Blood Homocysteine Levels
A Controlled Trial in Patients With Venous Thrombosis
and Healthy Volunteers
Martin den Heijer, Ingeborg A Brouwer, Gerard MJ Bös, Henk J Blom,
Nathalie MJ van der Put, Anja P Spaans, Frits R Rosendaal, Chns M G Thomas, Hans L Haak,
Pierre W Wyermans, Wim BJ Gemts
Abstract—Hyperhomocystememia is a nsk factor for atherosclerosis and thrombosis and is mversely related to plasma folate
and Vitamin B12 levels We assessed the effects of Vitamin Supplementation on plasma homocysteme levels m 89 patients with a history of recurrent venous thrombosis and 227 healthy volunteers Patients and hyperhomocysteinemic (homocysteme level >16 ;u.mol/L) volunteers were randomized to placebo or high-dose multiVitamin Supplements contaming 5 mg folic acid, 0 4 mg hydroxycobalamm, and 50 mg pyndoxme A subgroup of volunteers without hyperhomocystememia was also randomized into three additional regimens of 5 mg folic acid, 0 5 mg folic acid, or 0 4 mg hydroxycobalamm Before and after the Intervention penod, blood samples were taken for measurements of homocysteme, folate, cobalamm, and pyndoxal-5'-phosphate levels Supplementation with high-dose multivitamm preparations normalized plasma homocysteme levels (^16 μπιοΙ/L) m 26 of 30 mdividuals compared with 7 of 30 in the placebo group Also m normohomocystememic subjects, multivitamin Supplementation strongly reduced homocysteme levels (median reduction, 30%, ränge, —22% to 55%) In this subgroup the efFect of folic acid alone was sirmlar to that of multivitamin median reduction, 26%, ränge, —2% to 52% for 5 mg folic acid and 25%, ränge, —54% to 40% for 0 5 mg folic acid Cobalamm Supplementation had only a shght efFect on homocysteme lowenng (median reduction, 10%, ränge, —21% to 41%) Our study shows that combmed Vitamin Supplementation reduces homocysteme levels effectively in patients with venous thrombosis and m healthy volunteers, either with or without hyperhomocystememia Even Supplementation with 0 5 mg of folic acid led to a substantial reduction of blood homocysteme levels (Artertoscler Thromb
Vase Biol. 1998;18:356-361.)
Key Words: homocysteme · Vitamin Supplementation · venous thrombosis · folate · MTHFR
S
ubjects with hyperhomocystememia have a twofold to three-fold increase in nsk of developmg cardiovascular disease or venous thrombosis' 5 Reduction of plasma homocysteme levelsby Vitamins may therefore be of major climcal importance Several studies have investigated the homocysteme-lowenng properties of pyndoxine (vitamin B6), hydroxycobalamm (vitamin B12), or folic acid alone or in combinataon6 " However, some of diese
studies were not placebo controlled, and therefore, they cannot distmguish the extent to which the observed eiFects were due to regression to the mean Other studies were restncted to hyper-homocystememic subjects, healthy volunteers, or certam sub-groups (eg, elderly people, men, women, or patients with cardiovascular disease or renal fadure)
The aim of our study was to estimate and compare the homocys-teine-lowenng effect of vitamin Supplementation in patients with
