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NHLBI Workshop on Oxidative Stress/Inflammation and
Heart, Lung, Blood, and Sleep Disorders
Meeting
Summary Bethesda, Maryland
TABLE OF CONTENTS
Introduction
Normal metabolic processes generate potentially deleterious reactive
oxygen species, oxidative damage and inflammation, which increase with age
and may contribute to senescence. Oxidative stress and inflammation have
been implicated in many heart, lung, blood, and sleep (HLBS) disorders,
including atherosclerosis, hypertension, asthma, COPD, acute lung injury,
heart failure and sleep apnea. CRP and other measures of inflammation have
been incorporated into many population studies. However, there are many
components to these complex systems and it is unclear which are the most
predictive of disease, and how these measures can be integrated with
measures of oxidative stress. Both oxidative stress and inflammation
biomarkers, as well as anti-oxidants and anti-inflammatory factors, need
to be measured concurrently since the metabolic processes overlap and the
lack of balance between these stressors and protectors may lead to
development or progression of HLBS disorders. While there are many
biomarkers of inflammation, oxidative stress is difficult to measure in
vivo. Reliable surrogate biomarkers for use in large-scale population
studies need to be identified. The NHLBI thus convened a workshop on
August 18-19, 2004 with the following objectives: 1) assess the
opportunities for incorporating measures of oxidative stress and
inflammation into population studies; and 2) recommend future research
directions for studying the role of these risk factors in the development
of HLBS disorders.
The workshop began with recognition of the importance of studying
oxidative stress in population studies despite the failure of antioxidant
trials, and an overview of criteria to be considered in selecting
biomarkers for population studies. Laboratory/clinical perspectives on
several biomarkers and/or methods were next presented, followed by the
experience to date in utilizing these biomarkers in several NHLBI
observational studies and clinical trials. Integrating oxidative stress
and inflammation biomarkers and methodological issues were also addressed.
The workshop concluded with summary and recommendations on which
biomarkers were currently suited for use in large scale population
studies, and what additional data and/or resources were needed to continue
work in this field.
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Population Study Biomarkers and Evaluation Criteria
Dr. Nieto outlined issues regarding selecting biomarkers for large
scale population studies. Biomarkers may measure internal exposure, host
susceptibility, or early effects of disease. An individual biomarker may
be involved in several physiologic pathways. Biological validity,
sensitivity and specificity, standardization, and reproducibility, as well
as the need for special collection methods, must be evaluated during
planning. It is important to realize that strength of association does not
always equal predictive value and thus the contribution of some of these
novel markers to existing predictive equations based on traditional risk
factors might be modest. Biomarkers might prove most valuable in
furthering our understanding of the pathophysiology of complex conditions
by selecting those that most accurately reflect the stage of
pathophysiology or hypothesis to be studied.
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Laboratory/Clinical Perspectives on Biomarkers
F2-isoprostanes: Dr. Morrow reviewed the biochemistry of
F2-isoprostanes and their utility as oxidative stress biomarkers. Gas
chromatography/mass spectrometry measurement of F2-isoprostanes has been
shown to be robust and reliable using commercially available standards.
Some researchers also report that immunoassay methods are acceptable
although these antibodies were generated in individual investigator
laboratories. Commercially available immunoassay kits may present
difficulties with reproducibility and cross-reactivity issues. Evidence
that this class of biomarkers reflects in vivo lipid peroxidation was
presented. Some diurnal variation is seen in individuals but this is not
significant at the population level. Specimens for isoprostane analysis
must be analyzed immediately or stored at -70ºC to prevent artifact
generation, which is primarily a concern for plasma specimens.
F2-isoprostanes can be detected in all tissues and body fluids. Several
studies in which F2-isoprostanes levels have been associated with
cardiovascular risk factors were discussed. Weight and BMI in particular
have been strongly associated with elevations in F2-isoprostanes. While
vitamin E appears to be a relatively weak antioxidant, with sufficient
time lapse, a decrease in F2-isoprostanes in a small study of
hypercholesterolemic humans was eventually seen.
Reactive Oxygen and Nitrogen Species: Dr. Harrison discussed
several reactive oxygen and nitrogen species. Loss of endogenous nitric
oxide production is associated with hypercholestrolemia, diabetes,
hypertension, aging and heart failure. In vascular cells, the reduced form
of nicotinamide adenine dinucleotide phosphates, NADPH oxidases, are a
major source of reactive oxygen species. Electron spin resonance
measurement of xanthine and NADPH oxidases correlates with endothelial
dysfunction in patients with coronary artery disease, but is too difficult
to perform clinically or in population studies. Measurement of
intracellular antioxidant thiols, such as glutathione, may be more
feasible as they can be estimated using the free oxygen radical test
(FORT) via spectrophotometric techniques on specimens obtained from
fingersticks. FORT can be performed on frozen samples. Increased
glutathione redox ratios and increased FORT values have been associated
with increased IMT, microalbuminuria and depression.
