Hemodynamic changes in lipid emulsion therapy ( SMOFlipid ) for bupivacaine toxicity in swines 1 Alterações hemodinâmicas na terapia por emulsão lipídica ( SMOFlipid ) na intoxicação por bupivacaína em suínos

PURPOSE: To evaluate the hemodynamic changes following SMOFlipid emulsion therapy with after bupivacaine intoxication in swines. METHODS: Large White pigs were anesthetized with thiopental, tracheal intubation was performed and mechanical ventilation was instituted. Hemodynamic variables were recorded with invasive pressure monitoring and pulmonary artery catheterization (Swan-Ganz catheter). After a 30-minute resting period, 5 mg.kg-1 of bupivacaine by intravenous injection was administered and new hemodynamic measures were performed 1 minute later; the animals were than randomly divided into two groups and received 4 ml.kg-1 of saline solution or 4 ml.kg-1 of SMOFlipid emulsion 20%. Hemodynamic changes were then re-evaluated at 5, 10, 15, 20 and 30 minutes. RESULTS: Bupivacaine intoxication caused fall in arterial blood pressure, cardiac index, ventricular systolic work index mainly and no important changes in vascular resistances. SMOFlipid emulsion therapy was able to improve blood pressure mainly by increasing vascular resistance since the cardiac index had no significant improvement in our study. Hemodynamic results of the use of lipid emulsion in bupivacaine intoxication were better than the control group. CONCLUSION: The SMOFlipid emulsion is a option for reversing hypotension in cases of intoxication by bupivacaine.


Introduction
Locoregional anesthesia may require high doses of local anesthetics and there is always a potential risk of toxic reactions due to inadvertent intravascular injection.Bupivacaine is still the most widely used agent, owing to its good quality of anesthesia and prolonged duration of action 1,2 .However, in 1979, an editorial of Anesthesiology 3 on the severe cardiovascular effects produced by accidental intravascular injections led to a search for new agents with lower toxicity and antidotes to combat their effects.
Intravascular bupivacaine injection may result in severe toxicity of the central nervous and cardiovascular systems 4 which are the main targets.Damage to the nervous system precedes cardiac compromise with signs and symptoms including tinnitus, metallic taste, visual disturbance, perioral numbness, loss of consciousness, convulsions and death.Cardiotoxicity is characterized by a decrease in ventricular contractility, loss of vasomotor tonus, cardiovascular collapse, arrhythmia and asystole 5 .Until 1981 the incidence of systemic toxicity was 100 per 10,000 epidural anesthesias 6 ; safety measures such as frequent aspiration before injection, use of a test dose and epinephrine began to be adopted in an attempt to reduce complications 6 .In a review study conducted in 1995, Brown et al. 7 reported a rate of 1.2 complications per 10,000 epidural anesthesias, similar what was found by Auroy et al. 8 in France.New local anesthetics of long duration were synthetized and launched in the market such as ropivacaine and levobupivacaine which would be safer alternatives 9 .In 1998, Weinberg et al. 10 demonstrated that a lipid emulsion (LE) was efficient in the treatment of local anesthetic cardiotoxicity, increasing by 50% the mean lethal dose of bupivacaine, however these solutions had been used for parenteral nutrition since 1961!Rosenblatt et al. 11 and later Litz et al. 12 were the first to publish the successful use of LE in the treatment of cardiac arrest due to local anesthetics, when conventional resuscitative techniques had failed.There are now reports of the successful use of LE to treat toxicity due to these agents 13,14 .The aim of this study was to evaluate the hemodynamic changes after intravenous bupivacaine injection as a clinical scenario of toxicity and treatment with a 20% lipid solution named SMOFlipid 15,16 (Soybean oil + Mediumchain triglycerides + Olive oil + Fish oil).

