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Time Course Study of Liver Lipid, Mixed-Function Oxidase, and Estradiol Metabolism in Laying Hens Fed Different Diets1
Time Course Study of Liver Lipid, Mixed-Function Oxidase, and Estradiol Metabolism in Laying Hens Fed Different Diets1 KAZUAKI TAKAHASHI and LEO S. JENSEN Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication October 29, 1986)
1987 Poultry Science 66:1660-1664 INTRODUCTION
Studies in our laboratory have shown that feeding fish meal, alfalfa meal, and torula yeast to laying hens resulted in a reduction in liver lipid content and plasma estradiol (E2) concentration as compared with hens fed a corn-soy (CS) diet (Maurice et al., 1979; Akiba et al., 1982, 1983). Feeding similar diets to estrogenized male broiler chicks resulted in a significant reduction in liver lipid and plasma E2 concentration (Akiba et al., 1982; Takahashi and Jensen, 1984). Hepatic microsomes are a major site of degradation of steroids, including E2. Our previous report with growing birds (Takahashi and Jensen, 1984) showed that hepatic microsomal aniline hydroxylase activity and content of cytochrome P-450 were significantly greater in birds fed an FAY diet with and without E2 administration than in those fed a SC diet. These effects were associated with a significant reduction in liver fat and plasma estradiol concentration in estrogenized chicks fed the FAY diet. In
'Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Station of the University of Georgia. 2 To whom correspondence should be addressed.
in vitro studies, conversion of radioactive E2 into the water soluble fraction was significantly greater in microsomes from chicks fed the FAY diet than in those from chicks fed the CS diet. Brenes et al. (1985) observed significantly greater activity of hepatic microsomal aniline hydroxylase and aminopyrine N-demethylase in young chicks fed an FAY diet, and in those fed diets with other feed ingredients, compared with those fed a simple CS diet. The present study was conducted to determine if mixed-function oxidase (MFO) activity in laying hens fed the FAY diet would be modified compared with that in birds fed the CS diet. An additional objective was to determine if the previously reported relationship between MFO activity, plasma E2 concentration, and liver lipid in estrogenized chicks fed the FAY diet would be the same in laying hens fed a similar FAY diet. MATERIALS AND METHODS
White Leghorn hens 45 wk of age of a commercial strain were housed individually in wire cages, given feed and water ad libitum, and subjected to a daily photoperiod of 15 h light; 9 h dark. The hens were maintained on a regular University of Georgia (UGA) stock diet until the beginning of the experiment as described in
1660
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ABSTRACT The purpose of this investigation was to determine if hepatic mixed-function oxidase (MFO) activity, plasma estradiol, and estradiol metabolism changed in concert with liver lipid content when laying hens were fed two diets of different composition. White Leghorn laying hens were fed either a corn-soybean meal (CS) diet or CS plus fish meal, alfalfa meal, and torula yeast (FAY) for 24 days. Hens were sampled at the start of the experiment and at 4, 24, 10, 17, and 24 days after the dietary changes. By Day 17, liver weight per unit of body weight and liver lipid content were significantly lower for hens fed the FAY diet compared to those fed the CS diet. Plasma estradiol concentration was significantly higher for hens fed the FAY diet at 4 days, but a reversal occurred at later times and estradiol concentration tended to be higher for birds fed the CS diet for the remainder of the experiment. An in vitro estimate of the estradiol metabolism in the liver showed that by Day 17 the quantity of radioactive estradiol converted to water soluble metabolites was significantly increased in samples from hens fed the FAY diet. Feeding the FAY diet also significantly increased hepatic aminopyrine demethylation and aniline hydroxylation at Day 24. (Key words: diet, composition, estradiol metabolism, layers, liver lipid, mixed-function oxidase, plasma estradiol)
TIME COURSE STUDY ON LIVER LIPID T A B L E 1. Composition
1661
of diets
CS1
Corn, yellow S o y b e a n meal, dehulled Fish meal, m e n h a d e n Alfalfa meal Torula yeast P o u l t r y fat Limestone Dicalcium p h o s p h a t e Salt DL-Methionine Vitamin m i x 2 Trace mineral m i x 3 Calculated c o m p o s i t i o n Metabolizable energy, kcal/kg Crude protein, % Crude fat, % Crude fiber, % T o t a l sulfur amino acids, % Lysine, %
67.60 19.85
FAY1
•\">r
1
• -
2.00 8.22 1.48 .45 .10 .25 .05
65.99 7.20 5.00 5.00 5.00 2.59 7.80 .65 .41 .06 .25 .05
2,944 15.00 4.79 1.95 .62 .76
2,944 15.00 6.07 2.89 .62 .72
CS = Corn-soybean meal diet; F A Y = a diet containing fish meal, alfalfa meal, and torula yeast.
