EFFECTIVENESS OF CHONDROITIN IN PREVENTING GIZZARD EROSION IN CHICKS*

BY

H. R. BIRD,
the Department

J. J. OLESON, C. A. ELVEHJEM, E. B. HART

of Biochemistry, of Wisconsin, CQllege

AND

(From

of Agriculture,
12, 1938)
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University

Madison) September

(Received for publication,

The existence of a dietary factor necessary for the normal development of the gizzard lining in young chicks has been reported previously from this laboratory, and it has been designated the anti-gizzard erosion factor (l-3). Assays of various plant and animal materials showed pig lung, liver, and kidney to be good sources, and oats, bran, and middlings to be superior to other grain products and much superior to corn. Green plant material such as dried grass and alfalfa leaf meal proved to be poor sources. Attempts to extract the active substance with water, ethyl aIcohol, ether, and petroleum ether were uniformly unsuccessful, but dilute alkali removed the factor from lung tissue and acidification precipitated it along with the acid precipitable proteins. A reticulin preparation from lung tissue also proved to be potent, and this finding led to experiments with other connective tissues, among which cartilage proved most effective. Since chondroitin prepared from cartilage had been used in the treatment of stomach ulcer in humans by Crandall and Roberts (4), it appeared logical to try its effect on gizzard erosion. A preliminary report on its effectiveness has been presented previously (5). It is the purpose of this paper to describe these experiments in greater detail. Almquist and Stokstad (6) and Almquist (7) have also described gizzard lesions in chicks fed a ration of polished rice, ether-extracted fish meal, ether-extracted brewers yeast, salt, Wesson oil, and cod liver oil. They have reported prevention of erosion by the administration of hexane extracts of alfalfa leaf meal, kale,
* Published with the approval cultural Experiment Station. of the Director 671 of the Wisconsin Agri-

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Gizzard Erosion in Chicks

wheat bran, and hemp-seed. They stated further that the active substance was present in the saponifiable fraction of hexane extract of alfalfa leaf meal. Numerous attempts on our part to repeat these results have served merely to confirm our original conclusion that alfalfa leaf meal is a very poor source and that the factor is not extractable by hexane from wheat bran or from any of the other materials which we have found to be good sources. These experiments have been performed not only with basal rations developed here, but also with the ration used by Almquist. Recently Almquist (8) has reported that bile, cholic acid, desoxycholic acid, sodium glycocholate, and sodium taurocholate possess gizzard factor potency.
EXPERIMENTAL

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For the most part the details of the experimental procedure were the same as described previously, except that groups of five or six chicks were used, and all chicks were killed at 4 weeks of age. The practice of using small groups of chicks has been continued, since it permits a greater number of experiments; however, it necessitates the repetition of experiments yielding apparently significant results. Table I gives the basal rations used in these experiments. 5 per cent of granite grit was added to these rations, in some cases during the last 2 weeks, and in some cases during the last 3 weeks of the experiment. All chicks were irradiated under a quartz mercury vapor lamp at a distance of 18 inches once a week, and given 10 mg. of either oleum percomorphum or haliver oil per chick per week. The oleum percomorphum contained 60,000 and 8500 vitamin D units (u.s.P.) per vitamin A units (u.s.P.) gm. The haliver oil contained not less than 50,000 vitamin A XI) and not less than 850 vitamin D units (u.s.P. XI) units (u.s.P. per gm. The composition of Salts I and the method of preparing the casein have been described previously (9). Two liver extracts were used, both being by-products of the preparation of antipernicious anemia factor. They were obtained from The Wilson Laboratories. The brewers yeast was obtained locally and dried.
1 We are indebted to Dr. David Klein of The Wilson Laboratories, Chicago, for the liver extracts, cartilage, and chondroitin used in these experiments.

