Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A1B 3X9
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ABSTRACT |
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S-adenosylmethionine, formed by the adenylation of methionine via S-adenosylmethionine synthase, is the methyl donor in virtually all known biological methylations. These methylation reactions produce a methylated substrate and S-adenosylhomocysteine, which is subsequently metabolized to homocysteine. The methylation of guanidinoacetate to form creatine consumes more methyl groups than all other methylation reactions combined. Therefore, we examined the effects of increased or decreased methyl demand by these physiological substrates on plasma homocysteine by feeding rats guanidinoacetate- or creatine-supplemented diets for 2 wk. Plasma homocysteine was significantly increased (~50%) in rats maintained on guanidinoacetate-supplemented diets, whereas rats maintained on creatine-supplemented diets exhibited a significantly lower (~25%) plasma homocysteine level. Plasma creatine and muscle total creatine were significantly increased in rats fed the creatine-supplemented or guanidinoacetate-supplemented diets. The activity of kidney L-arginine:glycine amidinotransferase, the enzyme catalyzing the synthesis of guanidinoacetate, was significantly decreased in both supplementation groups. To examine the role of the liver in mediating these changes in plasma homocysteine, isolated rat hepatocytes were incubated with methionine in the presence and absence of guanidinoacetate and creatine, and homocysteine export was measured. Homocysteine export was significantly increased in the presence of guanidinoacetate. Creatine, however, was without effect. These results suggest that homocysteine metabolism is sensitive to methylation demand imposed by physiological substrates.
methionine; methyl balance; methyltransferases; hepatocytes; liver; kidney
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INTRODUCTION |
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A NUMBER OF EPIDEMIOLOGICAL STUDIES have confirmed a relationship between an increased plasma concentration of homocysteine and the development of vascular disease (for review, see Ref. 27). A meta-analysis performed by Boushey et al. (6) of 27 studies showed that homocysteine was an independent, graded risk factor for atherosclerotic disease. Total plasma homocysteine values of ~10 µM for men and ~8 µM for women are considered normal. However, as little as a 5-µM increase in total plasma homocysteine is associated with an increased risk of coronary artery disease of 60% for men and 80% for women. The mechanism by which homocysteine exerts pathological effects is currently unknown.
During the course of its metabolism, methionine is adenylated by the
enzyme S-adenosylmethionine synthase to form
S-adenosylmethionine (SAM), the methyl donor in virtually
all known biological methylation reactions (25). For
example, the methylation of DNA and RNA and the conversion of glycine
to sarcosine all require SAM as methyl donor. The end products of these
methyltransferase reactions are a methylated substrate and
S-adenosylhomocysteine (SAH), which is reversibly hydrolyzed
to homocysteine and adenosine. Homocysteine has several possible fates:
1) catabolism to cysteine via the pyridoxal
phosphate-dependent transsulfuration enzymes cystathionine -synthase
(C
S) and cystathionine
-lyase (C
L) (24),
2) remethylation to methionine via cobalamin-dependent
methionine synthase or betaine:homocysteine methyltransferase
(12), and 3) export to the extracellular space.
It is apparent, therefore, that biological methylations and homocysteine metabolism are intimately linked and that alterations in one may affect the other. Such an effect has been documented in studies of patients with Parkinson's Disease undergoing treatment with L-3,4-dihydroxyphenylalanine (L-Dopa). This disorder is characterized by an extreme depletion of nigro-striatal dopaminergic neurons that results in deficiencies of dopamine in the basal ganglia and of melanin in the substantia nigra. Treatment involves administration of L-Dopa alone or in combination with a peripheral decarboxylase inhibitor (9). The decarboxylation of L-Dopa in the brain alleviates the dopamine deficiency. In the course of treatment, a wasteful peripheral methylation of L-Dopa by catechol O-methyltransferase occurs with the production of 3-O-methyldopa, which is excreted. The result of this metabolic removal of L-Dopa is a therapy that requires very high doses of the drug. Consequently, methyl status is altered. Decreases in whole blood levels of SAM have been observed in L-Dopa-treated patients (9) as well as increases in plasma homocysteine in humans and rats (1, 23). It is clear that alterations in methylation demand by pharmacological agents can affect plasma homocysteine levels.
In light of this, an examination of the effects of creatine and
guanidinoacetate (GAA) on homocysteine metabolism is warranted, because
the methylation of GAA to creatine via GAA methyltransferase consumes more SAM than all other methylation reactions combined (34). Figure 1 describes the
interorgan metabolism of GAA and creatine and their interaction with
homocysteine metabolism. We undertook a series of experiments, in
vivo and in vitro, to determine whether manipulation of
methylation demand by the more physiological substrates, creatine and
GAA, could affect homocysteine metabolism. The results described herein
demonstrate that homocysteine metabolism is sensitive to the
methylation demands imposed by physiological substrates.
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MATERIALS AND METHODS |
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Animals, diets, and chemicals. Male Sprague-Dawley rats weighing between 250 and 300 g were used throughout the study. The animals were obtained from our institute's breeding colony and were housed and treated in accordance with guidelines of the Canadian Council on Animal Care (7). Memorial University's Institutional Animal Care Committee approved all procedures. All animals had free access to water and food. Rats were fed either chow or a 20% casein-based AIN-93 diet, designed to meet the nutritional requirements for growth of laboratory animals, for 2 wk before experimentation. Where indicated, diets were supplemented with 0.4% wt/wt creatine monohydrate or 0.36% wt/wt GAA. These levels were chosen because it has been previously shown that they downregulate the renal L-arginine:glycine amidinotransferase (21). Where diets were supplemented, an equivalent mass of cornstarch was omitted from the diet. Rats were housed at 22°C and exposed to a 12:12-h light-dark cycle, with the light cycle commencing at 0800. All experiments were performed immediately after the end of the dark cycle. All chemicals were purchased from Sigma Chemical (Oakville, ON, Canada), except where noted in the text.
