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HYPERHOMOCYSTEINEMIA FOLLOWING ORAL METHIONINE LOAD IS ASSOCIATED WITH INCREASED LIPID PEROXIDATION
ThrombosisResearch,Vol.87, No. 4,pp. 411416,1997 Copyright@ 1997EJsevierScienceLtd Printedinthe USA. Allnghtsxwewed 0049-3848/97$17.00+.00
P
PII SO049-3848(97)O0145-X
BRIEF COMMUNICATION
HYPERHOMOCYSTEINEML4 FOLLOWINGOIUL METHIONINELOADIS ASSOCIATEDWITHINCREASEDLIPIDPEROXIDATION Teresa B. Domagal~ MagdalenaLiburaand AndrzejSzczeklik Departmentof Medicine,JagiellonianUniversitySchoolof Medicine,Cracow, Poland. (Received25 February1997by EditorC. Cierniewski;revisecVaccepted2June 1997)
Over the last years, homocysteinehas been recognized as an independent risk factor for atherosclerotic vascular disease (l). The known reasons for an increased level of plasma homocysteineare genetic defects of cystathionine~-synthase(2) and methylenetetrahydrofolate reductase (3). An important role in developmentof hyperhomocysteinemiais also played by deficiencyof folic acid, vitaminsBcand BIz (4). Hyperhomocysteinemiais definedwhen plasma homocysteineconcentrationexceeds 16vmol/1(5). In clinicalsettingit is usefidnot onlyto assess homocysteinelevel in fastingblood samples,but also to measureit tier methionineloadingtest (6). The mechanism through which hyperhomocysteinemiapromotes development of atherosclerosisare not clear. Homocysteineis toxic to vascular endothelium(7), it promotes thrombosis(8) and potentates the oxidationof low densitylipoprotein(LDL) in vitro (9). Ithas been suggested lately that oxidationof LDL may be one of the main factors involvedin initial developmentof atheroscleroticlesions(10). The aim of our study was to assess the relationshipbetween hyperhomocysteinemiaand lipid peroxidationin vivo .The experimentalmodelwas the oral methionineloadingtest. SUBJECTSAND METHODS Subjects. We studied46 subjects:25 maleand 21 femaleat the averageage of 41 years. In all of them oral methionineloading test was performed. Subjectswere dividedinto two groups: 1) with normal (n=21) and 2) pathological loading test (n=25). The levels of total homocysteinein plasma Key Words:Homocysteine,lipidperoxidation,methionineloadindtest, atherosclerosis. Corresponding author: Professor A.Szczeklik,Department of Medicine, School of Medicine, JagiellonianUniversity,Skawinska8, 31-066Cracow,Poland.
411
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vol. 87, No. 4
discriminatingthe two groups were: in fastingstate = 13.lwmol/1,at the 4* hour 37.8wnol/l and at the 6* hour 42.l~mol/1(X+ 2SD for our entiregroup). Theseresultsare.similarto reported by other autors (6, 11, 12). Fifteen subjectsfrom group with pathologicalloading test had clinical signsof atherosclerosis,verifiedby angiographyor ultrasonography(3 - coronary artery disease, 6- periphera)arterialocclusivediseaseand 6- atheroticlesionsof numerousvascularbeds). In all of them the first symptoms of atherosclerosisoccured before the age of 50 years; distinct deficiencyof folate was present in 7 persons, of vitaminBlz in 2, and of both vitaminBU and folate in 3. The subjects with elevated levels of cholesterol (>6.5mmol/1),triglicerydes (>1.8mmol/1),diabetes, immunologicaldisorders, chronic renai disease and carcinoma were excludedfrom the study. Methods.
