Typical white muscle disease has been induced in lambs that were normal at weaning but thereafter fed natural dystrophogenic fodder for 2-4 months. Protection from the disease was given by selenium when it was administered regularly as an oral drench, or incorporated into an intraruminal heavy pellet placed in the rumen at weaning.Calcium and magnesium levels in serum were unaffected by the dystrophic condition, although urinary excretion of both ions was decreased. This lowered urinary excretion was associated with high levels of calcium in both heart and skeletal muscle, and to some extent associated with raised levels of calcium and magnesium in washed mitochondria isolated from the same tissues.The respiratory properties of the mitochondria were examined polarographically. No differences were found between heart muscle mitochondria isolated from normal and dystrophic hearts. Skeletal muscle mitochondria from dystrophic animals showed lowered respiratory rates with palmitoyl-DL-carnitine and acetyl-DL-carnitine as substrates. The smaller differences with pyruvate and succinate were not significant. Respiratory control ratios for the dystrophic skeletal muscle organelles were always 1 . 0, but for selenium-supplemented animals were always > 1 ·0.To some extent at least the abnormalities appear to be associated with the high levels of calcium in the tissues and mitochondria of dystrophic lambs.

Inorganic selenium, as Na275Se03, was given intraruminally to lactating ewes, and for 48 hr afterwards samples of blood and milk were taken regularly. The animals were then slaughtered and tissues taken for further analysis. Evidence is presented, based on isolation techniques using Sephadex chromatography and gas-liquid chromatography, that 75Se is incorporated into milk proteins as selenomethionine_ It is estimated that at least 3% of the selenium that enters the milk, from this inorganic source, is present as bound selenomethionine. This is a minimal value, since it has been shown that selenomethionine is easily degraded during a number of the isolation procedures.The steps followed in arriving at the conclusion were: (1) a large proportion of the 75Se activity in the milk was non-dialysable; (2) activity emerges with the milk protein on Sephadex and can be recovered as "soluble casein" by precipitation with ammonium sulphate; (3) Pronase digestion of the labelled protein produces fragments that behave like methionine and cysteine in column chromatography; and (4) gas-liquid chromatography, including collection of the effluent in fractions, has enabled positive identification of a [75SeJselenomethionine peak_Other selenoamino acid-like fractions are present but have not yet been identified.

The activity and distribution of the selenium-containing enzyme, glutathione peroxidase, has been determined in muscle fractions in normal adult rats and sheep. Skeletal and cardiac muscle have been examined, and in both types of muscle the major proportion of the enzyme appeared in the cytosol fraction. Enzyme activity was higher in cardiac muscle than in skeletal muscle in both species, and based on total protein present in fractions, it appears that rat muscle contains more enzyme activity than sheep muscle.In tissues from lambs born to selenium-deprived ewes the levels of enzyme were significantly depressed. Two sampling periods were selected, the first when the lambs were 2-3 weeks of age and the second at slaughter when they were 10 weeks old. Muscle, plasma and erythrocyte levels of the enzyme indicated that the most sensitive measure of selenium deficiency is likely to be that of the erythrocyte enzyme level.

Nutritional myopathy has been induced in both rats and lambs by feeding diets low in selenium. The distribution of 45Ca, administered as 45CaC12, has been examined firstly by autoradiography, and secondly by measuring the excretion of 45Ca in urine and faeces. Autoradiographs of skeletal muscle from unsupplemented animals showed radioactivity over discrete muscle fibres at a stage when no abnormalities were apparent using conventional staining techniques. Similar retention of 45Ca was found in some of the tubules in the kidneys of selenium-deficient rats.. Total excretion in urine and faeces of lambs, examined for 48 h after intravenous administration of 45CaC12, showed that in normal animals 18· 6 % of the dose was excreted, whereas in dystrophic lambs 12·0 % was lost. The difference was significant at the 2 % level.The respiratory rates of isolated skeletal muscle mitOChondria, measured polarographically in the presence of glutamate and pyruvate as substrates, were low for dystrophic rats. Respiratory control indices were 1· 0 for the same preparations but for supplemented rats they were all above 1· o. The differences in respiratory rates were significant at the 1 % level.The major conclusion drawn from the results of these experiments is that one of the first effects of selenium deficiency which can be visualized is the abnormal retention of calcium by individual muscle fibres.

The fate of selenium, given as Na275Se03, or [75Sejselenomethionine, and of [35Sjmethionine administered intravenously to ewes and lambs, has been examined. The main intention was to follow the incorporation of selenium into protein in a number of tissues, including liver and kidney, and to measure the extent of that incorporation of selenoamino acid, particularly with respect to the administration of selenite. The ewes chosen were lactating ewes with lambs at foot, and the lambs were animals which had been weaned on to fodder low in selenium and were recovering from white muscle disease with selenium therapy. These two experimental situations were chosen as they offered conditions under which selenium incorporation might be considered to be maximal.Entry of isotope into milk was rapid and was greater when 75Se was given as the selenoamino acid than as selenite. In both ewes and lambs greater amounts of activity, derived from selenite, were bound to plasma proteins than to the proteins of milk. This was particularly evident in samples taken some hours after administration. This ability of the plasma to bind selenium was demonstrated by alkaline dialysis.Small, though significant amounts of selenium, derived from Na2 75 Se03, were incorporated as selenoamino acids into the proteins of liver, kidney and pancreas, as well as into the proteins of milk and plasma. In ewes, both selenomethionine and selenocystine were identified chromatographically in enzyme digests of defatted liver and kidney. Some differences occurred in the distribution of labelled compounds in organs from lactating ewes and recovering lambs.The incorporation of selenium into protein is discussed briefly in relation to the recent findings of an association between selenium and the enzyme glutathione peroxidase.