Nutrient Metabolism

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synthase (MS), cystathionine synthase (CS) and betaine-homocysteine methyltransferase (BHMT) were studied in growing steers. Six Holstein steers (205 kg) ...
Nutrient Metabolism

Methionine Supply to Growing Steers Affects Hepatic Activities of Methionine Synthase and Betaine-Homocysteine Methyltransferase, but Not Cystathionine Synthase1,2 Barry D. Lambert, Evan C. Titgemeyer,3 Gerald L. Stokka, Brad M. DeBey* and Clint A. Lo¨est Department of Animal Sciences and Industry and *Department of Diagnostic Medicine, Kansas State University, Manhattan, KS 66506-1600 ABSTRACT The effects of supplemental methionine (Met), supplied abomasally, on the activities of methionine synthase (MS), cystathionine synthase (CS) and betaine-homocysteine methyltransferase (BHMT) were studied in growing steers. Six Holstein steers (205 kg) were used in a replicated 3 ⫻ 3 Latin square experiment. Steers were fed 2.6 kg dry matter daily of a diet containing 83% soybean hulls and 8% wheat straw. Ruminal infusions of 180 g/d acetate, 180 g/d propionate, 45 g/d butyrate, and abomasal infusion of 300 g/d dextrose provided additional energy. An amino acid mixture (299 g/d) limiting in Met was infused into the abomasum to ensure that nonsulfur amino acids did not limit growth. Treatments were infused abomasally and included 0, 5 or 10 g/d L-Met. Retained N (20.5, 26.9 and 31.6 g/d for 0, 5 and 10 g/d L-Met, respectively) increased (P ⬍ 0.01) linearly with increased supplemental Met. Hepatic Met, vitamin B-12, S-adenosylmethionine and S-adenosylhomocysteine were not affected by Met supplementation. Hepatic folates tended (P ⫽ 0.07) to decrease linearly with Met supplementation. All three enzymes were detected in hepatic tissue of our steers. Hepatic CS activity was not affected by Met supplementation. Hepatic MS decreased (P ⬍ 0.01) linearly with increasing Met supply, and hepatic BHMT activity responded quadratically (P ⫽ 0.04), with 0 and 10 g/d Met being higher than the intermediate level. Data from this experiment indicate that sulfur amino acid metabolism may be regulated differently in cattle than in other tested species. J. Nutr. 132: 2004 –2009, 2002. KEY WORDS:



cattle



methionine



liver



enzymes

Methionine (Met4) is the most limiting amino acid for growing cattle when ruminal microbial protein is the sole protein source (1), and Met also is the first limiting amino acid for growing cattle fed soybean hull– based diets (2). The postabsorptive fate of Met has been investigated relatively little in cattle. Available data indicate that Met metabolism may be regulated differently in cattle than in other species investigated. Under sulfur amino acid limiting conditions, cysteine (Cys) has been reported to spare up to half of the Met requirement in rats (3), pigs (4) and cats (5). However, we did not demonstrate improvements in N retention when Cys was supplemented to steers limited by sulfur amino acids (6,7). Based on this, we hypothesized that the hepatic enzymes



transsulfuration

involved in Met metabolism may be regulated differently in cattle than in other investigated species. Data concerning the presence and activity of the enzymes involved in Met metabolism in cattle are lacking. In rats, the Met metabolic pathways are partially regulated by S-adenosylmethionine (SAM) concentrations within the liver (8), which are positively correlated with Met intakes (9). Our objectives were to determine in growing steers supplemented with graded amounts of Met: 1) the hepatic activities of betaine-homocysteine methyltransferase (BHMT), methionine synthase (MS) and cystathionine synthase (CS); 2) the hepatic levels of key regulatory compounds and cofactors; and 3) the activities of BHMT, CS, and MS in several tissues. MATERIALS AND METHODS

1 Contribution no. 02-164-J, Kansas Agriculture Experimental Station, Manhattan, KS. 2 Data from this manuscript were presented in part at Kansas State University Cattlemen’s Day, Manhattan, KS, March 2002 [Lambert, B. D., Titgemeyer, E. C. & Lo¨est, C. A. (2002) Effect of methionine supplementation on methionine metabolism in growing cattle. Cattlemen’s Day, Report of Progress 890, Agricultural Experiment Station, Kansas State University, Manhattan, KS. pp. 17–19]. 3 To whom correspondence should be addressed. E-mail: [email protected]. 4 Abbreviations used: BHMT, betaine-homocysteine methyltransferase; CS, cystathionine synthase; Cys, cysteine; Met, methionine; MS, methionine synthase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; VFA, volatile fatty acid.

