Sex Differences in the Abundance of Endothelial Nitric Oxide in a Model of Genetic Hypertension
Abstract A deficiency of nitric oxide may be responsible for the increased vascular resistance associated with human essential hypertension and that seen in animal models of hypertension. Premenopausal females are relatively protected from hypertension and cardiovascular complications. Levels of superoxide can influence the availability of nitric oxide. We hypothesize that there are differences in nitric oxide availability between stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY) and that superoxide may be responsible for at least some of these differences. We studied vascular reactivity in endothelium-intact aortic rings from WKY and SHRSP. We measured nitric oxide synthase activity in endothelial cells removed from aortas and also measured circulating nitrite/nitrate levels. We found the response to NG-nitro-l-arginine methyl ester to be significantly greater in WKY compared with SHRSP (95% CI: 20 to 174; P=.015) and in females compared with males in WKY (95% CI: 143 to 333; P=.00004) and SHRSP (95% CI: 70 to 224; P=.0006). Endothelial nitric oxide synthase activity was significantly greater in SHRSP compared with WKY (95% CI: 2.3 to 17.6; P=.016). The EC50 for relaxation to carbachol was significantly greater in male rats compared with female rats (95% CI: −1.1 to −0.2; P=.003) within the SHRSP strain. The maximum relaxation to carbachol was significantly attenuated in stroke prone spontaneously hypertensive compared with Wistar-Kyoto rats (95% CI: 1.7 to 14.4; P=.015). Diethyldithiocarbamate had a significantly greater effect on the stroke prone spontaneously hypertensive rats’ carbachol response than that of Wistar-Kyoto rats (95% CI: 14.3 to 47.0; P=.0008). We conclude that superoxide may be responsible for strain differences in vascular reactivity, whereas nitric oxide availability may be responsible for sex differences independently of endothelial nitric oxide synthase activity and superoxide.
- rats, stroke-prone spontaneously hypertensive
- nitric oxide
- nitric oxide synthase
- superoxide anion
- superoxide dismutase
It is known that human cardiovascular disease is less common in women of reproductive age compared with age-matched males.1 There is also evidence from animal models for delayed and less severe hypertension2 and atherosclerosis3 in females compared with males. The mechanism of this difference is not known. NO, synthesized by NOS, and released from endothelial cells, regulates tone in normal vessels4 and is thought to protect against atherogenesis.5 There is evidence for sex differences in endothelial dysfunction in hypercholesterolemic humans6 and animal models of genetic hypertension.7 It has been suggested that sex differences in endothelial function are due to increased NO release in females, perhaps related to sex hormones,6 7 although this has never been proved.
There is evidence from animal models of genetic hypertension that sex hormones are important in the development of hypertension.2 8 9 10 There is also evidence from human studies that estrogen replacement can improve coronary risk factors11 and protect against coronary heart disease12 in postmenopausal women, and in animal models it can protect against atherosclerosis.13 Some of this sex hormone effect has been attributed to endothelial function in humans14 and animal models,15 16 17 although beneficial effects on lipid profiles13 and altered responses to other vasoactive molecules (including catecholamines,18 prostanoids,19 vasopressin,20 and endothelin 121 ) have also been reported.
There are theoretical reasons why estrogens may regulate eNOS expression. The 5′ flanking region of the bovine eNOS gene contains 15 copies of the half palindromic estrogen responsive element.22 In addition, 17β-estradiol has been shown to increase eNOS mRNA in cultured human endothelial cells23 and in rat aortas.24
The question of whether or not NO deficiency contributes to the pathogenesis of hypertension is still hotly debated. Studies of NO release in human essential hypertension25 26 27 and in animal models of genetic hypertension28 29 30 have been contradictory. An alternative theory is that NO may be released in normal or even greater amounts in humans or animals with apparent endothelial dysfunction, but the NO may be scavenged before it can activate guanylyl cyclase. One of the known scavengers is the superoxide radical (O2−), which reacts with NO to form peroxynitrite (ONOO−).31 Peroxynitrite is itself a weak vasodilator32 and has toxic effects of its own,33 but the major consequence of this reaction is to remove the potent vasodilator effect of NO. There is evidence that the apparently reduced NO availability demonstrated in the SHRSP can be exactly accounted for by increased levels of O2−.34 There is also evidence that O2− influences the vascular reactivity of human vessels35 and that it may contribute to the hypertension in the spontaneously hypertensive rat.36
We hypothesize that there are differences in NO availability between SHRSP and the normotensive reference strain WKY and/or between males and females within each strain. Furthermore, we aim to determine if any differences are due to reduced NO release or increased functional levels of O2−.
