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Capacity in the Liver of the Broiler Chick for Conversion of Supplemental Methionine Activity to L-Methionine
Capacity in the Liver of the Broiler Chick for Conversion of Supplemental Methionine Activity to L-Methionine JULIA J. DIBNER1 and F. J. IVEY Nomis International, Inc., Chesterfield, Missouri 63198 (Received for publication August 19, 1991)
1992 Poultry Science 71:700-708
INTRODUCTION
DL-HMB), and DL-methionine (DL-Met) are used as sources of L-methionine (LThe corn and soybean meal diets com- Met). The only form of methionine that monly used as broiler or layer rations can be used in intermediary metabolism or require the presence of supplemental me- incorporated into protein is L-Met; supplethionine activity for optimum bird per- mental sources must be converted before formance. To correct this deficiency, they can be used by the bird. Alimet®2 [an 88% aqueous solution of DLBiochemical conversion of DL-HMB to 2-hydroxy-4-(methylthio)butanoic acid, L-Met takes place in two steps (Dibner DL-HMB], MHA® (an 86% calcium salt of and Knight, 1984). The first reaction is the oxidation of the a carbon, yielding the intermediate 2-oxo-4-(methylthio)butanoic acid (keto-methionine). The molecular T To whom correspondence should be addressed. structures of both DL-HMB and DL-Met Novus International, Mail Zone BB21,700 Chesterfield contain one asymmetric carbon, resulting Parkway North, Chesterfield, MO 63198. 2 Alimet® is a registered trademark of Novus Inter- in the presence of equal amounts of the D and L stereoisomers in the commercially national, Inc.
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ABSTRACT The objective of the present experiments was to determine whether the levels of supplemental methionine sources currently used in practical diets exceed the capacity of the chick to convert the supplement to Lmethionine. Supplemental sources examined included DL-2-hydroxy-4(methylthio)butanoic acid (DL-HMB, Ahmet®, or MHA®) and DL-methionine (DL-Met). Two approaches were taken: first, the amount of enzyme activity available for conversion of the two supplemental methionine sources was determined using optimum reaction conditions for each and chick liver homogenate as the enzyme source. These experiments showed that total liver enzyme conversion activity was 564 umol/h for DL-HMB and 529 umol/h for DL-Met. The total activities for the two sources were not different when measured at saturating substrate concentration. Second, to address the question of whether the enzyme is limiting for either source under practical feeding conditions, birds were fed starter diets supplemented with DL-HMB or DL-Met at .25% of the diet for 3 wk. When hepatic levels of free HMB and methionine were determined, birds fed DL-HMB contained 7.6 nmol HMB/g of liver and 84.7 nmol methionine/g of liver. Birds fed DL-Met had levels of 7.6 nmol HMB/ g liver and 80.3 nmol methionine/g liver. These results indicate no accumulation of HMB or DL-Met, which might occur if conversion capacity were saturated. By calculation, a bird consuming 100 g/day of a diet supplemented with .25% activity would need to convert about 70 umol/h, indicating a sevenfold excess of enzyme in the liver alone. {Key words: DL-2-hydroxy-4-(methylthio)butanoic acid, methionine conversion, poultry, liver, Alimet®)
SUPPLEMENTAL METHIONINE CONVERSION
Although the biochemical pathway for the conversion of DL-HMB and DL-Met to keto-methionine has been described, little information exists relating enzyme capacity to demand for conversion of dietary supplemental sources by the broiler chick. A recent publication reported the use of a decarboxylation assay to determine the rate and total tissue capacity of the chick to produce keto-methionine from DLHMB and DL-Met (Dupuis et ah, 1989). The reaction conditions used, however, did not include a cofactor reported to be essential for one of the conversion enzymes (Dibner and Knight, 1984). In the studies reported here, the effect of this cofactor was examined using a spectrophotometric assay that permits examination of D-HADH activity alone. The decarboxylation assay was also compared with the spectrophotometric assay in terms of total activity obtained. Finally, the total liver enzyme capacity of 3- to 4-wk-old broiler chicks in relation to conversion requirements was studied.
^airview Farms, Remington, IN 47977. Ziggity Systems, Middlebury, IN 46540.
4
MATERIALS AND METHODS Animals Hubbard x Hubbard cockerel chicks 3 were fed a corn and soybean meal diet supplemented with L-Met, DL-Met, or DLHMB (Table 1) from 1 day of age through 4 wk. Birds were housed six per cage. Cages 51 cm wide x 69 cm long X 36 cm high were made of polyvinylchloride-coated wire mesh. Mesh size was 2.5 x 2.5 cm for sides and top and 1.25 cm for the floor. Feed was supplied in a galvanized trough feeder and water was supplied in sanitary plastic and stainless water nipples. 4 Thermoneutral temperatures were maintained starting at 33 C. After 3 days temperatures were decreased with a linear function to 24 C at 21 days and held constant thereafter. Complete exchange of room air with fresh air was provided 15 times/h. Fluorescent light with an intensity of 45 be was provided for 23 h / d a y . Chicks were euthanatized using carbon dioxide inhalation.
Experimental Design In the experiments illustrated in Figures 1 to 4, a randomized complete block design was used. In each experiment (block), a separate shipment of chicks was used. 3 After 3 w k on the diet described in Table 1, three randomly chosen chicks were used for each experiment and liver homogenate from them was pooled to generate the enzyme fraction. Duplicate enzyme reactions were run for each data point and one spectrophotometric assay or radiolabel count was performed for each duplicate. Data points in Figures 1 and 2 are the average of two values, one each from two replicate enzyme experiments (n = 2). Data points in Figures 3 and 4 are the average of three values, one each from three replicate enzyme experiments (n = 3). All references to treatment averages represent main effects of the experiment. N o significant treatment by experiment interactions were observed (P>.10). To determine the concentration of HMB and methionine in the livers of animals fed different supplemental methionine sources, three pens of six day-old birds were placed on the corn and soybean meal diet
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available product. Different enzymes catalyze the oxidation of the two stereoisomers of DL-HMB. Therefore, conversion of the two stereoisomers occurs simultaneously to produce the ketomethionine intermediate (Dibner and Knight, 1984). The enzyme specific for LHMB is L-2-hydroxy acid oxidase (LHAOX, EC 1.1.3.15) and has been studied in chick liver (Dibner and Knight, 1984), hog kidney (Robinson et ah, 1962), and rat l i v e r ( L a n g e r , 1965). The D-HMB stereoisomer requires the enzyme D2-hydroxy acid dehydrogenase (D-HADH, EC 1.1.99.6), which has been studied in the chick, where it occurs in all tissues tested, including liver, kidney, skeletal muscle, intestine, pancreas, spleen, and brain (Dibner and Knight, 1984). The conversion of D-Met to keto-methionine also requires a specific enzyme catalyst, D-amino acid oxidase (D-AAOX, EC 1.4.3.3). This enzyme, like L-HAOX, occurs primarily in the liver and kidney and is found in peroxisomes (Scott et al., 1969; Masters and Holmes, 1977).
701
702
DIBNER AND IVEY TABLE 1. Diet composition
0
2
4
6
8
10
D-HMB Concentration (mmol/L) FIGURE 1. The effect of the cofactor phenazine methosulfate (PM, 5 mmol/L) on the synthesis of keto-methionine (keto) from D-2-hydroxy-4(methylthio)butanoic acid (D-HMB, A). There is little activity of D-hydroxy acid dehydrogenase over the 60-min reaction period in the absence of this cofactor (•). Each point represents the x ± SEM (n = 2) of two sets of duplicate reactions. Liver homogenate from three birds was pooled for each experiment. Results from two experiments representing a total of six animals were combined. In each experiment, one spectrophotometric analysis was performed on duplicate enzyme reactions at each dose.
each diet were used to generate individual liver homogenates (n = 3). Two chromatographic analyses were performed per sample. Enzyme Preparation
described in Table 1 except supplemented with either DL-HMB, DL-Met, or L-Met at 2.5 g/kg diet. The DL-HMB was added to the diet as Alimet® (an 88% aqueous solution of DL-HMB5 in H 2 0). Alimet® is typically 65% monomelic DL-HMB, 23% polymeric DL-HMB, and 12% H2O (Lawson and Ivey, 1984). The DL-Met6 was added at 99.5% purity. The data in Table 2 represent one such study. Birds were fed the diets for 3 wk and three randomly chosen birds from
Monsanto, St. Louis, MO 63198. ^dne-Poulenc, Atlanta, GA 30350. 7 Mallinckrodt, St. Louis, MO 63147. ^igma Chemical Co., St. Louis, MO 63178-9916. Brinkmann Instruments, Westbury, NY 11590.
