Oxidative Stress Links Aging, Activity and Mobility Limitation
Phase: NA
Conditions: Aging
Sponsor: VA Office of Research and Development

University of Research Integrity and Compliance Office  
 
 
Protocol Title:  
Mechanisms of Adaptation to Exercise in Health & COPD:  
  
Oxidative stress links aging, activity and mobility 
 
Protocol:  
 
# IRB_00032404 
Approval date:  
10/30/17 
PI:  
 
 
Russell S. Richardson, Ph.D., D.Sc. 
 
 

METHODS 
Subjects and General Procedures. 42 young (25 ± 1 yrs) and 45 older (71 ± 1 yrs) healthy 
volunteers participated in the current study. All subjects were non-smokers, normotensive 
(<140/90 mmHg), and free of overt cardiovascular disease as determined by health history 
questionnaire and physical examination. Exclusion criteria included subjects with diagnosed 
cardiovascular disease, diabetes, hypercholesterolemia, hypertension, and women who were 
pregnant. Subjects were not taking any prescription medication, and were asked to abstain from 
vitamin supplements for 10 days prior to enrollment and during the course of the study. Subjects 
arrived to the laboratory at 0800 in a fasted state, having abstained from exercise and caffeine for 
12 hours prior, in accordance with recently published guidelines 24. Premenopausal females were 
studied within the first five days of their menstrual cycle, as FMD measurements of the brachial 
artery have been documented to fluctuate with menstrual phase. Protocol approval and written 
informed consent were obtained according to the University of Utah and Veteran Affairs 
Institutional Review Board requirements. All data collection took place with subjects supine, in a 
thermoneutral environment.  
 
Antioxidant Supplementation.  All subjects reported to the laboratory twice within one week (>48 
hrs apart) and received the antioxidant (AO) cocktail or placebo (PL) in a balanced, double-
blind, crossover design. Supplements were taken in two doses, separated by 30 minutes to 
improve absorption, consumed 90 and 60 minutes prior to the FMD protocol. The first dose 
consisted of 300 mg of alpha-lipoic acid, 500 mg Vitamin C, and 200 I.U. Vitamin E, and the 
second dose was 300 mg alpha-lipoic acid, 500 mg Vitamin C, and 400 I.U. Vitamin E. Placebo 
microcrystalline cellulose capsules of similar taste, color, and appearance were likewise 

consumed in two doses within the same time frame. We have previously reported the efficacy of 
this antioxidant cocktail to reduce carbon- and O2-centered free radical levels, as measured by 
electron paramagnetic resonance (EPR) spectroscopy, in both young 25 and older 26 subject 
populations. 
 
Measurements. Details of the FMD procedure have been described previously 27, and were 
performed in accordance with current recommendations 24. Briefly, a blood pressure cuff was 
placed on the right arm proximal to the elbow and distal to the placement of the ultrasound 
Doppler probe on the brachial artery (BA). The BA was insonated approximately midway 
between the antecubital and axillary regions, and measurements of BA diameter and blood 
velocity (Vmean) measurements were obtained continuously at rest and for 2 minutes following 
cuff deflation (Logiq 7, GE Medical Systems, Milwaukee, WI).  
 
Analyses.  Vmean was automatically calculated using commercially available software (Logiq 7). 
End-diastolic, ECG R-wave gated images were collected via video output from the Logiq 7 for 
off-line analysis of BA vasodilation using automated edge-detection software (Medical Imaging 
Applications, Coralville, Iowa). Flow-mediated vasodilation (FMD) was quantified as the 
maximal percent change in BA diameter following cuff release. Shear rate was calculated as: 
Shear Rate (s-1) = Vmean • 8/ vessel diameter. Blood flow was calculated as: Blood flow (ml • min) 
= ( Vmean • π • [Vessel Diameter/2]2 • 60). For both shear rate and blood flow, cumulative area-
under-curve (AUC) values were integrated with the trapezoidal rule and calculated as 
{yi[x(i+1) 
– xi] + (1/2)[y(i+1) – yi][x(i+1) – xi]}. Reactive hyperemia was quantified as cumulative BA blood 

flow for two minutes (AUC) following cuff occlusion. Normalized FMD was calculated by 
dividing FMD (%) by the cumulative shear rate AUC at the time of peak BA vasodilation. 
 
Assays.  In a subset of subjects (n = 18 young, n = 30 older), blood samples were obtained from 
the antecubital vein immediately prior to FMD testing on both PL and AO visits. Total AO 
capacity was assessed by the ferric reducing ability of plasma (FRAP) assay, and endogenous 
AO activity was assessed by determining superoxide dismutase (SOD, Cayman Chemical 
Company, Ann Arbor, MI). Plasma ascorbate concentration was also determined (Cosmo Bio, 
Carlsbad, CA). Quantitative determination of thiobarbituric acid reactive substances (TBARS) 
was performed to assess lipid peroxidation, a marker of oxidative stress (Bioassay Systems, 
Hayward, CA). Total nitrate and nitrite were measured using a standard colorimetric nitrate 
reductase/Greiss reaction assay (Cayman Chemical, Ann Arbor, MI). Measurement of 
nitrotyrosine (3-NT) was performed using competitive ELISA (Cell Biolabs, San Diego, CA). A 
lipid panel was obtained for all subjects by standard techniques. 
 
Statistics.  Statistics were performed with the use of commercially available software (SigmaPlot 
11, Systat Software Inc., Point Richmond, California, USA). Repeated measures two-way 
analysis of variance (ANOVA) was used to identify significant changes in measured variables 
between conditions and groups, with the Bonferroni test used for post-hoc analysis when a 
significant main effect was found. All group data are expressed as mean ± standard error. 
Significance was established at P<0.05.