J. Inher. Metab. Dis. 20 (1997) 316-325
`© SSIEM and Kluwer Academic Publishers. Printed in the Netherlands
`
`The role of vitamins in the pathogenesis and
`treatment of hyperhomocyst(e)inaemia
`
`J. B. UBBINK
`Department of Chemical Pathology, University of Pretoria, PO Box 2034, Pretoria 0001,
`
`South AJ?ica
`
`Summary: The relation between vitamin nutritional status and circulating plasma
`homocyst(e)ine concentrations is reviewed. Several studies have shown that plasma
`concentrations of folate, vitamin B 12 and pyridoxal 5’-phosphate are inversely asso-
`ciated with plasma total homocyst(e)ine concentrations. Of the three vitamins
`mentioned above, folate is the most powerful homocyst(e)ine lowering agent and a
`daily supplement of 0.65mg/day is sufficient to normalize moderate hyperhomo-
`cyst(e)inaemia in most individuals wifia normal renal function. In patients with
`severe renal failure, high doses of folate are required to treat hyperhomo-
`cyst(e)inaemia. Folic acid is ineffective in reducing plasma total homocyst(e)ine
`concentrations in patients with a vitamin B12 deficiency. Vitamin B6 supplementation
`has no effect on fasting plasma total homocyst(e)ine concentrations, but attenuates
`the post-methionine load plasma homocyst(e)ine peak.
`At least one report has shown that some individuals appear to be unable to main-
`tain plasma total homocyst(e)ine concentrations in the normal reference range by a
`dietary intake of folic acid only. Long-term vitamin supplementation may be
`indicated in flaese individuals. However, flae clinical benefit of vitamin supple-
`mentation has not yet been demonstrated and comxolled trials are urgently required.
`
`The sulphur-containing amino acid homocysteine stands at the intersection of two meta-
`bolic pafiaways, i.e. ~xanssulphuration and remethylation (Stipanuk 1986). In the transsul-
`phuration pathway, the condensation of homocysteine with serine to form cystathionine is
`catalysed by fiae enzyme cystathionine [3-synthase (EC 4.2.1.22). In a subsequent step,
`cystafiaionine is hydrolysed by the enzyme T-cystafiaionase (EC 4.4.1.1) to yield cysteine
`and c~-ketoglutarate. Both these reactions require fiae physiologically active form of vita-
`min B6, pyridoxal 5’-phosphate (PLP), as essential cofactor. The remethylation of homo-
`cysteine is catalysed by N-5-mefiayltetrahydrofolate:homocysteine methyltransferase (EC
`2.1.1.13) in a vitamin B 12-dependent reaction which transfers the methyl group from N-5-
`methyltetrahydrofolate to homocysteine resulting in fiae formation of methionine. The
`methyl group of N-5-mefiayltetrahydrofolate is in fact synthesized de novo when a carbon
`unit is transferred from a suitable source (e.g. serine) to te~xahydrofolate. This reaction
`produces methylenetetrahydrofolate which is subsequently reduced to N-5-mefiayltetra-
`hydrofolate by the riboflavin-dependent enzyme methylene tetrahydrofolate reductase
`
`316
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`Sandoz Inc.
`Exhibit 1041-0001
`
`

`
`Vitamins and homocysteine
`
`317
`
`(MTHFR; EC 1.1.1.68) (Brody 1991). Alternatively, homocysteine may acquire a methyl
`group from betaine in a reaction catalysed by betaine:homocysteine meflayltransferase
`(EC 2.1.1.5). Effective cellular homocysteine metabolism is therefore dependent on an
`adequate status of flae essential nutritional factors mentioned above.