After approval from the Animal Experimentation Ethics
Committee of the Biology Institute of UNICAMP, in compliance with the Council for International Organization of Medical Sciences (CIOMS) ethical code for animal experimentation, 20 Large-White pigs weighing between 19.5 and 23.5 kg were fasted overnight with free access to water.On the morning of the study the animals were weighed and body surface area was calculated by the formula 17 BS (m 2 ) = (9 x weight in grams 2/3 ) x 10 -4 introducing the results on the Engstron AS/3 hemodynamic monitor for index calculation.Venipuncture was performed in the animal ear and anesthesia was induced with 2.5 % sodium thiopental 25 mg.kg -1 .
After intubation, the animals were maintained under controlled ventilation in a partial rebreathing system, tidal volume of 15 ml.kg -1 and adequate respiratory rate to obtain P ET CO 2 between 32 and 34 mmHg.The pigs were ventilated with an air and O 2 mixture maintaining hemoglobin saturation higher than 97%, measured by a sensor placed on the tongue.A cardioscope was also used in DII lead.Isoflurane 1% expired fraction was used for maintenance of anesthesia.Local anesthesia with 5 ml of 1% lidocaine without vasoconstrictor was administered to the internal portion of the pig thigh for dissection of the femoral artery and vein for continuous measurement of arterial blood pressure and introduction of the 7F Swan-Ganz catheter located in a branch of the pulmonary artery by the morphologic aspect of the pressure curve obtained.The following variables were measured: cardiac output, heart rate, mean arterial blood pressure, central venous pressure, mean pulmonary artery pressure and pulmonary capillary pressure.The monitor, using classical formulas calculated vascular resistances, ventricular work and all their indexes.After 30 minutes of stabilization and rest, baseline standard hemodynamic measurements were taken (T 0 ), followed by 5 mg.kg. - of intravenous bupivacaine injection.One minute later, new hemodynamic measurements were taken (T 1 ).The animals were then randomly divided into two groups.In the first group 4 ml.kg - of saline solution were injected immediately after T 1 (CTRL group) and in the other 4 ml.kg - of SMOFlipid (SMOF group) were �injected.New hemodynamic measurements were taken at 5, 10, 15, 20 and 30 minutes after toxicity (T 5 to T 30 ).The numerical variables measured in a single time point were compared by the Student's t test.To study numerical variables in various time points Analysis of Variance for repeated measurements (ANOVA) was used, Duncan's test was used to compare the groups in each time point and the contrast profile test to analyze the progression between time points in each group.The significance level adopted was 5% (p < 0.05).

Results
Table 1 shows mean and standard deviations of weight and body surface areas.There were no statistic differences.There were no differences in hemodynamic parameters at rest as well.

Mean arterial blood pressure
As shown in Figure 1, after bupivacaine injection there was a significant and similar decline in mean arterial pressure in both groups.Immediately following injection of the lipid, pressure increased in SMOF and remained higher than CTRL until the end (p<0.001).In CTRL, pressure increased progressively until 15 minutes.After this time and until the end of the experiment, values returned to levels similar to those in T 0 (p<0.001).In SMOF, pressures increased significantly in each time period until T 15 and remained similar after that and always higher than at T o .

Heart rate
As shown in Figure 2 there was no changes in SMOF but the heart rate declined in CTRL and was lower than SMOF until T 5 (p=0.006).In CTRL values remained lower than T 0 till the end but increased from T 1 to T 10 (p=0.001).

Central venous pressure
As seen in Figure 3, there was an increase in values in both groups, in SMOF they were higher than CTRL until T 20 (p<0.001).In both groups the results did not return to levels similar at rest until the end of the experiment, although in SMOF results were successively different from previous values (p<0.001).

Mean pulmonary artery pressure
As observed in Figure 4, mean pulmonary artery pressure differed between groups after toxicity and until the end of the experiment (p<0.001).In SMOF, it increased significantly and remained higher than rest till the end with differences between T 1 and T 5 in addition to T 20 and T 30 .In CTRL, there were no differences relative to T 0 (p<0.001).

Pulmonary capillary wedge pressure
As observed in Figure 5, in SMOF values were higher than CTRL from T 1 to T 15 (p=0.002).In both groups, pressure values in all time periods were higher than in T 0 .However, in SMOF T 5 and T 15 were also higher than the values immediately prior to those time periods (p<0.001).

Cardiac index
As shown in Figure 6, the cardiac index decreased equally in both groups (p=0.004).In CTRL values only returned to those similar to rest in T 30 and T 10 was lower than T 15 .In SMOF, values did not return to levels similar to T 0 , although they increased from T 20 to T 30 (p<0.001).

Systemic vascular resistance index
As observed in Figure 7, the systemic vascular resistance index in SMOF was higher than in CTRL beginning at T 5 till the end (p<0.001).In CTRL there was an increase from T 10 to T 30 and T 1 was lower than T 5 .In SMOF all results were higher than T 0 and exhibited significant increases in T 5 , T 10 and T 30 compared to previous time (p<0.001).

Pulmonary vascular resistance index
As shown in Figure 8, there was a significant increase in pulmonary vascular resistance in SMOF that remained higher than in CTRL from T 5 to T 30 (p<0.001).In CTRL, there were no differences between time periods; in SMOF all values were higher than those at rest, T 1 was lower than T 5 and T 20 higher than T 30 (p<0.001).

