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Lack of Influence of Spermine or Diet on Calf Spermine Oxidase Formation
LACK OF INFLUENCE SPERMINE
O F S P E R M I N E OR D I E T ON C A L F OXIDASE FORMATION
E. UEYAMA, C. L. DAVIS, AND 5I..]. WOLIN Department of Dairy Science, Vniversity of Illinois, Urbana ABSTRACT
Studies were conducted to as(.ertain the changes in blood plasma spermine oxidase activity in the young dairy calf as the animal developed. Results show that there is a steady increase in plasma spermine oxidase activity up until 40-60 days of age with no further increase. Although the increase in the activity of this enzyme occurs when the rmnen is developing fastest, there appears to be no direct relationship between the two phenomena. Blaschko and Bonney (2) reached a similar conclusion from results obtained with developing kids.
fications. Manometric experiments were performed with Warburg flasks which contained 50 m:~I Na:HPO4-KH:P04 buffer, p H 7.0, 0.5 ml serum, and water to 2.8 ml in the main compartment; 0.15 ml of 20% K O H in the center well; and 0.2 ml of 0.1 M spermine HC1 (neutralized with NaOH) in the sidearm. The gas phase was air, and the temperature was 37.5C. Separate endogenous controls were run and the low oxygen uptake values were subtracted from the spermine-dependent oxygen uptake. Three feeding regimens were used for raising calves. Regimen A consisted of whole milk fed at a rate of 10% body weight per day, with the calves housed on rubber lnats without access to dry feeds. Regimen B was a commercial milk replacer formula reconstituted on the same dry matter basis as whole milk and fed at a rate of 10% of hod)" weight per day; the animals had free access to grain and alfalfa hay. Regimen C was a milk replacer forlnula in which fish meal was incorporated to supply the principal source of protein. Regimen C was reconstituted and fed in a manner identical with that used for Regimen B, including free access to grain and hay. Blood was taken from the jugular vein of bovines and from the heart of rabbits, allowed to clot at 4 C, and sera collected after centrifugation. When it was not possible to immediately assay serum samples, serum was stored at --15 C and assayed within four days. Separate experiments showed that serum spermine oxidase activity was unaffected by the storage 1)rocedm'e. Sera were not dialyzed before assay.
Blaschko and Hawes (2) and Blaschko and Bonnel (1) showed that spernfine oxidase was present in the sera of ruminants but absent from the sera of nonruminants, except for the hippopotaums and two hyracoid species. The spermine oxidase activity of young ruminant sera is low compared to adult sera and an increase in oxidase activity develops upon maturation of the animal (2). It was suggested (1, 2) that the unique presence of sperniine oxidase in rmninant sera is related, in an evolutionary sense, to development of the rumen. It was also postulated (1, 2) that the microbial population of the rumen produces spennine (or a related compound), and that the plasma enzyme acts on the amine which reaches the blood stream from the rumen. It is known that microorganisms produce polyamines such as spermine (5). It is also known that spernline is toxic in mammals (4). The present investigation was undertaken to determine whether a depression in rumen development in calves by manipulation of the diet would cause a decrease in the rate of spermine oxidase development during maturation of the animals, Whole-milk diets have been shown to decrease rumen development (6). Experin~ents were also performed to determine if spermine oxidase formation could be induced in young' calves or in a nonrmninant (rabbit) by injections of sperluine. ]'he over-all purpose of these expel'iments was to determine whether rumen development or spermine could trigger spermine oxidase formation in the y(mng ruminant. MATERIALS
AND
METI-IODS
Spermine oxidase was assayed by the niethod of Blasehko and Hawes (2) with slight modi-
R E S U L T S AND D I S C U S S I O N
Results shown in Table 1 do not indicate any depression in the spermine oxidase levels with
Received for publication September 14, 1964. 73
74
E.
UEYAMA,
C.
L.
DAVIS,
AND
3I. J .
