Effects of a single paracetamol injection on the sevoflurane minimum alveolar concentration in dogs

Abstract

This study aimed to determine the effect of a single injection of paracetamol on the sevoflurane minimum alveolar concentration (MAC) response to noxious mechanical stimulation. Seven healthy adult beagles were enrolled in a prospective, randomized, blinded, crossover experimental study. Anesthesia was induced with propofol [11.6 ± 2.4 mg/kg body weight (BW)] and maintained with sevoflurane. The MAC was determined before (MAC-1) and after (MAC-2) treatment with 15 mg/kg BW of intravenous (IV) paracetamol or saline over 15 minutes. Samples for plasma paracetamol determination were collected immediately after IV treatment administration and following MAC-2 determination (123 ± 27 minutes after starting paracetamol administration). The MAC-1 was similar between treatments (1.7% ± 0.4%). There were no differences between control and paracetamol groups at MAC-2 (2.0% ± 0.4% and 1.7% ± 0.5%, respectively; P = 0.285). Paracetamol plasma concentrations after paracetamol administration were 34.5 ± 9.9 μg/mL, decreasing at the end of the procedure (8.5 ± 4.2 μg/mL). In conclusion, 15 mg/kg BW of IV paracetamol did not significantly reduce sevoflurane MAC in healthy dogs.

Introduction

Paracetamol, also known as acetaminophen, is a synthetic non-opiate analgesic drug derived from p-aminophenol. Despite widespread use in the human clinical setting, its mechanism of action is complex and not fully understood (1). Paracetamol participates in the central and peripheral inhibition of cyclooxygenases (COX) (2) and probably affects centrally acting COX-3, also known as COX-1b (3). In addition, it may interact with the endogenous serotonergic, opioid, and cannabinoid systems (4,5). Paracetamol produces centrally acting analgesic and antipyretic effects, but unlike nonsteroidal anti-inflammatory drugs (NSAIDs), it has limited peripheral anti-inflammatory properties (1,6). As a result, gastrointestinal and renal side effects are rare and platelet function is not affected (4,7).

Concerns regarding adverse side effects of opioid drugs (e.g., respiratory and cardiovascular depression, altered thermoregulation, sedation, ileus, opioid dependence) have led to rising interest in other analgesic drugs (8,9). Adverse effects of paracetamol have been described in veterinary medicine, especially in cats (10). However, it has a high margin of safety in dogs (11) and its reported toxicity is associated with doses higher than those used in pharmacokinetic studies. Dogs administered up to 20 mg/kg body weight (BW) of paracetamol intravenously (IV) do not have side effects (12).

Few studies have assessed the efficacy of paracetamol in the postoperative period (13,14) and to the best of the authors’ knowledge, there are no reports assessing the effects of its intraoperative IV administration in dogs.

The perioperative efficacy of analgesic drugs under general anesthesia is generally assessed through reductions in the minimum alveolar concentration (MAC) of inhalational anesthetics (15,16). Several analgesic drugs commonly used intraoperatively such as opioids (17) or NSAIDs (18,19) may reduce the MAC in dogs. Previous studies in rats have provided contradictory results and whilst a single paracetamol dose did dose-dependently reduce the sevoflurane MAC by as much as 29% during several hours (20), a lack of effect has also been reported (4). Thus, the aim of this study was to determine the effect of a single paracetamol dose on the sevoflurane MAC in dogs. We hypothesized that paracetamol would decrease sevoflurane MAC in dogs.

Materials and methods

The study was approved by the Institutional Animal Care and Use Committee (Proex 032/16). The Animals in Research: Reporting In Vivo Experiments guidelines were followed.