The oral methionineloadingtest was performedusingmethionineat a dose of IOOmg/kgof body mass (6, 11). Venous blood were collectedto 5mlvacutainercontaining7.5rng EDTA (Becton DickinsonVacutainerSystemsEurope, England),the finalconcentrationof this anticoagulantwas 4.46mmol/l.Sampleswere taken beforeand at 4ti,6ti and 24* hour tier the loading.During the first 6 hours the subjectsremainedfasting. To 500u1 of plasma, 55u1 of 50% solution of sulfosalicylicacid (final concentration, 5’XO) containing500uM dithioerythritol(finalconcentration,50uM) was added. Precipitated protein was removedby centrifigation.Supernatantwas used for assessmentof fi-eehomocystehe. Plasma levels of total (tHcy) and flee (tltcy) homocysteinewere measured using High Petiormance Liquid Chromatographyaccording to method publishedby Mansaor et al (13). Instrumentationconsisted of: HPLC Hewlett Packard 1050 with autmw.mplerHewlett Packard 1100and HP Programmable Fluorescencedetector 1046A.Data were analysedby HP ChemStation (HP Vectra Xm). Sample of 25@ were injected into a 150x4.6mmcolumn packed with-3ym particles of Hypersil 120 ODS (Knauer,Germany).The temperaturewas 25°Cand the flow rate 1.5rnl/min.Elution solventA (lliter) was: IOmmoltetrabutylammoniumphosphate,2.5rnlglacial acetic acid dilutedwith distilledwater, pH adjustedto 3.8 with 2.OMNaOH. SolventB (1 liter) was: 200rnlacetonitrile,IOmmoltetrabutylammonium phosphate,2.5mlglacialacetic acid diluted with distilledwater, pH adjustedto 3.4 with 2.0 M NaOH; solventC was 75°/0acetonitrile.The elutionprofilewas as follows:O-13miR6-22°/oB;13.1-25min,22-40°/oB;25.1-30min,40-75°/0B; Afler each run, the column was washed for 5 min with solvent C to remove late-eluting fluorescentmaterial.The retentiontime for homocysteinewas 12.0 min. Sincethe concentration of the total homocysteine showed a marked positivelyskewed distribution, the results are presentedas geometricmean,as reportedotherby authors(14). In the same blood samples lipid peroxides were assessed by fluorimetricmethod (15) using LuminescenceSpectrometerLS 50B (Perkin-Elmer,USA),and expressed as thiobarbituricacid reactivesubstances(TBARS). To each studiedsamplebutylatedhydroxytoluene(BHT) at a final concentrationof 0.2mmol/lwas added to inhibitthe oxidativeprocess.In control experiments BHT at the concentrationused did not inteferewith TBARS estimations.Determinationswere performed only on fresh plasma, the results were expressedin nmol of malonylodialdehydehnl (MDA/ml)of plasmaand for statisticalanalysisas arithmeticmean* SD. All subjects had their plasma vit.Blz and folate levels tested using the isotopic method (Radioassaykit VitaminB~z[5’Co]/Folate[ 125Jl-Becton Dickinson). Statisticalanalyseswere performedusing the paired t-studenttest. Correlationswere calculated by Pearson test.
vol. 87,
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LIPID PEROXIDATION
413
RESULTS In the fastingstate plasmalevelsof TBARSwere similarin subjectswith normaland pathological loading test: 0.68 W.23rtmol/mland 0.70Ml.25nmolhnl,respectively.Plasma homocysteine showed inversed relationship to both folate (r = - 0.49, p= 0.001) and tit Blz levels (- 0.33, p= 0.03). Followingoral methionineload, levelsof TBARS reached signifkantlyhigher values at the 4* (@O.001)and 6ti hour (p
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MDA profilein plasma. — MDA in plasma total hornocysteinein plasma M: Subject$with normalmethkmheloadingtest Right: Subjectswith pathologicalmethionineloadingtest
L k SD
414
LIPID PEROXIDATION
vol.
87, No. 4
1,6
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Homocysteine [~mol/1]
FIG.2 Correlationbetween total homocysteineand MDAmeasured6 hourstier the loadingtest DISCUSSION Homocysteine,an independentrisk factor for atherothrombosis,might exert its ha.rmiideffects through a mechanisminvolvingoxidativedamage(16,17). Oxygen-derivedmoleculesgenerated during oxidation of homocysteine,could initiatelipid peroxidation,both at the endothelialcell surfaceand withinthe lipoproteinparticlesin plasma(18). Thisnotionis based on in vitro studies. Heinke et al (9), incubated LDL with increasingconcentrationsof homocysteineand assessed LDL peroxidationby TBARS. The degreeof lipidperoxidationdependedon the concentrationof added thiol and was maximalat concentrationof lmM ,whichexceedsseveral-foldplasmalevels of homocysteine.Similarobservationswere reported by Hirano et al. (19), who also showed the decrease in polyunsaturatedfatty acidsduringoxidationof LDL inducedby a homocysteine/iron mixture. However, until now evidence in vivo has been lacking. Measurement at fasting state performed by Blom and associates(20) showedno relationshipbetween plasma lipid peroxides, expressed as TBARS, and homocysteinelevels. Our results corroborate these findings. In a recent paper by the same group (21), fasting plasma MDA of hyperhomocysteinemic subjects was even lower then in a smrdl group of control;the latter, however, had unusually high MDA values (mean 5.0 +/- 2.0 nmolhnl). Using the diagnostic methionineloadingtest we created a simple experimentalmodel, which allowed to assess oxidativestress in vivo. We have demonstratedsignificantelevation of TBARS 4 and 6 hours followingmethionineloadingtest, which correlated with raised plasma concentrations of homocysteine. These results point to association between hyperhomocysteinemiaand increased lipid peroxidation, an association which might be of relevancefor developmentof atherosclerosis.