We studied 132 rats at 16 weeks of age in eight groups. For isometric tension recording we used WKY females (n=12), WKY males (n=17), SHRSP females (n=14), and SHRSP males (n=14). For assay of NOS activity we used WKY females (n=20), WKY males (n=15), SHRSP females (n=20), and SHRSP males (n=20).
These rats were obtained from WKY and SHRSP colonies established in Glasgow by brother-sister mating as previously described.37 These colonies were originally established from rats obtained from those maintained at the University of Michigan, which had obtained their breeding stocks from the National Institutes of Health (personal communication, D.F. Bohr, Department of Physiology, University of Michigan, 1991). All experiments were approved by the Home Office according to regulations regarding experiments in animals in the United Kingdom. These regulations are equivalent to those used by the American Physiological Society.
In the week before sacrifice, indirect blood pressure was measured in conscious, restrained rats habituated to the procedure by tail plethysmography as previously described.38 Rats were prewarmed to 34°C for 10 to 15 minutes before measurements.
Aorta: Isometric Tension Recording
The rats were killed by halothane overdose. The thoracic aorta was carefully removed, cleaned of adipose tissue, and cut into 2- to 3-mm rings. The rings were suspended under 1g tension in individual 10-mL muscle baths containing physiological saline solution of the following composition (mmol/L): NaCl 130, KCl 4.7, NaHCO3 14.9, KH2PO4 1.18, MgSO4·7H2O 1.17, CaCl2·2H2O 1.6, glucose 5.5, and CaNa2EDTA 0.03. The physiological saline solution was aerated with 95% O2/5% CO2, and indomethacin was added to a final bath concentration of 10−5 mol/L. Isometric tension was measured by a force transducer and recorded by a multichannel pen recorder.
Potassium chloride (KCl) was used as a receptor-independent vascular smooth muscle cell depolarizing agent. At 100 mmol/L maximal contraction is obtained. The noradrenaline analogue PE was used to constrict the rings via α-adrenoceptors. Carbachol, a stable analogue of acetylcholine, was used to relax the rings in an endothelium-dependent manner via muscarinic receptors, resulting in stimulated NO release. The NO donor SNP was used to relax the rings in an endothelium-independent manner. The NOS inhibitor L-NAME was used to inhibit basal eNOS activity. The resultant contraction is a measure of basal NO availability. The cell membrane permeable copper chelating agent DETCA was used to inhibit intracellular and extracellular copper zinc (Cu/Zn) SOD. Exogenous SOD was added to remove extracellular O2−. This form of SOD does not enter cells.
After a 1-hour equilibration period, four different protocols were followed on 4 to 17 rings from each group. Protocol 1 is illustrated in Fig 1a⇓. An initial response to KCl (100 mmol/L) was recorded. After washout a concentration-response curve for PE (10−8 to 10−5 mol/L) was obtained. After a further washout, the rings were preconstricted to the EC50 of PE, and a concentration-response curve for carbachol (10−8 to 10−5 mol/L) was obtained. The carbachol relaxation was expressed as a percentage of the PE EC50 in the conventional way to correct for any influence of body size on vessel structure. PE and carbachol were washed out and the rings were again preconstricted to the EC50 of PE, and a concentration-response curve for the endothelium-independent vasorelaxant SNP (10−8 to 10−5 mol/L) was obtained as a measure of vascular smooth muscle responsiveness to NO. Again the relaxations were expressed as a percentage of the PE EC50 to correct for any influence of body size on vessel structure.
Protocol 2 is illustrated in Fig 1b⇑. KCl, PE, and carbachol responses were obtained as above. After washing out the carbachol, the rings were preconstricted to the EC20 for PE, and the NOS inhibitor L-NAME was added to a final concentration of 10−4 mol/L. The resultant contraction, as a percentage of the PE EC20 contraction, is a measure of basal availability of NO. Again we express the contraction relative to the PE EC20 to correct for any influence of body size on vessel structure.29
Protocol 3 is illustrated in Fig 1c⇑. KCl, PE, and carbachol responses were obtained as above. Rings were then incubated with the SOD inhibitor DETCA (10 mmol/L) for 45 minutes. Concentration-response curves for PE and carbachol were again obtained as for protocol 1.
