Inhibitory effect of mannose on erythrocyte defense against oxidants.

The erythrocyte can phosphorylate a variety of hexoses. Since it can consume mannose and glucose equivalently in the hereditary deficiencies of hexokinase and phosphoglucose isomerase and since erythrocyte defense against oxidants is impaired in a variety of hereditary hemolytic anemias, we tested the hypothesis that mannose may be a significant alternative to glucose as a fuel for this defense system. Unexpectedly, mannose inhibited defense against oxidants as manifested by increased Heinz body formation when both normal and high-reticulocyte erythrocytes were incubated with acetylphenylhydrazine (APH). Using APH as the oxidant, mannose-incubated erythrocytes had decreased reduced glutathione stability and impaired hexose oxidation by the pentose shunt compared to glucose-incubated erythrocytes. After incubation with mannose and APH, normal erythrocytes showed a decrease in ATP content. Approximately 25% of the consumed mannose accumulated in the erythrocytes as mannose 6-phosphate. Erythrocytes incubated with mannose and APH displayed a significant loss of redox potential as manifested by decreased NADH/(NADH + NAD+) and NADPH/(NADPH + NADP+) ratios. Since phosphomannose isomerase is the rate-limiting step for mannose metabolism, our results suggest that mannose impairs erythrocyte defense against oxidants by causing ATP depletion and by impairing the regeneration of reduced pyridine nucleotides by the Embden-Meyerhof and pentose phosphate pathways.

Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency.

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di- and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl2 to promote PRPP synthetase subunit aggregation. Increasing the MgCl2 to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.

Red cell enzymopathies of the glycolytic pathway.

The delineation of specific erythrocyte glycolytic enzyme defects during the past three decades has clarified hitherto unexplained hereditary hemolytic syndromes. The glycolytic enzymopathies have proven to be important, not as a public health problem, but because the investigation of these experimental models of nature has provided information to increase our understanding of control of glycolysis and interrelationships of the Rapoport-Luebering shunt, mechanism of hemolysis, erythrocyte ageing, role of isozymes in various organs, and genetic control of enzyme structure/function. The application of ever improving techniques of recombinant DNA should yield a bonanza of new information to improve our comprehension of the pathogenesis and heterogeneity of these disorders as well as provide increased knowledge of regulation of these enzymes. It should be an exciting era.

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.

Effect of chloramphenicol and ethidium bromide on the level of ornithine carbamoyltransferase in Neurospora crassa.

The specific activity of the nuclear-gene-encoded, mitochondrial arginine biosynthetic enzyme ornithine carbamoyltransferase (EC 2.1.3.3) in Neurospora crassa was elevated in mycelia treated with chloramphenicol or ethidium bromide. The increase in specific activity was caused by an increase in the number of mature enzyme molecules rather than by the activation of a preexisting enzyme. Chloramphenicol and ethidium bromide appeared to act indirectly via arginine-mediated derepression. However, derepression did not appear to result from a drug-mediated decrease in the arginine pool.

Impaired erythrocyte NAD synthesis: a metabolic abnormality in thalassemia.

We have recently demonstrated that phosphoribosylpyrophosphate (PRPP) synthetase activity is decreased in RBC from individuals with thalassemia minor. Because NAD biosynthesis requires PRPP, the product of the PRPP synthetase reaction, we have investigated NAD synthesis in thalassemic RBC. NAD synthesis was measured in intact RBC both by following the accumulation of unlabeled NAD and by following the incorporation of 14C-nicotinic acid into NAD. Using both assay systems, we demonstrate that NAD synthesis is decreased significantly in thalassemic RBC compared to either normal or high reticulocyte red cells. Although this suggested that NAD content should be decreased in thalassemic RBC, no significant difference in NAD content was found among thalassemic, normal, or high reticulocyte red cells. Mechanisms for the lack of a significant decrease in NAD content in thalassemic RBC are discussed. These results indicate that NAD synthesis is impaired in thalassemic RBC possibly as a result of their decrease in PRPP synthetase activity. Our data provide evidence that thalassemic RBC have secondary metabolic abnormalities in addition to their primary defect in hemoglobin synthesis.

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.

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.

NAD kinase from Bacillus licheniformis: inhibition by NADP and other properties.

