Excretion of Bile Acids by Cockerels Fed Different Lipids1

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B . W . BlERER 1944. An electrophoretic study of serum and plasma from normal and leucosis-affected chickens. J. Biol. Chem. 155: 469-481. Sturkie, P. D., 1965. Avian Physiology, 2nd ed. Cornell University Press, Ithaca, New York 14850: 50. Stute, W. K., 1964. Paper electrophoresis studies on the blood serum of fowls: I, Composition of sera of healthy chicks from 1 day to 8 weeks. Arch. Gefluge. 28: 172-178. Sunderman, F. W., 1944. A rapid method for estimating serum proteins. J. Biol. Chem. 153: 139142. Turk, D. E., 1968. Personal communication, May 21, 1968. Clemson University, Clemson, S. C. 29631. Vanstone, W. E., W. A. Maw and R. H. Common, 1955. Levels and partitions of the fowl's serum proteins in relation to age and egg production. Canad. J. Biochem. Physiol. 33: 891-903.

Excretion of Bile Acids by Cockerels Fed Different Lipids1 O. B. LINDSAY 2 , JACOB BIELY AND B. E. MARCH 3 Department of Poultry Science, The University of British Columbia, Vancouver 8, B.C., Canada (Received for publication January 4, 1969)

T

HE mechanisms whereby hypo-or hypercholesterolemia is induced by the dietary fats fed in cholesterol-free diets are not completely understood. The effects of fats on blood cholesterol level may be related to differences in the rates of bile acid excretion induced by the fats. Gordon et al. (1957); Byers and Friedman (1958); Haust and Beveridge (1958); Srikantia and Gopalan (1960); Goldsmith et al. (1960); Moore et al. (1962); have all shown that unsaturated fats which reduce blood cholesterol levels also increase excretion of bile acids. On the other hand, Avigan and Steinberg (1965); Lindestedt 1 This research was supported by a grant from The National Research Council of Canada. 2 Present address: Simon Fraser University, Bumaby 2, B.C. 3 To whom requests for reprints should be addressed.

et al. (1965); Grundy et al. (1965); Ali et al. (1966); found no significant differences in bile acid excretion when fats of different degrees of saturation were fed. The above reports pertained to studies with human subjects, rats or rabbits. Comparatively few studies have been made of bile acid excretion in the chicken despite the considerable use which has been made of the cockerel in atherosclerosis research. Anderson et al. (1957) tabulated the bile acids which have been reported in the chicken. A dietary effect on bile acid excretion in birds was reported by Kummerow et al. (1960), who found bile acid excretion to be increased with a high protein diet. As with other species, plasma and liver cholesterol are reduced in chicks when bile acid excretion is promoted by feeding cholestyramine (Whiteside et al., 1965). However, because there

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Lynch, J. E., and H. J. Stafseth, 1953. Electrophoretic studies on the serum proteins of turkeys. Poultry Sci. 32: 1068-1073. Mclndoe, W. M., 1962. Occurrence of two plasma albumins in the domestic fowl. Nature, 195: 353354. McKinley, W. P., W. A. Maw, W. F. Oliver aad R. H. Common, 1954. The determination of serum protein fractions on filter paper electropherograms by the biuret reaction and some observations on the serum protein of the estrogenized immature pullet. Canad. J. Biochem. Physiol. 32: 189-199. Meltzer, T. H., 1968. Electrophoresis on cellulose acetate. Laboratory Management, 6: 20-21. Patterson, D. S. P., D. Sweasey, C. N. Herbert and W^R. A. B. Carnaghan, 1967. Comparative bioJllogical studies in hybrid chicks, 1. Brit. Poultry [Sci. 8: 273-278. Sanders, E., I. F. Huddleson and P. J. Schaible,

