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Biomedical subjects

K R Tanaka

Publications and source records attributed to K R Tanaka.

At least 37 records · Page 2Linked to original sources
PubMed1987

Spectrophotometric determination of oxidized and reduced pyridine nucleotides in erythrocytes using a single extraction procedure.

Several methods are available for the extraction and quantitation of oxidized and reduced pyridine nucleotides in erythrocytes. Enzymatic methods, however, are complicated by the presence of hemoglobin, which causes oxidation of NADH and NADPH during extraction. Although hemoglobin-mediated oxidation can be prevented by the addition of reducing agents, these interfere with spectrophotometric cycling assays for these nucleotides. Therefore, we have developed a method for determining oxidized and reduced NAD and NADP in human erythrocytes using a single extract. Our extraction method eliminates the need for reducing agents and thus allows the use of a spectrophotometric cycling assay. Using this method, we obtained full recovery of all added nucleotides with both normal and reticulocyte-enriched red blood cells. Our method is suitable for the determination of NAD+, NADH, NADP+, and NADPH in normal human erythrocytes and in red cells from patients with hemolytic anemia with a higher proportion of reticulocytes.

Erythrocytes↗
PubMed1987

Impaired antioxidant defense in hemoglobin E-containing erythrocytes: a mechanism protective against malaria?

Red blood cell (RBC) antioxidant defense was investigated in eight individuals with hemoglobin E (Six EE and two E-B(+) thalassemia) and compared to that in six individuals with thalassemia and ten normal subjects. Individuals with hemoglobin E had increased incubated Heinz body formation (68% +/- 18%; p less than 0.001) compared to normal and thalassemic RBC (10% +/- 2% and 11% +/- 5%, respectively). Stimulated pentose phosphate shunt activity was increased in the thalassemic and decreased in the hemoglobin E RBC as compared to normal. The 2,3-diphosphoglycerate (DPG) content of the EE RBC was increased to 5.59 +/- 0.69 mumol/ml RBC as compared to normal (4.51 +/- 0.77; p less than 0.001). In the EE RBC, there was a direct correlation between Heinz body formation and DPG content (r = 0.73). Ascorbic and dehydroascorbic acid (0.1 and 1.0 mM) were able to decrease the degree of Heinz body formation in the hemoglobin E RBC. Ascorbic acid (0.1 mM) prolonged the response of the pentose shunt. Thus impaired antioxidant defense may account for the persistence of the hemoglobin E gene in areas where malaria is endemic. Oxidant medications should be used with caution in individuals of Southeast Asian origin.

2,3-Diphosphoglycerate↗
PubMed1987

Dapsone-associated Heinz body hemolytic anemia in a Cambodian woman with hemoglobin E trait.

A Cambodian woman with hemoglobin E trait (AE) and leprosy developed a Heinz body hemolytic anemia while taking a dose of dapsone (50 mg/day) not usually associated with clinical hemolysis. Her red blood cells (RBCs) had increased incubated Heinz body formation, decreased reduced glutathione (GSH), and decreased GSH stability. The pentose phosphate shunt activity of the dapsone-exposed AE RBCs was increased compared to normal RBCs. Although the AE RBCs from an individual not taking dapsone had increased incubated Heinz body formation, the GSH content and GSH stability were normal. The pentose phosphate shunt activity of the non-dapsone-exposed AE RBCs was decreased compared to normal RBCs. Thus, AE RBCs appear to have an increased sensitivity to oxidant stress both in vitro and in vivo, since dapsone does not cause hemolytic anemia at this dose in hematologically normal individuals. Given the influx of Southeast Asians into the United States, oxidant medications should be used with caution, especially if an infection is present, in individuals of ethnic backgrounds that have an increased prevalence of hemoglobin E.

2,3-Diphosphoglycerate↗
PubMed1987

Pyrimidine nucleoside monophosphate kinase hyperactivity in hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency.