TABLE 1 Development of spermine oxidase in cahes fed different diets Calf
Feeding regimen "
Age of calf
Spermine oxidase
(DaBs)
(~l 0~/ 30 ~nin/0.5 ml semtm )
A A 7 9.7 A A 11 15.4 B A 14 39.0 B A 18 23.8 C B 4 8.9 C B 5 12.3 C B 9 11.3 D B 15 14.8 E C 15 14.8 E C 18 19.2 " A--ndlk, no ha)" or grain; B--milk replacer, hay and grain; C--milk replacer with fish meal protein, ha3', and grain. development of calves fed only on whole nfilk. The milk d i e t - f e d calves actually have slightly higher spernfine oxidase le-eels:at a given age than the animals fed the milk replacer diets plus hay and grain, although these differences are probably not significant. The main point, however, is that the diet expected to repress tureen development and function, i.e., the milk diet, did not repress the appearance of spernfine oxidase in the growing calves. This result does not rule out the possibility that bacterial development in the rumen is associated with plasma spennine oxidase development because bacteria are present in the undeveloped rumen of whole-milk-fed calves (3), although these bacteria are not necessarily representative oI' the bacteria of a functioning tureen. An experiment was p e r f o r m e d to determine whether injection in a growing calf would markedly affect the course of spernfine oxidase development. Two calves of a p p r o x i m a t e l y the same age on feeding Regnnen C were used in this experiment. One calf was injected in the j u g u l a r vein at 20, 21, 22, 27, and 29 days of age with 10 ml of a 1% sterile, neutral spermine solution. The other calf was k e p t as a control. Spermine oxidase levels of the control and i n j e c t e d calves at various periods between 19 and 30 days of age are shown in F i g u r e 1. S p e n n i n e injection did not a p p e a r to induce formation of large amounts of spermine oxidase. These results again are negative results and do not rule out the possibility of spermine induction of spernfine oxidase in the developing calf. Induction experiments were also attempted with a rabbit. The experiment was based on the possibility that the potential for forming
WOLIN
~ °~40
(-SPERMINE
_
,"
N
_z
~
/K 'SPERMtNE INJECTED:
--. 2 0 I
\%,
~START OF SPERMINE INJECTIONS
.% o 19
[
I
i
i
20
22
24
26
28
3(i)
AGE OF CALF (DAYS)
FIG. 1. Lack of influence of spernfine injection on serum spermine oxidase of developing calves. spermine oxidase was present in the rabbit, and the lack of f o n a a t i o n of plasma spennine oxidase was due to the lack of exposure to the potential inducer, spermine. Neutralized, sterile spermine was injected in the ear vein or intraperitoneally in an adult rabbit at a level of 0.01 mmole per kilogram of body weight. Samples of blood taken by heart puncture were assayed for spernfine oxidase, along with the hlood of a control untreated rabbit. Table 2 TABLE 2 Inability of spermine injection to induce a rabbit serum spermine oxidase Spernfine oxidase Time
In jettion
Spermine treated
Untreated
(M 0~/3'0 rain~ 0.5 ml semwn)
(Days)
o t)
I.V.
2• 4 (; 7 8 .~
I.V.
0.5
o.o
2.1 2.8
1.4 1.3
0.0
0.7
o.o
o.i;
i.P. I.P.
summarizes results obtained, and shows that spermine oxidase was not induced by spermine treatment of the rabbit. Addition of rabbit serum to adult bovine serum did not inhibit spernfine oxidase of the bovine serum nor was there any indication of a synergistic spermine oxidase activity. This result appears to rule out the possibility of an inhibitor of spermine oxidase in rabbit sera or
SPER3IINE
OXII)ASE
an activator in bovine sera for a possible latent spermine oxidase of the rabbit. Results of all of the above experiments argue against a direct effect of spermine or a product of microbial metabolism of the normally developing tureen on the presence of spernline oxidase in the bovine and on the increase in plasma levels of oxidase observed during the maturation of calves. We would emphasize, however, that these results do not completely rule out the possibility of an induction of enzyme activity by spermine produced by mieroorganisms in the rumen. It might be necessary to use genu-free ruminants to unequivocally rule out this possibility. There is no question of the relationship ot' spermine oxidase in plasma to ruminants because of the unique distribution of this enzyme in mammals. Except for the hippopota-
75
FOR3IAT1ON
nms and the two hyraeoid species studied only rulninant sera of all mammalian sera contain spermine oxidase (1, 2). Results presented in this paper would suggest that the capability for forming spermine oxidase is a constitutive expression of a unique genetic trait. The suggestion (1, 2) that spermine oxidase is an evolutionary adaptation to spermine production by microorganisms of special pouches developed for cellulose digestion is extremely interesting, but further experimental evidence is needed to relate the oxidase formation to rumen microbial activity. Finally, we would like to present the aceunmlated data of all of our experiments on the serum levels of bovines of different ages in Figure 2. No attempt has been made to distinguish between breeds or feeding treatments. These data of Figure 2 confirm results obtained
60
50
O O
C40
Oo
0
E
%
Oo
o
m
30 a~
o"
-%
¢°--
E o
0 20
I'0
O4 O
0
10
=
0
]*~IG. 2 .
