`
`Vitamin Requirements for the Treatment of
`Hyperhomocysteinemia in Humans
`JOHAN B. UBBINK,3 W. J. HAYWARD VERMAAK, ANNATJIE VAN OER MERWE,
`PIET J. BECKER, * RHENA DELPORT ANO HENDRIK C. POTGIETER
`
`Department of Chemical Pathology, Faculty of Medicine, University of Pretoria, 0001
`Pretoria, South Africa and *Institute for Biostatistics, Medical Research Council, Pretoria,
`South Africa
`
`ABSTRACT We have previously shown that a modest
`vitamin supplement containing folic acid, vitamin B-12
`and vitamin B-6 is effective in reducing elevated plasma
`homocysteine concentrations. The effect of supplemen-
`tation of the individual vitamins on moderate hyper-
`homocysteinemia has now been investigated in a
`placebo-controlled study. One hundred men with
`hyperhomocysteinemia were randomly assigned to five
`groups and treated with a daily dose of placebo, folic
`acid (0.65 mg), vitamin B-12 (0.4 mg), vitamin B-6 (10
`mg) or a combination of the three vitamins for 6 wk.
`Folic acid supplementation reduced plasma homocys-
`teine concentrations by 41.7% (P < 0.001), whereas the
`daily vitamin B-12 supplement lowered homocysteine
`concentrations by 14.8% (P < 0.01). The daily
`pyridoxine dose did not reduce significantly plasma
`homocysteine concentrations. The combination of the
`three vitamins reduced circulating homocysteine con-
`centrations by 49.8%, which was not significantly
`different (P -- 0.48) from the reduction achieved by
`folate supplementation alone. Our results indicate that
`folate deficiency may be an important cause of
`hyperhomocysteinemia in the general population. J.
`Nutr. 124: 1927-1933, 1994.
`
`INDEXING KEY WORDS:
`
`¯ humans ¯ homocysteine *
`¯ pyridoxine ¯ vitamin B-12
`
`folate
`
`Patients with premature vascular disorders often
`have elevated circulating total homocysteine concen-
`trations. Several retrospective studies have linked
`mild hyperhomocysteinemia to coronary heart
`disease IGenest et al. 1990, Israelsson et al. 1988,
`Ubbink et al. 1991aJ and cerebral {Brattstr6m et al.
`1984, Coull et al. 1990J and peripheral vascular dis-
`eases {Malinow et al. 1989, Taylor et al. 1991J.
`Prospective data from the Physicians Health Study
`also indicate that moderate hyperhomocysteinemia is
`a risk factor for premature vascular disorders
`
`(Stampfer et al. 1992). Participants in the above-men-
`tioned study who subsequently developed myocardial
`infarction had had significantly higher baseline
`plasma total homocysteine concentrations when
`compared with controls matched for age and smoking
`habits.
`Clinical observations support epidemiological
`findings that elevated plasma homocysteine concen-
`trations are involved in the pathogenesis of atheros-
`clerosis. Taylor et al. (1991} found that the
`progression of peripheral vascular disease, as assessed
`in a vascular laboratory, was more common in pa-
`tients with hyperhomocysteinemia than in patients
`with normal plasma homocysteine concentrations.
`Similarly, clinical progression of coronary heart
`disease, based on new occurrence of angina pectoris,
`myocardial infarction or congestive heart failure, oc-
`curred at a higher rate in patients with hyper-
`homocysteinemia {Taylor et al. 1991}. Malinow et al.
`11993} measured the thickness of the intimal-medial
`carotid walls in individuals free of clinical
`atherosclerotic disease and found significantly
`elevated plasma homocysteine concentrations in sub-
`jects with thickened intimal-medial carotid walls.
`Based on the assumption that carotid arterial wall
`thickening reflects atherosclerosis, the results from
`Malinow and co-workers suggest involvement of
`homocysteine in atherosclerotic plaque formation.
`The mechanisms by which homocysteine may
`promote atherogenesis include vascular endothelial
`
`1Supported by the Atherosclerosis Risk Factor Research
`
`Programme and Vesta Medicines Pry Ltd.
`~The costs of publication of this article were defrayed in part by
`the payment of page charges. This article must therefore be hereby
`marked "advertisement" in accordance with 18 USC section 1734
`solely to indicate this fact.
`3To whom correspondence and reprint requests should be ad-
`dressed.
`
`0022-3166/94 $3.00 © 1994 American Institute of Nutrition.
`Manuscript received 14 December 1993. Initial review completed 26 January 1994. Revision accepted 4 April 1994.
`
`1927
`
`Sandoz Inc.
`Exhibit 1039-0001
`
`
`
`1928
`
`UBBINK ET AL.
