Taysi et al Plasma homocysteine and liver tissue

Taysi et al Plasma homocysteine and liver tissue
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  Eur opean  Rev iew for  Med ical and  Pharmacol ogical  Sci ences 154  Abstract. –   OBJECTIVE:  The aim of thisstudy was to evaluate plasma homocysteine(Hcy), malondialdehyde (MDA), glutathione(GSH) levels, glutathione peroxidase (GSH-Px)and glutathione-S-transferase (GST) activitiesand liver tissue S-adenosylmethionine (SAM)and S-adenosylhomocysteine (SAH) levels incontrol and vitamin B6-deficient rats. MATERIALS AND METHODS: Thirty-two malerats with a weight of 65-75 g were used for theexperiment. The rats were divided into control(n=16) and vitamin B6-deficient groups. At theend of the experiment, the animals were anes-thetized with ketamine-HCl (Ketalar, 20 mg/kg,i.p.), and the blood was collected by cardiacpuncture after thoracotomy. Plasma Hcy, pyri-doxal phosphate (PLP), liver SAM, SAH levelsmeasured by an isocratic system with high per-formance liquid chromatography. Plasma GSH-Px, GST activities and GSH, MDA levels werecarried out using a spectrophotometer. RESULTS:  Plasma Hcy, MDA, liver tissue SAHlevels were significantly increased, whereas plas-ma GSH, PLP, liver tissue SAM levels, plasmaGST, GSH-Px activities and SAM/SAH ratio weredecreased compared to those of control group. CONCLUSIONS:  Vitamin B 6  deficiency causesan increase in plasma homocysteine levels.Thus, we think that vitamin B 6  supplementationcould be used for therapeutic purposes in hyper-homocysteinemia condition. Key Words:  Homocysteine, S-adenosylhomocysteine, S-adeno- sylmethionine, Vitamin B 6 -deficiency, Pyridoxal-5- phosphate, Free radical. Introduction Hyperhomocysteinemia is a risk factor for ath-erosclerosis 1,2 , which increases the effects of con- Plasma homocysteine and liver tissueS-adenosylmethionine, S-adenosylhomocysteinestatus in vitamin B 6 -deficient rats S. TAYSI 1 , M.S. KELES 2 , K. GUMUSTEKIN 3 , M. AKYUZ 4 , A. BOYUK  5 , O. CIKMAN 6 , N. BAKAN 2 1 Department of Medical Biochemistry, Gaziantep University, School of Medicine, Gaziantep, Turkey  2 Department of Biochemistry, Ataturk University, Medical School, Erzurum, Turkey  3 Department of Physiology, Abbant Izzet Baysal University, Medical School, Bolu, Turkey  4 Department of Chemistry, Science and Art Faculty, Kilis 7 Aralik University, Kilis, Turkey  5 Department of Surgery, Dicle University, Medical School, Diyarbakir, Turkey  6 Department of Surgery, Çanakkale Onsekiz Mart University, Medical School, Çanakkale, Turkey  Corresponding Author:   Seyithan Taysi, MD; e-mail: ventional risk factors such as arterial hyperten-sion, hypercholesterolemia, and aging 3-5 . Normalhuman plasma contains total concentrations of homocysteine (Hcy) and its derivative disulfidesclose to 10 µmol/L, although there is some varia-tion due to genetic factors, age, sex, menopausalstatus, and other physiological and lifestyle vari-ables. Hyperhomocysteinaemia (defined by val-ues of plasma Hcy > 15 µmol/L) is the presenceof an abnormally elevated concentration of plas-ma or serum Hcy 6 . Hcy is a sulfur-cantainingamino acid. It forms various disulphides by oxi-dation at physiological pH, since the pKa of thethiol group is 8.9. All Hcy found in body isformed by demethylation of the essential aminoacid methionine 7 .S-adenosylmethionine (SAM), the activatedform of methionine, is the principal methylgroup donor for a lot of methylation reactions.S-adenosylhomocysteine (SAH) is formed afterdemethylation, and irreversible hydrolysis leadsto homocysteine. Hcy is metabolized