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Anesthetic Pharmacology: Research Report

The Efficacy and Safety of Intravenous Emulsified Isoflurane in Rats

Zhou, Jian-Xin MD*; Luo, Nan-Fu MB; Liang, Xiao-Min MD; Liu, Jin MD

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doi: 10.1213/01.ane.0000189612.24147.07
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By circumventing the anesthesia circuitry and the lung's functional residual capacity, IV administration of volatile anesthetics can accelerate the induction of anesthesia. Unfortunately IV injection of liquid volatile anesthetics is usually lethal for humans (1,2) and animals (3). Several preliminary studies reported IV induction of anesthesia without adverse effects in animals using emulsified halothane and isoflurane (4–8). In some of these studies the concentrations of isoflurane or halothane in the lipid emulsion were as large as 10% (liquid anesthetic volume percentage) (4,7). We speculated that these concentrations might be super-saturated according to solubility data measured at 37°C by Taheri et al. (9) and temperature coefficients determined in our previous study (10). The primary determinant of maximal anesthetic concentration when preparing a volatile anesthetic lipid emulsion is their solubility in the lipid. Emulsified volatile anesthetics are usually prepared at room temperature. No study has been conducted to determine the solubility of volatile anesthetics in lipid at room temperature. Further, there are limited data on the pharmacological efficacy and safety of IV emulsified volatile anesthetics in experimental animals (6,8). In the present study, we measured the liquid/gas partition coefficient of desflurane, sevoflurane, isoflurane, enflurane, and halothane in 20% and 30% Intralipid at 20°C and calculated the saturated concentrations of these anesthetics in a lipid emulsion. Using emulsified isoflurane at a concentration smaller than the saturated concentration, we determined its median effective dose (ED50) and median lethal dose (LD50) for IV induction of anesthesia in rats and compared safety index (SI) and recovery time with those of propofol, a frequently used IV anesthetic.

Methods

This study was approved by the Committee of Scientific Research and the Committee of Animal Care of the West China Hospital, Sichuan University.

Liquid/gas partition coefficient (λL/G) of desflurane, sevoflurane, isoflurane, enflurane, and halothane in 20% or in 30% Intralipid (fat emulsion injection, Sino-Swed Pharmaceutical Corp. LTD, China) were made at 20°C using the syringe-flask two stage equilibration method described previously (9). The λL/G was determined 6 times for each anesthetic in each Intralipid. Approximately 7 mL of Intralipid was added to the glass syringe and anesthetic vapor mixture (containing 3.0% desflurane, 1.2% isoflurane and 1.0% halothane in air; or 1.8% enflurane and 2.2% sevoflurane in air) to the 18-mL mark, and the stopcock was closed. The syringe was shaken vigorously and immersed in a waterbath at 20°C. Every 15 min for 2 h, the syringe was shaken vigorously for 5–10 s. After the third shaking, the plunger of the syringe was withdrawn to the 20-mL scale with the stopcock closed, thereby creating a slightly negative pressure. After this 2-h period (the first equilibration stage), the concentrations of volatile anesthetics in the gas phase of the syringe were then analyzed by gas chromatography. After expelling all of the gas in the syringe, an aliquot of 3.15 mL of equilibrated liquid in the syringe was aspirated into an evacuated 311-mL glass flask, whose internal volume had been measured precisely by water displacement. The flask was fitted with a Teflon stopper pierced with 2 16-gauge metal needles each connected with a nylon stopcock. After transferring the liquid, the flask was placed in a waterbath at 20°C, and the vacuum in the flask was gradually reduced by briefly opening the stopcock to the atmosphere. The flask was shaken vigorously once every 15 min over a 2-h period to ensure equilibration of anesthetic between the liquid and gas phases. At the end of this 2-h period (the second equilibration stage), a 10-mL aliquot of room air was added to the flask, mixed thoroughly, and then slowly withdrawn. The concentrations of anesthetics in this gas sample were analyzed by gas chromatography.

The λL/G of anesthetic was calculated as follows:

where VF is the volume of the flask, VL is the volume of liquid sample transferred into flask, C1 is the concentration of anesthetic in the gas phase of the syringe measured at the end of first equilibration period, and C2 is the concentration of anesthetic in the gas phase of the flask measured at the end of second equilibration period. Ten milliliters are added to the numerator to account for the 10 mL of air added before removal of the sample for analysis. Volatile anesthetic concentrations were analyzed by a HP 4890 gas chromatograph, equipped with a 6-m long stainless steel column (0.32 cm in diameter) packed with chromosorb-P 60/80 mesh maintained at 70°C. A 15 mL/min nitrogen carrier stream flow was delivered through the column to a flame ionization detector supplied by hydrogen at 35 mL/min and by air at 350 mL/min. Output from gas chromatography was collected by a HP 3398 GC working station and peak areas were automatically integrated. Primary (glass flask) and secondary (compressed gas tank) standards were used to calibrate the gas chromatography by the method described in our previous study (11).

