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Effect of Raw and Heated Soybean Meal on Lipogenic Capacity of Chick Liver Slices
1239
RESEARCH NOTES
Moran, E. T. 1973. Unpublished observations. Simpson, G. G., A. Roe and R. C. Lewontin, 1960. Quantitative Zoology. Harcourt Brace & Co. New
York, pp. 172-305. Sweeny, P. R.,andR. G. Brown, 1972. Ultrastructural changes in muscular dystrophy. Cardiac tissue of
EFFECT OF RAW AND HEATED SOYBEAN MEAL ON LIPOGENIC CAPACITY OF CHICK LIVER SLICES
(Received for publication November 7, 1973)
ABSTRACT The incorporation of [1-14C] sodium acetate into cholesterol and fatty acids by liver slices from young chicks was enhanced by the feeding of raw soybean meal, compared to heated soybean meal. The hepatic fatty acid synthesizing capacity was also affected by age and by the amount of oil added to the diet. POULTRY SCIENCE 53: 1239-1241, 1974
INTRODUCTION
R
AW soybean meal (RS) has long been know to produce adverse effects on growth and protein utilization in experimental animals, compared to heated soybean meal (HS). In the young chicks, these effects are accompanied by a reduction in the apparent absorption of added dietary fat (Brambila et al., 1961), pancreatic hypertrophy (Chernick et al., 1948) and contraction of the gall bladders (Garlich and Nesheim, 1965). Pancreatic (Bielorai et al., 1973) and biliary (Sklan et al., 1973) secretions appear to be considerably increased. In an attempt to detect possible effects of RS on lipid metabolism we have investigated the lipogenic capacity of liver slices from chicks fed RS and HS. EXPERIMENTAL PROCEDURE Day-old New Hampshire x White Leghorn male chicks were maintained on a commercial starter diet. The experimental diets were given over a period of 6 d., starting either on the 7th, 14, or 35th day. The diets contained 50% of either raw or heated soybean meal, 4 or 15% soybean oil, a glucose-starch
mixture (equal parts by weight), minerals and vitamins (Nir and Ascarelli, 1966). At the end of the experimental feeding period, 10 chicks per treatment were killed by decapitation, and their livers were removed immediately. Liver slices weighing 0.5 g. were incubated for 2 h. at 37° C. in 5 ml. Krebs-Ringer phosphate buffer under 0 2 -C0 2 (95:5), in the presence of 0.5 ml. 10 mM sodium acetate and 0.1 nCi. [1-14C] sodium acetate (62 mCi./mole, The Radiochemical Centre, Amersham). Incubations were stopped by addition of 2 ml. 90% (w. / v . ) aqueous KOH, and the mixture was autoclaved for 30 min. at 1.5 atm. The unsaponifiable matter was extracted with petroleum ether (b.p. 60-80° C ) . The aqueous solution was acidified with HCI, and the fatty acids were extracted with petroleum ether. Samples of the waterwashed extracts of the unsaponifiable material and fatty acids were assayed for radioactivity in toluene containing 0.4% PPO and 0.01 POPOP, using a Packard Liquid Scintillation Counter. Evidence that the radioactivity of the unsaponifiable material was due essentially to the cholesterol was as follows: 95% of the total unsaponifiable radioactivity was found in the sterol fraction separated by TLC on Silica Gel G, and cholesterol made
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 19, 2015
D . SKLAN, I . ASCARELLI AND P . BUDOWSKI
Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel
1240
o
RESEARCH NOTES
is 2.0
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'
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i
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8 Z 6 LU
FATTY ACIDS
_I UJ *
0-
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^4 •4% 151. -13 d — I
4%
75%
—21 d —I
4%
&1 15%
1 — 41 d — I
FIG. 1. Incorporation of [1-14C] acetate into cholesterol and fatty acids by liver slices from chicks fed raw • or heated • soybean meal for six days. The height of each bar represents the mean incorporation determined for 10 chicks. Standard deviations are shown above the bars. The age of the chicks at killing and the percentage of oil in the diet are as indicated. For statistical treatment, see text. up 98% of the sterol fraction, as revealed by GLC. RESULTS AND DISCUSSION The results of the incubation tests are presented in Fig. 1. Factorial analysis of variance revealed that incorporation of acetate into cholesterol by liver slices was enhanced by RS, compared to HS (P < 0.01), whereas the other variables, namely dietary fat level and age of chicks, and interactions between them, produced no significant differences.
