Clinical Science (1996)
91. 79-86, FMT Loehrer et al.
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Effect of methionine loading on
5-methyltetrahydrofolate, S-adenosylmethionine and S-adenosylhomocysteine
in plasma of healthy humans
Franziska M. T. Loehrer, Walter E. Haefeli,
Christian P. Angst, Garry Browne, Greta Frick and Brian
Metabolic Unit, University Children's
Hospitol Basel, and Division of Clinical Pharmacology,
University Hospitol Basel, Basel, Switzerland
- Elevated plasma homocysteine concentration, either
in the fasting state or after methionine loading, is
an independent risk factor for vascular disease in man.
Methionine loading has been used to investigate impaired
methionine metabolism, especially of the trans-sulphuration
pathway, but most studies have focused on changes in
- We investigated the effect of methionine excess on
total plasma homocysteine, 5-methyltetrahydrofolate
(which is the active form of folate in the remethylation
of homocysteine to methionine), S-adenosylmethionine
(the first metabolite of methionine) and S-adenosylhomocysteine
(the demethylated product of S-adenosylmethionine) over
24h in 12 healthy subjects.
- As well as the expected increase in homocysteine
(from 8.0"1.3 to 32.6"10.3 Fmol/l, mean " SD, P<0.001),
S-adenosylmethionine showed a significant transient
increase (from 37.9 " 25.0 to 240.3 " 109.2 nmol/l,
P<0.001), which correlated well with homocysteine
(r2=0.92, P<0.001). 5-Methyltetrahydrofolate
values decreased significantly (from 23.2 " 7.2 to 13.1"2.9nmol/l,
P<0.01), and gradually returned to baseline levels
after 24h, No significant change over the time of measurement
was found for S-adenosylhomocysteine.
- The sequence of metabolic changes observed in this
study strongly suggests that a change in either homocysteine
or S-adenosylmethionine may cause a reduction in 5-methyltetrahydrofolate.
This must be considered in evaluating the relationship
between folate and homocysteine in vascular disease.
The metabolic relationships illustrated in this study
should be evaluated in the search for pathogenetic mechanisms
of mild hyperhomocysteinaemia and vascular disease.
The essential protein
amino acid methionine is converted to S-adenosylmethionine
by methionine adenosyltransferase (EC 184.108.40.206). S-adenosylmethionine
is the methyl donor in many important transmethylation
reactions that lead to the formation of the very short-lived
S-adenosylhomocysteine . Enzymic hydrolysis yields
homocysteine, which can be catabolized via the irreversible
transsulphuration pathway, the first step in which is
catalysed by the pyridoxal phosphate-dependent enzyme
cystathionine b-synthase (CS; EC 220.127.116.11). Alternatively,
it can be remethylated to methionine by 5-methytetrahydrofolate--homocysteine
methyltransferase (methionine synthase, MS; EC 18.104.22.168),
which requires vitamin B12, or by betaine-homocysteine
methyl-transferase (EC 22.214.171.124). Regulation of these pathways
depends on many factors [2-5], but a vital role of S-adenosylmethionine
in the coordinate control of remethylation and trans--sulphuration
seems evident from studies in vitro [6,7]. S-Adenosylmethionine
acts as an allosteric inhibitor of 5,10-methylenetetrahydrofolate
reductase (MTHFR; EC 126.96.36.199), which is crucial for 5-methyltetrahydrofolate
synthesis  and as an activator of CS  at micromolar
Disturbances of methionine metabolism are associated
with a variety of disease states. For example, inborn
errors due to CS, MTHFR and MS deficiencies lead to hyperhomocysteinaemia
and severe disease, including ocular, skeletal, neurological
and vascular pathology [1,8,9]. Furthermore, elevated
plasma homocysteine levels, either in the fasting state
or after methionine loading, have been found in a significant
proportion of patients with coronary artery [10-13], peripheral
arterial occlusive [14,15] or cerebral vascular disease
[15-17], pointing to mild hyperhomocysteinaemia as an
indep-endent risk factor for arteriosclerotic disease.
The exact cause of this is so far unknown, but moderate
deficiencies of MTHFR [18-20] and CS  and nutritional
deficiencies of vitamin B12, and folate [22,23]
have been implicated.
Administration of methionine followed by measurement
of sulphur amino acids in blood and urine has been used
to study homozygous and heterozygous CS deficiency [24-27].
Furthermore, Beers et al.  reported post-methionine
load increases in homo-cysteine similar to those in obligate
heterozygotes for CS deficiency in 36% of 25 patients
with peripheral and 28% of 25 patients with cerebrovascular
disease. Several studies have confirmed this abnormality
in patients with different forms of vascular disease [21,28-30].
It is thought that methionine loading mainly stresses
catabolism through homocysteine transsulphuration .
However, Clarke et al.  showed an inverse relation
between vitamin B12, or folate and post-load
homocysteine in patients with different forms of vascular
disease. On the other hand, in previous studies by Brattstrom
et al.  and Andersson et al. , no correlation
between post-load homocysteine and vitamin B12,
folate or pyridoxal phosphate was found in patients with
vascular disease. Previous studies on the relationship
between folate and homocysteine have all used measurement
of the total blood concentrations of the vitamin rather
than the methyl form, which specifically participates
in the remethylation reaction.
Until now methionine loading studies have concentrated
on changes in sulphur amino acid levels [33,34], and little
is known about the influence of methionine on levels of
the numerous other metabolites and cofactors in humans.
