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The effect of NO2 on erythrocyte glucose metabolism as related to changes in redox ratio [NAD+][NADH]
ENVIRONMENTAL
RESEARCH
12, 174- 179 (1976)
The Effect of NO, on Erythrocyte as Related to Changes in Redox STANLEY Department
M. CASSAN, BENJAMIN
GROSS’
Glucose Metabolism Ratio [NAD+]/[NADH] AND
DANIEL
Unir3ersity of California at Los Angeles. Los Angeles, Califarnia 90024
of Medicine,
H. School
SIMMONS of Medicine,
Received September 26. 1975 Glucose utilization of human erythrocytes increased to approximately 2.5 times control values when exposed to a mean NO, concentration of 336.6 ppm for 2 hours at 37°C and pH = 7.23. The fraction of glucose utilized by the pentose phosphate pathway remained constant so that actual glucose utilized by both this pathway and by glycolysis increased in proportion to the total glucose metabolized. The rate of total lactate plus pyruvate production remained unchanged. These data suggest that NO, may influence several loci of erythrocyte metabolism, including accumulation of glycolytic intermediates proximal to the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase, oxidation of NADH, NADPH, and GSH, and alteration of NADH dependent enzymes.
INTRODUCTION
Profound alterations in the redox ratio [NAD+]/[NADH] have previously been found in human erythrocytes exposed to NO, in concentrations exceeding 15 ppm (Cassan and Simmons, 1975a). Between 15 and 500 ppm NO2, a dose-response relationship was demonstrated, with progressive increases in redox ratio at higher NO, concentrations reflecting a more oxidized cellular state. The current study represents an attempt to correlate these redox changes with changes in glucose metabolism induced by NO, exposure. More specifically, we wished to know whether a redistribution of substrate between Embden-Meyerhof and pentose pathways during NO, exposure could explain the previously noted lack of change in the rate of lactate and pyruvate production accompanying redox alterations despite the generally accepted role of the nucleotide pair [NAD+]/[NADH] in the regulation of glycolytic rate. MATERIALS
AND METHODS
Venous blood was obtained from normal human volunteers using heparin anticoagulation. After initial centrifugation, plasma and buffy coats were removed by aspiration and red cells were resuspended in modified Ringers-Tris hydrochloride with the following composition: sodium, 147 mM/liter; potassium, 4 mM/liter; calcium, 4 mdliter; chloride, 115 mM/liter; Tris buffer, 39 mM/liter; hydrochloric acid, 0.15 M to bring pH to 7.4 at 37°C. Red cells were washed three times, each wash in a twofold volume of the above solution, followed by centrifugation and aspiration of supernatant. After the washes, the upper one-quarter of the packed cell volume was removed to assure elimination of leucocytes; 10 cc of the remaining red cells and 10 CC of modified Ringers-Tris solution Containing hM/liter glucose I Medical College of Wisconsin. 174 Copyright All rights
CI 1976 by Academic Press, Inc. of reproduction in any form reserved.
THE
EFFECT
OF
NO2
ON
RBC
GLUCOSE
METABOLISM
175
and 12 ,ul of [ l-Cl41 glucose (approximately 1 x lo6 dpm) were added to each of two 50 cc Erlenmeyer flasks containing 2 cc of CO, absorbing reagent (Phenethylamine) within a central well. Flasks were equilibrated with air by agitation for 5 minutes, after which the gas in one of the two flasks was diluted with an appropriate quantity of nitrogen dioxide to reach a range of 200 to 500 ppm (prepared from an E cylinder of 100% NO, by serial dilutions with air in a glass syringe), the other flask serving as a paired control. NO, concentration in the experimental flasks was measured using the Griess-Saltzmann reaction (Saltzmann, 1969) involving absorption of the gas in an azo-dye forming reagent and comparison of spectrophotometric absorption at wavelength 500 nm with a standard curve constructed from variable dilutions of sodium nitrate added to the azo-dye forming reagent. Control and NO,-containing flasks were incubated at 37°C for 2 hours in a Dubnoff metabolic shaker, following which samples were obtained from each flask for measurement of final pH and p0, (Radiometer gas analyzer, model PHM 72), glucose concentration (glucose oxidase method; Sigma Kit No. 510-DA), and lactate, and pyruvate concentrations (Vester modification of established techniques (Bergmeyer, 1963)). Changes in lactate and pyruvate concentrations were expressed as mM/liter red