NMR measurements of TAG turnover were performed on perfused hearts with sequential, proton-decoupled carbon-13 (13C) NMR spectra (2 min each) with 13C natural abundance correction, as previously reported (14 –15 (link)).
13C enrichment of TAG in the heart was monitored from the NMR signal at 30.5 ppm from the TAG methylene groups. TAG turnover was calculated from total TAG content and enrichment over time (15 (link)–18 (link)). Kinetic analysis of dynamic 13C-spectra from hearts was performed as previously reported (14 –15 (link), 17 (link)–18 (link)).
Metabolic flux was determined during 13C palmitate oxidation in the intact mouse heart using a previously described method for kinetic analysis of the progressive 13C enrichment of glutamate, as detected via NMR (14 , 20 (link), 22 (link)–24 ).
For kinetic analysis of oxidative rates, glutamate, aspartate, citrate malate and α-ketoglutarate contents in frozen myocardial samples were assayed spectrophotometrically and fluorometrically (19 –20 (link)). In vitro 13C NMR was performed on acid extracts of myocardium to determine fractional enrichment of [2-13C] acetyl CoA (21 (link)–22 (link)).
Lipid extracts were obtained from tissue and TAG quantified by colorimetric assay (Wako Pure Chemical Industries.) (15 (link)). The fractional 13C enrichment of TAG was assessed by liquid chromatography/mass-spectrometry (LC/MS) analysis. LCFA content in TAG, of carbon lengths 12–18, was determined by LCMS as a percentage of total LCFA. Total TAG turnover (nmoles TAG/min/mg protein) was quantified from 13C enrichment rates and the endpoint 13C enrichment (15 (link)–18 (link)).
Rates of palmitate unit turnover within the TAG pool were determined from TAG turnover rates and the percentage of acyl units represented by palmitate ([12C + 13C] palmitate) present in the TAG pool. LCMS analysis enabled determination of the percentage of each LCFA present in the TAG pool. From the stoichiometry of 3 fatty acyl groups per TAG molecule and the percentage of palmitate present in the TAG pool, TAG turnover rates were converted to rates of palmitate unit turnover within the TAG pool.
13C enrichment of TAG in the heart was monitored from the NMR signal at 30.5 ppm from the TAG methylene groups. TAG turnover was calculated from total TAG content and enrichment over time (15 (link)–18 (link)). Kinetic analysis of dynamic 13C-spectra from hearts was performed as previously reported (14 –15 (link), 17 (link)–18 (link)).
Metabolic flux was determined during 13C palmitate oxidation in the intact mouse heart using a previously described method for kinetic analysis of the progressive 13C enrichment of glutamate, as detected via NMR (14 , 20 (link), 22 (link)–24 ).
For kinetic analysis of oxidative rates, glutamate, aspartate, citrate malate and α-ketoglutarate contents in frozen myocardial samples were assayed spectrophotometrically and fluorometrically (19 –20 (link)). In vitro 13C NMR was performed on acid extracts of myocardium to determine fractional enrichment of [2-13C] acetyl CoA (21 (link)–22 (link)).
Lipid extracts were obtained from tissue and TAG quantified by colorimetric assay (Wako Pure Chemical Industries.) (15 (link)). The fractional 13C enrichment of TAG was assessed by liquid chromatography/mass-spectrometry (LC/MS) analysis. LCFA content in TAG, of carbon lengths 12–18, was determined by LCMS as a percentage of total LCFA. Total TAG turnover (nmoles TAG/min/mg protein) was quantified from 13C enrichment rates and the endpoint 13C enrichment (15 (link)–18 (link)).
Rates of palmitate unit turnover within the TAG pool were determined from TAG turnover rates and the percentage of acyl units represented by palmitate ([12C + 13C] palmitate) present in the TAG pool. LCMS analysis enabled determination of the percentage of each LCFA present in the TAG pool. From the stoichiometry of 3 fatty acyl groups per TAG molecule and the percentage of palmitate present in the TAG pool, TAG turnover rates were converted to rates of palmitate unit turnover within the TAG pool.