Liver tissue was homogenized into four volumes of ice-cold, 20 mmol/L potassium phosphate buffer7 (pH 7.5) containing .25 mol/L sucrose8 and .1 mmol/L phenylmethylsulfonyl fluoride8 using a Polytron tissue homogenizer.9 Liver weights were approximately 25 g per bird. Homogenates were stored at -20 C and were diluted 1:1 with potassium phosphate buffer for use in reaction mixtures. Enzymatic activity was stable for at least 30 days when homogenates were stored under these conditions. Protein levels in the homogenate were 270 to 300 mg/mL and were determined using a modified biuret protein assay (Ohnishi and Barr, 1978) with Folin and Ciocalteu's Phenol Reagent and using bovine serum albumin as the standard.8
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Ingredients and composition Percentage 54.90 Corn, yellow 35.20 Soybean meal 5.60 Soybean oil Calcium carbonate .60 2.80 Dicalcium phosphate .10 Vitamin premix1 .05 Mineral premix2 .08 Choline chloride 35 Supplemental methionine .05 L-lysine .37 Sodium chloride Nutrient composition Protein 20.80 Calcium .96 .86 Total phosphorus 1.43 Arginine .59 Methionine Methionine + cysteine .94 1.20 Lysine 3,174.5 ME, kcal/kg Vitamin premix provided per kilogram of diet: thiamin HQ» 1.1 mg; riboflavin, 8.8 mg; pantothenate, 13.2 mg; niacin, 38.5 mg; pyridoxine HO, 3.3 mg; folacin, .99 mg; biotin, .055 mg; cyanocobalamin, .013 mg; menadione dimethyl pyrimidinol, 2.64 mg; ethoxyquin, 125.0 mg; retinyl, 8,800 IU; cholecalciferol, 3,300 ICU; and DL-a-tocopherol acetate, 13.2 IU. Trace mineral premix provided in milligrams per kilogram of diet: MnS04-H20,64; zinc sulfate-HjO, 70; ferric citrate (ca) 5H2O, 50; copper sulfate-SHjO, 8; sodium selenite, .3; and iodine, .8. depending on the experiment, supplemental methionine was supplied as one of the following: Lmethionine, DL-methionine, or DL-2-hydroxy-4(methylthio)butanoic acid.
NoPM +PM
703
SUPPLEMENTAL METHIONINE CONVERSION No Cofaclor Phenazine Methosulfate 3.5 3.0 c
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30
40
50
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DL-HMB Concentration (mmol/L)
Reaction
Conditions
Reactions c o n t a i n e d .1 mL liver homogenate, .5 to 50 umol substrate, and potassium phosphate buffer (20 mmol/L) in a total volume of 1.0 mL. Substrates were either unlabeled DL-HMB 5 (sodium salt), unlabeled DL-Met, 8 [1-14C]DL-Met,10 or [114 C]DL-HMB 5 (sodium salt). The radiolabeled DL-HMB was entirely monomer, which results from the formation of the sodium salt. In some experiments phenazine methosulfate 8 (.5 mmol/L) was added. Dose-response curves with this cofactor showed dose-related increases in enzyme activity in the presence of .1 to .25 mmol/L, with a plateau from .5 to 1.0 mmol/L (data not shown).
lu
Amersham, Arlington Heights, IL 60005. Ultrospec Model 4050, Pharmacia LKB Biotechnology, Piscataway, NJ 08854. n
FIGURE 3. Activity toward the racemic mixture DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMB) in the presence (A) and absence (•) of phenazine methosulfate (.5 mmol/L). At all concentrations tested, the amount of keto-methionine (keto) produced from DL-HMB was approximately doubled in the presence of this cofactor. Each point represents the x+SEM (n = 3) of three sets of duplicate reactions. Liver homogenate from three randomly chosen birds was pooled for each experiment. Results from three experiments were combined. One spectrophotometric analysis was performed on duplicate enzyme reactions at each dose.
Reactions were incubated at 37 C on a rotating table (100 rpm) for 60 min. Previous experiments showed that the reactions were linear for over 3 h (Dibner and Knight, 1984). In experiments using unlabeled substrate, the reaction product, ketomethionine was detected using a dinitrophenylhydrazine 8 spectrophotometric 11 assay previously described (Dibner and Knight, 1984). For these experiments, reactions containing all components except substrate were subtracted as blank absorption values. The keto-methionine standard curve was generated by the addition of known concentrations of keto-methionine 8 into the complete reaction mixture, minus the substrate, and incubating these under reaction conditions for 60 min followed by the dinitrophenylhydrazine assay described above. In experiments using radiolabeled substrate, reactions were stopped with the addition of .5 mL .66 mol/L sulphosalicylic acid. 8 Radiolabeled keto-methionine was measured through the evolution of 14CC>2 initiated by adding .5 mL 30% hydrogen
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FIGURE 2. Synthesis of keto-methionine (keto) from s e p a r a t e i s o m e r s of D L - 2 - h y d r o x y - 4 (methylthio)butanoic acid (D-HMB and L-HMB) and DL-methionine (DL-Met), each tested at 10 mmol/L. In the presence of phenazine methosulfate (.5 mmol/L), D-HMB was converted to keto in amounts approximately equal to that of L-HMB. Half of commercially available DL-Met is in the D stereoisomeric configuration (D-Met) which must be converted to L-methionine (L-Met) before it can be used by the bird. Some keto is synthesized from L-Met by oxidation or transaminative degradation (Benevenga, 1983). Each bar represents the x ± SEM (n = 2) of two sets of duplicate reactions. Liver homogenate from three birds was pooled for each experiment. Results from two experiments were combined. One spectrophotometric analysis was performed on duplicate enzyme reactions for each substrate.
704
DIBNER AND IVEY TABLE 2. Content (x ± SEM) of methionine and DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMB) in the liver of chicks fed diets containing 25% DL-HMB, DL-methionine, or L-methionine1
DL-Met DL-HMB DL-HMB+PM
Dietary source DL-HMB DL-Methionine ^Methionine
0
5
10
15
20
Substrate Concentration (mmol/L)
Methionine (nmol/g) 7.64 ± 1.23 84.72 ± 6.92 7.63 ± 1.22 8028 ± 5.14 8.12 ± 1.93 95.51 ± 2.02
^ i r d s were fed diets supplemented with 35% DLHMB, DL-methionine, or L-methionine for 3 wk. Means ± SE (n = 3) were obtained from the General Linear Models procedure of base SAS® software (SAS Institute, 1985).
