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Analgesia: Research Report

The Effects of Lipid Infusion on Myocardial Function and Bioenergetics in l-Bupivacaine Toxicity in the Isolated Rat Heart

Stehr, Sebastian N., MD*; Ziegeler, Jörg C., BS*; Pexa, Annette, BS; Oertel, Reinhard, PhD; Deussen, Andreas, MD; Koch, Thea, MD*; Hübler, Matthias, MD, DEAA*

Section Editor(s): Horlocker, Terese T.

Author Information
doi: 10.1213/01.ane.0000248220.01320.58
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Weinberg et al. (1) demonstrated that pretreatment or resuscitation associated with a lipid infusion leads to positive changes in the dose–response of local anesthetic toxicity in a rat model and confirmed these findings in a whole dog model (2). On the basis of these experimental findings, a bolus lipid application (1 mL · kg−1 · min−1) and continuous infusion of 0.25 mL · kg−1 · min−1 of 20% lipid have been recommended for humans in cases of local anesthetic toxicity (3). Two major possible mechanisms have been discussed as a basis for the positive effects of lipid infusion: Lipids may function as a “sink” by binding local anesthetics, and therefore reducing free plasma levels. Conversely, beneficial metabolic effects might result from the application of lipids, as more than 70% of myocardial energy needs are supplied by mitochondrial oxidation of fatty acids (4). In this case, positive myocardial effects might also be expected in the absence of local anesthetics. One previous study has compared the effects of lipids and nanoparticle infusion on racemic bupivacaine-induced QRS interval prolongation in a guinea pig isolated heart model, without describing other hemodynamic changes (5).We hypothesized that lipids partially reverse the cardiac toxic effects of l-bupivacaine, independent of a “lipid sink” effect.

METHODS

Preparation of Isolated Hearts

An isolated perfused, nonrecirculating Langendorff rat heart preparation was used in our study. The investigation was performed in compliance with the Guide for the Care and Use of Laboratory Animals issued by the US National Institutes of Health and was approved by the local government authority (AZ 24-9168.24-1-2003-9). All experiments were conducted with Wistar rats (16–22 wk old; 215–230 g, ntotal = 30) purchased from Charles River (Sulzfeld, Germany). The animals were heparinized i.p. (1000 U/kg) to prevent the formation of intracoronary microthrombi (6) and were anesthetized with 150 mg/kg of i.p. thiopental. Hearts were rapidly excised, weighed, and then perfusion was performed in a retrograde manner via the aorta at a constant perfusion pressure of 90 mm Hg with a modified Krebs–Henseleit buffer (KHB) having NaCl 116 mmol/L, KCl 4.56 mmol/L, MgSO4 2.24 mmol/L, KH2PO4 1.18 mmol/L, NaHCO3 25.0 mmol/L, glucose 8.27 mmol/L, pyruvate 2.0 mmol/L, CaCl2 2.52 mmol/L. The solution was continuously bubbled with 95% oxygen and 5% carbon dioxide and pH was maintained at 7.35 ± 0.03. Arterial and effluent perfusate Po2 and Pco2 (sampled via the inflow line or via a catheter placed in the pulmonary artery, respectively) were measured at 10, 15, and 25 min (AVL 990, Medical Instruments, Bad Homburg, Germany). Myocardial oxygen consumption (Mvo2, μL min−1 · g−1) was calculated on the basis of the arterial-venous difference of Po2 according to Fick’s principle using Bunsen’s absorption coefficient (α = 0.036 μL · mm Hg−1 · mL−1) and AvDo2 (PartO2 −PvenO2) at 37°C as follows: Mvo2 (μL · min−1 · g−1) = AvDo2 × α × F, whereby F denotes coronary flow (mL · min−1 · g−1). All elements of the perfusion apparatus were water-jacketed and maintained at 37°C. In the spontaneously beating heart preparation, stable conditions were achieved in preliminary control experiments with minimal changes of inotropic variables, left ventricular pressure (LVP) and +dP/dt, in accordance with previous publications on the isolated heart model (7). Systolic LVP and its first derivative +dP/dt were continuously measured with a balloon catheter inserted into the left ventricle (Gould Inc. Instruments, Statham, USA) via the cut mitral valve. Diastolic LVP was adjusted to 5 mm Hg. Coronary flow and coronary perfusion pressure were continuously measured by an in-line flowprobe (Transonic Flowprobe, Transonic Systems, NY) and a pressure transducer (Gould Nicolet, Erlensee, Germany) attached to the perfusion cannula 2 cm above the orifice of the coronary vessels. Hemodynamic variables and derivatives (heart rate (HR), LVP, +dP/dt, coronary flow) and electrocardiogram data (PR, QRS intervals) were continuously sampled and documented by a software system (PoNeMah, P3 plus Version 4, Gould LDS Test and Measurement LLC, OH). The electrodes were consistently placed in a lead II position: one electrode in the right atrium and one epicardially at the apex of the heart. An indifferent electrode was connected to the KHB inflow-line. All electrocardiogram data were cross-checked manually offline to confirm correct assessment. Bi-atrial pacing was performed using a HSE Stimulator P (Hugo Sachs Elektronik, March, Germany) with a 2 ms/2 V amplitude. All infused compounds were applied through a stainless steel cannula placed into the aortic inflow line proximal to the flowprobe (Precidor, Infors AG, Basel, Switzerland). The experimental protocol was started when LVP, +dP/dt, and HR had reached stable baseline values, i.e., 20 min after artificial perfusion had been commenced.

