Acute hypoxic pulmonary vasoconstriction can be inhibited by high doses of the carbonic anhydrase inhibitor acetazolamide. This study aimed to determine whether acetazolamide is effective at dosing relevant to human use at high altitude and to investigate whether its efficacy against hypoxic pulmonary vasoconstriction is dependent on carbonic anhydrase inhibition by testing other potent heterocyclic sulfonamide carbonic anhydrase inhibitors. Six conscious dogs were studied in five protocols: 1) controls, 2) low-dose intravenous acetazolamide (2 mg·kg−1·h−1), 3) oral acetazolamide (5 mg/kg), 4) benzolamide, a membrane-impermeant inhibitor, and 5) ethoxzolamide, a membrane-permeant inhibitor. In all protocols, unanesthetized dogs breathed spontaneously during the first hour (normoxia) and then breathed 9–10% O2 for the next 2 h. Arterial oxygen tension ranged between 35 and 39 mmHg during hypoxia in all protocols. In controls, mean pulmonary artery pressure increased by 8 mmHg and pulmonary vascular resistance by 200 dyn·s·cm−5 (P <0.05). With intravenous acetazolamide, mean pulmonary artery pressure and pulmonary vascular resistance remained unchanged during hypoxia. With oral acetazolamide, mean pulmonary artery pressure increased by 5 mmHg (P < 0.05), but pulmonary vascular resistance did not change during hypoxia. With benzolamide and ethoxzolamide, mean pulmonary artery pressure increased by 6–7 mmHg and pulmonary vascular resistance by 150–200 dyn·s·cm−5 during hypoxia (P < 0.05). Low-dose acetazolamide is effective against acute hypoxic pulmonary vasoconstriction in vivo. The lack of effect with two other potent carbonic anhydrase inhibitors suggests that carbonic anhydrase is not involved in the mediation of hypoxic pulmonary vasoconstriction and that acetazolamide acts on a different receptor or channel.
- high altitude
- hypoxic pulmonary vasoconstriction
the oral carbonic anhydrase (CA) inhibitor acetazolamide is frequently used for prevention and treatment of acute mountain sickness (AMS) and to augment ventilation for high-altitude acclimatization (16, 18). Given the effectiveness of acetazolamide and other CA inhibitors in AMS, it was reasonable to consider whether CA inhibitors might also reduce high-altitude pulmonary edema (HAPE) by inhibition of hypoxic pulmonary vasoconstriction (HPV). Contained within an early study by Emery et al. (6) was a brief note that acetazolamide reduced HPV in isolated perfused lungs. This was confirmed in a more comprehensive study in the isolated blood-perfused rabbit lung by Deem et al. (4), who showed that acetazolamide slowed the kinetics and reduced the magnitude of HPV by roughly 50%. To extend this finding to the in vivo situation, we showed in live unanesthetized Beagle dogs that a dose of 10 mg/kg, sufficient to inhibit 98–99% of intracellular and extracellular CA, completely inhibited HPV (9). In parallel studies, we have also demonstrated that acetazolamide in isolated rat pulmonary artery smooth muscle cells inhibits the increase in intracellular calcium concentration ([Ca2+]i) upon exposure to 4% oxygen (13), a fundamental step in the initiation of HPV. The clinical relevance of these findings on HPV was explored in rats exposed to 24 h of hypobaric hypoxia (barometric pressure = 380 mmHg). Pretreatment with acetazolamide (20 mg/kg) prevented the alveolar protein leak typical of HAPE, as did nickel chloride, another HPV inhibitor (3).
In the present paper, we investigated whether acetazolamide at lower doses more typical of those used in humans at high altitude (2–5 mg/kg) would remain effective against HPV. In addition, to determine whether the effect of acetazolamide on HPV is primarily due to extra- or intracellular CA inhibition or whether the effect of acetazolamide might actually be independent of CA inhibition, we used benzolamide (a powerful, hydrophilic, cell membrane-impermeant CA inhibitor), which selectively inhibits the extracellular luminal membrane-bound CA isoenzyme IV, and ethoxzolamide (a potent lipophilic cell membrane-permeant CA inhibitor), which yields both intra- and extracellular enzyme inhibition (11). The structures and chemical characteristics of the three inhibitors are presented in Table 1.
