Title
Studies on the Roles of Folate and Betaine in the Metabolism of
Homocysteine( 本文（FULLTEXT) )
Author(s)
劉, 穎
Report No.(Doctoral
Degree)
博士(農学) 甲第577号
Issue Date
2012-03-13
Type
博士論文
Version
publisher
URL
http://repository.lib.gifu-u.ac.jp/handle/123456789/42963
※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
studies
on
Metabolism
(*t->x51J1
in the
of Folate and Betaine
of IIomocysteine
the Roles
>f%#8=B8J8*#tJ<9Jf
VL Bgla
>0@5g
8f9%)
2011
The
United
School
Graduate
Gifu
Science
of
of Agricultural
University
BiologicalResources
(Shizuoka University)
LIU
YING
Science,
ABBREVIATI
ONS
casein diet
10C
lO%
10CCD
choline-deprived
10CFD
folati-deprived1 0C
20C
20%
20CCDFD
choline
20CFD
folate-deprived
25S
25%
25SCD
choline-deprived
BHMT
betaine-homocy
CBS
cystathiomine
I)MG
N, N- dimethyl glycine
PC
pho sphatidylcholine
PE
phosphatidylethanol
MS
methionine
SAH
S-adenosylhomocy
SAM
S-adenosylmethionine
5-MTHF
5
VLDL
very low
-
10C
casein diet
and folate-deprived
e
20C
protein diet
-soybean
m
20C
25 S
steine S-methyltransferase
P-synthase
amine
synthase
steine
thy 1tetrahy drofol
ate
density lipoprotein
CONTENTS
ABBREVIATIONS
GENERAL
CHAPTER
INTRODUCTION
I
of Betaine
Effects
Folate
on
I
I.2
Matgrialsand
1.3
Results
Deficiency
in rats
14
Method
17
I.3.1 Effect
of betaine
supplementation
I.3.2 Effect
of choline
and
(experiment 1)
(experiment 2)
folate deprivation
17
24
28
Discussion
Effects
2.1
Choline
10
Introduction
Supplementation
of Dietary
with
folate
Hyperhomocysteinemia
Deficiency-induced
Choline
on
34
in rats
Introduction
2.2
Matirials
2.3
Results
2.3.I
Effect
and
on
deprivation
2.3.2
Effect
on
deprivation
2.4
Method
hyperhomocysteinemia
of 10C
induced
by choline
41
(experiment 1)
hyperhomocysteinemia
induced
by choline
48
(experiment 2)
of 25S
52
Discussion
56
CONCLUSION
AcKNOWLED
and
Hyperhomocysteinemia
Deficiency-induced
1.1
1.4
CHAPTER
Supplementation
59
GMENTS
60
REFERRENCES
3
GENERAL
INTRODUCTION
is
Homocysteine
differing
cysteine,
has been
plasma
1%
total, only
disulfides.
by
to 2%
disulfides,
occurs
defined
as
of the total is bound
hyperhomocysteinemia
increases
Homocysteine
NADH/NADPH
with
auto-oxidized
hydrogen
that potentially
affecting
of LDL
promote
metal
directly degrade
(low
events
cardiovascular
and
density
may
Decreased
decrease
NO
in the vascular
oxygen
the expression
bioavailability
the pfoliferation
was
of vascular
4
including
though
LDL
that
(1)
superoxide
shown
has many
induce
may
(NO).
as
such
has also been
NO
atherosclerosis
synthase
to be
enhances
and
predictive
to produce
muscle
to
characteristic
Homocysteine
shown
also shown
smooth
an
wall via
of endothelial
has been
It is
mechanisms:
species
(8-10). (3) Homocysteine
factor, nitric oxide
a
(7). (2) Homocysteine
dysfunction
This process
(ll-13). (4) Homocysteine
stimulate
by duplicate
,
lipoprotein). Oxidized
relaxing
be
It has been
process
reactive
and hydroxylradical.
disease,
vascular
(1 3-6).
production
ions, generating
atherogenesis
which
NO.
anion
is
(1).
might
disease
leads to endothelial
which
endothelia1-derived
lipid peroxidation
dysfunction
trace
peroxide
oxidation
atherosclerotic
superoxide
oxidase,
Alzheimer's
and
innuences
peripheral
and
protein
forms:
blood.
(>100uM)
that hyperhomocysteinemia
suggested
stroke, dementia
in human
severe
and
with
of other mixed
amounts
level of homocysteine
(30-100pM)
bonds
of
Hyperhomocysteinemia
(2).
disulfide
is the form
disulfide
minor
Of this
(1).
in non-protein-bound
more
and
acid
levels in
98%
remaining
through
disulfide,
risk factor for cardiovascular
atherosclerotic,
The
to protein
of the amino
individuals
in healthy
occurs
high
a
moderate
have
of studies
number
by
characterized
homologue
a
total homocysteine
the remainder
whereas
(15-30pM),
mild
independent
by
The
e.g., homocysteine-cysteinylglycine
condition
promote
group.
the thiol homocysteine.
as
75%
It is
of 5-15uM
to be in the range
homocysteine-cygteine
medical
anion,
methylene
additional
in albumin,
mainly
homocysteine,
an
acid.
amino
sulfur-containing
reported
Perhaps
cysteines,
A
a
of
endothelial
cells and
other
cells.
the formatioh
It is also increased
in vessel
sclerotic process
is
Homocysteine
by two
metabolized
a
walls
physiological,
clinical and
instance,
such
Furthern10re,
are
synthase
(1,3,4). We
also have
induces
of the deficient
due
hyperhomocysteinemia
A number
human
been
hyperhomocysteinemic
betaine
is based
concentration
betaine-BHMT
administration
on
and
reported
animal
BHMT
dietary
or
have
also have
and
supplementation
(18).
deficiency,
due
to betaine
are
number
reported
5
of choline,
(20).
to suppress
effect of betaine
(23-27).
The
hepatic
homocysteine
of studies
the aim
in
in
increases
folate with
with
a
by
also required
effects of betaine
there by stimulates
a
is
of homocysteine-metabolizing
the compound
been
such
deficiency
be easily prevented
treatments
also been
B-6
vitamin
that deprivation
demonstrated
can
vitamins
some
since folate and
activity and
mutations
The
(21, 22).
(28)
affect the
of
homocysteine-lowing
the plasma
by which
activity
There
defects
models
the mechanism
system.
or
to genetic
on
(MS)
deficiencies
but other
For
leads to hyperhomocysteinemia
hyperhomocysteinemia
vitamins,
of studies
have
subjects
cause
by vitamin
caused
deficiency
hyperhomocysteinemia,
of methionine
activity
B-6
nutritional
concentration.
that deficiencies
shown
co-factors
Hyperhomocysteinemia
enzymes.
has been
and
leads to hyperhomocysteinemia
of homocysteine
vitamin
compound,
administration
(MTHFR)
B- 12 and
factor of the CBS
vitamin-like
reports
nutritiona1,
(CBS)
Cystathionine-P-synthase
to
pathway
genetic,
hyperhomocysteinemia,
cause
may
which
dues
including
homocysteine
plasma
reductase
-to methionine,
Several
folate, vitamin
B-12
on
Plasma
(Fig. 1).
these factors, genetic
metabolism
of homocystinura
error
factors
(1,3,4). Of
innuence
in homocysteine
affect the transsulfuration
vitamin
a
greater
of homocysteine
inborn
an
(19).
co
a
the
promote
It is normally
(17).
transsulfuration
and
by various
5, 10-methylenetetrahydrofolate
as
which
as
defects
genetic
remethylation
And
to have
thought
are
or
activate
metabolism
of methionine
lifestyle factors
may
(14-16).
other tissues
is affected
which
matrix,
i.e. remethylation
pathways,
concentration
and
intermediate
normal
homocysteine
factors
of extracellular
on
efficacy
of
betaine
removal
the effect of
of decreasing
plasma
by the
homocysteine
by which
mechanism
yet been
not
in humans
concentration
homocysteine
but closely
connected
with
the MS
a
affect the BHMT
pathway.
increased
significantly
roles and
a
In fact, it has been
is currently
between
relationship
pathway
is
DMG
available
the BHMT
is not
Thus
we
which
might
was
concentration
deficiency,
B-12
reported
in the
DMG
of DMG,
serum
but not by vitamin
litter information
compensatory
that
are
(38,39).
of BHMT
consumption
reported
(37).
to
pathways
participate
acceptor
inhibitor
is catalyzed
It has been
(DMG).
methy1-group
an
two
that the BHMT
suggested
decreased
cause
might
by folate deficiency'
subjects(40,41). However,
specific
as
but also
reaction
that folate deficiency
The
pathway.
N, N-dimethylglycine
N-methylglycine
and
The
paid attention
we
folate, since tetrahydrofolate
requires
betaine.
or
Recently,
the MS
investigations
through
pathway
of the BHMT
product
Some
related.
system
of DMG
assumed
has
methionine
the latter reaction
and
(BHMT).
and
pathway
to generate
(5-MTHF)
(MS)
synthase
synthase
the BHMT
that the betaine-BHMT
metabolism
by methionine
S-methionine
between
separately
is remethylated
homocysteine
pathway'
is catalyzed
the relationship
only
the
of homocysteine
the concentration
reduces
of either 5-methyltetrahydrofolate
group
reaction
by betaine
(29-36). However,
models
clarified.
the methyl
former
animal
folate supplementation
In the remethylation
using
and
that the
concerning
pathway
in human
and
the MS
pathway.
Therefore,
metabolism
in the present
of homocysteine.
supplementation
and
For this purpose,
deficiency
choline
hyperhomocysteinemia
to determine
hyperhomocysteinemia
could
system.
of betaine
Since
investigated
we
study,
effect of dietary
anticipated
be suppressed
with
in the
the effects of betaine
folate deficiency-induced
by betaine
the hepatic
through
hyperhomocysteinemia
to differ depending
supplementation
firstly investigated
betaine
folate deficiency-induced
whether
folate deficiency-induced
were
on
we
the roles of folate and
on
betaine
dietary
at a high
6
and
casein
level
the supplemental
level,
(1%)
betaine-BHMT
we
investigated
in rats fed
effect
the
low
folate-deprived
(10%)
the effects of dietary
hyperhomocysteinemia
betaine-BHMT
folate status.
and
25%
soybean
investigated
induced
source
deprivation
(25S)
hyperhomocysteinemia
of Cl
units for 5-MTHF
secondly
of low
methionine
in obvious
supplementation
in rats fed 10C
with
and
(42).
7
25S
investigated
deprivation-induced
choline
pathway
hyperhomocysteinemia
diet resulted
the effects of dietary
on
we
the other remethylation
whether
impaired-induced
choline
diets. Then
casein
folate
with
to determine
protein
(20%)
standard
supplementation
pathway)
Since
and
10%
as
on
diet with
choline
or
casein
(10C)
(20),we
hyperhomocysteinemia
folate
by
be suppressed
could
diet such
(the
deprivation-
without
serine,
a
main
{
Homocysteine
cBSr
Ser
Cystathionine
i
cy/steine GSH
+
Tau,,ine
Fig.
I Metabolism
of methionine
s-methyltransferase
(EC
N-dimethylglycine;
FA,
5-methyltetrahydrofolate
S-adenosylhomocysteine;
serine ; THE,
and
2. l. 1.5); CBS,
folic acid; MS,
; PC,
SAM,
homocysteine.
cystathionine
methionine
phosphatidylcholine
S-adenosylmethionine;
tetrahydrofolate.
8
i.2_
BHMT,
betaine-homocysteine
b-synthase
synthase
; PE,
(EC
(EC
4.2.
1.22); DMG,
N,
2. 1. 1. 13); 5-MTHF,
phosphatidylethanolamine
Sar, sarcosine
; SAH,
(N-methlglycine)
; Ser,
I
CHAPTER
Effects
of Betaine
Supplementation
Deficiency-induced
and
Choline
Hyperhomocysteinemia
9
Deficiency
in rats
on
Folate
Introduction
l.1
is
Homocysteine
number
be
two
(MS)
synthase
(BHMT).
the transsulfuration
pathway,
homocysteine
and
deprivation
of choline,
deficiency
to suppress
The
efficacy
effect of betaine
hepatic
removal
in hyperhomocysteinemic
betaine
concentration
by the betaine-BHMT
on
and
in human
animal
the mechanism
BHMT
activity
system.