Vol.87, No.4
LIPID PEROXIDATION
415
REFERENCES 1. BOUSHEY, C.J., BERESFORD, S.A.A., OMENN, G.S. and MOTULSKY, A.G, A quantitative assessment of plasma homocysteineas a risk factor for vascular disease. JAMA 174,1049-1057,1995. 2. MUDD, S.H., LEVY H.L. and SKOSBYF. Disorders of transsulfhration.In: Z4e Metabolic andMolecukrBasesof InheritedDisease. SeriverC.R., BeaudetA.L., Sly W.S.,Vane D. (eds.), pp.1279-1327,McGraw-HillInc;New York (1995). 3. KANG, S.S., WONG P.W.K., BOCK H.G., HORWITZ, A. and GRIX, A. Intermediate hyperhomocysteinemiaresulting from compound heterozygosityof methylene-tetrahydrofolate reductasemutation.Am J Hum Genet 48, 546-551,1991. 4. KANG, S.S. and WONG, P.W.K. Genetic and nongenetic factors for moderate hyperhomocyst(e)inernia. Atherosclerosisf19, 135-138,1996. 5. MALINOW, M.R., KANG, S.S., TAYLORy L.M.,WONG, P.W.K., COULL, B. and IN~ T. Prevalence of hyperhomocyst(e)inemiain patients with peripheral arterial occlusive disease.Circulation79, 1180-1188,1989. 6. BOSTOM, A.G., JACQUES,P.F., NADEAU,M.R., WILLIAMS,R.R., ELLISON, R.C. and SELHUB J. Post-metbionineload hyperhomocysteinemiain persons with normal fasting total plasmahomocysteine:initialresults from [email protected], 147-151,1995. 7. HARKE~ L.A., ROSS, R., SLICHTE~ S.J. and SCOTT, C.R. Homocysteine-induced atherosclerosis:The role of endothelialcellinjuryand plateletresponsein its genesis.J Clin Invest 58,731-741, 1976. 8. REES, M.M. and RODGERS, G.M. Homocysteinemia:Associationof metabolic disorder with vasculardiseaseand thrombosis.ThrombRes 71,337-359, 1993. M., SUZUKI,L. and CHAIT, A. Oxidationof low density 9. HEINECKE, J.W., KAW~ lipoproteinby thiols. Superoxide-dependentand independent mechanism.,J Lipid Res 34, 345360, 1993. 10. WARE, J.A. and HEISTAD, D.D. Platelet endotheliuminteractions.New Engl J Med 328, 628-633, 1993. 11. ANDERSSON, A., BRATTSTROM, L., ISRAWLSSON B., ISAKSSON A. and HULTBERG B. The effect of excess daily methionineintake on plasma homocysteineafter a methionineloadingtest in humans.ClinChimActa 192,69-76, 1990. 12. MANSOO~ M.A., BERGMARK,C., SVARDAL,A.M., LONNING, P.E. and UELAND, P.M. Redox status and protein bindingof plasmahomocysteineand other aminothiolsin patients with early-onsetperipheralvasculardisease.ArteriosclThrombVase Biol 15, 232-240, 1995. 13. MANSOO~ M.A., SVARDAL, A.M. and UELAND, P.M. Determinationof the in vivo redox status of cysteine, cysteingIycine,homocysteineand glutathionein human plasma. Anal Biochem200,218-229, 1992. 14.NYGARD, O., REFSUM.H., UELAND, P.M., SVENSOLD, I., NORDREHAUG, J.E., GUNNARjK. and VOLLSET S.E. CoiTee consumption and plasma totaI homocysteine:The Hordaland HomocysteineStudy.AmerJClin Nutr 65, 136-143,1997. 15.WASOWICZ, W., NEVE, J. and PERETZ, A. Optimizedsteps in fluorirnetricdetermination of thiobarbituricacid-reactivesubstancein serum importanceof extraction pH and influenceof samplepreservationand storage.ClinChem39, 2522-2526,1993. 16.WALL, R.T., HARLm J.M., HARKE~ L.A. and STRIKER G.E. Homocyst(e)ine inducedendothelialcell injuryin vitro a modelfor the study of vascularinjury.Thromb Res J8, 113-121,1980.
