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Suppression of chick pancreatic xanthine dehydrogenase by unsaturated fatty acids
DEYELOPMENTALBIOLOGY
24, 54-60(1971)
Suppression
of Chick Pancreatic
Dehydrogenase
Xanthine
by Unsaturated
Fatty Acids’ W. D. WOODWARD AND J. R. FISHER Department
of
Chemistry and Institute of ikfolecukzr Biophysics, Florida State university, Tallahassee, Florida 32306 Accepted September 22, 1970 INTRODUCTION
Previously Fisher et al. (1967) have shown that the accumulation of pancreatic xanthine dehydrogenase in newly hatched chicks which occurs immediately after hatching is initiated by food intake. Subsequently, Woodward and Fisher (1967) presented evidence that this dietary response was not due simply to the intake of food but required specific kinds of dietary materials. Also in older chicks (raised on Startena for 3-4 weeks) starvation or feeding of egg as the exclusive diet causes xanthine dehydrogenase activity in the pancreas to decrease sharply and refeeding a normal diet causes a rapid accumulation of enzyme in the pancreas to a level double that in continuously fed controls. Subsequently this excess activity diminishes to the control level. Curtis and Fisher (1970) have shown that this dietary control is exerted at the level of enzyme synthesis. Originally (Woodward and Fisher, 1967) it was suggested that the normal diet of chicks includes a pancreatic xanthine dehydrogenase inducer and that egg is deficient in this material. Results presented in the present paper show that the decrease in egg-fed chicks is open to another interpretation. Extracts of boiled egg yolks suppress the developmental accumulation of this pancreatic enzyme as do unsaturated fatty acids. Since eggs contain sufficient quantities of these substances, it is suggested that the suppression induced by feeding egg and egg extracts is due to the presence of unsaturated fatty acids. ‘Supported in part by grants from the U.S. Public Health Service (HD-02579-03), National Science Foundation (GB-8557), and the Florida Division of the American Cancer Society. 54
CHICK
XANTHINE
DEHYDROGENASE
MATERIALS
AND
SUPPRESSION
55
METHODS
Babcock B-300 baby chicks were used throughout this series of experiments and Startena as the basic diet. This was modified in the various treatments by the addition of specified amounts of fatty acids or egg extracts. The fatty acids were selected from among those listed by Romanoff and Romanoff (1949) as being present in egg lipids. Additions to the basic diet were made on a weight-percentage basis and ranged from 2.5 to 15.0. The materials were carefully mixed to obtain complete homogeneity and fed ad libitum. The fatty acid preparations used in these studies were obtained from NBC. A commercial grade of powdered egg obtained locally was used as a diet for the chicks, and this same material was used to prepare alcohol and ether extracts. Xanthine dehydrogenase activities were measured as described by Curtis and Fisher (1970). Pancreatic material from chicks was extracted in the following manner. Ten-milliliter quantities of the homogenate were mixed with 10 ml of methyl alcohol containing 10% KOH. This mixture was refluxed for 30 minutes at 105°C for the purpose of hydrolyzing all fatty acid esters present. After cooling, 20 ml of water were added and the hydrolyzate extracted with 50 ml of n-hexane. The aqueous phase was acidified with HCl and successively extracted with three 50-ml quantities of ethyl ether. The ether extracts were pooled and the ether removed under vaccum. The residue containing fatty acids was treated with 1 ml of a solution containing 14% boron trifluoride in methyl alcohol for 5 minutes in a 80°C water bath. The methyl esters were then extracted with n-hexane and analyzed on a F and M Model 700 Gas Liquid Chromatograph (GLC). The column contained 10% diethylene glycol succinate on a support of 100/120-mesh Gas Chrom Q from Applied Science and was run at 140°C. RESULTS
AND
DISCUSSION
Since Woodward and Fisher (1967) showed that newly hatched chicks fed exclusively on egg exhibited a very low level of pancreatic xanthine dehydrogenase, and, since it appeared that this was due to the lack of inducer in egg material, it was concluded that whole egg would be a suitable neutral carrier to which substances could be added to test for their inducing ability. Using this approach, a series of studies were made in an effort to isolate the dietary inducer of
56
WOODWARD
o-
AND
5 % Ether
FISHER
IO
15
Extract
FIG. 1. The effect of an ether extract of boiled egg yolks on pancreatic dehydrogenase activity in chick. Newly hatched chicks were fed the various 7 days. Specific activity is in millimicromoles pteridine oxidized per minute gram of protein (Lowry et al., 1951). Assay conditions were the same as previously (Curtis and Fisher, 1970).