NAD kinase was purified 180-fold from Bacillus licheniformis to determine the role it plays in NADP turnover in this organism. The enzyme was found to have a pH optimum of 6.8 and an apparent Km for NAD of 2.7 mM. The ATP saturation curve was not hyperbolic; 5.5 mM ATP was required to reach half maximal activity. Both Mn2+ and Ca2+ could be substituted for Mg2+. Several compounds including nicotinic acid, nicotinamide, nicotinamide mononucleotide, quinolinic acid, NADPH, ADP, AMP and cyclic AMP did not affect NAD kinase activity. In contrast, the enzyme was inhibited by NADP at concentrations typically found in logarithmic cells of B. licheniformis. This inhibition was competitive with NAD and had a Ki of 0.13 mM. It is suggested that in vivo NAD kinase activity is highly dependent on the concentrations of NAD and ATP and the proportion of oxidized and reduced NADP.

Decreased erythrocyte phosphoribosylpyrophosphate synthetase activity and impaired formation in thalassemia minor: a mechanism for decreased adenine nucleotide content.

Adenosine triphosphate (ATP) and adenosine diphosphate levels are decreased in erythrocytes from individuals with beta-thalassemia minor. Because 5-phosphoribosyl-1-pyrophosphate (PRPP) is an essential precurosr of adenine nucleotides, we tested the hypothesis that impaired PRPP synthesis is a mechanism for the decreased adenine nucleotide content. Erythrocyte PRPP synthetase activity was significantly decreased, and the Michaelis-Menten constant (Km) for ribose-5-phosphate (R5P) was significantly increased in individuals with alpha-thalassemia minor and those with beta-thalassemia minor. Intact erythrocytes from individuals with alpha-thalassemia and those with beta-thalassemia minor also had an impaired rate of PRPP formation. Both the decrease in PRPP synthetase activity and the impaired PRPP formation were also found in erythrocytes with microcytosis resulting from iron deficiency, indicating that these phenomena may not be specific to thalassemia minor. In all individuals examined, the rate of PRPP formation correlated with ATP content, suggesting that either (1) PRPP synthetase activity is a determinant of ATP content or (2) ATP content is a determinant of PRPP synthetase activity. The depletion of ATP from normal erythrocytes did not affect PRPP synthetase activity, suggesting that ATP content is not a determinant of PRPP synthetase activity. However, a decrease in PRPP synthetase activity did cause an impairment in the rate of adenine nucleotide synthesis, suggesting that PRPP synthetase activity is a determinant of ATP content. Taken together, our results suggest that the decrease in PRPP synthetase activity and the resulting impairment in the rate of PRPP formation are mechanisms for the decreased adenine nucleotide content in thalassemic erythrocytes.

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.

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.

Impaired erythrocyte methemoglobin reduction in sickle cell disease: dependence of methemoglobin reduction on reduced nicotinamide adenine dinucleotide content.

We have examined aspects of methemoglobin (metHb) reduction in sickle and in thalassemic red blood cells (RBCs). NADH metHb reductase activity in sickle and thalassemic RBCs was significantly increased compared with normal RBCs. Because in vitro enzyme activity does not necessarily represent in vivo activity, we measured the rate of metHb reduction in intact RBCs. Intact thalassemic RBCs demonstrated a significantly increased rate of metHb reduction compared with normal RBCs. In contrast, intact sickle RBCs had a rate of metHb reduction that was similar to normal RBCs and significantly decreased relative to high reticulocyte RBCs of equivalent cell age. To determine the mechanism for the relative impairment of metHb reduction in sickle RBCs, we measured intraerythrocytic NADH, a cofactor in the metHb reduction reaction. Thalassemic RBCs had a significantly increased NADH content relative to normal RBCs. In contrast, sickle RBCs did not have an increase in NADH content. Furthermore, incubating normal RBCs under conditions that increase the NADH content resulted in an increased rate of metHb reduction. In contrast, conditions that decrease the NADH content in normal RBC resulted in a decreased rate of metHb reduction. These data and other results suggest that metHb reduction in intact RBCs is dependent on NADH content, and that the impaired metHb reduction rate in sickle RBCs may be a result of a lack of increase in NADH content. The dependence of metHb reduction on RBC NADH content and the ability to manipulate NADH content in vitro suggest a new strategy for decreasing oxidant damage to sickle RBCs in vivo.

