Response of oxidative stress biomarkers to a 16-week aerobic physical activity program, and to acute physical activity, in healthy young men and women

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Physical activity (PA) is associated with a reduced risk of coronary heart disease, and may favorably modify the antioxidant–prooxidant balance. This study assessed the effects of aerobic PA training on antioxidant enzyme activity, oxidized LDL
  Response of oxidati v e stress biomarkers to a 16-week aerobic physicalacti v ity program, and to acute physical acti v ity, in healthy young menand women R. Elosua a, *, L. Molina b , M. Fito a , A. Arquer a , J.L. Sanchez-Quesada c ,M.I. Co v as a,d , J. Ordon˜ez-Llanos c , J. Marrugat a a Unitat de Lipids i Epidemiologia Cardio v ascular, Institut Municipal d’In v estigacio´ Me`dica, Dr. Aiguader 80, Barcelona 08003, Spain b Ser v icio de Cardiologı´a, Hospital del Mar, Barcelona, Spain c Ser v icio de Bioquı´mica, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain d Laboratorio de Referencia, Barcelona, Spain Recei v ed 5 July 2002; recei v ed in re v ised form 23 December 2002; accepted 9 January 2003 Abstract Physical acti v ity (PA) is associated with a reduced risk of coronary heart disease, and may fa v orably modify the antioxidant    / prooxidant balance. This study assessed the effects of aerobic PA training on antioxidant enzyme acti v ity, oxidized LDLconcentration, and LDL resistance to oxidation, as well as the effect of acute PA on antioxidant enzyme acti v ity before and after thetraining period. Se v enteen sedentary healthy young men and women were recruited for 16 weeks of training. The acti v ity of superoxide dismutase in erythrocytes (E-SOD), glutathione peroxidase in whole blood (GSH-Px), and glutathione reductase inplasma (P-GR), and the oxidized LDL concentration and LDL composition, diameter, and resistance to oxidation were determinedbefore and after training. Shortly before and after this training period they also performed a bout of aerobic PA for 30 min. Theantioxidant enzyme acti v ity was also determined at 0 min, 30 min, 60 min, 120 min, and 24 h after both bouts of PA. Traininginduces an increase in GSH-Px (27.7%), P-GR (17.6%), and LDL resistance to oxidation, and a decrease in oxidized LDL (  / 15.9%).After the bout of PA, an increase in E-SOD and GSH-Px was obser v ed at 0 min, with a posterior decrease in enzyme acti v ity until30    / 60 min, and a tendency to reco v er the basal  v alues at 120 min and 24 h. Training did not modify this global response pattern.Regular PA increases endogenous antioxidant acti v ity and LDL resistance to oxidation, and decreases oxidized LDL concentration;30 min of aerobic PA decreases P-GR and B-GSH-Px acti v ity in the first 30    / 60 min with a posterior reco v ery. #  2003 Else v ier Science Ireland Ltd. All rights reserved. Keywords:  Antioxidants; Physical acti v ity; Free radicals; Lipoproteins 1. Introduction Regular physical acti v ity (PA) is associated with areduced risk of coronary heart disease (CHD) [1], andconsequently, physical inacti v ity has been considered arisk factor for CHD [2]. The mechanisms underlying thisprotecti v e effect are not fully established.On the other hand, oxidati v e stress has been found tobe linked to the de v elopment of se v eral chronic diseasesincluding atherosclerosis [3]. The oxidation of low-density lipoprotein (LDL) components is a cornerstoneof atherosclerosis [3]. The oxidati v e status is controlledby a wide spectrum of dietary exogenous antioxidantssuch as tocopherols, ascorbate, carotenoids, and phe-nolic compounds, and by endogenous antioxidants suchas the enzymes superoxide dismutase (SOD), glu-tathione peroxidase (GSH-Px), glutathione reductase(GR), and catalase among others [4]. The balancebetween free-radical generation and antioxidant acti v ityis critical in the pathogenesis of CHD. LDL resistance tooxidation, mainly dependent on its antioxidant contentand lipid particle composition, is also an importantfactor limiting this process. * Corresponding author. Tel.:   / 34-932211009; fax:   / 34-932213237. E-mail address: (R. Elosua).Atherosclerosis 167 (2003) 327    / 334www.else v$ - see front matter # 2003 Else v ier Science Ireland Ltd. All rights reserved.doi:10.1016/S0021-9150(03)00018-2  One of the fa v orable effects of regular PA could be tomodify the prooxidant/antioxidant balance, increasingendogenous antioxidant acti v ity, and LDL resistance tooxidation. On the other hand, acute PA increasesoxygen uptake and free-radical production, and conse-quently could induce lipid peroxidation [5,6]. The aims of this study were (1) to assess the effect of an aerobic PA training program on antioxidant enzymeacti v ity, oxidized LDL concentration, and LDL resis-tance to oxidation in young healthy subjects; and (2) toassess the effect of a single bout of acute PA onantioxidant enzyme acti v ity, before and after the train-ing period. 2. Subjects and methods  2.1. Subjects Se v enteen sedentary healthy young  v olunteers, se v enmen and 10 women, were recruited among medicalstudents. Subjects with pre v ious personal history of cardio v ascular disease, diabetes mellitus, dyslipemia,physical disability, or chronic respiratory disease, aswell as those with a body mass index (BMI) o v er 30 kg/m 2 , alcohol consumption greater than 40 g per day, orlong-term medication use, including mineral or  v itaminsupplements, were excluded. Another exclusion criterionwas regular PA for more than 2 h per week during thepreceding 3 months. The local research ethics committeeappro v ed the protocol and all participants pro v idedinformed consent.  2.2. Physical fitness assessment All the participants underwent a continuous, incre-mental cycling test on an electromagnetically brakedergocycle (EC 1200; Marquette Hellige Medical Sys-tems, Milwaukee, USA). The test began with a warm-upat 25 W (women) or 50 W (men) for 5 min, after whichthe power output was increased by 25 W e v ery 2 minuntil exhaustion. During the test, oxygen uptake (VO 2 ),minute  v entilation (VE), and respiratory exchange ratio(RER) were continuously measured using an OxiconAlpha (Jaeger, Wuerzburg, Germany). The metaboliccart and  v olume instruments were calibrated withknown calibration gases and with a 5-l calibrationsyringe, respecti v ely, before each test. VO 2  max wasdetermined by attaining at least two of the followingcriteria: RER abo v e 1.1, heart rate o v er 90% of thepredicted maximal rate, or a plateau in VO 2  despite anincrease in power output. VO 2  max was calculated as thea v erage of the three highest  V  O 2 v alues registered. The v entilatory anaerobic threshold (VT) was determined bythe  V  -slope method [7]. The maximal power output(MPO) was defined as the highest achie v ed powercompleted for at least 1 min. The maximal aerobicpower output (APO) was defined as the power achie v edat VT le v el.  2.3. Inter v ention A general schema of the inter v ention is presented inFig. 1.  2.3.1. Training period  Participants followed an indi v idualized and super- v ised aerobic PA training program during 16 weeks.Initially, the frequency of training sessions was four 30-min sessions per week. The frequency and duration of these sessions were progressi v ely increased to 5 days perweek and 50 min per day during the first 8 weeks, andcontinued at this le v el for another 8 weeks. The intensityof training was indi v idualized and adapted to the basalphysical fitness of each participant at 65    / 80% of his/hermaximal oxygen consumption. The training was per-formed in the Physical Acti v ity Ser v ice facilities of theUni v ersitat Auto`noma of Barcelona.  2.3.2. Bout of aerobic PA All the participants performed a bout of aerobic PAbefore and after the training period. One week aftercompleting a maximal effort test to assess fitness le v el,participants cycled in an ergometer for 30 min at a le v elcorresponding to his/her maximal aerobic power output.This aerobic PA was performed 3 h after lunch.Participants warmed up for 5 min at 25 or 50 W(women and men, respecti v ely), and achie v ed APO inthe following 5 min. This power was then sustained for25 min. The only dietary control was the requirementthat a participant eat the same diet in the 3 dayspreceding each of the two bouts of PA. Fig. 1. General schema of the inter v ention. R. Elosua et al. / Atherosclerosis 167 (2003) 327     /  334 328   2.4. Blood sampling  A blood sample was drawn (basal) prior to each boutof PA, immediately after exercising (0 min), at 30 min,and 1, 2, and 24 h thereafter.  