Left ventricular systolic work index
As observed in Figure 9, left ventricle systolic work index had a significant decline in both groups and this was more striking in CTRL until T 15 (p=0.049).In CTRL all values remained lower than standard levels until T 20 and T 15 and T 30 were higher than the previous values; in SMOF there was a progressive increase in T 5 , T 10 and T 20 when values returned to similar at rest (p<0.001).

Right ventricular systolic work index
As observed in Figure 10, the right ventricular systolic work index had a significant decrease in both groups, however, in SMOF values recovered and remained higher then CTRL from T 5 till the end (p<0.001).In CTRL, values returned to those similar to rest in T 15 and remained constant until the end.In SMOF, after T 5 and until the end these values were higher than those at rest (P<0.001).

Discussion
Cardiotoxicity of local anesthetics is still a concern for anesthesiologists.Until recently, it was a devastating complication without any specific treatment.Since 2006, with a publication on the successful use of lipid emulsion 11,12 to treat cardiotoxicity due to intravascular injection of local anesthetics, several formulations have been suggested for this purpose 13 .We chose to use the 20% SMOFlipid solution because there are few cases describing its use for this type of complication.Three hypotheses on the mechanism of action of lipids in local anesthetic toxicity have been formulated.
The first theory, known as 'lipid sink', suggests the establishment of new pharmacokinetics, with expansion of the lipemic phase that would sequester local anesthetic molecules, decreasing the free fraction and consequently toxicity of these agents 18 .This is a well-accepted theory since it was observed that toxicity of other lipophilic drugs, as some antipsychotics, barbiturates, antidepressants, organophosphates and antihypertensives, also responds to a lipid infusion.In corroboration with this theory, Turner-Laurence and Kerns demonstrated in vitro the binding capacity of lipid emulsions to local anesthetics in buffer solutions 19 .
In the plasma, bupivacaine binds to alfa-1 glycoprotein and albumin.The affinity for alfa-1 is greater than that for albumin, although albumin is more abundant.The binding capacity of lipid emulsions to bupivacaine is important, a 1% solution is equivalent to 1.5 to 4 times the binding capacity of human plasma.Thus 100 ml of a 20% lipid emulsion would have the same binding capacity to albumin contained in 7 to 12 liters of plasma 20 .The second theory states that lipid emulsions would overcome by mass effect, or by some other unknown mechanism, bupivacaine inhibition of carnitine-acetyltransferase.This enzyme is essential for fatty acid transportation into the mitochondria where they are used as substrate for energy production by the myocardium and other tissues 21 .And finally, the third theory states that an increase in fatty acids would increase the calcium levels in cardiac myocytes, promoting a positive inotropic effect 22 .In our study, we observed a rapid and significant improvement in arterial blood pressure in animals treated with SMOFlipid after local anesthetic toxicity.
We can infer that the reasons for this improvement may be an extraction of bupivacaine molecules by the lipid emulsion and an increase in the systemic and pulmonary vascular resistance indexes, since we did not observe great changes in cardiac index.
In 2011, Ok et al. 23 published the first study demonstrating that LE reverses vasodilation caused by local anesthetics in the isolated rat aorta, they suggested that the mechanism of this reversal may be blunting of inhibition mediated by local anesthetics in the calcium voltage-dependent channels in smooth vascular muscles and partially by endothelial release of nitric oxide 23 .It is worth noting that, if the reason for restoration of arterial blood pressure was only an increase in vascular resistance and the lipid emulsion did not exert this type of influence, the same results should have been achieved when vasoconstrictors were used.However, cardiac arrest due to this type of complication is often resistant to adrenergic therapy and may even lead to severe pulmonary edema after the use of epinephrine 24 .Weinberg studied cardiac arrest caused by bupivacaine toxicity in rats.The same author reported better results when LE rather than epinephrine was used for treatment 25 .Concerning the heart rate, in SMOF it was more stable than CTRL, a result similar to that found by Chen et al. 26 .We also observed a significant increase in pulmonary artery pressure in SMOF compared to CTRL.This alteration was described as one of the side effects of LE when a dose higher than 100 mg.kg -1 .hwas used or when LE was given to patients with previous pulmonary disease 27 .Finally, other side effects of lipid emulsions have been described: risk of infection, allergic reactions especially in soy oil emulsions, thrombophlebitis, increased amylase, hyperlipidemia, hemolytic anemia, thrombocytopenia and fat embolus 19 .

Conclusion
The SMOFlipid emulsion proved to be efficient in the reversal of hemodynamic alterations due to bupivacaine toxicity in pigs, suggesting that the early use of this lipid solution may help to prevent or mitigate the cardiotoxicity of local anesthetics.

TABLE 1 -
Weigh and body surface area.