2
Supplying per kilogram of feed: vitamin A, 5,000 IU; vitamin D 3 , 1,100 ICU; vitamin E, 11 IU; m e n a d i o n e sodium bisulfite, 1.1 m g ; riboflavin, 4.4 m g ; Ca p a n t o t h e n a t e , 12.0 mg; nicotinic acid, 4 4 . 0 m g ; choline chloride, 2 2 0 m g ; p y r i d o x i n e HCI, 2.2 m g ; folic acid, .55 mg; D-biotin, .11 m g ; thiamine m o n o n i t r a t e , 2.2 mg; vitamin B 1 2 , 6.6 Mg3
Supplying milligrams per kilogram of feed: manganese, 6 0 ; z i n c , 5 0 ; i r o n , 30; copper, 5 ; i o d i n e , 1.05.
the report of Akiba and Jensen (1983). Seventytwo hens were used in the study, eight of which were killed at the start of the experiment. Onehalf of the remainder were fed the CS diet and the other half the FAY diet (Table 1). Eight hens from each dietary treatment were randomly sampled at 4, 7, 14, and 24 days after start of the experiment. Heparinized blood samples were obtained from the wing vein 6 h after oviposition each sampling day for the determination of plasma estradiol concentration. Hens were then killed by cervical dislocation and the livers were removed and weighed. The liver homogenates (20% liver) were prepared in Krebs Ringer Phosphate Buffer (pH 7.4, 0 C) with a Potter-Elvehjem homogenizer. The homogenates were centrifuged at 9,000 x g for 10 min at 4 C. The E 2 metabolism was determined by a slight modification of the method of LaCroix andEechaute (1979). Instead of microsomes of homogenates, a .5 ml aliquot of the 9,000 x g supernatant containing about 850 mg protein was used in this assay. The activities of aniline hydroxylase and aminopyrine N-demethylase in the 9,000 x g
supernatant were determined by the method of Kato and Gillette (1965). Plasma estradiol concentration was determined using the method of Tojo and Huston (1980). Liver lipid was determined by the method of Folch et al. 1957). Data were analyzed using the general linear models procedure of Statistical Analysis System (1982). Means were separated by Duncan's multiple range test, with significance at P<.05 unless otherwise noted. RESULTS
Values for the various parameters obtained from the hens fed the two different diets and sampled at the end of the experiment (24 days) were compared with those obtained from hens sampled at the beginning of the experiment (Table 2). Neither diet significantly affected body weight of the hens but liver weight per unit of body weight was significantly increased in birds fed the CS diet. Liver lipid was also significantly increased by the CS diet. Hydroxylation of aniline, demethylation of aminopyrine,
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Ingredient
1662
TAKAHASHI AND JENSEN TABLE 2. Effect of corn-soybean meal (CS) and fish meal, alfalfa meal, and torula meal (FAY) diets fed to laying hens for 24 days on body weight, liver parameters, and plasma E2 0 days'
24 days FAY
CS
Parameter B o d y weight, g Liver weight, g / 1 0 0 g b o d y weight Liver lipid, % (wet weight basis) H y d r o x y l a t i o n of aniline 2 D e m e t h y l a t i o n of a m i n o p y r i n e 3 Plasma E 2 , p g / m L E 2 Metabolism 4
1,645 2.57 10.0 2.05 34.8 121 20.6
± 38a ± .06a ± .8a ± .19a ± 2.0a ± 10a ± .8a
1,698 3.03 16.8 2.34 35.1 179 20.0
±44a ± .18b ± 2.7b ± .18a ± 1.8 a ± 27a ± 1.4 a
1,699 2.50 10.9 3.26 51.9 145 29.2
± 53a ± .12a ± 1.2 a ± .05° ± 1.9 b ± 17a + ,9b
ab ' Values within a row with different superscripts are significantly different (P<.05).
2
/Vmoles P-aminophenol/20 min per milligrams of 9,000 X g supernatant protein.
3
Nmoles HCHO/20 min per milligrams of 9,000 X g supernatant protein.