`The relationship between homocysteine metabolism and cofactor status has been
`exploited in the treatment of homocyst(e)inuria due to inborn errors of metabolism. Homo-
`zygotes for cystathionine 13-synthase deficiency have been lxeated with high-dose pyri-
`doxine supplementation wifla varying degrees of success (Carson and Carre 1969; Mudd
`et al 1985), while patients with MTHFR deficiency may benefit from high-dose folate
`and/or vitamin B12 supplementation (Carey et al 1968; Harpey et al 1981). The numerous
`studies reported during the past decade indicating a possible role for milder forms of
`hyperhomocyst(e)inemia in premature cardiovascular disease have resulted in a renewed
`interest in vitamin nulxitional status as a determinant of circulating total homocyst(e)ine
`concentrations (Total homocyst(e)ine refers to the sum of flae concentrations of free
`homocysteine, protein-bound homocysteine, the disulphide homocystine, and the mixed
`disulphide homocysteine-cysteine.) (Arnesen et al 1995; Israelsson et al 1988; Stampfer et
`al 1992).
`
`VITAMIN NUTRITIONAL STATUS AS A DETERMINANT OF CIRCULATING
`TOTAL HOMOCYST(E)INE (tHcy) CONCENTRATIONS
`
`Several studies performed before 1990 indicated that subclinical deficiencies in folate,
`vitamin B6 and vitamin B 12 were associated with hyperhomocyst(e)inaemia. Lindenbaum
`and colleagues (1988) found that patients with vitamin B 12 deficiency, but without anaemia
`or macrocytosis, had markedly elevated total homocyst(e)ine 0ktcy) concentrations, while
`Stabler and colleagues (1988) reported that 77 patients out of 78 with vitamin B12
`deficiency had serum tHcy concentrations above the normal reference range. Similarly, at
`least 84% of individuals with subnormal serum folate concentrations also had hyper-
`homocyst(e)inaemia (Kang et al 1987), while an earlier report suggested that vitamin B6
`depletion resulted in increased urinary lktcy excretion (Park and Linkswiler 1970).
`In 1993, Ubbink and colleagues (1993a) reported that flae prevalence of suboptimal
`vitamin B6, BI~ and folate status in South African men wifla hyperhomocyst(e)inaemia was
`25.0%, 56.8% and 59.1%, respectively. This observation suggested a slxong relationship
`between poor vitamin nutritional status and elevated circulating tHcy concentrations, a
`finding which was confirmed when Selhub and coworkers (1993) published the result of
`their cross-sectional analysis of plasma tHcy concentrations and circulating vitamin con-
`centrations in elderly participants from flae Framingham study. These workers found a
`strong inverse association between plasma tHcy and folate concentrations. The mean
`plasma tHcy concentrations in subjects in the two lowest deciles of the plasma folate
`concentration frequency distribution were significantly higher compared to the mean tHcy
`concentration of individuals in the highest decile of the plasma folate concentration.
`Plasma tHcy concentrations showed similar inverse relations wifla plasma vitamin B 12 and
`PLP concentrations (Selhub et al 1993). Joosten and coworkers (1993) demonstrated that
`14.4% of flae variation in plasma tHcy concentrations in elderly people was explained by
`plasma folate concentrations. In contrast, only 4.4% of the variation in plasma tHcy
`
`J. ]nher. Metab. Dis’. 20 (1997)
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`
`

`
`318
`
`Ubbink
`
`concentrations could be explained by flae variation in plasma folate concentrations in
`younger people (Joosten et al 1993). Compared with younger people, the inverse relation
`between plasma folate and tHcy concentrations was thus more accentuated in elderly
`people.
`The inverse associations between plasma tHcy and vitamin concentrations were not
`only observed in apparently healthy individuals, but also in different patient groups.
`Hopkins and colleagues (1995) confirmed flae presence of an inverse association between
`plasma folate and tHcy concentrations as reported previously, but found that patients with
`early familial cardiovascular disease (CVD) displayed an increased sensitivity with respect
`to the effects of folate status on plasma tHcy concentrations. A cross-sectional survey of
`1041 elderly subjects from the Framingham Heart Study demonstrated that low plasma
`concentrations of folate and PLP as well as high tHcy levels were associated with an
`increased risk of extracranial carotid artery stenosis (Selhub et al 1995). Adjustment of
`plasma vitamin concentrations for tHcy concentrations diminished the strength of the
`association between vitamin status and carotid artery stenosis prevalence, indicating that
`the regulatory effect of these vitamins on plasma tHcy concentrations explains why a low
`vitamin status is linked to an increased risk for carotid artery stenosis (Selhub et al 1995).
