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Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611-0370
3To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • one-carbon metabolism • homocysteine • transsulfuration • vitamin B-6 • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Such a
relationship has been shown more recently in several epidemiological
studies. Verhoef et al. (1996)
reported that estimated
dietary intake of vitamin B-6 and folate and plasma concentration of
plasma pyridoxal (PL) 5'-phosphate
(PLP)4
and folate were lower in patients who had a myocardial infarction than
in controls. They also reported that the frequency of myocardial
infarction was negatively correlated with both folate and vitamin B-6
nutriture. Rimm et al. (1998)
confirmed these findings
in the Nurses’ Health Study with over 80,000 women. Those women in the
highest quintile of vitamin B-6, folate or folate plus vitamin B-6
intake had less coronary heart disease than those in the lowest
quintile of each intake category. Robinson et al. (1996)
reported the results of a multicenter trial
examining plasma homocysteine, plasma PLP concentration and risk of
vascular disease. In healthy control subjects, low folate or vitamin
B-6 status increased the risk of vascular disease. Risk of vascular
disease in low vitamin B-6 status was independent of plasma
homocysteine concentration, including fasting and postmethionine load
homocysteine values.
The mechanism by which low intake of vitamin B-6 is associated with
risk of vascular disease is not known; however, moderate elevation in
plasma homocysteine concentration is a clear risk factor
(Boushey et al. 1995
, Refsum et al. 1998
). Elevated plasma homocysteine has been observed in rats
and pigs with dietary deficiency of vitamin B-6 (Smolen and
Benevenga 1982
and 1984
, Smolin et al.
1983
). Vascular changes (degeneration and mural thickening of
renal arterioles) were observed in vitamin B-6-deficient pigs
(Smolin et al. 1983
). Miller et al.
(1994)
showed in rats that plasma homocysteine is elevated to a
greater extent in folate deficiency rather than vitamin B-6 deficiency,
while postmethionine load plasma homocysteine is greater in vitamin B-6
deficiency. Selhub et al. (1993)
determined intake and
nutritional status of vitamin B-6, B12 and folate in human subjects.
Low intake of vitamin B-6 as well as folate was associated with
elevated plasma homocysteine, as was low plasma PLP.
Steady-state concentrations of homocysteine in tissues and plasma are a
function of the rates of homocysteine formation, remethylation and
catabolism via the transsulfuration pathway. Remethylation can occur by
transfer of a methyl group from 5-methyltetrahydrofolate catalyzed by
methionine synthase or from betaine catalyzed by
betaine-homocysteine methyltransferase. The kinetic and regulatory
interrelationships governing these reactions have been recently
reviewed (Finkelstein 1997
). In studies with humans
conducted by Young and associates (1997)
, the basic
kinetics of methionine and homocysteine have been evaluated with
primary focus on individuals with apparently adequate micronutrient
status. The rates of the remethylation and transsulfuration reactions
in normal humans in postabsorptive state were 1.8 ± 0.4 and 4.0
± 0.4 µmol/(kg · h, respectively, while in fed state these
values were 5.7 ± 0.9 and 8.3 ± 0.6 µmol/(kg · h)
(Storch et al. 1988
). This study also indicated
that ~40% of homocysteine is remethylated, with the remainder
undergoing transsulfuration, and that over 50% of methyl groups used
for remethylation were derived from de novo synthesis. These figures
demonstrate the quantitative significance of both remethylation
and transsulfuration processes and imply that significant reduction in
a rate-limiting component of either would cause accumulation of
homocysteine. The relative contributions of 5-methyltetrahydrofolate
and betaine as methyl donors may change between postabsorptive and fed
states (Finkelstein 1997
, Finkelstein and Martin 1984
). Studies of vitamin B-6-deficient rats and pigs showed
that the deficiency caused an increase in plasma homocysteine
concentration along with a large reduction in plasma cysteine
concentration, which they interpreted as impairment in transsulfuration
(Smolin and Benevenga 1982
, Smolin et al. 1983
).
