Serum mitochondrial aspartate transaminase activity after isoflurane or halothane anaesthesia

J. R. Darling*,1, P. C. Sharpe2, E. K. Stiby1, J. A. McAteer1, G. P. R. Archbold2 and K. R. Milligan1

1Department of Anaesthesia, Musgrave Park Hospital, Belfast BT9 7JB, UK. 2Department of Clinical Chemistry, Belfast City Hospital, Belfast BT9 7AB, UK*Corresponding author

Accepted for publication: February 28, 2000


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
We examined the effect of halothane or isoflurane anaesthesia on hepatic function in 30 ASA I–III patients aged 18–70 yr undergoing lumbar discectomy. Hepatic function was assessed before anaesthesia, at the end of surgery, and at 3, 6, 24 and 48 h after surgery using routine enzyme tests of hepatic function and mitochondrial aspartate transaminase (mAST) activity. Although serum mAST activities increased after surgery in both groups of patients, these increases were statistically significantly greater in the group that received halothane. The groups were similar with regard to other tests of hepatic function. Calculation of the ratio of serum enzyme activities compared to baseline values suggested that mAST is a sensitive marker of anaesthetic-induced hepatic injury.

Br J Anaesth 2000; 85: 195–8

Keywords: anaesthetics, volatile, halothane; anaesthetics, volatile, isoflurane; liver, function


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Aspartate transaminase (AST) consists of two genetically distinct, structurally different isoenzymes known as mitochondrial and soluble (or cytosolic) AST. The plasma concentration of mitochondrial AST (mAST) is small (normal <4 u litre–1) despite its considerable activity in heart and liver tissues.1 mAST probably has a shorter half-life than cytosolic AST and makes up 80% of the total AST in liver tissue. It has been suggested that mAST is preferentially located in the perivenous hepatocytes which are more susceptible to damage by both alcohol and anoxia.2 3 Serum concentrations of mAST may be measured immunochemically and could be a marker of damage resulting from anaesthetic agents such as halothane.4

We compared the effect of halothane and isoflurane anaesthesia on serum mAST to determine if serum mAST is a more sensitive marker for anaesthetic-induced liver damage than AST, alanine transaminase (ALT), alkaline phosphate (ALP), {gamma}-glutamyl transferase ({gamma}GT) and serum bilirubin.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
With written informed consent and University Ethics Committee approval, 30 ASA grade I–III patients undergoing lumbar discectomy were enrolled in the study. All patients were aged 18–70 yr and had no history of hepatic disease or recent heavy alcohol intake (>21 u weekly for men and >14 u weekly for women). Any patient who had a general anaesthetic in the previous 6 months was excluded from the study.

Anaesthesia and analgesia
Patients were randomly allocated to receive either halothane or isoflurane by opening sealed envelopes after enrolment into the study. Premedication consisted of temazepam (20 mg) in all cases. Anaesthesia was induced with thiopentone (5 mg kg–1) and intubation of the trachea was facilitated with atracurium (0.5 mg kg–1). Analgesia was provided by morphine (10 mg) at induction and droperidol (2.5 mg) was administered intravenously as an anti-emetic. Patients were ventilated to maintain their end-tidal carbon dioxide concentration at 4.6–5.9 kPa, and placed in a modified tuck position for surgery.5 Patients in the halothane and isoflurane groups received halothane and isoflurane, respectively, in nitrous oxide and oxygen (FIO2=0.4). Initial expired concentrations of anaesthetic agent were 1.0 MAC that was reduced if systolic arterial pressure decreased by more than 30% of baseline values. Fluid therapy was Dextran 70 in 0.9% saline (500 ml) followed by Hartmann’s solution as required during surgery, and 0.18% saline in 4% dextrose (100 ml h–1) after anaesthesia. Heart rate and rhythm, non-invasive arterial pressure and peripheral oxygen saturation by pulse oximeter were monitored throughout anaesthesia. End-tidal carbon dioxide and anaesthetic concentrations were also recorded during anaesthesia. Bupivacaine 0.25% (20 ml) was applied to the wound before it was sutured and neuromuscular blockade reversed with neostigmine 2.5 mg and glycopyrrolate 0.5 mg. Postoperative analgesia consisted of patient-controlled analgesia (morphine sulphate: 1 mg bolus and 5 min lock-out).

Laboratory analysis
Venous blood samples were drawn immediately before induction of anaesthesia, at the end of surgery (time 0), and 3, 6, 24 and 48 h later. The samples were centrifuged and the serum assayed for bilirubin concentration and activities of creatine kinase (CK), ALT, ALP, {gamma}GT and AST using a multi-channel analyser (Prisma, Clinicon-AB). The remaining serum was stored at –20°C and assayed for mAST at a later date by an immunochemical method.4 Rabbit antibodies against human soluble AST were added to the serum in the presence of polyethylene glycol and the sample was centrifuged to precipitate the soluble AST. Residual mAST in the supernatant was then assayed in the presence of exogenous pyridoxal phosphate by coupling oxaloacetate production with NADH in reaction with malate dehydrogenase (absorbance change monitored at 340 nm (Prisma, Clinicon-AB)). The detection limit of the assay was less than 0.5 u litre–1 and the coefficient of variation was less than 3% of the residual mAST.

