Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions
Inhibition of Betaine-Homocysteine S-Methyltransferase Causes Hyperhomocysteinemia in Mice1,2 Michaela Collinsova,*3 Jana Strakova,* Jiri Jiracek,y and Timothy A. Garrow*4 * Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801 and yBiological Chemistry Department, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,16610 Prague 6, Czech Republic
KEY WORDS: betaine homocysteine dimethylsulfoniopropionate
In humans, insufficient intakes of vitamin B-6, vitamin B-12, and/or folic acid can cause mild-to-moderate hyperhomocysteinemia, a condition proposed to be an independent and graded risk factor for vascular disease and thrombosis (1– 3). Certain gene-nutrient interactions cause mild-to-moderate hyperhomocysteinemia; the best known of these is the interaction between the C677T polymorphism of methylenetetrahydrofolate reductase and folate and/or riboflavin status (4). Known causes of severe hyperhomocysteinemia, or homocystinuria, include the catastrophic loss of function of methylenetetrahydrofolate reductase, the proteins required for methylcobalamin biosynthesis (e.g., cblC or cblD gene
products), or most commonly, cystathionine-b-synthase (CBS).5 There is a growing body of evidence indicating that betainehomocysteine S-methyltransferase (BHMT) has a role in regulating plasma total homocysteine (tHcy) levels, and it is likely to be most important after the consumption of a proteincontaining meal. Many reports indicated that the daily consumption of gram quantities of betaine (Bet) dramatically lowers tHcy levels in homocystinurics, whether the disease is due to a remethylation or a transsulfuration defect (5,6). These studies indicate that the Bet/BHMT system is operating significantly below its capacity, at least in homocystinurics. More recently, fasting tHcy and plasma Bet levels in healthy humans were reported to be inversely related, and Bet or choline supplementation was shown to reduce fasting and postmethionine (Met) load tHcy levels (7–10; and references therein). In rats, an i.v.
1 Presented in part at Experimental Biology 06, April 2006, San Francisco, CA [Garrow TA, Collinsova M, Strakova J, Jiracek J. The role of betaine-homocysteine S-methyltransferase in the regulation of plasma total homocysteine (abstract). FASEB J. 2006;20: A859]. 2 This material is based upon work supported by the NIH (DK52501, T.A.G.), the Illinois Agricultural Research Station (50-352, T.A.G.), and by a grant from the Grant Agency of Czech Academy of Sciences (A4055302, J.J.) and Research Project (Z40550506, J.J.). 3 Present address: Biological Chemistry Department, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 16610 Prague 6, Czech Republic. 4 To whom correspondence should be addressed. Email:
[email protected].
5 Abbreviations used: AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine; Bet, betaine; BHMT, betaine-homocysteine S-methyltransferase; CBHcy, S-(d-carboxybutyl)-DL-homocysteine; CBHcy-sulfoxide, S-(d-carboxybutyl)-DL-homocysteine sulfoxide; CBS, cystathionine-b-synthase; DMSP, dimethylsulfoniopropionate; Hcy, homocysteine; IC50, 50% inhibitory concentration; MS, methionine synthase (cobalamin-dependent); SBD-F, 7-fluorobenzo-2-oxa-1,3diazole-4-sulfonic acid ammonium; tHcy, plasma total homocysteine (sum of oxidized and reduced forms).
0022-3166/06 $8.00 Ó 2006 American Society for Nutrition. Manuscript received 20 January 2006. Initial review completed 8 February 2006. Revision accepted 10 March 2006. 1493
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ABSTRACT Inhibitors and methyl donor substrates for betaine-homocysteine S-methyltransferase (BHMT) were used to study the role of this enzyme in the regulation of plasma total homocysteine (tHcy). Mice were administered an i.p. injection of S-(d-carboxybutyl)-DL-homocysteine (CBHcy; 1 mg), a specific and potent inhibitor of BHMT, and tHcy and hepatic BHMT protein and activity levels were monitored over a 24-h period. Compared with saline-injected control mice, at 2 h postinjection, the CBHcy-treated mice had 87% lower BHMT activity and a 2.7-fold increase (11.1 vs. 3.0 mmol/L) in tHcy, effects that lasted nearly 8 h but returned to normal by 24 h. The level of BHMT protein remained constant over the 24-h period. After 6 CBHcy (1 mg) injections (one every 12 h), the mice had 7-fold higher tHcy, a 65% reduction in the liver S-adenosylmethionine:S-adenosylhomocysteine ratio, and a marked upregulation of BHMT protein expression. At 2 h after injection of the sulfoxide derivative of CBHcy (10 mg) into mice, there was a modest reduction in BHMT activity and a 90% increase in tHcy. When given an injection of Met (3 mg) or Met plus CBHcy (1 mg), post-Met load tHcy levels were 2.2-fold higher (128 vs. 40 mmol/L) at 2 h postinjection in the mice given CBHcy. Like betaine, dimethylsulfoniopropionate was an effective tHcy-lowering agent when given with a Met load. These studies are the first to show that transient inhibition of BHMT in vivo causes transient hyperhomocysteinemia, and that dimethylsulfoniopropionate can reduce a post-Met load rise in tHcy. J. Nutr. 136: 1493– 1497, 2006.
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MATERIALS AND METHODS Chemicals and reagents. DMSP hydrochloride was purchased from TCI America. Bet hydrochloride, L-Met, L-Hcy thiolactone hydrochloride, and 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid ammonium (SBD-F) were purchased from Sigma-Aldrich. All other chemicals were of the highest purity available from commercial vendors. The synthesis of S-(d-carboxybutyl)-DL-homocysteine (CBHcy), and its sulfoxide derivative (CBHcy-sulfoxide) will be described elsewhere (Jiracek J., Collinsova M., Rosenberg I., Budesinsky M., Netusilova H., Garrow T. A. unpublished data). [14C(U)]-Ser (127 mCi/mmol) and [14C-methyl]-Bet (57 mCi/mmol) were obtained from Moravek Biochemicals. The barium salt of 5-14C-methyltetrahydrofolate (56 mCi/mmol) was purchased from Amersham Biosciences. Animal treatments and sample collection. Animal studies (n 5 5) were done using 6-wk-old Balb/C male mice (;22–23 g) obtained from Harlan. They were housed in groups of 3 or 4 in shoebox cages and consumed food (Diet 8626, Harlan) and water ad libitum. After the mice were given 3–5 d for acclimation to their new environment, they were administered i.p. injections (200 mL) of physiologic saline, CBHcy (1 mg), CBHcy-sulfoxide (10 mg), Met (3 mg), Bet (2 mg), DMSP (2.2 mg), or a combination of these reagents, as specified below. All chemicals were dissolved in physiologic saline and brought to pH 7.4 by the addition of sodium hydroxide, if required. Study 1 mice were administered a single injection of physiologic saline (n 5 30) or CBHcy (n 5 30). Study 2 mice received 6 injections (12 h apart) of either physiologic saline (n 5 7) or CBHcy (n 5 8). Study 3 mice were given a single injection of Met (n 5 6) or Met 1 CBHcy (n 5 6), and Study 4 mice were administered a single injection of either physiologic saline (n 5 4) or CBHcy-sulfoxide (n 5 4). Study 5 mice were given a single injection of Met (n 5 6), Met 1 Bet (n 5 6), or Met 1 DMSP (n 5 6). Except for Study 2, mice were deprived of food overnight (10 h) before injection. At the end of each study, mice were anesthetized by halothane inhalation, and while unconscious, blood was collected via cardiac puncture into EDTA-coated syringes. Mice were then killed by cervical dislocation. Except in Study 1, in which the mice were killed at various time points after injection (1, 2, 4, 8, and 24 h: n 5 6/ treatment for each time point), the mice in the other studies were
killed 2 h postinjection. After cervical dislocation, mouse livers were rapidly excised, frozen in liquid nitrogen, and stored at 2808C until analyzed. All animal procedures were approved by the University of Illinois Laboratory Animal Care and Use Committee. Enzyme assays. Mouse livers were homogenized in 4 volumes (wt:v) of ice-cold 50 mmol/L potassium phosphate buffer (pH 7.5) containing 2 mmol/L EDTA. The homogenates were centrifuged at 25,000 3 g for 45 min, and the supernatants were used for the methionine synthase (MS) and cystathionase assays. b-Mercaptoethanol was added (5 mmol/L final) to a portion of the resulting supernatants, and these extracts were used in the BHMT and CBS assays. For assays that used L-Hcy, the reduced thiol was freshly prepared from L-Hcy thiolactone hydrochloride as described by Garrow (15). For all enzyme assays that required the quantification of Hcy or cysteine (Cys), samples were derivatized with SBD-F, and the HPLC procedure of Garcia and Apitz-Castro (16) was used to separate and quantify the products. A unit of activity for each enzyme is defined as nanomole product per hour. Total liver protein concentration was measured by a Coomassie dyebinding assay using bovine serum albumin as a standard. The procedure used to measure MS activity was that described by Banerjee et al. (17). Notably, the standard reaction mixtures (500 mL) contained 500 mmol/L L-Hcy and 50 mL mouse liver extract. A concentration of 50 mmol/L L-Hcy was used when CBHcy (1 mmol/L) was tested for inhibition of MS activity (Table 1). The procedure used to assay CBS in the forward direction was that described by Lambert et al. (18), except that the separation of 14 C-cystathionine from 14C-Ser was done according to Taoka et al. (19). Notably, the standard assay mixture (400 mL) contained 0.38 mmol/L S-adenosylmethionine (AdoMet), 7.5 mmol/L L-Hcy, 10 mmol/L 14C-Ser (0.07 mCi), and 50 mL mouse liver extract. Higher specific activity 14C-Ser (0.7 mCi) and only 50 mmol/L L-Hcy were used when CBHcy (1 mmol/L) was evaluated for inhibition of CBS activity (Table 1). To determine whether CBS could convert CBHcy to Hcy by working in the reverse direction, the same assay conditions were employed, except that the mixtures (250 mL) were devoid of substrates (L-Hcy and 14C-Ser) and contained 10 mmol/L CBHcy. After incubation, the reactions were processed to determine whether any Hcy was produced. The cystathionase assay conditions were based on the method described by Vina et al. (20). The standard reaction mixtures (250 mL) contained 4 mmol/L L-cystathionine and 30 mL mouse liver extract. However, rather than using radiolabeled cystathionine and separating substrate from products by ion exchange on minicolumns, we quantified Cys by HPLC. To test whether cystathionase activity could be inhibited by CBHcy (1 mmol/L), the reaction mixtures contained 100 mmol/L cystathionine (Table 1). To test whether cystathionase could convert CBHcy to Hcy, the reaction cocktails were devoid of cystathionine but contained CBHcy (10 mmol/L), and after incubation, the samples were processed to determine whether any Hcy was produced.
Table 1 Effect of CBHcy on the Hcy-metabolizing enzymes of mouse liver1 Relative activity Enzyme
Extract alone
MS CBS Cystathionase
100 100 100
BHMT
100
Extract 1 CBHcy 103 94 94 0 0.04 0.55 4.4
[CBHcy], mmol/L 1000 1000 1000 1000 500 50 5
1 Values are the means of duplicate assays where 100 equals 3.2 U/mg liver protein for MS activity, 24 U/mg liver protein for CBS activity, 26.8 U/mg liver protein for cystathionase activity, and 46.7 U/mg liver protein for BHMT activity.
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bolus of Bet reduces fasting (11) and post-Met load tHcy (12). In mice, dietary choline has no effect on fasting tHcy, but reduces the post-Met load rise in tHcy in a dose-dependent manner (7). The percentage reduction in tHcy after choline or Bet treatment was greater after a post-Met load than in the fasted state. Together, these studies showed that the Bet/BHMT system of Hcy remethylation can be used to reduce tHcy levels in normal animals, but it remains uncertain to date whether a partial or complete loss of BHMT function would cause hyperhomocysteinemia or alter other tissue sulfur amino acid metabolites. No one has reported a mutation in the BHMT gene that was linked to fasting hyperhomocysteinemia, and it must be noted that a Met load test has not been performed on individuals harboring one of the 3 known mutations in the BHMT gene (13). Moreover, choline and Bet are abundant in human diets (14), essentially eliminating the possibility that hyperhomocysteinemia could result from a primary deficiency of these food constituents. The following question therefore remains: if flux through the BHMT reaction is dramatically reduced either pharmacologically or by a deleterious mutation, would it cause hyperhomocysteinemia after food deprivation or slow the recovery to normal tHcy levels after a Met load and therefore increase risk for Hcy-related diseases? The aim of the studies reported here was to determine whether the inhibition of BHMT in vivo by potent and specific inhibitors of the enzyme would elevate fasting and post-Met load tHcy levels, and whether the naturally occurring sulfonium analog of Bet, dimethylsulfoniopropionate (DMSP), would be an effective post-Met load tHcy-lowering agent.
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BHMT activities were measured as previously described (15). The standard assay (500 mL) contained 2.5 mmol/L L-Hcy, 2 mmol/L Bet (0.1 mCi), and 30 mL of mouse liver extract. Western blot analysis. Mouse liver extracts were probed for BHMT protein as described previously (21). Metabolite assays. tHcy was determined by a procedure of Garcia and Apitz-Castro (16), which includes the derivatization of plasma thiols using SBD-F, followed by their separation and quantification by reverse-phase HPLC with fluorescent detection. Liver AdoMet and S-adenosylhomocysteine (AdoHcy) levels were determined according to the method of Wang et al. (22). Statistics. For Studies 1–4, Student’s t test was used to test for differences in the means. For Study 5, data were analyzed using 1-way ANOVA, and when analysis gave a significant F-value (P , 0.05), treatment differences were evaluated using Fisher’s least-significant difference procedure. All variances are reported as SEM.
RESULTS
FIGURE 1 The effect of CBHcy on tHcy and hepatic BHMT activity. In Study 1, mice were administered a single injection of either physiologic saline or CBHcy and their tHcy (panel A) and hepatic BHMT activities (panel B) were measured at the indicated time points. Values are means 6 SEM. *Different from control, P , 0.05.
mmol/L Bet (0.1 mCi), BHMT activity did not differ between the groups. When BHMT activity was measured in these extracts using lower levels of L-Hcy (50 mmol/L) and higher specific activity Bet (250 mmol/L, 0.5 mCi), the level of activity in CBHcy-sulfoxide–treated mice was 53% lower than that of the control mice (21 6 6 vs. 45 6 2 U/mg; P , 0.05). Effect of different BHMT methyl donors on post-Met load tHcy. In Study 5, mice administered Met with isomolar levels of Bet or DMSP had 69–82% lower post-Met load tHcy concentration than those given Met alone (Fig. 4). DISCUSSION CBHcy is an S-alkylated Hcy derivative that was first synthesized by Awad et al. (23). It was designed to be a putative transition-state analog for the BHMT reaction as a way to confirm that the enzyme catalyzes a sequential reaction mechanism, which was suggested by earlier studies (24,25). Indeed, initial rate kinetics (23), ligand binding studies (26), and the crystal structure of the human BHMT-CBHcy complex (27) confirmed that CBHcy functions as a tight-binding bisubstrate inhibitor of BHMT. We recently synthesized a variety of derivatives of CBHcy, including its sulfoxide. The 50% inhibitory concentration (IC50) values of these compounds were determined using human recombinant BHMT (0.2 mmol/L) and a modified assay containing 500 mmol/L L-Hcy and 2 mmol/L Bet. The IC50 value of CBHcy-sulfoxide was 5.0 mmol/L, which is 55fold greater than that measured for CBHcy (IC50 ¼ 0.09 mmol/L;
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The specificity of CBHcy in vitro. CBHcy (1 mmol/L) did not inhibit MS, CBS, or cystathionase activity (Table 1). The concentration of Hcy used to test whether CBHcy could inhibit MS and CBS activity was much lower than the level normally used in standard assays (50 vs. 0.5 or 7.5 mmol/L, respectively). Similarly, the level of cystathionine used to test whether CBHcy could inhibit cystathionase was much lower than the concentration normally used in the standard assay (100 vs. 4 mmol/L). In contrast, a level of CBHcy as low as 5 mmol/L reduced BHMT activity ;95% in the standard assay that contained high levels (2.5 mmol/L) of L-Hcy (Table 1). Using standard cocktails and procedures for the CBS and cystathionase assays, except for being devoid of substrates and containing CBHcy (10 mmol/L), there was no evidence that either enzyme can convert CBHcy to Hcy. In addition, compared with untreated extracts, incubating (378C) EDTA-treated whole blood or crude liver extract with CBHcy did not affect the concentration of Hcy in these samples over a 4-h period. Effect of CBHcy on tHcy. In Study 1, CBHcy treatment significantly increased tHcy levels and reduced BHMT activity at the 1, 2, 4, and 8 h time points compared with saline-treated controls (Fig. 1). By 24 h, BHMT activity and tHcy levels did not differ between the 2 groups. The level of BHMT protein was not measurably affected by a single CBHcy treatment (Fig. 2). The levels of MS and CBS activities, as measured using the standard assays, did not differ among treatments. In Study 2, mice treated with CBHcy had 7-fold higher tHcy levels than the saline-treated controls (18.5 6 1.0 vs. 2.3 6 0.2 mmol/L; P , 0.05). The CBHcy-treated mice also had a 51% reduction in liver AdoMet (8.1 6 0.9 vs 16.4 6 2.0 nmol/g liver), a 28% increase in liver AdoHcy (19.4 6 1.1 vs. 15.1 6 1.5 nmol/g liver), and a 65% decrease in the liver AdoMet:AdoHcy ratio compared with saline-treated controls (Fig. 3). Interestingly, repeated injections of CBHcy caused liver BHMT protein to double (1.0 6 0.2 vs. 2.0 6 0.3; P , 0.05) (Fig. 2), but activity did not differ in the 2 groups (104 6 3 vs. 103 6 5 U/mg). The MS activity of the CBHcy-treated group did not differ from that of the saline-treated control group (4.8 6 0.2 vs. 4.9 6 0.2 U/mg), but their CBS activity was reduced 56% (Fig. 3). In Study 3, CBHcy-treated mice had .2-fold higher tHcy levels than mice administered Met alone (128 6 17 vs. 40 6 14 mmol/L; P , 0.05). As observed in Study 1, BHMT activity was dramatically inhibited in the CBHcy-treated mice compared with saline-treated controls (not shown). Effect of CBHcy-sulfoxide on tHcy. In Study 4, CBHcysulfoxide treated mice had 90% higher tHcy levels than salinetreated controls (7.5 6 1.5 vs 3.9 6 0.6 mmol/L; P , 0.05). Using the standard assay containing 2.5 mmol/L L-Hcy and 2
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FIGURE 2 The effect of CBHcy on hepatic BHMT protein content. A representative Western blot of mouse liver extracts probed for BHMT protein. Lane 1 (S1) shows the level of BHMT protein 1 h post injection of physiologic saline (Study 1). Lanes 2–6 (C1, C2, C4, C8, and C24) show the level of BHMT protein at 1, 2, 4, 8, and 24 h postinjection of CBHcy, respectively (Study 1). Lane 7 (C3d) shows the level of BHMT protein 2 h after the 6th injection of CBHcy (Study 2).
FIGURE 3 The effect of repeated CBHcy treatment on liver CBS activity, and liver AdoMet and AdoHcy concentrations. In Study 2, mice were administered multiple injections of physiologic saline or CBHcy. CBS activities, AdoMet, and AdoHcy concentrations, and the AdoMet: AdoHcy ratios of mice treated with saline or CBHcy are shown. Values are means 6 SEM (relative). A relative value of 1 equals 888 U/mg liver extract, 8.2 nmol/g liver, 7.5 nmol/g liver, and 1.26 for liver CBS activity, AdoMet concentration, AdoHcy concentration, and the AdoMet:AdoHcy ratio, respectively. *Different from control, P , 0.05.
significantly lower (38–45%) in the CBHcy-treated mice than in the saline-treated controls, but liver AdoHcy was not affected at any time point (data not shown). It is not known why the effects of CBHcy are transient. It is possible that the drug is metabolized, and we attempted to address its potential conversion to Hcy in this report (discussed below). It is also possible that CBHcy is filtered by the kidney and excreted in the urine. Another possibility is that other tissues might take up the drug more slowly than liver and retain it for a longer period, thereby slowly depleting the liver of CBHcy. These and other possibilities can be evaluated only by more thorough pharmacokinetic evaluations of the drug, studies that are certainly warranted. Prolonged treatment with CBHcy caused a 7-fold increase in tHcy, a significant reduction in the liver AdoMet:AdoHcy ratio (Fig. 3), and an induction of BHMT protein expression (Fig. 2). An upregulation of BHMT protein expression in CBHcytreated mice was also supported by the observation that BHMT activities did not differ in the saline- and CBHcy-treated groups, despite the injection with the drug only 2 h before being killed. The induction of BHMT expression was likely due to the reduction in AdoMet. AdoMet was shown to inhibit BHMT transcription in hepatoma cells (28), a response that was mapped to the 254 to 11 nucleotide region of the 59-flanking region of the human BHMT gene. Moreover, mice devoid of methionine adenosyltranferase-1 activity (Mat1a /) have low concentrations of AdoMet and elevated expression of BHMT mRNA compared with wild-type mice (29). Repeated exposure to CBHcy also caused a 56% reduction in liver CBS activity (Fig. 3). CBS mRNA or protein levels were not quantified in this study, but because activity was measured with saturating levels of AdoMet, an allosteric activator, it is reasonable to assume that this change in activity reflects a corresponding change in CBS protein content. It is possible that some of the increase in tHcy in the CBHcy-treated mice in Study 2 was due in part to the significant reduction in CBS activity (30), in addition to reduced flux through BHMT. Although we were unable to detect any capacity of liver or blood tissue to convert CBHcy to Hcy in vitro, it was possible that some of the increase in tHcy after CBHcy treatment was due to CBHcy metabolism that we were unable to detect. To address this concern, we evaluated the effects of CBHcy treatment on tHcy levels in mice given a Met load in Study 3. Our results provided clear evidence that CBHcy is functionally inhibiting BHMT at the 1-mg dose and that BHMT has a substantial role in attenuating the post-Met load rise in tHcy. Mice given CBHcy in combination with a Met load had tHcy
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Jiracek J., Collinsova M., Rosenberg I., Budesinsky M., Netusilova H., and Garrow T. A. Unpublished data). In this report, CBHcy potently inhibited mouse BHMT activity but had negligible effects on mouse MS, CBS, or cystathionase activity (Table 1). No evidence was obtained suggesting that CBS, cystathionase, or any other enzyme in liver or blood could convert CBHcy to Hcy, or that any nonenzymatic conversion of CBHcy to Hcy occurred in these tissues. The concentration of CBHcy in mouse liver has not been quantified in any study. Because the livers were not perfused with saline before homogenization, it is possible that some of the BHMT inhibition measured was due to CBHcy found in the blood and/or extracellular space that gained access to the enzyme during the preparation of the extract. If so, the true intracellular inhibition of BHMT could be overestimated. Conversely, preparing the extract resulted in at least a 4-fold dilution of the CBHcy, and the volume of extract added to the assay resulted in another 15.7-fold dilution. These dilutions could lead to an underestimation of the inhibition of BHMT in liver. Despite not knowing the true concentration of CBHcy in the hepatocyte and the resulting in vivo inhibition of BHMT activity, the metabolite measurements from Study 1 strongly suggested that CBHcy does enter the liver cell and inhibits the enzyme. A single injection of CBHcy elevated tHcy levels for 8 h (Fig. 1A). At the 2- and 4-h time points, liver AdoMet was
FIGURE 4 The ability of Bet or DMSP to attenuate the post-Met load rise in tHcy. In Study 5, mice were given a single injection of Met, Met 1 Bet, or Met 1 DMSP. Values are means 6 SEM. *Different from the Met treatment group, P , 0.05.
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ACKNOWLEDGMENT We thank Dr. Sandy Slow for her technical assistance and for her help preparing this manuscript.
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levels that were 88 mmol/L higher than those given Met alone. If BHMT inhibition were not taking place, there would be no reason to expect a rise in tHcy greater than that observed (6– 8.5 mmol/L) in Study 1, when the same amount of CBHcy was given without a Met load (Fig. 1). To confirm that inhibition of BHMT causes hyperhomocysteinemia in food-deprived mice, we tested the effects of CBHcysulfoxide on tHcy levels because this compound might be less likely to be converted, or converted more slowly to Hcy in vivo than CBHcy. CBHcy-sulfoxide is a 55-fold less potent inhibitor of BHMT than CBHcy; thus, we injected 10-fold more sulfoxide (10 mg vs 1 mg) in Study 4. As expected on the basis of its lower affinity and relative dose, it took higher specific activity 14C-Bet and lower Hcy in our assay cocktail to detect the inhibition of BHMT after CBHcy-sulfoxide treatment. However, mice treated with CBHcy-sulfoxide had 90% higher tHcy, which is impressive compared with the 2.7-fold increase in tHcy observed 2 h postinjection in Study 1 in which the much more potent CBHcy inhibitor was used (Fig. 1A). Moreover, in Study 1, we measured an 87% reduction in BHMT activity, and this inhibition was observed using the standard assay, which employs much higher substrate concentrations. Although the differences observed for BHMT inhibition and tHcy after injection of CBHcy (Study 1) or CBHcy-sulfoxide (Study 3) might be easily attributed to the differing affinities these compounds have for BHMT in combination with the doses given, it is also possible that there is a difference in the rate at which these compounds enter the liver. Nevertheless, taken together, Studies 1 and 4 indicate that BHMT does have a role in the regulation of tHcy levels in food-deprived mice. As discussed above for Study 3, injection of Met (3 mg) into mice caused a large increase in tHcy at 2 h. Using this methodology, we compared (Study 5) the ability of 2 methyl donor substrates (equimolar) for BHMT to lower post-Met load tHcy levels, i.e., Bet and DMSP. Both methyl donors significantly lowered tHcy levels (Fig. 4). DMSP is a compound made by plants, and is particularly high in marine algae. It has been known for decades that DMSP is a substrate for BHMT, although the catalytic constants were never reported. It should also be noted that i.v. injection of DMSP decreases tHcy levels in rats to a greater extent than Bet (11). In conclusion, using potent and selective inhibitors of BHMT, we reported the first evidence that a transient but significant reduction of BHMT activity causes a transient increase in both food-deprived and post-Met load tHcy in mice, and that BHMT is required to maintain normal levels of AdoMet in liver. These data suggest that if mutations in the BHMT gene are discovered that substantially reduce BHMT function, then individuals with 2 defective alleles will have some degree of hyperhomocysteinemia and increased risk for Hcy-related diseases. A lack of BHMT activity will predictably predispose the liver to fatty infiltration and other pathology, including hepatocellular carcinoma, due to the lack of AdoMet (29,31). In addition, we showed for the first time that a naturally occurring sulfonium analog of Bet, DMSP, is an effective post-Met load tHcy-lowering agent.
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