Tissue preparation and analysis.
Animals were anesthetized with pentobarbital sodium (65 mg/kg ip).
After a midline abdominal incision, blood samples were collected from
the abdominal aorta into heparinized syringes and placed on ice until
plasma was separated by centrifugation (15 min, 3,700 g).
Plasma was frozen at 20°C for later analysis. Kidneys and liver
were rapidly removed and homogenized in ice-cold 50 mM potassium
phosphate buffer (pH 6.9) with a Polytron (Brinkmann Instruments,
Toronto, ON, Canada) for 20 s at 50% output. The homogenates were
centrifuged at 18,000 g for 30 min at 4°C, and the
supernatant was retained. All enzyme assays were carried out on this
18,000-g supernatant. All enzyme assays were demonstrated to
be linear with time and with protein under the conditions employed.
Analytical procedures.
The following enzymes, responsible for the remethylation of
homocysteine, were measured in liver: methionine synthase
(19), methylenetetrahydrofolate reductase
(11), and betaine-homocysteine S-methyltransferase (32). The enzymes involved
in the catabolism of methionine to cysteine were also measured: CS
(22) and C
-L (29). For
betaine-homocysteine S-methyltransferase and C
S assays, methionine and cystathionine, respectively, were measured by HPLC (18). The L-arginine:glycine
amidinotransferase was assayed in kidney using the method of Van Pilsum
et al. (30), in which ornithine is detected by ninhydrin.
Protein concentration was determined using the Biuret method
(13) after solubilization with deoxycholate and with
bovine serum albumin as a standard (17).
Preparation and incubation of isolated hepatocytes.
Hepatocytes were isolated as previously described (5), and
viability was assessed by 0.2% trypan blue exclusion. Viability was
95% in all cases. Hepatocytes were preincubated for 20 min at
6-8 mg dry weight of cells/ml in a final volume of 1 ml in Krebs-Henseleit medium containing 1.25% bovine serum albumin. At the
end of the preincubation, substrates were added, and the incubation was
allowed to continue for an additional 30 min. Cells were gassed with
95% O2-5% CO2 at the beginning of the
incubation and at the addition of substrates.
Statistics. Data were analyzed by ANOVA followed by the Newman-Keuls multiple comparison posttest. In all cases, P < 0.05 was taken to indicate significant difference.
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RESULTS AND DISCUSSION |
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Effect of creatine and GAA supplementation.
Table 1 describes the effect of creatine
and GAA supplementation on muscle metabolites in hindlimb skeletal
muscle and on plasma creatine. Muscle creatine was increased by 39% in
GAA-supplemented animals and by 46% in the creatine-supplemented group
compared with control values. Phosphocreatine was unchanged. Plasma
creatine was about sixfold higher in both the GAA- and
creatine-supplemented groups. These data indicate that dietary
supplementation with creatine or GAA significantly alters both muscle
and plasma creatine levels.
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Effect of GAA and creatine supplementation on selected liver and
kidney enzymes.
Several enzymes of interest to homocysteine and GAA metabolism
were assayed (Table 2). The two enzymes
of the transsulfuration pathway, which together catalyze the catabolic
removal of homocysteine from the methionine cycle, were assayed in
liver. The activity of the first enzyme, CS, was 89% higher in the
GAA-supplemented group than in the creatine-supplemented group.
However, neither group differed significantly from the control group.
The activity of liver C
L, the final enzyme of the transsulfuration
pathway, was unchanged among groups.
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Effect of GAA and creatine on homocysteine export from isolated rat
hepatocytes.
To further characterize the effect of GAA and creatine on homocysteine
metabolism, we undertook a series of experiments in which we incubated
isolated primary rat hepatocytes in the presence and absence of GAA,
creatine, methionine, and serine. The results of these experiments are
shown in Table 3. In the presence of methionine, GAA significantly increased the rate of homocysteine export
(47% vs. methionine alone), but creatine was without effect. When
cells were incubated with methionine plus serine, homocysteine export
was decreased by 40% compared with cells incubated with methionine
alone. This "serine effect" was also evident in incubations containing methionine plus GAA. When serine was included in these incubations, homocysteine export was lowered by 51%, to the same level
of export as the methionine plus serine incubations. These data suggest
that alterations in plasma homocysteine imposed by methylation demand
may be partly due to effects on liver metabolism.
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ACKNOWLEDGEMENTS |
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We acknowledge the technical assistance of B. Hall and S. Lucas.
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FOOTNOTES |
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This study was supported by grants from the Canadian Diabetes Association, the Canadian Institutes of Health Research (CIHR), and the Canada-Newfoundland Cooperation Agreement on Economic Renewal. L. M. Stead was the recipient of a K. M. Hunter/CIHR Doctoral Research Award, and R. L. Jacobs was the recipient of a CIHR Doctoral Research Award.
Address for reprint requests and other correspondence: J. T. Brosnan, Dept. of Biochemistry, Memorial Univ. of Newfoundland, St. John's, Newfoundland, Canada A1B 3X9 (E-mail: jbrosnan{at}mun.ca).
1 "Total plasma homocysteine" refers to the sum of free and protein-bound homocysteine, as well as mixed disulfide forms.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 February 2001; accepted in final form 8 June 2001.
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