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Influence of Dietary Oil, Cholesterol, and Soysterols on the Fecal Neutral and Acidic Steroid Excretion in Laying Hens
Influence of Dietary Oil, Cholesterol, and Soysterols on the Fecal Neutral and Acidic Steroid Excretion in Laying Hens J. S. SIM, W. D. KITTS, and D. B. BRAGG
Department of Poultry Science, Faculty of Agricultural Sciences, University of British Columbia, Vancouver, British Columbia, Canada V6T 1W5 (Received for publication November 20, 1978)
INTRODUCTION The cholesterol metabolism in animals can be altered by a number of dietary factors. However, the mechanisms of action proposed by many investigators are not in complete accord. The reason for these differences appear to be the result of complex alteration in cholesterol absorption, synthesis, catabolism, and elimination from the body that may respond to the dietary influence under different experimental conditions. Cholesterol in the mammalian system is eliminated entirely by excretion into the feces, largely as neutral steroid and bile acids (Danielson and Tchen, 1969). Many studies have shown that the quantity or the patterns of the fecal sterols can be altered by various factors. Goldsmith et al. (1960) showed that excretion of bile acids in humans increased 20 to 25% concomitantly with a decrease in serum cholesterol when polyunsaturated fatty acids were fed. An increase in fecal output of total neutral sterols was also demonstrated in human studies when unsaturated fatty acids were included in the diet (Grundy and Ahrens, 1966; Moore et al, 1968; Wood et al, 1966; Connor et al, 1969). Cholesterol supplementation of the rat diet showed a significant increase in the fecal bile acid excretion (Portman and Stare, 1959; Wilson, 1964). Lindsey et al. (1969) reported
that feeding sitosterol to cockerels increased total bile acid excretion in feces. The following study was designed to examine the influence of dietary cholesterol levels, hydrogenated coconut oil, safflower oil, and soysterols on the excretion pattern of fecal neutral and acidic steroids in laying hens.
MATERIALS AND METHODS One hundred and twenty-eight Single Comb White Leghorn laying hens (30 weeks of age) were randomly divided into eight experimental groups of 16 birds and were fed eight experimental diets. Feed and water were provided ad libitum for the entire nine-week experimental period. The experimental diets (Sim and Bragg, 1977 and 1978) included two basal diets that contained 8.0% hydrogenated coconut oil (HCO) or 8.0% safflower oil (SFO). These basal diets were supplemented with 1.0% of cholesterol (CH), 2.0% of soysterols (ST) or a combination of both (CH + ST). Four birds per treatment group were selected on the basis of weekly egg production (55 to 65%) and body weight (1.7 to 2.2 kg) and transferred to individual cages. The eight experimental diets were mixed with a marker (.3% chromic oxide) and fed for six days. Feces were collected from individual birds
325
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 4, 2015
ABSTRACT The influence of dietary lipid factors (saturated and unsaturated oil, cholesterol, and plant sterols) upon the fecal neutral and acidic sterol excretion in the laying hen were investigated during a nine-week experimental period. Single Comb White Leghorn laying hens at thirty weeks of age were fed two basal diets containing 8.0% hydrogenated coconut oil or safflower oil, with or without supplementing 1.0% cholesterol, 2.0% soysterols, or combination of cholesterol and soysterols with each oil treatment. Feeding safflower oil increased the fecal excretion of bile acids and, to a lesser extent, catabolic neutral sterols as compared to hydrogenated coconut oil. The fecal excretion of sterol metabolites was further enhanced when soysterols and cholesterol were fed simultaneously. When soysterols were fed alone, endogenous cholesterol excretion in feces appeared to increase, but soysterol feeding with cholesterol did not retard the apparent absorption rate of cholesterol. 1980 Poultry Science 59:325-327
SIM ET AL.
