Some Selenium Responses in the Rat not Related to Vitamin E 1,2,3 KIM E. M. McCOY a n d PAUL H. WESWIG Department of Agricultural Chemistry, Oregon State University, Corvallis, Oregon ABSTRACT Rats fed a low selenium ration containing Torula yeast with adequate vitamin E grew and reproduced normally. Their offspring were almost hairless, grew more slowly and failed to reproduce. Supplemental di-methionine, sodium sulfate, or increased dosage of vitamins was without effect. Supplementing with 0.1 ppm selenium as sodium selenite restored haircoat, growth, and reproductive capabilities. Some usual biochemical tests for selenium or vitamin E deficiency status, or both, were incon­ clusive when compared with rats fed normal rations. Rats fed low selenium corn protein also had sparse hair and poor growth in the second generation. A low selenium ration from ewe muscle, however, supported rats for three generations without any abnormalities. The essentiality of selenium (Se) as a prevent WMD in the next generation. Mc­ nutrient with or without vitamin E has Connell and Roth (14) have shown a milk been debated (1 -5 ). The essential role of and placental selenium carryover in dogs selenium may be due to its analogue re­ using radiotracers. Recent evidence by placement of sulfur in naturally occurring Burk et al. (1 5) has shown that selenium compounds, or the formation of com­ is rapidly depleted from weanling rats on pounds not related to sulfur derivatives a Torula yeast diet, and that this loss is (6-8). Selenium may parallel the role of seemingly unaffected by vitamin E. vitamin E acting as an antioxidant against The object of the experiment was to lipid double bond peroxidation (9), or it determine whether rats fed a Torula yeast may have a sparing effect on vitamin E ration, or other low selenium protein ra­ itself (1 0 ). On a molar basis, selenium tions with added vitamin E and without compounds constitute the most potent fac­ added selenium, could grow and reproduce tors yet found to prevent death from liver through several generations. The results necrosis in the rat fed a ration which has would provide an insight into the carry­ Torula yeast as its source of protein (8 ). over effect of selenium in other animals Torula yeast4 is unique in that it is very and would present evidence that selenium low in sulfur amino acids, vitamin E and does have an essential function separate selenium, and high in unsaturated fatty from vitamin E. acids (11). Witting and Horwitt (12) have shown selenium growth responses in rats EXPERIMENTAL PROCEDURE supplementing a casein diet with a high Composition of rations used is listed in selenium (0.25 ppm) and a low a-tocoph- table 1. The Torula yeast ration (0.020 erol content. ppm Se) differs from that reported by It has been reported that ewes possess Schwarz and Foltz (8 ) by increasing a carryover effect from an adequate sele­ Torula yeast from 30 to 40% and sub-* 124 nium intake during the first pregnancy to Received for publication March 14, 1969. the second pregnancy when the dam is 1 Published with the approval of the Director as existing on a low selenium ration which Technical Paper no. 2646, Oregon Agricultural Experi­ ment Station. should otherwise produce lambs having 2 Supported in part by Public Health Research white muscle disease (W M D), a selenium- Grant no. HE 09228 from the National Institutes of H ealth. responsive myopathy (13). The pregnant s This report constitutes a portion of a thesis sub­ mitted by Kim E. M. McCoy in partial fulfillment ewe evidently possesses the metabolic of the requirements for the Ph.D. degree. ability of storing minute quantities of se­ 4 Lake States Yeast and Chemical Division 1959 Analysis of type B Torula yeast. St. Regis Paper lenium which it imparts to the lamb to Company, Rhinelander, Wise. J. N u t r it io n , 9 8 : 383-389. 3 8 3 384 KIM E. M. MCCOY AND PAUL H. WESWIG T A BLE 1 for 1 week/month. At 4 months of age, Composition of low selenium rations the animals were mated within each group and all were fertile. The offspring were Torula Ewe Corn 3 yeast1 muscle 2 fed their respective rations or with supple­ % % % ments as will be described. Selenium Protein source 40.0 35.0 85.0 analyses were made on all rations using Sucrose 41.5 51.5 3.0 the method of Allaway and Cary (17). Vegetable o il4 5.0 0.0 5.0 Protein was determined using AOAC pro­ HMW salt mixture 5 5.0 5.0 5.0 cedures (1 8). Blood was obtained by heart Vitamin mixture 6 1.0 1.0 1.0 Cellulose 7 7.5 7.5 0.0 puncture from rats anesthetized with car­ cZZ-Methionine 0.0 0.0 0.2 bon dioxide and the plasma was assayed Z-Lysine 0.0 0.0 0.5 according to Sigma for SGOT,7 LDH,8 and Selenium cone, ppm 0.020 0.027 0.030 CPK 9 enzymes. Selenium-75 red blood cell uptake from radioactive sodium selenite 10 1 Lake State Yeast, Rhinelander, Wise. 2 Freeze dried, low selenium. (specific activity =6.0 Ci/g Se) was de­ 3 Low selenium com was graciously supplied by termined by the method of Wright and Bell W. H. Allaway, Agricultural Research Service, U.S. Department of Agriculture, Ithaca, N. Y. (1 9 ), except that during incubation air 4 Wesson Oil, refined cottonseed oil, Wesson Sales Company, Fullerton, Calif. was used as previous results with air did s Hubbell, R. B., L. B. Mendel and A. J. Wakeman not differ from those using 95% N2 : 5% 1937 A new salt mixture for use in experimental diets. J. Nutr., 14: 273. C 02. Individual hearts and skin samples 6 Vitamin mixture contained: (in milligrams) thia- (0.5 g) were extracted by homogenizing mine-HCl, 40; Ca D-pantothenate, 200; menadione, 10; folic acid, 20; riboflavin, 25; pyridoxine-HCl, 20; bio­ with chloroform and methanol using the tin, 10; and (in grams) vitamin B1 2 , (1% trituration), 1; niacin, 1; choline chloride, 10; and lactose to make procedures of Bligh and Dyer (2 0 ). Fatty 100 g. Vitamin A acetate, 10 mg; d-a-tocopheryl ace­ acid esters were obtained by using 5% tate, 60 mg; and vitamin D2 , 100 fig were supplied in 95% ethanol/kg of diet. HC1 and anhydrous methanol. The fatty 7 BW 100 Solka Floe purified cellulose, Brown Company, New York, N.Y. acid methyl esters were chromatographed according to Lowry and Tinsley (21) using stituting cottonseed oil for stripped lard a chromatograph 11 fitted with a hydrogen as vitamin E was added to the ration. Low flame detector. The fatty acid methyl esters selenium ewe muscle protein was prepared were identified by the relative retention by freeze-drying selected lean muscle from times compared with methyl stearate (C 18:0) and by reference to standards from ewes which had been fed low selenium the Hormel Institute and from data in the alfalfa (0.010 ppm) for at least 1 year literature (22). and had produced WMD lambs. This muscle ration (0.027 ppm Se) was fed RESULTS AND DISCUSSION at a 20% protein level. A low selenium There was considerable difference in com ration (0.030 ppm) was prepared growth supported by these rations (table using ground whole kernels and adding 1), and between the first and second 0.2% dl-methionine and 0.5% 1-lysine. generations (fig. 1). Only females showed The protein content, even with the added decreased growth in generation 2 on the amino acids, was only 8.2%. A commer­ muscle ration (P < 0 .0 1 ), but the ration cial ration 5 constituted the control ration. 5 Purina Laboratory Chow, Ralston Purina Com­ Rats used were from our closed colony p an y, St. Louis, Mo. 6 Harr, J, R. 1968 Department of Veterinary Medi­ of OSU Browns. Four females with their cine, Oregon State University, personal communica­ litters (average of seven pups each) were tion. 7 The colorimetric determination of glutamic-oxalo­ fed Schwarz’s preweaning diet (16) until acetic and glutamic-pyruvic transaminases at 490-520 weaned at 21 days, when they were ran­ m fi in serum or other fluids. Sigma Chemical Com­ pany, St. Louis. Tech. Bull. no. 505, revised Septem­ domly assigned to one of the four dietary ber 1964. 8 The ultraviolet determination in serum at 340 m/i regimens. They were fed ad libitum with of lactic dehydrogenase. Sigma Chemical Company, weekly determinations of growth and feed St. Louis. Tech. Bull. no. 340—UV, June 1966. 9 The colorimetric determination of creatine phos- intake. As a prophylactic measure for the phokinase in serum and other fluids at 620-700 rn.fi. prevention of respiratory disease in long­ Sigma Chemical Company, St. Louis, Tentative Tech. Bull. no. 661, revised May 1967. term reproductive experiments, sulfa New England Nuclear Corporation, Boston, Mass. 11 Beckman model GC-2, Beckman Instruments, Inc., drugs 6 were added to the distilled water Fullerton, Calif. SOME SELENIUM RESPONSES IN THE RAT 385 Fig. 1 Growth curves for rats on vitamin E-adequate diets. Each line is an average of four females or three males. Parentheses indicate generation of rat. supported reproduction through three tion within 2 weeks and normal coverage generations. Both males and females at 4 weeks, whereas the other groups showed decreased growth in generation 2 showed no visible improvement (fig. 2). on the Torula yeast diet (P < 0 .0 1 ).