Reprintedfrom Trends in Pharmacological Sciences - October 1990
`
`Vol. II, No. 10
`
`411
`
`
`
`Clinical significance of
`pharmacological modulation of
`homocysteine metabolism
`
`Helga Refsum and Per Magne Ueland
`
`The metabolic fate of homocysteine is linked to vitamin B”, reduced folates,
`vitamin B.-, and sulfur amino acids. Clinical and experimental data suggest that
`elevated plasma homocysteine is an independent risk factor for premature
`vascular disease. This is particularly significant because plasma homocysteine
`levels are altered in several diseases,
`including folate and vitamin B”
`deficiencies, and because many commonly used drugs have now been shown to
`interfere with homocysteine metabolism.
`In summarizing the data, Helga
`Refsum and Per Ueland highlight the clinical implications for these metabolic
`changes.
`
`ubiquitous enzyme methionine
`synthase (Fig. 1). This enzyme re-
`quires vitamin B12 |methy|(l)cobal-
`amin] as a cofactor and 5-metl1yl-
`tetrahydrofolate as methyl donor;
`thus5-methyltetrahydrofolateenters
`the pool of reduced folates, and
`homocysteine is remethylated to
`methionine‘.
`There
`are
`
`cobalamin-
`
`two
`
`dependent enzymes in vertebrates:
`methionine synthase which util-
`izes methy|cob(I)alamin and is
`cytosolic,
`and methylmalonyl-
`CoA mutase which is a mitochon-
`drial enzyme containing aden-
`osylcobalamin. A major fraction of
`intracellular cobalamin is associ-
`ated with these two enzymes?
`Homocysteine remethylation is
`also catalysed by an alternative
`enzyme,
`betaine—homocysteine
`methyltransferase, requiring be-
`taine as methyl donor. However,
`this enzyme is generally confined
`to the liver.
`
`The metabolism of homocys-
`teine along the trans-sulfuration
`pathway is catalysed by two vit-
`amin B6-dependent enzymes. The
`first step is the cystathione [3-
`synthase reaction, where homo-
`cysteine is condensed with serine
`to
`form cystathionine. Cysta-
`thionine is then cleaved to <x-l<eto-
`butyrate and cysteine, catalysed
`by cystathionine lyasel.
`
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`Homocysteine is a sulfur amino
`acid which is not itself incorpor-
`ated into proteins, but is import-
`ant as an intermediate in the
`metabolism of methionine and
`cysteine, and because its metab-
`olism is linked to the function of
`some vitamins.
`In the late 1960s, inborn errors
`homocysteine metabolism
`of
`(homocystinuria) were demon-
`strated in patients with mental
`retardation,
`skeletal abnormali-
`ties, lens dislocation and prema-
`ture vascular disease‘. Research
`
`into the physiological and patho-
`logical roles of homocysteine was
`subsequently promoted. Since the
`pioneering work of Wilcken and
`coworkers in 1976, accumulated
`epidemiological and experimental
`evidence has shown that homo-
`
`cysteine may provoke vascular
`lesions, and that moderate homo-
`cysteinemia is an independent
`risk factor for premature vascular
`diseasez.
`im-
`During the last few years,
`proved analytical techniques have
`allowed
`the
`investigation
`of
`plasma homocysteine in healthy
`subjects and in disease states
`other than homocystinuria. Folate
`and cobalamin (vitamin B12) de-
`ficiencies cause very high plasma
`
`Helga Re[sum is Research Fellow (NAVF) and
`Per Magm: Ueland is Professor of Clinical
`Pharmacology and Head of
`the Clinical
`Pharmacology Unit at Haukelanrl Llniversity
`Hospital, N-5021 Bergen, Norway.
`
`levels of homocysteine, and plasma
`homocysteine has been estab-
`lished as a sensitive and respon-
`sive
`indicator
`of
`intracellular
`folate and cobalamin function3.
`Some drugs influence homo-
`cysteine metabolism and plasma
`levels. This may have some im-
`portant implications. First, plasma
`homocysteine may reflect pharma-
`codynamic effects of some drugs,
`as most clearly demonstrated with
`methotrexate and nitrous oxide
`(see below). Secondly,
`the in-
`creased plasma levels induced by
`some agents may have impli-
`cations
`for
`their
`side-effects.
`Finally, drugs decreasing plasma
`homocysteine may reduce the risk
`of vascular disease imposed by
`homocysteinemia3.
`
`Homocysteine and vascular
`disease
`Patients with homocystinuria
`suffer
`from premature vascular
`disease,
`localized to the central
`and peripheral arteries and large
`veins. This is the major cause of
`the high mortality (20—75% before
`the age of 30)
`in these patients.
`Thromboembolism may occur at
`any age and has even been de-
`Homocysteine metabolism
`scribed in children‘. Both clinical
`Homocysteine holds a unique
`and
`experimental
`evidence
`position in metabolic regulation.
