Minimum alveolar concentration (MAC) has been widely used in experimental and clinical studies to compare the potency of volatile anesthetics, and MAC of anesthetic at 1 atm produces immobility in 50% of those animals exposed to a noxious stimulus.1 Many factors, such as age, combined use of other anesthetics, body temperature, and acidosis, can affect MAC values2 – 5 and some pathologic conditions, such as diabetes and heart disease, may also alter MAC.6 – 8 In addition, homeostasis and metabolism of drugs change when liver function is damaged.9 A retrospective study has found that end-tidal isoflurane requirements were different among patients with various liver statuses when a preset target Bispectral Index (range, 45–55) was maintained.10 That study indicated that damaged liver function might have an effect on the MAC of volatile anesthetics.
Sevoflurane, an inhaled anesthetic used worldwide, has been found to affect both liver function and hepatic blood flow. Its effects on the liver enzymes were equal to or less than those of isoflurane.11 – 14 In an earlier study, we found prolonged low-flow sevoflurane anesthesia did not worsen hepatic function in rabbits with liver fibrosis.15 Thus, we postulate that sevoflurane may be more suitable for patients with damaged liver function or liver fibrosis, which may have an effect on the MAC of sevoflurane. This study was designed to determine the MAC of sevoflurane in New Zealand white rabbits with liver fibrosis.
Institutional approval for the study was granted by the Animal Care and Use Committee of Sichuan University. Thirty 6-month-old male New Zealand white rabbits weighing 2.25 to 2.75 kg were purchased from the animal laboratory of West China Medical School. All animals were housed in environmentally controlled units for 1 week before the study. After 1 week, the rabbits were divided randomly into 2 groups: fibrosis group (group F, n = 20) and a normal control group (group N, n = 10). Rabbits in the fibrosis group received a subcutaneous injection of 0.2 mL/kg carbon tetrachloride (CCl4) in corn oil (1:1 ratio) twice a week for 4 weeks, and 0.3 mL/kg CCl4 in corn oil for another 8 weeks. CCl4 was reduced to half the amount or stopped for 1 week when the rabbits' weight decreased >8% in 1 week or a severe skin ulcer occurred. Animals in the control group received subcutaneous injection of corn oil only.
Twelve weeks later, all surviving animals were tested for the response to stimulation with a tail clamp before anesthesia. Rabbits were excluded from the study if they had no reaction to stimulation.
Anesthesia and Monitoring
After the animals were fasted for 6 hours, marginal ear veins were cannulated and 3 mL of blood was sampled for determination of liver function including total protein, albumin, globulin, total bile acids, alanine aminotransferase (ALT), aspartame aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase, total bilirubin, direct bilirubin, and indirect bilirubin.
Rabbits were premedicated with atropine (0.02 mg/kg, IV). Anesthesia was induced by mask with 6% sevoflurane (Batch No. 9414; Maruishi Pharmaceutical Co., Ltd., Osaka, Japan) in 5 L/min oxygen, and a 3.0 endotracheal tube with a cuff was used for intubation after direct laryngoscopy following approximately 20 minutes when the depth of anesthesia was sufficient (animals did not react to intubation). Spontaneous breathing was maintained in all animals during the study. The base of the tail was shaved for the measurement of MAC, and the expired sevoflurane concentration was adjusted to a preselected level. Expired sevoflurane and CO2 concentrations were monitored continuously using infrared analyzers (M1026B; Philips Medizin Systeme Böblingen GmbH, Böblingen, Germany). The middle ear artery was cannulated for monitoring arterial blood pressure, and fluid was infused continually through the marginal ear veins. Rectal temperature was controlled to 38°C to 40°C using an electrical blanket. Electrocardiogram, arterial blood pressure, rectal temperature, respiratory rate, and pulse oximeter saturation of the tongue were monitored during the experiment using an electrocardiogram monitor (PM-9000; Cayman Mindray Medical Electronics Co., Ltd., Shenzhen, China).
