In experiments with rats and Peking ducklings it has been investigated to what extent the linoleic acid content of the diet is of influence on the requirement of vitamin E. This requirement was determined by adding D-α-tocopheryl acetate in increasing doses to vitamin E-free diets and to determine how much vitamin E was necessary to prevent deficiency symptoms, such as invitro
haemolysis in the rat and myopathy in the duckling. For these symptoms methods were developed with with the deficiency grade could be expressed by a number.
The erythrocytes of rats with a vitamin E deficiency haemolyse spontaneously in vitro. Use was made of this phenomenon to
determine the requirement of vitamin E. For this purpose, newly weaned male rats were placed on diets with increasing doses of vitamin E. After 2 weeks the haemolysis test was positive. The test was repeated weekly. Good results were obtained when a 0.01% erythrocyte suspension was incubated for 4 hours in a solution of physiological saline buffered with phosphate, at pH 7.4 and 38°C. It was found that there was a negatively linear relation between the logarithm of the vitamin E content of the food and the percentage invitro
haemolysis. By linear extrapolation it could be calculated how much vitamin E was necessary to prevent haemolysis. This is invitro
haemolysis, which cannot in itself be regarded as a deficiency symptom, was found, after prolonged vitamin E deficiency in rats, to show a clear relation with the degree of myopathy of the leg muscles.
The experiment with Peking ducklings was started with one-day-old chickens which were kept for 4 weeks on diets with increasing doses of vitamin E. In contrast with rats, in these ducklings the erythrocytes showed invitro
only a very slight spontaneous haemolysis. On the other hand, strong myopathy occurred. The strongest was the hyaline-degeneration of the
skeletal muscles, Moreover, necrocalcinosis was found in the smooth muscles of stomach and intestines and in the heart muscle. In the duckling, besides the degree of myopathy in the various organs, certain other criteria were investigated for their suitability for determining the requirement of vitamin E: weight increase, differential counting of leucocytes, activity of the serum enzymes creatine-phosphokinase, aspartate-aminotransferase and lactic acid dehydrogenase, differential determination of the serum proteins and the serum tocopherol level, All these criteria showed a clear connection with the vitamin E content of the food. The histologically determined degree of myopathy of the skeletal muscles was found to be the most suitable for the determination of the requirement of vitamin E. The degree of myopathy of a duckling was expressed as a score (0-24) which was determined by a microscopic assessment of the cross-sections of three pairs of leg muscles (score 0-4 per muscle). A linear relation was found between the myopathy score and the logaritm of the vitamin E content of the food. The amount of vitamin E needed to prevent myopathy could be calculated by linear extrapolation.
Myopathy in ducklings was found to occur only if the food contained little vitamin E at the same time as very little selenium. As little as 0.1 mg Se (as Na 2
) per kg food was sufficient to prevent myopathy completely, This amount has an anti-myopathic effect that is equal to at least 20 mg D-α-tocopheryl acetate,
All diets, of the rats as well as of the ducklings, contained 35 cal% fat. The linoleic acid content of the diet was varied by the use of fats with a low (lard and coconut fat) or with a high linoleic acid content (maize oil and safflower oil). Lard and coconut fat contain very little vitamin E; in the case of maize oil and safflower oil, the tocopherols were removed. Increasing doses of linoleic acid were obtained by the use of mixtures of coconut fat and safflower oil, Study of the effect of an increasing linoleic acid content of the food led to the following conclusions:
- Up to 10 cal% the linoleic acid content of the food has no influence on the requirement of vitamin E. This requirement is roughly 3.0 mg D-α-tocopherol per 1000 Kcal food.
- At a linoleic acid uptake of more than 10 cal% the requirement of vitamin E can increase to 6 mg D-α-tocopherol per 1000 Kcal food, This is probably the result of an increased breakdown of vitamin E in the gastro-intestinal tract.
- From the ratio between the content of vitamin E and linoleic acid it is not possible to deduce whether or not a diet contains sufficient vitamin E.
- Many investigators have observed an increase in the requirement of vitamin E after only very small doses of linoleic acid. However, they were working with diets which contained insufficient essential fatty acids (EFA). With these diets (0-2 cal% linoleic acid) linoleic acid uptake causes a proportional increase in the requirement of vitamin E, which no longer increases once the EFA-deficiency has been corrected. It seems probable that in biomembranes a critical ratio exists between vitamin E and polyunsaturated fatty acids.
- At a fat consumption of 30-40 cal% a dietary fat will need to contain 100 mg D-α-tocopherol (or an equivalent amount of other tocopherols) per kg fat to meet the requirement of vitamin E, This requirement can rise to 200 mg, if the fat contains more than 25% linoleic acid.
- In general, vegetable oils with a high linoleic acid content contain ample vitamin E. Strongly saturated fats often contain too little vitamin E.