Bioscience, Biotechnology, and Biochemistry

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Suppression of Methionine-Induced
Hyperhomocysteinemia by Glycine and Serine in
Rats

Shin-ichiro FUKADA, Yasuhiko SHIMADA, Tatsuya MORITA & Kimio SUGIYAMA

To cite this article: Shin-ichiro FUKADA, Yasuhiko SHIMADA, Tatsuya MORITA & Kimio
SUGIYAMA (2006) Suppression of Methionine-Induced Hyperhomocysteinemia by Glycine and
Serine in Rats, Bioscience, Biotechnology, and Biochemistry, 70:10, 2403-2409, DOI: 10.1271/
bbb.60130

To link to this article: https://doi.org/10.1271/bbb.60130

Published online: 22 May 2014.

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Biosci. Biotechnol. Biochem., 70 (10), 2403–2409, 2006

Suppression of Methionine-Induced Hyperhomocysteinemia

by Glycine and Serine in Rats
Shin-ichiro F UKADA, Yasuhiko S HIMADA, Tatsuya M ORITA, and Kimio S UGIYAMAy
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University,
836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan

Received March 10, 2006; Accepted May 31, 2006; Online Publication, October 7, 2006
[doi:10.1271/bbb.60130]

The hyperhomocysteinemia induced by a dietary Methionine

addition of 1% methionine was significantly suppressed DMG THF
by the concurrent addition of 1% glycine or 1.4% serine (4) SAM
Betaine Glycine
to the same degree. The methionine-induced increase in (1) CO2+ NH3
the hepatic concentration of methionine metabolites Sar
was significantly suppressed by glycine and serine, but THF 5,10-CH2-THF
SAH
(3) (5)
the hepatic cystathionine -synthase activity was not 5-CH3-THF THF
enhanced by these amino acids. When the methionine- Homocysteine
(6)
supplemented diet was changed to the methionine plus Serine
5,10-CH2-THF (2)
glycine or serine diet, the plasma homocysteine concen-
tration rapidly decreased during and after the first day. Cystathionine
The hyperhomocysteinemia induced by an intraperito-
neal injection with methionine was also suppressed by
Cysteine
concurrent injection with glycine or serine, although the
effect of serine was significantly greater than that of Taurine Sulfate
glycine. These results indicate that glycine and serine
were effective for suppressing methionine-induced hy- Fig. 1. Participation of Glycine and Serine in the Metabolism of
Methionine.
perhomocysteinemia: serine and its precursor glycine
DMG, N,N-dimethylglycine; SAH, S-adenosylhomocysteine;
are considered to have elicited their effects mainly by SAM, S-adenosylmethionine; Sar, sarcosine; THF, tetrahydrofolate.
stimulating cystathionine synthesis by supplying serine, Enzymes: (1) glycine N-methyltransferase [EC 2.1.1.20], (2) cys-
another substrate for cystathionine synthesis. tathionine
-synthase [EC 4.2.1.22], (3) methionine synthase [EC
2.1.1.13], (4) betaine:homocysteine S-methyltransferase [EC
2.1.1.5], (5) serine hydroxymethyltransferase [EC 2.1.2.1], (6)
Key words: homocysteine; methionine; glycine; serine;
5,10-methylenetetrahydrofolate reductase [EC 1.1.99.15].
rat

Homocysteine is an intermediate in the metabolism of
methionine, a sulfur-containing essential amino acid in
mammals (Fig. 1), but an elevated plasma homocysteine
concentration is recognized as an independent risk factor
for cardiovascular disease.1–4) It has been shown that the
plasma homocysteine concentration was affected by
genetic factors, physiological and lifestyle determinants,
nutritional and clinical conditions, and drugs.1–4) Of
these factors, genetic and nutritional factors are thought
to have the greatest influence on the plasma homocys-
teine concentration.4) Since the major part of plasma
homocysteine is derived from the liver, the hepatic
homocysteine concentration is thought to reflect the
plasma homocysteine concentration.5) The homocys-
teine concentration in the liver is affected by the rates of
the following processes: (i) production of homocysteine
from S-adenosylhomocysteine (SAH) and its precursor
S-adenosylmethionine (SAM), (ii) remethylation of
homocysteine to methionine, (iii) formation of cysta-
thionine, and (iv) export of homocysteine into blood
plasma. Glycine and serine participate in the metabolism
of methionine as a methyl-group acceptor and as a
substrate for cystathionine synthesis, respectively.6,7)
Based on these metabolic features, glycine and serine
have been shown to accelerate the metabolism of
methionine and thereby alleviate the adverse effects of
excess methionine, although the effect of glycine was
somewhat greater than that of serine.8,9) Some reports
have recently shown that serine had a hypohomocyste-
inemic effect.5,10) However, little information is avail-
able about the effect of glycine on the plasma homo-
cysteine concentration.

