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Fat Studies in Poultry
Fat Studies in Poultry 4. THE EFFECT OF TRITON WR-1339 ON TISSUE LIPID LEVELS IN COCKERELS B. MARCH AND J. BIELY Poultry Nutrition Laboratory], The University of British Columbia, Vancouver 8, B. C, Canada (Received for publication June 21, 1954)
I
EXPERIMENTAL AND RESULTS
Two preliminary experiments were carried out to determine if injections of Triton WR-1339 produced a toxic reaction in the fowl. Furthermore it was desired to ascertain if repeated injections of Triton were necessary and possible in order to obtain a prolonged effect. The birds used in the experiments were fed a commercial broiler ration. Experiment 1. In the first experiment a 12.5% solution of Triton WR-1339 was made up in a 0.9% saline solution buffered to pH 7.3 with Mcllvaine's buffer. Two ml. of this solution were injected into the wing veins of two six-week-old New
Hampshire cockerels. No immediate toxic reaction was observed to the injection of the surface active agent. Mention should be made of an edematous condition which was observed in some preliminary tests involving a course of daily injections (intravenous and subcutaneous) of Triton. Severe subcutaneous edema occurred apparently as the result of high levels of the surface active agent. The birds recovered when the injections were discontinued. Blood was drawn for fat analysis after 1, 2, 3, 4 and 6 days. Fat analysis was carried out according to the volumetric procedure of Allen (1938). It should be noted that the method does not measure phospholipids but includes neutral fat, sterols and sterol esters. The lipid levels are given in Table 1. Following a single intravenous injection of Triton, fat level in the blood reached its maximum after 2-3 days and then decreased. The average increase in fat level for the two birds was 125 mg./lOO cc. or 50%. Experiment 2. It was desired to study the effect of Triton injections upon fat deposition and consequently it was necesTABLE 1.—The effect of intravenous injection of 2 ml. of a 12.5% solution of Triton WR-1339 on blood lipid level in two cockerels
* Courtesy of Rohm and Haas Company, Philadelphia. A polymeric p-isooctyl polyoxyethylene. f Contribution No. 84.
293
Days
Cockerel #1, mg./lOO cc.
Cockerel #2, mg./lOO cc.
0 1 2 3 4 6
260 365 380 390 335 230
240 315 360 350 no detn. 300
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 18, 2015
N VIEW of the recent interest in the use of surface active agents in poultry nutrition it was considered of interest to determine what effect injection of a surface active agent might have upon fat deposition in the body of the fowl. It has been reported that injection of solution of the surface active agent, Triton WR-1339, into rabbits (Kellner, Correll and Ladd, 1951), mice (Cornforth et al., 1951), rats (Friedman and Byers, 1953) and ruminants (Chung and Shaw, 1951) resulted in an elevation of the lipid level in the blood. In the present experiments Triton WR1339* was the surface agent tested for its effect upon tissue lipid levels in cockerels.
294
B. MARCH AND J. BIELY
Experiment 3. Fifteen 8-week-old New Hampshire cockerels were used. All the birds were fed a commercial broiler ration throughout the experiment. Five of the birds were kept as controls, five received a single 2 ml. injection of Triton at the beginning of the experiment, and five birds received 1 ml. injections every 6 days (a total of seven injections) over the experimental period of 6 weeks. Injections TABLE 2.—The effect of subcutaneous injections of Triton WR-1339 on blood lipid level in cockerels over a period of 40 days Number of days on experiment 12 19 27 33 40 mg./lOO mg./lOO mg./lOO mg./lOO mg./lOO cc. cc. cc. cc. cc. Control
234
248
220
274
207
One injection a t beginning of experiment
363
435
427
310
215
Repeated injections a t 6-day-intervals
363
421
449
491
491
TABLE 3.—Moisture and fat levels in tissues from cockerels injected subcutaneously with Triton WR-1339 Av. wt. (gms.)
Wt. depot fat (gms.)