blood cells/hr incubation, assuming none was present at the start of the incubation. Samples of CO, absorbing reagent were also obtained following incubation for counting radioactivity of Cl4 (Hewlett-Packard Model 3003 Tricarb Scintillation Spectrophotometer; automatic control, model 527). The fraction of glucose utilized by the pentose pathway was determined by the number of [C14]02 counts in the absorbing reagent as a fraction of the total glucose counts utilized, since all CO, produced in the human red cell is derived solely from glucose 1-C in the pentose pathway. Total glucose counts utilized was calculated from the product of initial counts of radioactive glucose added to the incubate and the fraction of total glucose utilized: Initial glucose cont. - final glucose cont. Cl4 added x initial glucose cont. where the initial glucose concentration = 2.5 mM/liter total incubate. Background radiation and quench factors were incorporated in the count measurement. RESULTS
Since the NO, range of U-500 ppm produces consistent unidirectional changes in redox ratio, a single range of NO, exposure was chosen (200-500 ppm) in which unequivocal redox changes could be expected, the high concentrations of NOz producing redox changes larger than those at lower NO, concentrations (Cassan and Simmons, 1975a). The mean NO, concentration was 336.6 ppm (Table 1). Mean pH was identical in both control and NO,-exposed flasks. Mean p0, was 142.3 and 144.3 in NO,-exposed and control groups, respectively, corresponding to fully saturated hemoglobin. The sum of pyruvate and lactate produced was similar in both groups (Table 1) as had previously been noted (Cassan and Simmons, 1975a). A trend toward an
7.23 20.04 0.138 z-o.10
142.3 24.8 0.791 >O.lO
336.6 k89.l
Mean value + SD (II = 8) r P
7.23 to.03
PH
144.3 24.7
PO, (Tow)
UTILIZATION,
Mean value t SD (II = 8)
NO, (ppm)
Gr.ucos~
TABLE
AIR
2.008 >0.05
0.129 0.046
+ NO,
0.767 >O.lO
0.928 0.135
1.017 0.276
AIR 0.09 0.027
LACIATE
Lactate (mM/Yhr)
AND
1
Pyruvate (mM/l/hr)
PYKLIVAI.E
0.406 >O.lO
1.057 0.166
1.107 0.271
Pyruvate + lactate (mM/l/hr)
PR~DLTTION
4.332
24.9 8.36
IO.1 3.44
Total glucose utilized (%)
4.371
1.25 ,406
Sl .167
Actual glucose utilized m&l/hr
0.088 >o. IO
2.00 1.354
2.09 2.128
Percentage total glucose utilized-via Pentose shunt
s w
;
z i2
6 Fs
5 w
c z
THE
EFFECT
OF
NO2
ON
RBC
GLUCOSE
METABOLISM
177
increase in mean pyruvate and a decrease in mean lactate produced in NO, exposed samples was noted, but these changes were not statistically significant, perhaps due in the case of pyruvate to the unusually low value in experimental sample 2. Mean glucose utilization (Table 1) was significally increased to 24.9% of total glucose in the NO,-exposed erythrocytes compared with 10.1% in control samples. The mean actual glucose utilized in control samples of 0.51 m;\l/liter/hr corresponded well with the mean total lactate and pyruvate produced of 1.107 m&iter/hr. In NO,-exposed erythrocytes, however, the mean total lactate and pyruvate produced of 1.057 mM/liter/hr was less than half the quantity expected for the glucose utilized of 1.25 mM/liter/hr based on the production of 2 mole of lactate or pyruvate for each mole of glucose used. The fraction of glucose metabolized via the pentose pathway remained relatively constant, with mean values of 2.09 and 2.00% in control and NO,-exposed samples, respectively, so that actual glucose utilization via the pentose pathway increased proportionally to the increase in total glucose metabolized. DISCUSSION
Since the purpose of the study was to correlate NO,-induced changes in glucose metabolism with alterations in redox ratio previously described with NO, exposures for 2 hours (Cassan and Simmons, 1975a) and since redox ratio is calculated from momentary levels of glycolytic intermediates, the mean rate of glucose metabolism was also determined at 2 hours following incubation. The dynamics of the changes may involve a series of time-related alterations in glucose metabolism not reflected in these results. There may perhaps be several reactions with different half-times, of which this data is the net result. The increase in pentose pathway activity of the red cell during NO, exposure, as indicated by increased l-Cl4 utilization, is consistent with protective responses of erythrocytes exposed to other oxidants (Davidson and Tanaka, 1972). All oxidants, however, do not appear to act in the same way since only NO, appears to effect a change in cytoplasmic redox state in