y
To whom correspondence should be addressed. Fax: +81-54-238-4877; E-mail: acksugi@agr.shizuoka.ac.jp
2404 S. F UKADA et al.
To investigate regulation of the plasma homocysteine
concentration, appropriate animal models are useful.
One of the experimental hyperhomocysteinemia models
in rodents is the methionine-loading model; e.g., force-
feeding11) or an intraperitoneal injection12) of methio-
nine causes a transient increase in the plasma homo-
cysteine concentration. It has been shown that dietary
supplementation with methionine could also induce
persistent hyperhomocysteinemia,13) although the exper-
imental conditions for this model have not yet been fully
established.
We conducted a series of experiments in the present
study (1) to establish a model for dietary methionine-
induced hyperhomocysteinemia and (2) to assess the
comparative effects of glycine and serine on the plasma
homocysteine concentration in hyperhomocysteinemic
rats induced by both dietary supplementation and
intraperitoneal injection with methionine.
Materials and Methods
Animals and diets. Male six-week-old rats (120–
140 g) of the Wistar strain were obtained from Japan
SLC (Hamamatsu, Japan). They were individually
housed in hanging stainless-steel wire cages kept in an
isolated room at a controlled temperature (23–25
C)
and humidity (40–60%). Lighting was maintained on
a 12-h cycle (lights on from 0700 to 1900 h). Before
starting the experiments, the rats were acclimatized to
the facility for 4 or 5 d. The control diet (25C) consisted
of the following ingredients (g/100 g): casein, 25; corn
starch, 43.25; sucrose, 20; corn oil, 5; mineral mixture
(AIN-93), 3.5; vitamin mixture (AIN-93), 1; choline
bitartrate, 0.25; and cellulose, 2. Amino acids were
added to the diet at the expense of starch. Four separate
experiments were conducted in this study. In experiment
1-A, the rats were divided into four groups and fed on
the control diet or on a diet supplemented with L-
methionine at the level of 0.5, 1, or 2% for 10 d. In
experiment 1-B, the rats were fed on the control diet for
7 d and then injected intraperitoneally with saline alone
or with saline containing L-methionine at the level of
100, 200, 300, or 500 mg/kg of body wt. In experiment
2, the rats were divided into four groups and fed on
the control diet or on a diet supplemented with 1% L-
methionine with or without additional 1% glycine or
1.4% L-serine for 10 d. In experiment 3, the rats were fed
on the diet supplemented with 1% L-methionine for 7 d,
and seven of them were killed on the final day of the 7-d
period. The remaining rats were divided into three
groups; one group continued to be fed on the methio-
nine-supplemented diet, and the other groups of rats
were fed on a diet supplemented with 1% L-methio-
nine + 2.5% glycine or on a diet supplemented with 1%
L-methionine + 2.5% L-serine. In experiment 4, the rats
were divided into four groups after feeding the control
diet for 7 d and were then injected with saline alone or
with saline containing L-methionine (300 mg/kg) to-
gether with or without glycine (300 mg/kg) or serine
(420 mg/kg). The dose levels of glycine and serine were
adjusted on a molar basis, except for experiment 3. In
experiments 1-A, 2 and 3, the rats were killed by
decapitation between 1000 and 1030 h without prior
starvation. In experiments 1-B and 4, the rats were
injected with saline alone or with saline containing
amino acid(s) between 1100 and 1130 h, before being
killed 2 h after the injection. The experimental plan was
approved by the Laboratory Animal Care Committee of
the Faculty of Agriculture at Shizuoka University.
Biochemical analysis. Blood plasma was separated
from the heparinized whole blood by centrifugation at
2000  g for 15 min at 4
C and stored at 30
C until
needed for analyses. After collecting the blood, the
whole liver was quickly removed, rinsed in ice-cold
saline, blotted on filter paper, cut into two portions,
weighed, quickly frozen in liquid nitrogen and stored at
80
C until needed for analyses. One portion of the
liver was homogenized in 4 volumes (vol/wt) of an ice-
cold 25% perchloric acid solution and then centrifuged
at 10;000  g for 10 min at 4
C. The supernatant was
subjected to an assay of methionine metabolites. The
other portion of the liver was homogenized in 4 volumes
(vol/wt) of a 10 mM Tris–HCl buffer (pH 7.4) contain-
ing 150 mM KCl, and the homogenate was centrifuged
at 14;000  g for 10 min at 4
C. The supernatant was
subjected to an enzyme assay. The concentrations of
total homocysteine and cysteine in the plasma and liver
were measured by HPLC according to the method of
Durand et al.14) The concentrations of S-adenosylme-
thionine (SAM) and S-adenosylhomocysteine (SAH) in
the liver were measured by HPLC essentially according
to the method of Cook et al.15) with slight modifications
as described previously.16) The activity of cystathionine