xanthine diets for per millidescribed
pancreatic xanthine dehydrogenase. All attempts to isolate and identify the inducer by this means were totally unsuccessful. Results were not reproducible and response was nonlinear. This finally led us to suspect that the egg was not a neutral carrier but in some way was affecting the response of the chicks to the inducer. The first evidence of how an egg diet influences this induction was obtained when it was observed that alcoholic extracts of egg were capable of suppressing the developmental increase in pancreatic xanthine dehydrogenase when fed as a mixture with the ordinary Startena diet. Subsequently it was found that ether extracts of boiled egg yolks were equally effective (Fig. 1). In this experiment, newly hatched chicks were maintained for 7 days on normal Startena feed on which varying amounts of ether-extractable material was added. There is a decrease in the level of activity obtained in the chicks when even as little as 2.5% of the diet is made up of this extracted material. When 15% is added, activity is suppressed to approximately 20% of the control value. On the basis of these results, it can be seen that cooked egg yolks contain a substance or substances which are easily extractable that prevent the normal accumulation of pancreatic xanthine dehydrogenase in newly hatched chicks. A portion of the ether-soluble material used in one series of experiments was analyzed on the GLC and compared to the fatty acid pattern of pancreas from chicks which received this extract. There
CHICK
XANTHINE
DEHYDROGENASE
SUPPRESSION
57
was a very close correlation in fatty acid composition of the two extracts. In the case of two unidentified long-chain fatty acids present in the egg, there actually appeared to be an accumulation in the pancreas. It also may be of some significance that the relative height of these two peaks was inversely proportional to the xanthine dehydrogenase activity of the pancreas. Another outgrowth of these studies was the observation that cooked egg yolks as well as cooked and uncooked whole egg is a very unsatisfactory diet for chicks. In fact chicks maintained exclusively on egg diets die within 4 weeks after hatching. Because of these observations, care was taken to record the weights of the chicks that were fed ether-extracted material along with their normal diet (Fig. 1). In all these cases the chicks were completely healthy and showed normal weight gains during the course of these experiments. Therefore, it seems clear that the suppression of pancreatic xanthine dehydrogenase is not due to generally toxic factors in the extracts being tested. Nature of Egg Suppressors
In the course of studying the nature of the material in ether extracts of egg which suppress the developmental increase in pancreatic xanthine dehydrogenase, a group of fatty acids were surveyed. Re-
120-
0
I
I I I IO.0 5.0 % Linolcnlc Acid
I 15.0
FIG. 2. The relationships between level of linolenic acid in chick diet and pancreatic xanthine dehydrogenase activity. Newly hatched chicks were fed the various diets for 7 days. Results of three separate experiments are included. Bars represent the range of values obtained. Relative activities are ratios of experimental values to control values times 100.
58
WOODWARD
AND
FISHER
suits presented in Fig. 2 show that linolenic acid strongly suppresses this developmental change at relatively low concentrations in the diet. Results presented in Fig. 3 show that linoleic acid is equally effective with linolenic acid, oleic acid suppresses to a lesser extent, stearic acid induces a significant increase, and palmitic acid has no effect whatever on the level of pancreatic xanthine dehydrogenase. In these early experiments the linoleic acid was 75% pure and the linolenic acid was 55% pure. Results with highly purified preparations (99% pure) are shown in Table I. These highly purified preparations are more effective than those used in earlier studies. For example, the 13% value found with purified linolenic acid should be compared with 22% found with the earlier preparation (see Fig. 2). Fatty acid analysis of pancreatic material from chicks receiving
FIG. 3. Comparison of the influence of five fatty acids known to be present in egg lipids on pancreatic xanthine dehydrogenase. These materials were incorporated into the diet at a 5, 10, and 15% level. Numerals above bars indicate the number of independent measurements made with pooled samples from four chicks, and shaded areas give the range of values obtained. Relative activities are ratios of experimental values to control values times 100.
