Partition of porphyrins between cyclohexanone and aqueous sodium acetate as a function of pH. Determination of uroporphyrin and of hydrophilic porphyrin conjugates

1. The partition of uroporphyrins I and III, coproporphyrins I and III, haematoporphyrin IX, porphyrin c and a hydrophilic porphyrin–peptide fraction from variegate-porphyria faeces has been studied in systems of equal volumes of cyclohexanone and sodium acetate buffers of varying pH and concentration. 2. The concentration of acetate in the aqueous phase has little effect on the partition of porphyrin c, but markedly influences that of uroporphyrin. At 50% acetate saturation and pH4·5, only 5% enters the cyclohexanone phase whereas 60% of porphyrin c is extracted under similar conditions. 3. This circumstance forms the basis of a method for the determination of hydrophilic porphyrin–peptides in variegate-porphyria urine. Its reliability has been checked in model experiments. 4. At pH1·5 and an aqueous phase half-saturated with sodium acetate, an equal volume of cyclohexanone removes 95–97% of uroporphyrin and about 55% of porphyrin c. Uroporphyrin may therefore be determined as a second step in the method. 5. For the routine determination of uroporphyrin in systems free from other hydrophilic porphyrins, cyclohexanone extraction may be performed at any pH in the range 1·0–3·0.