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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 22, 2003 as doi:10.1096/fj.02-0456fje. |
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* Departments of Pediatrics, Human Genetics and Biology, McGill University-Montreal Children’s Hospital, Montreal, Canada;
Metabolic Unit, University Children’s Hospital, Düsseldorf, Germany;
Clinical Research Institute of Montreal, Montreal, Canada;
Department of Medicine, McGill University Health Center, Montreal, Canada;
|| Department of Nutrition, Schools of Public Health and Medicine, University of North Carolina, Chapel Hill, North Carolina USA; and
Department of Food Science and Human Nutrition, University of Illinois, Urbana, Illinois, USA
2Correspondence: Montreal Children’s Hospital, 4060 Ste. Catherine West, Room 200, Montreal, Canada H3Z 2Z3. E-mail: rima.rozen{at}mcgill.ca
SPECIFIC AIMS
The original aim of our study was to examine the interaction between homocysteine and choline/betaine metabolism and to determine whether betaine was effective in treating moderate hyperhomocystinemia, a risk factor for cardiovascular disease.
PRINCIPAL FINDINGS
1. Plasma levels of homocysteine and liver levels of betaine and phosphocholine, the intracellular storage form of choline, are strongly dependent on Mthfr genotype in mice
As a model for moderate and severe hyperhomocystinemia, we used our recently generated mice with a heterozygous (+/-) and homozygous (-/-) disruption of the gene for 5,10-methylenetetrahydrofolate reductase (Mthfr). This enzyme provides newly synthesized transferable methyl groups in the form of 5-methyltetrahydrofolate for remethylation of homocysteine to methionine by methionine synthase. An alternate methyl donor for homocysteine remethylation is betaine, derived from choline oxidation; betaine:homocysteine methyltransferase (BHMT) transfers the methyl group from betaine to homocysteine, resulting in demethylated betaine (dimethylglycine, DMG) and methionine. As an initial step in examining the interaction between homocysteine and betaine metabolism, we measured relevant plasma metabolites in mice on regular lab chow. Homocysteine levels were strongly positively correlated with the number of disrupted Mthfr alleles. Other significant changes were elevated cysteine and decreased DMG in Mthfr -/- mice; the decrease in betaine was not statistically significant. To evaluate the effect of methyl group intake, we used an amino acid-defined control (not supplemented with betaine) diet that was slightly reduced (by 20%) in transferable methyl groups, due to a decrease in choline. Homocysteine levels were still genotype dependent, but uniformly higher in all three genotype groups than the values from mice on lab chow. In liver, betaine, phosphocholine (PCho), and glycerophosphocholine (GPC) were lowest in Mthfr -/-, intermediate in +/-, and highest in +/+ mice. Specific activities of BHMT were not significantly different between Mthfr +/+ and Mthfr +/- mice, whereas Mthfr -/- mice had 1.5-fold higher activity. A highly significant negative correlation was found between BHMT activity and betaine concentration in liver. We identified gender differences for some of the choline metabolites in plasma, liver, and brain and for BHMT activity. Female mice tended to have higher metabolite levels than males; males had higher BHMT activity.
2. Betaine supplementation for 2 wk lowers plasma homocysteine in mice with normal Mthfr as well as in Mthfr-deficient mice
With a betaine supplement of 25 mmol/kg of the aforementioned diet, plasma homocysteine decreased significantly by 56%, 58%, and 50% in Mthfr +/+, +/-, and -/- mice, respectively. In Mthfr -/- mice, methionine increased by 25%. Liver betaine levels increased dramatically with the supplement; this increase appeared to be genotype dependent (7-fold, 17-fold, and 34-fold increases in Mthfr+/+, +/-, and -/- mice, respectively). Liver betaine levels correlated positively with plasma betaine (r=0.59, P<0.05) and plasma DMG (r=0.62, P<0.05) in mice on the betaine diet. PCho increased with betaine supplementation 2.9-fold, 2.3-fold, and 3.2-fold in Mthfr +/+, +/-, and -/- mice, respectively. Brain betaine, GPC, choline, and PCho levels were higher in nullizygotes on betaine supplementation than those on the control diet, although changes in the latter two metabolites were not statistically significant (Fig. 1
). BHMT activities remained essentially unchanged with betaine supplementation. The same degree of homocysteine lowering was observed with two other methyl donors used by BHMT, dimethylsulfonioacetate and dimethylsulfoniopropionate.
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We observed severe steatosis of the liver in seven and moderate steatosis in one of the Mthfr -/- mice on the control diet, whereas none of the four Mthfr -/- mice with the betaine supplement presented with severe steatosis; three had moderate and one had mild steatosis.
The 15 male and 15 female mice per treatment group responded similarly to the betaine supplement, but homocysteine plasma concentrations decreased more in males (64%) than females (54%). Methionine increased more in Mthfr -/- males (9-fold) than in Mthfr -/- females (2-fold).
