Reprintedfrom Trends in Pharmacological Sciences - October 1990
`
`Vol. ]1, 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 8 12 , reduced folates,
`vitamin 8 6 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 8]2
`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.
`
`Homocysteine is a sulfur amino
`acid which is not itself incorpor(cid:173)
`ated into proteins, but is import(cid:173)
`ant as an intermediate in the
`metabolism of methionine and
`cysteine, and because its metab(cid:173)
`olism is linked to the function of
`some vitamins.
`In the late 1960s, inborn errors
`homocysteine
`metabolism
`of
`(homocystinuria) were
`demon(cid:173)
`strated in patients with mental
`retardation,
`skeletal
`abnormali(cid:173)
`ties, lens dislocation and prema(cid:173)
`ture vascular disease l . Research
`into the physiological and patho(cid:173)
`logical roles of homocysteine was
`subsequently promoted. Since the
`pioneering work of Wi1cken and
`coworkers in 1976, accumulated
`epidemiological and experimental
`evidence has shown that homo(cid:173)
`cysteine may provoke vascular
`lesions, and that moderate homo(cid:173)
`cysteinemia is an independent
`risk factor for premature vascular
`disease2.
`im(cid:173)
`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 Bd de(cid:173)
`ficiencies cause very high plasma
`
`Helga Refslim is Research Fellow (NAVF) and
`Per Magne Ueland is Professor of Clinical
`the Clinical
`Pharmacology and Head of
`Pharmacology Unit at Hauke/and University
`Hospital, N-5021 Bergen, Norway.
`
`levels of homocysteine, and plasma
`homocysteine has been estab(cid:173)
`lished as a sensitive and respon(cid:173)
`sive
`indicator
`of
`intracellular
`folate and cobalamin function 3 .
`Some drugs
`influence homo(cid:173)
`cysteine metabolism and plasma
`levels. This may have some im(cid:173)
`portant implications. First, plasma
`homocysteine may reflect phamla(cid:173)
`codynamic effects of some drugs,
`as most clearly demonstrated with
`methotrexate and nitrous oxide
`(see below). Secondly,
`the in(cid:173)
`creased plasma levels induced by
`some agents may have
`impli(cid:173)
`cations
`for
`their
`side-effects.
`Finally, drugs decreasing plasma
`homocysteine may reduce the risk
`of vascular disease imposed by
`homocysteinemia 3 .
`
`Homocysteine metabolism
`Homocysteine holds a unique
`position in metabolic regulation.
`Its metabolism is linked to sulfur
`amino acids, reduced folates and
`vitamins 8 12 and 8 6 ,
`Its metab(cid:173)
`olism is summarized in Fig. 1.
`The only source of homocys(cid:173)
`teine in vertebrates is the hydroly(cid:173)
`sis of S-adenosylhomocysteine, an
`inhibitor and product of S-aden(cid:173)
`osylmethionine-dependent
`trans(cid:173)
`methy!ation 4 . The fate of
`intra(cid:173)
`cellular homocysteine is either
`salvage
`to methionine through
`remethylation, or conversion to
`cysteine via the trans-sulfuration
`pathway.
`In most
`tissues,
`the
`former reaction is catalysed by the
`
`enzyme methionine
`ubiquitous
`synthase (Fig. 1). This enzyme re(cid:173)
`quires vitamin Bl2 [methyl(I)cobal(cid:173)
`am in] as a cofactor and 5-methyl(cid:173)
`tetrahydrofolate as methyl donor;
`thus 5-methyltetrahydrofolate enters
`the pool of reduced folates, and
`homocysteine is remethylated to
`methionine l .
`cobalamin(cid:173)
`two
`There
`are
`dependent enzymes in vertebrates:
`methionine synthase which util(cid:173)
`izes methylcob(I)alamin and is
`cytosolic,
`and methyimalonyl(cid:173)
`CoA mutase which is a mitochon(cid:173)
`drial enzyme containing aden(cid:173)
`osylcobalamin. A major fraction of
`intracellular cobalamin is associ(cid:173)
`ated with these two enzymes5 .