B I L E ACID EXCRETION

appears to be no direct information regarding the effect of different dietary fats on the excretion of bile acids in the chicken the following experiments were conducted. EXPERIMENTAL

chromatography. T h e birds were weighed in order t h a t bile acid excretion could be related to body weight. T h e experimental diets containing the different oils were fed for a subsequent 17-day period. Corn oil, herring oil, and coconut oil respectively were added to the basal diet in place of 1 5 % of dextrose. Each of the fat-supplemented diets was fed to four groups of birds for two weeks. After this time plasma D P S , excretion rate of bile acids, and body weights were again determined. Experiment 2. The effects of lecithin and /3sitosterol on bile acid excretion and plasma sterol level. Single Comb White Leghorn cockerel chicks were fed a commercial chick-starting diet until they were three weeks of age at which time they were weighed and distributed into eight groups of eight birds of approximately similar average weight. Duplicate groups were then fed each of the respective experimental diets as follows: (1) basal diet; (2) 2 . 5 % crude soybean lecithin; (3) 0 . 5 % /3-sitosterol; (4) 2.5% crude soybean lecithin plus 0 . 5 % /3-sitosteroL T h e basal diet had the following composition: corn oil 8.0, ground yellow corn 51.4, soybean meal (48.5% protein) 31.46, dehydrated cereal grass 2.0, distillers' dried solubles 2.0, limestone 1.0, bone meal 3.0, iodized salt 0.5, choline chloride (37%) 0.22, DL-methionine 0.15 percent; manganese sulphate 220.0, folacin 1.1, riboflavin 6.6, calcium pantothenate 18.5, niacin 44 mg. per kg.; vitamin A 2000 I.U. and vitamin D 3 200 I.C.U. per kg. T h e lecithin and sitosterol were substituted for equal weights of corn oil in the basal diet. Sitosterol was added to the diet by dissolving it in diethyl ether and a minimum amount of chloroform and allowing the solution to drip into the diet during the mechanical mixing. T h e solvents were allowed to evaporate before

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Experiment 1. The effects of corn, herring, and coconut oils on bile acid excretion and plasma sterol level. Thirty-six adult Single Comb White Leghorn cockerels were individually caged and assigned a t random to twelve groups of three birds each. T h e birds were fed an all-vegetable basal diet during a pre-experimental period of 17 days. T h e basal diet had the following composition: dextrose 15.0, ground wheat 71.65, soybean meal ( 4 5 % protein), 8.85, dried distillers' solubles 2.0, bone meal 2.0 iodized salt 0.5 percent; manganese sulphate 110.0 mg. menadione 0.5 mg., vitamin A 4400 I.U. and vitamin D 3 200 I.C.U. per kg. At the end of the first and second week blood samples were taken for determination of digitonin-precipitable sterols ( D P S ) . Plasma from the three birds comprising each group was pooled for analysis. T h e sterols were extracted from the plasma by the method of Sperry and Webb (1950) and determined by the method of Vahouny et al. (1960). T h e concentrations of sterols found in the plasm a in the two sets of determinations were averaged to provide the base levels for the respective groups of birds. During the 48hour period following the second set of determinations the excreta voided by the birds were collected quantitatively. T h e excetra were lyophilized to constant weight, then ground and stored under nitrogen pending analysis for bile acids. T h e dried and ground excreta were pooled to give one sample for each four groups of birds. T h e pooled samples were analyzed in duplicate for bile acids by the method of Grundy et al. (1965). This method employs column, thin-layer, and gas-liquid

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0. B. LINDSAY, J. BIELY AND B. E. MARCH

RESULTS Experiment 1. The levels of DPS in the plasma of the cockerels fed a diet with and

without 15% of different fats are given in Table 1. Corn oil did not alter plasma DPS levels from those found when the birds were fed the low-fat basal diet. Herring oil slightly increased the level of plasma DPS in three of the four groups and the average sterol level was increased by 8.1 mg.%. The feeding of coconut oil consistently increased plasma DPS level. The average increase was 37.9 mg.%. The bile acids excreted were quantitated from the chromatograms by comparing the ratio of the total area of peaks attributed to bile acids with the area of the peak for the internal standard (5a-cholestane). Table 1 shows the amounts of bile

TABLE 1.—A Iterations in plasma sterol levels and bile acid excretion rates in response to feeding corn oil, herring oil, and coconut oil Group No.