Animals

Seven healthy adult beagles (6 males and 1 female) aged 6.6 ± 0.2 y and weighing 14.4 ± 2.1 kg were used. Dogs were obtained from the institutional colony, originally purchased from an authorized breeder (Isoquimen, Barcelona, Spain). Dogs were housed in groups of 2 to 3 animals per cage (3 × 6 m) using environmental enrichment with natural light at a relative humidity of 40% to 70% and 21°C ± 2°C ambient temperature. They were fed twice daily (Advanced Fitness Adult Medium; Hill’s Pet Nutrition, Madrid, Spain) and water was provided ad libitum. The dogs were judged to be in good health based on physical examination, complete blood (cell) count (CBC), and serum biochemical analysis.

Experimental design

The dogs were anesthetized to determine MAC on 2 occasions in a prospective, randomized, blinded, crossover design, with a minimum 2-week washout period between treatments. The treatment was a single IV injection of 15 mg/kg BW paracetamol (Paracetamol Kabi 10 mg/mL; Fresenius Kabi, Barcelona, Spain) administered over 15 min (6 mL/kg BW per hour) (PRC group) or the equivalent volume of saline (FisioVet 1.5 mL/kg BW, NaCl 0.9%; Braun, Barcelona, Spain), also administered over 15 min (CTL group) under general anesthesia. Doses were selected according to previous studies in dogs (12,21,22). Randomization of treatment allocation was performed with a random number generator (Excel 2007, Microsoft Office, Redmond, Washington, USA).

Anesthetic protocol

Food, but not water, was withheld beginning 12 h before the experiment. All studies were performed in the afternoon. A 20-gauge (G), 1 ¼ inch IV catheter (Surflo IV catheter; Terumo, Madrid, Spain) was placed in the cephalic vein and fluid therapy with lactated Ringer’s solution (Lactato-RingerVet; Braun) was started at a rate of 5 mL/kg BW per hour using an SK-600II infusion pump (Mindray Bio-Medical Electronics, Guangdong, China). Dogs were preoxygenated with 100% oxygen via facemask while anesthesia was induced with propofol IV titrated to effect (Propofol-Lipuro 10 mg/mL; Braun). The dose and time of propofol administration were recorded. Orotracheal intubation was performed using an appropriately sized orotracheal tube; dogs were connected to an anesthesia machine (Julian Anesthetic Workstation; Dräger, Madrid, Spain) and placed in left lateral recumbency. The dorsal pedal artery was catheterized with a 22G, 1-inch catheter (Surflo IV Catheter; Terumo) to measure invasive blood pressure. Anesthesia was maintained with sevoflurane (SevoFlo; Esteve, Barcelona, Spain). Initially set at 2.0% to 2.4%, end-tidal concentration of sevoflurane (ET_SEV) was delivered in a gas mixture of oxygen and air (FiO₂ 50%) in a continuous flow of 3 L/min, via a circle anesthetic rebreathing system. Dogs were allowed to breathe spontaneously but were mechanically ventilated when necessary (i.e., volume control using a tidal volume of 10 mL/kg, a 20% inspiratory pause, and a positive end-expiratory pressure of 5 cm H₂O) to maintain normocapnia [i.e., end-tidal carbon dioxide (ETCO₂) between 4.7 and 6 kPa). Mechanical ventilation was stopped when dogs started fighting the ventilator, usually after the application of the noxious stimuli.

Monitoring

During the procedure, systolic, diastolic, and mean arterial pressures, arterial oxygen hemoglobin saturation (SpO₂), heart rate using lead II electrocardiogram, respiratory rate (RR), ETCO₂, ET_SEV, and body temperature were continuously monitored using a monitoring system (Datex-Ohmeda S/5 Anesthesia Monitor; General Electric, Helsinki, Finland). The blood pressure was monitored invasively; alternatively, when arterial catheterization was not possible, the oscillometric method would be used by placing the cuff on the dog’s dorsal pedal artery.