Theoretically, when a solution is equilibrated with a saturated volatile anesthetic vapor, the anesthetic concentration in this solution represents the largest amount of anesthetic dissolved in solution without gross separation of liquid anesthetic. We named this concentration as saturated anesthetic concentration (Cs) and calculated Cs of desflurane, sevoflurane, isoflurane, enflurane, and halothane in anesthetic lipid emulsion at 20°C using the following equation:

where λL/G is liquid/gas partition coefficient at 20°C measured in this study; SVP, MW, and SG are saturated vapor pressure (mm Hg), molecular weight (g/mole), and specific gravity of liquid anesthetic (g/mL) at 20°C, respectively. The derivation of the above equation is described in the Appendix.

Solutions of 3.1% and 6.0% (liquid volatile anesthetic volume percent) emulsified isoflurane were prepared at room temperature (20°C) by adding 1.28 mL or 2.55 mL pure isoflurane to 40 mL 30% Intralipid in 50 mL gas-tight glass bottles fitted with Teflon stoppers. The bottles were vibrated on a vibrator at 50 Hz for 30 min. These concentrations were far below the lower limit of 95% confidential interval (CI) of Cs for isoflurane in 30% Intralipid (Table 1). No separation of emulsion was found during the entire study.

T1-21
Table 1:
Liquid/Gas Partition Coefficients and Saturated Concentrations of Volatile Anesthetics in 20 and 30 Intralipid at 20°C

For the dose-response measurements 112 female virgin Sprague-Dawley rats weighing approximately 200 g were studied. All rats were fasted for 4 h before the study but were given water to drink. Each rat was weighed and placed in an individual transparent plastic cylinder (internal diameter 6 cm, length 30 cm), where they were allowed to breathe pure oxygen spontaneously. A tail vein was cannulated with a 24-gauge catheter (SN 388614; Becton Dickinson, Franklin Lakes, NJ). In experiments to determine the ED, rats were allocated randomly to 7 groups each of 8 rats, and 3.1% emulsified isoflurane was injected as a single bolus via the tail vein by an infusion pump. The infusion volume of the pump was calibrated by water replacement before the animal study. The doses of emulsified isoflurane injected were 0.340, 0.374, 0.409, 0.443, 0.477, 0.512, and 0.546 mL in each of the 7 groups. Immediately after injection, rats were placed in supine position and observed for 15 s. The loss of the forepaw righting reflex (FRR) was accepted as successful induction of anesthesia. The time to return of the FRR was also recorded.

For the experiments for LD measurement, another 7 groups (each n = 8 rats) in which the electrocardiogram (ECG) was monitored, were studied. Emulsified 6.0% isoflurane was injected via the tail vein by an infusion pump. Doses of emulsified isoflurane were 0.546, 0.614, 0.683, 0.752, 0.820, 0.889, and 0.957 mL in each of the 7 groups. The disappearance of the ECG waveform was accepted as the death of the animal. All rats that survived these experiments were observed for 24 h afterwards for general well-being.

The dose-response relationship of IV propofol was investigated in 96 female virgin Sprague-Dawley rats weighing approximately 200 g each. The same experimental method was used as that in the emulsified isoflurane study. For measurement of the ED, 48 rats were randomly divided into 6 groups of 8. Propofol, diluted to 2.5 mg/mL with normal saline, was injected via the tail vein. The doses were 0.306, 0.374, 0.443, 0.512, 0.580, and 0.649 mL in each of the 6 groups. In the LD study, propofol was diluted to 5 mg/mL, and the doses were 0.546, 0.614, 0.683, 0.752, 0.820, and 0.889 mL in each of the 6 groups. Propofol maintained its emulsion when diluted with normal saline. The loss of the FRR and the disappearance of the ECG waveform were also accepted as successful induction of anesthesia and the death of the animal. The time to return of the FRR was recorded.