DPS mg. % in plasma Replicate Fed basal diet

Fed com oil

85.2 100.7 76.8 90.7

85.9 99.6 74.3 96.1

A B C D

88.3 A B C D

89.5 81.9 91.3 80.8

A B C D

Diet fed 2 weeks prior to and during 2-day fecal collection period

89.9 fed herring oil 99.6 95.8 102.7

77.7

85.9

94.0

83.9 92.1 90.7 73.1

fed coconut oil 124.8 119.2 133.7 113.6

84.9

122.8 Bile acids excreted

Av. mg./gm. of excreta

Mg./lOO gms. body wt./24 hrs.

Gm. excreta/100 gm. body wt./24 hrs.

1. basal diet diet with com oil

1.12 (1.16, 1.08)* 1.32(1.36, 1.28)

1.54 1.43

1.38 1.08

2. basal diet diet with herring oil

1.04(1.01, 1.07) 1.25 (1.26, 1.24)

1.58 1.27

1.51 1.01

3 . basal diet diet with coconut oil

1.23 (1.27, 1.19) 1.09 (1.11, 1.07)

91 18

.56 .08

* Figures in parentheses are values obtained in the duplicate determinations.

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the diets were fed. Lecithin was added to the diet in mixture with the corn oil in the diet. After the experimental diets had been fed for 15 days blood samples were drawn. The blood from each group was pooled and analyzed for DPS. Feed consumption was measured and the excreta were collected for analysis for bile acids as above during the last 48 hours of the experiment. The birds were weighed at the conclusion of the experiment.

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BILE ACID EXCRETION

T A B I E 2.—Plasma sterol level and bile acid excretion in response to feeding soybean lecithin and (3-sitosterc DPS mg. % in plasma Diet Replicates

Mean

116.7 128.0

basal 2 . 5 % lecithin 0.5%/3-sitosterol 2 . 5 % lecithin 0.5%/3-sitosterol

Bile acids excreted mg./gm. of excreta

Mg./lOO gm. body wt./24 hours

Replicates

Mean

Replicates

Mean

122.4

1.34 1.45

1.40

2.96 3.87

3.42

110.7 114.6

112.7

2.39 2.69

2.54

6.62 7.40

7.01

120.8 106.0

113.4

1.72 1.69

1.70

4.60 4.30

4.45

111.3 113.1

112 .2

3 .06 2 .85

2.96

8.16 7.76

7.97

Variance

Lecithin effect Sitosterol effect Interaction Error Significant at 5% level. Significant at 1% level.

DF

Plasma DPS

1 1 1 4

59.41 44.18 37.04 45.42

Bile acids mg./gm. excreta 2.8680** 0.2628* 0.0056 0.0184

Bile acids mg./lOO gm. body wt. 25.2405** 1.9701* O.0O37 0.2108

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acids excreted relative to the weight of matogram pattern of bile acids excreted. excreta voided and to the body weight of Experiment 2. The average plasma stethe birds. Because the inclusion of the oils rol concentration was slightly reduced in the diet depressed feed consumption, when lecithin and/or ^-sitosterol was fed the weights of the excreta voided per kg. in the diet (Table 2). body weight by the birds are given in orWhen lecithin was fed, bile acid excreder to explain the apparent discrepancies tion was increased by 8 1 % on the basis in the relative amounts of bile acids ex- of concentration in the feces and by creted on the different diets when the 105% on the basis of body weight of the amounts of bile acids excreted are ex- birds. Sitosterol also increased excretion pressed in the two ways. The feeding of of bile acids but to a lesser degree. The corn oil and herring oil increased the con- effect of lecithin and sitosterol together centration of bile acids in the excreta, was greater than that of either fed singly. whereas coconut oil decreased the concenThe amounts and relative proportions tration from that observed when the low- of different bile acids excreted by the birds fat basal diet was fed to the respective are given in Table 3. Those bile acids groups of birds. When calculated on the which appeared as a sequence of unresolvbasis of body weight, bile acid excretion ed peaks were grouped together. Although also varied depending on the nature of the the feeding of sitosterol increased the total oil which was fed. The birds fed corn oil excretion of bile acids, the chromatograms excreted the largest amount of bile acids of the control birds and those fed the basfollowed by those fed herring oil and coco- al diet with sitosterol were similar. The nut oil in that order. The feeding of the addition of lecithin to either the basal diet different oils had little effect on the chro- or to the basal diet with sitosterol in-

