title: Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa.
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 (13)C5,(15)N4-agmatine (synthesized by decarboxylation of uniformly labeled (13)C6,(15)N4-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.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN, 55455, USA, jdalluge@umn.edu.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Dalluge
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: McCurtain
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Gilbertsen
authors.forename: Kyle A
authors.initials: KA
authors.lastname: Kalstabakken
authors.forename: Bryan J
authors.initials: BJ
authors.lastname: Williams
mesh_terms: D000376:Agmatine / Q000032:analysis* / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003550:Cystic Fibrosis / Q000382:microbiology; D006801:Humans; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism; D007553:Isotope Labeling; D057230:Limit of Detection; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000376:Agmatine; D006867:Hydrolases; C019626:agmatine deiminase; D002262:Carboxy-Lyases; C019722:arginine decarboxylase