Submitted to: American Society for Mass Spectrometry
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2006
Publication Date: 6/1/2006
Citation: Adeuya, A., Whitehead, T.R., Price, N.P. 2006. Stable isotopes and mass spectrometry. Flux analysis of the fructose-6-p-phosphoketolase shunt pathway in bifidobacterium [abstract]. American Society for Mass Spectrometry. 17(5S):118S.
Technical Abstract: The enumeration of the human gut bacterium as Bifidobacterium is difficult; the most direct test is based on demonstration of fructose-6 phosphate phosphoketolase (F6PPK) activity in cellular extracts. This is the key enzyme of the so-called "Bifido shunt," a unique metabolic pathway that characterizes the genus. This enzymatic reaction results in a direct cleavage of fructose-6-phosphate to erythrose-4-phosphate and acetyl acetate. Here we show a preliminary study by means of isotopic labeling with [1-**13C]fructose and [U-**13C]fructose and isotopomer analysis of this predominant pathway for the degradation of carbohydrates by Bifidobacterium. In this study, time-depend growth of Bifidobacterium on [1-**13C]fructose and [U-**13C]fructose was carried out. The Bifidobacterium cell pellet for a given time period was hydrolyzed, and analyzed for fatty acid and monosaccharide compositions by using GC-MS. Prior to analysis the monosaccharide components were derivatized into their aldononitrile acetates, while the fatty acid components were converted into methyl esters and pyrrolidines by using reported methods. The derivatized species were analyzed on a Hewlett-Packard gas chromatograph equipped with an autoinjector and mass selective detector configured in positive ion detection mode. The fractional distribution of **13C into these metabolites was evaluated by analysis of their fragmentation pathways following electron impact ionization. Preliminary data confirm that in Bifidobacterium the bifido shunt is the predominant pathway for the degradation of carbohydrates, bypassing glycolysis catabolism and the pentose phosphate pathway. The acetyl phosphate generated from carbon-1,2 of fructose-6-P is reincorporated into cellular fatty acid metabolism. This, and the pattern of labeling in the pentose monosaccharides, are direct in vivo evidence for the present of the F6PPK shunt, and may be a quantifiable characteristic feature of the Bifidobacterium.