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Addendum to Chapter 9
Experimental Conditions for Nutritional Studies with Arsenic

EXPERIMENTS designed to induce deficiencies of trace elements in animals with very low requirements depend on strict control of contamination from diet, water, air, and caging (Smith and Schwarz 1967). Proteins and salts furnishing macrominerals are the major sources of dietary contaminants. Careful selection, backed by analysis, is routinely applied to the ingredients; the latter might have to be further purified by recrystallization or other means. Drinking water is of the highest attainable purity and is constantly monitored with resistance measurements. Animals are housed in plastic cages that are often isolated in laminar-flow hoods. These conditions of an "ultraclean environment" make it necessary to supply all essential micronutrients as supplements in an acceptable balance. Such complex diets have their own problems of contamination. Contamination from protein sources can be avoided by acid washing, by substituting chemically pure amino acid mixtures or by using ruminant animal species that can synthesize much of their protein needs from chemically pure urea.

Rats and Chicks

The animals are raised in plastic cages. In the earlier experiments, the cages were kept in laminar-flow hoods, but the extra protection was later found to be unnecessary when the arsenic content of the diets was consistently less than 20 ng/g. All ingredients are of the highest obtainable purity, but some sources of minerals, especially of potassium, calcium, and phosphate, require additional purification. The composition of representative diets, fed ad libitum, is given in Table A9-1.



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Page 302 Addendum to Chapter 9 Experimental Conditions for Nutritional Studies with Arsenic EXPERIMENTS designed to induce deficiencies of trace elements in animals with very low requirements depend on strict control of contamination from diet, water, air, and caging (Smith and Schwarz 1967). Proteins and salts furnishing macrominerals are the major sources of dietary contaminants. Careful selection, backed by analysis, is routinely applied to the ingredients; the latter might have to be further purified by recrystallization or other means. Drinking water is of the highest attainable purity and is constantly monitored with resistance measurements. Animals are housed in plastic cages that are often isolated in laminar-flow hoods. These conditions of an "ultraclean environment" make it necessary to supply all essential micronutrients as supplements in an acceptable balance. Such complex diets have their own problems of contamination. Contamination from protein sources can be avoided by acid washing, by substituting chemically pure amino acid mixtures or by using ruminant animal species that can synthesize much of their protein needs from chemically pure urea. Rats and Chicks The animals are raised in plastic cages. In the earlier experiments, the cages were kept in laminar-flow hoods, but the extra protection was later found to be unnecessary when the arsenic content of the diets was consistently less than 20 ng/g. All ingredients are of the highest obtainable purity, but some sources of minerals, especially of potassium, calcium, and phosphate, require additional purification. The composition of representative diets, fed ad libitum, is given in Table A9-1.

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Page 303 TABLE A9-1 Composition of Representative Diets for Rats and Chicksa Ingredient Rat Diet (g/kg) Chick Diet (g/kg) Milk powder   250 Casein, high protein 200 150 Ground corn, acid washed 673 230 Non-nutritive fiber   50 Dextrose, anhydrous   142.5 Corn oil   100 Soybean oil 70   CaCo3 15 14 KH2PO4 10 4 Choline bitartrate 2.5   A vitamin mix in cornstarch and a mineral mix in dextrose was added to the rat diet to furnish all known miconutrient requirements. The chick diet was slightly different to meet requirements of that species. Vitamins added to diet: (mg/kg): Niacin (30); d-pantothenic acid calcium (16); pyridoxin HCI (7); thiamin HCI (6); riboflavin (6); folic acid (2); d-biotin (0.2); vitamin B12(0.02); d,l-a-tocopherol acetate (120); retinyl palmitate (16); vitamin D3 (2.5); vitamin K, (0.75). Trace elements added to diet: (mg/kg): CaHPO4 (1,265); K2SO4 (1,632); CaCI2.2H2O (1,000); Na2CO3 (1,500); Mg(C2H3O2)2.4H2O (3,600); NaSiO3.9H2O (254); ferric citrate (220); zinc carbonate (58); manganese carbonate (22); copper acetate (11); KIO3 (0.35); Na2SeO4 (0.36 mg/kg); NH4MO7O24.4H2O (0.37); CrK(SO4)2.12 H2O (9.6); H3BO3 (2.9); NaF (2.2); NiCO3 (2.2); SnO (0.25); NH4VO3 (0.46). aData from Nielsen et al. 1978; Nielsen 1980; Uthus et al. 1983; Uthus 1992. Goats and Minipigs The animals are kept in plastic-lined wooden cages with cellulose as bedding material. An estimated 100 g per day of cellulose (arsenic at less than 15 ng/g) is eaten by goats. Cellulose contributes to their energy intake and dilutes the arsenic content from less than 35 ng/g in the semisynthetic diet to less than 20 ng/g in the total. Table A9-2 gives the composition of the rations fed ad libitum to both species.