Airway gas samples were obtained from a sampling port located between the proximal end of the endotracheal tube and the breathing system and directed into an infrared gas analyzer (Datex-Ohmeda S/5 Anesthesia Monitor; General Electric) to monitor RR, ETCO₂, and ET_SEV. The gas analyzer was calibrated at the start of the experiment using a reference gas (Quick Cal Calibration Gas; General Electric). Body temperature was monitored using an esophageal probe and maintained between 37.0°C and 38.5°C with a circulating warming water blanket (Heat Therapy Pump, Model TP-220; Gaymar, New York, USA) and a warm air blanket (Bair Hugger Model 505; 3M Health Care, Neuss, Germany), if necessary. Data were recorded 60 s before each tail clamping and values from the 4 clamping procedures employed for MAC determination were averaged to provide each MAC value.

Sevoflurane MAC determination

After a 20-minute equilibration period with ET_SEV kept at 2.0% to 2.4%, MAC-1 determination was initiated.

A noxious stimulus was applied employing the tail clamp method as previously described (16) using a Doyen intestinal clamp attached to the first ratchet lock onto the tail for 60 s or until a positive response immediately after acquiring the monitored data recording. The tail was always stimulated at different sites starting 12 cm distal to the tail base in an attempt to prevent potential pain tolerance or sensitization. A positive response was considered a gross purposeful movement of the head, limbs, or body. A negative response was considered to be the lack of movement or progressive withdrawal movement of limbs, swallowing, or tail flick. When a positive response was seen, ET_SEV was increased by 0.20% step increases until the positive response became negative. Similarly, when a negative response was seen, ET_SEV was reduced in decrements of 0.20% until the negative response became positive (23,24).

Thus, MAC was determined as the concentration mid-way between the highest concentration that permitted movement in response to the stimulus and the lowest concentration that prevented such movement. The MAC was calculated in duplicate and the mean value was considered the MAC value for that animal. Times from anesthetic induction with propofol until MAC determination as well as the duration of MAC determination were recorded. The same researcher (PGB), blinded to the treatment, was responsible for applying the noxious stimuli and assessing the response in all instances.

Drug administration and experimental procedure

After MAC-1 determination, there was a 20-minute equilibration period, whereby ET_SEV was maintained at that individual MAC-1 value. Dogs were randomly administered a single IV injection of either 15 mg/kg BW of 1% paracetamol over 15 min (6 mL/kg BW per hour) (PRC group) or 1.5 mL/kg BW of saline over 15 min (CTL group). Both treatments were administered using a syringe pump (Infusomat fmS; Braun). After paracetamol or saline administration, MAC was re-calculated (MAC-2).

At the end of each experiment, sevoflurane administration was discontinued, and dogs recovered from anesthesia (Figure 1). A CBC and serum biochemical analysis was repeated twice in each dog, 1 wk after each treatment.

Figure 1.

Experimental design.

MAC — minimum alveolar concentration; IV — intravenous.

Blood serum detection and quantification of paracetamol

Venous blood samples (6 mL) were drawn in sterile serum separator tubes (Corning 15 mL polypropylene centrifuge tubes; Merck KGaA, Madrid, Spain) from a peripheral vein twice: 1 to 2 min immediately after administering the treatment and just after determining MAC-2 (over 2 h after starting paracetamol administration). Blood was refrigerated and centrifuged to obtain the serum and stored at −80°C in Eppendorf tubes. Samples for plasma paracetamol determination were collected immediately after IV administration of treatment and following determination of MAC-2.

The Dimension RxL Max Integrated Chemistry System (Siemens Healthcare Diagnostics, Newark, Delaware, USA) and its ACTM Flex reagent cartridge were used to detect and quantify the level of paracetamol in 100 μL serum samples. The protocol assay is based on enzymatic hydrolysis-producing acetate and p-aminophenol. The level of p-aminophenol is determined colorimetrically by reaction with o-cresol and ammoniacal copper sulphate, producing indophenol with a wavelength absorbance of 600 nm. The amount of p-aminophenol produced and estimated by the analyzer is proportional to the blood concentration of acetaminophen. Data are expressed in μg/mL.