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O. B. LINDSAY, J. BIELY AND B. E. MARCH TABLE 3.—Levels of individual bile acids excreted* by cockerels in experiment 2

Relative retention time 1.15-1.26 1.64-2.39 2.67 2.98 3.43 9.16

Basal diet

/3-sitosterol

Lecithin

jS-sitosterol

+lecithin

%of total

Mg./gm. excreta

%of total

Mg./gm. excreta

%of total

Mg./gm. excreta

%of total

Mg./gm. excreta

13.7 9.8 35.4 1.5 18.9 20.7

192 137 496 21 265 290

13.0 8.9 36.4 2.6 18.3 20.8

221 151 619 44 311 354

9.3 6.4 34.4 9.2 31.5 9.2

236 163 874 234 800 234

8.9 6.5 32.9 9.6 34.2 7.9

263 192 974 284 1,012 234

creased the amounts excreted of all the bile acids except that with a relative retention time of 9.16. The relative proportions of the various bile acids were considerably altered when lecithin was fed. DISCUSSION The relative bile acid excretion of cockerels fed different oils agrees with reports cited earlier that unsaturated dietary oils tend to increase, and saturated dietary oils to decrease, levels of bile acids in the excreta of human subjects and rats. The reduction in the absolute amount of bile acids excreted when any of the oils was fed was due to the decrease in feed consumption and the resultant decrease in fecal excretion which occurred when the caloric density of the diet was raised by the inclusion of oil. The reduction in the amount of bile acids excreted when coconut oil was fed, however, was much greater than was the case when the other two oils were fed. Roels and Hashim (1962) reported similarly that when coconut oil was substituted isocalorically for corn oil sufficient to increase serum cholesterol, a marked reduction in daily excre-

tion of bile acids occurred in human subjects. Compounds in fats and oils, other than the component fatty acids, can affect blood cholesterol level. Fisher et al. (1961), for example, consider that the sterol fraction of corn oil is responsible for reducing avian aortic atherogenesis, but that the beneficial effect of the corn sterols is not due to interference with cholesterol absorption alone (1963). Higashi (1931) found that biliary excretion of bile acids is increased in dogs fed ergosterol. Soybean lecithin has been shown to lower serum cholesterol in rabbits (Kesten and Silbowitz, 1942) and in man (Steimer and Domanski, 1944). In experiment 2, therefore, the effects on bile acid excretion of feeding sitosterol and soybean lecithin were measured and both found to increase bile acid output. The rate of bile acid excretion per unit of body weight was greater with the growing birds employed in this experiment than with the adult birds in experiment 1. The effect of lecithin in experiment 2 in increasing bile acid excretion was greater than the effect of sitosterol or of the oils tested in experiment 1. Lecithin, furthermore, had a

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Chromatographed as the trimethyl silyl ethers of the methyl esters under the following conditions: Instrument: Hewlett Packard F and M Model 5750 gas chromatograph with dual flame detector Column: stainless steel 6'6"X0.25" Packing: 1% SE-30 on 100-120 Gas Chromosorb Q (Applied Science Laboratories, Inc.) Carrier gas: nitrogen 80 c.c./minute Temperature: column 245°C, injection port 275°C, detector 260°C.