`
`injury {Harker et al. 1976J, oxidative modification of
`low density lipoproteins {Olszewski and McCully
`1993} and enhanced binding of lipoprotein{al to fibrin
`in atherosclerotic plaque IHarpel et al. 1992}.
`Homocysteine may also perturb vascular coagulation
`mechanisms and thus promote a thrombotic tendency
`{Hajjar 19931.
`The evidence from epidemiological, clinical and
`biochemical studies indicating that an elevated
`homocysteine level is a risk factor for premature vas-
`cular disorders suggests that intervention trials are
`now required to determine whether the treatment of
`hyperhomocysteinemia will reduce the incidence of
`vascular disorders {Malinow 1990, Malinow et al.
`1993, Stampfer et al. 1992J. However, it is essential
`that the optimal therapy to normalize hyper-
`homocysteinemia should be established before
`clinical trials are initiated. We have previously shown
`that a modest vitamin supplement containing folio
`acid, vitamin B-12 and vitamin B-6 is effective
`in reducing plasma homocysteine concentrations
`{Ubbink et al. 1993J. We now report the results of a
`placebo-controlled study in which the effect of sup-
`plementation of the individual vitamins on hyper-
`homocysteinemia was investigated.
`
`SOBJECTS AI~ID /~"THODS
`
`Venous blood samples with sodium fluoride as
`preservative were obtained from 2788 fasting am-
`bulatory white men aged between 20 and 73 y, who
`were referred to the two major Pretoria pathology
`practices for routine medical investigations as are
`required for life insurance purposes. Blood samples
`were received within 8 h at our laboratory; plasma
`and cells were separated immediately after receipt
`and the plasma samples frozen at -70"C and analyzed
`within 1 wk for homocysteine.
`It has been established previously that plasma
`homocysteine increases when blood separation is
`delayed IUbbink et al. 1992J. This is presumably the
`result of continued methionine metabolism by blood
`cells, which can be partially inhibited by either
`chilling the blood samples in an ice bath or by the use
`of sodium fluoride as an enzyme inhibitor.
`Nevertheless, even in the presence of sodium
`fluoride, plasma homocysteine concentrations may
`increase by 36% within 8 h. Therefore, individuals
`with an initial plasma homocysteine concentration
`above the normal reference range {>16.3 ~mol/L} were
`requested to visit our laboratory for a repeat sampling.
`During this visit, blood samples with EDTA as an-
`ticoagulant were obtained and chilled on ice; plasma
`and blood cells were separated within 1 h by low
`speed centrifugation (1600 x g, 10 mini. At the first
`visit, the participants were weighed, length and blood
`pressures were recorded, and each participant com-
`
`pleted a questionnaire on his health status. Par-
`ticipants with confirmed hyperhomocysteinemia In =
`100J were randomly assigned to one of five groups [n =
`20 for each group]. Group A received placebo tablets,
`Group B was supplied with folic acid tablets [0.65
`mg}, Groups C and D received tablets containing
`pyridoxine HCI {12.2 mgJ and cyanocobalamin {0.4
`mg}, respectively, and Group E was treated with a
`tablet containing all three vitamins in the quantities
`mentioned above. All the tablets also contained 3.0
`mg of B-carotene, and the different tablets were
`therefore similar in appearance. The vitamin tablets
`were specially prepared and supplied by Vesta Medi-
`cines {Johannesburg, South Africa}.
`Participants were instructed to take one tablet
`daily after dinner and to refrain from using any other
`vitamin supplements during the study period. Venous
`samples were obtained from fasting subjects 3 and 6
`wk later. To monitor compliance, the 3-wk samples
`were analyzed for the vitamins supplemented. Data
`from two participants were omitted in the final
`analysis due to lack of compliance. Seven other par-
`ticipants opted to withdraw during the study. The
`participants were not aware of the identity of the
`tablets used in the study. The study was approved by
`the University Human Ethics Committee.
`Laboratory investigations. Plasma pyridoxal S’-
`phosphate (PLP) was determined by I-IPLC (Ubbink et
`al. 1985). Vitamin B-12 and folate concentrations
`were determined by a commercially available RIA kit
`(Simul TRAC-SNB, Becton Dickinson, Orangeburg~
`NY). Plasma homocysteine was derivatized with am-
`monium 7-fluoro 2-oxa-1,3 diazole-4-sulfonate [Wako,
`Neuss, Germany) and determined by HPLC as
`described previously {Ubbink et al. 1991b). This
`method measures total {free ÷ protein-bound) plasma
`homocysteine concentrations; homocystine, the
`mixed disulfide {cysteine-homocysteine} and protein-
`bound homocysteine are first quantitatively reduced
`before ammonium 7-fluoro 2-oxa-l,3 diazole-4-sul-
`fonate is added as derivatizing reagent.