Protocol 4 is illustrated in Fig 1d⇑. KCl, PE, and carbachol responses were obtained as above. Rings were treated with exogenous SOD (45 U/mL), and a concentration response curve for PE obtained and washed out. Exogenous SOD (45 U/mL) was again added and a concentration response curve obtained for carbachol carbachol as in protocol 1.
Citrulline Assay of NOS Activity
Measurement of NOS activity was as described.39 Fresh endothelial cells were obtained by carefully removing and cleaning the thoracic aorta as described above. The aorta was opened along its length and the endothelium removed into homogenizing buffer (pH 7.4, Tris 50 mmol/L, sucrose 3.2 mmol/L, dithiothreitol 1 mmol/L, leupeptin 10 μg/mL, soybean trypsin inhibitor 10 μg/mL, and aprotinin 2 μg/mL). The aortas from five rats of the same sex and strain were pooled to reduce variability before assaying NOS activity. The buffer containing the endothelial cells was snap-frozen and stored at −70°C until ready to assay.
On the day of the assay, the cells were thawed, sonicated three times for 3 seconds and centrifuged at 10 000g for 20 minutes. A 40 μL aliquot of the resultant supernatant containing both the soluble and particulate NOS protein was incubated at 37°C with the assay buffer (pH 7.2, 50 mmol/L KH2PO4, 1 mmol/L MgCl2, 0.2 mmol/L CaCl2, 50 mmol/L valine, 20 μmol/L l-citrulline, 20 μmol/L l-arginine, 1 mmol/L dithiothreitol, 100 μmol/L NADPH, 3 μmol/L tetrahydrobiopterin, 3 μmol/L flavin adenine dinucleotide, 3 μmol/L flavin mononucleotide, and 0.05 μCi ≈1 μmol/L L-[U-14C]-arginine). This was repeated in the presence of 3 mmol/L EGTA (a calcium chelator) and again in the presence of 1 mmol/L Nω-iminoethyl-l-ornithine a NOS inhibitor.40 After 20 minutes the re- action was terminated by the addition of 1:1 (vol/vol) Milli-Q water/activated Dowex-AG50W resin (200 to 400, 8% cross-linked, Na+ form), which removes arginine from the mixture. The resin was allowed to settle for 30 minutes, and the amount of labeled citrulline present in the supernatant was quantified in terms of counts per minute by a liquid scintillation counter. The soluble protein content of the supernatant was determined by the Coumassie blue binding method using Bio-Rad protein reagent with bovine serum albumin as standard. NOS activity is expressed as picomoles of citrulline produced per minute per milligram of protein. Total NOS activity was calculated by subtracting NOS-independent citrulline production (ie, that in the presence of Nω-iminoethyl-l-ornithine) from the overall activity and calcium-dependent NOS activity calculated as that which disappeared in the presence of EGTA.
Measurement of NOx
Serum NOx concentrations were measured in blood taken from a number of animals in each group when the animals were killed. NOx measurement was by a modified version of a previously published method41 that uses glycine buffer as the carrier solvent.42 The between-day coefficient of variation was less than 3%.
The concentration-response curves to PE, carbachol, and SNP were characterized by the Emax, and the concentration that produced 50% maximum effect (EC50). These parameters were calculated from the raw data for each pharmacological intervention for each aorta using Microsoft Excel. The response to L-NAME for each aorta was taken as the maximal contraction expressed as a percentage of the contraction to EC20 of PE.
The mean values and SEMs for each group were calculated, and the following comparisons were made by two-way ANOVA followed by unpaired t tests with Bonferroni correction for three comparisons: all SHRSP (males and females) versus all WKY (males and females), WKY females versus WKY males, and SHRSP females versus SHRSP males. Therefore, a value of P<.017 was considered to be significant. Because the EC50 values for carbachol and SNP relaxations are logarithmic and therefore not normally distributed, the data were normalized by log-transformation before calculating the mean and SEM for each group and comparing groups by unpaired t tests as above.
Mean systolic blood pressure (±SEM) and mean body weight (±SEM) for the four groups are shown in Table 1⇓.