Data were analysed using Wilcoxon ranked pairs within groups and Mann–Whitney U-test between groups. Ratios of enzyme activities compared to baseline values were calculated and analysed between groups using the Mann–Whitney U-test.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Fifteen patients were anaesthetized with isoflurane and 15 received halothane. The groups were similar in age, weight, gender, alcohol intake, duration of hypotension, minimum arterial pressure and dose of anaesthesia (Table 1). All patients had liver function test values of less than twice the upper limit of the reference range prior to anaesthesia. In both groups, serum concentration of bilirubin increased significantly above baseline at 24 and 48 h after anaesthesia (Table 2). Serum ALT activity decreased significantly (P<0.05) below baseline throughout the study period in the halothane group (Table 2). ALP and {gamma}GT activities significantly (P<0.05) decreased from baseline in both groups at the end of anaesthesia and in the case of {gamma}GT remained below baseline activities throughout the study period (Table 2).


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Table 1 Details of patients studied (median (range))
 

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Table 2 Serum bilirubin concentrations (µmol litre–1) and enzyme activities (u litre–1) for the groups receiving halothane (H) and isoflurane (I). Median (1st, 3rd quartiles). *P<0.05 difference from baseline activity (Wilcoxon rank sum)
 
Serum AST and mAST activity increased throughout the study period in both groups (Figs 1 and 2). In the halothane group this increase in serum activity reached significance in blood samples taken at more than 6 h after anaesthesia for AST and in all postoperative samples for mAST. In the isoflurane group, the increase in both AST and mAST reached significance at 24 and 48 h after anaesthesia.



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Fig 1 Serum aspartate transaminase (AST) activity (u litre–1) before and after halothane or isoflurane anaesthesia. Values are median and interquartile range. Normal range: 17–45 u litre–1. *P<0.05 difference between groups using Mann–Whitney U-test.

 
The groups were statistically different (P<0.05) with regard to AST in all samples taken after operation (Fig. 1). This difference was maximal (P<0.001) at 48 h after operation. The groups were also statistically different with regard to mAST activity in all samples taken after 6 h (Fig. 2). This difference was maximal (P<0.0001) at 48 h after anaesthesia. No significant difference was noted between the groups with regard to any of the other tests of hepatic function.



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Fig 2 Serum mitochondrial aspartate transaminase (mAST) activity (u litre–1) before and after halothane or isoflurane anaesthesia. Values are median and interquartile range. Normal range: 2–4 u litre–1. *P<0.05 difference between groups using Mann–Whitney U-test.

 
The baseline activity of AST and ALT was greater in the halothane group before operation. Although this did not reach statistical significance (P<0.05, Fig. 1), it was felt necessary to compare the change in the activities of AST and mAST from baseline activities. There was no statistical difference between the halothane and isoflurane groups when the increase in AST was considered as a ratio (Fig. 3). When a similar plot was carried out for the ratio of mAST activity to baseline (Fig. 4), there remained a statistical difference (P<0.001) between the halothane and isoflurane groups at 6, 24 and 48 h after surgery.



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Fig 3 Ratio of serum aspartate transaminase (AST) activity after halothane or isoflurane anaesthesia to baseline activity. Values are median and interquartile range.

 


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Fig 4 Ratio of serum aspartate transaminase (AST) activity after halothane or isoflurane anaesthesia to baseline activity. Values are median and interquartile range. **P<0.001 difference between groups using Mann–Whitney U-test.

 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
These data show an increase in serum activity of both AST and mAST in patients after lumbar discectomy. This increase is significantly greater after anaesthesia with halothane than isoflurane and is in agreement with previous work that found an increase in serum concentrations of glutathione S-transferase (GST) after a single, brief exposure to halothane.6 The conclusion of these previous studies suggested a loss of hepatocellular integrity following halothane, whereas isoflurane is free of this effect.7

Serum AST activity increased significantly from baseline for all samples drawn more than 6 h after surgery in the halothane group and 24 h after surgery in the isoflurane group. Serum AST activity was non-significantly greater in the halothane group prior to anaesthesia or surgery (P=0.09). Although the difference between the groups was significant (P<0.05) in all of the postoperative samples, we aimed to reduce the influence of the baseline difference by expressing the activities of AST after surgery and anaesthesia as a ratio of baseline activity (Fig. 3). This ratio did not show a statistical difference between the groups at any time after surgery, and therefore these data do not support the use of serum AST activity as an indicator of subclinical hepatic injury found after halothane anaesthesia. Other studies have provided conflicting data on the value of serum AST as an indicator of hepatic injury following halothane anaesthesia. The surgical procedure performed may be important as it has been found that serum AST activity decreases significantly after body surface or minimally invasive surgery,8 but is significantly increased after gastric or biliary surgery.9

There was no difference between the groups before surgery in terms of serum mAST activity. However, after anaesthesia and surgery, serum mAST was significantly greater in the halothane group of patients (Fig. 2). A similar pattern was found when mAST activities after anaesthesia were expressed as ratios of baseline activity (Fig. 4).