The present investigation was performed during 1 h of normoxia and 2 h of hypoxia in conscious, spontaneously breathing dogs in whom the systemic acid-base changes with acute CA inhibition are present and all humoral and neural regulatory mechanisms affecting acute HPV remain intact. In this integrative approach, we not only measured hemodynamics, but also arterial blood gases and electrolytes, plasma vasoregulatory hormones, renal function, and urine output in an attempt to better understand the full dose-response relationship of acetazolamide against HPV in vivo in relationship to its inhibition of CA.
MATERIALS AND METHODS
Animal maintenance and diet.
A total of 30 experiments were performed on 6 purebred female Beagle dogs (body wt 14.3 ± 0.3 kg). The dogs were obtained from the Central Animal Facilities of the Humboldt-University in Berlin. They were tested for their social behavior, tolerance to urinary bladder catheterization, and intravascular cannulae. Permission to perform experiments was obtained from the Governmental Animal Protection Committee (AZ VB103 - G 0084/04).
The dogs were kept under highly standardized conditions: an air-conditioned animal room during the day and large individual kennels (5 m2) during the night (21°C, 55% humidity). General status, body temperature, and weight were checked daily. The dogs were trained to lie quietly on their right side on a padded animal table for at least 3 h. During the entire experimental period, the dogs were attended by a technician, who knew the animals well and could provide a calming influence by voice and petting.
Five days before the experiments, the dogs were begun on a standardized diet. The diet contained 91 ml of water, 2.5 mmol sodium, and 3.5 mmol potassium (all values given per kg body wt and per day). The calories supplied with this diet (277 kJ·kg body wt−1·day−1) were sufficient to keep the dogs' body weight constant. The food mash was offered once a day at 2 PM. Eight days before an experiment, 50 ml of the dog's own blood was collected via puncture of a foreleg vein and stored in a blood bag at 4°C (Biopack; Biotrans, Dreieich, Germany). The blood served to replace the blood withdrawn for analysis during the experiments.
After completion of the studies, the dogs were adopted by private persons with the assistance of our university veterinarians.
Procedures during the experiments.
Preparation of the dogs started at 7:30 AM. Body weight and temperature were recorded. The urinary bladder was catheterized with a self-retaining Foley catheter. After local anesthesia (1% lidocaine; Braun, Melsungen, Germany), an arterial line (20 G, No. 4235-8; Ohmeda, Erlangen, Germany) was advanced into the abdominal aorta via the femoral artery, and a pulmonary artery catheter (5 F, No. 132F5; Baxter, Unterschleissheim, Germany) was inserted via the right external jugular vein. The catheters were used for continuous systemic and pulmonary blood pressure monitoring, cardiac output measurements, and blood sampling. After catheter insertion, the dogs were placed on a padded animal table and positioned on their right side. The pressure transducers were adjusted to the level of the right atrium. The distance between transducer and table was recorded and also used for the next experiment on this individual dog. Finally, a face mask was attached, which enclosed the dog's snout in a leakproof fashion. This custom made mask was equipped with a 2.5-cm inner diameter connector to connect it to a ventilator set to continuous positive airway pressure mode (3–4 cmH2O, Servo 900 C; Siemens-Elema, Erlangen, Germany). This permitted spontaneous breathing through a low-resistance circuit. Thereafter, the conscious dogs were given 30 min to adjust to the experimental situation.
The six dogs were studied five times in randomized order: 1) control (no medication); 2) acetazolamide intravenously (iv acetazolamide): bolus injection of 2 mg/kg acetazolamide (Diamox; Lederle, Wolfratshausen, Germany) 15 min before the start of the experiment, followed by a continuous infusion of 2 mg·kg−1·h−1; 3) acetazolamide orally (po acetazolamide): dogs received 5 mg/kg acetazolamide orally at two times, 12 h and 1 h before the start of the experiment; 4) benzolamide intravenously: bolus injection of 2 mg/kg benzolamide 15 min before the start of the experiment, followed by a continuous infusion of 2 mg·kg−1·h−1. This dosage inhibits only renal and vascular endothelial extracellular CA without affecting red blood cell CA (19); and 5) ethoxzolamide intravenously: bolus injection of 1 mg/kg ethoxzolamide (Sigma Aldrich, Steinheim, Germany) 15 min before the start of the experiment, followed by a continuous infusion of 1 mg·kg−1·h−1. This somewhat lower dosage compared with iv acetazolamide and benzolamide was chosen for the fact that ethoxzolamide is a 5- to 10-fold more potent CA inhibitor (Ki value 0.7 × 109) (19), and its easy penetrance into the brain may induce more severe central nervous system (CNS) acid-base changes and greater hyperventilation if used at equivalent doses.
The interval between experiments on the same dog was at least 14 days to permit complete washout of the drugs, restoration of acid-base balance, and resolution of any hypoxic and diuretic effects.
In control experiments, the dogs breathed room air (21% O2, 79% N2; normoxia) for 1 h, followed by breathing a gas mixture containing 10% O2 and 90% N2 for 2 h (hypoxia). The time course was the same for the CA inhibitor experiments, but FiO2 was reduced to 0.09 during the hypoxia period to match the arterial and alveolar Po2 of the control dogs, which otherwise would have been higher on 10% O2 as a result of the known ventilatory stimulant effect of all CA inhibitors (9).
Mean arterial blood pressure (MAP), heart rate, central venous pressure (CVP), mean pulmonary artery pressure (MPAP), pulmonary capillary wedge pressure (PCWP), and minute ventilation (using the flow transducer in the ventilator) were measured continuously, and the data were stored on a computer. Cardiac output was measured using the thermodilution technique (5-ml injection volume at 5–10°C, Vigilance; Baxter Edwards Critical Care, Unterschleissheim, Germany). Five consecutive measurements were performed. The highest and lowest values were rejected. The mean cardiac output was calculated from the remaining three determinations and taken for calculation of systemic (SVR) and pulmonary (PVR) vascular resistance by standard formulas.
At the end of each experimental hour, blood samples were drawn to determine arterial blood gases, actual bicarbonate, base excess, plasma electrolytes, and plasma hormones. The blood withdrawn was immediately replaced with an equal amount of the dog's own stored blood.
Urine flow and urinary concentrations of sodium, potassium, and creatinine were determined for the normoxia and hypoxia period. Exogenous creatinine clearance (priming dose 1.4 g over 30 min before the start of experiments, maintenance infusion 4.7 mg/min) was calculated by the standard formula to assess glomerular filtration rate (GFR).
Measurement of urinary and plasma values.
Urinary and plasma sodium and potassium concentrations were measured by flame photometry (Photometer Eppendorf, Hamburg, Germany). Creatinine was determined with a creatinine analyzer (modified Jaffé reaction; Beckmann Instruments). Blood gas analysis and acid-base measurements were performed at hourly intervals (ABL 505; Radiometer, Copenhagen, Denmark). Blood samples for plasma hormone analysis were cooled and centrifuged at 4°C and stored at −20°C until analysis by commercial kits: plasma renin activity (DiaSorin, Diezenbach, Germany), plasma endothelin-1 concentration (Biomedica, Vienna, Austria), and plasma acetazolamide concentration (HPLC, Laboratoriumsmedizin Dortmund, Germany).
Values are given as means ± SE (n = 6). For intragroup comparisons (treatment and time), a general linear model of analysis of variance for repeated measures was applied (NCSS97, PASS 2000; Saugus, MA). Post hoc testing of means was performed with Student's t-test. Level of significance for error of first order was adjusted according to Holm's procedure. Statistical significance was assumed at P < 0.05.
Arterial blood gases, ventilation, and acid-base status.
During normoxia, PaO2 was greater and PaCO2 lower in iv acetazolamide, po acetazolamide, and ethoxzolamide protocols (P < 0.05) compared with both control and benzolamide-treated groups. These changes in blood gases are reflective of the higher ventilation in these groups. During hypoxia, PaO2 decreased to 35–39 mmHg in all dogs (P < 0.05, Table 2), and PaCO2 decreased from 38 to 32 mmHg during normoxia to 24–31 mmHg during hypoxia (P < 0.05, Table 2). Minute ventilation increased during hypoxia in all protocols (Table 2).
Changes in acid-base status are depicted in Table 3. With po acetazolamide and ethoxzolamide, base excess was about −4 to −5 mmol/l lower than in controls (P < 0.05), but due to hyperventilation and consequently low PaCO2 values (Table 2), plasma pH remained between 7.34 and 7.40 during normoxia and increased to 7.40–7.46 during hypoxia (Table 3) in conjunction with an increase in minute ventilation (Table 2).
Pulmonary and systemic hemodynamics.
With iv acetazolamide, MPAP and PVR did not change during hypoxia (Fig. 1, Table 4), whereas MPAP and PVR both increased in controls, as well as with benzolamide and ethoxzolamide (P < 0.05, Fig. 1, Table 4). With po acetazolamide, MPAP increased slightly after 2 h of hypoxia, whereas PVR did not change (Table 4). Figure 2 combines the pulmonary hemodynamic data with acetazolamide in this study with that from our previous study with 10 mg/kg iv (9) to better show the full drug dose-response relationship.
Heart rate increased slightly during hypoxia in all protocols (Table 4). Cardiac output, MAP, SVR, CVP, and PCWP did not change during hypoxia in any protocol and were not different between the groups (Table 4).
Plasma hormones, electrolytes, and renal function.
Plasma renin activity increased from 2.9–3.2 to 5–5.6 ng·ml−1·h−1 during hypoxia in both acetazolamide protocols, but did not change in controls or with benzolamide and ethoxzolamide. Endothelin-1 plasma concentrations were 0.4–0.57 pg/ml during normoxia and increased to a maximum of 1.5 ± 0.2 during hypoxia (P < 0.05). There were no differences between the protocols.
Plasma sodium concentration varied between 139 and 143 mmol/l in all protocols and did not change during the experiments. Plasma potassium concentration was lower in all protocols in which CA inhibitors were applied (P < 0.05, Table 3).
In controls, urine output, sodium, and potassium excretion did not change after 2 h of hypoxia. In all protocols in which CA inhibitors were applied, urine output, sodium, and potassium excretion were greater compared with controls during normoxia as well as during hypoxia (P < 0.05, Table 5). GFRs were significantly lower with all CA inhibitors with the exception of iv acetazolamide where there was a nonsignificant 12% reduction. The lower GFR with drug treatment was sustained throughout the hypoxic exposure (Table 5).
Acetazolamide plasma concentration.
Representative acetazolamide plasma concentrations were measured in two individual dogs with iv and po acetazolamide. During normoxia, acetazolamide plasma concentrations were comparable in both protocols (4.65 ± 0.7 μg/ml po acetazolamide vs. 5.3 ± 0.6 μg/ml iv acetazolamide). During hypoxia, acetazolamide concentrations remained stable with po acetazolamide (4.35 ± 0.75 μg/ml), whereas they increased with iv acetazolamide (7.4 ± 0.2 μg/ml) as a consequence of the maintenance infusion.
The principal findings of this study are that acetazolamide inhibits acute HPV with a dose-response relationship yielding reduction of HPV in concentrations and dosing relevant to humans taking conventional and tolerable doses (2–5 mg/kg). Furthermore, for the first time we show in live animals that acetazolamide does not inhibit acute HPV by CA inhibition since two other potent CA inhibitors are ineffective against HPV.
In control dogs, MPAP and PVR increased with hypoxia (Fig. 1). This is consistent with earlier results from our laboratory (8, 9) and many studies in humans and animals (1, 7). The stimulus for HPV is the decrease in oxygen tension of the pulmonary artery smooth muscle cells, and to a smaller extent, venular vascular smooth muscle cells, determined by O2 tensions primarily in the alveolar gas (5). It has been shown that hypoxia leads to a rise in the [Ca2+]i in pulmonary artery smooth muscle cells via several pathways (1) that initiate and sustain smooth muscle contraction.