2.5. Laboratory methods 2.5.1. Enzymatic assays E-SOD acti v ity in erythrocytes followed that of McCord and Frido v ich [8] (Ransel RS 125; RandoxLaboratories, Crumlin, UK), and was expressed in U/gof hemoglobin. B-GSH-Px acti v ity was measured by amodification of the method of Plagia and Valentine [9](Ransel RS 505; Randox Laboratories, Crumlin, UK),and expressed in U/l. The catalytic concentration of plasmatic glutathione reductase (P-GR) was measuredby following the oxidation of NADPH to NADP  during the reduction of oxidized glutathione (RanselGR 2368; Randox Laboratories, Crumlin, UK) [10], andexpressed in U/l. Intra-run imprecision was 4.7, 3.6, and3.5% for E-SOD, B-GSH-Px, and P-GR, respecti v ely.Between-run imprecision was 5.6, 5.4, and 4.4% for E-SOD, B-GSH-Px, and P-GR, respecti v ely.  2.5.2. LDL preparation, isolation, and composition Blood from  v olunteers was collected in tubes contain-ing 1 g/l of EDTA. Plasma was separated by centrifuga-tion at 1000  /  g   at 4  8 C for 15 min. LDL isolation wasperformed by sequential flotation ultracentrifugation[11]. Nati v e LDL was desalted by chromatography of molecular size exclusion in a G-25 Sephadex column(Pharmacia, Uppsala, Sweden) with 2.7 ml of 0.01 Mphosphate buffered saline, pH 7.4. Protein content wasdetermined by the red pyrogallol method (Sigma, St.Louis, MO). Total and free cholesterol, triglyceride,apoB (Roche Diagnostics), and phospholipid (WakoChemicals GmbH) content of LDL were determined bycommercial methods in a Hitachi 911 autoanalyzer, andthe results were expressed as percentage of LDL mass.  2.5.3. Copper-mediated LDL oxidation Dialyzed LDL (0.05 g protein/l) was incubated withcupric sulfate (5  m M) in PBS at a final  v olume of 1 ml.Absorbance at 234 nm was continuously monitored at 2min inter v als for 5 h at 30  8 C [12] using a spectro-photometer (Hewlett-Packard, Palo Alto, USA). Thediene  v ersus time profile was di v ided into three con-secuti v e phases: lag phase, propagation phase, anddecomposition phase [12]. Intra-run imprecision was2.3, 5.3, and 1.5% for lag phase, oxidation rate, andmaximum amount of dienes, respecti v ely. Between-runimprecision was 7.1, 7.9, and 7.0%, respecti v ely.  2.5.4. Antioxidant content a -Tocopherol,  a -carotene,  b -carotene, and lycopenecontent in LDL were quantified by re v erse-phase HPLC(Ultrasphere ODS 4.6  / 25  / 5 mm 3 column, SystemGold, Beckman) with a diode-array detector (Detec-tor168, Beckman), as described pre v iously [13].  2.5.5. LDL size LDL particle size was determined by polyacrilamidegradient gel electrophoresis (2    / 16%) according to Ni-chols et al. [14] with modifications.  2.5.6. Oxidized LDL Oxidized LDL concentration was determined inserum by an enzyme-linked immunosorbent assay usingtwo antibodies against the antigenic determinants of oxidized apolipoprotein B molecule (ox-LDL; MercodiaAB, Uppsala, Sweden). Values were expressed in U/l.The intra- and inter-assay coefficients of   v ariation were2.8 and 10.7%, respecti v ely.Antioxidant enzyme acti v ity and oxidized LDL con-centration were adjusted for plasma  v olume changes, asdescribed elsewhere [15].  2.6. Other measurements Weight and height were measured on a calibratedbalance. BMI was calculated as the weight (in kilogram)di v ided by the squared height (in meters). Smokers weredefined as those smoking one or more cigarettes per dayduring the pre v ious 3 months.  2.7. Sample size Accepting an  a  risk of 0.05 and a  b  risk of 0.20 in atwo-sided test, the sample of 17 subjects allows us torecognize as statistically significant a difference greaterthan or equal to 0.70  / standard de v iation units betweenthe initial and the final measurements.  