ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 10, No. 5 Copyright © 1980, Institute for Clinical Science, Inc. Clinical Consequences of Enzyme Deficiencies in the Erythrocyte MICHAEL L. NETZLOFF, M.D. Department of Pediatrics and Human Development, Michigan State University College of Human Medicine, East Lansing, Ml 48824 ABSTRACT The anueleate mature erythrocyte also lacks ribosomes and mitochondria and thus cannot synthesize enzymes or derive energy from the Krebs citric acid cycle. Nevertheless, the red blood cell is metabolically active and contains numerous residual enzymes and their products which are essential for its survival and normal functioning. Enzyme deficiencies in the Embden-Myerhoff glycolytic pathway can result in nonspherocytic hemoly­ tic anemia (NSHA), and some are also associated with neuromuscular or neurologic disorders. Glucose-6-phosphate dehydrogenase deficiency in the hexose monophosphate shunt also results in hemolytic anemia, espe­ cially following exposure to various drugs. Defects in glutathione synthesis and pyrimidine 5'-nucleotidase deficiency also cause NSHA, as does in­ creased adenosine deaminase activity. Glutathione synthetase deficiency which is not limited to the red cell also presents as oxoprolinuria with neurologic signs. All red cell enzyme defects appear as single gene errors, in most cases recessive in inheritance, either autosomal or X-linked. Clinical Consequences of Enzyme tains over 40 different enzymes, many of Deficiencies in the Erythrocyte which are essential for its survival or nor­ mal functioning. The purpose of this re­ Prior to maturity, the nucleated red port is to review some inherited enzyme blood cell (rbc) can perform a variety of deficiencies of the erythrocyte which metabolic functions, including active en­ compromise its viability in the circulation zyme synthesis. The mature erythrocyte or its capacity to transport oxygen, result­ has no nucleus or ribosomal apparatus, ing in clinical disease. and thus cannot synthesize protein. How­ ever, it is not metabolically inert and con- Red Blood Cell Metabolism Reprinted requests to: Michael L. Netzloff, M.D., In addition to the metabolic restrictions Department of Pediatrics and Human Development, B-240 Life Sciences, Michigan State University, on the mature erythrocyte previously East Lansing, MI 48824. noted, its energy sources are also some­ 414 0091-7370/80/0900-0414 $01.80 © Institute for Clinical Science, Inc. SIGNS OF RED CELL ENZYME DEFICIENCIES 415 what limited by the loss of its mitochon­ H M P may be used in the synthesis of nuc­ dria. Without these organelles, the rbc leotides or rearranged to the 3-and cannot metabolize pyruvate through the 6-carbon sugars used in glycolysis for Krebs citric acid cycle. Other energy sub­ production of additional 2,3-DPG and strates can be used by the red cell, but the ATP. major source is normally glucose through Some common hereditary diseases of either the Embden-Meyerhoff or hexose the rbc which involve alterations in shape monophosphate pathways. The energy (e.g., spherocytosis, elliptocytosis, etc.) thus derived has several essential uses:3,24 are likely membrane defects and may in­ (1) the m aintenance of sodium and potas­ volve enzyme deficiencies not yet iden­ sium gradients across the cell membrane; tified.24 This review will be limited to (2) the prevention of calcium accumula­ known enzyme deficiency states, many of tion in the red cell membrane; (3) mainte­ which cause nonspherocytic hemolytic nance of the integrity and shape of the rbc anemia. m em brane; (4) reduction of m ethem oglo- bin and oxidized gluthathione; (5) inacti­ Red Blood Cells Enzyme Deficiencies vation of peroxides and other oxidants in the Embden-Meyerhoff Pathway which denature hemoglobin; and (6) P y r u v a t e K i n a s e D e f i c i e n c y maintenance of levels of organic phos­ phates, e.g., 2,3 diphosphoglycerate Although the recognition of non­ (2,3-DPG) and adenosine triphosphate spherocytic hemolytic anemia (NSHA) (ATP), affecting hemoglobin-oxygen was made as recently as 1953, it was not affinity. until 1961 that Valentine et al32 discov­ The energy derived from the ered pyruvate kinase (PK) deficiency, the Embden-Meyerhoff glycolytic pathway is most common cause of NSHA. PK defi­ primarily stored in the high energy phos­ ciency is second to glucose-6-phosphate phate of ATP. The latter is used to drive dehydrogenase (G-6-PD) deficiency as the sodium and potassium pumping sys­ the most common inherited rbc enzyme tems which oppose the passive diffusion defect and is an autosomal recessive dis­ of these ions and water across the cell order. Although it has been suggested that membrane. If unopposed, these natural heterozygotes may infrequently have processes