more complex cells (Simons et al., 1974). As in the case of hyperoxic exposure, the increase in pentose pathway activity in this study is limited and represents only a small fraction of the potential pentose pathway activity which can be induced with methylene blue (Davidson and Tanaka, 1972; Imarisio et nl., 1969). The mechanism by which the pentose shunt is stimulated by NO, exposure could include direct or indirect oxidation of NADPH or glutathione (GSH) (Menzel et al., 1972; Jaffe, 1971; Willis and Kratzing, 1972). In more complex cells, exposure to oxidants is associated with significant increases in activity of pentose pathway related enzymes (Tierney et al., 1973). Glucose utilization by the Embden-Meyerhof pathway was also increased by NO, exposure. The reason for this is not clear. In previous studies on the effect of anaerobiosis on erythrocyte glycolysis (Hamasaki et al., 1970; Cassan ef al., 1972), decreases in red cell [NAD+]/[NADH] ratio determined indirectly (Krebs and Veech, 1969) and based on mass action equilibria were inversely related to glycolytic rate. Since [NAD+]/[NADH] ratio in NO,-exposed erythrocytes is significantly increased (Cassan and Simmons, 1975a), it was expected that glucose utilization by the Embden-Meyerhof pathway might be concomitantly diminished, but this was
178
CASSAN,
GROSS
AND
SIMMONS
not the case. An increase in [NAD+]/[NADH] ratio might be expected to increase glycolysis, as found in this study, by improving the availability of oxidized nucleotide at the glyceraldehyde-3-phosphate dehydrogenase step. Although the latter enzyme is not rate limiting under these experimental conditions, substrate availability for subsequent reactions in glycolysis would be affected. On the other hand, reduction in [NAD+]/[NADH] ratio in the presence of high glycolytic rates (Rose, 1971), and high nucleotide ratios accompanying reduced glycolysis (Omachi et nl., 1969) have been described by others and are related to variations in the pool of 2,3 DPG. Thus, glycolytic rate may be largely independent of the redox ratio, which is determined primarily by relative concentrations of glycolytic intermediates. Alternately, the apparent discrepancies between redox ratio and glycolytic rate may represent evidence for the absence of mass action equilibrium conditions upon which indirect calculation of apparent redox ratios are based. In the present study, NO, exposure resulted in increased glucose utilization, but the total lactate and pyruvate production did not increase. This suggests a metabolic block in the Embden-Meyerhof pathway leading to accumulation of glycolytic intermediates proximal to these products. The increase in [NAD+]/ [NADH] ratio suggests this locus to be proximal to the glyceraldehyde-3-phosphate dehydrogenase step. Such an NO, effect, perhaps on phosphofructokinase, could be similar to that previously described for a pH-independent effect of CO, in canine erythrocytes (Zborowska-Sluis and Klassen, 1972). However, other NO, effects altering the [NAD+]/[NADH] ratio, including a direct NO, oxidant effect on NADH or alteration of NADH dependent enzymes (Vassallo et al., 1973) cannot be excluded, although stoichiometric considerations suggest that NO, is not present in sufficient quantities to explain redox alterations on the basis of direct NADH oxidation alone. An elucidation of effects on glucose metabolism and the associated redox changes with NOz exposure may assist in defining the mechanism of NO* induced cellular damage, particularly since lipid peroxidation hitherto invoked as the essential mechanism of cell damage (Roehm, 1971) and redox alterations (Mintz, 1972), appears not to be involved in the production of these effects in normal human erythrocytes (Cassan and Simmons, 1975b; Cassan and Simmons, in press). Although morphologic studies were not performed in these latter studies, evidence of hemolysis which is known to accompany lipid peroxidation was not observed despite the high NO, concentrations used. ACKNOWLEDGMENTS The authors wish to thank Jane Christoferson for laboratory assistance and Julie Taylor for typing of manuscripts.
REFERENCES Bergmeyer, H. U., Ed. (1963). “Methods of Enzymatic Analysis.” Academic Press, New York. Cassan, S. M., and Simmons, D. H. (1975a). The effect of vitamin E on NO, induced redox changes in the human erythrocyte. Environ. Physiol. Biochern. 5, 201-207. Cassan, S. M., and Simmons, D. H. (1975b). The effect of NO, on redox state of the human erythrocyte. Arch.