Experimental Protocol—Isolated Rat Heart

Spontaneously beating hearts were randomized by lot to one of four groups (five hearts per group): 1) Perfusion with KHB (Control), 2) Perfusion with KHB and lipid infusion (0.25 mL · kg−1 · min−1) after 15 min (Control Lipid), 3) Switch to 5 μg/mL l-bupivacaine after 10 min (l-Bupi), 4) Switch to 5 μg/mL l-bupivacaine after 10 min and lipid infusion (0.25 mL · kg−1 · min−1) after 15 min (l-Bupi Lipid).

The amount of lipid was based on a recommendation by Weinberg (3). Body weight was used to calculate the amount of lipid needed to affect an infusion of 0.25 mL · kg−1 · min−1. For instance an animal weighing 230 g would receive 0.0575 mL of lipid solution per min via the arterial inflow.

To elucidate the effects of a constant HR on myocardial energy status in l-bupivacaine-induced myocardial depression and lipid application two further experimental groups (five hearts per group, l-Bupi Pacing and l-Bupi Lipid Pacing) were evaluated. The protocol was similar to the l-Bupi and l-Bupi Lipid groups, except that hearts were constantly paced at 300 bpm.

At the end of each protocol hearts were freeze-clamped and stored at −60°C for further analysis.

Local Anesthetics/Lipid

The commercially available local anesthetic solution l-bupivacaine hydrochloride (Chirocain ™, Abbott GmbH & CO KG, Wiesbaden, Germany) and lipid solution (Structolipid ™ 20%, Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany) were used. Structolipid is a structured triacylglycerol emulsion with a medium-chain to long-chain fatty acid ratio of 1:1. We evaluated the dose-dependent effects of l-bupivacaine in three pilot experiments. A concentration of 5 μg/mL (15.4 μM) l-bupivacaine consistently led to myocardial depression (HR, +dP/dt, systolic pressure, flow >−30%).

Effluent Lipid Concentration Measurement

To evaluate the exact effluent lipid concentration used in our experiments, effluent perfusate samples were measured in the two pacing groups. In brief, samples were collected at 20 and 25 min and diluted with KHB. The standard curve was fitted using known samples with 1:1000 to 1:10000 of lipid solution using KHB with 5 μg/mL l-bupivacaine measured at 210 nm with a Büchi 901 photometer (Büchi Labortechnik GmbH, Essen, Germany) and corrected for l-bupivacaine background. The correlation was linear (r = 0.99).

Sample Preparation for Adenine-Nucleotide Measurements

Frozen hearts were lyophilized with a Christ alpha 1-2-lyophilisator (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany) and homogenized in 0.5 M perchloric acid. Precipitated proteins were separated by centrifugation and supernatant was neutralized with 1 M K3PO4. For fluorescence detection of adenine-nucleotides derivatization with chloracetaldehyde to their 1,N6-etheno-analogs was performed. The neutralized sample (150 μL) was mixed with 2000 μL KHB and 770 μL citrate-phosphate-buffer. The reaction was started with 80 μL chloracetaldehyde. Samples were incubated for 40 min at 80°C and then immediately cooled to stop the reaction. Samples were stored at −20°C until analysis was performed.