would result in eventual clinical findings, most of these examples hemolysis. Anaerobic glycolysis also gen­ are confounded by the simultaneous oc­ erates reducing power by converting currence of other hemolytic disorders.3 nicotinamide adenine dinucleotide Large numbers of heterozygotes have (NAD+) to NADH, which reduces been found to have no hemolytic disease, methemoglobin to hemoglobin. Synthesis and it is doubtful that the heterozygous of 2,3-DPG also occurs in the glycolytic condition itself causes hemolysis.3 pathway. Considerable heterogeneity in bio­ Normally, the hexose monophosphate chemical properties of mutant PK enzymes pathway (HMP) accounts for only 10 per­ has been observed and may explain cent of the glucose metabolism of the red the marked variation in hemolysis and cell.3 This percentage is greatly increased anemia in affected patients. Disease oc­ during oxidative stress with the produc­ curs in true homozygotes who have inher­ tion of NADP+ from NADPH. The HMP ited two identical mutant alleles, as well maintains this conversion in favor of as in double heterozygotes, or genetic NADPH, which keeps gluthathione in its compounds, with two different mutant al­ reduced form, in turn protecting the rbc leles affecting different parts of the PK from peroxidation. Pentose formed in the enzyme. The latter condition could give 416 NETZLOFF rise to two different abnormal amino acid Earlier studies and those recently re­ substitutions with the formation of two ported by Bowman and Oski5 have variant enzymes or their hybrid enzyme. suggested that the spleen is the first organ Since PK is known to be an allosteric en­ to trap and destroy susceptible PK defi­ zyme affected by fructose diphosphate cient red cells, principally reticulocytes. with marked pH dependency, a mutant These observations help to explain the re- allele might result in enzyme products ticulocytosis which occurs in all red cell with modifications of either the catalytic PK-deficient patients after splenectomy. or the allosteric site. Gherardi et al11 has When the spleen is present, its low oxy­ recently reported data on a family suggest­ gen tensions prevent mitochondrial ATP ing an alteration of the PK allosteric site. production in reticulocytes both in normal In addition to the hereditary PK defi­ and PK-deficient patients. The later ciency, the enzyme defect can be caused would also suffer from impaired by a wide variety of hematologic disor­ glycolysis, with further reduction of avail­ ders.3 These include, most commonly, able ATP. The ATP-dependent cation acute leukemia, as well as cytopenias, pump appears defective in red cells from anemias, and aplasias. The acquired vs. such patients, and this might affect the hereditary forms of PK deficiency can be external calcium pump and cause inter­ differentiated by observing the return of membrane calcium concentration, induc­ enzym e activity to norm al once the u nder­ ing membrane ridigity. This sequence lying disorder is treated, or by family could be the pathogenesis of the abnormal studies. The latter rely on the facts that cell morphology21 and deformability22 parents of patients with hereditary PK de­ reported in erythrocyte populations in ficiency are obligate heterozygotes with PK-deficiency anemia following diminished enzyme levels intermediate splenectomy. between normal and affected, and sib­ Because of the biochemical hetero­ lings of patients with hereditary PK defi­ geneity of PK-deficiency, variability of ciency may also have demonstrable PK clinical expression is expected.3 Anemia disorders. may be so severe that multiple transfu­ Since some investigators have reported sions or splenectomy is life-saving, or the no relationship between in vitro proper­ patient may remain asymptomatic for ties of variant PK enzymes and the sever­ years. Other signs are also not distinctive ity of hemolysis, the hypothesis has been for PK-deficiency disease. Patients may advanced that hemolysis in PK deficient have jaundice, bilirubinuria, splenome­ red cells is caused by another defect in the galy, and sometimes gallstones. Jaundice cell membrane.36 However, interpretation usually presents in the neonatal period of biochemical results is hampered by and kernicterus has been reported and can several factors. First, inheritance of two be fatal. Hemolysis increases during in­ different mutant alleles allows formation tercurrent infection and following use of of five different types of the tetramer en­ oral contraceptives.18 PK-deficiency is a zyme. Also, studies utilizing crude cause of failure to thrive in severely af­ hemolysates are hampered by the actions fected children. Radiographs show the of other enzymes, and purification of PK changes secondary to a hypertrophic bone itself can change