`Statistical analysis. The five groups were com-
`pared with respect to plasma concentrations of
`homocysteine, PLP, folate and vitamin B-12 in a one-
`way ANOVA. Pairwise comparisons were done with
`an appropriate t test.
`Within each group, plasma homocysteine and
`vitamin concentrations measured after the 6-wk
`treatment period were also compared to the basal
`concentrations by using Student’s paired t test. In the
`pyridoxine-treated group, the data for folate did not
`meet the assumption required for Student’s paired t
`test, and the comparison was made by Wilcoxon’s
`matched-pairs signed ranks test. In view of the rela-
`tively small samples, outcomes of the Student, s
`paired t test in all the other comparisons were also
`confirmed by Wilcoxon’s matched-pairs signed ranks
`test {Conover 1971). Differences were considered sig-
`nificant at P < 0.05.
`
`Sandoz Inc.
`Exhibit 1039-0002
`
`
`
`VITAMIN REQUIREMENTS AND HYPERHOMOCYSTEINEMIA
`
`1929
`
`TABLE 1
`
`Hea/th pro~/e o[ the study part~dpaats1
`
`Group
`
`A
`
`B
`
`C
`
`D
`
`E
`
`n
`
`17
`
`19
`
`18
`
`17
`
`20
`
`Age
`
`y
`
`40.6
`114.5)
`40.0
`
`34.6
`113.7)
`35.0
`
`42.3
`(9.9)
`
`Blood pressure
`
`Prevalence (%1 of
`
`BMI
`
`Diastolic
`
`Sistolic
`
`Smoking
`
`Obesity Hypertension Angina or MI
`
`l~g/m2
`
`27.0
`13.7)
`27.1
`14.21
`25.5
`(3.61
`26.1
`(3.6}
`26.7
`(3.6)
`
`mm Hg
`121.2
`(16.3]
`125.8
`(9.4}
`122.4
`(13.3}
`119.4
`(10.4}
`116.5
`(14.0}
`
`78.8
`{11.8]
`80.5
`(10.71
`75.2
`{8.91
`76.9
`{7.7~
`79.8
`(10.7)
`
`41.1
`
`47.4
`
`44.4
`
`41.2
`
`55.0
`
`64.7
`
`63.2
`
`55.5
`
`58.8
`
`64.9
`
`11.8
`
`5.3
`
`5.5
`
`5.9
`
`10.0
`
`17.6
`
`0
`
`11.1
`
`11.8
`
`5.0
`
`The five groups did not differ significantly with respect to age, body mass index (BMI) or blood pressure (P > 0.05
`IValues are means (SD).
`for each variable, ANOVAL /VII - myocardial irdarction.
`
`The relationship between changes in plasma
`homocysteine and folate concentrations in the
`placebo-treated group was tested for significance with
`Fisher’s exact test {Conover 1971].
`
`RESCJLTS
`
`Table 1 summarizes the characteristics of the study
`participants. One-way ANOVA indicated that the five
`groups did not differ significantly from each other
`with respect to age, body mass index or blood
`pressure. More than 50% of the participants in each
`
`group had a body mass index of >25.0 kg/m2, and,
`using the criteria of Garrow and Webster 11985}, these
`participants were classified as obese. The prevalences
`of smoking, obesity, hypertension (diastolic blood
`pressure >90 mm Hg) and ischemic heart disease were
`similar in the five groups.
`Table 2 summarizes the effect of the different
`vitamin supplements on circulating homocysteine
`concentration. A between-group comparison demon-
`strated that at the start of the study Iwk 01, the five
`groups did not differ with respect to plasma concen-
`trations of homocysteine, PLP, cobalamin or folate.
