5.4 Erythroenzymopathies

Erythroenzymopathies are hereditary diseases due to a defect of RBC metabolism, in general an enzyme deficiency, that can be associated with an haemolytic crisis, a chronic haemolytic anaemia (HHA), neonatal cyanosis with metahaemoglobinemia or c) hereditary erythrocytosis. An association between enzymatic defect and HHA has been described in 14 of the 38 enzymes that make up the erythrocyte metabolism (Table 4). Some enzymatic defects produce haemolysis only under cellular stress produced by infections, oxidizing drugs or by the intake of fava beans. Other enzymatic deficiencies are associated with chronic nonspherocytic haemolytic anaemia (CNSHA). On some occasions, the expression of the deficiency is not restricted to the erythrocyte, but extends to other tissues, mainly the neurological, hepatic or muscular tissues, and a neuropathy, hepatopathy or myopathy, respectively, associated with anaemia appears. The RBC is especially sensible to enzymopathies because, unlike other cells, it cannot resynthesize the deficient enzymes because it lacks a nucleus and ribosomes. Enzymatic deficiencies occur when one of the enzymes of the reduced RBC metabolism is unstable and disappears very quickly, or has lost catalytic functionality and lacks activity. In the mature RBC there are two fundamental metabolic pathways: (1) the anaerobic glycolysis, by which glucose is used to generate ATP and (2) the aerobic pentose phosphate pathway (PPP), by which it eliminates any oxidative aggression to generate NADH and NADPH (Figure 10). ATP is used to meet energy requirements and NADH to reduce methaemoglobin. The NADPH is used to reduce the oxidized glutathione (GSH) which, in turn, is required to maintain the sulfhydryl groups of proteins in a reduced state and to detoxify hydrogen peroxide


#### Table 4.

Erythroenzymopathies of the glycolytic pathway.

(Figure 11). Enzymopathies have been described in both metabolic pathways. Glucose-6-phosphate dehydrogenase (G6PD, OMIM 300908, 134,700) is the most frequent antioxidant system, and shows hereditary transmission linked to sex. It is especially frequent in Africa, Asia and in the Mediterranean region (Greece and Italy, mainly) and, due to its polymorphic nature, it has many variants, among which the G6PD A-, predominant in black people, and the G6PD () Mediterranean, predominant in Caucasians. In G6PD A-forms, the enzyme is unstable but enzymatic activity is almost normal in reticulocytes, whereas in Mediterranean G6PD variants, the enzyme is even more unstable, and its activity is very low even in reticulocytes. This explains that in G6PD A-carriers, the acute haemolytic episode is self-limited and the recovery of the anaemia is faster than in the carriers of Mediterranean G6PD variants. Clinically, the G6PD deficiency occurs with haemolytic crisis sometimes associated with severe anaemia, in general triggered by the intake of oxidant substances, like fava beans (favism), or certain oxidizing drugs. Due to this, the carriers of a G6PD deficiency can remain asymptomatic for many years, until a contact occurs with the substances that may trigger the haemolytic crisis. Among the drugs that can induce haemolysis in G6PD deficiency, can be mentioned certain analgesics, sulphonamides, antimalarials, and antibiotics [31]. Favism, a severe haemolytic anaemia induced by fava beans ingestion or exposure to pollen from the plant, is the most frequent clinical manifestation in caucasians bearing the deficient G6PD Mediterranean variant, but also the G6PD A () variant (G66PD Betica). There are also ultrarare forms of G6PD deficiency that do not obey to polymorphic variants but to

#### Figure 10.

Anaerobic glycolysis and antioxidant metabolic pathways of red blood cells. Abbreviations: BPG, bisphosphoglyceric acid; DHAP, dihydroxyacetone phosphate; F6P, fructose 6-phosphate; FDP, fructose-1,6 diphosphate; G3P, glycerol 3-phosphate; G3PD, glyceraldehyde 3-phosphate dehydrogenase; G6P, glucose 6 phosphate; G6PD, glucose-6-phosphate dehydrogenase; GCS, glutamylcysteine synthetase; GPI, glucosephosphate isomerase; GS, glutathione synthetase; GSH, glutathione; GSSG, glutathione disulfide; HK, hexokinase; LD, lactate dehydrogenase; NADP, nicotinamide adenine dinucleotide phosphate; PEP, phosphoenolpyruvic acid; PFK, phosphofructokinase; PG, phosphoglyceric acid; PGK, phosphoglycerate kinase; PK, pyruvate kinase; Ru5P, ribose-5-phosphate isomerase.

#### Figure 11.

Anti-oxidant system present in RBC to protect the cells against the oxidant threat generated by the ingestion of oxidant drugs of fava beans (favism).

sporadic variants that present with a chronic haemolytic syndrome (CSSHA). Other factors that can induce haemolysis in the G6PD deficiency are viral infections, especially influenza and hepatitis, diabetic ketoacidosis, and other different situations called of "stress." The diagnosis of G6PD deficiency is based on the clinical history, and the exclusion of the autoimmune mechanism through the negativity of the direct antiglobulin test (DAGT) or Coombs test. During the haemolytic crisis, the observation of the smear shows the presence of eccentrocytes or RBCs subjected to oxidative stress where haemoglobin is pushed off to one part of the cytoplasm (Figure 12). For screening purposes, the fluorescent spot test is used based on demonstrating the formation of NADPH (fluorescent) from NADP (non-fluorescent) in the Beutler's G6PD fluorescence spot test or the reduction of methaemoglobin in the presence of methylene blue.

Figure 12. Excentrocytes present in the blood of a patient with G6PD deficiency and haemolytic crisis.

The pyruvate kinase deficiency (PKD, OMIM 266200) is the most frequent enzyme of anaerobic glycolysis (Embden-Meyerhof pathway) and the most frequent cause of hereditary haemolytic anaemia (HHA), after hereditary spherocytosis [32]. It is an enzymopathy much less frequent than G6PD deficiency and its diagnosis requires the quantification of enzymatic activity in patient's haemolysates. Since haemolytic anaemia in PK deficiency is always due to mutations in the PKLR gene, leukocytes have a normal PK activity, so if they are not eliminated well when preparing the haemolysate, they can geopardise the result and mask the existence of a RBC PK deficiency (PKD). To avoid this important cause of error (false negatives), blood treated with anticoagulant must be filtered through a column of celulose-micro celulose in order to eliminate leukocytes and platelets. Also the presence of a high number of circulating reticulocytes, very frequent in newborns and children, can also give false negatives because the PK activity of the reticulocytes is much higher than that of the mature RBCs. Therefore, if in the presence of intense reticulocytosis a normal PK activity is obtained, the result should be confirmed by determining the quotient between PK activity and hexokinase (HK). HK is an enzyme whose activity also increases in the presence of reticulocytosis and, therefore, if this increase is much greater than that of PK, this enzyme deficiency is evidenced by a significant decrease in the PK / HK ratio [33].

Very recently a concise guide to PK deficiency for primary care providers, but also for haematologists, healthcare providers and medical students has been published [34]. There, the underlying PKD defect is explained in great detail together with its mode of inheritance, clinical manifestations, diagnostic procedures and an attempt for medical treatment.
