Robert S. Katz 1,2 and David H. Baker

University o f Illinois, Urbana, 618013 et al., 1970; Stekol and Szaran, 1962: Daniel

SUMMARY

Five growth assays were conducted with young chicks to study relative toxicities of various organic sulfur compounds. Evaluation of the dietary requirements for glycine and threonine indicated that .52% threonine and .51% glycine were the minimum requirements for maximal gain. To provide a margin of safety .55% threonine and .60% glycine were chosen as levels to use in subsequent assays. A level of 1.25% excess DL-methionine resulted in close to a 40% reduction in growth rate and was chosen as the level to use in later assays. Consumption of excess methionine, calcium methionine h y d r o x y analogue (OH-M(Ca)) or ethionine resulted in reduced rate and efficiency of gain as well as a lowered concentration of hepatic ATP. DL-homocysteine was equally as toxic as an equimolar level of DL-methionine, but D-methionine was less growth depressing than either L- or DL-methionine. Chicks consuming diets with excess methionine exhibited much greater growth depressions than those fed diets with an equimolar concentration of either OH-M (Ca) or cystine. Homocysteine accumulation in plasma and tissues is thought to be one of several possible factors responsible for lesions associated with methionine toxicity. (Key Words: Methionine, Chick, Threonine, Glycine, Ethionine, Homocysteine.) INTRODUCTION

Effects of consumption of excess methionine have been studied extensively (Hardwick 1Present address: The Quaker Oats Co., 617 W. Main St., Barrington, IL. 60010. z Part of a thesis submitted to the Graduate College of the University of Illinois in partial fulfillment of the requirements for the Ph.D. degree. 3Department of Animal Science.

and Waisman, 1969). The underlying mechanism by which excess methionine exerts its pathogenicity, however, is not well understood, although numerous theories prevail. Hardwick et al. (1970) proposed the depletion of hepatic ATP as an explanation for methionine toxicity. Depletion of methyl acceptors in the conversion of S-adenosylmethionine to S-adenosylhomocysteine has also been investigated. This explanation has proved untenable, however, since little benefit has been observed from supplementing the diet with methyl-accepting compounds such as guanidoacetic acid (Sauberlich, 1961 ; Cohen et al., 1958) or nicotinamide (Klain et al., 1963 ; DeBey et al., 1952). More recently, the metabolism of the labile methyl group of methionine has been proposed as a means by which methionine toxicity is exerted (Benevenga, 1974). It is suggested that a pathway which is competitively inhibited by S-methyl-L-cysteine accounts for the majority of the methionine catabolized when high levels of methionine are fed. It is postulated that an intermediate in this pathway is the toxic product in methionine toxicity. Observations are conflicting regarding the relative toxicity of homocysteine and methionine. In rat studies, homocysteine has been found equally as toxic as methionine l~y some (Cohen et al., 1958) b u t less toxic by others (Benevenga and Harper, 1967). The studies described herein were conducted to determine the optimum dietary conditions in which to study a methionine-induced toxicity and to investigate the relative toxicities of various organic sulfur compounds. EXPERIMENTAL PROCEDURE

Diets. The basal diet used for all assays (table 1) has been described previously (Sasse and Baker, 1973). The amino acid mixture 1355 JOURNAL OF A~IMAL SCIENCE, Vol. 41, No. 5,197'~a

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TOXICITY OF VARIOUS ORGANIC SULFUR COMPOUNDS FOR CHICKS FED CRYSTALLINE AMINO ACID DIETS CONTAINING THREONINE AND GLYCINE AT THEIR MINIMAL DIETARY REQUIREMENTS FOR MAXIMAL GROWTH

KATZ AND B A K E R

1356

TABLE 1. COMPOSITION OF BASAL DIET AND CRYSTALLINE AMINO ACID MIXTURE Basal diet

% 20.34 15.00 5.37 3.00 1.00 .20 .20 +

Antioxidant d (125 ppm) Cornstarch

+ to

100.00

g/20.34g

L-arginine .HC1 L-histidine-HC1. H 2 0 Lqysine.HC1 L-tyrosine L-tryptophan L-phenylalanine DL-methionine L-cystine L-thteonine Lqeucine L-isoleucine L-valine Glycine L-proline L-glutamic acid