14
peroxide.8 The reactions were incubated for an additional 90 min and the 14CC>2 was trapped using .3 mL phenylethylamine8 in a well suspended over the reaction mixture. Longer incubation times (up to 4 h) did not yield additional radioactivity (data not shown). The entire contents of the well were scintillation-counted12 using Instagel12 as the scintillant. Blank values were generated by adding all of the reaction components, including the radiolabeled substrate at either 5, 10, or 20 mmol/L, to a vial containing enzyme denatured in a boiling water bath for 10 min. The amount of radiolabel from the denatured reactions was subtracted from the total radiolabel
12 ' Model 4640, Packard Tri-Carb, Downers Grove, IL 60515. 13 Sorvall Model RC3B, Sorvall Centrifuges, Dupont, Wilmington, DE 19898. 4 " B&J Solid Phase System, Baxter Healthcare Co., Muskegon, MI 49442. 15 Acrodisc, Gehnan Sciences, Ann Arbor, MI 48106.
evolution at the same substrate concentration. Liver DL-2-Hydroxy-4-(Methylthio)Butanoic Acid and DL-Methionine Levels Liver tissue (5 g) was homogenized into 5 mL deionized H2O. A 30-mL volume of 2:1 methanol:chloroform was added and extraction phases were separated with 20 mL each of chloroform and deionized H2O. Following lyophilization, the dried residue from the aqueous phase was resuspended in 5 mL deionized H2O. A 250-uL sample was extracted using an equal volume of a mixture of chloroform:methanol:water (1:2: .8). Phase separation was achieved by changing the solvent ratios to 2:2:1.8 through the addition of more chloroform and water. Following phase separation, the mixture was centrifuged13 for 5 min at 750 x g. The aqueous phase was collected and passed through an aminopropyl 100-mg column.14 The organic phase was carefully washed with water (1 mL) and the washing passed through the column. Finally, the column was washed with another 1.5 mL of water to ensure elution of the metabolites. This mixture was dried under a stream of nitrogen, lyophilized, reconstituted in 500 (J.L of water, and filtered using a .2 um filter.15 Spiked samples showed >90% recovery of DL-HMB and DL-Met following these steps. Metabolites were quantified using a modification of an high-performance liquid
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FIGURE 4. Keto-methionine (keto) synthesis from C-DL-2-hydioxy-4-(methylthio)butanoic add (DLHMB) as estimated with the CO2 evolution assay in the presence (•) and absence (A) of phenazine methosulfate (PM, .5 mmol/L). The cofactor PM increased 14 C02 evolution from DL-HMB about twofold, as would be predicted by the data shown in Figure 3. Total keto synthesis from DL-HMB (•) was comparable to that with 14C-DL-methionine (DL-Met). Each point represents the x±SEM (n = 3) of three sets of duplicate reactions. Liver homogenate from three randomly chosen birds was pooled for each experiment. Results from three experiments were combined. In each experiment, one radiolabel scintillation analysis was performed per reaction.
HMB
SUPPLEMENTAL METHIONINE CONVERSION
c h r o m a t o g r a p h y assay previously described (Dibner, 1983). The Waters chromatograph16 was equipped with an NH2 column 1 'with phosphoric acid6 (.01 mol/L, pH 3.2) and acetonitrile18 (Optima Grade) as the elution buffer. Retention times for DL-HMB and methionine were determined by the addition of standards into water and were confirmed by co-chromatography of standards added into experimental samples. Using this method, the detection limit for DL-HMB is .5 jig/mL and for methionine is 5 ug/mL. Statistical Methods
RESULTS Figure 1 shows the results of experiments designed to evaluate the requirement of D-HADH for the cofactor phenazine methosulfate. This cofactor was found to be the most effective of those tested in earlier studies in which synthesis of ketomethionine was detected using a dinitrophenylhydrazine assay (Dibner and Knight, 1984). In order to evaluate the cofactor requirement, D-HMB was tested as the substrate using chick liver homogenate as the enzyme source. As is clear from Figure 1, the crude enzyme mixture has a very limited capacity for keto-methionine synthesis from D-HMB in the absence of this cofactor. When the D and L stereoisomers of DLHMB and DL-Met were tested individually at 10 mmol/L, each isomer was observed to yield some keto-methionine (Figure 2). The two isomers of DL-HMB were similar to one another and when added together were similar in magnitude to activity toward D-Met. There was ketomethionine synthesis from L-Met, which represents the product of oxidation (Lamino acid oxidase, EC 1.4.3.2) or trans-
16
Waters Associates, Milford, MA 01757. Alltech, Deerfield, IL 60015. Fisher Scientific, St. Louis, MO 63132.
17
18
amination (EC 2.6.1) of L-Met to ketomethionine (Livesey and Lund, 1980). The racemic mixture of DL-HMB was also tested for keto-methionine synthesis in the presence and absence of phenazine methosulfate. Figure 3 shows the results of triplicate dose response experiments. The reaction mixture containing no phenazine methosulfate gave activities less than those containing the cofactor at every dose tested. When the identical reaction conditions were tested using the radiolabeled CO2 evolution assay, results were obtained that are illustrated in Figure 4. Clearly, the addition of phenazine methosulfate resulted in a significant increase in radiolabeled CO2 evolution from DL-HMB at each concentration tested. The CO2 evolution from DL-HMB in the presence of phenazine methosulfate was not different from that seen using DL-Met as the substrate. A comparison of the data in Figures 3 and 4 shows that the two assay methods give comparable values for keto-methionine synthesis by chick liver homogenates. In order to evaluate the possibility that enzyme capacity could be limiting under normal dietary situations, livers from birds fed .25% supplemental DL-HMB, DL-Met, or L-Met were compared in terms of methionine and HMB content. When livers from birds fed the three methionine sources were analyzed (Table 2), there were no significant differences in methionine or HMB content. DISCUSSION Phenazine methosulfate has been used as an artificial electron acceptor in biochemical studies of purified D-HADH (Dhydroxy acid dehydrogenase) from a number of animals (Cammack, 1969); however, it is not a naturally occurring molecule. The identity of the physiological cofactor in this reaction has not been determined, although use of menadione (Dibner and Knight, 1984) or ubiquinone (Cammack, 1969) as acceptors yields about 75% of the activity seen with artificial electron acceptors. Naturally occurring acceptors that have been tested in chick liver include flavin-adenine dinucleotide, flavin mononucleotide, nicotinamide-
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Means and standard errors were obtained from the General Linear Models procedure of base SAS® software (SAS Institute, 1985). Details of the experimental design were given previously.
705
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DIBNER AND IVEY
Figures 3 and 4 illustrate the increase in the conversion of the racemic mixture, DLHMB, by the addition of phenazine methosulfate. When no cofactor is added,
activity is not reduced to zero because the oxidative conversion of L-HMB does not require it (Figure 3). The cofactor requirement is obvious only when D-HMB is tested in the absence of L-HMB (Figure 1). When the racemic mixture is used, oxidase activity toward the L-HMB stereoisomer obscures the lack of activity toward the DHMB stereoisomer. These results are compatible with the hypothesis that activity toward the racemic mixture in the absence of phenazine methosulfate is due to the action of L-HAOX only; i.e., activity toward the L-stereoisomer only. Using optimum reaction conditions and saturating substrate concentrations, calculations based on the experiments shown in Figure 4 indicate that total liver enzyme conversion activity was essentially equal for the two sources, i.e., 564 |xmol/h for DL-HMB and 529 p m o l / h for DL-Met at a substrate concentration of 20 mmol/L. A related question is whether enzyme ever can be limiting at even high commercial supplementation levels. Calculations based on the data shown in Figure 4 indicate that the liver of a 3-wk-old broiler could convert about 500 u m o l / h of either DL-HMB or DL-Met if substrate were present at saturating concentrations. If a 3-wk-old bird were consuming a diet supplemented with .25% of DL-HMB or DL-Met, the amount of methionine activity that would have to be converted in the liver (based on 100 g of feed intake/day) would be about 1,600 umol/day, or about 70 umol/h. This calculation suggests that the liver alone contains a sevenfold excess of enzyme capacity. The suggestion is further supported by the data shown in Table 2, where no accumulation of HMB was observed in the livers of birds supplemented with DL-HMB in comparison with those fed DL-Met or L-Met. Such an accumulation might occur if enzyme capacity were exceeded. The presence of HMB in chick liver (Dibner et ah, 1990), rat liver (Trackman and Abeles, 1981), and in plants (Miyazake and Yang, 1987), has been reported, and the biochemical pathway for natural HMB identified (Dibner and Ivey, 1990; Dibner et a/., 1990) in the chick. Recent evidence suggests that both HMB and keto-methionine are methionine precur-
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adenine dinucleotide, menadione, coenzyme Q, and oxygen (Dibner and Knight, 1984). The activity obtained with these cofactors was higher than that observed with no cofactor, but was below the activity seen with phenazine methosulfate (Dibner and Knight, 1984). Chick feeding studies and cellular protein synthesis studies support the observation reported here that enzyme activity toward D-HMB is approximately equal that toward L-HMB. First, Baker and Boebel (1980) reported that D-HMB gave chick performance superior to L-HMB when fed in crystalline amino acid diets. In addition, when the separate isomers of DL-HMB were tested for activity in m e t h i o n i n e - d e f i c i e n t p r i m a r y chick hepatocyte cultures, L-HMB and D-HMB supported equal incorporation of radiolabeled leucine into protein across a wide range of concentrations (Lawson and Ivey, 1984). Figure 2 shows the synthesis of ketom e t h i o n i n e from each i n d i v i d u a l stereoisomer of HMB and methionine. Separation of the isomers yielded some interesting information about these reactions. From the data in Figure 2, it is clear that the two isomers of HMB were converted to keto-methionine at similar rates under these saturating conditions. Activity toward the two stereoisomers of methionine was very dissimilar. Activity toward the D-Met isomer was much greater than that toward the L-Met isomer, as might be expected. Activity toward the L-Met isomer could be the result of the action of L-arnino acid oxidase or of a transaminase. The synthesis of ketomethionine from L-Met by transamination is the first step in its degradation (Livesey and Lund, 1980; Benevenga, 1983; Jones and Yeaman, 1986) and thus would reduce rather than increase the amount of L-Met available for protein synthesis or intermediary metabolism. Thus the total radiolabeled carbon dioxide evolution from DL-Met shown in Figure 4 could include some keto-methionine representing L-Met catabolism rather than its synthesis.
