Ž .Comparative Biochemistry and Physiology Part A 128 2001 99]104

Role of exercise and ascorbate on plasma antioxidantcapacity in thoroughbred race horses

Allan Whitea,U, Manuel Estradaa, Katherine Walker a, Pamela Wisniaa,Gonzalo Filgueiraa, Fernando Valdesa, Oscar Aranedaa, Claus Behna,´

Ramon Martınezb´ ´aBiomedical Sciences Institute, Faculty of Medicine, Uni ersity of Chile, P.O. Box 70005, Santiago 7, Chile

bDepartment of Animal Biologic Sciences, Faculty of Veterinary and Pecuary Sciences, Uni ersity of Chile, Santiago 7, Chile

Received 2 April 1998; received in revised form 8 September 2000; accepted 21 September 2000

Abstract

During exercise, the oxygen consumption and the production of free radicals increase and can lead to oxidative stresswith a deleterious effect on cellular structures involved in physical activity. To evaluate the oxidative stress produced byexercise and the role of ascorbate as an antioxidant, venous blood samples were obtained from 44 thoroughbredracehorses, before and after a 1000"200-m race at maximum velocity. Fourteen of these horses were treated

Ž .intravenously with 5 g of ascorbate before running. Antioxidant capacity PAOC , endogenous and exogenous ascorbateŽ .concentration, total antioxidant reactivity TAR , urate concentration, creatine kinase activity, protein concentration

Ž .and thiobarbiturate reactive substances TBAR as oxidative stress indicators were measured in the plasma of some ofthese horses. PAOC, TAR and TBAR increased after the race, while plasma ascorbate and urate concentrations

Ž .remained unchanged. Total plasma protein TPP concentrations increased in line with antioxidant capacity. Aspredicted, both the plasma ascorbate concentration and PAOC increased immediately after ascorbate administration,but was not modified after the race, such as TBAR. However, in both groups plasma creatine kinase activity increasedafter the race. These results would suggest that the administration of ascorbate could nullify the oxidative stressproduced by exercise in thoroughbred racehorses, but could not prevent muscular damage. Q 2001 Elsevier Science Inc.All rights reserved.

Keywords: Antioxidant capacity; Ascorbate; Urate; Free radicals; Oxidative stress; Lipid peroxidation; Exercise; Thoroughbred horse

1. Introduction

Free radicals are active chemical structures thatare in constant production in the organism.

U Corresponding author. Tel.: q56-2-678-6424; fax: q56-2-777-6916.

Ž .E-mail address: awhite@machi.med.uchile.cl A. White .

Among the most important are the reactive oxy-gen species produced in biological systemsŽCheeseman and Slater, 1993; Aruoma, 1994; Ji,

.1995 . Increased oxygen consumption in mam-mals such as thoroughbred race horses after phys-

Ž .ical exercise Jones and Lindstedt, 1993 , togetherwith leakage of electrons produced in the mito-

Žchondrial transport chain Jenkins, 1988; Witt et.al., 1992 , and ischemia-reperfusion processes

1095-6433r01r$ - see front matter Q 2001 Elsevier Science Inc. All rights reserved.Ž .PII: S 1 0 9 5 - 6 4 3 3 0 0 0 0 2 8 6 - 5

( )A. White et al. r Comparati e Biochemistry and Physiology Part A 128 2001 99]104100

Ž .Granger, 1988 , are accompanied by the produc-tion of free radicals, leading to an unbalanced

Žsituation known as oxidative stress Aruoma, 1994;.Alessio et al., 1997 .

Efficient systems of intracellular and extracellu-lar antioxidant protection exist to suppress free

Ž .radicals Sies et al., 1992 . Plasma antioxidantŽ .capacity PAOC includes the action of free radi-

cal-trapping compounds such as urate and ascor-bate, including proteins such as ceruloplasmin

Žand transferrin Halliwell and Gutteridge, 1990;.Wayner et al., 1987 , whose action complexing

transition metal ions avoid acceleration of lipidŽ .peroxidation processes Halliwell, 1994 .

