Colloquium: Homocyst(e)ine, Vitamins and
`Arterial Occlusive Diseases
`
`Vitamins as Homocysteine-Lowering AgentsI
`
`Department of Medicine, County Hospital, S-391 85 Kalmar, Sweden
`
`ABSTRACT Moderate hyperhomocysteinemia is, to-
`day, considered an established risk factor for cardio-
`vascular disease. A waded dose-response relationship
`between plasma homocysteine concentratkm over its
`full range and cardiovascular risk strongly supports
`causality. Therefore, intervention studies with homo-
`cystaine-lowerinfl vitamins are needed. Thls mlnl re-
`view shows that supplementation with foIIc acid not
`only markedly reduces elevated plasma homocysteine
`concentrations but also reduces normal homocysteine
`concentrations. FoIIc acid doses of < I mg/d may be
`effective. Suppiementatinn with a combination of folic
`acid and cyanocobalamin w~I secure full homocys-
`teine.lowerin~ effect end prevent occurrence of vitamin
`B- 12 deficiency durin~ the course of therapy. J. Nutr.
`126: 1276S-1280S, 1996.
`
`INDEXING KEY WORDS:
`
`¯ homocysteine * [olate * folicacid
`¯ vitamin B-12 ¯ vitamin
`
`There is rapidly accumulatin£ evidence that moder-
`ate hyperhomocysteinemi:~ is an independent risk factor
`for cardiovascular disease (Boushey et al. 1995, Selhub
`et el. 1995, Stampfer et al. 1992, Ueland et al. 1992}. To
`date, all but a few of over 75 studies, including a total
`of more than 15,000 investigated patients and controls,
`support this issue {rid1 reference List on request}. Both
`basal hyperhomocysteinemia and hyperhomocysteine-
`mia unmasked by a methionine load are markers for
`increased cardiovascular risk {Ueland et el. 1992}. More-
`over, the findings of a dose-response relationship be-
`tween plasma homocysteine concentration, over its full
`range, and the relative risk for IAmesen et el. 1995, Marl-
`now et al. 1993, Panchanmiti et el. 1994, Robinson et
`el. 1995, Perry et el. 1995) the prevalence of ISelhub et
`el. 1995} or the severity of cardiovasc.~d-r disease {Ub-
`bink et al. 1991} strongly supports causality. Now, we
`must focus on intervention studies to establish whether
`homocysteine lowering with vit~min.~ reduces cardio-
`vascular risk {Stampfer and M~]inow 1995}.
`
`0022-3166/96 $3.00 ~ 1996 American Institute of Nutrition.
`
`The dietary vitamins B-6, B-12 and folate and their
`synthetic oral counterparts, pyridoxine hydrochloride,
`cyanocobelamin and folic acid, serve as precursors of
`the cofactors for homocysteine metabolism, pyridoxel
`5-phosphate, methylcobalamin and methyltetrahydro-
`folate, respectively {Ueland et al. 1992}. In humans,
`vitamin B-6 deficiency does not result in basel hyperho-
`mocysteinemia (Miller et el. 1992}. In contrast, folate
`and vitamin B-12 deficiency may result in considerable
`hyperhomocysteinemia, which is rapidly normalized
`after replenishment with the deficient vitamin {Allen
`et al. 1990, BrattstrOm et el. 1988a, Kang et el. 1987,
`Stabler et al. 1988}. Even within thei~ normal ranges,
`the levels of serum or red cell folate and serum vitamin
`B-12 are strong determinants of plasma homocysteine
`concentration {Andersson et al. 1992, BrattstrOm et el.
`1994, Selhub et el. 1994, Ueland et al. 1993).
`In untreated young cases of genetically caused severe
`hyperhomocysteinemias {homocystinurias}, li~e-threat-
`ening cardiovascular events are frequent {Erbe 1986,
`Mudd et el. 1989}. In most cases, treatment with cofac-
`tots for homocysteine metabolism result in consider-
`able decreases of plasma homocysteine concentration
`[Mudd et el. 1989, Ueland et el. 1992}. In pyridoxine-
`responsive hyperhomocysteinemia it was statistically
`confirmed that homocysteine lowering reduces the
`number of cardiovascular events (Mudd et el. 1985}.
`The lack of reports on vascular events in cases of non-
`pyridoxine-responsive hyperhomocysteinemias on ef-
`fective homocysteine-lowering therapy with betaine,
`folic acid and/or vitamin B-12 suggests that homocys-
`teine lowering also in these cases reduces cardiovascu-
`lar risk.