Statistical analysis

The sample size calculation was performed for a 2-tailed Student’s t-test with a power of 80% and an alpha error of 0.05 to detect a difference of 0.5% in sevoflurane MAC. This determined a minimum of 7 dogs per group.

Data were tested for normality employing the Kolmogorov- Smirnov test. Monitored parameters at MAC-1 and MAC-2 included arterial blood pressure, heart and respiratory rates, ETCO₂, SpO₂, esophageal temperature, and time from propofol administration to MAC-1 and MAC-2 determination. Propofol induction dose, time required to determine MAC-1 and MAC-2, and paracetamol plasma concentrations were analyzed using the 2-tailed Student’s paired t-test. To assess the effect of the treatment on MAC, the Student’s t-test was also used to compare the MAC-1 and MAC-2 from each treatment group. The correlation between baseline MAC and time from propofol administration was analyzed with the Pearson test.

Data are shown as mean ± standard deviation and a P-value < 0.05 was set to indicate statistical significance. All statistical analyses were performed using SPSS for Windows (IBM SPSS Statistics V22.0).

Results

The MAC-1 was similar in both groups (1.7% ± 0.5% and 1.6% ± 0.3% in CTL and PRC groups, respectively; P = 0.525). The MAC-2 value was 13% lower in the PRC group than in the CTL group, but there were no statistically significant differences in MAC-2 between treatments (2.0% ± 0.4% and 1.7% ± 0.5% in CTL and PRC groups, respectively; P = 0.285). The MAC-2 was significantly higher than MAC-1 in the CTL group (P = 0.025) and no significant differences were observed between MAC-1 and MAC-2 in the PRC group (P = 0.444).

No adverse effects were noted when administering paracetamol to the dogs. There were no significant differences in arterial blood pressure, heart and respiratory rates, ETCO₂, SpO₂, and temperature between treatment groups (Table I).

Table I.

Monitored parameters from 7 healthy beagles during minimum alveolar concentration (MAC) determination. Data are expressed as mean ± standard deviation.

	Treatment
	Paracetamol		Saline
	MAC-1	MAC-2	MAC-1	MAC-2
Systolic arterial pressure (mmHg)	123 ± 17	130 ± 11	118 ± 12	122 ± 22
Diastolic arterial pressure (mmHg)	72 ± 15	78 ± 13	80 ± 10	73 ± 5
Mean arterial pressure (mmHg)	88 ± 11	94 ± 10	93 ± 10	88 ± 9
Heart rate (beats/min)	114 ± 23	117 ± 30	108 ± 16	115 ± 22
Respiratory rate (breaths/min)	42 ± 28	32 ± 16	29 ± 16	25 ± 14
ETCO2 (kPa)	4.3 ± 1.0	4.4 ± 0.5	4.7 ± 0.5	4.5 ± 0.2
SpO2 (%)	95 ± 3	96 ± 2	97 ± 2	98 ± 3
Esophageal temperature (°C)	37.5 ± 0.6	37.5 ± 0.6	37.2 ± 0.5	37.4 ± 0.5

ETCO₂ — end-tidal carbon dioxide; SpO₂ — arterial oxygen hemoglobin saturation.

The time needed to determine MAC-1 was shorter in the PRC than in the CTL group (83 ± 18 min and 122 ± 38 min, respectively; P = 0.037) but similar for MAC-2 (86 ± 24 min and 94 ± 29 min, respectively; P = 0.590).

The propofol dose required for intubation in all non-premedicated dogs was 11.6 ± 2.4 mg/kg BW and did not differ between treatment groups (11.7 ± 2.7 mg/kg BW and 11.4 ± 2.4 mg/kg BW in PRC and CTL groups, respectively; P = 0.811). The time from propofol induction until MAC determination was similar in both groups (MAC-1: 125 ± 17 min and 165 ± 42 min in PRC and CTL groups, respectively; P = 0.051 and MAC-2: 255 ± 33 min and 295 ± 35 min in PRC and CTL groups, respectively; P = 0.051). A positive correlation between the MAC and time to propofol administration in MAC-1 was observed (P = 0.027) (Figure 2).