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17.STARKEBAUM, G. And HARLAM, J.M. Endothelialcell injury due to cooper-catalyzed hydrogenperoxidegenerationfrom homocysteine.J ClinInvest 77, 1370-1376, 1986. 18. SCHONEICH, C., DILLINGE~ U., BRUCHAUSEN,F. and ASMUS, K.D. Oxidationof polyunsaturatedfatty acids and lipidsthrough thiyl and sulfonylradicals:reaction kinetics, and influenceof oxygenand structureof thiolradicals.ArchBiochemBiophys292, 456-467, 1992. 19. HIRANO, K., OGT., MIKI, M., YASUD& H., TAMAI, H., KAW~ N., and MINO, M. Homocysteineinducesiron-catalyzedlipidperoxidationof low-densitylipoprotein that is preventedby alpha-tocopherol.Free RadicalRes 21,267-276, 1994. 20.BLOM.H.J., ENGELEN, D.P.E., BOERS, G.H.J., STADHOUDERS, A.M., SENGERS, R.C.A., DE ABREU, R., TEPOELE-POTHOFF, M.T.W.B., and TRIJBELS, J.M.F. Lipid peroxidationin homocysteinaemia.J InherMetab 15,419-422, 1992. 21. BLOM, H.J., KLEINVELD, H.A., BOERS, G.H.J., DEMACKE~ P.M.N., HAKLEMMERS, H.L.M.,TE POELE-POTHOFF, M.T.W.B. and TRIJBELS, J.M.F. Lipid peroxidation and susceptibility of low-density lipoprotein to in vitro oxidation in hyperhmocysteinemia.Eur J ClinInvest25, 149-154,1995.
P
PII SO049-3848(97)O0145-X
BRIEF COMMUNICATION
HYPERHOMOCYSTEINEML4 FOLLOWINGOIUL METHIONINELOADIS ASSOCIATEDWITHINCREASEDLIPIDPEROXIDATION Teresa B. Domagal~ MagdalenaLiburaand AndrzejSzczeklik Departmentof Medicine,JagiellonianUniversitySchoolof Medicine,Cracow, Poland. (Received25 February1997by EditorC. Cierniewski;revisecVaccepted2June 1997)
Over the last years, homocysteinehas been recognized as an independent risk factor for atherosclerotic vascular disease (l). The known reasons for an increased level of plasma homocysteineare genetic defects of cystathionine~-synthase(2) and methylenetetrahydrofolate reductase (3). An important role in developmentof hyperhomocysteinemiais also played by deficiencyof folic acid, vitaminsBcand BIz (4). Hyperhomocysteinemiais definedwhen plasma homocysteineconcentrationexceeds 16vmol/1(5). In clinicalsettingit is usefidnot onlyto assess homocysteinelevel in fastingblood samples,but also to measureit tier methionineloadingtest (6). The mechanism through which hyperhomocysteinemiapromotes development of atherosclerosisare not clear. Homocysteineis toxic to vascular endothelium(7), it promotes thrombosis(8) and potentates the oxidationof low densitylipoprotein(LDL) in vitro (9). Ithas been suggested lately that oxidationof LDL may be one of the main factors involvedin initial developmentof atheroscleroticlesions(10). The aim of our study was to assess the relationshipbetween hyperhomocysteinemiaand lipid peroxidationin vivo .The experimentalmodelwas the oral methionineloadingtest. SUBJECTSAND METHODS Subjects. We studied46 subjects:25 maleand 21 femaleat the averageage of 41 years. In all of them oral methionineloading test was performed. Subjectswere dividedinto two groups: 1) with normal (n=21) and 2) pathological loading test (n=25). The levels of total homocysteinein plasma Key Words:Homocysteine,lipidperoxidation,methionineloadindtest, atherosclerosis. Corresponding author: Professor A.Szczeklik,Department of Medicine, School of Medicine, JagiellonianUniversity,Skawinska8, 31-066Cracow,Poland.