Environ.
Health
30, 502-506.
Cassan, S. M., and Simmons, D. H. Anerobiosis Emiron. Res., in press.
and the effect of NO2 on the human erythrocyte.
THE
EFFECT
OF
NO2
ON
RBC GLUCOSE METABOLISM
179
Cassan, S. M., Theodore, J., and Robin, E. D. (1972). The effect of low 0, tensions on red cell glycolysis, NAD+/NADH ratios and transmembrane potential (TMP)-The Pasteuroid effect. C/in. Res. 20, 574.
Davidson. W. D., and Tanaka, K. R. (1972). Factors effecting pentose phosphate pathway activity in human red cells. hit. J. Hematol. 23, 371-385. Hamasaki, N., Asakura, T., and Minakami, S. (1970). Effect of oxygen tension on glycolysis in human erythrocytes. J. Biochem. 68, 157-161. Imarisio. J. J., Gendelman, B., and Strother, J. A. (1969). Red cell in vitro metabolism in approximate steady state. Metabolism 18, 1033-1047. Jaffe, E. R. (1971). Introduction to discussion of red cell GSH metabolism. The functions of reduced glutathione in human erythrocytes. Exp. Eye Res. 11, 306309. Krebs, H. A., and Veech, R. L. (1969). Equilibrium relations between pyridine nucleotides and their roles in the regulation of metabolic processes. Adv. Enzm. Reg. 7, 397-413. Menzel, D. B., Roehm, J. N., and Lee, S. D. (1972). Vitamin E: The biological and environmental antioxidant. J. Agric. Food Chem. 20, 481-486. Mintz. S. (1972). Nitrogen dioxide toxicity in alveolar macrophages-a mitochondrial lesion. Chest 62, 382. Omachi, A., Scott, C. B., and Parrey, T. E. (1969). Influence of glycolysis on NADH content in human erythrocytes. Amer. .I. Physiol. 216, 527-530. Roehm, J. N. (1971). Oxidation of unsaturated fatty acids by ozone and nitrogen dioxide, a common mechanism of action. Arch. Environ. Health 23, 142-148. Rose, I. A. (1971). Regulation of human red cell glycolysis: A review. Exp. Eye Res. 11, 264-272. Saltzmann. B. E. (1969). Tentative method of analysis for nitrogen dioxide content of the atmosphere (Griess-Saltzmann Reaction). Healih Lab. Sci. 6, 106113. Simons, J. R., Theodore, J., and Robin, E. D. (1974). Common oxidant lesion of mitochondrial redox state produced by NO, ozone and high 0, in alveolar macrophages. Chest 66 (Suppl.) 95. Tierney, D., Ayers, L., Herzog, S., and Yang, J. (1973). Pentose pathway and production of reduced nicotinamide adenine dinucleotide phosphate. A mechanism that may protect lungs from oxidants. Amer. Rev. Resp. Dis. 108, 1348-1351. Vassallo, C. L., Domm, B. M., Roe, R. H., Duncombe, M. L., and Gee, B. G. (1973). NO, gas and NO, effects on alveolar macrophage phagocytosis and metabolism. Arch. Environ. Health 26,270-274. Willis, R. J., and Kratzing, C. C. (1972). Changes in levels of tissue nucleotides and glutathione after hyperbaric oxygen treatment. Aust. J. Exp. Biol. Med. Sci. 50, 725-729. Zborowska-Sluis, D. T.. and Klassen, G. A. (1972). The effect of carbon dioxide and H+ on canine erythrocyte glycolysis. Resp. Physiol. 1.5, 96-103.
RESEARCH
12, 174- 179 (1976)
The Effect of NO, on Erythrocyte as Related to Changes in Redox STANLEY Department
M. CASSAN, BENJAMIN
GROSS’
Glucose Metabolism Ratio [NAD+]/[NADH] AND
DANIEL
Unir3ersity of California at Los Angeles. Los Angeles, Califarnia 90024
of Medicine,
H. School
SIMMONS of Medicine,
Received September 26. 1975 Glucose utilization of human erythrocytes increased to approximately 2.5 times control values when exposed to a mean NO, concentration of 336.6 ppm for 2 hours at 37°C and pH = 7.23. The fraction of glucose utilized by the pentose phosphate pathway remained constant so that actual glucose utilized by both this pathway and by glycolysis increased in proportion to the total glucose metabolized. The rate of total lactate plus pyruvate production remained unchanged. These data suggest that NO, may influence several loci of erythrocyte metabolism, including accumulation of glycolytic intermediates proximal to the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase, oxidation of NADH, NADPH, and GSH, and alteration of NADH dependent enzymes.
