Determination of porphobilinogen deaminase activity in human erythrocytes:pertinent factors in obtaining optimal conditions for measurements

Determination of porphobilinogen deaminase (PBGD; EC 4.3.1.8) activity in e rythrocytes can contribute to the identification of patients suspected of a cute intermittent porphyria. PBGD catalyses the polymerization of four mole cules of porphobilinogen (PBG) to the highly unstable 1-hydroxymethylbilane . The 1-hydroxymethylbilane is transformed into uroporphyrinogen III by uro porphyrinogen III synthase. When this enzyme is inactivated, 1-hydroxymethy lbilane cyclizes non-enzymatically to uroporphyrinogen I, which can be oxid ized to uroporphyrin I. PBGD activity can be measured by quantitation of ur oporphyrin I formed from PEG under conditions where this is the only end pr oduct. The purpose of the present study was to define the optimal condition s for quantitating PBGD activity in human erythrocytes. The preanalytical f actors examined were: anticoagulants and methods for disruption of the eryt hrocytes. The analytical factors examined were: duration of preincubation, reaction time, reaction temperature, pH, ionic strength and conditions for the oxidation of uroporphyrinogen I to uroporphyrin I. Based on the results , we propose an optimized method for determination of PBGD activity in eryt hrocytes.