Experimental protocols were approved by Kansas State University’s Institutional Animal Care and Use Committee. Six Holstein steers (initial body weight ⫽ 205 ⫾ 18 kg) fitted with ruminal cannulas were housed in individual metabolism crates in a temperature-controlled room (22°C) under constant lighting. The experiment was a replicated 3 ⫻ 3 Latin square design, and treatments were continuous abomasal infusions of 0, 5 or 10 g/d L-Met. Abomasal infusions were accomplished by a peristaltic pump connected to polyvinyl chloride tubing (0.24 cm i.d.) that was passed through the ruminal cannula and reticulo-omasal orifice. The tube

0022-3166/02 $3.00 © 2002 American Society for Nutritional Sciences. Manuscript received 2 April 2002. Initial review completed 11 April 2002. Revision accepted 17 April 2002. 2004

METHIONINE METABOLISM IN CATTLE

terminated in the abomasum and was held in place by a rubber flange (9 cm diameter). The diet (Table 1) used in this study was previously used to investigate Met metabolism in growing cattle (6,7) and contained 83% pelleted soybean hulls and 8% wheat straw (dry basis). Steers received 2.6 kg/d of the diet (dry basis) fed twice daily at 12-h intervals. The diet and restricted level of intake were to minimize microbial and dietary nondegradable protein supply to the small intestine. To ensure that Met was clearly first limiting for steers not receiving supplemental Met, an amino acid mixture was infused abomasally with all treatments. The mixture contained (g/d): Lglutamate (150), glycine (50), L-valine (10), L-leucine (15), L-isoleucine (10), L-lysine䡠HCl (20), L-histidine䡠HCl䡠H2O (5), L-arginine (10), L-threonine (10), L-phenylalanine (15) and L-tryptophan (4). Amino acids were prepared daily as follows. Branched-chain amino acids were dissolved in 1 kg of water containing 70 g of 6 mol/L HCl. Once the branch-chained amino acids were solubilized, the remaining amino acids, except glutamic acid were added to the mixture. The glutamic acid was dissolved separately in 500 g of water containing 32 g NaOH. After all amino acids were dissolved, the two solutions were mixed, and 300 g of dextrose was added to provide additional energy to the steers. Water was added to bring the total volume of the mixture to 4 kg. To increase energy supply to the steers, without increasing protein supply, volatile fatty acids (VFA) were continually infused into the rumen by a peristaltic pump through polyvinyl chloride tubing (0.24 cm i.d.). The VFA infused were acetate (180 g/d), propionate (180 g/d) and butyrate (45 g/d), which were mixed with water to a final solution of 4 kg/d. Steers were adapted to their diet (Table 1) for 2 wk before initiation of treatments. Before onset of the experiment, steers were adapted to infusions and metabolism crates for 4 d. Experimental periods were 7 d in length and consisted of 3 d of adaptation and 4 d of fecal and urine collection. Fecal material was collected daily in pans under the metabolism crates, composited by period, and a representative sample (10%) was stored at ⫺20°C for later analysis. Urine was collected daily into buckets containing 250 mL 6 mol/L HCl. A representative sample (1%) was collected, composited by period and stored at ⫺20°C for later analysis. Samples of diet were collected each day, composited by period and stored at ⫺20°C until later analysis. Dietary, urinary and wet fecal samples were analyzed for Kjeldahl N.

