Qp2010

For GCMS separation, the instrument consisted of Shimadzu 2010 plus gas chromatograph with an injector (split/splitless), MS detector (QP2010), column (non-polar Rxi-5 MS capillary column; Restek Corporation) with dimensions of 30 m × 0.25 mm, 1.00 μm, whereas, Helium at a flow rate of 1.5 mL/min was used as a carrier gas. The operating condition for GC-oven consisted of: an initial temperature of 50 °C (2 min) → 150 °C (1 min) ramped at 4 °C/min → 250 °C (3 min) ramped at 8 °C/min. A temperature of 250 °C was maintained for the ion source whereas; 280 °C for the mass transfer line and mass spectra (33–450 m/z) were recorded after a 6.5 min solvent delay. For data acquisition and processing, the software used was Shimadzu GCMS Solution^® (version 4.52). Moreover, the area normalization process (% content) was applied for semi-quantification whereas, the NIST11 mass spectral Library database was utilized for the identification of the volatile components.

Total lipids were extracted using the Folch extraction procedure and quantified using the sigma colorimetric assay kit as previously described^{31 (link)}. Briefly, heart tissues were weighed and homogenized in 2:1 chloroform:methanol, incubated on ice for 30 min, then centrifuged at 3000 rpm for 12 minutes. The top layer was discarded, the bottom layer was dried under N2 gas and reconstituted in 1% Triton X-100:Isopropanol. Individual lipid classes were extracted as described above were loaded onto an NH2 column (Agilent BondElut) preconditioned with hexane and neutral lipids were extracted with 2:1 Chlorofom:Isopropanol, dried under N2 gas, reconstituted in hexane and loaded onto another preconditioned NH2 column^{4 (link),32 (link)}. TG were eluted from the column by the addition of 1% diethyl ether:10% methylene chloride in hexane and DAG with 15% ethyl acetate in hexane dried under N2 gas. DAG was quantified using the triglyceride assay kit with a DAG standard curve. Fatty acid composition and ¹³C labeling patterns was assessed using a gas chromatograph mass spectrometer (GCMS, Shimadzu QP2010) with a 0.25 mm inner diameter column (Restek Rtx-225) on fatty acid methyl esthers (FAMES) made from the triglyceride fraction obtained using solid phase separation as previously described^{33 (link)}.