`Homocysteine remethylation is
`also catalysed by an alternative
`enzyme,
`betaine-homocysteine
`methyltransferase,
`requiring be(cid:173)
`taine as methyl donor. However,
`this enzyme is generally confined
`to the liver.
`The metabolism of homocys(cid:173)
`teine along the trans-sulfuration
`pathway is catalysed by two vit(cid:173)
`amin B6 -dependent enzymes. The
`first step is
`the cystathione ~­
`synthase reaction, where homo(cid:173)
`cysteine is condensed with serine
`to
`form cystathionine. Cysta(cid:173)
`thionine is then cleaved to lX-keto(cid:173)
`butyrate and cysteine, catalysed
`by cystathionine lyase1 .
`
`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(cid:173)
`scribed in children 1. Both clinical
`and
`experimental
`evidence
`suggest
`that high homocysteine
`levels cause the vascular lesions
`(see Ref. 1).
`Even moderate homocystein(cid:173)
`emia may provoke venous throm(cid:173)
`bosis
`and
`premature
`vascular
`lesions in the cerebral, peripheral
`and coronary arteries (L. Bratt(cid:173)
`strom, Thesis, University of Lund,
`1989). Such a relation was suggested
`10 years ago from clinical studies
`based on measurement of acid(cid:173)
`soluble mixed disulfides in plasma
`from a small number of patients 2 .
`This has later been confirmed in
`several
`investigations, most of
`
`© 1990, Elsevier Science rublishe"s Ltd. (UK)
`
`0165 - 6147Nll/$02.110
`
`ACCORD EX 1008
`
`

`

`412
`
`Protein
`
`~\
`
`Methionine
`;""I/UI"')
`
`Serine
`
`AdoMet
`
`Methionine
`
`R
`
`Dimethyl-
`glycine
`
`.
`Betame
`
`THF
`~
`• .~dTMP Glycine
`dUMP
`5,lO-CH2-THF
`~
`
`DHF
`
`AdoHcy
`~ Homocysteine
`
`~
`5 - CH3 -THF ~ 5-CHO-THF
`
`Cy,::r.:;~ ~
`f- ine ~ Folic acid
`B6
`Homocysteine
`Cysteine
`(extracellular)
`I. Homocysteine metabolism. AdoHcy, S-adenosylhomocysteine; Adomet,
`Fig.
`S-adenosylmethionine; S,10-CH2-THF, 5,IO-methylene THF; S-CH3-THF, 5-methyl(cid:173)
`tetrahydrofolate; S-CHO-THF, 5-formyltetrahydrofolate (folinate); CL, cystathionine
`lyase; CS, cystathionine /3-synthase; DHF, dihydrofolate; DR, dihydrofolate reductase;
`MET, methionine; MS, methionine synthase (5-methyl- THF homocysteine methyltrans(cid:173)
`ferase); SA, S-adenosylhomocysteine hydrolase; SH, serine hydroxymethyltransfer(cid:173)
`ase; THF, tetrahydrofolate.
`
`which determined total plasma
`homocysteine3•6- 1O . Furthermore,
`an increased incidence of hetero(cid:173)
`zygous homocystinuria has been
`demonstrated in patients with
`early onset vascular disease7 .
`Other clinical and experimental
`data support an association be(cid:173)
`tween raised homocysteine levels
`and vascular lesions (reviewed in
`Ref.
`Plasma
`homocysteine
`3).
`levels show age- and sex-depen(cid:173)
`dent variations resembling those
`described
`in
`arteriosclerotic
`disease. Men and postmenopausal
`women
`have
`higher
`plasma
`homocysteine levels during fast(cid:173)
`ing and after methionine loading
`than
`young women.
`Plasma
`homocysteine is significantly in(cid:173)
`creased in chronic renal
`failure,
`severe psoriasis, and in some
`patients with cancer. These are
`conditions
`associated with in(cid:173)
`creased risk of vascular disease
`not adequately explained by risk
`factors like smoking, lipid abnor(cid:173)
`malities, hypertension or other
`known predisposing conditions3 .