10
number
as
folate,
models
by which
and
(1,3,4).Furthermore,
deficiencies
but other treatments
defects
or
of studies
mutations
due
been
have
also been
reported
are
be
also
of
(21,22).
reported
stimulates
The
(23-27).
increases
the compound
thereby
can
to
the plasma
on
have
subjects
(28)
these
plasma
hyperhomocysteinemia
by vitamin
vitamins,
a
on
such
vitamins
factors
co
to genetic
of
(1,3,4). Of
since these vitamins
caused
due
factors
innuence
of certain
of the
out
cysteine
by various
greater
also induces
Results
enzymes.
is based
a
enzyme
of the deficient
hyperhomocysteinemia
of betaine
as
compound,
administration
homocysteine-lowering
effects of betaine
of homocysteine
by
the first step of
toward
lifestyle factors
hyperhomocysteinemia,
cause
of either
is catalyzed
catalyzes
is affected
deficiencies
group
pathway,
by betaine-homocysteine
flows
to have
Hyperhomocysteinemia
homocysteine-metabolizing
reaction
is catalyzed
thought
are
vitamin-like
(20).
by
easily prevented
a
former
clinical and
For instance,
B- 12
in the metabolism
the methyl
concentration
factors
nutritional
vitamin
and
using
sulfur of methionine
homocysteine
concentration.
B-6
participate
required
by which
has
In the remethylation
(CBS)
Cystathionine-P-synthase
nutritiona1, physiological,
genetic,
factors, genetic
betaine
Plasma
cycle.
The
a
concentration
(1,3,4). Homocysteine
disease
methionine
betaine.
or
homocysteine
transsulfuration.
and
(17) (Fig. 1. 1),but
metabolism
plasma
the latter reaction
and
s-methyltransferase
methionine
elevated
to generate
(5-MTHF)
5-methyltetrahydrofolate
methionine
of methionine
risk factor for cardiovascular
is remethylated
homocysteine
vitamin
an
rates, i.e.,remethylation
metabolic
including
that
suggested
independent
an
intern1ediate
normal
have
of studies
might
a
homocysteine
BHMT
THF
5-MT-HF
SAM
Betaine
+
+
5-MTHF
T:cH.F
Choline
+
PC.+
cH3-:]^[Pi
sfH
THF+
T
+/
Diet
FA+Diet
Homocysteine
Serine
i
CBS
Cystathionine
+
G[utathione
Fig. l.I
Metabolism
of methionine
S-methyltransferase
(EC
N-dimethylglycine.,
FA,
and
2. 1. l.5).,
CBS,
folic acid; MS,
5-methyltetrahydrofolate;
PC,
S-adenosylhomocysteine;
SAM,
THF,
Cysteine
++
+
,+
Taurine,
homocysteine.
BHMT,
cystathionine
b-synthase
methionine
phosphatidylcholine;
S-adenosylmethionine;
tetrahydrofolate.
ll
synthase
PE,
SO42-
betaine-homocysteine
(EC
(EC
4.2.
1.22); DMG,
N,
2. 1. l. 13);5-MTHF,
phosphatidylethanolamine;
Sar, sarcosine
SAH,
(N-methlglycine)
;
one
of the representative
folate deficiency
hyperhomocysteinemia
homocysteine
due
to
decrease
a
to
duplicate
decreasel
a
in hepatic
of CBS
activator
by
due
However,
(45).
decrease
the betaine-BHMT
system,
of the 5-MTHF-MS
system
issue appears
(DMG)
to be
folate, since
requires
deficiency
might
deficiency,
increased
by DMG
on
DMG
In the present
study,
the function
investigated
increased
Of
inhibitor
an
of BHMT
et al.
(48)
renal failure patients
these findings,
they postulated
that folate deficiency
might
However,
system.
and
investigated
hyperhomocysteinemia
of the hep?tic
not
the effect of dietary
only
l2
status
whether
system.
with
DMG
serum
B-12
DMG
was
a
significant
BHMT
activity due
to
renal failure
the 5-MTHF-MS
on
system
the significance
hyperhomocysteinemia.
determine
supplementation
a
only
In fact,
in chronic
the effect of betaine
betaine-BfMT
is not
total homocysteine
plasma
there is little information
to
that folate
that there
that reduced
in folate deficiency-induced
system
that plasma
reported
and
impair
(46,47).
but not by vitamin
to hyperhomocysteinemia
contribute
we
(38,39).
by folate deficiency,
McGregor
the capacity
of N,N-dimethylglycine
interest is that DMG
concentration
concentration
deficiency-induced
impairs
but also
than
(37), suggesting
acceptor
DMG
might
suggests
in the metabolism
pathway.
plasma
but also the betaine-BHMT
of hepatic
thgBHMT
in.chronic
between
Based
This
patients.
methyl-group
subjects(40,41).
correlation
inhibition
a
significantly
was
concentration.
as
by
be fully compensated
the fact that the betaine-BHMT
participates
an
folate
activities in the liver of rats
their enzyme
at least in part by
solved
reaction
in human
concentration
positive
judging from
is
which
of why
to be greater
is thought
of which
of
transsulfuration
(SAM),
cannot
metabolism
a
induces
(2) decreased
and
is
remethylation
raise the question
explanations
the capacity
also affect
was
concentration
concentration
of S-adenosylmethionine
these
tetrahydrofolate
of the BHMT
product
(1,43):(1) disturbed
in homocysteine
N-methylglycine
and
in 5-MTfiF
in rodents
models
that folate deficiency
thought
mechanisms
concentration
deficiency-induced
This
It has been
(43,44).
model
hyperhomocysteinemia
experimental
For
betaine
on
folate
folate deficiency
this purpose,
at a high
level
actually
we
(1%)
in rats
low
fed folate-deprived
we
investigated
folate-deprived
(10%)
and
standard
the effect of choline
standard
casein
diets
(20%)
deprivation
casein
(experiment 1).
in rats fed folate-sufficient
(experiment 2).
13
diets
and
In addition,
I.2
Materials
Method
and
1.2.I Chemicals
Betaine
folic acid
and
succinylsulfathiazole
were
chemicals
were
were
was
ingredients
of the diet
1.2.2 Animals
and
Six-week-old
Japan).
in
room
isolated
an
Lighting
was
and
25%
a
rats
They
kept
a
were
12:h
were
two
separate
(10CFD),
10CFD
(20C),
folate-deprived
20C
(20CFD),
and
572.26;
cornstarch,
mixture
mg/g),
was
casein
folate-free
four
consisted
200,
sucrose,
corn
succinylsulfathiazo16,
raised
lactose
diet groups:
to 200
was
20C,
g/kg
used.
+
Yeast
were
obtained
from
from
mixture
(Tokyo).
humidity
and
to 19:00
six diet groups:
betaine
1%
+
ingredients
at the expense
In experiment
20C
of cornstarch.
2, rats
were
(20CCD),
14
20CFD,
assigned
and
conducted.
In
10%
diet
casein
diet
casein
of the
casein,
100;
(33.3
vitamin-free
In folate-deprived
randomly
to water
folate
containing
In 20C,
starting
35; vitamin
(AIN-93G),
20.
powder,
cages
access
One
(g/kg):vitamin-free
bitartrate, 2.5; lactose
cellulose
20%
(20CFDB).
mixture
Before
free
were
(10CFDB),
wire
SLC
(40-60%).
h).
given
experiments
betaine
Japan
stainless-steel
07:00
animal
oil, 50; mineral
lO; and
choline-deprived
(23-25oC)
1%
20CFD
of the following
(AIN-93, folate-free),10., choline
0.24;
Oriental
in hanging
to the following
assigned
lOC
(lOC)
vitamin
from
to the facility for 5 d and
acclimated
folate-deprived
diets
housed
(lightson
cycle
randomly
strain
temperature
(10C),
control
and
Wako.
the Wistar
individually
diet. In this study,
1, rats
experiment
were
on
all rats
casein
Sigma-Aldrich
or
(AIN-93G),
mixture
purchased
from
(l20-140 g) of
at a controlled
maintained
the experiments,
were
purchased
(Osaka, Japan)
mineral
All other
(Irvine,Cal).
diets
male
(Hamamatsu,
Chemical
powder
were
Biomedicals
casein,
cellulose
(St.Louis, Mo).
Sigma-Aldrich
MP
Pure
Vitamin-free
(AIN-93, folate-free),and
Other
Wako
from
grade.
of analytical
from
purchased
purchased
from
purchased
diets,
to the following
choline-deprived
and
folate-deprived
and
20C
increased.
was
cornstarch
by intestinal bacteria
and
20CCD)
to
according
folate at
contained
a
to the experimental.diets
access
10:00
did not affect lasting
treatment
was
study
were
1.2.3 Tissue
Blood
blood,
the whole
cut into two
ice-cold
The
was
liver
One
containing
at 4oC.
The
rinsed
frozen
of the liver
portion
acid solution
betaine,
in 4 volumes
KCl,
between
(50).
Use
This
the animals
and
of Laboratory
concentration,
w'as
lipids
extracted
an
was
and
by the method
a
10 mM
sodium
to enzyme
et al.
15
phosphate
centrifuged
assays.
of the liver homogenate
(51).
other
(vo1/wt) of
g for 10 min
x
buffer
for
(pH 7.4)
at 14,000
lyophilized,
at
of the liver
portion
For the assay
was
of
until
-80oC
subjected
The
x
filter paper,
to assays
was
serine.
was
on
at
stored
at lO,000
then centrifuged
homogenate
of Folch
and
at 2,000
collection
in 4 volumes
homogenized
and
After
saline, blotted
in ice-cold
subjected
aliquot
for analysis.
liver homogenate
(vo1/wt) of
by centrifugation
in liquid nitrogen,
dimethylglycine
the resulting
and
supernatant
triglyceride
were
given
that dietary
shown
and
blood
whole
until needed
of the deproteinized
metabolites,
0. 15 M
at -30oC
quickly
trichloroacetic
homogenized
were
University,
for the Care
20C,
to make
in humans
of Shizuoka
heparinized
quickly removed,
weighed,
supernatant
methionine
from
stored
was
portions,
0.3 M
(10C,
fractionation
and
separated
and
for analysis.
needed
min
was
at 4oC
Committee
diets
killed by decapitation
and
concentration
the "Guidelines
with
folate synthesis
University.
collection
plasma
g for 15 min
was
in accordance
of Shizuoka
Animals"
homocysteine
Use
vitamin-free
Rats
(49).
report
omitted
was
level of AIN-93,
since it has been
deprivation,
plasma
by the Animal
approved
maintained
4oC.
prior food
1 l:00 h without
and
for 4 wk
water
and
four-fold
a
to a previous
according
and
Folate-sufficient
(49).
report
level of 8 mg/kg,
a
clear the effect of folate deficiency
free
previous
mixture
vitamin
in the diet to suppress
included
was
succinylsulfathiazole
casein, antibiotic
to folate-free
In addition
bitartrate
diets, choline
In choline-deprived
(20CCDFD).
x
g for 10
of hepatic
and
total
1.2.4 Biochemical
analysis
The
concentrations
cysteine
in the plasma
The
(52).
liver
and
concentration
deproteinized
and
in the liver
(DMG)
The
activity of BI-IMT
was
used
in the assay
assav
I
of the reaction
triglyceride
E-Test
(60)
was
bovine
using
The
Wako).
serum
in the liver
protein
as
albumin
was
concentration
a
standard.
mean
j=
(55)
a
using
(Model
Huang
et a1.
(56).
(58), but
HPLC
was
(59).
et a1.
The
(55).
et a1.
kit
commercial
measured
by
et al. (57), but HPLC
Laryea
Einarsson
et al.
the
and
following
Finkelstein
following
Cook
N,N-dimethylglycine
and
acid autoanalyzer
et a1.
and
by HPLC
measured
amino
Mudd
enzymatically
following
et a1.
following
following
cystathionine,
were
measured
DMG,
of SAM
Laryea
following
product,
measured
an
was
measured
measured
product,
concentration
Wako,
was
by
subtracting
concentrations
of betaine
following
by
by HPLJC
liver
and
measured
was
of the reaction
in the liver
activity of CBS
the
in the liver
The
et al.
using
measured
estimated
measured
concentrations
was
activity of MS
Hitachi). The
were
by HPLC
measured
of serine in the liver
concentration
L-8500;
were
was
in the plasma
et a1. (54). The
of Shimoda
was
and
of Durand
the method
using
total homocysteine.
of 5-MTHF
concentrations
the method
by HPLC
homocysteine
in the liver
(SAH)
homocysteine
non-protein-bound)
homocysteine
from
homocysteine
plus
measured
protein-bound
s-adenosylhomocysteine
The
were
of non-protein-bound
plasma,
non-protein-bound
(53).
