Volatile anesthetics, including sevoflurane, cause a dose-dependent decrease in cardiac contractility1 and systemic vascular resistance, which results in hypotension.2 Thus, it may be advantageous to reduce the concentration of the volatile anesthetic administered by adding injectable drugs with MAC-decreasing properties and with less depressant cardiovascular effects. In research dogs, fentanyl3 and lidocaine4 caused a dose-dependent decrease in the MAC of inhalation anesthetics, and both drugs are commonly used as components of partial IV anesthetic protocols because of their MAC-sparing and analgesic effects.5–7
The objective of the study reported here was to determine the effects of fentanyl and lidocaine, alone and in combination, on the MACNM of sevoflurane in dogs. It was hypothesized that fentanyl and lidocaine would each cause a decrease in the MACNM of sevoflurane and that the fentanyl-lidocaine combination would cause a greater decrease in MACNM than would either drug alone.
Materials and Methods
Animals
Six healthy adult (3- to 5- year-old) purpose-bred sexually intact male Beagles were used in the study. Mean ± SD body weight of the dogs was 10.3 ± 1.7 kg. All dogs were up-to-date with regard to parasite prevention treatments and vaccinations and were considered healthy on the basis of results of physical examination and PCV, total solids concentration, and blood glucose values. The study was approved by an institutional animal care and use committee.
Experimental design
Dogs were anesthetized 3 times, with a 1-week interval between successive anesthetic episodes. The experiments were performed at an altitude of 270 m, and the approximate barometric pressure was 755 mm Hg.
Anesthesia
Food was withheld from dogs for 12 hours prior to each anesthetic episode, but access to water was allowed. Anesthesia was induced with sevofluranea (4 vol% to 5 vol%) delivered in oxygen (2 L/min) by use of a face mask attached to a circle system until endotracheal intubation was possible. After dogs were tracheally intubated, they were positioned in right lateral recumbency, and anesthesia was maintained with sevoflurane in oxygen (1 L/min) delivered via an anesthesia machine.b
Controlled ventilation was instituted by use of a pressure-limited, time-cycled ventilatorb with a mean tidal volume of 15 mL/kg and respiratory rate of 8 to 12 breaths/min to maintain the end-tidal partial pressure of CO2 between 35 and 45 mm Hg. Values for Fesevo and end-tidal partial pressure of CO2 were monitored continuously with an infrared gas analyzerc by use of samples collected from the proximal end of the endotracheal tube at a rate of 200 mL/min. The monitor was calibrated at the start and again approximately midway through each experiment by use of the calibration gases supplied by the manufacturer (1% sevoflurane, 5% CO2, and 60% N2O).c An 18-gauge catheterd was aseptically placed in the left jugular vein for collection of blood samples (3 mL) used to determine plasma concentrations of fentanyl and lidocaine. A 20-gauge catheterd was aseptically placed in the right cephalic vein for infusion of fentanyl, lidocaine, or the fentanyl-lidocaine combination and lactated Ringer solutione (3 mL/kg/h). Blood pressure was monitored indirectlyc by use of an oscillometric technique and an appropriately sized cuff (width, approx 40% of the circumference of the limb)8 placed over the right dorsal pedal artery in the midmetatarsal area. Heart rate and ECG were monitored continuously.c Body temperature was monitored via an esophageal probe,c and a circulating warm water blanketf and warm air blanketg were used to maintain body temperature within reference limits (37.5° to 38.5°C). The urinary bladder was expressed intermittently during equilibration periods to prevent stimulation from overdistention.
MAC NM-B determination
Determination of MACNM-B was initiated approximately 45 minutes after induction of anesthesia. The Fesevo was held constant at 2.4 vol% for at least 20 minutes before MACNM-B determination. A noxious stimulus (50 V at 50 Hz for 10 milliseconds) was delivered by use of a square-wave voltage output electrical stimulatorh via two 25-gauge needle electrodes placed 5 cm apart in the subcutaneous tissues on the lateral aspect of the left forelimb. The stimulus consisted of 2 single stimuli followed by 2 continuous stimuli of 5 seconds’ duration, with a 5-second interval between subsequent stimuli.9 Withdrawal or twitching of the nonstimulated limbs, movement of the head, chewing, licking, blinking, or swallowing was considered a positive response; however, twitching of the stimulated limb was not considered a positive response. If the response was positive, Fesevo was increased by 0.1 vol%; conversely, if the response was negative, Fesevo was decreased by 0.1 vol%. The noxious stimulus was reapplied after a 15-minute equilibration period. The MACNM was considered to be the minimum Fesevo that abolished movements in response to noxious stimulation. The MACNM-B was determined in duplicate, and the mean value was used as the MACNM-B for that dog. If the difference between the 2 MACNM-B values was > 10%, a third MACNM-B was obtained, and the mean of the 3 values was used as the MACNM-B. Time to MACNM-B was recorded as the interval from endotracheal intubation to completion of MACNM-B determination (in duplicate or triplicate).