BILE ACID EXCRETION

marked effect on the chromatographic pattern of the bile acids present in the excreta, whereas the bile acids excreted by the birds fed the other lipids appear to be similar to those excreted by the birds fed the basal diets. SUMMARY

REFERENCES Ali, S. S., A. Kuksis and J. M. R. Beveridge, 1966. Excretion of bile acids by three men on corn oil and butterfat diets. Can. J. Biochem. 44: 13771388. Anderson, I. G., G. A. D. Haslewood and I. D. P. Wootton, 1957. Comparative studies of bile salts. 10. Bile salts of the king penguin Aptenodites patagonica. Biochem. J. 67:323-328. Avigan, J., and D. Steinberg, 1965. Sterol and bile acid excretion in man and the effects of dietary fat. J. Clin. Invest. 44: 1845-1856. Byers, S. O., and M. Friedman, 1958. Bile acids, dietary fats and plasma cholesterol levels. Proc. Soc. Exp. Biol. Med. 98: 523-526. Fisher, H., H. S. Weiss and P. Griminger, 1961. Influence of fatty acids and sterols on atherosclerosis in the avian abdominal aorta. Proc. Soc. Exp. Biol. Med. 106:61-63. Fisher, H., H. S. Weiss and P. Griminger, 1963.

Corn sterols and avian atherosclerosis. Proc. Soc. Exp. Biol. Med. 113: 415-418. Goldsmith, G. A., J. G. Hamilton and O. N. Miller, 1960. Lowering of serum lipid concentrations. Mechanism used by unsaturated fats, nicotinic acid and neomycin: excretion of sterols and bile acids. Arch. Internal Med. 105: 512-517. Gordon, H., B. Lewis, L. Eales and J. F. Brock, 1957. Fecal elimination of bile acids and other lipids. Lancet, 2: 1299-1306. Grundy, S. M., E. H. Ahrens, Jr. and T. Miettinon, 1965. Quantitative isolation and gas liquid chromatographic analysis of total fecal bile acids. J. Lipid Res. 6: 397-410. Haust, H. L., and J. M. R. Beveridge, 1958. Effect of varying type and quantity of dietary fat on the fecal excretion of bile acids in humans subsisting on formula diets. Arch. Biochem. Biophys. 78: 367-375. Higashi, S., 1930. Arb. Med. Univ. Okayama 1: 582. As cited by T. Fukase, and K. Fuziwara, 1932. Uber die Gallinsaurebildung (v). Uber den Einfluss der Nahrung auf den Gallensauregehalt der Galle. J. Biochem. (Tokyo) 15: 193-196. Kesten, H. D., and R. Silbowitz, 1942. Experimental atherosclerosis and soya lecithin. Proc. Soc. Exp. Biol. Med. 49: 71-73. Kummerow, F. A., A. Uene, T. Nishida and M. Kokatnur, 1960. Unsaturated fatty acids and plasma lipids. Am. J. Clin. Nutrition, 8: 62-67. Lindestedt, S., J. Avigan, De W. S. Goodman, S. Sjovall and D. Steinberg, 1965. The effect of dietary fat on the turnover of cholic acid and on the composition of the biliary bile acids in man. J. Clin. Invest. 44:1754-1765. Moore, R. B., J. T. Anderson, A. Keys and I. D. Franz, Jr., 1962. Effect of dietary fat on the fecal excretion of cholesterol and its degradation products in human subjects. J. Lab. Clin. Med. 60: 1000. Moore, R. B., J. T. Anderson, H. L. Taylor, A. Keys and I. D. Frantz, Jr., 1968. Effect of dietary fat on the fecal excretion of cholesterol and its degradation products in man. J. Clin. Invest. 47: 1517-1534. Roels, O. A., and S. A. Hashim, 1962. Influence of fatty acids on serum cholesterol. Fed. Proc. 21: Suppl. 11:71-76. Sperry, W. M., and M. Webb, 1950. A revision of the Schoenheimer-Sperry method for cholesterol determination. J. Biol. Chem. 187: 97-106. Spritz, N., E. H. Ahrens, Jr. and S. Grundy, 1965. Sterol balance in man as plasma cholesterol concentrations are altered by exchanges of dietary fats. J. Clin. Invest. 44: 1482-1493.

