crystalline amino acids (flee amino acid diet) by a constant proportion, e.g., to a dietary crude protein (Kjeldahl N x 6.25) level of, say, 5%. The key to such a procedure is that the diet arrived at must, when adequately supplemented with sulfur amino acids, facilitate a consumption level that will allow both weight maintenance and a nitrogen retention of at least zero. Sulfur requirements for maintenance of adult animals are impacted importantly by the continued growth of keratoid tissues such as skin, hair, and feathers. These tissues are high in cyst(e)ine and relatively low in methi0nine. Thus, cyst(e)ine may be capable of furnishing up to 70% of the total need for the sulfur amino acids of adult animals compared with the 50% value that is common in young animals whose sulfur requirement is dominated by soft tissue somatic growth.
 P r o d u c t i o n o f S e l e n i u m D e f i c i e n c y in t h e R a t
By RAYMOND F. BURK
Selenium is an essential nutrient for animals, and diets deficient in the element have been in use for a number of years. Most studies of selenium deficiency have been carried out in rats, and this chapter will focus on that species. It is common practice to feed mice and rats the same diet. Selenium-deficient diets for chickens, I pigs, 2 sheep, 3 fish, 4 guinea pigs, 5 monkeys, 6 and human beings 7 have been described but will not be presented here.
Assessing Severity of Selenium Deficiency An integral part of producing selenium deficiency for research purposes is an assessment of its severity. Although clinical signs occur, biochemical measurements are more commonly used in characterizing the
G. F. C o m b s , Jr., C. H. Liu, Z. H. Lu, and Q. Su, J. Nutr. 114, 964 (1984). z G. R. Ruth and J. F. V a n Vleet, Am. J. Vet. Res. 35, 237 (1974). 3 p. D. Whanger, P. H. Weswig, J. A. Schmitz, and J. E. Oldfield, J. Nutr. 107, 1288 (1977). 4 D. M. Gatlin III, and R. P. Wilson, J. Nutr. 114, 627 (1984). R. F. Burk, J. M. Lane, R. A. L a w r e n c e , and P. E. Gregory, J. Nutr. 111, 690 (1981). M. A. Beilstein and P. D. W h a n g e r , J. Nutr. 113, 2138 (1983). 7 O. A. L e v a n d e r , B. Sutherland, V. C. Morris, and J. C. King, Am. J. Clin. Nutr. 34, 2662 (1981).
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TABLE 1 SUGGESTED CLASSIFICATION OF SELENIUM DEFICIENCY IN THE RAT BY ITS SEVERITY
Degree of severity Mild Moderate Severe
Liver glulathione peroxidase activity" (% of control)
Effects on drugmetabolizing e n z y m e s and plasma glutathione ~'
5-50% <5% <5%
Absent Absent Present
" T h e 5% dividing line is based on a s s a y of the s e l e n o e n z y m e using H202 as substrate. The 5% figure was chosen because of the variety of a s s a y s used in different laboratories. In our experience (see Table Ill) it could be set at 1-2%. h Described in Burk 9 and Reiter and W e n d e l ? °
deficiency. Activity of the selenoenzyme glutathione peroxidase (EC 220.127.116.11) decreases as selenium deficiency developsf Hepatic activity is very sensitive to selenium intake and is the most commonly used index of selenium deficiency; other tissues lose glutathione peroxidase activity more slowly than liver when rats are fed a selenium-deficient diet. In recent years it has become apparent that selenium has biochemical functions other than glutathione peroxidase. Other selenoenzymes have not been identified, but effects on hepatic heine metabolism, hepatic drugmetabolizing activities, and plasma glutathione appear after hepatic glutathione peroxidase activity has fallen to very low levels. 9,1°This suggests that indices in addition to glutathione peroxidase activity could be useful in assessing severity of selenium deficiency states. Suggested guidelines for assessing selenium deficiency in the rat are shown in Table I.