INTRODUCTION
Profound alterations in the redox ratio [NAD+]/[NADH] have previously been found in human erythrocytes exposed to NO, in concentrations exceeding 15 ppm (Cassan and Simmons, 1975a). Between 15 and 500 ppm NO2, a dose-response relationship was demonstrated, with progressive increases in redox ratio at higher NO, concentrations reflecting a more oxidized cellular state. The current study represents an attempt to correlate these redox changes with changes in glucose metabolism induced by NO, exposure. More specifically, we wished to know whether a redistribution of substrate between Embden-Meyerhof and pentose pathways during NO, exposure could explain the previously noted lack of change in the rate of lactate and pyruvate production accompanying redox alterations despite the generally accepted role of the nucleotide pair [NAD+]/[NADH] in the regulation of glycolytic rate. MATERIALS
AND METHODS
Venous blood was obtained from normal human volunteers using heparin anticoagulation. After initial centrifugation, plasma and buffy coats were removed by aspiration and red cells were resuspended in modified Ringers-Tris hydrochloride with the following composition: sodium, 147 mM/liter; potassium, 4 mM/liter; calcium, 4 mdliter; chloride, 115 mM/liter; Tris buffer, 39 mM/liter; hydrochloric acid, 0.15 M to bring pH to 7.4 at 37°C. Red cells were washed three times, each wash in a twofold volume of the above solution, followed by centrifugation and aspiration of supernatant. After the washes, the upper one-quarter of the packed cell volume was removed to assure elimination of leucocytes; 10 cc of the remaining red cells and 10 CC of modified Ringers-Tris solution Containing hM/liter glucose I Medical College of Wisconsin. 174 Copyright All rights
CI 1976 by Academic Press, Inc. of reproduction in any form reserved.
THE
EFFECT
OF
NO2
ON
RBC
GLUCOSE
METABOLISM
175
and 12 ,ul of [ l-Cl41 glucose (approximately 1 x lo6 dpm) were added to each of two 50 cc Erlenmeyer flasks containing 2 cc of CO, absorbing reagent (Phenethylamine) within a central well. Flasks were equilibrated with air by agitation for 5 minutes, after which the gas in one of the two flasks was diluted with an appropriate quantity of nitrogen dioxide to reach a range of 200 to 500 ppm (prepared from an E cylinder of 100% NO, by serial dilutions with air in a glass syringe), the other flask serving as a paired control. NO, concentration in the experimental flasks was measured using the Griess-Saltzmann reaction (Saltzmann, 1969) involving absorption of the gas in an azo-dye forming reagent and comparison of spectrophotometric absorption at wavelength 500 nm with a standard curve constructed from variable dilutions of sodium nitrate added to the azo-dye forming reagent. Control and NO,-containing flasks were incubated at 37°C for 2 hours in a Dubnoff metabolic shaker, following which samples were obtained from each flask for measurement of final pH and p0, (Radiometer gas analyzer, model PHM 72), glucose concentration (glucose oxidase method; Sigma Kit No. 510-DA), and lactate, and pyruvate concentrations (Vester modification of established techniques (Bergmeyer, 1963)). Changes in lactate and pyruvate concentrations were expressed as mM/liter red blood cells/hr incubation, assuming none was present at the start of the incubation. Samples of CO, absorbing reagent were also obtained following incubation for counting radioactivity of Cl4 (Hewlett-Packard Model 3003 Tricarb Scintillation Spectrophotometer; automatic control, model 527). The fraction of glucose utilized by the pentose pathway was determined by the number of [C14]02 counts in the absorbing reagent as a fraction of the total glucose counts utilized, since all CO, produced in the human red cell is derived solely from glucose 1-C in the pentose pathway. Total glucose counts utilized was calculated from the product of initial counts of radioactive glucose added to the incubate and the fraction of total glucose utilized: Initial glucose cont. - final glucose cont. Cl4 added x initial glucose cont. where the initial glucose concentration = 2.5 mM/liter total incubate. Background radiation and quench factors were incorporated in the count measurement. RESULTS
