Lipid Oxidation Products and Chick Nutritional Encephalopathy P. BUDOWSKI, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel, and I. BARTOV, Division of Poultry Science, Agricultural Research Organization, The Volcani Center, Rehovot, Israel, and Y. DROR, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel and E.N. FRANKEL, Northern Regional Research Center, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, 1 Peoria, Illinois 61604 ABSTRACT Safflower oil and its distilled methyl esters were thermally oxidized and fed to young chicks in a vitamin E deficient diet. At a dietary level of 10%, the oxidized lipids caused more severe nutritional encephalopathy (NE) than the unoxidized methyl esters, indicating that factors other than dietary linoleic acid and vitamin E affect the development of NE. A polar lipid extract from oxidized methyl[ esters accelerated the induction of NE, as did the synthetic methyl esters of keto-octadecenoic and keto-octadecadienoic acids. Dicumarol exerted a protective action against NE. The possibility is dis.cussed that conjugated keto-polyenoic fatty acids, provided by oxidized oils or formed endogenously in vitamin E deficiency, may play a role in causing NE. INTRODUCTION

diets c o n t a i n e d 4 or 10% lipids, and the c o m p o sition was adjusted so as to ensure a c o n s t a n t ratio o f m e t a b o l i z a b l e energy to p r o t e i n . The linoleic acid in these diets was p r o v i d e d by distilled s a f f l o w e r m e t h y l esters or by t h e r m a l l y o x i d i z e d s a f f l o w e r oil or m e t h y l esters, prep a r e d as described below. The diets c o n t a i n e d 0.005%, 2,6-di-tert.-butyl-4-methylphenol (BHT), a level o f a n t i o x i d a n t w h i c h e n s u r e d the stability o f the dietary linoleic acid and ~-tocop h e r o l for over a week at r o o m t e m p e r a t u r e . During the e x p e r i m e n t s , the diets were k e p t at -18 C and dispensed daily.

N u t r i t i o n a l e n c e p h a l o p a t h y (NE), b e t t e r k n o w n as e n c e p h a l o m a l a c i a , is i n d u c e d in y o u n g chicks by diets deficient in a - t o c o p h e r o l and c o n t a i n i n g p o l y u n s a t u r a t e d f a t t y acids (1). The disease is c h a r a c t e r i z e d by degenerative changes mainly in the c e r e b e l l u m , a c c o m p a n i e d by ataxia, p r o s t r a t i o n , and death. Ultrastructural changes in the cerebella o f chicks a f f e c t e d w i t h NE have been described by various a u t h o r s (for a brief review, see ref. 2). The dietary lipid causing NE is linoleic acid or its esters, whereas derivatives o f linolenic acid are inactive (3-5). Autoxidized polyunsaturated oils have occasionally b e e n used to i n d u c e NE in chicks (4,5), arm ~ve f o u n d t h e r m a l l y o x i d i z e d safflower oil t o be very effective for t h a t p u r p o s e (2,6,7). 'The ~ - t o c o p h e r o l level o f such oil is greatly r e d u c e d w h i l e the linoleic acid c o n t e n t is still high. Since the t r e a t m e n t o f t h e oil results in the a c c u m u l a t i o n o f o x i d a t i o n p r o d u c t s , the q u e s t i o n a r i s e s w h e t h e r or n o t s o m e o f these p r o d u c t s m a y play an active role in causing NE. The evidence p r e s e n t e d in this r e p o r t p o i n t s in this direction.