TABLE 1 Composition of experimental diet fed to steers Ingredient

Dry matter g/100 g

Pelleted soybean hulls Wheat straw Cane molasses Dicalcium phosphate Sodium bicarbonate Calcium carbonate Urea Magnesium oxide Trace mineralized salt1 Vitamin A, D, E2 Sulfur Bovatec-683

83.3 7.6 3.7 2.0 1.0 1.0 0.49 0.40 0.29 0.10 0.10 0.02

Nutrient Organic matter Nitrogen

87.3 2.2

1 Composition (g/100 g): NaCl (95 to 99), Mn (⬎ 0.24), Cu (⬎ 0.032), Zn (⬎ 0.032), I (⬎ 0.007), and Co (⬎ 0.004). 2 Supplied per kg of dry matter: 2.7 mg vitamin A, 37.5 ␮g vitamin D and 40 mg vitamin E. 3 Supplied 33 mg lasalocid per kg of dry matter.

2005

Blood samples were collected 7 h after the morning feeding on the last day of each period by jugular venipuncture. Samples were collected into heparinized tubes, placed on ice immediately after collection and centrifuged (5000 ⫻ g). Equal volumes of plasma and 100 g/L sulfosalicylic acid containing 1 mmol/L norleucine as an internal standard were mixed, cooled on ice for 30 min and then centrifuged (13,800 ⫻ g). The resulting supernatant was stored at ⫺20°C for later amino acid separation by cation exchange chromatography and fluorimetric quantification after postcolumn o-phthalaldehyde derivitization (Beckman System Gold; Beckman Instruments, Palo Alto, CA). Liver biopsies were collected from all steers on the final day of each of the first two periods 8 h after the morning feeding, by the method of Chapman et al. (10). Biopsies were obtained either through the 11th or 12th intercostal space. On the final day of the final period, steers were stunned by captive bolt and killed by exsanguination. A sample (250 g) was obtained from the right lobe of the liver, immediately frozen in liquid N2 and stored under liquid N2 until analysis. After killing, tissues samples were also collected from the following: lung (⬃200 g from the ventral one third of the right middle lobe), small intestine (a 20-cm section of the distal jejunum), large intestine (a 10-cm section of the proximal loop of the ascending colon), kidney (⬃250 g from the anterior kidney), skeletal muscle (proximal semimembranosus and semitendinosus), cardiac muscle (⬃50 g of the apex of the heart), spleen (approximately 8 cm from the ventral extremity), pancreas (⬃ 150 g from the right lobe), rumen (⬃50 g from the dorsal sac), skin (⬃100 g from the caudal femoral area) and blood (⬃500 mL obtained from femoral artery). Upon collection, extrahepatic tissues were immediately frozen in liquid N2 and stored at ⫺80°C for later analysis. Hepatic concentrations of vitamin B-12 and folate (period 3 samples only) were determined by the method of Stangl et al. (11). This method used a competitive binding radioimmunoassay (no. 06B257117; ICN, Costa Mesa, CA) that contained an extracting reagent to release vitamin B-12 from transcobalamines. Before analysis, a homogenate was prepared in a 1 mol/L borate buffer (pH 9.2). Hepatic SAM and S-adenosylhomocysteine (SAH) concentrations (period 3 samples only) were determined by HPLC (12). Before analysis, hepatic tissue samples were weighed, homogenized in 5 volumes of 0.4 mol/L perchloric acid and centrifuged at 10,000 ⫻ g for 20 min. The resulting supernatant was filtered through a 0.45-␮m filter and stored in liquid N2 until SAM and SAH quantification. For hepatic amino acid determination, liver samples were homogenized in 4 volumes of 100 g/L sulfosalicylic acid containing 1 mmol/L norleucine as an internal standard, cooled on ice for 30 min and then centrifuged (13,800 ⫻ g). The resulting supernatant was stored at ⫺20°C until analysis. Hepatic amino acid concentrations were determined as described for plasma amino acid. Enzyme activity determinations. All samples were minced with scissors, weighed and homogenized immediately in 4 volumes of 0.04 mol/L potassium phosphate buffer (pH 7.4) containing 1 mmol/L EDTA and 10 mmol/L mercaptoethanol. The homogenate was centrifuged at 0°C for 30 min at 3000 ⫻ g, and the supernatant fraction was used in all enzyme assays and protein concentration determinations. For all enzyme assays, values from duplicate blanks containing the homogenizing buffer in place of tissue extract were routinely subtracted. Linearity of all enzyme assays was verified for incubation times and levels of tissue extract used. Protein concentrations in tissue extracts were determined by the use of a colorimetric assay kit (no. 610-A; Sigma-Aldrich, St. Louis, MO). The assay for BHMT was based on Finkelstein and Mudd (13). [Methyl-14C]betaine was synthesized by incubation of [methyl-14C] choline chloride (Sigma-Aldrich) with choline oxidase (14). DLHomocysteine was prepared fresh daily from homocysteine thiolactone hydrochloride (15). The following components were incubated for 60 min at 37°C in a volume of 505 ␮L: 175 ␮L of 200 mmol/L potassium phosphate buffer (pH 7.4); 70 ␮L of 100 mmol/L DLhomocysteine; 110 ␮L of 18 mmol/L betaine–HCl containing 150 ⫻ 103 dpm of 14CH3-betaine; and 150 ␮L of tissue extract. After the reaction, labeled reaction products were separated from betaine (15) and measured by liquid scintillation. Methionine synthase activity was determined based on Mudd et al. (16). DL-Homocysteine was prepared fresh daily from homocys-