by twomajor pathways. Hcy obtains a methyl goupfrom 5-methyl-tetrahydrofolate to form methio-nine in the remethylation pathway. It condenseswith serine to form cystathionine in transsulfu-ration pathway. The pyridoxal-5-phosphate(PLP)-dependent enzyme cystathione- β -synthase catalyzes this irreversible reaction.Cysteine and ketobutyrate are formed by the ac-tion of   γ -cystathionase, in the presence of PLPas a cofactor 8 .Vitamin B 6  has been shown to be important fornormal cognitive function and in lowering the in-cidence of coronary heart disease among the el-derly 9-12 . In addition, Vitamin B 6  supplementation 2015; 19: 154-160  has been shown to reduce diabetic complicationsand the occurrences of neurodegenerative dis-eases in varying degrees 9,13 .Vitamin B 6  refers to three primary forms of water-soluble vitamins: pyridoxine, pyridoxalphosphate, and pyridoxamine. Some studies havebeen reported antioxidant activities of vitaminB 6 . Vitamin B 6 -deficiency decreases the activityof   γ -cystathionase markedly, resulting in the ac-cumulation of cystathionine 14 . Methionine me-tabolism was found to be altered in rats fed a vit-amin B 6 -deficient diet, as indicated by accumula-tion of SAH in liver and accordingly a concomi-tant decrease in SAM/SAH ratio. The SAM toSAH ratio in liver cells is important in the regula-tion of transmethylation reaction 8,15 .Cells have developed different antioxidant sys-tems and various antioxidant enzymes to defendthemselves against free radical attacks. Glu-tathione-dependent antioxidant system consistingof reduced glutathione and an array of function-ally related enzymes plays a fundamental role incellular defense against reactive free radicals andother oxidant species. Of these enzymes, glu-tathione peroxidase (GSH-Px) is a selenoproteinthat reduces hydroperoxides as well as H 2 O 2 while oxidizing glutathione. A number of poten-tially toxic electrophilic xenobiotics (such as cer-tain carcinogens, bromobenzene, chlorobenzene)are conjugated to the nucleophilic glutathione byglutathione S-transferases (GSTs) present in highamounts in cell cytosol. GST can also catalyzereactions reducing peroxides like GSH-Px. Re-duction of oxidized glutathione (GSSG) to glu-tathione (GSH)is mediated by the widely distrib-uted enzyme glutathione reductase (GRD) thatuses NADPH as the reductant 16,17 .In the present study, we aimed to investigateplasma GST, GSH-Px, Hcy, reduced GSH, mal-ondialdehyde (MDA) pyridoxal phosphate (PLP)and liver tissue SAM and SAH levels in vitaminB 6 -deficient rats, and relationship among theseparameters in vitamin B 6 -deficient rats. Material and Methods  Animals  Thirty-two male rats (4 weeks old, Sprague-Dawley strain) with a weight of 65-75 g wereused for the experiment. All animals received hu-mane care compliance with the guidelines of Ataturk University Research Council’s criteria.The composition of the diet was done as de-scribed 18 . After 4 weeks of feeding, the animalswere anesthetized with ketamine-HCl (Ketalar,20 mg/kg, i.p.), and the blood was collected bycardiac puncture after thoracotomy. Blood sam-ples were collected in vacutainer tubes with K 3 -EDTA as anticoagulant. They were centrifuged at2000xg for 10 min and plasma was removed by aPasteur pipette. Liver tissues were removed andwashed out from contaminated blood with coldwater, and homogenized 5-fold percloric acid(mol/L) solution by using a homogenizer (OmniAcceory Pack International omogenizer, Warren-ton, VA, USA). The homogenate was centrifugedat 10,000 g for 20 min to remove debris. Theplasma and homogenates were kept in –80°C un-til biochemical determinations. Biochemical Measurements  Liver tissue SAM and SAH levels were mea-sured by HPLC following the method of She etal 19 . The total plasma Hcy level was measured byhigh performance liquid chromatography(HPLC) pump (flow rate 1.7 mL/min), injector(injection volume 20 mL, analytical run time 4-6min) and fluorescence detector (Ex: 385 nm, Em:515 nm). Plasma PLP, the bioactive form of vita-min B 6 , level was measured by HPLC pump(flow rate 1.3 mL/min), injector (injection vol-ume 50 mL, analytical run time 12 min) and flu-orescence detector (Ex: 370 nm, Em:470 nm)(HP 1100). Plasma GSH-Px, GST activities,GSH and MDA levels were measured as de-scribed, respectively 20-23 . Statistical Analysis  The findings were expressed as the mean ±SD. Statistical and correlation analyses were un-dertaken using the Mann-Whitney U-test andSpearman’s rank correlation test, respectively.Significance (p) values less than 0.05 were con-sidered significant. Statistical analysis was per-formed with Statistical Package for the SocialSciences for Windows (SPSS, version 10.0,Chicago, IL, USA). Results The results from vitamin B 6 -deficient rats andcontrol group the parameters measured in thestudy were summarized in Tables I and II, re-spectively. As seen from Tables, vitamin B 6 -defi-cient rats caused a significant loss of bodyweight. Plasma Hcy, MDA, liver tissue SAH lev- 155 Homocysteine status in vitamin B 6  deficient rats  156 S. Taysi, M.S. Keles, K. Gumustekin, M. Akyuz, A. Boyuk, O. Cikman, N. Bakan Control group (n=16) Vitamin B 6 -deficient group (n=16) Hcy (µmol/L) 9.6 ± 2.8 18.6 ± 6.9 c PLP (µg/L) 104.9 ± 18.2 32.6 ± 7.0 c MDA (nmol/ml) 1.2 ± 0.6 2.2 ± 1.02 c GSH (nmol/L) 61.1 ± 6.9 52.5 ± 7.1 b GST (U/L) 18.5. ± 8.9 10.4 ±2.2 d GSH-Px (IU/L) 2.8 ± 0.58 2.3 ± 0.93 d Final body weight 183.8 ± 17.3 103.5 ±10.2 c Initial body weight 66.8 ± 2.6 67.4 ± 2.8 els were significantly increased, whereas plasmaGSH, GST, GSH-Px activities, PLP levels, livertissue SAM levels, and SAM/SAH ratio were de-creased compared to those of control group.Correlation analyses were shown in Table III.Serum Hcy level negatively correlated withserum PLP (r = –0.69,  p  < 0.005), GST andGSH-Px activities (r = –0.52,  p  < 0.05 and r = –0.54,  p  < 0.05, respectively) in vitamin B 6 -defi-cient group while there was a positive correlationbetween serum Hcy and MDA levels in thegroup. There were significant negative correla-tion between GSH-Px and MDA and positivecorrelation between GST and GSH-Px in vitaminB 6 -deficient group. However, no other correlationcould be found among the parameters in controlsgroup. Discussion Vitamin B6 deficiency is rare in humans, butoccurs in association with low concentrations of other B-complex vitamins such as vitamin B 12 and folate. For instance: populations most com-monly at risk for vitamin B 6  deficiency includethe elderly, alcoholics, renal patients undergoingdialysis, individuals with malabsorption condi-tions such as celiac disease, Crohn’s disease orulcerative colitis, and those with autoimmunedisorders. Low vitamin B 6  levels have also beenlinked to various conditions including cardiovas-cular disease, cancer and cognitive function. Vit-amin B 6  deficiency can lead to anemia, dermati-tis, glossitis, depression, confusion, and weak-ened immune function 24 . Pyridoxal phosphate is,the biologically active form of vitamin B 6,  acoenzyme in various metabolic pathways includ-ing degrading of Hcy.Homocysteine has been emerged as a poten-tial risk factor for premature cardiovascular dis-ease, promoting many processes that