METABOLISM AND NUTRITION Effect of Dietary Aflatoxin on Vitamin D3 Metabolism in Chicks W. M. BRITTON and R. D. WYATT Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication April 21, 1977)
INTRODUCTION
MATERIALS AND METHODS
T h e relationship between aflatoxicosis and vitamin metabolism has recently been reviewed by Hamilton ( 1 9 7 5 ) . Lipid-soluble vitamin D has been s h o w n to interact with aflatoxin (Hamilton et al, 1974). When 0.625 p . p . m . aflatoxin was added t o a vitamin D 3 deficient diet for chicks, t h e deficiency was increased as measured by a further reduction in b o d y weight. When a d e q u a t e vitamin D 3 was present, n o effect of aflatoxin on g r o w t h was observed. Research reviewed by DeLuca ( 1 9 7 4 ) has shown t h a t vitamin D 3 as ingested or generated in t h e skin is t r a n s p o r t e d t o t h e liver where it is converted t o 25-hydroxyvitamin D 3 . T h e 2 5 - h y d r o x y vitamin D 3 is t h e n t r a n s p o r t e d to the kidney where it is converted to 1,25-dihydroxy-vitamin D 3 or 24,25-dihydroxyvitamin D 3 . T h e 1,25-dihydroxyvitamin D 3 is considered t h e active m e t a b o l i t e t h a t effects calcium absorption from t h e gut or in t h e presence of p a r a t h y r o i d h o r m o n e reabsorption of calcium from the bone. T h e physiological role of 24,25-dihydroxyvitamin D 3 , if any, has n o t been established.
Male broiler chicks used in this s t u d y were maintained free from natural light in a h e a t e d b a t t e r y b r o o d e r with raised wire floor with feed and water supplied ad lib. A t o t a l of 4 0 day-old chicks were equally divided into 2 groups and fed a commercial t y p e diet (control) or this diet containing 2.5 p . p . m . of aflatoxin ( t o x i n ) for 4 weeks. T h e diet is presented in Table 1. At four weeks of age, 8 chicks from t h e control and 8 from t h e t o x i n g r o u p were injected intravenously with 1.4 £iCi. of vitamin D 3 [ 1 , 2 - 3 H ] (specific activity 12.3 Ci/m.mol.) in 0.05 ml. of ethanol. T w e n t y - f o u r hours after injection, blood samples were o b t a i n e d b y cardiac p u n c t u r e and plasma collected and frozen until ready for analysis. T w o o t h e r groups of chicks (8 controls and 8 toxin) were injected intravenously with 1.4 juCi. of 25-hydroxycholecalciferol [26(27)m e t h y l - 3 H ] (specific activity 6.9 Ci/m.mol.) in 0.05 ml. of ethanol. Six h o u r s after injection, b l o o d samples were o b t a i n e d and handled as described above. T h e radioactivity in the plasma was extracted using a p r o c e d u r e similar t o t h e o n e described b y L u n d and DeLuca ( 1 9 6 6 ) . T h e plasma sample (5 t o 1 0 ml.) was c o m b i n e d with
This s t u d y was c o n d u c t e d to c o m p a r e t h e ability of chicks fed dietary aflatoxin or a control diet t o metabolize vitamin D 3 . 163
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on April 28, 2015
ABSTRACT Male broiler chicks were fed diets containing either 0 (control) or 2.5 p.p.m. aflatoxin (toxin) for four weeks. A group of eight birds fed each diet was injected intravenously with radioactive vitamin D 3 (D 3 ) and a second group with radioactive 25-hydroxy vitamin D 3 (25-0H D 3 ). Plasma was obtained 24 hr. after dosing with D 3 and 6 hr. after dosing with 25-OH D 3 . The vitamin D metabolites were extracted from the plasma and chromatographed on Sephadex LH-20 for separation. The four peaks of radioactivity separated corresponded to D 3 , 25-OH D 3 , 24,25-dihydroxy vitamin D 3 (24,25-(OH) 2 D 3 ) and 1,25-dihydroxy vitamin D 3 (l,25-(OH) 2 D 3 ). Percentages of radiation in each peak were (1) controls given D 3 — 6.55, 64.30, 5.94 and 4.04; (2) toxin given D 3 - 10.05, 56.96, 8.95 and 4.68; (3) control given 25-OH D 3 - 2.16, 85.80, 4.11 and 2.04; and (4) toxin given 25-OH D 3 - 1.53, 79.84, 5.56 and 2.14. The only significant differences between the control and toxin groups were in D 3 and 24,25-(OH) 2 D 3 in chicks given D 3 . Even these changes were small and the data would suggest that feeding 2.5 p.p.m. aflatoxin for four weeks does not greatly alter vitamin D metabolism.