`Moderate homocysteinemia should
`be considered as a possible cause
`of vascular disease in those cases
`(15-30%) when other risk factors
`cannot be identified. Down syn(cid:173)
`drome, on the other hand,
`is an
`abnormality
`characterized
`by
`low plasma homocysteine levels,
`
`probably due to increased gene
`dosage for the enzyme cystathio(cid:173)
`nine
`residing
`on
`~-synthase
`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(cid:173)
`66 years12 .
`direct
`also more
`is
`There
`that
`high
`levels
`of
`evidence
`homocysteine
`mediate
`the
`thrombogenesis and accelerated
`atherogenesis observed in homo(cid:173)
`cystinuria, and that homocysteine
`may also be responsible for
`the
`vascular disease associated with
`moderate homocysteinemia. Homo(cid:173)
`cysteine damages human endo(cid:173)
`thelial cells in culture, possibly by
`producing hydrogen peroxide in
`an
`oxygen-dependent
`reaction.
`Moreover, endothelial cells from
`patients heterozygous for cysta(cid:173)
`thionine
`~-synthase gene
`de(cid:173)
`ficiency may have an increased
`susceptibility to injury by homo(cid:173)
`cysteine. Mechanisms linking mild
`homocysteinemia
`and
`vascular
`effects could also involve produc(cid:173)
`tion of free radicals and oxidation
`of low-density lipoprotein. Con(cid:173)
`flicting data exist on possible roles
`of platelet sequestration in the
`development
`of
`atherosclerotic
`lesions
`under
`conditions
`of
`
`TiPS - October 1990 [Vol. 11]
`
`elevated plasma homocys(cid:173)
`teine levels2 .
`
`Folate and cobalamin
`deficiency
`Cobalamin deficiency may
`increase plasma homocys(cid:173)
`teine to levels observed in
`homocystinurics (high micro(cid:173)
`molar range); there is a nega(cid:173)
`tive
`correlation
`between
`serum cobalamin and total
`plasma homocysteine. Homo(cid:173)
`cysteine levels may also be
`elevated
`in
`cobalamin(cid:173)
`deficient patients devoid of
`typical
`signs
`like· anemia,
`macrocytosis
`and
`reduced
`serum cobalamin. Levels are
`normalized following cobal(cid:173)
`amin therapy3.13.
`Similarly, folate deficiency
`is a common cause of el(cid:173)
`evated plasma homocysteine
`levels, and a close negative
`correlation with serum folate
`has
`been
`demonstrated;
`again, folate therapy normal(cid:173)
`izes levels. Moderate elevation
`of plasma homocysteine is
`also observed in subjects with low
`but normal serum folate levels,
`suggesting that
`increased homo(cid:173)
`cysteine levels in these subjects
`may reflect an intracellular folate
`content
`insufficient
`for optimal
`folate-dependent remethylation of
`homocysteine3.
`Measurement of plasma homo(cid:173)
`cysteine is therefore a promising
`laboratory
`test
`for
`evaluating
`cobalamin or
`folate
`deficiency
`states. It may be particularly use(cid:173)
`ful when used in conjunction with
`serum methylmalonic acid, which
`is a specific measure of disturb(cid:173)
`ances of cobalamin metabolism 13 .
`
`Agents decreasing homocysteine
`concentrations
`co(cid:173)
`as
`serving
`Compounds
`factors
`in homocysteine catab(cid:173)
`olism or remethylation may en(cid:173)
`hance homocysteine metabolism
`and thereby reduce plasma homo(cid:173)
`cysteine levels in inherited en(cid:173)
`zymic defects. Thus, vitamin 66
`reduces plasma homocysteine in
`homocystinurics with
`residual
`cystathionine ~-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 ~-syn-
`
`

`

`TiPS - October 1990 [Vol. 111
`
`thase deficiency who are un(cid:173)
`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(cid:173)
`out overt folate deficiency. Treat(cid:173)
`ment of healthy subjects with folic
`acid for 14 days significantly re(cid:173)
`duced
`plasma
`homocysteine,
`especially in persons with high
`pretreatment levelsl4.
`The marked effect of high doses
`of folic acid on the concentration
`of homocysteine in plasma is im(cid:173)
`portant. Since moderate homo(cid:173)
`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-effectsI4.
