Over the past year an additional 4 subjects were enrolled in the CHOP component of this study, which involves the development of a novel method that measures the in vivo rate of ureagenesis by administering urea precursors (15NH4Cl ) to human beings and then utilizing gas chromatography-mass spectrometry to measure the formation of [15N]urea. Subjects are given the isotope orally at T-O. Then blood samples were taken at times (minutes) 30, 60, 90, 120, 150, 180 and 240. After appropriate processing deproteinization of samples, the t-butyldimethylsilyl derivative of urea is prepared. This species then is injected onto the gas chromatograph-mass spectrometer system. Selected ion monitoring of the m/z 231/232 fragment is used to monitor 15N isotopic labeling in urea. Only the M+1 species is monitored because of the very low probability that a doubly labeled (M + 2) species would be generated, given the relatively limited amount of isotope that is given in the first instance. With this experimental regimen, we find that [15N]urea in blood typically reaches a peak of labeling between 30 and 90 minutes, after which the concentration recedes. By normalizing the [15N]urea label (atom % excess) to total blood urea (15N + 14N), we can determine reliably the absolute [15N]urea (measured in terms of mmol/liter).
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