After MAC determination, the animals were killed and their livers were removed for the measurement of wet liver weight and pathologic examination. Small samples were taken from the left lobe and fixed in 10% neutral buffered formalin. Histologic sections of all removed tissues were prepared and stained with hematoxylin and eosin. Liver sections were examined and graded for the degree of fibrosis and superimposed acute hepatotoxic damage, according to previously established criteria.16,17
The MAC of sevoflurane was determined by the classic tail-clamp and “up and down” methods.18 After the rabbits were equilibrated for at least 15 minutes at a preset end-tidal concentration of sevoflurane of 3.70%,18 a clamp was placed around the tail and closed to the second ratchet. The clamp was kept in place for 10 seconds or until purposeful movement developed. Purposeful movement was defined as substantial movement of the head or extremities and did not include coughing, chewing, swallowing, or head nodding. When the rabbit exhibited purposeful movement, the end-tidal concentration of sevoflurane was increased by 10% (0.40% sevoflurane), and the animal was retested after 15 minutes of re-equilibration. If the rabbit did not move, the end-tidal concentration of sevoflurane was decreased by 10% and the animal would be retested after 15 minutes of re-equilibration. The MAC of a rabbit was calculated as the value midway between the end-tidal concentration preventing and allowing purposeful movement in response to tail clamp.
Statistical analysis was performed with the Social Program for Statistical Sciences (SPSS version 11.0; IBM, Armonk, NY). Results are represented as mean ± SD. Nonpaired t tests were used to detect any differences between the 2 groups (group F and group N) for each recorded variable. Correlation between the MAC of sevoflurane and the indexes of liver function was analyzed using multiple linear regression analysis. A P value <0.05 was considered significant.
Twelve weeks after CCl4 administration, 14 rabbits survived in the fibrosis group and 9 survived in the control group. Three rabbits in the fibrosis group were excluded, 1 for other diseases, 1 for no response to tail-clamp stimulation before anesthesia, and 1 with liver fibrosis that did not react to tail clamp during anesthesia and was found to have developed metabolic acidosis (pH = 7.233, PCO2 = 40.6 mm Hg, PO2 = 388 mm Hg, base excess = −11 mEq/L, HCO3− = 21.1 mEq/L). At the end of the study, there were 11 rabbits in group F and 9 in group N.
Body weights of CCl4-treated rabbits were slightly less, but without statistical difference, than those of untreated rabbits. The globulin, aspartame aminotransferase, and γ-glutamyl transpeptidase in CCl4-treated rabbits significantly increased compared with control animals, whereas albumin and alkaline phosphatase were significantly lower in group F than in group N. Total protein, albumin/globulin, ALT, aspartame aminotransferase, total bile acids, total bilirubin, direct bilirubin, and indirect bilirubin were comparable in both groups (Table 1).
The wet liver weight of rabbits with fibrosis was not statistically different compared with rabbits with healthy livers (60.12 ± 7.98 g vs 65.83 ± 9.97 g). Histologic examination revealed that liver fibrosis was moderate to severe in group F and normal in group N (Fig. 1).
No difference was found in mean arterial blood pressure, heart rate, end-tidal PCO2, pulse oximeter saturation, and temperature between groups (Table 2). MAC of sevoflurane was 4.10% ± 0.50% in normal rabbits and 3.52% ± 0.23% in rabbits with liver fibrosis. Sevoflurane MAC was significantly less in rabbits with liver fibrosis (4.10% ± 0.50%) than in healthy animals (3.52% ± 0.23%).
Multiple linear regression analysis showed that the effect of any indexes of liver function on MAC of sevoflurane was not significant. The only factor that affected the MAC of sevoflurane was liver fibrosis (P = 0.004).
Several methods of induction of fibrosis have been described. Of those, CCl4 intoxication is probably the most widely studied and it can cause hepatic degenerative pathogenesis, including cellular injury, necrosis, and fibrosis.19,20 The mechanisms of CCl4-induced hepatotoxicity include metabolism to free radicals by cytochrome P450 enzyme isoform 2E1 (CYP2E1), increased lipid peroxidation, interference of calcium homeostasis, and sustained regenerative and proliferative changes in the liver.21 – 24 A rabbit model of liver fibrosis was established successfully by using CCl4 in our study. After 12 weeks of CCl4 treatment, the biochemical index changed significantly, and histologic examination showed that all animals had developed moderate to severe liver fibrosis. Rabbits can recover from fibrosis spontaneously after cessation of CCl4. In a CCl4/ethanol rat model, the incidence of liver cirrhosis decreased from 40% to 14% 12 months after the cessation CCl4/ethanol.25 In another study, the authors also found that serum ALT activity was decreased to normal 4 weeks after the cessation of the CCl4 treatment, and necrosis in mice liver disappeared, although fibrotic changes were still observed.20 The same result was found in our rabbit fibrosis model.15 Therefore, we continued to administer CCl4 on the 13th study week and the determination of MAC was done after 24 to 36 hours of the CCl4 treatment.