BRITTON AND WYATT
164 TABLE 1.—Experimental diet
g./100g.
Ground yellow corn Soybean meal (dehulled) Poultry by-product meal Poultry fat Dehydrated alfalfa Ground limestone Defluorinated phosphate NaCl Methionine Trace element mix 1 Vitamin mix2
57.25 30.50 5.00 3.00 1.00 0.65 1.75 0.40 0.15 0.05 0.25
1 Trace mineral mix provides (mg./kg. of diet): Mn., 60;Zn., 50; Fe., 30;Cu., 5; I., 1.05 and Ca., 75. 2 Vitamin mix provides (per kg. of diet): vitamin A, 4400 I.U.; vitamin D 3 , 880 I.C.U.; vitamin E, 11 I.U.; riboflavin, 4.4 mg.; Ca pantothenate, 9.6 mg.; nicotinic acid, 44 mg.; choline chloride, 220 mg.; vitamin B I 2 , 6.6 Mg-; pyridoxine, 2.2 mg.; menadione sodium bisulfite, 3.49 mg.;folic acid, 55 mg.;biotin, 0.11 mg.; thiamine mononitrate, 2.2 mg.jand ethoxyquin, 125 mg.
one p a r t chloroform and t w o parts m e t h a n o l and magnetically stirred overnight at 4 C. One part of chloroform was stirred and followed by 0.1 part of water. The m i x t u r e was allowed t o stand for 2 hr. at 4 C. T h e lo'wer chloroform phase was removed and t h e u p p e r a q u e o u s phase was re-extracted twice with c h l o r o f o r m . T h e chloroform extract was c o n c e n t r a t e d u n d e r N 2 to a b o u t 0.3 ml. T h e radioactive extracts were t h e n chrom a t o g r a p h e d using a p r o c e d u r e similar t o t h a t described by Holick and DeLuca ( 1 9 7 1 ) . T h e column was prepared by making a slurry of 20 g. of Sephadex LH-20 (Pharmacia Fine Chemi-
RESULTS AND DISCUSSION T h e chicks fed for 4 weeks t h e diet containing 2.5 p . p . m . aflatoxin had significantly lower b o d y weights. T h e control and toxin groups given radioactive vitamin D 3 weighed 601 g. and 4 9 7 g., respectively. The control and toxin groups given radioactive 25-hydroxyvitamin D 3 weighed 6 0 5 g. and 4 5 6 g. This level of toxin did n o t cause m o r t a l i t y , however, typical lesions of aflatoxicosis including an enlarged yellow liver, increased kidney size and bile duct hyperplasia were evident. F o u r peaks of radioactivity corresponding t o
TABLE 2.—Plasma metabolites 24 hr. after radioactive vitamin D 3 I. V. injection in chicks fed 2.5 p.p.m. aflatoxin for 4 weeks Vitamin D 3 metabolites
Dietary treatment Control
Toxin (% of recovered H 3 ) 1
D3 25-OH D 3 24-25-(OH) 2 D 3 l,25-(OH) 2 D 3
6.55 64.30 5.94 4.04
+ 0.87^ ± 3.20^ ± 0.41 a ± 0.90*
10.05 56.96 8.95 4.68
1
Mean ± S.E. of 8 samples.