20
40
60
80
age of calves ( d a y s ) "Increase of ,serum spermine oxidqse levels in deve}oping calves.
tOO
76
E. UEYAMA. C. L. DAVIS, AND M. J. WOLIN
by B l a s c h k o a n d H a T e s on the i n c r e a s e of s p e r m i n e oxidase in calf s e r u m w i t h increasi n g age (2).
REFERENCES (1) BLASCHKO, H., AND BO~NEY, R.
Spcrmine Oxidase and Benzylamine Oxidase. Distribution, Development and Substrate Specificity. Proc. Royal Soc. London, Series B, 156: 268. 1962.
(2) BLASCI-IKO, H., AND HA\VES, R.. Observatio~ls on Spermine Oxidase of Mammalian Plasma. J. Physiol., 145: 124. 1959. (3) BRYANT, M. P., SI~IALL, W., BoLivIA, C., .\ND ROBINSON, I. Studies on the Composition
of the Ruminal Flora and F a u n a of Young Calves. J. Dairy Set., 41: 1747. 1958. (4) TABOR, C. W., AND ROSENTHAL, S. M. Pharmacology of Spermine and Spermidine. Some Effects on Animals and Bacteria. J. Pharmacol., 116: 139. 1956.
(5) TABOR, H., TABOR, C. W., AND R.OSENTHAL, S. M. The Biochemistry of Polyamines: Spermine and Spermidine. Ann. Rev. Biochem., 30: 579. 1961.
(6) TA~IATE, H., MCGILLIARD, A. D., ,~ACOBSON, X. L., AND GETTY, :[~. Effect of Various Dietaries oll the Anatomical Development of the Stomach in the Calf. J. Dairy Sci., 45: 498. 1962.
O F S P E R M I N E OR D I E T ON C A L F OXIDASE FORMATION
E. UEYAMA, C. L. DAVIS, AND 5I..]. WOLIN Department of Dairy Science, Vniversity of Illinois, Urbana ABSTRACT
Studies were conducted to as(.ertain the changes in blood plasma spermine oxidase activity in the young dairy calf as the animal developed. Results show that there is a steady increase in plasma spermine oxidase activity up until 40-60 days of age with no further increase. Although the increase in the activity of this enzyme occurs when the rmnen is developing fastest, there appears to be no direct relationship between the two phenomena. Blaschko and Bonney (2) reached a similar conclusion from results obtained with developing kids.
fications. Manometric experiments were performed with Warburg flasks which contained 50 m:~I Na:HPO4-KH:P04 buffer, p H 7.0, 0.5 ml serum, and water to 2.8 ml in the main compartment; 0.15 ml of 20% K O H in the center well; and 0.2 ml of 0.1 M spermine HC1 (neutralized with NaOH) in the sidearm. The gas phase was air, and the temperature was 37.5C. Separate endogenous controls were run and the low oxygen uptake values were subtracted from the spermine-dependent oxygen uptake. Three feeding regimens were used for raising calves. Regimen A consisted of whole milk fed at a rate of 10% body weight per day, with the calves housed on rubber lnats without access to dry feeds. Regimen B was a commercial milk replacer formula reconstituted on the same dry matter basis as whole milk and fed at a rate of 10% of hod)" weight per day; the animals had free access to grain and alfalfa hay. Regimen C was a milk replacer forlnula in which fish meal was incorporated to supply the principal source of protein. Regimen C was reconstituted and fed in a manner identical with that used for Regimen B, including free access to grain and hay. Blood was taken from the jugular vein of bovines and from the heart of rabbits, allowed to clot at 4 C, and sera collected after centrifugation. When it was not possible to immediately assay serum samples, serum was stored at --15 C and assayed within four days. Separate experiments showed that serum spermine oxidase activity was unaffected by the storage 1)rocedm'e. Sera were not dialyzed before assay.
