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Antioxidant Levels in MS Besler 02

Antioxidant Levels in MS Besler 02

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Published by Rama Shakti
Antioxidant Levels in MS Besler 02
Antioxidant Levels in MS Besler 02

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Published by: Rama Shakti on Jun 20, 2013
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04/07/2014

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Serum Levels of Antioxidant Vitamins and LipidPeroxidation in Multiple Sclerosis
HALI˙T TANJU BESLER
a,
*, SELC¸UK C¸OMOG˘LU
 b
and ZEKI˙OKC¸U
c
a
Division of Nutritional Sciences, Department of Nutrition and Dietetics, School of Health Technology, Hacettepe University, Sihhiye, 06100-Ankara,Turkey;
b
Department of Neurology, Ankara Numune Hospital, Samanpazari, Ankara, Turkey;
c
Department of Physical Theraphy and Rheumatology, Ankara Rehabilitation Center Hospital, Sihhiye, Ankara, Turkey(Received 23 July 2001; Revised 31 August 2001; In final form 13 December 2001)
We determined serum levels of ascorbic acid, beta-carotene, retinol and alpha tocopherol and lipidperoxidation (as estimated by thiobarbituric acid react-ing substances (TBARS) generation) in 24 multiplesclerosis (MS) patients and 24 healthy sex- and age-matched person as control. The levels of four antioxidantvitamins were significantly lower in MS patients com-pared to controls (
 p
<
0.05). TBARS levels were signifi-cantly higher in the patients of MS compared to thecontrols (
 p
5
0.001). In MS patients, the levels of beta-carotene, alpha tocopherol and ascorbic acid correlatedsignificantly with each other (
2
5
0.689
2
0.779). Itappeared that there was inverse correlation between theserum levels of ascorbic acid or beta-carotene, but not ofalpha tocopherol or retinol, and TBARS levels in MS.The present study indicates that antioxidant vitamins(alpha tocopherol, beta-carotene, retinol and ascorbicacid) are decreased in sera of MS patients during anattack, and that this decrease may well be dependent onthe increased oxidative burden as reflected by lipidperoxidation products. The role of antioxidant vitaminsupplementation in prevention and/or treatment of MSremains to be explored.
Keywords
: Alpha tocopherol; Ascorbic acid; Beta-carotene;Multiple sclerosis; Retinol; Thiobarbituric acid reactive substances
INTRODUCTION
Multiple sclerosis (MS) is an inflammatory demye-linating disease of unknown origin (Karg
et al.
, 1999;Noseworthy
et al.
, 2000). Genetic susceptibility andenvironmental influences are the risk factorsinvolved in MS. Relapsing–remitting MS—the typepresent in 80% of patients—typically begins in thesecond or third decade of life and has a femalepredominance of approximately 2:1. Persistent signsof central nervous system dysfunction may developafter a relapse, and the disease may progress between relapses (secondary progressive MS).Twenty percent of the affected patients have primaryprogressive MS, which is characterized by agradually progressive clinical course and a similarincidenceamongmenandwomen(Noseworthy
etal.
,2000). The risk of developing MS in the generalpopulation is approximately 0.2%. However, the riskto members of families who have a father, mother,sister, or brother with MS ranges from 1.0 to 6.0%. Ingeneral, rates increase with latitude, although thereare some noteworthy exceptions to this geographicgradient (McMichael and Hall, 2001).Perturbation of the cellular oxidant/antioxidant balance has been suggested to be involved in theneuoropathogenesis of several disease states, includ-ing stroke, MS, Parkinson disease, Alzheimer’sdisease as well as “normal” physiological aging(Calabrese
et al.
, 2000; Noseworthy
et al.
, 2000). It has been suggested that there is a possible role of freeradicals, including nitric oxide, in the pathogenesisof MS (Hunter
et al.
, 1985; Bo¨
et al.
, 1994; Calabrese
et al.
, 2000). Increased lipid peroxidation has beenobserved both in the cerebrospinal fluid and in the blood of MS patients. In addition to the antioxidantenzymes, namely catalase, superoxide dismutase,glutathione peroxidase and glucose-6-phosphatedehydrogenase, the blood and some other tissues
ISSN 1028-415X print/ISSN 1476-8305 online
q
2002 Taylor & Francis LtdDOI: 10.1080/10284150290029205
*Corresponding author. Tel.:
þ
90-312-3052465. Fax:
þ
90-312-3091310. E-mail: htbf@hacettepe.edu.tr
Nutritional Neuroscience
, 2002
Vol.
5 (3), pp. 215–220
 