Effect of L-NAME
When L-NAME was added to the rings at the EC20 of PE, the resultant contraction, relative to the initial PE EC20 contraction (% of PE±SEM), was significantly greater in WKY (379±31) compared with SHRSP (282±23) (95% CI, 20 to 174; P=.015). Furthermore, the contraction in rings from WKY females (519±37) was significantly greater than WKY males (280±26) (95% CI, 143 to 333; P=.00004). Similarly, the contraction in rings from SHRSP females (356±29) was significantly greater than SHRSP males (209±24) (95% CI, 70 to 224; P=.0006) (Fig 2⇓).
Effect of Carbachol
Fig 3⇓ shows the effect of adding carbachol at the EC50 of PE. The Emax for carbachol relaxation (% of PE±SEM) of WKY rings (94.4±1.6) was significantly greater than that of SHRSP rings (86.3±2.7) (95% CI, 1.7 to 14.4; P=.015). There was no significant difference in the mean maximum relaxation when WKY females were compared with WKY males or when SHRSP females were compared with SHRSP males (Table 2⇓).
There was no significant difference in the log-transformed EC50 for carbachol relaxation (log mol/L±SEM) when the concentration-response curves for WKY rings were compared with SHRSP rings or when WKY females were compared with WKY males (Table 2⇑). However, within the SHRSP strain, there was a significant difference in the mean log-transformed EC50 when females (−0.39±0.14) were compared with males (0.29±0.15) (95% CI, −1.1 to −0.2; P=.003) (Fig 3c⇑).
Effect of SNP
Fig 4⇓ shows the effect of adding SNP at the EC50 of PE. There was no significant difference in the mean Emax for SNP relaxation (% of PE±SEM) when WKY rings (100±1.7) were compared with SHRSP rings (100±1.9) (95% CI, −5.7 to 5.4; P=.95). Similarly, there was no significant difference when WKY females (99.3±3.4) were compared with WKY males (100±1.3) (95% CI, −9.6 to 6.6; P=.69) or when SHRSP females(100±0.5) were compared with SHRSP males (100±4.1) (95% CI, −9.6 to 10.6; P=.91).
There was no significant difference in the mean log-transformed EC50 for SNP relaxation (log mol/L±SEM) when WKY rings (1.59±0.45) were compared with SHRSP rings (2.40±0.54) (95% CI, −2.3 to 0.7; P=.27); when WKY females (1.17±0.8) were compared with WKY males (1.93±0.6) (95% CI, −3.0 to 1.5; P=.44) or when SHRSP females (2.72±0.3) were compared with SHRSP males (2.14±1.0) (95% CI, −2.3 to 3.5; P=.60).
Effect of DETCA on Carbachol Responses
Fig 5⇓ shows the effect of preincubation with the SOD inhibitor DETCA on WKY and SHRSP rings. As shown in Table 3⇓, DETCA attenuated the relaxation to carbachol in WKY and SHRSP males and females. DETCA attenuated the relaxation (% change) to carbachol of SHRSP (59±5.1) significantly more than WKY (29±6.0) (95% CI, 14.3 to 47.0; P=.0008). The attenuation by DETCA on rings from WKY females did not differ significantly from WKY males. Similarly, the attenuation in rings from SHRSP females did not differ significantly from SHRSP males after DETCA preincubation.
As shown in Table 3⇑, DETCA preincubation tended to increase the EC50 (×10−7 mol/L) for carbachol relaxation. However, there was no significant difference in this increase (% change) between strains or between sexes within each strain.
Effect of Exogenous SOD on Carbachol Relaxations
Table 3⇑ shows the effect of coadministration of exogenous SOD to the rings on their response to carbachol. Exogenous SOD tended to improve the relaxation to carbachol, especially in SHRSP. However, the improvement (% change) did not differ significantly between strains or between sexes within each strain.
Exogenous SOD tended to reduce the EC50 (×10−7 mol/L) of the carbachol response curves, again especially in SHRSP. However, when the % change in EC50 was compared between strains or between sexes within each strain, there was no significant difference.
Effect of DETCA on Contraction to PE
The effect of preincubation of the rings with DETCA on the contraction to PE is shown in Table 4⇓. When the rings were preincubated with DETCA the mean Emax and EC50 to PE (relative to KCl) were increased in all groups. However, there was no significant difference in the % change when strains or sexes within each strain were compared.