(protein-bound
of total
The
used
hepatic
(Triglyceride
to Lowry
according
et a1.
1.2.5 Statistical analysis
Each
ANOVA
value
(experiment l) or
experimental
When
is expressed
groups
variances
logarithmically
Tokei-Kaiseki
were
among
the
two-way
Data
ANOVA
the experimental
before
(version
SEM.
were
were
groups
ANOVA.
1.5; Esumi,
test
whei
not
16
a
one-way
differences
the F value
homogeneous,
Statistical analysis
Tokyo).
by
analyzed
(experiment 2), and
by the Tukey
analyzed
transformed
software
as
was
was
among
the
significant.
data
were
pel-formed
in
with
Mac
I.3
Results
I.3.1 Effect
of betaine
Body
gain
weight
did n.t
differ among
(93.5%) (Fig. 1.2,
was
increase
10C
the extent
20C,
(48.5%)
than
(Fig. 1.2,
homocysteine
significantly
betaine
an
SAH
of dietary
was
significantly
irrespective
tended
by betaine
lower
betaine
rats fed
or
10C
to increase
increased
(Fig. l3,
(Fig. 1.3,
by folate deprivation
of folate deprivation
concentration,
(Fig. 1.2,
fed 20C
and
17
diets
and
(Fig. 1.3,
B).
was
Hepatic
this increase
was
Hepatic
A).
and
ratio
was
was
diets
slightly increased
supplementation
homocysteine
by
restored
panel
SAM:SAH
Hepatic
E).
by folate deprivation
panel
betaine
C).
measured
panel
diets than in rats fed the control
although
panel
was
in rats fed folate-deprived
to be increased
supplementation
the ratio
those
of either
to that of total
by folate deprivation
and
tended
supplementation,
5-MTHF
fed
rats
supplementation
concentration
supplementation
decreased
in rats fed folate-deprived
of betaine
significantly
significantly
increased
decreased
greatly
betaine
irrespective
Plasma
D).
panel
level and
in both
supplementation
increased
(61).,was
10C
in both
in rats fed 10C
similarly
total cysteine
in rats fed
than
folate
exists in the form
and
while
in rats fed 20C
suppressed
smaller
deprivation
Plasma
C).
(Fig. 1.2,
casein
was
concentration
further
or
in rats fed 20C
concentration
betaine
B and
was
of homocysteine
types
fed 20C,
concentration
homocysteine
Plasma
than
significantly
was
concentration
those
and
(126.7%)
suppression
(2). Folate
of both
of folate status
irrespective
of increment
panels
10C
10C
total homocysteine
(69.7%).
supplementation
index
SAM
higher
in rats fed
supplementation
non-protein-bound
the concentrations
affected
as
in rats fed 20C
or
protein-bound
and
while
rats fed
intake
food
whereas
total homocysteine
in both
than in rats
in rats fed 20C
supplementation,
1. 1). Plasma
in plasma
deprivation-induced
and
betaine
or
greater
Betaine
A).
panel
higher
significantly
by folate deprivation
of the increase
the magnitude
were
(Table
the groups
increased
significantly
liver weight
and
of folate deprivation
irrespective
fed 10C
(experiment 1)
supplementation
concentration
suppressed
or
tended
was
to
by betaine
be suppressed
Hepatic
betaine
MS
activity
rats fed
10C
a
Folate
the magnitude
of CBS,
was
Hepatic
DMG
l.5, panel
B).
that of hepatic
panel
of MS,
was
higher
lower
(Fig. 1.5,
relatively
greatly
A).
panel
in rats fed 20C
Hepatic
increased
Betaine
Since
the profile of plasma
DMG
concentration,
D).
panel
(Fig. 1.4,
activity, although
a
of serine,
concentration
(Fig. I.4,
by folate deprivation
substrate
F).
panel
in both
rats
fed 10C
suppressed
significantly
of the effect
partial. Betaine:DMG
the
supplementation
was
diets without
the correlation
coefficient
positive
C).
l8
betaine
correlation
ratio
(Fig.
supplementation
was
concentration
was
a
by folate
the enzyme
total homocysteine
significant
of betaine,
than in rats fed 10C
10C
in
supplementation
(Fig. 1.4,
in rats fed
was
activity
concentration
supplementation
than
BHMT
to be decreased
tended
or
small.
but the extent
a
Hepatic
to decrease
in rats fed folate-deprived
was
by betaine
tended
lower
There
C).
panel
Hepatic
A).
panel
by folate deprivation
Hepatic
B).
panel
increased
in rats fed 20C
was
concentration,
or
decreased
by
unaffected
and
(Fig. 1.4,
fed 20C
greatly
by betaine
decreased
was
by folate deprivation
those
decreased
significantly
D).
panel
(Fig. 1.4,
(Fig. 1.4,
increased
concentration
of six groups.
(Fig. 1.5,
was
significantly
markedly
and
significantly
significantly
of the effect
in DMG
increase
and
fed 20C
deprivation
fed 20C
th.se
10C
supplementation
greatly
activity
rats fed
substrate
those
was
was
and
CBS
banelE).
values
and
of BHMT,
deprivation
was
significantly
by folate deprivation
substrate
and
decreased
by betaine
unaffected
Hepatic
was
of 5-MTHF,
unaffected
both
(Fig. 1.3,
in both
supplementation
concentration
and
supplementation
estimated
between
similar
using
the two
to
mean
variables
Table
Body
intake
gain, food
weight.
diets
experimental
of rats fed the
liver weight
and
(experiment 1)
Body
Diet
1.I
wt
intake
Food
gain
Liver
d
g/28
wt
g body
g/100
4b,1
460
j=
17
3.55
j=
0.07b
4b
436
i
13
3.69
j=
0.04b
60t4b
408
i
17
3.64
i
0.04b
20C
100i3a
461
i
12
4.28
j=
0.06a
20CFD
lO7
19
4.31
iO.l2a
16
4.25
j=
75
10C
66
1 0CFD
10CFD
20CFD
lEach
j=
+
+
1%
l%
Bet
is the
value
letter differ, P
<
diet, respectively;
respectively;
95
Bet
mean
0.05.
10C
10CFD
j=
j=
4a
448i
j=
5a
410
i
SEM,
and
and
20C,
n
20CFD,
=
8.
j=
without
casein
diet and
10%
Values
folate-deprived
Bet, betaine.
19
lOC
wt
0.06a
a common
20%
and
casein
20C,
I
BED
E23 F=D+1%Bet
gO
E
a
a
I
i
O
d)
E
n
a
10C
2OC
20
J
a
>=
O
O
E
3
E
15
5
A
O
A
I
I
E=
=
O
-A
10
100
0
O
J3
I)
dl
E
E
ul
5
u)
cO
A
50
a.
EL
i
10C
20C
lOC
20C
200
0
E
E=
LL
150
E
=
Lb
∼
loo
E
q)
a
EL
20C
10C
Fig. 1.2
and
n
-
Effects
other variables
8,
20%
Means
casein
homocy
in rats fed lO%
in apanel
Hcy,
and
20%
Bet, betaine;
homocysteine;
casein
diets with
5-MTHF,
bound
betaine
diets (experiment
letterdiffer,
withoutacommon
diet, respectn,ely:
folate-deprived;
of folate-depr"ed
of supplementation
Hey,
P
<0.()5.
10C
protein-bound
5-methyltetrahydrofolate;
steine.
20
( l%)
I). Each
and
on
plasma
is die
value
20C,
homocystehe;
unbound
concentration
10%
mean
casein
j=
SEM,
dietand
FD.
Hey,
protein-unbound
I control
DFD
FDB+1%Bet
5tD
0
O
5
0
E
E
E=
E:
=
I
u)
u)
<
<
4J
>
d>
J
.2
IJ
0
∼
d1
5tD
O
E
=
<
E:
u)
<
>
i)
I
O
Ll
E
4)
>
4I
>
=
=
Fig.
I.3
Effects
fed 10%
without
and
20%
a common
See the legend
diets with
offolate-deprived
of supplementation
of S-adenosylmethionine
1 0C
20C
1 0C
diets (experiment
letter differ, P
ofFig.
<
0.05,
I). Each
SAH,
betaine (l%)
hepatic
on
(B), their ratio (C), and homocysteine
(A), S-adenosylhon10CySteine
casein
20C
value
is themean
S-adenosylhomocysteine;
2 for other abbreviations.
21
j=
SEM,
SAM,
n
8. Means
-
concentrations
(D)
in rats
in apanel
S-adenosylmethionine.
Il control
DFD
Cg
FD+1%BBt
20
-
tn
5
0
E
5
1S
LL.
I
5.o
0
>
6
=
10C
20C
>
'5;
O
d)
E
I
a)
d)
>
J
<tn
>
a
O
P
a)
A
5
3
=>
0
E=
2
=
d>
O
U
0
>
JJ
0
>
=
10C
Fig. 1.4
enzymes
and
common
Effects
of homocysteine
20%casein
diets
p-synthase, MS,
metabolism
and
(experiment 1). Each
letter differ, P
<
methionine
0.05.
1 0C
20C
BHMT,
synthase,
hepatic
value
concentrat10nS
is the
meant
betaine-homocysteine
See the legend
22
20C
diets with betaine
offolate-deprived
ofsupplementation
1
ofFig.
(l%)
Of enzyme
SEM,
n
-
on
hepatic
substrates
8.
Means
S-methyltransferase;
activities of
in rats fed IO%
in apanel
CBS.
2 for other abbreviationsL
withouta
cystathionine
A Control
DFD
g FD+1%Bet
0
∼
a)
L
U
=
E)
d}
E:
'jio
J)
0
>
'=
10C
20C
a-o
E
-I
5
,r
>
O
I
//+
d}
E
∼)
q
a
r=0.929
P<0.Ol
o
0.2
0.6
0.4
Liver DMG
Fig. 1L5
plasma
value
is the
Means
in
a
offolate-deprived
(A). betaine.DMG
homocysteine
panel
ratm
concentration
SEM,
meanj=
(A
and
n
-
8
1
OLmOVg)
Effects ofsupplementation
concentration
0.8
(B), and
(C)
banels
B) without
relationship
in rats fed I O%
Aand
a common
diets with betaine (1%)
B).
between
2O%
and
h. panel
23
casein
C, each
letter differ. P
<
hepatic
DMG
diets
DMG,
hepatic
DMG
and
concentration
(experlment 1).
represents
value
0,05-
on
Each
the meanvalue.
N,N-dimethylglycine.
1.3.2 Effect
The
was
of choline
deprivation
effect of choline
in order
investigated
choline
deprivation
weight
gain
food
and
homocysteine
homocysteine
total cysteine
folate-deprived
SAM
Hepatic
alone
SAH
was
and
Consequently'
deprivation.
Hepatic
was
further
Hepatic
was
concentration
SAM:SAH
homocysteine
increased
MS
decreased
by choline
decreased
by folate deprivation
did
not
choline
differ among
deprivation
irrespective
the four groups.
deprivation.
Hepatic
CBS
deprivation.
Hepatic
serine
activity
by choline
was
concentration
and
was
deprivation.
choline
folate.
and
deprivation
Hepatic
folate
or
alone
folate
and
and
folate
by folate deprivation
alone
decreased
increased
by choline
was
significantly
was
further
further
was
Hepatic
was
concentration
decreased
decreased
significantly
24
and
was
concentration
deprivation.
of choline
and
alone
5-MTHF
Hepatic
Iiepatic betaine
folate deprivation
and
of both
by folate deprivation
folate deprivation.
and
The
folate deprivation.
and
decreased
was
and
concentration
deprivation
choline
was
plasma
enhanced
of choline
markedly
concentration
by choline
activity
was
ratio
total
in rats fed the choline-
5-MTHF
increased
significantly
plasma
to that of total homocysteine.
by deprivation
further decreased
was
in rats fed the
increased
markedly
diets irrespective
by
was
did not affect plasma
lower
diets. Plasma
1.2. Body
weight
non-protein-bound
similar
decreased
significantly
deprivation.
and
in rats fed other
Liver
diet than
alone
significantly
in rats fed folate-deprived
concentration
deprivation
were
in Table
significantly
profiles of plasma
was
concentration
alone
effect between
summarized
are
folate deprivation
concentrations
diet than
lower
markedly
The
concentration.
interacting
an
the four groups.
deprivation
and
hyperhomocysteinemia
folate-deprived
and
choline
Choline
concentration.
total homocysteine
results
folate deprivation
concentration,
protein-bound
The
in rats fed the choline-
total homocysteine
there exists
whether
did not differ among
diet. Although
choline-deprived
plasma
to detetmine
intake
higher
significantly
folate deprivation-induced
on
folate deprivation.
and
(experiment 2)
folate deprivation
and
higherin
BHMT
activity
decreased
by choline
by choline
markedly
and
and
by
folate
folate
rats fed the choline-
and
significandy
by folate deprivation
increased
folate deprivation.
folate-deprived
measured.