Since the NO, range of U-500 ppm produces consistent unidirectional changes in redox ratio, a single range of NO, exposure was chosen (200-500 ppm) in which unequivocal redox changes could be expected, the high concentrations of NOz producing redox changes larger than those at lower NO, concentrations (Cassan and Simmons, 1975a). The mean NO, concentration was 336.6 ppm (Table 1). Mean pH was identical in both control and NO,-exposed flasks. Mean p0, was 142.3 and 144.3 in NO,-exposed and control groups, respectively, corresponding to fully saturated hemoglobin. The sum of pyruvate and lactate produced was similar in both groups (Table 1) as had previously been noted (Cassan and Simmons, 1975a). A trend toward an
7.23 20.04 0.138 z-o.10
142.3 24.8 0.791 >O.lO
336.6 k89.l
Mean value + SD (II = 8) r P
7.23 to.03
PH
144.3 24.7
PO, (Tow)
UTILIZATION,
Mean value t SD (II = 8)
NO, (ppm)
Gr.ucos~
TABLE
AIR
2.008 >0.05
0.129 0.046
+ NO,
0.767 >O.lO
0.928 0.135
1.017 0.276
AIR 0.09 0.027
LACIATE
Lactate (mM/Yhr)
AND
1
Pyruvate (mM/l/hr)
PYKLIVAI.E
0.406 >O.lO
1.057 0.166
1.107 0.271
Pyruvate + lactate (mM/l/hr)
PR~DLTTION
4.332
24.9 8.36
IO.1 3.44
Total glucose utilized (%)
4.371
1.25 ,406
Sl .167
Actual glucose utilized m&l/hr
0.088 >o. IO
2.00 1.354
2.09 2.128
Percentage total glucose utilized-via Pentose shunt
s w
;
z i2
6 Fs
5 w
c z
THE
EFFECT
OF
NO2
ON
RBC
GLUCOSE
METABOLISM
177
increase in mean pyruvate and a decrease in mean lactate produced in NO, exposed samples was noted, but these changes were not statistically significant, perhaps due in the case of pyruvate to the unusually low value in experimental sample 2. Mean glucose utilization (Table 1) was significally increased to 24.9% of total glucose in the NO,-exposed erythrocytes compared with 10.1% in control samples. The mean actual glucose utilized in control samples of 0.51 m;\l/liter/hr corresponded well with the mean total lactate and pyruvate produced of 1.107 m&iter/hr. In NO,-exposed erythrocytes, however, the mean total lactate and pyruvate produced of 1.057 mM/liter/hr was less than half the quantity expected for the glucose utilized of 1.25 mM/liter/hr based on the production of 2 mole of lactate or pyruvate for each mole of glucose used. The fraction of glucose metabolized via the pentose pathway remained relatively constant, with mean values of 2.09 and 2.00% in control and NO,-exposed samples, respectively, so that actual glucose utilization via the pentose pathway increased proportionally to the increase in total glucose metabolized. DISCUSSION
Since the purpose of the study was to correlate NO,-induced changes in glucose metabolism with alterations in redox ratio previously described with NO, exposures for 2 hours (Cassan and Simmons, 1975a) and since redox ratio is calculated from momentary levels of glycolytic intermediates, the mean rate of glucose metabolism was also determined at 2 hours following incubation. The dynamics of the changes may involve a series of time-related alterations in glucose metabolism not reflected in these results. There may perhaps be several reactions with different half-times, of which this data is the net result. The increase in pentose pathway activity of the red cell during NO, exposure, as indicated by increased l-Cl4 utilization, is consistent with protective responses of erythrocytes exposed to other oxidants (Davidson and Tanaka, 1972). All oxidants, however, do not appear to act in the same way since only NO, appears to effect a change in cytoplasmic redox state in more complex cells (Simons et al., 1974). As in the case of hyperoxic exposure, the increase in pentose pathway activity in this study is limited and represents only a small fraction of the potential pentose pathway activity which can be induced with methylene blue (Davidson and Tanaka, 1972; Imarisio et nl., 1969). The mechanism by which the pentose shunt is stimulated by NO, exposure could include direct or indirect oxidation of NADPH or glutathione (GSH) (Menzel et al., 1972; Jaffe, 1971; Willis and Kratzing, 1972). In more complex cells, exposure to oxidants is associated with significant increases in activity of pentose pathway related enzymes (Tierney et al., 1973). Glucose utilization by the Embden-Meyerhof pathway was also increased by NO, exposure. The reason for this is not clear. In previous studies on the effect of anaerobiosis on erythrocyte glycolysis (Hamasaki et al., 1970; Cassan ef al., 1972), decreases in red cell [NAD+]/[NADH] ratio determined indirectly (Krebs and Veech, 1969) and based on mass action equilibria were inversely related to glycolytic rate. Since [NAD+]/[NADH] ratio in NO,-exposed erythrocytes is significantly increased (Cassan and Simmons, 1975a), it was expected that glucose utilization by the Embden-Meyerhof pathway might be concomitantly diminished, but this was