The fatty acid synthesizing capacity of liver slices was influenced significantly (P < 0.01) by the three variables: fat level, age and type of soybean meal. Incorporation decreased with increasing age and dietary fat level. On the other hand, RS caused an increase in acetate incorporation into fatty acids, the effect being particularly pronounced in 13day-old chicks, when RS more than doubled the incorporation, compared to HS. Furthermore, a significant interaction (P < 0.05) was found between dietary fat level and age, due to a greater depression of fatty acid synthe-
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 19, 2015
go,
X
V
RESEARCH NOTES
ACKNOWLEDGMENT Supported in part by a grant from the Central Research Fund of the Hebrew University of Jerusalem; technical assistance by Ofra Kedar. REFERENCES Bielorai, R. M., M. Tamir, E. Alumot, A. Bar and S. Hurwitz, 1973. Digestion and absorption of pro-
tein in the intestinal segments of chicks fed raw and heated soybean meal. J. Nutr. 103: 1291-1298. Brambila, S., M. C. Nesheim and F. W. Hill, 1961. Effect of trypsin supplementation on the utilization by the chick of diets containing raw soybean oil meal. J. Nutr. 75: 13-20. Chernik, S. S., S. Lepkovsky and I. J. Chaikoff, 1948. A dietary factor regulating the enzyme content of the pancreas: Changes induced in size and proteolytic activity of the chick pancreas by the ingestion of raw soybean meal. Am. J. Physiol. 155: 33-41. Garlich, J. D., and M. C. Nesheim, 1965. Effect of sodium taurocholate on fat malabsorption induced by feeding unheated soybean proteins. Proc. Soc. Exp. Biol. Med. 118: 1022-1025. Nir, I., and I. Ascarelli, 1966. Effect of protein level and thyroxine on vitamin A depletion in chicks. Br. J. Nutr. 20: 40-53. Sabine, J. R., H. McGrath and S. Abraham, 1969. Dietary fat and inhibition of hepatic lipogenesis in the mouse. J. Nutr. 98: 312-318. Sklan, D., P. Budowski, I. Ascarelli and S. Hurwitz, 1973. Lipid secretion and absorption in the chick: effect of raw soybean meal. J. Nutr. 103: 1299-1305. piglets deprived of vitamin E and selenium. Amer. J. Path. 68: 479-486. Sweeny, P. R., J. G. Buchanan-Smith, F. Demille, J. R. Pettit and E. T. Moran, 1972. Ultrastructure of muscular dystrophy. II. A comparative study in lambs and chickens. Amer. J. Path. 68: 493-504.
JUNE 11-13. 8TH ANNUAL CONFERENCE ON TRACE SUBSTANCES IN ENVIRONMENTAL HEALTH, MEMORIAL UNION, UNIVERSITY OF MISSOURI, COLUMBIA, MISSOURI. JUNE 12-15. SPECIAL CONFERENCE ON THE ANALYSIS OF LIPIDS AND LIPOPROTEINS, AMERICAN OIL CHEMISTS' SOCIETY, SHERATON PARK HOTEL, WASHINGTON, D.C. JUNE 16-21. 25TH ANNUAL MEETING, AMERICAN INSTITUTE OF BIOLOGICAL SCIENCES, ARIZONA STATE UNIVERSITY, TEMPE. JUNE 21-22. DELMARVA CHICKEN FESTIVAL AND DELMARVA CHICKEN COOKING CONTEST, SEAFORD, DELAWARE. JULY 16-17. HATCHERY BREEDER FLOCK MANAGEMENT CLINIC, SOUTHEASTERN POULTRY AND EGG ASSOCIATION, ALL AMERICA INN, AUBURN, ALABAMA. JULY 28-31. 66TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF ANIMAL SCIENCE, UNIVERSITY OF MARYLAND, COLLEGE PARK, MARYLAND. AUGUST 4-8. ANNUAL MEETING OF AGRICULTURAL INSTITUTE OF CANADA AND AFFILIATED SOCIETIES. LAVAL UNIVERSITY, QUEBEC, QUEBEC.
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 19, 2015
sizing capacity with age at the low fat level. There was also an interaction between type of soybean meal and dietary fat level (P < 0.05), due to the greater influence of RS at the low fat level. Other interactions were not significant. The depression of hepatic acetate incorporation into fatty acids observed with the high dietary oil level parallels a similar effect reported for mice receiving dietary oils rich in linoleic acid (Sabine et al., 1969). The probable relationship between the effects of RS on the lipogenic capacity of chicken liver, as found in the present study, and the bilary secretion of lipids and bile acids, reported previously (Sklan etal., 1973), is being examined.