Serine serves as a major source of one-carbon units for de novo
generation of methyl groups in methionine regeneration and as a
substrate in the transsulfuration pathway (Wagner 1995
).
Cystathionine ß-synthase catalyzes the irreversible condensation of
homocysteine and serine, which commits homocysteine to the catabolic
transsulfuration pathway. Both cystathionine ß-synthase and
-cystathionase, the second enzyme in this pathway, require PLP as
coenzyme. Cystathionine ß-synthase is a tetrameric heme protein that
has been shown recently to bind four PLP molecules per tetramer with
tight, but nonequivalent binding affinities (Taoka et al. 1999
). Studies of PLP binding based on short-term
incubation with the apoenzyme indicated a single apparent
Kd of 0.7 µmol/L, characteristic of an enzyme
that binds PLP tightly (Kery et al. 1999
). It is unclear
whether this in vitro observation would predict tightness of in vivo
PLP binding and susceptibility to loss of cystathionine ß-synthase
activity during vitamin B-6 deficiency. On the basis of these in vitro
data, we predict that cystathionine ß-synthase would be somewhat more
resistant to inactivation in vitamin B-6 deficiency than enzymes with
weaker binding of PLP. Because serine hydroxymethyltransferase binds
PLP less tightly, with a dissociation constant of 27.5 µmol/L
(Jones and Priest 1978
), we propose that serine
hydroxymethyltransferase (SHMT) would be much more sensitive to effects
of vitamin B-6 deficiency. However, because of complex regulatory
interactions such as the inhibitory effect of
S-adenosylmethionine (SAM) on methyl group formation and the
stimulation by SAM of cystathionine ß-synthase, the full effects of a
vitamin B-6 deficiency may not be predictable solely on the basis of
coenzyme availability. -Cystathionase, the second enzyme in the
transsulfuration pathway, has been reported to have an association
constant for PLP of 7 x 105 L ·
mol-1 (Oh and Churchich 1973
),
which corresponds to a dissociation constant of 1.4 µmol/L and
suggests that it binds PLP tightly.
Stable isotopically labeled amino acids have enabled the determination
of basic kinetics of methionine, homocysteine and cysteine kinetics in
humans (reviewed by Young et al. 1997
). Radiolabeled
serine (3-14C) has been used to investigate the
overall kinetics of one-carbon acquisition and labeling (via
5-methyltetrahydrofolate) of the methionine methyl group pool
(Schalinske and Steele 1989
and 1996
). The protocol
reported here is an extension of the Schalinske-Steele procedure in
which we use stable isotope-labeled tracers and analysis by gas
chromatography-mass spectrometry to allow the simultaneous
examination of several aspects of one-carbon metabolism.
The main objectives of this study were to determine by using stable isotopic procedures the impact of vitamin B-6 deficiency in rats on: i) the in vivo rate of homocysteine remethylation with one-carbon units derived from exogenous serine, ii) the in vivo rates of reactions constituting the transsulfuration pathway and iii) the relationships between vitamin B-6 intake, hepatic and plasma PLP concentration and the activities of hepatic SHMT and cystathionine ß-synthase. Our primary hypotheses were that vitamin B-6 depletion would reduce the in vivo rate of remethylation with serine-derived methyl groups and the rate of conversion of serine to cysteine via transsulfuration in proportion to the relative losses of SHMT and cystathionine ß-synthase activities. The observed influence of vitamin B-6 status on protein turnover was not anticipated. It should be noted that we designed this study before the quantitative roles of both mitochondrial and cytosolic metabolism were identified as sources of one-carbon units for mammalian remethylation of homocysteine. Thus, this study focused primarily on one-carbon metabolism through the cytosolic form of SHMT. Separately reported rat and human studies address the contributions of cytosolic and mitochondrial compartments in methyl group synthesis (Cuskelly and Gregory, unpublished).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thirty-two male weanling Sprague-Dawley rats (~50 g; Charles
River Laboratories, Wilmington, MA) were randomly assigned to two
groups. One group was fed AIN-93M purified diet (Reeves et al. 1993
) containing PN at 7 mg/kg (Dyets, Bethlehem, PA). The
other group was fed the same diet containing 0.1 mg pyridoxine (PN)/kg
(Dyets) to induce vitamin B-6 depletion as performed extensively in
previous protocols in this laboratory. The rats were given free access
to food and water for 3 wk. They were housed in wire-mesh stainless
steel cages and were maintained on a 12-h light cycle. All rats were
weighed weekly and food consumption monitored daily. This protocol was
reviewed and approved by the University of Florida Institutional Animal
Care and Use Committee.