`Between-group comparisons after 6 wk of vitamin
`
`Effects Of differ~lit Vitamin suppleme~tts on plasma conc~ttrations o[ homo~:yst~ine,, ,cobalm’ni.;
`pyridoxal-5’-phosphate (PLP) and [olate in mat with hyperhom~emia¯
`
`TABLE 2
`
`Group
`
`Vitamin
`supplement
`
`A
`
`B
`
`C
`
`D
`
`E
`
`Placebo
`
`Folate
`
`Pyridoxine
`
`Cobalamin
`
`Combination
`
`n
`
`17
`
`19
`
`18
`
`17
`
`20
`
`Week
`
`Homocysteine
`
`Folate
`
`Cobalamin
`
`PLP
`
`0
`6
`0
`6
`0
`6
`0
`6
`0
`6
`
`ttraol/L
`
`nmol/L
`
`pmol/L
`
`nmol/L
`
`30.6 {24.21
`30.7 (24.0}
`28.8 (14.5}
`16.8 {7.6}ad
`29.1 115.0}
`27.8 (14.5}
`30.5 (16.9}
`26.0 (11.8}c
`27.1 (8.3}
`13.6 (4.9}bd
`
`37.7 122.4}
`44.5 128.0]
`44.8 (30.4]
`38.0 (28.0)
`45.8
`(25.9}L
`
`217.2 {75.71
`5.7 {3.61
`210.6 {76.5}
`6.1 15.6}
`6.0 (4.4]
`180.3 (72.1]
`17.4 {10.4)M 203.6 {89.8}
`6.8
`13.0) 214.5 (93.5}
`5.8
`228.1 (73.4}
`(2.4)c
`188.9 (101.5)m
`4.1
`48.2 {23.4)
`(1.0)
`217.6 {122.6}
`4.1
`(1.3}
`378.5 {188.8}M
`45.5 116.0}
`5.2
`261.1 (124.0)
`45.4 (14.8)
`(3.0)
`18.4 (11.1)bd 389.1 (127.71bd 213.7 180.2)M
`
`IValues are means (SD). Plasma concentrations of vitamins and homocysteine aher 6 wk of vitamin supplementation were compared with
`plasma concentrations after 6 wk of placebo supplementation: si~tmificantly different from results achieved with placebo supplement, ap ¯
`0.01, bp ¯ 0.001. Plasma concentrations of vitamins and homocysteine aher 6 wk of supplementation were also compared with basal levels
`Iwk 0! for each supplementation group: significantly di~erent from corresponding value for wk 0: cp ¯ 0.05, dp ¯ 0.001.
`
`Sandoz Inc.
`Exhibit 1039-0003
`
`
`
`UBBINK ET AL.
`
`1930
`
`2.2
`
`A
`
`D
`
`1.8
`
`1.6
`
`1.4
`
`1.2
`
`plasma homocysteine concentration {49.8%, P <
`0.0011. The effect obtained by the vitamin combi-
`nation was not significantly different from that
`achieved by folate supplementation {P = 0.48,
`ANOVA}.
`Figure 1 depicts the individual responses to the
`different vitamin supplements used in this trial.
`Group E {combined supplement} was the only group
`in which all the participants responded with a
`reduction in circulating homocysteine concentra-
`tions. Plasma homocysteine concentrations from two
`individuals failed to respond to the folate supplement
`{Fig. 1B}. The reductions observed with vitamin B-19.
`therapy were considerably less {Fig. 1D}, with at least
`five participants showing no decline or even showing
`an increase in plasma homocysteine concentration.
`The individual changes in plasma homocysteine con-
`centrations during placebo and pyridoxine sup-
`plementation were erratic {Fig. 1A, C} and may reflect
`small changes in the dietary folic acid intake.
`Figure 2 depicts the changes in plasma
`homocysteine concentrations from wk 0 to wk 6 in
`participants receiving the placebo treatment as a
`function of the change in plasma folic acid concentra-
`tions over the same period. In nine participants the
`
`n=9
`
`n=2
`
`1o
`
`+
`
`÷ +
`
`+
`
`n=l
`
`÷
`
`n=5
`
`-I0
`-4
`
`0
`
`CMBSO
`
`’ ’
`2
`4
`
`6
`
`8
`
`FIGURE P. The change in plasma total homocysteine
`concentrations in placebo-treated subjects as function of the
`change in plasma folic acid concentrations. The individual
`changes in plasma folate concentrations were associated
`with opposite changes in total homocysteine concentrations
`{p < 0.09., Fisher’s exact testl.
`
`Sandoz Inc.
`Exhibit 1039-0004
`
`FIGURE I The effect of different vitamin supplements
`on plasma total homocysteine concentrations. Hyper-
`homocysteinemic men were supplemented daily for 6 wk
`with {A) placebo, {B) 0.65 mg folic acid, {CI 10 mg
`pyridoxine, {D} 0.4 mg cyanocobalamin or IEI a combination
`of the three vitamins. Significant declines in plasma
`homocysteine concentrations were observed in men sup-
`plemented with folic acid {P < 0.001}, cyanocobalamin IP <
`0.01) and the vitamin combination IP < 0.0011.
`
`supplementation demonstrated that treatment with
`folate and with the combined vitamin preparation
`resulted in significantly lower plasma homocysteine
`concentrations when compared with placebo
`treatment {P - 0.004 and P < 0.001, respectively}.