Hepatic
and
between
DMG
the four experimental
and
triglyceride
folate-deprived
concentration
groups
was
concentration
and
plasma
(Fig. 1.6).
25
diets.
was
fatty infiltration
triglyceride
significant
total homocysteine
choline
lower
significantly
positive
and
in rats fed
was
concentration
increased
significantly
a
by
increased
Since
diet, hepatic
folate-deprived
diet. There
was
was
concentration
further
ratio
diets than in rats.fed folate-sufficient
fed the cholinehepatic
DMG
DMG
was
and
alone
the betaine:
Consequently,
visible in rats fed the cholinewas
Hepatic
diet than in rats fed other diets.
folate-deprived
only
in rats
correlation
concentration
among
Table
Effects
of choline
rats fed a 20%
folate deprivation
and/or
diet
casein
on
1.2
homocysteine
plasma
and other variables
concentration
in
(experiment 2)
ANOVA2
20CCD
20 C
Body
wt
Food
intake, g/28
Liver
wt,
98
d
gain, g/28
d
107i=
5
105j=3
18
467j=
l4
452
4.l9iO.lob
4.27iO.07ab
wt
20CFD
41
i=
452j=
g body
g/100
20 CCDFD
j=
CF
99i3
l9
454
j=
8
4.36i=0.13ab
4.62iO.08a
F
Plasma:
Total
Hey,
Hey,
Unbound
Bound
Hey,
Unbound
FLmOl/i
Hey,
Total
FLmOl/L,
Hey,
Bound
13.5
FLmOl/i,
Cys,
%
%
0.5c
26.6
i
0.8b
45.4
j=
0.7a
C, F, CF
6.6
i
0.3c
10.3
i
0.2b
25.4
j=
0.8a
C, F, CF
8.0
j=
0.5c
16.3
j=
0.8b
20.0
j=
1.Oa
F, CF
j=
1.3ab
56.Oj=
1.8a
C, F
13ab
44.Oj=
1.8c
C, F
2c
C,F
0.5c
C,F,
CF
CF
l4.6
4.3 j= 0.3d
9.l iO.3c
32.1
j=
1.9c
45.3
j=
2.2b
39.0
67.9
j=
l.9a
54.7
j=
2.2b
6l.Oj=
146
j=
3a
131
j=
2b
136i
J210
j=
4.1a
198
j=
43b
6.4
FLmOl/L,
5-MTHF,
j=
0.4c
j=
nmol/i,
3ab
119j=
j=
0.4c
6.6
j=
Liver:
SAM,
nmol/g
88.4
j=
1.3a
49.2
j=
2.4b
46.4
j=
0.9b
21.7j=
1.Oc
C,F,
SAH,
nmol/g
17.3
j=
0.4bc
l5.0
j=
0.4c
l9.1
j=
0.5b
29.8
1.2a
C, F, CF
5.13
j=
0.12a
3.I)_7j= 0.lob
2.44
j=
0.09c
o.73j=0.05d
i
0.2b
j=
0.003b
SAM:SAH
Hey,
MS
ratio
3.I i:0.1c
nll-lOl/g
0.218
activity3
BHMT
CBS
1.36i
activity3
6.02
activity3
-i
0.20l
i
0.005a
o.145
7.4
l.29
i
0.03
1.37
j=
0.03
1.39
0.24a
5.26
i
0.24a
5.29
j=
0.17a
3.29iO.13b
0.4a
l5.I
j=
0.5b
FLmOl/g
1.72
j=
0.12a
1.32
j=
0.08b
l.24
j=
0.07b
0.84
Serine,.LLmOl/g
o.43
j=
0.02b
o.52
i
0.07b
o.62
j=
0.06b
DMG,
0.20
i
0.02c
0.19
j=
0.03c
o.79
j=
9.38i
1.12a
8.16i
l.28a
1.69
23.l.i
0.4b
25.7
0.5b
26.3
Betaine:DMG
ratio
FLmOl/g
Triglyceride,
lEach
casein
value
is the
diet; 20CCD,
folate-deprived
2Two-way
<
0.05., CF,
3Expressed
mean
j=
n
=
ANOVA.,
See
the legend
C, affected
interactively
affected
nmo1/(min. mg
8. Values
20C;
choline-deprived
20C.
as
SEM,
20CFD,
of Table
by
i
without
1.1 j=0.lc
a
1 for other
deprivation,
P
by choline
deprivation
and
26
0.07
c,F,CF
C,F
C, F
I.07i
0.10a
C, F, CF
0.08b
I.54j=
0.05a
C, F, CF
j=
0.2lb
o.55j=0.03b
F
i
0.3b
61.3
C,F,
differ, P
<
20C., 20CCDFD,
j=
i=
I.9a
0.05.
choline
20C,
20%
and
abbreviations.
choline
protein).
j=
0.04c
commonletter
folate-deprived
C,F,CF
1.OiO.1c
j=
FLmOl/g
C, F, CF
0.4a
0.05
16.5
Betaine,
j=
c,F
0.090j=0.004c
nmol/g
5-MTHF,
P
0.06a
i
4.5
3.1 j=0.1c
j=
<
0.05; F, affected
folate deprivation,
by folate deprivation,
P
<
0.05.
CF
I
I
I
<^
I
I
I
-o
I
JJ
E
I
I
I
1
I
∼
I,
>
O
I
I
I
I
a
,I.
I
I
E
I-
O
a
-i
r=
0.998
P<0,01
0.5
0
Liver
Fig. 1.6
Relationship
concentration
value
between
hepatic
in rats fed the experimental
of the group.
DMG,
1
DMG
N,N-dimethylglycine
diets
1,5
(FJmOL/g)
concentration
(experiment 2). Each
N,N-dimethylglycine.
27
and
plasma
homocysteine
closed circle represents
the
mean
1.4 Discussion
In the present
we
study'
is defined
hyperhomocysteinemia
100
Hence,
(1).
pM)
present
was
study
Furthermore,
elevation
guanidinoacetic
dietary
plasma
homocysteine
10CFD
casein
might
a
contain
small
of folate than
plasma
5-MTHF
of three enzymes
did rats fed
cysteine
plasma
through
the enhancement
decreasing
suppressing
deficiency
homocysteine
rats fed
However,
because
higher
increased
(65).
levels of casein
thought
of 5-MTHF,
remethylation
to induce
a
In any
are
not
be
a
larger
since
Another
to increase
tended
The
possible
the activities
third possible
in rats fed 20CFD
(1). The
case,
thereby
and
the present
effect
study
suggests
to folate deficiency.
hyperhomocysteinemia
present
in
casein
major reason
rat groups.
in rats
higher
two-fold
a
less susceptible
methyl-group
28
being
about
of
than
elicits its hypohomocysteinemic
cysteine
cysteine
or
by
also suppressed
of homocysteine.
was
concentration
the two
protein
(62,63).
in rats fed 20CFD
this may
increased
the metabolism
soybean
for the phenomenon
ingested
20CFD
this,
with
is that vitamin-free
reason
folate
against
of homocysteine,
possible
10CFD,
with
is generally
the concentration
One
10CFD.
of plasma
that rats fed diets containing
lower
significantly
in the
20C,
and
or
was
reasons
possible
several
did not differ between
compared
homocysteine,
reduced
concentrations
the sole precursor
folate and
amount
that participate.in
is that plasma
Folate
are
being
of Met,
concentration
is that 20CFD,
reason
There
(64).
the intake
amount
reason
level
than in rats fed 10CFD.
rats fed 20CFD
homocysteine
hyperhomocysteinemia
acid-induced
concentration
despite
levels of casein
(>
severe
and
Consistent
concentration.
higher
that diets containing
plasma
10C
level led to resistance
casein
of
magnitude
observed
in rats fed
(26.5 yM)
mild
homocysteine
of plasma
The
(30-100 yM)
moderate
and
mixture
hyperhomocysteinemia
dietary
but rather decreased
raising
fed
and
that increasing
demonstrated
did not increase
(15-30 yM),
mild
(34.I pM)
moderate
deprivation-induced
previously
as
vitamin
folate deficiency.
convenient
folate deprivation-induced
indicating
respectively,
we
to induce
succinylsulfathiazole
antibiotic
casein, folate-free
vitamin-free
used
mainly
donor
for MS,
study
demonstrated
and
by
thereby
that folate
decreased
deprivation
not
that the 5-MTHF-MS
indicating
folate deficiency
severe
in rats.
This
1.23
hepatic
0.08 ymo1/g
j=
hepatic
has not been
the increased
o.76
by
hepatic
0.07
j=
0. 19
DMG
concentration,
in
a
similar
SAH
group,
is known
as
here, for the first time
impair
hepatic
not
only
DMG
to our
system
of rat BHMT
and
in the 10CFD
ymo1/g
inhibit BHMT
0. 14
of various
knowledge,
supporting
some
significance
types
but also the betaine-BHMT
of the
objectivesof
the present
study
was
29
to examine
Hence,
group
In addition
whether
to
in the BHMT
Thus,
markedly
that folate deprivation
system
and
in vivo,
of methyltransferase.
that folate deprivation
the concept
was
transmethylation
and
hyperhomocysteinemia.
One
was
in the 10C
high.
also considerably
also have
Km
for DMG
reaction
0.01 pmo1/g
i
and
that the
of BHMT,
ratio in SAM-dependent
inhibitor
an
demonstrated
at 1 mM.
0.04
were
group'
group
by90%
groups,
ratio may
concentration,
the 5-MTHF
j=
10CFD
of rat BHMT
mM,
by
betaine
that the reaction
strongly
to the SAM:SAM
we
increased
0.90
in control
the betaine:DMG
where
the Ki value
atO.1
might
in-the 20C
reactions,
report
by76%
decreased
the reported
inhibitor
an
also
et a1. (38) reported
concentrations
fashion
and
than
&e
the other hand,
product
concentrations,
0.02 jimO1/g
j=
and
On
that
showed
in the
higher
stillone-order
is
by folate deprivation,
0.07 ymo1/g
+-
Although
atO.02mM,
DMG
DMG
group
reaction
19%
1.55
(67).
reaction
in the 20CFD
ymol/g
hepatic
although
120 pM
Finkelstein
reported,
study
in hepatic
the decrease
whether
that
showed
since choline
to be
tended
or
activity,
phosphocholine
and
concentration,
decreased
were
group'
a
choline
In fact, the present
concentrations,
by folate deprivation.
by DMG
inhibited
betaine
betaine
(66)
et al.
of hyperhomocysteinemia
to the -induction
of DMG,
increased
markedly
it is uncertain
for betaine,
concentration
in rats.
significantly
in the 20CFD
of rat BHMT
value
was
of hepatic
for hepatic
especially
However,
contributed
since decreased
depletion
secondary
concentration
folate deprivation.
concentration
caused
Kim
depressed.
greatly
MS
but also hepatic
concentration
was
system
to betaine,
betaine
5-MTHF
to be also the case
appears
easily metabolized
hepatic
hepatic
only
folate
thereby
might
induce
hyperhomocysteinemia
deficiency-induced
An
important
finding
of the present
low
a
diet. It appears
casein
hyperhomocysteinemia
at a level of 0.34%
supplementation
hyperhomocysteinemia
o.28%
to be
to
liver. This
concentration
BHMT
and
between
activity being
hepatic
hand,
the present
DMG
folate deprivation-induced
increased
decreased
synthesis
hepatic
or
SAH
tended
concentrations
groups
study
(Fig. 1.5,
showed that
decrease
to be decreased
The
betaine
while
C)
panel
hepatic
(Fig.1.3). These
system
might
supports
partial
increase
of
hepatic
a
such
a
in hepatic
significant
concentrations
On
possibility.
and
homocysteine
concentration
enhanced
One
betaine
completely
results suggest
or
be fully
not
concentration
be significantly
30
may
homocysteine
supplementation
SAM
insufficient.
was
restored
significantly
was
that methionine
by betaine
is
betaine
activity in the
not
despite
existence
and plasma
in hepatic
concentration,
by the betaine-BHMT
was
in vivo
the effect of betaine,
enhanced.