SUPPLEMENTAL METHIONINE CONVERSION
ACKNOWLEDGMENTS The authors gratefully acknowledge technical assistance by D. C. Williams, chromatography by I. R. Putnam, and animal care by M. E. Wehmeyer. REFERENCES Baker, D. H., and K. P. Boebel, 1980. Utilization of the D- and L-isomers of methionine and methionine hydroxy analogue as determined by the chick bioassay. J. Nutr. 110559-964. Boebel, K. P., and D. H. Baker, 1983. Blood and liver concentrations of sulfur-containing amino acids in chicks fed deficient, adequate, or excess levels of dietary cysteine. Proc. Soc. Exp. Med. 172: 498-501. Benevenga, N. J., 1983. Evidence for alternative pathways of methionine metabolism. Adv. Nutr. Res. 6:1-18. Cammack, R., 1969. Assay, purification and properties of mammalian D-2-hydroxy acid dehydrogenase. Biochem. J. 115:55-64. DeMoraes, G.H.K., 1980. Studies on Non-Specific Nitrogen and D-Amino Acid Metabolism in the Chick, Ph.D. dissertation. Purdue University, Lafayette, IN. Dibner, J. J., 1983. Utilization of supplemental methionine sources by primary cultures of chick hepatocytes. J. Nutr. 1132116-2123. Dibner, J. J., R. C. Durley, J. G. Kostelc, and F. J. Ivey, 1990. 2-Hydroxy-4-(methylthio)butanoic acid is a naturally occurring methionine precursor in the chick. J. Nutr. 120553-560. Dibner, J. J., and F. J. Ivey, 1990. Hepatic protein and amino acid metabolism in poultry. Poultry Sci. 69:1188-1194. Dibner, J. J., and C. D. Knight, 1984. Conversion of 2-hydroxy-4-(methylthio)butanoic acid to Lmethionine in the chick: A stereospecific pathway. J. Nutr. 114:1716-1723. Dibner, J. J., and C. D. Knight, 1985. Age-dependent changes in the metabolism of DL-2-hydroxy4-(methylthio)butanoic acid (HMB) and DLMethionine. Poultry Sci. 64(Suppl. l):14.(Abstr.) Dupuis, L., C. L. Saunderson, A. Puigserver, and P. Brachet, 1989. Oxidation of methionine and 2 - h y d r o x y - 4 - m e t h y l t h i o b u t a n o i c acid stereoisomers in chicken tissues. Br. J. Nutr. 62: 63-75. Finkelstein, J. P., W. E. Kyle, B. J. Harris, and J. J. Martin, 1982. Methionine metabolism in mammals: concentration of metabolites in rat tissues. J. Nutr. 112:1011-1018. Jones, S.M.A., and S. J. Yeaman, 1986. Oxidative decarboxylation of 4-methylthio-2-oxobutyrate by branched-chain 2-oxo acid dehydrogenase complex. Biochem. J. 237:621-623. Langer, B. W., 1965. The biochemical conversion of 2-hydroxy-4-methylthiobutyric acid into methionine by the rat in vitro. Biochem. J. 95:683-687. Lawson, C. Q., and F. J. Ivey, 1986. Hydrolysis of 2-hydroxy-4-(methylthio)butanoic acid diner in two model systems. Poultry Sci. 65:1749-1752. Livesey, G., and P. Lund, 1980. Methionine metabo-
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sors in a naturally occurring methionine salvage pathway (Dibner et al., 1990). It is not known whether the biochemical synthesis of HMB results in one or both isomers and therefore no isomeric configuration is indicated for liver HMB. The levels of methionine shown in Table 2 are slightly higher than values reported by other investigators (Finkelstein et ah, 1982; Boebel and Baker, 1983). Several observations suggest that the relative excess of enzyme capacity to conversion requirement increases with the age of the broiler. First, required supplemental methionine levels decrease with age in a corn and soybean meal commercial diet. Second, the liver continues to grow with continued body growth and therefore total hepatic enzyme capacity would increase. Third, hepatic L-HAOX (Scott et ah, 1969; Dibner and Knight, 1985) and D-HADH (Dibner and Knight, 1985) activities generally increase as a function of age. Although there is a decrease in Damino acid oxidase activity over the first 3 to 4 wk of life (DeMoraes, 1980; Dibner and Knight, 1985), specific activity remains stable after that period (DeMoraes, 1980). Thus, the enzyme activities observed at 3 w k of age may be considered a relative nunimum in relation to supplemental methionine requirements. Finally, the additional enzyme present in renal peroxisomes and that contributed by the mitochondria of virtually every organ indicates that supplemental methionine levels would not exceed conversion capacity even at the high levels required in diets containing a minimal amount of intact protein. In summary, data presented in this report indicate that conversion rates for DL-HMB and DL-Met were comparable under conditions of substrate saturation. Further, the data indicate that birds fed DL-Met, DL-HMB, and L-Met contain similar amounts of both methionine and HMB in the liver, with no accumulation of excess HMB in birds fed DL-HMB. Finally, calculations of liver enzyme capacity in chicks 3 w k of age indicate that there is a sevenfold excess of enzyme capacity in the liver alone, assuring that growing broilers have more than enough biochemical capability to convert practical supplemental levels of DL-HMB or DL-Met to L-Met.
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lism via the transamination pathway in rat liver. Biochem. Soc. Trans. 8:540-541. Masters, C , and R. Holmes, 1977. Peroxisomes: new aspects of cell physiology and biochemistry. Physiol. Rev. 57:816-882. Miyazake, J. H., and S. F. Yang, 1987. Metabolism of 5-methylthioribose to methionine. Plant Physiol. 84:277-281. Ohnishi, S. T., and J. K. Barr, 1978. A simplified method of quantitating proteins using the biuret and phenol reagents. Anal. Biochem. 86:193-197. Robinson, J. C, L. Keay, R. Molinare, and I. W. Sizer,
1962. Lr2-Hydroxy acid oxidases of hog renal cortex. J. Biol. Chem. 2372001-2010. SAS Institute, 1985. SAS® Users Guide: Statistics. Version 5 Edition. SAS Institute Inc., Cary, NC. Scott, P. J., L. P. Visenttn, and J. M. Allen, 1969. The enzymatic characteristics of peroxisomes of amphibian and avian liver and kidney. Ann. N.Y. Acad. Sci. 168:244-264. Trackman, P. C, and R. H. Abeles, 1981. The metabolism of l-phospho-5-methyl-thioribose. Biochem. Biophys. Res. Commun. 103: 1238-1244.
