M e t a b o l i s m o f A r g i n i n e a n d O r n i t h i n e in t h e C o w a n d Rabbit Mammary
Tissue 1 J. H. CLARK,
ABSTRACT
R. G. DERRIG, C. L. DAVIS, and H. R. SPIRES Department of Dairy Science University of Illinois Urbana 61801
specific uptake mechanism. However, recent studies by Derrig et al. (4) and Bickerstaffe et al. (2) indicate that the bovine mammary gland extracts arginine in excess of the amount secreted in milk protein. Therefore this study was to determine uptake by m a m m a r y gland of arginine and ornithine and to determine the metabolism of these amino acids in lactating mammary tissues of the cow and rabbit.
Amino acid uptake by the bovine mammary gland was determined b y arteriovenous difference. Extraction of arginine from the plasma by the lactating bovine mammary gland was in excess of requirements for milk protein synthesis. Ornithine and citrulline also were extracted by the gland but are not in milk protein. Incubations of slices of lactating mammary tissue from cows and rabbits indicate that nonessential amino acids, especially proline and glutamate, are the major end products of arginine and ornithine metabolism in the lactating mammary gland.
MATERIALS AND METHODS Arteriovenous Differences
INTRODUCTION
Measurements of arteriovenous differences to determine metabolite uptake by the lactating mammary gland indicate an adequate uptake of essential amino acids for milk protein synthesis but a deficient uptake of several nonessential amino acids (2, 4, 12, 14, 17). In these studies, amino acids classified as essential for the mammary gland were assumed to be those established as essential for growth of rats. Halfpenny et al. (9) concluded that synthesis of milk protein may be limited by the supply in plasma of nonessential amino acids, especially glutamate and proline. Uptake of ornithine, an amino acid not in milk protein, by the mammary gland was significant in the cow (17), goat (14), and sow (12). Uptake of arginine has been in excess of output in milk protein for the mammary gland of the goat (14) and sow (12) but not for the cow (14, 17). These differences were attributed to a species difference in the
Received September 30, 1974. 1Supported in part by the Illinois Agricultural Experiment Station and the National Soybean Processors Association.
Blood samples were drawn from the internal iliac artery and subcutaneous abdominal vein of five lactating Holstein cows averaging 33 kg of milk per day. The cows were bled 4 to 5 h post-feeding and post-milking at two sampling times 14 days apart. Plasma samples were prepared for amino acid analysis as described by Derrig et al. (4). Free amino acids of plasma were determined by Phoenix Amino Acid Analyzer, Model 8000KC. Calculations for determining uptake of amino acids by the gland and output in milk protein were as described b y Derrig et al. (4). Tissues
Mammary tissue was obtained at slaughter from four cows, approximately 6 m o lactating, producing 14 to 15 kg of milk per day and from two lactating New Zealand white rabbits, 12 to 20 days lactating. Mammary tissue was obtained approximately 15 to 20 rain postslaughter. The tissue for incubation studies was placed in iso-osmotic Tris-sucrose buffer (pH 7.3; 30 mM-tris, .3 M-sucrose, 1 mM-glutathione, 1 mM-EDTA) at 4 C prior to slicing and incubation (approximately 10 rain). Mammary and liver tissues for enzyme studies were maintained in ice-cold distilled water as recommended by Schimke (16) prior to the assays (approximately 30 min).
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ARGININE AND ORNITHINE METABOLISM I ncubation Studies
Incubations of tissue slices were according to Bauman et al. (1). Tissue slices weighing 120 to 180 mg (wet weight) were prepared with a Stadie-Riggs hand microtome and were incubated in Erlenmeyer flasks containing 3.0 ml of Krebs-Ringer bicarbonate buffer (pH 7.3). Glucose was dissolved in the media to a final concentration of 20 mM. Each flask contained .3 unit of insulin and either .5 or 1.0 /aCi of radioactive substrate. Concentrations of unlabeled ornithine, proline, and arginine were 6 mM when added. All incubations were for 1 h in a shaking water bath at 37 C under an atmosphere of O2 + CO2 (95:5). At the end of each incubation, .1 ml of 25% KOH (wt/vol) was injected through the rubber serum cap of each flask onto a 2 x 2 cm piece of filter paper suspended in a plastic bucket. Tissue metabolism was terminated by injecting .25 ml of .5 N H2SO4 into the media. Flasks were incubated for an additional 30 min to trap the CO2. The filter paper containing trapped CO2 was removed and air-dried for 12 h. Radioactivity on the paper was determined by liquid scintillation counting (5 g 2,5-diphenyloxazole/liter toluene). Counting efficiencies of all scintillation cocktails were determined by external standard ratios. Isolation of Metabolites
The incubation media containing the tissue slice was homogenized in a Sorvall Omni-Mixer equipped with a micro attachment (two bursts of 30 s each), sonified with a Branson sonifier (Model 575; Power setting 7), centrifuged (17,300 x g), and the supernatant was decanted. The pellet was resuspended in .2 M sodium citrate buffer (pH 2.2), centrifuged (17,300 x g), and the supernatant was removed and combined with the first supernatant. The sample was diluted to 10 mt with citrate buffer, and 2 ml of polyethylene glycol (M.W. 400) were added to each sample. The acidic and neutral amino acids were separated by Technicon Automatic Amino Acid Analyzer (Model TSM) equipped with a stream-splitting apparatus and fraction collector attachment. A standard mixture of amino acids was added to each sample at the time of analysis to visualize each peak. Effluent from the column was assayed for radioactivity by
