d
`
`
`
`,
`American Heart
`Associations
`
`Arteriosclerosis,
`Thrombosis’ and
`Vascular Biology
`
`
`
`
`
`Vitamin Supplementation Reduces Blood Homocysteine Levels : A Controlled
`Trial in Patients With Venous Thrombosis and Healthy Volunteers
`Martin den Heijer, Ingeborg A. Brouwer, Gerard M. J. Bos, Henk J. Blom, Nathalie M.
`J. van der Put, Anja P. Spaans, Frits R. Rosendaal, Chris M. G. Thomas, Hans L.
`Haak, Pierre W. Wijermans and Wim B. J. Gerrits
`
`Arterioscler Thromb Vasc Biol 1998, l8:356-361
`doi: 10.1161/0l.ATV.l8.3.356
`Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association.
`7272 Greenville Avenue, Dallas, TX 725 I4
`Copyright O 1998 American Heart Association. All rights reserved. Print ISSN: 1079-5642. Online
`ISSN: 1524-4636
`
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`
`Vitamin Supplementation Reduces
`Blood Homocysteine Levels
`A Controlled Trial in Patients With Venous Thrombosis
`
`and Healthy Volunteers
`
`Martin den l-leijer, Ingeborg A. Brouwer, Gerard M._[. Bos, I-Ienk _]. Blom,
`Nathalie M._]. van der Put, Anja P. Spaans, Frits R. Rosendaal, Chris M.G. Thomas, Hans L. l-laak,
`Pierre W. Wijennans, Wirn B._I. Gerrits
`
`/lbstract—Hyperhomocysteinemia is a risk factor for atherosclerosis and thrombosis and is inversely related to plasma folate
`and vitamin B12 levels. We assessed the cfi’ects of vitamin supplementation on plasma homocysteine levels in 89 patients
`with a history of recurrent venous thrombosis and 227 healthy volunteers. Patient: and hyperhomocysteinemic
`(homocysteine level >16 ltmol/L) volunteers were randomized to placebo or high-dose multivitamin supplements
`containing 5 mg folic acid, 0.4 mg hydroxycobalamin, and 50 mg pyridoxine. A subgroup of volunteers without
`hyperhomocysteinemia was also randomized into three additional regimens of 5 mg folic acid, 0.5 mg folic acid. or 0.4
`mg hydroxycobalamin. Before and after the intervention period, blood samples were taken for measurements of
`homocysteine. folate, cobalarnin, and pyridoxal-5'-phosphate levels. Supplementation with high-dose multivitamin
`preparations nomtalized plasma homocysteine levek (S 16 umol/L) in 26 of30 individuals compared with 7 of 30 in the
`placebo group. Also in normohomocysteinemic subjecs, multivitamin supplementation strongly reduced homocysteine
`levels (median reduction, 30%; range, -22% to 55%). In this subgroup the efiect of folic acid alone was similar to that of
`multivitamin: median reduction, 26%; range. -2% to 52% for 5 mg folic acid and 25%; range. -54% to 40% for 0.5 mg
`folic acid. Cobalamin supplementation had only a slight eH'ect on homocysteine lowering (median reduction, 10%; range,
`-21% to 41%). Our study shows that combined vitamin supplementation reduces homocysteine levels effectively in
`patients with venous thrombosis and in healthy volunteers, either with or without hypcrhomocysteinemia. Even
`supplementation with 0.5 mg offolic acid led to a substantial reduction ofblood homocysteine levels. (Arteriosder Thromb
`Vasc Biol. 1998;18:356-361.)
`
`Key Words: homocysteine I vitamin supplementation I venous thrombosis I folate I MTHFR
`
`ubjects with hyperhomocysteinernia have a twofold to three-
`fold increase in risk of developing cardiovuculat disease or
`venous thrombosis.” Reduction of plasma homocysteine levels
`by vitamins may therefore be ofmajor clinical importance. Several
`studies have investigated the homocysteine-lowering properties of
`pyridoxine (vitamin B6), hydroxycobalarnin (vitamin B12), or
`folic acid alone or in combination.““ However, some of these
`studies were not placebo controlled. and therefore, they cannot
`the extent to which the observed efects were due to
`regression to the mean. Other studies were restricted In hyper-
`homocysteinemic subjects, healthy volunteers, or certain sub-
`groups '(eg, elderly people, men, women, or patients with
`cardiovascular disease or renal failure).
