
 multiple sclerosis ( ms ) is an inflammatory immune mediated demyelinating disease of the central nervous system ( cns ) .
an energy deficient state and oxidative and nitrative stress have been implicated in the degeneration of axons in multiple sclerosis [ 14 ] . in chronic lesions ,
axonal degeneration correlates with the extent of inflammation and leads to axonal loss through a slow burning process .
mickel   proposed that a lipid peroxidation disturbance caused by free radicals production is involved in the breakdown of the myelin sheath .
since then several studies have demonstrated the role of increased free radical production and/or a decreased antioxidant defense in cns as causal factors of ms [ 1 , 4 , 7 , 8 ] .
following demyelination the axonal membrane undergoes a number of changes including an increase in number of sodium channel within demyelinated part of the axon [ 911 ] .
the maintenance of intra - axonal ionic balance and resting membrane potential following the influx of sodium through the increased sodium channels relies on the largest consumer energy in the central nervous system , na / k atpase . in noninflammatory environments
this increase in energy demand of axons lacking a healthy myelin sheath is apparent by the changes in density and activity of energy producing organelles mitochondria , the most efficient producers of energy [ 2 , 13 , 14 ] . though the principal site of ms pathology is the cns , the lipid status and the membrane properties in the platelets and erythrocytes in the peripheral blood are also altered .
increased lipid peroxide levels have been observed both in the cerebrospinal fluid and in the blood of ms patients [ 7 , 8 , 15 ] .
lack of sufficient vitamin a and e in the diet has been suggested to be a risk factor for the onset of the disease [ 7 , 16 ] .
however , other studies have found that the plasma levels of these vitamins are similar in ms patients and in controls [ 7 , 17 ] .
the present study examined serum levels of vitamin a ( -carotene ) , vitamin e ( - and -tocopherol ) isomers , and coenzyme q10 ( antioxidant , which is an indicator of bioenergetic state ) in ms patients in relation to the cerebrospinal fluid levels of purine nucleotides degradation products ( adenosine , inosine , hypoxanthine , xanthine , and uric acid ) , which are known to be produced during energy deficiency .
furthermore , we were interested to discover which of these lipophilic antioxidants is associated the most with lipid peroxidation and degradation of purine nucleotides in ms patients .
blood serum and cerebrospinal fluid ( csf ) samples from 42 patients diagnosed with multiple sclerosis ( ms ) according to mcdonald 's rule were analyzed .
test group consisted of 32 females and 10 males with average age of 36.3  11.97 years .
each patient had relapsing - remitting form and was out of relapse at the time . according to our knowledge ,
the control group was created from neurological patients with noninflammatory diseases of the central nervous system ( n = 34 , 8 males , 26 females ) with an average age of 36.06  11.92 years , who had routine csf analysis and biochemical parameters within the physiological values .
ethical committee statement was not necessary since the examination of parameters was indicated by neurologist and was a component of diagnostic process .
none of the patients had demyelinating disease or any other diseases associated with an increase of oxidative stress and degradation of purine nucleotides .
aliquots of csf and serum samples were centrifuged , coded , and immediately stored at 70c in polypropylene tubes until being assayed .
csf levels of uric acid ( ua ) , hypoxanthine ( hyp ) , xanthine ( xan ) , inosine ( ino ) , and adenosine ( ado ) were measured by isocratic hplc method ( high - performance liquid chromatography ) with uv detection ( 254  nm ) .
deproteinized and centrifuged samples were loaded ( 20  l ) onto a lichrocart 250 - 4 column filled with lichrospher 100  rp-18 ( 5  m ) ( merck , germany ) .
the chromatographic column was connected to an hplc apparatus consisting of a lachrom l-7100 pump system and l-7400 uv detector ( merck - hitachi , germany ) .
data acquisition and analysis were performed by a pc using the csw v1.7 , ( dataapex ltd . ,
czech ) . mobile phase included 60  mmol / l kh2po4 , 2% methanol ( v / v ) , ph 2.9 .
serum - and -tocopherol ( t and t ) , -carotene ( c ) , and coenzyme q10 ( coq10 ) were measured by modified isocratic hplc method with uv detection at 275  nm ( coq10 ) , 295  nm ( tocopherols ) , and 450  nm ( -carotene ) [ 18 , 19 ] .