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liquid scintillation counting (2 parts toluene to 1 part triton X-IO0 vol/vol; 5 g 2,5-diphenyloxazole plus 200 mg 1,4 bis-(2-(5-phenyloxazolyl))-benzene per liter of triton-toluene). Counting efficiency was approximately 70 to 75%. Basic amino acids were separated by the method described above or by means of a Phoenix Amino Acid Analyzer, Model 8000KC. Bray's scintillation fluid (Napthalene, 600 g; 2,5-diphenyloxazole, 4 g; 1,4 bis-(2-(5-phenyloxazolyl))-benzene, 200 mg; methanol, 100 ml; ethylene glycol, 20 ml; and P-dioxane to make 1 liter) was used to assay for radioactivity. The efficiency of this counting system was determined by internal standardization. Enzyme Assays
Mammary and liver tissues were homogenized in two and four volumes of ice-cold distilled water, respectively. The initial homogenization was for two bursts of 30 s each in a Sorvall Omni-Mixer followed by several strokes in a Potter-Elvehjen Teflon-glass homogenizer. The preparation was filtered through two layers of cheesecloth. Arginase (EC 3.5.3.1), ornithine carbamyltransferase, (EC 2.1.3.3), and argininosuccinase (EC 4.3.2.1) were assayed according to Schimke (16). The arginase assay mixture contained .7 /amol of manganese sulfate, 178 /amol of arginine, and homogenate in a total volume of 1.4 ml. Manganese sulfate and tissue homogenate were pre-incubated for 5 min at 55 C. Arginine (pH 9.7) was then added, and the mixture was incubated for an additional 10 min at 37 C. The reaction was terminated by addition of 15% perchloric acid (vol/vol). Perchloric acid was added to the controls before incubation. The precipitated protein was removed by centrifugation (17,300 x g), and the amount of urea formed was determined colorimetrically with 1-phenyl-1, 2-propanedione-2-oxime. The assay mixture for ornithine carbamyltransferase contained 34 /lmol glycylglycine (pH 8.0), 10 /2mol L-ornithine'hydrochloride, 14 /amol dilithium carbamylphosphate, and tissue homogenate in a total volume of 1.45 ml. Control assays were either with perchloric acid added before incubation or without ornithine and carbamylphosphate included in the media. The assay mixture and tissue homogenate were Journal of Dairy Science Vol. 58, No. 12
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CLARK ET AL.
incubated for 15 min at 37 C at which time 15% perchloric acid was added to stop the reaction. Upon termination of the reaction, the mixture was centrifuged (17,300 × g), and the supernatant assayed for formation of citrulline with 2,3-butanedione-2-oxime. The assay media for argininosuccinate cleavage enzyme contained 9 /~mol potassium phosphate (pH 7.4), 2.6/lmol argininosuccinate (potassium salt), 170 /ag arginase, and tissue homogenate in a total volume of .4 ml. Media and tissue homogenate were incubated at 37 C for 30 min at which time 15% perchloric acid was added to stop the reaction. Controls were assayed with perchloric acid added at the onset of incubation. Urea formed was measured colorimetrically as described for arginase. Enzyme activities are expressed as /amol of product formed per h per mg protein. Protein was determined with bovine serum albumin as the standard (13). RESULTS Arteriovenous Differences
Arginine was extracted by the lactating bovine mammary gland in quantities exceeding output in milk by approximately 4-fold (Table 1). Uptake of ornithine and citrulline by the gland was apparent. Although not shown, uptakes of aspartate and glutamate were not sufficient to supply the amount required for milk protein synthesis. Uptake of proline was not determined due to difficulties in analysis for this amino acid.
Arginine and Ornithina Metabolism
Results of bovine tissue incubations with L-[U-14C] arginine show that about 46% of the radioactivity isolated in the products of arginine metabolism was in proline (Table 2). Approximately 36% of the label was in ornithine with the remainder being distributed between glutamate, citrulline, CO2, urea, and aspartate. Urea losses during analysis resulted in a lower percent of radioactivity recovered in this product than theoretically possible based on the stoichiometry of the reaction. Incubations using rabbit mammary tissue with L-[U14C]arginine resulted in 38% and 33% of the recovered radioactivity in proline and ornithine with small amounts of activity detected in glutamate, urea, CO2, aspartate, and citrulline (Table 2). In other incubations, nonlabeled ornithine was included in the incubation medium with labeled arginine to trap the ornithine that was formed from degradation of arginine. In these incubations, recovery of radioactivity was 2fold greater (77 vs. 36%) than when nonlabeled ornithine was included in the incubation mixture. The remainder of the radioactivity was recovered in proline, citrulline, glutamate, aspartate, urea, and CO2. Following incubation of bovine mammary tissue slices with L-[U-laC]ornithine, 72% of the total activity was in proline with the remainder being distributed among glutamate, aspartate, CO2, and citrulline (Table 2). No radioactivity was in arginine or urea. Results were similar in rabbit mammary tissue.
TABLE 1. Uptake of arginine, omithine, and citmlline by the bovine mammary gland and output in milk proteina.
Amino acid
Uptake from plasma
Output in milk protein
Excess uptake
Arginine Ornithine Citrulline
127 50 44
(g/24 h) 33 ...b . . .b
94 50 44
aBlood samples were prepared and assayed for plasma free amino acids as described by Derrig et al. (4). The arteriovenous difference corrected for differences in packed cell volume was multiplied by the estimated blood flow for 24 h. Blood flow was estimated by the regression equation of Kronfeld et al. (11). Amino acid output in milk was determined by calculation based on the amino acid composition of milk (10) and daily output of milk protein. Values represent the mean of five cows bled 4 to 5 h post feeding and post milking at two sampling times 14 days apart. bNot in milk proteins. Journal of Dairy Science Vol. 58, No. 12
T A B L E 2. P e r c e n t d i s t r i b u t i o n of r a d i o a c t i v i t y in v a r i o u s m e t a b o l i t e s f o l l o w i n g i n c u b a t i o n s o f L-[U -14 C] a r g i n i n e a n d L - [ U -~ 4 C] o r n i t h i n e w i t h m a m m a r y t i s s u e f r o m c o w a n d r a b b i t a.
Substrate
L-[U -14C] A R G . + n o n l a b e l e d ORN.
L_[U_~ 4 C] A R G .