Measurement of Adenine-Nucleotides in Heart Tissue

High performance liquid chromatography for 1,N6-etheno-analogs of adenine-nucleotides was performed on a Waters Alliance 2690 (Waters Corp., Milford, MA) coupled to a Merck-Hitachi (Hitachi, Tokyo, Japan) F 1050 fluorescence detector (λex = 280 nm, λem = 410 nm) as previously reported by Haink and Deussen (8). In brief, separations were performed on a Waters XTerra MS C18, 4.6 × 50 mm2 ID column, with a particle size of 5 μm and a 125 Å pore size. Samples (10 μL) were injected applying an autosampler and compounds were eluted with a flow rate of 1.5 mL/min using a binary tetrabutylammoniumhydrogensulfate/acetonitrile gradient. Eluent A contained 6% acetonitrile in tetrabutylammoniumhydrogensulfate buffer, and eluent B was a mixture of 32% eluent A and 68% acetonitrile. Initial conditions were 100% eluent A, linearly changed to 66% A within 2.80 min. This condition was maintained for 0.70 min before initial conditions (100% eluent A) were re-established within 0.1 min and the column was equilibrated for the next run for 3.40 min. Equally treated external standards of known concentrations were used to check retention times and to permit sample quantification based on the analysis of peak area. Millenium software (Waters) was used to report data.

In Vitro Plasma-Lipid Effects

After approval by the ethics committee of the Medical Faculty of the Technical University (EK146072004) expired fresh frozen plasma was obtained from the Red Cross Blood Bank, Saxony (Fiedlerstr. 23, 01307 Dresden, Germany). Measurement of plasma protein concentration, albumin, and α-1-acid glycoprotein was performed in the Department of Clinical Chemistry of our faculty. Effects of lipid solution on plasma concentrations of l-bupivacaine were measured in two different groups:

Group 1: 5 μg/mL l-bupivacaine and 0, 1, 5, 10, 100, and 500 μL of lipid in 1 mL human plasma.

Group 2: 5 μg/mL l-bupivacaine and 0, 1, 5, 10, 100, and 500 μL of lipid in 1 mL KHB.

l-Bupivacaine (5 μg/mL) was added to human plasma and KHB. Lipid was added and aliquots of 1 mL were vortexed and shaken gently for 1 h at 38°C and centrifuged at 10,000g (g-force, where 1 g = 9.80665 m/s2) for 10 min. The lipid phase was discarded and the lower clear aqueous phase was used for l-bupivacaine measurement. Experiments for each concentration were performed in triplicates (36 experiments in all). Data were corrected for dilution.

Measurement of l-Bupivacaine

A liquid chromatography-tandem mass spectrometric method with a rapid and simple sample preparation was developed and validated for the detection of l-bupivacaine. A detailed description of the exact methodology has been described elsewhere (9). The mass spectrometer was operated in the multiple reaction monitoring mode. Because of the high sensitivity of the method a simple protein precipitation was sufficient as sample preparation. The liquid chromatography-tandem mass spectrometric system used was a Quattro micro (Waters) equipped with an electrospray interface. The chromatographic separation was performed on a Synergy 4 μ Polar-RP 80A, 150 mm × 2 mm column. A mobile phase gradient was applied with a mixture of acetonitrile, ammonium acetate in water and formic acid. To achieve high-quality analytical data for samples with low levels of analytes originally in biological fluids, sufficient chromatographic retention of the analyte is preferred to minimize signal suppression and other matrix effects. Retention time for l-bupivacaine was 4.5 min. A good linear response rate was found from 1 to 200 ng/mL. Higher concentrated samples were diluted.

Statistical Analysis

All data are presented as mean ± sd. For between-group comparisons of hemodynamic variables values from 0 to 10 min were averaged and compared with a 30 s average at 15 or 25 min respectively. Inter-group significances were calculated using Student’s t-test for unpaired data. Statistical analyses were performed using SPSS software for MS Windows (Release 11.0, SPSS, Chicago, IL). All variables were tested for normal distribution with the Kolmogorov–Smirnoff test and were found to have normal distribution. ANOVA for repeated measures and Bonferroni post hoc adjustment was performed. A P < 0.05 was taken to indicate a statistical significance.