serum levels of thiobarbituric acid reactive substances ( tbars ) were determined spectrophotometrically at 532  nm according to janero and burghardt .
state of blood - brain barrier ( bbb ) of patients was evaluated via qa - index , as ratio between albumin concentration in csf and in blood serum multiplied by 1000 .
intrathecal igg production was evaluated via igg index ( igg index = iggcsf / albumincsf  :  iggs / albumins ) and reiber 's index ( rig ) .
all statistical analyses were carried out using statsdirect statistical software , version 2.7.2 ( statsdirect sales , sale , cheshire m33 3uy , united kingdom ) .
the means and standard deviations were shown and student 's t - test for comparing two independent samples was used . for non - gaussian distribution , the median values and 2575% interquartile range ( iqr ) were shown and independent variables were compared using the nonparametric mann - whitney u test . the linear relationship between continuous variables was evaluated using the spearman 's correlation coefficient .
selected basic biochemical parameters in the serum and cerebrospinal fluid ( csf ) are shown in table 1 characterizing the control group and the group of patients with multiple sclerosis ( ms ) in comparison to the reference values .
the group with multiple sclerosis patients ( n = 42 , the average age of 36.49  11.97 years ) was characterized by pathologically high csf levels of immunoglobulin igg , with evidence of increased intrathecal synthesis of immunoglobulin igg expressed by elevated values of igg index and reiber 's index ( rig ) ( table 1 ) .
average qa values of ms patients ( that characterize the integrity of blood - brain barrier ) were within the physiological range but in comparison to controls were significantly higher ( p = 0.0293 ) . in table 2
are shown csf levels of purine nucleotide degradation products adenosine ( ado ) , inosine ( ino ) , hypoxanthine ( hyp ) , xanthine ( xan ) , and uric acid ( ua ) in comparison with a control set of neurological patients with noninflammatory cns diseases .
compared to control group , ms patients had a significantly elevated values of all purine nucleotide metabolites , except of adenosine , which were significantly lower ( 0.20  0.16 
serum lipophilic antioxidants -tocopherol ( t ) , -tocopherol ( t ) , the ratio of /-tocopherol ( /t ) , -carotene ( c ) , and coenzyme q10 ( coq10 ) in the vast majority of cases were lower than normal or were in the close proximity to the bottom border of the physiological values ( table 3 ) .
the greatest incidence of antioxidants deficiency occurred at t levels , where up to 71.4% of ms patients had subliminal serum values of t and t values of the rest of the ms patients were in the bottom border of the t physiological values ( figure 1 ) .
the ratio between the isomers of vitamin e , - and -tocopherols ( /t ) , was reduced compared to the reference values of all analyzed ms patients .
we observed deficit status in serum levels of -carotene in approximately 30% and in coq10 levels in 45% of ms patients ( table 3 ) .
serum levels of tbars ( thiobarbituric acid reactive substances ) and marker of lipid peroxidation were increased by 81% in the ms patients ( table 3 ) .
these values correlated negatively with serum levels of -carotene ( r = 0.329 ; p = 0.048 ) ( figure 2 ) .
in addition , -carotene negatively correlated with igg index ( r = 0.465 , p = 0.003 ) and rig ( r = 0.504 , p = 0.0017 ) ( figure 2 ) .
spearman rank correlations among lipophilic antioxidants and intrathecal synthesis of igg expressed by igg index and reiber 's index rig ( figure 2 ) showed also significant relationship between coq10 and t ( r = 0.355 , p = 0.02 ) and between t and igg index ( r = 0.314 , p = 0.04 ) .
serum levels of t significantly correlated with levels of -tocopherol as well ( r = 0.314 , p = 0.04 ) ( figure 2 ) . out of the measured antioxidants , -tocopherol correlated the most with csf levels of purine nucleotide degradation products ( figure 3 ) .
serum levels of t correlated positively with csf levels of adenosine ( r = 0.355 , p = 0.05 ) , metabolic turnover of adenosine to inosine ( r = 0.449 , p = 0.01 ) , csf levels of hypoxanthine ( r = 0.391 , p = 0.03 ) , and xanthine ( r = 0.411 , p = 0.02 ) and negatively with csf levels of uric acid ( r = 0.407 , p = 0.02 ) and metabolic turnover of hypoxanthine to uric acid ( r = 0.352 , p = 0.05 ) ( figure 3 ) . with regard to the occurrence of not uniform serum deficit of lipophilic antioxidants ms patients
, we analyzed our set of patients divided into 4 subgroups ( table 4(a ) ) .
