Quantitative analysis of ¹⁵N‐labeled positional isomers of glutamine and citrulline via electrospray ionization tandem mass spectrometry of their dansyl derivatives

Abstract

The enteral metabolisms of glutamine and citrulline are intertwined because, while glutamine is one of the main fuel sources for the enterocyte, citrulline is one of its products. It has been shown that the administration of (15)N-labeled glutamine results in the incorporation of the (15)N label into citrulline, but it is not clear which of the three nitrogen groups of citrulline is actually labeled. To determine the (15)N-enrichment of the positional isomers of glutamine and citrulline, a rapid liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed. The amino acids were analyzed as their dansyl derivatives. The product ion resulting from the loss of NH(3) from the omega carbon allows for the determination of the enrichment of the ureido (citrulline) or amido groups (glutamine). The protonated pyrrolidine (citrulline) or 5-oxopyrrolidine (glutamine) product ion contains the 2-N (amino group) and is used to determine its enrichment. The method described showed no ion suppression and a wide dynamic range ranging from 1.3 picomoles to 2 nanomoles for citrulline. Background samples and standards resulted in enrichments not different from those theoretically expected. The enrichment curves for the different glutamine and citrulline isotopomers were linear (R(2) > 0.998) over the range of enrichments studied. The method developed provides an additional insight into the metabolism of glutamine and citrulline tracing the precursor-product relationship between these two amino acids.

Alterations in glutamine metabolism and its conversion to citrulline in sepsis

“…The transitions observed were precursor ion m/z 409 to product ion m/z 392 at 14 eV for citrulline and precursor ion m/z 365 and product ion m/z 170 for leucine. Plasma isotope enrichments of glutamine were also measured by tandem LC-MS as well as the location of the 15 N positional isomers of glutamine and citrulline, as previously described (23). Briefly, high and low collision energies were applied to the protonated DANS-citrulline derivative, with multiple reactions monitoring of transitions m/z 410 to 392 to identify labeling at the ureido-N position of citrulline, 410 to 71 for labeling at the ␣-N position, and 410 to 393 for labeling at the ␣-N or ␦-N position of citrulline.…”

Reply to GC Ligthart-Melis et al

“…The use of this nitrogen tracer and its shortcomings have already been mentioned in our article's Discussion (1). We agree that, although our study provides qualitative information on the glutamine to citrulline and arginine pathway by using this [2][3][4][5][6][7][8][9][10][11][12][13][14][15] N]glutamine, quantitative results should be interpreted with caution because the outcome may overestimate the contribution of glutamine to citrulline and arginine formation (1,6).…”

“…We recently confirmed using the same analytic approach these findings in postprandial surgical patients who received alanyl- [2-15 N]glutamine as a stable isotope to investigate the precursor relation between glutamine, citrulline, and arginine (GC Ligthart-Melis GC, unpublished results, 2014). Had Buijs et al used a similar LC-MS/MS analytic approach that allows for the measurement of the positional isomers of glutamine and citrulline (10), they would have observed this same phenomenon. For this reason and because every single assumption on the use of [2-15 N]glutamine to determine precursor-product relations is violated, the conversion data generated by Buijs et al cannot be interpreted.…”

Extrarenal citrulline disposal in mice with impaired renal function

“…Arginine and citrulline plasma concentrations and enrichments were determined by liquid chromatography-tandem mass spectrometry as their dansyl derivatives as described elsewhere (26). Tissue was homogenized, and protein was precipitated with perchloric acid (PCA) to determine the incorporation of 14 C into protein.…”