Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa

Joseph J. Dalluge, Jennifer L. McCurtain, Adam J. Gilbertsen, Kyle A. Kalstabakken, Bryan J. Williams

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

Abstract A method has been developed for the direct determination of agmatine in bacterial culture supernatants using isotope dilution ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (UPLC-MS/MS). Agmatine determination in bacterial supernatants is comprised of spiking culture or isolate supernatants with a fixed concentration of uniformly labeled 13C5,15N4-agmatine (synthesized by decarboxylation of uniformly labeled 13C6,15N4-arginine using arginine decarboxylase from Pseudomonas aeruginosa) as an internal standard, followed by derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBDF) to improve the reversed-phase chromatographic retention characteristics of agmatine, as well as the selectivity and sensitivity of UPLC-MS/MS detection of this amine in complex biologically derived mixtures. Intrasample precisions for measurement of agmatine in culture supernatants average 4.1 % (relative standard deviation). Calibration curves are linear over the range 5 nM to 10 μM, and the detection limit is estimated at 1.5 nM. To demonstrate the utility of the method, agmatine levels in supernatants of overnight cultures of wild-type (UCBPP-PA14), as well as arginine decarboxylase and agmatine deiminase mutant strains of P. aeruginosa strain UCBPP-PA14 were measured. This method verified that the mutant strains are lacking the specific metabolic capabilities to produce and metabolize agmatine. In addition, measurement of agmatine in supernatants of a panel of clinical isolates from patients with cystic fibrosis revealed that three of the P. aeruginosa isolates hyper-secreted agmatine into the supernatant, hypothesized to be a result of a mutation in the aguA gene. Because agmatine has potential inflammatory activities in the lung, this phenotype may be a virulence factor for P. aeruginosa in the lung environment of cystic fibrosis patients.

Original languageEnglish (US)
Article number8724
Pages (from-to)5513-5519
Number of pages7
JournalAnalytical and Bioanalytical Chemistry
Volume407
Issue number18
DOIs
StatePublished - Jul 28 2015

Keywords

  • Biomarker
  • Disease
  • Metabolite
  • Metabolite profiling

Fingerprint

Dive into the research topics of 'Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa'. Together they form a unique fingerprint.

Cite this