4
Percentage of 14 C activity in aqueous medium after extraction with ether/100 mg 9,000 X g supernatant protein per 40 min.
and E2 metabolism were significantly increased by feeding the FAY diet as compared with the CS diet. Although the plasma E2 concentration was higher for the hens fed the CS diet, the
value was not significantly different from that for hens fed the FAY diet. Temporal measurements of the various parameters are presented in Figures 1,2, and 3. Both liver weight per unit of body weight and liver lipid of chickens fed the two diets because significantly different by 17 days after the start
210 180 Pd
150
o e E ^ g S 120 90
24 31 r E " £
•2 •- 5 o o _ -§2 S
27
lis23 o S
.t-
10
17
24
Time (day)
Time (day) FIGURE 1. Effect of dietary composition on liver weight and lipid in laying hens (values ± standard error). * indicates significant difference between dietary treatments (P<.05).
FIGURE 2. Effect of dietary composition on plasma E, and hepatic E2 metabolism on laying hens (values ± standard error). * indicates significant diffferences between dietary treatments (P<.05).
Downloaded from http://ps.oxfordjournals.org/ at Mount Royal College on June 9, 2015
'Means ± standard error of the mean (8 observations). Hens were fed the University of Georgia layer breeder stock diet prior to start of the experiment.
TIME COURSE STUDY ON LIVER LIPID
10 Time (day)
17
24
FIGURE 3. Effect of dietary composition on activity of aniline hydroxylase (A) and aminopyrine N-demethylase in laying hens (B) (values ± standard error). * indicates significant difference between dietary treatments (P<.05).
of the experiment (Figure 1). Plasma E2 concentration sampled at four days was significantly higher for birds fed the FAY diet than for birds fed the CS diet. This pattern was reversed during subsequent measurements, with higher values being observed for birds fed the CS diet. However, differences were not statistically significant (P>.05) at any other sampling period (Figure 2). In vitro estimation of E2 metabolism showed a significant increase by 17 days for birds fed the FAY diet when compared with values for birds fed the CS diet and the difference continued to widen at the 24-day sampling period (Figure 2). Aniline hydroxylase activity was consistently higher for birds fed the FAY diet but the difference was statistically significant only at the 24-day sampling period (Figure 3). A similar trend was observed for aminopyrine N-demethylase activity and the differences were significant at both the 10 and 24day sampling periods (Figure 3). DISCUSSION
The results of this experiment with laying hens fed CS or FAY diets are similar to those observed with estrogenized chicks (Takahashi and Jensen, 1984). A period of about 10 to 17 days on the experimental diets was required be-
fore differences became significant. Although statistically significant differences in plasma estradiol concentrations were not observed in hens fed the two different diets, differences observed in mean values were similar to those previously observed in other studies in our laboratory. For example, Akiba and Jensen (1983) observed a significantly higher estradiol concentration in the plasma of hens fed a CS diet for 7 days than in hens fed a diet with 10% fish meal. A relationship between the appearance of excess lipid deposition in the liver with increased plasma E2 concentration, and decreased estrogen metabolism and MFO activity is indicated when results of feeding the different diets are compared. A comparison however, of these values and those in birds fed the control diets fails to demonstrate a definite correlation between MFO activity and lipid accumulation (Table 2). Although feeding the CS diet increased hepatic lipid content, it did not decrease values below those in controls. In order to explain the effect of dietary changes on liver lipid accumulation in laying hens, (Jensen et al., 1984) hypothesize that optimum activation of the microsomal MFO system does not occur in hens fed a simple corn-soybean meal diet. Various feed ingredients included in diets which reduce liver lipid accumulation may contain a factor (or factors) which stimulates the MFO system to degrade the more rapidly circulating estradiol. The reduced level of estradiol in turn would result in reduced hepatic lipogenesis and less liver lipid accumulation. Results of this study with the FAY diet and other investigations with chicks (Takahashi and Jensen, 1984; Brenes et al., 1985) indicate increased activity of the MFO system resulting from feeding of the more complex diets. Although the activities of aniline hydroxylase and aminopyrine demethylase have been used as general indicators of the state of activation of the MFO system, they do not necessarily indicate that the enzymes involved in the estradiol degradation are specifically activated. The estradiol metabolism studies with liver microsomes from chicks (Takahashi and Jensen, 1984) and liver supernatant in this study support the suggestion that estradiol is metabolized more rapidly in birds fed the more complex diets. The nature of the factor or factors in fish meal, alfalfa meal, and torula yeast that result(s) in changes in MFO activity remains to be determined. There are a number of dietary factors that have been shown to affect microsomal MFO
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TAKAHASHI AND JENSEN
REFERENCES Akiba, Y., and L. S. Jensen, 1983. Temporal effect of change in diet composition on plasma estradiol and thyroxine concentrations and hepatic lipogenesis in laying hens. J. Nutr. 113:2178-2184. Akiba, Y., L. S. Jensen, C. R. Barb, and R. R. Kraeling, 1982. Plasma estradiol, thyroid hormones and liver lipid content in laying hens fed different isocaloric diets. J. Nutr. 112:299-308. Akiba, Y., L. S. Jensen and C. X. Mendonca, 1983. Laboratory model with chicks for assay of nutritional factors affecting hepatic lipid accumulation in laying hens. Poultry Sci. 62:143-161. Anderson, K. E., A. H. Connery, and A. Kappas, 1982. Nutritional influence on chemical biotransformations in humans. Nutr. Rev. 40:161-171.