-synthase (CBS) in the liver was measured according to
the method of Mudd et al.,17) but using HPLC for the
assay of the reaction product, cystathionine, according to
the method of Einarsson et al.18) Protein was measured
according to the method of Lowry et al.,19) using bovine
serum albumin as the standard.
Statistical analysis. Each value is expressed as the
mean  SEM. Data were analyzed by a one-way analy-
sis of variance, and the difference between mean values
was tested by the Tukey test when the F value was
significant. A statistical analysis was performed with
Mac Toukei-Kaiseki ver. 1.5 software (Esumi, Tokyo,
Japan). A P value of 0.05 or less is considered
significant.
Results
Experiment 1
The body weight gain, food intake and relative liver
weight of the rats were not affected by methionine when
added to the control diet at levels of 0.5% and 1%, but
 Suppression of Hyperhomocysteinemia by Glycine and Serine 2405
Table 1. Body Weight Gain, Food Intake and Relative Liver Weight of Rats Fed on the Experimental Diets1

Diet Body wt. gain Food intake Liver wt.

(g/10 d) (g/10 d) (% of body wt)
Experiment 1-A:
25C (6) 44  1a 115  4a 4:67  0:12a
25C + 0.5% L-Met (6) 46  2a 115  3a 4:61  0:11a
25C + 1.0% L-Met (6) 43  2a 114  3a 4:68  0:05a
25C + 2.0% L-Met (6) 19  1b 83  1b 4:47  0:04a

Experiment 2:
25C (8) 46  2 125  2 4:77  0:03
25C + 1.0% L-Met (25CM) (7) 42  2 123  3 4:68  0:10
25CM + 1.0% Gly (6) 42  1 123  4 4:76  0:08
25CM + 1.4% L-Ser (6) 43  2 123  7 4:93  0:08
1
Each value is the mean  SEM for the number of rats indicated in the parentheses; values in each experiment with different superscripts are significantly different at
p < 0:05. 25C, 25% casein diet.

80
to the same degree (Fig. 3A). The plasma cysteine
175 A B
a a a concentration, which was measured for comparison with
150 a homocysteine, was slightly decreased by glycine and
Plasma Hcy (nmol/ml)

Plasma Hcy (nmol/ml)

