesterol, and glucose were measured using the Vitros 250 system (GMI, Ramsey, MN). Enzymatic assay kits were used to determine serum levels of NEFA (Wako). Serum levels of insulin were measured using an enzyme-linked immunosorbent assay (ELISA) assay (Crystal Chem, Downers Grove, IL).
5
Plasma Fatty Acid Methyl Ester Analysis
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Plasma FA methyl esters were prepared as described previously (Moser et al., 1999) [24 (link)]. Briefly, 200 μl plasma and 10 μg C13:0 as an internal standard were mixed with 1 ml methanol/methylene chloride 3:1 (v/v). Subsequently, 200 μl of acetyl chloride, were added and the samples were placed in a 75°C water bath for 1 h. After cooling down, 4 ml of 7% potassium carbonate and 2 ml hexane were added, and samples were mixed and centrifuged for 10 min at 3000 rpm at room temperature. The plasma sample in hexane layer was dried using nitrogen gas and analyzed with Agilent 7820A GC using flame ionization detection on a SP-2560 polar fused silica capillary column (100 m x 0.25 mm x 0.2 μm, Supelco Inc.) with nitrogen as carrier gas. The oven temperature program was initially set to 60°C for 1 min, then increased by 25°C per minute to 160°C, then increased by 2°C per minute to 240°C for 10 min, and finally increased by 5°C per minute to 245°C for 5 min. The FA peaks were identified by comparing retention times from our samples with retention times of a standard mixture of GLC-68A, GLC-481, GLC-532, GLC-744 (Nu-Chek Prep), 37 FAME, cis/trans 18:2n-6 and cis/trans 18:3n-3, (all from SUPELCO). The FA composition was expressed as the weight of a percentage of the total weight of carbon 12 to carbon 24 FAs (wt%). http://dx.doi.org/10.17504/protocols.io.h6hb9b6
6
Fatty Acid Profiling and Desaturase Activity
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Total lipids were extracted from the plasma samples and converted to their methyl ester equivalents as described previously (12 (link), 22 (link)), and analyzed using an Agilent 7820A GC using flame ionization detection on a SP-2560 polar fused silica capillary column (100 m × 0.25 mm × 0.2 μm; Supelco Inc., PA, USA) with nitrogen as the carrier gas (12 (link)). The FA peaks were identified by comparing retention times with those of a standard mixture of GLC-68A, GLC-481B, GLC-532, GLC-744 (Nu-Chek Prep), 37 FAME, trans 16:1n-7, trans 18:1n-7, trans 18:1n-9, and cis/trans 18:2n-6 (all obtained from Supelco Inc., or Sigma). 13:0 free fatty acid (10 μg) was added as an internal standard. FA composition was expressed as the weight of a percentage of the total weight of carbon-12 to carbon-24 FAs (wt%). The estimated desaturase activity was calculated using the ratio of FA in plasma (11 (link), 17 (link), 23 (link)); SCD1(16) activity = (16:1n-7/16:0), SCD1(18) activity = (18:1n-9/18:0), D5D activity = (20:4n-6/20:3n-6), and D6D activity = (20:3n-6/18:2n-6).
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