Article, Cardiology

Intravenous lipid emulsion prolongs survival in rats intoxicated with digoxin

a b s t r a c t

Background: Intravenous lipid emulsion eliminates the toxicity-related symptoms of several drugs. We hypoth- esized that intravenous lipid emulsion prolongs the survival time in digoxin-intoxicated rats.

Methods: Electrocardiograms of 14 anesthesized Wistar rats were monitored. All of the rats received digoxin in- fusion at a rate of 12 mL/h (0.25 mg/mL). Five minutes after the start of digoxin infusion, animals were treated either with 12.4 mL/kg intravenous lipid emulsion (group L) or saline (group C). The primary outcome variable was time elapsed until asystole development. Cumulative dose of digoxin required to induce asystole was also recorded.

Results: Mean time until asystole development in groups C and L were 21.28 +- 8.61 and 32.00 +- 5.41 minutes, respectively (Pb .05). The mean lethal doses of digoxin in the groups C and L were 3.97 +- 1.54 and 6.09 +-

0.96 mg/kg, respectively (Pb .05). Conclusion: Intravenous lipid emulsion prolonged the time until asystole development and increased cumulative lethal dose in rats intoxicated with digoxin.

(C) 2016

  1. Introduction

Treatment of local anesthetic toxicity with the aid of intravenous lipid emulsion (ILE) became a standard of care in recent years [1]. At the first years of the discovery, it was thought that ILE forms a “lipid sink” in the intravenous compartment and that this lipid sink incorporates highly lipid-soluble bupivacaine inside itself so that bupivacaine is removed from the tissues where it exerts its toxic effects [2]. This hypothesis has led to an idea that ILE therapy could have similar therapeutic effect at the toxic conditions caused by highly lipid-soluble drugs. Thereafter, suc- cessful treatment reports of cardiovascularly collapsed or central nervous system disoriented patients owing to drug intoxications have appeared in the literature [3-7]. Experimental animal models of cardiac arrest due to drug toxicity with verapamil, clomipramine, and amitriptyline have also yielded supporting evidence for ILE therapy [8-10]. Thus, ILE has become a promising therapy for drug toxicities [11,12].

Digoxin is one of the oldest drugs used for the treatment of heart fail- ure. Although digoxin is prescribed frequently, its therapeutic margin of

? Financial support: none.

?? Conflict of interest: none.

* Corresponding author at: Dokuz Eylul Universitesi, Tip Fakultesi, Dokuz Eylul Universitesi Hastanesi, Ameliyathane, Kat: 1, Narlidere, Izmir, Turkey. Tel.: +90 535 887

17 23, +90 232 412 28 01.

E-mail addresses: [email protected], [email protected] (B.S. Yurtlu).

safety is narrow. It is a lipid-soluble cardiac glycoside that is readily absorbed from the gastrointestinal tract [13]. Because its therapeutic index is narrow, dose adjustment is a major concern in the elderly [14]. Patients with digoxin overdose can be seen at emergency and in- tensive care unit services. Mortality rate during the hospital course can be as high as 7% in patients intoxicated with digoxin [14]. Because digoxin is lipid soluble, we have hypothesized that ILE therapy may delay cardiac arrest due to digoxin intoxication. To test this hypothesis, digoxin-intoxicated rats were treated with either ILE or saline. The pri- mary outcome variable was determined as time until asystole.

  1. Methods

We used a model that is an established way of studying drug toxicity [8,15]. The study protocol was approved by the Animal Ethics Commit- tee of the School of Medicine, Dokuz Eylul University (date 04/03/2014, number: 03/05). The study was carried out at the Dokuz Eylul Universi- ty Faculty of Medicine, Department of Laboratory Animals. All of the rats included in the study were obtained from institution’s Laboratory Ani- mals Department, all fed with standard rat pellets and housed in temperature- and humidity-controlled (22?C-24?C and 60% relative hu- midity) rooms that were lit on a daily schedule (12:12 hours light/dark) until the day of experiment. During the experimental period, the care of the laboratory animals was in accord with international guidelines.