Blaschko and Hawes (2) and Blaschko and Bonnel (1) showed that spernfine oxidase was present in the sera of ruminants but absent from the sera of nonruminants, except for the hippopotaums and two hyracoid species. The spermine oxidase activity of young ruminant sera is low compared to adult sera and an increase in oxidase activity develops upon maturation of the animal (2). It was suggested (1, 2) that the unique presence of sperniine oxidase in rmninant sera is related, in an evolutionary sense, to development of the rumen. It was also postulated (1, 2) that the microbial population of the rumen produces spennine (or a related compound), and that the plasma enzyme acts on the amine which reaches the blood stream from the rumen. It is known that microorganisms produce polyamines such as spermine (5). It is also known that spernline is toxic in mammals (4). The present investigation was undertaken to determine whether a depression in rumen development in calves by manipulation of the diet would cause a decrease in the rate of spermine oxidase development during maturation of the animals, Whole-milk diets have been shown to decrease rumen development (6). Experin~ents were also performed to determine if spermine oxidase formation could be induced in young' calves or in a nonrmninant (rabbit) by injections of sperluine. ]'he over-all purpose of these expel'iments was to determine whether rumen development or spermine could trigger spermine oxidase formation in the y(mng ruminant. MATERIALS
AND
METI-IODS
Spermine oxidase was assayed by the niethod of Blasehko and Hawes (2) with slight modi-
R E S U L T S AND D I S C U S S I O N
Results shown in Table 1 do not indicate any depression in the spermine oxidase levels with
Received for publication September 14, 1964. 73
74
E.
UEYAMA,
C.
L.
DAVIS,
AND
3I. J .
TABLE 1 Development of spermine oxidase in cahes fed different diets Calf
Feeding regimen "
Age of calf
Spermine oxidase
(DaBs)
(~l 0~/ 30 ~nin/0.5 ml semtm )
A A 7 9.7 A A 11 15.4 B A 14 39.0 B A 18 23.8 C B 4 8.9 C B 5 12.3 C B 9 11.3 D B 15 14.8 E C 15 14.8 E C 18 19.2 " A--ndlk, no ha)" or grain; B--milk replacer, hay and grain; C--milk replacer with fish meal protein, ha3', and grain. development of calves fed only on whole nfilk. The milk d i e t - f e d calves actually have slightly higher spernfine oxidase le-eels:at a given age than the animals fed the milk replacer diets plus hay and grain, although these differences are probably not significant. The main point, however, is that the diet expected to repress tureen development and function, i.e., the milk diet, did not repress the appearance of spernfine oxidase in the growing calves. This result does not rule out the possibility that bacterial development in the rumen is associated with plasma spennine oxidase development because bacteria are present in the undeveloped rumen of whole-milk-fed calves (3), although these bacteria are not necessarily representative oI' the bacteria of a functioning tureen. An experiment was p e r f o r m e d to determine whether injection in a growing calf would markedly affect the course of spernfine oxidase development. Two calves of a p p r o x i m a t e l y the same age on feeding Regnnen C were used in this experiment. One calf was injected in the j u g u l a r vein at 20, 21, 22, 27, and 29 days of age with 10 ml of a 1% sterile, neutral spermine solution. The other calf was k e p t as a control. Spermine oxidase levels of the control and i n j e c t e d calves at various periods between 19 and 30 days of age are shown in F i g u r e 1. S p e n n i n e injection did not a p p e a r to induce formation of large amounts of spermine oxidase. These results again are negative results and do not rule out the possibility of spermine induction of spernfine oxidase in the developing calf. Induction experiments were also attempted with a rabbit. The experiment was based on the possibility that the potential for forming
WOLIN
~ °~40
(-SPERMINE
_
,"
N
_z
~
/K 'SPERMtNE INJECTED:
--. 2 0 I
\%,
~START OF SPERMINE INJECTIONS
.% o 19
[
I
i
i
20
22
24
26
28
3(i)
AGE OF CALF (DAYS)
FIG. 1. Lack of influence of spernfine injection on serum spermine oxidase of developing calves. spermine oxidase was present in the rabbit, and the lack of f o n a a t i o n of plasma spennine oxidase was due to the lack of exposure to the potential inducer, spermine. Neutralized, sterile spermine was injected in the ear vein or intraperitoneally in an adult rabbit at a level of 0.01 mmole per kilogram of body weight. Samples of blood taken by heart puncture were assayed for spernfine oxidase, along with the hlood of a control untreated rabbit. Table 2 TABLE 2 Inability of spermine injection to induce a rabbit serum spermine oxidase Spernfine oxidase Time
In jettion
Spermine treated
Untreated
(M 0~/3'0 rain~ 0.5 ml semwn)
(Days)
o t)
I.V.