Relationship between the nicotinamide adenine dinucleotide redox potential and the 2,3-diphosphoglycerate content in the erythrocyte in sickle cell disease.

The percentage of nicotinamide adenine dinucleotide (NAD) in the oxidized form [NAD+/(NAD+ and NADH); i.e. the NAD+/NADT ratio] is increased in the red cell (RBC) in sickle cell disease. We tested the hypothesis that the increased NAD+/NADT ratio was a determinant of the increased 2,3-diphosphoglycerate (DPG) content of the SCD RBC. Using normal subjects and individuals with sickle cell disease or autoimmune haemolytic anaemia (AIHA), we observed an inverse relationship between the packed cell volume (PCV) and the RBC DPG concentration (r = -0.69) and a direct relationship between the RBC NAD+/NADT ratio and the DPG concentration (r = 0.74). When the effect of the PCV on DPG was removed using analysis of covariance [DPGady(PCV)], the NAD+/NADT ratio had a significant relationship with the DPGadj(PCV) (r = 0.50, P less than 0.001). In in vitro incubation studies, increasing the NAD+/NADT ratio significantly increased the DPG content of both normal and AIHA RBC. Conversely, decreasing the NAD+/NADT ratio decreased the DPG content of normal, AIHA and SCD RBC. Thus, the increased DPG content in the SCD RBC appears to be due, in part, to the increased NAD+/NADT ratio and is not purely a physiologic response to decreased oxygen carrying capacity.

Partial purification and properties of nicotinamide adenine dinucleotide synthetase from human erythrocytes: evidence that enzyme activity is a sensitive indicator of lead exposure.

We have examined properties of nicotinamide adenine dinucleotide (NAD) synthetase from human erythrocytes. The enzyme was found to be cold labile and extremely unstable in crude hemolysate, with complete loss of activity occurring after 24 hours at 4 degrees C. However, maintenance of crude hemolysate at 20 to 25 degrees C in the presence of EDTA and KCl increased NAD synthetase stability substantially (half-life = 10 days). Using these conditions, NAD synthetase was purified 3,100-fold with a 29% yield using DEAE-cellulose column chromatography, ammonium sulfate fractionation, and dialysis. The apparent Michaelis-Menten constants for nicotinic acid adenine dinucleotide (NAAD), adenosine triphosphate, Mg2+, glutamine, and K+ were 0.108, 0.154, 1.36, 2.17, and 8.32 mmol/L, respectively. The pH optimum ranged between 6.8 and 7.4, and the molecular weight was estimated to be 483 +/- 5 Kd. The enzyme was markedly inhibited by Pb2+ and Zn2+, with concentrations necessary for 50% inhibition of activity of 1.3 and 2.0 mumol/L, respectively. The incubation of intact red blood cells with lead followed by rigorous washing to remove lead abolished nearly all NAD synthetase activity. In contrast, glucose-6-phosphate dehydrogenase activity, which is not sensitive to lead, was unaffected, whereas pyrimidine 5'-nucleotidase activity, which is sensitive to lead, was decreased 30% to 50% under these conditions. More importantly, patients with lead overburden (34 to 72 micrograms Pb2+/dL blood) all had markedly decreased NAD synthetase activity. These data together with other results suggest that erythrocyte NAD synthetase activity is a sensitive indicator of lead exposure in humans.

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.

Decreased erythrocyte nicotinamide adenine dinucleotide redox potential and abnormal pyridine nucleotide content in sickle cell disease.

RBCs from individuals with sickle cell disease are more susceptible to oxidant damage. Because key antioxidant defense reactions are linked to the pyridine nucleotides nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), we tested the hypothesis that the RBC redox potential as manifested by the NADH/[NAD+ + NADH] and NADPH/[NADP+ + NADPH] ratios is decreased in sickle erythrocytes. Our data demonstrate that sickle RBCs have a significant decrease in the NADH/[NAD+ + NADH] ratio compared with normal RBCs (P less than .00005). Interestingly, sickle RBCs also had a significant increase in total NAD content compared with normal RBCs (P less than .00005). In contrast, although sickle RBCs had a significant increase in the total NADP content compared with normal RBCs (P less than .00005), sickle RBCs had no significant alteration in the NADPH/[NADP+ + NADPH] ratio. High reticulocyte controls demonstrated that these changes were not related to cell age. Thus, sickle RBCs have a decrease in NAD redox potential that may be a reflection of their increased oxidant sensitivity. The changes in these pyridine nucleotides may have further metabolic consequences for the sickle erythrocyte.