play a rolein vascular and endothelial cell damage 25,26 .Therefore, a progressive increase in the plasmaHcy concentration raises the risk of coronaryartery disease 26,27 . Normal levels of Hcy in fast-ing conditions are of 5-15 µmol/L. In hyperho-mocysteinaemia it may reach values of 16-30µmol/L (moderate), 31-100 µmol/L (medium) orgreater than 100 µmol/L (severe), values as highas 500 µmol/L being found in patients with ho-mocystinuria 26,28 . The mechanisms underlyingendothelial injury promoted either by Hcy aloneor in combination with other risk factors are notunderstood well.  In vitro  studies provide evi-dence that supraphysiological Hcy levels have a Control group (n=16) Vitamin B 6 -deficient group (n=16) S-adenosylmethionine (SAM) (nmol/g of liver) 163.7 ± 14.2 120.8 ± 5.1 a S-adenosylhomocysteine (SAH) (nmol/g of liver) 119.0 ± 6.5 176.2 ± 9.1 a SAM/SAH ratio 1.4 ± 0.2 0.7 ± 0.05 a Table I.  Mean ± SD of liver tissue S-adenosylmethionine and S-adenosylhomocysteine levels in control and vitamin B 6 -defi-cient groups. A  p  < 0.001, vs. control group. Table II.  Mean ± SD of plasma homocysteine, GSH, MDA levels, GST, GSH-Px activities and initial and final body weightof control and vitamin B6-deficient groups. b  p  < 0.01,  c  p  < 0.001,  d  p  < 0.0001, vs. control group.  free sulfhydryl group of albumin, cysteine or ho-mocysteine, since reduced free Hcy contains afree sulfhydryl group. It is apparent that the plas-ma level of reduced free Hcy affects and en-hances oxidative stress. The endogenous attack on DNA by ROS including hydrogen peroxidemay generate many DNA adducts in human cells.Of the over 100 oxidative lesions in mammalianDNA, 8-hydroxyguanine is one of the most abun-dant lesion 31,35,36 .Important metabolic indicators of the cellularmethylation capacity are the intracellular concen-trations of SAM and SAH, as the substrate andproduct of essential cellular methyltransferase re-actions 36 . A chronic increase in SAH, secondaryto the homocysteine-mediated reversal of theSAH hydrolase reaction, may also have signifi-cant, indirect, pathologic consequences, despitethe emphasis has been placed on the toxicity of increased Hcy and depressed SAM concentra-tions in metabolic pathology of various dis-eases 29,34,36,37 . Because, SAH can bind to and in-hibit multiple cellular methyltransferases, an in-crease in SAH can lead to DNA hypomethylationand altered chromatin configuration, reducedmembrane phosphatidylcholine concentrations,reduced protein and RNA methylation, and re-duced neurotransmitter synthesis 31,38 . These alter-ations may lead to inappropriate gene expression,altered signal transduction, immune deficiency,and cytotoxicity 31,39 . Nguyen et al 15 and She etal 19 reported that an marked increase in SAH anda decrease SAM levels were in liver tissues of vi-tamin B 6 -deficient rats. We found that vitaminB 6 -deficiency for 4 weeks caused a marked re-duction of the SAM/SAH ratio, accumulation of liver tissue SAH, reduction of SAM level in vita-min B 6 -deficient rats. The increase in SAH, de-crease in SAM levels and SAM/SAH ratio in ratliver induced by vitamin B 6  deficiency are inagreement with previous studies 15,19 .GSH shows antioxidant properties itself. GSHacts as an antioxidant molecule protecting sulph-hydryl groups. Results demonstrated that GSHlevels in plasma of vitamin B 6 -deficient rats weresignificantly lower than in the control group.GSH synthesis depends on the availability of cysteine, which is synthesized by vitamin B 6 -de-pendent enzymes cystathionine- β -synthase andcystathionine- γ -lyase from methionine. Cysteinesynthesis appears to be reduced in vitamin B 6  de-ficient rats because of the altered activity of theseenzymes, which leads to decreased GSH synthe-sis 22 . The results confirm this hypothesis. 