Liver HiO
fat
H,0
fat
Control
2,707
5.1
71.6
12.2
77.0
10.0
One injection at the beginning of experiment
2,580
3.7
73.0
14.2
77.8
8.0
Repeated injections at 6day-intervals
2,370
0.7
73.5
19.1
77.4
5.6
%
Tibial muscle
%
%
were made subcutaneously in the neck at the back of the head. Blood samples were taken for analysis at intervals as indicated in Table 2. Following the single 2 ml. injection of Triton the blood lipid level rose to approximately 430 mg./lOO cc. at between 2 and 3 weeks and decreased between 3-4 weeks after injection. In birds receiving the repeated injections, on the other hand, hyperlipemia was maintained until the end of the experiment. When the birds which had had the single injection had returned to normal all the birds on experiment were sacrificed. The liver and left tibial muscles (freed of adhering fat) were analyzed for their fat and moisture contents. The abdominal depot fat was also removed and weighed. The results of these analyses are summarized in Table 3. It will be seen that injection of Triton WR-1339 resulted in a marked increase in the amount of fat deposited in the liver. Repeated injections resulted in a greater increase in fat deposition than did a single injection. The amount of fat in the abdominal depots and in the tibial muscles was lowered by injection of the surface active agent. The greatest effect occurred as the result of repeated injections. DISCUSSION
Injection of Triton increased the lipid levels in the blood in cockerels. Kellner, Correll and Ladd (1951) and Cornforth
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 18, 2015
sary to use a method of administration which would give a more prolonged effect. Repeated intravenous injection is exceedingly difficult. Accordingly subcutaneous injections using undiluted Triton WR-1339 were tested. An eight-weekold cockerel was given 2 ml. injections subcutaneously at the back of the head on three successive days, without apparent toxic reaction. Blood lipid levels during this period were unchanged for 4 days after the first injection. After 7 days blood lipids had risen from 260 mg./lOO cc. to 380 mg./lOO cc. At 9 days the level was >500 mg./lOO cc. It was therefore concluded that, although response to subcutaneous injection of undiluted Triton was not so rapid as to intravenous injection of a Triton solution, the former method could be used to obtain a more prolonged response. In the next experiment, therefore, administration of Triton WR-1339 was by subcutaneous injection of the undiluted compound.
EFFECT OF SURFACTANT ON BLOOD LIPID
The lipemia which resulted from Triton injection was apparently quite different in its causative mechanism to lipemia induced by estrogen administration. Whereas prolonged lipemia from the latter is accompanied by increased fat deposition through the muscle tissue and in the abdominal depots, lipemia from Triton injection had the reverse effect. Even a single injection of Triton lowered the muscle and abdominal fat. The effect on tissue fat does not seem to be the same as for mice. With mice, Cornforth el al. (1951) found that Triton did not have any apparent effect on the fat depots although it was noted that the adrenal cortex was stripped of fat. These investigators found some decrease of fat in the liver. This latter observation is contrary to the findings with cockerels in the present experiment where there was a marked increase in the fat content of the livers as a result of Triton injections. Injection of Triton, while producing lipemia, decreased the deposition of fat in
cockerels. The association of lipemia and decreased fat deposition in the present experiment cannot be attributed to withdrawal of fat from the depots because fat so mobilized would have appeared in the blood in the form of phospholipids which were not detected by the analytical method used. On the contrary the mechanism by which the blood lipid levels was increased apparently involved the obstruction of removal of fat from the blood. SUMMARY
Triton WR-1339 has been injected subcutaneously into six-week-old cockerels. The effects on the amount of fat present in the blood, muscle, liver and abdominal fat depots were as follows: (1) A single injection produced temporary lipemia, repeated injections maintained an elevated blood level. (2) Liver fat was increased. (3) Tibial muscle fat was decreased. (4) Abdominal depot fat was decreased. ACKNOWLEDGMENT
The authors acknowledge the technical assistance of Miss Freda Morel in carrying out the above experiments. REFERENCES Allen, N. N., 1938. Blood fat of dairy cattle. Minnesota Tech. Bull. 130. Chung, A. C , and J. C. Shaw, 1951. Sustained elevation of blood lipides and effect upon milk production of ruminants given a surface active agent intravenously. J. Dairy Sci. 34: 11801185. Cornforth, J. W., P. D'A. Hart, R. J. W. Rees and J. A. Stock, 1951. Antituberculous effect of certain surface active polyoxyethylene ethers in mice. Nature, 168: 150-153. Friedman, M., and S. O. Byers, 1953. The mechanism responsible for the hypercholesteremia induced by Triton WR-1339. J. Exptl. Med. 97: 117-130. Kellner, A., J. W. Correll and A. T. Ladd, 1951. Sustained hyperlipemia induced in rabbits by means of intravenously injected surface active agents. J. Exptl. Med. 93: 373-383.
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 18, 2015
el al. (1951) found that with rabbits and mice respectively, intravenous injection of Triton was followed immediately by convulsions which, however, were transitory and appeared to leave no after-effects. In the present experiments using cockerels no toxic reactions of this type were observed. With reference to the subcutaneous edema occurring upon frequent administration of Triton, Cornforth el al. (1951) have likewise reported edema in mice when injected with Triton. Friedman and Byers (1953) noted that the dry weight of tissues in Triton-injected rats was consistently smaller than normal. In the case of the cockerels in the present experiment, however, injection with Triton did not alter the moisture content of the tissues analyzed in spite of the differences in fat content which are usually accompanied by differences in moisture content.