326
RESULTS AND DISCUSSION
The amount of cholesterol detected in the feces from hens fed the cholesterol-free diets was less than 10 mg per hen/day (Fig. 1), indicating that laying hens do not eliminate large amounts of endogenous cholesterol in the feces when no cholesterol is provided in the diet. However, soysterol incorporation into the cholesterol-free basal diets caused an increase in the fecal cholesterol excretion (6.2 mg to 19.2 mg with HCO diet and 9.4 mg to 17.0 mg with SFO diet, respectively). The type of dietary oil affected the quantity of fecal cholesterol excreted when cholesterol was incorporated into the diet. Birds fed hydrogenated coconut oil excreted 422 and 375 mg/hen/day with HCO + CH and HCO + CH + ST, respectively, whereas hens fed diets containing safflower oil excreted only 255 and
BASAL
CH
CM HIT) niFT
S
BASAL
CH
ST
CH + ST
SFO DIET
FIG. 1. Effect of dietary oil, choelsterol, and soysterols on the fecal steroid excretion.
243 mg/hen/day with SFO + CH and SFO + CH + ST respectively. Apparent retention rates of dietary cholesterol were 75.6% and 58.5% for birds fed SFO + CH and HCO + CH diets, respectively. Data (Fig. 1) clearly indicate that laying hens fed cholesterol with safflower oil retained more cholesterol than those fed dietary cholesterol with hydrogenated coconut oil. Neither the amounts of cholesterol excreted in feces nor the rate of dietary cholesterol retention shows any indication that the dietary presence of soysterols interfered with cholesterol absorption. It is difficult to reconcile these results with the idea that plant sterols inhibit cholesterol absorption (Davis, 1955). Birds receiving no added soysterols in the diet excreted a small amount of plant sterols (7.6 and 13.0 mg/hen/day with HCO and SFO basal diets, respectively). The fecal output of plant sterols increased to more than 400 mg/hen/day when 2% of soysterols were incorporated into diets. The dietary presence of cholesterol further accelerated the fecal output of plant sterols (more than 30%), irrespective of the type of oil source used. The apparent absorption of soysterol in laying hens fed HCO + ST or SFO + ST was 77%. Results obtained clearly demonstrated that plant sterols used in this study were absorbed by laying hens in relatively large amounts and the absorbability was comparable to that of cholesterol. Birds fed safflower oil excreted fecal bile acids at a greater rate than birds fed hydrogenated coconut oil irrespective of sterol treatments. The amount of bile acids excreted by hens fed the SFO diet was almost four-fold that of hens fed the HCO diet. These results are in accord with reports that dietary unsaturated fats increase and saturated fats decrease the fecal output of acidic sterols in human subjects (Goldsmith et al, 1960) and in chickens (Lindsey et al, 1969). The rate of bile acid output in feces, however, was markedly altered by the presence of dietary sterols. The addition of soysterols to either SFO or HCO diets increased fecal bile acid excretion. The influence of dietary soysterols was greatest when fed simultaneously with cholesterol. Feeding soysterols in combination with cholesterol (SFO + CH + ST and HCO + CH + ST) increased the fecal bile acid output more than 40% as compared to feeding soysterol alone (SFO + ST and HCO + ST). No change, however, occurred in the amount of bile acids
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 4, 2015
for three consecutive days after a three-day adjustment period on the chromic oxide food. The three-day fecal collection was homogenized in a Waring blender, lyophilized, and subjected to the quantitative analysis of neutral steroids and total bile acids by the methods of Miettinen et al. (1965) and Grundy et al. (1965), respectively. Chromic oxide in the feed and feces samples was determined by the method described by Williams etal. (1962) using an atomic absorption spectrophotometer (Jarrell Ash 82-500). Daily fecal excretion of cholesterol, plant sterols, bile acids, and unidentifiable steroid metabolites were computed using daily feed intake and the ratio of chromic oxide in the dried feed and feces (Fig. 1).