Bird, Oleson, Elvehjem,

and Hart

673

Most of the experiments reported here involved the feeding of Ration 465. Ration 466 was formulated in an attempt to secure better growth without reducing the severity of gizzard erosion. Previously published results were based on experiments with rations very similar to Ration 465, except that they contained 10 per cent of peanuts and no alfalfa leaf meal. Experience showed that peanuts were quite variable in their content of the antigizzard erosion factor, so the use of these rations was discontinued. Table II gives a summary of the results obtained. Both the cartilage and the chondroitin were furnished by Dr. David Klein of The Wilson Laboratories. The chondroitin was a mixture of
TABLE

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I of Rations
Ration 465

Composition
Ingredients

-___ Ration ____466

Dextrinized corn-starch ............... Reprecipitated casein ................. Salts I ................................ Brewers yeast ........................ Liver extract .......................... Yellow corn ........................... Wheat bran, heated at 120 for Alfalfa leaf meal ...................... Soy bean oil ...........................

64 18 5 1 2

.
24 hrs. 10

57 18 5 1 2 5 5 5 2

chondroitin and chondroitinsulfuric acid containing some protein and mineral impurities. It strongly reduced Fehlings solution, probably indicating the presence of breakdown products of chondroitin. From this material the calcium chondroitin sulfate preparation was made by first treating with fullers earth a solution of chondroitin in 0.2 N HCI, removing the fullers earth by centrifuging, precipitating the chondroitin with 2 volumes of 95 per cent ethyl alcohol, filtering, redissolving in water, and subsequently precipitating with 1 volume of alcohol in the presence of acetic acid and excess of calcium acetate. The product was washed with successive portions of 50 per cent, 95 per cent, and absolute alcohol, and anhydrous ether. Later it was found advantageous to convert the material to the calcium salt before

674
Results

Gizzard Erosion in Chicks

TABLE II

of Assays

for

Anti-Gizzard

Erosion

Factor

I a i Bi

Supplement

Basal Ration 465

gm.

!Jm.

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8 0

None 10% cartilage None Hexane* extract 3 10% cartilage I residue z 10% None 3yo chondroitin (Wilson) None 5% chondroitin (Wilson) None Hexane* extract XJ 5% chondroitin residue E 5% IL None 3% chondroitinsulfuric acid (Meyer) None 3% Ca chondroitin sulfate preparation 0.2 gm. per day Ca chondroitin sulfate preparation 2% chondrosamine None 3% galactose Basal Ration
9

82 123 80 81 114 82 97 82 86 74 71 88 67 83 72 84 87 59 80 83 466 74 92 87 101 102

80

103 158 99 107 147 105 122 104 112 100 94 116 81 99 85 110 107 74 99 105

2 13 0 0 6 2 6 8 16 0 1 6 2 6 2 6 4 2 0 2

4 4 0 4 2 11 3 5 2 0 5 3 2 0 2 3 4 1

4 0 2 2 0 2 0 3 0 1 3 0 3 1 2 0 0 1 2 3

-.

10

1% cod liver oil + 3yo alcoholic extract of rice bran Same + 3yo chondroitin (Wilson) None 3yo chondroitin (Wilson) 2yo aldobionic acid preparation from gum arabic 1% sodium glycocholate taurocholate 1% 0.5% cholic acid Ox bile E 2% solids B.