2.8. Statistical analysis Paired Student’s  t -test was used to compare means of continuous  v ariables before and after the trainingperiod; when the  v ariables departed from normality,the Wilcoxon rank test was used instead. To assesschanges of antioxidant enzymes or lipid peroxides afterthe bout of acute PA with respect to the basal, pairedStudent’s  t -test was also used, taking into account,howe v er, the Bonferroni correction for multiple com-parisons. Linear general models for repeated measureswere used to assess the effect of training, acute aerobicPA, and the modifier effect of training on antioxidantenzyme acti v ity. R. Elosua et al. / Atherosclerosis 167 (2003) 327     /  334  329  A  P  - v alue lower than 0.05 was considered as statis-tically significant. The SPSS program was used forstatistical analysis. 3. Results Se v enteen participants were enrolled in the study.Mean age was 19.5 (standard de v iation: 1.2). Fi v e(29.4%) were smokers, and mean BMI was 23.6 kg/m 2 (standard de v iation: 2.1). No statistically significantdifferences between men and women, and betweensmokers and non-smokers, were detected in antioxidantenzyme acti v ity, LDL resistance to oxidation, or oxi-dized LDL concentration. Men had a higher VO 2  maxthan women, 44.5 (5.7) and 32.9 (4.2) ml/kg, respecti v ely( P  B / 0.01). Women had higher high-density lipoprotein(HDL) cholesterol than men, 54.0 (13.1)  v ersus 40.4(11.4) mg/dl, respecti v ely ( P   / 0.04). 3.1. Effects of PA training program The obser v ed changes in physical fitness parametersand lipid le v els are presented in Table 1. Statisticallysignificant increases in VO 2 max, anaerobic threshold,and aerobic power output were obser v ed, along with amarginally significant increase in maximal power out-put. No statistically significant changes in total choles-terol, LDL cholesterol, triglycerides, and HDLcholesterol were obser v ed.In Table 2, changes in antioxidant enzyme acti v ity,LDL resistance to oxidation, LDL particle character-istics (components, antioxidant content, and diameter),and oxidized LDL concentration are presented. Astatistically significant increase in the acti v ity of B-GSH-Px and P-GR was obser v ed, ranging from 15 to23% with respect to the basal le v el. No statisticallysignificant change in E-SOD acti v ity was obser v ed afterthe training period.A significant increase in LDL resistance to oxidationwas obser v ed, with an increase in the lag-time duration,a decrease in the maximal oxidation rate, and a margin-ally significant decrease in the maximal amount of produced dienes in LDL.No changes were obser v ed in LDL characteristics(components, antioxidant content, and diameter). How-e v er, a significant decrease in oxidized LDL concentra-tion after training was obser v ed. 3.2. Effects of a bout of aerobic PA before and aftertraining on antioxidant enzyme acti  v ity The effect of 30 min of aerobic PA practice ondifferent antioxidant enzymes (P-GR, E-SOD, and B-GSH-Px), before and after the training program, ispresented in Figs. 2    / 4. Before training, a late (24 h) andsignificant increase in E-SOD acti v ity was obser v ed afterthe bout of PA. After training, no significant changes inE-SOD acti v ity were obser v ed. E-SOD acti v ity wassignificantly higher after than before training at 30, 60,and 120 min, and 24 h.Before training, B-GSH-Px acti v ity showed an abruptincrease just after finishing PA, followed by a decreaseat 30 and 60 min, with a posterior steep tendency tonormalization (Fig. 3). After training, the patternpresented a similar decrease at 30 and 60 min, with aposterior normalization, but without the initial increasein GSH-Px acti v ity. A higher GSH-Px acti v ity wasobser v ed after training at times basal, 120 min, and 24 h.Before training, P-GR showed an abrupt and sig-nificant acti v ity decrease 30 min after finishing PA; aslow tendency to reco v er the basal  v alue was obser v edafterwards (Fig. 4). After the training period, a differentpattern was obser v ed with a light and non-significantdecrease in P-GR acti v ity after the acute PA bout.Consequently, higher P-GR acti v ity at