L _ [ U 1 4 C] O R N .
L-[U -14C] O R N , + nonlabeled ARG.
L-[U -1aC] O R N . + n o n l a b e l e d A R G . a n d PRO.
Animal
Cow
Rabbit
Cow
Cow
Rabbit
Cow
Cow
No. animals:
4
2
4
4
2
1
1
t~
Z Metabolites: CO 2 Urea Aspartate Glutamate Proline Citrulline Ornithine Arginine
2.5 2.4 1.3 8.3 45.7 4.1 35.9 .
-+ .8 +- .9 ± .3 ± 2.2 ± 6.2 +- .4 +- 3.2 . .
4.5 9.3 2.6 11.5 37.5 1.8 32.7 . . .
.3 .5 .9 1.7 13.0 6.4 77.2 .
.
.
-+ .1 ± .3 +- .7 -+ .5 +- 1.7 +- 2.4 -+ 4.1
3.2 0 5.8 18.3 72.2 .6 . 0
±
.5
+- 1.7 ± 7.7 ± 8.7 ± .4 .
.
5.6 0 11.9 23.8 58.3 .4 . . 0
.
.
1.5 0 13.0 11.0 74.5 0 . . 0
.9 0 8.3 52.9 37.9 0 .
.
60
60
67
78
0 O [-
78
82.0
aTissue slices ( 1 2 0 t o 1 8 0 mg) w e r e i n c u b a t e d in 3.0 m l o f K r e b s - R i n g e r b i c a r b o n a t e b u f f e r (pH 7.3) w i t h r a d i o a c t i v e s u b s t r a t e for 1 h at 37 C, a n d m e t a b o l i t e s were i s o l a t e d . R e s u l t s are r e p o r t e d as t h e m e a n or as t h e m e a n ± SEM. b T i s s u e a n d m i l k p r o t e i n were n o t h y d r o l y z e d ; thus, a n y 24 C a r g i n i n e i n c o r p o r a t e d i n t o these p r o t e i n s was n o t recovered.
< O
.o
7~
z
.
Percent recovery of t o t a l r a d i o a c t i v i t yb 57
O
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CLARK ET AL.
Tissue slices from one cow were incubated in medium containing L-[U J 4 C ] ornithine and either 6 mM concentration of nonlabeled arginine or nonlabeled arginine plus nonlabeled proline to trap any labeled arginine formed if an active urea cycle was in the mammary tissue. Results indicate that ornithine cannot be metabolized to arginine in the mammary gland. Furthermore, argininosuccinate synthetase or lyase activity is low or absent. Total radioactivity from labeled ornithine appearing in products was reduced 75% when nonlabeled arginine was included in the medium. This can be explained by the dilution of L - [ U J a C ] o r n i t h i n e by nonlabeled ornithine from the cleavage of arginine. The decrease in activity recovered in proline coupled with the increase in radioactivity in glutamate when nonlabeled proline was in the medium suggests that the synthesis of these two products occurs via a common pathway as proposed by Greenberg (7).
Enzyme Assays
Measurements of enzyme activity show that arginase and ornithine carbamyltransferase were in cow mammary tissue; however, activities were low compared to liver (Table 3). Argininosuccinate lyase activity could not be detected in cow and rabbit mammary tissue. The presence of ornithine carbamyltransferase which catalyzes the formation of citrulline needs further investigation. In liver, the equilibrium constant of the reaction strongly favors citrulline formation (3). The formation of citrulline from labeled ornithine also suggests the presence of carbamylphosphate synthetase, an enzyme which has been found in rat mammary tissue and is thought to be important in pyrimidine biosynthesis (19).
DISCUSSION
Data in Table 1 indicate uptake of arginine by the lactating bovine mammary gland was four-fold greater than secretion in milk protein. Similar results have been reported for the cow (2, 4), goat (14), and sow (12). In contrast, Verbeke and Peeters (17) and Mephan and Linzell (14) reported that uptake of arginine by the bovine mammary gland was approximately equal to the amount secreted in milk protein. Ornithine and citrulline also were extracted by the lactating bovine mammary gland but are not in milk protein (Table 1). Data of Derrig et al. (4) and Verbeke and Peeters (17) also indicate a significant uptake of ornithine by the lactating bovine mammary gland. Citrulline uptake by the mammary gland of the goat was not significant, but uptake of ornithine was large (14). Uptake of several nonessential amino acids was variable and often not adequate to supply the amino acids required for milk protein synthesis in the lactating cow (2, 4, 17), goat (14), and sow (12) mammary glands. Results of incubations of mammary tissue of both cow and rabbit with [U -14C] arginine and [U J a C] ornithine indicate that they serve as precursors for the synthesis of proline and other nonessential amino acids in the lactating gland (Table 2). Our findings in these species are similar to results from perfusion experiments with goat and sheep m a m m a r y glands. Mepham and Linzell (15) used L - [ u J a c ] arginine to perfuse the goat mammary gland and recovered 14 C in proline and arginine of casein; urea, proline, and ornithine of plasma; very slight in CO2 ; and none in citrulline. Perfusing sheep mammary glands with D L - [ 2 J a C ] ornithine and DL-[5 J 4 C] arginine resulted in considerable 14C incorporation into proline with trace amounts of ~4C incorporation into gluta-
TABLE 3. Activities of urea cycle enzymes in bovine mammary and liver tissuea.