1.15 .41 1.14 .45 .15 .50 .35 .35 .55 1.00 .60 .69 .60 .40 12.00 20.34

asalt mixture, as percent of the diet: CaCO 3 , .3 ; Ca 3 (PO 4 )2,2.8 ; K 2 HPO 4 , .9 ; NaCI, .88; MgSO 4 97H 2 O, .35 ; MnSO 4 .H 2 O, .065 ; ferric citrate, .05 ; ZnCO3, .01 ; CuSO 4 95H 2 O, .002 ; H 3 BO3, .0009 ; Na 2 MoO 4 .2H 2 O, .0009 ; KI, .004; CoSO 4 .7H20 , .0001 ; Naz SeO3, .00002 ; total 5.37. bSolka Floc, Brown Company, Chicago, Illinois. CVitarnins per kilogram diet: Thiamine-HCl, 100 rag; niacin, 100 nag; riboflavin, 16 mg; CA-pantothenate, 20 rag; vitamin B 12, .02 mg; pyridoxine-HCl, 6 mg; biotin, .6 mg; folic acid, 4 mg; inositol, 100 mg; para-aminobenzoic acid, 2 mg; menadione, 5 mg;ascorbic acid, 250 mg; vitamin A acetate (250,000 IU/g), 10,000 IU; vitamin D 3 (200,000 IU/g), 600 I.C.U. dsantoquin, Monsanto Co., St. Louis, Missouri.

c o n t a i n s t h e t e n classical i n d i s p e n s i b l e a m i n o acids plus p r o l i n e a n d g l y c i n e - b o t h o f w h i c h are s y n t h e s i z e d t o s o m e e x t e n t b y t h e c h i c k b u t n o t in s u f f i c i e n t q u a n t i t y to a l l o w m a x i m a l g r o w t h . B o t h m e t h i o n i n e a n d c y s t i n e serve as a source o f s u l f u r a m i n o acids; p h e n y l a l a n i n e a n d t y r o s i n e p r o v i d e a r o m a t i c a m i n o acids. T h e levels o f t h e a m i n o acids are at a m i n i m u m requirement for optimal growth, and glutamic acid provides all n i t r o g e n r e q u i r e d f o r b i o s y n thesis o f dispensible a m i n o acids. T h e diet c o n t a i n s 4 , 2 0 0 kcal M E / k g a n d 14.8% p r o t e i n e q u i v a l e n t (i.e., N • 6.25). Experimental Care of Cbicks. Chicks origin a t i n g f r o m t h e cross o f N e w H a m p s h i r e males a n d C o l u m b i a n females were used in all assays. F r o m day 1 t o day 7 t h e y were fed a 24% p r o t e i n c o r n - s o y b e a n m e a l diet. F e e d was w i t h d r a w n f r o m 9 : 0 0 a m t o 1 : 0 0 p m o n day 7 a f t e r w h i c h birds were allowed free access t o b o t h feed a n d w a t e r for 2 h r at w h i c h t i m e b o t h feed a n d w a t e r were r e m o v e d u n t i l 8 : 0 0 a m t h e n e x t day. O n d a y 8, t h e chicks were weighed t o t h e nearest g r a m a n d c h e c k e d visually for o b v i o u s defects. T h e y were t h e n a l l o t t e d to e x p e r i m e n tal g r o u p s such t h a t t h e average s t a r t i n g w e i g h t

a n d w e i g h t range f o r each g r o u p were similar. Chicks were c o n f i n e d in electrically h e a t e d t h e r m o s t a t i c a l l y c o n t r o l l e d b a t t e r i e s in an airc o n d i t i o n e d r o o m c o n t a i n i n g u n i f o r m artificial light a n d m a i n t a i n e d at 24 C. All assays were o f 6 days d u r a t i o n . Assay Procedures. Since serine is i n t i m a t e l y involved in t h e c o n v e r s i o n o f m e t h i o n i n e t o cystine, a n d t h r e o n i n e is h y p o t h e s i z e d t o b e a n t a g o n i z e d b y excess m e t h i o n i n e (Katz a n d Baker, 1 9 7 5 ; G i r a r d - G l o b a et al., 1 9 7 2 ) , it was d e e m e d necessary t o r e e v a l u a t e t h e m i n i m u m d i e t a r y r e q u i r e m e n t s for t h e s e t w o a m i n o acids. In t h e basal diet glycine serves as a s o u r c e (i.e., p r e c u r s o r ) o f serine, a n d t h u s t w o assays (assays 1 a n d 2) were c o n d u c t e d to d e t e r m i n e t h e d i e t a r y r e q u i r e m e n t s f o r t h r e o n i n e a n d glycine. G l y c i n e was c o n t a i n e d in t h e basal diet a t .60% o f t h e diet (Sasse a n d Baker, 1 9 7 3 ) for t h e t h r e o n i n e r e q u i r e m e n t s t u d y a n d t h e glycine s t u d y was c o n d u c t e d in t h e p r e s e n c e o f t h e e s t i m a t e d r e q u i r e m e n t for t h r e o n i n e e s t a b l i s h e d in assay 1. T h e r e q u i r e m e n t s were e s t i m a t e d b y fitting a c o n t i n u o u s b r o k e n line to t h e gain d a t a for each assay b y least s q u a r e analysis (Steel a n d Torrie, 1 9 6 0 ) . T h r e e replicate pens o f e i g h t