0735-6757/(C) 2016

Fourteen Wistar rats weighing between 245 and 291 g were anesthesized with intraperitoneal injection of 60 mg/kg ketamine. Elec- trocardiogram was applied to monitor the heart rate, and 2 intravenous cannulas were placed at the lateral tail veins. A venous blood sample from the tail vein was drawn to control blood pH and gas tensions. One liter per minute of oxygen was supplied to all the rats. Digoxin (0.25 mg/mL), at a rate of 12 mL/h, was started to be infused to all of the rats at time zero (T0). Digoxin infusion continued until rat’s death (defined as asystole lasting 1 minute together with absence of respirato- ry efforts). This infusion rate of digoxin was taken from a previous ex- periment to enable death of animals within a reasonable time frame [15]. At the fifth minute of infusion (T5), rats were administered either

12.4 mL/kg 20% ILE (Clinoleic 20% Lipid Emulsiyonu, Eczacibasi-Baxter, Istanbul) or saline at an equal volume over 5 minutes through the sec- ond venous route [8]. Duration of the time elapsed between start of di- goxin infusion and death of the rat was defined as time until asystole and recorded in minutes. Times of first appearance of dysrhythmia (atrio- ventricular conduction delay) and widening of QRS complex were re- corded in minutes. The total dose of digoxin infused until asystole development was recorded.

Statistical analysis

Data were analyzed by a statistician who was blinded to treatment groups. SPSS 15.0 program was used for the analysis of data. Data were expressed as mean +- SD. Mann-Whitney U test was used for the analysis of baseline and toxicologic characteristic properties of the groups. A P value less than .05 was considered as statistically significant.

  1. Results

The mean age and weight of the rats in all the groups included in the study and their mean heart and respiratory rates before drug infusion were similar (Table 1). In control blood gases analyses, none of the ani- mals had developed respiratory acidosis, hypoxia, or hypercarbia. In all animals, the terminal event was respiratory arrest, most often preceded by primary apnea and then gasping respirations. The T5 heart rates of groups C and L before lipid infusion were determined as 372.42 +-

45.69 and 361.00 +- 50.57, respectively, and there were no statistical dif- ference between them (PN .05).

Time until asystole at groups C and L were determined as 21.28 +-

8.61 and 32.00 +- 5.41 minutes, respectively. Time until asystole in group L was significantly longer than that in group C (Pb .05) (Table 2, Figs. 1 and 2). Whereas 4 rats in the group L survived until the 30th min- ute, only 1 rat survived longer than 30 minutes in group C (Figs. 1-3).

The mean lethal doses of digoxin in control and lipid groups were

3.97 +- 1.54 and 6.09 +- 0.96 mg/kg, respectively. The mean lethal dose of digoxin was significantly higher in group L than in group C (Pb .05).

Heart rate tended to decrease in both groups during the digoxin in-

fusion; however, the rate of decrease was faster in group C in compari- son with group L (Fig. 4).

Electrocardiographic analysis showed that the dominant rhythm was Sinus bradycardia together with atrioventricular conduction block, and widening of the QRS complex followed that later on. Some of the rats had junctional, idioventricular rhythms or varying degrees of heart block before asystole.

Table 1

Baseline characteristics of groups (mean +- SD)


Control (n = 7)

Lipid (n = 7)


Age (d)

126.00 +- 9.89

127.00 +- 10.24


Weight (g)

266.28 +- 15.46

262.57 +- 16.02


Heart rate (beat/min)

388.28 +- 35.59

387.14 +- 62.37


Respiratory rate (breath/min)

86.14 +- 5.08

85.00 +- 4.32


Mann-Whitney U test.