The pyrimidine nucleoside triphosphates (CTP, UTP) increase in the pyrimidine 5'-nucleotidase (P5N) deficient red blood cell (RBC) to a greater degree than do the pyrimidine nucleoside monophosphates (CMP, UMP). Pyrimidine nucleoside monophosphate (PNMP) kinase phosphorylates CMP and UMP to their respective phosphodiesters. We tested the hypothesis that increased PNMP kinase activity contributes to the disproportionate increase in CTP and UTP in the P5N deficient RBC. CMP and UMP kinase activities were increased in high reticulocyte (4.4 +/- 2.1 and 8.5 +/- 3.3 mumol/ml RBC per minute) compared to normal RBC (2.8 +/- 1.0 and 6.0 +/- 2.5 mumol/ml RBC per minute). P5N deficient RBC (n = 2) had significantly increased CMP and UMP kinase activities (14.0 and 26.5 mumol/ml RBC per minute). UMP and CDP-ethanolamine were able to increase the activity of CMP kinase in crude haemolysate and the activity of partially purified enzyme. Since the Km for CMP of CMP kinase was 33 mumol/l in P5N deficient RBC and since the CMP concentration is 25-90 mumol/l in the P5N deficient RBC, the enzyme should be nearly saturated with CMP in the P5N deficient RBC. Thus, PNMP kinase hyperactivity appears to contribute to the disproportionate increase in CTP and UTP in the P5N deficient RBC.

5'-Nucleotidase↗
PubMed1987

Impaired nicotinamide adenine dinucleotide synthesis in pyruvate kinase-deficient human erythrocytes: a mechanism for decreased total NAD content and a possible secondary cause of hemolysis.

Erythrocytes from individuals with pyruvate kinase (PK) deficiency have approximately half the total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD) of normal erythrocytes. In order to elucidate the mechanism(s) for the decrease in total NAD, we examined NAD synthesis in intact erythrocytes. It is demonstrated that NAD synthesis is impaired in PK-deficient erythrocytes to a degree that is dependent on the PK activity and adenosine 5'-triphosphate (ATP) concentration of these cells. After incubation in the presence of fluoride, which simulates the characteristics of PK deficiency by inhibiting enolase, normal erythrocytes had impaired NAD synthesis and decreased ATP concentrations. Fluoride did not inhibit NAD synthesis in a hemolysate system that is not dependent on glycolysis for ATP generation. These data suggest that fluoride does not inhibit the enzymes of NAD synthesis and that impairment of NAD synthesis by fluoride is mediated by decreased ATP formation. Thus, it is concluded that impaired NAD synthesis in PK-deficient erythrocytes is caused by decreased ATP formation due to the PK deficiency. Since the rate of glycolysis is limited by the availability of NAD+, it is suggested that impaired NAD synthesis causes further ATP depletion and thereby may enhance hemolysis in PK-deficient erythrocytes.

Adenosine Triphosphate↗
PubMed1986

Regulation of red cell 2,3-DPG and Hb-O2-affinity during acute exercise.

Reports from the literature and our own data on red cell 2,3-DPG and its importance for unloading O2 from Hb to the tissues during exhaustive exercise are contradictory. We investigated red cell metabolism during incremental bicycle ergometry of various durations. Furthermore changes in blood composition occurring during exercise were simulated under in vitro conditions. The effect of a moderate (11.2 mmol X l-1 lactate, pH = 7.127) and severe (18 mmol X l-1 lactate, pH = 6.943) lactacidosis on red cell 2,3-DPG concentration was compared with the effect of similar acidosis induced by HCl. Our data indicate that the concentration of 2,3-DPG in red cells depends on the degree of lactacidosis, but not on the duration of exercise. During moderate lactacidosis red cell 2,3-DPG remains unchanged. This can be explained by an interruption of red cell glycolysis on the PK and GAP-DH step caused by a lactate and pyruvate influx into the erythrocyte, as well as an intraerythrocytic acidosis and a drop in the NAD/NADH ratio. During severe lactacidosis and HCL-induced acidosis a decrease in 2,3-DPG due to an inhibition of 2,3-DPGmutase and other glycolytic enzymes can be found. Mathematical correction of the observed P-50 value for the decrease in 2,3-DPG occurring during severe lactacidosis showed that a decrease in Hb-O2-affinity during strenuous exercise depends on the degree of lactacidosis and temperature elevation.