In our study, the MAC of sevoflurane was 4.10% ± 0.50% in healthy rabbits, which is different from 3.70% ± 0.16% determined by Scheller et al.18 Here, we used New Zealand white rabbits and the same MAC-determined methods as Scheller et al. In our study, rabbits were 9 months old, but the rabbits' ages were not specified in the Scheller et al. study. Previous relevant studies demonstrated that age was an important factor that can affect MAC. Another methodologic difference between the 2 studies is that mechanical ventilation was performed in the study by Scheller et al. and spontaneous breathing was maintained in our study. Both mechanical ventilation and surgery can interfere with splanchnic perfusion.26,27 Docquier et al.28 showed that spontaneously breathing rats had a larger MAC of sevoflurane than that of mechanically ventilated rats. Whether mechanical ventilation has an effect on the MAC of inhaled anesthetics is unclear. The animal strains and environment in which animals were fed may have also contributed to the discrepancy in the 2 studies, which requires further study.
Our study has shown that the rabbits with liver fibrosis have a decreased volatile anesthetic requirement compared with animals with normal liver function, indicated by decreased sevoflurane MAC (3.52% vs 4.10%, P = 0.018). Sevoflurane MAC of rabbits with liver fibrosis was only 85% of that of healthy rabbits. In our study, factors that could affect MAC, including age4 and temperature,3 were similar in both groups. Normoxia and normocapnia were maintained during anesthesia. Hypotension, which occurred after sevoflurane inhalation, because of its depressant effects on cardiovascular function, has been considered an important factor determining the MAC of inhaled anesthetics.29 However, there was no statistical difference in mean arterial blood pressure and heart rate between groups. No other anesthetic was used in this study. Therefore, the discrepancy in sevoflurane MAC between the 2 groups is the result of liver fibrosis. Correlation analysis confirmed our speculation; however, the mechanisms for this result are unclear.
To our knowledge, increased bilirubin is very common in many liver diseases and damaged liver function in patients. In previous studies, patients with obstructive jaundice had an increased sensitivity to isoflurane, and the MAC-awake of desflurane for those patients decreased significantly.30,31 These studies indicated that bilirubin, which can induce central nervous system injury, had an effect on the requirement of inhaled anesthetic. However, the discrepancy between those studies and our liver fibrosis model is that bilirubin was in the normal range and did not increase significantly in our study (Table 1).
Sevoflurane biotransformation occurs at approximately 3% of that absorbed in the body, through cytochrome P-450 2E1 in the liver.32 The concentration of cytochrome P-450 is reduced in livers of patients with alcoholic cirrhosis and rats with CCl4-induced cirrhosis.33 – 35 It is possible that the biotransformation of sevoflurane may decrease in animals with liver fibrosis and the requirement of sevoflurane therefore may decrease.
Donovan et al.36,37 showed that the requirement for analgesic decreased in human and animal recipients after liver transplantation. In their study, the metenkephalin plasma levels, which modulated pain, were significantly increased both before and after liver transplantation when compared with an operation in the control group. Pain threshold can affect the anesthetic MAC, but whether the patient or animal with damaged liver function has a higher pain threshold is not clear.
Cerebrum disorder may decrease the MAC of inhaled anesthetics.8 A damaged liver can induce mental illness or hepatic encephalopathy (HE). In our previous study, a rabbit with liver fibrosis displayed disordered behavior when biting itself and other rabbits.15 It may have signified that mental illness or HE had developed in the rabbit. The presence of ammonia is the consequence of hepatic failure and it is a major neurotoxin implicated in HE. Brosnan et al.38 found that ammonia infusion to rats could decrease the MAC of isoflurane. This could have been the reason why the MAC of sevoflurane decreased in rabbits with liver fibrosis.
Another factor affecting MAC that should be considered is metabolic acidosis.1 One rabbit with liver fibrosis did not react to tail clamp and was excluded from the study during sevoflurane exposure. Metabolic acidosis was confirmed in that animal. It is unclear how metabolic acidosis developed and whether it correlated with the liver fibrosis. It is also not clear whether other liver-fibrosis rabbits had metabolic acidosis because gas analysis was not performed for each animal.