the first group consisted of patients with deficiency of -tocopherol only ( t , n = 16 ) .
the second group consisted of patients with deficiency and thresholds to be deficit t and coq10 ( tcoq10 ; n = 16 ) , the third group consisted of patients with deficiency and thresholds to be deficit t and c ( tc ; n = 10 ) , and the fourth group consisted of patients with deficiency and thresholds to be deficit t , c , and coq10 ( tcoq10c ; n = 10 ) . in accordance with the correlation relationships ( figure 3 ) , the groups of ms patients with deficiency of -carotene ( tc and tcoq10c ) had higher values of tbars and higher intrathecal synthesis of igg ( iggindex , rig ) compared to groups t and tcoq10 ( table 4(a ) ) .
serum levels of coq10 in ms patients significantly interfere with the metabolism of purine nucleotides in csf ( table 4(b ) ) .
coq10 deficiency leads to increased degradation of adenosine to inosine ( ino / ado ) , which is manifested by significantly lower csf levels of adenosine ( group 2 ; ado = 0.12 ( 0.070.17 )  mol / l ) in comparison to ms patients with t deficiency ( group 1 ; ado = 0.17 ( 0.130.54 )  mol / l , p = 0.028 ) .
all ms subgroups had significantly lower csf levels of adenosine and significantly increased degradation of adenosine to inosine ( ino / ado ) and to hypoxanthine ( hyp / ado ) compared to the control group ( table 4(b ) ) .
the group of ms patients with -carotene deficiency had the increased degradation of the hypoxanthine to the xanthine ( xan / hyp ) , as well as significantly higher csf levels of uric acid compared to the control group ( 20.09 ( 17.8827.54 ) versus 15.07 ( 14.1216.02 )  mol / l , p = 0.024 ) . in comparison to the control group , significantly elevated csf levels of xanthine were observed in patients with t deficiency and t + coq10 deficiency only ( table 4(b ) ) .
the results presented suggest that , in patients with multiple sclerosis ( ms ) alteration in the metabolism of purine nucleotides ( table 2 ) , reduced antioxidant and neuroprotection ( table 3 ) occur , and is associated with increased intrathecal synthesis of igg .
ms patients may suffer from a cell energy metabolism deficit that can be documented in biological fluids ( cerebrospinal fluid and serum ) .
the profile of compounds directly ( csf adenosine , inosine , hypoxanthine , xanthine , and uric acid ) or indirectly ( serum coenzyme q10 ) reflects the imbalance between adenosine triphosphate ( atp ) production and consumption . increased purine nucleotide degradation observed in our ms patients ( table 2 ) is activated in situations that are associated with a decrease in the amount of atp and the related rise of adenosine monophosphate ( amp ) levels .
amp can be metabolized in the cells in two ways : ( a ) by deamination to inosine monophosphate ( imp ) followed by dephosphorylation of imp to inosine or ( b ) by dephosphorylation to adenosine and its deamination to inosine ( figure 3 ) . at physiological concentrations of atp deamination of amp to imp
is preferred . in experimental autoimmune encephalomyelitis ( eae ) , the animal model of ms , it has been effectively shown that efficiency in neuronal atp biosynthesis is decreased due to mitochondrial malfunctioning , leading to cell energy state imbalance [ 27 , 28 ] .
if this occurs in ms patients too , a continuous outflow of atp catabolites , including uric acid and its precursors , is expected from cerebral tissue of the ms lesions into the extracellular space .
the picture of an energetic alteration in ms patients indirectly is reinforced by data referring to the coenzyme q10 decrease in serum of ms patients ( table 3 ) .
coenzyme q10 is essential for the energy production of the cells as an electron transporter in the mitochondrial respiratory chain . in the process of oxidative phosphorylation in mitochondria coenzyme q10 transfers electrons from complex
i ( nadh coq reductase ) to complex iii ( cytochrome bc1 complex ) or complex ii ( succinate dehydrogenase ) on the complex iii .
coq10 deficiency during the time of increased energy requirements causes a decrease in production of atp and activates the processes that lead to the degradation of atp to amp , its dephosphorylation to adenosine , and its subsequent degradation to inosine and hypoxanthine ( table 4(b ) ) .