178
CASSAN,
GROSS
AND
SIMMONS
not the case. An increase in [NAD+]/[NADH] ratio might be expected to increase glycolysis, as found in this study, by improving the availability of oxidized nucleotide at the glyceraldehyde-3-phosphate dehydrogenase step. Although the latter enzyme is not rate limiting under these experimental conditions, substrate availability for subsequent reactions in glycolysis would be affected. On the other hand, reduction in [NAD+]/[NADH] ratio in the presence of high glycolytic rates (Rose, 1971), and high nucleotide ratios accompanying reduced glycolysis (Omachi et nl., 1969) have been described by others and are related to variations in the pool of 2,3 DPG. Thus, glycolytic rate may be largely independent of the redox ratio, which is determined primarily by relative concentrations of glycolytic intermediates. Alternately, the apparent discrepancies between redox ratio and glycolytic rate may represent evidence for the absence of mass action equilibrium conditions upon which indirect calculation of apparent redox ratios are based. In the present study, NO, exposure resulted in increased glucose utilization, but the total lactate and pyruvate production did not increase. This suggests a metabolic block in the Embden-Meyerhof pathway leading to accumulation of glycolytic intermediates proximal to these products. The increase in [NAD+]/ [NADH] ratio suggests this locus to be proximal to the glyceraldehyde-3-phosphate dehydrogenase step. Such an NO, effect, perhaps on phosphofructokinase, could be similar to that previously described for a pH-independent effect of CO, in canine erythrocytes (Zborowska-Sluis and Klassen, 1972). However, other NO, effects altering the [NAD+]/[NADH] ratio, including a direct NO, oxidant effect on NADH or alteration of NADH dependent enzymes (Vassallo et al., 1973) cannot be excluded, although stoichiometric considerations suggest that NO, is not present in sufficient quantities to explain redox alterations on the basis of direct NADH oxidation alone. An elucidation of effects on glucose metabolism and the associated redox changes with NOz exposure may assist in defining the mechanism of NO* induced cellular damage, particularly since lipid peroxidation hitherto invoked as the essential mechanism of cell damage (Roehm, 1971) and redox alterations (Mintz, 1972), appears not to be involved in the production of these effects in normal human erythrocytes (Cassan and Simmons, 1975b; Cassan and Simmons, in press). Although morphologic studies were not performed in these latter studies, evidence of hemolysis which is known to accompany lipid peroxidation was not observed despite the high NO, concentrations used. ACKNOWLEDGMENTS The authors wish to thank Jane Christoferson for laboratory assistance and Julie Taylor for typing of manuscripts.
REFERENCES Bergmeyer, H. U., Ed. (1963). “Methods of Enzymatic Analysis.” Academic Press, New York. Cassan, S. M., and Simmons, D. H. (1975a). The effect of vitamin E on NO, induced redox changes in the human erythrocyte. Environ. Physiol. Biochern. 5, 201-207. Cassan, S. M., and Simmons, D. H. (1975b). The effect of NO, on redox state of the human erythrocyte. Arch.
Environ.
Health
30, 502-506.
Cassan, S. M., and Simmons, D. H. Anerobiosis Emiron. Res., in press.
and the effect of NO2 on the human erythrocyte.
THE
EFFECT
OF
NO2
ON
RBC GLUCOSE METABOLISM
179
Cassan, S. M., Theodore, J., and Robin, E. D. (1972). The effect of low 0, tensions on red cell glycolysis, NAD+/NADH ratios and transmembrane potential (TMP)-The Pasteuroid effect. C/in. Res. 20, 574.
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