Indeed,
BHMT
is that folate deprivation-induced
interfere with
the six experimental
among
other
might
both.
or
of folate deficiency.
the condition
under
is needed
effect of betaine
concentration
by BHMT
catalyzed
level of
study
experiment
and
level of betaihe
homocysteine
plasma
supplementation
for the phenomenon
reason
concentration
on
in the present
activity,
concentration
that l'% supplementation
that the actual reaction
by betaine
stimulated
betaine
a
at
supplementation
used
BHMT
concentration,
both
the effect of betaine
suggesting
correlation
betaine
by betaine
dose-response
a
is solely due
that guanidinoacetic
that the hypohomocysteinemic
assume
increased
also suggests
Nevertheless,
limited,
in hepatic
to
although
in
deprivation-induced
level of betaine
to elicit its effect,
suppress
partial, especially
suppressed
by betaine
suppressed
was
effect of betaine
completely
that choline
and
supplementation
markedly
supplementation
DMG
1%
high enough
increase
an
possible
completely
this. It is reasonable
to confirm
due
Therefore,
(20).
appears
was
(27)
could
demonstrated
previously
almost
supplementation.
supplementation
that the limited
We
was
by betaine
but the effect
unlikely
level of betaine.
to the supplementation
acid-induced
is that betaine
study
hyperhomocysteinemia,
folate deficiency-induced
rats fed
be suppressed
can
the
Hence,
supplementation.
effect of betaine
2,
is determined
not
the choline
synthesize
SAM:SAH
ratio
deprivation
did not
10C
25%
induction
and
a
while
condition.
folate deprivation
homocysteine
deprivation
The
present
deprivation
homocysteine
diet
protein
results clearly
caused
alone
deprivation
caused
and
appeared
study
under
we
rats
We
used
(20).
increase
In other words,
concentrations,
MS
several
and
CBS
variables
considered
effect under
deprivation
in rats fed 20C,
concentration
enhanced
This
the
and
deprivation.
deprivation
in the liver, e.g., SAM,
DMG
indicates
effect of choline
by choline
of choline
and
plasma
and, conversely,
reinforced
activities, and
31
it was
of choline
the hyperhomocysteinemic
that the combination
diet to avoid
(26.6 pM).
of folate deprivation
diets
methionine
interacting
homocysteine
or
diet,
casein
(45.4 yM)
synergistically
was
intake
the control
an
is
N-methylation
25%
a
alone, since
hyperhomocysteinemia
mild
also demonstrated
as
give rise to
plasma
folate deprivation
affected
20C
SAM
that choline
fed low
hyperhomocysteinemia
the condition
fed
The
using
to methionine
were
deprivation
may
the PE
and
were
(68).
N-methylation
that the combination
showed
did not
only
when
choline
since choline
intake
demonstrated
rats
when
cause
not
methionine,
responds
previously
effect of folate deprivation
synergistically
(PC)
which
choline
moderate
alone
concentration.
hyperhomocysteinemic
The
of this,
folate deprivation
and
deprivation
choline
that choline
In support
soybean
folate deprivation
and
concentration,
hyperhomocysteinemia
deprivation
that choline
such
SAM
does
(PE)
of phosphatidylcholine
folate
on
but also by methionine
intake
hyperhomocysteihemiaby
of
deprivation
levels of dietary
high
hyperhomocysteinemia
cause
marked
as
such
(69-71).
it caused
while
moiety
by hepatic
mainly
regulated
deprivation
is that phosphatidylethanolamine
basis of this phenomenon
can
by choline
only
studies.
Choline
bf relatively
the condition
under
be
cannot
concentration
the effect of choline
hyperhomocysteinemia.
deprivation-induced
status
by further
the insufficient
underlying
homocysteine
plasma
investigated
we
mechanism
of another
to be clarified
remains
In experiment
deficiency
on
supplementation
This
excluded.
the possibility
SAH
triglyceride
and
and
folate
Some
concentrations.
homocysteine
plasma
inhibition
of BHMT
the betaine
fed 20CCDFD,
BHMT
for betaine
decreased
and, conversely,
increased
in such
decreased
and
rats.
This
in rats fed 20CCDFD
deprivation,
a
treatment
supplementation
hepatic
but it seems
significant
positive
homocysteine
The
when
hepatic
DMG
as
DMG
a
of
be
not
markedly
significantly
consumption
high
level in rats
BHMT
reaction
betaine
whereas
in experiment
shown
is associated
mechanism
The
the
with
fact that there existed
concentrations
DMG
1. The
is currently
concentration
that hepatic
by choline
reinforced
concentration,
homocysteine
of plasma
might
inhibited
was
folate deprivation.
and
system
value
plasma
and
is
concentration
concentration,
a
one
of
the
under
especially
of folate deficiency.
combination
choline
a
that such
(Fig. 1.6)suggests
for alterations
fatty liver, while
of both
between
correlation
concentrations
variables
influences
the Km
in rats
It is of interest that folate
concentration
to assume
deprivation
betaine
DMG
b'etaine status
reasonable
effect of choline
condition
decreased
betaine
concentration
concentration
hepatic
than
at relatively
maintained
DMG
DMG
also decreased
concentrations
in rats fed 20CFD.
as
well
that decreased
synergistic
the key
hepatic
in hepatic
significantly
by which
mechanism
as
increase
deprivation-induced
was
concentration
are
results in
concentration
that hepatic
suggests
It is likely that higher
fed 20CCDFD.
SAM
in
activities
concentration
still higher
homocysteine
and
assumption
betaine
thereby
SAH
CBS
and
concentration
was
hepatic
since
the increase
with
the betaine-BHMT
However,
above.
hepatic
betaine
0.84 ymol/g,
in rats fed 20CCDFD,
in vivo
uncertain,
hepatic
concentration,
described
as
functional
in vivo
Although
MS
DMG
increased
and
metabolism,
reaction.
decreased
For instance,
concentration.
for homocysteine
unfavorable
to be associated
thought
are
of these changes
deprivation
single deprivation
and
relatively
infiltrations
of choline
caused
of choline
folate synergistically
high
and
enhances
level of methionine
by nutritional
or
treatments,
folate deprivation
folate did not,
hepatic
is contained
indicating
triglyceride
deficiency,
development
of
that deprivation
even
concentration
in the diet. Most
e.g., choline
32
induced
are
of the hepatic
due
to PC
fatty
deficiency
(62).
lipoprotein
(VLDL),
which
lipoprotein
particle.
Therefore,
(71).
The
of PC
CDP-choline
Hence,
on
and
pathway
the PE
synthesis
ratio and
Although
PE
via the PE
an
exclusively
there
increase
when
that marked
N-methylation
decreases
and
pathway
liver.
33
density
for PC
is not
synthesis,
PC
supplied
in hepatic
of VI-DL
SAM
cause
by the
i.e., the
synthesis
from
development
mainly
the diet.
concentration
in rats fed 20CCDFD
thereby
of the
phospholipid
also explained
(69,70), hepatic
choline
concentration
to be
pathways
pathway
pathway
in SAH
appears
two
surface
major
low
of very
is essential for the secretion
of PC
are
N-methylation
to assume
an
as
active synthesis
N-methylation
it is reasonable
SAM:SAH
pc
synthesis.
PC
in rats fed 20CCDFD
fatty liver observed
depression
depends
contains
the liver in the form
from
is secreted
Triglyceride
might
and
suppress
of fatty
I
CHAPTER
Effect
of Dietary
Supplementation
-induced
with
Folate
Hyperhomocysteinemia
34
on
Choline
in rats
Deficiency
2.I
Introduction
A
concentration
is
homocysteine
is also
and
factors
genetic
(MS)
the methyl
using
cystathionine
is catalyzed
some
a co
diets also
methionine
is formed
which
(1,3,4).We
factor of CBS
when
from
considering
impairment
of
with
supplementation
deprivation
effect
The
was
of homocysteine,
betaine
that betaine
or
partial
results suggested
have
been
with
folate with
a
number
limited
on
(lO%)
we
significantly
despite
the aim
on
(lOC)
suppressed
could
or
plasma
investigated
(20%)
standard
level being
be fully
homocysteine
35
cause
B-6
vitamin
of betaine,
the effect of dietary
by dietary
casein
dietary
folate
diet. The
but the
relatively
high
(1%).
by betaine.
overcome
or
whether
by stimulating
the hyperhomocysteinemia,
not
shown
of low
to the deficiency
be compensated
the effect of administration
of decreasing
and
deprivation
induced
betaine
supplemented
that folate deficiency
of studies
can
previously
diet
B-6
vitamin
it is of interest to know
the hyperhomocysteinemia
casein
It has been
(20).
choline
pathways
to this line,
of betaine.
group
factors of MS
due
mainly
the metabolism
remethylation
co
synthase
by
or
and
that choline
compound
in rats fed low
results showed
demonstrated
the vitamin-like
According
the other pathway.
have
B-12,
are
to
either by methionine
P-synthase(CBS).
B- 12
vitamin
concentration,
serine
with
the methyl
folate, vitamin
as
such
and
the concentration
on
(5-MTHF)
using
hyperhomocysteinemia
cause
of the two
one
condensed
(BHMT)
since folate and
hyperhomocysteinemia,
or
by cystathionine
vitamins
innuence
greater
is remethylated
S-methyltransferase
of
a
to methionine
Homocysteine
impairment
homocysteine
plasma
of 5-methyltetrahydrofolate
group
synthesis
that deficiencies
to have
thought
is remethylated
betaine-homocysteine
is
are
(1,3,4). Plasma
disease
of cognitive
factors affecting
Of the many
(72).
homocysteine
plasma
risk factor for cardiovascular
(Fig. 2. 1) (17).
cystathionine
elevated
an
risk factor for the development
a
Homocysteine
(73).
that
suggested
independent
an
disease
Alzheimer7s
nutritional
have
of studies
number
There
supplementation
concentration,
especially
in
humans
However,
(29-34).
deficiency-induced
Therefore,
folate
soybean
5-MTHF
on
protein
on
the effect of folate
betaine
on
hyperhomocysteinemia.
in this study
choline
there is littleinformation
we
investigated
deprivation-induced
diet
(42), the
(25S).
Since
the effects of dietary
in rats fed lOC
hyperhomocysteinemia
serine is thought
effect of serine alone
or
serine-in
investigated.
36
to be
a
main
combination
with
supplementation
source
with
of Cl
folate
and
25%
units for
was
also
DMG
BHMT
Betaine
+
:][pi:<
+
tc.
+
CH3-X
pc+
THF
+
+
FA+
->
sfH
Diet
ChoLine
i
+/
Diet
Homocysteine
SerlL ne
i
CBS
Cystathionine
+
cystleine
+
Taurine,
Fig. 2.1
of methiomine
and homocysteine.
(EC 2. 1. 1.5);CBS,
cystathionine
Metabolism
s-methyltransferase
SO42-
N-dimethylglycine;
FA,
5-methyltetrahydrofolate.,
S-adenosylhomocysteine;
folic acid; MS,
PC,
methiomine
phosphatidylcholine;
SAM,
BHMT,
P-synthase(EC 4.2.l.22);DMG,
synthase
(EC
THF,
N,
2.1. 1.13);5-MTHF,
PE, phosphatidylethanolamine;
S-adenosylmethiomine;
37
betaine-homocysteine
tetrahydrofolate.
SAH,
Materials
2.2
Method
and
Chemicals
2.2.1
Folic
acid and
choline
All other
chemicals
were
sigma-Aldrich
and
(AIN-93G),
vitamin
yeast
(Tokyo).
Other
diets
male
rats
Japan).
They
(Hamamatsu,
isolated
Lighting
kept
room
was
the experiments,
and
a
25%
diet. In this study,
I, forty rats
were
rats
25S
(5)
and
were
10C
(2) choline-deprived
L-serine,
10CCD
randomly
folate
+
(20
(g/kg): vitamin-free
(AIN-93G),
powder,
20.
In 25S,
choline-deprived
folate
+
(20
(20
diet)+
2.5%
animal
+
was
mg/kg
at
a
to 19:00
The
582.5;
level of 250
was
2.5%
omitted
38
were
10C,
2.5%
+
2, forty
L-Serine,
of 10C
corn
and
as
was
(5)
follows
oil, 50., mineral
bitartrate, 2.5; cellulose
g/kg at the expense
with
(1)
In experiment
2.5%
200;
10; choline
In
conducted.
diet),(4) 10CCD
composition
sucrose,
to water
(1) 25S, (2) choline-deprived
+
increase
in
starting
access
five diet groups:
L-Serine.
