Nutrition classics from The Journal of Biological Chemistry 144:135-138, 1942. A green pigment-producing compound in urine of pyridoxine-deficient rats.

While determining the bisulfite-binding substance of urine from pyridoxine-deficient rats and their paired pyridoxine-fed controls, E. J. Wickson, a student assistant, noticed that when slightly alkaline the urine of the pyridoxine-deficient rat, had a green color, making the iodometric titration in the determination of bisulfite-binding substances impossible. The green pigment was found only in the urine of rats deficient in pyridoxine. It was not excreted as such, but as a precursor which became green upon contact with the iron of the metabolism cages. This color could also be readily developed by the addition of ferric ammonium sulfate and other iron salts to the urine. The pigment was green only while the pH was near the neutral point. The green color disappeared upon the addition of either acid or alkali and reappeared upon neutralization. Administration of pyridoxine to the deficient rat caused the disappearance from its urine in 6 to 10 hours of most of the precursor of the green pigment, indicating a close relationship between it and pyridoxine deficiency. The green pigment was concentrated by chromatographic procedures with filter paper as the packing in the column. It acted as an indicator, being light yellow in strongly acid solution, green when near neutrality, and yellow in strongly alkaline solution. Qualitative tests of the compound indicated the presence of carbon, hydrogen, nitrogen, and iron. Care of Animals-The animals were fed the pyridoxine-deficient diet previously employed (1) with the following changes: Factor 2 concentrates were discontinued and in their place were used 200 y daily of Ca pantothenate’ (Merck) and a rice bran preparation to supply unknown filtrate factors. It was prepared by extracting a rice bran concentrate (Vitab rice bran concentrate,2 Nopco) with acetone. The acetone extract was concent,rated and the sugar was fermented out of the concentrate with yeast. The alcohol was removed by distillation in vacua, and the concentrate made strongly acid with H&304 (Congo red) and extracted continuously with ether for 1 week. The extracted concentrate was diluted