contain nonenzymatic antioxidants, namely alphatocopherol, beta-carotene, retinol and ascorbic acid,among others. The data regarding the relationship between antioxidant vitamins and MS is limited.Warren (1982) found that low dietary intake of vitamin A, vitamin E and selenium but not of beta-carotene or ascorbic acid, are associated to be a riskfactor for the onset of the disease. Further, twostudies have found that the plasma levels of alphatocopherol, beta-carotene, retinol and ascorbic acidare similar in MS patients and in controls (Wikstro¨m
et al.
, 1976; Wong
et al.
, 1993). The data, however, arescarce and conflicting. The aim of this study was,therefore, to investigate the possible changes inantioxidant vitamin levels and their relationship tothe oxidative stress produced by MS. For thispurpose, we measured the serum levels of anti-oxidant vitamins, alpha tocopherol, beta-carotene,retinol and ascorbic acid and of thiobarbituric acidreactive substances (TBARS) in a group of secondaryprogressive MS patients who first experienceexacerbation, and compared them to levels obtainedfrom healthy controls.
MATERIALS AND METHODSPatients and Controls
Twenty-four patients (35.9
^
7.3 years) with second-ary progressive MS were included in the presentstudy. The diagnosis of definite MS was confirmedaccording to the clinical and laboratory diagnosticcriteria of the Poser Committee (1983), and 24healthy sex- and age-matched person (36.8
^
4.1years) without any diseases, who also had normalphysical examination findings, served as controlgroup. Secondary progressive MS patients who firstexperience exacerbations were recruited from out-patients making the first visit to the departments of neurologyor the physical therapy and rheumatologyof two hospitals based in the region of Ankara.Neurologic status and progression of disease wereevaluated by the Kurtzke disability status scoring.The patients and healthy controls had receivedneither steroid therapy nor vitamin supplemen-tation, and none were smokers. Informed consentaccording to the declaration of Helsinki wasobtained from each subject at the time of recruitmentto the study, which was approved by the ResearchEthical Committee of the Hospitals, Ankara.
Sample Collection and Preservation
From each patient and healthy age- and sex-matchedperson,10mlofbloodfromtheantecubitalvein weretaken. Blood was always collected while fastingduring the morning hours, and centrifuged at 4
8
Cfor 10min at 2500rpm to obtain serum. The serumsampleswerefreshlyfrozenundernitrogenandstoredat
2
70
8
C until analysis. Samples were not storedlonger than six months. The samples were thawed atroom temperature only once at the time of assay.
Determination of Vitamins and Lipid PeroxideLevels
Serum alpha tocopherol concentration wasmeasured by using the method described by Desai(1984). In short, the mixture containing 0.5mlvolume of serum, 0.5ml of ethanol and 0.25ml of 25% ascorbic acid was preincubated at 70
8
C for5min. Following incubation, 0.3ml of saturatedpotassium hydroxide was added and the mixturewas further incubated at 70
8
C for 30min. Tubes werecooled immediately in an ice bath. A4.0ml of hexanewas added into tubes. Duplicate samples wereextracted in glass-stoppered centrifuge tubes, fol-lowed by vigorous vortex mixing for 1min andcentrifugation at 1500rpm for 5–10min. Afterseparation of phases, hexane extracts were used toestimate the concentration of the vitamin level on aspectrofluorometer (Jasco FP-750). Excitation andemission wavelengths were 286 and 330nm, respect-ively. Alpha tocopherol concentration was obtaineddirectly from the standard curve. The intra- andinter-assay of coefficient variation for the measure-ment were 1.9 and 3.1%, respectively. Beta-caroteneand retinol were determined by using the method of Neeld and Pearson (1963). Briefly, duplicate serumsamples (0.5ml) were mixed with 1.0ml of coldethanol and then extracted with 2.0ml of hexane,followed by being vortex mixed for 2min. Themixture was centrifuged at 800
 g
for 10min. Beta-carotene was determined spectrophotometrically at450nm (Unicam 8700 series). Retinol was extractedinto petroleum ether in the presence of ethanol andwas reacted with trifluoracetic acid, which leads tothe dehydrated anhydrovitamin, which was thenmeasuredspectrophotometricallyat620nm(Unicam8700 series). Both beta-carotene and retinol concen-trations were obtained directly from their standardcurves.Intra- andinter-assays ofcoefficient variationin beta-carotene measurements were calculated as1.6 and 1.7%, and retinol measurements were2.6 and3.1, respectively. Serum ascorbic acid was measuredspectrophotometrically at 520nm after its reactionwith 2,6-dichlorophenolindophenol (Kalay
et al.
,2000). The intra- and inter-assay variations for thevitamin C assay in this study were 2.1 and 3.2%,respectively.Serum lipid peroxide levels were determinedmeasuring thiobarbituric acid (TBA) reactivity asdescribed by Wade and van Rij (1988). Briefly, 200
m
lof trichloroacetic acid (TCA) (25g TCA in 10mldistilled water) was added to 1ml of serum. The
BESLER
et al.
216
 