RESULTS

With spontaneously beating hearts, baseline variables for HR (272 ± 46 min−1), coronary flow (12.7 ± 2 mL/min), PR interval (30 ± 8 ms), QRS interval (54 ± 15 ms), systolic pressure (102 ± 9 mm Hg), and +dP/dt (2275 ± 257 mm Hg/s) showed no significant between-group differences. Perfusate oxygenation values and pH were comparable at baseline. The application of 5 μg/mL l-bupivacaine led to a significant decrease in Mvo2 (216 ± 53 in controls versus 146 ± 31 μL · min−1 · g−1 in l-bupivacaine-treated hearts, P < 0.05), whereas AvDo2 remained stable (491 ± 50 vs 497 ± 58 mm Hg, not significant).

The application of 5 μg/mL l-bupivacaine at 10 min into the experiment led to a significant decrease in HR (Fig. 1A, P < 0.05) and coronary flow (Fig. 1B, P < 0.05). Lipid infusion at 15 min had no effect on these variables in controls and in l-bupivacaine-treated hearts. PR and QRS intervals were significantly increased by l-bupivacaine and also were not influenced by lipid application. l-Bupivacaine led to a significant decrease of +dP/dt (Fig. 2A, P < 0.05) and systolic pressure (Fig. 2B, P < 0.05). Lipid application led to a reversal of decreased +dP/dt and systolic pressure to baseline values within 10 min in the l-Bupi Lipid group (Tables 1 and 2).

Figure 1.
Figure 1.:
Heart rate (bpm) (Fig. 1A) and coronary flow (mL/min) (Fig. 1B) over the time course of the experiment for the four different groups without pacing: Control, l-Bupi (5 μg/mL at 10 min), Control Lipid (0.25 mL · kg−1 · min−1 at 15 min), and l-Bupi Lipid (l-bupivacaine 5 μg/mL at 10 min and 0.25 mL · kg−1 · min−1 lipid at 15 min).
Figure 2.
Figure 2.:
+dP/dt (mm Hg/s) (Fig. 2A) and left ventricular systolic pressure (mm Hg) (Fig. 2B) over the time course of the experiment for the four different groups without pacing: Control, l-Bupi (5 μg/mL at 10 min), Control Lipid (0.25 mL · kg−1 · min−1 at 15 min), and l-Bupi Lipid (l-bupivacaine 5 μg/mL at 10 min and 0.25 mL · kg−1 · min−1 lipid at 15 min).
Table 1
Table 1:
ANOVA and post hoc Bonferroni Results for Control, l-Bupi (l-Bupivacaine 5 μg/mL at 10 min), Control Lipid (0.25 mL/kg/min at 15 min) and l-Bupi Lipid (l-Bupivacaine 5 μg/mL at 10 min and lipid 0.25 mL/kg/min at 15 min)
Table 2
Table 2:
ANOVA and post hoc Bonferroni Results for Control, l-Bupi (l-Bupivacaine 5μg/mL at 10 min), Control Lipid (0.25 mL/kg/min at 15 min) and l-Bupi Lipid (l-Bupivacaine 5μg/mL at 10 min and Lipid 0.25 mL/kg/min at 15 min)

In hearts that were continuously paced at 300 bpm baseline variables were comparable (systolic pressure 106 ± 10 mm Hg, +dP/dt 2866 ± 326 mm Hg/s, coronary flow 10.3 ± 1.4 mL/min, PR 32 ± 6 ms and QRS 63 ± 6 ms). As previously seen in spontaneously beating hearts lipid infusion also resulted in an inotropic effect leading to a significant increase of systolic pressure from 77 ± 8 to 89 ± 11 mm Hg and of +dP/dt from 1770 ± 270 to 2180 ± 419 mm Hg/s (Figs. 3A and B, P < 0.05), while coronary flow, PR, and QRS intervals remained unchanged (7.0 ± 1 mL/min, 75 ± 11 ms and 92 ± 12 ms, respectively). AvDo2 remained comparable during the course of the pacing experiments (491 ± 50 and 540 ± 14 at 10 min, 493 ± 27 and 529 ± 14 mm Hg at 15 min, and 546 ± 28 and 566 ± 32 at 25 min for l-Bupi Pacing and l-Bupi Lipid Pacing, respectively, not significant). Mvo2 was again significantly reduced after l-Bupi infusion in both paced groups (159 ± 33 and 196 ± 22 at 10 min, 119 ± 21 and 140 ± 6 μL · min−1 · g−1 at 15 min for l-Bupi Pacing and l-Bupi Lipid Pacing, respectively, P < 0.05). After lipid infusion Mvo2 was significantly increased in the l-Bupi Lipid Pacing group (153 ± 9 vs 124 ± 20 μL · min−1 · g−1 in the l-Bupi Pacing group, P < 0.05).