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2006

teine thiolactone hydrochloride (15). The following were incubated at 37°C for 15 min under N2 in a final volume of 150 ␮L: 20 ␮L of 1 mol/L potassium phosphate buffer (pH 7.5); 15 ␮L of 2 mmol/L S-adenosylmethionine; 10 ␮L of 10 mmol/L cyanocobalamin; 10 ␮L of 10 mmol/L L-Met; 5 ␮L of 6 ␮mol/L N5-methyltetrahydrofolate containing 150 ⫻ 103 dpm of N5-[14C]-methyltetrahydrofolate (Amersham Pharmacia Biotech, Piscataway, NJ); 5 ␮L of 8 mol/L 2-mercaptoethanol; 5 ␮L of 10 mmol/L DL-homocysteine; 30 ␮L of deionized water; and 50 ␮L of tissue extract. The reaction was terminated by the addition of 400 ␮L of ethanol, and the mix was centrifuged at 20,000 ⫻ g for 10 min. A 250-␮L aliquot of the supernatant was added to 2 mL of deionized water and placed onto a 1-mL column bed of AG-1 (Cl⫺), and Met was eluted from the column with 2 mL of deionized water (unreacted 5-methyltetrahydrofolate was retained on the column) and assayed for radioactivity by liquid scintillation. Cystathionine synthase activity was determined based on Mudd et al. (17). DL-Homocysteine was prepared fresh daily from homocysteine thiolactone hydrochloride (15). The following were incubated at 37°C for 30 min in a total volume of 400 ␮L: 60 ␮L of 1 mol/L Tris (pH 8.4); 10 ␮L of 100 mmol/L EDTA; 10 ␮L of 25 mmol/L propargylglycine; 40 ␮L of 3.5 mmol/L L-cystathionine; 50 ␮L of 100 mmol/L DL-homocysteine; 10 ␮L of 100 mmol/L L-serine containing 150 ⫻ 103 dpm of 14C-L-serine (Sigma-Aldrich); 150 ␮L of deionized water; 20 ␮L of 10 mmol/L pyridoxal phosphate (PLP); and 50 ␮L of tissue extract. The reaction was terminated by the addition of 400 ␮L of 100 g/L trichloroacetic acid, and labeled cystathionine was separated from serine (17) and measured by liquid scintillation. Statistical analyses. Nitrogen balance, hepatic enzyme activities and plasma amino acid data were analyzed using the MIXED procedure of SAS (SAS Institute, Cary, NC). The model contained the effects of level of Met supplementation and period, and steer was included as a random variable. Effects of Met supplementation on period 3 extrahepatic enzyme activities and period 3 hepatic concentrations of amino acids, SAM, SAH, vitamin B-12 and folic acid were analyzed as a completely randomized design using the MIXED procedure of SAS. The model contained the effects of level of Met supplementation. Linear and quadratic effects of Met supplementation were tested using contrasts for equally spaced treatments. Significance was declared at P ⬍ 0.05.