DIETARY OIL, CHOLESTEROL, SOYSTEROLS, FECAL STEROIDS
Evidence described above indicates t h a t t h e anti-cholesterogenic function of plant sterols in laying hens (Sim and Bragg, 1 9 7 7 ) is d u e t o an influence o n cholesterol catabolism rather t h a n cholesterol a b s o r p t i o n . This factor appears t o increase t h e degradation followed b y excretion of degraded cholesterol in feces as bile acids and neutral sterol metabolites. ACKNOWLEDGMENTS T h e a u t h o r s wish t o express their appreciation t o N. E m b r e e , Distillation P r o d u c t s Industries, Rochester, NY for supplying t h e soysterols used in this s t u d y . S u p p o r t from t h e National Research Council of Canada and t h e Canadian Egg Marketing Agency is acknowledged w i t h appreciation.
REFERENCES Connor, W. E., D. B. Stone, and M. L. Armstrong, 1969. Cholesterol balance and fecal neutral sterols and bile acid excretion in normal man fed dietary fats of different fatty acid composition. J. Clin. Invest. 48:1363-1375. Danielson, H., and T. T. Tchen, 1969. Page 1 1 7 - 1 6 8 in Steroid metabolism in metabolic pathways. Vol. II. D. M. Greenberg, ed. 3rd ed. Academic Press, New York.
Davis, W. W., 1955. The physical chemistry of cholesterol and B-sitosterol related to the intestinal absorption of cholesterol. Trans. New York Acad. Sci. 18:123-134. 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. Int. Med. 105:512-517. Grundy, S. M., and E. H. Ahrens, 1966. An evaluation of the relative methods for measuring the balance of sterols in man: Isotopic balance versus chromatographic analysis. J. Clin. Invest. 45:1503 — 1515. Grundy, S. M., E. H. Ahrens, and T. A. Miettinen, 1965. Quantitative isolation and gas-liquid chromatographic analysis of total fecal bile acids. J. Lipid. Res. 6:397-410. Lindsey, O. B., J. Biely, and B. E. March, 1969. Excretion of bile acids by cockerels fed different lipids. Poultry Sci. 48:1216-1222. Miettinen, T. A., E. F. Ahrens, and S. M. Grundy, 1965. Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral steroids. J. Lipid Res. 6:411—424. 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. Portman, O. W., and F. J. Stare, 1959. Dietary regulation of serum cholesterol levels. Physio. Revs. 39:407. Sim, J. S., and D. B. Bragg, 1977. Effect of dietary factors on serum and egg yolk cholesterol levels of laying hens. Poultry Sci. 56:1616-1621. Sim, J. S., and D. B. Bragg, 1978. Effect of dietary oil, cholesterol and soysterols on the lipid concentration and fatty acid composition of egg yolk, liver and serum of laying hens. Poultry Sci. 5 7 : 4 6 6 472. Williams, C. H., D. J. David, and O. Ismaa, 1962. The determination of chromic oxide in feces samples by atomic absorption spectrometry. J. Agr. Sci. 59:381-385. Wilson, J. D., 1964. The quantification of cholesterol excretion and degeneration in the isotopic steady state in the rat: the influence of dietary cholesterol. J. Lipid Res. 5:409-417. Wood, P. D., R. Shioda, and L. W. Kinsell, 1966. Dietary regulation of cholesterol metabolism. Lancet 2:604-607.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 4, 2015
excreted w h e n cholesterol was fed w i t h o u t dietary soysterols. Fecal o u t p u t of unidentifiable n e u t r a l sterol o t h e r t h a n cholesterol and plant sterols (termed as degraded steroids) was almost negligible w h e n hens were fed t h e basal diets. Soysterol i n c o r p o r a t i o n into these diets p r o d u c e d a t r e m e n d o u s increase in t h e fecal o u t p u t of degraded neutral sterols and t h e i n c r e m e n t s were a p p r o x i m a t e l y d o u b l e d w h e n hens were fed cholesterol and soysterols simultaneously. T h e dietary c o m b i n a t i o n of cholesterol and plant sterols p r o d u c e d a synergistic effect o n t h e fecal excretion of bile acids and degraded neutral steroids in laying hens.
