Hereditary Hemolytic Anemia with Human Erythrocyte Pyrimidine 5′-Nucleotidase Deficiency
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Hereditary Hemolytic Anemia with Human Erythrocyte Pyrimidine 5′-Nucleotidase Deficiency William N. Valentine, … , Susan R. Harris, William S. Adams J Clin Invest. 1974;54(4):866-879. https://doi.org/10.1172/JCI107826. Research Article A severe deficiency of a red cell pyrimidine 5′-nucleotidase was found to be associated with hereditary hemolytic anemia in four members of three kindreds. The syndrome was characterized by marked increases above normal in red cell basophilic stippling, total nucleotides, and GSH and by a fairly severe deficiency of ribosephosphate pyrophosphokinase (EC 2.7.6.1.). Patient erythrocytes uniquely contained large amounts of pyrimidine 5′-ribonucleotides. In earlier studies, these were erroneously considered to be adenosine phosphates, since all previous investigations of the nucleotides of human red cells and reticulocytes have shown 97% or more to contain adenine. Total nucleotides in patient cells were present in amounts 3-6 times greater than normal, and approximately 80% contained pyrimidine. The ultraviolet spectral curves of deproteinized red cell extracts exhibited a shift in maximum absorbance from the usual 256-257 nm to approximately 266-270 nm, and absorbance at 250, 270, 280, and 290 nm, expressed as a ratio of that at 260 nm, differed greatly from normal. The spectral characteristics of extracts provide the basis of a readily performed screening procedure, which does not require enzyme assay. The nucleotidase activity in deficient red cells assayed less than 14%, and usually less than 10%, of normal and much less in terms of reticulocyte-rich blood, where it was consistently found to be increased. The enzyme has a pH optimum of […] Find the latest version: https://jci.me/107826/pdf Hereditary Hemolytic Anemia with Human Erythrocyte Pyrimidine 5'-Nucleotidase Deficiency WILLIAM N. VALENTINE, KAY FINK, DONALD E. PAGLIA, SusAN R. HARms, and WILLIAM S. ADAMS From the Departments of Medicine and Pathology, University of California, Center for the Health Sciences, the Wadsworth General Hospital, Veterans Administration Center, Los Angeles, California 90024 and from the Department of Medicine, Albert Einstein College of Medicine, Bronx Municipal Hospital Center, Bronx, New York 10461 A B S T R A C T A severe deficiency of a red cell pyrimi- mode of transmission of the deficiency. The pyrimidine dine 5'-nucleotidase was found to be associated with 5'-ribonucleotides are presumably derived from RNA hereditary hemolytic anemia in four members of three degradation and, not being diffusible, accumulate when kindreds. The syndrome was characterized by marked the enzyme catalyzing their dephosphorylation is defi- increases above normal in red cell basophilic stippling, cient. It is postulated that the prominent basophilic total nucleotides, and GSH and by a fairly severe defi- stippling results from retarded ribosomal RNA degra- ciency of ribosephosphate pyrophosphokinase (EC 2.7. dation secondary to accumulation of degradation prod- 6.1.). Patient erythrocytes uniquely contained large ucts, namely pyrimidine 5'-ribonucleotides. Ribose- amounts of pyrimidine 5'-ribonucleotides. In earlier phosphate pyrophosphokinase deficiency is considered studies, these were erroneously considered to be adeno- to be an epiphenomenon. The mechanism responsible sine phosphates, since all previous investigations of the for increased red cell GSH is unknown. nucleotides of human red cells and reticulocytes have shown 97% or more to contain adenine. Total nucleo- tides in patient cells were present in amounts 3-6 times INTRODUCTION greater than normal, and approximately 80% contained In 1972-73, this laboratory reported four subjects with pyrimidine. The ultraviolet spectral curves of depro- hereditary nonspherocytic hemolytic anemia associated teinized red cell extracts exhibited a shift in maximum with increased red cell adenine nucleotides GSH, and absorbance from the usual 256-257 nm to approximately basophilic stippling, and a fairly severe, though partial, 266-270 nm, and absorbance at 250, 270, 280, and 290 deficiency of ribosephosphate pyrophosphokinase (RPK,1 nm, expressed as a ratio of that at 260 nm, differed 5-phosphoribosyl-1-pyrophosphate synthetase, EC 2.7.