Dietary Lipids R e f i n e d edible s a f f l o w e r oil was p u r c h a s e d TABLE I Composition of Diets Ingredient Lipid a Extracted soybean meal Cellulose D,L-methionine Mineral mix b Vitamin mix c BHT d Glucose monohydrate

MATERIALS A N D METHODS Animals and Feeds Day-o~d c r o s s b r e d New H a m p s h i r e X White L e g h o r n male chicks were h o u s e d in t h e r m o statically h e a t e d b a t t e r y b r o o d e r s with raised wire floors and had free access to w a t e r and feed. The c o m p o s i t i o n s o f the t w o vitamin E deficient diets are p r e s e n t e d in Table I. The 1Mention of firm names or trade p r o d u c t s d o e s imply e n d o r s e m e n t or recommendation by the Department of Agriculture over other firms or similar products not mentioned. not

Percent 4.00 52.00 1.00 0.14 4.00 0.50 0.005 38.36

10.00 5(5.00 3.00 0.15 4.00 0.50 0.005 26.35

aSafflower oil or safflower methyl esters. bSupplying per kg feed: dicalcium phosphate 28 g; l i m e s t o n e 7 g; NaCI 3.5 g; MnSO4"H20 370 mg; ZnCO 3 145 rag; ferric citrate 165 rag; CuSO4-5H20 11.8 rag; KI 2.35 mg; and CoCl2~ 1.21 mg. CSupplying per kg feed: vitamin A 3000 U; vitamin D 3 200 U; menadione sodium bisulfite 1 mg; thiamine 3.6 mg; riboflavin 7.2 mg; Ca pantothenate 20 mg; niacin 55 mg; pyridoxine 6 rag; biotin 0.2 mg; folie acid 2.4 rag; vitamin B12 0.02 mg-choline chloride 1.3 g. These amounts were premixed with 21.6 g glucose monohydrate. d2,6-di-tert, butyl-4-methylphenol.

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TABLE II Effect of Thermally Oxidized Safflower Oil (SO-24) a and Freshly Distilled Safflower Methyl Esters (ME-0) on Nutritional Encephalopathy b Dietary lipids

Analysis of lipids a-Tocopherol Linoleic acid P,g/g

Incidence per 20 chicks Ataxia

Mortality

% At 3 weeks

SO-24 4% ME-SO 4%

10-20 5-12

60-65 70-76

7 15

5 9 At 2 weeks

SO-24 10% ME-0 10%

12 8

11 1

aSafflower oil aerated at 145 C for 24 hr. bThe lipids were fed from hatching at the levels indicated. from Shemen Ltd., Halfa and from Teth-Beth Ltd., Petah-Tikva. Different batches contained from 71 to 76% linoleic acid and ca. 350 pg a-tocopherol/g. Methyl esters were prepared from the oil by a modification of the transm e t h y l a t i o n procedure of Hartman (8), followed by vacuum distillation. Thermal oxidation of safflower oil and distilled m e t h y l esters was done by heating batches of 1-2 kg to 145 C -+ 2 C under a stream of air (0.5 1/min). The length of the thermal t r e a t m e n t was 24 hr for the oil and 3 hr for the esters. The following abbreviations were used: SO-24 for the oxidized oil; ME-3 for the oxidized m e t h y l esters; and ME-0 for the fresh m e t h y l esters. A crude extract of polar lipids was prepared from thermally oxidized m e t h y l esters by repeated partition between hexane and 90% (v/v) ethanol, using six separatory funnels arranged in c o u n t e r c u r r e n t fashion. The final ethanolic extract was concentrated under reduced pressure and extracted with ethyl ether. The yield of polar lipids averaged 6%. Methyl esters of conjugated keto-octadecenoic acid and keto-octadecadienoic acid were prepared from m e t h y l oleate and linoleate, respectively, as described elsewhere (9). The oleate-derived p r o d u c t contained 96% conjugated k e t o esters consisting of an isomeric mixture of m e t h y l 8-, 9-, 10-, and l l - o x o octadecenoate. The linoleate-derived p r o d u c t contained 91% keto-dienes consisting mainly of methyl 13-oxo-9,11- and 9-oxo-10,12-octadecadienoate. F o r testing in chicks, lipid fractions or synthetic products were dissolved in safflower m e t h y l esters in the amounts indicated. Quantitative Expression of NE