`
`~ Presented as part of the colloquium "Homocyst{e}ine, Vitarnin.~
`and Arterial Occlusive Diseases" given at the EX-l~imental Biology
`’95 meeting, Atlanta, GA, on April 13, 1995. This symposium was
`sponsored by the American Institute of Nutrition. Guest ecfitors ~or
`the symposium were M. R. N~alinow, Oregon Regional Primate Re-
`search Center, Beaverton, OR, and M. J. Stamp~er, Haxvard School
`of Public Health, Cambridge, MA.
`
`1276S
`
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`Exhibit 1020-0001
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`JOINT 1020-0001
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`
`VITAMINS AS HOMOCYSTEINE-LOWERING AGENTS
`
`1277S
`
`Vitamins for lowering basal homocystelne
`concentration
`
`Renal insufiqciency results both in moderate hyperho-
`mocysteinemia and accelerated atherosclerosis (Wflcken
`et al. 1988). Several studies have consistendy shown that
`oral treatment with folic acid (5- I0 mg/dy) reduces renal
`hyperhomocysteinemia by a mean of 30-60% (Amadot-
`tier et al. 1993, Chauveau et al. 1994, Janssen et al. 1994,
`Wflcken et al. 1981, Wilcken et al. 1988). Oral pyridoxine
`has no homocysteine-lowering effect {Amadottir et al.
`1993, Wflcken et al. 1981).
`In two studies, including a total of 28 nonvitamin-
`deficient healthy subjects with mostly normal plasma
`homocysteine concentrations, we tested the homocys-
`teine-lowering effect of folic acid {5 mg/d for 2-4 wkl
`(BrattstrOm et al. 1985, BrattstrOm et al. 1988b). All
`but two with low homocysteine concentrations re-
`sponded to foIic acid, with reductions on average >30%
`and were most marked in those with high homocys-
`teine concentrations. Oral treatment over 2 wk with
`pyridoxine (40 rag/d) or cyanocobalamin {I mg/d) had
`no homocysteine-lowering effect (BrattstrOm et al.
`1988b). In another study, pyridoxine (120 mg/d for 6
`wk) had no effect on plasma homocysteine concentra-
`tion in 16 healthy subiects (BrattstrOm and Hultberg
`unpublished). On the basis of these observations, we
`proposed that the homocysteine-lowering effect of folic
`acid in nonfolate-deficient subjects is that excess folic
`acid after conversion to methyltetrahydrofolate in-
`creases the rate by which homocysteine is remethyl-
`ated to methionine. In contrast, excess vitamin B-12
`and pyridoxine will not decrease plasma homocysteine
`unless deficiency is present because these vitamins
`serve as coenzymes and not as cosubstrates as does
`methyltetrahydrofolate {Brattstr6m et al. 1988b}.
`Subsequently, we studied the effect of folic acid and
`pyridoxine in 20 moderately hyperhomocysteinemic
`patients with cardiovascular disease (BrattstrOm et al.
`1990). After pyridoxine {240 rag/d, for 2 wk) plasma
`homocysteine tended to increase, but aher another 2
`wk on pyridoxine with the addition of folic acid (10
`rag/d) all patients showed reduced homocysteine con-
`centrations, with 57% mean reduction. We also failed
`to show a homocysteine-lowering effect of high dose
`pyridoxine (300 mg/d for 12 wk) in 37 stroke patients
`(Lindgren, BrattstrOm and Hultberg unpublished). In
`two recent studies of patients with vascular disease
`and hyperhomocysteinemia (Glueck et al. 1995, van
`den Berg et al. 1994) and in one study of normal normo-
`homocysteinemic subiects [Haglund et al. 1993), the
`combination of pyridoxine (100-250 mg/d} and folic
`acid (5-10 mg/d) reduced plasma homocysteine by a
`mean of 51, 38, and 30%, respectively.
`In groups of consecutive patients with acute myocar-
`dial infarction of whom most were normohomocys-
`teinemic and all of whom had normal serum folate
`concentrations, we found that 2.5 and 10 mg of folic
`
`acid over 6 wk had similar homocysteine-lowering
`fect~ in both groups plasma homocysteine was reduced
`by a mean of 27% (Landgren et al. 19951. Reductions
`were seen in all but two patients, both with low homo-
`cysteine values. With a few exceptions the response to
`folic acid was proportional to the pretreatment homo-
`cysteine levels. These exceptional patients were hyper-
`homocysteinemic and had low or low normal serum
`vitamin B-12 concentrations. In one with a subnormal
`vitamin B-12 concentration and a partial response to
`folic acid, oral treatment with cyanocobalamin {2 rag/
`d for 2 wk) normalized plasma homocysteine.