Brenes, A., L. S. Jensen, and H. M. Cervantes, 1985. Activation of hepatic microsomal mixed function oxidase system in broiler chicks by diet changes. Poultry Sci. 64:963-968. Folch, J., M. Lee, and G. H. Sloane-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Haghighi-Rad, F., and D. Polin, 1982a. The unidentified factor for alleviating fatty liver-hemorrhagic syndrome. Poultry Sci. 61:2075-2082. Haghighi-Rad, F., and D. Polin, 1982b. Lipid alleviates fatty liver hemorrhagic syndrome. Poultry Sci. 61:2465-2472. Hathcock, J. N., and J. Coon, 1978. Nutrition and Drug Interactions. Academic Press, New York, NY. Jensen, L. S., A. Brenes, and K. Takahashi, 1984. Association of reduced plasma estrogen and hepatic lipids with increased activity of the microsomal mixed function oxidase system in chickens. Pages 348-350 in Proc. World's Poult. Cong., Helsinki, Finland. Kato, R., and J. R. Gillette, 1965. Sex differences in the effects of abnormal physiological states on metabolism of drugs by rat liver microsomes. J. Pharmacol. Exp. Ther. 150:285-291. LaCroix, E., and W. Eechaute, 1979. Estradiol metabolism by liver homogenates and microsomes of normal and cirrhotic male rats. Horm. Res. (Basel) 11:69-80. Maurice, D. V., and L. S. Jensen, 1978. Liver lipid deposition in caged layers as influenced by fermentation by-products and level of dietary fat. Poultry Sci. 57:1690-1695. Maurice, D. V., L. S. Jensen, andH. Tojo, 1979. Comparison of fish meal and soybean meal in prevention of fatty liver-hemorrhagic syndrome in caged layer. Poultry Sci. 58:864-870. Statistical Analysis System, 1982. SAS User's Guide. SAS Institute, Inc. Raleigh, NC. Takahashi, K., and L. S. Jensen, 1984. Effect of dietary composition and estradiol implants on hepatic microsomal mixed function oxidase and lipid metabolism in growing chicks. Poultry Sci. 63:2217-2224. Tojo, H., and T. M. Huston, 1980. Effects of environmental temperature on the concentration of serum estradiol, progesterone and calcium in maturing female domestic fow. Poultry Sci. 59:2797-2802. Wade, A. E., and W. P. Norred, 1976. Effect of dietary lipid on drug-metabolizing enzymes. Fed. Proc. 35:2475-2479.
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activity in other species (Hathcock and Coon, 1978; Takahashi and Jensen, 1984; Brenes et al., 1985). In many cases, deficiencies of specific nutrients have been shown to reduce enzyme activity. Both the CS and FAY diets contain similar nutrient profiles and were adequate in nutrients to maintain performance of laying hens. Haghighi-Rad and Polin (1982a,b) proposed that the addition of lipid (corn oil) to the diet reduced liver lipid content and hemorrhages in laying hens. Wade and Norred (1976) showed that essential fatty acids were necessary for maximal activation of the MFO system in rats. More recent studies by Anderson etal., (1982), however, failed to provide evidence for an important influence of dietary lipid content on hepatic drug oxidations. The FAY diet contained slightly more total crude fat than the CS diet in the present investigation, but previous results in our laboratory demonstrated that a significant reduction in liver fat was still observed in birds fed a more complex diet compared to those fed CS when the diets contained equal levels of fat (Maurice and Jensen, 1978).
