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Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa.
- Source :
- Analytical & Bioanalytical Chemistry; Jul2015, Vol. 407 Issue 18, p5513-5519, 7p
- Publication Year :
- 2015
-
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 C,N-agmatine (synthesized by decarboxylation of uniformly labeled C,N-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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 16182642
- Volume :
- 407
- Issue :
- 18
- Database :
- Complementary Index
- Journal :
- Analytical & Bioanalytical Chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 103383361
- Full Text :
- https://doi.org/10.1007/s00216-015-8724-0