In conscious dogs, iv acetazolamide applied at full CA-inhibiting concentrations (10 mg·kg−1·h−1) totally abolished HPV (9). When an 80% lower dose was applied (2 mg·kg−1·h−1), the increase in MPAP and PVR during acute hypoxia was still inhibited (Figs. 1 and 2, Table 3). The values for arterial pH before and during hypoxia were not as low as those found with the high-dose iv acetazolamide (9), supporting the known dose-response relationship of acetazolamide on acid-base status in the dog. The effects of acetazolamide on HPV in live dogs are in line with results in rat pulmonary artery smooth muscle cells obtained by Shimoda et al. (14). They demonstrated the functional presence of CA in these cells in the regulation of basal intracellular pH under normal conditions. During hypoxia, [Ca2+]i increases, and acetazolamide inhibits this rise, by a mechanism(s) other than changes in the membrane potential or blockade of voltage-sensitive membrane L-type calcium channels. Inhibition of the increase in [Ca2+]i by acetazolamide in the pulmonary arterial vasculature may well explain the lack of HPV in our acetazolamide-treated dogs. Although our results in the live dog are consistent with an action of acetazolamide on pulmonary vascular smooth muscle, we recognize the possibility that acetazolamide may also affect HPV in vivo by actions on the vascular endothelium. Pulmonary vascular endothelial cells release several vasoactive mediators known to alter HPV, thus it is conceivable that acetazolamide might reduce the generation of vasoconstricting substances or stimulate the release of vasodilating agents. Because vascular endothelial cells also contain CA, future work in this area will similarly need to determine whether or not the action of acetazolamide on endothelial cell responses to hypoxia is via CA.
To determine whether intra- and/or extracellular CA inhibition is involved in the suppression of HPV by CA inhibitors, we used benzolamide, a powerful, hydrophilic, nonpermeant CA inhibitor, to inhibit only extracellular CA. Benzolamide did not inhibit the increase in MPAP and PVR during hypoxia (Fig. 1, Table 4). This left intracellular CA as a possible target. To test whether intracellular CA inhibition was involved in HPV inhibition, we used ethoxzolamide, a cell membrane-permeant CA inhibitor. Similar to benzolamide, ethoxzolamide did not prevent the increase of MPAP and PVR during hypoxia (Fig. 1, Table 4). Ethoxzolamide differed from acetazolamide and benzolamide in reducing normoxic PVR with a trend to a lower MPAP (Table 4). Partly, the calculated lower normoxic PVR with ethoxzolamide resulted from a slightly higher cardiac output and PCWP, two of the three integral parameters in the calculation of PVR. Despite this baseline normoxic vasodilation of the pulmonary circulation, hypoxia still elicited a strong hypoxic vasoconstrictor action, in contrast to acetazolamide, but similar to benzolamide. It is not readily obvious why dogs treated with ethoxzolamide had a lower normoxic PVR, but they also had a lower SVR and slightly higher cardiac output. All of these would suggest a hyperdynamic state induced by ethoxzolamide with nonspecific vasodilation in the dogs, possibly arising in response to a likely greater degree of respiratory acidosis in the CNS owing to the drug's extreme lipid solubility (see ether partition coefficients for these drugs in Table 1) and high diffusibility across the blood-brain barrier (11). A greater respiratory acidosis in the CNS due to more complete neuronal CA inhibition than with the other two CA inhibitors may have led to greater activation of the sympathetic nervous system. In addition to any central effects on vascular resistance in the systemic circulation, there may have been direct effects of ethoxzolamide on the systemic vasculature itself. Pickkers et al. (12) showed in isolated noradrenaline-preconstricted mesenteric resistance vessels that ethoxzolamide relaxed these systemic vessels at 100 times the potency of acetazolamide and 10 times that of benzolamide.
Our findings in the live animal mirror the results of Shimoda et al. (14) obtained in isolated pulmonary artery smooth muscle cells, showing that benzolamide and ethoxzolamide do not prevent the hypoxia-related increase in [Ca2+]i. Despite the failure to prevent HPV, benzolamide and ethoxzolamide clearly exhibited potent CA-inhibiting properties, reflected by the lower plasma bicarbonate concentrations and base excess during the normoxia and hypoxia period (Tables 3 and 4), increased urinary sodium and potassium excretion, and reduction in GFR compared with controls (Table 5). These all are well-known effects of CA inhibitors caused by renal bicarbonate excretion and by tissue CO2 retention (9, 11, 16, 17) and indicate the efficacy of both drugs, as far as CA inhibition is concerned.
Our results in the pulmonary circulation with these three CA inhibitors differ from the results in systemic vessels by Pickkers et al. (12) alluded to above. What they found was that all three CA inhibitors could relax isolated preconstricted vessels with a rank order of potency equivalent to their inhibition of CA and intracellular penetration (ethoxzolamide > benzolamide > acetazolamide). These findings are entirely consistent with a role of CA in systemic vascular regulation. In marked contrast, we find that with respect to a constricted pulmonary vasculature (i.e., by hypoxia), the results with the three CA inhibitors do not follow a pattern of effect predicted by their ability to inhibit CA.
We tested the effect of 5 mg/kg po acetazolamide, given twice, 12 h and 1 h before the hypoxia exposure. The time points and dosage were chosen to reflect the use of acetazolamide, by mountaineers or those traveling to a high altitude, for AMS prevention. The effect on HPV as measured by MPAP was less pronounced compared with iv acetazolamide, but it nevertheless had an almost equal effect on PVR during hypoxia (Fig. 1, Table 4). The lesser effect of po acetazolamide on HPV and MPAP is likely due to the lower plasma concentrations during hypoxia compared with iv acetazolamide (4.35 ± 0.75 vs. 7.4 ± 0.2 μg/ml) and to a slightly higher cardiac output. In addition, these dogs had greater metabolic acidosis (Table 5) due to a longer time of action of the drug on the kidney and more cumulative urinary bicarbonate loss. Metabolic acidosis is known to augment HPV (10), and this may have also acted to blunt the effectiveness of the drug.
Systemic circulation and ventilatory response.
The systemic hemodynamic parameters of heart rate, MAP, cardiac output, and SVR were similar in all protocols during the normoxia period (Table 4). The increases in heart rate and MAP during hypoxia in controls and during CA inhibition are comparable to former studies of our group (8, 9). Cardiac output, SVR, and CVP did not change in any of the protocols (Table 4), indicating that in the time frame of the study, CA inhibition caused no significant volume depletion, even when acetazolamide was given orally 12 h before the experiment.
In controls, acute hypoxia increased minute ventilation by 35%, inducing a significant respiratory alkalosis (Tables 2 and 3). In normoxia, acetazolamide (both po and iv) and ethoxzolamide increased ventilation as reflected in lower PaCO2 values and increased minute ventilation. Benzolamide, however, did not appear to increase ventilation. With all CA inhibitors, there was increased ventilation with hypoxia, but the increase was least with benzolamide, reflecting the inability of benzolamide to penetrate to intracellular sites of CA (15). The increase in minute ventilation in the present acetazolamide protocols (partial CA inhibition) was less pronounced than in the former study in which a fully inhibitory dose of acetazolamide was administered (9).
Renal and acid-base effects.
In all protocols with CA inhibition, urine volume, sodium, and potassium excretion was greater than in controls during normoxia as well as during hypoxia (Table 5). This is caused by renal CA inhibition increasing urinary excretion of the anion bicarbonate which for reasons of electroneutrality must be coupled with an increased excretion of the cations sodium and potassium (9). The somewhat lower excretion rates for sodium and potassium during po acetazolamide may be due to the lower dose of acetazolamide (lower plasma concentrations) but may also reflect counterregulatory processes engaged to protect the body's sodium and potassium stores over the 12 h following the first dose.
In summary, we found that acetazolamide reduces acute hypoxic pulmonary vasoconstriction in a dose-dependent manner and is effective at dosing relevant to human use. The efficacy of acetazolamide as a HPV inhibitor does not appear to be related to CA inhibition, since neither intracellular enzyme inhibition by ethoxzolamide nor extracellular enzyme inhibition by benzolamide had an effect on HPV in our conscious dogs. With recent evidence in abstract form by Balanos et al. (2), they could show that acetazolamide (250 mg 3 times a day) reduces HPV by 50% in humans; there are solid grounds for a clinical trial of acetazolamide in HAPE prevention. Further studies should address whether a molecule lacking CA inhibitory activity and thus devoid of the unwanted systemic acid-base effects, but otherwise possessing equivalent molecular characteristics to acetazolamide, will have the same effect on HPV and perhaps on other forms of pulmonary hypertension.
This study was supported by Deutsche Forschungsgemeinschaft Grant Ho-3295/2-1 (to C. Höhne) and by National Heart, Lung, and Blood Institute Grant HL-24163 (to E. R. Swenson).
The authors are grateful to Rainer Mohnhaupt for help with the statistics and to Birgit Brandt and Daniela Bayerl for expert technical assistance.
Part of this work was presented at the Hypoxia Symposium 2005 Meeting, Lake Louise, Canada, and at the ATS Meeting 2006, San Diego, CA.
↵* C. Höhne and P. A. Pickerodt contributed equally to this work.
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