Massive Glutamine Cyclization to Pyroglutamic Acid in Human Serum Discovered Using NMR Spectroscopy

Abstract

Glutamine is one of the most abundant metabolites in blood and is a precursor as well as end product central to numerous important metabolic pathways. A number of surprising and unexpected roles for glutamine, including cancer cell glutamine addiction discovered recently, stress the importance of accurate analysis of glutamine concentrations for understanding its role in health and numerous diseases. Utilizing a recently developed NMR approach that offers access to an unprecedented number of quantifiable blood metabolites, we have identified a surprising glutamine cyclization to pyroglutamic acid that occurs during protein removal. Intact, ultrafiltered and protein precipitated samples from the same pool of human serum were comprehensively investigated using 1H NMR spectroscopy at 800 MHz to detect and quantitatively evaluate the phenomenon. Interestingly, although glutamine cyclization occurs in both ultrafiltered and protein precipitated serum, the cyclization was not detected in intact serum. Strikingly, due to cyclization, the apparent serum glutamine level drops by up to 75% and, concomitantly, the pyroglutamic acid level increases proportionately. Further, virtually under identical conditions, the magnitude of cyclization is vastly different for different portions of samples from the same pool of human serum. However, the sum of glutamine and pyroglutamic acid concentrations in each sample remains the same for all portions. These unexpected findings indicate the importance of considering the sum of apparent glutamine and pyroglutamic acid levels, obtained from the contemporary analytical methods, as the actual blood glutamine level for biomarker discovery and biological interpretations.

NMR-Guided Mass Spectrometry for Absolute Quantitation of Human Blood Metabolites

“…Here, MS concentrations for one serum sample were set equal to those derived from NMR (serum 1, Table S3) and used as the references for the quantitation of the same metabolites in the remaining serum samples. The poor correlations of glutamine and pyroglutamic acid are as anticipated on the basis of massive glutamine cyclization discovered recently by NMR 15,25 as well as MS. 26 …”

“…For glutamine and pyroglutamic acid, the observed correlations were in accordance with the known phenomena of glutamine cyclization to pyroglutamic acid. 15,25,26 Glutamine cyclization is shown to occur during sample preparation. 25 Cyclization also occurs in the electrospray-ionization (ESI) source during LC-MS analysis; the magnitude of cyclization in the ESI source can range from 33 to 100% and depends on the fragmentor voltage.…”

“…15,25,26 Glutamine cyclization is shown to occur during sample preparation. 25 Cyclization also occurs in the electrospray-ionization (ESI) source during LC-MS analysis; the magnitude of cyclization in the ESI source can range from 33 to 100% and depends on the fragmentor voltage. 26 In view of such effects by the ESI source, as well as those caused by sample preparation, 15,25 it is important to be careful with the general assumption that the vast majority of metabolites are stable when evaluated in metabolomics.…”

“…25 Cyclization also occurs in the electrospray-ionization (ESI) source during LC-MS analysis; the magnitude of cyclization in the ESI source can range from 33 to 100% and depends on the fragmentor voltage. 26 In view of such effects by the ESI source, as well as those caused by sample preparation, 15,25 it is important to be careful with the general assumption that the vast majority of metabolites are stable when evaluated in metabolomics. These findings also open new avenues for the identification of potentially unstable metabolites during MS analysis, the knowledge of which is potentially important for improved biomarker discovery and biological interpretations.…”

“…Glutamine and pyroglutamic acid were omitted because of errors arising from glutamine cyclization. 15,25,26 Unless otherwise noted, the MS data were obtained using an AB Sciex QTrap 5500 mass spectrometer.…”