411
e
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vol. 87, No. 4
discriminatingthe two groups were: in fastingstate = 13.lwmol/1,at the 4* hour 37.8wnol/l and at the 6* hour 42.l~mol/1(X+ 2SD for our entiregroup). Theseresultsare.similarto reported by other autors (6, 11, 12). Fifteen subjectsfrom group with pathologicalloading test had clinical signsof atherosclerosis,verifiedby angiographyor ultrasonography(3 - coronary artery disease, 6- periphera)arterialocclusivediseaseand 6- atheroticlesionsof numerousvascularbeds). In all of them the first symptoms of atherosclerosisoccured before the age of 50 years; distinct deficiencyof folate was present in 7 persons, of vitaminBlz in 2, and of both vitaminBU and folate in 3. The subjects with elevated levels of cholesterol (>6.5mmol/1),triglicerydes (>1.8mmol/1),diabetes, immunologicaldisorders, chronic renai disease and carcinoma were excludedfrom the study. Methods.
The oral methionineloadingtest was performedusingmethionineat a dose of IOOmg/kgof body mass (6, 11). Venous blood were collectedto 5mlvacutainercontaining7.5rng EDTA (Becton DickinsonVacutainerSystemsEurope, England),the finalconcentrationof this anticoagulantwas 4.46mmol/l.Sampleswere taken beforeand at 4ti,6ti and 24* hour tier the loading.During the first 6 hours the subjectsremainedfasting. To 500u1 of plasma, 55u1 of 50% solution of sulfosalicylicacid (final concentration, 5’XO) containing500uM dithioerythritol(finalconcentration,50uM) was added. Precipitated protein was removedby centrifigation.Supernatantwas used for assessmentof fi-eehomocystehe. Plasma levels of total (tHcy) and flee (tltcy) homocysteinewere measured using High Petiormance Liquid Chromatographyaccording to method publishedby Mansaor et al (13). Instrumentationconsisted of: HPLC Hewlett Packard 1050 with autmw.mplerHewlett Packard 1100and HP Programmable Fluorescencedetector 1046A.Data were analysedby HP ChemStation (HP Vectra Xm). Sample of 25@ were injected into a 150x4.6mmcolumn packed with-3ym particles of Hypersil 120 ODS (Knauer,Germany).The temperaturewas 25°Cand the flow rate 1.5rnl/min.Elution solventA (lliter) was: IOmmoltetrabutylammoniumphosphate,2.5rnlglacial acetic acid dilutedwith distilledwater, pH adjustedto 3.8 with 2.OMNaOH. SolventB (1 liter) was: 200rnlacetonitrile,IOmmoltetrabutylammonium phosphate,2.5mlglacialacetic acid diluted with distilledwater, pH adjustedto 3.4 with 2.0 M NaOH; solventC was 75°/0acetonitrile.The elutionprofilewas as follows:O-13miR6-22°/oB;13.1-25min,22-40°/oB;25.1-30min,40-75°/0B; Afler each run, the column was washed for 5 min with solvent C to remove late-eluting fluorescentmaterial.The retentiontime for homocysteinewas 12.0 min. Sincethe concentration of the total homocysteine showed a marked positivelyskewed distribution, the results are presentedas geometricmean,as reportedotherby authors(14). In the same blood samples lipid peroxides were assessed by fluorimetricmethod (15) using LuminescenceSpectrometerLS 50B (Perkin-Elmer,USA),and expressed as thiobarbituricacid reactivesubstances(TBARS). To each studiedsamplebutylatedhydroxytoluene(BHT) at a final concentrationof 0.2mmol/lwas added to inhibitthe oxidativeprocess.In control experiments BHT at the concentrationused did not inteferewith TBARS estimations.Determinationswere performed only on fresh plasma, the results were expressedin nmol of malonylodialdehydehnl (MDA/ml)of plasmaand for statisticalanalysisas arithmeticmean* SD. All subjects had their plasma vit.Blz and folate levels tested using the isotopic method (Radioassaykit VitaminB~z[5’Co]/Folate[ 125Jl-Becton Dickinson). Statisticalanalyseswere performedusing the paired t-studenttest. Correlationswere calculated by Pearson test.
vol. 87,
4
LIPID PEROXIDATION
413
RESULTS In the fastingstate plasmalevelsof TBARSwere similarin subjectswith normaland pathological loading test: 0.68 W.23rtmol/mland 0.70Ml.25nmolhnl,respectively.Plasma homocysteine showed inversed relationship to both folate (r = - 0.49, p= 0.001) and tit Blz levels (- 0.33, p= 0.03). Followingoral methionineload, levelsof TBARS reached signifkantlyhigher values at the 4* (@O.001)and 6ti hour (p
t
tH [
[
[ 1 -
[
7
“
*\
.