Drug administration
After MACNM-B was determined, dogs were assigned by use of a random-number generator to receive 1 of 3 treatments. Each of the treatments was administered IV as a loading dose followed by a CRI. The 3 treatments were as follows: fentanyli (loading dose, 15 μg/kg; CRI, 6 μg/kg/h), lidocainej (loading dose, 2 mg/kg; CRI 6, mg/kg/h), and the fentanyl-lidocaine combination (each drug at the aforementioned doses). Solutions for the loading dose and CRI were created by the use of physiologic saline (0.9% NaCl) solution and administered with syringe pumps.k The loading dose was a solution that provided 1 mL/kg and was administered over a 20-minute period. The CRI was a solution that provided 1 mL/kg/h. The loading dose and CRI were started simultaneously.
Determination of MACNM-T began 90 minutes after start of the CRI. The Fesevo was held constant at each dog's MACNM-B for at least 15 minutes before delivery of the noxious stimulus. The same method as described for determination of MACNM-B was used to determine MACNM-T. A jugular blood sample (3 mL) was collected into a lithium-heparin tube immediately after each of the duplicate measurements for MACNM-T determination. Time to MACNM-T was the interval from determination of MACNM-B until determination of MACNM-T (in duplicate or triplicate).
Equal volumes of the plasma harvested from the 2 blood samples collected at the time of each dog's MACNM-T determination were combined for analysis. Plasma was stored at −80°C until analyzed.
Analysis of drug concentrations
Fentanyl plasma concentrations were determined by use of liquid chromatography and triple-quadrupole mass spectrometry,1 as described elsewhere.10 Briefly, 1 mL of plasma was added to 0.1 mL of fentanyl d5 (internal standard) and 1 mL of 0.1N sodium hydroxide; the solution was mixed in a vortex device for 5 seconds. Drug was extracted from plasma by use of solid-phase extraction.m The extracted material was conditioned with 1 mL of methanol followed by 1 mL of deionized water. The plasma mixture was loaded into a solid-phase extraction cartridge and rinsed with 1 mL of 5% methanol; fentanyl was then eluted with 1 mL of methanol. The eluate was evaporated to dryness at 40°C under an airstream and then reconstituted with 200 μL of 50% methanol. The injection volume was 50 μL. Fentanyl was monitored at m/z 337→188, and fentanyl d5 was monitored at m/z 341.4→105. Separation was achieved by use of a C18 column.n The mobile phase consisted of 40% acetonitrile and 60% formic acid (0.1%). Flow rate was 0.3 mL/min. The plasma standard curve was linear (r > 0.99) between 0.05 and 10 ng/mL, and all measured concentrations were within 15% of actual concentrations. The lower limit of quantification was 0.05 ng/mL, and the upper limit of quantification was 10 ng/mL. Mean ± SD accuracy of the assay was 101 ± 5%, which was determined by use of 5 replicates for each of 3 concentrations (0.05, 1, and 10 ng/mL). Coefficient of variation was 6%, 2%, and 3% for 5 replicates for each of 3 concentrations (0.05, 1, and 10 ng/mL, respectively).