Biomarkers in Exhaled Breath: Dr. Erzurum reviewed the
biomarkers of systemic and pulmonary disease that can be measured
non-invasively in exhaled breath and exhaled breath condensate using
chemiluminescent analyzers. Increased exhaled nitric oxide is associated
with upper respiratory tract infections, asthma and lung cancer, while
decreased exhaled nitric oxide is associated with smoking, glucocorticoid
steroids, pulmonary hypertension and cystic fibrosis. Collection of
exhaled nitric oxide can be done in participants of all ages including
children, and in a clinic visit or offsite via collection of breath into
balloons that can be stored and shipped. There is good correlation among
analyzer equipment and good reproducibility within subjects. Exhaled
nitric oxide may be a good biomarker of lower airway inflammation and
suitable for monitoring anti-inflammatory therapy in asthmatic patients or
exacerbations in disease activity. Genetic variants in nitric oxide
synthase have been identified. Relatively little is known about the
reliability of exhaled breath condensate measurement.
Mass Spectrometry Methods: Dr. Hazen discussed the use of mass
spectrometry to create molecular fingerprints of oxidized tyrosine species
that result from NADPH and NOS pathways. These biomarkers are not affected
by freeze/thaw cycles and assays can be performed on de-paraffinized
tissues. The coefficient of variation is low for both intra- and
inter-assays. Normal levels have been established for several gender, age,
and race/ethnic groups in healthy subjects. Bromotyrosine is associated
with FEV1 and airways obstruction in asthmatics. Nitrotyrosine is
associated with endothelial dysfunction and coronary artery disease.
Evidence of the antioxidant effects of statins on inhibiting formation of
superoxide and oxidized tyrosines was presented. Myeloperoxidase was found
to oxidize apoA1 in subjects with cardiovascular disease, and to be an
independent predictor of future adverse outcome in troponin-negative
patients. Methylated arginine species (ADMA), which are stable to storage
and freeze/thaw cycles, have been associated with the metabolic syndrome.
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Observational Studies/Clinical Trial Experience
Framingham Heart Study: Oxidative Stress and Inflammation: Dr.
Benjamin began by reiterating the desired characteristics of a population
study biomarker: measurable via an assay that performs well in the low
range likely to be found in a community-based study; feasible on a large
scale; and utilize a minimal amount of specimen. ELISA assays are often
practical but do not have high throughput. Decisions must be made on
whether to measure a biomarker in the entire cohort or within a
case-referent study. The Framingham Heart Study has experience with
several oxidative stress and inflammation biomarkers. HSP-70 assays were
not fruitful. BMI was found to be an independent risk factor for urinary
F2-isoprostane levels. CRP levels were significantly associated with
hyperemia, but not flow-mediated dilation, after adjustment for other risk
factors. The heritabilities of several inflammatory biomarkers were
presented, and ranged from 14%-44% after multivariate adjustments.
Challenges such as multiple-statistical testing and accessibility of data,
particularly in light of negative publication biases, were discussed.
CARDIA Study: F2 -Isoprostanes: Dr. Gross described how
endogenous and exogenous stressors result in oxidative stress, but need
not lead to disease if the stressors are absorbed without damage, or a
sufficient adaptive increase in antioxidant pools can be induced.
Temporality versus causation is also an issue; the goal is to determine
whether oxidative damage precedes the development of clinical events.
F2-isoprostanes are likely to be a systemic biomarker of oxidative stress,
and can be measured in either plasma or urine. The plasma assay requires a
larger sample but has been validated more than the urine assay. Artifact
formation can be avoided in both specimen types. Gas chromatography/mass
spectrometry measurement is more costly than immunoassays, but more
specific, sensitive, precise and reliable. Whole blood specimens were
stable with slight hemolysis or single freeze-thaw cycles, but
F2-isoprostanes levels increased with storage at -20ºC. In the CARDIA
Study, elevated levels of F2-isoprostanes were associated with female
gender, smoking, triglycerides, HDL, alcohol and BMI. Decreased levels
were associated with blood antioxidant levels, exercise, education and
African-American race.
