I. Quantitative Measurement of Coproporphyrin and Total Goproporphyrin I Excretion in Normals

Abstract

Coproporphyrin I is excreted in the urine and feces under normal and most pathological circumstances. 1 , 2 , 3 In order to determine the relations between normal and abnormal pigment construction and destruction it has been necessary to develop an adequate method for the exact quantitative separation and measurement of porphyrins. Urine and feces were collected for one- to 3-day periods and kept in the dark. After careful mixing aliquot portions were analyzed. Not less than 50 gamma of coproporphyrin should be available for measurement; normally this amount is present in about half the daily amount of urine and about one-quarter of the daily feces. The remaining urine and feces were extracted for determination of melting points.

The reaction of the aliquot was made negative to Congo red by 10% sodium acetate. One-third of the volume of glacial acetic acid was added and the mixture shaken 3 times with 3 volumes of ether. The combined ether extracts were washed twice with dilute sodium acetate solution and the total porphyrin extracted quantitatively with 5% HCl. With sodium acetate and acetic acid the porphyrin was driven again into ether and extracted repeatedly and quantitatively from the ether after washing with dilute sodium acetate by equal volumes of 0.5% HCl. The 0.5% HCl was diluted to 0.2%, shaken repeatedly with alcohol-free chloroform until the chloroform was colorless. In the 0.2% solution only coproporphyrin is present. This is driven quantitatively into ether by means of sodium acetate and acetic acid. The ether was washed twice, extracted 3 times with small quantities of 5% HCl. These extracts were combined and brought to volume. A volume was chosen which gave a concentration of porphyrin about the same as the standard solution.