`In a case-control study involving 101 white men with angiographically demonstrated
`CVD, Pancharuniti and colleagues (1994) also found flaat the plasma folate concentration
`was related to CVD risk. After adjustment for plasma tHcy concentrations, the risk was
`abolished, indicating that a low plasma folate concentration is not an independent CVD
`risk factor, but that the effect of folate status on CVD is mediated through its effect on
`plasma lktcy concentrations. Similarly, Dalery and colleagues (1995) found in 150 French-
`Canadian subjects with angiographically documented CVD a significant inverse relation
`between plasma folate and tHcy concentrations, again indicating that folate status may
`influence CVD progression by its effect on plasma tHcy concentrations.
`To summarize: Observational studies have shown that vitamin nutritional status is a
`strong determinant of plasma tHcy concentrations. In particular, a compromised folate
`status has consistently been linked to hyperhomocyst(e)inaemia. These observations have
`spurred further research to define the optimum method of vitamin supplementation in
`order to reduce elevated circulating tHcy concentrations.
`
`THE EFFECT OF VITAMIN SUPPLEMENTATION ON PLASMA tHcy
`CONCENTRATIONS
`
`Folic acid: Several studies on various population groups have been performed to demon-
`strate the effect of vitamin supplementation on plasma tHcy concentrations. From the data
`summarized in Table 1, it is apparent that folate is the most powerful tHcy-lowering agent.
`Folate has been used in daily doses ranging from 0.65 to 10mg/day, and it seems that in
`apparently healthy volunteers a low daily dose of 0.65mg or less may be sufficient to
`maintain plasma tHcy concentrations within flae normal reference range (Ubbink et al
`1994). This low folate dose may, however, be insufficient in various pathological con-
`ditions predisposing towards coronary heart disease. In patients wifla severe chronic
`kidney failure, 10mg of folate per day administered for 3 months failed to reduce plasma
`tHcy concentrations to normal in all the participants (Chauveau et al 1996), while 5mg of
`
`J. Inher. Metab. Dis’. 20 (1997)
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`Sandoz Inc.
`Exhibit 1041-0003
`
`

`
`continued
`
`Franken et al (1994a)
`
`Patients had documented arterial
`
`occlusive disease
`
`hypohomocyst(e)inemia
`individuals with moderate
`trial on apparently healthy
`
`Ubbink et al (1994)
`
`Randomized, placebo-controlled
`
`Arnadottir et al (1993)
`
`Dialysis patients; open trial
`
`-53%, p< 0.006
`
`-13%, NS
`+a%, NS
`
`-49.8%, p<0.001
`-41.7%, p<0.001
`-14.8%; p<0.05
`
`-4.5%, NS
`
`-33.4%, p=0.001
`+ 23.2%, p< 0.05b
`
`Ubbink et al (1993 a)
`
`Placebo-controlled trial on
`
`- 59.8%, p < 0.001
`
`hyperhomocyst(e)inaemic men
`
`volunteers
`
`- 5%, NS
`+7%, NS
`
`BrattstrOm et al (1988)
`
`Open study on healthy
`
`-52%, p<O.O1
`
`Reference
`
`Remarks
`
`tHcy concentration
`
`in circulating
`
`Percentage change
`
`10
`10
`10
`
`20
`19
`17
`18
`
`18
`lg
`
`13
`
`15
`14
`
`13
`
`n
`
`6 weeks
`6 weeks
`6 weeks
`
`6 weeks
`6 weeks
`6 weeks
`6 weeks
`
`4 months
`4 months
`
`6 weeks
`
`2 weeks
`2 weeks
`
`2 weeks
`
`period
`
`Eolate 5 mg, combined
`Pyridoxine 250mg
`Pyridoxine 250 mg
`Pyridoxine 50 mg
`
`Combination of above
`Eolate 0.65 m g
`Cyanocobalamin 0.4rag