Stable isotope-labeled tracers, protocol and rationale.
The isotopic tracers included:
[2,3,3-2H3]-L-serine,
[5,5,5-2H3]-L-leucine, and
[1-13C]-L-methionine.
[2H3]Serine and
[2H3]leucine were obtained from Cambridge
Isotope Laboratories (Andover, MA), while
[1-13C]methionine was from Isotec (Miamisburg, OH). The
amino acids were dissolved in isotonic saline and sterilized by
filtration through a 0.2 um pore size filter (Nalge, Rochester, NY).
Doses were 36.7, 9.86 and 9.86 µmol/kg body weight for
[2H3]serine,
[2H3]leucine, and
[1-13C]methionine, respectively, administered as a single
bolus dose by intraperitoneal injection in a protocol based on the
flooding dose procedure of Garlick et al. (1989)
. All
injections were performed at the end of the 3-wk dietary treatment
after an overnight period in which food was withheld. After injections
the rats were returned to their cages. Because of the need to obtain
both blood and liver samples, repeated sampling from each rat could not
be done. Thus, in this protocol each animal constituted a single timed
observation. We killed rats at time 0 (i.e., no injection) and at 30,
60 and 120 min after injection (n = 4 rats per time
point for each diet treatment) by exsanguination into a heparinized
syringe via open-heart puncture under methoxyflurane anesthesia
(Metofane, Schering-Plow, Union, NJ). Plasma was obtained by
centrifugation at ~1000 x g for 15 min
immediately after blood collection. Livers were excised immediately
after blood collection. Each liver was wrapped and quick-frozen in
a dry ice-acetone slurry then stored at -80°C until analysis.
We selected these tracers [2,3,3-2H3]serine, [3,3,3-2H3]leucine, and [1-13C]methionine for the following reasons.
i) The C-3 of serine enters one-carbon metabolism via cytosolic
SHMT or via mitochondrial SHMT. As shown in Figure 1
, the passage of [2,3,3-2H3]serine through
cytosolic SHMT, 5,10-methylene-tetrahydrofolate reductase yields
5-[2H2]methyl-tetrahydrofolate that yields
[2H2]methionine via the methionine synthase
reaction. Any reduction in the rate of generation of
[2H2]methionine provides a direct indication
of reduced flux through cytosolic SHMT.
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ii) [2,3,3-2H3]Serine also permits
measurement of transsulfuration kinetics. The primary indicators of
transsulfuration flux were [2H3]cystathionine
and [2H3]cysteine (Fig. 1)
.
iii) [3,3,3-2H3]Leucine was used as an indicator of overall rate of protein turnover. Because of the absence of dietary leucine influx during this protocol, dilution of the tracer would occur solely by the appearance of leucine from protein turnover.
iv) [1-13C]Methionine was used for two reasons. Because of the absence of dietary methionine, dilution of this tracer would occur by either protein turnover or remethylation of homocysteine. This tracer also yields labeling of homocysteine as [1-13C]homocysteine produced through the methionine cycle. Aside from providing an indicator of the production of homocysteine from methionine, this tracer also provides a second indicator of transsulfuration flux because it yields via [1-13C]homocysteine the formation of [1-13C]cystathionine.
Enzyme activity assays.
The activities of total SHMT (cysotolic plus mitochondrial isozymes)
cystathionine ß-synthase were measured using HPLC to provide a
specific measurement of glycine and cystathionine formation,
respectively. For SHMT activity, incubation mixtures (1 mL) contained
10 mmol/L serine and 340 µmol/L (6RS)-tetrahydrofolate in 0.1 mol/L
potassium phosphate buffer at pH 7.4 containing 10 mmol/L
2-mercaptoethanol (Ogawa and Fujioka 1981
) with
no exogenous PLP. Incubations were conducted for 1 h at 37°C and
reactions were stopped by immersion in a boiling water bath for 3 min.