`Mean plasma homocysteine concentrations after 6 wk
`of pyridoxine or vitamin B-12 supplementation were
`not significantly different when compared with con-
`centrations of placebo-supplemented subjects. Com-
`pared with the placebo-supplemented subjects,
`plasma concentrations of the vitamins supplemented
`increased significantly {P < 0.0011 within the 6-wk
`study period.
`A within-group comparison showed that the
`modest folic acid supplement lowered the plasma
`total homocysteine concentration by 41.7% IP <
`0.0011 when compared with the basal Iwk 0) concen-
`tration ITable 2}. Similarly, vitamin B-12 supplemen-
`tation reduced the circulating homocysteine concen-
`tration significantly {P < 0.01} by 14.8%; however, the
`4.5% reduction in homocysteine concentration
`achieved by pyridoxine supplementation was not
`statistically significant. The supplement containing
`all three vitamins caused the largest reduction in
`
`
`
`VITAMIN REQUIREMENTS AND
`
`HYPERHOMOCYSTEINEMIA
`
`1931
`
`observed increase in plasma homocysteine concentra-
`tions was associated with a decline in plasma folate
`concentrations, whereas only one participant showed
`a decline in both homocysteine ahd folic acid concen-
`trations. Similarly, five individuals had lower
`homocysteine concentrations and higher folate con-
`centrations, whereas only two participants showed an
`increase in both homocysteine and folate concentra-
`tions. Among those who had decreased folate concen-
`trations, a significantly larger proportion had in-
`creased homocysteine concentrations than amongst
`the individuals with increased folate concentrations
`[P = 0.02, Fisher’s exact test). There were no sig-
`nificant relationships between changes in plasma
`homocysteine concentrations and changes in plasma
`PLP or cobalamin concentrations in the placebo-
`treated group [results not shown}.
`
`DISCUSSION
`
`Intracellular homocysteine is either remethylated
`to methionine in a reaction that requires methyltetra-
`hydrofolate and vitamin B-12 or is condensed with
`serine in a reaction catalyzed by the PLP-dependent
`cystathionine-fl-synthase [EC 4.2.1.22). Deficiencies
`in the cofactors required for homocysteine metab-
`olism may result in hyperhomocysteinemia, which
`can be successfully treated with a modest daily
`vitamin supplement [Ubbink et al. 1993). The results
`from the current study confirm that a combined
`vitamin preparation may be used to lower elevated
`circulating homocysteine concentrations. The aim of
`this study was to assess the ability of each individual
`vitamin component to lower the plasma homocys-
`teine concentration.
`Our results suggest that folic acid is a very
`powerful homocysteine-lowering agent; in fact, the
`combination of the three vitamins was only slightly
`more effective compared with folic acid. Folate sup-
`plementation has been used by others to treat
`hyperhomocysteinemia in renal insufficiency
`(Wilcken et al. 1988) as well as premature vascular
`disorders [Brattstr0m et al. 1990, Dudman et al. 1993).
`The daily doses of folate used in these studies ranged
`between 5 and 10 mg; in contrast, our results were
`obtained by an appreciably lower daily supplement
`[0.65 mg, or 3.25 x the Recommended Daily Al-
`lowance (RDA) for folate].
`In view of the high success rate obtained with
`folate therapy, the obvious question is whether the
`other two vitamins are required at all to control
`plasma homocysteine concentrations. Compared with
`placebo treatment, the homocysteine-lowering effect
`of vitamin B-12 was not statistically significant (P -
`0.31, ANOVA). However, a within-group comparison
`showed that vitamin B-12 supplementation resulted
`in a modest but significant decline in the mean
`
`plasma homocysteine concentration (Table 2J. This
`reduction in basal plasma homocysteine concen-
`t.ration after vitamin B-12 supplementation was
`notably less than the results obtained by Lindenbaum
`and co-workers 11988). This difference is explained by
`the fact that Lindenbaum et al. supplemented diag-
`nosed vitamin B-12-deficient patients with cobala-
`min, whereas in our study the hyperhomocysteinemic
`men were randomized into the different treatment
`groups without prior knowledge of vitamin nutri-
`tional status or any possible genetic aberrations. The
`pre-treatment plasma vitamin B-12 concentrations in
`our study were higher than those reported by Lin-
`denbaum et al. (Fig. 3}. Obviously, some of our par-
`ticipants receiving vitamin B-12 were not really deft-
`cient in this vitamin, and this may explain why
`vitamin B-12 supplementation had only a small
`homocysteine-lowering effect in our study.
`Folic acid supplementation in patients with a
`chronic vitamin B-12 deficiency may eventually
`result in neuropathy due to failure to recognize the
`vitamin B-12 deficiency (Beck 1991}. Moreover, Allen
`et al. 11990~ have recently shown that folate sup-
`plementation will not correct hyperhomocysteinemia
`that is primarily the result of a vitamin B-12 deft-
`ciency. It is therefore essential that vitamin B-12 and
`folate be combined to treat hyperhomocysteinemia.