the energy deficit of coq10 ( group 2 , table 4(a ) ) reduces the csf levels of neuroprotective adenosine due to increased degradation of adenosine to inosine and hypoxanthine .
moreover , coenzyme q10 is one of the most important lipophilic antioxidants , preventing the generation of free radicals as well as oxidative modifications of proteins , lipids , and dna and can also regenerate the other powerful lipophilic antioxidant , -tocopherol
. decreased levels of coq10 in humans are observed in many pathological conditions ( e.g. , cardiac disorders , neurodegenerative diseases , aids , and cancer ) associated with intensive generation of free radicals and their action on cells and tissues . a crucial role in all these processes
is played by nad(p)h - dependent reductase(s ) acting as the plasma membrane to regenerate the reduced ubiquinol form of coq10 , contributing to the maintenance of its antioxidant properties .
moreover , correlations of serum levels of coenzyme q10 and vitamin e isomers ( particularly with -tocopherol ) in ms patients show that coq10 is involved to the mechanisms that lead to alteration in the purine nucleotides metabolism , as well as in the processes of regeneration of vitamin e ( figure 2 ) .
serum -tocopherol levels were in ms patients within the physiological values , but serum -tocopherol levels were reduced . up to 71.4% of ms patients had subliminal serum values of t and the rest of the ms patients were in the bottom border of the t physiological values ( figure 1 ) .
serum levels of t significantly correlated with levels of -tocopherol ( r = 0.314 , p = 0.04 ) ( figure 2 ) .
vitamin e collectively refers to 8 different structurally related tocopherols and tocotrienols that all possess antioxidant activity .
the antioxidant activity of vitamin e is derived primarily from t and t , of which t is the most biologically active and the predominant form found in blood . during lipid oxidation
this reaction produces oxidized tocopheroxyl radicals that can be recycled back to the active reduced form through reduction by vitamin c. without reduction of vitamin e by vitamin c , vitamin e can act as ros donor .
in addition to scavenging ros , t in contrast to t also react with nitrogen species such as peroxynitrite , forming 5-nitro--tocopherol . it has been accepted that the molecules of peroxynitrite are the final molecules responsible for pathological processes in neurodegenerative diseases and ms ; 3-nitrotyrosine is an indicator of increased formation of peroxynitrite and the most important molecule considered to be in charge of demyelination .
significantly increased plasma levels of 3-nitrotyrosine were reported in ms patients [ 15 , 34 ] .
low levels of -tocopherol and /-tocopherol ratio in our ms patients might indirectly point out to its high consumption through quenching reactive nitrogen species .
serum levels of t showed positive significant relationship between coq10 ( r = 0.355 ,  p = 0.02 ) and t and igg index ( r = 0.314 ,  p = 0.04 ) , indicator of intrathecal synthesis igg . while both tocopherols exhibit anti - inflammatory activity in vitro and in vivo , supplementation with mixed ( -enriched ) tocopherols seems to be more potent than supplementation with -tocopherol alone .
cook - mills   reported that supplementation with physiological levels of purified natural forms of the vitamin e isoforms t and t has opposing regulatory functions during inflammation such that t is anti - inflammatory and t is proinflammatory
. positive correlation of t with igg index could indicate proinflammatory effect of t in ms patients .
out of the measured antioxidants , t correlated the most with csf levels of purine nucleotide degradation products ( figure 3 ) .
serum levels of t correlated positively with csf levels of adenosine , hypoxanthine , xanthine , and metabolic turnover of adenosine to inosine and negatively with csf levels of uric acid and metabolic turnover hypoxanthine to uric acid ( figure 3 ) .
participation of t in the process of purine nucleotides degradation is probably connected with its ability effectively to scavenge no and other free nitrogen radicals and with scavenging activity of uric acid , which effectively scavenges peroxynitrite formed by the reaction of no with superoxide . due to its significant correlation with coq10 , -carotene , and igg index , it can be assumed that its effect on the metabolism of purine nucleotides has more complex ( synergistic ) nature .
levels of -carotene in serum ( 0.64  0.48 ( 0.271.06 )  mol / l ) in ms patients were in the lower range of the reference value ( 0.33.0  mol / l ) .
we observed deficit status of -carotene at approximately 30% of ms patients ( table 3 ) .