`Its metabolism is linked to sulfur
`suggest
`that high homocysteine
`levels cause the vascular lesions
`amino acids, reduced folates and
`(see Ref. 1).
`vitamins B12 and B6.
`lts metab-
`Even moderate homocystein-
`olism is summarized in Fig. 1.
`emia may provoke venous throm-
`The only source of homocys-
`teine in vertebrates is the hydroly-
`bosis
`and premature
`vascular
`lesions in the cerebral, peripheral
`sis of S-adenosylhomocysteine, an
`and coronary arteries (L. Brati-
`inhibitor and product of S-aden-
`strom, Thesis, University of Lund,
`osylmethionine-dependent trans-
`1989). Such a relation was suggested
`methylation‘. The fate of
`intra-
`cellular homocysteine is either
`10 years ago from clinical studies
`based on measurement of acid-
`salvage to methionine through
`soluble mixed disulfides in plasma
`remethylation, or conversion to
`cysteine via the trans-sulfuration
`from a small number of patientsz.
`This has later been confirmed in
`pathway.
`In most
`tissues,
`the
`former reaction is catalysed by the
`several
`investigations, most of
`©1990, Elsovior Science Publishers Ltd. (UK)
`0165 - (-1-I7I‘l0l$u2.ti:J
`
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`

`
`412
`
`Protein
`
`Melhlonlne
`(extracellular)
`
`Serine
`
`/\AdoMet
`
`Methionine
`
`THF
`
`Dlmettlyl-
`glycine
`
`Betaine
`
`Bu @ “IMP
`
`d1‘MP
`
`DHF
`
`_
`
`Glycine
`
`5,10-CH2-THF
`
`AdoHcy
`W_ l-lomocystelne
`
`we
`
`Cystnthionine
`
`5 - CH3-THF 4—— 5-CHO-’I'Hl-‘
`
`./
`'3
`
`Follc acld
`
`..;l
`
`TiP$ — October 1990 (Vol. 717
`
`elevated plasma homocys-
`teine levelsz.
`
`Folate and cobalamin
`deficiency
`Cobalamin deficiency may
`increase plasma homocys-
`teine to levels observed in
`homocystinurics (high micro-
`molar range); there is a nega-
`tive
`correlation
`between
`serum cobalamin and total
`plasma homocysteine. Homo-
`cysteine levels may also be
`elevated
`in
`cobalamin-
`deficienl patients devoid of
`typical signs
`like vanemia,
`macrocytosis
`and
`reduced
`serum cobalamin. Levels are
`normalized following cobal-
`amin therapy3-'3.
`Similarly, folate deficiency
`is a common cause of el-
`evated plasma homocysteine
`levels, and a close negative
`correlation with serum folate
`has
`been
`demonstrated;
`again, folate therapy normal-
`izes levels. Moderate elevation
`
`Be
`
`Homocysteine
`(extracellular)
`Cysteine
`I.
`Fig.
`Homocysteine metabolism. AdoHcy, S~adenosyIhomocysteine; Adomot,
`S-adenosylmethionine; 5.10-CH,-THF. 5,10-methylene THF; 5-CH,-THF. 5-melhyI-
`tetlahydrololate; 5-CHO-THF, 5-Iormyltetrahydrololate (lalinate); CL, cystathionine
`Iyase: OS, cystathionine B-synthase: DHF, dlhydrololate; DH. dihydrololate reductase;
`MET, methionine; MS, methionine synthase (5-methyl-THF homocysteine methyItrans-
`lerase); SA. S-adenosylhomocysteina nydrolase: SH, serine nydroxymeIhyIIransIer-
`ase; THF, tetrahydrololate.
`
`which determined total plasma
`homocysteine3'°'1°. Furthermore,
`an increased incidence of hetero-
`zygous homocystinuria has been
`demonstrated in patients with
`early onset vascular disease7.
`Other clinical and experimental
`data support an association be-
`tween raised homocysteine levels
`and vascular lesions (reviewed in
`Ref.
`3). Plasma homocysteine
`levels show age- and sex-depen-
`dent variations resembling those
`described
`in
`arteriosclerotic
`disease. Men and postmenopausal
`women
`have
`higher
`plasma
`homocysteine levels during last-
`ing and alter methionine loading
`than
`young women.
`Plasma
`homocysteine is significantly in-
`creased in chronic renal failure,
`severe psoriasis, and in some
`patients with cancer. These are
`conditions associated with in-
`creased risk of vascular disease
`not adequately explained by risk
`factors like smoking, lipid abnor-
`malities, hypertension or other
`known predisposing conditions’.
`Moderate homocysteinemia should
`be considered as a possible cause
`of vascular disease in those cases
`(15-30°/o) when other risk factors
`cannot be identified. Down syn-
`drome, on the other hand, is an
`abnormality
`characterized
`by
`low plasma homocysteine levels,
`
`probably due to increased gene
`dosage for the enzyme cystathio-
`nine B-synthase
`residing
`on
`chromosome 21 (Ref. 11). In 1977
`Murdoch and co-workers suggested
`this state as an atheroma-free
`model because of the remarkable
`absence of arteriosclerotic lesions
`observed in five patients aged 44-
`66years12.
