Return to study ST000600 main page
MB Sample ID: SA033135
| Local Sample ID: | LM-27 |
| Subject ID: | SU000623 |
| Subject Type: | Animal |
| Subject Species: | Rattus norvegicus |
| Taxonomy ID: | 10116 |
| Species Group: | Mammal |
Select appropriate tab below to view additional metadata details:
Sample Preparation:
| Sampleprep ID: | SP000630 |
| Sampleprep Summary: | Plasma non-esterified fatty acids (NEFAs) were isolated as previously described by Smedes (1). and Gladine et al. (2). Specifically, plasma aliquots (100 mL) were enriched with 5 mL 0.2 mg/ml butylated hydroxytoluene/EDTA in 1:1 methanol:- water, and a suite of extraction surrogates, which included deuterated-tri-palmitoyl glycerol (d31-16:0-TG; CDN Isotopes, Pointe-Claire, Quebec, Canada), deuterated distearoylphosphotidylcholine (d35-18:0-PC; Avanti Polar Lipids, Alabaster, Alabama), dodeca-(9E)-enoyl cholesterylesters (22:1n9-CE; NuChek Prep, Elysian MN) and dodecatrienoic acid (22:3n3; NuChek Prep). Lipids were then extracted with 10:8:11 cylcohexane: 2- propanol:ammonium acetate. Briefly, enriched samples were mixed with cyclopropane/2-propanol, phases were split with ammonium acetate, the organic phase was isolated and the aqueous phase was re-extracted with cyclohexane. The combined organic total lipid extract was reduced to dryness and reconstituted in 200 µL of 1:1 methanol/toluene and the total lipid extract was used to quantify plasma fatty acids as methyl esters by gas chromatography-mass spectrometry (GC-MS). It was derivitized by adding 45 μL 2M (trimethylsilyl) diazomethane in hexanes and spiked with 15:1n5 free acid to track methylation efficiency. Next, it was brought to a final volume of 200 mL with 90:10 methanol/toluene (v/v) and left at room temperature for 30 min, before being brought to dryness. The remaining fatty acid methyl esters (FAMEs) were re-constituted in 300 mL Hexane plus 10 uL of 44 mM tricosanoate methyl ester (23:0; NuChek Prep), vortexed, and 100 uL was transferred to a GC-MS Vial for analysis. |
| Sampleprep Protocol Filename: | NEFA_Plasma_Newman_Data_Report.docx |