96 125 113 128 131 106 116 140 129

0 4 1 5 5 5 3 5 5

5 2 2 1 0 1 2 0 0

2 0 1 0 0 0 0 0 0

83 101 93

* Skelly-solve

Bird, Oleson, Elvehjem,

and Hart

675

treatment with fullers earth. The resulting preparation gave no biuret test and only a very slight Fehlings test. The results of analysis were as follows: N 2.85, S 5.33, Ca 5.73, ash 24.43 per cent. Nitrogen was determined by the Gunning modification of the Kjeldahl method, sulfur by sodium peroxide fusion and subsequent conversion to BaS04, and calcium by the method of the Association of Official Agricultural Chemists for calcium in a mineral feed. The calculated composition of chondroitinsulfuric acid completely converted to the calcium salt is: N 2.77, S 6.31, Ca 7.90, ash 26.8 per cent. The results indicate a mixture of the calcium salts of chondroitinsulfuric acid and chondroitin, relatively free of protein and mineral impurities. The chondrosamine was prepared according to the method of Levene (10) from a barium salt prepared in a manner similar to that for the calcium salt described above. The aldobionic acid preparation was made from gum arabic by the method of Heidelberger and Kendall (II), except that the acid was not crystallized. The product was reprecipitated twice and then fed as the syrup, which probably contained besides the glucuronogalactose some free glucuronic acid and some higher polymers. Chondroitinsulfuric acid was prepared from cartilage by the method of Meyer and Smyth (12), except that the final precipitation in glacial acetic acid was omitted. Some difficulty was encountered in obtaining sufficient material for feeding.
DISCUSSION

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The data in Table II afford ample support for the conclusion that chondroitin possesses gizzard factor potency. The difficulty of preparing very pure chondroitin makes it equally difficult to rule out the possibility of impurities accounting for the activity, but the following evidence may be cited as bearing on this possibility. In the first place, the protein-free calcium salt was more effective than the crude chondroitin from which it was prepared; secondly, extraction with hexane removed no potent material; and thirdly, two methods of preparation of chondroitin from cartilage yielded active material. Aside from the possibility of impurities, there is also the ques-

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Gizzard Erosion in Chicks

tion as to whether a part of the chondroitin molecule would be effective. Chondrosamine may be ruled out on the basis of the data given in Table II, and the related sugar, galactose, also proved inactive. The glucuronic acid portion of the molecule has not been adequately tested. Prior to the experiments described here, glucuronic acid was fed at a level of 0.2 per cent of the ration, because it was known to be a constituent of various connective tissues. This relatively low level showed no activity, and higher levels have not been tried. The possibility that free glucuronic acid added to the ration may be destroyed before being assimilated by the chick requires consideration, since this is a rather labile compound. Of particular interest are the results of the feeding of the crude aldobionic acid fraction from gum arabic. This finding requires further investigation with a larger number of chicks, but the very definite effect obtained here would seem to indicate that glucuronic acid is involved in the gizzard erosion problem. Here also the possibility of impurities must be considered, but it does not seem very likely that the same impurities would be present in this preparation as in chondroitin. It is of interest to note that Crandall and coworkers (4, 13, 14) have pointed out the likelihood that glucuronic acid is the effective portion of the chondroitin molecule in alleviating gastric ulcer in humans and in promoting weight gains and improved nutrition in Eck fistula dogs. Manville et al. (15) have produced a glucuronic acid deficiency in rabbits by administration of menthol and have stated that, Ulcerative and hemorrhagic defects were commonly found in the stomach, duodenum, gall bladder, and small and large intestine. The apparent potency of the aldobionic acid preparation is also of interest in that it offers a possible explanation of the effectiveness of such plant materials as oats and bran in preventing gizzard erosion. The potency of the animal tissues which have been tried could be explained on the basis of their chondroitin content, but so far as we know, chondroitin has not been reported in plants. The limit,ed amount of data available on bile compounds is presented in confirmation of Almquists recent report. Aside from the similarity in the lesions observed these data constitute the first evidence for the identity of the deficiencies observed by the California workers and by us. The data are not sufficiently