2
Values within a metabolite not followed by the same superscript are significantly different (P
INTRODUCTION
MATERIALS AND METHODS
T h e relationship between aflatoxicosis and vitamin metabolism has recently been reviewed by Hamilton ( 1 9 7 5 ) . Lipid-soluble vitamin D has been s h o w n to interact with aflatoxin (Hamilton et al, 1974). When 0.625 p . p . m . aflatoxin was added t o a vitamin D 3 deficient diet for chicks, t h e deficiency was increased as measured by a further reduction in b o d y weight. When a d e q u a t e vitamin D 3 was present, n o effect of aflatoxin on g r o w t h was observed. Research reviewed by DeLuca ( 1 9 7 4 ) has shown t h a t vitamin D 3 as ingested or generated in t h e skin is t r a n s p o r t e d t o t h e liver where it is converted t o 25-hydroxyvitamin D 3 . T h e 2 5 - h y d r o x y vitamin D 3 is t h e n t r a n s p o r t e d to the kidney where it is converted to 1,25-dihydroxy-vitamin D 3 or 24,25-dihydroxyvitamin D 3 . T h e 1,25-dihydroxyvitamin D 3 is considered t h e active m e t a b o l i t e t h a t effects calcium absorption from t h e gut or in t h e presence of p a r a t h y r o i d h o r m o n e reabsorption of calcium from the bone. T h e physiological role of 24,25-dihydroxyvitamin D 3 , if any, has n o t been established.
Male broiler chicks used in this s t u d y were maintained free from natural light in a h e a t e d b a t t e r y b r o o d e r with raised wire floor with feed and water supplied ad lib. A t o t a l of 4 0 day-old chicks were equally divided into 2 groups and fed a commercial t y p e diet (control) or this diet containing 2.5 p . p . m . of aflatoxin ( t o x i n ) for 4 weeks. T h e diet is presented in Table 1. At four weeks of age, 8 chicks from t h e control and 8 from t h e t o x i n g r o u p were injected intravenously with 1.4 £iCi. of vitamin D 3 [ 1 , 2 - 3 H ] (specific activity 12.3 Ci/m.mol.) in 0.05 ml. of ethanol. T w e n t y - f o u r hours after injection, blood samples were o b t a i n e d b y cardiac p u n c t u r e and plasma collected and frozen until ready for analysis. T w o o t h e r groups of chicks (8 controls and 8 toxin) were injected intravenously with 1.4 juCi. of 25-hydroxycholecalciferol [26(27)m e t h y l - 3 H ] (specific activity 6.9 Ci/m.mol.) in 0.05 ml. of ethanol. Six h o u r s after injection, b l o o d samples were o b t a i n e d and handled as described above. T h e radioactivity in the plasma was extracted using a p r o c e d u r e similar t o t h e o n e described b y L u n d and DeLuca ( 1 9 6 6 ) . T h e plasma sample (5 t o 1 0 ml.) was c o m b i n e d with
This s t u d y was c o n d u c t e d to c o m p a r e t h e ability of chicks fed dietary aflatoxin or a control diet t o metabolize vitamin D 3 . 163
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on April 28, 2015
ABSTRACT Male broiler chicks were fed diets containing either 0 (control) or 2.5 p.p.m. aflatoxin (toxin) for four weeks. A group of eight birds fed each diet was injected intravenously with radioactive vitamin D 3 (D 3 ) and a second group with radioactive 25-hydroxy vitamin D 3 (25-0H D 3 ). Plasma was obtained 24 hr. after dosing with D 3 and 6 hr. after dosing with 25-OH D 3 . The vitamin D metabolites were extracted from the plasma and chromatographed on Sephadex LH-20 for separation. The four peaks of radioactivity separated corresponded to D 3 , 25-OH D 3 , 24,25-dihydroxy vitamin D 3 (24,25-(OH) 2 D 3 ) and 1,25-dihydroxy vitamin D 3 (l,25-(OH) 2 D 3 ). Percentages of radiation in each peak were (1) controls given D 3 — 6.55, 64.30, 5.94 and 4.04; (2) toxin given D 3 - 10.05, 56.96, 8.95 and 4.68; (3) control given 25-OH D 3 - 2.16, 85.80, 4.11 and 2.04; and (4) toxin given 25-OH D 3 - 1.53, 79.84, 5.56 and 2.14. The only significant differences between the control and toxin groups were in D 3 and 24,25-(OH) 2 D 3 in chicks given D 3 . Even these changes were small and the data would suggest that feeding 2.5 p.p.m. aflatoxin for four weeks does not greatly alter vitamin D metabolism.