hyperhomocysteinerma-related disease and in healthy volunteers with or without elevated homocysteme levels Therefore, we studied the eflects of an 8-week dady combmed admirustration of 5 mg folic acid 0 4 mg hydroxycobalamm, and 50 mg pyndoxine versus placebo on blood homocysteme levels in patients with a history of recurrent venous thrombosis and healthy volunteers We also compared this high-dose multivitamin regimen with single-vitarmn regimens of folate or hydroxycobalamm to assess which vitamin at which dose was the most effecüve in lowenng homocysteme levels For reasons of sample size, we restncted this "drug- and dose-finding study" to volunteers with normohomocysteinemia Finally, we analyzed the influence of initial homocysteme and vitamin concentrations and of the 677C—»T mutaoon in the methylenetetrahydrofokte reductase (MTHFR) gene on the homocysteme-lowenng efiect of multivita-min supplementaflon
Received August 5 1997, revmon accepted October 29 1997
From the Departments of Hematology (M den H I A B HLH P W W W B J G ) and Climcal Chemistry (A P S) Leyenburg Hospital The Hague the Department of Hematology Daniel den Hoed Clmic Rotterdam (G M J B ) the Department of Pediatncs Laboratorv of Pediatncs and Neurology (H J B , N MJ van der P ) and the Department of Obstetncs and Gynecology Laboratory of Endocnnology and Reproduction (C M G T ) Umveraty Hospital Nijmegen Nymegen and Departments of Climcal Epidemiology and Hematology (F R R1 Umversity Hospital Leiden Leiden the Netherlands
Correspondence to Martin den Heijer MD PhD Department of General Intemal Mediane Umversity Hospital Nymegen PO Box 9101 6bOO HB Nymegen the Netherlands
E mad m denheijer@aig azn nl © 1998 Amencan Heart Association Ine
den Heijer et al
357Methods
The study group was recruited from subjects vvho had participated m a previous case-control study4 In the current study, 92 ot the 185
patients with recurrent venous thrombosis and 230 of the 500 volunteers from the general population agreed to participate m an 8-week daily Vitamin supplementation tnal Six participants (three from each group) withdrew dunng the study All participants were asked not to take ("self-prescnbed") vitarmn Supplements for at least 2 months pnor to the Start of the current study
Both patients and hyperhomocystemermc healthy volunteers were randomtzed to either a placebo or a high-dose multiVitamin schedule Each multivitamin tablet contamed 5 mg folic acid, 0 4 mg hydroxy-cobalarrun, and 50 mg pyndoxine (Randomization of the volunteers had been stratified by homocysteme level m a previous study4 [cutoff
pomt, 16 μηιοΙ/L] ) Volunteers with previous homocysteme levels £16 μηιοΙ/L were randomized to placebo, multivitamin, or smgle-vitamin regimens (5 mg folic acid, 0 5 mg fohc acid, or 0 4 mg hydroxycobalamm) These three additional subregimens were re-stncted to normocystememic volunteer group because the other subgroups were too small to allow randomization mto more than two schedules Randomization was performed by usmg the last digit of each patient's number So all hyperhomocystememic subjects and normocystememic patients with recurrent venous thrombosis with an odd or even number were assigned to the multivitamin or placebo group, respectively In the normohomocystememic healthy volun-teers, subjects with a last digit of 0 or l were assigned to placebo, 2 and 3, to multiVitamins, 4 and 5, to 5 mg folic acid,, 6 and 7, to 0 5 mg fohc acid, and 8 and 9, to 0 4 mg Vitamin B12 All subjects were asked to take l tablet per day for 56 days The tnal was kept double-bhnd Before and after the supplementation penod, blood was collected after an overrught fast for homocysteme, folate, cobalamm, and pyndoxal-5'-phosphate measurements
For homocysteme and Vitamin measurements, blood samples were taken from the antecubital vem and collected mto EDTA-contaimng tubes Whole blood was stored at —70°C for pyndoxal-5'-phosphate determination For the other determmations, EDTA-treated samples were immediately placed on ice and centnfuged within half an hour at