`direct
`also more
`is
`There
`that high
`levels
`of
`evidence
`homocysteine
`mediate
`the
`thrombogenesis and accelerated
`atherogenesis observed in homo-
`cystinuria, and that homocysteine
`may also be responsible for the
`vascular disease associated with
`moderate homocysteinemia. Homo-
`cysteine damages human endo-
`thelial cells in culture, possibly by
`producing hydrogen peroxide in
`an oxygen-dependent
`reaction.
`Moreover, endothelial cells from
`patients heterozygous for cysta-
`thionine
`[3-synthase gene de-
`ficiency may have an increased
`susceptibility to injury by homo-
`cysteine. Mechanisms linking mild
`homocysteinemia
`and vascular
`effects could also involve produc-
`tion of free radicals and oxidation
`of low~density lipoprotein. Con-
`flicting data exist on possible roles
`of platelet sequestration in the
`development
`of
`atherosclerotic
`lesions
`under
`conditions
`of
`
`of plasma homocysteine is
`also observed in subjects with low
`but normal serum folate levels,
`suggesting that
`increased homo-
`cysteine levels in these subjects
`may reflect an intracellular folate
`content
`insufficient
`for optimal
`folate-dependent remethylation of
`homocysteine3.
`Measurement of plasma homo-
`cysteine is therefore a promising
`laboratory
`test
`for
`evaluating
`cobalamin or
`folate deficiency
`states. lt may be particularly use-
`ful when used in conjunction with
`serum methylmalonic acid, which
`is a specific measure of disturb-
`ances of cobalamin metabolism“.
`
`Agents decreasing homocysteine
`concentrations
`
`co-
`serving as
`Compounds
`factors
`in homocysteine catab-
`olism or remethylation may en-
`hance homocysteine metabolism
`and thereby reduce plasma homo-
`cysteine levels in inherited en-
`zymic defects. Thus, vitamin B6
`reduces plasma homocysteine in
`homocystinurics with
`residual
`cystathionine fi—synthase activity,
`and vitamin B12 acts similarly in
`some mutations of
`cobalamin
`metabolism. Betaine and folic acid
`have been shown to efficiently
`reduce plasma homocysteine in
`patients with cystathionine l3-syn-
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`
`TiPS — October 1990 I Vol. 11]
`
`thase deficiency who are un-
`responsive to vitamin B6 (Ref. 1).
`
`Folic acid
`Folic acid (5 mg daily) efficiently
`decreases plasma homocysteine
`levels. It reduces elevated plasma
`homocysteine in renal transplant
`recipients and even in those with-
`out overt folate deficiency. Treat-
`ment of healthy subjects with folic
`acid for 14 days significantly re-
`duced
`plasma
`homocysteine,
`especially in persons with high
`pretreatment levels“.
`The marked effect of high doses
`of folic acid on the concentration
`of homocysteine in plasma is im-
`portant. Since moderate homo-
`cysteinemia may provoke vascular
`lesions,
`folic acid may prevent
`atherosclerotic disease in selected
`subjects. This
`intervention is
`particularly
`attractive
`because
`folic acid intake has essentially no
`side-effects“.
`Interestingly,
`the
`effect of folic acid suggests that
`the intracellular
`folate content
`is
`insufficient
`for an optimal
`remethylation of homocysteine.
`This may be a more common state
`than hitherto recognized, as
`it
`may not be detected by estab-
`lished
`laboratory
`procedures,
`including determination of folate
`in serum or erythrocytes’.
`Folic acid probably decreases
`plasma homocysteine levels by in-
`creasing the availability of intra-
`cellular 5-methyltetrahydrofolate,
`thereby enhancing homocysteine
`remethylation (Fig. 2). This re-
`action
`forms
`tetrahydrofolate,
`which enters the pools of reduced
`folates carrying one-carbon units
`via
`the
`serine hydroxymethyl
`transferase reaction (Figs 1 and 2).
`In this reaction, serine is con-
`sumed and glycine formed, as
`would be
`expected
`from the
`moderate reduction in plasma
`serine
`levels and increase
`in
`plasma glycine levels that follow
`folic acid administration".
`
`D-Penicillamine
`D-Penicillamine (D-[3,[3-dimethyl-
`cysteine) is currently used for the
`treatment of heavy metal poison-
`ing, rheumatoid arthritis, hepato-
`lenticular degeneration, cystinuria
`and scleroderma. It is metabolically
`stable, chelates heavy metals, pro-
`duces disulfides and forms a thi-
`azolidine ring with aldehydes and
`ketones. In plasma, penicillamine
`forms symmetrical penicillamine
`
`disulfides and mixed disulfides
`with cysteine, homocysteine and
`plasma proteins. The low mol-
`ecular weight disulfides undergo
`rapid renal excretion, which ex-
`plains the short plasma half-life
`and
`the
`therapeutic effect
`in
`cystinuria“.