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Bile acid excretion in chickens was measured in response to feeding different lipids. Adult cockerels fed 15% of coconut oil excreted lesser amounts of bile acids than did cockerels fed 15% of either corn oil or herring oil. The differences in bile acid excretion were evident both in the percentages of bile acids in the excreta and in the daily excretion per kg. of body weight. Both 0.5% of /3-sitosterol and 2.5% of crude soybean lecithin increased bile acid excretion in growing cockerels. The increase in excretion of bile acids induced by the sitosterol was 20 to 30 percent depending upon the basis for calculation, and the increase resulting from feeding lecithin was 80 to 100 percent. The feeding of lecithin resulted in appreciable alteration of the proportions of different bile acids excreted.

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Srikantia, S. G., and C. Gopalan, 1960. Effects of different dietary fats on the fecal excretion of bile acids. Indian J. Med. Res. 48: 503-506. Steimer, A., and B. Domanski, 1944. Effect of feeding "soya" lecithin on serum cholesterol. Proc. Soc. Exp. Biol. Med. 55: 236-238. Vahouny, G. V., C. R. Borja, R. M. Mayer and

C. R. Treadwell, 1960. A rapid quantitative determination of total and free cholesterol with anthrone reagent. Anal. Biochem. 1: 371-381. Whiteside, C. H., H. B. Fluckiger and H. P. Sarrett, 1965. Plasma and liver cholesterol levels in chicks fed medium chain triglycerides and cholestyramine. Proc. Soc. Exp. Biol. Med. 18: 77-79.

A. W. ADAMS, j . L. WEST 1 AND A. J. KAHRS Departments of Dairy and Poultry Science and Pathology, Parasitology and Public Health, Kansas Slate University, Manhattan, Kansas 66502 (Received for publication January 7, 1969)

INTRODUCTION

E

XPERIMENTAL evidence was presented in a previous report suggesting the poult is more susceptible to nitrate toxicity than the chick and laying hen (Adams et.al., 1966). Arends et al. (1967) reported that sodium nitrate, at a level equivalent to 675 p.p.m. nitrate, caused heavier body weights at subsequent ages, when added to the drinking water of two strains of Broad Breasted Bronze to four weeks of age. The effect was more pronounced in males than females. However, a similar response was not obtained from poults receiving an equivalent level of nitrate as potassium nitrate in the water or sodium nitrate in the feed equivalent to 1,000 p.p.m. nitrate. More recently Marrett and Sunde (1968) reported that chicks up to five weeks old were more tolerant to nitrate and nitrite in the feed than poults. They also found that mortality and rate of respiration were increased and growth reduced when high levels of nitrate and nitrite were fed in the presence of marginal levels of vitamin A. 1 Present address: UT-AEC Agricultural Research Laboratory, Oak Ridge, Tennessee 37832.

The data reported here describe the effects of various levels of nitrate, administered continuously in the drinking water, on poults of various ages. EXPERIMENTAL Experiments 1 and 2. These experiments were conducted for the purpose of obtaining information on the lethal level of nitrates for young poults and to determine if toxicity symptoms exhibited by poults on high levels of sodium nitrate are due to an excess of nitrate and/or sodium ions. Day-old Kimber Broad Breasted Bronze poults were randomly distributed into an electrically heated battery brooder. Duplicate groups of 10 and 25 straight run poults for experiments 1 and 2, respectively, were given well water to which various levels of sodium nitrate and sodium acetate were added. For experiment 1, sodium nitrate was added to the water at levels equivalent to 0, 2,660, 3,990, 5,320, 6,650 and 7,980 p.p.m. nitrate. For the second experiment sodium nitrate was added to the water at levels equivalent to 0, 3,325, 3,990, and 4,655 p.p.m. nitrate. An additional treatment consisted of adding sodium in the form of sodium

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Some Effects on Turkeys of Nitrate in the Drinking Water