There are several limitations in this study. First, we did not perform gas analysis on each rabbit. Nonetheless, no evidence indicated that metabolic acidosis had a straightforward relationship to the liver fibrosis. Second, the animals' mental status assessments were not evaluated before sevoflurane MAC determination. We also did not measure the amount of ammonia, nor did we identify the presence of HE in the animals. However, it is difficult to define and evaluate the mental status of an animal.
In conclusion, the MAC of sevoflurane of liver-fibrosis rabbits was determined to be 3.52% ± 0.23%, which is significantly different from animals with normal liver function. Damaged liver function seems to contribute to the decrease in the requirement for sevoflurane. The outcome of our study indicates that the amount of inhaled anesthetic to the patient with damaged liver function should be decreased. However, whether this finding also applies to other animal species, other anesthetics, or humans is unknown. Further studies need to be performed.
Name: Yan Yin, MD.
Contribution: This author helped analyze the data and write the manuscript.
Attestation: Yan Yin has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Ming Yan, MD.
Contribution: This author conducted the study.
Attestation: Ming Yan has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Tao Zhu, MD.
Contribution: This author helped design the study and write the manuscript.
Attestation: Tao Zhu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Marcel Durieux, MD, PhD.
1. Eger EI II, Saidman LJ, Brandstar B. Minimum alveolar anesthetic concentration: a standard of anesthetic potency. Anesthesiology 1965; 26: 756–63
2. Doherty T, Redua MA. Effect of intravenous lidocaine and ketamine on the minimum alveolar concentration of isoflurane in goats. Vet Anaesth Analg 2007; 34: 125–31
3. Liu M, Hu X, Liu J. The effect of hypothermia on isoflurane MAC in children. Anesthesiology 2001; 94: 429–32
4. Mapleson WW. Effect of age on MAC in humans: a meta-analysis. Br J Anaesth 1996; 76: 179–85
5. Bridges BE Jr, Eger EI II. The effect of hypocapnia on the level of halothane anesthesia in man. Anesthesiology 1966; 27: 634–7
6. Brian JE Jr, Bogan L, Kennedy RH, Seifen E. The impact of streptozotocin-induced diabetes on the minimum alveolar anesthetic concentration (MAC) of inhaled anesthetics in the rat. Anesth Analg 1993; 77: 342–5
7. Barbry T, Le Guen M, De Castro V, Coriat P, Riou B, Vivien B. Minimum alveolar concentration of halogenated volatile anaesthetics in left ventricular hypertrophy and congestive heart failure in rats. Br J Anaesth 2007; 99: 787–93
8. Frei FJ, Haemmerle MH, Brunner R, Kern C. Minimum alveolar concentration for halothane in children with cerebral palsy and severe mental retardation. Anaesthesia 1997; 52: 1056–60
9. Sonne J. Drug metabolism in liver disease: implication for therapeutic drug monitoring. Ther Drug Monit 1996; 18: 397–401
10. Wang CH, Chen CL, Cheng KW. Bispectral index monitoring in healthy, cirrhotic, and end-stage liver disease patients undergoing hepatic operation. Transplant Proc 2008; 40: 2489–91
11. Bito H, Ikeda K. Renal and hepatic function in surgical patients after low-flow sevoflurane or isoflurane anesthesia. Anesth Analg 1996; 82: 173–6
12. Fukuda H, Kawamoto M, Yuge O, Fujii K. A comparison of the effects of prolonged (>10 hour) low-flow sevoflurane, high-flow sevoflurane, and low-flow isoflurane anaesthesia on hepatorenal function in orthopaedic patients. Anaesth Intensive Care 2004; 32: 210–8
13. Kharasch ED, Frink EJ Jr, Artru A, Michalowski P, Rooke GA, Nogami W. Long-duration low-flow sevoflurane and isoflurane effects on postoperative renal and hepatic function. Anesth Analg 2001; 93: 1511–20
14. Nishiyama T, Fujimoto T, Hanaoka K. A comparison of liver function after hepatectomy in cirrhotic patients between sevoflurane and isoflurane in anesthesia with nitrous oxide and epidural block. Anesth Analg 2004; 98: 990–3