SLC
cages
wire
Before
free
given
mg/kg
diet),(4) 25SCD
(AIN-93),
diets, cholinel bitartrate
(20
Japan
(40-60%).
h).
experiments
five diet groups:
L-Serine.
mixture
used
folate
diet)+
100; cL-COrnStarCh,
35; vitamin
07:00
to the following
lOCCD
mg/kg
folate
+
mg/kg
SPI
(3)
to the following
casein,
mixture
separate
from
humidity
to the facility for 5 d and
two
(10CCD),
from
(lightson
Fuji Oil
stainless-steel
and
Oriental
Wako.
purchased
in hanging
(23-25oC)
randomlyassigned
assigned
(25SCD), (3) 25SCD
25SCD
12-h cycle
acclimated
were
all rats
casein
experiment
a
from
were
strain
by
supplied
purchased
housed
temperature
controlled
on
maintained
were
from
purchased
kindly
or
mixture
mineral
were
powder
the Wistar
individually
were
casein,
(SPI, Fujipro)was
g) of
(Osaka, Japan)
Chemical
cellulose
of the diet
(120-140
a
at
Pure
(St.Louis, Mo).
Sigma-Aldrich
Vitamin-free
and
isolate
ingredients
and
six-week-old
an
protein
Wako
grade.
(AIN-93),
mixture
from
purchased
from
of analytical
Soybean
Animals
were
purchased
were
(Izumisano, Japan).
2.2.2
bitartrate
of cornstarch.
in cornstarch.
The
In
level of folate
supplementation
Rats
mg/kg).
were
shown
that non-fasting
dietary
treatment
for the Care
and
2.2.3 Tissue
blood,
in 4 volumes
the resulting
supernatant
analysis
2.2.4
,
HPLC
homogenate
was
subjected
and
according
to manufacture.s'
the "Guidelines
with
in ice-cold
After
and
at 14,000
The
isolated
x
third portion
using
a
x
kit, ISOGEN
at
for
assays
homogenized
was
at 4oC.
of the liver
until
g for lO min
(pH 7.4) containing
g for 10 min
filter paper,
(vo1/wt) of
subjectedto
of the liver
portion
x
of
at -80oC
in 4 volumes
was
buffer
on
stored
then centrifuged at 10,000
phosphate
collection
saline, blotted
homogenized
Another
assays.
was
for analysis.
liver homogenate
at 2,000
was
(Nippon
0.15
M
KC1,
The
to
subjected
Gene,
instructions.
analysis
of homocysteine
the method
S-adenosylmethionine
by
Committee
by centrifugation
in liquid nitrogen,
and
centrifuged
to enzyme
blood
whole
was
serine.
sodium
total mRNA
concentrations
using
and
10 mM
Biochemical
The
acid solution
a
of mRNA,
Tokyo)
by
was
of the liver
betaine
(vo1/wt) of
University.
frozen
quickly
of the deproteinized
metabolites,
of Shizuoka
rinsed
removed,
portion
trichloroacetic
in accordance
until needed
at -30oC
quickly
One
maintained
heparinized
from
stored
was
Use
by the Animal
approved
killed
since it has been
liable to be affected
was
(2
fractionation
weighed,
supernatant
methionine
and
liver
were
for 14 d and
water
deprivation,
prior food
was
study
level of AIN-93
ten-fold
diets and
concentration
Animals"
and
was
and
for analysis.
The
This
the animals
separated
portions,
0.3 M
1 1:00 h without
a
as
to the experimental
of Laboratory
at 4oC
determined
was
homocysteine
(50).
and
was
the whole
ice-cold
and
collection
cut into three
4oC.
Use
plasma
needed
10:00
in humans
University,
g for 15 min
access
plasma
of Shizuoka
Blood
free
given
between
by decapitation
(20 mg/kg)
of Durand
(SAM)
and
et a1.
cysteine
(52).
The
and S-adenosylhomocysteine
39
in the
liver
were
measured
in the liver
were
measured
plasma
and
concentrations
of
(SAH)
by HPLC
following
following
HPLC
were
measured
in the liver
was
was
Laryea
et al.
by HPLC
using
the method
measured
by
amino
following
measured
reaction
measured
following
following
Mudd
described
method,
P-actinin
(60)
the liver
were
Foster
HPLC
Th; amount
probe
and
The
serum
primer
for MS
TaqMan
Gene
protein
albumin
for MS
of mRNA
as
standard.
i=
SEM.
product,
for BHMT
real-time
PCR
was
Assay
and
by the
same
number:
products
(Applied
according
measured
CBS
as
analysis
also measured
Expression
was
measured
(assay identification
concentration
a
was
of mRNA
was
of the
in the liver
of the reaction
amounts
in the liver
in the assay
in the liver
by quantitative
measured
used
liver
and
serine concentration
activity of MS
The
in the assay
used
et a1. (59). The
were
CA).
(55).
was
activity of CBS
was
pre-designated
City,
bovine
using
The
(56).
HPLC
The
by
measured
activity of BHMT
The
acid autoanalyzer.
(57), but
(54).
et a1.
was
in the plasma
of 5-MTHF
of Shimoda
et a1.
in the liver
concentration
concentrations
et a1.
Einarsson
the validated
where
Biosystems,
(58), but
betaine
Laryea
et al.
(74).
previ.usly
RnOO578368_m1)
et a1.
Huang
following
cystathionine,
an
follow-ing
et al.
The
(55).
Finkelstein
DMG,
product,
relative to
et a1. (53). The
Cook
to Lowry
2.2.5 Statistical analysis
Each
ANOVA,
when
is expressed
value
and
differences
the F value
Tokei-Kaiseki
was
software
as
the
among
mean
the experimental
Data
groups
statistical analysis
significant.
(version
l.5; Esumi,
Tokyo).
40
were
was
were
by
analyzed
analyzed
performed
with
a
one-way
by the Tukey
Mac
test
2.3
Results
Effect
2.3.1
on
Body
gain, food
weight
choline
deprivation
choline
deprivation-induced
i:
Plasma
respectively.
10CCD
was
25.72
0.46, and
of
cysteine
irrespective
significantly
decreased
significantly
increased
hepatic
homocysteine
or
serine
SAH
were
activities of BHMT
supplementation
increase
with
folate supplementation
significantly
concentration
Choline
a
deprivation
Choline
substrate
(Fig. 2.i,
panel B).
increased
of serine,
a
and
Hepatic
SAH
substrate
of CBS,
was
in SAM
41
and
increase
SAH
A,
C and
concentration
was
hepatic
tended
or
to
hepatic
slightly suppressed
of 5-MTHF,
(Fig. 2.4,
panel
increased
and
E).
decreased
this decrease
folate,
concentrations
increased
markedly
in hepatic
with
decreased
significantly
panels
serine
deprivation
by supplementation
significantly
with
(61),
ratio and, conversely,
(Fig.2.3). The
by folate supplementation
the body
within
Choline
SAM:
in rats
Plasma
B).
panel
supplementation
significantly
deprivation
of BHMT,
(Fig. 2.2,
i
46.2%,
to be higher
tended
C).
(Fig.2.4,
of serine
29.6, 37.8, and
or
panel
suppressed
but not that of MS
folate irrespective
of betaine,
and
changes
suppressed
increment
of
were
although
concentrations
significantly
activities.
extents
of folate status
(Fig.2.2,
deprivation-induced
CBS
and
index
an
concentration
unaffected.
these enzyme
concentration
was
was
concentration
ratio
SAM
homocysteine
and
both, but choline
SAM:SAH
as
the concentration
hepatic
The
lOC
by folate supplementation,
also slightly increased
alone
is measured
which
increased
in rats fed
than
by
The
A).
panel
significantly
was
slightly higher
was
increased
folate plus serine to levels of 28.79
and
respectively.
concentration
of supplements
concentration,
(Fig. 2.2,
pmo1/L
and folate plus serine
alone,
serine
the experimental
significantly
homocysteine
alone,
0.47pmo1/L,
i
0.59
i=
of plasma
folate alone, serine
with
was
concentration
to 34.28
0.38
j=
elevation
by folate alone,
suppression
5-MTHF
15.75
did not differ among
liver weight
and
homocysteine
from
by supplementation
o.73, 27.28
intake
2. 1). Plasma
(Table
groups
MS,
deprivation
by choline
(experiment 1)
lOC
fed
induced
hyperhomocysteinemia
a
D).
substrate
Hepatic
by serine
by
of
supplementation
significantly
(Fig. 2.4,
increased
relative levels of mRNA
panel
F).
The
relative level of mRNA
by folate supplementation,
for BHMT
and
CBS
there
among
42
was
no
for MS
in the liver
significant
difference
the experimental
groups
was
in
(Fig.2.5).
Table
Body
gain, food
weight
intake
2.I
of rats fed the experimental
liver weight
and
diets
Body
Diet
wt
Food
gain
Liver
d
g/14
Experiment
intake
g/100
wt
g body
wt
1
10C
42
10CCD
39j=3
4.08
j=
0.04
239i8
4.15
i
0.06
234
21
+_
i:
7
10CCD
+
FA2
43 j=4
239i7
4.18
10CCD
+
SeI3
42i3
235i4
3.94i
10CCD
+
FA2
40 j=2
232i9
3.98
j=
0.08
25S
50j=3
219j=7
3.89
i
0.06b
25SCD
57 +_2
230
4.17
i
0.07a
Experiment
+
set
j=
0.09
0.l2
2
j=
6
25SCD
+
FA2
53 j=3
229i6
4.10
i
0.05ab
25SCD
+
Sef
49i2
212i5
4.04
j=
0.05ab
25SCD
+
FA2
48 j=3
221
4.04
i
0.05ab
lEach
differ, P
25%
+
is the
value
<
0.05.
soybean
ser3
mean
10C,
protein
lO%
j=
SEM,
casein
diet; 25SCD,
2supplemented
at
3supplemented
at a level of 25
3supplemented
at
a
a
n
j=6
8. Values
=
diet; 10CCD,
without
diet.
g/kg.
level of 25 g/kg diet.
43
letter
common
choline-deprived
choline-deprived
level of 20 mg/kg
a
25S., FA,
10C;
25S,
folic acid.
I
D
EnoccD.Ser
FA
EB 10CCD.
10C
10CCD
10CCD
+
'
Ser
FA
s[
0
E
5
>
tO
O
>
I
O
4
∼
E
E
0
a
0
6
a
a
s!O
E
E
LJ-
=
I
=
I
Lr)
∼
E
0
dl
E
Fig. 2.2
serine
on
Effects
of supplementation
plasma
concentrations
(experiment
<
o.o5.
lOC,
supplemented
I). Each
lO%
to
value
casein
the diet
of choline-deprived
of homocysteine
is the
mean
diet; lOCCD,
at
levels of 20
j=
SEM,
(A), cysteine
n
=
8,
choline-deprived
mg/kg
iO%
Means
lOC;
diet and 2.5%,
44
casein
diets With folate, sehne
(B) and 5-methylteral1ydro[olale
in
FA,
a
panel
without
folic acid.
respectively,
folate plus
(C)
in
rats
letter differ, P
a common
Folic acid
or
and
serine
were
L
D
U
a
10C
10CCD
10CCD
+
10CCD
+
Ser
FA
10CCD.
'
Ser
FA
0
STD
-o
5:2
E
E
L1
J=
5
I
=
<
<
e1
0
u)
L
4J
>
0
>
-=
=
0
0
tD
5
0
-
∼
LI
I
E
<
E=
u)
>
=
L}
<
I
u)
I_
4)
>
LIt
0
>
=
=
Fig. 2.3
serine
on
Effects
of supplementatlOn
hepatlC
concentrations
and hotnocysteine
without
See
a common
the legend
(D)
Of choline-deprived
or S-adenosylnlethion.ne
in rat_q (experinlent i). FACh
letter differ, P
of Fig. 2 for other
<
0.05.
SAH,
abbreviations
value
10%
casein
45
folate, serine
is the
mean
j=
SEM,
SAM,
n
=
or
folate plus
(B), 1Jleir ratio
(A), S-adenosylhomocysteine
SJdenosylhonmcysteine.,
and note.
diet With
8.