2000g for 10 minutes The plasma was separated and stored at — 20°C
The EDTA-treated samples for folate and cobalamm measurements were stored at — 70°C and analyzed within 2 months Folate and cobalamm concentration:, were measured with a Dualcount SPNB (sohd phase no boil) radioassay kit (Diagnostic Products Corp) Determination of pyndoxal-5'-phosphate was performed by high-performance liquid chromatography techniques accordmg to Schnjver et al12 with some modifications 13 Total homocysteme concentrations
were measured accordmg to the method descnbed by Fiskerstrand et al14 with some modifications '5 Mutation analysis was carned out by
means of polymerase-cham reaction and restnction enzyme digestion
äs descnbed elsewhere 1δ
In the analysis we first looked at normalization rates of homocys-teme levels after multivitamin or placebo supplementation Therefore, we calculated the fraction of hyperhomocystememic subjects (homo-cysteme >16 μιηοΙ/L in the present study) who became normoho-mocystememic (homocysteme £16 μπιοΙ/L) after the supplementa-tion penod The cutofF pomt was a rounded value based on the 80th percentile m our previous study 4 Second, we calculated the percent
homocysteme reduction for each subject and compared the median reduction in the different Vitamin supplementation groups with respect to the correspondmg placebo group To compare the homo-cysteme-lowenng effects m paaents and volunteers, we later stratified the patients mto a hyperhomocystememic and a normohomocysteme-mic group accordmg to their homocysteme levels äs determmed in our
previous study (cutoffpomt, 16 μηιοΙ/L) Fmally, we studied
deter-mmants of the homocysteme-lowenng effect of multivitamin supple-mentation by calculatmg the median reduction in men and women, in subjects under or above 53 years of age (median age of healthy volunteers), and for several strata (tertiles) of initial homocysteme, folate, cobalamm, and pyndoxal-5'-phosphate concentrations, äs well
äs for the three different MTHFR-genotypes (677C—>T) To evaluate the difference m median reduction, we used the Mann-Whitney U test for unpaired cases (SPSS Software) All participants gave their
tHcy (pmol/L) after Intervention tHcy (μί-nol/L) after Intervention
A
0 10 20 30 tHcy (μπιοΙ/L) before Intervention
tHcy (pmol/L) after Intervention
30 20 10
B
|4» l) > · / · «Λ\. . <69C|> ' 0tHcy (pmol/L) before Intervention
tHcy (pmol/L) after Intervention
D
0 10 20 30 40 tHcy (pmol/L) before Intervention
0 10 20 30 40 tHcy (μηιοΙ/L) before Intervention
Total plasma homocysteme (tHcy) levels before (x axis) and after
<y axis) 8 weeks of daily multivitamin or placebo
supplementa-tion m patients with a history of recurrent venous thrombosis (A, placebo group, B, multivitamin group) and in healthy volunteers (C, placebo group, D, multivitamin group) The multivitamin tab-lets contamed 5 mg folic acid, 0.4 mg hydroxycobalamm, and 50 mg pyndoxine
mformed, wntten 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 (ränge, 31 to
89) and of the volunteers, 53 years (ränge, 23 to 82) The median homocysteme concentration of the patient group was 13 6 μπιοΙ/L (ränge, 7 2 to 69) and of the volunteer group 128 μηιοΐ/ΐ, (ränge, 4 7 to 49 8)
The Figure shows the homocysteme concentrations before and after Intervention for the placebo and mgh-dose tamin groups of both patients and volunteers In the multivi-tamin group, 11 of 14 hyperhomocystememic patients with thrombosis had a normalized value after Intervention (cutoff pomt, 16 μηιοΙ/L) compared with only 4 out 10 in the placebo