-
\ / /
1
I
.
\
I
-
-
\
I
-
\
/ 0
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\
/ ‘
-
~
P
T
T
-..
MDA profilein plasma. — MDA in plasma total hornocysteinein plasma M: Subject$with normalmethkmheloadingtest Right: Subjectswith pathologicalmethionineloadingtest
L k SD
414
LIPID PEROXIDATION
vol.
87, No. 4
1,6
0
0 0
1,4
0
0 1.2
0°0 0 0
a
o
o
00
0 000
O? (DO
0
0
o
Ezl
o
0,4
0,2 o
40
120
80
160
200
Homocysteine [~mol/1]
FIG.2 Correlationbetween total homocysteineand MDAmeasured6 hourstier the loadingtest DISCUSSION Homocysteine,an independentrisk factor for atherothrombosis,might exert its ha.rmiideffects through a mechanisminvolvingoxidativedamage(16,17). Oxygen-derivedmoleculesgenerated during oxidation of homocysteine,could initiatelipid peroxidation,both at the endothelialcell surfaceand withinthe lipoproteinparticlesin plasma(18). Thisnotionis based on in vitro studies. Heinke et al (9), incubated LDL with increasingconcentrationsof homocysteineand assessed LDL peroxidationby TBARS. The degreeof lipidperoxidationdependedon the concentrationof added thiol and was maximalat concentrationof lmM ,whichexceedsseveral-foldplasmalevels of homocysteine.Similarobservationswere reported by Hirano et al. (19), who also showed the decrease in polyunsaturatedfatty acidsduringoxidationof LDL inducedby a homocysteine/iron mixture. However, until now evidence in vivo has been lacking. Measurement at fasting state performed by Blom and associates(20) showedno relationshipbetween plasma lipid peroxides, expressed as TBARS, and homocysteinelevels. Our results corroborate these findings. In a recent paper by the same group (21), fasting plasma MDA of hyperhomocysteinemic subjects was even lower then in a smrdl group of control;the latter, however, had unusually high MDA values (mean 5.0 +/- 2.0 nmolhnl). Using the diagnostic methionineloadingtest we created a simple experimentalmodel, which allowed to assess oxidativestress in vivo. We have demonstratedsignificantelevation of TBARS 4 and 6 hours followingmethionineloadingtest, which correlated with raised plasma concentrations of homocysteine. These results point to association between hyperhomocysteinemiaand increased lipid peroxidation, an association which might be of relevancefor developmentof atherosclerosis.
Vol.87, No.4
LIPID PEROXIDATION
415
REFERENCES 1. BOUSHEY, C.J., BERESFORD, S.A.A., OMENN, G.S. and MOTULSKY, A.G, A quantitative assessment of plasma homocysteineas a risk factor for vascular disease. JAMA 174,1049-1057,1995. 2. MUDD, S.H., LEVY H.L. and SKOSBYF. Disorders of transsulfhration.In: Z4e Metabolic andMolecukrBasesof InheritedDisease. SeriverC.R., BeaudetA.L., Sly W.S.,Vane D. (eds.), pp.1279-1327,McGraw-HillInc;New York (1995). 3. KANG, S.S., WONG P.W.K., BOCK H.G., HORWITZ, A. and GRIX, A. Intermediate hyperhomocysteinemiaresulting from compound heterozygosityof methylene-tetrahydrofolate reductasemutation.Am J Hum Genet 48, 546-551,1991. 4. KANG, S.S. and WONG, P.W.K. Genetic and nongenetic factors for moderate hyperhomocyst(e)inernia. Atherosclerosisf19, 135-138,1996. 5. MALINOW, M.R., KANG, S.S., TAYLORy L.M.,WONG, P.W.K., COULL, B. and IN~ T. Prevalence of hyperhomocyst(e)inemiain patients with peripheral arterial occlusive disease.Circulation79, 1180-1188,1989. 