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The Effect of Calcium Carbonate and Sodium Bicarbonate on the Toxicity of Gossypol»

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The Effect of Calcium Carbonate and Sodium Bicarbonate on the Toxicity of Gossypol» THE EFFECT OF CALCIUM CARBONATE AND SODIUM BICARBONATE ON THE TOXICITY OF GOSSYPOL» By WILLIS D. GALLUP, assistant chemist, and RUTH REDER, associate chemist, Department of Agricultural Chemistry Research, Oklahoma Agricultural Experi- ment Station INTRODUCTION In a previous study of the influence of certain dietary constituents on the response of rats to gossypol ingestion {2) ^ the authors showed that the toxicity of diets containing known amounts of gossypol was materially reduced when the diets were made basic by the addition of calcium carbonate and sodium bicarbonate. When the diets were made acidic by the addition of calcium chloride, only indirect evidence of a slight decrease in toxicity was obtained. Decreased toxicity was observed also when the protein content of the diet was increased from 13 percent to 35 percent. The conclusion was drawn that diets of high protein content and basic diets of high calcium content are favorable to the detoxication of gossypol. Considerable importance is attached to the results since calcium and protein are variable ingredients in diets used for the bio-assay of gossypol. The importance of these constituents in feeding cottonseed products to livestock has not been fully determined, although the value of supplementary protein in cottonseed-meal rations for pigs has recently been pointed out by Robison (5). In view of the favorable results obtained with sodium bicarbonate and calcium carbonate, it was deemed desirable to determine the pro- portion of these salts which would offer the greatest degree of pro- tection against gossypol injury and to determine the value of each salt in the presence of moderate amounts of thç other, as the principal elements of both are requisite for a normal nutritive condition. The present paper reports the results of this study. The results of other experiments, in which calcium altered the inhibiting effect of gossypol on the hydrolysis of fat by lipase, are also reported. The results of these studies are offered as further evidence of the important part played by calcium in the detoxication of gossypol. MATERIAL AND METHODS The manner of conducting the feeding experiments, in which albino rats were used as experimental animals, has been described in a previous paper (^). The percentage composition of the basal diet to which varying amounts of sodium bicarbonate and calcium carbonate were added was as follows: Cottonseed 10, starch 49, casein 24, yeast 5, cod-liver oil 3.5, salt mixture {S) 3.5, and Crisco 5. The cottonseed contained 0.60 percent of gossypol. Control diets were made up with gossypol-free 1 Received for publication Aug. 19, 1935; issued February, 1936. A preliminary report of this work was presented before the American Society of Biological Chemists in Detroit, Mich., Apr. 10-13,1935. * Reference is made by number (italic) to Literature Cited, p. 72. Journal of Agricultural Research, Vol. 52, no. 1 Washington, D. C. Jan. 1, 1936 Keyno. Okla.-24 (65) 49563—36 5 66 Journal of Agricultural Research voi. 52, no. 1 cottonseed. Fresh diets were prepared weekly, at which time the prescribed amounts of calcium carbonate and sodium bicarbonate were added. In one experiment sodium bicarbonate was added to the basal diet at the time of feeding, and any food residues from the previous day were discarded. This procedure was followed as a pre- caution against the possible destruction of the gossypol, by contact with the alkali, before ingestion. In the lipase studies the general procedure of Willstätter, Wald- schmidt-Leitz, and Memmen (7) was followed. A glycerol extract of pig^s pancreas was used as a source of lipase. The fresh tissue was dried with acetone and ether and ground in a mortar with 16 times its weight of 80-percent glycerol. After standing at room temperature for 4 hours or longer, with occasional shaking, the solution was filtered through filter paper and stored in an electric refrigerator. Neutral olive oil was prepared by shaking a high-grade commercial oil with an equal volume of 1-percent sodium hydroxide in a separatory funnel at 60° C. After the aqueous layer had been removed, the oil was thoroughlv washed with hot water and dried in the electric oven at 100° C. for'^e hours. A 2-percent solution of egg albumin was prepared by dissolving the dry powder in an excess of ammonium hydroxide and, after this had been shaken for several hours, removing the excess ammonia with a current of warm air. Two buffer solutions were prepared. One was composed of 66 parts of N/1 ammonium hydroxide and 134 parts of N/1 ammonium