CHICK
XANTHINE
DEHYDROGENASE
59
SUPPRESSION
TABLE I RESULTS OBTAINED FROM THE FEEDING OF PURIFIED (99%) LINOLENIC AND LINOLEIC ACIDS TO NEWLY HATCHED CHICKS FOR 7 DAYS Diet 7.5% linoleic
7.5% linolenic
Specific activity” 17.0 15.2 13.5 17.3 21.8 12.0
Startena Control
“Each specific activity pancreases pooled. b Normalized values.
Average”
12
13 loo
represents the value obtained
with a sample from 4 chick
either linoleic or linolenic acids showed striking correlations between the acid administered and the level of that acid in the pancreas. Results indicated approximately a 2-fold increase in the level of pancreatic linoleate compared to controls and a 6-fold increase in pancreatic linolenate. In addition, there were differences in overall fatty acid composition of pancreas which were common to both groups of treated chicks but not to the controls. Romanoff and Romanoff (1949) have summarized our present knowledge of the fatty acid composition of eggs. They report that 50% of the fatty acid material in eggs is oleic, 27% is palmitic, 11% is linoleic, and 6% is stearic. Palmitoleic, linolenic, clupanodonic, and myristic acids make up 6% of the total. Also whole dried egg is 45% lipid and 26% of this material is true fat. Eighty percent of the true fat is fatty acid, and 66% of the fatty acids are unsaturated. On this basis about 15% of an exclusive egg diet is composed of unsaturated fatty acids. These results are consistent with the interpretation that the suppressive activity of egg and egg extracts is due to the presence of unsaturated fatty acids. So far we have been unable to obtain sufficient quantities of clupanodonic to test the possibility that this highly unsaturated fatty acid would suppress activity. In conclusion we would like to point out that although unsaturated fatty acids suppress pancreatic xanthine dehydrogenase development in the chick, it is by no means clear that these substances act directly on the pancreas itself. In fact, we favor the possibility that
60
WOODWARD
AND
FISHER
they act by eliciting the presence of some other agents in the chick which in turn act in controlling the level of this enzymatic activity. SUMMARY
A study of pancreatic xanthine dehydrogenase levels in chicks immediately after hatching has revealed that the sharp developmental increase normally occurring can be suppressed by maintenance on whole egg (Fisher et al., 1967; Woodward and Fisher, 1967). Further work has shown that ether extracts of egg yolks are equally effective in supressing this developmental increase. Of the lipid components present, four have been found to affect the accumulation of pancreatic xanthine dehydrogenase: linolenic, linoleic, and oleic acids suppress and stearic acid enhances. It seems reasonable to believe that the suppressing activity of ether extracts of eggs is due to the presence of unsaturated fatty acids. The administration of specific fatty acids leads to increased concentrations of these materials in the lipids of the pancreas. REFERENCES CURTIS, J. L., and FISHER, J. R. (1970). Control
of pancreatic xanthine dehydrogenase synthesis in the chick. Biochim. Biophys, Acta 201,26. FISHER, J. R., CURTIS, J. L., and WOODWARD, W. D. (1967). Developmental changes and control of xanthine dehydrogenase in developing chicks. Deuelop. Biol. 15, 289. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. ROMANOFF, A. L., and ROMANOFF, A. J. (1949). “The Avian Egg.” Wiley, New York. WOODWARD, W. D., and FISHER, J. R. (1967). Control of xanthine dehydrogenase in the chick pancreas. Develop. Biol. 16, 282.