6. BOSTOM, A.G., JACQUES,P.F., NADEAU,M.R., WILLIAMS,R.R., ELLISON, R.C. and SELHUB J. Post-metbionineload hyperhomocysteinemiain persons with normal fasting total plasmahomocysteine:initialresults from [email protected], 147-151,1995. 7. HARKE~ L.A., ROSS, R., SLICHTE~ S.J. and SCOTT, C.R. Homocysteine-induced atherosclerosis:The role of endothelialcellinjuryand plateletresponsein its genesis.J Clin Invest 58,731-741, 1976. 8. REES, M.M. and RODGERS, G.M. Homocysteinemia:Associationof metabolic disorder with vasculardiseaseand thrombosis.ThrombRes 71,337-359, 1993. M., SUZUKI,L. and CHAIT, A. Oxidationof low density 9. HEINECKE, J.W., KAW~ lipoproteinby thiols. Superoxide-dependentand independent mechanism.,J Lipid Res 34, 345360, 1993. 10. WARE, J.A. and HEISTAD, D.D. Platelet endotheliuminteractions.New Engl J Med 328, 628-633, 1993. 11. ANDERSSON, A., BRATTSTROM, L., ISRAWLSSON B., ISAKSSON A. and HULTBERG B. The effect of excess daily methionineintake on plasma homocysteineafter a methionineloadingtest in humans.ClinChimActa 192,69-76, 1990. 12. MANSOO~ M.A., BERGMARK,C., SVARDAL,A.M., LONNING, P.E. and UELAND, P.M. Redox status and protein bindingof plasmahomocysteineand other aminothiolsin patients with early-onsetperipheralvasculardisease.ArteriosclThrombVase Biol 15, 232-240, 1995. 13. MANSOO~ M.A., SVARDAL, A.M. and UELAND, P.M. Determinationof the in vivo redox status of cysteine, cysteingIycine,homocysteineand glutathionein human plasma. Anal Biochem200,218-229, 1992. 14.NYGARD, O., REFSUM.H., UELAND, P.M., SVENSOLD, I., NORDREHAUG, J.E., GUNNARjK. and VOLLSET S.E. CoiTee consumption and plasma totaI homocysteine:The Hordaland HomocysteineStudy.AmerJClin Nutr 65, 136-143,1997. 15.WASOWICZ, W., NEVE, J. and PERETZ, A. Optimizedsteps in fluorirnetricdetermination of thiobarbituricacid-reactivesubstancein serum importanceof extraction pH and influenceof samplepreservationand storage.ClinChem39, 2522-2526,1993. 16.WALL, R.T., HARLm J.M., HARKE~ L.A. and STRIKER G.E. Homocyst(e)ine inducedendothelialcell injuryin vitro a modelfor the study of vascularinjury.Thromb Res J8, 113-121,1980.
416
LIPID PEROXIDATION
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17.STARKEBAUM, G. And HARLAM, J.M. Endothelialcell injury due to cooper-catalyzed hydrogenperoxidegenerationfrom homocysteine.J ClinInvest 77, 1370-1376, 1986. 18. SCHONEICH, C., DILLINGE~ U., BRUCHAUSEN,F. and ASMUS, K.D. Oxidationof polyunsaturatedfatty acids and lipidsthrough thiyl and sulfonylradicals:reaction kinetics, and influenceof oxygenand structureof thiolradicals.ArchBiochemBiophys292, 456-467, 1992. 19. HIRANO, K., OGT., MIKI, M., YASUD& H., TAMAI, H., KAW~ N., and MINO, M. Homocysteineinducesiron-catalyzedlipidperoxidationof low-densitylipoprotein that is preventedby alpha-tocopherol.Free RadicalRes 21,267-276, 1994. 20.BLOM.H.J., ENGELEN, D.P.E., BOERS, G.H.J., STADHOUDERS, A.M., SENGERS, R.C.A., DE ABREU, R., TEPOELE-POTHOFF, M.T.W.B., and TRIJBELS, J.M.F. Lipid peroxidationin homocysteinaemia.J InherMetab 15,419-422, 1992. 21. BLOM, H.J., KLEINVELD, H.A., BOERS, G.H.J., DEMACKE~ P.M.N., HAKLEMMERS, H.L.M.,TE POELE-POTHOFF, M.T.W.B. and TRIJBELS, J.M.F. Lipid peroxidation and susceptibility of low-density lipoprotein to in vitro oxidation in hyperhmocysteinemia.Eur J ClinInvest25, 149-154,1995.