chloride and had a pH of 8.9 at 30° C. The other buffer solution was composed of equal parts of N/2 acetic acid and N/2 sodium acetate and had a pH of 4.7 at 30°. A 1-percent solution of calcium chloride was made up with the chemically pure salt. Gossypol was prepared and purified according to methods described by Clark (1). Determinations of the rate of hydrolysis of olive oil by lipase in the presence of calcium and gossypol were made in the following manner (4). One-half cubic centimeter of lipase extract, 3 cubic centimeters of olive oil containing various amounts of gossypol, 2 cubic centimeters of the alkaline buffer solution, 1 cubic centimeter of calcium chloride solution, and sufficient amounts of a dilute glycerol solution to keep the total volume of the water' 11 cubic centimeters and the total volume of glycerol 2 cubic centimeters, were shaken together in a small Erlenmeyer flask. One cubic centimeter of egg-albumin solution was then added, and after being skaken for 3 minutes, the flask was placed in a constant-temperature water bath held at 30° C ± 0.2 for 2 hours and 57 minutes. At the end of this time the contents of the flask were washed into a 250-cubic centimeter Erlenmeyer flask with 112 cubic centimeters of 95-percent alcohol. Twenty cubic centimeters of ether was added and the fatty acids titrated with N/10 alcoholic sodium hydroxide, thymolphthalein being used as an indicator. Blank determinations were carried out without the addition of the enzyme. Determinations in which calcium and albumin were omitted were made in the manner described above. In another series of experiments the gossypol was dissolved in the buffer instead of in the oil. In a final series of experiments hydrolysis was carried out in an acid medium with 2 cubic centimeters of the acid buffer solution replacing the alkaline buffer solution. The gossypol was dissolved in the oil. Jan. 1,1936 Efcct of Dietary Constituents on Toxicity of Gossypol 67 EXPERIMENTAL RESULTS FEEDING EXPERIMENTS WITH CALCIUM CARBONATE AND SODIUM BICARBONATE Data obtained with rats which received diets containing 0.06 per- cent of gossypol and varying amounts of calcium carbonate and sodium bicarbonate, together with their controls, are shown in table 1. Table 1 shows a conspicuous difference between the food intake of animals receiving gossypol and the intake of their controls. That of the rats on gossypol was noticeably increased by the addition of sodium bicarbonate to the diet. The same result was obtained with combinations of this salt with calcium carbonate, although the latter by itself tended to decrease the food intake of rats on gossypol diets. The average food intake of the gossypol rats on diets containing 3 percent of sodium bicarbonate slightly exceeded that of their controls, being 599 grams as compared with 585 grams. Similarly there was very little difference between the food intake of the controls and that of the gossypol rats on a diet to which a combination of 2 percent of sodium bicarbonate and 2 percent of calcium carbonate had been added. The increase in food consumption on the part of the rats on gossypol is taken as an indication of improvement in the nutritive condition of the animals. The average gains in weight of rats on the gossypol diets likewise increased with increasing percentages of sodium bicarbonate in the diet. At the 3-percent level, sodium bicarbonate was particularly effective in producing an increase in their gains. The greatest average gain for the rats on gossypol, 118 grams, was made on a diet containing 2 percent of calcium carbonate and 2 percent of sodium bicarbonate, although the average gains made by gossypol rats on diets containing 3 percent of sodium bicarbonate or a combination of 2 percent of calcium carbonate and 1 percent of sodium bicarbonate approached this value. TABLE 1.—Effect of gossypol on growth of rats as influenced by the addition of varioua amounts of sodium bicarbonate and calcium carbonate to the diet Average Average Difference Gossypol food con- gain in Salt additions to basal diet content of Rats sumed in weight in in gains in diet 60 days i 60 daysi weight Percent Number Grams Grams Grams f 0.06 12 468 88 82 None.-- 1 .00 7 629 170 / .06 14 530 92 71 1 percent of NaHCOa 1 .00 6 703 163 / .06 12 527 99 50 2 percent of NaHCOa 1 .00 6 659 149 / .06 6 530 94 55 2percentofNaHC03 2 t .00 6 659 149 / .06 7 599 106 44 3 percent of NaHCOs ( .00 7 585 150 / .06 6 413 87 76 2 percent of CaCO» - I .00 6 678 163 r .06 7 624 118 28 2 percent of CaCOs; 2 percent of NaHCOs-- 1 .00 6 630 146 102 2 percent of CaCOsU percent of NaHCOs-- r .06 9 500 58 1 .00 9 621 160 1 The sum of the averages obtained with males and females divided by 2. 2 2 percent of sodium bicarbonate added daily to gossypol diet.
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