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Method Development for the Micromolar Analysis of Pyocyanin (PYO) in Blood and Urine via Mass Spectrometry (MS)
Author(s) -
Mawhinney Thomas Patrick,
Chance Deborah L.,
Waters James K.,
Mossine Valeri V.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.658.9
Subject(s) - pyocyanin , chemistry , phenazine , reactive oxygen species , mass spectrometry , viability assay , virulence , chromatography , biochemistry , cell , gene , quorum sensing
The virulence factor, pyocyanin (PYO), is one of several toxins that are produced by the opportunistic Gram negative microbe Pseudomonas aeruginosa (PA) and is capable of affecting a broad range of cellular constituents and pathways. It is a soluble, redox‐active blue phenazine pigment that is zwitterionic and, at the pH of blood, freely moves through the cytoplasmic membrane and has been shown to be important in PA virulence. Intracellularly, PYO increases reactive oxygen species (ROS) which can directly and/or indirectly cause cell damage. Its cytotoxicity had been demonstrated on numerous cell lines which can result in morphological changes that include PYO‐induced cell vacuolation, along with a drop in the cellular viability, metabolic mitochondrial activity, and decreased transcriptional and translational activity. A major difficulty in research moving forward is the measurement of PA‐derived PYO in blood and urine from patients with bacteremia, urinary tract infections, cystic fibrosis, and others. In this study, we address the need for a more sensitive method of analysis by synthesizing internal standards (IS) of PYO with stable isotopes to be used for MS analysis. Initial experiments demonstrated that by reacting phenazine methosulfate (PMS) with H 2 O 18 (98% purity), PYO‐O 18 ( m/z , 213 M+H + ) was produced in near quantitative yields. In a parallel fashion, by reacting phenazine with dimethyl sulfate‐D6, crystalline PMS(D3) was produced. When this product was individually reacted with H 2 O 16 and H 2 O 18 , PYO‐D3 ( m/z , 214 M+H + ), and PYO‐D3O 18 ( m/z , 216 M+H + ) were formed, respectively. With this library of isotopically labeled PYO analogues, differing in atomic mass units (amu) of 2, 3 and 5 (i.e., PYO‐O 18 , PYO‐D3 and PYO‐D3O 18 , respectively) from PYO ( m/z , 211 M+H + ), an analytical scheme was devised. Bovine serum, fortified with PYO, and spiked with PYO‐D3 as the IS, was first used to assist in the development of a PYO isolation scheme amenable to MS analysis (Orbitrap) via direct infusion, and by LC‐MS analysis. Preliminary results indicated that PYO, with a level of detection of ≤40 ng/mL, is achievable. Current research demonstrates that by utilizing both PYO‐D3 and PYO‐D3O 18 the unambiguous identification and accurate measurement of bacterial exotoxin PYO in both human serum and urine is possible in the low ng/mL range. Support or Funding Information Supported by the Leda J. Sears Trust Fund, the Cystic Fibrosis Association of Missouri, and the University of Missouri Agricultural Experiment Station Laboratories.