Tissue
ArgJnase
Ornithine carbamyltransferase
Argininosuccinate lyase
Mammary Liver
3.2 -+ 2.2 153.0 -+ 6.9
.2 -+ .1 24.6 -+ 7.2
b .4 -+ .2
avalues represent the mean of three animals +-SEM. Activities are expressed as #tool of product forrned/h/mg protein. bNot detectable. Journal of Dairy Science Vol. 58, No. 12
ARGININE AND ORNITHINE METABOLISM m a t e , aspartate, alanine, serine, a n d CO2 (18). Citrulline was n o t labeled. Arginase has b e e n d e m o n s t r a t e d in liver (7), m a m m a r y tissue (5, 6, 8), small i n t e s t i n e a n d k i d n e y (8) of t h e rat. Results f r o m this s t u d y s h o w arginase in b o v i n e m a m m a r y tissue. However, activity was m u c h l o w e r for m a m m a r y tissue t h a n liver. Urea cycle e n z y m e s argininos u c c i n a t e s y n t h e t a s e or lyase c o u l d n o t b e d e t e c t e d in m a m m a r y tissue i n d i c a t i n g t h e lack of a c o m p l e t e u r e a cycle. This f i n d i n g agrees w i t h o u r t r a c e r studies ( T a b l e 2). Yip a n d K n o x (19) have r e p o r t e d o r n i t h i n e a m i n o t r a n s f e r a s e , a n e n z y m e i n v o l v e d in b i o s y n t h e s i s o f p r o l i n e f r o m o r n i t h i n e , in m a m m a r y tissue o f t h e rat. T h e y i n d i c a t e t h a t o r n i t h i n e is c o n v e r t e d i n t o g l u t a m i c s e m i a l d e h y d e via o r n i t h i n e a m i n o t r a n s f e r a s e a n d s u b s e q u e n t l y i n t o proline. D a t a f r o m this s t u d y i n d i c a t e similar r e a c t i o n s in m a m m a r y tissue o f c o w a n d r a b b i t . Arginase a n d o r n i t h i n e a m i n o t r a n s f e r a s e activity in b o vine m a m m a r y tissue c o u p l e d w i t h a n excess u p t a k e of a r g i n i n e a n d o r n i t h i n e b y t h e g l a n d in r e l a t i o n t o o u t p u t in m i l k p r o t e i n provides f o r t h e b i o s y n t h e s i s of proline, g l u t a m a t e , a n d possibly o t h e r n o n e s s e n t i a l a m i n o acids w h i c h are n o t e x t r a c t e d in s u f f i c i e n t a m o u n t s to m e e t r e q u i r e m e n t s f o r s y n t h e s i s of m i l k p r o t e i n . If m a m m a r y gland e x t r a c t i o n a n d de n o v o s y n t h e sis fail to m e e t t h e r e q u i r e m e n t s for t h e s e n o n e s s e n t i a l a m i n o acids, t h e n t h e y m a y l i m i t s y n t h e s i s of m i l k p r o t e i n .
REFERENCES
1 Bauman, D. E., R. E. Brown, and C. L. Davis. 1970. Pathways of fatty acid synthesis and reducing equivalent generation in mammary gland of rat, sow, and cow. Arch. Biochem. Biophys. 140:237. 2 Bickerstaffe, R., E. F. Annison, and J. L. Linzell. 1974. The metabolism of glucose, acetate, lipids and amino acids in lactating dairy cows. J. Agr. Sci. 82:71. 3 Cohen, P. P., and H. J. Sallach. 1961. Nitrogen metabolism of amino acids. Page 1 in Metabolic pathways. Vol. 2. D. M. Greenberg, ed. Academic Press, London and New York. 4 Derrig, R. G., J. H. Clark, and C. L. Davis. 1974. Effect of abomasal infusion of sodium caseinate on milk yield, nitrogen utilization and amino acid
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nutrition of the dairy cow. J. Nutr. 104:151. 5 FoUey, S. J., and A. L. Greenbaum. 1947. Changes in the arginase and alkaline phosphatase contents of the mammary gland and liver of the rat during pregnancy, lactation and mammary involution. Biochem. J. 41:261. 6 Graham, W. R., O. B. Houchin, and C. W. Turner. 1937. The production of urea in the mammary gland. J. Biol. Chem. 120:29. 7 Greenberg, D. M. 1961. Carbon catabolism of amino acids. Page 80 in Metabolic pathways. Vol. 2. D. M. Greenberg, ed. Academic Press, London and New York. 8 Greengard, O., M. K. Sahib, and W. E. Knox. 1970. Developmental formation and distribution of arginase in rat tissue. Arch. Biochem. Biophys. 137:477. 9 Halfpenny, A. F., J. A. F. Rook, and G. H. Smith. 1969. Variations with energy nutrition in the concentrations of amino acids of the blood plasma in the dairy cow. British J. Nutr. 23:547. 10 Jacobson, D. R., H. H. Van Horn, and C. J. Sniffen. 1970. Lactating ruminants. Fed. Proe. 29:35. 11 Kronfeld, D. S., F. R a i l , and C. F. Ramberg, Jr. 1968. Mammary blood flow and ketone body metabolism in normal, fasted, and ketotic cows. Amer. J. Physiol. 215:218. 12 Linzell, J. L., T. B. Mepham, E. F. Annison, and C. E. West. 1969. Mammary metabolism in lactating sows: Arteriovenous differences of milk precursors and the mammary metabolism of [14Clglucose and [14 C] acetate. British J. Nutr. 23:319. 13 Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265. 14 Mepham, T. B., and J. L. Linzell. 1966. A quantitative assessment of the contribution of individual plasma amino acids to the synthesis of milk proteins by the goat mammary gland. Biochem. J. 101:76. 15 Mepham, T. B., and J. L. Linzell. 1967. Urea formation by the lactating goat mammary gland. Nature 214:507. 16 Schimke, R. T. 1962. Adaptive characteristics of urea cycle enzymes in the rat. J. Biol. Chem. 237:459. 17 Verbeke, R., and G. Peeters. 1965. Uptake of free plasma amino acids by the lactating cow's udder and amino acid composition of udder lymph. Biochem. J. 94:183. 18 Verbeke, R., G. Peeters, A. M. Massart-leen, and G. Cocquyt. 1968. Incorporation of DL-[5 "14 C] arginine in milk constituents by the isolated lactating sheep udder. Biochem. J. 106:719. 19 Yip, C. M., and W. E. Knox. 1972. Function of arginase in lactating mammary gland. Biochem. J. 127:893.