serum levels of -carotene significantly negatively correlated with serum tbars levels ( r = 0.3295 , p = 0.048 ) and with indicators of intrathecal synthesis of igg - igg index ( 0.4653 , p = 0.0033 ) and rig ( r = 0.5038 , p = 0.0017 ) ( figure 2 ) .
after the division of ms patients , according to their vitamin deficiency status into 4 groups ( table 4 ) , ms patients with -carotene deficiency ( group 3 ) in comparison to patients with t deficiency ( group 1 ) and coq10 deficiency ( group 2 ) had significantly higher values of igg index and rig and in comparison to group 1 ( t ) they had higher levels of tbars ( table 4(a ) ) .
these results show that -carotene in ms patients participates significantly in the neutralization of lipid peroxidation processes running in this disease . among the analyzed serum antioxidants ,
beta - carotene in comparison to -tocopherol is more lipophilic and quenches radicals in lipophilic compartments more effective than -tocopherol .
carotenoids are best known for their antioxidant activities including quenching - free radicals , reducing damage from reactive oxidant species , and inhibiting lipid peroxidation .
carotenoids also facilitate cell - to - cell communication which regulates cell growth , differentiation , and apoptosis , and some carotenoids convert to vitamin a .
carotenoids play a pivotal role in prevention of many degenerative diseases mediated by oxidative stress including neurodegenerative diseases .
low levels of -carotene observed in our group of ms patients may be due to a degradation of -carotene in its scavenging activity .
it was found that , during the oxidation attacks , carotenoid breakdown products are formed ( cbps ) , including highly reactive aldehydes and epoxies .
stimulated neutrophils are able to breakdown the -carotene and form a number of cbps , which inhibit the mitochondrial respiration .
this is accompanied by a reduction in the protein sulfhydryl content , a reduction of glutathione ( gsh ) levels and redox status , and increased accumulation of malonydialdehyde ( mda ) .
changes in the mitochondrial membrane potential can lead to deterioration in function of adenine nucleotides translocator .
an inflammatory stimulus , such as ifn- , activates macrophages to produce various proinflammatory cytokines ( tnf , il-1 ) and inflammatory mediators , which are synthesized by cyclooxygenase ( pge2 ) and by induced no synthase .
expression of these cytokines and genes may be regulated by activation of transcription factor nf-b .
the association between inflammation and a decrease in -carotene also showed van herpen - broekmans , who found a negative correlation between serum levels of -carotene and inflammatory marker crp .
the results of the work show that patients with multiple sclerosis in the early stage of the disease are characterized by reduced antioxidant , immunoregulatory , and neuroprotective ability , which are reflected by the increased metabolism of purine nucleotides , reduced csf adenosine levels , low serum levels of lipophilic antioxidants -tocopherol , -carotene , coq10 , and elevated levels of serum tbars . serum levels of coq10 ( an indicator of bioenergetic state ) and t ( isomer of vitamin e ) significantly interfere with the metabolism of purine nucleotides in csf , while -carotene is rather associated with intrathecal synthesis of igg and with neutralization of lipid peroxidation processes running in this disease . due to the fact that one of the possible causes of the axonal degeneration and disability may be an energy deficiency by increased energy requirements for axonal remyelination , demyelination and lipid peroxidation disturbance caused by free radicals production , decreased serum levels of coq10 , and lipophilic antioxidants
 in the pathogenesis of demyelinating diseases including multiple sclerosis ( ms ) an important role is played by oxidative stress . increased energy requirements during remyelination of axons and mitochondria failure is one of the causes of axonal degeneration and disability in ms . in this context , we analyzed to what extent the increase in purine catabolism is associated with selected blood lipophilic antioxidants and if there is any association with alterations in serum levels of coenzyme q10 . 
 blood serum and cerebrospinal fluid ( csf ) samples from 42 patients with diagnosed ms and 34 noninflammatory neurologic patients ( control group ) were analyzed . 
 compared to control group , ms patients had significantly elevated values of all purine nucleotide metabolites , except adenosine . 
 serum lipophilic antioxidants -tocopherol , -carotene , and coenzyme q10 for the vast majority of ms patients were deficient or moved within the border of lower physiological values . 
 serum levels of tbars , marker of lipid peroxidation , were increased by 81% in the ms patients . 
 the results indicate that the deficit of lipophilic antioxidants in blood of ms patients may have a negative impact on bioenergetics of reparative remyelinating processes and promote neurodegeneration . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions