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Methionine as a methyl group donor in growing cattle¹

C. A. Löest^*, E. C. Titgemeyer^*,2, G. St-Jean D. C. Van Metre and J. S. Smith

^* Department of Animal Sciences and Industry and and Department of Clinical Sciences, Kansas State University, Manhattan 66506-1600

² Correspondence:
132 Call Hall (E-mail:
etitgeme{at}oznet.ksu.edu).

	Abstract

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Holstein steers were used in two 5 x 5 Latin square experiments to evaluate the sparing of methionine by alternative sources of methyl groups (betaine and choline). Steers were housed in metabolism crates and limit-fed a soybean hull-based diet high in rumen degradable protein. To increase energy supply, ruminal infusions of volatile fatty acids and abomasal infusions of glucose were provided. An amino acid mixture, limiting in methionine, was infused abomasally to ensure that nonsulfur amino acids did not limit protein synthesis. Treatments for Exp. 1 were abomasal infusion of 1) water, 2) 2 g/d L-methionine, 3) 1.7 g/d L-cysteine, 4) 1.6 g/d betaine, and 5) 1.7 g/d L-cysteine + 1.6 g/d betaine. Treatments for Exp. 2 were abomasal infusion of 1) water, 2) 2 g/d L-methionine, 3) 8 g/d betaine, 4) 16 g/d betaine, and 5) 8 g/d choline. In both experiments, nitrogen retention increased in response to methionine (P < 0.05), demonstrating a deficiency of sulfur amino acids. Responses to cysteine, betaine, and choline were all small and not significant. The lack of response to cysteine indicates that the response to methionine was not due to transsulfuration to cysteine or that cysteine supply did not alter the flux of methionine through transsulfuration. The lack of response to betaine suggests that the steers’ needs for methyl groups were met by the dietary conditions or that betaine was relatively inefficient in increasing the remethylation of homocysteine to methionine and, thereby, reducing the synthesis of cysteine from homocysteine. Under our experimental conditions, responses to methionine were likely due to a correction of a deficiency of methionine per se rather than of methyl group donors.

Key Words: Betaine • Choline • Cysteine • Methionine • Steers

	Introduction

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Methionine is an essential amino acid that often limits ruminant growth (Richardson and Hatfield, 1978). Campbell et al. (1997) and Greenwood and Titgemeyer (2000) identified methionine as a limiting amino acid for growing cattle fed restricted amounts of soybean hull-based diets. Methionine functions as a precursor for protein synthesis, and a deficiency of this amino acid may result in inefficient use of dietary protein for protein deposition. Methionine has many functions in the body in addition to its direct contribution to protein synthesis. It can be activated to S-adenosylmethionine (SAM), which plays an important role as a methyl group donor for many transmethylation reactions in the body (Eloranta, 1977; Finkelstein, 1990). These SAM-dependent reactions include the synthesis of polyamines, as well as the methylation of phospholipids, proteins, nucleic acids, and many other molecules (Lobley, 1992; Chiang et al., 1996). Methionine also has an important function in the synthesis of cysteine via homocysteine, which is a product of the SAM-dependent methylation reactions and a critical branch point between the transsulfuration to cysteine and remethylation to methionine (Finkelstein, 1998).

Research suggests that at least half of the methionine requirements of rats (Shannon et al., 1972), cats (Teeter et al., 1978), dogs (Hirakawa and Baker, 1985), pigs (Chung and Baker, 1992), and chicks (Baker et al., 1996) can be replaced by cysteine. Research with cattle has indicated that cysteine did not effectively spare methionine (Campbell et al., 1997). The lack of response to cysteine may have been due to a deficiency of methyl groups for the remethylation of homocysteine to methionine, resulting in transsulfuration rates that exceeded cysteine requirements. We hypothesized that supplementation of methyl groups might improve methionine utilization. Our objective was to evaluate the sparing of methionine by two methyl group sources (betaine and choline).

	Materials and Methods

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Experimental protocols were approved by Kansas State University’s Institutional Animal Care and Use Committee.

Experiment 1
Five ruminally cannulated Holstein steers averaging 156 kg at the beginning of the experiment were maintained in metabolism crates (to facilitate total collection of feces and urine) in a room with constant temperature (21°C) and continuous lighting. Steers were allowed free access to fresh water and received a soybean hull-based diet (Table 1) that was formulated to contain little undegradable intake protein to minimize the basal supply of dietary amino acids to the small intestine. Steers were fed 2.4 kg/d (DM basis) of diet in equal portions twice daily (0600 and 1800). Additional energy was supplied to steers without increasing ruminal microbial growth through a continuous infusion of volatile fatty acids (180 g/d acetate, 180 g/d propionate, and 45 g/d butyrate) into the rumen and a continuous infusion of 300 g/d glucose into the abomasum. Also, to ensure that nonsulfur amino acids did not limit tissue protein synthesis, 12 amino acids (Table 2), in a profile similar to that used by Campbell et al. (1997), were continuously infused into the abomasum. The daily supply of infused L-methionine was restricted to 2 g/d. This was to maintain conditions where sulfur amino acids were limiting, but also where more than 50% of the total sulfur amino acid supply came from methionine; basal supplies of methionine and cysteine were predicted to be 2.4 g/d and 2.1 g/d, respectively (Campbell et al., 1997). Amino acids and glucose were infused together in solution. To prepare this solution, branched-chain amino acids were dissolved in 1.7 kg of water containing 90 g of 6 N HCl. Then, the remaining amino acids (excluding glutamic acid) were added and allowed to dissolve. The glutamate was dissolved in 700 g of water containing 45 g of NaOH in a separate container; the glutamate solution plus 300 g of dextrose was then mixed with the other amino acids before adding water to bring the total weight to 4 kg. The entire solution was stirred to allow the glucose to dissolve completely and then refrigerated until use. Ruminal infusions were made by placing flexible polyvinylchloride tubing (2.4 mm i.d.) through the rumen cannula, and abomasal infusions were made by extending a similar tube through the reticulo-omasal orifice and into the abomasum. A rubber flange, 8 cm in diameter, was used to hold the tubing in the abomasum. This model created methionine-limiting conditions in previous research (Campbell et al., 1997).

On d 3 through d 7 of each period, daily urinary output was collected into buckets containing 300 mL of 6 N HCl (to prevent the loss of NH₃). Urine weights for each steer were recorded, and a representative sample (1% of daily urine) was saved and composited by period. Total fecal output was collected on d 3 through d 7, weighed, and a representative sample (10% of daily feces) was saved and composited for each steer by period. Urine and wet fecal samples were frozen and later analyzed for N by Kjeldahl analysis (AOAC, 1990). Fecal samples were analyzed for DM by drying at 105°C for 24 h and for OM by ashing at 450°C for 8 h. Feed samples and feed refusals, if any, were collected (d 3 through d 7 of each period) and composited by period. Feed samples and orts were dried at 55°C in a forced-air oven, allowed to air-equilibrate before being ground through a 1-mm screen, analyzed for DM, OM, and Kjeldahl N. Urine samples were analyzed for N-methylhistidine concentration by automated fluorimetry (Murray et al., 1981) using a Technicon Auto Analyzer II (Technicon Industrial Systems, Tarrytown, NY).

To calculate protein synthesis and degradation, whole-body protein turnover was measured using the ¹⁵N-glycine single-dose urea end-product method (Assimon and Stein, 1992; Wessels et al., 1997). A single dose of 0.4 g ¹⁵N-glycine (98 atom percent excess; Sigma-Aldrich Chemical Company, St. Louis, MO) dissolved in 100 mL of water was infused abomasally as a pulse dose to each steer 48 h prior to the end of each period. Urine excreted the day prior to the infusion of ¹⁵N-glycine was used to determine ¹⁵N background enrichment in urinary urea. After administration of ¹⁵N-glycine, urine collections for 48 h were used for measurement of ¹⁵N excretion in urinary urea (Wessels et al., 1997). Whole-body protein turnover (Q) was calculated as the urinary urea N excreted (g/d) divided by the fractional recovery of the ¹⁵N dose in urinary urea. Protein synthesis (PS) and protein degradation (PD) were calculated using the relationship: Q = PS + urinary N = PD + absorbed N.

Methionine flux was measured on the last day of each period by continuously infusing labeled methionine (methyl-²H₃) into the abomasum and subsequently measuring plasma enrichment of methionine. Infusions commenced at feeding time and contained 0.4 g of ²H₃-methionine (98%, Cambridge Isotope Laboratories, Andover, MA), which replaced an equimolar amount of unlabeled methionine that was present in infusates designed to last for 12 h. Blood samples were collected by jugular venipuncture into heparinized syringes after 9 and 11 h of infusion of the labeled methionine. Background enrichment of ²H₃-methionine in plasma was measured in samples collected 2 h prior to initiation of label infusion (i.e., 10 h after feeding). Isotopic enrichment of ²H₃-methionine in plasma was measured by gas-liquid chromatography/mass spectrometry analysis (H/5890 gas chromatograph with H/5970 Mass Selective Detector; Avondale, PA) of t-butyldimethylsilyl derivatives (Calder and Smith, 1988) separated on a HP-5MS column (30 m x 0.25 mm x 0.25 µm film thickness; Agilent Technologies, Wilmington, DE). The retention time of the t-butyldimethylsilyl derivative of methionine was approximately 21.5 min. Fragment ions were monitored at m/z of 323 and 320. Calculations were as described by Lobley et al. (1996) with the average enrichment of the 9- and 11-h samples being used for calculation of irreversible loss rate.

For analysis of plasma amino acids, blood samples collected at 10 h after feeding (i.e., background samples from methionine flux measures) were used. Blood samples were immediately chilled on ice and processed. Blood was centrifuged for 20 min at 5,000 x g, and plasma was mixed with an equal volume of 10% (wt/vol) sulfosalicylic acid containing 1 mM norleucine as an internal standard for amino acid analysis, cooled on ice for 30 min, and centrifuged for 20 min at 25,000 x g. Deproteinized plasma was stored frozen until later analysis of amino acids by cation exchange chromatography with postcolumn o-phthalaldehyde derivatization and fluorimetric quantification (Beckman System Gold, Beckman, Palo Alto, CA).

All data were analyzed statistically using the General Linear Models procedure of SAS (SAS System for Windows Release 6.11; SAS Inst. Inc., Cary, NC). The statistical model included effects of steer, period, and treatment. Mean separation was by t-tests for all possible pair-wise comparisons when the treatment F-test was significant. Only four observations were obtained for the 1.7 g/d L-cysteine + 1.6 g/d betaine treatment because of a problem with the infusion for a steer assigned to this treatment.

Experiment 2
Five ruminally cannulated Holstein steers (158 kg initial BW) were maintained under experimental conditions similar to Exp. 1, except feed (Table 1) was provided to steers at 2.5 kg/d (DM basis). The study was a 5 x 5 Latin square similar to Exp. 1 with treatments being continuous abomasal infusions of 1) water (control), 2) 2 g/d additional L-methionine, 3) 8 g/d betaine, 4) 16 g/d betaine, and 5) 8 g/d choline. Betaine was provided in amounts that were five and 10 times that in Exp. 1 to test the efficiency of betaine as a methyl group donor. Choline was infused to examine its role as an alternative methyl group donor. Protein turnover was not measured in this experiment. All other data were collected as described in Exp. 1.

	Results and Discussion

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Experiment 1
Nitrogen retentions and diet digestibilities are presented in Table 3. Apparent total tract digestion of DM and OM were similar across treatments and averaged 72.7% and 74.4%, respectively. These digestibilities are consistent with those reported by Campbell et al. (1996, 1997) for a similar diet.

Abomasal infusion of cysteine resulted in nitrogen balance responses that were less dramatic than those for methionine with only a slight numerical (P = 0.16) increase in nitrogen retention relative to the control. A small response to cysteine is consistent with the findings of Campbell et al. (1997), who reported no increases in nitrogen retention for steers abomasally infused with either 1.62 or 3.25 g/d of cysteine. Using plasma methionine as a response criterion, Towns and Bergen (1979) similarly observed no responses to intraperitoneal injections of cysteine in steers. In contrast, Ahmed and Bergen (1983) observed higher plasma methionine concentrations when methionine was injected with cysteine than without. The lack of response to cysteine supplementation demonstrates that the steers’ requirements for methionine were not effectively spared by cysteine. This may indicate that the responses in this experiment to additional methionine supplementation were not dependent on transsulfuration of methionine to cysteine. These results for cattle are somewhat surprising, as there are many reports showing that cysteine can effectively replace at least half of the dietary requirements of methionine in nonruminants (Shannon et al., 1972; Finkelstein et al., 1988; Chung and Baker, 1992; Baker et al., 1996). Although it has been assumed that methionine metabolism is regulated similarly for all mammals, Campbell et al. (1997) hypothesized that transsulfuration in cattle may be poorly regulated. The relatively low efficiency of methionine use (43%) may explain the lack of sparing of methionine by cysteine in our calves. If most of the unutilized methionine was metabolized to cysteine through transsulfuration, control steers probably would have an adequate physiological supply of cysteine.

In addition to cysteine and protein synthesis, methionine plays a central role in methyl group metabolism (Lobley, 1992; Chiang et al., 1996), which may differ considerably in ruminants vs nonruminants due to ruminal microbial activity. Dietary choline, a major preformed methyl group nutrient, is extensively degraded by ruminal microbes (Atkins et al., 1988; Sharma and Erdman, 1989). Betaine, which is the active methylating form of choline, is also rapidly degraded by ruminal microbes (Mitchell et al., 1979). Consequently, ruminants obtain fewer methyl group-containing nutrients from the diet than monogastrics, and Campbell et al. (1997) concluded that the lack of a response to cysteine could be due to a low availability of methyl groups, leading to inefficient remethylation of homocysteine and, thus, loss of methionine via transsulfuration. This was supported by Finkelstein et al. (1988), who reported that cysteine could not spare methionine if rats had a methyl group deficiency.

Methylation reactions involving SAM yield S-adenosylhomocysteine which, in turn, is converted to homocysteine. Homocysteine is a critical branch point in the metabolism of methionine, and it can be either transsulfurated to form cysteine or remethylated to conserve methionine (Finkelstein, 1998). There are two enzymes responsible for yielding methionine from homocysteine. The enzyme, 5-methyltetrahydrofolate-homocysteine methyltransferase (5-MHMT), requires 5-methyltetrahydrofolate to donate a methyl group to homocysteine, whereas betaine-homocysteine methyltransferase (BHMT) yields methionine by methylating homocysteine using betaine as a methyl donor (Smolin and Benevenga, 1989; Finkelstein, 1990). The availability of a source of methyl groups, such as betaine, could be important for the sparing of methionine.

Nitrogen balance responses were small from abomasal infusions of betaine alone or in combination with cysteine (Table 3). Abomasal infusion of betaine numerically (P = 0.15) increased nitrogen retention, a response over the control that was 20% ([21.04 - 19.65]/[26.61 - 19.65] x 100%) as large as that for methionine. Research evaluating betaine in cattle is limited, although several studies have demonstrated that supplementation of betaine (unprotected from ruminal degradation) to growing (Löest et al., 2001) and finishing (Löest, 1999; Goodall and Brethour, 1999) diets had little effect on cattle performance. Puchala et al. (1998) observed increases in ADG of goats supplemented with a rumen-protected betaine source.

Treatments had little effect on protein synthesis and protein degradation (Table 3). Steers supplemented with 2 g/d of methionine had a higher (P < 0.05) efficiency of protein deposition because of the increase in nitrogen retention but not protein synthesis. Urinary excretion of N-methylhistidine, which is reflective of the breakdown of skeletal muscle (Gopinath and Kitts, 1984), was not greatly affected by treatment, and none of the supplements led to amounts significantly different than the control. Using ¹⁵N-glycine techniques with steers, Wessels et al. (1997) reported increased protein accretion in response to supplementation with a mixture of amino acids and observed an increase in both protein synthesis and protein degradation. Increases in both protein synthesis and protein degradation, with the increase in protein synthesis being greater than that for degradation, are typical for protein-deficient animals receiving amino acid supplementation (Wessels et al., 1997). The lack of increase in protein synthesis and protein degradation for our steers supplemented with methionine suggests that the nitrogen balance response may not occur due to mechanisms that typically are observed when amino acid deficiencies are corrected. It is also possible that the relatively large supply of glycine from the basal infusions may have led to glycine metabolism being less than completely reflective of the total amino acid pool within the body. In our study, protein synthesis accounted for a much lower proportion and oxidation a greater proportion of flux than in the study of Wessels et al. (1997); this probably was a result of the glycine supplementation in our study.

Irreversible loss rate (flux) of methionine was not significantly affected by treatment. Because our label was present on the methyl group, transmethylation of methionine would be measured as part of the methionine flux. The lack of change in methionine flux in response to betaine supplementation would suggest that betaine did not greatly alter the cycling of methionine through transmethylation reactions with subsequent resynthesis of methionine from homocysteine. However, we did not measure the proportion of methionine flux that was independent of transmethylation, so we were unable to directly determine if recycling of methionine was altered. Several studies with nonruminants have demonstrated an increase in the remethylation of methionine from homocysteine in response to supplemental betaine (Finkelstein et al., 1983; Emmert et al., 1996, 1998). Increases in remethylation of homocysteine might reduce the competing conversion of homocysteine to cystathionine. Thus, the lack of change in the methionine flux in response to betaine supplementation fits well with the lack of change in nitrogen retention when betaine was supplied.

Plasma levels of serine (Table 4) decreased (P < 0.05) in response to methionine supplementation, a response also reported by Campbell et al. (1996, 1997). Finkelstein and Martin (1986) demonstrated that increasing methionine in rat diets resulted in increased activity of cystathionine synthase, an enzyme that uses serine and homocysteine to synthesize cystathionine in the transsulfuration process of methionine to cysteine. Thus, it is likely that decreases in plasma serine levels in response to methionine were due to an increase in transsulfuration. Abomasal infusion of methionine also decreased (P < 0.05) plasma valine levels, which appears to be a typical response for plasma branched-chain amino acids (Titgemeyer and Merchen, 1990; Campbell et al., 1996, 1997), and this probably reflects increases in use for protein deposition. Abomasal infusion of cysteine did not significantly alter plasma levels of most amino acids, but did lower (P < 0.05) plasma serine and valine concentrations. Lower plasma serine concentrations were also observed by Campbell et al. (1997), an observation that is in contrast to expected decreased use of serine due to depression in the activity of cystathionine synthase with added cysteine (Finkelstein and Mudd, 1967; Finkelstein et al., 1988; Yamamoto et al., 1995). Abomasal infusion of betaine alone or in combination with cysteine had little effect on plasma amino acids (Table 4). In contrast, Puchala et al. (1998) observed increases in plasma methionine concentrations when betaine was infused into the duodenum of calves. Puchala et al. (1994, 1997) also observed increases in plasma methionine levels when goats received jugular infusions of betaine. These discrepancies in plasma methionine responses to supplemental betaine may be explained by the methionine status of the animal. Plasma amino acid levels will increase only after the requirement for that amino acid has been met (Bergen, 1979). Therefore, under methionine-limiting conditions created by our model, plasma methionine concentrations were not likely to increase even if betaine increased remethylation of homocysteine to methionine.

Experiment 2
Nitrogen balance and diet digestibilities are shown in Table 5. Abomasal infusion of methionine, betaine, or choline did not significantly alter apparent total tract digestion of DM and OM when compared to the control. These digestibilities averaged 75.3% and 77.2% for DM and OM, respectively, and are similar to those observed in Exp. 1.