`re-
`efficiently
`Penicillamine
`duces both free and protein-
`bound plasma homocysteine in
`homocystinurics“
`and
`total
`plasma homocysteine in patients
`with rheumatoid arthritis, a con-
`dition with normal homocysteine
`pretreatment
`levels". This
`re-
`duction in plasma levels may be
`associated with an intracellular
`homocysteine
`depletion
`suf-
`ficiently pronounced to decrease
`homocysteine remethylation and
`thereby induce methionine de-
`ficiency and secondary effects on
`folate metabolism. If such effects
`occur, penicillamine may act as an
`antifolate agent and may therefore
`interact adversely with metho-
`trexate used in low doses in the
`management of
`rheumatoid ar-
`thritis (see below)“.
`A further clinical implication of
`this work is that penicillamine
`may be a useful means to reduce
`plasma homocysteine.
`
`Adenosine (nucleoside) analogs
`The
`cleavage of S-adenosyl-
`homocysteine to adenosine and
`homocysteine, catalysed by S-
`adenosylhomocysteine hydrolase
`(Figs 1 and 2), is the only known
`source of homocysteine in ver-
`tebrates. Several nucleoside ana-
`logs block this reaction by serving
`either as an inactivator or
`in-
`
`hibitor of the enzyme. In addition,
`some analogs act as substrate and
`are thus converted to the corre-
`sponding S-adenosylhomocysteine
`analogue‘.
`Inhibition
`of
`S-
`adenosylhomocysteine hydrolase
`leads to massive accumulation of
`S-adenosylhomocysteine in iso-
`lated cells, whole animals, and in
`patients. This is important for the
`antiviral effects of
`this class of
`compound‘‘'‘‘’.
`immediate conse-
`The other
`quence of S-adenosylhomocys-
`teine hydrolase inhibition,
`re-
`duction
`of homocysteine
`for-
`mation (Fig. 2), has been studied
`only
`recently.
`Homocysteine
`depletion and inhibition of homo-
`cysteine export have been demon-
`strated in isolated cells exposed to
`nucleoside analogs’°'“. A reduc-
`
`413
`
`tion in plasma homocysteine was
`found in patients with acute
`leukemia treated with 2-deoxy-
`coforrnycin, which indirectly in-
`activates S-adenosylhomocysteine
`hydrolasezzl’.
`lnhibition
`of
`homocysteine formation plays an
`important role in the cytostatic
`action of some nucleoside analogs
`against some“-25 but not all2"'2°
`cell types, and probably mediates
`the differentiation of HL-60 cells
`induced by adenosine dialde-
`hyde”. The consequences of cellular
`homocysteine deficiency are two-
`fold. First, nucleoside analogs may
`induce severe methionine de-
`ficiency, since homocysteine sal-
`vage is
`a significant source of
`methionine in humans3°v3‘. Sec-
`ondly, lack of homocysteine may
`trap reduced folate as 5-methyl-
`tetrahydrofolate because homo-
`cysteine is the methyl acceptor in
`the methionine synthase reaction
`catalysing
`the
`conversion
`of
`5-methyltetrahydrofolate to tetra-
`hydrofolate. In this way, lack of
`tetrahydrofolate may ensue, and
`thereby inhibit folate-dependent
`purine and thymidylate synthesis.
`Both mechanisms
`have
`been
`demonstrated in cultured cells?‘-33.
`lnduced deficiencies of meth-
`ionine
`and
`folates might be
`avoided clinically by appropriate
`supplementation which would be
`expected to
`reduce
`associated
`side-effects. Moreover,
`adverse
`interactions of adenosine ana-
`logues with drugs that also inter-
`fere with folate or methionine
`metabolism such as nitrous oxide
`or methotrexate should be con-
`sidered. This has been suggested
`for the antiviral agent vidarabine
`(9-fl-D-arabinofuranosyladenine)
`and high-dose methotrexate with
`folinic acid rescue-"’°. Reduction in
`plasma homocysteine levels might
`however have beneficial vascular
`effects.
`
`Inhibitors of homocysteine
`remethylation and degradation
`Several important drugs interfere
`with homocysteine metabolism.
`
`Nitrous oxide
`The anesthetic agent nitrous
`oxide was used for a century be-
`fore it was discovered that long-
`term exposure caused megalo—
`blastic and aplastic bone marrow
`changes, anemia and myelopathy.
`
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`
`Folic acid
`
`Folic acid
`
`/
`5- CH3 -THF
`
`Hey
`
`Bu
`
`Met
`
`Nitrous oxide
`
`DHF\W—> THF
`5.10-CH,-THF /
`5- CH 3-THF
`/$3"
`
`Folic acid
`
`M3‘
`
`BIZ
`CH3oob(l)alamin
`c‘‘‘ \ \
`Hcy-SH
`(extracellular)
`
`Ado
`
`H9)’
`9‘.