`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(cid:173)
`lished
`laboratory
`procedures,
`including determination of folate
`in serum or erythrocytes3.
`Folic acid probably decreases
`plasma homocysteine levels by in(cid:173)
`creasing the availability of intra(cid:173)
`cellular 5-methyltetrahydrofolate,
`thereby enhancing homocysteine
`remethylation (Fig. 2). This re(cid:173)
`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(cid:173)
`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 administration14.
`
`D-Penicillamine
`D-Penicillamine (D-~,~-dimethyl­
`cysteine) is currently used for the
`treatment of heavy metal poison(cid:173)
`ing, rheumatoid arthritis, hepato(cid:173)
`lenticular degeneration, cystinuria
`and scleroderma. It is metabolically
`stable, chelates heavy metals, pro(cid:173)
`duces disulfides and forms a thi(cid:173)
`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(cid:173)
`ecular weight disulfides undergo
`rapid renal excretion, which ex(cid:173)
`plains the short plasma half-life
`and
`the
`therapeutic
`effect
`in
`cystinuria15.
`re(cid:173)
`efficiently
`Penicillamine
`and protein(cid:173)
`duces both free
`bound plasma homocysteine in
`homocystinurics16
`and
`total
`plasma homocysteine in patients
`with rheumatoid arthritis, a con(cid:173)
`dition with normal homocysteine
`levels1?
`pretreatment
`This
`re(cid:173)
`duction in plasma levels may be
`associated with an intracellular
`homocysteine
`depletion
`suf(cid:173)
`ficiently pronounced to decrease
`homocysteine remethylation and
`thereby induce methionine de(cid:173)
`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(cid:173)
`trexate used in low doses in the
`management of
`rheumatoid ar(cid:173)
`thritis (see below)l8.
`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(cid:173)
`homocysteine to adenosine and
`homocysteine,
`catalysed by S(cid:173)
`adenosylhomocysteine hydrolase
`(Figs 1 and 2), is the only known
`source of homocysteine in ver(cid:173)
`tebrates. Several nucleoside ana(cid:173)
`logs block this reaction by serving
`either as an inactivator or
`in(cid:173)
`hibitor of the enzyme. In addition,
`some analogs act as substrate and
`are thus converted to the corre(cid:173)
`sponding S-adenosylhomocysteine
`analogue4.
`Inhibition
`of
`S(cid:173)
`adenosylhomocysteine hydrolase
`leads to massive accumulation of
`S-adenosylhomocysteine in iso(cid:173)
`lated cells, whole animals, and in
`patients. This is important for the
`antiviral effects of
`this class of
`compound4,19.
`conse(cid:173)
`immediate
`The other
`quence
`of S-adenosylhomocys(cid:173)
`teine hydrolase
`inhibition,
`re(cid:173)
`duction
`of
`homocysteine
`for(cid:173)
`mation (Fig. 2), has been studied
`only
`recently.
`Homocysteine
`depletion and inhibition of homo(cid:173)
`cysteine export have been demon(cid:173)
`strated in isolated cells exposed to
`nucleoside analogs20,21. A reduc-
`
`413
`
`tion in plasma homocysteine was
`found
`in patients with acute
`leukemia treated with 2-deoxy(cid:173)
`coformycin, which indirectly in(cid:173)
`activates S-adenosylhomocysteine
`hydrolase22,23.
`Inhibition
`of
`homocysteine formation plays an
`important
`role in the cytostatic
`action of some nucleoside analogs
`against some24,25 but not alJ26--28
`cell types, and probably mediates
`the differentiation of HL-60 cells
`induced by adenosine
`dialde(cid:173)
`hyde29. The consequences of cellular
`homocysteine deficiency are two(cid:173)
`fold. First, nucleoside analogs may
`induce
`severe methionine
`de(cid:173)
`ficiency, since homocysteine sal(cid:173)
`vage is a significant source of
`methionine in humans30,31. Sec(cid:173)
`ondly, lack of homocysteine may
`trap reduced folate as 5-methyl(cid:173)
`tetrahydrofolate because homo(cid:173)
`cysteine is the methyl acceptor in
`the methionine synthase reaction
`catalysing
`the
`conversion
`of
`5-methyltetrahydrofolate to tetra(cid:173)
`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 cells24,32.