group A very similar observaüon was made m the healthy
volunteers
In the multivitamin group, 15 of 16 hyperhomocystememic subjects had a normalized value (cutoff pomt, 16 μιτιοΙ/L)
compared with only 3 of 20 m the placebo group So for all hyperhomocystememic mdividuals together, 26 of 30 subjects had normalized homocysteme levels (<16 μιηοΙ/L) after supplementation with multiVitamins compared w'th only 7 of 30 m the placebo group
TABLE 1. Homocysteine-Lowering Effect of Multivitamin or Placebo Supplementation in Patients With Recurrent Venous Thrombosis and in Healthy Volunteers
Study Subjects
Patients with recurrent venous thrombosis Homocysteme >16 μΐηοΙ/L* Placebo Multivitaminf Homocysteme <16 μmol/L* Placebo Multivitammt Healthy volunteers Homocysteme >16 μΓηοΙ/L* Placebo Multivitammt Homocysteme <16 μηιοΙ/L* Placebo Multivitammt Smgle-vitamm regimen n 89 34 15 19 55 28 27 227 50 27 23 177 36 34 107
Mediän tHcy Before Intervention, μπτοΙ/L (Range) 158(106-315) 165(107-690) 128(81-175) 118(72-481) 180(99-498) 166(110-375) 115(64-178) 118(71-193)
Mediän tHcy After Intervention, ju.mol/L (Range) 148(126-263) 107(68-175) 122(81-484) 86(49-193) 174(92-258) 107(70-214) 114(70-214) 85(55-115) Mediän Reduction, % (Range) -3 (-30-27) 36(4-83)t -1 (-193-45) 20(-38-60)Φ 0 (-27-53) 36(-46-70)ί 3 (-72-61) 30 (-22-55)Φ tHcy indicates total plasma homocysteme The homocysteme Iowermg effect
homocysteme concentration alter Intervention
'Stratified on homocysteme levels m the previous study
tContammg 5 mg folic acid 04 mg hydroxycobalamm and 50 mg pyndoxme ΦΡ< 001 compared with the correspondmg placebo group
is expressed äs the median percent reduction m
In Table 2 we compared the homocysteme-lowenng effect of several smgle-vitamm regimens in volunteers who were normohomocysteinermc in a previous study From these data we may conclude that the effect of 5 mg folic acid, even a low dose of 0 5 mg, is nearly äs effective äs the multivitamm regimen In contrast, Vitamin B12 only slightly decreased the homocysteme concentration
In Table 3 we analyzed the effects of age and sex with respect to the homocysteme-lowenng effect of multivita-min Supplementation For reasons of homogeneitv, we restncted this analysis to volunteers who had been random-ized mto either the placebo or the multivitamm group (n= 120) We found a similar homocysteme-lowenng efFect in men and women and m subjects under and above 53 years
of age
In Table 4 we stratified the homocysteme-lowenng effect on tertiles of initial homocysteme, folate, cobalarrun, and pyndoxal-5'-phosphate levels We found a stronger teme-lowenng effect in subjects with high initial homocys-teme levels However, even m subjects with an initial homo-cysteme level of <11 8 μ,ηιοΙ/L, we still found a median reduction of 21% (ränge, —22 to 41%) An mverse effect was seen with respect to initial vitamm concentrations The ho-mocysteme-lowenng effect was strengest in subjects with low folate, cobalarrun, or pyndoxal-5'-phosphate concentrations
Six of the 92 patients with recurrent venous thrombosis were homozygous for the 677C—»T mutation versus 22 of the 230 control subjects (odds ratio, 0 7, 95% confidence interval, 0 3 to l 7) In Table 4 we also show that the homocysteme-lowenng effect of multivitamm Supplementation was not
TABLE 2. Homocysteine-Lowering Effect of Several Vitamin Regimens or Placebo in Normohomocysteinemic Healthy Volunteers