157 Homocysteine status in vitamin B 6  deficient rats direct toxic effect in endothelial cells, probablyby free-radical generation through oxidation of the Hcy sulphydryl group 25,29 . Alvarez-Maquedaet al 26 found that Hcy significantly increasedO 2•− in a dose- and time-dependent manner.Lipid peroxidation is a well-established mecha-nism of oxidative damage caused by reactiveoxygen species (ROS), and the measurement of MDA provides an important index of lipid per-oxidation 22,30 . In our study, we found that MDAlevels in plasma of vitaminB 6 -deficient ratswere significantly increased compared to thoseof control group. Increased MDA levels in vita-min B 6  deficient rats were increased in agree-ment with previous reports 22 . This confirms thepresence of increased oxidative stress in vitaminB 6  deficient rats.Furthermore, increased hydrogen peroxide(H 2 O 2 ) production accompanies increased O 2•− re-lease induced by Hcy. Different sources andpathways produce H 2 O 2  in eukaryotic cells. Forexample, mitochondria, microsomes, peroxi-somes and cytosolic enzymes are effective gener-ators of ROS, which yield H 2 O 2  after dismuta-tion. In mitochondria, H 2 O 2  is derived from thedismutation of O 2•− generated by minor side ef-fects from the sequential reduction of O 2•− viarespiratory carriers 31 .In some studies, it has been reported that plas-ma total Hcy levels were significantly increasedin vitamin B 6 -deficient rats 32-34 . We found that vi-tamin B 6 -deficiency led to a marked reduction of the plasma PLP level, accumulation of plasmaHcy in vitamin B 6 -deficient rats. Our results arein agreement with these studies 32-34 .Overproduction of ROS generated from theoxidation of Hcy may cause endothelial injuryand DNA damage. Free radicals including hydro-gen peroxide can be generated upon oxidation of homocysteine, forming a disulfide linkage with r =  p   < Hcy-PLP –0.69 0.005Hcy-GST –0.52 0.05Hcy-GSH-Px –0.54 0.05Hcy-MDA 0.53 0.05GSH-Px-MDA –0.87 0.001GST-MDA –0.77 0.001GST-GSH-Px 0.57 0.05 Table III.  Spearman’s rank correlation coefficients betweenplasma GSH-Px, GST activities, Hcy, PLP, MDA levels invitamin B 6 -deficient group.  Conclusions We showed that vitamin B 6 -deficiency causeda marked reduction of the plasma PLP, GSH lev-els, GST, GSH-Px activities, accumulation of plasma Hcy and liver tissue SAH levels and re-duction of SAM level, SAM/SAH ratio, and anincrease in plasma MDA level in vitamin B 6 -defi-cient rats. The increase in plasma Hcy and livertissue SAH levels and the reduction of SAM/SAH ratio may be due to a block in themethionine catabolism via the transsulfurationpathway. These may lead to inhibition of trans-methylation reactions of DNA, RNA and protein,and result in liver damage. By increasing the for-mation of MDA, an indicator of lipid peroxida-tion, and reducing the GSH levels and GSH-de-pendent enzymes activities, vitamin B 6  deficien-cy causes an increase in plasma Hcy levels andoxidative stress. Thus, the results presented heresuggest the vitamin B 6  supplementation can bereconsidered and be determined for therapeuticpurpose in hyperhomocysteinaemia.