In thoroughbred race horses, as in other speciessubjected to physical activity, gives rise to animbalance between free radical production and

Žantioxidant agents leading to oxidative stress Sies,.1991; Sies et al., 1992 . This stress can produce

Ždamage in several biomolecules Natta et al., 1992;.Brent and Rumack, 1993 creating metabolic al-

terations affecting physical performance.Ascorbate is an effective antioxidant in biologi-

Ž .cal fluids Sies et al., 1992; Rokitzki et al., 1994 .Also, it is a reducing agent and traps peroxyl

Ž .radicals Wefers and Sies, 1988 . Furthermore, itdirectly inhibits the propagation of the lipid-de-gradation chain reactions in cellular membranesŽ .Hornsby and Crivello, 1983 and helps to regen-erate vitamin E from the alpha-tocopheryl radicalŽ .Gohil et al., 1986; Kanter et al., 1993 .

PAOC of biological fluids can indicate an or-ganism’s capacity to withstand oxidative stressŽ .Lissi et al., 1995 and diet supplement with ex-ogenous antioxidants, like ascorbate, when plasmaPAOC is diminished or is exceeded, as commonly

Žoccurs during intensive physical exercise Snow,.1990 .

In this study, we evaluated the oxidative stressproduced in thoroughbred race horses during asimulated race, with and without previous intra-venous administration of ascorbate.

2. Materials and methods

Forty four healthy thoroughbred race horsesŽ .Equs caballus , males, females and castratedmales, between 2 and 5 years old, weighing450]500 kg, feeding oats and hay, without supple-ments, were studied. All horses were trained andcompeted regularly in Chilean racetracks.

Basal venous blood samples were collected inless than 20 s by jugular puncture, at stall, fromthe fasting horses at 08.00 h, in racetracks locatedat approximately 250 m of altitude. Fourteen ofthese horses, randomly allocated, were treatedwith 5 g of ascorbate, by intravenous administra-tion of 20 ml of a 23% aqueous solution, pH 7.30Ž W .Cevet , Veterquımica Ltd. , within 3 min collect-´ing basal samples and 15 min before a simulated

Žrace of 1000"200 m at maximum speed approx..16 mrs , allowing 200 m beforehand to reach this

speed as for all horses. Two minutes after treat-ment additional blood samples were taken. Fi-nally, post-exercise blood samples were obtainedfrom all the horses 5 min after racing. Treatedhorses acted as their own controls.

In an aliquot of each sample, EGTA-glutath-ione was used as an anticoagulant to determinethe plasma ascorbate concentration by enzymaticreduction with ascorbate oxidase, reaction witho-phenylenediamine and high pressure liquid

Ž .chromatography measurements Shearer, 1986 .In the remaining blood, heparin was used as ananticoagulant and plasma was obtained by cen-trifugation at 1000=g, at 08C, for 15 min. PlasmaPAOC, was determined by two methods. For thefirst method, a lipid peroxidation model was usedto evaluate the plasma capacity to inhibit thespontaneous autoxidation of rat-brain homo-genate produced by incubation for 30 min at 378C

Žin the presence of an aliquot of plasma Stocks et.al., 1974; Lissi et al., 1986 . The second experi-

mental procedure used ‘Total Antioxidant Reac-Ž . Ž .tivity’ TAR Lissi et al., 1995 . This method was

adapted from the determination of TotalŽ .Radical-Trapping Antioxidant Parameter TRAP

Ž .Lissi et al., 1992 .Plasma malondialdehyde concentration, ex-

pressed as thiobarbiturate reactive substancesŽ . ŽTBAR was determined Ohkawa et al., 1979;

.Holley and Cheeseman, 1993 using te-tramethoxypropane as a standard. The total

Ž .plasma protein TPP concentration was mea-Ž .sured Lowry et al., 1951 , using bovine serum

albumin as a standard. Commercial kits wereused to determine plasma urate concentrationŽ .Randox Laboratories Ltd. and creatine kinase

Ž W .activity CK NAC-act. , Human, Germany .A different number of horses were considered

in each kind of analysis and comparison betweenŽ .the results mean"S.E.M obtained in horses

submitted to different experimental conditions

( )A. White et al. r Comparati e Biochemistry and Physiology Part A 128 2001 99]104 101

were performed using variance analysisŽ .ANOVA . The Bonferroni post-test was used toevaluate the significance of the differences, with

ŽP-0.05 considered statistically different Milton,.1994 .