RESULTS AND DISCUSSION Nitrogen balance and plasma amino acids. Nitrogen retention increased linearly (P ⬍ 0.01) with Met supplementation (Table 2). Based on the data of Campbell et al. (6), our basal absorbable intestinal supplies of Met and Cys were 2.6 and 2.3 g/d, respectively. Thus, total supplies of Met ranged from 2.3 g/d for control to 12.3 g/d for steers receiving 10 g/d supplemental Met. Based on previous experiments with similar steers and nutrient supplies (6), we expected the Met requirement of our steers to be about 5 g/d supplemental Met. We had hoped that the broad range of Met supplied would allow us to detect changes in hepatic Met metabolism at Met supplies both below and above the steers’ requirements. However, based on linear increases of N balance, 10 g/d supplemental Met did not greatly exceed the requirement of our steers. Plasma Met concentrations (Table 2) increased linearly (P ⬍ 0.001), whereas plasma serine decreased linearly (P ⬍ 0.001) with Met supplementation. Decreases in plasma serine are commonly observed when steers limited by sulfur amino acids receive supplemental Met (6,7). Aside from use in protein synthesis, serine is necessary for the synthesis of Cys from homocysteine, and Met supplementation leads to increased use of serine for both of these purposes. Hepatic enzymes and metabolite concentrations. Case and Benevenga (18) reported that SAM-independent pathways of Met catabolism exist, and Gill and Ulyatt (19) suggested that Met transamination was quantitatively important in sheep. However, these pathways are thought to be relatively minor under normal conditions, as discussed by Finkelstein (8). For this reason, we limit our discussion to the SAMdependent transsulfuration and transmethylation pathways. Betaine-homocysteine methyltransferase, MS and CS all were present in hepatic tissue of cattle (Table 3). Enzyme activities as measured are maximal rates with saturating concentrations of substrates and cofactors. These values may differ

TABLE 2 Effect of level of methionine supplementation on nitrogen balance and concentrations of plasma and hepatic metabolites in growing cattle Methionine, g/d Item Nitrogen, g/d1 Total intake Fecal Urinary Retained Plasma concentrations, ␮mol/L1 Methionine Taurine Serine Hepatic concentrations, nmol/g wet liver2 Vitamin B-12 Total folates SAM3 SAH3 Hepatic concentrations, mmol/L2 Methionine Taurine Serine

0

5

94.2 21.7 52.0 20.5

94.7 20.7 47.1 26.9

12.1 22.8 184.1

22.9 20.9 109.3

1.10 43.5 95.7 12.8 0.177 2.76 4.94

1 n ⫽ 6. 2 n ⫽ 2. 3 SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine.

0.95 35.8 66.0 12.3 0.126 2.40 3.43

P-value 10

SEM

Linear

95.1 19.9 43.7 31.6

— 0.84 2.0 2.1

— 0.16 0.01 ⬍ 0.01

— 0.92 0.77 0.72

29.7 29.6 104.9

1.2 2.3 9.7

⬍ 0.001 0.07 ⬍ 0.001

0.13 0.09 ⬍ 0.001

0.62 29.9 114.1 12.3

0.34 3.6 17.0 0.68

0.39 0.07 0.50 0.62

0.83 0.85 0.16 0.78

0.52 0.06 0.28

0.31 0.12 0.37

0.151 5.46 3.68

0.025 0.64 0.68

Quadratic

METHIONINE METABOLISM IN CATTLE

2007

TABLE 3 Hepatic activities of cystathionine synthase (CS), methionine synthase (MS), and betaine-homocysteine methyltransferase (BHMT) in growing cattle supplemented with graded amounts of methionine1 Methionine, g/d Enzyme

0

P-value

5

10

SEM

Linear

Quadratic

19.6 7.6 21.9

1.8 0.68 2.3

0.46 ⬍ 0.01 0.96

0.11 0.03 0.04

nmol product 䡠 h⫺1 䡠 mg protein⫺1 CS MS BHMT

17.7 11.5 21.8

15.1 7.4 17.1

1 n ⫽ 6.