Chicks were inspected twice daily and the

times at which the first signs of ataxia were observed and when death occurred, were recorded. Inspection of the cerebella always c o n f i r m e d that the affected chicks were stricken with NE. Results were expressed as n u m b e r of chicks affected per total n u m b e r of chicks per t r e a t m e n t at the age indicated. Alternatively, curves representing the cumulative incidence of ataxia or death have been plotted. Analytical Determinations

a - T o c o p h e r o l in the dietary lipids was determined by saponification, fractionation of the unsaponifiables by thin layer chromatography on Silica Gel G with h e x a n e / e t h y l ether (8:2), and colorimetric reaction of the a-tocopherol fraction with ferric chloride and bathophenanthroline (10). The fatty acid c o m p o s i t i o n of the lipids was determined after t r a n s m e t h y l a t i o n of the samples with 3% (w/v) H2SO 4 in m e t h a n o l at reflux temperature for 1 hr and extraction of the met])yl esters with hexane. The esters were s u b m i t t e d to isothermal gas liquid chrom a t o g r a p h y at 180 C on Gas Chrom W coated with 15% DEGS. All materials were obtained from Packard Ltd., Jerusalem. Methyl esters prepared for feeding experiments were injected directly into the chromatograph. Glyceryl t r i h e p t a d e c a n o a t e and m e t h y l h e p t a d e c a n o a t e were added as internal standards to the oil and m e t h y l ester samples, respectively, for calculation of the true linoleic acid c o n t e n t of the oxidized samples. RESULTS

In a first trial, two e n c e p h a l o p a t h o g e n i c diets were compared: thermally oxidized safflower oil, SO-24, and freshly distilled LIPIDS, VOL. 14, NO. 9

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TABLE III Effect of Fresh and Thermally Oxidized Safflower Methyl Esters on Nutritional Encephalopathy

Dietary lipids b ME-0 ME-3

Analysis of lipids c~-Tocopherol Linoleic acid /.tg/g 5.0 0.35

% 75.9 68.5

Vitamin Ea added to feed

Incidence per 20 chicks at 19 days Ataxia Mortality

Isg/g 0

14

6

1

13

4

0

18 19

14 14

1

aD,L-~-Tocopheryl acetate. bME-0, freshly distilled safflower methyl esters; ME-3, methyl esters aerated at 145 C for 3 hr. The lipids were fed as 10% of the diet from the 8th day, after the chicks received 4% ME-0 during the first week. safflower m e t h y l esters, ME-0. The o x i d i z e d oil h a d less linoleic acid b u t n o less c~-tocopherol t h a n the fresh esters (Table II). W h e n these lipids were fed as 4% of the diet, ME-0 caused a greater i n c i d e n c e o f ataxia a n d m o r t a l i t y t h a n did SO-24. However, at the 10% level, the o x i d i z e d oil was m o r e active t h a n t h e fresh m e t h y l esters. A similar c o m p a r i s o n was m a d e b e t w e e n fresh a n d o x i d i z e d m e t h y l esters, ME-0 and ME-3. Table III shows t h a t ME-3, w i t h less linoleic acid, nevertheless p r o d u c e d a m o r e severe i n c i d e n c e o f NE, c o m p a r e d to ME-0. This g r e a t e r activity o f ME-3 was n o t due to the l o w e r c~-tocopherol c o n t e n t o f t h e o x i d i z e d vs. the fresh esters, since the difference in tocop h e r o l c o n t e n t b e t w e e n t h e t w o diets was n o m o r e t h a n 0.5 /ag t o c o p h e r o l / g diet, whereas even the a d d i t i o n of 1 /ag D L ~ - t o c o p h e r y l a c e t a t e / g diet h a d virtually n o effect on NE (Table III). E x t r a c t s of polar lipids were p r e p a r e d f r o m ME-3 b y r e p e a t e d p a r t i t i o n b e t w e e n h e x a n e a n d 90% e t h a n o l . The polar lipids were a d d e d to e n c e p h a l o p a t h o g e n i c diets and t h e i r influence o n NE was s t u d i e d . T h e results o f one such e x p e r i m e n t , i l l u s t r a t e d in Figure 1, s h o w t h a t the polar lipids increased the i n c i d e n c e o f NE. T w o s y n t h e t i c f a t t y acid o x i d a t i o n p r o d u c t s were t e s t e d in t h e same c h i c k m o d e l : a k e t o m o n o e n e p r e p a r e d f r o m m e t h y l oleate and a k e t o diene o b t a i n e d f r o m m e t h y l linoleate. B o t h c o m p o u n d s accelerated the i n d u c t i o n of NE (Figs. 2 a n d 3). E b r i n clots have previously b e e n o b s e r v e d in cerebellar capillaries of chicks a f f e c t e d w i t h N F (2). The p r e s e n t c h i c k m o d e l was used t o s t u d y t h e effect o f d i c u m a r o l o n t h e i n c i d e n c e of NE. Table IV s h o w s t h a t t h e a n t i c o a g u l a n t e x e r t e d a p r o t e c t i v e effect w h i c h increased in direct r e l a t i o n t o its c o n c e n t r a t i o n in the diet. In this LIPIDS, VOL. 14, NO. 9