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1992 Poultry Science 71:700-708
INTRODUCTION
DL-HMB), and DL-methionine (DL-Met) are used as sources of L-methionine (LThe corn and soybean meal diets com- Met). The only form of methionine that monly used as broiler or layer rations can be used in intermediary metabolism or require the presence of supplemental me- incorporated into protein is L-Met; supplethionine activity for optimum bird per- mental sources must be converted before formance. To correct this deficiency, they can be used by the bird. Alimet®2 [an 88% aqueous solution of DLBiochemical conversion of DL-HMB to 2-hydroxy-4-(methylthio)butanoic acid, L-Met takes place in two steps (Dibner DL-HMB], MHA® (an 86% calcium salt of and Knight, 1984). The first reaction is the oxidation of the a carbon, yielding the intermediate 2-oxo-4-(methylthio)butanoic acid (keto-methionine). The molecular T To whom correspondence should be addressed. structures of both DL-HMB and DL-Met Novus International, Mail Zone BB21,700 Chesterfield contain one asymmetric carbon, resulting Parkway North, Chesterfield, MO 63198. 2 Alimet® is a registered trademark of Novus Inter- in the presence of equal amounts of the D and L stereoisomers in the commercially national, Inc.
700
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ABSTRACT The objective of the present experiments was to determine whether the levels of supplemental methionine sources currently used in practical diets exceed the capacity of the chick to convert the supplement to Lmethionine. Supplemental sources examined included DL-2-hydroxy-4(methylthio)butanoic acid (DL-HMB, Ahmet®, or MHA®) and DL-methionine (DL-Met). Two approaches were taken: first, the amount of enzyme activity available for conversion of the two supplemental methionine sources was determined using optimum reaction conditions for each and chick liver homogenate as the enzyme source. These experiments showed that total liver enzyme conversion activity was 564 umol/h for DL-HMB and 529 umol/h for DL-Met. The total activities for the two sources were not different when measured at saturating substrate concentration. Second, to address the question of whether the enzyme is limiting for either source under practical feeding conditions, birds were fed starter diets supplemented with DL-HMB or DL-Met at .25% of the diet for 3 wk. When hepatic levels of free HMB and methionine were determined, birds fed DL-HMB contained 7.6 nmol HMB/g of liver and 84.7 nmol methionine/g of liver. Birds fed DL-Met had levels of 7.6 nmol HMB/ g liver and 80.3 nmol methionine/g liver. These results indicate no accumulation of HMB or DL-Met, which might occur if conversion capacity were saturated. By calculation, a bird consuming 100 g/day of a diet supplemented with .25% activity would need to convert about 70 umol/h, indicating a sevenfold excess of enzyme in the liver alone. {Key words: DL-2-hydroxy-4-(methylthio)butanoic acid, methionine conversion, poultry, liver, Alimet®)
SUPPLEMENTAL METHIONINE CONVERSION
Although the biochemical pathway for the conversion of DL-HMB and DL-Met to keto-methionine has been described, little information exists relating enzyme capacity to demand for conversion of dietary supplemental sources by the broiler chick. A recent publication reported the use of a decarboxylation assay to determine the rate and total tissue capacity of the chick to produce keto-methionine from DLHMB and DL-Met (Dupuis et ah, 1989). The reaction conditions used, however, did not include a cofactor reported to be essential for one of the conversion enzymes (Dibner and Knight, 1984). In the studies reported here, the effect of this cofactor was examined using a spectrophotometric assay that permits examination of D-HADH activity alone. The decarboxylation assay was also compared with the spectrophotometric assay in terms of total activity obtained. Finally, the total liver enzyme capacity of 3- to 4-wk-old broiler chicks in relation to conversion requirements was studied.
^airview Farms, Remington, IN 47977. Ziggity Systems, Middlebury, IN 46540.
4
MATERIALS AND METHODS Animals Hubbard x Hubbard cockerel chicks 3 were fed a corn and soybean meal diet supplemented with L-Met, DL-Met, or DLHMB (Table 1) from 1 day of age through 4 wk. Birds were housed six per cage. Cages 51 cm wide x 69 cm long X 36 cm high were made of polyvinylchloride-coated wire mesh. Mesh size was 2.5 x 2.5 cm for sides and top and 1.25 cm for the floor. Feed was supplied in a galvanized trough feeder and water was supplied in sanitary plastic and stainless water nipples. 4 Thermoneutral temperatures were maintained starting at 33 C. After 3 days temperatures were decreased with a linear function to 24 C at 21 days and held constant thereafter. Complete exchange of room air with fresh air was provided 15 times/h. Fluorescent light with an intensity of 45 be was provided for 23 h / d a y . Chicks were euthanatized using carbon dioxide inhalation.
Experimental Design In the experiments illustrated in Figures 1 to 4, a randomized complete block design was used. In each experiment (block), a separate shipment of chicks was used. 3 After 3 w k on the diet described in Table 1, three randomly chosen chicks were used for each experiment and liver homogenate from them was pooled to generate the enzyme fraction. Duplicate enzyme reactions were run for each data point and one spectrophotometric assay or radiolabel count was performed for each duplicate. Data points in Figures 1 and 2 are the average of two values, one each from two replicate enzyme experiments (n = 2). Data points in Figures 3 and 4 are the average of three values, one each from three replicate enzyme experiments (n = 3). All references to treatment averages represent main effects of the experiment. N o significant treatment by experiment interactions were observed (P>.10). To determine the concentration of HMB and methionine in the livers of animals fed different supplemental methionine sources, three pens of six day-old birds were placed on the corn and soybean meal diet
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available product. Different enzymes catalyze the oxidation of the two stereoisomers of DL-HMB. Therefore, conversion of the two stereoisomers occurs simultaneously to produce the ketomethionine intermediate (Dibner and Knight, 1984). The enzyme specific for LHMB is L-2-hydroxy acid oxidase (LHAOX, EC 1.1.3.15) and has been studied in chick liver (Dibner and Knight, 1984), hog kidney (Robinson et ah, 1962), and rat l i v e r ( L a n g e r , 1965). The D-HMB stereoisomer requires the enzyme D2-hydroxy acid dehydrogenase (D-HADH, EC 1.1.99.6), which has been studied in the chick, where it occurs in all tissues tested, including liver, kidney, skeletal muscle, intestine, pancreas, spleen, and brain (Dibner and Knight, 1984). The conversion of D-Met to keto-methionine also requires a specific enzyme catalyst, D-amino acid oxidase (D-AAOX, EC 1.4.3.3). This enzyme, like L-HAOX, occurs primarily in the liver and kidney and is found in peroxisomes (Scott et al., 1969; Masters and Holmes, 1977).
701
702
DIBNER AND IVEY TABLE 1. Diet composition
0
2
4
6
8
10
D-HMB Concentration (mmol/L) FIGURE 1. The effect of the cofactor phenazine methosulfate (PM, 5 mmol/L) on the synthesis of keto-methionine (keto) from D-2-hydroxy-4(methylthio)butanoic acid (D-HMB, A). There is little activity of D-hydroxy acid dehydrogenase over the 60-min reaction period in the absence of this cofactor (•). Each point represents the x ± SEM (n = 2) of two sets of duplicate reactions. Liver homogenate from three birds was pooled for each experiment. Results from two experiments representing a total of six animals were combined. In each experiment, one spectrophotometric analysis was performed on duplicate enzyme reactions at each dose.
each diet were used to generate individual liver homogenates (n = 3). Two chromatographic analyses were performed per sample. Enzyme Preparation
described in Table 1 except supplemented with either DL-HMB, DL-Met, or L-Met at 2.5 g/kg diet. The DL-HMB was added to the diet as Alimet® (an 88% aqueous solution of DL-HMB5 in H 2 0). Alimet® is typically 65% monomelic DL-HMB, 23% polymeric DL-HMB, and 12% H2O (Lawson and Ivey, 1984). The DL-Met6 was added at 99.5% purity. The data in Table 2 represent one such study. Birds were fed the diets for 3 wk and three randomly chosen birds from
Monsanto, St. Louis, MO 63198. ^dne-Poulenc, Atlanta, GA 30350. 7 Mallinckrodt, St. Louis, MO 63147. ^igma Chemical Co., St. Louis, MO 63178-9916. Brinkmann Instruments, Westbury, NY 11590.