24, 54-60(1971)
Suppression
of Chick Pancreatic
Dehydrogenase
Xanthine
by Unsaturated
Fatty Acids’ W. D. WOODWARD AND J. R. FISHER Department
of
Chemistry and Institute of ikfolecukzr Biophysics, Florida State university, Tallahassee, Florida 32306 Accepted September 22, 1970 INTRODUCTION
Previously Fisher et al. (1967) have shown that the accumulation of pancreatic xanthine dehydrogenase in newly hatched chicks which occurs immediately after hatching is initiated by food intake. Subsequently, Woodward and Fisher (1967) presented evidence that this dietary response was not due simply to the intake of food but required specific kinds of dietary materials. Also in older chicks (raised on Startena for 3-4 weeks) starvation or feeding of egg as the exclusive diet causes xanthine dehydrogenase activity in the pancreas to decrease sharply and refeeding a normal diet causes a rapid accumulation of enzyme in the pancreas to a level double that in continuously fed controls. Subsequently this excess activity diminishes to the control level. Curtis and Fisher (1970) have shown that this dietary control is exerted at the level of enzyme synthesis. Originally (Woodward and Fisher, 1967) it was suggested that the normal diet of chicks includes a pancreatic xanthine dehydrogenase inducer and that egg is deficient in this material. Results presented in the present paper show that the decrease in egg-fed chicks is open to another interpretation. Extracts of boiled egg yolks suppress the developmental accumulation of this pancreatic enzyme as do unsaturated fatty acids. Since eggs contain sufficient quantities of these substances, it is suggested that the suppression induced by feeding egg and egg extracts is due to the presence of unsaturated fatty acids. ‘Supported in part by grants from the U.S. Public Health Service (HD-02579-03), National Science Foundation (GB-8557), and the Florida Division of the American Cancer Society. 54
CHICK
XANTHINE
DEHYDROGENASE
MATERIALS
AND
SUPPRESSION
55
METHODS
Babcock B-300 baby chicks were used throughout this series of experiments and Startena as the basic diet. This was modified in the various treatments by the addition of specified amounts of fatty acids or egg extracts. The fatty acids were selected from among those listed by Romanoff and Romanoff (1949) as being present in egg lipids. Additions to the basic diet were made on a weight-percentage basis and ranged from 2.5 to 15.0. The materials were carefully mixed to obtain complete homogeneity and fed ad libitum. The fatty acid preparations used in these studies were obtained from NBC. A commercial grade of powdered egg obtained locally was used as a diet for the chicks, and this same material was used to prepare alcohol and ether extracts. Xanthine dehydrogenase activities were measured as described by Curtis and Fisher (1970). Pancreatic material from chicks was extracted in the following manner. Ten-milliliter quantities of the homogenate were mixed with 10 ml of methyl alcohol containing 10% KOH. This mixture was refluxed for 30 minutes at 105°C for the purpose of hydrolyzing all fatty acid esters present. After cooling, 20 ml of water were added and the hydrolyzate extracted with 50 ml of n-hexane. The aqueous phase was acidified with HCl and successively extracted with three 50-ml quantities of ethyl ether. The ether extracts were pooled and the ether removed under vaccum. The residue containing fatty acids was treated with 1 ml of a solution containing 14% boron trifluoride in methyl alcohol for 5 minutes in a 80°C water bath. The methyl esters were then extracted with n-hexane and analyzed on a F and M Model 700 Gas Liquid Chromatograph (GLC). The column contained 10% diethylene glycol succinate on a support of 100/120-mesh Gas Chrom Q from Applied Science and was run at 140°C. RESULTS
AND
DISCUSSION
Since Woodward and Fisher (1967) showed that newly hatched chicks fed exclusively on egg exhibited a very low level of pancreatic xanthine dehydrogenase, and, since it appeared that this was due to the lack of inducer in egg material, it was concluded that whole egg would be a suitable neutral carrier to which substances could be added to test for their inducing ability. Using this approach, a series of studies were made in an effort to isolate the dietary inducer of
56
WOODWARD
o-
AND
5 % Ether
FISHER
IO
15
Extract
FIG. 1. The effect of an ether extract of boiled egg yolks on pancreatic dehydrogenase activity in chick. Newly hatched chicks were fed the various 7 days. Specific activity is in millimicromoles pteridine oxidized per minute gram of protein (Lowry et al., 1951). Assay conditions were the same as previously (Curtis and Fisher, 1970).