Table 2

Toxicological characteristics of groups (mean +- SD)


Control (n = 7)

Lipid (n = 7)


Dysrhythmia duration (min)

14.28 +- 6.99

22.28 +- 8.69


QRS change duration (min)

16.71 +- 8.63

21.57 +- 7.95


Time until asystole (min)

21.28 +- 8.61

32.00 +- 5.41


Lethal dose of digoxin (mg/kg)

3.97 +- 1.54

6.09 +- 0.96


* Pb .05 compared with control group, Mann-Whitney U test.

  1. Discussion

The results of the current study demonstrated that administration of ILE before a catastrophic cardiac event increases the dosage of digoxin required to produce asystole. In addition, ILE therapy increases the time until asystole development. To the best of our knowledge, these re- sults are the first findings in the literature giving clues about the poten- tial role of ILE therapy for digoxin intoxication.

Digoxin has a long history in the treatment of chronic heart failure. Although new therapeutic options have emerged for the treatment of heart failure during the last decades, digoxin remains as an important tool, and intoxication reports with this drug are still frequently encoun- tered in the literature. Because the therapeutic index of digoxin is small and overdose is a common problem, digoxin-binding antibodies were developed as an antidote. Thus, most of the previous studies focus on the effects of digoxin-binding antibodies. The other examples focus on the effect of glucose-Insulin infusion, anticalin administration, or nanomagnet-based electrochemical immunosensor technology to remove digoxin molecules from the plasma [16-19]. Unfortunately, it is not possible to make direct comparisons between the results of this study and those previous ones because of methodologic heterogeneities. The improvement in survival with ILE therapy in drug toxicity other than a local anesthetic was first demonstrated by Krieglstein et al [20]. They had shown that rabbits which have been intoxicated with chlor- promazine had improved survival if they were pretreated with ILE [20]. Two decades later, the therapeutic efficacy of ILE for local anesthet- ic systemic toxicity was established both with experimental studies and with case reports. The first experimental evidence of ILE therapy came up with the lipophilic local anesthetic bupivacaine, and clinical reports have confirmed this finding later on [21-23]. Because the drug’s lipo- philicity is suggested as a key point for the efficacy of ILE, it is thought that the same therapy could have beneficial effects on toxidromes with other lipophilic agents. However, accumulated experimental evi- dences about drugs other than local anesthetics are limited at the mo- ment. Most of the other drugs investigated for ILE therapy’s effect belong to the cardiovascular group of drugs such as ?-blockers and cal- cium channel blockers. One of these studies was conducted with verap- amil. Tebbutt et al [8] administered intravenous 37.5-mg/(kg h) verapamil infusion to the rats, and then at the fifth minute of infusion, rats were treated with either ILE or saline. They found that the survival time in ILE-treated rats was almost twice longer than that in the saline- treated rats (44 +- 21 vs 24 +- 9 minutes). They also found out that there was also a similar difference in the mean lethal dose (27.4 vs 14.7 mg) of verapamil for both groups. The results of the current study demonstrate a similar increase in both survival time (32.00 vs 21.28 minutes) and the mean lethal doses of digoxin (3.97 vs 6.09 mg) for saline- or ILE-treated rats, respectively. The findings of Tebbutt et al [8] were later replicated by another study in which dogs were used and resuscitated [24]. In the later study, it was demonstrated that while resuscitation has survived 100% of animals which were treated with ILE, resuscitation was success- ful only in 14% of animals which received saline [24]. The results of our study gave the initial clues about the role of ILE for digoxin intoxication. In case of life-threatening toxidromes, it does not seem ethical to con- duct a randomized controlled study on volunteers, so our findings

need a similar confirmation and advancement with a resuscitation


Image of Fig. 1

Fig. 1. Box-and-whisker plot of asystole duration by lipid and control group status. Median indicated by black line, and upper and lower quartiles indicated by box edges.