For cystathionine ß-synthase assays, liver samples were homogenized
in 3 vol of ice-cold 0.01 mol/L potassium phosphate (pH 7.5), 0.15
mol/L KCl and 0.5 mol/L ethylenediaminetetraacetic acid, as described
by Asagi et al. (1996)
, except with no added PLP, then
centrifuged at 10,000 x g for 15 min at 2°C.
Reaction mixtures (1 mL) contained 10 mmol/L L-serine and
10 mmol/L L-homocysteine in 0.1 mol/L
tris-hydroxymethylaminomethane HCl buffer, pH 8.6 (Kraus 1987
), with no exogenous PLP, and reactions were stopped as
described above. Preliminary studies showed that each reaction was
linear for at least 60 min and that rates were a linear function of
enzyme concentration.
Reverse-phase HPLC was used for measurement of amino acid products
(glycine and cystathionine, respectively) following reaction with
DANSYL chloride (Tapuhi et al. 1981
). This separation
was performed at ambient temperature using an octadecylsilyl column
(UltraMex 5 C18, 250 x 4.6 mm i.d.; Phenomenex, Torrance, CA)
with a mobile phase comprised of 2.5 mmol/L sodium acetate and 1.5 mL/L
phosphoric acid (final pH 3.5) and a nonlinear gradient from 200–600
mL/L acetonitrile in 30 min. Protein concentration was measured by a
spectrophotometric method (Markwell et al. 1978
).
Metabolite analyses.
Plasma PLP was measured by reverse-phase fluorometric HPLC as the
semicarbazone derivative using a minor modification of the method of
Ubbink et al. (1985)
. PLP in liver was similarly
measured by a minor modification of the method of Gregory (1980)
. SAM and S-adenosylhomocysteine (SAH) in
liver were determined by reverse-phase HPLC as described by
Miller et al. (1994)
. Total plasma homocysteine
concentration was determined by the fluorometric HPLC method of
Gilfix (1997)
. For determination of total homocysteine
in liver, samples were extracted as described by Svardal et al. (1986)
then analyzed by a minor modification of the
Gilfix method in which a gradient elution was employed to improve
resolution.
Determination of isotopic enrichment of liver and plasma amino acids.
Liver samples were prepared for analysis using a modification of
methods described by Garlick et al. (1980)
and
Reeds et al. (1992)
. Portions of liver (0.5 g) were each
homogenized in 5 vol of 0.02 kg/L HClO4 and centrifuged at
3500 x g for 15 min at 4°C. A 100-µL portion
of supernatant was acidified with 200 µL of 2 mol/L acetic acid; then
amino acids were purified by cation exchange chromatography
(Reeds et al. 1992
) and dried under N2 gas.
To each sample, 100 µL of methoxylamine in pyridine (40 mg/mL) was
added, incubated at 100°C for 60 min in an aluminum heating block,
then evaporated to dryness under N2 gas with gentle
heating. The dried samples were derivatized by incubation with 50 µL
of N-methyl-bis(trifluoroacetamide) (Aldrich Chemicals,
Milwaukee, WI), 100 µL of acetonitrile and 10 µL of ethanethiol for
60 min at 100°C. The ethanethiol was necessary as a reductant to
improve the detection of cysteine. After this derivatization, the
samples were suitable for analysis by gas chromatography-mass
spectrometry (GC-MS).
GC-MS was conducted in the electron impact ionization mode with a Hewlett-Packard Model 5890 gas chromatograph interfaced with a Model 5989A mass spectrometer (Hewlett-Packard, Palo Alto, CA) equipped with a 25 m x 0.20 mm i.d. DB 17 column. Selected-ion monitoring was performed for the derivatives of serine (m/z 362–365), leucine (m/z 302–305), methionine (m/z 320–322), cysteine (m/z 406–409). Isotopic enrichments (i.e., molar ratios of labeled and nonlabeled species) were calculated by solving simultaneous equations in a multiple regression procedure that corrected for natural abundance of mass isotopomers essentially as described by Storch et al. (1990).
