Pyrimidine 5′-nucleotidase (P5′N) partial deficiency has been described in several hematological disorders and also in the β-thalassemic trait. To check if the P5’N deficiency in thalassemia was acquired we used thalassemic red cells (from either homo- or heterozygous subjects), whose P5′N activity was lower than in control cells. After separation on a density gradient, activity in lighter cells was similar to controls and less than 50% in denser cells. The most probable mechanism for this faster inactivation involves enzyme -SH groups modification by oxidation and reaction with monofunctional aldehydes produced by membrane lipid peroxidation. In vitro challenge of thiol enzymes as pyruvate kinase (PK), adenylate kinase (AK) and P5′N with increasing concentrations of GSSG, hexanal (HEX) and 4-hydroxynonenal (HNE), showed that HNE is the most powerful among the enzyme inhibitors tested and that P5′N activity is a more sensitive index of -SH groups damage, when compared to PK and AK.

Recent reports have suggested that haemolytic anaemia in pyrimidine 5′ nucleotidase (P5′N) deficiency might be due to impaired erythrocyte hexose monophosphate shunt (HMS). To investigate the relationship between pyrimidine accumulation, HMS impairment and shortened red‐cell survival, we tested glucose 6‐phosphate deydrogenase (G‐6PD), HMS, P5′N activities and the UV spectrum in whole red cells and in red cells of different age from 2 P5′N‐deficient patients with different degrees of haemolytic anaemia. In whole red cells we found a reduction of both GdPD and stimulated HMS activity in the presence of a variable amount of pyrimidine nucleotides (37.79 and 17.88 pmol/gHb respectively). A drastic inhibition of stimulated HMS activity was already present in the lightest red‐cell fractions from patient 1, who presented a more severe haemolytic anaemia. The variable degree of pyrimidines found among red cell fractions, with a minor accumulation in the older red cells, supports the hypothesis that pyrimidine accumulation and HMS impairment occur in the younger erythrocytes of P5′N‐deficient patients.