Several reports demonstrated that supplemental betaine may stimulate betaine-dependent homocysteine methylation, thus increasing methionine remethylation in nonruminants, including humans (Finkelstein et al., 1983; Dudman et al., 1996; Emmert et al., 1996). However, due to extensive ruminal degradation of methyl group-containing nutrients (Mitchell et al., 1979; Atkins et al., 1988; Sharma and Erdman, 1989), ruminants have adapted to the low availability of those nutrients. Snoswell and Xue (1987) concluded that sheep synthesize most of their choline (a methyl group-containing nutrient) using SAM as a methylating agent. Xue and Snoswell (1985) compared enzyme activities involved in methionine metabolism in sheep (ruminants) and rats (nonruminants), and they demonstrated that sheep had relatively low hepatic BHMT activity and high hepatic 5-MHMT activity when compared to rats. Xue and Snoswell (1986) also compared enzyme activities in preruminant lambs vs mature sheep and observed a decrease in BHMT and a compensatory increase in 5-MHMT when lambs reached a ruminant state.

Nitrogen retention was not significantly altered by abomasal supplementation with 8 g/d of choline (Table 5). Puchala et al. (1998) similarly observed no responses to choline when infused via the duodenum of calves. These observations are in contrast to those of Bindel et al. (2000) and Drouillard et al. (1998), who demonstrated that finishing cattle respond positively to supplementation of ruminally protected choline. One function of choline is methyl group donation via betaine. Choline, however, functions in several ways and plays an important role in synthesis of phosphatidylcholine (Mookerjea, 1971), lipid digestion, and transport of triglycerides from the liver (Haines and Mookerjea, 1965; Lombardi et al., 1968). Contradicting results in cattle may be due to differences in physiological state. Finishing cattle may respond to choline due to requirements for lipid synthesis, whereas growing cattle may require less choline due to a lower need for lipid synthesis.

Urinary excretion of N-methylhistidine was not greatly affected by treatment, and none of the supplements led to amounts significantly different than control. This supports the conclusion that supplementation of methionine alone does not increase both protein synthesis and degradation as a means of increasing protein deposition.

Irreversible loss rate (flux) of methionine was not significantly affected by treatment. Methionine flux, which would include transmethylation reactions, was actually numerically lower when either 8 or 16 g/d of betaine was supplemented. Thus, betaine, even in relatively large amounts, did not impact methionine metabolism in our experiment. The lack of change in methionine flux and presumably the lack of change in remethylation of homocysteine to methionine would explain why betaine was unable to greatly alter protein deposition in our calves.

Although there were no large differences, plasma taurine levels (Table 6) tended (P = 0.19) to be lower for supplemental methionine and tended (P < 0.13) to be higher for supplementation of 16 g/d of betaine and 8 g/d of choline. Campbell et al. (1997) also observed lower plasma taurine levels when steers were infused with methionine. Taurine is a product of cysteine metabolism (Hayes, 1988) and would be expected to increase with supplemental methionine. However, nitrogen retention increased in response to methionine supplementation and more cysteine may have been used for protein synthesis rather than taurine production, which may explain lower plasma taurine levels. Research has demonstrated that methyl groups (betaine) may increase homocysteine remethylation (Finkelstein et al., 1983; Dudman et al., 1996; Emmert et al., 1996). Therefore, plasma taurine levels would be expected to be lower when betaine is supplemented. However, in addition to methyl group donation, betaine may play a significant role in osmoregulation (Dragolovich, 1994) and could potentially displace taurine in tissues. As in Exp. 1, plasma serine levels decreased (P < 0.05) when 2 g/d of L-methionine was infused abomasally.

	Implications

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Large increases in nitrogen retention in response to postruminal supplementation of methionine demonstrated that conditions for steers were limiting in sulfur amino acids as intentionally created by our research model. Small responses were observed with postruminal supplementation of betaine, and this suggests that there was an inefficient replacement of methionine by betaine. Higher levels of betaine, however, did not overcome this inefficiency. The lack of response to choline demonstrates that it did not spare methionine. Under our experimental conditions, responses to methionine were likely due to a correction of a deficiency of methionine per se rather than its role as a methyl group donor.

	Footnotes

 
¹ Contribution Number 02-332-J, Kansas Agric. Exp. Sta., Manhattan.

Received for publication February 22, 2002. Accepted for publication April 26, 2002.

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