`Hyperhomocysteinemia due to vitamin B-12 defi-
`ciency does not respond to folic acid therapy IAllen et
`al. 1990}. It is likely, that even in subjects with low
`normal vitamin B-12 concentrations ~ response to
`folic acid cannot be achieved unless vitamin B-12 is
`given concomitantly (Landgren et al. 1995). This view
`is supported by recent studies by Ubbink et al. 11993a,
`1993b, 19941. It was shown that men with moderate
`hypethomocysteinemia (>16.3/zmol/ll in most cases
`had suboptimal plasma vitamin B- 12 { <200 pmol]l} and
`folate {<5 nmol/ll concentrations {Obbink et al. 1993a}.
`Such men were in a 6-wk trim given either folic acid
`{0.65 mg]dJ, pyridoxine {10 mg]d}, cyanocobalamin 10.4
`mg/d) or the combination of these vitamins {Ubbink et
`al. 1994). Most but not all responded to folic acid, with
`the mean homocysteine concentration decreased from
`28.8 to 16.8 /~mol/1 (-42%), a posttreatment value,
`however, still above normal. Pyridoxine had no homo-
`cysteine-lowering effect, whereas cyanocobalamin de-
`creased plasma homocysteine by a mean of 15%. In
`contrast, all responded to the combination by a mean
`homocysteine reduction of 50% although homocys-
`teine values were not normalized in all subjects during
`this short trial. Because the majority of these men prob-
`ably had suboptimal vitamin B-12 status, homoeys-
`teine lowering could have been better if a higher cyano-
`cobalmin dose had been used or ff the treatment period
`had been extended for several weeks. There are recent
`results showing that high dose parenteral administra-
`tion of cobalamin decreases plasma homocysteine in
`subjects with normal vitamin B-12 levels (Arald et al.
`1993, Nilsson et al. 1994}.
`
`Vitamins for lowering postmethlonlne load
`hyperhomocystelnemla
`
`Several studies have shown that patients with pre-
`mature cardiovascular disease frequently respond to
`oral methionine loading tests {100 mg/kg body weight}
`with abnormally high increases in plasma homocys-
`teine concentrations (Ueland et al. 1992). There is’evi-
`dence to suggest that an abnormal response to methio-
`nine loading indicates impaired pyridoxal 5-phos-
`phate-dependent homocysteine catabolism, whereas
`an abnormally high basal homocysteine concentration
`
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`Exhibit 1020-0002
`
`JOINT 1020-0002
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`

`
`1278S
`
`SUPPLEMENT
`
`mainly reflects impaired vitamin B-12 and folate-de-
`pendent homocysteine remethylation {BrattstrOm et al.
`1990, Christensen and Ueland 1993, Miller et al. 1994}.
`In accordance with this and in contrast to the lack of
`effect of pyridoxine on basal homocysteine concentra-
`tions, several studies have shown that pyridoxine (100-
`250 mg]d) improves abnormal methionine loading
`tests in many but not all patients IBrattstrOm et al.
`1990, Dudman et al. 1993, Franken et al. 1994}. How-
`ever, when the combination of pyridoxine [100-250
`mg]dJ and folic acid {5-10 mg]d} was administered, all
`patients responded and the abnormality was mostly
`normalized {BrattstrOm et al. 1990, Dudman et al. 1993,
`van den Berg et al. 1994}. It has recently been demon-
`strated that methionine-rich meals normally cause
`slight increases in plasma homocysteine concentration
`(Guttormsen et al. 1994]. It is quite possible that sub-
`jects with abnormal methionine loading tests also re-
`spond abnormally to methionine-rich meals leading to
`transient periods of hyperhomocysteinemia, which
`could be normalized with combined pyridoxine and fo-
`lic acid therapy. This, however, warrants further study.
`
`Study cohort, Selhub et al. {1994} found a mean of 5.3
`/amol]l lower {-36%} plasma homocysteine concentra-
`tions in those with a high dietary intake of vitamins
`B-6, B-12 and folate than in those with a low intake of
`these vitamins.