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Page 304 TABLE A9-2 Composition of Representative Diets for Goats and Minipigsa Ingredient Goat Diet (g/kg) Minipig Diet (g/kg) Potato starch 483 570 Beet sugar 320 147 Casein 100 200 Urea 30   Sunflower oil 30 20 CaCO3 10 10 KH2PO4 13.4 22 Micronutrients added to goat diet: NaCI (3.5 g); A12(SO4)3 (2 g); MgO (2.2 g); K2SO4(3.5 g); ZnSO4.7H2O(0.5g); FeSO4.7H2O(0.45 g); MnSO4.7H2O(0.4 g); S (0.35 g); LiCO3 (106 mg); CuSO4.5H2O(40 mg); KBr (30 mg); Ni2SO4.7H2O (34 mg); Cr2(SO4)3.18H2O(6 mg); (NH4)VO3 (4.6 mg); NaF (2.2 mg); NaWO4.H2O(1.8 mg); KJ(1.0 mg); (NH4)6Mo7O24.4H2O (0.92 mg); SeO2 (0.8 mg); CoSO4.7H2O(0.8 mg); CdC12.H2O (0.36 mg); vitamin A (20 mg); vitamin D3 (4 mg); vitamin E (200 mg). Micronutrients added to minipig diet: MgSO4.7H2O(5.0 g); NaCI (5.2 g); K2SO4(12.4 g); FeSO4.7H2O(0.5 g); MnSO4.4H2O(0.25 g); ZnSO4.7H2O(50 mg); CuSO4.5H2O(40 mg); Ni2SO4.7H2O (48 mg); NaHSO3 (1.6 mg); Ni2SO.7H2O (34 mg); Cr2(SO4)3.18H2O (6 mg); KI (0.6 mg); CoSO4.7H2O(0.5 mg); NaWO4.2H2O(1.8 mg); NaF (2.2 mg); KBr (30 mg); (NH4)Mo7O24.4H2O(0.92 mg); choline (2 g); vitamin C (80 mg); vitamin E (100 mg); Ca pantothenate (80 mg); Niacin (80 mg); vitamin E (20 mg); vitamin D (40 mg); vitamin B1 (4 mg); vitamin B2 (6 mg); vitamin B6 (4 mg); vitamin B12(0.05 mg); folic acid (2 mg); ethoxyquinolin (200 mg). aData from Anke 1986, 1991; Anke et al. 1976. References Anke, M. 1986. Arsenic. Pp. 347-372 in Trace Elements in Human and Animal Nutrition, Vol. 2, 5th Ed., W. Mertz, ed. Orlando, Fla.: Academic. Anke, M. 1991. The essentiality of ultra trace elements for reproduction and pre-and postnatal development. Pp. 119-144 in Trace Elements in Nutrition of Children—II, R.K. Chandra, ed. New York: Raven. Anke, M., M. Grün, and M. Partschefeld. 1976. The essentiality of arsenic for animals.  Pp. 403-409 in Trace Substances in Environmental Health—X, Proceedings of the University of Missouri's Tenth Annual Conference on Trace Substances in Environmental Health, D.D. Hemphill, ed. Columbia, Mo.: University of Missouri Press. Nielsen, F.H. 1980. Evidence of the essentiality of arsenic, nickel, and vanadium and their possible nutritional significance. Pp. 157-172 in Advances in Nutritional Research, Vol. 9, H.H. Draper, ed. New York: Plenum.

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Page 305 Nielsen, F.H., D.R. Myron, and E.O. Uthus. 1978. Newer trace elements—Vanadium (V) and arsenic (As) deficiency signs and possible metabolic roles. Pp. 244-247 in Trace Element Metabolism in Man and Animals, Vol. 3, M. Kirchgessner, ed. Freising-Weihenstephan, Germany: Technische Universitat Munchen Uthus, E.O. 1992. Evidence for arsenic essentiality. Environ. Geochem. Health 14:55-58. Uthus, E.O., W.E. Cornatzer, and F.H. Nielsen. 1983. Consequences of arsenic deprivation in laboratory animals. Pp. 173-189 in Arsenic: Industrial, Biomedical, Environmental Perspectives, W.H. Lederer and R.J. Fensterheim, eds. New York: Van Nostrand Reinhold. Smith, J.C., and K. Schwarz. 1967. A controlled environment system for new trace element deficiencies. J. Nutr. 93:182-188.

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