1241
RESEARCH NOTES
Moran, E. T. 1973. Unpublished observations. Simpson, G. G., A. Roe and R. C. Lewontin, 1960. Quantitative Zoology. Harcourt Brace & Co. New
York, pp. 172-305. Sweeny, P. R.,andR. G. Brown, 1972. Ultrastructural changes in muscular dystrophy. Cardiac tissue of
EFFECT OF RAW AND HEATED SOYBEAN MEAL ON LIPOGENIC CAPACITY OF CHICK LIVER SLICES
(Received for publication November 7, 1973)
ABSTRACT The incorporation of [1-14C] sodium acetate into cholesterol and fatty acids by liver slices from young chicks was enhanced by the feeding of raw soybean meal, compared to heated soybean meal. The hepatic fatty acid synthesizing capacity was also affected by age and by the amount of oil added to the diet. POULTRY SCIENCE 53: 1239-1241, 1974
INTRODUCTION
R
AW soybean meal (RS) has long been know to produce adverse effects on growth and protein utilization in experimental animals, compared to heated soybean meal (HS). In the young chicks, these effects are accompanied by a reduction in the apparent absorption of added dietary fat (Brambila et al., 1961), pancreatic hypertrophy (Chernick et al., 1948) and contraction of the gall bladders (Garlich and Nesheim, 1965). Pancreatic (Bielorai et al., 1973) and biliary (Sklan et al., 1973) secretions appear to be considerably increased. In an attempt to detect possible effects of RS on lipid metabolism we have investigated the lipogenic capacity of liver slices from chicks fed RS and HS. EXPERIMENTAL PROCEDURE Day-old New Hampshire x White Leghorn male chicks were maintained on a commercial starter diet. The experimental diets were given over a period of 6 d., starting either on the 7th, 14, or 35th day. The diets contained 50% of either raw or heated soybean meal, 4 or 15% soybean oil, a glucose-starch
mixture (equal parts by weight), minerals and vitamins (Nir and Ascarelli, 1966). At the end of the experimental feeding period, 10 chicks per treatment were killed by decapitation, and their livers were removed immediately. Liver slices weighing 0.5 g. were incubated for 2 h. at 37° C. in 5 ml. Krebs-Ringer phosphate buffer under 0 2 -C0 2 (95:5), in the presence of 0.5 ml. 10 mM sodium acetate and 0.1 nCi. [1-14C] sodium acetate (62 mCi./mole, The Radiochemical Centre, Amersham). Incubations were stopped by addition of 2 ml. 90% (w. / v . ) aqueous KOH, and the mixture was autoclaved for 30 min. at 1.5 atm. The unsaponifiable matter was extracted with petroleum ether (b.p. 60-80° C ) . The aqueous solution was acidified with HCI, and the fatty acids were extracted with petroleum ether. Samples of the waterwashed extracts of the unsaponifiable material and fatty acids were assayed for radioactivity in toluene containing 0.4% PPO and 0.01 POPOP, using a Packard Liquid Scintillation Counter. Evidence that the radioactivity of the unsaponifiable material was due essentially to the cholesterol was as follows: 95% of the total unsaponifiable radioactivity was found in the sterol fraction separated by TLC on Silica Gel G, and cholesterol made
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 19, 2015
D . SKLAN, I . ASCARELLI AND P . BUDOWSKI
Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel
1240
o
RESEARCH NOTES
is 2.0
O <>
i 1
T
1
^ l IV> l _ l _ O 1 L.IA ^ l T
'
1.
i
/•
T
1
i1
8 Z 6 LU
FATTY ACIDS
_I UJ *
0-
A
^4 •4% 151. -13 d — I
4%
75%
—21 d —I
4%
&1 15%
1 — 41 d — I
FIG. 1. Incorporation of [1-14C] acetate into cholesterol and fatty acids by liver slices from chicks fed raw • or heated • soybean meal for six days. The height of each bar represents the mean incorporation determined for 10 chicks. Standard deviations are shown above the bars. The age of the chicks at killing and the percentage of oil in the diet are as indicated. For statistical treatment, see text. up 98% of the sterol fraction, as revealed by GLC. RESULTS AND DISCUSSION The results of the incubation tests are presented in Fig. 1. Factorial analysis of variance revealed that incorporation of acetate into cholesterol by liver slices was enhanced by RS, compared to HS (P < 0.01), whereas the other variables, namely dietary fat level and age of chicks, and interactions between them, produced no significant differences.