`Cystathioninc
`$6
`Cysteine
`
`414
`
`TiPS — October 1990 lVol. 11]
`
`Methotrexate
`
`DI1:w?7w
`
`s.mcn,.-nu=
`i
`5. cu ,.m=
`
`HCY
`
`B 12
`
`Adenosine analogues
`
`AdoHcy
`
`Cystathionine
`)6
`Cysteine
`
`D-Penicillamine
`(P-SH)
`
`R-S-S-R 4
`
`5- CH3 -THFTHF'
`
`ctI,uos(tI)ul.msn Mu
`inactive B P
`
`excretion
`
`(extracellular)
`R-S-S-R \£
`Hey-S-S-R
`
`Fig. 2. Proposed or established mechan-
`isms for the effect of various agents on
`the metabolism and plasma level of
`homocystelne. The figure tn the center
`indicates normal metabolism and the six
`panels in the boxes show the effects of
`various drugs on different pathways.
`Sizes of type and arrows indicate the
`concentration of a particular metabolite
`and the flux through the pathway, re-
`spectively. Folic acid probably increases
`the cellular content of 5-melhyltetrahydrofolate (5-CH3-THF), which increases the homocysleine (Hey) remelhylation catalysed by
`methionine synthase (MS). n-Penicillamine (P-SH) forms a mixed disulfide with homocysteine (Hey-Sps-P): this disulfide has a high renal
`clearance. Adenosine analogs are inhibitors of S-adenosylhomocysteine hydrolase (SA). Nitrous oxide oxidizes methylcobtltalamin to
`melhylcob(ll)alamin and thereby irreversibly inhibits methionine synthase (MS). Methotrexale inhibits dihydrofolate reductase (DR),
`thereby inhibiting regeneration of letrahydrololate (THF) from dihydrofolate (DHF). Tetrahydrofofate supplies 5-methyltetrahydrofolate via
`5.10-melhylenetefrahydrofolale (5.10-CH,-Tl-IF). Methotrexate may therefore induce depletion ol 5-methyltetrahydrololate. and inhibit
`homocysteine remethylation catalysed by methionine synthase. Azauridine is a vitamin B6 antagonist, and may inhibit some vitamin B5-
`dependenl enzymes. including cystalhianine ti-synlhase (CS).
`Hey, homocysleine; Met, methionine; Ado. adenosine; P-SH, o-penicillamlne reduced form; Hey-S-S-R, homocysteine mixed disulfide;
`Hcy-S-S-P. a mixed disullide between homocysteine and o-penicillamina: P-S-S-R. o-penicillamine mixed disulfide.
`For other abbreviations, see Fig. 1.
`
`These side-effects are similar to
`the
`symptoms of vitamin 8,2
`deficiency-33.
`Nitrous oxideoxidizes the cob-
`alamine
`species methylcob(I)-
`alamine,
`inactivating specifically
`the enzyme methionine synthase,
`
`without affecting the adenosyl—
`cobalamine cofactor of methyl-
`malonyl-CoA mutase33.
`Inacti-
`vation of methionine synthase
`causes a cascade effect on folate
`metabolism including trapping of
`reduced folates such as 5-methyl-
`
`loss of folate in
`tetrahydrofolate,
`the urine, and reduction in tissue
`folate levels. There is
`a
`sub-
`sequent decrease of folate-depen-
`dent
`purine
`and
`thymidylate
`synthesis, as demonstrated by the
`deoxyuridine suppression (dU)
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`
`TiPS — October 1990 IVol. 11 I
`
`test, which reveals changes in
`human bone marrow within 5-6
`hours. In humans, 50% of meth-
`ionine synthase is inactivated in
`about two hours, and there is a
`reduction in plasma methionine
`after 8-24 hours of exposure.
`Megaloblastic
`bone
`marrow
`changes can be detected after 12-
`24 hours of exposure”.
`Data on the effect of nitrous
`oxide on homocysteine metab-
`olism are sparse, but this drug has
`been reported to increase the
`urinary excretion of homocysteine
`in sheep, and increase plasma
`homocysteine levels in fruit bats3.
`We have recently demonstrated
`that nitrous oxide
`induces
`a
`marked increase in plasma homo-
`cysteine within 90 minutes, with a
`concurrent
`increase in urinary
`homocysteine excretion. In patients
`receiving nitrous oxide for 3-6
`hours, plasma homocysteine re-
`mained above normal for at least 7
`days (Ermens, A. A. M. et al., un-
`published data). The homocysteine
`response evolved before other
`early signs of nitrous oxide-
`induced cobalamine inactivation”.