`For intervention studies in cardiovascular disease
`patients, a combination of 1 mg folic acid and 0.4 mg
`cyanocobalamin is probably sufficient for effective ho-
`mocysteine lowering. This combination will be an in-
`nocuous means that not only normalizes hyperhomo-
`cysteinemia in most patients but also will reduce nor-
`mal homocysteine values, leading to a shift of the
`entire homocysteine distribution toward lower values.
`The latter is important because results of several stud-
`ies have shown a dose-response relationship between
`plasma homocysteine concentration over its full range
`and risk for cardiovascular disease. At present, there
`are not sufficient data to recommend intervention also
`against postmethionine load hyperhomocysteinemia
`with high dose pyridoxine therapy.
`
`Discussion and recommendations
`
`LITERATURE CITED
`
`What doses and what combination of vitamins
`should be recommended for long-term homocysteine
`lowering.Z For several reasons, it seems wise to combine
`folic acid and cyanocobalamin. First, folic acid seems to
`reduce almost all but low homocysteine levels. Second,
`cyanocobalamin will probably secure full folic acid re-
`sponsiveness. Third, in vitamin B-12 deficiency, erro-
`neous treatment with folic acid may correct the hema-
`tological abnormalities but elicit and deteroriate vita-
`min B-12 neuropathy {Chanarin 1994). Therefore,
`before start of therapy, vitamin B-12 deficiency must
`be excluded, and the combination must contain a dose
`of cyanocobalamin high enough to prevent the occur-
`rence of vitamin B-12 deficiency, even if complete in-
`trinsic factor deficiency develops during the course of
`therapy. Of oral administered cyanocobalamin only
`about 1% is passively absorbed to the blood (Berlin et
`al. 1968]. The normal daily intrinsic factor receptor-
`mediated uptake of vitamin B-12 is <2 /zg, which
`means that at least 0.2 mg of cyanocobalamin have to
`be administered {Adams et al. 1971}.
`There are recent data that suggest that modest doses
`of folic acid (< 1 rng]d} are sufficient for homocysteine
`lowering {Ubbink et al. 1994}. We found that subjects
`regularly taking multivitamins containing, among
`other vitamins, only 0.2-0.4 mg folic acid had si£nifi-
`candy lower plasma homocysteine levels {-22%} than
`subjects not taking multivitamins [Brattstr6m et al.
`1994]. Hitherto, unpublished results from the European
`Community Concerted Action Project on Homocys-
`teinemia and Vascular Disease are confirmative. More-
`over, in survivors of the ori#nal Framingham Heart
`
`Adams, I. F., Ross, S. K., Mervyn, L., Boddy, L. & King, P. (1971}
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`
`Sandoz Inc.
`Exhibit 1020-0003
`
`JOINT 1020-0003
`
`

`
`VITAMINS AS HOMOCYSTEINE-LOWERING AGENTS
`
`1279S
`
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`R. (1993bl Hyperhomocysteinemia and the response to vitamin
`supplementation. Clin. Invest. 71: 993-998.
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`Ueland, P. M., Refsum, H., Stabler, S. P., Ma!i~ow, M. R., Andersson,
`
`0
`
`0
`
`Sandoz Inc.
`Exhibit 1020-0004
`
`JOINT 1020-0004
`
`

`
`1280S
`
`SUPPLEMENT
`
`A. & Allen, R. H. (1993) Total homocysteine in plasma or serum:
`methods and clinical applications. Clin. Chem. 39: 1764-1779.
`van den Berg, M., l~ranken, D. G., Boers, G. H. J., Blom, H. J.,
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`bined vitamin B6 plus folic acid therapy in young patients with
`arteriosclerosis and hyperhomocysteinemia. J. Vasc. Surg. 20:
`933-940.
`
`Wilcken, D. E. L., Dudman, N. P. B., Tyrrell, P. A. & Robertson,
`M.R. 11988). l~olic acid lowers elevated plasma homocysteine in
`ckronic renal insufficiency: possible implications for prevention
`of vascular disease. Metabolism 37: 697-701.
`Wilcken, D. E. L., Gupta, V. J. & Betts, A. K. 11981) Homocysteine
`in the plasma of renal transplant recipients: effects of cofactors
`for methionine metabolism. Clin. Sci. 61: 743-749.
`
`Sandoz Inc.
`Exhibit 1020-0005
`
`JOINT 1020-0005