Journal of Dairy Science Vol. 58, No. 12
Tissue 1 J. H. CLARK,
ABSTRACT
R. G. DERRIG, C. L. DAVIS, and H. R. SPIRES Department of Dairy Science University of Illinois Urbana 61801
specific uptake mechanism. However, recent studies by Derrig et al. (4) and Bickerstaffe et al. (2) indicate that the bovine mammary gland extracts arginine in excess of the amount secreted in milk protein. Therefore this study was to determine uptake by m a m m a r y gland of arginine and ornithine and to determine the metabolism of these amino acids in lactating mammary tissues of the cow and rabbit.
Amino acid uptake by the bovine mammary gland was determined b y arteriovenous difference. Extraction of arginine from the plasma by the lactating bovine mammary gland was in excess of requirements for milk protein synthesis. Ornithine and citrulline also were extracted by the gland but are not in milk protein. Incubations of slices of lactating mammary tissue from cows and rabbits indicate that nonessential amino acids, especially proline and glutamate, are the major end products of arginine and ornithine metabolism in the lactating mammary gland.
MATERIALS AND METHODS Arteriovenous Differences
INTRODUCTION
Measurements of arteriovenous differences to determine metabolite uptake by the lactating mammary gland indicate an adequate uptake of essential amino acids for milk protein synthesis but a deficient uptake of several nonessential amino acids (2, 4, 12, 14, 17). In these studies, amino acids classified as essential for the mammary gland were assumed to be those established as essential for growth of rats. Halfpenny et al. (9) concluded that synthesis of milk protein may be limited by the supply in plasma of nonessential amino acids, especially glutamate and proline. Uptake of ornithine, an amino acid not in milk protein, by the mammary gland was significant in the cow (17), goat (14), and sow (12). Uptake of arginine has been in excess of output in milk protein for the mammary gland of the goat (14) and sow (12) but not for the cow (14, 17). These differences were attributed to a species difference in the
Received September 30, 1974. 1Supported in part by the Illinois Agricultural Experiment Station and the National Soybean Processors Association.
Blood samples were drawn from the internal iliac artery and subcutaneous abdominal vein of five lactating Holstein cows averaging 33 kg of milk per day. The cows were bled 4 to 5 h post-feeding and post-milking at two sampling times 14 days apart. Plasma samples were prepared for amino acid analysis as described by Derrig et al. (4). Free amino acids of plasma were determined by Phoenix Amino Acid Analyzer, Model 8000KC. Calculations for determining uptake of amino acids by the gland and output in milk protein were as described b y Derrig et al. (4). Tissues
Mammary tissue was obtained at slaughter from four cows, approximately 6 m o lactating, producing 14 to 15 kg of milk per day and from two lactating New Zealand white rabbits, 12 to 20 days lactating. Mammary tissue was obtained approximately 15 to 20 rain postslaughter. The tissue for incubation studies was placed in iso-osmotic Tris-sucrose buffer (pH 7.3; 30 mM-tris, .3 M-sucrose, 1 mM-glutathione, 1 mM-EDTA) at 4 C prior to slicing and incubation (approximately 10 rain). Mammary and liver tissues for enzyme studies were maintained in ice-cold distilled water as recommended by Schimke (16) prior to the assays (approximately 30 min).
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ARGININE AND ORNITHINE METABOLISM I ncubation Studies
Incubations of tissue slices were according to Bauman et al. (1). Tissue slices weighing 120 to 180 mg (wet weight) were prepared with a Stadie-Riggs hand microtome and were incubated in Erlenmeyer flasks containing 3.0 ml of Krebs-Ringer bicarbonate buffer (pH 7.3). Glucose was dissolved in the media to a final concentration of 20 mM. Each flask contained .3 unit of insulin and either .5 or 1.0 /aCi of radioactive substrate. Concentrations of unlabeled ornithine, proline, and arginine were 6 mM when added. All incubations were for 1 h in a shaking water bath at 37 C under an atmosphere of O2 + CO2 (95:5). At the end of each incubation, .1 ml of 25% KOH (wt/vol) was injected through the rubber serum cap of each flask onto a 2 x 2 cm piece of filter paper suspended in a plastic bucket. Tissue metabolism was terminated by injecting .25 ml of .5 N H2SO4 into the media. Flasks were incubated for an additional 30 min to trap the CO2. The filter paper containing trapped CO2 was removed and air-dried for 12 h. Radioactivity on the paper was determined by liquid scintillation counting (5 g 2,5-diphenyloxazole/liter toluene). Counting efficiencies of all scintillation cocktails were determined by external standard ratios. Isolation of Metabolites
The incubation media containing the tissue slice was homogenized in a Sorvall Omni-Mixer equipped with a micro attachment (two bursts of 30 s each), sonified with a Branson sonifier (Model 575; Power setting 7), centrifuged (17,300 x g), and the supernatant was decanted. The pellet was resuspended in .2 M sodium citrate buffer (pH 2.2), centrifuged (17,300 x g), and the supernatant was removed and combined with the first supernatant. The sample was diluted to 10 mt with citrate buffer, and 2 ml of polyethylene glycol (M.W. 400) were added to each sample. The acidic and neutral amino acids were separated by Technicon Automatic Amino Acid Analyzer (Model TSM) equipped with a stream-splitting apparatus and fraction collector attachment. A standard mixture of amino acids was added to each sample at the time of analysis to visualize each peak. Effluent from the column was assayed for radioactivity by