The fatty acid synthesizing capacity of liver slices was influenced significantly (P < 0.01) by the three variables: fat level, age and type of soybean meal. Incorporation decreased with increasing age and dietary fat level. On the other hand, RS caused an increase in acetate incorporation into fatty acids, the effect being particularly pronounced in 13day-old chicks, when RS more than doubled the incorporation, compared to HS. Furthermore, a significant interaction (P < 0.05) was found between dietary fat level and age, due to a greater depression of fatty acid synthe-
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 19, 2015
go,
X
V
RESEARCH NOTES
ACKNOWLEDGMENT Supported in part by a grant from the Central Research Fund of the Hebrew University of Jerusalem; technical assistance by Ofra Kedar. REFERENCES Bielorai, R. M., M. Tamir, E. Alumot, A. Bar and S. Hurwitz, 1973. Digestion and absorption of pro-
tein in the intestinal segments of chicks fed raw and heated soybean meal. J. Nutr. 103: 1291-1298. Brambila, S., M. C. Nesheim and F. W. Hill, 1961. Effect of trypsin supplementation on the utilization by the chick of diets containing raw soybean oil meal. J. Nutr. 75: 13-20. Chernik, S. S., S. Lepkovsky and I. J. Chaikoff, 1948. A dietary factor regulating the enzyme content of the pancreas: Changes induced in size and proteolytic activity of the chick pancreas by the ingestion of raw soybean meal. Am. J. Physiol. 155: 33-41. Garlich, J. D., and M. C. Nesheim, 1965. Effect of sodium taurocholate on fat malabsorption induced by feeding unheated soybean proteins. Proc. Soc. Exp. Biol. Med. 118: 1022-1025. Nir, I., and I. Ascarelli, 1966. Effect of protein level and thyroxine on vitamin A depletion in chicks. Br. J. Nutr. 20: 40-53. Sabine, J. R., H. McGrath and S. Abraham, 1969. Dietary fat and inhibition of hepatic lipogenesis in the mouse. J. Nutr. 98: 312-318. Sklan, D., P. Budowski, I. Ascarelli and S. Hurwitz, 1973. Lipid secretion and absorption in the chick: effect of raw soybean meal. J. Nutr. 103: 1299-1305. piglets deprived of vitamin E and selenium. Amer. J. Path. 68: 479-486. Sweeny, P. R., J. G. Buchanan-Smith, F. Demille, J. R. Pettit and E. T. Moran, 1972. Ultrastructure of muscular dystrophy. II. A comparative study in lambs and chickens. Amer. J. Path. 68: 493-504.
JUNE 11-13. 8TH ANNUAL CONFERENCE ON TRACE SUBSTANCES IN ENVIRONMENTAL HEALTH, MEMORIAL UNION, UNIVERSITY OF MISSOURI, COLUMBIA, MISSOURI. JUNE 12-15. SPECIAL CONFERENCE ON THE ANALYSIS OF LIPIDS AND LIPOPROTEINS, AMERICAN OIL CHEMISTS' SOCIETY, SHERATON PARK HOTEL, WASHINGTON, D.C. JUNE 16-21. 25TH ANNUAL MEETING, AMERICAN INSTITUTE OF BIOLOGICAL SCIENCES, ARIZONA STATE UNIVERSITY, TEMPE. JUNE 21-22. DELMARVA CHICKEN FESTIVAL AND DELMARVA CHICKEN COOKING CONTEST, SEAFORD, DELAWARE. JULY 16-17. HATCHERY BREEDER FLOCK MANAGEMENT CLINIC, SOUTHEASTERN POULTRY AND EGG ASSOCIATION, ALL AMERICA INN, AUBURN, ALABAMA. JULY 28-31. 66TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF ANIMAL SCIENCE, UNIVERSITY OF MARYLAND, COLLEGE PARK, MARYLAND. AUGUST 4-8. ANNUAL MEETING OF AGRICULTURAL INSTITUTE OF CANADA AND AFFILIATED SOCIETIES. LAVAL UNIVERSITY, QUEBEC, QUEBEC.
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 19, 2015
sizing capacity with age at the low fat level. There was also an interaction between type of soybean meal and dietary fat level (P < 0.05), due to the greater influence of RS at the low fat level. Other interactions were not significant. The depression of hepatic acetate incorporation into fatty acids observed with the high dietary oil level parallels a similar effect reported for mice receiving dietary oils rich in linoleic acid (Sabine et al., 1969). The probable relationship between the effects of RS on the lipogenic capacity of chicken liver, as found in the present study, and the bilary secretion of lipids and bile acids, reported previously (Sklan etal., 1973), is being examined.
1241