`Furthermore, it is also important that the cyano-
`cobalamin content of the vitamin supplement should
`
`+
`
`0.5
`
`0.4
`
`0.!
`
`Oob. ,
`
`FIGURE 3 The frequency distribution of plasma vitamin
`B-12 concentrations in this study compared with the study
`reported by Lindenbaum et al. (1988~.
`
`Sandoz Inc.
`Exhibit 1039-0005
`
`
`
`1932
`
`UBBINK ET AL.
`
`be sufficient to allow adequate absorption of vitamin
`B-12 from the gastrointestinal tract, even in patients
`with pernicious anemia. Large oral doses of vitamin
`B-12 can be used to treat pernicious anemia, because a
`very small portion of the cobalamin will be absorbed
`even in the absence of intrinsic factor
`IDoscherholmen et al. 1957). We used a daily vitamin
`B-12 supplement of 400 ~g 1200 x RDAI, because it
`has been calculated that up to 1% of an oral
`400-/zg vitamin B-12 dose may be absorbed in subjects
`with pernicious anemia {Ellenbogen and Cooper
`19911. This implies that even in cases with intrinsic
`factor deficiency, -4 ~g 12x RDA] will be absorbed
`from a daily 400-/~g vitamin B-12 dose. We therefore
`suggest that effective treatment of hyperhomocys-
`teinemia should include at least vitamin B-12 and
`folic acid supplementation; the vitamin B-12 sup-
`plement should be sufficient to satisb] the require-
`ments of patients suffering from intrinsic factor
`deficiency.
`Due to its dramatic effect in cystathionine-B syn-
`thase deficiency {Mudd et al. 19891, pyridoxine may
`be regarded as the obvious choice of treatment for
`moderate hyperhomocysteinemia. However, our
`results indicate that a daily 10-rag 15x RDAI
`pyridoxine supplement did not significantly reduce
`plasma homocysteine concentration. The data
`presented in Fig. I C suggest that some individuals
`responded to pyridoxine therapy, whereas others did
`not. However, placebo supplementation had a similar
`effect, with some individuals apparently responding
`to placebo treatment. These changes in circulating
`homocysteine concentrations are presumably ex-
`plained by small changes in folic acid nutritional
`status during the study period IFig. 2}. It therefore
`seems that a folate deficiency is far more common
`and important in causing hyperhomocysteinemia in
`the general population. Our results support evidence
`presented by Miller et al. [1992~ as well as from
`BrattstrOm’s laboratory [Brattstr0m et al. 19901 that
`pyridoxine supplementation does not affect basal
`homocysteine concentration. The intriguing question
`of why only vitamin B-12 and folate, but not vitamin
`B-6, may modulate basal plasma homocysteine con-
`centration has recently been addressed by Selhub and
`Miller [19921. These authors postulate that a folate
`and/or vitamin B-12 deficiency results in low S-
`adenosylmethionine (an activator for the enzyme cys-
`tathionine B-synthasel concentrations, thus causing
`diminished cystathionine B-synthase activity in ad-
`dition to impaired remethylation. This then results in
`homocysteine accumulation. During a vitamin B-6
`deficiency, no depletion of S-adenosylmethionine is
`expected to occur, and homocysteine is removed by
`the remethylation pathway {Selhub and Miller 1992}.
`Although pyridoxine supplementation fails to
`lower basal plasma homocysteine concentration,
`vitamin B-6 modulates the homocysteine peak after
`
`the oral methionine load test (Brattstr6m et al. 1990,
`Dudman et al. 19931. BrattstrOm et al. 11990! found
`that 240 mg of pyridoxine decreased the mean post-
`methionine load homocysteine elevation by 26%,
`whereas Dudman and co-workers 11993) reported
`similar results with a smaller I100 mg/d) pyridoxine
`supplement. Several studies have found that post-
`prandial hyperhomocysteinemia is related to in-
`creased vascular disease risk IBoers et al. 1985, Clarke
`et al. 19911. Vitamin B-6 may be particularly effective
`in limiting the plasma homocysteine increase after
`methionine loading, and it may be prudent to include
`pyridoxine in intervention trials designed to evaluate
`homocysteine as a causative agent in vascular disease.
`However, because high doses of pyridoxine may
`induce neuropathy ISchaumburg et al. 1988), it should
`be established whether a smaller daily vitamin B-6
`dose would not be equally effective in preventing
`postprandial hyperhomocysteinemia.