1987 Poultry Science 66:1660-1664 INTRODUCTION
Studies in our laboratory have shown that feeding fish meal, alfalfa meal, and torula yeast to laying hens resulted in a reduction in liver lipid content and plasma estradiol (E2) concentration as compared with hens fed a corn-soy (CS) diet (Maurice et al., 1979; Akiba et al., 1982, 1983). Feeding similar diets to estrogenized male broiler chicks resulted in a significant reduction in liver lipid and plasma E2 concentration (Akiba et al., 1982; Takahashi and Jensen, 1984). Hepatic microsomes are a major site of degradation of steroids, including E2. Our previous report with growing birds (Takahashi and Jensen, 1984) showed that hepatic microsomal aniline hydroxylase activity and content of cytochrome P-450 were significantly greater in birds fed an FAY diet with and without E2 administration than in those fed a SC diet. These effects were associated with a significant reduction in liver fat and plasma estradiol concentration in estrogenized chicks fed the FAY diet. In
'Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Station of the University of Georgia. 2 To whom correspondence should be addressed.
in vitro studies, conversion of radioactive E2 into the water soluble fraction was significantly greater in microsomes from chicks fed the FAY diet than in those from chicks fed the CS diet. Brenes et al. (1985) observed significantly greater activity of hepatic microsomal aniline hydroxylase and aminopyrine N-demethylase in young chicks fed an FAY diet, and in those fed diets with other feed ingredients, compared with those fed a simple CS diet. The present study was conducted to determine if mixed-function oxidase (MFO) activity in laying hens fed the FAY diet would be modified compared with that in birds fed the CS diet. An additional objective was to determine if the previously reported relationship between MFO activity, plasma E2 concentration, and liver lipid in estrogenized chicks fed the FAY diet would be the same in laying hens fed a similar FAY diet. MATERIALS AND METHODS
White Leghorn hens 45 wk of age of a commercial strain were housed individually in wire cages, given feed and water ad libitum, and subjected to a daily photoperiod of 15 h light; 9 h dark. The hens were maintained on a regular University of Georgia (UGA) stock diet until the beginning of the experiment as described in
1660
Downloaded from http://ps.oxfordjournals.org/ at Mount Royal College on June 9, 2015
ABSTRACT The purpose of this investigation was to determine if hepatic mixed-function oxidase (MFO) activity, plasma estradiol, and estradiol metabolism changed in concert with liver lipid content when laying hens were fed two diets of different composition. White Leghorn laying hens were fed either a corn-soybean meal (CS) diet or CS plus fish meal, alfalfa meal, and torula yeast (FAY) for 24 days. Hens were sampled at the start of the experiment and at 4, 24, 10, 17, and 24 days after the dietary changes. By Day 17, liver weight per unit of body weight and liver lipid content were significantly lower for hens fed the FAY diet compared to those fed the CS diet. Plasma estradiol concentration was significantly higher for hens fed the FAY diet at 4 days, but a reversal occurred at later times and estradiol concentration tended to be higher for birds fed the CS diet for the remainder of the experiment. An in vitro estimate of the estradiol metabolism in the liver showed that by Day 17 the quantity of radioactive estradiol converted to water soluble metabolites was significantly increased in samples from hens fed the FAY diet. Feeding the FAY diet also significantly increased hepatic aminopyrine demethylation and aniline hydroxylation at Day 24. (Key words: diet, composition, estradiol metabolism, layers, liver lipid, mixed-function oxidase, plasma estradiol)
TIME COURSE STUDY ON LIVER LIPID T A B L E 1. Composition
1661
of diets
CS1
Corn, yellow S o y b e a n meal, dehulled Fish meal, m e n h a d e n Alfalfa meal Torula yeast P o u l t r y fat Limestone Dicalcium p h o s p h a t e Salt DL-Methionine Vitamin m i x 2 Trace mineral m i x 3 Calculated c o m p o s i t i o n Metabolizable energy, kcal/kg Crude protein, % Crude fat, % Crude fiber, % T o t a l sulfur amino acids, % Lysine, %
67.60 19.85
FAY1
•\">r
1
• -
2.00 8.22 1.48 .45 .10 .25 .05
65.99 7.20 5.00 5.00 5.00 2.59 7.80 .65 .41 .06 .25 .05
2,944 15.00 4.79 1.95 .62 .76
2,944 15.00 6.07 2.89 .62 .72
CS = Corn-soybean meal diet; F A Y = a diet containing fish meal, alfalfa meal, and torula yeast.