xanthine diets for per millidescribed
pancreatic xanthine dehydrogenase. All attempts to isolate and identify the inducer by this means were totally unsuccessful. Results were not reproducible and response was nonlinear. This finally led us to suspect that the egg was not a neutral carrier but in some way was affecting the response of the chicks to the inducer. The first evidence of how an egg diet influences this induction was obtained when it was observed that alcoholic extracts of egg were capable of suppressing the developmental increase in pancreatic xanthine dehydrogenase when fed as a mixture with the ordinary Startena diet. Subsequently it was found that ether extracts of boiled egg yolks were equally effective (Fig. 1). In this experiment, newly hatched chicks were maintained for 7 days on normal Startena feed on which varying amounts of ether-extractable material was added. There is a decrease in the level of activity obtained in the chicks when even as little as 2.5% of the diet is made up of this extracted material. When 15% is added, activity is suppressed to approximately 20% of the control value. On the basis of these results, it can be seen that cooked egg yolks contain a substance or substances which are easily extractable that prevent the normal accumulation of pancreatic xanthine dehydrogenase in newly hatched chicks. A portion of the ether-soluble material used in one series of experiments was analyzed on the GLC and compared to the fatty acid pattern of pancreas from chicks which received this extract. There
CHICK
XANTHINE
DEHYDROGENASE
SUPPRESSION
57
was a very close correlation in fatty acid composition of the two extracts. In the case of two unidentified long-chain fatty acids present in the egg, there actually appeared to be an accumulation in the pancreas. It also may be of some significance that the relative height of these two peaks was inversely proportional to the xanthine dehydrogenase activity of the pancreas. Another outgrowth of these studies was the observation that cooked egg yolks as well as cooked and uncooked whole egg is a very unsatisfactory diet for chicks. In fact chicks maintained exclusively on egg diets die within 4 weeks after hatching. Because of these observations, care was taken to record the weights of the chicks that were fed ether-extracted material along with their normal diet (Fig. 1). In all these cases the chicks were completely healthy and showed normal weight gains during the course of these experiments. Therefore, it seems clear that the suppression of pancreatic xanthine dehydrogenase is not due to generally toxic factors in the extracts being tested. Nature of Egg Suppressors
In the course of studying the nature of the material in ether extracts of egg which suppress the developmental increase in pancreatic xanthine dehydrogenase, a group of fatty acids were surveyed. Re-
120-
0
I
I I I IO.0 5.0 % Linolcnlc Acid
I 15.0
FIG. 2. The relationships between level of linolenic acid in chick diet and pancreatic xanthine dehydrogenase activity. Newly hatched chicks were fed the various diets for 7 days. Results of three separate experiments are included. Bars represent the range of values obtained. Relative activities are ratios of experimental values to control values times 100.
58
WOODWARD
AND
FISHER
suits presented in Fig. 2 show that linolenic acid strongly suppresses this developmental change at relatively low concentrations in the diet. Results presented in Fig. 3 show that linoleic acid is equally effective with linolenic acid, oleic acid suppresses to a lesser extent, stearic acid induces a significant increase, and palmitic acid has no effect whatever on the level of pancreatic xanthine dehydrogenase. In these early experiments the linoleic acid was 75% pure and the linolenic acid was 55% pure. Results with highly purified preparations (99% pure) are shown in Table I. These highly purified preparations are more effective than those used in earlier studies. For example, the 13% value found with purified linolenic acid should be compared with 22% found with the earlier preparation (see Fig. 2). Fatty acid analysis of pancreatic material from chicks receiving
FIG. 3. Comparison of the influence of five fatty acids known to be present in egg lipids on pancreatic xanthine dehydrogenase. These materials were incorporated into the diet at a 5, 10, and 15% level. Numerals above bars indicate the number of independent measurements made with pooled samples from four chicks, and shaded areas give the range of values obtained. Relative activities are ratios of experimental values to control values times 100.