MeaJIS
in
a
panel
S-adenosylmeth10nine-
(C),
I
EZ310CCD.Ser
10C
10CCD
10CCD
FA
+
Ser
+
FA
10CCD+
5P
O
E
5
0
E:
d7
S
A)
0
>
=
5D)
>
0
>
E
=
'B:
LL
I
>
u)
=
=
4)
>
Lb
0
>
J
J
>
5tD
O
>
E
i
5
a
0
E
a)
a)
O
0
0
4J
>
dJ
>
=
J
Fig. 2.4
serine
on
Effects
hepatic
cystathlOmine
serine
common
(F) in
of supplementation
actlVitleS Of betaine-homocysteine
P-synthase(E)
rats
(experlment
letter dlffer, P
B-synthase; MS.
meth10nlne
<
and
hepatic
1). Each
0.05.
10%
of choline-deprived
BHMT,
SynthaSe;
is the
Or
mean
j=
betalne-homocysteine
5-MTHF,
diet with
S-ruethyltransferase
concentrations
value
casein
betaine
SEM,
(A), meth"nine
or
rotate plus
Synthase
(A), 5-ethyltetral1ydrofolate
n
=
8. Means
S-methyltransferase;
5-methyltetral1ydrofolate.
46
llolate. serine
in
a
(C)
and
(D) and
a
panel
without
CBS,
cystath10nlne
%10CCD
a 10CCD
lL 10C
u 10CCD
10CCD
+
'Ser
'
FA
'Ser
FA
<
Z
Z
J=
EE
E
E
=
u)
=
=
I
EE)
JN
d)
>
Ll
q)
>
J
=
<
Z
EE
E
u)
EE)
O
L-
0
>
.J
0<
Effects
Fig. 2.5
on
plus serine
meth10nlne
SEM,
of supplementation
Of hepatlC
relative amounts
(B)
SynthaSe
8. Means
mean
j=
2 and
4 for abbreviations
n
=
and
or choline-deprlVed
cystath10nlne
in
a
panel
mRNA
cascin
diet with
for betaine-hoJnOCySteine
P-synthase(C)
without
lO%
a common
and note.
47
in rats
folate
or
(A),
S-methyltransrerase
(experiment
letter differ, P
folate, serinc
<
I), Each
0-05.
See
value
is the
the legends
of Figs.
2.3.1 Effect
Body
weight
gain
homocysteine
0.80
i
of
serine alone,
(Fig. 2.6,
was
significantly
folate plus serine
The
27.2, 36.6, and 42.8%,
were
concentration
concentration
did not differ among
SAM
was
concentration
supplementation
was
with
concentration
homocysteine
decreased
panels
supplementation
both
with
A, C and
with
concentration
(Fig.2.8,
panel
folate supplementation
supplementation
hepatic
E).
CBS
The
increased
or
this decrease
Hepatic
and
hepatic
panels
induced
both.
was
unaffected
serine
D
and
of 5-MTHF
concentration
F).
48
was
was
and
although
panel
SAH
and
deprivation
the increase
markedly
supplementation
was
in hepatic
MS
activities
by
decreased
with
significantly
significantly
were
deprivation
Choline
and
C).
hepatic
slightly suppressed
deprivation
by
0.71
Plasma
B).
decreased
affect BHMT
activity
Choline
concentration
panel
by choline
by supplements.
in CBS
i
cysteine
hepatic
both, although
or
suppressed
decrease
B).
(Fig.2.8,
significantly
activity, but it did not
folate, serine,
and
changes
j=
serine alone,
(Fig. 2.6,
the concentration
folate, serine,
to be
tended
concentration
significantly
betaine
(Fig.2.7). These
26.l2
by folate supplementation,
deprivation
14.79
profile of plasma
(Fig. 2.6,
ratio and, conversely,
by supplementation
homocysteine
1, choline
SAM:SAH
concentrations
affected
(Fig. 2.8,
and
increased
Plasma
folate alone,
with
l. Plasma
groups
1).
elevation
0.71, and
i
The
whereas
from
by folate alone,
respectively.
slightly decreased
to the results in experiment
0.58, 27.35
suppression
the experimental
significantly
deprivation
by supplementation
i
2.
(Table
deprivation-induced
to that in experiment
similar
serine alone
by choline
choline
suppressed
of increment
extents
homocysteine
Similar
The
A).
folate plus serine to levels of 29.2l
and
respectively.
5-MTHF
panel
groups,
in rats fed
than in rats fed 25S
increased
significantly
the experimental
to be higher
tended
or
of supplements
was
pmol/L
homocysteine
of plasma
higher
significantly
concentration
to 34.61
pmol/L,
was
did not differ among
intake
diets irrespective
choline-deprived
o.58
food
and
relative liver weight
or
deprivation
by choline
(experiment 2)
25S
not
induced
hyperhomocysteinemia
on
hepatic
folate, serine,
increased
increased
by
by serine
25S
25SCD
+
Ser
25SCD
25SCD
+
FA
25SCD
+
Ser
+
FA
i
i
0
O
E
i
5
0
>
O
I
>
O
4
dl
E
E
0
4
0
a
a
a
J
∼
-o
E
E
u
I
I
=
Ln
dl
E
0
6
a
Fig. 2r6
folate plus
Effects
of supplementation
on
serine
panel
without
a
common
of homocysleine
concentrations
plasma
5-methyltetral1ydrofolate
of choline-deprived
(C) in
rats
(experiment
letter dlffer, P
<
0.05.
2), Each
See
25%
soybean
(A), cyLqteine (B)
value
the legend
49
protein
is the
mean
j=
diet with
folate, serine
or
and
SEM,
n
=
of Fig. 2 for abbreviations
8.
Means
and
note.
in
a
L
D
a
g
25S
25SCD
25SCD
25SCD
+
Ser
FA.Ser
25SCD.
FA
+
BED
0
E
E:
I
<
W
20
0
>
'3
10
0
∼
∼
5tD
0
L
I
<
u)
E
i
>
O
<
u)
I
E
I_
0
>
4)
>
=
lJ
Fig. 2.7
Effects
folate plus serine
of supplementation
on
hepatic
Means
in
a
abbreviations
panel
and
without
a common
(D) in
rats
(experiment 2).
letter dlffer. P
0.O5.
<
note.
50
Each
See
Protein
dlet With
folate-qerine
(A), S-adenosylhonwcysteine
or S-adenosylmethlOn.ne
concentrations
their ratio (C), and homocysteine
I)-5%.qoybeiul
of choline-deprived
value
is the
the legends
meaJl
i
SEM,
or Figs- 2 and
or
(B),
=
n
3 Iror
8.
)
D
u
g
25S
25SCD
25SCD
+
25SCD
25SCD+
0
i3
･i
d)
E=
gFg
-Z3
e
5
0
E
=i}5
E
LL
10
I
gf
i
BSE
LL}
4)
>
E
J
E
Q)
i
-i
:-;
O
E
A
1
N
I)
E
4)
u)
B
E
!.I;
3
d)5
>
0
=
E
4)
>
E
8-
Means
lJ
Effects of supplementation
a
panel
P-synthase (E) and
(D)
withotlt
25%
of choline-deprived
activities of betaine-homocysteine
hepatic
(c) and cystathioninc
in
15
I)
E=
5methyltetrahydrofolate
1
J
>4)
synthase
2
i
nf!
on
Ser
5
5a
folate plus serine
FA.
D
>.=4)
or
Ser
FA
:E
Fig. 2.8
+
and
a
serine
COmmOn
(F) in
rats
hepatlC
51
<
0.05.
protein
diet with
S-methyltransferase
CC.nCentratlOnS
(expehment
letter differ, P
soybean
2), Each
Of
betaine
value
is the
folate,
serlne
(A), methm.1lne
(B),
mean
j=
SEM,
n
=
2.4 Discussion
our
10C
study
previous
and
therefore
25S
in hyperhomocysteinemia
resulted
both
used
10C
deprivation, because
of choline
hepatic
concentration
increases
(PC),
phosphatidylcholine
which
dietary
Thus,
of the present
BHMT
MS
pathway,
serine, since
serine is
hyperhomocysteinemia
folate alone,
concentrations
folate and/or
dependent
lOCCD
on
with
study
were
similar
on
serine
dietary
supplemented
be
a
in rats fed 10C
choline
protein
elevation
source
demonstrated
suppressed
and
The
levels.
In
our
of plasma
homocysteine
in the liver. The
objective
To
in the
the
stimulate
in combination
or
units for 5-MTHF
(42).
The
deprivation-induced
by dietary
25S,
(69,70).
depression
supplementation
indicating
experiment,
diet suppressed
concentration
with
homocyseine
of plasma
that the effects of
hyperhomocysteinemia
preliminary
52
of Cl
profiles
rats fed
20 mg/kg
of PE
folate alone
with
of
via the
inhibits the reaction
pathway.
that choline
deprivation-induced
folate at levels of 5, 10, and
deprivation-induced
major
folate plus serine.
or
of the MS
conversely,
endogenously,
deprivation-induced
choline
be significantly
could
serine alone,
were
thought to
in the present
results obtained
deficiency
by stimulation
diets
choline-deprived
betaine
and,
of the synthesis
betaine
SAH
and
Under
concentration.
since the reaction
concentration
serine
homocysteine
decreases
further
and
pathway,
whether
be compensated
can
betaine
results in depression
choline
accelerates
to determine
was
study
pathway
with
deprivation
choline
SAM
by decreased
caused
concentration
This
(75).
with
that choline
in hepatic
SAM
N-methylation
hepatic
on
decrease
a
provides
(PE)
phosphatidylethanolamine
depends
to
by choline
in combination
or
as
we
study,
hyperhomocysteinemia
It appears
is mainly
hepatic
deficiency,
the condition
N-methylation
level.
protein
diets such
methionine
In the present
(20).
rats
effect of folate alone
due
pathway
of low
diets to induce
hyperhomocysteinemia
via the BHMT
SAH
in
basal
as
the dietary
on
deprivation-induced
removal
25S
and
the supplemental
differ depending
may
deprivation
that choline
showed
were
supplementation
not
of
choline
in
a
dose-dependent
diet may
mg/kg
bring
have
harmful
a
(4-10
times
was
significant
difference
experiments
1 and
homocysteine
actually
0.7 mM
serine, about
It should
It has been
of rats
stimulated
&mbination of
This
conditions.
the BHMT
diets when
folate and
is also the
of the MS
pathway.
pathway
This
these
case
enzyme
one
or
lower
was
activities
for the present
be
was
to be
saturated
Km
not
increased
uhcertainwhether
the reported
serine
than
value
53
reasons
partial
to exhibit
or
serine
with
of CBS
for
in
even
effect.
in the liver
by dietary
the concept
is far lower
limited
the maximal
the activity of BHMT
supporting
metabolism
of the
only
also innuenced
were
study,
was
tended
serine, which
for homocysteine
might
plasma
concentration
appears
and
into consideration.
that the activity of MS
shown
since CBS
reaction,
that the effect of folate
(78,46,47), although
capacity
CBS
it is
MS
with
rather than by
serine concentration
However,
effect
concentration,
together
5-MTHF
in both
in Figs. 2.4 and
decrease
might
no
was
little additive
homocysteine
plasma
since hepatic
serine.
(77), is taken
be stressed
of the
case
with
alone
the results shown
serine supplementation
concentration,
serine had
concentration
increasing hepatic
by
5-MTHF
5-MTHF
in rats fed serine-unsupplemented
even
serine
hepatic
the other hand,
supplementation
concentration
the
On
hepatic
by
decreased
that folate supplementation
concentration
increasing
increased
cohcentration. Judging from
partially, by increasing
activities.
that folate and
results indicate
serine
the
effect of
the effect of folate alone, there
greater than
to be
is considered
in
Hence,
(76).
the hypohomocysteinemic
the effects of folate plus serine and
between
2. These
2.8, it is probable
only
Although
range.
significantly
diet, which
to
of folate
the carcinogenesis
level of 20 mg/kg
a
a
doses
modest
than 20
diet)tended
40 mg/kg
in rats, while
diet)suppressed
mg/kg
folate_ at
homocysteine
the plasma
CBS
8-20
that
shown
the requirement,
times
carcinogenesis
the nutritional
within
folate plus serine
(e.g.,20
it has been
However,
fu;thereffect.
colorectal
used
we
study'
dose
though
on
effect
level of folate higher
that supplementation
suggests
of folate
the requirement,
the present
maximal
a
about
dose
supraphysiological
on
This
(data not shown).
mannel-
that the
than the capacity
for the insufficient
effect of
of
folate deficiency
of why
MS
folate alone
with
supplementation
being
pathway
elevation
homocysteine
the plasma
concentration
p,.duct.f
BHMT
activities of both
previous
not
and
BHMT
and
based
DMG
bi dietary
It has been
support
MS
and
and
addition
homocysteine
concentration
GAA-induced
hyperhomocysteihemia:
homocysteine
due
deficiency
to decreased
latter mechanism
resembles
fact, GAA-induced
supplementation
in rats
to compulsive
due
cannot
choline
or
betaine
mechanisms
are
considered
via the PE
N-methylation
suppleh.entation(unpublished data). These
not
results, together
54
by
and
plasma
for the
and
(ii)betaine
(27,64).