Plasma concentrations of lidocaine were measured by use of high-performance liquid chromatography with UV detection, as described elsewhere.11 Briefly, the system consisted of a separation module,° reverse-phased C18 columnp (5-μm particle size; 4.6 × 150 mm), guard column,q absorbance detector,r and computer equipped with software.s The mobile phase was an isocratic mixture of 0.02M potassium dihydrogen phosphate (pH, 6) with concentrated phosphoric acid and acetonitrile (84:16 [vol/vol]). It was prepared fresh daily with double-distilled, deionized water filtered through a 0.22-μm filter and degassed before use. Flow rate was 1.0 mL/min, and UV absorbance was measured at 205 nm. Frozen plasma samples were thawed and mixed in a vortex device. An aliquot (1 mL) of each plasma sample was placed in a 15-mL screw-cap tube, and 25 μL of trimethoprim (50 μg/mL solution [internal standard]) was added. Then, 200 μL of a 1M NaOH solution was added followed by 5 mL of methylene chloride. Tubes were placed on a rocker for 15 minutes and then centrifuged at 1,050 × g for 15 minutes. The bottom layer was placed in a clean tube and evaporated under a stream of nitrogen gas. Samples were reconstituted with 1 mL of mobile phase, and a 100-μL aliquot of sample was injected into the liquid chromatograph.
Standard curves for plasma analysis were prepared by fortifying untreated plasma with lidocaine, which yielded a linear concentration range of 50 to 7,000 ng/mL. Calibration samples were treated in the exact same manner as plasma samples. Recovery ranged from 83% to 100% for lidocaine. Interassay and intra-assay variability ranged from 4.3% to 7.6%. Lower limit of quantification was 50 ng/mL.
Statistical analysis
Percentage change in MAC was calculated as ([MACNM-B – MACNM-T]/MACNM-B) × 100. The effects of treatment, time to measurement, and body weight on MACNM-B, MACNM-T, fentanyl and lidocaine concentrations measured at the time of MACNM-T determination, and time to extubation were analyzed by use of a mixed-model ANOVA.t Dog, treatment, and week were included as class variables. Independent variables were treatment, time to MACNM-B, time to MACNM-T, and body weight. Dog and week were included as random variables. A similar model was used to assess the effect of treatment on time to MACNM-B and time to MACNM-T. Time to MACNM-B and time to MACNM-T were used as dependent variables, and treatment, body weight, and MACNM-B or MACNM-T were independent variables. A Tukey multiple range test was used to distinguish among the 3 treatments. The −2 log-likelihood ratio was used to assess fit of the model to the data. The assumption that residuals from the model approximated a normal distribution was examined by use of the Shapiro-Wilk test. Data were reported as least squares mean ± SEM. Significance was set at values of P < 0.05.
Results
Mean ± SEM overall MACNM-B for all treatments was 2.70 ± 0.27 vol%. The MACNM-B did not differ significantly (P > 0.40) among treatments (Table 1). The MACNM decreased from MACNM-B to MACNM-T by a mean of 39.2 ± 4.1%, 21.4 ± 4.3%, and 55.3 ± 4.2% for the fentanyl, lidocaine, and fentanyl-lidocaine combination treatments, respectively. The percentage change in MACNM-B was significantly different between fentanyl and lidocaine (P = 0.02), fentanyl and the fentanyl-lidocaine combination (P = 0.02), and lidocaine and the fentanyl-lidocaine combination (P < 0.001). The greatest percentage decrease in MACNM-B was for the fentanyl-lidocaine combination (mean decrease, 55.3 ± 4.2%). Plasma fentanyl concentration was 3.25 and 2.94 ng/mL for fentanyl and the fentanyl-lidocaine combination, respectively; the concentrations did not differ significantly (P = 0.38) between these treatments. Plasma lidocaine concentration was 2,570 and 2,417 ng/mL for lidocaine and the fentanyl-lidocaine combination, respectively; the concentrations did not differ significantly (P = 0.50) between these treatments.
Effect of IV administration of fentanyl (loading dose, 15 μg/kg; CRI, 6 μg/kg/h), lidocaine (loading dose, 2 mg/kg; CRI, 6 mg/kg/h), and a fentanyl-lidocaine combination (each drug at the aforementioned doses) on sevoflurane MACNM in dogs (n = 6).