125
60
100
40
b significantly suppressed by glycine and serine to the
75
50 c 20 c
25 c
0 0
0 0.5 1.0 2.0
Met added to diet (%)
Fig. 2. Effects of Dietary Addition (A) and Intraperitoneal Injection
(B) of Graded Levels of Methionine on the Plasma Homocysteine
Concentration in Rats (Experiment 1).
Each value is the mean  SEM for 6 rats. a{c Values with different
letters are significantly different at p < 0:05.
an addition level of 2% significantly depressed the
growth and food intake (Table 1). Dietary supplemen-
tation with methionine increased the plasma homo-
cysteine concentration in a dose-dependent manner,
although the effect of 0.5% methionine was not statisti-
cally significant (Fig. 2A). The intraperitoneal injection
with methionine also increased the plasma homocysteine
concentration in response to the amount of methionine
injected and nearly reached a plateau at a dose level of
300 mg/kg of body weight (Fig. 2B). Based on these
data, we used dose levels of methionine with 1%
addition and 300 mg/kg injection in experiments 2 to 4
to make the effects of glycine and serine clear.
serine (Fig. 3B). A dietary addition of 1% methionine
markedly increased the hepatic concentrations of SAM,
b
SAH and homocysteine, and these increases being
same degree (Figs. 3C, D and E). The activity of CBS in
the liver tended to be increased by methionine addition,
and this increase was significantly suppressed by serine
(Fig. 3F).
0 0.1 0.2 0.3 0.5
Met injected (g/kg) Experiment 3
When the diet was changed from one with 1%
methionine added to one with 1% methionine + 2.5%
glycine or 1% methionine + 2.5% serine added on d 0,
the plasma homocysteine concentration decreased sig-
nificantly on and after d 1 to the same degree and
reached a nadir (Fig. 4). The hepatic concentrations of
SAM, SAH and homocysteine were also affected in a
similar manner by the diet change. The activity of CBS
in the liver tended to be decreased by the dietary
addition of serine and glycine, although no statistical
significance could be detected.
Experiment 4
Methionine (300 mg/kg) injected intraperitoneally in
combination with glycine (300 mg/kg) or serine (420
mg/kg) to rats that were killed 2 h after the injection
resulted in the enhancement of plasma homocysteine
concentration being significantly suppressed by both
glycine and serine, although the effect of serine was
significantly greater than that of glycine (Fig. 5).

Experiment 2 Discussion
Addition of 1% glycine or 1.4% serine to the 1%
methionine-supplemented control diet did not affect the The present study demonstrated that methionine in
growth, food intake or relative liver weight of the rats the range of 0.5–1% could induce hyperhomocysteine-
(Table 1). The methionine-induced increase in plasma mia in rats without growth retardation when added to
homocysteine concentration was significantly sup- a 25% casein diet. This dietary methionine-induced
pressed by the concurrent addition of glycine or serine hyperhomocysteinemia might be useful as a convenient
2406 S. F UKADA et al.

25C 25CM + 1.0% Gly 25C + 1% Met

25C + 1% Met + 2.5% Gly
25C + 1.0% Met 25C + 1% Met + 2.5% Ser
(25CM) 25CM + 1.4% Ser
100 250
a A B
80 A 200 B a

Plasma Hcy (µmol/L)

80 ab

Plasma Cys (µmol/L)

200
b

Plasma Cys (nmol/ml)

Plasma Hcy (nmol/ml)

a
60 150 ab b b 60 150

40 100 40 100
b b
c c
20 50
20 c 50 c c
0 0
0 1 2 3 0 1 2 3
0 0
600 80
C D

Liver SAM (nmol/g liver)

a

Liver SAH (nmol/g liver)

500 80
C D ab a a a
a a 60
b
400 400
Liver SAM (nmol/g)

Liver SAH (nmol/g)

60

300 40
40 b 200 b
b b c b
200 c 20
bc c c
c 20 c b b
100
0 0
0 1 2 3 0 1 2 3
0 0
30
E F
20 50 15 a

[pmol/(min.mg protein)]
Liver Hcy (nmol/g liver)

E F a
(pmol/min/mg of protein)

a a
40 20

CBS activity
Liver Hcy (nmol/g)

a a
15
Liver CBS activity

10
ab b
30
10 10
b 5
b 20
b
b
5
c 10 b b
0 0
0 1 2 3 0 1 2 3
0 0 Days after diet change Days after diet change