3. Results

Plasma ascorbate concentrations in horsestreated and untreated with this antioxidant, did

Ž .not change as a result of the race Table 1 .A significant increase in plasma TBAR was

observed in the exercised animals as comparedwith those in basal conditions not treated with

Ž .ascorbate P-0.01 , while in treated horses,TBAR was not modified. Nevertheless, plasma

Ž .creatine kinase CK activity after the race wasŽ .higher in ascorbate treated P-0.01 and un-

Ž . Ž .treated animals P-0.001 Table 2 .A significant increment in PAOC with respect

Ž .to basal conditions P-0.005 was also foundwhen the effect of physical exercise was measuredin ascorbate untreated horses by the spontaneousoxidation of rat-brain homogenate method. Nev-ertheless, in treated animals, PAOC did notchange after the race, with respect to the pre-raceascorbate post-administration condition. A simi-lar response was obtained when evaluating total

Ž .antioxidant reactivity TAR in some of thesehorses. With both methods, higher post-race val-ues were observed in animals previously treated

Ž .with ascorbate Table 3 .A significantly higher post-race, total plasma

Ž .protein concentration P-0.05 was observed inascorbate treated and untreated animals. Mean-while, plasma urate concentration in these horses

Table 1Plasma ascorbate concentration in thoroughbred race horsessubmitted to exercise and treated previously with 5 g of

aascorbate

Ž .Plasma ascorbate concentration mgrl

Before race After race

Basal After administrationof ascorbate

Ž .Untreated 10 7.5"1.3 ] 8.1"1.2b bŽ .Treated 10 9.6"1.1 75.9"3.5 73.0"2.2

a Values are expressed as mean"S.E.M. Number of horsesis given in parentheses.

bCorrespond to P-0.001, in comparison with basal value.

Table 2Ž .Plasma creatine kinase CK activity and thiobarbiturate

Ž .reactive substances TBAR in thoroughbred race horsessubmitted to exercise and treated previously with 5 g of

aascorbate

Basal After race

UntreatedbŽ . Ž . Ž .CK UIrl 95.0"33.0 13 187.0"45.0 13

Ž . Ž . Ž .TBAR nmolesrl 1.7"0.1 16 2.2"0.2 16

TreatedcŽ . Ž . Ž .CK UIrl 70.0"10.0 5 219.0"22.0 5

Ž . Ž . Ž .TBAR nmolesrl 1.6"0.1 14 1.7"0.1 14

a Values are expressed as mean"S.E.M. Number of horsesis given in parentheses.

bP-0.001 compared to basal values.cP-0.01 compared to basal values.

was very low in basal conditions and did notchange significantly as a consequence of the raceŽ .Table 4 .

4. Discussion

The increase in plasma malondialdehyde con-centration, measured as TBAR, in thoroughbredhorse after a race can be attributed to oxidativedamage due to free radicals produced as a conse-quence of exercise. Simultaneously, an increase inplasma antioxidant capacity was observed for bothmethods used. This can be attributed to a com-pensatory response to the increased amount ofpro-oxidant species.

ŽIn accordance with other horse studies Snow,.1990; McMeniman and Hintz, 1992 , the basal

plasma ascorbate concentration determined in thiswork did not increase with the race. Conse-quently, the increase of PAOC in thoroughbredhorses produced by short races at maximal veloc-ity should not be attributable to endogenous vari-ations in the concentration of this antioxidant.Nevertheless, it could be possible that, even inthe absence of a net change in the total plasmaascorbate concentration, variations in the ascor-baterdehydroascorbate ratio could result afterthe race, thus affecting PAOC.

The increase of PAOC in these animals couldbe due, at least in part, to the increase in plasmaprotein concentration due to hemoconcentration,caused by water transfer from vascular compart-ment to hyperosmotic muscle during exercise

( )A. White et al. r Comparati e Biochemistry and Physiology Part A 128 2001 99]104102

Table 3Ž . Ž .Effect of exercise and ascorbate administration on plasma antioxidant capacity PAOC and total antioxidant reactivity TAR of

aluminol in thoroughbred race horses

Basal Basal qascorbate After race

UntreateddŽ . Ž . Ž .PAOC % inhibition 49.9"2.8 30 60.6"2.8 30bŽ . Ž . Ž .TAR mmoles troloxrl 52.2"5.9 19 70.2"8.7 19

Treatedc cŽ . Ž . Ž . Ž .PAOC % inhibition 47.5"3.7 14 70.0"4.0 6 69.8"3.2 14

b cŽ . Ž . Ž . Ž .TAR mmoles troloxrl 63.3"9.2 11 180.5"14.1 4 166.2"24.2 11

a Values are expressed as mean"S.E.M. Number of horses is given in parentheses.bP-0.05 compared to basal values.cP-0.01 compared to basal values.dP-0.005 compared to basal values.