Figure 2.

Positive correlation between sevoflurane MAC-1 and time from propofol induction (P = 0.027).

MAC — minimum alveolar concentration.

Paracetamol plasma concentrations immediately after the administration of paracetamol were significantly higher in the PRC group (34.5 ± 9.9 μg/mL) than the CTL group (3.8 ± 1.7 μg/mL) (P = 0.000). These concentrations decreased when MAC-2 was determined (123 ± 27 min from starting paracetamol administration) and were significantly higher than in the CTL group (8.5 ± 4.2 μg/mL and 1.6 ± 0.9 μg/mL in the PRC and CTL groups, respectively; P = 0.027).

Discussion

Paracetamol 15 mg/kg BW, IV was not successful at reducing the sevoflurane MAC in dogs. Previous studies had determined a sevoflurane MAC reduction of 30% in rats for at least 4 h following a single intraperitoneal dose (300 mg/kg BW) (20). Different species, but also pharmacokinetics, dose, and administration route, may account for these apparently contradictory results. However, another study using a single dose of 300 mg/kg BW, IV in rats reported a lack of effect on the MAC and no MAC reduction of isoflurane (4). Different routes of administration, anesthetic gases used, and equilibration time from drug administration to MAC determination, may account for these apparently contradictory results.

The MAC reduction produced by paracetamol (13%) here was not statistically significant and may reflect a limited effect by paracetamol, similar to that produced by other drugs, including NSAIDs. Robenacoxib, a selective COX-2 inhibitor, decreased the sevoflurane MAC for blunting adrenergic response by 17% in dogs (18). Similarly, carprofen and meloxicam decreased the sevoflurane MAC by 11% and 13%, respectively (19). However, a lack of effect from orally administered carprofen was reported in the isoflurane MAC, in which a non-statistically significant MAC reduction of 9% was detected (25).

Although paracetamol might not have a relevant anesthetic-sparing effect, if any, it may still provide clinically useful perioperative analgesia (13,14). The reduced side effects of paracetamol could support its perioperative administration in patients with gastrointestinal or renal impairment and when NSAIDs are contraindicated. All data from the physical examination and hematological and biochemical analyses in this study showed values within the physiological range and no side effects were noted.

The baseline sevoflurane MAC (MAC-1) is lower than in previous studies that reported values between 2.4% ± 0.3% (15) and 2.7% ± 0.2% (21), and similar to MAC studies employing propofol as the anesthetic induction agent (1.8% ± 0.1%) (26). Factors affecting variability in MAC values include the type of noxious stimulus, the anatomical site of stimulation, and subjectivity in the interpretation of purposeful movement. Other factors such as ETCO₂, body temperature, and arterial blood pressure were controlled in this study (27). Finally, the age of the dogs included in the study (6 y) may also affect the MAC value, since the sevoflurane MAC is 17% lower in older dogs (8 to 10 y) than in dogs under 2 y (24). Sevoflurane MAC of 1.9% ± 0.3% has been reported in dogs aged 8 to 10 y (28). Additionally, the effects of an anesthetic induction using propofol on sevoflurane MAC should be considered. Propofol clearance is slower in unpremedicated elderly dogs, perhaps due to age-related physiological changes such as an increase in the lipid compartment, where lipophilic drugs like propofol distribute, which would result in slower body clearance (29).