Mean and standard deviations of λL/G, and mean and 95% CIs of Cs were calculated. Dose-response relationship data were analyzed by probit analysis (SPSS 10.0 software package; SPSS, Chicago, IL). The Pearson goodness-of-fit χ2 test was used to analyze the fitness of regression between anesthetic doses and experimental animals' responses (anesthetized or died). ED50, ED99, LD50, and LD1, and respective 95% confidential limit (95%CL) were calculated. SI = LD50/ED50 and certain safety factor (CSF = LD1/ED99) of emulsified isoflurane and propofol, which represented margin of pharmacodynamic safety, were also calculated. Student's t-test was used to compare the time to return of FRR for emulsified isoflurane and propofol in the ED study. A value of P < 0.05 was accepted as statistically significant.

Results

λL/G and Cs of the 5 volatile anesthetics in 20% and 30% Intralipid at 20°C are given in Table 1. λL/G in 30% Intralipid was approximately 1.5 times larger than that in 20% Intralipid. Raw data of dose-response relationship determination of emulsified isoflurane are shown in Table 2. Induction of anesthesia occurred within 5 s after bolus infusion. No excitatory phenomena were observed, and spontaneous respiration was maintained in all rats participating in the ED response study. In the LD study (n = 56), 27 rats died immediately after IV infusion, and a further four died within 3–5 min after the infusion. Dose-response curves for loss of FFR and lethal dose for emulsified isoflurane are shown in Figure 1. The corresponding curves for propofol are shown in Figure 2. ED50, ED99, LD50 and LD1 of IV emulsified isoflurane were 0.072 (95% confidence limit: 0.068–0.076), 0.092 (0.085–0.113), 0.216 (0.201–0.230), and 0.154 (0.116–0.173) mL pure isoflurane/kg body weight, respectively (Fig. 1). SI and CSF were 3.0 and 1.7, respectively.

T2-21
Table 2:
Grouped Data in Determination of Dose-Response Relationship of Intravenous Injection of Emulsified Isoflurane
F1-21
Figure 1.:
Dose-response curve of emulsified isofluraneThe abscissa shows the volume of pure isoflurane infused (volume of emulsion infused multiplied by the volume percentage of isoflurane emulsion). The ordinate is the percentage of rats with loss of forepaw righting reflex (FRR) (closed circles) or dying (open circles). The solid lines represent the fitted curves; dotted lines represent the 95% confidence limits (CLs). For the effective dose study (closed circles), χ2 = 1.014, df = 5, and P = 0.96. For the lethal dose study (open circles), χ2 = 1.514, df = 5, and P = 0.91.
F2-21
Figure 2.:
Dose-Response Curve of PropofolThe abscissa shows the dose of propofol infused (mg/kg). The ordinate is the percentage of rats with loss of forepaw righting reflex (FRR) (closed circles) or dying (open circles). The solid lines represent the fitted curves; dotted lines represent the 95% confidence limits (CLs). For the effective dose study (closed circles), χ2 = 0.190, df = 5, and P = 0.996. For the lethal dose study (open circles), χ2 = 0.975, df = 4, and P = 0.914.

The raw data from the dose-response relationship experiments with propofol are shown in Table 3. Loss of FRR occurred within 5 s after bolus infusion and no excitatory phenomena were observed. ED50, ED99, LD50, and LD1 of propofol were 5.89 (95% confidence limits: 5.30–6.51), 9.53 (8.01–15.63), 18.19 (16.85–19.79), and 12.38 (8.61–14.16) mg/kg body weight, respectively (Fig. 2). SI and CSF were 3.1 and 1.3, respectively.

T3-21
Table 3:
Grouped Data in Determination of Dose-Response Relationship of Intravenous Injection of Propofol

Time to return of FRR was significantly shorter for emulsified isoflurane (38 s ± 18s, 95% CI: 31–45 s) than that of propofol (101 s ± 62 s, 95% CI: 73–128 s) (P < 0.05). Recovery from anesthesia was smooth in all animals in the ED study of emulsified isoflurane. In contrast, in the propofol study, all rats exhibited abnormal paw extension and irregular respiratory patterns (cough, swallow, transient dyspnea, and tachypnea) before return of FRR.

Discussion

Although IV injection of liquid volatile anesthetics is invariable fatal (1,3), studies in a variety of animals have shown that IV administration of lipid emulsions of isoflurane or halothane are safe and effective for induction of anesthesia (4–8). Our present study confirms these findings for emulsified isoflurane in rats.