Liver tissue was homogenized into four volumes of ice-cold, 20 mmol/L potassium phosphate buffer7 (pH 7.5) containing .25 mol/L sucrose8 and .1 mmol/L phenylmethylsulfonyl fluoride8 using a Polytron tissue homogenizer.9 Liver weights were approximately 25 g per bird. Homogenates were stored at -20 C and were diluted 1:1 with potassium phosphate buffer for use in reaction mixtures. Enzymatic activity was stable for at least 30 days when homogenates were stored under these conditions. Protein levels in the homogenate were 270 to 300 mg/mL and were determined using a modified biuret protein assay (Ohnishi and Barr, 1978) with Folin and Ciocalteu's Phenol Reagent and using bovine serum albumin as the standard.8
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Ingredients and composition Percentage 54.90 Corn, yellow 35.20 Soybean meal 5.60 Soybean oil Calcium carbonate .60 2.80 Dicalcium phosphate .10 Vitamin premix1 .05 Mineral premix2 .08 Choline chloride 35 Supplemental methionine .05 L-lysine .37 Sodium chloride Nutrient composition Protein 20.80 Calcium .96 .86 Total phosphorus 1.43 Arginine .59 Methionine Methionine + cysteine .94 1.20 Lysine 3,174.5 ME, kcal/kg Vitamin premix provided per kilogram of diet: thiamin HQ» 1.1 mg; riboflavin, 8.8 mg; pantothenate, 13.2 mg; niacin, 38.5 mg; pyridoxine HO, 3.3 mg; folacin, .99 mg; biotin, .055 mg; cyanocobalamin, .013 mg; menadione dimethyl pyrimidinol, 2.64 mg; ethoxyquin, 125.0 mg; retinyl, 8,800 IU; cholecalciferol, 3,300 ICU; and DL-a-tocopherol acetate, 13.2 IU. Trace mineral premix provided in milligrams per kilogram of diet: MnS04-H20,64; zinc sulfate-HjO, 70; ferric citrate (ca) 5H2O, 50; copper sulfate-SHjO, 8; sodium selenite, .3; and iodine, .8. depending on the experiment, supplemental methionine was supplied as one of the following: Lmethionine, DL-methionine, or DL-2-hydroxy-4(methylthio)butanoic acid.
NoPM +PM
703
SUPPLEMENTAL METHIONINE CONVERSION No Cofaclor Phenazine Methosulfate 3.5 3.0 c
n
2.5
a,
§ ?n C
r. 1 1
ri Q
.0
U
X, t
L-Met
1
I-
\
,,
I
D-Mcl
1 ,
i
L-HMB
J
I
i
D-HMB
().() 11
1
1
1
1
1
IT
10
20
30
40
50
W
DL-HMB Concentration (mmol/L)
Reaction
Conditions
Reactions c o n t a i n e d .1 mL liver homogenate, .5 to 50 umol substrate, and potassium phosphate buffer (20 mmol/L) in a total volume of 1.0 mL. Substrates were either unlabeled DL-HMB 5 (sodium salt), unlabeled DL-Met, 8 [1-14C]DL-Met,10 or [114 C]DL-HMB 5 (sodium salt). The radiolabeled DL-HMB was entirely monomer, which results from the formation of the sodium salt. In some experiments phenazine methosulfate 8 (.5 mmol/L) was added. Dose-response curves with this cofactor showed dose-related increases in enzyme activity in the presence of .1 to .25 mmol/L, with a plateau from .5 to 1.0 mmol/L (data not shown).
lu
Amersham, Arlington Heights, IL 60005. Ultrospec Model 4050, Pharmacia LKB Biotechnology, Piscataway, NJ 08854. n
FIGURE 3. Activity toward the racemic mixture DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMB) in the presence (A) and absence (•) of phenazine methosulfate (.5 mmol/L). At all concentrations tested, the amount of keto-methionine (keto) produced from DL-HMB was approximately doubled in the presence of this cofactor. Each point represents the x+SEM (n = 3) of three sets of duplicate reactions. Liver homogenate from three randomly chosen birds was pooled for each experiment. Results from three experiments were combined. One spectrophotometric analysis was performed on duplicate enzyme reactions at each dose.
Reactions were incubated at 37 C on a rotating table (100 rpm) for 60 min. Previous experiments showed that the reactions were linear for over 3 h (Dibner and Knight, 1984). In experiments using unlabeled substrate, the reaction product, ketomethionine was detected using a dinitrophenylhydrazine 8 spectrophotometric 11 assay previously described (Dibner and Knight, 1984). For these experiments, reactions containing all components except substrate were subtracted as blank absorption values. The keto-methionine standard curve was generated by the addition of known concentrations of keto-methionine 8 into the complete reaction mixture, minus the substrate, and incubating these under reaction conditions for 60 min followed by the dinitrophenylhydrazine assay described above. In experiments using radiolabeled substrate, reactions were stopped with the addition of .5 mL .66 mol/L sulphosalicylic acid. 8 Radiolabeled keto-methionine was measured through the evolution of 14CC>2 initiated by adding .5 mL 30% hydrogen
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FIGURE 2. Synthesis of keto-methionine (keto) from s e p a r a t e i s o m e r s of D L - 2 - h y d r o x y - 4 (methylthio)butanoic acid (D-HMB and L-HMB) and DL-methionine (DL-Met), each tested at 10 mmol/L. In the presence of phenazine methosulfate (.5 mmol/L), D-HMB was converted to keto in amounts approximately equal to that of L-HMB. Half of commercially available DL-Met is in the D stereoisomeric configuration (D-Met) which must be converted to L-methionine (L-Met) before it can be used by the bird. Some keto is synthesized from L-Met by oxidation or transaminative degradation (Benevenga, 1983). Each bar represents the x ± SEM (n = 2) of two sets of duplicate reactions. Liver homogenate from three birds was pooled for each experiment. Results from two experiments were combined. One spectrophotometric analysis was performed on duplicate enzyme reactions for each substrate.
704
DIBNER AND IVEY TABLE 2. Content (x ± SEM) of methionine and DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMB) in the liver of chicks fed diets containing 25% DL-HMB, DL-methionine, or L-methionine1
DL-Met DL-HMB DL-HMB+PM
Dietary source DL-HMB DL-Methionine ^Methionine
0
5
10
15
20
Substrate Concentration (mmol/L)
Methionine (nmol/g) 7.64 ± 1.23 84.72 ± 6.92 7.63 ± 1.22 8028 ± 5.14 8.12 ± 1.93 95.51 ± 2.02
^ i r d s were fed diets supplemented with 35% DLHMB, DL-methionine, or L-methionine for 3 wk. Means ± SE (n = 3) were obtained from the General Linear Models procedure of base SAS® software (SAS Institute, 1985).