`Induced deficiencies of meth(cid:173)
`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(cid:173)
`logues with drugs that also inter(cid:173)
`fere with folate or methionine
`metabolism such as nitrous oxide
`or methotrexate should be con(cid:173)
`sidered. This has been suggested
`for the antiviral agent vidarabine
`(9-~-D-arabinofuranosyladenine)
`and high-dose methotrexate with
`folinic acid rescue30. 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(cid:173)
`fore it was discovered that long(cid:173)
`term exposure
`caused megalo(cid:173)
`blastic and aplastic bone marrow
`changes, anemia and myelopathy.
`
`

`

`414
`
`TiPS - October 1990 [Va/. 11J
`
`MethOtrex~ate
`DHF
`.
`
`/
`
`5, IO-CII 2-11IP
`
`5-CH)-THF
`
`THF
`
`Mel
`
`,,'
`t
`~~IF
`Hey
`B12
`DHF -m,......--..~ THF
`
`AdOH)CCH~::(_~HF
`
`Folic acid
`
`Ado ~
`Hey
`Met
`B 12
`
`Cystathionine
`86~
`~
`Cysteine
`
`"
`
`Hey-SH
`(extracellular)
`
`"
`
`,
`
`""
`
`....
`
`" /
`"6 ~H3COb(J)ala~i~".
`R-S-S-R i
`
`D-Penicillamine
`(P-SH)
`
`~ Hey-S-S-P
`
`Adenosine analogues
`
`AdoHeyJ<Ado
`
`Hcy
`
`6-Azauridine triacetate
`
`~ Hey
`~
`Cystathionine
`~
`Cysteine
`
`Folic acid
`
`~ Folie aeid
`/
`
`5- CH -THF
`3
`~HF
`Met
`Bn
`
`Hey
`
`Nitrous oxide
`
`)
`
`'",
`
`Mel
`
`5- CH3 -THF
`~THF'
`"'"
`Hcy-S-S-R ~
`Hey ClI 3cdb(ll)alamin
`inaclivc B 12
`
`P-S-S-R -1 ~
`R-S-S-Ri
`
`Hey-SH
`(ext racellular)
`
`ReM/
`excretion
`
`Fig. 2. Proposed or established mechan(cid:173)
`isms for the effect of various agents on
`the metabolism and plasma level of
`homocysteine. The figure in 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-methyltetrahydrofolate (5-CH 3-THF), which increases the homocysteine (Hey) remethylation catalysed by
`methionine synthase (MS). D-Penicillamine (P-SH) forms a mixed disulfide with homocysteine (Hey-S-S-P); this disulfide has a high renal
`clearance. Adenosine analogs are inhibitors of S-adenosylhomocysteine hydrolase (SA). Nitrous oxide oxidizes methylcob(l)alamin to
`methylcob(lJ)alamin and thereby irreversibly inhibits methionine synthase (MS). Methotrexate inhibits dihydrofolate reductase (DR),
`thereby inhibiting regeneration of tetrahydrofolate (THF) from dihydrofolate (DHF). Tetrahydrofolate supplies 5-methyltetrahydrofolate via
`5, IO-methylenetetrahydrofolate (5,10-CH 2-THF). Methotrexate may therefore induce depletion of 5-methyltetrahydrofolate, and inhibit
`homocysteine remethylation catalysed by methionine synthase. Azauridine is a vitamin 8 6 antagonist, and may inhibit some vitamin 86 (cid:173)
`dependent enzymes, including cystathionine f3-synthase (CS).
`Hey, homocysteine; Met, methionine; Ado, adenosine; poSH, o-penicillamine reduced form; Hey-S-S-A. homocysteine mixed disulfide;
`Hey-S-S-P, a mixed disulfide between homocysteine and o-penicillamine; P-S-S-R, o-penicillamine mixed disulfide.
`For other abbreviations, see Fig. I.
`
`Hey-S-S-R
`
`These side-effects are similar to
`the
`symptoms
`of vitamin 8 12
`deficiency33.