Regimens Placebo Multivitamm* Folie acid 5 mg Folie acid 05 mg Hydroxycobalamm 0 4 mg n 36 34 35 36 36
Median tHcy Before Intervention, μΐηοΐ/ί (Range) 115(64-178) 118(71-193) 118(70-221) 122(47-223) 126(64-184)
Median tHcy After Intervention, μΐηοΙ/L (Range) 114(70-180) 85(55-115) 87(59-138) 100(28-138) 110(67-159) Median Reduction, % (Range) 3 (-72-61) 30(-22-55)t 26(-2-52)t 25(-54-40)t 10(-21-41)t tHcy indicates total plasma homocysteme The homocysteme-lowenng effect is expressed äs the median percent reduction m homocysteme concentration alter Intervention
'Containing 5 mg folic acid 0 4 mg hydroxycobalamm and 50 mg pyndoxme tP< 001 compared with the placebo group
den Heijer et ai
359
TABLE 3. Homocysteme-Lowering Effect of Multivitamin or Placebo Supplementation in
Men and Women and in Subjects <53 or s=53 Years Old
Study Subjects Men Placebo Multivitamin* Women Placebo Multivitamin* Volunteers <53 years Placebo Multivitamin* Volunteers s53 years Placebo Multivitamin* n 24 26 39 31 27 23 36 34
Mediän tHcy Betöre Intervention, μΐηοΙ/L (Range) 148(74-498) 140(72-375) 12 4 (6 4-21 8) 121(71-348) 120(64-498) 131(71-348) 147(74-251) 138(95-375)
Mediän tHcy After Intervention, μπιοΐ/ί (Range) 132(70-253) 101(76-214) 127(70-258) 87(55-168) 109(70-258) 8 3 (5 5-21 4) 139(95-253) 95(63-168) Mediän Reduction, % (Range) 12 (-69-61) 33 (-46-70) -1 (-72-25) 31 (-22-64) 5 (-72-61) 31 (-46-64) -1 (-69-19) 32 (3-70)
tHcy mdicates total plasma homocysteme The homocysteme lowermg effect is expressed äs the median percent reduction m homocysteme concentration after Intervention
*Contammg 5 mg folic acid 0 4 mg hydroxycobalamm and 50 mg pyndoxme
impaired in subjects homozygous for the 677C—»T mutation and m fact, rrught even be stronger
Discussion
We investigated the effect of multivitarmn supplementation on homocysteme levels m patients with recurrent venous throm-bosis and healthy volunteers firom the general population We found that combmed supplementation with folic acid, hy-droxycobalamm, and pyndoxme effectively reduced and nor-mahzed homocysteme levels in patients with recurrent venous thrombosis äs well äs in healthy volunteers For a subgroup of normohomocystememic volunteers, folic acid at a dose of 5 mg or even 0 5 mg seemed to be almost äs effective äs the high-dose multivitamm supplementation Oral cobalamm sup-plementation had only a moderate effect on homocysteme levels
In 1985 Brattstrom et al6 reported a substantial
homocys-teme reduction m 15 volunteers who received 5 mg folic acid per day for 4 weeks Wilcken et al7 reported a
homocysteme-lowermg effect of folic acid supplementation m patients with chromc renal msufHciency Franken et al8 and van den Berg et
al9 reported sigruficant reductions in postmethionme-loading
homocysteme concentrations with Vitamin B6 folic acid, or a combmaüon of both m patients with vascular disease Al-though these studies were performed in large groups of patients, they were not placebo controlled and were restncted to hyperhomocystememic subjects, which charactenstics make them rather sensitive to regression to the mean Ubbink et al10
studied the effects of l mg folic acid, 0 4 mg cyanocobalamm, and 122 mg pyndoxal HC1 alone or m combination m a placebo-controlled study in subjects with hyperhomocysteme-mia High-dose multivitamm admmistration resulted in a 49 8% reduction of the mean homocvsteme level Naurath et al" studied the effect of mtramuscular Vitamin supplementation with folate, vitamm B6, and Vitamin B12 m elderly subjects with blood vitamm concentrations in the normal ränge