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liquid scintillation counting (2 parts toluene to 1 part triton X-IO0 vol/vol; 5 g 2,5-diphenyloxazole plus 200 mg 1,4 bis-(2-(5-phenyloxazolyl))-benzene per liter of triton-toluene). Counting efficiency was approximately 70 to 75%. Basic amino acids were separated by the method described above or by means of a Phoenix Amino Acid Analyzer, Model 8000KC. Bray's scintillation fluid (Napthalene, 600 g; 2,5-diphenyloxazole, 4 g; 1,4 bis-(2-(5-phenyloxazolyl))-benzene, 200 mg; methanol, 100 ml; ethylene glycol, 20 ml; and P-dioxane to make 1 liter) was used to assay for radioactivity. The efficiency of this counting system was determined by internal standardization. Enzyme Assays
Mammary and liver tissues were homogenized in two and four volumes of ice-cold distilled water, respectively. The initial homogenization was for two bursts of 30 s each in a Sorvall Omni-Mixer followed by several strokes in a Potter-Elvehjen Teflon-glass homogenizer. The preparation was filtered through two layers of cheesecloth. Arginase (EC 3.5.3.1), ornithine carbamyltransferase, (EC 2.1.3.3), and argininosuccinase (EC 4.3.2.1) were assayed according to Schimke (16). The arginase assay mixture contained .7 /amol of manganese sulfate, 178 /amol of arginine, and homogenate in a total volume of 1.4 ml. Manganese sulfate and tissue homogenate were pre-incubated for 5 min at 55 C. Arginine (pH 9.7) was then added, and the mixture was incubated for an additional 10 min at 37 C. The reaction was terminated by addition of 15% perchloric acid (vol/vol). Perchloric acid was added to the controls before incubation. The precipitated protein was removed by centrifugation (17,300 x g), and the amount of urea formed was determined colorimetrically with 1-phenyl-1, 2-propanedione-2-oxime. The assay mixture for ornithine carbamyltransferase contained 34 /lmol glycylglycine (pH 8.0), 10 /2mol L-ornithine'hydrochloride, 14 /amol dilithium carbamylphosphate, and tissue homogenate in a total volume of 1.45 ml. Control assays were either with perchloric acid added before incubation or without ornithine and carbamylphosphate included in the media. The assay mixture and tissue homogenate were Journal of Dairy Science Vol. 58, No. 12
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CLARK ET AL.
incubated for 15 min at 37 C at which time 15% perchloric acid was added to stop the reaction. Upon termination of the reaction, the mixture was centrifuged (17,300 × g), and the supernatant assayed for formation of citrulline with 2,3-butanedione-2-oxime. The assay media for argininosuccinate cleavage enzyme contained 9 /~mol potassium phosphate (pH 7.4), 2.6/lmol argininosuccinate (potassium salt), 170 /ag arginase, and tissue homogenate in a total volume of .4 ml. Media and tissue homogenate were incubated at 37 C for 30 min at which time 15% perchloric acid was added to stop the reaction. Controls were assayed with perchloric acid added at the onset of incubation. Urea formed was measured colorimetrically as described for arginase. Enzyme activities are expressed as /amol of product formed per h per mg protein. Protein was determined with bovine serum albumin as the standard (13). RESULTS Arteriovenous Differences
Arginine was extracted by the lactating bovine mammary gland in quantities exceeding output in milk by approximately 4-fold (Table 1). Uptake of ornithine and citrulline by the gland was apparent. Although not shown, uptakes of aspartate and glutamate were not sufficient to supply the amount required for milk protein synthesis. Uptake of proline was not determined due to difficulties in analysis for this amino acid.
Arginine and Ornithina Metabolism
Results of bovine tissue incubations with L-[U-14C] arginine show that about 46% of the radioactivity isolated in the products of arginine metabolism was in proline (Table 2). Approximately 36% of the label was in ornithine with the remainder being distributed between glutamate, citrulline, CO2, urea, and aspartate. Urea losses during analysis resulted in a lower percent of radioactivity recovered in this product than theoretically possible based on the stoichiometry of the reaction. Incubations using rabbit mammary tissue with L-[U14C]arginine resulted in 38% and 33% of the recovered radioactivity in proline and ornithine with small amounts of activity detected in glutamate, urea, CO2, aspartate, and citrulline (Table 2). In other incubations, nonlabeled ornithine was included in the incubation medium with labeled arginine to trap the ornithine that was formed from degradation of arginine. In these incubations, recovery of radioactivity was 2fold greater (77 vs. 36%) than when nonlabeled ornithine was included in the incubation mixture. The remainder of the radioactivity was recovered in proline, citrulline, glutamate, aspartate, urea, and CO2. Following incubation of bovine mammary tissue slices with L-[U-laC]ornithine, 72% of the total activity was in proline with the remainder being distributed among glutamate, aspartate, CO2, and citrulline (Table 2). No radioactivity was in arginine or urea. Results were similar in rabbit mammary tissue.
TABLE 1. Uptake of arginine, omithine, and citmlline by the bovine mammary gland and output in milk proteina.
Amino acid
Uptake from plasma
Output in milk protein
Excess uptake
Arginine Ornithine Citrulline
127 50 44
(g/24 h) 33 ...b . . .b
94 50 44
aBlood samples were prepared and assayed for plasma free amino acids as described by Derrig et al. (4). The arteriovenous difference corrected for differences in packed cell volume was multiplied by the estimated blood flow for 24 h. Blood flow was estimated by the regression equation of Kronfeld et al. (11). Amino acid output in milk was determined by calculation based on the amino acid composition of milk (10) and daily output of milk protein. Values represent the mean of five cows bled 4 to 5 h post feeding and post milking at two sampling times 14 days apart. bNot in milk proteins. Journal of Dairy Science Vol. 58, No. 12
T A B L E 2. P e r c e n t d i s t r i b u t i o n of r a d i o a c t i v i t y in v a r i o u s m e t a b o l i t e s f o l l o w i n g i n c u b a t i o n s o f L-[U -14 C] a r g i n i n e a n d L - [ U -~ 4 C] o r n i t h i n e w i t h m a m m a r y t i s s u e f r o m c o w a n d r a b b i t a.
Substrate
L-[U -14C] A R G . + n o n l a b e l e d ORN.
L_[U_~ 4 C] A R G .
L _ [ U 1 4 C] O R N .