Strategies for Producing Selenium Deficiency Feeding a selenium-deficient diet is the key for the production of selenium deficiency. Since animals can conserve their selenium stores when deprived of the element, it is customary to begin depletion in weanlings weighing about 50 g. Male rats are generally preferred because they have a higher selenium requirement than females. 8 D. G. H a f e m a n , R. A. Sunde, and W. G. Hoekstra, J. Nutr. 104, 580 (1974). 9 R. F. Burk, Annu. Rev. Nutr. 3, 53 (1983). ~0 R. Reiter and A. Wendel, Biochem. Pharmacol. 33, 1923 (1984).
To produce very severe selenium deficiency, investigators have fed low-selenium diets to female rats from weaning and then to their offspring. These "second generation" selenium-deficient rats grow very slowly, have sparse hair, and cannot reproduce. 11,12 Another approach to producing very severe deficiency is to use an amino acid diet. Attempts have been made to induce selenium deficiency by administering substances which interfere with selenium utilization. Silver can exacerbate the effects of selenium deficiency, 13 but this approach has not been generally adopted because of effects unrelated to selenium. The Selenium-Deficient Diet The selenium-deficient diet currently used in our laboratory (Table II) is often modified to optimize specific experiments. Therefore, a discussion of the major components of the diet is provided. Protein. Since the vast majority of selenium in feedstuffs is present in protein, this is the most critical ingredient of the selenium-deficient diet. Dried Toruhl yeast supplied by Rhinelander Paper Co. is the most reliable low-selenium protein source available. It contains 45-50% protein and is commonly used as 30-40% of the diet. The yeast protein is deficient in sulfur-containing amino acids, requiring the addition of methionine to obtain maximum growth rates. The selenium content of the yeast is specified to be less than 0.05 mg per kg. Assay of a recent batch received by us yielded a value of 0.015 mg selenium per kg.14 Assay of the complete diet, mixed as in Table II, yielded a value of 0.008 mg selenium per kg, i.e., the Torula yeast accounted for over half the selenium in the diet. Other protein sources have been used. Amino acid diets can produce more severe selenium deficiency than diets based on Torula yeast ~2,~5but are very expensive. Casein from low-selenium areas can be used 7 but careful monitoring of selenium content is necessary. Fat. Most investigators use corn oil or lard as the fat source with this diet. If a vitamin E-deficient diet is desired, vitamin E-stripped corn oil or lard (Eastman Organic Chemicals, Rochester, NY 14650) is used. Minerals. The mineral mix in Table II is suitable for use with Torula yeast, which contains approximately 130 mg zinc per kg. If the protein Ji K. E. M. McCoy and P. H. Weswig, J. Nutr. 98, 383 (1969). ~2 H. D. Hurt, E. E. Cary, and W. J. Visek, J. Nutr. 101, 761 (1971). ~3 R. P. Peterson and L. S. Jensen, Poult. Sci. 54, 795 (1975). ~4The analyses were performed by Dr. Ivan Palmer of South Dakota State University, Brookings, South Dakota. ~5 G. Siami, A. R. Schulert, and R. A. Neal, J. Nutr. 102, 857 (1972).
T A B L E II COMPOSITION OF SELENIUM-DEFICIENT DIET FOR RATS Component Torulu yeast" Sucrose Corn oil Mineral mix ~' Vitamin mix" DL- Methionine Vitamin E d
Percentage by weight 311 56.99 6.67 5 l 0.3 0.04
" Lake States Torula Dried Yeast, U.S.P. X1X, Type B, supplied by Lake States Division, Rhinelander Paper Co., Rhinelander, WI 54501. b Contains (in g per kg) CaCO3, 543; KH2PO4, 225.2; KCI, 104.8; NaCI, 59.69; MgCO3, 25; MgSO4, 16; ferric ammonium citrate, 20.5; MnSOa-H20, 3.44; NaF, I; CuSO4, (I.9; CrCI3 - 6H30, 0. I ; KI, 0.08. ' Contains (per 100 g) sucrose, 88 g; choline chloride, 10 g; niacin, 1 g; calcium D-pantothenate, 200 rag; thiamin. HCI, 40 mg; riboflavin, 25 mg; pyridoxine. HCI, 20 mg; menadione sodium bisulfite, 20 rag; Colic acid, 20 mg; biotin, 10 mg; vitamin Be, 1 mg; retinyl palmitate, water dispersible dry form to provide 15,000 IU per kg diet; ergocalciferol to provide 240 IU per kg diet. ,t Dt.-~-Tocopherol powder to providc 100 IU per kg diet.