2
Supplying per kilogram of feed: vitamin A, 5,000 IU; vitamin D 3 , 1,100 ICU; vitamin E, 11 IU; m e n a d i o n e sodium bisulfite, 1.1 m g ; riboflavin, 4.4 m g ; Ca p a n t o t h e n a t e , 12.0 mg; nicotinic acid, 4 4 . 0 m g ; choline chloride, 2 2 0 m g ; p y r i d o x i n e HCI, 2.2 m g ; folic acid, .55 mg; D-biotin, .11 m g ; thiamine m o n o n i t r a t e , 2.2 mg; vitamin B 1 2 , 6.6 Mg3
Supplying milligrams per kilogram of feed: manganese, 6 0 ; z i n c , 5 0 ; i r o n , 30; copper, 5 ; i o d i n e , 1.05.
the report of Akiba and Jensen (1983). Seventytwo hens were used in the study, eight of which were killed at the start of the experiment. Onehalf of the remainder were fed the CS diet and the other half the FAY diet (Table 1). Eight hens from each dietary treatment were randomly sampled at 4, 7, 14, and 24 days after start of the experiment. Heparinized blood samples were obtained from the wing vein 6 h after oviposition each sampling day for the determination of plasma estradiol concentration. Hens were then killed by cervical dislocation and the livers were removed and weighed. The liver homogenates (20% liver) were prepared in Krebs Ringer Phosphate Buffer (pH 7.4, 0 C) with a Potter-Elvehjem homogenizer. The homogenates were centrifuged at 9,000 x g for 10 min at 4 C. The E 2 metabolism was determined by a slight modification of the method of LaCroix andEechaute (1979). Instead of microsomes of homogenates, a .5 ml aliquot of the 9,000 x g supernatant containing about 850 mg protein was used in this assay. The activities of aniline hydroxylase and aminopyrine N-demethylase in the 9,000 x g
supernatant were determined by the method of Kato and Gillette (1965). Plasma estradiol concentration was determined using the method of Tojo and Huston (1980). Liver lipid was determined by the method of Folch et al. 1957). Data were analyzed using the general linear models procedure of Statistical Analysis System (1982). Means were separated by Duncan's multiple range test, with significance at P<.05 unless otherwise noted. RESULTS
Values for the various parameters obtained from the hens fed the two different diets and sampled at the end of the experiment (24 days) were compared with those obtained from hens sampled at the beginning of the experiment (Table 2). Neither diet significantly affected body weight of the hens but liver weight per unit of body weight was significantly increased in birds fed the CS diet. Liver lipid was also significantly increased by the CS diet. Hydroxylation of aniline, demethylation of aminopyrine,
Downloaded from http://ps.oxfordjournals.org/ at Mount Royal College on June 9, 2015
Ingredient
1662
TAKAHASHI AND JENSEN TABLE 2. Effect of corn-soybean meal (CS) and fish meal, alfalfa meal, and torula meal (FAY) diets fed to laying hens for 24 days on body weight, liver parameters, and plasma E2 0 days'
24 days FAY
CS
Parameter B o d y weight, g Liver weight, g / 1 0 0 g b o d y weight Liver lipid, % (wet weight basis) H y d r o x y l a t i o n of aniline 2 D e m e t h y l a t i o n of a m i n o p y r i n e 3 Plasma E 2 , p g / m L E 2 Metabolism 4
1,645 2.57 10.0 2.05 34.8 121 20.6
± 38a ± .06a ± .8a ± .19a ± 2.0a ± 10a ± .8a
1,698 3.03 16.8 2.34 35.1 179 20.0
±44a ± .18b ± 2.7b ± .18a ± 1.8 a ± 27a ± 1.4 a
1,699 2.50 10.9 3.26 51.9 145 29.2
± 53a ± .12a ± 1.2 a ± .05° ± 1.9 b ± 17a + ,9b
ab ' Values within a row with different superscripts are significantly different (P<.05).
2
/Vmoles P-aminophenol/20 min per milligrams of 9,000 X g supernatant protein.
3
Nmoles HCHO/20 min per milligrams of 9,000 X g supernatant protein.
