2006 Progress Report: Oxidative Stress Responses to PM Exposure in Elderly Individuals With Coronary Heart Disease

EPA Grant Number: R832413C004
Subproject: this is subproject number 004 , established and managed by the Center Director under grant R832413
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Southern California Particle Center
Center Director: Froines, John R.
Title: Oxidative Stress Responses to PM Exposure in Elderly Individuals With Coronary Heart Disease
Investigators: Delfino, Ralph , Vaziri, Nosratola D , Staimer, Norbert , Neuhausen, Susan
Current Investigators: Delfino, Ralph , Vaziri, Nosratola D , Gillen, Dan , Staimer, Norbert , Neuhausen, Susan , Gastanaga, Victor
Institution: University of California - Irvine
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2012)
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Health Effects , Air


The overall goal of this study is to advance knowledge on the importance of particle size and composition to the induction of oxidative stress responses in a high-risk population of elderly people with coronary artery disease. We hypothesize that biomarkers of oxidative stress responses will be associated with indoor and outdoor home PM mass and total particle number concentration, which will support the view that PM leads to systemic inflammatory responses. We further hypothesize that biomarkers will be more strongly associated with predicted indoor exposure to PM of outdoor origin (from source tracer analyses). We will also evaluate effects of exposure to specific metals, elemental and organic carbon, and specific organic components used as source tracers. We further hypothesize that biomarker associations with ultrafine and fine PM will be better explained by chemical assays that measure reactive oxygen species and electrophilic activity.

Progress Summary:


Over a period of seven months (July 10, 2005 to February 17, 2006), 32 subjects were followed with weekly blood draws. Venous peripheral blood samples were drawn at the retirement homes using anti-coagulant Vacutainer tubes. The blood samples were taken to a mobile field laboratory (equipped with a Class II Type A biological safety cabinet and a refrigerated benchtop clinical centrifuge) next to the retirement homes, and rapidly separated (< 30 min) into erythrocytes and plasma. After centrifugation, each fraction was aliquoted, coded, and transported frozen on dry ice from the field to the UCI core laboratory and stored at ~80°C until tested. Further, whole blood was aliquoted into microcentrifuge vials before centrifugation and immediately put on dry ice as well. So far, around 1,500 Vacutainer blood samples (separated into 3,240 fractions of whole EDTA blood, EDTA plasma, CTAD plasma, RBC lysates, and 4,680 EDTA plasma aliquots) have been collected.


To date, all of the collected erythrocyte lysates (358 samples) have been assayed for Glutathione Peroxidase-1 (GPx-1) and Superoxide Dismutase (SOD) activity (Cayman Chem., cat. #703102 and #706002). In addition, the concentration of hemoglobin in blood cell hemolysate was determined colorimetrically at 540 nm (cyanmethemoglobin method; Eagle Diagnostics, cat# 6200) to express the units of erythrocyte GPx-1 and SOD activities in relationship to the hemoglobin concentration of each sample (U/gHb). The mean enzyme activities (U/gHb) were 14.8 ± 5.9 for GPx-1 and 5281 ± 1746 for SOD (n = 358). Both GPx-1 and SOD activities were in the range previously reported for elderly subjects (Karolkiewicz, et al., 2003; Kedziora-Kornatowska, et al., 2004)

Further, Myeloperoxidase (MPO) was added to the oxidative stress marker panel, because it has antioxidant functions and is an important indicator of age-dependent cardiovascular risk (Bekesi, et al., 2001). So far, all of the human EDTA plasma samples (358 samples in total) have been analyzed for Myeloperoxidase using a commercially available 96-well immunoassay kit (Alpco Diagnostics; cat# 30-6631). The mean MPO concentration (ng/mL) was 34.0 (SD 48.1) and is similar to EDTA plasma concentrations found in healthy blood donors (Bekesi, et al., 2001).

All of the samples were run in duplicate and all of the repeated measurements (up to 12 per subject) were analyzed on one plate in order to circumvent inter-assay variation. A temperature controlled, monochromatic microplate reader (VERSAmaxTM; wavelength range: 340-850 nm) was used for high sample throughput.