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Bird, Oleson, Elvehjem,

and Hart

677

extensive to warrant conclusions as to the comparative effectiveness of these compounds. The use of bile in the treatment of peptic ulcer has been discussed recently by Emery and Schnitker (16). They found that it relieved the symptoms in a number of cases but concluded that their data were such as to exclude the use of desiccated ox bile as a specific cure for this disease. The possibility of a physiological relationship explaining the activity both of chondroitin and of bile would appear to be a very interesting one from the standpoint of further research. The effect of chondroitin in promoting weight gains in Eck fistula dogs observed by Crandall et al. may indicate such a relationship. Also it is well known that the liver plays a part in glucuronic acid metabolism, at least to the extent that detoxication by conjugation with glucuronic acid occurs there. As might be expected, the chicks receiving bile and bile compounds gave evidence of marked stimulation of bile secretion. The gallbladders were much enlarged and distended with bile. Further, it is common knowledge that bile contains mucin, which may explain on a chemical basis the protective activity of chondroitin and bile or bile salts. The growth of the chicks in these experiments, as indicated in Table II, appears worthy of some comment. Almquist and Stokstad have made the statement that gizzard erosion does not affect growth. Our previous results led us to the opposite conclusion, although there was a possibility that growth differences were affected by complicating factors. Table II shows that, with the exception of the sodium glycocholate and taurocholate, every supplement preventing gizzard erosion also improved growth, although in some cases t,he differences were small and perhaps not significant. It seems likely that improvement in the basal ration to eliminate the possibility of other deficiencies would result in greater growth differences. A number of cases of the type of paralysis we have associated with vitamin Bd deficiency were observed (17) and in nearly all cases these chicks were very much stunted in growth. As an example the group receiving Ration 466 + 3 per cent chondroitin may be cited. The average weight at 4 weeks given in Table II is 128 gm.; but if one chick weighing 55 gm. and exhibiting severe paralysis be eliminated, the average is raised to 143 gm. It is also of interest that

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Gizzard Erosion in Chicks

this was the one chick erosion. in the group
SUMMARY

showing

marked

gizzard

Chondroitin preparations are effective in preventing gizzard erosion in chicks. Such animal tissues as have been found effective probably owe their potency to their chondroitin content. The chondrosamine portion of the chondroitin molecule is not effective. The evidence that glucuronic acid is the active portion of the molecule is presented and discussed. Confirmation is offered for Almquists recent report on the activity of bile and bile compounds.
BIBLIOGRAPHY

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1. Bird, H. R., Elvehjem, C. A., and Hart, E. B., Proc. Am. Sot. Biol. Chem., J. Biol. Chem., 114, p. x (1936). 2. Bird, H. R., Kline, 0. L., Elvehjem, C. A., Hart, E. B., and Halpin, J. G., J. Nutrition, 12,571 (1936). 3. Bird, H. R., Oleson, J. J., Elvehjem, C. A., Hart, E. B., and Halpin, J. G., Poultry SC., 16, 238 (1937). 4. Crandall, L. A., and Roberts, G. M., Proc. Sot. Exp. Biol. and Med., 30, 704 (1933). 5. Bird, H. R., and Oleson, J. J., Proc. Am. Sot. Biol. Chem., J. Biol. Chem., 123, p. xi (1938). 6. Almquist, H. J., and Stokstad, E. L. R., Nature, 137, 581 (1936); J. Nutrition, 13, 339 (1937). 7. Almquist, H. J., J. Nutrition, 14, 241 (1937); Poultry SC., 17, 155 (1938). 8. Almquist, H. J., Science, 87, 538 (1938). 9. Kline, 0. L., Bird, H. R., Elvehjem, C. A., and Hart, E. B., J. Nutrition, 11, 515 (1936). 10. Levene, P. A., Hexosamines and mucoproteins, Monographs on biochemistry, London and New York, 113 (1925). 11. Heidelberger, M., and Kendall, F. E., J. Biol. Chem., 84,639 (1929). 12. Meyer, K., and Smyth, E. M., J. Biol. Chem., 119,507 (1937). 13. Crandall, L. A., Roberts, G. M., and Gibbs, J. W., Proc. Sot. Exp. Biol. and Med., 29, 1082 (1932). 14. Crandall, L. A., Roberts, G. M., and Snorf, L. D., Am. J. Digest. Dis. and Nutrition, 3,289 (1936). 15. Manville, I. A., Bradway, E. M., and McMinis, A. S., Am. J. Digest. Dis. and Nutrition, 3, 570 (1936). 16. Emery, E. S., and Schnitker, M. A., Ann. Int. Med., 11, 2007 (1938). 17. Bird, H. R., and Oleson, J. J., Proc. Sot. Exp. Biol. and Med., 38, 870 (1938).