Fig. 3. Effects of Dietary Addition of 1% Methionine with or without Fig. 4. Effects of Dietary Glycine and Serine on the Plasma
1% Glycine or 1.4% Serine on the Plasma Concentrations of Concentrations of Homocysteine (A) and Cysteine (B), the Hepatic
Homocysteine (A) and Cysteine (B), the Hepatic Concentrations Concentrations of S-Adenosylmethionine (C), S-Adenosylhomocys-
of S-Adenosylmethionine (C), S-Adenosylhomocysteine (D) and teine (D) and Homocysteine (E), and the Activity of Cystathionine
Homocysteine (E), and the Activity of Hepatic Cystathionine
-
-Synthase (F) in Rats (Experiment 3).
Synthase (F) in Rats (Experiment 2). Each value is the mean  SEM for 6 or 7 rats. a{c Values with
Each value is the mean  SEM for 6 to 8 rats. a{c Values with different letters are significantly different at p < 0:05. CBS,
different letters are significantly different at p < 0:05. 25C, 25% cystathionine
-synthase; SAH, S-adenosylhomocysteine; SAM, S-
casein diet; CBS, cystathionine
-synthase; SAH, S-adenosylhomo- adenosylmethionine.
cysteine; SAM, S-adenosylmethionine.

was also shown that the methionine-induced enhance-

and chronic hyperhomocysteinemia model. Hyperhomo- ment of plasma homocysteine concentration could be
cysteinemia in humans is a measure of the plasma total rapidly decreased by a dietary addition of glycine or
homocysteine concentration above 15 mM and is defined serine, even with only one day of feeding, although
as being moderate (15–30 mM), intermediate (30–100 glycine or serine was added to the diet at a relatively
mM) or severe (> 100 mM).20) Therefore, the hyperhomo- high (2.5%) level (experiment 3). The hypohomocyste-
cysteinemia model used in the present study to assess inemic effect of serine observed here is consistent with
the effects of glycine and serine was at the intermediate that in some previous reports.5,10) For instance, Stead
level. The results of experiment 2 clearly demonstrate et al.5) have demonstrated that a high (60%) casein diet
that methionine-induced hyperhomocysteinemia could increased the plasma homocysteine concentration in rats
be effectively suppressed by the concurrent addition of a compared with a 20% casein diet and that this increase
relatively low level of glycine (1%) or serine (1.4%). It in plasma homocysteine concentration was significantly
 Suppression of Hyperhomocysteinemia by Glycine and Serine
80 a
Plasma Hcy (nmol/ml)
60
b
40
20 d metabolism of SAM to SAH by the glycine N-methyl-
0 spite being a methyl-group acceptor. The exclusively
Saline
+ Gly
Met
Met
Fig. 5. Effect of an Intraperitoneal Injection of Methionine (300 mg/
kg) with or without Glycine (300 mg/kg) or Serine (420 mg/kg) on
the Plasma Homocysteine Concentration in Rats (Experiment 4).
Each value is the mean  SEM for 6 rats. a{d Values with different
letters are significantly different at p < 0:05.
suppressed by dietary supplementation with 3% serine.
Verhoef et al.10) have also demonstrated that serine
supplementation suppressed the increase in plasma
homocysteine concentration due to one-day ingestion
of a methionine-fortified meal in humans. On the other
hand, to our knowledge, this is the first demonstration of
the hypohomocysteinemic effect of glycine. An experi-
ment in another study of ours demonstrated that dietary
supplementation with 1% glycine or 1.4% serine did
not decrease the plasma homocysteine concentration
when added to the control (25% casein) diet (unpub-
lished data), suggesting that these amino acids might
act to normalize hyperhomocysteinemia, rather than to
decrease the normal range of plasma homocysteine
concentration.
The rate-limiting step of methionine metabolism in
animals loaded with an excess of methionine is thought
to be the demethylation of SAM, since methionine
loading decreases the hepatic concentration of glycine,
an exclusively major methyl-group acceptor, thereby
hampering the metabolism of SAM. Glycine and serine
can be mutually converted in a reaction catalyzed by
serine hydroxymethyltransferase, but the reaction is
known to favor the synthesis of serine. This might be
the reason why the alleviating effect of glycine on
methionine toxicity is somewhat greater than that of
serine.8) In contrast, cystathionine synthesis appears to
be more critical than the demethylation of SAM for
maintaining the hepatic homocysteine concentration at a
low level, since cystathionine synthesis is directly linked
to the removal of homocysteine. This warrants the
potential efficacy of serine in reducing the homocysteine
concentration, since serine participates in cystathionine