Effect of Exogenous SOD on Contraction to PE
Table 4⇑ also shows the effect of coadministration of exogenous SOD to the rings on their response to PE. Exogenous SOD reduced the maximum contraction to PE and increased the EC50. This effect (% change) did not differ significantly between strains or between sexes within each strain.
Endothelial NOS Activity
Endothelial NOS activity (pmol/min per mg protein) is significantly greater in the aortas from SHRSP (24.6±3.1) compared with those from WKY (14.6±1.3) (95% CI, 2.3 to 17.6; P=.016). Endothelial NOS activity tends to be greater in WKY males compared with WKY females and similarly in SHRSP males compared with SHRSP females, but these differences do not reach significance (Fig 6⇓).
When corrected for triple comparison, there was no significant difference in NOx (μmol/L±SEM) between WKY (10.6±0.66, n=11) and SHRSP (13.3±1.2, n=14) (95% CI, −55.5 to 0.1; P=.06); WKY females (12.2±0.64) and WKY males (9.32±0.74) (95% CI, 0.62 to 5.14; P=.02); or SHRSP females (12.4±1.3) and SHRSP males (13.9±1.7) (95% CI, −6.2 to 3.3; P=.52).
We have, for the first time, demonstrated reduced basal NO availability in aortas of SHRSP compared with those from WKY by examining the response to the NOS inhibitor L-NAME. This is in accordance with Malinski et al28 who have looked at stimulated NO release in response to bradykinin in cultured aortic endothelial cells isolated from SHRSP and WKY. In addition, we have demonstrated reduced basal NO availability in males compared with females in both strains. This sex difference in endothelial function has been described after acetylcholine stimulation,7 in pregnant spontaneously hypertensive rats,16 rats, and rabbits treated with exogenous sex hormones,15 17 and under basal conditions in untreated rabbits,43 but there are no previous reports describing the effects of sex and blood pressure on basal and stimulated NO availability in the same model.
We also examined stimulated NO release in response to the acetylcholine analogue carbachol. We demonstrated a strain difference in carbachol Emax, with SHRSP rings relaxing to a significantly lesser extent than WKY rings. We also found that within the SHRSP strain, males are less sensitive to carbachol because they have a significantly greater carbachol EC50 than females. Despite finding a sex difference in basal NO in WKY, we found no such sex difference in the carbachol EC50 within the WKY strain. This suggests a different mechanism for regulation of basal and stimulated NO availability. Such a difference has been suggested by others.44 45 The presence of a sex effect within the SHRSP strain, but not within the WKY strain, may be related to the large sex difference in blood pressure in SHRSP, whereas WKY show only a small sexdifference. It is not clear whether this relationship between blood pressure and endothelial function is cause or effect.7
We found no strain or sex differences in the response to SNP, suggesting that the responsiveness of the vascular smooth muscle to NO is not altered. The absence of a sex difference in the response of aortic rings of SNP has been described before.7 One would not expect differences in luminal or endothelial O2− to affect the response to SNP, which is an endothelium-independent vasodilator, and does not give up its NO until it reaches the smooth muscle.46
The methods we used examine only the functional levels of basal or stimulated NO that are available to the underlying vascular smooth muscle. The reduced availability of basal and stimulated NO in SHRSP may be due to reduced production (as was commonly thought), increased scavenging, or both. In an attempt to answer this question, we measured NOS activity directly in endothelial cells isolated from the aortas of SHRSP and WKY rats. We found increased NOS activity in SHRSP compared with WKY, suggesting that NO production is increased rather than decreased in the hypertensive animals. This may represent a compensatory mechanism to some other blood pressure increasing–factor or –factors. Others have found impaired endothelium-dependent vasorelaxation in aortas of rats with genetic hypertension that is not due to reduced eNOS activity as measured by inhibition of endothelin contractile response.47 It therefore seems likely that the reduced NO availability in SHRSP is due to increased scavenging. In addition, we have demonstrated a small reduction in eNOS activity in females compared with males of both strains. Although this sex difference does not reach statistical significance, it is contrary to reports that 17β-estradiol increases eNOS message23 24 but in agreement with a report that showed no effect of estrogen on eNOS activity.48 Perhaps there is posttranscriptional downregulation of eNOS or inhibition of the enzyme in females of these strains. In both strains the males have higher blood pressure than the females. The increased eNOS activity in males is further evidence that NO production is increased as a compensatory mechanism.