`'I‘heaimofourstudywastostimateandcornparethehomocys-
`oeine-lowering e&'cct of vitamin supplementation in patients with
`
`hyperhomocysteinerrria-related disuse and in healthy volunteers with
`or without elevated homocysteine levek 'l1'ierefore, we studied the
`effects ofan 8-week daily combined administration 05 mg folic acid,
`0.4 mg hydroxycobalamin. and 50 mg pyridoxine versus placebo on
`blood homocysteine levels in patients with a history of recurrent
`venous thrombosis and healthy volunteers. We also compared this
`high-dose multivitamin’'
`regimen with singe-vitamin tegtmeru' of
`fblare or hydroxycobalamin no asess which vitamin at which dose
`was the most eflective in lowering homocysteine levels. For twons
`ofsample size, we restricted club "drug- and dose-finding study" to
`volunteers with nomrohomocysteinernia. Finally, we analyzed the
`influence of homocysteine and vitamin concentration and of
`the 677C-IT mutation in the methylerleteuahydrofolate reductase
`(M11-IFR) gene on the homocysteine-lowering eflect ofmultivita-
`min supplementation.
`
`
`
`Received August 5. I997: revision accepted October 29, 1997.
`From the Departments ofHernatology (M. den H., l.A.B., H.L.H., P.\V.W., V/.B.].G.) and Clinical Chemistry (A.P.$.), Leyenburg Hospital. The Hague;
`the Department of Hematology, Daniel den Hoed Clinic, Rotterdam (G.M.].B.); the Department of Pediatrics. Laboratory of Pediatrics and Neurology
`(H.].B., N.M._]. van der P.). and the Department of Obstetrics and Gynecology. Laboratory of Endocrinology and Reproduction (C.M.G.T.). Univerlity
`Hospital Nijmegen, Nijmegen; and Departments ofClinical Epidemiology and Hematology (F.R.R.), University Hospital Leiden, Leiden. the Netherlands.
`Correspondence to Martin den Heijcr. MD. PhD. Depamnent of General Internal Medicine. University Hospital, Nijmegen, PO Box 9101, 6500 HB
`Nijmegen. the Netherlands.
`E-mail rn.denheijer@aig.azn.nl
`0 I998 American Heart Association, lnc.
`
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`
`den Herjer el al
`
`357
`
`Methods
`The study group was recruited from subjects who had participated in
`a previous case-control study.‘ In the current study. 92 of the 185
`patients with recurrent venous thrombosis and 230 of the 500
`volunteers from the general population agreed to participate in an
`8-week daily Vitamin supplementation trial. Six participants (three
`from each group) withdrew during the study. All participants were
`asked not to take ("self-prescribed") vitamin supplements for at least 2
`months prior to the start of the current study.
`Both patients and hyperhomocysteinemie healthy volunteers were
`randomized to either a placebo or a high-dose multivitamin schedule.
`Each multivitamin tablet contained 5 mg folic acid. 0.4 mg hydroxy-
`cobalamin. and 50 mg pyridoxine. (Randomization of the volunteers
`had been stratified by homocysteine level in a previous study‘ [cutoff
`point, I6 itmol/L].). Volunteers with previous homocysteine levels
`516 ll-mol/L were randomized to placebo, multivitamin, or single-
`vitamin regimens (5 mg folic acid, 0.5 mg folic acid. or 0.4 mg
`hydroxycobalamin). These three additional subregimens were re-
`stricted to norrnocysteinemic volunteer group because the other
`subgroups were too small to allow randomization into more than two
`schedules. Randomization was perfonncd by using the last digit of
`each pau'ent's number. So all hyperhomocysteinemic subjects and
`normocysteinemic patients with recurrent venous thrombosis with an
`odd or even number were assigned to the multivitamin or placebo
`youp. respectively. In the normohomocysteinemic healthy volun-
`teers. subjeco with a last digit of0 or I were assigned to placebo; 2 and
`3. to multivitamins; 4 and 5. to 5 mg folic acid;. 6 and 7. to 0.5 mg
`folic acid; and 8 and 9, to 0.4 mg vitamin B12. All subjects were asked
`to take 1 tablet per day for 56 days. The trial was kept double-blind.
