Lipid Emulsion Reverses Bupivacaine-induced Asystole in Isolated Rat Hearts: Concentration-Response and Time-Response Relationships

Adult male Sprague Dawley rats weighing 300–350 g were obtained from the animal center of Wenzhou Medical College (Zhejiang, China). All work was approved by the college's Animal Care and Use Committee.

Rats were anesthetized by 350 mg/kg chloral hydrate intraperitoneal injection. Hearts were rapidly excised after systemic heparinization and cannulation through the ascending aorta with suspension from a Langendorff apparatus (ML870B2; ADInstruments, Sydney, Australia). Retrograde perfusion was at a constant pressure of 120 mmHg with Krebs-Henseleit buffer comprised of 118 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO⁴, 1.2 mm KH²PO⁴, 25.0 mm NaHCO³, 10.0 mm glucose, and 2.5 mm CaCl². The solution was continuously bubbled with 95% oxygen and 5% carbon dioxide and its pH was maintained at 7.40 in a 37°C countercurrent flow water bath. A balloon was inserted into the left ventricle and inflated with water to achieve a constant left ventricular end-diastolic pressure of 4–10 mmHg and pressure was transduced and analyzed continuously (Powerlab Data Analysis System, Chart 5.5.6; ADInstruments). After Krebs-Henseleit buffer had been perfused for 25 min (baseline time, designated Tb), a 100 μm bupivacaine infusion was initiated and sustained for 180 s beyond asystole.

The control group was then perfused with 40 μm bupivacaine. Seven experimental groups were perfused with 40 μm bupivacaine along with lipid emulsions (20% intralipid; Huarui Pharmaceuticals Co., Ltd., Wuxi, China) at concentrations of 0.25, 0.5, 1, 2, 4, 8 and 16%. There were six hearts in each group. Group assignment was randomized with investigators blinded to group assignments. All groups were perfused for 45 min after asystole (the final time designated Te). The cardiac apex of each heart was then dissected and immediately frozen in liquid nitrogen and stored at −70°C for further analysis.

The time from initiation of bupivacaine infusion to asystole (designated Ts) and the time from the end of 100 μm bupivacaine infusion to cardiac recovery (designated Tr) were recorded. The following cardiac function parameters were recorded/calculated: left ventricular developed pressure (LVdevP = systolic − diastolic pressure), maximum change rate of LVdevP fall (−dP/dt^max), heart rate (HR), and rate-pressure product (RPP; = HR × LVdevP). Parameters of cardiac function were continuously monitored until the end of reperfusion, and were recorded at the following times: the balance, asystole, 1 min after restoration of cardiac rhythm (1 min), every 5 min from restoration for 40 min. The ratio of the highest RPP (RPPh) during the recovery to baseline RPP (RPPr) was also recorded. Finally, the ratio of RPP at Te to baseline RPP (RPPe) was documented.

Frozen hearts were homogenized with perchloric acid 0.4 M. Precipitated proteins were separated by centrifugation at 3,000g  for 15 min. The supernatant was neutralized with KOH 2 M. Samples were then centrifuged for another 15 min and the supernatant was stored at −70°C until analysis was performed.

A liquid chromatography-tandem mass spectrometric method with a rapid and simple sample preparation was developed and validated for the detection of bupivacaine. The liquid chromatography-tandem mass spectrometric system (Bruker Esquire; Bruker Company, Karlsruhe, Germany) was equipped with an electrospray interface. A Zorbax SB-C18 column (150 × 2.1 mm, 5 μm; Agilent Technologies, Inc., Santa Clara, CA) was used with the mobile phase of methanol and formic acid 0.1% solution (55:45 v) and lidocaine was used as the internal standard. Electrospray ionization source was applied and operated in positive ion mode. Multiple-reaction monitoring mode was used to quantify bupivacaine. The mass-to-charge ratio (m/z ) for detected ions were m/z  289→140 for bupivacaine and m/z  235→86 for the internal standard. It was linear and ranged from 0.01 to 20.0 μg/g for bupivacaine in myocardium. The limit of detection for bupivacaine was 4 ng/g. Retention time in bupivacaine was 2.3 min. Recovery of bupivacaine from myocardium ranged from 66.45 to 74.47% with intraday and interday relative standard deviations less than 7%. We performed a regression analysis between cardiac bupivacaine concentrations and the corresponding RPPe to determine their relationship.

We used the following equation to generate the concentration-response curve for lipid emulsion to reverse bupivacaine-induced cardiac arrest in isolated rat heart:

where Y = RPPr and X = the logarithm of lipid concentration.