Figure 3.
Figure 3.:
+dP/dt (mm Hg/s) (Fig. 3A) and left ventricular systolic pressure (mm Hg) (Fig. 3B) over the time course of the experiment for the two groups that were paced at 300 bpm: l-Bupi Pacing (5 μg/mL at 10 min) and l-Bupi Lipid Pacing (l-bupivacaine 5 μg/mL at 10 min and 0.25 mL · kg−1 · min−1 lipid at 15 min).

Measurement of effluent lipid concentration during lipid infusion in the pacing experiments revealed a lipid concentration of 8.9 ± 0.4 μL/mL.

Comparison of energy charge (EC) [(ATP + 0.5 × ADP) × (ATP + ADP + AMP)−1] revealed no significant differences among the four groups (Control 0.87 ± 0.02, Control Lipid 0.86 ± 0.02, l-Bupi 0.87 ± 0.02, l-Bupi Lipid 0.88 ± 0.01). This was also the case for EC measurements of the hearts paced at 300 bpm (0.95 ± 0.01 for l-Bupi Pacing and 0.95 ± 0.002 l-Bupi Lipid Pacing, not significant).

In vitro measurement of the effects of lipid on the concentration of l-bupivacaine in KHB and human plasma are shown in Figure 4. The plasma protein concentration was 59.9 g/L, albumin 44.9 g/L, and 0.51 g/L α-1-acid glycoprotein. The addition of lipid led to a significant decline in l-bupivacaine concentrations at 100 μL/mL of lipid in the KHB group and at 500 μL/mL of lipid in the plasma group.

Figure 4.
Figure 4.:
l-Bupivacaine concentrations after addition of 0, 1, 5, 10, 100, and 500 μL to 1 mL of human plasma or Krebs–Henseleit Buffer (KHB) with 5 μg/mL of l-bupivacaine. *P < 0.05 versus same lipid concentration in human plasma (Student’s t-test). †P < 0.05 versus all other concentrations in the respective group (ANOVA, post hoc Bonferroni).

DISCUSSION

In our experiments, the application of l-bupivacaine led to a significant decrease in HR, +dP/dt, and systolic pressure, and prolongation of PR and QRS intervals. These changes reflect the two main myocardial toxic effects of local anesthetics, i.e., negative inotropy and myocardial conduction block (10). Direct negative inotropic effects of local anesthetics have been described as a result of Ca2+ channel blockade (11), sarcoplasmic reticulum Ca ATPase blockade (12) and even actin–myosin interaction (13). In addition, we observed a significant decrease in coronary flow, recently described as a direct coronary vascular effect of l-bupivacaine, possibly via KATP channel blockade, in nonbeating isolated hearts (14). High concentrations of local anesthetics have been shown to influence myocardial energy status due to inhibition of mitochondrial oxidation (15,16). For characterization of the energy state of the adenylate system (ATP + ADP + AMP) the parameter EC [EC = (ATP + 0.5 × ADP) × (ATP + ADP + AMP)−1] ranging from 1 to 0 (complete discharge) was developed by Atkinson and Walton in 1967 (17). In our experiments, no changes in EC were found among the different experimental groups. We therefore do not consider inhibition of mitochondrial processes to be of importance in negative inotropy observed at low, but toxic, local anesthetic concentrations.