`Pyridoxine 10rag
`
`Eolate
`Pyfidoxine 300 mg
`
`Vitamin B 12 (0.4 lilg)
`Pyridoxine (10 rag)
`Folate (1.0rag)
`
`Combination:
`
`Pyridoxine 40 mg
`Cyanocobalamin 1 mg
`
`Folate 5rag
`
`Vitamins and dose~
`
`Supplementation
`
`Table 1 A sunnnary of intervention trials to lower circulating, fasting tHcy concentrations
`
`

`
`NS = not significant
`compared with individuals (n = 53) with an initial high tHcy concentration
`aTwo significantly different clusters were observed: individuals with an initial low they concentration (n= 182) showed a smaller decrease upon folate supplementation
`~Calculated from graph
`bThe increase in plasma tHcy concentration is explained by an insufficient washout period prior to the start of the study
`SUnless specified, the daily dose was administered orally
`
`(1996)
`Rasmussen et al
`
`- 16.2 to -33.6%, p<0.0011a 109 men, all apparently healthy
`Open study on 126 women and
`
`-4.8%; p<0.05
`
`Chauveau et al (1996)
`
`Open trial
`renal failure
`Nondialyzed patients with chronic
`
`-33 to -40%, p<0.001
`
`0-18.4%, NS
`
`Naurath et al (1995)
`
`trial on elderly subjects
`Placebo-controlled randomized
`
`- 31.3 %, p < 0.001°
`
`Landgren et al (1995)
`
`acute MI
`Open study on patients with
`
`-27%, p<0.001
`
`(1994)
`Van den Berg et al
`
`premature vascular disease
`Open study on patients with
`
`Reference
`
`Remarks
`
`-51%
`
`tHcy concentration
`
`in circulating
`
`Percentage change
`
`235
`235
`
`37
`
`37
`
`88
`
`33
`
`72
`
`n
`
`1 week
`1 week
`
`Folate 10mg
`Cyanocobalamin 2rag
`
`3 months
`
`Folate 10rag
`
`3 months
`
`Pyridoxine 70 mg
`
`over 3 weeks
`
`injections
`
`intramuscular
`
`Eight
`
`Vitamin B12 (1 nlg)
`Pyridoxine (Stag)
`Folate (1.1 rag)
`
`Combination:
`
`6 weeks
`
`Folic acid, 2.5 or 10rag
`
`6 weeks
`
`period
`
`Supplementation
`
`Folate 5 rag, combined
`Pyridoxine 250rag
`
`Vitamins and dosea
`
`Table 1 Continued
`
`

`
`Vitamins and homocysteine
`
`321
`
`folate per day was insufficient to normalize hyperhomocyst(e)inaemia observed in dialysis
`patients (Arnadottir et al 1993). Low daily doses of folic acid have not yet been tested in
`patients with premature vascular disease and it may be possible that this patient group will
`also require higher daily folic acid doses to maintain plasma tHcy concentrations in the
`normal range.
`
`Vitamin B12: Although folic acid is the most powerful tHcy-lowering agent, this does not
`imply that vitamin B12 and vitamin B6 may be omitted in the treatment of moderate
`hyperhomocyst(e)inemia. Vitamin B 12 supplementation has a small, but significant effect
`on circulating tHcy concentxations (Ubbink et al 1994; Rasmussen et al 1996). Moreover,
`it has been shown that folic acid supplementation is ineffective in reducing tHcy concen-
`trations in patients with a vitamin B12 deficiency (Allen et al 1990). In my opinion, the
`optimum vitamin supplement to treat hyperhomocyst(e)naemia will contain at least
`400gg of vitamin B12 per day. At this high daily dose, even patients with intrinsic
`factor deficiency will absorb a sufficient amount of vitamin B12 by passive diffusion
`(Doscherholmen and Hagen 1957). Vitamin B12 supplementation at high doses is innoc-
`uous (Ellenbogen and Cooper 1991) and will eliminate the risk that folic acid
`supplementation may mask an underlying vitamin B12 deficiency.