295
I
EXPERIMENTAL AND RESULTS
Two preliminary experiments were carried out to determine if injections of Triton WR-1339 produced a toxic reaction in the fowl. Furthermore it was desired to ascertain if repeated injections of Triton were necessary and possible in order to obtain a prolonged effect. The birds used in the experiments were fed a commercial broiler ration. Experiment 1. In the first experiment a 12.5% solution of Triton WR-1339 was made up in a 0.9% saline solution buffered to pH 7.3 with Mcllvaine's buffer. Two ml. of this solution were injected into the wing veins of two six-week-old New
Hampshire cockerels. No immediate toxic reaction was observed to the injection of the surface active agent. Mention should be made of an edematous condition which was observed in some preliminary tests involving a course of daily injections (intravenous and subcutaneous) of Triton. Severe subcutaneous edema occurred apparently as the result of high levels of the surface active agent. The birds recovered when the injections were discontinued. Blood was drawn for fat analysis after 1, 2, 3, 4 and 6 days. Fat analysis was carried out according to the volumetric procedure of Allen (1938). It should be noted that the method does not measure phospholipids but includes neutral fat, sterols and sterol esters. The lipid levels are given in Table 1. Following a single intravenous injection of Triton, fat level in the blood reached its maximum after 2-3 days and then decreased. The average increase in fat level for the two birds was 125 mg./lOO cc. or 50%. Experiment 2. It was desired to study the effect of Triton injections upon fat deposition and consequently it was necesTABLE 1.—The effect of intravenous injection of 2 ml. of a 12.5% solution of Triton WR-1339 on blood lipid level in two cockerels
* Courtesy of Rohm and Haas Company, Philadelphia. A polymeric p-isooctyl polyoxyethylene. f Contribution No. 84.
293
Days
Cockerel #1, mg./lOO cc.
Cockerel #2, mg./lOO cc.
0 1 2 3 4 6
260 365 380 390 335 230
240 315 360 350 no detn. 300
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 18, 2015
N VIEW of the recent interest in the use of surface active agents in poultry nutrition it was considered of interest to determine what effect injection of a surface active agent might have upon fat deposition in the body of the fowl. It has been reported that injection of solution of the surface active agent, Triton WR-1339, into rabbits (Kellner, Correll and Ladd, 1951), mice (Cornforth et al., 1951), rats (Friedman and Byers, 1953) and ruminants (Chung and Shaw, 1951) resulted in an elevation of the lipid level in the blood. In the present experiments Triton WR1339* was the surface agent tested for its effect upon tissue lipid levels in cockerels.
294
B. MARCH AND J. BIELY
Experiment 3. Fifteen 8-week-old New Hampshire cockerels were used. All the birds were fed a commercial broiler ration throughout the experiment. Five of the birds were kept as controls, five received a single 2 ml. injection of Triton at the beginning of the experiment, and five birds received 1 ml. injections every 6 days (a total of seven injections) over the experimental period of 6 weeks. Injections TABLE 2.—The effect of subcutaneous injections of Triton WR-1339 on blood lipid level in cockerels over a period of 40 days Number of days on experiment 12 19 27 33 40 mg./lOO mg./lOO mg./lOO mg./lOO mg./lOO cc. cc. cc. cc. cc. Control
234
248
220
274
207
One injection a t beginning of experiment
363
435
427
310
215
Repeated injections a t 6-day-intervals
363
421
449
491
491
TABLE 3.—Moisture and fat levels in tissues from cockerels injected subcutaneously with Triton WR-1339 Av. wt. (gms.)
Wt. depot fat (gms.)