327
Department of Poultry Science, Faculty of Agricultural Sciences, University of British Columbia, Vancouver, British Columbia, Canada V6T 1W5 (Received for publication November 20, 1978)
INTRODUCTION The cholesterol metabolism in animals can be altered by a number of dietary factors. However, the mechanisms of action proposed by many investigators are not in complete accord. The reason for these differences appear to be the result of complex alteration in cholesterol absorption, synthesis, catabolism, and elimination from the body that may respond to the dietary influence under different experimental conditions. Cholesterol in the mammalian system is eliminated entirely by excretion into the feces, largely as neutral steroid and bile acids (Danielson and Tchen, 1969). Many studies have shown that the quantity or the patterns of the fecal sterols can be altered by various factors. Goldsmith et al. (1960) showed that excretion of bile acids in humans increased 20 to 25% concomitantly with a decrease in serum cholesterol when polyunsaturated fatty acids were fed. An increase in fecal output of total neutral sterols was also demonstrated in human studies when unsaturated fatty acids were included in the diet (Grundy and Ahrens, 1966; Moore et al, 1968; Wood et al, 1966; Connor et al, 1969). Cholesterol supplementation of the rat diet showed a significant increase in the fecal bile acid excretion (Portman and Stare, 1959; Wilson, 1964). Lindsey et al. (1969) reported
that feeding sitosterol to cockerels increased total bile acid excretion in feces. The following study was designed to examine the influence of dietary cholesterol levels, hydrogenated coconut oil, safflower oil, and soysterols on the excretion pattern of fecal neutral and acidic steroids in laying hens.
MATERIALS AND METHODS One hundred and twenty-eight Single Comb White Leghorn laying hens (30 weeks of age) were randomly divided into eight experimental groups of 16 birds and were fed eight experimental diets. Feed and water were provided ad libitum for the entire nine-week experimental period. The experimental diets (Sim and Bragg, 1977 and 1978) included two basal diets that contained 8.0% hydrogenated coconut oil (HCO) or 8.0% safflower oil (SFO). These basal diets were supplemented with 1.0% of cholesterol (CH), 2.0% of soysterols (ST) or a combination of both (CH + ST). Four birds per treatment group were selected on the basis of weekly egg production (55 to 65%) and body weight (1.7 to 2.2 kg) and transferred to individual cages. The eight experimental diets were mixed with a marker (.3% chromic oxide) and fed for six days. Feces were collected from individual birds
325
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 4, 2015
ABSTRACT The influence of dietary lipid factors (saturated and unsaturated oil, cholesterol, and plant sterols) upon the fecal neutral and acidic sterol excretion in the laying hen were investigated during a nine-week experimental period. Single Comb White Leghorn laying hens at thirty weeks of age were fed two basal diets containing 8.0% hydrogenated coconut oil or safflower oil, with or without supplementing 1.0% cholesterol, 2.0% soysterols, or combination of cholesterol and soysterols with each oil treatment. Feeding safflower oil increased the fecal excretion of bile acids and, to a lesser extent, catabolic neutral sterols as compared to hydrogenated coconut oil. The fecal excretion of sterol metabolites was further enhanced when soysterols and cholesterol were fed simultaneously. When soysterols were fed alone, endogenous cholesterol excretion in feces appeared to increase, but soysterol feeding with cholesterol did not retard the apparent absorption rate of cholesterol. 1980 Poultry Science 59:325-327
SIM ET AL.