In this study the effect of oral methionine on plasma
levels of key compounds involved in the transmethylation
pathway were studied over 24 h. In particular, and in
addition to the often measured homocysteine, we determined
the levels of 5-methyltetrahydrofolate, the form of folate
active in remethylation of homocysteine, and the intermediate
methionine metabolites S-adenosylmethionine and S-adenosylhomocysteine.
Additionally, the heat stable activity of MTHFR in lymphocytes
|Fig. 2. Concentrations of total homocysteine
(a), S-adenosyl-methionine (b), S-adenosylhomocysteine
(c) and 5-methyltetra-hydrofolate (d) in plasma after
methionine loading in 12 healthy subjects (closed
symbols) and without methionine administration in
three control subjects (open symbols) over a period
of 24 hours. Results are expressed as mean " SEM.
This study set out to investigate the effects
of methionine loading on S-adenosylhomocysteine, S-adenosylmethionine
and 5-methyltetrahydrofolate and their relation to homocysteine
in normal subjects.
That the 12 subjects handled methionine normally was
suggested by the finding of normal fasting homocysteine
values, which increased to a mean of 32.6" 10.3Fmol/l,
which compares with the mean of 35"6Fmol/l found by Mansoor
et al. . However, this does not completely exclude
the heterozygous state for CS deficiency in all individuals
as some overlap of post-methionine homo-cysteine levels
between control subjects and obligate heterozygotes has
been reported . The variable time to peak values in
different subjects in our study emphasizes individual
variation in such metabolic studies in humans. It helps
to explain the better discrimination of obligate heterozygotes
from control subjects observed when several post-load
samples were analysed than in simplified tests using a
single post-load measurement. Such variable peak level
times have important implications both for the studies
in vascular disease patients and for investigations of
metabolic inter-relationships as in this study.
The major conclusions from this study are that methionine
loading in normal human subjects leads to simultaneous
increases in homocysteine and S-adenosylmethionine, without
significant changes in S-adenosylhomocysteine, and to
a later fall of 5-methyltetrahydrofolate. These changes
must be consequent to methionine loading and do not simply
reflect circadian variations, as demonstrated by the lack
of such changes in three control subjects who received
no methionine. The increase in S-adenosylmethionine probably
reflects liver metabolism of methionine and increases
in S-adenosylmethionine concentration as seen in rats
injected with a single dose of methionine . This is
supported by studies in mammals, which showed that adaptation
to an excess of methionine occurs in liver as only the
liver-specific isoenzyme of methionine adenosyltransferase
can adapt immediately to changes in methionine concentrations
. The absence of a change in S-adenosylhomocysteine
concentration after methionine loading despite the large
increase in homocysteine is surprising. It was shown by
Hoffman et al.  that in isolated rat liver the administration
of homocysteine results in an accumulation of S-adenosylhomocysteine
if homocystene is not removed immediately. However, Guttormsen
et al.  reported no change in S-adenosyl-homocysteine
concentration after homocysteine loading, which resulted
in similar concentrations of homocysteine to those found
after meth ionine loading. If the lack of increase in
S-adenosylhomocysteine in plasma does indeed reflect tissue
levels, this could be explained simply by a transient
increase in homocysteine to concentrations below those
required to inhibit S-adenosylhomocysteine hydrolase 
and by a fully active trans-sulphuration pathway owing
to normal enzyme activities in normal subjects.
The decrease in 5-methyltetrahydrofolate found in this
study could result from an increase in homocysteine levels,
which may result in an enhanced turnover of the remethylation
reaction, thereby depleting the co-substrate 5-methyltetrahydrofolate.
Alternatively, elevated S-adenosylmethionine could lead
to allosteric inhibition of MTHFR, as shown previously
in vitro , at the concentrations of S-adenosylmethionine
obtained in rat liver by injection of similar doses of
methionine . It is possible that the two effects may
operate in combination. The significant positive correlation
between fasting level and both the maximum change and
the AUC(t0-24) in 5-methyltetrahydrofolate
indicates smaller decreases in 5-methyltetrahydrofolate
in subjects with lower baseline values. This suggests
that the extent of remethylation in the presence of homocysteine
excess may also depend on the availability of 5-methyltetrahydrofolate.
The one exceptional subject (VIII) who showed no decrease
in 5-methyltetrahydrofolate but the highest post-load
level of S-adenosylhomocysteine may reflect the extreme
of normal variation. However, these findings could also
result from a disturbed remethylation pathway, preventing
normal conversion of 5-methyltetra-hydrofolate to methionine,
and subsequent stress of the trans-sulphuration pathway
reflected by increased S-adenosylhomocysteine. However,
this subject exhibited 36% heat-stable activity of MTHFR,
which is well above the range of 2.5-4.5 SDs below the
mean value, which was defined by Kang et al.  as indicating
the thermolabile MTHFR mutation.
An additional finding in this study was that the S-adenosylmethionine/S-adenosylhomocyste
ratio in fasting human plasma, at 1.2, is lower than that
reported for erythrocytes (3.3)  and cerebrospinal
fluid (6.9) , indicating variation of this ratio between
different tissues and compartments. Previous workers have
shown that this ratio and not S-adenosylmethionine alone
is a critical factor in the influence of methyltransferases
The finding that methionine loading leads to decreases
in 5-methyltetrahydrofolate subsequent to increases in
homocysteine and S-adenosylmethionine indicates that a
change in either homocysteine or S-adenosylmethionine
may cause reductions in 5-methyltetra-hydrofolate. This
must be considered in evaluating the relationship between
folate and homocysteine in vascular disease. The metabolic
relationships illustrated in this study should be evaluated
in the search for pathogenetic mechanisms of mild hyperhomocysteinaemia
and vascular disease.
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