`Before and after the supplementation period, blood was collected after
`an overnight fast for homocysteine. folate. cobalamin, and pyridoxal-
`5'-phosplnte measurements.
`For homocysteine and vitamin measurements, blood samples were
`taken from the antecubital vein and collected into EDTA-containing
`tubes. Whole blood was stored at -70°C for pyridoxal-5'-phosphate
`determination. For the other detenninarions. EDTA-treated samples
`were immediately placed on ice and centrifuged within half an hour at
`2000g for 10 minutes. The plzma was separated and stored at -20°C.
`The EDTA-treated samples for folate and cobalamin measurements
`were stored at -70°C and analyzed within 2 months. Folate and
`cobalamin concentrations were measured with a Dualcount SPNB
`(solid phase no boil)
`radioassay ltit (Diagnostic Products Corp).
`Detemiination of pyridoxal-5'-phosphate was performed by high-
`performance liquid chromatography techniques according to Schrijver
`et al" with some modifications." Total homocysteine concentrations
`were measured according to the method described by Fiskerstl-and er
`al“ with some modifications." Mutation analysis was carried out by
`means ofpolymelase-chain reaction and restriction enzyme digestion
`as described elsewhere."
`In the analysis we first looked at nomialiution rates of homocys-
`teine levels after multivitamin or placebo supplementation. Therefore.
`we calculated the fraction of hyperhomocysteinemic subjecu (homo-
`cysteine >16 umol/L in the present study) who became normoho-
`mocysteinemic (homocysteine S16 umol/L) after the supplementa-
`tion period. The cutofl' point was a rounded value based on the 80th
`percentile in our previous study.‘ Second, we calculated the percent
`homocysteine reduction {or each subject and compared the median
`reduction in the di£'erent vitamin supplementation groups with
`respect to the corresponding placebo group. To compare the homo-
`cysteine-lowering efl'ects in patients and volunteers, we later su-atified
`the patients into a hyperhomocysteinemic and a normohomocysteine-
`mic group according to their homocysteine levels as determined in our
`previous study (cutofpoint. 16 pmol/L). Finally, we studied deter-
`minants of the homocysteine-lowering efl'cct of multivitamin supple-
`mentation by calculating the median reduction in men and women: in
`subject: under or above 53 years of age (median age of hmlthy
`volunteers); and for several strata (tertiles) of initial homocysteine.
`folate. cobalamin. and pyridoxal-5'-phosphate concentrations. as well
`as for the three difierenr MTHFR-genotypes (677C-OT). To evaluate
`the difference in median reduction. we used the Mann-Wlumey U
`test for unpaired cases (SPSS software). All participants gave their
`
`
`
`‘o
`
`so
`so
`to
`Ileyunuutahruirunrlht
`
`do
`
`°o
`
`3°
`3°
`'0
`I-It.7(wIdI|.)ha!oIIlItarvuIIn
`
`40
`
`Total plasma homocyslelne (tHcy) levels beiore (x axis) and alter
`(y axis) 8 weeks of daily multivitamin or placebo supplementa-
`tion In patients with a history of recurrent venous thrombosis (A.
`placebo group; B. multivitamin group) and in henllhy volunteers
`(C, placebo group: D. mullivllamln group). The multivitamin tab-
`lets contained 5 mg folio acid, 0.4 mg hydroxycobalarnin. and
`50 mg pylidoxine.
`
`infomied. written consent. and the study protocol was approved by
`the medical ethics committee of Leyenburg Hospital.