2• 4 (; 7 8 .~
I.V.
0.5
o.o
2.1 2.8
1.4 1.3
0.0
0.7
o.o
o.i;
i.P. I.P.
summarizes results obtained, and shows that spermine oxidase was not induced by spermine treatment of the rabbit. Addition of rabbit serum to adult bovine serum did not inhibit spernfine oxidase of the bovine serum nor was there any indication of a synergistic spermine oxidase activity. This result appears to rule out the possibility of an inhibitor of spermine oxidase in rabbit sera or
SPER3IINE
OXII)ASE
an activator in bovine sera for a possible latent spermine oxidase of the rabbit. Results of all of the above experiments argue against a direct effect of spermine or a product of microbial metabolism of the normally developing tureen on the presence of spernline oxidase in the bovine and on the increase in plasma levels of oxidase observed during the maturation of calves. We would emphasize, however, that these results do not completely rule out the possibility of an induction of enzyme activity by spermine produced by mieroorganisms in the rumen. It might be necessary to use genu-free ruminants to unequivocally rule out this possibility. There is no question of the relationship ot' spermine oxidase in plasma to ruminants because of the unique distribution of this enzyme in mammals. Except for the hippopota-
75
FOR3IAT1ON
nms and the two hyraeoid species studied only rulninant sera of all mammalian sera contain spermine oxidase (1, 2). Results presented in this paper would suggest that the capability for forming spermine oxidase is a constitutive expression of a unique genetic trait. The suggestion (1, 2) that spermine oxidase is an evolutionary adaptation to spermine production by microorganisms of special pouches developed for cellulose digestion is extremely interesting, but further experimental evidence is needed to relate the oxidase formation to rumen microbial activity. Finally, we would like to present the aceunmlated data of all of our experiments on the serum levels of bovines of different ages in Figure 2. No attempt has been made to distinguish between breeds or feeding treatments. These data of Figure 2 confirm results obtained
60
50
O O
C40
Oo
0
E
%
Oo
o
m
30 a~
o"
-%
¢°--
E o
0 20
I'0
O4 O
0
10
=
0
]*~IG. 2 .
20
40
60
80
age of calves ( d a y s ) "Increase of ,serum spermine oxidqse levels in deve}oping calves.
tOO
76
E. UEYAMA. C. L. DAVIS, AND M. J. WOLIN
by B l a s c h k o a n d H a T e s on the i n c r e a s e of s p e r m i n e oxidase in calf s e r u m w i t h increasi n g age (2).
REFERENCES (1) BLASCHKO, H., AND BO~NEY, R.
Spcrmine Oxidase and Benzylamine Oxidase. Distribution, Development and Substrate Specificity. Proc. Royal Soc. London, Series B, 156: 268. 1962.
(2) BLASCI-IKO, H., AND HA\VES, R.. Observatio~ls on Spermine Oxidase of Mammalian Plasma. J. Physiol., 145: 124. 1959. (3) BRYANT, M. P., SI~IALL, W., BoLivIA, C., .\ND ROBINSON, I. Studies on the Composition
of the Ruminal Flora and F a u n a of Young Calves. J. Dairy Set., 41: 1747. 1958. (4) TABOR, C. W., AND ROSENTHAL, S. M. Pharmacology of Spermine and Spermidine. Some Effects on Animals and Bacteria. J. Pharmacol., 116: 139. 1956.
(5) TABOR, H., TABOR, C. W., AND R.OSENTHAL, S. M. The Biochemistry of Polyamines: Spermine and Spermidine. Ann. Rev. Biochem., 30: 579. 1961.
(6) TA~IATE, H., MCGILLIARD, A. D., ,~ACOBSON, X. L., AND GETTY, :[~. Effect of Various Dietaries oll the Anatomical Development of the Stomach in the Calf. J. Dairy Sci., 45: 498. 1962.