The Mechanism of Gossypol Detoxification by Ruminant Animals1

RAYMOND REISER AND HWEI C. FU Department of Biochemistry and Nutrition, Texas Agricultural Experiment Station, College Station, Texas

It has been known for many years that cottonseed meal containing free gossypol produces toxic symptoms if fed to swine (Withers and Carruth, '15, '18) or chicks (Couch et al., '55; Rigdon et al., '58) at significant levels, and that gossypol has been demonstrated to be toxic to other ani mals (Withers and Carruth,'18; Schwartze, '24; Eagle, '50). But it has also been dem onstrated that relatively large amounts of gossypol contained in cottonseed meal may be fed to ruminant animals with no sign of toxicity (Cranford, '10; Jones et al., '41; Ramsey and Miles, '53). The mechanism whereby gossypol is de toxified by the ruminant animal has re mained unknown. Since most differences in metabolism between ruminant and nonruminant animals may be traced to the activity of rumen microorganisms, the pos sibility was considered that these organ isms might also act upon gossypol to de stroy it in some manner. Previous work in this laboratory has shown that dietary polyunsaturated fatty acids may be hydrogenated by rumen liquor,2 or in the rumen in vivo (Reiser and Reddy, '56). This suggested that gossypol might also be reduced in the highly re ducing environment of the rumen.
EXPERIMENTAL

Preparation of soluble gossypol. About 50 mg of crystalline gossypol3 were dis solved in 15 ml of alcohol. Twenty milliliters of glycerol were added and the alcohol evaporated on a steam bath under reduced pressure. The product is a clear sol which may be mixed with water in any proportion without precipitation. Determination of free and bound gossy pol. Gossypol assays, free and bound,
J. NUTRITION, 76: "62

were determined by the method of Pons and Hoffpauir ('57). Determination of free t-amino group. This assay was made by the method of Baliga et al. ('59). Preparation of rumen liquor. Rumen liquor was obtained from freshly slaugh tered beefs, then strained through cheese cloth and used as soon as possible. Aerobic and anaerobic incubation. To determine whether anaerobic or aerobic organisms have a different influence on gossypol, 3.5 ml of the strained liquor were mixed with 1.25 ml of gossypol sol. In one set of experiments 0.5 ml of 4% glu cose and in another set 0.5 ml of 95% alcohol were added as a source of energy. The results of the study are given in table 1. There was no loss of total gos sypol, but the free gossypol was signifi cantly reduced, indicating a binding effect but no destruction. There was probably no significant difference between the aero bic and anaerobic cultures, bringing into question bacterial participation in the free gossypol disappearance. Nevertheless glu cose apparently stimulated more activity than alcohol. However, different rumen liquors were used and there was no con trol run without glucose or alcohol. Effect of energy source. The influence of the nature of the added energy source was further studied by anaerobic incuba tion. The results (table 2) do not support the superior activity of glucose. This sugReceived for publication September 18, 1961. 1 Supported, in part, by a grant from the Oscar Johnston Foundation. - Reiser, R. 1951 Hydrogénation of polyunsatu rated fatty acids by the ruminant. Federation Proc., 10: 236 (abstract). 3 Gossypol acetate was sent to us from the Southern Utilization Research and Development Division, USDA, New Orleans, Louisiana by Dr. T. H. Hopper whose kindness we gratefully acknowledge. 215

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216

RAYMOND

REISER

AND HWEI

C. FU

TABLE 1

Effect of aerobic and anaerobic incubation of rumen fluid and gossypol on the disappearance of total and free gossypol
gossypolIncubation conditions^Anaerobic Disappearance of

AerobicTestFree%

62 ± 4.9s 0 75±2.1JÕ.3Total% 0TestFree%

29 ±2.6 0 36±4.123,4Total% 0
1.25 ml. 1.25 ml.

1 Incubation at 37°C for 48 hours. 2 Test 1. Rumen liquor, 3.5 ml; 4% glucose, 0.5 ml; and gossypol solution, 3 Each value is the result of 4 replicate tests. 4 Test 2. Rumen liquor, 3.5 ml; 95% alcohol, 0.5 ml; and gossypol solution, •• Standard deviation.