Lipid infusion has been shown to be beneficial in treating experimental local anesthetic toxicity (1,2). However, the exact mechanisms responsible for these effects remain unclear. In our experiments, lipid application did not significantly alter HR or coronary flow in hearts pretreated with l-bupivacaine. We therefore consider a lipid effect on membrane channel local anesthetic blockade unlikely. In a letter Morey et al. (5) described a reduction of QRS interval prolongation after nanoparticle and lipid infusion in a guinea pig isolated heart model, but little information concerning the exact methodology of these experiments was presented. In an abstract, the same group (18) described the effects of 0.4 mL/kg intralipid infusion on QRS prolongation in a whole rat bupivacaine intoxication model and reported no significant effect. In contrast to HR and coronary flow, inotropic variables, i.e., systolic pressure and +dP/dt, significantly increased in our experiments after lipid application in hearts pretreated with l-bupivacaine, returning to baseline values. This could indicate a direct positive inotropic effect of lipid infusion. Conversely, an indirect inotropic effect by some form of reduction of local anesthetic-induced inotropic impairment could be possible.

To test the latter hypothesis, we measured the lipid effect on perfusate and plasma concentrations of l-bupivacaine. At concentrations of 100 μL/mL of the perfusion buffer, a significant effect of lipids on free l-bupivacaine concentration was noted (reducing free l-bupivacaine concentration from 5 to 2.2 μg/mL). Varshney et al. (19) examined the effects of microemulsions on free bupivacaine concentrations and also noted that 10 μL/mL of a specific microemulsion (Pluronic F127) had little effect on extracted bupivacaine (0.8% of free bupivacaine). At a concentration of 100 μL/mL, this same emulsion managed to extract 40% of free bupivacaine. In our experiments, lipid extracting effects were not as pronounced in plasma: here 500 μL/mL of lipids significantly decreased l-bupivacaine concentrations (Fig. 4), probably due to free plasma protein binding. In the experiments with the isolated hearts lipids were applied at a mean concentration of 9 μL/mL in the coronary perfusate—a concentration that had no significant effect on in vitro perfusate-free l-bupivacaine concentrations. We did not measure intracellular local anesthetic concentration changes, and therefore cannot exclude a change in the intracellular l-bupivacaine concentration, but previous work on distribution kinetics of local anesthetics have shown that extracellular concentrations (20) and pH (21) are decisive factors for intracellular concentrations, all controlled in our setup. We can also not exclude that in in vitro experiments lower concentrations of bupivacaine may result in different scavenging effects of lipids.

If lipid infusion had a direct positive inotropic effect, one would expect to see an effect under control circumstances. In our experiments a (nonsignificant) positive inotropic tendency was observed in control hearts (Fig. 2). To control the observed reduction in HR after local anesthetic infusion, the lipid intervention group experiments were repeated while pacing hearts at 300 bpm. Although no differences in EC were found, we did now see a significant increase in Mvo2 in paced, as opposed to spontaneously beating, hearts.

Although Sztark et al. (22) found that enantiomers do not seem to have differential effects concerning mitochondrial toxicity differences have been found for other aspects of myocardial toxicity, e.g., coronary vascular resistance (14), channel interaction (23), arteriovenous conduction (24), and arrhythmogenic effect (25). We therefore chose the single enantiomer l-bupivacaine so that we could ignore the possible differential effects resulting from the application of racemic bupivacaine. The isolated, perfused Langendorff rat heart preparation used in our study has limitations in the assessment of local anesthetic toxicity, e.g., hearts are denervated during the preparation process. Nevertheless, the isolated heart has been widely used to study local anesthetic-induced myocardial effects (26).

CONCLUSION

In summary, l-bupivacaine application led to a significant decrease in HR, +dP/dt, systolic pressure, and coronary flow, and to an increase in PR and QRS intervals. Neither local anesthetic, nor lipid infusion had a significant effect on EC. Lipid application in l-bupivacaine-induced cardiovascular depression significantly increased +dP/dt and systolic pressure, which we would attribute to a direct inotropic effect rather than to an indirect, local anesthetic plasma-binding effect of lipids in an isolated heart model. Our study results further underscore the possible efficiency of lipid infusion as an alternative therapeutic approach for local anesthetic-induced cardiovascular depression.

ACKNOWLEDGMENTS

The authors thank Bianca Müller (Institute of Physiology) and Ute Mann (Institute of Clinical Pharmacology) for excellent technical assistance.

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