Final Report: Oxidation Stress Makers

EPA Grant Number: R827355C011
Subproject: this is subproject number 011 , established and managed by the Center Director under grant R827355
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Airborne PM - Northwest Research Center for Particulate Air Pollution and Health
Center Director: Koenig, Jane Q.
Title: Oxidation Stress Makers
Investigators: Simpson, Chris , Kavanagh, Terrance J , Luchtel, Daniel L.
Institution: University of Washington
EPA Project Officer: Chung, Serena
Project Period: June 1, 1999 through May 31, 2004 (Extended to May 31, 2006)
RFA: Airborne Particulate Matter (PM) Centers (1999) RFA Text |  Recipients Lists
Research Category: Air Quality and Air Toxics , Particulate Matter , Air


The aims of this project are to assess oxidative stress in vivo in humans and animals exposed to diesel exhaust through the measurement of 3-nitrotyrosine and F2-isoprostanes in biological samples.

Summary/Accomplishments (Outputs/Outcomes):

The oxidative stress markers project was a new pilot scale project that was established in year 6 of the PM center. In the first year we acquired isotopically labeled standards for the F2-isoprostane assay, and we have completed synthesis, purification and evaluation of the isotopically labeled standards for the nitrotyrosine assay. Optimization of the Agilent XCT Trap for analysis of nitrotyrsoine has been completed, and validation of our sample preparation protocols for measurement of free nitrotyrosine in urine and plasma, and protein-bound nitrotyrosine in plasma is complete. Blood and urine samples have been collected and archived from human volunteers exposed to diesel exhaust.

The seventh year has been devoted exclusively towards trying to develop a sensitive, reliable and robust LC/MS/MS method in order to determine 3-nitrotyrosine in human urine, plasma filtrate and bound to plasma proteins.

  1. Synthesis of the 13C labeled compounds for the nitrotyrosine assay proved more challenging than we had anticipated. We had to develop our own synthetic protocols as the yields from the literature methods were unacceptably low. The new protocols we developed proved to be successful, and we have now prepared and substantial quantities of 13C6-nitrotyrosine, 13C6-3-nitro-4-hydroxyphenylacetate (NHPA) and 13C8-4-hydroxyphenylacetate. These standards are an invaluable resource that will facilitate the accurate, artifact-free determination of nitrotyrosine in biological samples.
  2. We developed a one step SPE extraction procedure based on ion exchange chromatography, in order to enrich and purify 3-nitrotyrosine from human samples. While analyte recoveries using this procedure were good, sensitivity was unacceptably poor as a result of ion suppression effects in the HPLC/MS interface.
  3. A two step SPE extraction procedure was then developed, using C18 and chrom-P cartridges, in order to remove the extraneous material in the biological samples that was responsible for the ion suppression. Although this two-step procedure dramatically reduced the ion suppression we observed in infusion studies, the ion suppression was still too severe to permit measurement of 3-nitrotyrosine at the low levels present in human samples. Furthermore, an interfering compound was present in the urine samples that prevented accurate quantification of 3-nitrotyrosine.
  4. Following the procedure of Delatour et al, we are now derivatizing the 3-nitrotyrosine to its butyl-ester. The butyl ester elutes at a later retention time, and appears to be relatively (but not completely) resistant to the ion suppression effects that have plagued this assay (at least for urine and plasma filtrate samples). The procedure does not appear to be effective for determining 3-nitrotyrosine adducted to plasma proteins. We shall continue to evaluate the reliability and reproducibility of the butyl ester procedure.
  5. Finally, we propose to develop a dual column HPLC separation procedure in order to try and overcome the problems of ion suppression and co-eluting interferences that have thus far prevented us from reliably measuring 3-nitrotyrosine in human biological samples.

It is disappointing that we were not able to develop a protocol for analysis of nitrotyrosine with the time frame of the research funded by the EPA NW PM Center. As work progresses on this objective, we will credit the Center for its support.

Supplemental Keywords:

RFA, Health, Scientific Discipline, Air, particulate matter, air toxics, Environmental Chemistry, Health Risk Assessment, Risk Assessments, Biochemistry, automobile exhaust, air pollution, pariculate matter, particle exposure, diesel exhaust, human exposure, PM, airborne urban contaminants, human health risk

Progress and Final Reports:

Original Abstract
  • 1999
  • 2000
  • 2001
  • 2002
  • 2003
  • 2004 Progress Report

  • Main Center Abstract and Reports:

    R827355    Airborne PM - Northwest Research Center for Particulate Air Pollution and Health

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R827355C001 Epidemiologic Study of Particulate Matter and Cardiopulmonary Mortality
    R827355C002 Health Effects
    R827355C003 Personal PM Exposure Assessment
    R827355C004 Characterization of Fine Particulate Matter
    R827355C005 Mechanisms of Toxicity of Particulate Matter Using Transgenic Mouse Strains
    R827355C006 Toxicology Project -- Controlled Exposure Facility
    R827355C007 Health Effects Research Core
    R827355C008 Exposure Core
    R827355C009 Statistics and Data Core
    R827355C010 Biomarker Core
    R827355C011 Oxidation Stress Makers