| Treatment | MACNM-B | MACNM-T | Change (%) | Time to MACNM-B (min) | Time to MACNM-T (min) | Plasma fentanyl (ng/mL) | Plasma lidocaine (ng/mL) |
|---|---|---|---|---|---|---|---|
| Fentanyl | 2.63 ± 0.11 | 1.62 ± 0.09a | −39.2 ± 4.1a | 136 ± 14 | 162 ± 8 | 3.25 ± 0.17 | NA |
| Lidocaine | 2.68 ± 0.12 | 2.09 ± 0.09b | −21.4 ± 4.3b | 157 ± 14 | 161 ± 14 | NA | 2,570 ± 126 |
| Combination | 2.79 ± 0.12 | 1.20 ± 0.09c | −55.3 ± 4.2c | 135 ± 14 | 143 ± 13 | 2.94 ± 0.18 | 2,417 ± 126 |
Values are expressed as least squares mean ± SEM. There was a 1-week interval between successive anesthetic episodes and treatments. Percentage change in MAC was calculated as ([MACNM-B - MACNM-T]/MACNM-B) × 100. Time to MACNM-B was the interval from endotracheal intubation to completion of MACNM-B determination (in duplicate or triplicate). Time to MACNM-T was the interval from completion of MACNM-B determination until completion of MACNM-T determination (in duplicate or triplicate). Plasma concentrations were determined by use of 2 venous blood samples obtained from each dog at the time of each MACNM-T determination on each day; equal volumes of plasma were combined for analysis.
NA = Not applicable.
Values in the same column with different superscript letters differ significantly (P < 0.05).
Mean arterial blood pressure was > 60 mm Hg at all times. Heart rate was > 60 beats/min at all times, and no arrhythmias were observed during the study. Time to MACNM-B or time to MACNM-T did not differ significantly (P > 0.40) among treatments. Mean ± SEM time to extubation was 7.3 ± 1.2 minutes, 7.5 ±1.2 minutes, and 9.5 ± 1.2 minutes for fentanyl, lidocaine, and the fentanyl-lidocaine combination, respectively, and did not differ significantly (P = 0.32) among treatments. Recovery from anesthesia was uncomplicated for all dogs for all anesthetic episodes, and dogs were able to walk unaided within 45 minutes after extubation.
Discussion
The mean MACNM-B value of 2.70 vol% in the study reported here is comparable to the values of 2.65 vol%10 and 2.66 vol%12 reported in other studies conducted by our laboratory group by use of the same experimental design. The administration of fentanyl, lidocaine, and the fentanyl-lidocaine combination caused significant and clinically important decreases in sevoflurane MACNM. Drug infusion rates were based on those used in previous studies.12,13 In the present study, MACNM was used as an endpoint to evaluate the interaction of fentanyl and lidocaine with sevoflurane, and it is the authors' opinion that MACNM determination is less subjective than is the traditional MAC determination and that MACNM provides more clinically relevant information. The method for MACNM determination was based on experiments conducted by our laboratory group, and the starting Fesevo of 2.4 vol% was chosen because it is the approximate value for the MACNM of sevoflurane.10,12
The administration of lidocaine at a CRI of 6 mg/kg/h was associated with a 21% decrease from the MACNM-B of sevoflurane in the present study. Comparable plasma lidocaine concentrations have been associated with a 29% decrease in the MAC of sevoflurane in dogs.13 In another study,14 lidocaine administered at a CRI of 6 mg/kg/h decreased the MAC of sevoflurane by 21%; however, plasma lidocaine concentrations were not reported in that study. In dogs undergoing ovariohysterectomy, lidocaine CRI at 3 mg/kg/h reduced the required end-tidal concentration of isoflurane by 16.8%,5 and when lidocaine was used in combination with fentanyl (CRI, 3.6 μg/kg/h) and ketamine (CRI, 0.6 mg/kg/h), the MAC of isoflurane was decreased by 97%.7 However, plasma lidocaine concentrations were not reported in either of those studies. The effect of lidocaine on MAC seems to be a dose-dependent phenomenon, and a CRI at 12 mg/kg/h, with associated plasma lidocaine concentrations of approximately 4,500 ng/mL, resulted in a decrease of 43% in the MAC of isoflurane.4 In another study,15 lidocaine infused at a rate of 12 mg/kg/h decreased MAC of sevoflurane by 37%. Although no adverse effects were associated with the administration of lidocaine in the study reported here, it was in the aforementioned study15 that 75% of dogs given an infusion of lidocaine at a rate of 12 mg/kg/h vomited during recovery; however, vomiting was not reported in another study4 that used the same infusion rate (12 mg/kg/h).