14
peroxide.8 The reactions were incubated for an additional 90 min and the 14CC>2 was trapped using .3 mL phenylethylamine8 in a well suspended over the reaction mixture. Longer incubation times (up to 4 h) did not yield additional radioactivity (data not shown). The entire contents of the well were scintillation-counted12 using Instagel12 as the scintillant. Blank values were generated by adding all of the reaction components, including the radiolabeled substrate at either 5, 10, or 20 mmol/L, to a vial containing enzyme denatured in a boiling water bath for 10 min. The amount of radiolabel from the denatured reactions was subtracted from the total radiolabel
12 ' Model 4640, Packard Tri-Carb, Downers Grove, IL 60515. 13 Sorvall Model RC3B, Sorvall Centrifuges, Dupont, Wilmington, DE 19898. 4 " B&J Solid Phase System, Baxter Healthcare Co., Muskegon, MI 49442. 15 Acrodisc, Gehnan Sciences, Ann Arbor, MI 48106.
evolution at the same substrate concentration. Liver DL-2-Hydroxy-4-(Methylthio)Butanoic Acid and DL-Methionine Levels Liver tissue (5 g) was homogenized into 5 mL deionized H2O. A 30-mL volume of 2:1 methanol:chloroform was added and extraction phases were separated with 20 mL each of chloroform and deionized H2O. Following lyophilization, the dried residue from the aqueous phase was resuspended in 5 mL deionized H2O. A 250-uL sample was extracted using an equal volume of a mixture of chloroform:methanol:water (1:2: .8). Phase separation was achieved by changing the solvent ratios to 2:2:1.8 through the addition of more chloroform and water. Following phase separation, the mixture was centrifuged13 for 5 min at 750 x g. The aqueous phase was collected and passed through an aminopropyl 100-mg column.14 The organic phase was carefully washed with water (1 mL) and the washing passed through the column. Finally, the column was washed with another 1.5 mL of water to ensure elution of the metabolites. This mixture was dried under a stream of nitrogen, lyophilized, reconstituted in 500 (J.L of water, and filtered using a .2 um filter.15 Spiked samples showed >90% recovery of DL-HMB and DL-Met following these steps. Metabolites were quantified using a modification of an high-performance liquid
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FIGURE 4. Keto-methionine (keto) synthesis from C-DL-2-hydioxy-4-(methylthio)butanoic add (DLHMB) as estimated with the CO2 evolution assay in the presence (•) and absence (A) of phenazine methosulfate (PM, .5 mmol/L). The cofactor PM increased 14 C02 evolution from DL-HMB about twofold, as would be predicted by the data shown in Figure 3. Total keto synthesis from DL-HMB (•) was comparable to that with 14C-DL-methionine (DL-Met). Each point represents the x±SEM (n = 3) of three sets of duplicate reactions. Liver homogenate from three randomly chosen birds was pooled for each experiment. Results from three experiments were combined. In each experiment, one radiolabel scintillation analysis was performed per reaction.
HMB
SUPPLEMENTAL METHIONINE CONVERSION
c h r o m a t o g r a p h y assay previously described (Dibner, 1983). The Waters chromatograph16 was equipped with an NH2 column 1 'with phosphoric acid6 (.01 mol/L, pH 3.2) and acetonitrile18 (Optima Grade) as the elution buffer. Retention times for DL-HMB and methionine were determined by the addition of standards into water and were confirmed by co-chromatography of standards added into experimental samples. Using this method, the detection limit for DL-HMB is .5 jig/mL and for methionine is 5 ug/mL. Statistical Methods
RESULTS Figure 1 shows the results of experiments designed to evaluate the requirement of D-HADH for the cofactor phenazine methosulfate. This cofactor was found to be the most effective of those tested in earlier studies in which synthesis of ketomethionine was detected using a dinitrophenylhydrazine assay (Dibner and Knight, 1984). In order to evaluate the cofactor requirement, D-HMB was tested as the substrate using chick liver homogenate as the enzyme source. As is clear from Figure 1, the crude enzyme mixture has a very limited capacity for keto-methionine synthesis from D-HMB in the absence of this cofactor. When the D and L stereoisomers of DLHMB and DL-Met were tested individually at 10 mmol/L, each isomer was observed to yield some keto-methionine (Figure 2). The two isomers of DL-HMB were similar to one another and when added together were similar in magnitude to activity toward D-Met. There was ketomethionine synthesis from L-Met, which represents the product of oxidation (Lamino acid oxidase, EC 1.4.3.2) or trans-
16
Waters Associates, Milford, MA 01757. Alltech, Deerfield, IL 60015. Fisher Scientific, St. Louis, MO 63132.
17
18
amination (EC 2.6.1) of L-Met to ketomethionine (Livesey and Lund, 1980). The racemic mixture of DL-HMB was also tested for keto-methionine synthesis in the presence and absence of phenazine methosulfate. Figure 3 shows the results of triplicate dose response experiments. The reaction mixture containing no phenazine methosulfate gave activities less than those containing the cofactor at every dose tested. When the identical reaction conditions were tested using the radiolabeled CO2 evolution assay, results were obtained that are illustrated in Figure 4. Clearly, the addition of phenazine methosulfate resulted in a significant increase in radiolabeled CO2 evolution from DL-HMB at each concentration tested. The CO2 evolution from DL-HMB in the presence of phenazine methosulfate was not different from that seen using DL-Met as the substrate. A comparison of the data in Figures 3 and 4 shows that the two assay methods give comparable values for keto-methionine synthesis by chick liver homogenates. In order to evaluate the possibility that enzyme capacity could be limiting under normal dietary situations, livers from birds fed .25% supplemental DL-HMB, DL-Met, or L-Met were compared in terms of methionine and HMB content. When livers from birds fed the three methionine sources were analyzed (Table 2), there were no significant differences in methionine or HMB content. DISCUSSION Phenazine methosulfate has been used as an artificial electron acceptor in biochemical studies of purified D-HADH (Dhydroxy acid dehydrogenase) from a number of animals (Cammack, 1969); however, it is not a naturally occurring molecule. The identity of the physiological cofactor in this reaction has not been determined, although use of menadione (Dibner and Knight, 1984) or ubiquinone (Cammack, 1969) as acceptors yields about 75% of the activity seen with artificial electron acceptors. Naturally occurring acceptors that have been tested in chick liver include flavin-adenine dinucleotide, flavin mononucleotide, nicotinamide-
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Means and standard errors were obtained from the General Linear Models procedure of base SAS® software (SAS Institute, 1985). Details of the experimental design were given previously.
705
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DIBNER AND IVEY
Figures 3 and 4 illustrate the increase in the conversion of the racemic mixture, DLHMB, by the addition of phenazine methosulfate. When no cofactor is added,
activity is not reduced to zero because the oxidative conversion of L-HMB does not require it (Figure 3). The cofactor requirement is obvious only when D-HMB is tested in the absence of L-HMB (Figure 1). When the racemic mixture is used, oxidase activity toward the L-HMB stereoisomer obscures the lack of activity toward the DHMB stereoisomer. These results are compatible with the hypothesis that activity toward the racemic mixture in the absence of phenazine methosulfate is due to the action of L-HAOX only; i.e., activity toward the L-stereoisomer only. Using optimum reaction conditions and saturating substrate concentrations, calculations based on the experiments shown in Figure 4 indicate that total liver enzyme conversion activity was essentially equal for the two sources, i.e., 564 |xmol/h for DL-HMB and 529 p m o l / h for DL-Met at a substrate concentration of 20 mmol/L. A related question is whether enzyme ever can be limiting at even high commercial supplementation levels. Calculations based on the data shown in Figure 4 indicate that the liver of a 3-wk-old broiler could convert about 500 u m o l / h of either DL-HMB or DL-Met if substrate were present at saturating concentrations. If a 3-wk-old bird were consuming a diet supplemented with .25% of DL-HMB or DL-Met, the amount of methionine activity that would have to be converted in the liver (based on 100 g of feed intake/day) would be about 1,600 umol/day, or about 70 umol/h. This calculation suggests that the liver alone contains a sevenfold excess of enzyme capacity. The suggestion is further supported by the data shown in Table 2, where no accumulation of HMB was observed in the livers of birds supplemented with DL-HMB in comparison with those fed DL-Met or L-Met. Such an accumulation might occur if enzyme capacity were exceeded. The presence of HMB in chick liver (Dibner et ah, 1990), rat liver (Trackman and Abeles, 1981), and in plants (Miyazake and Yang, 1987), has been reported, and the biochemical pathway for natural HMB identified (Dibner and Ivey, 1990; Dibner et a/., 1990) in the chick. Recent evidence suggests that both HMB and keto-methionine are methionine precur-
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adenine dinucleotide, menadione, coenzyme Q, and oxygen (Dibner and Knight, 1984). The activity obtained with these cofactors was higher than that observed with no cofactor, but was below the activity seen with phenazine methosulfate (Dibner and Knight, 1984). Chick feeding studies and cellular protein synthesis studies support the observation reported here that enzyme activity toward D-HMB is approximately equal that toward L-HMB. First, Baker and Boebel (1980) reported that D-HMB gave chick performance superior to L-HMB when fed in crystalline amino acid diets. In addition, when the separate isomers of DL-HMB were tested for activity in m e t h i o n i n e - d e f i c i e n t p r i m a r y chick hepatocyte cultures, L-HMB and D-HMB supported equal incorporation of radiolabeled leucine into protein across a wide range of concentrations (Lawson and Ivey, 1984). Figure 2 shows the synthesis of ketom e t h i o n i n e from each i n d i v i d u a l stereoisomer of HMB and methionine. Separation of the isomers yielded some interesting information about these reactions. From the data in Figure 2, it is clear that the two isomers of HMB were converted to keto-methionine at similar rates under these saturating conditions. Activity toward the two stereoisomers of methionine was very dissimilar. Activity toward the D-Met isomer was much greater than that toward the L-Met isomer, as might be expected. Activity toward the L-Met isomer could be the result of the action of L-arnino acid oxidase or of a transaminase. The synthesis of ketomethionine from L-Met by transamination is the first step in its degradation (Livesey and Lund, 1980; Benevenga, 1983; Jones and Yeaman, 1986) and thus would reduce rather than increase the amount of L-Met available for protein synthesis or intermediary metabolism. Thus the total radiolabeled carbon dioxide evolution from DL-Met shown in Figure 4 could include some keto-methionine representing L-Met catabolism rather than its synthesis.
