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Human nutrition and gossypol

Ricardo Bressani, J.E. graham, and L.G. Elias
Division of Agricultural and Food Sciences, Institute of Nutrition of Central America and Panama, Guatemala City, Guatemala

EDITOR'S NOTE

Two articles published in an earlier issue of the Food and Nutrition Bulletin {volume 2, number 2) discussed the conceptual basis, evolution, and current status of Incaparina, a prototype of relatively low-cost processed vegetable mixtures for the complementary and mixed feeding of infants and young children. Because no other concentrated source of protein was available in Central America at low cost, cottonseed flour (28 per cent) was combined with whole ground corn (56 per cent) and other ingredients to give a weaning food with the approximate protein value of milk and a balanced complement of other nutrients except for vitamin C. Since this was the first major use of cottonseed flour for human consumption, at /east in modem times, there was concern for the effects of the pigment gossypol which it contains on both protein quality and toxicity. This issue was taken up by the Protein Advisory Group of the UN, which concluded that cottonseed flour could be used for this purpose provided that the total gossypol consent did not exceed 0.95 per cent and free gossypol was not more than 0.07 per cent, with an epsilon-lysine con ten t of not less than 3.6 per cent of the total protein.

The following review paper by Dr. Ricardo Bressani completes the Incaparina story by describing the extensive laboratory and animal testing that preceded the use of cottonseed flour in clinical trials. Recent research in China has demonstrated that an alcohol extract of gossypol in pure form, given orally, acts as a ma/e contraceptive by inhibiting spermatogenesis. For this reason the trials in experimental animals conducted by Dr. Bressani in the 1950s and early 1960s which indicated that cottonseed flour as processed for Incaparina did not have such activity take on added significance.

INTRODUCTION

The Institute of Nutrition of Central America and Panama (INCAP) in 1955 began a research programme for the development of foods of high nutritive value for feeding children and other people suffering from protein/calorie malnutrition (1, 2). Various formulations were developed; one based on cottonseed protein, Vegetable Mixture 9, was chosen because it was the only vegetable protein source available in the country. A level of 38 per cent cottonseed flour was selected to provide a dry mixture supplying 27.5 9 protein per 100 9 to complement the cereal beverages and starchy gruels commonly used as weaning foods. The composition of this formula and its clinical evaluation have been described previously (1), and the commercial history of the formula, marketed as Incaparina, appeared in volume 2, number 2, of the Bulletin.

The cottonseed flour chosen, as shown in table 1, contained 50 per cent protein, 2 per cent oil, 3 to 4 per cent crude fibre, and 3.6 9 available Iysine per 16 g N. The free gossypol level was 0.55 per cent, with a total gossypol content of 0.95 per cent. The cottonseed flour utilized came from a pre-press solvent extraction process with operating conditions established at the processing sites. Emphasis was placed on choosing those processing conditions leading to the retention of the highest available Iysine level with the minimum level of free and total gossypol.

On the basis of the quality standards for edible flour and from direct chemical analysis, the composition of the protein food is shown in table 2. Protein content varies between 26 and 27 per cent free gossypol from 0.017 to 0.021 per cent, total gossypol from 0.35 to 0.38 per cent, and available Iysine 4.2 9/16 g N.

TABLE 1. Incaparina Formula

TABLE 2. Chemical Specifications of Cottonseed Flour (CSF) and of Incaparina (INCAP VP 9)

TABLE 3. Effect of Cooking on the Free and Total Gossypol Content of INCAP VP 9

Source: Bressani et al. (5)

TABLE 4. Effect of Cooking on Changes in Several Components of Vegetable Mixture 9*

* Formula: corn, 58%; cottonseed flour, 38%; torula yeast, 3%; CaCO₃, 1%. All analytical values expressed on a dry-weight basis,

a. Before water was added
b. Cottonseed flour contained 45 mg free gossypol/100 g flour
c. Cottonseed flour contained 60 mg free gossypol/100 9 flour
Source: Bressani et al. (7)

With this as background information, the results of various studies carried out before conducting tests with children will be discussed briefly.

EFFECT OF COOKING FOR CONSUMPTION

The food described above was made from ingredients that need additional cooking before consumption. This process was also recommended for sanitary reasons, since the water supplies used in the home would be of doubtful sanitary quality. Furthermore, the food was to be consumed as a thin or thick gruel. Some representative results of cooking are shown in table 3. The most significant observation is that free gossypol levels decreased by about 55 per cent of the original value. On the other hand, total gossypol remains the same. Lower free gossypol levels are obtained if the mixture is cooked in metal containers, particularly those made of iron. Additional information is shown in table 4, which confirms previous findings. In one case the initial free gossypol level was around 16 mg and dropped to about 3 mg, while in a second case the decrease was from 21 to 10 mg/100 9. As before, total gossypol does not change, and available Iysine also remains the same throughout cooking time.

The decrease in free gossypol could also be caused by its reaction with other organic substances; however, it was found later that calcium ions present in the formula as calcium carbonate are capable of destroying, or otherwise inactivating, free gossypol, as shown by representative data in table 5. This information was obtained by chemical tests and confirmed biologically by means of swine-feeding tests carried out in the course of determining the wholesomeness of the food. It should also be indicated that ferrous salts such as FeSO₄ also decrease gossypol toxicity. The mixture cooked for various periods of time was fed to dogs in order to detect any loss in protein quality induced by the reaction of free gossypol and Iysine. Some representative results are shown in table 6. The last line shows nitrogen retention to be essentially the same, except at 30 minutes of cooking time, which resulted in a lower value. Protein digestibility was not affected significantly, varying between 72.3 per cent at 0 minutes cooking time and 69.9 per cent at 30 minutes. In addition to dogs, rats, swine, and poultry were used for such studies. Other studies showed the mixture to have a good supplementary effect to the poor quality diets typically consumed by many people in developing countries.