`
`Results
`
`The median age of the patient group was 62 years (range. 31 to
`89) and of the volunteers. 53 years (range. 23 I0 82). The
`median homocysteine concentration of the patient group was
`13.6 umol/L (range, 7.2 to 69) and of the volunteer group
`12.8 umol/L (range, 4.7 to 49.8).
`The Figure shows the homocysteine concentrations before
`and after intervention for the placebo and high-dose multivi-
`tamin groups of both patients and volunteers. In the.multivi-
`tarnin group, 11 of 14 hyperhomocysteinemic patients with
`thrombosis had a normalized value after intervention (curofl'
`
`point, 16 pmol/L) compared with only 4 out 10 in the placebo
`group. A very similar observation was made in the healthy
`volunteers.
`
`In the multivitamin group, 15 of 16 hyperhomocysteinemic
`subjects had a nonnalized value (cutofl' point. 16 ).tmol/L)
`compared with only 3 of 20 in the placebo group. So for all
`hyperhomocysteinemic individuals together, 26 of 30 subjects
`had normalized homocysteine levels (<16 nmol/L) after
`supplementation with multivitamins compared with only 7 of
`30 in the placebo group.
`Table 1 shows that there was no clear difference between
`
`patients with recurrent venous dirombosis and healthy volun-
`teers with respect to their homocysteine-lowering response
`due to multivitamin supplementation. ln both groups there
`was also a substantial effect in the norrnohomocysteinemic
`subjects.
`
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`
`358
`
`Vitamin Supplementation and Homocysteine
`
`llomocysteine~l.owerlng Ellecl ol Multivitamin or Placebo Supplementation in Patients with
`TABLE 1.
`Recurrent Venous Tlrromllls and in Healthy Volunteers
`
`Median lllcy Alter
`Median lllcy Ielore
`Intervention. umol/I.
`lnlervenllon. urnolll
`Median Badoellon.
`
`study subiecls
`n
`(nanoe)
`(flange)
`%(l1anoe)
`Falienrs Vlllll recurrent venous thrombosis
`89
`
`Homocyslelne >16 y.moVI.'
`Placebo
`Mullivlramlni
`l-lotnotysleine 516 urnovlf
`Placebo
`Mullivitanuni
`Healthy volunteers
`llomocysteine >18 umollL'
`Placebo
`Multivitamin?
`Homooystelne 516 urnolllf
`Placebo
`Mullivilaminf
`single-vilamln reglmen
`
`34
`15
`19
`55
`28
`27
`227
`50
`27
`23
`177
`36
`34
`107
`
`15.8 (10.6-31.5)
`18.5 (10.7-69.0)
`
`14.8 (125-263)
`10.7 (6.8-17.5)
`
`-3 (-30-27)
`35 (4-83):
`
`12.8 (01-175)
`11.8 (72-481)
`
`122 (81-484)
`8.8 (4.9-19.3)
`
`-1 (-193-45)
`20 (-38-60):
`
`18.0 (99-498)
`16.8 (11.0-37.5)
`
`11.5 (6.4-17.8)
`11.8 (7.1-10.3)
`
`17.4 (9.2-25.8)
`10.7 (7.0-21.4)
`
`11.4 (7.0—21.4)
`8.5 (5.5-11.5)
`
`0 (-2743)
`36 (-46-70):
`
`3 (-72-61)
`30 (—22—55)t
`
`indicates total plasma homocystelne. The homocystelne-lowering ellect
`ll-Icy,
`homooystelne concentration alter intervention.
`‘stratllled on homocyslelne levels in the previous study.
`tconlainlng 5 mg lolio acid. 0.4 mg hydroxyoobalamln. and 50 mg pyridoxine.
`tP<.0o1 compared with the corresponding placebo group.
`
`is expressed as the median oercent reduction In
`
`in Table 2 we compared the homocysteine-lowering efi'ect
`of several single-viurnin regimens in volunteers who were
`normohomocysteinernic in a previous study. From these data
`we may conclude that the efi'ect of 5 mg folic acid, even a low
`dose of 0.5 mg.