SUPPLEMENTAL METHIONINE CONVERSION
ACKNOWLEDGMENTS The authors gratefully acknowledge technical assistance by D. C. Williams, chromatography by I. R. Putnam, and animal care by M. E. Wehmeyer. REFERENCES Baker, D. H., and K. P. Boebel, 1980. Utilization of the D- and L-isomers of methionine and methionine hydroxy analogue as determined by the chick bioassay. J. Nutr. 110559-964. Boebel, K. P., and D. H. Baker, 1983. Blood and liver concentrations of sulfur-containing amino acids in chicks fed deficient, adequate, or excess levels of dietary cysteine. Proc. Soc. Exp. Med. 172: 498-501. Benevenga, N. J., 1983. Evidence for alternative pathways of methionine metabolism. Adv. Nutr. Res. 6:1-18. Cammack, R., 1969. Assay, purification and properties of mammalian D-2-hydroxy acid dehydrogenase. Biochem. J. 115:55-64. DeMoraes, G.H.K., 1980. Studies on Non-Specific Nitrogen and D-Amino Acid Metabolism in the Chick, Ph.D. dissertation. Purdue University, Lafayette, IN. Dibner, J. J., 1983. Utilization of supplemental methionine sources by primary cultures of chick hepatocytes. J. Nutr. 1132116-2123. Dibner, J. J., R. C. Durley, J. G. Kostelc, and F. J. Ivey, 1990. 2-Hydroxy-4-(methylthio)butanoic acid is a naturally occurring methionine precursor in the chick. J. Nutr. 120553-560. Dibner, J. J., and F. J. Ivey, 1990. Hepatic protein and amino acid metabolism in poultry. Poultry Sci. 69:1188-1194. Dibner, J. J., and C. D. Knight, 1984. Conversion of 2-hydroxy-4-(methylthio)butanoic acid to Lmethionine in the chick: A stereospecific pathway. J. Nutr. 114:1716-1723. Dibner, J. J., and C. D. Knight, 1985. Age-dependent changes in the metabolism of DL-2-hydroxy4-(methylthio)butanoic acid (HMB) and DLMethionine. Poultry Sci. 64(Suppl. l):14.(Abstr.) Dupuis, L., C. L. Saunderson, A. Puigserver, and P. Brachet, 1989. Oxidation of methionine and 2 - h y d r o x y - 4 - m e t h y l t h i o b u t a n o i c acid stereoisomers in chicken tissues. Br. J. Nutr. 62: 63-75. Finkelstein, J. P., W. E. Kyle, B. J. Harris, and J. J. Martin, 1982. Methionine metabolism in mammals: concentration of metabolites in rat tissues. J. Nutr. 112:1011-1018. Jones, S.M.A., and S. J. Yeaman, 1986. Oxidative decarboxylation of 4-methylthio-2-oxobutyrate by branched-chain 2-oxo acid dehydrogenase complex. Biochem. J. 237:621-623. Langer, B. W., 1965. The biochemical conversion of 2-hydroxy-4-methylthiobutyric acid into methionine by the rat in vitro. Biochem. J. 95:683-687. Lawson, C. Q., and F. J. Ivey, 1986. Hydrolysis of 2-hydroxy-4-(methylthio)butanoic acid diner in two model systems. Poultry Sci. 65:1749-1752. Livesey, G., and P. Lund, 1980. Methionine metabo-
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sors in a naturally occurring methionine salvage pathway (Dibner et al., 1990). It is not known whether the biochemical synthesis of HMB results in one or both isomers and therefore no isomeric configuration is indicated for liver HMB. The levels of methionine shown in Table 2 are slightly higher than values reported by other investigators (Finkelstein et ah, 1982; Boebel and Baker, 1983). Several observations suggest that the relative excess of enzyme capacity to conversion requirement increases with the age of the broiler. First, required supplemental methionine levels decrease with age in a corn and soybean meal commercial diet. Second, the liver continues to grow with continued body growth and therefore total hepatic enzyme capacity would increase. Third, hepatic L-HAOX (Scott et ah, 1969; Dibner and Knight, 1985) and D-HADH (Dibner and Knight, 1985) activities generally increase as a function of age. Although there is a decrease in Damino acid oxidase activity over the first 3 to 4 wk of life (DeMoraes, 1980; Dibner and Knight, 1985), specific activity remains stable after that period (DeMoraes, 1980). Thus, the enzyme activities observed at 3 w k of age may be considered a relative nunimum in relation to supplemental methionine requirements. Finally, the additional enzyme present in renal peroxisomes and that contributed by the mitochondria of virtually every organ indicates that supplemental methionine levels would not exceed conversion capacity even at the high levels required in diets containing a minimal amount of intact protein. In summary, data presented in this report indicate that conversion rates for DL-HMB and DL-Met were comparable under conditions of substrate saturation. Further, the data indicate that birds fed DL-Met, DL-HMB, and L-Met contain similar amounts of both methionine and HMB in the liver, with no accumulation of excess HMB in birds fed DL-HMB. Finally, calculations of liver enzyme capacity in chicks 3 w k of age indicate that there is a sevenfold excess of enzyme capacity in the liver alone, assuring that growing broilers have more than enough biochemical capability to convert practical supplemental levels of DL-HMB or DL-Met to L-Met.
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lism via the transamination pathway in rat liver. Biochem. Soc. Trans. 8:540-541. Masters, C , and R. Holmes, 1977. Peroxisomes: new aspects of cell physiology and biochemistry. Physiol. Rev. 57:816-882. Miyazake, J. H., and S. F. Yang, 1987. Metabolism of 5-methylthioribose to methionine. Plant Physiol. 84:277-281. Ohnishi, S. T., and J. K. Barr, 1978. A simplified method of quantitating proteins using the biuret and phenol reagents. Anal. Biochem. 86:193-197. Robinson, J. C, L. Keay, R. Molinare, and I. W. Sizer,
1962. Lr2-Hydroxy acid oxidases of hog renal cortex. J. Biol. Chem. 2372001-2010. SAS Institute, 1985. SAS® Users Guide: Statistics. Version 5 Edition. SAS Institute Inc., Cary, NC. Scott, P. J., L. P. Visenttn, and J. M. Allen, 1969. The enzymatic characteristics of peroxisomes of amphibian and avian liver and kidney. Ann. N.Y. Acad. Sci. 168:244-264. Trackman, P. C, and R. H. Abeles, 1981. The metabolism of l-phospho-5-methyl-thioribose. Biochem. Biophys. Res. Commun. 103: 1238-1244.
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