TABLE 5. Effect of Calcium Hydroxide and Cooking on Free and Total Gossypol of VP 9

Source: Bressani et al. (7)

TABLE 6. Nitrogen Balance in Young Dogs Fed INCAP VP 9 Cooked for Various Periods of Time

Source: Bressani et al. 181

TABLE 7. Short-Term Reproduction Trials in Rats Fed INCAP VP 9

* 38% cottonseed-flour diets containing 25% protein and 18 mg free gossypol/100 9

TABLE 8. Average Weight Gain of Male and Female Rats at 14 Months of Age

Source: Bressani et al. (3)

LONG-TERM FEEDING TESTS

Even though the levels of free gossypol in the mixture were low, it was felt necessary to run long-term feeding tests to detect any harmful physiological effects caused by gossypol that were not detected in the short-term studies. When these studies were carried out in 1969 (3), there was no thought to find out if there was interference with reproductive performance when animals were fed cottonseed flour with small amounts of free and total gossypol. Actually, the purpose was to measure lactation performance of the animals. Several short term trials were run and the results from one of the first are shown in table 7. In this case, two male and two female 30-day-old rats weighing 60 9 were fed Vegetable Protein Mixture 9 supplemented with additional calories, vitamins, and minerals. The average weight gains of the rats at the end of the 14-month study are shown in table 8, which also shows the free gossypol content of the diets used, as well as the level of protein. The weight gained was essentially the same among groups, except for the males on cottonseed flour B showing a slightly lower weight gain. The male animals consumed an average of 22.5 9 of diet per day over the whole period; therefore, free gossypol intake varied from 2.4 mg/rat/day in diet K to 6.2 mg/rat/ day in diet group C. For females, average consumption was 19 9, with corresponding lower intakes of free gossypol.

Overall breeding performance is shown in table 9. Except for one male from the control group and one female from diet group P that died at 198 and 96 days, respectively, after the initiation of the study, fertility was relatively high, with rats showing normal birth weight. At the end of the study, organ weight was normal and no pathology was detected. Actually, breeding performance was relatively good, which may have been because of the high level of protein fed, a factor that may introduce some protection against the anti-physiological effects of gossypol.

These, as well as other results in experimental animals, suggested that the food was safe to be fed to human subjects.

METABOLIC STUDIES IN EXPERIMENTAL ANIMALS

The question of the fate of the small levels of gossypol ingested remains largely unanswered. In the results shown in figure 1, dogs were fed, for four days, a casein diet to which gossypol was added in the form of crude oil as found in cottonseed. Faecal samples were collected before gossypol feeding and every two days after feeding for a total of six additional days. The results show a peak

FIG. 1. Faecal Excretion of Gossypol in Dogs Fed a Casein Diet and Free Gossypol (Average Two Dogs per Group)

TABLE 9. Breeding Performance of Rats Fed for 14 Months on Diets Containing Various Levels of Free Gossypol

Source: Bressani et al. (31

TABLE 10. Free and Total Gossypol Balance in Young Pigs

Source: Braham et al (4)

TABLE 11. Gossypol Balance in Children Fed INCAP VP 9

excretion value that decreased with time. The total recovery was zero on the sixth day. The identity of gossypol in faeces was established by chemical means and by feeding faeces to hens.

In other studies (4), swine were fed gossypol as found in cottonseed flour for one hour and then the animals were sacrificed at hourly intervals to collect and analyse the intestinal contents. Both free and total gossypol were analysed. The results of some studies are shown in table 10. They reveal changes in gossypol that correlate with gastric movement. One problem that arose, however, was that free gossypol levels in the faeces were always higher than the levels fed. Recoveries ranged from 91 to 273 per cent, figures also found in rat and dog studies.

The lower section of table 10 shows that total gossypol levels in the stomach decrease with a simultaneous decrease in the intestinal content. The total amount tallied with the levels ingested. Recoveries in this case varied from 83 to 105 per cent. It was suggested at the time that some hydrolysis of bound gossypol took place and that free gossypol was absorbed through the intestine, being transported to the liver and excreted back again through the bile into the gastrointestinal tract. Total gossypol analysis includes free gossypol; therefore one would expect total gossypol recoveries such as those indicated for free gossypol. The easiest explanation is to blame the analytical methodology used, which attempted to measure mg quantities of free gossypol; however, the free gossypol was identified by physico-chemical analysis as well as by feeding

TABLE 12. Gossypol Balance in Children Fed INCAP VP 9 Made with Low Free Gossypol Cottonseed Flour (11 mg/100 9)*

Total gossypol, 67 mg/100 9

TABLE 13. Gossypol Balance in Children Fed INCAP VP 9 Made with Low Free Gossypol Cottonseed Flour (11 mg/100 9)*

* Total gossypol. 0.36 g/100 g faecal extracts to hens. The yolk of the eggs laid became dark upon exposure to ammonia, a biological indication that the pigment was gossypol.

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