`
`Vitamin 1~6: Owing to its dramatic effect in cystathionine ~3-synthase deficiency (Mudd
`et al 1989) pyridoxine may be regarded as the obvious choice in the treatment of hyper-
`homocyst(e)inaemia. However, even high-dose pyridoxine supplementation did not lower
`fasting plasma tHcy concentxations significantly (Table 1). Selhub and Miller (1992)
`recently addressed the intriguing question why folic acid and vitamin B12 supplementation,
`but not pyridoxine supplementation, will reduce elevated, fasting circulating tHcy concen-
`trations. It has been postulated that a low folate and/or vitamin B12 status results in low
`S-adenosylmethionine concentxations. S-Adenosylmethionine, however, is required to
`activate the enzyme cystathionine ~3-synthase. The supplementation of only vitamin B6
`(without folate and vitamin B12) does not appear to activate the txanssulphuration pathway,
`as the essential activator S-adenosylmethionine will remain low owing to the inadequate
`folate and vitamin B 12 status. Only when the latter two vitamins are present in abundance
`will remethylation proceed unimpeded with a subsequent increase in S-adenosylmeth-
`ionine concentrations and activation of the transsulphuration pathway (Selhub and Miller
`1992).
`Although pyridoxine supplementation has no effect on fasting plasma tHcy
`concentrations, it lowers the post-methionine load tHcy peak (Dudman et al 1993; Franken
`et al 1994a,b; Ubbink et al 1996a). This phenomenon is also explained by Selhub and
`Miller’s (1992) hypothesis. The high post-methionine load S-adenosylmethionine con-
`centrations will inhibit remethylation and stimulate transsulphuration, but activation of
`transsulphuration cannot proceed during a vitamin B6 deficiency. When vitamin B6 is
`supplemented, transsulphuration can proceed unimpeded and thus methionine loading will
`result in lower peak plasma tHcy concentrations (Selhub and Miller 1992). This hypoth-
`esis assumes that the enzyme cystathionine ~3-synthase is sensitive towards vitamin B6
`depletion, which may not be true for all population groups. For example, African Blacks
`have a genetically determined lower vitamin B6 status (Vermaak et al 1987) owing to a low
`
`J. ]nher. Metab. Dis’. 20 (1997)
`
`Sandoz Inc.
`Exhibit 1041-0006
`
`

`
`322
`
`Ubbink
`
`pyridoxal kinase activity (Chem and Beutler 1975). Pyridoxal kinase is required to
`phosphorylate pyridoxal to its physiologically active form, PLP (McCormick et al 1961).
`However, Blacks do not present with high plasma tHcy concentrations when challenged
`with methionine. In fact, the post-methionine load tHcy peak is significantly lower in
`Blacks compared with Whites, despite the poorer vitamin B6 status of Blacks (Ubbink et
`al 1995). These observations in Blacks are still unexplained, but may relate to a
`cystaflaionine 13-synthase polymorphism where Blacks may commonly possess a form of
`cystaflaionine 13-synthase with a high affinity for PLP.
`In caucasian populations it has been shown that a normal fasting plasma tHcy
`concentration is not synonymous with a normal methionine load test (Bostom et al 1995).
`Elevated post-methionine load tHcy concentrations are independently associated with
`diverse pathological conditions, for example premature vascular disease (Clarke et al
`1991) and neural tube defects (Steegers-Theunissen et al 1994). It is flaerefore prudent that
`vitamin therapy should normalize both fasting and post-methionine load tHcy concen-
`trations. Since pyridoxine supplementation attenuates the post-methionine load tHcy peak
`(Dudman et al 1993; Franken et al 1994a,b; Ubbink et al 1996a), this vitamin should be
`included in the trealxnent of hyperhomocyst(e)inaemia. Initial studies used very high daily
`doses of pyridoxine (70-300mg) to lower flae post-methionine load tHcy peak (Dudman
`et al 1993; Franken et al 1994a,b). However, these high doses are undesirable as patients
`may develop pyridoxine-induced sensory neuropathy (Schaumburg et al 1983). We do not
`yet know the minimum daily pyridoxine dose required for the optimal reduction in post-
`methionine load plasma tHcy concentrations. We recently found that a pyridoxine dose of
`only 20mg/day administered for a 6-week period significantly reduced the post-
`methionlne load tHcy peak in asthma patients with a theophylline induced vitamin B6
`deficiency (Ubbink et al 1996a). Despite pyridoxine supplementation, the post-methionlne
`load plasma tHcy concentrations in these formerly vitamin B6-deficient patients remained
`significantly higher compared with healthy controls. This may indicate flaat 20mg pyri-
`doxine per day is not adequate for certain patient populations and higher doses may be
`required.
