Kronos Reperfusion Oxidative Stress Study

Oxidative Stress in Response to Forearm Ischemia/Reperfusion


Oxidative stress, defined as an imbalance between production and activity of oxidant molecules and antioxidant capacity, is believed to be a key mechanism in the aging process and a variety of age-related chronic diseases.

In recent years, oxidative stress in vivo has most commonly been measured by determining the levels of plasma F₂-isoprostanes, stable end-products of arachidonic acid oxidation. A recent comparative study has validated isoprostane measurement as the most sensitive and reproducible indicator of a variety of techniques for estimating oxidative stress in an experimental model. Plasma and/or urine F₂-isoprostanes levels are increased in humans during chronic oxidative conditions such as obesity, hypercholesterolemia, diabetes, and chronic smoking. In contrast, F₂-isoprostane levels do not appear to increase with healthy aging in humans, although significant increases have been shown in older rats.

These findings suggest that there may be a diminution in the capacity to resist oxidative stress that is not apparent under normal conditions, but could become apparent when challenged by additional oxidant stresses. With progressive diminution into extreme old age, as with the rats, this effect may manifest itself even under baseline conditions.

Development of a method to test total capacity to resist oxidative stress in humans is a challenge because the oxidant stress induced must not be such that it leads to significant tissue damage. One potential method to measure oxidative stress resistance capacity is to remove plasma from an individual, and measure the resistance of plasma to chemical oxidation.

Such a strategy has been utilized and has revealed that the plasma of patients with end-stage renal disease had significantly higher levels of F₂-isoprostanes after exposure of the plasma to a free radical initiator (SIN-1) than the plasma of normal individuals, despite no significant difference in basal plasma F₂-isoprostane levels.

A major drawback of this strategy is that it only tests the oxidative resistance of small molecule antioxidants in the plasma, not the total systemic capacity to resist oxidative stress. For instance, increased expression of antioxidant enzymes in various organs could increase systemic oxidative resistance, but is unlikely to influence oxidation of plasma ex vivo.

A potentially more attractive method is to produce acute transient oxidative stress in a limited area and measure systemic resistance to such stress. Induced regional ischemia/reperfusion may provide a valid model. This can be accomplished by the timed inflation of a blood pressure cuff above arterial pressure on a participant's arm followed by deflation.

Ischemia/reperfusion is a well-established oxidative stress with the extent of the oxidation being determined by the duration and extent of the ischemic event. Pilot data from our collaborators at Vanderbilt University using forearm ischemia/reperfusion, in a protocol approved by their local IRB, determined that the ideal duration of cuff inflation to induce significant increases in free F₂-isoprostanes and yet be well tolerated, was three sequential 10-minute periods of cuff inflation, with a 2-minute deflation in between.

This method appears to integrate contributions of multiple resistance mechanisms to oxidative stress, including small molecular antioxidants, antioxidant enzymes, downregulation of oxidant generating signaling enzymes, and modulators of leukocyte activation. Increased levels of F₂-isoprostanes have been demonstrated in response to a number of acute oxidative stresses including carbon tetrachloride poisoning, diquat poisoning, endotoxin exposure and kainic acid exposure. Therefore, plasma F₂-isoprostanes should provide an excellent measure of any oxidative stress that might result from prolonged blood pressure cuff inflation.

Because reduced capacity to resist oxidative stress may be an important factor contributing to increased morbidity and mortality from a number of diseases in the elderly, it could be extremely valuable to identify individuals at increased risk. Moreover, the forearm ischemia/reperfusion test proposed could be used to evaluate diet, lifestyle and other therapeutic approaches to increase resistance capacity.

For example, supplementation with high levels of vitamin E does not lower basal F₂-isoprostane levels in healthy adults, but does lower elevated F₂-isoprostane levels in individuals with hypercholesterolemia. The lack of an effect of such dietary antioxidants in healthy adults had led some to hypothesize that they are of no benefit in increasing functional life span in apparently normal individuals.

However, if elderly individuals supplemented with vitamin E or other dietary antioxidants showed significant decreases in the levels of F₂-isoprostanes formed after local ischemia-reperfusion stress in comparison to non-supplemented individuals, it would suggest that such supplementation may in fact have significant impact on resistance to oxidation and might therefore affect morbidity.

The primary objective of this study was to examine the effects of aging on the pattern of responses to localized oxidative stress as measured by changes in free F₂-isoprostane levels over time after a forearm arterial occlusion-reperfusion protocol. The secondary objective is to examine whether gender differences exist in the response to this stress.

KLRI recruited 32 healthy, non-smoking adult men and women, with 16 participants between the 20-35 years of age and an additional 16 participants ages 60 and above.

The exclusion criteria included

• Use of anti-oxidant supplements, in excess of standard multi-vitamins (1 tablet/day)
• Current hormone replacement therapy
• Any history of significant chronic disease
• Uncontrolled hypertension: (systolic BP > 140 or diastolic BP > 85 mm Hg on at least 2 measurements, at least 10 minutes apart)
• Body mass index (BMI) > 30 kg/m2
• High physical activity level