For analysis of plasma amino acids, we found that GC-MS plasma
amino acids as n-propyl ester, heptafluorobutyramide
derivatives, prepared as described by Reeds et al. (1992)
except with the addition of ethanethiol at each step in
derivatization, yielded superior sensitivity and cleaner samples.
Analyses were conducted in ECNI mode using a Finnigan Voyager GCMS
instrument (Finnigan Corp., Schaumburg, IL) equipped with a DB-5 MS
column (30 m x 0.25 mm i.d.; J & W. Scientific, Folsom, CA).
Selected-ion monitoring was conducted for serine,
m/z 519–522; leucine, m/z 349–352;
cysteine, m/z 535–538; homocysteine, m/z
549–552; methionine, m/z 367–371; cystathionine,
m/z 678–682.
Kinetic and statistical analysis.
For each labeled form of each amino acid, the isotopic enrichment observed in each rat was plotted against time after injection. For each labeled compound evaluated, the area under the curve was calculated using Sigma Stat Version 1.02 (Jandel Scientific, San Rafael, CA) as the area of the polygon. The standard errors for the total area estimates were determined and treatment effects evaluated as follows:
i) For each mass isotopomer of interest in each diet treatment, ANOVA was conducted on isotopic enrichment data as a function of time after injection.
ii) The mean square error of each ANOVA constituted an estimate of the variance of enrichment at each time point. The variance of the area under the curve was calculated from this value as the sum of variances for each component of the total area (i.e., of each trapezoid generated).
iii) Differences in total area between treatments were assessed by calculating the z score as follows: Z = [Estimated area(1) - Estimated area(2)]/[sqrt((Var(area(1)) + (Var(area(2)].
Differences in ratios of area-under-the-curve values (e.g.,
[2H2]methionine/[1-13C]methionine)
were also evaluated by this Z-score procedure. For statistical
analysis of all other variables, two-way ANOVA was conducted with
dietary vitamin B-6 intake and time after injection as variables after
data were log-transformed to equalize variance. Since time after
injection was found not to be significant, all statistical results
reported refer to the effect of dietary vitamin B-6 intake. For both
ANOVA and the Z-score test above, dietary effects were considered
significant at the 
level of 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The dietary treatment used in this study caused substantial differences
in vitamin B-6 status. PLP concentration in liver and plasma was
significantly lower in rats fed the low-PN diet (Table 1
). Growth also differed between dietary groups (final body weight 165
± 14.9 and 130 ± 13.7 for adequate and deficient diets,
respectively, P < 0.0001).
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. Vitamin B-6 deficiency caused a small but significant reduction in
hepatic SAM concentration (Table 1)
. The ratio of SAM/SAH tended to be
lower in rats consuming the low-PN diet (P = 0.122;
Table 1
). The mean activity of total SHMT (i.e., cytosolic and
mitochondrial) was depressed ~40% (P < 0.05) while
that of cystathionine ß-synthase was not significantly affected
(Table 1)
. Kinetic results.
Time-course plots of isotopic enrichment vs. time for plasma and liver
amino acids indicated that uptake of the intraperitoneally injected
tracers into liver occurred rapidly, with a slower appearance of
injected tracers as free amino acids in plasma. The appearance of
labeling in hepatic and plasma amino acid pools indicates the
effectiveness of peritoneal absorption, as has been shown previously by
others (Albert et al. 1984
, Gilsdorf et al. 1985
, LeLeiko et al. 1983
, Pessa et al. 1988
).
Plots of isotopic enrichment vs. time for
[2H3]leucine in liver and
plasma free amino acid pools (Fig. 2
) and area-under-the-curve values (Table 2
) indicate greater enrichment in vitamin B-6-deficient rats. This
indicates that the dilution of tracer with nonlabeled leucine from
turnover of body protein is reduced in vitamin B-6 deficiency. A
similar effect was seen for
[2H3]serine (Fig. 3
).