At the beginning of MAC-1 determination, 30 to 60 min after anesthetic induction, propofol plasma concentrations of at least 0.95 μg/mL were expected (29). Lower values of at least 0.3 μg/mL were expected once MAC-1 was determined, 2 to 3 h after anesthetic induction (29). Propofol decreases MAC in a dose-dependent manner; thus, the propofol blood levels in our study may have reduced the MAC by at least 23% and 16%, at the beginning and end of MAC-1 determination, respectively, as suggested by studies using propofol under a continuous infusion rate (30). We found a positive correlation between MAC-1 and the time required for its determination, since longer times favored propofol clearance and thus a higher MAC value. A longer equilibration time before the MAC baseline determination could have reduced this effect. In addition, residual propofol in plasma may explain why MAC-2 increased significantly in the CTL group, which had received no other co-administered drugs. However, confirming this requires assessing propofol plasma concentrations, which was not done here. To prevent the interference of propofol or any other drug in the MAC, dogs should ideally use an anesthetic mask for inhalational induction. There are 3 reasons for using a propofol induction: i) it produces a less stressful anesthetic induction, ii) propofol administration better mimics the clinical situation where mask induction is rarely employed, and iii) induction with propofol avoids atmospheric contamination inside the operating room. Similarly, previous experimental (26,31,32) and clinical (33,34) MAC studies determining the inhalational anesthetic sparing action of analgesics, anesthetics, and sedatives used propofol to induce anesthesia in dogs; although, lower induction doses or longer equilibration times were used in those studies.

Paracetamol plasma concentrations were 34.5 ± 9.9 μg/mL before the second MAC determination, approximately 2 h after propofol anesthetic induction. Time required to determine MAC-2 could have affected the paracetamol plasma concentration and therefore, the MAC reduction observed in each dog. Lower plasma concentrations of 7 to 8 μg/mL have been observed in greyhounds 1 h after 8 to 23 mg/kg of paracetamol was administered orally (22,35). A mean maximum serum concentration of approximately 45 μg/mL has been determined after an oral dose of 100 mg/kg BW and 90 μg/mL after a dose of 200 mg/kg BW (11). The differences may reflect the administration route of the drug. Previous studies have not reported on paracetamol plasma concentrations using IV administration nor have they correlated the plasmatic concentration of paracetamol with analgesic efficacy in dogs. A similar correlation has been observed in humans with postoperative dental pain (36).

As expected, the paracetamol plasma concentration decreased at the end of the study. Paracetamol is rapidly absorbed and eliminated within 1 h in dogs (22,35) and humans, with a rapid onset of analgesic effect within 5 min (1). Therefore, a higher MAC reduction might be expected just after paracetamol administration and an intraoperative continuous infusion would have provided an improved effect. As a limitation of the study, it should be noted that low levels of paracetamol were measured in the CTL group, probably as a consequence of phenolic compound cross-detection by the analytic method employed (37). The resulting measured levels were too low (4 ± 2 μg/mL) to have modified the observed results.

There are additional limitations to this study. First, the reduction in the sevoflurane MAC does not necessarily reflect an analgesic effect, although this has been suggested (36), as with opioids. Second, the lack of a paracetamol effect on MAC may be due to the low statistical power; a higher sample size could have made a significant difference. To achieve a power of 80%, a larger sample size of 8 to 10 dogs per group (1- and 2-tailed, respectively) would have provided higher MAC variation and made a statistically significant difference. Furthermore, additional paracetamol doses could have been studied to determine a potential dose-dependent sevoflurane-sparing effect. Finally, the use of propofol might have modified the sevoflurane MAC and affected paracetamol plasma measurements. Longer equilibration times before the MAC baseline determination and lower propofol doses could have been used to minimize this effect. However, it was decided to use an anesthetic induction agent such as propofol since it is widely used and better mimics a clinical setting, whilst inhalational anesthetic induction is rarely used and is banned in many clinical practices because of personnel safety concerns.

Despite the limitations to the study, the relatively small anesthetic sparing effect of paracetamol is of limited clinical value when MAC reductions above 20% are considered more desirable. This decrease is higher than the expected inter-individual variability of MAC (27), making it unnecessary to increase the number of dogs.

In conclusion, 15 mg/kg BW of IV paracetamol did not significantly reduce sevoflurane MAC in healthy dogs.