Evaluation of Blood Sample Preparation Methods for the GSH/GSSG Assay

We evaluated the potential use of whole blood collected in the field laboratory for the accurate measurement of the GSH/GSSG ratio as an indicator of oxidative stress. The following sample preparation method was tested (recommended test kit protocol; OxisResearch, cat# 21040):

Whole blood aliquots for the GSH sample were obtained from 13 subjects with six consecutive weekly field blood draws per subject. In order to prevent oxidation of GSH to GSSG, ex vivo whole blood aliquots for the GSSG samples were added into microcentrifuge tubes pre-loaded with a M2VP thiol-scavenging reagent and stored at –70°C. GSH/GSSG analysis was characterized by unusual high peaks in GSSG concentrations within the repeated measurements (up to 250 times above expected values) strongly indicating ex vivo oxidation of GSH after the blood draws. The observed phenomenon may only be circumvented by pre-loading the Vacutainer tubes with the scavenging reagent, resealing, and then reproducing the negative pressure. As a result, accurate determinations of the GSH/GSSG ratio in frozen whole blood samples collected in the field are not feasible at this time given the need for our phlebotomist to collect multiple tubes preloaded by the manufacturer with reagents.

Evaluation of the 8-Isoprostane (8-iso PGF2α) EIA Test Kit

Blood samples were spiked with 12 pg/mL 8-isoprostane standard and then analyzed by using the 8-Isoprostane EIA kit (Cayman Chem; cat # 51351) resulting in a recovery of 108%. Preliminary tests indicate that 8-isoprostane can be accurately quantified in EDTA plasma samples collected in the field by following an affinity sorbent purification step.

Future Activities:

Further evaluation of the 8-Isoprostane (8-iso PGF2α) EIA test kit will be carried out in order to confirm preliminary recovery studies. Based on the results, routine procedures for the measurement of 8-isoprostane in EDTA plasma samples will be established. We will also test methods for the determination of extracellular superoxide dismutase (SOD) activity in plasma aliquots. We will also evaluate less expensive and noninvasive methods to assess oxidative stress using exhaled hydrocarbons to estimate in vivo lipid peroxidation, and compare this with blood data from Projects 2 and 4. MPO, SOD, and GPx-1 analysis will resume as soon as the weekly blood draws in the second year panel are completed (Jul 2005-Feb 2007). We will conduct preliminary analyses of year one data to evaluate the relationship of biomarkers to indoor and outdoor home air pollutants as proposed.


Karolkiewicz J, Szczesniak L, Deskur-Smielecka E, et al. Oxidative stress and antioxidant defense system in healthy, elderly men: relationship to physical activity. The Aging Male 2003;6:100-105.

Kedziora-Kornatowska K, Czuczejko J, Pawluk H, et al. The markers of oxidative stress and activity of the antioxidant system in the blood of elderly patients with essential arterial hypertension. Cellular & Molecular Biology Letters 2004;9:635-641.

Bekesi G, Kakucs R, Sandor J, et al. Plasma concentration of myeloperoxidase enzyme in pre- and post-climacterial people: related superoxide anion generation. Experimental Gerontology 2001;37:137-148.

Journal Articles:

No journal articles submitted with this report: View all 35 publications for this subproject

Supplemental Keywords:

health effects, human health, sensitive populations, dose-response, enzymes, genetic polymorphisms, particulates, epidemiology, environmental chemistry, modeling,, RFA, Health, Scientific Discipline, Air, particulate matter, Health Risk Assessment, Risk Assessments, Biochemistry, Ecology and Ecosystems, elderly adults, particulates, atmospheric particulate matter, human health effects, PM 2.5, airway disease, cardiovascular vulnerability, airborne particulate matter, air pollution, human exposure, vascular dysfunction, cardiovascular disease, human health risk

Relevant Websites:

http://www.scpcs.ucla.edu/ Exit

Progress and Final Reports:

Original Abstract
  • 2007 Progress Report
  • 2008 Progress Report
  • 2009 Progress Report
  • 2010 Progress Report
  • 2011
  • Final Report

  • Main Center Abstract and Reports:

    R832413    Southern California Particle Center

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R832413C001 Contribution of Primary and Secondary PM Sources to Exposure & Evaluation of Their Relative Toxicity
    R832413C002 Project 2: The Role of Oxidative Stress in PM-induced Adverse Health Effects
    R832413C003 The Chemical Properties of PM and their Toxicological Implications
    R832413C004 Oxidative Stress Responses to PM Exposure in Elderly Individuals With Coronary Heart Disease
    R832413C005 Ultrafine Particles on and Near Freeways