`Thus, significant cobalamine oxi-
`dation and methionine synthase
`inactivation
`occurs
`even after
`short-term exposure, which pre-
`viously has not been regarded as
`harmful. The resultant increase in
`plasma homocysteine observed in
`some patients may in itself be
`detrimental.
`Furthermore,
`the
`homocysteine response may re-
`flect loss of functional cobalamine
`and folate, and may enhance sen-
`sitivity towards antifolate drugs
`such as methotrexate-"‘
`(Ermens,
`A. A. M., PhD Thesis, University
`of Rotterdam, 1990). Since an in-
`crease in plasma homocysteine
`levels is both an early and sensi-
`tive measure of cobalamine oxi-
`dation,
`plasma
`homocysteine
`monitoring could be useful in the
`detection of such effects in the
`clinic.
`
`Methotrexate
`Methotrexate is an antifolate
`drug which has been used exten-
`sively in intermediate and high
`doses in the treatment of leukemia
`and some solid tumors. Low-dose
`methotrexate
`is
`used
`in
`the
`management of some non-malig-
`nant diseases such as rheumatoid
`arthritis and psoriasis.
`Methotrexate acts by inhibiting
`dihydrofolate reductase,
`thereby
`
`blocking the regeneration of tetra-
`hydrofolate
`from dihydrofolate
`(see Ref. 35). This leads to deple-
`tion of reduced folates, including
`5-methyltetrahydrofolate3°-37. Thus,
`methotrexate may also inhibit the
`folate-dependent remethylation of
`homocysteine catalysed by methio-
`nine synthase. This would explain
`the increased homocysteine export
`from cultured cells exposed to
`methotrexate, and the methotrex-
`ate-induced homocysteinemia and
`urinary homocysteine excretion in
`patients”.
`Low-dose methotrexate (25mg
`daily) given to psoriatics induced
`increased plasma homocysteine
`levels, which maximized after
`about two days, and normalized
`within one week”. This shows
`that plasma homocysteine is a
`sensitive measure of the antifolate
`effect.
`(1—13.6g)
`lntermediate doses
`given
`to
`patients with
`solid
`tumors, induced a rapid increase
`in plasma homocysteine within
`hours, which was reversed on
`administration of folinic acid 24
`hours after start of infusion. This
`response was observed following
`several methotrexate doses in a
`single patient‘°. High doses of
`methotrexate (8—33.6 m—2) given to
`children gave a similar response,
`i.e. a rapid increase a few hours
`after start of administration and
`a
`decline
`following
`‘rescue’
`therapy“. This is analogous to the
`results obtained with cultured
`cells”.
`in
`Basal homocysteine levels
`patients with acute lymphoblastic
`leukemia were often above normal
`before treatment, and declined
`markedly
`following
`treatment
`with cytotoxic agents including
`methotrexate (Refsum, H. et al.,
`unpublished). This may be due to
`eradication of proliferating cells
`exporting large amounts of homo-
`cysteine.
`The
`high-dose methotrexate
`regimen also induced a transient
`but marked reduction in plasma
`methionine“ which may contrib-
`ute to the killing of tumor cells or
`toxicity of methotrexate. Plasma
`homocysteine
`response
`and
`methionine depletion may corre-
`late with the therapeutic as well as
`the side-effects of methotrexate,
`including liver toxicity“ and an
`increased incidence of thrombo-
`
`embolism“; plasma homocysteine
`measurements could provide a
`
`415
`
`to serum metho-
`useful adjunct
`trexate
`determination
`in
`the
`management
`of methotrexate
`therapy.
`
`Vitamin B5 antagonists
`Azauridine is an antimetabolite
`interfering with de novo synthesis
`of uridine-5’-monophosphate.
`It
`was initially used for the treat-
`ment of refractory psoriasis, but
`was withdrawn by the FDA in
`1976 because its use was associ-
`ated with an increased incidence
`
`of vascular episodes“. This may
`be due to effects on homocysteine
`metabolism. Azauridine causes
`homocysteinemia, abnormal homo-
`cysteine excretion and a signifi-
`cant increase in serum methionine
`
`levels in patients. Studies in rab-
`bits suggest that it functions as a
`pyridoxal 5’-phosphate antagonist
`and causes homocysteinemia by
`inhibiting vitamin B6-dependent
`cystathionine
`synthesis“. This
`suggests
`that
`supplementing
`vitamin B6 would prevent the in-
`hibition of homocysteine catab-
`olism; determination of plasma
`homocysteine
`may
`identify
`patients at risk of vascular epi-
`sodes.
`Several other drugs also inter-
`fere with the function of vitamin
`B6:
`isoniazid,
`cycloserine, hy-
`dralazine, penicillamine, phenel-
`zine and procarbazine“. Pertur-
`bation of homocysteine metab-
`olism in patients has been demon-
`strated with isoniazid“. In one
`out of six patients given 300mg
`isoniazid daily for one month,
`urinary homocysteine excretion
`was fivefold higher than normal.
