Charles R. Zerez

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.

Oral L-glutamine therapy for sickle cell anemia: I. Subjective clinical improvement and favorable change in red cell NAD redox potential.

Previously, we demonstrated that there is an increased utilization of glutamine by intact sickle red blood cells (RBC) in conjunction with nicotinamide adenine dinucleotide (NAD) metabolism in vitro. In this report, we describe the in vivo effect of L‐glutamine supplementation on total NAD, nicotinamide adenine dinucleotide reduced (NADH), and NAD redox potential of sickle RBC. Seven adult sickle cell anemia patients participated in this study. The exclusion criteria were pregnancy, previous or current use of hydroxyurea, and transfusion within 3 months of initiation of the study. After proper consent, L‐glutamine was started at a dose of 30 g/day administered orally. Fasting blood samples were drawn at baseline and after 4 weeks of therapy by routine phlebotomy for evaluation of RBC total NAD and NADH levels. We found significant changes in both the NADH level and NAD redox potential (ratio of NADH to NAD+ + NADH). NAD redox potential increased from 47.2 ± 3.7% to 62.1 ± 11.8% (P < 0.01). The NADH level increased from 47.5 ± 6.3 to 72.1 ± 15.1 nmol/ml RBC (P < 0.01). The total NAD level demonstrated an upward trend (from 101.2 ± 16 to 116.4 ± 14.7 nmol/ml RBC) but this was not statistically significant. Our data show that oral L‐glutamine can significantly increase the NAD redox potential and NADH level in sickle RBC. These changes may decrease oxidative susceptibility of sickle RBC and result in clinical benefit. Am. J. Hematol. 58:117–121, 1998.

Increased red cell glutamine availability in sickle cell anemia: Demonstration of increased active transport, affinity, and increased glutamate level in intact red cells

Sickle red blood cells (RBCs) have been shown to have an increase in total nicotinamide adenine dinucleotide (NAD) content by an as-yet-unknown mechanism. Because glutamine is an essential precursor in NAD biosynthesis, we have examined the rates of active RBC glutamine transport and glutamine transport kinetics with Michaelis-Menten constant (K[m]) and maximum velocity (V[max]) in RBCs from patients with sickle cell disease, patients with high reticulocyte counts, and normal volunteers. In addition, plasma and RBC levels of glutamine and glutamate in the three groups were analyzed. The rate of active glutamate transport in sickle RBCs increased threefold over that in high-reticulocyte RBCs and increased 15-fold over that in normal RBCs. Glutamine transport K(m) in sickle RBCs was decreased fivefold in comparison with that in the high-reticulocyte group and that in normal control subjects. Glutamine transport V(max) for sickle RBCs was twofold and eightfold higher in comparison with those in the high-reticulocyte RBCs and normal control RBCs, respectively. Finally, the level of RBC glutamate (a byproduct of glutamine in NAD synthesis) in the sickle group was significantly increased in comparison with that in the high-reticulocyte group, whereas the RBC glutamine level was not. The higher glutamate level in sickle cells may suggest a higher glutamine turnover in these cells. These data suggest that sickle RBCs have an increased glutamine availability and affinity that may facilitate the increase in total NAD in sickle RBCs.

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 μmol/ml RBC per minute) compared to normal RBC (2.8±1.0 and 6.0±2.5 μmol/ml RBC per minute). P5N deficient RBC (n= 2) had significantly increased CMP and UMP kinase activities (14.0 and 26.5 ±mol/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 μmol/1 in P5N deficient RBC and since the CMP concentration is 25‐90 μmol/1 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.

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

Summary. 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 < 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.

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.