In our study we were able to compare the effects in patients with homocysteme-related disease (venous thrombosis) with those in healthy volunteers These effects were quite sirmlar We also found about the same effects in men compared with women and m subjects under and above 53 years of age The strongest homocysteme-lowering effect of vitamm supplemen-tation was seen m subjects with high initial homocysteme and/or low initial vitamm concentrations However, we also observed a moderate reduction m homocysteme levels m subjects with homocysteme and vitamm levels within the normal ränge This observation raises the question of "normal" homocysteme and vitamm levels Our defimtion of hyperho-mocystmemia at the 80th percentile of the distnbution in the general population is arbitrary Other suggested defimtions are based on mean concentrations m populations without cardio-vascular disease " at the flat plateau of the homocysteme-folate plot18 We thmk that the best defimtion should be based on
clmical Intervention studies the lowest concentration at which vitamm supplementation reduces the nsk of vascular disease However, data on clmical studies are not yet available
In a subgroup of normohomocystememic volunteers, we found approximately the same homocysteme-lowenng effect of folic acid at a dose of 0 5 mg äs with a dose of 5 mg This means that considerably low doses of folic acid supplementa-tion are effective m lowenng homocysteme levels Further studies are needed to see whether doses lower than 0 5 mg may also be effective
TABLE 4. Homocysteine-Lowering Effect of Multivitamin ur Placebo Supplementation Stratified Over Tertiles of Homocysteine, Folate, Cobalamin, and Pyridoxal-5'-Phosphate Levels in 120 Healthy Volunteers
Placebo Homocysteine <11 8 μΓΠθΙ/L 118-157 μπποΙ/L >157 μηηοΙ/L Folate <104 nmol/L 104-143 nmol/L >143 nmol/L Vitamin B12 <207 pmol/L 207-296 pmol/L a296 pmol/L Vitamin B6 <37 nmol/L 37-47 nmol/L >47 nmol/L MTHFR 677 C-»T +/+ + / - < / Multivitamin* Homocysteine <11 8 μπιοΙ/L 118-157μΐτιοΙ/ί >15 7 μΐηοΙ/L Folate <10 4 nmol/L 104-1 4 3 nmol/L >14 3 nmol/L Vitamin B12 <207 pmol/L 207-296 pmol/L a296 pmol/L Vitamin B6 <37 nmol/L 37-47 nmol/L >47 nmol/L MTHFR 677C^T +/+ +/- -/-n 21 20 22 19 23 21 20 23 20 18 21 24 5 25 33 18 21 18 19 18 20 19 18 20 14 24 19 8 21 28
Mediän tHcy Betöre Intervention, μπιοΐ/ί (Range) 99(64-116) 141(118-156) 189(157-498) 166(93-498) 125(64-251) 120(74-251) 140(91-498) 136(74-218) 144(64-251) 119(64-218) 146(84-246) 150(74-498) 164(90-498) 146(93-251) 124(64-251) 103(71-117) 132(118-155) 185(157-375) 157(99-375) 122(72-188) 124(71-244) 147(99-375) 127(76-244) 120(71-189) 147(71-348) 133(88-375) 123(72-244) 164(109-375) 126(71-348) 131(88-244)
Mediän tHcy After Intervention, μΐτιοΙ/L (Range) 107(70-127) 132(78-154) 195(70-258) 162(70-258) 127(88-253) 125(78-221) 129(92-253) 138(78-258) 125(70-232) 115(84-233) 12 7 (7 0-25 8) 136(70-253) 133(70-232) 137(70-258) 126(78-221) 78(55-107) 9 0 (7 0-21 4) 110(70-258) 102(58-214) 87(63-118) 89(55-138) 10 3 (5 8-21 4) 86(63-168) 86(55-138) 89(63-137) 85(55-168) 9 3 (7 8-21 4) 9 7 (7 0-13 7) 86(63-214) 88(55-168) Mediän Reduction, % (Range) -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) 2, ^-46-64) 31 (7-57)
tHcy mdicates tal plasma homocysteme, MTHFR, methylenetetrahydrofolate reductase The homocysteme-lowenng effect is expressed äs the percent reduction m homocysteme concentration after Intervention