`
`LONG~TERM VITAMIN REQUIREMENTS IN MODERATE
`HYPERHOMOCYST(E)INAEMIA
`
`Most studies on vitamin supplementation and plasma tHcy concentrations were conducted
`for 6-week periods and give no information on whether long-term supplementation is
`required to maintain plasma tHcy concentrations in the normal range. We recently studied
`a group of men (n = 22) wifla hyperhomocyst(e)inaemia over a year (Ubbink et al 1993b).
`Participants in the study had a plasma tHcy concenlxation > 16.3 gmol/L and were treated
`for 6 weeks with a multivitamin preparation containing 1.0mg folic acid, 50gg vitamin
`B12 and 10mg pyridoxine. Mean (±SD) plasma tHcy concentrations declined from
`30.9 ± 14.4 gmol/L to 14.0± 7.9 gmol/L. Vitamin supplementation was then discontinued
`for 18 weeks. During this period, the mean (±SD) plasma tHcy concentration increased to
`25.1 ± 14.7 gmol/L. Thirteen participants had plasma tHcy concentrations > 16.3 gmol/L
`and they were again treated with the vitamin formulation described above, which resulted
`in a mean (±SD) tHcy concentration of ll.0±4.0gmol/L after 6 weeks of supple-
`
`J. ]nher. Metab. Dis’. 20 (1997)
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`Sandoz Inc.
`Exhibit 1041-0007
`
`

`
`Vitamins and homocysteine
`
`323
`
`mentation. Vitamin therapy was subsequently discontinued and the participants were
`supplied with dietary guidelines to increase their folate intake. Eighteen weeks later these
`subj ects again had elevated plasma tHcy concentration (mean(± SD)= 28.6 ± 16.1 gmol/L).
`We had no control over the extent to which the participants adhered to the high-folate diet
`as recommended by a dietician. However, participant plasma folate concentrations at the
`end of the study period were significantly higher compared to baseline concentrations,
`suggesting flaat the participants paid attention to their diets. These results suggest that
`some individuals with hyperhomocyst(e)inaemia may be unable to maintain normal
`plasma tHcy concentrations from dietary folate alone and that long-term supplementation
`may be required.
`
`BENEFITS OF VITAMIN SUPPLEMENTATION
`
`Several epidemiological criteria suggesting that hyperhomocyst(e)inaemia causes
`premature vascular disease have already been fulfilled. The data describing the relation-
`ship between plasma tHcy concentrations and vascular disease are consistent; flae stxength
`of the association is of a similar order as for lipid risk factors and vascular disease; and
`prospective studies suggest temporality (Boushey et al 1995). However, it is still unknown
`whether vitamin supplementation aimed to lower plasma tHcy concentrations will reduce
`the incidence of cardiovascular disease. A recent meta-analysis suggests that 9.7% of
`cardiovascular-related deaths in American men are the result of elevated plasma tHcy
`concentrations and that up to 50000 deaflas may be prevented annually by improving the
`daily folate intake of the US population (Boushey et al 1995). The above calculation may
`represent a gross overestimation of the real achievable benefit of vitamin supplementation
`(Ubbink et al 1996b). Nevertheless, the study of Boushey and colleagues (1995) should
`serve as a stimulus to design and perform contxolled clinical trials to put to test the
`hypothesis that treatment of hyperhomocyst(e)inaemia will decrease cardiovascular
`disease incidence.
`
`REFERENCES
`
`Allen RH, Stabler SP, Savage DG, Lindenbaum J (1990) Diagnosis of cobalamin deficiency 1:
`usefulness of serum methylmalonic acid and total homocysteine concentrations. Am J Hematol
`34:90 98.