The decrease of 39% from the sevoflurane MACNM-B for the fentanyl treatment is comparable to the previously reported decreases in sevoflurane MACNM of 35%12 and 37%,10 which were determined by use of the same infusion rate and experimental design. In dogs undergoing ovariohysterectomy, fentanyl administered at a CRI of 10 μg/kg/h reduced the end-tidal concentration of isoflurane necessary to maintain a surgical plane of anesthesia by 35.3%, but fentanyl plasma concentrations were not reported.5 There appears to be a ceiling effect for fentanyl on MAC reduction because increasing the fentanyl infusion rate from 6 to 12 μg/kg/h was not associated with a significantly greater decrease in MACNM, despite a 2-fold difference in fentanyl plasma concentration.12 Nevertheless, in another study,3 larger doses of fentanyl were associated with a nonlinear decrease in the MAC of enflurane, although a ceiling effect was detected at a decrease of approximately 65%. It is also of interest that comparable plasma concentrations of fentanyl were associated with a 53% decrease in the MAC of enflurane,3 a decrease in the sevoflurane MACNM of only 39% in the present study, and a decrease in sevoflurane MACNM of 35% in another study.12 This may reflect a true difference in the interaction of fentanyl with enflurane and sevoflurane or differences in study methods.
In the present study, the fentanyl-lidocaine combination decreased MACNM by a mean of 55%, and this value was slightly less than, but within the experimental error for, the sum of the effects for each drug administered separately. In contrast, it has been reported that the addition of lidocaine at a CRI of 3 mg/kg/h did not augment the effect of fentanyl on the required end-tidal concentration of sevoflurane in dogs undergoing ovariectomy.16 The most likely reason for the lack of effect of lidocaine in that study16 was its extremely low plasma concentrations. The median plasma lidocaine concentration of 280 ng/mL in the aforementioned study16 would not have been expected to exert a substantial anesthetic-sparing effect and was much less than the mean value of 2,417 ng/mL for the present study. Other factors that may have contributed to the lack of an influence of lidocaine for augmenting the effect of fentanyl in that study16 included differences in study design (eg, use of a surgical nociceptive stimulus) and the inclusion of female dogs rather than male dogs.
In the present study, fentanyl, lidocaine, and the fentanyl-lidocaine combination were administered as a loading dose and CRI. At the doses used, they caused clinically important and significant decreases in the MACNM of sevoflurane in dogs.
Acknowledgments
Presented in abstract form at the Association of Veterinary Anaesthetists Annual Conference, Nottingham, England, April 2014.
ABBREVIATIONS
| CRI | tinuous rate infusion |
| Fesevo | Fractional concentration of sevoflurane in expired gas |
| MAC | Minimum alveolar concentration |
| MACNM | Minimum alveolar concentration preventing motor movement |
| MACNM-B | Minimum alveolar concentration preventing motor movement measured at baseline |
| MACNM-T | Minimum alveolar concentration preventing motor movement measured after treatment |
Footnotes
Sevoflo, Abbott Laboratories, North Chicago, Ill.
North American Drager, Telford, Pa.
Datex-Ohmeda, Planar Systems, Hillsboro, Ore.
Jelco Protectiv, Smiths Medical, Saint Paul, Minn.
Abbott Laboratories, North Chicago, Ill.
Allegiance Healthcare Corp, Waukegan, Ill.
Bair Hugger, Arizant, Saint Paul, Minn.
S48 stimulator, Natus Neurology Inc, Grass Technologies, Warwick, RI.
Fentanyl citrate USP, Hospira, Lake Forest, Ill.
Lidocaine, Vedco Inc, St Joseph, Mo.
Medfusion 2010, Medox Inc, Duluth, Ga.
API 2000, Applied Biosystems, Foster City, Calif.
SPE, Varian Bond Elut C18, Varian, Palo Alto, Calif.
Supelco discovery C18, 150 mm × 2.1 mm × 5 μm, Sigma-Aldrich Corp, St Louis, Mo.
2695 separation module, Waters Corp, Milford, Mass.
Xterra RP18 column, Waters Corp, Milford, Mass.
Xterra guard column, Waters Corp, Milford, Mass.
2487 absorbance detector, Waters Corp, Milford, Mass.
Empower 3, Waters Corp, Milford, Mass.
PROC MIXED, SAS, version 9.4, SAS Institute Inc, Cary, NC.
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