`is nearly as efl'ective as the multivitamin
`regimen. In contrast, vitamin B12 only slightly decreased the
`homocysceine concentration.
`In Table 3 we analyzed the effects of age and sex with
`respect to the homocysreine-lowering etfect of multivita-
`min supplementation. For reasons of homogeneity, we
`restricted this analysis to volunteers who had been random-
`ized into either the placebo or the multivitamin group
`(n= 120). We found a similar homocysteine-lowering effect
`in men and women and in subjects under and above 53 years
`of age.
`
`In Table 4 we stratified the homoeysteine—lowering eflect
`on tertiles of initial homocysteine,
`folate, cobalarnin. and
`pyridoxal-5'-phosphate levels. We found a stronger homocys-
`teine-lowering efi'ect in subjects with high initial homocys-
`teine levels. However, even in subjects with an initial homo-
`cysteine level of <l1.8 pmol/L, we still found a median
`reduction of 21% (range. -22 to 41%). An inverse eflect was
`seen with respect to initial vitamin concentrations. The ho-
`mocysteine-lowering efl'ect was strongest in subjects with low
`folate, cobalamin, or pyridoxal-5'-phosphate concentrations.
`Six of the 92 patients with recurrent venous thrombosis
`were homozygous for the 677C->1‘ mutation versus 22 of the
`230 control subjects (odds ratio, 0.7; 95% confidence interval,
`0.3 to 1.7). In Table 4 we also show that the homocysteine-
`lowering efi'ect of multivitamin supplementation was not
`
`TABLE 2. Homosystoine-towering Elieot cl several vitamin Regimens or Placebo In
`llormollomooystelnemlc Healthy Volunteers
`
`Median ltley Altar
`Median lllcy flelota
`lnletvenllon. pmolll.
`Intervention. pmol/I.
`Median Reduction.
`
`Regimens
`n
`(Range)
`(Range)
`so (flange)
`Placebo
`38
`11.5 (8.4-17.8)
`11.4 (7.0-18.0)
`3 (- 72-81)
`MlllllVll3lTlllI'
`34
`11.8 (7.1-19.3)
`85(5.5-11.5)
`30(—22—55)1
`Folic acid 5 mg
`35
`11.8 (7.0-22.1)
`8.7 (59-118)
`26 (-2-5211
`Folic acid 0.5 mg
`38
`12.2 (4.7-22.3)
`10.0 (28-188)
`25 (-54-40)1
`
`Hydroxycobalamin 0.4 mg 10 (-21-41): 38 12.6 (8.4-18.4) 11.0 (6.7-15.9)
`
`
`
`lllcy Indicates total nlasma homocyslelne. The nomocysleine-lowering alien! is expressed as the median pertant reurcllon In homocystelne
`concenttallon alter intervention.
`‘containing 5 mg tollc acid. 0.4 mg hydrcxycobalamln. and 50 mg oyridoxlne.
`fP<.001 compaed with he placebo group.
`tP=.14.
`
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`

`
`den I-Ietjer et al
`
`359
`
`ttomocystelne-Lowering Eitect ol Multivitamin or Placebo supplementation in
`TABLE 3.
`Men and women and in Subleets <53 or 253 Years Old
`
`Median titty Alter
`Median titty some
`Median Reduction.
`Intervention. uflllllll.
`Intervention. pinoill
`
`
`
`
`Men
`
` Study Submit 11 (Range) (Range) % (Range)
`
`Plaeebo
`Multivitamin‘
`women
`
`24
`26
`
`14.8 (7.4-49.8)
`14.0 (72-375)
`
`13.2 (7.0-25.3)
`10.1 (16-21 .4)
`
`12 (-69-61)
`33 (-46-70)
`
`39
`31
`
`27
`23
`
`12.4 (6.4-213)
`12.1 (7.1-3413)
`
`12.0 (64-493)
`13.1 (7.1-3413)
`
`12.7 (7.0—25.8)
`e.1(5.5-15.3)
`
`10.9 (7.0—25.8)
`8.3 (5.5—21.4)
`
`-1 (-72-25)
`31 (-22-64)
`
`5 (—72—61)
`31 (—46—64)
`
`Plaeebo
`Multivitamin‘
`Volunteers <53 years
`Placebo
`Multivitamin’
`Volunteers 253 years
`-1 (-69-19)
`13.9 19.5-25.3)
`14.7 (7.4-25.1)
`36
`Placebo
`32 (3-70)
`9.5 (6.3-16.8)
`13.8 (95-375)
`34
`MuItivIamin'
`it-icy indiutts total plasma homocysteine. The homocysteine-towering ellect is expressed as the median percent reduction
`in homocysteine concentration alter intervention.