den Heijer et al
361
not study doses higher than 50 mg pyndoxme because higher doses may cause sensory neuropathy 2U 2I
Recently, we found the 677C—>T mutation in the MTHFR gene162223 This mutation is associated with elevated
homocysteme levels, but it is unclear whether this mutation is also associated with artenal vascular disease 24 In this study the
prevalence of the 677C—>T mutation m the recurrent venous thrombosis group did not really difFer from that m the control group This finding is in accordance with the results in a study of first-time venous thrombosis 2D We found that the
homo-cysteme-lowenng efFect 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 Malmow et al,26
These results suggest that the efFect of a mutated MTHFR might be "compensated" by a higher folate mtake
In conclusion, our study shows that combmed supplemen-tation with fohc acid, cobalamin, and pyndoxme reduces homocysteme levels by =«30% compared with placebo within 8 weeks in patients with recurrent venous thrombosis äs well äs m healthy volunteers. Whether the reduction in homocysteme levels by vitamm supplementation will lead prevention of artenal vascular disease and venous thrombosis is a major task for further climcal research.27 28
Acknowledgments
This work was supported by grants from the Prevention Fund (to G.MJ.B.) (28-2263-1). We thank G E Th Ferguson, pharmacist, for prepanng the Vitamin and placebo tablets and C Postma, Υ Lenstra,
M.T W B te Poele-Pothoff, A de Graaf-Hess, D van Oppenraaij-Emmerseel, M.F.G. Segers, L M.F Geelen, and J Beunk for thetr excellent laboratory assistance We wish thank R Clarke, MD, MRCP, for his valuable comments of a previous Version of the manuscnpt Fmally, we thank all participants for their kind contnbu-aon the study
References
1 Ueland PM, Refsum H, Brattstrom L Plasma homocysteme and cardio-vascular disease In Francis RJ3, Jr, ed Atherosdemtu Cardiocardio-vascular Disease,
Hemostasis, and Endothelwl Funclwn New York/Basel/Hong Kong Marcel
Dekker Ine, 1993 183-236
2 Rees MM, Rodgers GM Homocystememia association of a metabohc disorder with vascular disease and thrombosis Thromb Res 1993,71 337-359
3 Boushey CJ, Beresford SAA, Omenn GS, Motulsky AG A quantitative assessment of plasma homocysteme äs a nsk factor for vascular disease probable benefits of increasmg folic acid mtakes JAMA 1995,274 1049-1057
4 den Heijer M, Blom HJ, Geräts WBJ, Rosendaal FR, Haak HL, Wijermans PW, Bös GMJ Is hyperhomocystemaerma a nsk factor for recurrent venous thrombosis' Lancet 1995,345 882-885
5 den Heyer M, Koster T, Blom HJ, Bös GMJ, Bnet E, Reitsma PH,
Vandenbroucke JP, Rosendaal FR Hyperhomocysteinemia äs a nsk factor for deep-vem thrombosis N EnglJ Med 1996,334759-762
6 Brattstrom LE, HultbergBL, HardeboJE Folie acid responsive postmeno-pausal homocystememia Metabohsm 1985,341073-1077
7 Wilcken DEL, Dudman NPB, Tyrrell PA, Robertson MR Folie acid lowers elevated plasma homocysteme m chromc renal msufficiency possible rmphcations for prevention of vascular disease Metabohsm 1988, 37 697-701
8 Franken DG, Boers GHJ, Blom HJ, Tnjbels FJM, Kloppenborg PWC Treatment of mild hyperhomocystememia m vascular disease patients
Artenosder Thromb 1994,14465-470
9 \an den Berg M, Franken DG Boers GHJ Blom HJ, Jakobs C Stehouwer CDA, Rauwerda JA Combmed \itamm B6 plus folic acid therapv in young patients with artenosclerosis and hyperhomocystememia J Vase
Surg 1994,20933-940
10 Ubbmk JB, Vermaak WJH, van der Merwe A, Becker P] Delport R, Potgieter HC Vitamin requirements for the treatment of hyperhomocys-tememia m humans J Nulr 1994,124 1927-1933