Some investigators (4,7) used lipid emulsion containing concentrations of isoflurane or halothane of 10% (liquid anesthetic volume percentage). We speculated that these concentrations might be super-saturated. Theoretically, when a solution is equilibrated with a saturated volatile anesthetic vapor, the anesthetic concentration in this solution represents the largest amount of anesthetic dissolved in solution without gross separation of liquid anesthetic. This concentration, which we named “Cs,” is determined mainly by anesthetic solubility, saturated vapor pressure of the anesthetic, and atmospheric pressure and temperature during equilibration. Our results show that the Cs of isoflurane and halothane in 20% Intralipid at 20°C were 5.64% and 9.46%, respectively. In other words, the maximum volume of pure liquid anesthetic that can be dissolved at room temperature in 100 mL 20% Intralipid would be 5.64 mL for isoflurane and 9.46 mL for halothane. At larger concentrations than these, there is a danger that the liquid volatile anesthetic can separate from the emulsion, with the risk that pure liquid anesthetics be directly injected into the circulation. The data in Table 1 should be considered when preparing emulsified volatile anesthetics at room temperature.

We have shown that the λL/G in 30% Intralipid were approximately 1.5 times more than those in 20% Intralipid. For the 5 different anesthetics we studied, the Cs of halothane and isoflurane were larger than those of the other 3. This facilitates a wider margin of safety for anesthetic emulsion preparation and a smaller volume of drug injection. Because halothane is associated with hepatitis, we choose to study isoflurane and 30% Intralipid as the carrier solution for IV injection. Isoflurane lipid emulsion was prepared at 3.1% and 6.0%, well below the lower 95% CI of Cs for isoflurane in 30% Intralipid. No separation of emulsion occurred during the study. We demonstrated that anesthesia was successfully induced in rats with IV injection of isoflurane lipid emulsion. The calculated SI in our study (3.1) agreed with the value of 3.2 reported by Eger and MacLeod (6) and is comparable to that of propofol in the present study.

The time to return of FRR in rats given emulsified isoflurane (38 ± 18 s) was significantly shorter than that of propofol (101 ± 62 s) (P < 0.05). This indicates a rapid redistribution and immediate elimination of the anesthetic through the lungs. Recovery from a single bolus infusion of emulsified isoflurane was smooth. In contrast, all rats given propofol exhibited abnormal paw extension and irregular respiration before return of FRR.

In conclusion, the solubility of volatile anesthetics in lipid emulsion was determined and Cs was calculated. These calculations will provide basic data for further study of IV-administered emulsified volatile anesthetics. We also showed that IV-emulsified isoflurane produced a predictable and rapidly reversible anesthesia in rats. The safety margin for IV emulsified isoflurane is comparable to that of propofol. The recovery of anesthesia from emulsified isoflurane was rapid and smooth, and this might be a favorable property for single IV injection of isoflurane.

Appendix. Derivation of the Equation for Calculation of Saturated Concentration

λL/G is defined as ratio of the amount of anesthetic present in liquid phase compared with that in gas phase, the two phases being of equal volume and being in equilibrium. The amount of anesthetic in a liquid solution (AL) after equilibration can be expressed as:

where AL and AG are volume of anesthetic vapor in liquid solution and gas phase, respectively.

Assuming 100 mL of solution and 100 mL of saturated anesthetic vapor being equilibrated at 1 atm at sea level, AL (volume in liter) can be expressed as:

where SVP is saturated vapor pressure (mm Hg) and 760 is atmosphere pressure expressed as mm Hg at sea level.

According to the universal gas equation (PV = nRT), the number of moles of the anesthetic vapor in 100 mL of solution (n) can be calculated as:

where 0.08206 is the universal gas constant, and T is the absolute temperature, 273 + ºC. Because P = 1, it is not shown in Equation 3.

Weight of liquid anesthetic (WL, g) in 100 mL of solution can be calculated as n times molecular weight (MW, g/mole):

Volume of liquid anesthetic (VL, mL) in 100 mL of solution can be calculated as WL divided by specific gravity (SG, g/mL) of liquid anesthetic:

Re-arranging equation 5 yields:

Because we assumed that 100 mL of solution was equilibrated with saturated anesthetic vapor at 1 atm, we named the concentration of liquid anesthetic in this solution as theoretical saturated concentration (Cs, liquid anesthetic volume percent) and expressed Cs as:

Combining Equations 6 and 7 yields:

We used Equation 8 to calculate Cs of desflurane, sevoflurane, isoflurane, enflurane, and halothane in anesthetic lipid emulsion at 20°C, according to λL/G at 20°C measured in this study, data of MW, SVP, and SG at 20°C shown in textbook (12).

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