4
Percentage of 14 C activity in aqueous medium after extraction with ether/100 mg 9,000 X g supernatant protein per 40 min.
and E2 metabolism were significantly increased by feeding the FAY diet as compared with the CS diet. Although the plasma E2 concentration was higher for the hens fed the CS diet, the
value was not significantly different from that for hens fed the FAY diet. Temporal measurements of the various parameters are presented in Figures 1,2, and 3. Both liver weight per unit of body weight and liver lipid of chickens fed the two diets because significantly different by 17 days after the start
210 180 Pd
150
o e E ^ g S 120 90
24 31 r E " £
•2 •- 5 o o _ -§2 S
27
lis23 o S
.t-
10
17
24
Time (day)
Time (day) FIGURE 1. Effect of dietary composition on liver weight and lipid in laying hens (values ± standard error). * indicates significant difference between dietary treatments (P<.05).
FIGURE 2. Effect of dietary composition on plasma E, and hepatic E2 metabolism on laying hens (values ± standard error). * indicates significant diffferences between dietary treatments (P<.05).
Downloaded from http://ps.oxfordjournals.org/ at Mount Royal College on June 9, 2015
'Means ± standard error of the mean (8 observations). Hens were fed the University of Georgia layer breeder stock diet prior to start of the experiment.
TIME COURSE STUDY ON LIVER LIPID
10 Time (day)
17
24
FIGURE 3. Effect of dietary composition on activity of aniline hydroxylase (A) and aminopyrine N-demethylase in laying hens (B) (values ± standard error). * indicates significant difference between dietary treatments (P<.05).
of the experiment (Figure 1). Plasma E2 concentration sampled at four days was significantly higher for birds fed the FAY diet than for birds fed the CS diet. This pattern was reversed during subsequent measurements, with higher values being observed for birds fed the CS diet. However, differences were not statistically significant (P>.05) at any other sampling period (Figure 2). In vitro estimation of E2 metabolism showed a significant increase by 17 days for birds fed the FAY diet when compared with values for birds fed the CS diet and the difference continued to widen at the 24-day sampling period (Figure 2). Aniline hydroxylase activity was consistently higher for birds fed the FAY diet but the difference was statistically significant only at the 24-day sampling period (Figure 3). A similar trend was observed for aminopyrine N-demethylase activity and the differences were significant at both the 10 and 24day sampling periods (Figure 3). DISCUSSION
The results of this experiment with laying hens fed CS or FAY diets are similar to those observed with estrogenized chicks (Takahashi and Jensen, 1984). A period of about 10 to 17 days on the experimental diets was required be-
fore differences became significant. Although statistically significant differences in plasma estradiol concentrations were not observed in hens fed the two different diets, differences observed in mean values were similar to those previously observed in other studies in our laboratory. For example, Akiba and Jensen (1983) observed a significantly higher estradiol concentration in the plasma of hens fed a CS diet for 7 days than in hens fed a diet with 10% fish meal. A relationship between the appearance of excess lipid deposition in the liver with increased plasma E2 concentration, and decreased estrogen metabolism and MFO activity is indicated when results of feeding the different diets are compared. A comparison however, of these values and those in birds fed the control diets fails to demonstrate a definite correlation between MFO activity and lipid accumulation (Table 2). Although feeding the CS diet increased hepatic lipid content, it did not decrease values below those in controls. In order to explain the effect of dietary changes on liver lipid accumulation in laying hens, (Jensen et al., 1984) hypothesize that optimum activation of the microsomal MFO system does not occur in hens fed a simple corn-soybean meal diet. Various feed ingredients included in diets which reduce liver lipid accumulation may contain a factor (or factors) which stimulates the MFO system to degrade the more rapidly circulating estradiol. The reduced level of estradiol in turn would result in reduced hepatic lipogenesis and less liver lipid accumulation. Results of this study with the FAY diet and other investigations with chicks (Takahashi and Jensen, 1984; Brenes et al., 1985) indicate increased activity of the MFO system resulting from feeding of the more complex diets. Although the activities of aniline hydroxylase and aminopyrine demethylase have been used as general indicators of the state of activation of the MFO system, they do not necessarily indicate that the enzymes involved in the estradiol degradation are specifically activated. The estradiol metabolism studies with liver microsomes from chicks (Takahashi and Jensen, 1984) and liver supernatant in this study support the suggestion that estradiol is metabolized more rapidly in birds fed the more complex diets. The nature of the factor or factors in fish meal, alfalfa meal, and torula yeast that result(s) in changes in MFO activity remains to be determined. There are a number of dietary factors that have been shown to affect microsomal MFO
Downloaded from http://ps.oxfordjournals.org/ at Mount Royal College on June 9, 2015
0
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REFERENCES Akiba, Y., and L. S. Jensen, 1983. Temporal effect of change in diet composition on plasma estradiol and thyroxine concentrations and hepatic lipogenesis in laying hens. J. Nutr. 113:2178-2184. Akiba, Y., L. S. Jensen, C. R. Barb, and R. R. Kraeling, 1982. Plasma estradiol, thyroid hormones and liver lipid content in laying hens fed different isocaloric diets. J. Nutr. 112:299-308. Akiba, Y., L. S. Jensen and C. X. Mendonca, 1983. Laboratory model with chicks for assay of nutritional factors affecting hepatic lipid accumulation in laying hens. Poultry Sci. 62:143-161. Anderson, K. E., A. H. Connery, and A. Kappas, 1982. Nutritional influence on chemical biotransformations in humans. Nutr. Rev. 40:161-171.