Effect of incubating

TABLE 2 gossypol and rumen liquor anaerobically
of

with glucose or alcohol
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gossypolTest Mediai 1»Free% no.i»2»3«Disappearance

22Free%

±9.0*70±1.881 85 000Test ±4.6Total%
1 Incubation at 37°Cfor 2 Each value is the result 3 Medium no. 1. Rumen 4 Standard deviation. 5 Medium no. 2. Rumen 6 Medium no. 3. Rumen 48 hours. of 4 replicate tests. liquor, 5 ml; gossypol solution,

5.233 80 ± 4.454±10Total% ±

000

0.2 ml.

liquor, 5 ml; gossypol solution, 0.2 ml; and 2% glucose, 2 ml. liquor, 5 ml; gossypol solution, 0.2 ml; and 95% alcohol, 1 ml. TABLE 3

Effect of centrifugation

of rumen fluid at various speeds on the formation of the bound gossypol

gossypolTest of free Centrifugation speed 2»30±0«33 3»36 4*67±9.167±369±664±0Tests»59±059±059±059±0 I»%49485855Test (20 min.)None5001,0002,0002,5004,5005,0006,0006,50010,00010,500Disappearance

±2.229 ±2.548 ±2.439 ±2.530±545±5Test ±8.331±3.1Test

1 Test 1. Rumen liquor, 10 ml; gossypol solution, 0.2 ml; incubated aerobically at 37°C for 24 hours; one tube at each speed. 2 Test 2. Rumen liquor, 5 ml; gossypol solution, 0.2 ml; and 1% glucose, 2 ml; incubated an aerobically at 37" C for 48 hours, in duplicate. 3 Test 3. Rumen liquor, 5 ml and gossypol solution, 0.2 ml; incubated anaerobically at 37°Cfor 48 hours, in triplicate. *Test 4. Rumen liquor, 5 ml and gossypol solution, 0.4 ml; heated for 20 minutes at 70°C in a water bath, in duplicate. 5 Same as test 4, except that a different sample of rumen fluid was used, in duplicate. 6 Standard deviation.

GOSSYPOL

DETOXIFICATION

217

gests that the reaction is probably not due to a reduction of the gossypol to a colorless derivative by bacteria and again suggests that the disappearance of free gossypol is not due to microbial action. Activity of centrifugea rumen liquor. To determine whether the gossypol is bound by bacteria or by soluble protein, samples of rumen liquor were centrifuged at various speeds up to 10,500 rpm and then incubated with gossypol sols for 48 hours at 37°C.The results (table 3) show clearly that centrifugation had no influ ence on the percentage of gossypol that was bound. Although bacteria are diffi cult to remove completely from rumen liquor by centrifugation, there are rela tively few remaining in the supernatant after 20 minutes at 10,500 rpm, and one should expect a graded decrease in num bers from uncentrifuged liquor. If the gossypol had been bound by the bacteria one should expect a decrease in the degree of binding in the centrifuged specimens. Because no decrease occurred, the tenta tive conclusion was made that the gossypol was bound by soluble protein. Nonbacterial nature of the reaction. A second test to determine whether the gos sypol is bound by rumen bacteria or solu ble protein was made by adding gossypol sol to the liquor and immediately immers ing in a hot water bath at 70°Cfor 10, 20, 30, or 60 minutes. The results (table 4) show clearly that the binding effect cannot be due to the met abolic action of bacteria because the reac tion was completed at 70° in 10 minutes C or less. The reaction is thus chemical.
TABLE 4

Comparison of bacterial incubation and hot water temperatures. To obtain some comparison between the hot water bath and incubation reactions, 10-ml samples of rumen liquor were mixed with 4 ml of gossypol sol and either held at 70°Cfor 20 minutes, aerobically, or incubated at 37°Cfor 48 hours anaerobically. Dupli cate tests gave identical results, the 70°C reaction binding somewhat more gossypol than the 37°C incubation reactions (table 5). Relation between binding of free gos sypol and disappearance of lysine ¿-amino groups. A final test of whether the gos sypol is bound by rumen liquor protein is the simultaneous disappearance of free gossypol and lysine e-amino groups. Sam ples of rumen liquor were incubated at 37°C 48 hours with the ratios of rumen for to gossypol sol of 5 to 0.4 and 10 to 0.4. One sample was heated at 70°Cfor 20 minutes with the ratio of rumen to gos sypol sol of 5 to 0.4. The milligrams of free gossypol and lysine c-amino groups, that disappeared were determined (table 6). The mole ratio of bound gossypol to bound lysine was 1:2 in three of the 4 tests. Influence of proteolytic enzymes on the stability of rumen liquor bound gossypol. To test the effect of digestion upon the stability of the lysine-gossypol complex, 5 ml of rumen liquor were incubated for 24 hours at 37°Cwith 0.2 ml of gossypol sol. One milligram of the enzymes was added and incubation continued for an additional 24 hours. The percentage of free gossypol that disappeared was then determined. The addition of enzymes had no influence on the gossypol binding ac tivity of the liquor (table 7).
TABLE 5

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Effect of heating gossypol-rumen fluid solutions for varying periods of time at 70°C on the disappearance of total and free gossypol Heating periodminutes10203060Disappearance
gossypol'-2Free81±1.7379 of

Comparison of bound gossypol formation different types of incubation
Types of incubation Disappearance Free

under

of gossypol'-2 Total

±2.580 ±4.482 ±7.4Total0000 1 Determined in triplicate. 2 The test solutions contained 0.4 ml of gossypol solution and 5 ml of rumen fluid. 3 Standard deviation.