Refrigerated storage of lyophilized and rehydrated, lyophilized human red cells.

Human red cells (RBCs) were collected in CPDA-1 and then freeze-dried in lyoprotective solution. The lyophilized RBCs were then stored at -20 degrees C for 7 days. At the end of the storage period, the lyophilized RBCs were rehydrated and washed in dextrose saline. The washed, reconstituted, lyophilized RBCs were resuspended in final wash solutions of ADSOL, CPDA-1, or a special additive solution containing glucose, citrate, phosphate, adenine, and mannitol, and then they were stored at 4 degrees C for an additional 7 days. The main purpose of this study was to determine whether human RBCs can be lyophilized in such a manner that normal metabolic, rheologic, and cellular properties are maintained during rehydration and subsequent storage in standard blood bank preservative solutions. Our results show that reconstituted, lyophilized RBCs maintained levels of ATP, 2,3 DPG, lactate, and cellular properties that are equal to or better than those in control nonlyophilized RBCs stored for a comparable period in CPDA-1. Reconstituted, lyophilized RBCs stored at 4 degrees C after rehydration also show better maintenance of ATP, 2,3 DPG, and lactate than do control RBCs stored in the same preservative solutions for comparable periods.

Decreased pyrimidine nucleoside monophosphate kinase activity in sickle erythrocytes.

We have previously shown that physiologic concentrations of hemin cause marked inhibition of several red blood cell (RBC) enzymes. Because endogenous heme content is elevated in sickle RBCs, we have examined the activity of hemin-sensitive enzymes in these RBCs. One of the hemin-sensitive enzymes, pyrimidine nucleoside monophosphate kinase (PNMK), was shown to have decreased activity in sickle RBCs relative to RBCs of equivalent cell age. The other hemin-sensitive enzymes, including adenylate kinase (AK), pyrimidine 5'-nucleotidase (P5N), 6-phosphogluconate dehydrogenase (6PGD), and aldolase, had activities that were appropriate for cell age. We have also examined the affinity of the hemin-sensitive enzymes to hemin. Using two different methods, PNMK was shown to have the highest binding affinity to hemin. The exquisite sensitivity of PNMK to inhibition by hemin, coupled with the enzyme's high affinity to hemin, may account for the decrease in PNMK activity and the lack of significant decrease in the other hemin-sensitive enzymes in sickle RBCs. These results suggest that the increased endogenous heme content in sickle RBCs may be responsible for the decrease in PNMK activity. Whether the increased endogenous heme content of sickle RBCs can cause hemolysis indirectly by inhibiting RBC enzymes remains to be determined.

Preservation of metabolic activity in lyophilized human erythrocytes.

Normal human erythrocytes (RBC) were freeze-dried under conditions that caused minimal modification in normal RBC metabolic activities. Because of the known effects of long-term storage on metabolic activities, we studied the effects of our lyophilization process on RBC metabolism. Of all the metabolic enzymes studied, only triosephosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1), enolase (2-phospho-D-glyceratehydro-lyase, EC 4.2.1.11), and pyruvate kinase (ATP:pyruvate O2-phosphotransferase, EC 2.7.1.40) were decreased when compared with fresh control nonlyophilized RBC. The activities of these enzymes did not differ significantly from those of blood bank RBC. Concentrations of high-energy intermediates, ATP, and 2,3-diphosphoglycerate, along with lactate and ATP production were decreased in lyophilized RBC. No enzymes of the pentose phosphate shunt were altered during lyophilization. In addition, our data show that lyophilized RBC possess an intact capacity to (i) synthesize adenine nucleotides and (ii) reduce MetHb to Hb and, thus, maintain the Hb in a functional physiologic state similar to fresh nonlyophilized RBC. The present study demonstrates the possibility of lyophilizing RBC in a manner that maintains normal metabolic and enzymatic function upon rehydration.