Brenes, A., L. S. Jensen, and H. M. Cervantes, 1985. Activation of hepatic microsomal mixed function oxidase system in broiler chicks by diet changes. Poultry Sci. 64:963-968. Folch, J., M. Lee, and G. H. Sloane-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Haghighi-Rad, F., and D. Polin, 1982a. The unidentified factor for alleviating fatty liver-hemorrhagic syndrome. Poultry Sci. 61:2075-2082. Haghighi-Rad, F., and D. Polin, 1982b. Lipid alleviates fatty liver hemorrhagic syndrome. Poultry Sci. 61:2465-2472. Hathcock, J. N., and J. Coon, 1978. Nutrition and Drug Interactions. Academic Press, New York, NY. Jensen, L. S., A. Brenes, and K. Takahashi, 1984. Association of reduced plasma estrogen and hepatic lipids with increased activity of the microsomal mixed function oxidase system in chickens. Pages 348-350 in Proc. World's Poult. Cong., Helsinki, Finland. Kato, R., and J. R. Gillette, 1965. Sex differences in the effects of abnormal physiological states on metabolism of drugs by rat liver microsomes. J. Pharmacol. Exp. Ther. 150:285-291. LaCroix, E., and W. Eechaute, 1979. Estradiol metabolism by liver homogenates and microsomes of normal and cirrhotic male rats. Horm. Res. (Basel) 11:69-80. Maurice, D. V., and L. S. Jensen, 1978. Liver lipid deposition in caged layers as influenced by fermentation by-products and level of dietary fat. Poultry Sci. 57:1690-1695. Maurice, D. V., L. S. Jensen, andH. Tojo, 1979. Comparison of fish meal and soybean meal in prevention of fatty liver-hemorrhagic syndrome in caged layer. Poultry Sci. 58:864-870. Statistical Analysis System, 1982. SAS User's Guide. SAS Institute, Inc. Raleigh, NC. Takahashi, K., and L. S. Jensen, 1984. Effect of dietary composition and estradiol implants on hepatic microsomal mixed function oxidase and lipid metabolism in growing chicks. Poultry Sci. 63:2217-2224. Tojo, H., and T. M. Huston, 1980. Effects of environmental temperature on the concentration of serum estradiol, progesterone and calcium in maturing female domestic fow. Poultry Sci. 59:2797-2802. Wade, A. E., and W. P. Norred, 1976. Effect of dietary lipid on drug-metabolizing enzymes. Fed. Proc. 35:2475-2479.
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activity in other species (Hathcock and Coon, 1978; Takahashi and Jensen, 1984; Brenes et al., 1985). In many cases, deficiencies of specific nutrients have been shown to reduce enzyme activity. Both the CS and FAY diets contain similar nutrient profiles and were adequate in nutrients to maintain performance of laying hens. Haghighi-Rad and Polin (1982a,b) proposed that the addition of lipid (corn oil) to the diet reduced liver lipid content and hemorrhages in laying hens. Wade and Norred (1976) showed that essential fatty acids were necessary for maximal activation of the MFO system in rats. More recent studies by Anderson etal., (1982), however, failed to provide evidence for an important influence of dietary lipid content on hepatic drug oxidations. The FAY diet contained slightly more total crude fat than the CS diet in the present investigation, but previous results in our laboratory demonstrated that a significant reduction in liver fat was still observed in birds fed a more complex diet compared to those fed CS when the diets contained equal levels of fat (Maurice and Jensen, 1978).