time basal and 30min was statistically significant when comparing beforeand after training status. Table 1Physical fitness and lipids before and after the training period (mean and standard de v iation)Before training After training %Change  P  - v alue Physical fitness VO 2  max (ml/kg) 37.43 (7.66) 46.19 (11.02) 23.41  B / 0.001VT (%VO 2  max) 63.36 (13.39) 77.02 (8.48) 19.33  B / 0.001APO (W) 131.25 (46.99) 159.37 (63.82) 21.43 0.009MPO (W) 206.25 (55.15) 220.31 (72.01) 6.82 0.057BMI (kg/m 2 ) 23.51 (2.07) 23.47 (2.51)   / 0.01 0.875 Lipids Total cholesterol (mmol/l) 4.41 (0.73) 4.08 (0.84)   / 7.45 0.125HDL cholesterol (mmol/l) 1.25 (0.36) 1.31 (0.34) 4.69 0.390LDL cholesterol (mmol/l) 2.54 (0.67) 2.23 (0.81)   / 12.48 0.177Triglycerides (mmol/l) a 1.02 (0.87    / 1.40) 1.15 (0.83    / 1.35)   / 11.36 0.756VO 2  max, maximal oxygen uptake; VT,  v entilatory anaerobic threshold; APO, aerobic power output; MPO, maximum power output. a Median (25    / 75 percentile). Wilcoxon rank test. R. Elosua et al. / Atherosclerosis 167 (2003) 327     /  334 330  3.3. Global effects of training, acute PA, and theinteraction between training and acute PA on antioxidantenzyme acti  v ity The effects of training, acute PA, and the interactionbetween them on antioxidant enzyme acti v ity wereassessed by general linear models for repeated measures(MANOVA) (Table 3). Considering not only the basalacti v ity, but all measures after the bout of PA, trainingincreased the acti v ity of E-SOD, B-GSH-Px, and P-GR.On the other hand, acute PA significantly reduced theacti v ity of B-GSH-Px and P-GR.Training did not significantly modify the globalresponse of antioxidant enzyme acti v ity to acute aerobicPA. 4. Discussion This experimental study found an increase in physicalfitness, B-GSH-Px acti v ity, P-GR acti v ity, and LDLresistance to oxidation, and a decrease in oxidized LDLconcentration after 16 weeks of aerobic training. Theeffect of 30 min of aerobic PA on antioxidant enzyme Table 2Antioxidant enzyme acti v ity, LDL resistance to oxidation, LDL main components and characteristics, and oxidized LDL concentration, before andafter the training period (mean and standard de v iation)Before training After training %Change  P  - v alue Antioxidant enzyme acti  v ity E-SOD (U/g Hb) 859.25 (155.68) 995.69 (337.78) 15.88 0.100B-GSH-Px (U/l) 5531.69 (1160.04) 6802.12 (1313.60) 22.97 0.016P-GR (U/l) 48.47 (9.95) 55.56 (11.97) 14.64 0.033 LDL resistance to oxidation Lag-phase duration (min) 111.53 (13.81) 126.80 (12.57) 13.69 0.007Maximal oxidation rate ( m mol/(min/g)) 11.28 (2.29) 8.18 (2.45)   / 27.51 0.002Maximal amount ( m mol/g) 825.42 (177.22) 717.67 (159.84)   / 13.05 0.073 LDL mass components Free cholesterol (%) 9.00 (0.57) 9.13 (0.52) 1.44 0.404Sterified cholesterol (%) 30.51 (1.95) 30.95 (1.77) 1.44 0.404Triglycerides (%) 6.56 (1.03) 7.08 (0.83) 7.98 0.080Phospholipids (%) 27.41 (2.19) 26.41 (1.70)   / 3.67 0.245Apoprotein B (%) 26.52 (1.02) 26.43 (0.90)   / 0.33 0.785 LDL antioxidant content Lycopene (mol/mol apoB) 0.28 (0.18) 0.45 (0.26) 58.79 0.062 a -Carotene (mol/mol apoB) 0.07 (0.04) 0.07 (0.06) 3.90 0.874 b -Carotene (mol/mol apoB) 0.31 (0.13) 0.30 (0.16) 0.74 0.947Tocopherol (mol/mol apoB) 7.77 (0.85) 7.78 (1.23) 0.04 0.988LDL diameter (nm) 26.64 (0.58) 26.72 (0.58) 0.32 0.102Oxidized LDL (U/l) 48.80 (16.21) 41.06 (10.55)   / 15.9 0.043E-SOD, erythrocytary superoxide dismutase; B-GSH-Px, blood glutathione peroxidase.Fig. 2. Time response pattern of catalytic acti v ity of SOD inerythrocytes to acute aerobic PA before and after training. Adjustedfor plasma  v olume changes. * P  B / 0.05 when comparing before andafter training acti v ity;   P  B / 0.05 when comparing to basal  v alue.Fig. 3. Time response pattern of catalytic acti v ity of GSH-Px in wholeblood to acute aerobic PA before and after training. Adjusted forplasma  v olume changes. * P  B / 0.05 when comparing before and aftertraining acti v ity;   P  B / 0.05 when comparing to basal  v alue. R. Elosua et al. / Atherosclerosis 167 (2003) 327     /  334  331
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