L-[U -14C] O R N , + nonlabeled ARG.
L-[U -1aC] O R N . + n o n l a b e l e d A R G . a n d PRO.
Animal
Cow
Rabbit
Cow
Cow
Rabbit
Cow
Cow
No. animals:
4
2
4
4
2
1
1
t~
Z Metabolites: CO 2 Urea Aspartate Glutamate Proline Citrulline Ornithine Arginine
2.5 2.4 1.3 8.3 45.7 4.1 35.9 .
-+ .8 +- .9 ± .3 ± 2.2 ± 6.2 +- .4 +- 3.2 . .
4.5 9.3 2.6 11.5 37.5 1.8 32.7 . . .
.3 .5 .9 1.7 13.0 6.4 77.2 .
.
.
-+ .1 ± .3 +- .7 -+ .5 +- 1.7 +- 2.4 -+ 4.1
3.2 0 5.8 18.3 72.2 .6 . 0
±
.5
+- 1.7 ± 7.7 ± 8.7 ± .4 .
.
5.6 0 11.9 23.8 58.3 .4 . . 0
.
.
1.5 0 13.0 11.0 74.5 0 . . 0
.9 0 8.3 52.9 37.9 0 .
.
60
60
67
78
0 O [-
78
82.0
aTissue slices ( 1 2 0 t o 1 8 0 mg) w e r e i n c u b a t e d in 3.0 m l o f K r e b s - R i n g e r b i c a r b o n a t e b u f f e r (pH 7.3) w i t h r a d i o a c t i v e s u b s t r a t e for 1 h at 37 C, a n d m e t a b o l i t e s were i s o l a t e d . R e s u l t s are r e p o r t e d as t h e m e a n or as t h e m e a n ± SEM. b T i s s u e a n d m i l k p r o t e i n were n o t h y d r o l y z e d ; thus, a n y 24 C a r g i n i n e i n c o r p o r a t e d i n t o these p r o t e i n s was n o t recovered.
< O
.o
7~
z
.
Percent recovery of t o t a l r a d i o a c t i v i t yb 57
O
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CLARK ET AL.
Tissue slices from one cow were incubated in medium containing L-[U J 4 C ] ornithine and either 6 mM concentration of nonlabeled arginine or nonlabeled arginine plus nonlabeled proline to trap any labeled arginine formed if an active urea cycle was in the mammary tissue. Results indicate that ornithine cannot be metabolized to arginine in the mammary gland. Furthermore, argininosuccinate synthetase or lyase activity is low or absent. Total radioactivity from labeled ornithine appearing in products was reduced 75% when nonlabeled arginine was included in the medium. This can be explained by the dilution of L - [ U J a C ] o r n i t h i n e by nonlabeled ornithine from the cleavage of arginine. The decrease in activity recovered in proline coupled with the increase in radioactivity in glutamate when nonlabeled proline was in the medium suggests that the synthesis of these two products occurs via a common pathway as proposed by Greenberg (7).
Enzyme Assays
Measurements of enzyme activity show that arginase and ornithine carbamyltransferase were in cow mammary tissue; however, activities were low compared to liver (Table 3). Argininosuccinate lyase activity could not be detected in cow and rabbit mammary tissue. The presence of ornithine carbamyltransferase which catalyzes the formation of citrulline needs further investigation. In liver, the equilibrium constant of the reaction strongly favors citrulline formation (3). The formation of citrulline from labeled ornithine also suggests the presence of carbamylphosphate synthetase, an enzyme which has been found in rat mammary tissue and is thought to be important in pyrimidine biosynthesis (19).
DISCUSSION
Data in Table 1 indicate uptake of arginine by the lactating bovine mammary gland was four-fold greater than secretion in milk protein. Similar results have been reported for the cow (2, 4), goat (14), and sow (12). In contrast, Verbeke and Peeters (17) and Mephan and Linzell (14) reported that uptake of arginine by the bovine mammary gland was approximately equal to the amount secreted in milk protein. Ornithine and citrulline also were extracted by the lactating bovine mammary gland but are not in milk protein (Table 1). Data of Derrig et al. (4) and Verbeke and Peeters (17) also indicate a significant uptake of ornithine by the lactating bovine mammary gland. Citrulline uptake by the mammary gland of the goat was not significant, but uptake of ornithine was large (14). Uptake of several nonessential amino acids was variable and often not adequate to supply the amino acids required for milk protein synthesis in the lactating cow (2, 4, 17), goat (14), and sow (12) mammary glands. Results of incubations of mammary tissue of both cow and rabbit with [U -14C] arginine and [U J a C] ornithine indicate that they serve as precursors for the synthesis of proline and other nonessential amino acids in the lactating gland (Table 2). Our findings in these species are similar to results from perfusion experiments with goat and sheep m a m m a r y glands. Mepham and Linzell (15) used L - [ u J a c ] arginine to perfuse the goat mammary gland and recovered 14 C in proline and arginine of casein; urea, proline, and ornithine of plasma; very slight in CO2 ; and none in citrulline. Perfusing sheep mammary glands with D L - [ 2 J a C ] ornithine and DL-[5 J 4 C] arginine resulted in considerable 14C incorporation into proline with trace amounts of ~4C incorporation into gluta-
TABLE 3. Activities of urea cycle enzymes in bovine mammary and liver tissuea.