from physiological activities because of the effects of substrate, inhibitor and/or cofactor concentrations. It is also important to note that tissue homogenates were used in the determination of enzyme activities, eliminating the cellular compartmentalization that exists physiologically. However, our assays were essentially identical to those used in studies with other species, which allows for ready comparisons with existing data. Cystathionine synthase activity was not affected by Met supplementation. Radcliffe and Egan (20) reported no change in hepatic CS activity of sheep when abomasal Met supply was increased from 1.4 to 4.2 g/d. This is in contrast to results reported by Finkelstein and Martin (9) with rats, where increasing dietary Met from 0.3 to 1.0% increased CS activity by 35%. Finkelstein et al. (21) also demonstrated an increase in hepatic CS activity when rats were fed increasing levels of dietary casein. Although we planned to provide excess Met to our steers, N balance data indicated that we did not greatly exceed the steers’ Met requirements; it is possible that greater Met intakes would have affected CS activity. Overall, the activities of hepatic CS of our steers were ⬃25% of those measured in rats (3,22). Finkelstein (23) reported that preincubation of partially purified rat liver CS with SAM increased activity in a dose-dependent manner. Hepatic SAM represents a logical regulatory compound for Met metabolism, given that its concentration is coupled to Met supply (9,24). As Met supply increases, it is less critical that Met be conserved and more important that it be metabolized. In rats this is accomplished by an increase in CS and a concomitant decrease in MS (8). Methionine synthase activity decreased 35% when steers received 5 or 10 g/d of supplemental Met (linear, P ⬍ 0.01; quadratic, P ⫽ 0.03). Overall, MS activity was similar to previously reported values in sheep (14) but was two to three times that reported for rats (14). Finkelstein and Martin (9) reported a decrease in hepatic MS of 32% when dietary Met was increased from 0.3 to 1.0% in rats. Betaine-homocysteine methyltransferase activity responded quadratically (P ⫽ 0.04) to increasing Met supplementation, with 0 or 10 g/d resulting in higher activity than that with 5 g/d. Overall, the activities of hepatic BHMT were approximately twofold those reported in sheep and half of those in rats (14). A quadratic response in BHMT to Met supplementation was previously observed in rats (25), wherein it was suggested that BHMT activity was high at low Met supply to conserve Met and high at high Met intake to prevent homocysteine accumulation. Finkelstein and Martin (26) further elucidated a possible mechanism by which the quadratic effect of Met supply on BHMT activity occurs. Their experiment included preincubation of a partially purified BHMT with SAM. They

observed that BHMT, when preincubated with SAM, had a marked loss of activity. However, when either SAH or homocysteine was added to the preincubation medium, the inactivation of BHMT did not occur. Increasing Met supply increases hepatic SAM concentration in rats (9,24). In our steers, hepatic SAM concentration (Table 2) averaged 91.9 nmol/g and was not changed by Met supply, although variation was large. Changes in BHMT and MS activities, which are regulated by hepatic SAM in rats, would indicate that at least some regulatory mechanism was at work in our steers. Hepatic SAH concentration (Table 2) averaged 12.5 nmol/g in our steers and was not affected by Met supply. The presence of BHMT activity might indicate that betaine could be used as a source of methyl groups in cattle. However, Lo¨ est (7) reported no significant response in N balance when betaine was supplied postruminally to steers similar to those in our experiment. Hepatic taurine (Table 2) tended to increase (P ⫽ 0.06) linearly with Met supplementation. Because taurine is a product of sulfur amino acid metabolism, it is not surprising that the hepatic concentration of taurine was increased with Met supplementation. Hepatic Met and serine concentrations were not affected by Met supplementation. The Met cycle relies on several B-vitamins as cofactors; 5-methyltetrahydrofolate, vitamin B-12 and vitamin B-6 all play roles in the Met metabolic cycle. Because of the limited feeding protocol used and the subsequently low microbial flow to the small intestine, B-vitamin status could have been compromised in our steers. Hepatic vitamin B-12 concentrations (Table 2) averaged 0.89 nmol/g, and responses to Met supplementation were not detected because of large variation. Reference ranges for hepatic vitamin B-12 in cattle are limited. Kennedy et al. (27) induced vitamin B-12 deficiency by decreasing dietary cobalt. They reported hepatic vitamin B-12 concentrations of 0.08 and 0.65 nmol/g for cobalt vitamin B-12– deficient and control cattle, respectively. We measured (our unpublished results) hepatic vitamin B-12 concentrations in steers under similar conditions, and steers had hepatic vitamin B-12 concentrations of 0.74 and 1.00 nmol/g before and after supplementation with 100 ␮g/d vitamin B-12, respectively. With average hepatic vitamin B-12 concentrations of 0.89 nmol/g, it does not appear that our steers were greatly deficient in vitamin B-12. Hepatic folate concentration averaged 36.4 nmol/g and tended (P ⫽ 0.07) to be decreased by Met supplementation. Hepatic folate concentrations in similar steers (our unpublished results) were 38.5 and 61.6 nmol/g folic acid before and after postruminal folic acid supplementation (10 mg/d), respectively. Dumoulin et al. (28) reported hepatic folate con-

