Preparation of crystalline porphyrin esters from bovine porphyria urine

Abstract

Urine from a porphyric steer was collected and acidified with HC1 to a pH of 2. The porphyrins were adsorbed on talc or crude Kieselguhr and extracted by suspension in N HCl-acetone (1:9, v/v). The combined HCl-acetone eluates were precipitated by the addition of concentrated aqueous NH3 solution to about pH 8.5. The dried precipitate was pulverized and treated overnight with . H2SO4-methanol (5:95, v/v). After standing overnight the residue was stirred and extracted on a Buchner funnel with CHC13. The pooled methanol-CHCl3 extracts were neutralized with saturated Na acetate and sufficient water was added to bring about separation. The aqueous phase was extracted with CHC13 and the pooled extracts were washed with water. The CHC13 extract was dried with CaCl2 and the CHC13 removed at room temperature. Chromatographic purification was performed on 2-3 cm columns of MgO. The porphyrin esters were added to the column in pharmacopoeia quality CHC13, which was also used for development. The ethanol present in the CHC13 is of importance in this procedure. Chromatographic fractionation on 7 x 2 cm columns of Al2O3 was performed using benzene: CHC13 mixtures 9:1, 7:3, 5:5, 3:7, v/v, pure CHC13 and finally absolute methanol in CHC13. The purified ester solutions were evaporated to dryness at room temperature. The dry ester was dissolved in as little CHC13 as possible and then the uroporphyrins were crystallized by adding 3-4 volumes of absolute methanol. More rapid and complete crystallization of coproporphyrin was obtained by using light petroleum (BP below 50[degree] C) instead of methanol. About 10% of the porphyrin present in the urine is recovered as crystalline esters. Besides uro-and coproporphyrin 1, a 6-carboxyl porphyrin was constantly present in minor quantities in bovine porphyrin urine.