11 Naurath HJ, Joosten E, Riezler R, Stabler SP, Allen RH, Lindenbaum J Effects of vitamm B12, folate, and Vitamin B6 Supplements m elderly people with normal serum vitamm concentrations Lancet 1995,346 85-89
12 Schnjver J, Speek AJ, Schreurs WHP Semi-automated fluorometnc deter-mination of pyndoxal-5'-phosphate (vitamm B6) m whole blood by high-performance liquid chromatography (HPLC) Int J Vttam Nutr Res 1981, 51 216-222
13 Steegers-Theumssen RPM, Boers GHJ, Steegers ΕΑΡ, Tnjbels FJM, Thomas CMG, Eskes TKAB Effects of sub-50 oral contraceptives on homocysteme metabolism a prehminary study Conlrcufpnon 1992,45 129-139
14 Fiskerstrand T, Refsum H, Kvalheim G, Ueland PM Homocvsteme and other thiols m plasma and unne automated determmation and sample stability Chn Chem 1993,39263-271
15 te Poele-Pothoff MTWB, van der Berg M, Franken DG, Boers GHJ, Jacobs C, de Kroon IFI, Eskes TAKB, Tnjbels JMF, Blom HJ Three different methods for the determmation of tal homocysteme m plasma Ann
Clm Biochem 1995,32 218-220
16 Fresst P, Blom HJ, Milos R, Go>ette P, Sheppard CA, Matthews RG, Boers, GHJ, den Heijer M, Kluytmans LAJ, van den Heuvel LP, Rozen R A candidate genetic nsk factor for vascular disease a common mutation m methylene-tetrahydrofolate reductase Nat Genet 1995,10111—113 17 Ubbmk JB, Venrnak WJH, Bennett JM, Becker PJ, van Staden DA,
Bissbort S The prevalence of homocystememia and hypercholesterolemias m angiographically defined coronary heart disease Kim Wochenschr 1991, 69 527-534
18 Seihub J.Jaques PF, Wilson PWF.Rush D, Rosenberg IH Vitamm Status and mtake äs pnmary determmants of homocystememia m an elderly populanon JAMA 1993,270 2693-2698
19 Lederle FA Oral cobalamin for pemicious anemia medicme's best kept secret'_//lA£4 1991,26594-95
20 Parry GJ, Bredensen DE Sensory neuropathy with low dose pyndoxme
Neimbgy 1985,35 1466-1468
21 Schaumberg H, KaplanJ, Wmdebanke A, Vick N, Rasmus S, Pleasure D, Brown MJ Sensory neuropathy from pyndoxme abuse N Eng] J Med 1983,309 445-448
22 Engbertsen AMT, Franken DG, Boers GHJ, Stevens EMB, Tnjbels FJM, Blom HJ Thermolabile 5,10-methylenetetrahydrofolate reductase äs a cause of mild hyperhomocystememia Am J Hunt Genet 1995,56142—150 23 Kluytmans LAJ, van den Heuvel LPWJ, Boers GHJ, Frosst P, Stevens
EMB, van Oost BA, den Heijer M, Tnjbels FJM, Rozen R, Blom HJ Molecular genetic analysis in mild hyperhomocystememia a common mutation in the methylenetetrahydrofolate reductase gene is a genetic nsk factor for cardiovascular disease Am J Hum Genet 1996,5835-41 24 Kluytmans LAJ, KastelemJJP, LmdemansJ, Boers GHJ, Heil SG, Bruschke
AVG, Jukema JW, van den Heuvel LPWJ, Tnjbels JMF, Boerma GJM, Verbeugt FWA, Willems F, Blom HJ Thermolabile methylenetetrahydro-folate reductase m coronary artery disease Ctrculanon 1997,962573—2577 25 Kluytmans LAJ, den Heyer M, Reitsma PH, Heil SH, Blom HJ, Rosendaal
FR Thermolabile methylenetetrahydrofolate reductase and factor V Leiden in the nsk of deep-vem thrombosis Tliromb Haemost In press 26 Malmow MR, Nieto FJ, Kruger WD, Duell PB, Hess DL, Gluckman RA,
Block PC, Holzgang, Anderson PH, Seltzer D, Upson B, Lm QR The effects of folic acid supplementation on plasma tal homocysteme are mod-ulated by multiVitamin use and methylenetetrahydrofolate reductase geno-rypes Anmovkr Thromb Vase Biol 1997,171157-1162
27 Stampfer MJ, Rimm EB Folate and cardiovascular disease whv we need a mal now JAMA 1996,275 1929-1930