`In conclusion, we have shown that the
`homocysteine-lowering effect of a multivitamin com-
`bination containing folate, vitamin B-12 and
`pyridoxine is mainly due to its folic acid content.
`Vitamin B-12 had a modest ability to lower basal
`plasma homocysteine concentration, whereas
`pyridoxine had no effect on basal homocysteine con-
`centration. Treatment of hyperhomocysteinemia
`should at least include a folic acid-vitamin B-12 com-
`bination~ the requirement for vitamin B-6 needs
`further investigation.
`
`ACKNOWLEDGMENTS
`
`A. Schnell and L. Goddard rendered excellent tech-
`nical assistance. We thank C. Niehaus and A. Botha
`as well as Du Buisson and partners for obtaining
`blood specimens from 2788 men for homocysteine
`determinations. ¯
`
`UTERATURE CITED
`
`Allen, R. H., Stabler, S. P., Savage, D. G. & Lindenbaum, J. {1990)
`Diagnosis of cobalamin deficiency. 1: Usefulness of serum
`methylmalonic acid and total homocysteine concentrations.
`Am. 1. Hematol. 34: 90-98.
`Beck, W. S. {1991} Diagnosis of megaloblastic anemia. Annu. Rev.
`Med. 42: 311-322.
`Boers, G.H.I., Smals, A.G.H., Trijbels, F.LM., Fowler, B., Bakkeren,
`I.A.I.M., Schoonderwaldt, H. C., Kleijer, W. 1. & Kloppenborg~
`P.W.C. {1985J Heterozygosity for homocystinuria in premature
`peripheral and cerebral occlusive arterial disease. N. Engl. I.
`Med. 313: 709-715.
`BrattstrOm, L. E., Hardebo, J. E. & Hultberg, B. L. {1984} Moderate
`homocysteinemia--a possible risk factor for arteriosclerotic
`cerebrovascular disease. Stroke 15: 1012-1016.
`BrattstrOm, L., Israelsson, B., Norrving, B., Bergqvist, D., ThOrne,
`Hultberg, B. & Hamfelt, A. {1990} Impaired homocysteine me-
`tabolism in early-onset cerebral and peripheral occlusive arterial
`disease. Atherosclerosis 81: 51-60.
`
`Sandoz Inc.
`Exhibit 1039-0006
`
`
`
`VITAMIN REOUIREMENTS AND HYPERHOMOCYSTEINEMIA
`
`1933
`
`Clarke, R., Daly, L., Robinson, K., Naughten, E., Cahalane, S.,
`Fowler, B. & Graham, I. 11991) Hyperhomocysteinemia: an in-
`dependent risk factor for vascular disease. N. Engl. J. Med. 324:
`1149-1155.
`Conover, W. J. 11971) Practical Non-parametric Statistics, 2nd ed.,
`pp. 167 and 280. John Wiley & Sons, New York, NY.
`Coull, B. M., Malinow, M. R., Beamer, N., Sexton, G., Nordt, F. &
`De Garmo, P. (1990) Elevated plasma homocyst(eJine concen-
`tration as a possible independent risk factor for stroke. Stroke
`21: 572-576.
`Doscherholmen, A., Hagen, P. S., Lui, M. & Olin, L. (1957} A dual
`mechanism of vitamin B-12 plasma absorption. J. Clin. Invest.
`36: 1151-1157.
`Dudman, N.P.B., Wilcken, D.E.L., Wang, J., Lynch, J. F., Macey, D.
`& Lundberg, P. {19931 Disordered methionme/homocysteine
`metabolism in premature vascular disease: its occurrence,
`cofactor therapy, and enzymology. Arterioscler. Thromb. 13:
`1253-1260.
`Ellenbogen, L. & Cooper, B. A. (1991! Vitamin B-12. In: Handbook
`of Vitamins {Machlin, L. J., ed.L 2nd ed., pp. 491-536. Marcel
`Dekker, New York, NY.
`Garrow, J. S., & Webster, J. (1985! Quetelet’s Index as a measure of
`fatness. Int. J. Obes. 9: 147-153.
`Gen.est, J. J., McNamara, J. R., Salem, D. N., Wilson, P.W.F.,
`Schaefer, E. J. & Malinow, M. R. {1990) Plasma homocyst(eJine
`concentrations in men with premature coronary artery disease.
`J. Am. Coll. Cardiol. 16: 1114-1119.
`Hajjar, K. A. (1993} Homocysteine-induced modulation of tissue
`plasminogen activator binding to its endothelial cell membrane
`receptor. J. Clin. Invest. 91: 2873-2879.
`Harker, L. A., Ross, R., Schlichter, S. I. & Scott, C. R. {1976}
`Homocystine induced arteriosclerosis: the role of endothelial
`cell injury and platelet response in its genesis. J. Clin. Invest. 58:
`731-741.