mixture was centrifuged at 1000
 g
for 10min, and theprecipitate was reacted with 1ml of 0.67% TBA(w/v). The samples were heated at 100
8
C for 30min.After centrifugation, the absorption of malondialde-hyde (MDA)–TBA chromogen was measured at532nm. Tetramethoxy propane was used as MDAstandard. TBA reactivity was calculated as micro-mole MDA per liter (
m
mol/l). Intra- and inter-assayof coefficient variation averaged 5.1 and 6.2%,respectively.
Statistical Analysis
All statistical analyses were conducted using “SPSS10.0 for Windows(SPSS Inc., USA) statisticalprogram. Mann–Whitney U test was used, andcomparison of MS patients and controls. Corre-lations between parameters were studied usingPerson’s test. Values are expressed as mean
^
standard deviation (SD). A significance level (
 p
value) of 5% was used unless stated otherwise.
RESULTS
Mean ages of the patients and healthy controls were35
:
9
^
7
:
3 and 36
:
8
^
4
:
1 years, respectively. Nosignificant differences appeared between the groups.The results are shown in Table I.Serum levels of ascorbic acid, alpha tocopherol,retinol and beta-carotene were found to be lower inMS patients during exacerbation than in the healthyage- and sex-matched control group
ð
 p
,
0
:
05
Þ
:
MSpatients had significantly higher serum TBARSlevels
ð
3
:
94
^
0
:
12
m
mol
=
l
Þ
compared to the controlgroup
ð
1
:
81
^
0
:
15
m
mol
=
l
Þ ð
 p
¼
0
:
001
Þ
:
The possi- bility of gender based differences in the antioxidantvitamins and a marker of lipid peroxidationproducts, TBARS in MS patients was also examined by Wilcoxon signed rank test. It appeared that therewere no differences in the levels of antioxidantvitamins and TBARS of the patients with MS whenthe data was dissected by gender (data not shown).Interestingly, in MS patients the levels of  beta-carotene, alpha tocopherol and ascorbic acidcorrelated significantly with each other, with
r
2
values ranging between 0.689 and 0.779 (Fig. 1a–e).It appeared that there was inverse correlation between the serum levels of TBARS and theantioxidant vitamins namely ascorbic acid or beta-carotene in MS patients (Fig. 2a and b). Nocorrelation was observed between the serum levelsof TBARS and alpha tocopherol or retinol (data notshown).
DISCUSSION
In the present study, the results confirm previousobservations that there is an oxidative stress inpatients with MS and further extend the concept thatthis oxidative stress is associated with significantlydecreased levels of all four antioxidant vitamins(ascorbic acid, alpha tocopherol, retinol and beta-carotene), and increased levels of TBARS.Endogenous antioxidant defenses in body werenot completely effective for neutralization of oxi-dative stress (Gey, 1998). Dietary antioxidants suchas vitamins appear to be of great importance forthe control of the effects of reactive oxygen species.In our study, the levels of all four antioxidantvitamins were significantly lower in MS patientscompared to controls. One possible explanation forthe low levels of vitamins is that MS may havecaused decreased dietary intake vitamins as waspartly in the study of Warren (1982). This seemsunlikely, as none of the patients had any evidence of nutritional deficiency. A more likely explanationwould be that these vitamins are consumed at anincreased rate as defense mechanisms of theorganism against the ongoing oxidative burden.This explanation may well be partly satisfactory ascorrelation was observed between the serum levelsof vitamins and of TBARS. However the results of this study do not elucidate a definitive cause–effectrelationship between the antioxidant vitamins andoxidative stress in MS. Alpha tocopherol is the majorlipid-soluble antioxidant which is capable of break-ing the lipid peroxidation chain reaction in cellmembranes, thereby preventing the formation of 
TABLE I Characteristics and some analytical data of patients with MS and control groupsParameters MS patients
ð
n
¼
24
Þ
Control
ð
n
¼
24
Þ
CharacteristicsAge (years) 35.9
^
7.336.8
^
4.1Gender male/female (
n
) 16/8 16/8Analytical dataRetinol (
m
mol/l) (Ranges) 2.07
^
0.21* (1.392.48) 2.53
^
0.26 (1.67–3.29)Beta carotene (
m
mol/l) (Ranges) 0.41
^
0.13* (0.120.72) 0.63
^
0.08 (0.34–0.87)Ascorbic acid (
m
mol/l) (Ranges) 31.19
^
3.87* (22.3040.28) 49.05
^
3.69 (35.94–58.32)Alpha tocopherol (
m
mol/l) (Ranges) 22.05
^
2.05* (16.7425.31) 26.20
^
1.99 (21.44–33.28)TBARS (
m
mol/l) (Ranges) 3.93
^
0.12* (3.634.23) 1.89
^
0.03 (1.46–2.20)
*
 p
,
0
:
05 MS patients vs. the controls. †
^
SD
:
ANTIOXIDANT VITAMINS IN MULTIPLE SCLEROSIS 217

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