The FA within total lipids was analyzed after saponification to account for esterified and nonesterified FAs in postmortem tissues. Chow diet, brain (temporal lobe) and liver tissue FAs were measured as FAME by GC/FID. FAME were prepared following a modification of the protocol by Metcalfe et al. [40 , 41 (link)]. Briefly, tissue samples were homogenized at 100 mg tissue/mL in distilled water. Triheptadecanoin (100 μg; a triglyceride of C17:0; NuChek Prep, Elysian MN, USA,) was added to homogenates as an internal standard and the mixture exposed to boron trifluoride to form fatty acid methyl esters. FAME were separated using an Agilent J&W DB-23 column (30 m × 0.25 mm ID, film thickness 0.25 μm) on an HP 5890 GC with a flame ionization detector. The FAMEs were identified by their elution times relative to authenticated methylated FA, and quantities were determined by their abundance relative to the added internal standard. On the GC-FID system used for these studies, evaluation of equal weight FAME mixtures produced nearly identical response factors over a range of chain lengths and fatty acid masses indicating that the array of FAMEs generate equivalent peak area at equivalent mass. Approximately 23 and 24 FAs for brain and liver, respectively, were routinely identified and these accounted for ~99% of the FA peaks.
Triheptadecanoin
Manufactured by Nu-Chek Prep
Sourced in United States
Triheptadecanoin is a triglyceride compound containing three heptadecanoic acid (C17:0) fatty acid chains. It is a laboratory standard used for various analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
J w db 23 column, by Agilent Technologies (3 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Luna c8 column, by Phenomenex (1 mentions)
Hp 7890, by Agilent Technologies (1 mentions)
Agilent 7890a, by Agilent Technologies (1 mentions)
J w db 23, by Agilent Technologies (1 mentions)
8 protocols using triheptadecanoin
1
Comprehensive Fatty Acid Profiling in Tissues
2
Triglyceride Quantification in Liver
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Triglycerides (TG) were extracted from ~15 mg liver powder with 200 μl ethyl acetate spiked with 21.2 pmol triheptadecanoin (Nu-Chek Prep., Inc.) as internal standard. Samples were vortexed and centrifuged for 5 min at 15,000 g at 4 °C. Supernatant was diluted in acetonitrile/ethylacetate (50/50, v/v) and TG were analyzed by HPLC-High Resolution (HR)-MS/MS as previously described [18 (link)]. Briefly, HPLC-HR-MS/MS analysis of TG was performed using a C8 Luna column (2 × 150 mm, 5 μm, Phenomenex) with a flow rate of 0.4 ml/min and mobile phases of acetonitrile/water (9:1, v/v) 0.1% ammonium acetate (solvent A) and isopropanol/ acetonitrile 0.1% ammonium acetate (7:3, v/v) (solvent B). The gradient program was the following: 35–100% solvent B (0.1–10 min), 100% solvent B (10–13 min), followed by 4 min of re-equilibration to initial conditions. A Q-Exactive hybrid quadrupoleorbitrap mass spectrometer (ThermoFisher) was used in positive mode with the following parameters: auxiliary gas heater temperature 250 °C, capillary temperature 300 °C, sheath gas flow rate 20, auxiliary gas flow rate 20, sweep gas flow rate 0, spray voltage 4 kV, S-lens RF level 60 (%). Full mass scan analysis ranged from 700 to 1500 m/z at 17,500 resolution.
3
Fatty Acid Profiling of Prostate Tissue
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Fatty acids (FAs) were extracted from PCA tissue and measured as fatty acid methyl esters (FAMEs). FAMEs were prepared following a modification of the protocol by Metcalfe et al. 22 , as previously described 20 , 23 . Briefly, prostate tissue samples were thawed and homogenized in distilled water at 100 mg tissue/ml. One hundred microgram (100 μg) of triheptadecanoin (a triglyceride of C17:0; NuChek Prep, Elysian, MN) was added to homogenates as an internal standard. Total fatty acids were extracted by base hydrolysis and then derivatized under alkaline conditions in methanol in the presence of boron trifluoride (5 min, 100°C) to form FAMEs. FAMEs were analyzed on an Agilent J&W DB‐23 column (30 m × 0.25 mm ID, film thickness 0.25 µm) using an HP 5890 gas chromatography (GC) with a flame ionization detector (FID). Individual FAs were identified by their elution times relative to authenticated FA standards. Twenty‐four FAs, which account for 99% of the total FAs within the extract, were routinely identified from these tissue samples. FA quantities were determined by their relative abundance to that of the added C17:0 internal standard. Individual FA concentrations were expressed as the percentage of each FA relative to the total FA concentration within each sample.