formation as a substrate. In fact, the hypohomocystei-
nemic effect of serine was significantly greater than that
of glycine in a short-term (2 h) experiment (Fig. 5).
2407
The most important finding of this study is that the
potency of the hypohomocysteinemic effect of glycine
was equivalent to that of serine when these amino acids
were separately added to the diet. It has been reported
that direct or indirect acceptors of the methyl group of
SAM, such as guanidinoacetic acid21) and nicotinic
c acid,22) brought about an increase in the plasma homo-
cysteine concentration, probably by accelerating the
conversion of SAM to SAH. Glycine also stimulates the
transferase system.23) Therefore, it should be noted that
glycine exhibited a hypohomocysteinemic effect, de-
+ Ser different feature of glycine from other methyl-group
Met
acceptors is that glycine can be converted to serine,
suggesting that glycine might elicit its homocysteine-
reducing effect mainly after being converted to serine.
The result that the methionine-induced increase in
the hepatic concentration of such methionine metabo-
lites as SAM, SAH and homocysteine was significantly
suppressed by both glycine and serine indicates that the
metabolism of methionine proceeded smoothly when
glycine or serine was exogenously provided (Figs. 3 and
4). Although homocysteine has two major fates in the
liver, i.e., cystathionine formation and remethylation,
cystathionine formation is thought to predominate under
the condition of methionine loading.24) It seems reason-
able to consider that serine stimulated cystathionine
synthesis as a substrate for CBS and thereby increased
homocysteine removal, although the dietary addition of
serine did not increase the enzyme activity, but rather
decreased it (Fig. 3). In support of this, we have
previously demonstrated that the alleviating effect of
dietary glycine on methionine toxicity was primarily
elicited by restoration of the hepatic glycine and serine
concentrations, rather than by any increase in the
activities of methionine-metabolizing enzymes.25) It is
thus likely that serine and its precursor, glycine, elicit
their effects mainly by stimulating cystathionine syn-
thesis by supplying serine, a substrate for cystathionine
synthesis.
Both glycine and serine can provide a one-carbon unit
which is finally converted to 5-methyltetrahydrofolate
(Fig. 1). This raises the possibility that glycine and
serine may increase the formation of 5-methyltetrahy-
drofolate and thereby stimulate the remethylation of
homocysteine catalyzed by methionine synthase. It has
been shown that the activity of methionine synthase
declined with increasing dietary methionine from 0.3%
to 1.0%.24) Furthermore, it is known that SAM, which
increases in response to the amount of methionine
ingested, inhibits 5,10-methylenetetrahydrofolate reduc-
tase, a key enzyme in the formation of 5-methyltetrahy-
drofolate.26) These facts suggest that the 5-methyltetra-
hydrofolate-dependent remethylation of homocysteine is
depressed under the condition of methionine loading.
Since glycine and serine both suppressed the methio-
nine-induced increase in hepatic SAM concentration, the
2408 S. F UKADA et al.
possibility that these amino acids may stimulate the
formation of 5-methyltetrahydrofolate and thereby en-
hance the removal of homocysteine cannot be com-
pletely excluded. However, it remains to be elucidated
whether glycine and serine actually affect the remeth-
ylation of homocysteine by methionine synthase. On the
other hand, there is currently no information about
the effects of glycine and serine on the remethylation
of homocysteine catalyzed by betaine:homocysteine S-
methyltransferase.
It has been shown that the addition of methionine
increased the plasma cholesterol concentration27–29) and
that this increase could be effectively suppressed by the
concurrent addition of glycine or serine, although the
effect of glycine was greater than that of serine.30) In
addition, the present study has provided data which
indicate that methionine-induced hyperhomocysteine-
mia could also be effectively suppressed by dietary
glycine and serine to the same degree. Since increased
concentrations of plasma cholesterol and homocysteine
are both risk factors for cardiovascular disease, it is
likely that both glycine and serine might be effective in
suppressing the pathogenesis of cardiovascular disease
by counteracting such deleterious effects of methionine
as methionine-induced hypercholesterolemia and hyper-
homocysteinemia.
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