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An Antithyrotoxic Assay Based upon the Metabolic Rate Response^1,2,

Department of Biochemistry, State University of New York Upstate Medical Center, Syracuse, New York

(1) The antithyrotoxic activity of various dietary supplements was compared with a standard liver residue by an assay procedure in which weanling male rats were fed a purified 30% casein diet containing 0.1% of iodinated casein (1% thyroxine equivalent) with or without the test supplement for 19 to 28 days; metabolic rates were determined after 19 days and compared with the metabolic rate decreases produced by the inclusion of liver residue in the diet. The average metabolic rates which constituted the assay curve were 7.2 (liters of O₂ consumed per square meter body surface per hour) for the nonthyroidal basal group, 13.5 for the unprotected group fed 0.1% of iodinated casein, 10.2 when 5% of liver residue was included, and 8.2 for the 10% liver residue diet.

(2) Female rats were less satisfactory for the assay but responded to the test supplements like the males; the inhibition of the normal development of the ovaries and uterus produced by feeding iodinated casein was overcome by all those substances which effectively reduced the metabolic rate.

(3) A similar assay procedure in which a diet containing 0.8 mg/kg of triiodothyronine (T₃) was fed to weanling male rats for 16 to 28 days before determining the metabolic rate was less satisfactory than the use of iodinated casein because of the relatively high residual metabolic rate that remained in the presence of a large excess of the antithyrotoxic factor. In general, the same substances were active against both T₃ and iodinated casein, but the test with T₃ was more rigorous.

(4) The adrenal hypertrophy that resulted from the thyroid feeding was reduced by the various test substances to the same degree that these substances reduced the metabolic rate.

(5) The following substances were good sources of antithyrotoxic activity (equal to or better than 5% of liver residue; 50 units or more) by all the criteria studied: 10% of liver residue, 5% of liver residue fat 1% of liver residue concentrate, 5% of hemoglobin, 10% of cottonseed meal or fermentation residue, 20% of lactalbumin or fibrin, and 0.2% of deoxycholic acid, dehydrocholic acid or sodium glycocholate. Moderate activity (approximately 35 units) was exhibited in all tests by 20% of soy protein and in some tests by 10% of distiller's solubles, 10% of egg yolk, 0.2% of cholic acid or 10% of brewer's yeast. The 6-propyl-thiouracil also had a moderate-to-good effect in reducing the increased metabolic rate produced by iodinated casein or T₃, but had little or no effect on growth. The following substances had little or no antithyrotoxic activity by any criterion: 10% of dried grass, whey, wheat germ, casein, dry fish solubles or corn oil, 20% of egg albumin or 1% of cholesterol. Lithocholic and hyodeoxycholic acids were also inactive in the standard metabolic rate assay procedure, as were 10% of lard, 20% of gelatin and a variety of miscellaneous substances. Procaine penicillin gave erratic results, but along with other antibiotics was generally inactive.

(6) No substance tested gave a good growth response without also reducing the metabolic rate.

² Materials used in this study were obtained as follows: Protamone and Cerophyl: Cerophyl Laboratories, Kansas Ctiy, Missouri; triiodothyronine: Warner-Lambert, Morris Plains, New Jersey; casein (vitamin free), egg yolk, brewer's yeast, lactalbumin, fibrin, egg albumin, hemoglobin, methionine and AET: Nutritional Biochemicals Corporation, Cleveland; liver residue: Wilson and Company, Chicago; fermentation residue (Omafac): E. R. Squibb and Sons; fish solubles: Philip R. Park, Incorporated, San Pedro, California; distiller's solubles: Schenley Distillers, Louisville; whey: Western Condensing Company, Appleton, Wisconsin; soy protein (ADM C-1, Assay Protein): Archer-Daniels-Midland Company, Minneapolis; cottonseed meal: Eufaula Cotton Oil Company, Eufaula, Alabama; wheat gluten: General Biochemicals Inc., Chagrin Falls, Ohio; fish meal: Viobin, Monticello, Illinois; gelatin: Baker and Adamson, New York; bile acids: Steraloids, Queens, New York; Guanethidine; Ciba, Summit, New Jersey; thiouracila: Mann Laboratories, New York; dibenzyline: Smith, Kline and French, Philadelphia; reserpine: California Biochemicals, Los Angeles; dihydroergotamine: Sandoz, Hanover, New Jersey; antibiotics: Bristol Laboratories, Syracuse, New York.

Manuscript received 6 August 1962.