Liver HiO
fat
H,0
fat
Control
2,707
5.1
71.6
12.2
77.0
10.0
One injection at the beginning of experiment
2,580
3.7
73.0
14.2
77.8
8.0
Repeated injections at 6day-intervals
2,370
0.7
73.5
19.1
77.4
5.6
%
Tibial muscle
%
%
were made subcutaneously in the neck at the back of the head. Blood samples were taken for analysis at intervals as indicated in Table 2. Following the single 2 ml. injection of Triton the blood lipid level rose to approximately 430 mg./lOO cc. at between 2 and 3 weeks and decreased between 3-4 weeks after injection. In birds receiving the repeated injections, on the other hand, hyperlipemia was maintained until the end of the experiment. When the birds which had had the single injection had returned to normal all the birds on experiment were sacrificed. The liver and left tibial muscles (freed of adhering fat) were analyzed for their fat and moisture contents. The abdominal depot fat was also removed and weighed. The results of these analyses are summarized in Table 3. It will be seen that injection of Triton WR-1339 resulted in a marked increase in the amount of fat deposited in the liver. Repeated injections resulted in a greater increase in fat deposition than did a single injection. The amount of fat in the abdominal depots and in the tibial muscles was lowered by injection of the surface active agent. The greatest effect occurred as the result of repeated injections. DISCUSSION
Injection of Triton increased the lipid levels in the blood in cockerels. Kellner, Correll and Ladd (1951) and Cornforth
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 18, 2015
sary to use a method of administration which would give a more prolonged effect. Repeated intravenous injection is exceedingly difficult. Accordingly subcutaneous injections using undiluted Triton WR-1339 were tested. An eight-weekold cockerel was given 2 ml. injections subcutaneously at the back of the head on three successive days, without apparent toxic reaction. Blood lipid levels during this period were unchanged for 4 days after the first injection. After 7 days blood lipids had risen from 260 mg./lOO cc. to 380 mg./lOO cc. At 9 days the level was >500 mg./lOO cc. It was therefore concluded that, although response to subcutaneous injection of undiluted Triton was not so rapid as to intravenous injection of a Triton solution, the former method could be used to obtain a more prolonged response. In the next experiment, therefore, administration of Triton WR-1339 was by subcutaneous injection of the undiluted compound.
EFFECT OF SURFACTANT ON BLOOD LIPID
The lipemia which resulted from Triton injection was apparently quite different in its causative mechanism to lipemia induced by estrogen administration. Whereas prolonged lipemia from the latter is accompanied by increased fat deposition through the muscle tissue and in the abdominal depots, lipemia from Triton injection had the reverse effect. Even a single injection of Triton lowered the muscle and abdominal fat. The effect on tissue fat does not seem to be the same as for mice. With mice, Cornforth el al. (1951) found that Triton did not have any apparent effect on the fat depots although it was noted that the adrenal cortex was stripped of fat. These investigators found some decrease of fat in the liver. This latter observation is contrary to the findings with cockerels in the present experiment where there was a marked increase in the fat content of the livers as a result of Triton injections. Injection of Triton, while producing lipemia, decreased the deposition of fat in
cockerels. The association of lipemia and decreased fat deposition in the present experiment cannot be attributed to withdrawal of fat from the depots because fat so mobilized would have appeared in the blood in the form of phospholipids which were not detected by the analytical method used. On the contrary the mechanism by which the blood lipid levels was increased apparently involved the obstruction of removal of fat from the blood. SUMMARY
Triton WR-1339 has been injected subcutaneously into six-week-old cockerels. The effects on the amount of fat present in the blood, muscle, liver and abdominal fat depots were as follows: (1) A single injection produced temporary lipemia, repeated injections maintained an elevated blood level. (2) Liver fat was increased. (3) Tibial muscle fat was decreased. (4) Abdominal depot fat was decreased. ACKNOWLEDGMENT
The authors acknowledge the technical assistance of Miss Freda Morel in carrying out the above experiments. REFERENCES Allen, N. N., 1938. Blood fat of dairy cattle. Minnesota Tech. Bull. 130. Chung, A. C , and J. C. Shaw, 1951. Sustained elevation of blood lipides and effect upon milk production of ruminants given a surface active agent intravenously. J. Dairy Sci. 34: 11801185. Cornforth, J. W., P. D'A. Hart, R. J. W. Rees and J. A. Stock, 1951. Antituberculous effect of certain surface active polyoxyethylene ethers in mice. Nature, 168: 150-153. Friedman, M., and S. O. Byers, 1953. The mechanism responsible for the hypercholesteremia induced by Triton WR-1339. J. Exptl. Med. 97: 117-130. Kellner, A., J. W. Correll and A. T. Ladd, 1951. Sustained hyperlipemia induced in rabbits by means of intravenously injected surface active agents. J. Exptl. Med. 93: 373-383.
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 18, 2015
el al. (1951) found that with rabbits and mice respectively, intravenous injection of Triton was followed immediately by convulsions which, however, were transitory and appeared to leave no after-effects. In the present experiments using cockerels no toxic reactions of this type were observed. With reference to the subcutaneous edema occurring upon frequent administration of Triton, Cornforth el al. (1951) have likewise reported edema in mice when injected with Triton. Friedman and Byers (1953) noted that the dry weight of tissues in Triton-injected rats was consistently smaller than normal. In the case of the cockerels in the present experiment, however, injection with Triton did not alter the moisture content of the tissues analyzed in spite of the differences in fat content which are usually accompanied by differences in moisture content.
295