Detection of acyl-coenzyme A thioester intermediates of fatty acid beta-oxidation as the N-acylglycines by negative-ion chemical ionization gas chromatography-mass spectrometry.
An analytical method for the separation and quantitation of acyl-CoA thioesters by gas chromatography-mass spectrometry is described. The method utilizes glycine aminolysis of the acyl-CoA thiolesters, esterification with pentafluorobenzyl bromide followed by gas chromatographic separation, and detection by negative chemical ionization mass spectrometry of the N-acylpentafluorobenzyl glycinates. The glycine aminolysis provides over 100-fold discrimination against oxygen esters and obviates the difficulty of removing trace contaminants of free fatty acids. The limit of detection of the described methodology for palmitoyl-CoA has been found to be 300 fmol, which improves at shorter chain lengths. Baseline separation was obtained for a standard mixture of seven acyl-CoAs (60 pmol injected) containing butyryl-CoA, hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA. The above procedure is also applicable to the alpha-beta unsaturated and 3-hydroxyacyl-CoA derivatives, making it possible to quantify all of the intermediates in fatty acid oxidation, except the 3-ketoacyl-CoAs, in a single procedure.
Tamvakopoulos
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CS
Department of Chemistry, Brown University, Providence, Rhode Island 02912.
Anderson
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VE
D000214:Acyl Coenzyme A / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D002250:Carbon Radioisotopes; D005227:Fatty Acids / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D005998:Glycine / Q000031:analogs & derivatives*; D007202:Indicators and Reagents; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010171:Palmitoyl Coenzyme A; D011865:Radioisotope Dilution Technique; D051381:Rats; D013329:Structure-Activity Relationship; D014316:Tritium
D000214:Acyl Coenzyme A; D002250:Carbon Radioisotopes; D005227:Fatty Acids; D007202:Indicators and Reagents; D014316:Tritium; D010171:Palmitoyl Coenzyme A; D005998:Glycine