2,3-Diphosphoglycerate↗
PubMed1986

Antioxidants in sickle cell disease: the in vitro effects of ascorbic acid.

The authors examined the ability of antioxidants to prevent in vitro oxidant damage to the sickle red blood cell (RBC). One millimolar ascorbic acid and alpha-mercaptopropionylglycine significantly (p less than 0.005) protected against RBC Heinz body formation during incubation with acetylphenylhydrazine, while cysteine, cysteamine, and methionine did not. The effect of ascorbic acid was concentration dependent with concentrations as low as 0.1 mM having significant antioxidant effects. Ascorbic acid protected the RBC against hydrogen peroxide induced hemolysis as well (p less than 0.05). Ascorbic acid had a significant stimulatory effect on the rate of glucose oxidation by the pentose phosphate shunt (PPS), especially in the sickle RBC. Ascorbic acid did not protect the RBC from a patient with chronic hemolytic anemia due to G6PDTorrance from Heinz body formation, suggesting that an intact PPS is necessary for ascorbic acid to express its antioxidant properties. These data suggest that clinical trials should be undertaken to examine the efficacy of ascorbic acid in the treatment of SCD.

Adult↗
PubMed1986

Red cell metabolism in hereditary pyrimidine 5'-nucleotidase deficiency: effect of magnesium.

Since pyrimidine nucleotides avidly bind magnesium, we tested the hypothesis that the haemolytic anaemia in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency is due to a state of functional magnesium depletion in the red cell (RBC). In haemolysates from normal subjects, cytidine triphosphate (CTP) inhibited the activity of pyruvate kinase in a competitive manner for magnesium. The CTP Ki was 0.4 mmol/l. CTP inhibited the activity of hexokinase in a competitive manner for ATP (Mg-ATP2-) with a Ki of 4 mmol/l. The inhibitory effect of CTP on both enzymes was overcome by increasing the magnesium content of the test system. Since CTP appeared to inhibit enzymes which required magnesium as a cofactor or Mg-ATP2- as a substrate, we tested the effect of exogenous magnesium on the metabolism of P5N deficient RBC. The autohaemolysis test, the incubated Heinz body assay and the rate of glucose oxidation by the pentose phosphate shunt were abnormal in the intact RBC from a patient with hereditary P5N deficiency. The addition of MgCl2 (6-10 mmol/l) did not improve these abnormal in vitro measures of metabolism in the P5N deficient RBC. This lack of effect of exogenous magnesium may be due to the slow uptake of magnesium by the human RBC.

5'-Nucleotidase↗
PubMed1986

Effect of triethyltin on enzyme activity in human adult and cord red cells.

Since organotin compounds represent an environmental health hazard, we determined the effect of triethyltin bromide (TTB) on red blood cell (RBC) enzyme activity. TTB produced a concentration-dependent inhibition of hexokinase and pyrimidine 5'-nucleotidase for both adult and cord RBC. D-Glucose, but not ATP or MgCl2, prevented the hexokinase inhibition by TTB. Glucose-6-phosphate dehydrogenase, adenylate kinase, and hypoxanthine-guanine phosphoribosyltransferase were also inhibited by TTB. Cord RBC enzymes were more resistant to the effects of TTB than were adult RBC enzymes. Although TTB is a potent inhibitor of hexokinase, physiologic concentrations of glucose appear to protect the RBC during clinical tin intoxication.

5'-Nucleotidase↗
PubMed1986

Decrease in subunit aggregation of phosphoribosylpyrophosphate synthetase: a mechanism for decreased nucleotide concentrations in pyruvate kinase-deficient human erythrocytes.