The effect of ILE therapy for lipophilic ?-blockers is not similar to the results obtained with verapamil. For example, propranolol and meto- prolol are lipophilic ?-blockers, and if “lipid sink” is the sole mechanism of ILE therapy, theoretically, their toxicity would have benefit from ILE. However, in several Animal experiments, supportive evidence for ILE therapy in lipophilic ?-blocker toxicities could not be identified [25-28]. On the other hand, ILE therapy resulted in better recovery scores when compared with standard bicarbonate therapy in lipophilic

clomipramine toxicity model [29]. To clarify the underlying mechanism of this extraordinary success of ILE in clomipramine toxicity, the same authors conducted a new study in which they administered ILE therapy either alone or in combination with plasma exchange [30]. They found that there was no difference in survival time between sole ILE and ILE plus plasma exchange treatment groups. According to these results, the authors concluded that it cannot be just lipid sink explaining the ac- tion of ILE therapy [30]. These findings together with the demonstrated

Image of Fig. 2

Fig. 2. Dot plot of lethal dose by digoxin in lipid and control groups status.

Lipid Control





Number of live rats







0 1 2 3 4 5 6 7 8 9 10

Digoxin Dose (mg/kg)

Fig. 3. Dose-response curves for lipid and control groups. *Pb .05 compared with control group, ?2 test.

inefficacy of ILE for lipophilic ?-blockers suggest that a mechanism other than “lipid sink” exists. Possible mechanisms of effect are still a subject of investigation without a conclusive end [20,21,30,31]. Contro- versial issues about the underlying mechanisms of ILE therapy were summarized in a recent article [31]. The authors stated that lipid sink theory is not the sole mechanism and it is not enough to explain the suc- cess/failure dilemma of several lipophilic drugs [31]. Lipid sink theory or one of the other proposed mechanisms could be responsible for the delay in asystole duration observed in this current experiment. The present study does not propose any underlying mechanism of the ob- served effect but rather establishes a connection between digoxin over- dose and lipid emulsions.

The results of this study should be interpreted carefully in that these results do not present evidence about the return of spontaneous circu- lation after digoxin intoxication. The rats in the study were not resusci- tated to enable return of spontaneous circulation because we had no piece of evidence signaling that such a resuscitation could be helpful. In addition, there are opposite findings about epinephrine inclusion in Resuscitation protocols of ILE-treated Cardiovascular collapse models, which could be important for the success of resuscitation after digoxin toxicity [32,33].

Digoxin intoxications are continuous features of emergency services and intensive care units. According to the results of a recent retrospec- tive analysis, there was a 7% mortality rate during the hospital course of definitely “digoxin intoxication”-diagnosed patients [14]. On the other hand, results of a hospital-based questionnaire conducted at a de- veloped location (Ontario, Canada) revealed that only 9% of the acute care hospitals had an adequate supply of life-saving digoxin immune Antibody fragments in their stores [34]. The availability of lipid

emulsions elsewhere makes ILE therapy a valuable alternative where the other therapeutic choices are unavailable and the time is limited to increase survival.


Firstly, researchers conducted this study by themselves, so the study was single blinded; however, analysis of the data was performed by another re- searcher (VH) who was not directly involved in experiments and was unaware of the group assignments. Secondly, rats had spontaneous ventilation in the model, and this can be a confounding factor, as acid-base status and thus surviv- al duration of the rats could be affected by their ventilation patterns. However, there was no statistical difference at the initial blood gas measurements of the groups before the digoxin infusion, reflecting a similar baseline acid-base status. On the other hand, this situation mimics the condition in the real life where pa- tients have spontaneous breathing during the toxin intake.

In conclusion, intravenous lipid emulsion increases time required to asystole development and cumulative lethal dose in digoxin-intoxicated rats. These results exhibit the potential of ILE therapy in cardiovascularly col- lapsed digoxin overdose patients when specific antibodies are unavailable. Supporting further evidence is necessary to make conclusive statements.


The authors would like to thank Dokuz Eylul University, Department of Laboratory Animals Science, for their supply of the animals to conduct this study.

Image of Fig. 4

Fig. 4. Heart rate vs time for control and lipid groups. *Pb .05 compared with control group, Mann-Whitney U test.


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