326
RESULTS AND DISCUSSION
The amount of cholesterol detected in the feces from hens fed the cholesterol-free diets was less than 10 mg per hen/day (Fig. 1), indicating that laying hens do not eliminate large amounts of endogenous cholesterol in the feces when no cholesterol is provided in the diet. However, soysterol incorporation into the cholesterol-free basal diets caused an increase in the fecal cholesterol excretion (6.2 mg to 19.2 mg with HCO diet and 9.4 mg to 17.0 mg with SFO diet, respectively). The type of dietary oil affected the quantity of fecal cholesterol excreted when cholesterol was incorporated into the diet. Birds fed hydrogenated coconut oil excreted 422 and 375 mg/hen/day with HCO + CH and HCO + CH + ST, respectively, whereas hens fed diets containing safflower oil excreted only 255 and
BASAL
CH
CM HIT) niFT
S
BASAL
CH
ST
CH + ST
SFO DIET
FIG. 1. Effect of dietary oil, choelsterol, and soysterols on the fecal steroid excretion.
243 mg/hen/day with SFO + CH and SFO + CH + ST respectively. Apparent retention rates of dietary cholesterol were 75.6% and 58.5% for birds fed SFO + CH and HCO + CH diets, respectively. Data (Fig. 1) clearly indicate that laying hens fed cholesterol with safflower oil retained more cholesterol than those fed dietary cholesterol with hydrogenated coconut oil. Neither the amounts of cholesterol excreted in feces nor the rate of dietary cholesterol retention shows any indication that the dietary presence of soysterols interfered with cholesterol absorption. It is difficult to reconcile these results with the idea that plant sterols inhibit cholesterol absorption (Davis, 1955). Birds receiving no added soysterols in the diet excreted a small amount of plant sterols (7.6 and 13.0 mg/hen/day with HCO and SFO basal diets, respectively). The fecal output of plant sterols increased to more than 400 mg/hen/day when 2% of soysterols were incorporated into diets. The dietary presence of cholesterol further accelerated the fecal output of plant sterols (more than 30%), irrespective of the type of oil source used. The apparent absorption of soysterol in laying hens fed HCO + ST or SFO + ST was 77%. Results obtained clearly demonstrated that plant sterols used in this study were absorbed by laying hens in relatively large amounts and the absorbability was comparable to that of cholesterol. Birds fed safflower oil excreted fecal bile acids at a greater rate than birds fed hydrogenated coconut oil irrespective of sterol treatments. The amount of bile acids excreted by hens fed the SFO diet was almost four-fold that of hens fed the HCO diet. These results are in accord with reports that dietary unsaturated fats increase and saturated fats decrease the fecal output of acidic sterols in human subjects (Goldsmith et al, 1960) and in chickens (Lindsey et al, 1969). The rate of bile acid output in feces, however, was markedly altered by the presence of dietary sterols. The addition of soysterols to either SFO or HCO diets increased fecal bile acid excretion. The influence of dietary soysterols was greatest when fed simultaneously with cholesterol. Feeding soysterols in combination with cholesterol (SFO + CH + ST and HCO + CH + ST) increased the fecal bile acid output more than 40% as compared to feeding soysterol alone (SFO + ST and HCO + ST). No change, however, occurred in the amount of bile acids
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 4, 2015
for three consecutive days after a three-day adjustment period on the chromic oxide food. The three-day fecal collection was homogenized in a Waring blender, lyophilized, and subjected to the quantitative analysis of neutral steroids and total bile acids by the methods of Miettinen et al. (1965) and Grundy et al. (1965), respectively. Chromic oxide in the feed and feces samples was determined by the method described by Williams etal. (1962) using an atomic absorption spectrophotometer (Jarrell Ash 82-500). Daily fecal excretion of cholesterol, plant sterols, bile acids, and unidentifiable steroid metabolites were computed using daily feed intake and the ratio of chromic oxide in the dried feed and feces (Fig. 1).