`Inhibition
`of
`cystathionine
`metabolism in these patients is
`supported by increased excretion
`of
`this compound after meth-
`ionine loading“.
`
`Otheragents
`Premenopausal women have
`lower plasma homocysteine than
`men
`and
`postmenopausal
`women“, and plasma levels are
`low during pregnancy. However
`there is no conclusive evidence
`that homocysteine metabolism
`and plasma homocysteine levels
`are under the influence of estro-
`gens. Preliminary data in women
`given contraceptive steroids or the
`antiestrogen tamoxifen suggest a
`polymorphic response.
`In some
`women, altered estrogen status
`may cause a decrease, and in
`
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`
`416
`
`others, an increase in plasma
`homocysteine levels. Because of
`the widespread use of contracep-
`tives, and the suggested use of
`tamoxifen as prophylactic inter-
`vention in healthy women at high
`risk of developing breast cancer.
`the effect on plasma homocysteine
`from altered estrogen status is a
`question of major concern to
`public health’.
`drugs,
`Various
`antiepileptic
`particularly phenytoin but also
`phenobarbital, primiclone, carba-
`mazepine, and valproic acid, may
`induce folate deficiency”. The
`activity of methylenetetrahydro-
`folate
`reductase,
`the
`enzyme
`producing
`5-methy|tetrahydro-
`folate,
`is altered in mouse liver
`following exposure to these drugs.
`Preliminary data
`suggest
`that
`phenytoin and possibly carbama-
`zepine may
`increase
`plasma
`homocysteine, but its relation to
`overt
`folate deficiency has not
`been established3.
`Several other drugs are known
`to interfere with folate metab-
`olism or function. These include
`some phenothiazines and tricyclic
`antidepressants, oral contracep-
`tives, possibly some tuberculo-
`static drugs, and antifolate drugs
`such as trimethoprim“. Thiols or
`disulfide forming drugs, such as
`cysteamine and N-acetylcysteine
`are actual candidates as modifiers
`of plasma homocysteine levels,
`due to a possibly unique dis-
`position of the mixed disulfides
`with homocysteine, as demon-
`strated with penicillamine
`in
`humans“.
`
`El
`
`E1
`
`U
`
`Various pharmacological agents
`have been shown to enhance
`homocysteine remethylation and
`urinary
`excretion,
`or
`inhibit
`homocysteine
`production,
`re-
`methylation or catabolism thereby
`affecting plasma homocysteine
`levels. Modulation of homocys-
`teine metabolism and plasma
`concentrations may be an import-
`ant component of drug action.
`
`Agents that reduce plasma homo-
`cysteine (e.g. folic acid, penicill-
`amine) may prevent
`vascular
`disease, while agents increasing
`plasma homocysteine (e.g. nitrous
`oxide, methotrexate, azauridine)
`may provoke vascular episodes.
`Also for some drugs (e.g. nitrous
`oxide and methotrexate), elevation
`of plasma homocysteine is an
`early and sensitive measure of
`drug action and plasma homocys-
`teine has been shown to be use-
`ful in the diagnosis and follow-up
`of some diseases,
`in particular
`homocystinuria, folale deficiency
`and cobalamine deficiency.
`References
`1 Mudd, S. H., Levy, H. L. and Skovby, F.
`(1989) in The Metabolic Basis for Inherited
`Diseases (Scriver, C. R. at al., eds), pp.
`693-734, McGraw-Hill
`2 Wilcken, D. E. L. and Dudman, N. P. B.
`(1989) Hnetnostasis 19 (suppl. 1), 14-23
`3 Ueland, P. M. and Relsum. H.
`(1989)
`I. Lab. Clin. Med. 114, 473-501
`4 Ueland, I’. M. (1982) Pharmacnl. Rev. 34,
`223-253
`5 Kolhouse, ]. F. and Allen, R. H. (1977)
`Proc. Natl Acad. Sci. USA 74, 921-925
`6 Brattstrom,
`L.,
`lsraelsson, B.
`and
`Hultberg, B.
`(1989) Haernostasis
`19
`(suppl. 1), 35-44
`7 Boers, G. H. J. (1989) Haemoslasis 19
`(suppt. 1), 29-34
`8 Kang, S-S.,Wong, P. W. l<.,Cool<,H. Y.,
`Norusis, M. and Messer, J. V.
`(1986)
`I. Clin. invest. 77, 1482-1486
`9 Mallnow, M. R. et nl. (1988) Circ. Res. 79,
`1180-1188
`10 Araki, A. et al. (1989) Atherosclerosis 79,
`139-146
`11 Chadefaux, B. et al. (1988) Lancet ii, 741
`12 Murdoch, J. C., Rodger,J. C., Rao, 5. S.,
`Fletcher, C. D. and Dunnigan, M. G.
`(1977) Br. Med. I. 2, 226-228
`13 Allen, R. H., Stabler, 5. l’., Savage, D. G.
`and Lindenbaum,
`J.
`(1990) Am.