––––––––––––––––––––  Acknowledgements This study has been supported by The Research Foundationof theAtaturk University, Erzurum, Turkey. References 1)  M C C ULLY   KS . Homocysteine, folate, vitamin B 6 , andcardiovascular disease. JAMA 1998; 279: 392-393.3)  T  EHLIVETS  O . Homocysteine as a risk factor for ath-erosclerosis: Is its conversion to s-adenosyl-l-homocysteine the key to deregulated lipid metab-olism? J Lipids 2011; 2011: 702853.3)  K  IM  H, O H  S  J , K  WAK   H C , K  IM  J K  , L  IM  C H , Y   ANG  J S ,P  ARK   K, K  IM  S K  , L  EE  MY  .The impact of intratracheal-ly instilled carbon black on the cardiovascularsystem of rats: Elevation of blood homocysteineand hyperactivity of platelets. J Toxicol EnvironHealth A 2012; 75: 1471-1483.4)  T   AYSI  S, S  ARI  R  A  , D URSUN  H, Y  ILMAZ  A, K  ELES  M, C  AYIR K, A  KYUZ  M, U YANIK   A, G UVENC  A  . Evaluation of ni-tric oxide synthase activity, nitric oxide, and ho-mocysteine levels in patients with active behcet'sdisease.Clin Rheumatol 2008; 27: 1529-1534.5)  M EMISOGULLARI  R, Y  UKSEL   H, C OSKUN  A, Y  UKSEL   H K  ,Y   AZGAN  O, B ILGIN  C.  High serum homocysteinelevels correlate with a decrease in the bloodflow velocity of the ophthalmic artery in highwaytoll collectors. Tohoku J Exp Med 2007; 212:247-252. We found that GSH-dependent enzyme activi-ties (GSH-Px and GST) in plasma of vitamin B 6 -deficient rats were significantly decreased whencompared to those of control group. GSH has animportant function both as a cosubstrate for GSTand as a substrate for GSH-Px. The low GSH-Pxactivities may be directly explained by the lowGSH content found in vitaminB 6 -deficient rats,since GSH is a substrate of this enzyme, whichhas been mentioned above. Therefore, low GSHcontent implies low GSH-Px activity, which mayincrease the propensity for oxidative stress. In thestudy, decrease in GST activities in vitaminB 6 -deficient rats may be the accumulation of the O 2•– which inactivates that by reacting with the activesite of this enzyme.Examining correlation analysis results fromTable III, it can be seen that plasma Hcy con-centration negatively correlated with plasmaPLP, GST, GSH-Px in vitamin B 6 -deficientgroup. These negative correlations confirm thepresence of increased oxidative stress in vita-min B 6  deficient rats. Robinson et al 40 reportedthat low PLP level confers an independent risk for coronary artery disease (CHD). Rimm etal 41 has also reported that folate and vitamin B 6 levels may be important in the primary preven-tion of CHD. Endo et al 14 reported that vitaminB 6  deficiency induced the oxidant stress whichaccelerates atherosclerosis and the antioxidantactivity of vitamin B 6  appears to suppress ho-mocysteine-induced atherosclerosis. Theyshowed that the oxidative stress was caused bya low level of vitamin B6 accelerates the devel-opment of homocysteine-induced atherosclero-sis in rats. Endo et al 14 also reported that as vit-amin B 6  has antioxidant activity apart from itsrole as coenzyme, the antioxidant activity of vi-tamin B 6  may suppress the homocysteine-in-duced atherosclerosis independent of homocys-teine action itself.Selhub et al 42 showed an association betweeninsufficient PLP status and carotid arteriosclero-sis, though this reduced after adjustment for ho-mocysteine. They also reported that the majorityof persons with elevated plasma homocysteineconcentrations have insufficient concentrationsof folate, vitamin B 12 , or vitamin B 6 . In additionto, some studies have been demonstrated that in-nocuous regimens of vitamin supplementation(including folate, vitamin B 12 , and vitamin B 6 )effectively lower moderately elevated plasma ho-mocysteine concentrations to the normalrange 43,44 . 158 S. Taysi, M.S. Keles, K. Gumustekin, M. Akyuz, A. Boyuk, O. Cikman, N. Bakan
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