Many of the patients in this study were receiving medication, including TylexTM (paracetamol 500 mg and codeine phosphate 30 mg), diazepam and diclofenac that may cause some hepatic injury. The position of the patient during surgery may also be important because posture alters total hepatic blood flow and blood flow through the portal vein in particular.10 The tuck position may reduce blood flow through the portal vein. The hepatic arterial response to reduced portal venous blood flow is abolished in rats anaesthetized with halothane, but maintained in the presence of isoflurane.11 Therefore, a reduction in blood flow through the portal vein may be compensated in the isoflurane group but not in the halothane group. Patients undergoing more minor superficial surgery in the supine position may therefore allow a clearer examination of differences in liver function related to choice of anaesthetic agent.

The source of AST and mAST is not clear as both are found in liver, heart, kidney, brain and skeletal muscle.12 It may therefore be possible that the leakage of enzyme into the serum came from muscle damage at the time of surgery.9 13 However, the changes in serum bilirubin concentration and CK activity were similar in both groups of patients indicating similar severity of surgical tissue damage. As both groups of patients were similar in all other ways, it is most likely that the increased serum activities of mAST are due to the anaesthetic agents used and are hepatic in origin. Therefore, this study provides further evidence that a short exposure to halothane results in a greater loss of liver cell integrity than similar exposure to isoflurane.

In summary, mAST indicates hepatic dysfunction after anaesthesia with halothane. Limitations of the assay for mAST include a lack of organ specificity and the labour-intensive nature of the assay. However, the assay may become much easier in the future as automated techniques utilizing proteinases which inactivate cytosolic AST but have no effect on mAST become available.14 Although the use of halothane is diminishing in the western world, this assay may be useful in evaluating the effects of newer anaesthetic agents on hepatic function.


    Acknowledgement
 
We would like to thank Dr Robert Rej for his donation of antibody directed against cytosolic aspartate transaminase.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
1 Rej R. Aspartate aminotransferase activity and isoenzyme proportions in human liver tissues. Clin Chem 1978; 24: 1971–9[Abstract/Free Full Text]

2 Lumeng L. New diagnostic markers of alcohol abuse. Hepatology 1986; 6: 742–5[ISI][Medline]

3 Sharpe PC, McBride R, Archbold GPR. Biochemical markers of alcohol abuse. Q J Med 1996; 89: 137–44[Abstract]

4 Rej R. An immunochemical procedure for determination of mitochondrial aspartate aminotransferase in human serum. Clin Chem 1980; 26: 1694–700[Abstract/Free Full Text]

5 Wayne SJ. A modification of the tuck position for lumbar spine surgery: a 15-year follow-up study. Clin Orthopaedics 1984; 184: 212–6

6 Allan LG, Hussey AJ, Howie J, Beckett GJ, Smith AF, Hayes JD, Drummond GB. Hepatic glutathione S-transferase release after halothane anaesthesia: open randomised comparison with isoflurane. Lancet 1987; i: 771–4

7 Hussey AJ, Aldridge LM, Paul D, Ray DC, Beckett GJ, Allan LG. Plasma glutathione S-transferase concentration as a measure of hepatocellular integrity following a single general anaesthetic with halothane, enflurane or isoflurane. Br J Anaesth 1988; 60: 130–5[Abstract]

8 Darling JR, Murray JM, McBride DR, Trinick TR, Fee JPH. Serum glutathione S-transferase concentrations and creatinine clearance after sevoflurane anaesthesia. Anaesthesia 1997; 52: 121–6[ISI][Medline]

9 Clarke RSJ, Doggart JR, Lavery T. Changes in liver function after different types of surgery. Br J Anaesth 1976; 48: 119–28[Abstract]

10 Brown HS, Halliwell M, Qamar M, Read AE, Evans JM, Wells PNT. Measurement of normal portal venous blood flow by Doppler ultrasound. Gut 1989; 30: 503–9[Abstract]

11 Gelman S, Fowler KC, Smith LR. Liver circulation and function during isoflurane and halothane anaesthesia. Anesth Analges 1984; 61: 726–30

12 Sada E, Tashiro S, Morino Y. The significance of serum mitochondrial aspartate transaminase activity in obstructive jaundice: experimental and clinical studies. Jpn J Surg 1990; 20: 392–405[Medline]

13 Panteghini M, Malchiodi A, Calarco M, Bonora R. Clinical and diagnostic significance of aspartate aminotransferase isoenzymes in sera of patients with liver diseases. J Clin Chem Clin Biochem 1984; 22: 153–8[ISI][Medline]

14 Watazu Y, Okabe H, Sugiuchi H, Uji Y, Shirashe Y, Kaneda N. Proteolytic measurement of mitochondrial aspartate aminotransferases in human serum. Clin Biochem 1990; 23: 127–30[ISI][Medline]





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