3. There is a threshold dose for betaine intake beyond which additional reductions in plasma homocysteine cannot be achieved; this phenomenon is not related to induction of BHMT activity
In a dose-response study of Mthfr +/- mice, we increased betaine in the drinking water from 0 to 6.37g/kg body weight per day. Betaine supplementation resulted in a significant decline of plasma homocysteine which did not further decrease above an intake of 53 mg/kg body weight. Even with a 120-fold increase over this intake of betaine, homocysteine levels remained at 
40% of the initial level and were still 1.5-fold elevated over +/+ mice. BHMT activity remained unchanged until a betaine intake of 327 mg/kg body weight, then was induced to 335% of initial activity. Liver betaine rose sevenfold with increasing betaine intake from 0 to 480 mg/kg body weight.
4. Plasma homocysteine levels in mice and humans with cardiovascular disease are significantly negatively correlated with plasma betaine, suggesting that betaine may be useful in homocysteine lowering in humans with moderate hyperhomocystinemia
Homocysteine correlated negatively with plasma betaine in Mthfr +/+ mice (r=-0.148, n=23), +/- mice (r=-0.363, n=28), and -/- mice (r=-0.590, n=14) on lab chow, but significance was achieved only in the last group. Betaine and homocysteine concentrations in 121 human plasma samples showed a weak, but highly significant, negative correlation with a linear correlation factor r = -0.254 (P<0.005) (Fig. 2
).
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CONCLUSIONS AND SIGNIFICANCE
The dependence of plasma homocysteine and liver choline metabolite levels on Mthfr genotype demonstrates that a decrease in MTHFR activity results in an inability to maintain homocysteine homeostasis with consequent disturbances of choline/betaine metabolism. The increased homocysteine is observed in the presence of increased flux through the catabolic (transsulfuration) pathway for homocysteine, as indicated by higher cysteine levels in Mthfr -/- mice, and in the presence of enhanced flux through the alternate remethylation pathway, as suggested by decreased levels of betaine and DMG. Hyperhomocystinemia seems to promote conversion of choline to betaine to enhance homocysteine outflow through BHMT, thereby leading to depletion of betaine and other choline metabolites in liver. The substantial decrease in choline metabolites in Mthfr -/- mice was associated with severe steatosis in liver. Our findings in these mice have relevance for human populations, in which mild MTHFR deficiency is common due to a polymorphism at bp 677 that results in decreased enzyme activity and moderate hyperhomocystinemia if folate status is low.
Since wild-type Mthfr mice were as sensitive as Mthfr +/- or Mthfr -/- mice to a change in methyl intake, it appears that even a fully functional folate-dependent remethylation pathway cannot compensate for mildly impaired betaine-dependent remethylation caused by lower choline intake. This is a new finding and suggests that mild choline deficiency might be another important cause of moderate hyperhomocystinemia, in addition to deficiency of folate and other vitamins. Betaine supplementation prevented severe steatosis of the liver in Mthfr -/- mice, even though homocysteine levels were not normalized, providing indirect evidence for a causal relationship between choline deficiency and steatosis. The gender-related differences in metabolite concentrations may be explained by different rates of trans- and remethylation, caused by different metabolic needs and enzyme activities in males and females. Male gender and MTHFR deficiency may be associated with greater sensitivity toward choline deficiency and betaine supplementation, at least in mice.
There is a threshold dose for betaine beyond which additional homocysteine lowering cannot be observed irrespective of an induction of BHMT, the catabolic pathway of betaine. This threshold effect is not due to product inhibition of BHMT by DMG, since alternate methyl donors showed the same degree of homocysteine lowering as betaine. Supplementation of betaine resulted in a maximal homocysteine lowering effect at 40% of the value without betaine and did not result in normalization of homocysteine in Mthfr +/- mice to the values observed in wild-type mice. This finding has also been described in humans with homocystinuria, and might be caused by tissue-specific differences in production and elimination of homocysteine.
Our murine studies emphasize the close interrelationship between homocysteine, folate, and choline metabolism (Fig. 3
). In mice on lab chow, we found a negative correlation between homocysteine and betaine in plasma, influenced by the Mthfr genotype, and between liver betaine and BHMT activity, indicating that betaine levels correlate negatively with the flux of betaine through the BHMT pathway. In humans, this interrelationship has not been extensively investigated. In our sample of patients with cardiovascular disease, we found a surprisingly strong negative correlation between homocysteine and betaine in plasma.
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Choline requirements in humans, especially adults, are ill-defined. It is possible that some nutritional habits, e.g., avoiding eggs and meat in a strict low-cholesterol diet or increased choline demands, such as during pregnancy or infancy, could lead to a moderate choline deficiency. As with the use of methylmalonic acid or homocysteine to detect subclinical cobalamin or folate deficiency, respectively, plasma betaine and homocysteine may reflect subclinical choline deficiency in humans. Our findings suggest that betaine may be useful for lowering plasma homocysteine in humans with hyperhomocystinemia and that maintenance of adequate dietary choline may be particularly important when folate-dependent homocysteine remethylation is disturbed.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0456fje; to cite this article, use FASEB J. (January 22, 2003) 10.1096/fj.02-0456fje 
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