CHICK
XANTHINE
DEHYDROGENASE
59
SUPPRESSION
TABLE I RESULTS OBTAINED FROM THE FEEDING OF PURIFIED (99%) LINOLENIC AND LINOLEIC ACIDS TO NEWLY HATCHED CHICKS FOR 7 DAYS Diet 7.5% linoleic
7.5% linolenic
Specific activity” 17.0 15.2 13.5 17.3 21.8 12.0
Startena Control
“Each specific activity pancreases pooled. b Normalized values.
Average”
12
13 loo
represents the value obtained
with a sample from 4 chick
either linoleic or linolenic acids showed striking correlations between the acid administered and the level of that acid in the pancreas. Results indicated approximately a 2-fold increase in the level of pancreatic linoleate compared to controls and a 6-fold increase in pancreatic linolenate. In addition, there were differences in overall fatty acid composition of pancreas which were common to both groups of treated chicks but not to the controls. Romanoff and Romanoff (1949) have summarized our present knowledge of the fatty acid composition of eggs. They report that 50% of the fatty acid material in eggs is oleic, 27% is palmitic, 11% is linoleic, and 6% is stearic. Palmitoleic, linolenic, clupanodonic, and myristic acids make up 6% of the total. Also whole dried egg is 45% lipid and 26% of this material is true fat. Eighty percent of the true fat is fatty acid, and 66% of the fatty acids are unsaturated. On this basis about 15% of an exclusive egg diet is composed of unsaturated fatty acids. These results are consistent with the interpretation that the suppressive activity of egg and egg extracts is due to the presence of unsaturated fatty acids. So far we have been unable to obtain sufficient quantities of clupanodonic to test the possibility that this highly unsaturated fatty acid would suppress activity. In conclusion we would like to point out that although unsaturated fatty acids suppress pancreatic xanthine dehydrogenase development in the chick, it is by no means clear that these substances act directly on the pancreas itself. In fact, we favor the possibility that
60
WOODWARD
AND
FISHER
they act by eliciting the presence of some other agents in the chick which in turn act in controlling the level of this enzymatic activity. SUMMARY
A study of pancreatic xanthine dehydrogenase levels in chicks immediately after hatching has revealed that the sharp developmental increase normally occurring can be suppressed by maintenance on whole egg (Fisher et al., 1967; Woodward and Fisher, 1967). Further work has shown that ether extracts of egg yolks are equally effective in supressing this developmental increase. Of the lipid components present, four have been found to affect the accumulation of pancreatic xanthine dehydrogenase: linolenic, linoleic, and oleic acids suppress and stearic acid enhances. It seems reasonable to believe that the suppressing activity of ether extracts of eggs is due to the presence of unsaturated fatty acids. The administration of specific fatty acids leads to increased concentrations of these materials in the lipids of the pancreas. REFERENCES CURTIS, J. L., and FISHER, J. R. (1970). Control
of pancreatic xanthine dehydrogenase synthesis in the chick. Biochim. Biophys, Acta 201,26. FISHER, J. R., CURTIS, J. L., and WOODWARD, W. D. (1967). Developmental changes and control of xanthine dehydrogenase in developing chicks. Deuelop. Biol. 15, 289. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. ROMANOFF, A. L., and ROMANOFF, A. J. (1949). “The Avian Egg.” Wiley, New York. WOODWARD, W. D., and FISHER, J. R. (1967). Control of xanthine dehydrogenase in the chick pancreas. Develop. Biol. 16, 282.