`‘containing 5 mo tolic acid. 0.4 mg llydroxycnbalamin. and 50 mo uyriiioxine.
`
`impaired in subjects homozygous for the 677C->T mutation
`and in fact. might even be stronger.
`
`Discussion
`
`We investigated the efl’ect of multivitamin supplementation on
`homocysteine levels in patients with recurrent venous throm-
`bosis and healthy volunteers from the general population. We
`found that combined supplementation with folic acid, hy-
`droxycobalamin, and pyridoxine effectively reduced and nor-
`malized homocysteine levels in patients widl recurrent venous
`thrombosis as well as in healthy volunteers. For a subgroup of
`nor-mohomocysteinemic volunteers, folic acid at a dose of 5
`mg or even 0.5 mg seemed to be almost as efiective as the
`high-dose multivitamin supplementation. Oral cobalamin sup-
`plementation had only a moderate efi'ect on homocysteine
`levels.
`
`In 1985 Brattstriim et all‘ reported a substantial homocys-
`teine reduction in 15 volunteers who received 5 mg folic acid
`per day for 4 weeks. Wilcken et al7 reported a homocysteine-
`lowering effect of folic acid supplementation in patients with
`chronic renal insuficiency. Franken et al' and van den Berg et
`al’ reported significant reductions in postrnethionine-loading
`homocysteine concentrations with vitamin B6 folic acid, or a
`combination of both in patients with vascular disease. Al-
`though these studies were performed in large groups of
`patients. they were not placebo controlled and were restricted
`to hyperhomocysteinemic subjects, which characteristics make
`them rather sensitive to regression to the mean. Ubbink et al”
`studied the effects of 1 mg folic acid. 0.4 mg cyanocobalamin.
`and 12.2 mg pyridoxa] HCI alone or in combination in a
`placebo-controlled study in subjects with hyperhomocysteine-
`rnia. High-dose multivitamin administration resulted in a
`49.8% reduction of the mean homocysteine level. Naurath et
`al" studied the effect ofintramuscular vitamin supplementation
`with folate, vitamin B6, and vitamin B12 in elderly subjects
`with blood vitamin concentrations in the normal range.
`
`In our study we were able to compare the effects in patients
`with homocysteine-related disease (venous thrombosis) with
`those in healthy volunteers. These effects were quite similar.
`We also found about the same e&'ects in men compared with
`women and in subjects under and above 53 years of age. The
`strongest homocysteine-lowering effect of vitamin supplemen-
`tation was seen in subjects with high initial homocysteine
`and/or low initial vitamin concentrations. However, we also
`observed a moderate reduction in homocysteine levels in
`subjects with homocysteine and vitamin levels within the
`normal range. This observation raises the question of "normal"
`homocysteine and vitamin levels. Our definition of hyperbo-
`mocystinemia at the 80th percendle of the distribution in the
`general population is arbitrary. Other suggested definitions are
`based on mean concentrations in populations without cardio-
`vascular disease," at the flat plateau of the homocysteine-folate
`plot." We think that the best definition should be based on
`clinical intervention studies: the lowest concentration at which
`
`vitamin supplementation reduces the risk of vascular disease.
`However, data on clinical studies are not yet available.