`I.
`Hematol. 34, 90-98
`14 Anon. (1989) Nutr. Rev. 47, 247-249
`15 Joyce, D. A. (1989) Pharmacol. Ther. 42,
`405-427
`16 Kang, 5-S., Wong, 1’. W. K. and Curley,
`K. (1982) Perliatr. Res. 16. 370-372
`17 Kang, 5-S., Wong, P. W. K., Glickman,
`P. B., MacLeod, C. M. and Jaffe, l. A.
`(1986) I. Clin. Pharmacol. 26, 712-715
`18 Wilke, W. S. and Mackenzie, A. H.
`(1986) Drugs 32, 103-113
`19 De Clercq, E. (1987) Biochem. Pharmacol.
`36, 2567-2575
`20 Svardal, A. M.. Djurhuus, R. and
`Ueland, P. M. (1986) Mol. Pharmacol. 30,
`154-158
`21 Svardal, A. M., Djurhuus, R., Relsum,
`H. and Ueland, P. M. (1986) Cancer Res.
`
`I. 242.
`
`TiPS - October 1990 [VOL 11]
`46. 5095-5100
`22 Kredich, N. M. et al. (1981) Clin. Res. 29,
`541A
`23 Hershfield, M. 5. (1984) Cancer Treat.
`Syrup. 2, 29-32
`24 Kim, l-K., Aksamit, R. R. and Cantoni,
`C.
`L.
`(1932)
`I. Biol. Chem.
`257,
`14726-14729
`25 Wolfson, C., Chisholm, J., Tashjian,
`A. H. J., Fish, 5. and Abeles. R. H. (1986)
`I. Biol. Chem. 261, 4492-4498
`26 Djurhuus, R., Svardal, A. M. and
`Ueland, P. M. (1989) Clncer Res. 49,
`324-330
`27 De Clercq, E., Cools, M. and Ba|1arini,J.
`(1989)
`Biachem.
`Pharmacol.
`38,
`1771-1778
`28 Cools, M., Hasobe, M., De Clercq, E.
`and Borchardt, R. T.
`(1990) Biocitem.
`Pharmacol. 39, 195-202
`29 Pilz, R. 3., Van den Berghe, C. and Boss,
`G. R. (1987) Blood 70, 1161-1164
`30 Cantoni, G. L., Aksamit, R. R. and Kim,
`I-K. (1982) N. Engl. I. Med. 307, 1079
`31 Boss, C. R. and Pilz, R. B. (1984) I. Clin.
`Invest. 74, 1262-1268
`32 Boss, G. R.
`(1987) Biachem.
`425-431
`33 Nurm,J. F. (1987) Br. I. Amreslh. 59, 3-13
`34 Ermens, A. A. M., Schoester, M.,
`Spijkers, L. J. M., Lindemans, J. and
`Abels,
`J.
`(1989) Cancer Res.
`49.
`6337-6341
`35 Matherly, L H., Seither, R. L. and
`Goldman. l. D. (1987) Pharmncal. Ther.
`35. 27-56
`36 Allegra, C. J., Fine, R. L., Drake, J. C.
`and Chabner, B. A. (1986) I. Biol. Chem.
`261, 6478-6485
`37 Baram, J., Allegra, C. J., Fine, R. L. and
`Chabner, B. A. (1987) J. Clin. Invest. 79,
`692-697
`38 Ueland, P. M., Relsum, H., Male, R. and
`Lillehaug, J. R. (1986) I. Natl Cancer inst.
`77, 283-289
`39 Refsum, H., Helland, S. and Ueland,
`P. M. (1989) Clin. Phnrmacol. Tiler. 46.
`510-520
`and
`P. M.
`40 Relsum, H., Ueland,
`Kvinnsland, S.
`(1986) Cancer Res. 46,
`5385-5391
`41 Broxson, E. H., Stork, L. C., Allen, R. H.,
`Stabler, S. P. and Kolhouse, J. F. (1989)
`Cancer Res. 49, 5858-5862
`and
`l.
`42 Barak, A. T., Tuma, D.
`Beckenhauer, H. C. (1984) I. Am. Coll.
`Nuir. 3, 93-96
`43 Brattstriim, L., Ueland, P. M. and
`Refsum, H. (1988) N. Engl. I. Med. 319,
`443-444
`44 Drell, W. and Welcli, A. D.
`(1989)
`Pharmacol. Ther. 41, 195-206
`45 Krishnaswamy, K. (1974) lnt. J. Vitam.
`Nulr. Res. 44, 457-465
`46 Boers, G. H. J. (1988) in Genetic Suscepti-
`bility to Environmental Factors 1 Chal-
`lenge for Public Intervention (Smith, U.,
`Eriksson,S. and Lindgirde, F., eds), pp.
`35-42, Almqvist & Wiksell International
`47 Lambie, D. C. and Johnson, R. H. (1985)
`Drugs 30, 145-155
`
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