BRITTON AND WYATT
164 TABLE 1.—Experimental diet
g./100g.
Ground yellow corn Soybean meal (dehulled) Poultry by-product meal Poultry fat Dehydrated alfalfa Ground limestone Defluorinated phosphate NaCl Methionine Trace element mix 1 Vitamin mix2
57.25 30.50 5.00 3.00 1.00 0.65 1.75 0.40 0.15 0.05 0.25
1 Trace mineral mix provides (mg./kg. of diet): Mn., 60;Zn., 50; Fe., 30;Cu., 5; I., 1.05 and Ca., 75. 2 Vitamin mix provides (per kg. of diet): vitamin A, 4400 I.U.; vitamin D 3 , 880 I.C.U.; vitamin E, 11 I.U.; riboflavin, 4.4 mg.; Ca pantothenate, 9.6 mg.; nicotinic acid, 44 mg.; choline chloride, 220 mg.; vitamin B I 2 , 6.6 Mg-; pyridoxine, 2.2 mg.; menadione sodium bisulfite, 3.49 mg.;folic acid, 55 mg.;biotin, 0.11 mg.; thiamine mononitrate, 2.2 mg.jand ethoxyquin, 125 mg.
one p a r t chloroform and t w o parts m e t h a n o l and magnetically stirred overnight at 4 C. One part of chloroform was stirred and followed by 0.1 part of water. The m i x t u r e was allowed t o stand for 2 hr. at 4 C. T h e lo'wer chloroform phase was removed and t h e u p p e r a q u e o u s phase was re-extracted twice with c h l o r o f o r m . T h e chloroform extract was c o n c e n t r a t e d u n d e r N 2 to a b o u t 0.3 ml. T h e radioactive extracts were t h e n chrom a t o g r a p h e d using a p r o c e d u r e similar t o t h a t described by Holick and DeLuca ( 1 9 7 1 ) . T h e column was prepared by making a slurry of 20 g. of Sephadex LH-20 (Pharmacia Fine Chemi-
RESULTS AND DISCUSSION T h e chicks fed for 4 weeks t h e diet containing 2.5 p . p . m . aflatoxin had significantly lower b o d y weights. T h e control and toxin groups given radioactive vitamin D 3 weighed 601 g. and 4 9 7 g., respectively. The control and toxin groups given radioactive 25-hydroxyvitamin D 3 weighed 6 0 5 g. and 4 5 6 g. This level of toxin did n o t cause m o r t a l i t y , however, typical lesions of aflatoxicosis including an enlarged yellow liver, increased kidney size and bile duct hyperplasia were evident. F o u r peaks of radioactivity corresponding t o
TABLE 2.—Plasma metabolites 24 hr. after radioactive vitamin D 3 I. V. injection in chicks fed 2.5 p.p.m. aflatoxin for 4 weeks Vitamin D 3 metabolites
Dietary treatment Control
Toxin (% of recovered H 3 ) 1
D3 25-OH D 3 24-25-(OH) 2 D 3 l,25-(OH) 2 D 3
6.55 64.30 5.94 4.04
+ 0.87^ ± 3.20^ ± 0.41 a ± 0.90*
10.05 56.96 8.95 4.68
1
Mean ± S.E. of 8 samples.
2
Values within a metabolite not followed by the same superscript are significantly different (P