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) showed greater enrichment during vitamin B-6 deficiency (Table 2)
of a
magnitude that was similar to the change in leucine area under the
curve values. These findings suggest that most of the observed M + 1
methionine isotopomer is
[1-13C]methionine-derived from the injected
methionine tracer rather than
[2H1]methionine produced
metabolically from
[2H3]serine. Again, this
is evidence of reduced protein turnover in vitamin B-6 deficiency.
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,
similar area-under-the-curve values were observed for hepatic
[2H2]methionine in
vitamin B-6-adequate and -deficient rats (Table 2)
, while in
deficient rats
[2H2]methionine
area-under-the-curve was significantly reduced. However, because
the appearance of nonlabeled methionine from protein breakdown was
depressed which altered the kinetics of the methionine pool in vitamin
B-6 deficiency, direct comparison of the areas-under-the-curve for
[2H2]methionine would not
directly reflect the rate of
[2H2]methionine
formation. To compensate for these differences and allow comparison of
[2H2]methionine
production between dietary treatments, we examined the ratio of area
under the curve values of M + 2/M + 1 methionine isotopomers (i.e.,
[2H2]methionine/[1-13C]methionine).
This ratio indicated a large reduction in the relative rate of both
hepatic and plasma
[2H2]methionine
production in vitamin B-6 deficiency (84 and 93% reduction,
respectively). Similarly, large effects of vitamin B-6 deficiency were
seen on the ratio of
[2H2]methionine/[2H3]serine
(Table 3
), with 81 and 88% reductions in liver and plasma, respectively. These
findings indicate impaired generation of methyl groups from serine
during vitamin B-6 deficiency.
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. Hepatic cystathionine was not detected in the electron-impact
GC-MS analysis of the silyl derivative, while the n-propyl
ester heptafluorobutyryl derivative was well suited for ECNI–GC-MS
determination of plasma cystathionine and all other amino acids. The
area-under-the-curve values for
[2H3]cystathionine and
[2H3]cysteine in plasma
were both significantly reduced in deficient rats, although the effect
on [2H3]cystathionine was
small (Table 2)
. In contrast, the areas-under-the-curve for
[1-13C]homocysteine and
[1-13C]cystathionine were significantly greater
in vitamin B-6-deficient rats (Table 2)
.
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. Using this
approach, we could examine effects of vitamin B-6 deficiency with two
indicators of cystathionine production
([2H3]cystathionine/[2H3]serine
and
[1-13C]cystathionine/[1-13C]homocysteine)
(Table 3)
. Vitamin B-6 deficiency significantly reduced
[2H3]cystathionine
production from
[2H3]serine (54.3%
reduction). Similarly, the production of
[1-13C]cystathionine from
[1-13C]homocysteine declined 28.9%, although
this was not statistically significant (P > 0.1).
Vitamin B-6 deficiency yielded significantly greater hepatic
[2H3]cysteine/[2H3]serine
ratio, suggestive of enhanced net transsulfuration flux in the liver.
In contrast are the plasma observations indicative of whole-body
transsulfuration. Ratios of plasma
[2H3]cysteine/[2H3]serine
and
[2H3]cysteine/[2H3]cystathionine
were reduced in vitamin B-6 deficient rats by 96.5 and 90.8%,
respectively (Table 3)
. This observation is consistent with impaired
overall whole-body transsulfuration in spite of the increase in
hepatic cysteine production in vitamin B-6 deficiency. These data also
suggest that the impact of vitamin B-6 deficiency is greater on the
conversion of cystathionine to cysteine, i.e., the -cystathionase
reaction of transsulfuration, that appears to become rate-limiting. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Dalery et al. 1995
, Rimm et al. 1998
, Robinson et al. 1998
, Verhoef et al. 1996
). Plasma total
homocysteine concentration is a function primarily of the rate and
extent of methyl group generation (as 5-methyltetrahydrofolate), rate
of synthesis of homocysteine, rates of homocysteine catabolism and
remethylation processes, and renal excretion of homocysteine
(Finkelstein 1997
). As seen in this study and others
(Miller et al. 1992
and 1994a
,
Smolin and Benevenga 1982
, Ubbink et al. 1996
), fasting plasma homocysteine concentration is increased
in vitamin B-6 deficiency, although changes are rather small.