All data were analyzed using SAS for Microsoft Windows (version 9.2; SAS Institute, Inc., Cary, NC) and are presented as mean ± SD. Baseline cardiac function parameters, weight, Ts, Tr, RPPr, RPPe, and myocardial bupivacaine concentration among the groups were analyzed with one-way analysis of variance and a two-sided t  test. A linear mixed model was used for the cardiac factors during recovery in lipid-treated groups (7 groups × 9 times). Cardiac function parameters between lipid groups during the recovery phase and RPP ranged from 5 to 40 min compared with 1 min were analyzed using a linear mixed model with two-tailed P  values that were adjusted by Holm method. A P  value of less than 0.05 was considered statistically significant. Curve-fitting for the cardiac bupivacaine and lipid concentration effects were performed (Prism version 5.0; GraphPad Software Inc., San Diego, CA).

There were no differences in baseline weight and cardiac function among the eight groups in the study (table 1).

All hearts developed asystole after 100 μm bupivacaine infusion. The time from initiation of the 100 μm bupivacaine infusion to asystole (Ts) did not vary among the eight groups (table 2). Cardiac recovery was defined in advance of the study as a RPP greater than 10% of the baseline value for more than 1 min. Control group hearts did not recover although all hearts in lipid-treated groups did so. The time of recovery, Tr, for the 1–16% lipid concentration groups was less than for the 0.25–0.5% lipid groups (table 2). Lipid-treated groups demonstrated no significant differences in LVdevP at any time after asystole (fig. 1). Comparison among lipid groups in the recovery phase demonstrated that the −dp/dt^maxin the 2 and 4% groups was greater than in the 0.25 and 0.5% groups whereas the 16% group was greater than the 0.5% group (fig. 2). During recovery phase, HR in the 2–16% lipid groups was greater than in the 0.25–1% lipid groups. Further delineation demonstrated that HR was better maintained by increasing lipid concentrations: 16% > 2%, 4%, and 8%; also, 4% and 8% > 2% (fig. 3). During the recovery phase, RPP in the 2–16% lipid groups was significant greater than that observed for the 0.25–1% groups. In addition, RPP in the 16% lipid group was greater than that of the 2–8% groups. Among the 4–16% lipid groups, RPP was lower at 15–40 min than at 1 min, with the time-response relationship showing a decreasing tendency during recovery phase. However, in the 0.25%–2% lipid groups, it did not differ at any time during recovery when data were compared at 1 min (fig. 4).

The concentration of myocardial bupivacaine in all lipid-treated groups was significantly lower than in the control group. Furthermore, the myocardial bupivacaine concentration in 2–16% lipid groups was lower than in 0.25–1% lipid groups. In addition, myocardial bupivacaine concentration in the 16% lipid group was lower than in 2–8% groups (table 2).

RPPr demonstrated a concentration-dependent relationship among lipid groups, with cardiac function significantly improved when lipid concentration was 2% or more (table 2). However, lipid concentrations higher than 8% did not result in improved recovery, thereby demonstrating a “plateau effect” in the benefit of lipids (fig. 5).

RPPe in the 2–16% lipid groups were significant greater than in the 0.25–0.5% lipid groups, with the 8% group greater than the 1% group. The relationship between the concentration of lipid emulsion and myocardial bupivacaine and RPPe is shown in table 2and figure 6.

Studies7,8from several laboratories have established the isolated heart model as a powerful tool for studying the pathophysiology, mechanisms, and treatment of bupivacaine-related cardiac toxicity. Animal studies have found that bupivacaine peaks in the blood and myocardium within seconds after intravenous injection. It rapidly declines within 3–5 min but remains in the blood and myocardium in low concentrations for a long time.9,10Previous isolated heart model studies did not add bupivacaine to reperfusion fluid because the existence of residual plasma bupivacaine concentrations after toxic insult was questionable. In the current study, 40 μm bupivacaine was added as a background concentration to the reperfusion fluid to better approximate common clinical situations. Our results indicate that even low concentrations of lipid emulsion can reverse bupivacaine-induced asystole, demonstrating a concentration-effect and time-effect relationship between lipid emulsion and the recovery of cardiac function.