It is known that NO can be scavenged by the free radical O2− to form peroxynitrite.31 Although it is a weak vasodilator,32 the formation of peroxynitrite would reduce the availability of NO. There is evidence that the apparently reduced NO availability demonstrated in the SHRSP can be exactly accounted for by increased levels of O2−.34 There is also evidence that O2− influences the vascular reactivity of human vessels35 and may contribute to the hypertension in some animal models.36 An excess of O2− is therefore a likely candidate for reducing basal and/or stimulated NO availability in SHRSP. To investigate this possibility we studied the effects of exogenous SOD and the SOD inhibitor DETCA.
As one would expect, by inhibiting SOD and thereby increasing O2− levels, DETCA attenuated the carbachol relaxation in WKY and SHRSP and also in males and females within both strains. We have previously demonstrated in male animals that the attenuation by DETCA is significantly greater in SHRSP than WKY.34 Here we have demonstrated that when males and females of both strains are considered, the attenuation of the carbachol response by DETCA is still significantly greater in SHRSP compared with WKY. This could mean that SOD is more active or present in higher concentrations in the aorta of SHRSP and that it therefore inhibits the enzyme and produces a greater effect; or it could mean that when SOD is completely inhibited, O2− accumulates faster in SHRSP and thus it impairs relaxation to carbachol to a greater extent. We observed no sex differences in the attenuation of carbachol relaxation by DETCA. This would suggest that some other mechanism is responsible for sex differences in NO availability.
DETCA increased the mean Emax and mean EC50 of the PE concentration-response curves in all groups as one would expect from its action of indirectly removing the vasodilator tone of NO. The changes in individual animals were variable, and no significant strain or sex effects were detected.
The addition of exogenous SOD to the rings tended to augment the carbachol relaxations. This effect was most marked in SHRSP males who had the poorest relaxation to begin with. Nonetheless, the control relaxations were so good (>85% of PE in most cases) that there was very little room for improvement. The responses to SOD were consequently relatively small and quite variable, resulting in no detectable strain or sex differences. Again, as one might expect, exogenous SOD attenuated the contraction to PE in all groups. As before, the effects were relatively small and variable, resulting in no detectable strain or sex differences. Exogenous SOD added to the water-bath is unable to enter the cells and therefore only removes extracellular O2−. This may also partly explain the relatively small effect. Others have encountered the same problem and attempted to overcome this by using polyethylene-glycolated SOD,49 but such compounds are not widely available.
We detected no significant strain or sex differences in serum NOx. This may be because the diet was not standardized for NOx intake. In addition, all isoforms of NOS, not just eNOS, will contribute to the serum NOx, thus confounding any underlying difference in NO production by eNOS.
We conclude that excess intracellular O2− may be responsible for reduced endothelium-dependent vascular relaxation in SHRSP. The reason for this excess is not clear. One may speculate that increased intracellular O2− production in SHRSP may be responsible. Laursen et al have shown increased O2− production in angiotensin II–induced compared with norepinephrine-induced hypertension.50 Our data would support a compensatory increase in SOD activity and eNOS activity in SHRSP that is insufficient to correct the NO/O2− imbalance. We further conclude that differences in NO availability, although apparently not due to differences in eNOS activity or O2− levels, are responsible for sex differences in vascular reactivity. Increased NO availability may contribute to the relative cardiovascular protection in females.
Selected Abbreviations and Acronyms
|eNOS||=||endothelial nitric oxide synthase|
|L-NAME||=||NG-nitro-l-arginine methyl ester|
|NOS||=||nitric oxide synthase|
|SHR||=||spontaneously hypertensive rats|
|SHRSP||=||stroke-prone spontaneously hypertensive rats|
This work was supported by a Scottish Hospital Endowments Research Trust Jean Baxter Fellowship to M.M. and British Heart Foundation Grants PG95123 and FS92035. A.F.D. is a British Heart Foundation Senior Research Fellow. We wish to acknowledge the assistance of Professor Nigel Benjamin and Dr Mahesh Patel in performing the NOx assay.
- Received June 12, 1997.
- Revision received June 16, 1997.
- Accepted June 16, 1997.
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