`In a subgroup of normohomocysteinemic volunteers. we
`found approximately the same homocysteine-lowering effect
`of folic acid at a dose of 0.5 mg as with a dose of 5 mg. This
`means that considerably low doses of folic acid supplementa-
`tion are efl'ective in lowering homocysteine levels. Further
`studies are needed to see whether doses lower than 0.5 mg may
`also be effective.
`
`Although folic acid supplementation seems to be the cor-
`nerstone in the treatment of hyperhomocysteinemia, there are
`some reasons for adding cobalamin and pyridoxine. First. this
`combination may have a stronger efl'ect in subjects with low
`cobalamin or pyridoxine levels. Second, folatc administration
`alone might mask vitamin B12 deficiency. Addition of cobal-
`amin in suflicient dose prevents the complications of vitamin
`B12 deficiency, such as subacute combined degeneration ofthe
`spinal cord. even in the case of pernicious anemia." We did
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`360
`
`Vitamin Supplementation and I-lomocysteine
`
`TABLE 4. Homocyslelne-lowulnn Eflact of Multivitamin or Placebo Supplemenlallon
`Stratified Ovet Taniles 01 Itomocystelnu. Folale. cohalamin, and Pyrldoxal-5'-Phosphate
`
`Levels In 120 Healthy Volunleels
`
`Median may A1131
`Modlan may Baton
`Median Reduction,
`lnlomnliun, umolll
`Immemlon. pmolll
`
` 11 (Range) (flangu) 96 (Range)
`
`
`Placebo
`
`I-lomocystelne
`<11.8 umovL
`11.8-15.7 )um1/L
`215.7 ,l.moIIL
`Folale
`
`<10.4 nmo|IL
`10.4-14.3 nmol/L
`214.3 nrnoVL
`vilamin B12
`
`<207 pmoIIL
`207-296 pmom.
`2296 prnolll.
`vitamin B6
`
`<37 nmol/L
`37-47 nmolll.
`>47 nmol/L
`MTHFR 677 c-.1
`
`+/+
`+/—
`-I-
`MuIlIvlIam1n'
`
`Homocysteine
`<11.8 pIfl0|/L
`11.8-15.7 umol/L
`215.7 umolll.
`Folate
`
`<10.4 nmolIL
`1.04-14.3 nmollt
`214.3 nmol/L
`Vitamin B12
`
`<207 pmovt
`207-296 pmol/L
`2296 pmolit
`wlamin 86
`
`<37 nmol/L
`37-47 nmol/L
`>47 nmol/L
`M1111-‘R 677c—>T
`
`21
`20
`22
`
`19
`23
`21
`
`20
`23
`20
`
`18
`21
`24
`
`5
`25
`33
`
`18
`21
`18
`
`19
`18
`20
`
`19
`16
`20
`
`14
`24
`19
`
`9.916.-1-11.6)
`14.1 (11.8-15.6)
`189 (157-498)
`
`16.6 (9.3—49.8)
`12.5 (6.4—25.1)
`12.0 (7.4-25.1)
`
`14.0 (9.1-49.8)
`13.6 (7.4-21.8)
`14.4 16.4-25.1)
`
`11.9 (6.4-21.8)
`14.6 (8.4—24.6)
`15.0 (7.4-49.8)
`
`164 (90-498)
`14.6 (93-25))
`12.4 (6.4-25.1)
`
`103 (7.1-11.7)
`132 (118-155)
`18.5 (15.7-37.5)
`
`.