`Nitrous oxide oxidizes the cob(cid:173)
`alamine
`species methylcob(l)(cid:173)
`alamine,
`inactivating specifically
`the enzyme methionine synthase,
`
`without affecting the adenosyl(cid:173)
`cobalamine cofactor of methyl(cid:173)
`malonyl-eoA mutase33 .
`Inacti(cid:173)
`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
`is
`a sub(cid:173)
`levels. There
`folate
`sequent decrease of folate-depen(cid:173)
`dent
`purine
`and
`thymidylate
`synthesis, as demonstrated by the
`deoxyuridine
`suppression (dU)
`
`

`

`TiPS - October 1990 [Vol. 11}
`
`in
`test, which reveals changes
`human bone marrow within 5-6
`hours. In humans, 50% of meth(cid:173)
`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(cid:173)
`24 hours of exposure33.
`Data on the effect of nitrous
`oxide on homocysteine metab(cid:173)
`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(cid:173)
`cysteine within 90 minutes, with a
`concurrent
`increase
`in urinary
`homocysteine excretion. In patients
`receiving nitrous oxide for 3-6
`hours, plasma homocysteine re(cid:173)
`mained above normal for at least 7
`days (Ermens, A. A. M. et aI., un(cid:173)
`published data). The homocysteine
`response
`evolved before other
`early
`signs
`of nitrous
`oxide(cid:173)
`induced cobalamine inactivation33.
`Thus, significant cobalamine oxi(cid:173)
`dation and methionine synthase
`inact~'/ation occurs
`even
`after
`short-term exposure, which pre(cid:173)
`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(cid:173)
`flect loss of functional cobalamine
`and folate, and may enhance sen(cid:173)
`sitivity towards antifolate drugs
`such as methotrexate34 (Ermens,
`A. A. M., PhD Thesis, University
`of Rotterdam, 1990). Since an in(cid:173)
`crease in plasma homocysteine
`levels is both an early and sensi(cid:173)
`tive measure of cobalamine oxi(cid:173)
`dation,
`plasma
`homocysteine
`monitoring could be useful in the
`detection of such effects in the
`clinic.
`
`Methotrexate
`Methotrexate is an anti folate
`drug which has been used exten(cid:173)
`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(cid:173)
`nant diseases such as rheumatoid
`arthritis and psoriasis.
`Methotrexate acts by inhibiting
`dihydrofolate reductase,
`thereby
`
`blocking the regeneration of tetra(cid:173)
`hydrofolate
`from dihydrofolate
`(see Ref. 35). This leads to deple(cid:173)
`tion of reduced folates, including
`5-methylteh'ahydrofolate36.37. Thus,
`methotrexate may also inhibit the
`folate-dependent remethylation of
`homocysteine catalysed by methio(cid:173)
`nine synthase. This would explain
`the increased homocysteine export
`from cultured cells exposed to
`methotrexate, and the methotrex(cid:173)
`ate-induced homocysteinemia and
`urinary homocysteine excretion in
`patients38.
`Low-dose methotrexate (25 mg
`daily) given to psoriatics induced
`increased plasma homocysteine
`levels, which maximized after
`about
`two days, and normalized
`within one week39. This shows
`that plasma homocysteine is a
`sensitive measure of the antifolate
`effect.
`(l-13.6g)
`doses
`Intermediate
`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 patient40. 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'
`therapy41. This is analogous to the
`results obtained with cultured
`cells38.
`Basal homocysteine levels
`in
`patients with acute lymphoblastic
`leukemia were often above normal
`before
`treatment, and declined
`markedly
`following
`treatment
`with cytotoxic agents including
`methotrexate (Refsum, H. et aI.,
`unpublished). This may be due to
`eradication of proliferating cells
`exporting large amounts of homo(cid:173)
`cysteine.