Tissue
ArgJnase
Ornithine carbamyltransferase
Argininosuccinate lyase
Mammary Liver
3.2 -+ 2.2 153.0 -+ 6.9
.2 -+ .1 24.6 -+ 7.2
b .4 -+ .2
avalues represent the mean of three animals +-SEM. Activities are expressed as #tool of product forrned/h/mg protein. bNot detectable. Journal of Dairy Science Vol. 58, No. 12
ARGININE AND ORNITHINE METABOLISM m a t e , aspartate, alanine, serine, a n d CO2 (18). Citrulline was n o t labeled. Arginase has b e e n d e m o n s t r a t e d in liver (7), m a m m a r y tissue (5, 6, 8), small i n t e s t i n e a n d k i d n e y (8) of t h e rat. Results f r o m this s t u d y s h o w arginase in b o v i n e m a m m a r y tissue. However, activity was m u c h l o w e r for m a m m a r y tissue t h a n liver. Urea cycle e n z y m e s argininos u c c i n a t e s y n t h e t a s e or lyase c o u l d n o t b e d e t e c t e d in m a m m a r y tissue i n d i c a t i n g t h e lack of a c o m p l e t e u r e a cycle. This f i n d i n g agrees w i t h o u r t r a c e r studies ( T a b l e 2). Yip a n d K n o x (19) have r e p o r t e d o r n i t h i n e a m i n o t r a n s f e r a s e , a n e n z y m e i n v o l v e d in b i o s y n t h e s i s o f p r o l i n e f r o m o r n i t h i n e , in m a m m a r y tissue o f t h e rat. T h e y i n d i c a t e t h a t o r n i t h i n e is c o n v e r t e d i n t o g l u t a m i c s e m i a l d e h y d e via o r n i t h i n e a m i n o t r a n s f e r a s e a n d s u b s e q u e n t l y i n t o proline. D a t a f r o m this s t u d y i n d i c a t e similar r e a c t i o n s in m a m m a r y tissue o f c o w a n d r a b b i t . Arginase a n d o r n i t h i n e a m i n o t r a n s f e r a s e activity in b o vine m a m m a r y tissue c o u p l e d w i t h a n excess u p t a k e of a r g i n i n e a n d o r n i t h i n e b y t h e g l a n d in r e l a t i o n t o o u t p u t in m i l k p r o t e i n provides f o r t h e b i o s y n t h e s i s of proline, g l u t a m a t e , a n d possibly o t h e r n o n e s s e n t i a l a m i n o acids w h i c h are n o t e x t r a c t e d in s u f f i c i e n t a m o u n t s to m e e t r e q u i r e m e n t s f o r s y n t h e s i s of m i l k p r o t e i n . If m a m m a r y gland e x t r a c t i o n a n d de n o v o s y n t h e sis fail to m e e t t h e r e q u i r e m e n t s for t h e s e n o n e s s e n t i a l a m i n o acids, t h e n t h e y m a y l i m i t s y n t h e s i s of m i l k p r o t e i n .
REFERENCES
1 Bauman, D. E., R. E. Brown, and C. L. Davis. 1970. Pathways of fatty acid synthesis and reducing equivalent generation in mammary gland of rat, sow, and cow. Arch. Biochem. Biophys. 140:237. 2 Bickerstaffe, R., E. F. Annison, and J. L. Linzell. 1974. The metabolism of glucose, acetate, lipids and amino acids in lactating dairy cows. J. Agr. Sci. 82:71. 3 Cohen, P. P., and H. J. Sallach. 1961. Nitrogen metabolism of amino acids. Page 1 in Metabolic pathways. Vol. 2. D. M. Greenberg, ed. Academic Press, London and New York. 4 Derrig, R. G., J. H. Clark, and C. L. Davis. 1974. Effect of abomasal infusion of sodium caseinate on milk yield, nitrogen utilization and amino acid
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nutrition of the dairy cow. J. Nutr. 104:151. 5 FoUey, S. J., and A. L. Greenbaum. 1947. Changes in the arginase and alkaline phosphatase contents of the mammary gland and liver of the rat during pregnancy, lactation and mammary involution. Biochem. J. 41:261. 6 Graham, W. R., O. B. Houchin, and C. W. Turner. 1937. The production of urea in the mammary gland. J. Biol. Chem. 120:29. 7 Greenberg, D. M. 1961. Carbon catabolism of amino acids. Page 80 in Metabolic pathways. Vol. 2. D. M. Greenberg, ed. Academic Press, London and New York. 8 Greengard, O., M. K. Sahib, and W. E. Knox. 1970. Developmental formation and distribution of arginase in rat tissue. Arch. Biochem. Biophys. 137:477. 9 Halfpenny, A. F., J. A. F. Rook, and G. H. Smith. 1969. Variations with energy nutrition in the concentrations of amino acids of the blood plasma in the dairy cow. British J. Nutr. 23:547. 10 Jacobson, D. R., H. H. Van Horn, and C. J. Sniffen. 1970. Lactating ruminants. Fed. Proe. 29:35. 11 Kronfeld, D. S., F. R a i l , and C. F. Ramberg, Jr. 1968. Mammary blood flow and ketone body metabolism in normal, fasted, and ketotic cows. Amer. J. Physiol. 215:218. 12 Linzell, J. L., T. B. Mepham, E. F. Annison, and C. E. West. 1969. Mammary metabolism in lactating sows: Arteriovenous differences of milk precursors and the mammary metabolism of [14Clglucose and [14 C] acetate. British J. Nutr. 23:319. 13 Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265. 14 Mepham, T. B., and J. L. Linzell. 1966. A quantitative assessment of the contribution of individual plasma amino acids to the synthesis of milk proteins by the goat mammary gland. Biochem. J. 101:76. 15 Mepham, T. B., and J. L. Linzell. 1967. Urea formation by the lactating goat mammary gland. Nature 214:507. 16 Schimke, R. T. 1962. Adaptive characteristics of urea cycle enzymes in the rat. J. Biol. Chem. 237:459. 17 Verbeke, R., and G. Peeters. 1965. Uptake of free plasma amino acids by the lactating cow's udder and amino acid composition of udder lymph. Biochem. J. 94:183. 18 Verbeke, R., G. Peeters, A. M. Massart-leen, and G. Cocquyt. 1968. Incorporation of DL-[5 "14 C] arginine in milk constituents by the isolated lactating sheep udder. Biochem. J. 106:719. 19 Yip, C. M., and W. E. Knox. 1972. Function of arginase in lactating mammary gland. Biochem. J. 127:893.
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