- greatly from normal. The spectral characteristics of ex- 6.1.) (1, 2). In these studies, AMP, ADP, and ATP tracts provide the basis of a readily performed screening were measured by a conventional enzymatic procedure procedure, which does not require enzyme assay. The (3). The assay had been considered reliable, since nucleotidase activity in deficient red cells assayed less normally 97% or more of the nucleotides of both human than 14%, and usually less than 10%, of normal and red cells and reticulocytes are adenosine phosphates much less in terms of reticulocyte-rich blood, where it (3-11). Nucleotides containing guanosine may range mn was consistently found to be increased. The enzyme has amount from less than 1 to 3%, and pyrimidine-contain- a pH optimum of 7.5-8.0, is inhibited by EDTA, and does not utilize purine 5'-ribonucleotides or 8-glycero- 'Abbreviations used in this paper: AK, adenylate kinase; EU, enzyme units; LDH, lactate dehydrogenase; NDPK, phosphate as substrates. While comparatively few nucleoside diphosphate kinase; NMPK, nucleoside mono- family members have been available thus far for study, phosphate kinase; PCA, perchloric acid; PEP, P-enol- initial data are compatible with an autosomal, recessive pyruvate; PGK, phosphoglycerate kinase; PK, pyruvate kinase; PRPP, 5-phosphoribosyl-l-pyrophosphate; RBC, Received for publication 22 April 1974 and in revised erythrocytes; RPK, ribosephosphate pyrophosphokinase; form 31 May 1974. TCA, trichloroacetic acid; TEA, tetraethylammonium 866 The Journal of Clinical Investigation Volume 54 October 1974- 866-879 ing nucleotides are present in negligibly small amounts. phosphate kinase (NMPK) and bacterial (E. coli) alkaline In the assay procedures, however, nonadenosine nucleo- phosphatase were obtained from Sigma Chemical Co., St. Louis, Mo. Lactate dehydrogenase (LDH), phosphoglyce- tides would react but relatively slowly. In recent investi- rate kinase (PGK), and 3-phosphoglycerate were purchased gations of a new patient with the hemolytic syndrome from Calbiochem, LaJolla, Calif. Pyruvate kinase (PK), described above, we became aware of the probable pres- adenylate kinase (myokinase, AK), glyceraldehyde-3-phos- ence of significant concentrations of such "slow-reacting" phate dehydrogenase, and nucleoside diphosphate kinase nucleotides in the deproteinized blood extracts. Reexami- (NDPK) were obtained from Boehringer Mannheim Corp., New York. Plasmagel is manufactured by Roger Bellon nation of the recorded assays of the earlier patients Laboratories, Neville, France. Each 100 ml contains fluid revealed identical findings, which were absent in he- gelatin 3 g, NaCl 0.7 g, CaCl2 2H20 0.2 g. 4 mg heparin molysate extracts of all other hemolytic anemias studied was added per 100 ml. in this laboratory over a period of several years. This report documents subsequent investigations indicating Experiments (a) the red cells of subjects with this hereditary he- General. Blood collected in heparin or EDTA was molytic syndrome uniquely contain very large amounts transported to the Los Angeles laboratory in ice. Leuko- of cytidine and uridine nucleotides, (b) the latter are cytes were separated with Plasmagel as the erythrocyte- partially reflected in the enzymatic assays for nucleo- sedimenting agent. The red cells were washed thrice in tides and were the basis for our initial misinterpretation saline and adjusted to a concentration of about 3 X 106/,A, the erythrocytes and contaminating white cells enumerated, that adenine nucleotides were increased in patient eryth- and the reticulocyte percentage was determined. The sus- rocytes (1, 2), and (c) the genetically determined lesion pensions were frozen and thawed three times if hemolysates is a severe deficiency of a pyrimidine-specific 5'-nucleo- were required and were deproteinized and neutralized before tidase, an enzyme readily demonstrable in normal eryth- analysis for content of nucleotides or glycolytic metabo- rocytes, that exhibits increased activity in reticulocytes. lites. GSH was measured as described by Beutler, Duron, and Kelly (12). As will be discussed, RPK deficiency is believed to be Enzyme assays. All the enzymatic activities of the Emb- an epiphenomenon, secondary to the relatively enormous den-Meyerhof pathway, the two-pentose shunt dehydrogen- concentrations of pyrimidine nucleotides in circulating ases, glutathione peroxidase, and glyoxalase I and II were erythrocytes and presumably in marrow nucleated pre- assayed in hemolysates as previously described (13-18). cursor cells. The activities of enzymes of the distal pentose shunt, including those of transketolase and transaldolase, were screened in terms of the conversion of ribose-5-phosphate METHODS to fructose-6-phosphate. RPK (PRPP synthetase) and adenine phosphoribosyltransferase (EC 2.4.2.7) activities Case material were assayed by methods described elsewhere (19). AK Patient R. has not been previously reported. This 24-yr- activity was measured essentially as described by Haslam old woman of Jewish ancestry was first seen at the Albert and Mills (20) and ATPase was assayed according to Einstein College of Medicine in 1972. There was no history Brewer (21). of neonatal jaundice, but active hemolytic anemia was di- NMPK (EC 2.7.4.4.) activity was assayed at 370C in