c o m e s from a different source, zinc should be added to the mineral mix. Other c o m p l e t e mineral mixes can be used, provided they do not contain selenium. Vitamins. The vitamin mix in Table II is one we have used for many years j6 with s o m e modifications. It omits vitamin E, which we add separately. Other vitamin mixes can be used. Many investigators find it convenient to purchase the A I N Vitamin Mixture 7617 from a commercial supplier; the formulation can be prepared without vitamin E. It does not contain choline, so that this nutrient must be added separately. ~6 R. F. Burk, R. Whitney, H. Frank, and W. N. Pearson, J. Nutr. 95, 420 (1968). ~7 AIN Committee on Standards for Nutritional Studies, J. Nutr. 110, 1726 (1980).
Vitamin E. This diet is often used to produce vitamin E deficiency, either alone or combined with selenium deficiency. To do so, the fat source must be stripped of vitamin E and no exogenous vitamin E is provided. Selenium. The control diet is the selenium-deficient diet with selenium added. The nutritional requirement of the rat for selenium is satisfied by 0.1 mg selenium per kg of diet, or less, for most commonly used forms of the element. Inorganic selenium compounds such as selenate or selenite are generally added to control diets to give a final concentration of 0.1-0.5 mg selenium per kg. We prefer to use selenate because of evidence that dietary selenite causes lipid peroxidation. ~8,J9The selenium concentration we use is 0.5 mg per kg diet. Na2SeO4 is desiccated and 358.4 mg is added to 2 kg of sucrose in ajar. The jar is sealed and its contents are thoroughly mixed, preferably by the use of a rolling mill for several hours. This s e l e n i u m - s u c r o s e mixture is added as 0.67% of the diet (40 g per 6 kg batch) at the expense of sucrose. Organic forms of selenium such as selenomethionine are occasionally used but are subject to dietary influences which can confound interpretation of experimental r e s u l t s ) ° Mixing. Investigators who mix their own diets can alter them to optimize experimental design and can directly control the quality of the diet. in addition, cost is less than when diets are purchased from commercial suppliers. Torula yeast is a fine powder which makes a dust cover necessary for the mixer. We use a commercial mixer with a 20-quart capacity and make 6-kg batches of diet. All the ingredients except the corn oil are added into the mixing bowl and blended slowly for 1-2 rain. Then, the corn oil is added without stopping the mixer and mixing is continued for an additional 5 min. Overmixing can lead to destruction of vitamins. It is our practice to mix selenium-deficient diets first and to remove them from the mixing room before using any selenium-sucrose. The diet is stored at 4 ° and can be kept for a month unless it is vitamin E-deficient. Vitamin E-deficient diets must be used within 1-2 weeks. Animal Care and Feeding The diet is the only substantial source of selenium to the animal. Water and air generally contain very small amounts of the element. It is our practice to supply tap water for drinking. Since, however, water has been found to have a significant selenium content in a few locations, it ~8 A. S. Csallany, L.-C. Su, and B. Z. Menken, J. Nutr. 114, 1582 (1984). 19 R. F. Burk and J. M. Lane, Toxicol. Appl. Pharmacol. 50, 467 (1979). 2o R. A. Sunde, G. E. Gutzke, and W. G. Hoekstra, J. Nutr. 111, 76 (1981).