e x p e r i m e n t , the diet c o n t a i n e d 4% ME-(), b u t similar results were o b t a i n e d w i t h diets cont a i n i n g 10% o x i d i z e d safflower oil. DISCUSSION

Several a u t h o r s (4,5) have r e p o r t e d t h a t the i n c i d e n c e of NE in v i t a m i n E d e f i c i e n t chicks is directly related to the a m o u n t of linoleic acid c o n s u m e d b y the chicks. This is seen to be t h e case in t h e first e x p e r i m e n t in w h i c h SO-24 a n d ME-0 were fed at the 4% level (Table II). However, the reversal o f activities of the t w o lipids at t h e 10% level does n o t agree w i t h this c o n c e p t . One possible e x p l a n a t i o n for the a p p a r e n t l y c o n t r a d i c t o r y results is t h a t SO-24 c o n t a i n s o x i d a t i o n p r o d u c t s w h i c h are e n c e p h a l o p a t h o g e n i c and w h i c h , at t h e h i g h e r dietary level, are a b s o r b e d in sufficient a m o u n t s to o v e r c o m e the o p p o s i t e r e a c t i o n e x p e c t e d f r o m t h e l o w e r linoleic acid c o n t e n t of SO-24 vs. ME-0. T h e above e x p l a n a t i o n receives s u p p o r t f r o m t h e o b s e r v a t i o n t h a t t h e o x i d i z e d esters, in spite of t h e i r l o w e r linoleic acid c o n t e n t , caused a m o r e severe i n c i d e n c e o f NE t h a n t h e fresh esters (Table III), while m o r e direct evidence for t h e i n v o l v e m e n t o f lipid o x i d a t i o n p r o d u c t s is p r o v i d e d b y results o b t a i n e d w i t h polar lipids e x t r a c t e d f r o m o x i d i z e d m e t h y l esters (Fig. 1). T h e effects o f the t w o s y n t h e t i c k e t o e n o i c f a t t y acid esters (Figs. 2 a n d 3) are of i n t e r e s t for the following reasons: 12-oxo-9-eis-octad e c e n o i c acid was previously r e p o r t e d to increase t h e severity of NE w h e n given orally w i t h s t r i p p e d c o r n oil (11), b u t n o t a f t e r i n j e c t i o n (12). S u b s e q u e n t l y , this c o m p o u n d was s h o w n to possess s t r o n g p r o o x i d a n t activity in v i t r o (13) a n d e v e n t u a l l y its activity o n NE was ascribed, a c c o r d i n g to a c o m m u n i c a t i o n f r o m t h e same l a b o r a t o r y (14), to d e s t r u c t i o n o f residual t o c o p h e r o l in t h e diet d u r i n g t h e

LIPID OXIDATION AND ENCEPHALOPATHu

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