Vitamin B-6 provides the necessary coenzyme, PLP, for the production of
methyl groups via both cytosolic and mitochondrial SHMT reactions and
for the catabolism of homocysteine via the transsulfuration pathway. As
seen in the present study, elevation occurred in the concentration of
both hepatic and plasma total homocysteine (Table 1)
while the
activities of total SHMT and cystathionine ß-synthase were depressed
(Table 1)
. The kinetic results of this study indicate that both
remethylation and transsulfuration phases of homocysteine metabolism
are depressed in vitamin B-6 deficiency. Further studies with
additional degrees of vitamin B-6 deficiency and supplementation are
needed to define more completely the relationship between vitamin B-6
status and these aspects of homocysteine metabolism.
The in vitro measurement of hepatic cellular enzyme activities
showed that the vitamin B-6 deficiency induced in this protocol caused
a greater loss of SHMT activity than that of cystathionine
ß-synthase. However, the in vivo kinetic data indicate major
reductions in the in vivo rates of both remethylation and
transsulfuration processes. The observed approximately threefold
increase in hepatic production of
[2H3]cysteine from
[2H3]serine indicates
that the ~20% reduction in cystathionine ß-synthase did not yield
a rate-limiting in vivo reduction in hepatic
transsulfuration, nor did any possible change in
-cystathionase activity. In contrast, the large reduction seen in
plasma for [2H3]cysteine appearance
from both [2H3]serine and
[2H3]cystathionine is
evidence that large reductions in whole-body transsulfuration flux
occurred during vitamin B-6 deficiency. The plasma data indicate a
greater impact of vitamin B-6 deficiency on in vivo activity of
-cystathionase than cystathionine ß-synthase. This is consistent
with the observation of Ubbink et al. (1996)
that plasma
cystathionine concentration increases in vitamin B-6 deficiency.
We anticipated that vitamin B-6 deficiency could alter the in vivo regulation of cystathionine ß-synthase, possibly by the reduction in SAM concentration, which could lead to a greater extent than could be explained by the reduction in hepatic PLP concentration. We also recognize that measurements of hepatic cystathionine ß-synthase activity and PLP concentration may not fully predict the effects of deficiency because of the role of other tissues in cystathionine production. As judged by both tracers (i.e., 13C or 2H), cystathionine production was reduced in vitamin B-6 deficiency, possibly to a greater extent than would be predicted solely on the loss of hepatic activity measured in vitro.
It is possible that the de novo production of methyl groups via SHMT
and the catabolism of homocysteine through the transsulfuration pathway
exhibit different sensitivities to vitamin B-6 deficiency. Thus, the
degree of impairment of each aspect of homocysteine regulation may
differ at different extents of vitamin B-6 deficiency. Purified SHMT
from beef liver has been shown to exhibit a dissociation constant of
27.5 µmol/L for its coenzyme PLP (Jones and Priest 1978
), which indicates a substantially lower affinity for PLP
than exhibited by cystathionine ß-synthase (Kery et al. 1999
, Taoka et al. 1999
). The observed greater
loss of SHMT activity in vitamin B-6 deficiency is consistent with
these in vitro data regarding PLP binding. However, the kinetic data
clearly indicate large reductions in the in vivo rates of both
remethylation and transsulfuration reactions. Thus, the deficiency that
occurred in this study appears to have had a similar large in vivo
impact on cytosolic SHMT but little or no effect on cystathionine
ß-synthase during a vitamin B-6 deficiency of this magnitude. We did
not conduct in vitro assays of hepatic -cystathionase activity
because we incorrectly assumed that cystathionine ß-synthase activity
would be rate-limiting in transsulfuration. The kinetic results of
this study show that the -cystathionase reaction was reduced to a
much greater extent than cystathionine ß-synthase in vivo. This is
consistent with the report of Sato et al. (1996)
that
vitamin B-6 deficiency greatly increases the proportion of
-cystathionase in apoenzyme form in rat liver. These authors also
observed that vitamin B-6 deficiency also increases the rate of
catabolism of the enzyme.
