| Background: Lipid emulsion (LE) therapy can rapidly restore circulation after local anesthetic–induced cardiovascular collapse. Experimental models of local anesthetic toxicity have shown accelerated return of spontaneous cardiac function after lipid infusion in both intact animals and the isolated heart. Several case reports have extended these findings to the clinical setting, where patients in cardiac arrest after regional anesthesia were quickly resuscitated with lipid. Two case have been reported that lipid reversal of central nervous system symptoms of local anesthetic intoxication. The LE mechanism of action is still unclear now. One of possible mechanisms is reversal of the inhibition of mitochondrial fatty acid transport which is called the metabolic mechanism, the other is the possibility of a'lipid sink'mechanism, where the offending agent is removed from affected tissues by partitioning into a plasma lipid phase created by the infusion of lipid. The effect of LE post-treatment on bupivacaine pharmacokinetics is unclear. The present study was designed to try and solve these questions. Section One: The effect of LE pretreatment on cardiac toxicity by bupivacaine intoxication and the main mechanismObjective: To investigate the effect of lipid emulsion pretreatment on cardiac toxicity of bupivacaine and the main mechanisms.Methods: Fourteen New Zealand rabbits were pretreated with lactate ringer solution (group A, n=7) or 20% Intralipid (group B, n=7), followed by 0.75% bupivacaine until the arterial wave turning to a straight line. Duration until adverse cardiac events and cardiac bupivacaine concentration was compared. Blood pressure (BP), electrocardiogram (ECG) and end-tidal CO2 (ETCO2) were all measured continuously. Arterial blood gas analysis was done. High performance liquid chromatography (HPLC) determined bupivacaine concentration in heart tissue. Bupivacaine was extracted by liquid-liquid extraction method and extraction liquid included equal amount of n-hexane and tert-butyl. The mobile phase consisting of a mixture of methanol and triethylamine buffer and pH was adjusted to 3.0 with phosphoric acid. Flow rate was 1.0ml/min. Chromatographic analysis was performed by a Dima C18 chromatographic column at 30?C and wavelength was set at 215nm. After clipping 0.5g heart sample into fragment and homogenate in 2ml 50% methanol. 200μl homogenate and 100μl 4% NaOH was mixed by vortex for 30 seconds and then mixed with 3ml extraction liquid for 3 minutes. The mixture was then centrifuged at 3000rpm for 10min and 2ml of organic phase was collected in a centrifuge tube. This was followed by evaporating of the solution to dryness in a water bath at 35℃under a gentle stream of nitrogen. The dried residue was reconstituted with 100μl mobile phase, followed by mixing by vortex for 30s and centrifuged at 12000rpm for 10min. The supernatant was transfer into a 250μl vial-insert and a volume of 25μl was drawn by the HPLC system automatically for each analysis.Results: Basic hemodynamic data (MAP, HR) was similar between group A and group B. Mean arterial BP was 94.4±5.9mmHg in group B and 96.4±4.2mmHg in group A (P=0.482); HR was 273±17bpm in group B and 278±15bpm in group A (P=0.522). The onset of ventricular arrhythmia or cardiac stand-still was significantly delayed in group B (P<0.001). The concentration of bupivacaine in the myocardial tissue was comparable between the 2 groups (P=0.108), But the total dose of intra-venous bupivacaine needed to achieve this cardiac muscle concentration was significantly higher in group B (P<0.001). Conclusions: Lipid emulsion pretreatment reduces cardiac toxicity of bupivacaine. The main mechanism may be the"lipid pool"effect.Section two: The effect of LE post-treatment on bupivacaine pharmacokinetics and mechanism studyObjective: To investigate the effect of lipid emulsion post-treatment on bupivacaine concentration in circulation and cardiac tissue, as well as the possible mechanism.Methods: Fourteen rabbits were infused with 2mg/kg bupivacaine, followed by ringer solution (group C, n=7) or 20% Intralipid (group D, n=7). Circulating bupivacaine concentration on 10 min, 20 min, 30 min, 40 min, 50 min and 60 min and cardiac bupivacaine concentration at 65 min after bupivacaine infusion were compared between the 2 groups. Electrocardiogram, arterial pressure and ETCO2 were monitored continuously and arterial blood gas was measured. The determination of Bupivacaine concentration in heart tissue is same with section one. High performance liquid chromatography determined bupivacaine concentration in plasm. 200μl plasma and 100μl 4% NaOH was mixed by vortex for 30 seconds and then mixed with 3ml extraction liquid for 3 minutes. The mixture was then centrifuged at 3000rpm for 10min and 2ml of organic phase was collected in a centrifuge tube. This was followed by evaporating of the solution to dryness in a water bath at 35℃under a gentle stream of nitrogen. The dried residue was reconstituted with 100μl mobile phase, followed by mixing by vortex for 30s and centrifuged at 12000rpm for 10min.The supernatant was transfer into a 250μl vial-insert and a volume of 25μl was drawn by the HPLC system automatically for each analysis. Standard curve of blood bupivacaine level: Different amounts of bupivacaine standard solution were added into blank rabbit plasma so as to prepare standard sample of different concentration (20, 50, 100, 200, 500, 1000, 2000 and 5000ng/ml). The standard samples were analyzed in HPLC system as described in blasm bupivacaine determination and standard curve of peak area was constructed by linear regression analysis.Results: Hemodynamic data (MAP, HR) at basic level and after sub-toxic dose of bupivacaine infusion was shown no statistical significant difference between group C and group D. Determination of bupivacaine concentration by HPLC analysis as described in the method part was accurate and reproducible, as the peak area and plasma bupivacaine concentration were linearly correlated (Y= 58.839X + 395.12,n=5, R2=0.9998). Plasma bupivacaine concentration of each time point was significantly higher in group D than in group C (P<0.01), while bupivacaine concentration in the myocardial tissue was significantly lower in group D compared to that in the group C (176±50ng/g vs 304±61ng/g respectively, P=0.004).Conclusions: Lipid emulsion increases bupivacaine concentration in plasm, while reduces bupivacaine concentration in heart tissue by"lipid pool"effect. It may be the main reason that lipid can fast reverse cardiac arrest induced by local anesthetic.
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