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2008

TABLE 4 Tissue activities of cystathionine synthase (CS), methionine synthase (MS), and betaine-homocysteine methyltransferase (BHMT) in growing cattle Enzyme1 Tissue

CS

MS

BHMT

nmol product 䡠 h⫺1 䡠 mg protein⫺1 Blood Cardiac muscle Kidney Large intestine Lung Skeletal muscle Rumen Small intestine Skin Spleen Pancreas

ND2 ND 6.2 ⫾ 1.03 ND ND 0.4 ⫾ 0.54 20.8 ⫾ 1.8 7.2 ⫾ 1.8 ND 5.1 ⫾ 1.4 52.8 ⫾ 11

ND 14.0 ⫾ 1.7 5.3 ⫾ 0.30 13.1 ⫾ 2.9 8.3 ⫾ 0.45 8.6 ⫾ 1.2 10.2 ⫾ 0.54 35.5 ⫾ 3.0 0.3 ⫾ 0.34 36.8 ⫾ 5.3 13.9 ⫾ 2.6

ND ND ND ND ND ND ND ND ND 1.2 ⫾ 0.504 26.1 ⫾ 6.7

1 Mean ⫾ SEM of six observations, with two from each of three levels of methionine supplementation (0, 5 and 10 g/d). 2 Not detectable (average value across steers ⱕ 0). 3 Activity was significantly affected by methionine supply (linear and quadratic, P ⱕ 0.01). Activities were 5.6, 3.9 and 9.0 (SEM ⫽ 0.45) nmol product 䡠 h⫺1 䡠 mg protein⫺1 for 0, 5 and 10 g/d supplemental methionine. 4 Not different from 0 (P ⬎ 0.05).

centrations of 68 nmol/g for young calves maintained on either ad libitum consumption or restricted feeding regimens. Stangl et al. (11) reported that cobalt-deficient cattle had lower hepatic folate concentrations, which were 20 and 57 nmol/g liver for cobalt-deficient and -sufficient cattle, respectively. Given the limited amount of data to compare hepatic folate concentrations, it is unclear whether our steers were deficient in folates, although hepatic concentrations were low enough to suggest the possibility. Reasons for decreases in hepatic folates in response to Met supplementation are unknown, but Krebs et al. (29) suggested that when one-carbon metabolism is altered, such as during a cobalamine deficiency, tissue uptake and retention of folates may be altered as well. Methionine supplementation certainly may have altered one-carbon metabolism and, thus, hepatic folate concentrations by these mechanisms. Extrahepatic enzymes. Extrahepatic tissue enzyme activities (pooled across supplemental Met levels) are presented in Table 4. Data for tissue activities of these enzymes in cattle are lacking in the literature. With the exception of renal CS, methionine supply did not affect extrahepatic enzyme activities. None of the enzymes was detected in blood or skin. Overall, our data agree with available data from sheep (14) and rats (14,17), in that BHMT was present only in select tissues, whereas MS was in many tissues. Other than liver, we detected high BHMT activity only in pancreatic tissue. Xue and Snoswell (14) detected BHMT in kidney and pancreas of both rats and sheep. Methionine synthase activity was present in all tissues tested except blood and skin. These findings are similar to results with rats and sheep (14,17), although these authors did not evaluate blood or skin. Cystathionine synthase activity was present in several tissues including kidney, rumen, small intestine, spleen, and pancreas. In a review of several published manuscripts, Finkelstein (8) reported that CS had been detected in liver, small intestine, pancreas, adipose, brain, kidney, and spleen in rats.