T A B I . E 111 DEVELOPMENT OF SELENIUM DEFICIENCY IN RATS FED THE SELENIUM-DEFICIENT DI F,T"
Percentage of control Live, glutathione
Weeks on diet
17 6 2 1 1 1
99 91 114 133 131 131
2 3 4 5 6
" Diet described in T a b l e 11. V a l u e s are derived from means of three male S p r a g u e - D a w l e y rats per diet at each time point. The rats were w e a n l i n g s at lhe start of the e x p e r i ment. ;' A s s a y s p e r l o r m e d on 105,000 ,k' s u p e r n a t a n l fluid. T h e subs t r a t e used tbr the glutalhione p c r o x i d a s e a s s a y w a s H3(), 22 and that for the glutathione S - t r a n s f e r a s e a s s a y w a s Ic h l o r o - 2 , 4 - d i n i t r o b e n z e n e . -'~
should be tested before routine use. This is conveniently done by comparing liver glutathione peroxidase activity in rats fed a selenium-deficient diet and given tap water with that of similar rats given distilled water. When attempts are being made to produce very severe selenium deficiency, the use of highly purified water is advisable. Many nutritional deficiencies decrease food intake, requiring pairfeeding for their study. Selenium deficiency causes a mild decrease in food intake 21 except in second generation animals in which the decrease is severe. Because of this, and the long period over which the diets must be fed, pair feeding is seldom used in studies of selenium deficiency. It should be used, however, when very severe deficiency is produced as in second generation selenium-deficient animals. If possible, selenium-deficient and control rats should be housed in separate cage racks. We house animals in wire-bottom cages. When the animals are fed, the selenium-deficient diet should be fed first; the supply should then be returned to the refrigerator before the control diet is opened. This prevents contamination of the deficient diet. 3~ R. C. E w a n , J. Nulr. 106, 702 (1976).
INTRACELLULAR DELIVERY OF CYSTEINE
Development of Selenium Deficiency Selenium deficiency is readily produced with the diet in Table II. Table III shows 22,z3 results from a recent experiment in our laboratory in which this diet was used. 24 Liver glutathione peroxidase activity had fallen to 2% of control by 3 weeks, and a rise in glutathione S-transferase activity followed, indicating progression of the deficiency to the severe category (Table I). Acknowledgments The author is grateful to Mr. J a m e s M. Lane for help in developing the diet and procedures described here, and to Mrs. R e b e c c a E. Ortiz for typing the manuscript. This work has been supported by the National Institutes of Health, Grant ES 02497. 22 R. A. L a w r e n c e and R. F. Burk, Biochem. Biophys. Res. Commun. 71, 952 (1976). 23 W. H. Habig, M. J. Pabst, and W. B. Jakoby, J. Biol. Chem. 249, 7130 (1974). _,4 K. E. Hill, R. F. Burk, and J. M, Lane, J. Nutr. 117 (1987).
 I n t r a c e l l u l a r D e l i v e r y of C y s t e i n e By M A R Y
E. ANDERSON and ALTON MEISTER
Introduction Intracellular cysteine is required for the synthesis of proteins, glutathione, and a number of other metabolites that are considered in this volume. Animals obtain cysteine from dietary peptide-bound cyst(e)ine and methionine. These amino acids are transported across the intestinal mucosa, both as the corresponding free amino acids and in the form of peptides. ~ Cysteine and cystine, which are present in the blood plasma, are transported into cells; the relative rates of uptake of cysteine and cystine vary in different cells. Conversion of methionine sulfur to cysteine sulfur by the cystathionine pathway constitutes a significant source of cysteine; this pathway functions chiefly in the liver but apparently also to some extent in other cells. Under normal conditions, these processes suffice to supply the amounts of cysteine moieties needed for protein synthesis and other metabolic purposes. I Pcptide Transport and Hydrolysis, Ciba Found. Syrup. [N.S.] 50 (1977}.
METHODS IN ENZYMOLOGY,VOL. 143
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