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Amino acid mixture Corn oil Salt mixture a Celluloseb NaHCO 3 Choline chloride Vitamins c a-tocopheryl acetate (20 ppm)

Amino acid mixture

ORGANIC SULFUR COMPOUNDS FOR CHICKS I00

80

40

c-

20

.4

.6

.5

$ dietary

.7

.8

thveonine

Figure 1. Broken-line plot of gain vs % dietary threonine (assay 1). ment for maximal gain of .52% (figure 1). To provide a margin of safety, .55% was chosen as the level to use in subsequent assays. The glycine requirement for maximal gain was estimated as .51% of the diet (figure 2). However, since previous investigations at the University of Illinois had indicated a growth response up to .60% of the diet, this level was chosen for subsequent assays. It was considered important that glycine not be deficient in the assay diets, because it was deemed essential that any response to supplemental threonine when added to a methionine-imbalanced diet could be attributed to a threonine deficiency per se 7570-

t

65 0)

55 s r

50

45

R ESU LTS Assays 1 and 2. The results of assay 1 suggested a minimum dietary threonine require-

aN/Error mean square No~ pens/treatment

9 '1

:2

. '3

.

. '6

dietary

glycine

.7'

.'8

Figure 2. Broken-line plot of gain vs % dietary glycine (assay 2).

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male chicks per treatment were placed on test in assay 1; three replicate pens of seven were employed in assay 2. Assay 3 was conducted to determine the level of excess methionine necessary to depress chick growth by 30 to 40%. It was felt that this decrement in growth was such that a response to reverse this effect could easily be detected. Triplicate groups of eight male chicks were employed. The fourth assay involved triplicate groups of seven male chicks and was designed to investigate the relative toxicities o f the three isomers of methionine (L-, D- and DL) and the ethyl and h y d r o x y analogues of methionine. Each compound was added at a level equimolar to 1.25% methionine, except for ethionine which was added at one-tenth this level due to its extreme toxicity. After the growth assay was completed, livers were removed by freezedamping with liquid nitrogen and assayed for ATP according to procedures outlined by Strehler and Totter (1954). Assay 5 was conducted to confirm the growth-depressing effects of the L-, D- and DL-isomers of methionine. Duplicate groups of eight female chicks were used. Equimolar concentrations of methionine h y d r o x y analogue (Ca), homocysteine, homocystine, cysteine and cystine were also evaluated. All compounds were tested at a level isosulfurous to 1.25% methionine, and in the case of the h y d r o x y analogue, a level twice that amount was also tested. Assay 6 was designed to evaluate gain of chicks at an equal intake of feed. The regression of cumulative gain on cumulative feed intake was used to adjust gain to an equal intake of feed (i.e., 200 g) for chicks fed both control and methionine-imbalanced diets. This procedure has been explained previously (Sasse and Baker, 1974). Triplicate groups of seven male chicks were employed for each of the two dietary treatments. Data were subjected to analysis of variance (Steel and Torrie, 1960): Single degree-of-freedom comparisons were made where appropriate. Pooled standard errors 4 were calculated for each response criterion.

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KATZ A N D B A K E R

TABLE 2. EFFECT OF EXCESS METHIONINE ON CHICK GROWTH (ASSAY 3) a Level of excess DL-methionine (%)

1.50

2.00 Pooled SE

Feed consumption (g)

Gain/feed

73.5 64.5 47.7 29.8 22.5

105 96 79 64 52

.70 .68 .60 .47 .43

2.00

2.7

.01

aAverage of triplicate groups of eight male chicks for the period 8 to 14 days posthatching; average initial weight was 74 grams. bBasal diet contained .35% DL-methionine and .35% L-cystine. ~

rather than to formation of glycine from threonine via the threonine aldolase pathway (Baker et al., 1972). Assay 3. As can be seen from the results of assay 3, gain, feed consumption and gain/feed decreased linearly (P