The
hyperhomocysteinemia.
be effectively
it was
to SAH
pathway
deprivation-induced
previous
removal
increased
(38,39)
(27), but
our
(GAA)
to creatine
could
between
acid
of GAA
that of choline
DMG
mutually.
of SAM
synthesis
our
effect of
hepatic
for homocysteine
conversion
hyperhomocysteinemia
with
Thus,
be fully compensated
At least two
In
at a relatively
correlation
concentrations.
(i)accelerated
metabolism
PC
even
by increasing
positive
of guanidinoacetic
(79,80).
that
for the insufficient
reaction
pathways
a
(TfIF)is
(37),indicating
betaine
reasons
significantly
that the two
pathways,
that dietary
shown
a
is
hyperhomocysteinemia
with
homocysteine
plasma
the notion
BHMT
was
DMG
subjects(41).
by folate deficiency.
innuenced
BHMT
increases
Tetrahydrofolate
(38).
acceptor
of the
impaired
the fact that there
concentrations
remethylation,
in human
supplementation
One
but also the
pathway
that folate deprivation-induced
(unpublished data).
on
studies
the MS
only
methy1-group
are
of the
likely
the most
proposed,
of BHMT
pathways
be that folate deficiency
might
present
a
as
the capacity
the fact that folate deficiency
on
inhibitor
an
despite
been
not
is based
of DMG
demonstrated
in rats
concentration,
hepatic
but also
be fully suppressed
level, l%,
betaine
the MS
we
study,
have
impair
might
there is the question
so,
for the folate deficiency-induced
ofN,∼-dimethylglycine(DMG)
reaction
If
serine.
mechanisms
concentration
mechanism
for the metabolism
required
high
This
(41).
pathway
could
several
is that folate deficiency
mechanism
BHMT
Although
with
hyperhomocysteinemia
causes
generally
small.
of plasma
in combination
or
suppressed
suppressed
with
by dietary
by folate
the results in the present
In
study,
that folate deficiency
suggest
has
supplementation
no
hyperhomocysteinemia,
There
have
of the MS
been
several
the Km
Another
difference
BHMT
increased
the dietary
restriction
also increased
decreased
as
the dietary
to dietary
level
the enzyme
activity
methiohinelevel
in response
facts, Finkelstein
et al. (28,46,78,83) have
pathway
homocysteine
catabolism
should
not
pathway
might
and
be ignored
that the MS
Cl
units, which
provides
BHMT
pathway
not
betaine
of choline
These
could
by_the
features
appear
and
in addition
pathway
are
to removal
regenerates
by THE
accepted
by another
with
function
hepatic
(28).
Based
THF
as
level and
a
pathway
in the metabolism
MS
these
by
that
for
Furthermore,
the BHMT
and, conversely,
or
on
MS
BHMT
remethylation
of homocysteine.
the fact that
pathway.
55
Furthermore,
betaine
of the MS
roles characteristic
to bee reconciled
be fully compensated
might
of hepatic
the activity
of the basal methionine
pathway
of BHMT.
although methionine
that homocysteine
postulated
BHMT
or
e.g.,
value
in rats,
(46).
normal
activity of hepatic
The
In contrast,
levels of choline
low,
(81),
Under
(38).
for
value
1.7 pM
was
than the Km
increased
the roles
and
is the Km
12 pM
level.
increased
to maintenance
contribute
remethylation
sarcosine.
or
to dietary
difference
was
lower
was
of
BHMT
in rats is relatively
(46,83).
was
and
for homocysteine
methionine
methionine
types
several
of MS
striking
MS
folate
whereas
hyperhomocysteinemia.
for homocysteine
activity increased
the MS
most
is considerably
is the response
as
The
concentration
(82), which
4 nmo1/g
approximately
on
effect
the distinct features
BHMT
homocysteine
the hepatic
conditions,
limited
of hepatic
value
of hepatic
value
or
pathway.
In rats, the Km
homocysteine.
partial
on
reports
BHMT
and
a
for folate deficiency-induced
except
pathway
whereas
than
more
obvious
hyperhomocysteinemia,
causes
pathway
of DMG
impaihnent of
and
one
and
the BHMT
pathway
it
CONCLUSION
In this study,
homocysteine.
For this purpose,
decrease
in homocysteine
system.
Then
we
there is
a
hyperhomocysteinemia
in
decrease
we
rats
investigated
fed 10%
The
by
1%
o.25%
present
Even
hepatic
supplementation,
betaine
is high
deprivation-induced
of betaine,
The
Hence,
hepatic
of DMG,
deficiency-induced
ih hepatic
betaine
an
and
was
partial
level of betaine
concentration
and
of BHMT,
hepatic
DMG
In fact,
limited.
study
was
high
is that folate
interfere with
activity being
to N-methylglycine
it is likely that folate deficiency
or
in the present
might
the BHMT
increased
hyperhomocysteinemia
for the phenomenon
reason
the hepatic
and
activity markedly
folate deficiency-induced
concentration
inhibitor
1%
by the betaine
of 5-MTHF
the BHMT
of suppression
DMG
diets with
of folate-deprived
that folate deficiency-induced
concentration
possible
in
decrease
system.
the concentration
supplementation
One
increase
despite
metabolism
1%
We
deficiency.
deficiency-induced
be fully compensated
demonstrated
to suppress
enough
choline
find out that folate deficiency-induced
to
cannot
the extent
and
folate
on
diet to determine
casein
by the 5-MTHF-MS
by decreasing
betaine
betaine
to elicit its effect.
choline
choline
diets
casein
study
mainly
(unpublished data). Thus
enough
20%
remethylation
hyperhomocysteinemia
activity.
whether
on
the effect of supplementation
and
in homocysteine
supplementation.
MS
folate deficiency
be compensated
can
metabolism
Firstly,
betaine
to determine
deprivation
in rats fed standard
the effects of folate status
folate
on
stauts
by the betaine-BHMT
the effect of choline
effect between
synergtic
thirdly investigated
homocysteine
investigated
of
folate deficiency-induced
whether
be fully compentated
can
hyperhomocysteinemia
deficiency-induced
whether
to determine
metabolism
secondly
the effect of betaine
firstly investigated
we
hyperhomo6ysteinemia
deficiency-induced
in the metabolism
the roles of folate and betaine
investigated
we
requires
concentration
the effect
enhanced.
folate
and
(37).
resultant
yincreased
inhibition
of betaine-BHMT
system
by DMG
could
56
not
be fully mitigated
even
by betaine
supplementation.
Then
investigated
we
in rats fed standard
hyperhomocysteinemia
deprivation
folate deprivation
and
does
deprivatibn
cause
diet, but choline
casein
folate-deprived
standard
also reinforced
under
combination
which
deprivation
that homocysteine
suggested
markedly
concentrations
significantly
be functional
significantly
are
changes
deprivation
is also
single deprivation
while
choline
and
concentration
triglyceride
precursor
impairs
important
an
choline
choline
was
deficiency.
homocysteine
hepatic
SAH
hepatic
and
These
hepatic
deficiency
as
DMG
increase
that only
findings
not
only
index
in plasma
might
casein
diet.
PC
Since
These
homocysteine
of choline
increased,
that deprivation
the concept
by the MS
activity.
concentration
to represent
support
betaine
and
system
the combination
triglyceride
decreased,
concentration
of the BHMT
in standard
an
SAM
several
homocysteine
and
folate did not, indicating
or
measured
metabolism
finding
hepatic
that betaine-BHMT
suggested
that the
affected
activities significantly
unfavorable,
of
demonstrated
synergistically
CBS
the standard
hyperhomocysteinemia
study
present
under
the condition
induced
the synergistic
induced
of choline
folate induced
which
with
folate deprivation
and
under
mLight result in inhibition
which
There
concentration.
deficiency
are
homocysteine,
to be associated
thought
deficiency
and
conversely
increased,
to remethylate
increased,
MS
metabolism
decreased,
concentrations
not
hepatic
instance,
choline
diet. The
that choline
homocysteine
plasma
folate deprivation
and
to find out
(20C)
folate deprivation
deprivation
the choline
in the liver. For
variables
choline
folate deprivation-induced
on
enhanced
cause
not
diet and
casein
of choline
diet
casein
synergistically
deprivation
Choline
concentration.
deprivation
the effect of choline
of both
hepatic
deficiency
its
or
that folate deficiency
but also by the BHMT
pathway
pathway.
Finally,
both
on
25%
soybean
investigated
we
the effects of dietary
deprivation-induced
choline
protein
(25S)
supplementation
hyperhomocysteinemia
diet to find out that choline
57
with
in rats fed
deprivation-induced
folate, serine,
10%
casein
or
(10C)
depression
in
or
the BHMT
in the present
we
study,
serine had
folate and
little additive
of supplementation
MS
activities
DMG
The
combination
plasma
deficiency
system.
of choline
homocysteine
might
was
Furthermore,
supplemented
system
removal
closely
compared
deficiency
not
only
it has been
with
and
the MS
related, the impairn1ent
Of
these enzyme
be
one
fTolate deficiency
findings
effectiveness
hyperhomocysteinemia.
synergistically
the concept
support
system
increased
for incomplete
reason
in the
betaine
enhanced
that fTolate
but also the betaine-BIiMT
found that the effect of fTolate supplementation
betaine-BHMT
was
because
pathway
and
pathway
even
of the lower
of 5-MTIq-MS
58
two
the BHMT
could
pathway.
on
also partial or limited
The
system.
one
effect
that the activity of
was
the roles of folate and
the 5-MTIq-MS
serine, probably
by remethylation,
5-MTHF
that folate deprivation markedly
hyperhomocysteinemia
with
serine
folate deficiency-induced
These
concentration.
impair
deprivation-induced
investigated
in suppressing
of betaine supplementation
even
conditions.
finding might
This
concentration.
with
and
range,
for the insufficient
in the liver of rats, although
demonstrate
to
hepatic
reason
possible
In fact,
pathway.
concentration,
increased
in combination
or
study
of homocysteine
One
activity.
by dietary
the present
homocysteine
the plasma
on
of the MS
the nutritional
within
serine significantly
the MS
also innuenced
were
metabolism
with
dose
maximal
effect
folate alone
with
In conclusion,
hepatic
with
a
than the activity df BHMT
lower
was
or
alone
together
concentration
folate at
used
folate supplementation
by stimulation
be fully compensated
cannot
pathway
not
capacity
pathways
pathway
when
choline
folate
for homocysteine
are
separately
be fully compensated
but
by another
ACKNOWLEDGMENTS
I would
Department
like to express
whose
of Applied
expertise,
Morita
A
very
special
a
I could
difference
I would
University
thanks
not
in my
like to thank
and
students
also like to thank
particular,
I must
assistance,
I would
my
goes
added
and
skill in many
out
Mori,
of Agriculture,
Shizuoka
and
areas,
at all levels
to study.
to my
considerably
Prof. Takashi
to Prof. Jian
go abroad
Sugiyama
Feng,
Hayakawa
without
experience.
in writing
and
reports.
Prof. Tatsuya
project.
whose
Prof. Jian Fens
University,
graduate
his assistance
for the research
of
is the
motivation
one
and
teacher
who
truly
life.
students
of Department
of Chengdu
family
acknowledge
not
Faculty
patience,
provided
Prof. Kimio
supervisor
and
Prof. Makoto
they
to my
Chemistry,
knowledge
for the assistance
encouragement
made
Biological
like to thank
I also would
gratitude
understanding
his vast
I appreciate
my
have
University
for the support
my
of Applied
they
of Traditional
Chinese
Medicine.
me
through
my
provided
friends, without
finished
this thesis.
59
BiologicalChemistry,
whose
encouragement
Shizuoka
I would
entire life and
and
editing
in
REFERRENCE
S
1) Shlhub
J. 1999.
2) Ueland
PM.
1995.
Annu
metabolism.
Homocysteine
as
species
RevNutr
components
19: 217-246.
redox thiol
ofplasma
Clin
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