4
Quantification of Fatty Acid Profiles
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Serum was isolated from fasting whole blood and fatty acid methyl esters (FAME) were prepared [51 (link)] after saponification of duplicate samples (100 μl) in the presence of an internal standard (triheptadecanoin: Nuchek Prep, Elysian, MN, USA) as previously described [52 (link)]. A panel of 28 fatty acids was quantified by gas chromatography with flame ionization detection and individual fatty acids are expressed as percent of total fatty acids in each sample.
The fatty acid composition of the oil supplements (Table3 ) was determined in aliquots of oil diluted in hexane and processed as described above. Individual fatty acids were expressed as percent of total fatty acids and as grams fatty acid/grams oil in order to calculate the fatty acid doses for each intervention arm.
The fatty acid composition of the oil supplements (Table
5
Quantitative Hepatic Lipid Profiling
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Lipids were extracted from each hepatic construct following exposure to LA and HSA vehicle control using 500 μl of 3:2 hexane:isopropanol overnight. The solvent was removed and evaporated under nitrogen before lipids were converted to FAME and quantified using GC-FID, as previously described [37 (link),38 (link)]. Samples were analyzed by GC-FID on a HP 7890 (Agilent Technologies, Inc., Santa Clara, CA) with a DB-23 column as described above using triheptadecanoin (100μg; NuChek Prep) as an internal standard. Fatty acids were cleaved from complex lipids and converted to methyl esters in duplicate samples when possible, utilizing a modification of the protocol developed by Metcalfe et al [39 (link)] and Sergeant et al [37 (link)] Fatty acids in samples were identified based on retention times of commercially available internal standards. Approximately 12–25 peaks were identified and accounted for >99% of the total fatty acids in the sample. Fatty acid data are presented as the total mass in sample (μg) normalized to protein content (mg).
6
Fatty Acid Profiling of 3D Hepatic Constructs
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The fatty acid profile, including evaluation of complex lipids, was assessed in 3D hepatic constructs cultured in both control and LA-enriched media. Briefly, 25 μl of media was subjected to a total lipid extraction using an acidified Bligh-Dyer method (10 μg of triheptadecanoin, NuChek Prep, Elysian, MN), subjected to base hydrolysis, and the resultant fatty acids were derivatized to methyl esters (FAME). FAME were quantified using gas chromatography with flame ionization detection (GC-FID) and identified using a Hewlett Packard 7890 instrument system with an Agilent J&W DB-23 column (30 m, 0.25 mm ID, 0.25 μm film) fitted with an inert pre-column (1 m, 0.53 mm ID) for cool-on column injection as previously described [37 (link)].
7
Comprehensive Fatty Acid Profiling
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Total fatty acids (TFA) were extracted from 100 mg freeze‐dried tissue as previously described (Vanhercke et al., 2013 ). Solvent of the lipid extract was evaporated under a N2 flow and total lipids were resuspended in 2 μL chloroform per mg leaf DW. Total lipids were separated by thin layer chromatography in hexane:diethylether:acetic acid (70:30:1, v:v:v) as previously described (Vanhercke et al., 2013 ). Fatty acid methyl esters (FAME) of total lipids (15 mg leaf DW) or TAG (30 mg leaf DW) were prepared in 750 μL 1N methanolic‐HCl in the presence of triheptadecanoin (Nuchek Prep, Inc.) as an internal standard (Vanhercke et al., 2013 ). FAME analysis was performed on an Agilent 7890A gas chromatography system with flame ionization detection (GC‐FID). Chromatograms were recorded and peaks were identified against retention times of authentic FAME standards (NuChek Prep, Inc).
8
Comprehensive Fatty Acid Profiling Protocol
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Total circulating (cohort 1: serum, cohort 2: plasma) fatty acids were assessed as fatty acid methyl esters (FAME) analyzed by gas chromatography with flame ionization detection (GC-FID) using a Hewlett Packard 5890 instrument system with an Agilent J&W DB-23 column (30 m, 0.25 mm ID, 0.25 μm film) fitted with an inert pre-column (1 m, 0.53 mm ID) for cool on-column injection as previously described [27 (link)]. Independent experiments from our lab and others have shown that the fatty acid profiles of serum and plasma from anti-coagulated blood (heparin or EDTA) are comparable [28 (link), 29 (link)]. Fatty acids were cleaved from complex lipids and converted to methyl esters in duplicate samples (100 μl) utilizing a modification of the protocol developed by Metcalfe et al. [30 (link)] and Sergeant et al. [27 (link)]. Fatty acids in samples were identified based on retention times of commercially available standards. Triheptadecanoin (100 μg; NuChek Prep, Elysian, MN) was used as an internal standard. Fatty acid peaks (23–29 peaks) were identified and accounted for > 99% of the total fatty acids in the sample. Fatty acid data are presented as the mass percent of total fatty acids from serum (cohort 1) or plasma (cohort 2).
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