Aerobic water bath at 70°C 20 minutes for Anaerobic at 37°C for 48 hours75

±03 64±000

1 In duplicate. 2 The test samples contained 10 ml of rumen liquor and 4 ml of gossypol solution. 3 Standard deviation.

218
The simultaneous

RAYMOND REISER AND HWEI C. FU TABLE 6 disappearance of the free c-amino group of lysine and of free gossypol during incubation of rumen fluid-gossypol solution ratio of bound of free gossypolMole gossypol

Test' :boundlysine1»2«3»4«mg0.53 of free lysine c-amino groupDisappearance no.Disappearance

±0.08"1.24 1.2: ±0.260.98 ±0.161.58±01.75±0moles0.00310.00440.00310.00331111: 1.9: ±0.0450.99 2.1: ±0.0045moles0.00340.00840.00670.0068mg1.48±0.112.28 2.0 1All tests were run in duplicate. 2Test 1. Five milliliters of rumen liquor incubated anaerobically with 0.4 ml of gossypol-glycerol solution at 37°C 48 hours. for 3Standard deviation. 4Test 2. Ten milliliters of rumen liquor incubated anaerobically with 0.8 ml of gossypol solution at 37°C 48 hours. for 5Test 3. Ten milliliters of rumen liquor incubated anaerobically with 0.4 ml of eossypol solution at 37°C 48 hours. for ' Test 4. Ten milliliters of rumen liquor incubated aerobically with 0.4 ml of gossypol solution at 70°C water bath for 20 minutes. in
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LITERATURE CITED Baliga, B. P., M. E. Bayliss and C. M. Lyman 1959 Determination of free lysine amino groups in cottonseed meals and preliminary studies on relation to protein quality. Arch. Biochem. Biophys., 84: 1. Enzymes added Couch, J. R., W. Y. Chang and C. M. Lyman 1'No Test 1955 The effect of free gossypol on chick growth. Poultry Sci., 34: 178. enzymePancreatinRhozyme-6Rhozyme-pfTest ±2.538±6.125±079 Crawford, A. C. 1910 A poisonous principle in certain cottonseed meals. J. Pharmacol. Exper. Therap., 1: 519. Eagle, E. 1950 Effect of repeated doses of 2«No gossypol on the dog. Arch. Biochem., 26: 68. enzymePepsinTrypsin32±4.1338±1.684 Jones, J. H., J. M. Jones and J. J. Bayles 1941 ±2.977 Utilization of home grown feeds in fattening ±4.9 steers in the trans-pecos region. Texas Agr. Exp. Sta. Bull. 604, College Station, Texas. 1The basic incubation medium in each tube was Pons, W. A., Jr., and C. L. Hoffpauir 1957 5 ml incubation for and 0.2 at 37°C, ne milligram After of rumen fluid 24 hours ml of gossypol solution. o Determination of free and total gossypol in of the enzyme was added to the medium and incuba mixed feeds containing cottonseed meals. J. tion continued for an additional 24 hours. Assoc. Official Agr. Chemists, 40: 1068. 2In duplicate. ' Standard deviation. Ramsey, D. S., and J. T. Miles 1953 Cotton 4 In triplicate. seed vs. cottonseed meal and corn as protein source in a concentrate mixture for dairy cows. SUMMARY AND CONCLUSION J. Dairy Sci., 36: 1308. H. G. Reddy The indifference of the degree of gos Reiser, R., and dietary R.unsaturated1956 The hydroJ. génationof fatty acids. Amer. Oil Chemists' Soc., 33: 155. sypol binding of rumen liquor to aerobic Rigdon, R. H., G. Cross, T. M. Feguson and J. R. or anaerobic incubation, high or low tem Couch 1958 Effects of gossypol in young perature, centrifugation or proteolytic en chicks with the production of a ceroid like zymes, and the simultaneous disappear pigment. A. M. A. Arch. Pathol., 65: 228. ance of two moles of lysine e-amino groups Schwartze, E. W. 1924 Pharmacology of gos sypol, J. Agr. Res., 28: 191. to each mole of gossypol, demonstrates Withers, W. A., and F. E. Carruth 1915 Gos convincingly that the mechanism of rumi sypol, the toxic substance in cottonseed. J. nant detoxification of gossypol is by bind Agr. Res., 12: 83. ing to soluble proteins, and that the bond 1918 Gossypol, the toxic substance in cottonseed. Ibid., 12: 83. is permanent during protein digestion. Effect of proteolytic enzymes on the disappearance of free gossypol bound in rumen fluid-gossypol solution1

TABLE 7

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