15. Yin Y, Zhang W, Zhu T. The effects of prolonged low-flow sevoflurane anesthesia on hepatic function in rabbits with liver fibrosis. J West China Univ Med Sci 2008; 39: 1046–8
16. Cameron GK, Karunaratne WAN. Carbon tetrachloride cirrhosis in relation to liver regeneration. J Pathol Bacteriol 1936; 193: 265–75
17. Hepatic Fibrosis Group, Chinese Society of Hepatology. Common understanding of diagnosis and effect of hepatic fibrosis. Chin J Hepatol 2002; 10: 327–8
18. Scheller MS, Saidman LJ, Partridge BL. MAC of sevoflurane in humans and the New Zealand white rabbit. Can J Anaesth 1988; 35: 153–6
19. Proctor E, Chatamra K. High yield micronodular cirrhosis in the rat. Gastroenterology 1982; 83: 1183–90
20. Chen X, Wu T, Hu Y, Yu H, Zhou N. Long term observation of CCl4
-induced liver cirrhosis in rat. J Clin Hepatol 2006; 9: 195–7
21. Johnston DE, Kroening C. Mechanism of early carbon tetrachloride toxicity in cultured rat hepatocytes. Pharmacol Toxicol 1998; 83: 231–9
22. Comporti M. Biology of disease: lipid peroxidation and cellular damage in toxic liver injury. Lab Invest 1985; 53: 599–623
23. Agarwal AK, Mehendale HM. Effect of chlordecone on carbon tetrachloride-induced increase in calcium uptake in isolated perfused rat liver. Toxicol Appl Pharmacol 1986; 83: 342–8
24. Agarwal AK, Mehendale HM. Excessive hepatic accumulation of intracellular Ca2+
in chlordecone potentiated CCl4
toxicity. Toxicology 1984; 30: 17–24
25. Jiang Y, Liu J, Waalkes M, Kang YJ. Changes in the gene expression associated with carbon tetrachloride-induced liver fibrosis persist after cessation of dosing in mice. Toxicol Sci 2004; 79: 404–10
26. Gelman S. General anesthesia and hepatic circulation. Can J Physiol Pharmacol 1987; 65: 1762–79
27. Gelman SI. Disturbances in hepatic blood flow during anesthesia and surgery. Arch Surg 1976; 111: 881–3
28. Docquier MA, Lavand P, Ledermann C, Collet V, De Kock M. Can determining the minimum alveolar anesthetic concentration of volatile anesthetic be used as an objective tool to assess antinociception in animal? Anesth Analg 2003; 97: 1033–9
29. Tanifuji Y, Eger EI II. Effect of arterial hypotension on anaesthetic requirement in dogs. Br J Anaesth 1976; 48: 947
30. Song JG, Cao YF, Yang LQ, Yu WF, Li Q, Song JC, Fu XY, Fu Q. Awakening concentration of desflurane is decreased in patients with obstructive jaundice. Anesthesiology 2005; 102: 562–5
31. Yang LQ, Song JC, Irwin MG, Song JG, Sun YM, Yu WF. A clinical prospective comparison of anesthetics sensitivity and hemodynamic effect among patients with or without obstructive jaundice. Acta Anaethesiol Scand 2010; 54: 871–7
32. Kharasch ED, Thummel KE. Identification of cytochrome P450 2E1 as the predominant enzyme catalyzing human liver microsomal defluorination of sevoflurane, isoflurane, and methoxyflurane. Anesthesiology 1993; 79: 795–803
33. Villenueve JP, Wood AJ, Shand DG, Rogers L, Branch RA. Impaired drug metabolism in experimental cirrhosis in the rat. Biochem Pharmacol 1978; 27: 2577–81
34. Farrell GC, Cooksley WGE, Powell LW. Drug metabolism in liver disease: activity of hepatic microsomal drug metabolising enzymes. Clin Pharmacol Ther 1979; 26: 483–92
35. Farrell GC, Zaluzny L. Microsomal protein synthesis and induction of cytochrome P-450 in cirrhotic rat liver. Aust J Exp Biol Med Sci 1984; 62: 291–301
36. Donovan KL, Janicki PK, Striepe VI, Stoica C, Franks WT, Pinson CW. Decreased patient analgesic requirements after liver transplantation and associated neuropeptide levels. Transplantation 1997; 63: 1423
37. Donovan KL, Janicki PK, Franks WT, Striepe VI, Pinson CW. Liver transplantation is associated with increased met-enkephalin levels in the pig. Acta Anaesthesiol Scand 1996; 40: 1161
38. Brosnan RJ, Yang L, Milutinovic PS, Zhao J, Laster MJ, Eger EI II, Sonner JM. Ammonia has anesthetic properties. Anesth Analg 2007; 104: 1430–3