Isolation and Quantitation of Long-Chain Acyl-Coenzyme A Esters in Brain Tissue by Solid-Phase Extraction

Deutsch, Joseph; Grange, Eric; Rapoport, S; Purdon, A. D.

doi:10.1006/abio.1994.1344

Cited by 113 publications

(107 citation statements)

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“…Acyl-CoA species were repurified, when needed, using an oligonucleotide purification cartridge (Deutsch et al, 1994). A.…”

Section: Methodsmentioning

confidence: 99%

“…The different molecular species of the cerebral acyl-CoA pool then were isolated and quantified by the method of Deutsch et al (1997). The procedure was similar to that reported previously (Deutsch et al, 1994), including solubilization and solid-phase extraction with an oligonucleotide cartridge (Applied Biosystems, Foster City, CA, U.S.A.). However, the extract was passed three times through the cartridge.…”

Section: Acyl-coa Analysismentioning

confidence: 99%

“…In this study, we compare values for the dilution factor X before and after 5 mm of ischemia-reperfusion in the gerbil, using steady-state infusion of radiolabeled fatty acids in plasma and isolation of the acylCoA species from brain (Deutsch et al, 1994;Washizaki et al, 1994;Grange et al, 1995). We then estimate by Eq.…”

Section: Jfa = C~r(t)/xf(cp*iicpi)dtmentioning

confidence: 99%

“…The eluate was dried down under nitrogen and redissolved in 60 p1 of isopropanol! 1 mM glacial acetic acid, and all acyl-CoA molecular species, including the polyunsaturated molecular species, were separated by HPLC (Deutsch et al, 1994Rabin et al, 1996). The fractions corresponding to radiolabeled acyl-CoA molecular species were collected, and the coincident /3 radioactivity was quantified for palmitoylCoA or arachidonoyl-CoA peaks.…”

Section: Acyl-coa Analysismentioning

confidence: 99%

“…Although monitoring incorporationof radiolabeled fatty acids by autoradiography can localize brain areas responsive to pathology, the underlying kinetics of reincorporation cannot be defined by this method alone. To overcome this limitation, we have developed a fatty acid model (Robinson et al, 1992) that utilizes procedures for the isolation and quantification of molecular species of acyl-CoA (Deutsch et al, 1994 for determining rates of incorporation of unlabeled fatty acids into brain membranes.…”

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confidence: 99%

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Selective Acceleration of Arachidonic Acid Reincorporation into Brain Membrane Phospholipid Following Transient Ischemia in Awake Gerbil

Rabin

Chang

Grange

et al. 1998

Journal of Neurochemistry

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Awake gerbils were subjected to 5 mm of forebrain ischemia by clamping the carotid arteries for 5 mm and then allowing recirculation. Radiolabeled arachidonic or palmitic acid was infused intravenously for 5 mm at the start of recirculation, after which the brains were prepared for quantitative autoradiography or chemical analysis. Dilution of specific activity of the acyl-C0A pool was independently determined for these fatty acids in control gerbils and following 5 mm of ischemia and 5 mm of reperfusion. Using a quantitative method for measuring regional in vivo fatty acid incorporation into and turnover within brain phospholipids and determining unlabeled concentrations of acyl-CoAs following recirculation, it was shown that reperfusion after 5 mm of ischemia was accompanied by a threefold increase compared with the control in the rate of reincorporation of unlabeled arachidonate that had been released during ischemia, whereas reincorporation of released palmitate was not different from the control. Selective and accelerated reincorporation of arachidonate into brain phospholipids shortly after ischemia may ameliorate specific deleterious effects of arachidonate and its metabolites on brain membranes. Key Words: lschemia-Fatty acid metabolism-Phospholipid metabolism-Acyl-C0A-Brain-Gerbil-.Reperfusion-Recovery-Arachidonic acid-Palmitic acid.