`
`15.7 (9.9-37.5)
`122(7.2—18.8)
`12.4 (7.1-24.4)
`
`14.7 (9.9-37.5)
`12.7 (7.6-24.4)
`12.0(7.1-18.9)
`
`14.7 (7.1-34.9)
`13.3 (8.6-37.5)
`12.3 (7.2-24.4)
`
`10.7 (70-127)
`13.2 (7.9-15.4)
`195 (70-258)
`
`162 (7.0-25.8)
`127 (6.8-25.3)
`12.5 (78-221)
`
`12.9 (92-253)
`13.8 (7.8—25.8)
`12.5 (70-232)
`
`115(8.4-233)
`12.7 (7.0-25.8)
`13.6 (7.0-25.3)
`
`13.3 (7.0-23.2)
`13.7 (7.0-25.8)
`126 (18-21)
`
`78(5.5-10.7)
`900.0-21.4)
`110(7.0-25.8)
`
`10.2 (5.8—21.4)
`8.7 (6.3-11.8)
`8.9 (55-138)
`
`10.3 (5.8—21.4)
`8.6 (6.3-16.8)
`8.6(5.5-138)
`
`8.9 (6.3—13.7)
`8.5 (55-169)
`9.3 (7.8-21.4)
`
`-3 (—72—25)
`9 (-19-35)
`-1 (—27—61)
`
`0 ( —21—61)
`-1 (-72-21)
`5 (-69-35)
`
`-6 (-25-53)
`-2 (-69-35)
`9 (-72-61)
`
`-2 (—72—18)
`0 (-21-25)
`9 (-69-61)
`
`12 (2-61)
`0 (-27—25)
`-1 (-72-35)
`
`21 (-22-41)
`31 (-46-47)
`44 (13-70)
`
`39 (-46-70)
`27 (-8-51)
`27 (-22-44)
`
`34 (-46-70)
`31 (-22-55)
`26 (-8-57)
`
`40 (0-74)
`34 (7-70)
`20 (-46-51)
`
`49 (3-70)
`9.7 (7.0-13.7)
`16.4 (10.9-37.5)
`8
`+/+
`25 (—46-64)
`8.6 (6.3-21.4)
`12.6 (7.1-34.8)
`21
`+I-
`
`-I— 31 (7-57) 28 13.1 (8.8-24.4) 8.6 (5.5—16.8)
`
`
`
`may indlmles lal plasma homocyslainsz MIHFH. mallylenetetranydrololate ledudase. The homocyslelne-lowerlno eflecl
`is exptessed as the percent reduaion in homocystelne oanoematlon alter Intervamlon.
`‘comalnlng 5 my tone add. 0.4 mg hynlroxycobalamin, and 50 mo pyrldoxina.
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`

`
`den I-Ierjer et al
`
`361
`
`not study doses higher than 50 mg pyridoxine because higher
`doses may cause sensory neuropathy.“""
`Recently, we found the 677C-tT mutation in the
`MTHFR gene"“n"’ This mutation is associated with elevated
`homocysteine levels, but it is unclear whether this mutation is
`also associated with arterial vascular disease.“ In this study the
`prevalence of the 6TlC->T mutation in the recurrent venous
`thrombosis group did not really differ from that in the control
`group. This finding is in accordance with the results in a study
`of first-time venous thrombosis.” We found that the homo-
`cysteine-lowering elfcct
`in subjects with this mutation en
`might be even stronger than in those without this mutation.
`This finding is in accordance with the study of Malinow et al,“
`These results suggest that the efiect of a mutated MTHFR
`might be "compensated" by a higher folate intake.
`_ In conclusion. our study shows that combined supplemen-
`tation with folic acid, cobalamin, and pyridoxine reduces
`homocysteine levels by ~30% compared with placebo within
`8 weeks in patients with recurrent venous thrombosis as well as
`in healthy volunteers. Whether the reduction in homocysteine
`levels by vitamin supplementation will
`lead prevention of
`arterial vascular disease and venous thrombosis is a major task
`for further clinical rcsearch.”'"
`
`Acknowledgments
`This work was supported by grants from the Prevention Fund (to
`G.M._].B.) (28 -2263-1). We thank G.E.Th. Ferguson, pharmacist. for
`preparing the vitamin and placebo tablets and C. Postma. Y. Lenstra.
`M.T.W.B te Poele-Pothofll A. de Graaf-Hess, D. van Oppenraaij-
`Emmerseel, M.F.G. Segers, L.M.F. Geelen. and J. Beunk for their
`excellent laboratory assistance. We wish thank R. Clarke, MD,
`MRCP, for his valuable comments of a previous version of the
`manuscript. Finally, we thank all participants for their kind contribu-
`tion the study.
`
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