Isotopomer enrichment assay for very short chain fatty acids and its metabolic applications.
The present work illustrated an accurate GC/MS measurement for the low isotopomer enrichment assay of formic acid, acetic acid, propionic aicd, butyric acid, and pentanoic acid. The pentafluorobenzyl bromide derivatives of these very short chain fatty acids have high sensitivity of isotopoic enrichment due to their low natural isotopomer distribution in negative chemical ionization mass spectrometric mode. Pentafluorobenzyl bromide derivatization reaction was optimized in terms of pH, temperature, reaction time, and the amount of pentafluorobenzyl bromide versus sample. The precision, stability, and accuracy of this method for the isotopomer analysis were validated. This method was applied to measure the enrichments of formic acid, acetic acid, and propionic acid in the perfusate from rat liver exposed to Krebs-Ringer bicarbonate buffer only, 0-1mM [3,4-(13)C(2)]-4-hydroxynonanoate, and 0-2mM [5,6,7-(13)C(3)]heptanoate. The enrichments of acetic acid and propionic acid in the perfusate are comparable to the labeling pattern of acetyl-CoA and propionyl-CoA in the rat liver tissues. The enrichment of the acetic acid assay is much more sensitive and precise than the enrichment of acetyl-CoA by LC-MS/MS. The reversibility of propionyl-CoA from succinyl-CoA was confirmed by the low labeling of M1 and M2 of propionic acid from [5,6,7-(13)C(3)]heptanoate perfusates.
Department of Nutrition, Case Western Reserve University, 10900 Euclid Avenue, W-G48, Cleveland, OH 44106-4954, USA.
Kristyen
K
Tomcik
Rafael A
RA
Ibarra
Sushabhan
S
Sadhukhan
Yong
Y
Han
Gregory P
GP
Tochtrop
Guo-Fang
GF
Zhang
D000085:Acetates / Q000737:chemistry; D000818:Animals; D002247:Carbon Isotopes / Q000737:chemistry; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D005232:Fatty Acids, Volatile / Q000737:chemistry* / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006538:Heptanoic Acids / Q000737:chemistry / Q000378:metabolism; D007536:Isomerism; D008297:Male; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley
D000085:Acetates; D002247:Carbon Isotopes; D005227:Fatty Acids; D005232:Fatty Acids, Volatile; D006538:Heptanoic Acids; C008776:pelargonic acid