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“…Acyl-CoA species were repurified, when needed, using an oligonucleotide purification cartridge (Deutsch et al, 1994). A.…”

Section: Methodsmentioning

confidence: 99%

Section: Acyl-coa Analysismentioning

confidence: 99%

Section: Jfa = C~r(t)/xf(cp*iicpi)dtmentioning

confidence: 99%

Section: Acyl-coa Analysismentioning

confidence: 99%

mentioning

confidence: 99%

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Selective Acceleration of Arachidonic Acid Reincorporation into Brain Membrane Phospholipid Following Transient Ischemia in Awake Gerbil

Rabin

Chang

Grange

et al. 1998

Journal of Neurochemistry

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Measuring long‐chain acyl‐coenzyme A concentrations and enrichment using liquid chromatography/tandem mass spectrometry with selected reaction monitoring

Błachnio-Zabielska

Koutsari

Jensen

2011

Rapid Comm Mass Spectrometry

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Long chain acyl-CoA's (LCACoA) are the activated form of long chain fatty acids and serve as key lipid metabolites. Excess accumulation of intracellular LCACoA, diacylglycerols (DAGs) and ceramides may create insulin resistance with respect to glucose metabolism. We present a new method to measure LCACoA concentrations and isotopic enrichment of palmitoyl-CoA ([U-13C]16-CoA) and oleoyl-CoA ([U-13C]18:1-CoA) using UPLC/MS/MS to quantitate 7 different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on reverse-phase UPLC column using a binary gradient with ammonium hydroxide (NH4OH) in water and NH4OH in ACN. The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate and oleate were monitored as [M+2+H]+ and the [U13C]16-CoA and [U13C]18:1-CoA were monitored as [M+16+H]+ and [M+18+H]+ respectively. The method is simple, sensitive and efficient (run time as short as 5 min) and allowed us to measure the concentration and detect enrichment in intramyocellular [U13C]16-CoA and [U13C]18:1-CoA during a low dose intravenous infusion of [U13C]palmitate and [U13C]oleate in adults undergoing either a saline control experiment or an insulin/glucose infusion experiment. This technique should allow investigators to measure the trafficking of extracellular fatty acids to the intracellular LCACoA pool.

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Stable isotope dilution liquid chromatography/mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-coenzyme A thioesters

Snyder

Basu

Zhou

et al. 2014

Rapid Commun. Mass Spectrom.

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RATIONALE Acyl-Coenzyme A (CoA) thioesters are the principal form of activated carboxylates in cells and tissues. They are employed as acyl carriers that facilitate the transfer of acyl groups to lipids and proteins. Quantification of medium-chain and long-chain acyl-CoAs represents a significant bioanalytical challenge because of their instability. METHODS Stable isotope dilution-liquid chromatography-selected reaction monitoring-mass spectrometry (LC-SRM/MS) provides the most specific and sensitive method for the analysis of CoA species. However, relevant heavy isotope standards are not available and they are challenging to prepare by chemical synthesis. Stable isotope labeling by essential nutrients in cell culture (SILEC) developed originally for the preparation of stable isotope labeled short-chain acyl-CoA thioester standards has now been extended to medium-chain and long-chain acyl-CoAs and used for LC-SRM/MS analyses. RESULTS Customized SILEC standards with > 98 % isotopic purity were prepared using mouse Hepa 1c1c7 cells cultured in pantothenic-free media fortified with [13C315N1]-pantothenic acid and selected fatty acids. A SILEC standard in combination with LC-SRM/MS was employed to quantify cellular concentrations of arachidonoyl-CoA (a representative long-chain acyl-CoA) in two human colon cancer cell lines. A panel of SILEC standards was also employed in combination LC-SRM/MS to quantify medium- and long-chain acyl-CoAs in mouse liver. CONCLUSION This new SILEC-based method in combination with LC-SRM/MS will make it possible to rigorously quantify medium- and long-chain acyl-CoAs in cells and tissues. The method will facilitate studies of medium- and long-chain acyl-CoA dehydrogenase deficiencies as well as studies on the role of medium- and long-chain acyl-CoAs in cellular metabolism.

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Isolation and Quantitation of Long-Chain Acyl-Coenzyme A Esters in Brain Tissue by Solid-Phase Extraction

Cited by 113 publications

References 0 publications

Selective Acceleration of Arachidonic Acid Reincorporation into Brain Membrane Phospholipid Following Transient Ischemia in Awake Gerbil

Selective Acceleration of Arachidonic Acid Reincorporation into Brain Membrane Phospholipid Following Transient Ischemia in Awake Gerbil

Measuring long‐chain acyl‐coenzyme A concentrations and enrichment using liquid chromatography/tandem mass spectrometry with selected reaction monitoring

Stable isotope dilution liquid chromatography/mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-coenzyme A thioesters

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