Pyruvate kinase (PK)-deficient RBCs have several unexplained metabolic abnormalities, such as decreased concentrations of total adenine nucleotides (AMP, ADP, and ATP) and total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD). Because 5-phosphoribosyl-1-pyrophosphate (PRPP) is an intermediate in the synthesis of adenine nucleotides and NAD, we investigated PRPP synthetase (PRPPS), the enzyme responsible for PRPP synthesis. This enzyme is regulated, in part, by changes in its state of subunit aggregation. The proportion of aggregated PRPPS can be altered in vitro by ATP and 2,3-diphosphoglycerate (DPG). Because PK-deficient RBCs have decreased ATP and increased DPG concentrations, we examined the state of subunit aggregation of PRPPS in RBCs from normal and PK-deficient subjects, using gel permeation chromatography. Young normal RBCs have more aggregated PRPPS than do older RBCs. In contrast, due to their decreased ATP and increased DPG concentrations, PK-deficient RBCs contain less aggregated PRPPS than do RBCs of comparable age without PK deficiency. These data suggest that PRPPS should be less active in vivo in PK-deficient RBCs. This may play a key role in mediating the decreases in total adenine nucleotide and total NAD concentrations in these RBCs.

Adenine Nucleotides↗
PubMed1985

Increased Heinz body formation and impaired erythrocyte pentose phosphate shunt function during pregnancy.

The erythrocytes of 90 pregnant women were evaluated for the presence of in vivo or in vitro oxidant damage. The reduced glutathione (P less than 0.005) and the membrane reduced sulfhydryl (P less than 0.001) concentrations were decreased in fresh erythrocytes. Following incubation with acetylphenylhydrazine, Heinz body formation was significantly increased (P less than 0.001). Both the increase in Heinz body formation and the reduction in membrane reduced sulfhydryl content correlated strongly with duration of pregnancy. Glucose consumption was significantly decreased before, but not after, new methylene blue stimulation. Pentose phosphate shunt activity was impaired both before (P less than 0.05) and after (P less than 0.001) stimulation. No changes were observed in pentose phosphate recycling. The only alteration observed in the activity of the enzymes of the pentose shunt was an elevation of 6-phosphogluconate dehydrogenase activity. Although the clinical significance of these findings remains to be determined, medications with an oxidant potential should be used judiciously during gestation.

Erythrocyte Aging↗
PubMed1985

Mitral valve prolapse in sickle cell disease: manifestation of a generalized connective tissue disorder.

Previous studies have shown an association of sickle cell disease with generalized connective tissue disorders such as pseudoxanthoma elasticum. We recently documented an unexpectedly high prevalence of mitral valve prolapse, a connective tissue disorder, in sickle cell disease. To investigate this association, skin biopsies were analyzed from 32 sickle cell disease patients, 11 of whom had mitral prolapse. Total and type III collagen, collagen solubility, and uronic acid were not different between the patients with or without mitral prolapse (p greater than 0.05). Computerized morphometric quantitation of the volume fraction of elastic fibers was greater in sickle cell disease patients than in 10 normals (3.1 +/- 0.1 mean +/- SEM vs 2.0 +/- 0.3%; p less than 0.01) but less than in three patients with pseudoxanthoma elasticum (9.7 +/- 0.6%; p less than 0.001). Desmosine radioimmunoassay (an index of elastic fibers) was greater in sickle cell disease patients with mitral prolapse than those without (239.3 +/- 9.3 vs 171.7 +/- 25.4 ng/mg wet weight; p less than 0.02). Histopathologic grading showed a similar trend (p = 0.07). The combined probabilities of these three independent tests of elastic fiber quantity showed an increased elastic fiber concentration in mitral prolapse patients compared to those without mitral prolapse (p less than 0.02). Thus, there is no evidence for a specific collagen defect; rather, sickle cell disease appears to be associated with a spectrum of elastic tissue disorders, a feature that could predispose to mitral valve prolapse.

Adolescent↗
PubMed1985

A continuous spectrophotometric assay for pyrimidine-5'-nucleotidase.

A simple method is presented for the determination of pyrimidine-5'-nucleotidase activity using a continuous spectrophotometric assay system. Activity is determined by measuring inorganic phosphate generation using a linked indicator system that produces uric acid in the presence of inosine, purine nucleoside phosphorylase, and xanthine oxidase. This method has several advantages over any of the methods currently in use.

5'-Nucleotidase↗
PubMed1985

Pyrimidine nucleotides do not affect the enzymes of glutathione biosynthesis.