Measures of contractility, such as left ventricular systolic pressure, are highly dependent on and inversely related to HR. Therefore, we used RPP as a measure of recovery because it incorporates contractility with a correction for HR. RPPr is more useful as an indicator of recovery. RPPr was improved with the increasing concentrations of lipid emulsion in a concentration-dependent manner. Postulated mechanisms of lipid emulsion in the reversal of local anesthetic toxicity include either: (1) lipid emulsion extraction of lipophilic local anesthetics from aqueous plasma or cardiac tissues2,3,11(“lipid sink” theory), and/or (2) counteraction of local anesthetic inhibition of myocardial fatty acid oxidation.12Our results showed that even low lipid concentrations could lead to a reversal of LVdevP, whereas increasing lipid concentration did not result in increased myocardial contractility. In addition, left ventricle diastolic function was improved in higher lipid concentration groups, although a concentration-dependent relationship was not demonstrated. The trend for RPP paralleled that for recovery of HR (fig. 3 and 4). Enhanced RPPr was mainly a result of improved HR. Lipid treatment may produce a concentration-related restoration of cardiac function primarily by counteracting bupivacaine inhibition of myocardial conduction.

A key finding of our study is the relationship between the concentration of lipid emulsion and myocardial bupivacaine concentration, and the resulting recovery of the heart from asystole. The myocardial bupivacaine concentration-effect curve (fig. 6) indicated lipid treatment hastened the removal of bupivacaine from myocardial tissue when demonstrating some degree of cardiac recovery. When lipid concentrations were 2% or more, myocardial bupivacaine concentration significantly decreased when compared with lower concentration lipid groups—thereby confirming improved cardiac function and better recovery when the lipid concentration was higher. These findings support the lipid sink theory—or at least support the premise that lipid infusion accelerates the loss of bupivacaine from myocardial tissue. In addition, our results demonstrate that higher lipid concentrations result in a larger loss, and that this loss is contemporaneous with better recovery of cardiac function.

Our study time-response analysis demonstrated that, when lipid emulsion concentrations are 4% or higher, RPP peaks and then trends downward during recovery. This result may be related to the structural characteristics of lipid emulsion; high-dose lipid emulsion infusion in a short period of time may have fat embolism effects, especially when part of emulsified fat is greater than 5 μm in diameter.13,14Hiller et al.  15found microscopic abnormalities in the lung when giving high concentrations (60 ml/kg) of 20% lipid emulsion to rats in vivo . Our study instead used an isolated rat heart model, without the effect of the “filtration” properties of an in vivo  lung. It may be that future studies will link the use of high concentrations of lipid emulsions to cardiac fat embolism. The application of lipid emulsion as an antidote in the setting of bupivacaine toxicity should be done under strict monitoring conditions with paramount vigilance on the part of the physician during and after resuscitation.

Stehr et al.  16found that lipid application in l-bupivacaine–induced cardiac depression resulted in improved inotropy in isolated rat hearts, but that HR did not change. The amount of lipid in their study was based on a recommendation by Weinberg.17The effluent lipid concentration used was 8.9 ± 0.4 μl/ml, or a 0.18% concentration. Our results showed HR during recovery in the 2–16% lipid groups was higher than in the 0.25% group. This finding suggests that higher concentrations of lipid emulsion than those investigated in previous studies may demonstrate larger beneficial effects on cardiac function. It is noteworthy that, in our study, cardiac function was significantly improved in the 2% lipid group although a decreasing tendency during recovery was not observed. This result suggests an optimal concentration of lipid emulsion should exist in its application for use in local anesthetic toxicity.

Our study demonstrated that the effect of lipid emulsion to reverse bupivacaine cardiac toxicity was exact, showing—within specified limits—a concentration-dependent relationship during the early recovery phase. An appropriate increase in the current recommended dose of lipid emulsion may improve cardiac function after resuscitation. However, safe use of increasing concentrations of fat emulsion in patients with bupivacaine toxicity requires further study.

The authors thank Roger Dzwonczyk, M.S.B.M.E., Associate Professor, Department of Anesthesiology, Ohio State University, Columbus, Ohio, for editing this manuscript. They also acknowledge Lianbo Yu, Ph.D., Biostatistical Scientist, Center for Biostatistics, Columbus, Ohio, and Hongying Shi, Ph.D., Assistant Professor, Department of Preventive Medicine, Wenzhou Medical College, Zhejiang, China, for data analysis.

• ❖ Intravenous administration of lipid emulsion reverses cardiotoxicity from local anesthetics, but the concentration-response and time-response relationships for this treatment are unclear.

• ❖ In isolated rat hearts, lipid emulsions reversed bupivacaine-induced asystole with increasing efficacy from 1% to 8% concentrations, paralleled by decreasing bupivacaine concentrations in the myocardium.