`The
`high-dose methotrexate
`regimen also induced a transient
`but marked reduction in plasma
`methionine41 which may contrib(cid:173)
`ute to the killing of tumor cells or
`toxicity of methotrexate. Plasma
`homocysteine
`response
`and
`methionine depletion may corre(cid:173)
`late with the therapeutic as well as
`the side-effects of methotrexate,
`including liver toxicity42 and an
`increased incidence of thrombo(cid:173)
`embolism43; plasma homocysteine
`measurements could provide a
`
`415
`
`to serum metho(cid:173)
`useful adjunct
`trexate
`determination
`in
`the
`management
`of methotrexate
`therapy.
`
`Vitamin B6 antagonists
`Azauridine is an antimetabolite
`interfering with de novo synthesis
`It
`of uridine-5'-monophosphate.
`was initially used for
`the treat(cid:173)
`ment of refractory psoriasis, but
`was withdrawn by the FDA in
`1976 because its use was associ(cid:173)
`ated with an increased incidence
`of vascular episodes44. This may
`be due to effects on homocysteine
`metabolism. Azauridine
`causes
`homocysteinemia, abnormal homo(cid:173)
`cysteine excretion and a signifi(cid:173)
`cant increase in serum methionine
`levels in patients. Studies in rab(cid:173)
`bits suggest that it functions as a
`pyridoxal 5' -phosphate antagonist
`and causes homocysteinemia by
`inhibiting vitamin B6-dependent
`synthesis44. This
`cystathionine
`suggests
`that
`supplementing
`vitamin B6 would prevent the in(cid:173)
`hibition of homocysteine catab(cid:173)
`olism; determination of plasma
`homocysteine
`may
`identify
`patients at risk of vascular epi(cid:173)
`sodes.
`Several other drugs also inter(cid:173)
`fere with the function of vitamin
`isoniazid,
`cycloserine,
`hy(cid:173)
`B6:
`dralazine, penicillamine, phenel(cid:173)
`zine and procarbazine44. Pertur(cid:173)
`bation of homocysteine metab(cid:173)
`olism in patients has been demon(cid:173)
`strated with isoniazid45. In one
`out of six patients given 300 mg
`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(cid:173)
`ionine loading45.
`
`Other agents
`have
`Premenopausal women
`lower plasma homocysteine than
`men
`and
`postmenopausal
`women46, 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(cid:173)
`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
`
`

`

`416
`
`TiPS - October 1990 [Vol. 11]
`
`in plasma
`an increase
`others,
`homocysteine levels. Because of
`the widespread use of contracep(cid:173)
`tives, and the suggested use of
`tamoxifen as prophylactic inter(cid:173)
`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 health3 .
`drugs,
`Various
`antiepileptic
`particularly phenytoin but also
`phenobarbital, primidone, carba(cid:173)
`mazepine, and valproic acid, may
`induce folate deficiency47. The
`activity of methylenetetrahydro(cid:173)
`folate
`reductase,
`the
`enzyme
`producing
`5-methyltetrahydro(cid:173)
`folate,
`is altered in mouse liver
`following exposure to these drugs.
`Preliminary
`data
`suggest
`that
`phenytoin and possibly carbama(cid:173)
`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(cid:173)
`olism or function. These include
`some phenothiazines and tricyclic
`antidepressants, oral contracep(cid:173)
`tives, possibly some tuberculo(cid:173)
`static drugs, and antifolate drugs
`such as trimethoprim47. 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(cid:173)
`position of the mixed disulfides
`with homocysteine, as demon(cid:173)
`strated with
`penicillamine
`in
`humans 16.
`o
`
`o
`
`o
`
`Various pharmacological agents
`have been shown to enhance
`homocysteine remethylation and
`urinary
`excretion,
`or
`inhibit
`homocysteine
`production,
`re(cid:173)
`methylation or catabolism thereby
`affecting
`plasma
`homocysteine
`levels. Modulation of homocys(cid:173)
`teine metabolism and
`plasma
`concentrations may be an import(cid:173)
`ant component of drug action.
`
`Agents that reduce plasma homo(cid:173)
`cysteine (e.g. folic acid, penicill(cid:173)
`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(cid:173)
`teine has been shown to be use(cid:173)
`ful in the diagnosis and follow-up
`of some diseases,
`in particular
`homocystinuria, folate deficiency
`and cobalamine deficiency.
`
`(1989) Haemostasis 19
`
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