`Arnadottir M, Brattstr6m L, Simonsen O, et al (1993) The effect of high dose pyridoxine and folic
`acid supplementation on serum lipid and plasma homocysteine concentrations in dialysis patients.
`Clin Nephro140: 236 240.
`Arnesen E, Refsum H, Bonaa KH, Ueland PM, Forde OH, Nordrehaug JE (1995) Serum total
`homocysteine and coronary heart disease. IntJEpidemiol 24:704 709.
`Brattstr6m LE, Israelsson B, Jeppson JO, Hultberg BL (1988) Folic acid an innocuous means to
`reduce plasma homocysteine. ScandJ Clin Lab Invest 48:215 221.
`Bostom AG, Jacques PF, Nadeau MR, Williams RR, Ellison RC, Selhub J (1995) Post-methionine
`load hyperhomocysteinemia in persons with normal fasting total plasma homocysteine: initial
`results from the NHLBI Family Heart Study. Atherosclerosis 116:147 151.
`Boushey C J, Beresford SAA, Omenn GS, Motulsky AG (1995) A quantitative assessment of plasma
`homocysteine as a risk factor for vascular disease. JAmMedAssoc 274:1049 1057.
`Brody T (1991) Folic acid. In Machlin LJ, ed. Handbook of Vitamins. New York: Marcel Dekker,
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`J. ]nher. Metab. Dis’. 20 (1997)
`
`Sandoz Inc.
`Exhibit 1041-0008
`
`

`
`324
`
`Ubbink
`
`Carey MC, Fennelly JJ, Fitzgerald O (1968) Homocystinuria: subnormal serum folate levels,
`increased folate clearance and effects of folic acid therapy. AmJMed 45:26 30.
`Carson NAJ, Carre IJ (1969) Treatment of homocystinuria with pyridoxine. A preliminary study.
`Arch Dis Child 44:387 392.
`Chauveau P, Chadefaux B, Coude M, Aupetit J, Kamoun P, Jungers P (1996) Long-term folic acid
`(but not pyridoxine) supplementation lowers elevated plasma homocysteine level in chronic renal
`failure. Miner Electrolyte Metab 22:106 109.
`Chern CJ, Beutler E (1975) Pyridoxal kinase: decreased activity in red blood cells of Afro-
`Americans. Science 187:1084 1086.
`Clarke R, Daly L, Robinson K, et al (1991) Hyperhomocysteinemia: an independent risk factor for
`vascular disease. NEngl JMed 324:1149 1155.
`Dalery K, Lussier-Cacan S, Selhub J, Davignon J, Latour Y, Genest J (1995) Homocysteine and
`coronary artery disease in French Canadian subjects: relation with vitamins B12, B~ pyridoxal
`phosphate and folate. Am J Cardio175:1107 1111.
`Doscherholmen A, Hagen PS (1957) A dual mechanism of vitamin B12 plasma absorption. JClin
`Invest36:1551 1557.
`Dudman NPB, Wilcken DEL, Wang N, Lynch JF, Macey D, Lundberg P (1993) Disordered
`methionine/homocysteine metabolism in premature vascular disease. Its occurrence, cofactor
`therapy, and enzymology. Arterioscler Thromb 13:1253 1260.
`Ellenbogen L, Cooper BA (1991) Vitamin B12. In Machlin L J, ed.Handbook of Vitamins. New York:
`Marcel Dekker, 491 536.
`Franken DG, Boers GHJ, Blom H J, Trijbels JMF (1994a) Effect of various regimens of vitamin B6
`and folic acid on mild hyperhomocysteinaemia in vascular patients. J Inher Metab Dis 17:
`159 162.
`Franken DG, Boers GHJ, Blom HJ, Trijbels FJM, Kloppenborg PWC (1994b) Treatment of mild
`hyperhomocysteinemia in vascular disease patients. Arterioscler Thromb 14:465 470.
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`J. ]nher. Metab. Dis’. 20 (1997)
`
`Sandoz Inc.
`Exhibit 1041-0009
`
`

`
`Vitamins and homocysteine
`
`325
`
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`
`J. ]nher. Metab. Dis’. 20 (1997)
`
`Sandoz Inc.
`Exhibit 1041-0010