Extrahepatic tissues could be important sites of Cys synthesis. In our steers, several tissues had CS activities that were greater than those in liver. Renal CS activity was affected (P ⬍ 0.01) by supplemental Met, with activity being increased when the highest level of methionine was supplemented [activities were 5.6, 3.9, and 9.0 (SEM ⫽ 0.45) nmol product䡠h⫺1䡠mg protein⫺1 for 0, 5 and 10 g/d supplemental methionine, respectively]. Thus, regulatory mechanisms in the kidneys of our steers were similar to those in the liver of rats (9,21). The presence of regulatory mechanisms for renal CS would suggest that the kidneys are potentially important for the synthesis of Cys when excess Met is available to cattle. Lobley et al. (30) observed substantial Met cycling in sheep kidney, supporting the idea that the kidney may play an important role in Met metabolism. In our steers, we were unable to detect in the skin any of the three enzymes that we measured. This was surprising based on the reports of Liu et al. (31), who reported in sheep that a proportionally higher rate of transsulfuration occurred in the skin of young lambs than that in the whole body. Pisulewski and Buttery (32) reported in sheep that approximately 70% of Cys-sulfur in wool originated from Met, regardless of Met supply. Because plasma Cys-sulfur originating from Met was much lower, this indicated the occurrence of transsulfuration in the skin of sheep. Species differences between sheep and cattle could be related to the need for Cys to support wool growth. General discussion. In previous experiments in our laboratory (6,7), we were unable to demonstrate the Met-sparing effect of Cys in Holstein steers, and the lack of response of CS activity to supplemental Met could explain these results (6,7). In rats, where the Met-sparing effect of Cys is well documented, CS activity increases in response to increasing Met supply, but remains low during low Met supply (8,9). The lack of an enzymatic control point could account for at least a part of the lack of Met sparing by Cys in cattle. Because total CS activity was low and MS activity was high relative to activities in rats, it is possible that in cattle, remethylation of homocysteine predominates, independent of Cys supply, and regulation of CS is less important than in rats. However, in vitro assays do not account for all physiologically relevant modifiers of activity and may not yield true partitioning between the transsulfuration and transmethylation reactions. In our steers, hepatic CS activity was unresponsive to Met supply, which is different from that in other species investigated. From our data, the quantitative importance of this lack of control of CS activity by Met supply is unclear. More data on hepatic enzyme regulation are needed to further the understanding of sulfur amino acid metabolism in cattle. LITERATURE CITED 1. Richardson, C. R. & Hatfield, E. E. (1978) The limiting amino acids in growing cattle. J. Anim. Sci. 46: 740 –745. 2. Greenwood, R. H. & Titgemeyer, E. C. (2000) Limiting amino acids for growing Holstein steers limit-fed soybean hull-based diets. J. Anim. Sci. 78: 1997–2004. 3. Finkelstein, J. D., Martin, J. J. & Harris, B. J. (1988) Methionine metabolism in mammals. The methionine-sparing effect of cystine. J. Biol. Chem. 263: 11750 –11754. 4. Chung, T. K. & Baker, D. H. (1992) Maximal portion of the young pig’s sulfur amino acid requirement that can be furnished by cysteine. J. Anim. Sci. 70: 1182–1187. 5. Teeter, R. G., Baker, D. H. & Corbin, J. E. (1978) Methionine and cysteine requirements of the cat. J. Nutr. 108: 291–295. 6. Campbell, C. G., Titgemeyer, E. C. & St-Jean, G. (1997) Sulfur amino acid utilization by growing steers. J Anim. Sci. 75: 230 –238. 7. Lo¨est, C. A. (1999) Methionine and Betaine for Growing and Finishing Cattle. Ph.D. Dissertation, Kansas State University, Manhattan, KS.

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