Erythrocytes from patients with hereditary pyrimidine 5'-nucleotidase (P5N, EC 3.1.3.5) deficiency accumulate large quantities of several pyrimidine nucleotides and their derivatives. In addition, the reduced glutathione (GSH) concentration is elevated in erythrocytes from patients with this enzyme deficiency. In the present study, we were unable to demonstrate any effect of various pyrimidine nucleotides and their derivatives on enzymes of glutathione biosynthesis and metabolism. Thus, elevation of GSH levels in erythrocytes from P5N patients is not a result of modulation of these enzymes by pyrimidine nucleotides and their derivatives.

5'-Nucleotidase↗
PubMed1985

Hemolytic anemias and erythrocyte enzymopathies.

The human erythrocyte generates high-energy adenosine triphosphate by anaerobic glycolysis and cycles oxidized and reduced nicotinamide adenine dinucleotide phosphate by the aerobic pentose phosphate shunt pathway. Certain enzymopathies of the pentose phosphate shunt are associated with hemolysis resulting from oxidative denaturation of hemoglobin. Glucose-6-phosphate dehydrogenase deficiency, an X-chromosome-linked disorder, is the prototype of these diseases and is genetically and clinically polymorphic. Six enzymopathies of anaerobic glycolysis cause hemolytic anemia; lactate dehydrogenase deficiency does not. In 2,3-diphosphoglycerate mutase deficiency, 2,3-diphosphoglycerate is greatly reduced and asymptomatic polycythemia is noted. Pyrimidine-5'-nucleotidase deficiency, an enzymopathy of nucleotide metabolism, is characterized by intracellular accumulations of pyrimidine-containing nucleotides, marked basophilic stippling on the stained blood film, splenomegaly, and hemolysis. Lead inhibits the nucleotidase and an identical syndrome occurs during severe lead poisoning. Hemolysis also accompanies an unusual enzymopathy characterized by a 40- to 70-fold increase (not decrease) in adenosine deaminase activity.

5'-Nucleotidase↗
PubMed1985

Mitral valve prolapse in sickle cell disease. Presumptive evidence for a linked connective tissue disorder.

To determine the prevalence of mitral valve prolapse in sickle cell disease, M-mode echocardiography was performed on 57 patients with sickle cell disease and 35 patients with chronic anemia of end-stage renal disease (anemic control group). In 25% (14/57) of patients with sickle cell disease, unequivocal mitral valve prolapse was diagnosed by echocardiography; all these patients had a mobile systolic click and/or late systolic murmur. This figure was significantly greater than the reported 5% to 6% prevalence in the general adult population, the 1% to 3% prevalence in the black population, and the 3.0% prevalence (1/35) in the anemic control group. The association of mitral valve prolapse and sickle cell disease cannot be explained on the basis of left ventricular size, systolic function, ischemic left ventricular or papillary muscle dysfunction, or chronic anemia. Therefore, a linked connective tissue defect in these two diseases is a hypothesis worthy of further investigation.

Adolescent↗
PubMed1984

Hemolytic anemia in hereditary pyrimidine 5'-nucleotidase deficiency. II. Effect of pyrimidine nucleotides and their derivatives on glycolytic and pentose phosphate shunt enzyme activity.

We evaluated the glycolytic intermediate concentrations from the erythrocytes of a patient with hereditary pyrimidine 5'-nucleotidase (P5'N) deficiency. Conclusive evidence for a metabolic block was not found. We evaluated the effects of the pyrimidine (cytidine and uridine) tri- and diphosphate nucleotides (CTP, CDP, UTP, UDP) and the choline and ethanolamine derivatives of CDP (CDP-choline, CDP-ethanolamine) on the activities of key enzymes of the Embden-Meyerhof pathway. CTP and UTP inhibited fructose-6-phosphate competitively for phosphofructokinase and phosphoenolpyruvate competitively for pyruvate kinase. In both cases, the Ki of the pyrimidine nucleotide and Km of the glycolytic substrate were above their intraerythrocytic concentrations. CTP was a competitive inhibitor of ADP for pyruvate kinase with a Ki near its intraerythrocytic concentration. CDP-choline and CDP-ethanolamine had no effect on the activities of Embden-Meyerhof or pentose phosphate shunt enzymes. Thus, the nature of the hemolytic anemia in hereditary P5'N deficiency remains enigmatic.

5'-Nucleotidase↗