Intravenous Lipid Emulsion Increased Muscles Power and Survival Time of Phenobarbital in Intoxicated Rats

Introduction
Phenobarbital (PHB) is an anticonvulsant, which causes acute poisoning due to easy availability ^1,2. Suicidal actions are the major cause of phenobarbital intoxication ¹. PHB poisoning leads to stupor or coma and in high dose induces severe respiratory depression and cardiovascular collapse. These may result fatal outcome up to 7% of intoxicated patients ¹. Authors of literatures suggest no especial antidote; however, they recommended some methods such as alkaline diuresis, activated charcoal and hemodialysis.
Administration of Intravenous Lipid Emulsion (ILE) to severe intoxicated patients could reverse poisoning and rescue their lives ³. Sirianni and his colleagues reported the use of ILE following 70 min of unsuccessful standard cardiopulmonary resuscitation in a 17-year-old girl who developed seizures and cardiovascular collapse after intentionally ingesting an overdose of bupropion, lamotrigine and amphetamine. Her cardiovascular status has been improved and she recovered with near-normal neurologic function ⁴.
In past ten years, there are several animal studies that reveled antidotal effect of ILE against drug intoxication ^3,5-9. Moshiri et al reported that ILE could reversed the hypotension, hypothermia, catalepsy and miosis of haloperidol intoxicated rabbits sooner than normal saline ^5,6. His colleagues evaluated the ILE effect on tramadol induced seizure and hypotension in rabbit and found similar effects ⁷. We evaluated the effect of ILE on PHB poisoning in this issue.

Materials and Methods
Ten male rats weighting 200-250 g were housed in cages with a 12 hr light/dark cycle and easy access to food and water. Animals were divided into two groups consisting of five rats.
Ampoules of PHB (200 mg/ml, Chemidarou, Iran) were diluted and administrated to all animals. They also received ILE 20% [Intralipid Emulsion (Fresenius Kabi AB) Spain].
We applied 18.6 ml/kg ILE 20% or 18.6 ml/kg Normal Saline (NS), as a control, 30 min after 100 mg/kg PHB injection. ILE or NS were infused over 15 min. Rats received the injections through the vein cannula.
The animals were monitored before PHB injection (zero time), 0.5 hr (before ILE 20% or NS infusion) and 1, 3, and 6 hr after PHB injection. The muscular powers of animals were evaluated by scoring scale as Grade 4: normal mobility; Grade 3: ataxic gait; Grade 2: stretch movements after tail stimulation; Grade 1: standing after tail stimulation; Grade 0: no voluntary movements after tail stimulation, four limb paralysis ⁹. All animals were evaluated by an individual unaware of the groups. Blood pressures of all animals were measured using tail-cuff method (Power lab, Data Acquisition Systems, US). Mortality rates and survival times were also recorded.
The data were expressed as the mean ± SEM. Unpaired T test was used to determine the significance of a difference in blood pressure and survival times between groups. Non parametric tests were used if necessary. Fisher’s exact test was used to evaluate differences in mortality rate of different groups. The alpha level was set at 0.05. All statistical analyses were performed in SPSS 11.5.

Results
Half an hour after PHB toxic dose administration, the blood pressure of animals could not be detectable and animals were hypotensive. We found no significant difference between blood pressure of NS and ILE treated groups at all evaluating times, and there was no difference in returning to normal blood pressure between two groups (Figure 1).
There was no significant difference in animal muscular power scores before PHB administration. The scores significantly increased 3 and 6 hr after ILE infusion compare with control group (p= 0.007 and 0.0371, respectively), (Figure 2).
There was no statistically significant difference between mortality rates of ILE treated and control groups (0% and 60% respectively; p=0.167), (Table 1). However, the mean survival time of ILE group was higher than NS group (Table 1).

Discussion
PHB is a relative lipid-insoluble barbiturate that at high doses induces cardiovascular depression with hypotension, nystagmus, ataxia, severe respiratory depression, coma and death ¹⁰. Blocking the sympathetic ganglion and inhibition of medullar vasomotor centers by PHB at high doses induce vasodilatation, hypotension, bradycardia and low cardiac output ¹¹. ILE could reverse hypotension induced by lipophilic-drug overdose such as haloperidol, methamphetamine and propranolol ^6,8,12. However, it is not able to reveres non lipophylic drug induced hypotension, such as atenolol ¹³. In our study, the administration of ILE could not reverse PHB induced hypotension sooner than normal saline, as volume expander.
The most important hypothesized mechanism of ILE therapy in lipophilic drug induced toxicity is lipid sink. Based on this theory, ILE relieves the toxicity of lipophilic xenobiotics via redistribution from action site and receptors in tissue to plasma or inert lipid induced compartment ^14-21. It has been shown that the lipid solubility of drug has a direct relationship with ILE detoxification capacity ^16,17. PHB is relatively lipid-insoluble barbiturate with volume of distribution of 0.5-1 mg/L ²². It seems that the ILE effect on survival and muscles power of PHB intoxicated rats are not explained by lipid sink theory and some other mechanisms might be involved ^5,7,18-21.
PHB induces Central Nervous System (CNS) depression by enhancing GABAergic transmission ²². There are some evidence on cross talking of N-methyl-D-aspartate (NMDA) and GABAA receptors on neurons, two important excitatory and inhibitory receptors ²³. NMDA receptor activation cross-inhibits GABAA receptor in a concentration dependent manner ²⁴. Cong et al reported that the inhibitory effect of NMDA receptor activation on GABAA receptor mediated by calcium influx through NMDA receptors ²⁴. Stelzer and coworkers evaluated the effect of intralipid on cell membrane of mice cultured cortical neurons, by patch clamp technique. They revealed that intralipid activates NMDA receptor channels in a concentration-dependent manner in cortical neurons ²³. So it seems that part of effect of ILE on PHB toxicity is related to reversing GABAA receptor inhibition through cross talking of NMDA activation by lipid. However, it has been stated by Weigt et al that ILE has a dual effect on NMDA receptor channels. According to their research projects ILE can decrease ^25,26 and increase ²⁷ NMDA receptor activity.
PHB is excreted unchanged through kidney and due to its low pKa (7.24), urine alkalinization to pH of 7.5-8.0 can increase PHB excretion up to 5-10 folds ^11,22,28. Intralipid has alkaline pH (pH=8) ²⁹. The blood volume of a male rat is 68.6 ml/kg ³⁰ and we administrated ILE in 27% of total rat blood volume (18.6/68.6). It is suspected that ILE by raising the blood pH can increase the PHB renal excretion. However, we have not recorded rat’s blood pH or urine pH. On the other hand, our control group received normal saline without bicarbonate, and we could not able to evaluate this hypothesis. Fox et al evaluated pH of premature infants after continues administration of 5 ml/kg (1 g/kg) intralipid and they found no difference on pH of their patients at 4 hours later ³¹. We used much higher dose and infusion rate.
Nutritional theory may be another suggested mechanism to explain rising muscles activity score of ILE group. However, this theory is expressed for cardiac muscles ³, there are some evidences that ILE has similar effects on skeletal muscles. Free fatty acid oxidation is an energy source of skeletal muscles cells. This process is limited by two steps, the availability of free fatty acid and the abundance of fatty acid transports into mitochondria ^32-34. The infusion of high dose ILE raises the serum level of free fatty acid secondary to activation of lipoprotein lipase ³⁵, so it seems that improving rat muscles power after ILE infusion is partly due to free fatty acid availability to skeletal muscles.

Conclusion
In conclusion, ILE could reverse the reduction of muscles power and improve survival time of PHB intoxicated rats. However, further detailed research needed to confirm this finding and to reveal the exact mechanisms.

Acknowledgments
The authors wish to thank the Vice Chancellor of Research, Mashhad University of Medical Sciences for their financial support.

Conflict of Interest
The authors report no declarations of interest.