`Harpel, P. C., Chang, V. T. & Borth, W. {1992} Homocysteine and
`other sulfhydryl compounds enhance the binding of
`lipoprotein{a} to fibrin: a potential biochemical link between
`thrombosis, atherogenesis, and suIfhydryl compound metab-
`olism. Proc. Natl. Acad. Sci. U.S.A. 89: 10193-10197.
`Israelsson, B., Brartstr6m, L. E. & Hultberg, B. L. {1988}
`Homocysteine and myocardial infarction. Atherosclerosis 71:
`227-233.
`Lindenbaum, J., Healton, E. B., Savage, D. G., Brust, J.C.M., Garrett,
`T. J., Podell, E. R., Marcell, P. D., Stabler, S. P. & Allen, R. H.
`(1988) Neuropsychiatric disorders caused by cobalamin defi-
`ciency in the absence of anemia or macrocytosis. N. Engl. I-
`Med. 318: 1720-1728.
`Malinow, M. R. 11990} Hyperhomocyst{eJinemia: a common and
`easily reversible risk factor for occlusive atherosclerosis. Circu-
`lation 81: 2004-2006.
`Malinow, M. R., Kang, S. S., Taylor, L. M., Wong, P.W.K., Coull, B.,
`Inahara, T., Mukerjee, D., Sexton, G. & Upson, B. ~1989J Preva-
`lence of hyperhomocystie}inemia in patients with peripheral
`arterial occlusive disease. Circulation 79: 1180-1188.
`
`Malinow, M. R., Nieto, J., Szklo, M., Chambless, L. E. & Bond, G.
`{1993J Carotid artery intimal-medial wall thickening and plasma
`homocyst{eJine in asymptomatic adults. Circulation 87:
`1107-1113.
`Miller, J. W., Ribaya-M~rcado, J. D., Russell, R. M., Shepard, D. C.,
`Morrow, F. D., Cochary, E. F., Sadowski, I. A., Gershoff, S. N. &
`Selhub, I. {1992} Effect of vitamin B-6 deficiency on fasting
`plasma homocysteine concentrations. Am. J. Clin. Nutr. $5:
`1154-1160.
`Mudd, S. H., Levy, H. L. & Skovby, F. 119891 Disorders of transsul-
`furation. In: The Metabolic Basis of Inherited Disease (Scriver,
`C. R., Beaudet, A. L., Sly, W. S. & Valle, D., eds.}, vol. 1, 6th ed.,
`pp. 693-734. McGraw-Hill, New York, NY.
`Olszewski, A. l- & McCully, K. S. ~19931 Homocysteine metabolism
`and the oxidative modification of proteins and lipids. Free
`Radical Biol. & Med. 14: 683-693.
`Schaumburg, H., Kaplan, J., Windebank, A., Vick, N., Rasmus, S.,
`Pleasure, D. & Brown, M. I. {1983J Sensory neuropathy from
`pyridoxine abuse. N. Engl. J. Med. 309: 445-448.
`Selhub, J. & Miller, J. W. ~1992) The pathogenesis of
`homocysteinemia: interruption of the coordinate regulation by
`S-adenosylmethionine of the remethylation and transsulfuration
`of homocysteine. Am. J. Clin. Nutr. 55: 131-138.
`Stampfer, M. I., Malinow, R., Willet, W. C., Newcomer, L. M.,
`Upson, B., Ullmann, D., Tishler, P. V. & Hennekens, C. H.
`(1992} A prospective study of pl~ma homocyst(eJine and risk of
`myocardial infarction in US physicians. JAMA 268: 877-881.
`Taylor, L. M., DeFrang, R. D., Hams, E. J. & Porter, J. M. (1991) The
`association of elevated plasma homocyst(eline with progression
`of symptomatic peripheral arterial disease. J. Vasc. Surg. 13:
`128-136.
`Ubbink, J. B., Serfontein, W. J. & de Villiers, L. S. (1985} Stability of
`pyridoxal-S’-phosphate semicarbazone: application in plasma
`vitamin B-6 analysis and population surveys of vitamin B-6
`nutritional status. J. Chromatogr. 342: 277-284.
`Ubbink, J. B., Vermaak, W.J.H., Bennett, J. M., Bocker, P. I-, van
`Staden, D. A. & Bissbort, S. (1991a} The prevalence of
`homocysteinemia and hypercholesterolemia in angiographieally
`defined coronary heart disease. FAin. Wochenschr. 69: 527-534.
`Ubbink, J. B., Vermaak, W.J.H. & Bissbort, S. {1991b} Rapid high-
`pe~ormance liquid chromato