In the present study, hepatic PLP concentration was 14.6 ± 4.2 nmol/g in adequately nourished rats and 8.85 ± 1.11 nmol/g in vitamin B-6-deficient rats, although these values represent total, not free PLP (note that the concentration of free PLP relative to the dissociation constant would primarily govern the extent of coenzyme binding to SHMT). Because total PLP concentrations were lower than the dissociation constant for PLP, it is predicted that SHMT activity in vivo would be highly sensitive to vitamin B-6 status. It is emphasized that the cytosolic generation of methyl groups was being assessed in this protocol in which the M + 2 labeling of the methionine pool is derived from [2H3]serine via the cytosolic SHMT pathway. We have recently shown in stable isotopic studies with similar protocols in rats and humans that production of M + 1 methionine occurs from the [2H3]serine tracer, which reflects the generation of methyl groups from one-carbon units produced from [2H3]serine entering the mitochondrial SHMT pathway or by shuttling reversibly through cytosolic SHMT and 5,10-methenyltetrahydrofolate dehydrogenase (Cuskelly and Gregory, unpublished). The sensitivity of mitochondrial SHMT to inadequate vitamin B-6 status requires further study. The fact that hepatic SAM concentration was reduced in vitamin B-6 deficiency suggests that in vivo formation of methyl groups via both cytosolic and mitochondrial forms of SHMT was impaired. However, the fact that SAM concentration was reduced by only 10%, in comparison to the large reduction in the in vivo production of [2H2]methionine, suggests that the mitochondrial SHMT isozyme was impaired to a lesser extent than that of the cytosolic isozyme under the conditions of this experiment. Further studies are needed to evaluate this issue.
As shown here, vitamin B-6 deficiency causes a small but detectable
reduction in hepatic SAM concentration. Selhub and Miller (1992)
have proposed that hyperhomocysteinemia is accompanied
by a reduction in the regulatory effects of SAM (i.e., its inhibition
of methylenetetrahydrofolate reductase and its stimulation of
cystathionine ß-synthase). In a test of this hypothesis in
folate-deficient rats, the increase in hepatic homocysteine
concentration and decrease in hepatic SAM concentration far exceeded
those seen in this study (Miller et al. 1994b
). Thus, it
is unclear whether the changes of SAM concentration of the magnitude
induced by vitamin B-6 deficiency in this study would yield
physiologically important changes in this coordinate regulation.
The metabolic significance of the observed reduction in rate of hepatic
and whole-body protein turnover in vitamin B-6 deficiency is
unclear. Although the relationship between dietary protein intake is
well-documented (Leklem 1991
), to our knowledge an
influence of vitamin B-6 status on the kinetics of protein turnover has
not been previously reported.
In summary, the results of this study have shown that inadequate intake of vitamin B-6 has substantial effects on one-carbon metabolism and overall protein turnover kinetics. The results of this study also demonstrate the applicability of this basic protocol to the study of various aspects of one-carbon metabolism.
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FOOTNOTES |
|---|
2 Previously presented in part as: Martinez, M.,
Toth, J. P., Williamson, J. & Gregory, J. F. (1999). Effects
of vitamin B-6 deficiency on one-carbon metabolism using stable
isotope infusion of serine. FASEB J. 13: A227 (abstr. 208.1).
4 Abbreviations used: GC-MS, gas
chromatography-mass spectrometry; PLP, pyridoxal 5'-phosphate; PN,
pyridoxine; SAM, S-adenosylmethionine; SAH,
S-adenosylhomocysteine; SHMT, serine
hydroxymethyltransferase.
Manuscript received October 13, 1999. Initial review completed November 13, 2000. Revision accepted December 22, 1999.
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