DIETARY OIL, CHOLESTEROL, SOYSTEROLS, FECAL STEROIDS
Evidence described above indicates t h a t t h e anti-cholesterogenic function of plant sterols in laying hens (Sim and Bragg, 1 9 7 7 ) is d u e t o an influence o n cholesterol catabolism rather t h a n cholesterol a b s o r p t i o n . This factor appears t o increase t h e degradation followed b y excretion of degraded cholesterol in feces as bile acids and neutral sterol metabolites. ACKNOWLEDGMENTS T h e a u t h o r s wish t o express their appreciation t o N. E m b r e e , Distillation P r o d u c t s Industries, Rochester, NY for supplying t h e soysterols used in this s t u d y . S u p p o r t from t h e National Research Council of Canada and t h e Canadian Egg Marketing Agency is acknowledged w i t h appreciation.
REFERENCES Connor, W. E., D. B. Stone, and M. L. Armstrong, 1969. Cholesterol balance and fecal neutral sterols and bile acid excretion in normal man fed dietary fats of different fatty acid composition. J. Clin. Invest. 48:1363-1375. Danielson, H., and T. T. Tchen, 1969. Page 1 1 7 - 1 6 8 in Steroid metabolism in metabolic pathways. Vol. II. D. M. Greenberg, ed. 3rd ed. Academic Press, New York.
Davis, W. W., 1955. The physical chemistry of cholesterol and B-sitosterol related to the intestinal absorption of cholesterol. Trans. New York Acad. Sci. 18:123-134. 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. Int. Med. 105:512-517. Grundy, S. M., and E. H. Ahrens, 1966. An evaluation of the relative methods for measuring the balance of sterols in man: Isotopic balance versus chromatographic analysis. J. Clin. Invest. 45:1503 — 1515. Grundy, S. M., E. H. Ahrens, and T. A. Miettinen, 1965. Quantitative isolation and gas-liquid chromatographic analysis of total fecal bile acids. J. Lipid. Res. 6:397-410. Lindsey, O. B., J. Biely, and B. E. March, 1969. Excretion of bile acids by cockerels fed different lipids. Poultry Sci. 48:1216-1222. Miettinen, T. A., E. F. Ahrens, and S. M. Grundy, 1965. Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral steroids. J. Lipid Res. 6:411—424. 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. Portman, O. W., and F. J. Stare, 1959. Dietary regulation of serum cholesterol levels. Physio. Revs. 39:407. Sim, J. S., and D. B. Bragg, 1977. Effect of dietary factors on serum and egg yolk cholesterol levels of laying hens. Poultry Sci. 56:1616-1621. Sim, J. S., and D. B. Bragg, 1978. Effect of dietary oil, cholesterol and soysterols on the lipid concentration and fatty acid composition of egg yolk, liver and serum of laying hens. Poultry Sci. 5 7 : 4 6 6 472. Williams, C. H., D. J. David, and O. Ismaa, 1962. The determination of chromic oxide in feces samples by atomic absorption spectrometry. J. Agr. Sci. 59:381-385. Wilson, J. D., 1964. The quantification of cholesterol excretion and degeneration in the isotopic steady state in the rat: the influence of dietary cholesterol. J. Lipid Res. 5:409-417. Wood, P. D., R. Shioda, and L. W. Kinsell, 1966. Dietary regulation of cholesterol metabolism. Lancet 2:604-607.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 4, 2015
excreted w h e n cholesterol was fed w i t h o u t dietary soysterols. Fecal o u t p u t of unidentifiable n e u t r a l sterol o t h e r t h a n cholesterol and plant sterols (termed as degraded steroids) was almost negligible w h e n hens were fed t h e basal diets. Soysterol i n c o r p o r a t i o n into these diets p r o d u c e d a t r e m e n d o u s increase in t h e fecal o u t p u t of degraded neutral sterols and t h e i n c r e m e n t s were a p p r o x i m a t e l y d o u b l e d w h e n hens were fed cholesterol and soysterols simultaneously. T h e dietary c o m b i n a t i o n of cholesterol and plant sterols p r o d u c e d a synergistic effect o n t h e fecal excretion of bile acids and degraded neutral steroids in laying hens.
327