DASH Studies: Dietary Patterns and Oxidative Stress: Dr. Miller
reviewed the apparent paradox between the role of antioxidants in reducing
oxidative stress and the failure of many antioxidant supplementation
trials, and described the findings of the DASH and DASH-sodium trials. The
Oxygen Radical Absorbing Capacity, or ORAC, antioxidant assay was
utilized. ORAC values are primarily derived from thiol proteins and uric
acid, with smaller contributions from vitamins A, C and E, folic acid and
bilirubin. The DASH trials featured controlled nutrient intake and
repeated measurements, allowing for longitudinal analyses of biomarkers
and traditional CVD risk factors. Those participants following the DASH
diet had increases in serum antioxidant and ORAC levels, and reduced
breath ethane levels. Similarly in the DASH Sodium Trial, those on the
DASH diet had increases in serum antioxidant and ORAC levels, lowered
F2-isoprostane levels and increased antibodies to oxidized LDL. Thus
modulation of oxidative stress biomarkers was demonstrated to be
achievable with dietary changes.
San Antonio Family Heart Study: Genetic Influences on Oxidative
Stress and Inflammation Biomarkers: Dr. Mahaney reported heritability
estimates from the San Antonio Family Heart Study of eight biomarkers of
oxidative stress and ten biomarkers of inflammation, along with the
coefficients of variation for the assays used. Particularly high
heritabilities were found for advanced glycation endproduct (ACE, 0.91)
and paranoxase (PON, 0.84). Plasma nitrotyrosine levels did not differ in
diabetics compared to non-diabetics, nor were the levels associated with
fasting or 2-hour glucose, smoking or BMI. Levels were higher in women
than men. Evidence for pleiotropy between plasma total antioxidant
activity (TAS) and other plasma CVD risk factors, including fasting
insulin and glucose, CRP, VCAM and fibrinogen, was presented. These data
suggest a common genetic basis for interindividual variation in redox
homeostasis and these other plasma CVD risk factors.
Genotype-by-environment interactions were found for the biomarker genotype
with sex and smoking. No significant evidence of chromosomal quantitative
trait loci for TAS has been found to date, possibly due to the composite
nature of the measure; however, some of the additive genetic effect
appears due to a mitochondrial locus. Valid biomarkers are necessary for
all types of studies; in statistical genetic studies accuracy is also
desired, but precision is essential. Genetic association studies should
only be done in populations in which there is some evidence of genetic
influence on the phenotype/trait of interest.
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Integrating Biomarkers/Methological Considerations
Integrating Oxidative Stress and Inflammation Biomarkers: Dr.
Tracy discussed plasma biomarkers of inflammation related to
atherosclerosis and how they represent the underlying physiology or
pathophysiology in a graded and continuous manner; however, any individual
biomarker may or may not be in the causal pathway of any particular health
outcome. Inflammation biomarkers have been associated with multiple
outcomes and may have different meanings across the age span. CRP, a
systemic biomarker, is only weakly associated with ethnicity, gender, age,
and hypertension and moderately associated with glucose tolerance status.
However, CRP is strongly associated with obesity and a number of
components of the metabolic syndrome; associations which begin early in
life and persist throughout the life span. Interestingly, CRP is not
consistently associated with IMT or coronary calcification, but has been
shown to co-localize with the NADPH oxidase subunit p22phox in
atherosclerotic plaque. Oxidized LDL, a marker of oxidative stress is also
strongly associated with the metabolic syndrome and, like CRP and other
inflammation markers, predicted incident MI in the Health ABC Study. It
appears likely that inflammation and oxidative stress biomarkers may
reflect different aspects of the underlying pathophysiology. The concept
of "antagonistic pleiotropy" was introduced, in which genetics, early life
"programming" and current physiological state may result in short-term
benefit to an individual but, due to longer spans, long-term damage. Due
to the way defense mechanisms are integrated with basic metabolism, larger
panels of individual biomarkers or metabolic processes may need to be
measured in population studies. These approaches; however, will result in
large amounts of data to be interpreted. Thus, new conceptual frameworks
and data analytic tools for understanding oxidative stress and
inflammation, particularly in population studies, will be needed.
Methodological Considerations: Dr. Schisterman stated that there
are a number of biomarkers available to measure different phases of
oxidative stress status. However, less is known about appropriate choice
of biomarkers to use in a study, and in particular, which combination of
biomarkers might distinguish between those with and those without a
disease such as coronary heart disease. Methods and issues discussed
included: 1) use of ROC curves and AUC (area under the curve) values; 2)
measurement error corrections; 3) pooling biomarkers, which is
cost-effective and particularly useful in pilot studies; and 4) linear
combination assessments. AUC can be used to evaluate discrimination
ability of biomarkers. Five biomarkers of oxidative stress or antioxidant
status were demonstrated to be independently associated with CVD, however;
using AUC- linear combination assessments, TBARS were found to contribute
the most discriminatory ability. Ultimately, the choice of biomarkers
should be made based on biological relevance, not statistical
relevance.