Ž .McArdle et al., 1994 . The role of urate as anantioxidant in horses is negligible because theirplasma concentrations are very low. The contribu-tion of pulmonary ventilatory dehydration andsweating does not appear to be important, be-cause these races are run in a relatively short

Ž .time approx. 60 s . The increase in plasma pro-tein concentration could well explain the increasein PAOC as determined by different methodsŽ .Halliwell, 1988 .

A high value using the rat brain homogenatemethod, could be obtained due to the proteincapacity of complexing transition metals. Whilst ahigh value for the TAR method could be due tooxidation of protein sulfhydryl groups. Thesegroups could be acting as first-line antioxidants,avoiding the propagation of the chain of reactions

Ž .producing free radicals Wayner et al., 1987 .Nevertheless, this phenomenon could partly ex-plain the observed increase in PAOC. The partic-ipation of additional antioxidant mechanisms oran increase in the efficiency of other known reac-tions is possible. Furthermore, the antioxidantcontribution of endogenous compounds such asthe proteins does not appear to be adequate forcontrolling oxidative stress during exercise, con-sidering that the increase in PAOC did not pre-vent an increase in the generation of lipid peroxi-dation products measured as TBAR. The increasein the plasma creatine kinase activity, in bothgroups of animals, could be mainly due to aselective increase of muscle membrane perme-

Ž .ability Anderson, 1975 , produced by peroxida-tion phenomena occurring during strong exerciseŽ .Matsuki et al., 1991 . This would mean that

increased PAOC does not necessarily prevent da-mage at the muscle cell level.

The administration of ascorbate produces anincrease of basal PAOC that is more prominentwhen the TAR method is used. This could be dueto the antioxidant action of ascorbate based onthe predominance of the peroxy-radicals trapping

Žcapacity of this compound Wayner et al., 1987;.Lissi et al., 1995 . It has been also described that

Žascorbate could act as a pro-oxidant agent Lissi.et al., 1986 ; this could occur when ascorbate in

aqueous solution finds favorable conditions toq2 Ž .react with Fe Halliwell and Gutteridge, 1990 .

Nevertheless, plasma substances that can complexmetal ions, could participate in these experimentsenhancing the antioxidant action of ascorbate.TBAR remained unchanged after exercise inascorbate treated horses, indicating that the dosesof ascorbate used were adequate to avoid the

Table 4Ž .Plasma urate and total protein TPP concentration in

thoroughbred horses submitted to exercise and treated previ-aously with 5 g of ascorbate

Basal After race

UntreatedbŽ . Ž . Ž .TPP grdl 6.64"0.48 10 8.16"0.40 10

Ž . Ž . Ž .Urate mgrdl 0.68"0.12 10 0.78"0.13 10

TreatedbŽ . Ž . Ž .TPP grdl 7.76"0.32 6 9.76"0.88 6

Ž . Ž . Ž .Urate mgrdl 0.76"0.10 6 0.85"0.08 6

a Results are expressed as the mean"S.E.M. Number ofhorses is given in parentheses.

bCorresponds to P-0.05, in comparison with basal values.

( )A. White et al. r Comparati e Biochemistry and Physiology Part A 128 2001 99]104 103

oxidative stress generated by this type of exercise.The small contribution of endogenous antioxidantin this situation was counterbalanced by the an-tioxidant effect of ascorbate. Nevertheless, in spiteof its inhibitory effect on measured oxidativestress produced by exercise, it was not possible toavoid apparent muscle membrane alterations, aswas shown by the rise in plasma CK activity.

Our results indicate that PAOC in horses couldbe enhanced by the administration of exogenousantioxidants, inhibiting the oxidative stresses pro-duced by exercise. However, in order to knowwhether the inhibition of oxidative processes dur-ing exercise benefits physical performance, itwould be necessary to study the effect of antioxi-dants such as ascorbate. It would also be neces-sary to simultaneously measure parameters relat-ing to work capacity such as blood lactate, heartrate and oxygen consumption, both in basal con-ditions and at different times after a standardizedrace.

Acknowledgements

Supported by FONDECYT: Grant N8 1940486

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