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MB Sample ID: SA177844
| Local Sample ID: | 0060_LC_20190903_sample_0033 |
| Subject ID: | SU001997 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Male and female |
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Sample Preparation:
| Sampleprep ID: | SP002003 |
| Sampleprep Summary: | Male and female, humanized PPARa mice (hPPARa) were generated from mouse PPARa-null, human PPARa-heterozygous breeding pairs (generously provided by Dr. Frank Gonzalez, NCI). At weaning, mice were provided a custom diet based on the What we eat in America (NHANES 2013/2014) analysis (Research Diets, New Brunswick, NJ) (USDA, 2018): 51.8% carbohydrate, 33.5% fat (soybean oil, lard and butter, with cholesterol at 224 mg/1884 kcal), and 14.7% protein, as a % energy intake. Fats are in the form of . Vehicle (VH) and treatment water were prepared from NERL High Purity water (23-249-589, Thermo Fisher Scientific), prepared with PFAS removal. Mice were administered vehicle (0.5% sucrose) drinking water or PFOA (8 mM +0.5% sucrose) drinking water ad libitum for 6-7 weeks. Food and water consumption were determined on a per cage basis each week and previously reported. Body weight was measured weekly. Aliquots of liver for lipidomics were flash frozen in liquid nitrogen and stored at -80?C. A total of 11 female mice (5 VH and 6 PFOA) and 12 male mice (7 VH and 5 PFOA) were analyzed. Liver tissues were first homogenized with cryo-homogenization (Covaris, CryoPrep CP02, Massachusetts, USA) and weighted (ca. 5 mg). 20 µL of internal standard mixture 1A was added. This mixture contained PC(17:0/0:0), PC(17:0/17:0), PE(17:0/17:0), PG(17:0/17:0)[rac], Cer(d18:1/17:0), PS(17:0/17:0) and PA(17:0/17:0) (Avanti Polar Lipids, Inc., Alabaster, AL) as well as MG(17:0/0:0/0:0)[rac], DG(17:0/17:0/0:0)[rac] and TG(17:0/17:0/17:0). The lipids were extracted using a mixture of HPLC-grade chloroform and methanol (2:1; 400 µL). 50 µl of 0.9% NaCl was added and the lower phase (200 µL) was collected and 20 µL of an internal standard mixture containing labeled PC (16:1/0:0-D3), PC(16:1/16:1-D6) and TG(16:0/16:0/16:0-13C3) was added. The extracts were analyzed on a Waters Q-Tof Premier mass spectrometer combined with an Acquity Ultra Performance LCTM. The column (at 50 °C) was an Acquity UPLCTM BEH C18 2.1 × 100 mm with 1.7 µm particles. The solvent system included A. ultrapure water (1% 1 M NH4Ac, 0.1% HCOOH) and B. LC/MS grade acetonitrile/isopropanol (1:1, 1% 1M NH4Ac, 0.1% HCOOH). The gradient started from 65% A / 35% B, reached 80% B in 2 min, 100% B in 7 min and remained there for 7 min. The flow rate was 0.400 ml/min and the injected amount was 2.0 µL (Acquity Sample Organizer, at 10 °C). Reserpine was used as the lock spray reference compound. The lipid profiling was carried out using electrospray ionization mode and the data were collected at a mass range of m/z 300-1200 with a scan duration of 0.2 sec. The data processing included alignment of peaks, peak integration, normalization and identification. Lipids were identified using an internal spectral library. The data were normalized using one or more internal standards representative of each class of lipid present in the samples: the intensity of each identified lipid was normalized by dividing it with the intensity of its corresponding standard and multiplying it by the concentration of the standard. All monoacyl lipids except cholesterol esters, such as monoacylglycerols and monoacylglycerophospholipids, were normalized with PC(17:0/0:0), all diacyl lipids except ethanolamine phospholipids were normalized with PC(17:0/17:0), all ceramides with Cer(d18:1/17:0), all diacyl ethanolamine phospholipids with PE(17:0/17:0), and TG and cholesterol esters with TG(17:0/17:0/17:0). Other (unidentified) molecular species were normalized with PC(17:0/0:0) for retention times < 300 s, PC(17:0/17:0) for a retention time between 300 s and 410 s, and TG(17:0/17:0/17:0) for longer retention times. Quality control of the method showed that the day-to-day repeatability of control serum samples, and the relative standard deviation (RSD) for values identified was on average below 25% and 20% for discovery and validation sECs, respectively. The internal standards added to all samples in the study had an average RSD of 25% and 13 % in the discovery and validation sECs. |
| Processing Storage Conditions: | Described in summary |
| Extract Storage: | -80? |