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Summary and Recommendations
As evidenced in the workshop presentations, oxidative stress and
inflammation biomarkers are associated with many HLBS conditions and risk
factors in population and clinical studies including participants across a
broad spectrum of age, race/ethnicity, gender, geographical regions.
Genetic and environmental influences on the biomarker levels were also
demonstrated. Clearly, this work is still in the early stages of
investigation. The Workshop Members recognized that ideally, biomarkers
for population studies need to meet criteria related to reproducibility,
biological variability, analytic variability, sensitivity and specificity
in a healthy population, as well as large-scale study feasibility.
However, it was also recognized that the data necessary to fully evaluate
these criteria are not yet available for most biomarkers. The Workshop
Members are building on their own experience as well as compiling
published information related to these evaluation criteria for several
biomarkers, which will be presented in a forthcoming report.
While there are many useful inflammation biomarkers, there are few
markers of oxidative stress. The most studied oxidative stress biomarkers
to date are the F2-isoprostanes, which meet the criteria for use in
large-scale population studies; however, other markers, in particular
those found in exhaled breath, may also be suitable.
Additional biomarkers are certainly needed, as Workshop Members agreed
that no one biomarker is likely to serve as a general marker of oxidative
stress. The same biomarker measured in different specimens (i.e., plasma,
urine, exhaled breath, etc) provides information on different aspects of
the underlying physiology and pathophysiology. A combination of biomarkers
will be needed to accurately reflect oxidative stress and inflammation
status across the range of normal physiology and HLBS disorders. In
addition, it was noted that many of the available assays are technically
quite challenging and only a few laboratories have the resources and/or
skills necessary to perform them. Stronger collaboration between
laboratory scientists and epidemiologists is critical to further advance
the application of new biomarker techniques to large-scale population
studies.
The following recommendations were made:
- Biomarkers of oxidative
stress status and inflammation have been associated with many HLBS
conditions and risk factors; additional examination in population studies
is important to elucidate which are most predictive of disease.
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Further standardization of specimen collection, storage, processing and
assaying conditions would facilitate evaluation of the biomarkers for use
in large scale studies and comparison of disease/risk factor associations
across studies.
- If existing specimen resources in the ongoing
or completed population studies are not sufficient or have not been
collected appropriately to enable this evaluation and hypothesis testing,
additional biological specimen repositories should be established, along
with appropriate funding to enable their use.
- Less technically
challenging assays that can be more easily performed in general research
laboratories, particularly with high throughput, should be developed. For
assays where this is not feasible, funding of core laboratories to serve
multiple studies is encouraged.
- Methods for combining and
integrating multiple or large panels of biomarkers of oxidative stress and
inflammation into analyses should be developed.
- Dissemination
of negative results, which are often not published but do provide useful
information to other researchers, should be encouraged. Abstracts of these
results could be posted on study websites, although peer-review is also
important.
- While some intervention studies examining the
effects of diet and exercise on biomarkers of oxidative stress and
inflammation have been done, more studies are needed to better understand
the underlying biology and help direct future development of therapies.
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Workshop Members
Co-Chairs:
David G. Harrison, MD, Division of Cardiology,
Emory University School of Medicine F. Javier Nieto, MD, PhD,
Department of Population Health Sciences, University of Wisconsin Medical
School
Members:
Emelia J. Benjamin, MD, ScM, Framingham
Heart Study, Boston University School of Medicine Serpil C. Erzurum,
MD, Pulmonary and Critical Care Medicine, Cleveland Clinic Foundation
Myron D. Gross, PhD, Department of Laboratory Medicine and Pathology,
University of Minnesota Stanley L. Hazen, MD, PhD, Department of Cell
Biology, Cleveland Clinic Lerner Research Institute Michael C.
Mahaney, PhD, Department of Genetics, Southwest Foundation for Biomedical
Research Edgar R. Miller, MD, PhD, Departments of Medicine and
Epidemiology, Johns Hopkins Medical University Jason D. Morrow, MD,
Department of Clinical Pharmacology, Vanderbilt University Medical Center
Enrique F. Schisterman, Epidemiology Branch, National Institute of
Child Health and Human Development, NIH, DHHS Russell P. Tracy, PhD,
Departments of Pathology and Biochemistry, University of Vermont
Maurizio Trevisan, MD, PhD, Department of Social and Preventive
Medicine, State University of New York at Buffalo
Last Updated June 2011
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