HEPATIC GLUTATHIONE S-TRANSFERASE RELEASE AFTER HALOTHANE ANAESTHESIA: OPEN RANDOMISED COMPARISON WITH ISOFLURANE

771

however, favour the two-step approach since not all patients will need a myelogram and in our experience myelography is more distressing than a lumbar puncture to the patient, but the final decision rests with the individual doctor. What is clear, though, is that an abnormal VER is not sufficient reason for withholding myelography. The major policy implication of our recommendations would be to reduce the number of invasive investigations and associated hospital costs. VER can be done as an outpatient procedure and lumbar punctures would require only a brief hospital stay. In some cases a repeat admission may be needed for myelography, but this is preferable to and more cost-effective than doing myelograms on all patients. Our findings also demonstrate how decision analysis allows a rational appraisal of the contributions made by investigations in the management of common clinical problems. This procedure also pinpoints areas where data are inadequate, so that the necessary information can then be collected. We no longer need to assess the value of newer investigations in everyday patient management intuitively when rational methods are available.

HEPATIC GLUTATHIONE S-TRANSFERASE RELEASE AFTER HALOTHANE ANAESTHESIA: OPEN RANDOMISED COMPARISON WITH ISOFLURANE AMANDA J. HUSSEY LAUREN G. ALLAN GEOFFREY J. BECKETT JANE HOWIE ALISTAIR F. SMITH JOHN D. HAYES GORDON B. DRUMMOND

University Departments of Anaesthetics and Clinical Chemistry, Royal Infirmary, Edinburgh

Plasma

of concentrations hepatic S-transferase (GST) are a glutathione more sensitive measure of acute hepatic damage than aminotransferase activity. Plasma GST concentrations have been measured by radioimmunoassay in an open randomised study after halothane or isoflurane anaesthesia. The concentration of GST was significantly increased after anaesthesia in patients who received halothane in 30% oxygen/70% nitrous oxide (n=37) and in patients who received halothane in 100% oxygen (n = 17). The frequency of abnormal GST concentrations, defined as 4µg/l or more, was 35% and 24%, respectively. GST concentrations usually reached a peak 3-6 h after the end of anaesthesia. In 17 patients who received isoflurane in 30% oxygen/70% nitrous oxide, there was no significant rise in GST concentration and no patient had a concentration above 4 µg/l. No patient in any of the groups had a significant increase in alanine aminotransferase. In clinically identical situations, anaesthesia with halothane but not isoflurane leads to demonstrable impairment of hepatocellular

Summary

integrity. Introduction THE frequency of unexplained hepatitis after halothane administration has not been established. The incidence of fulminant hepatic failure may be as high as 1 in 7000 with a 20-50% mortality rate.2 First described in 1958,3 the nature,

incidence, mechanism,

and

predisposing

factors for this

Correspondence should be addressed to R. I., Department of Neurology, Hospital, St Kilda Road, Melbourne, Australia 3004.

Prince Henry’s

REFERENCES

RA, Hillman BJ, McLennan JE, Strand RD, Kaufinan SM. Sequelae of metrizamide myelography in 200 examinations. AJR 1978, 130: 499-502. 2. Mastaglia FL, Black JL, Cala LA, Collins DWK. Electrophysiology and avoidance of invasive neuroradiology in multiple sclerosis. Lancet 1980; i: 144. 3. Iansek R, Balla JI. Decision analytical approach to the role of VER and CSF abnormalities in the management of singular spinal sclerosis. Clin Exp Neurol 1985; 21: 249-55. 4. McAlpine D, Lumsden CE, Acheson ED. Multiple sclerosis: an appraisal. Edinburgh: Churchill Livingston, 1972. 5. Merrill CR, Goldman D, Van Keuren ML. Simplified silver protein detection and image enhancement methods in polyacrilamide gels. Electrophoresis 1982, 3: 17-23. 6. Marshall J. Spastic paraplegia of middle age. Lancet 1955; i: 643-46 7. Bartel DR, Markand ON, Kolar OT. The diagnosis and classification of multiple sclerosis: evoked responses and spinal electrophoresis. Neurology 1983; 33: 611-17. 8. Paty DW, Blume WT, Brown WF, Taaloul N, Kerstesz A, McInnis W. Chronic progressive myelopathy: investigation with CSF electrophoresis, evoked potentials and CAT scan. Trans Am Neurol Assoc 1978, 103: 110-12. 9. Assalman P, Chadwick DW, Marsden CD. Visual evoked responses in the diagnosis and management of patients suspected of multiple sclerosis. Brain 1975; 98: 1. Baker

261-82. 10. Blumhardt LD, Barrett G, Halliday AM. Clinical application of evoked potentials in neurology. In: Courjon J, ed. The pattern evoked potential in the clinical assessment of undiagnosed spinal cord disease. New York: Raven Press, 1980. 11. Latchaw RE, Hirsch WL, Horton JA, Bissonette D, Shaw DD, Iohexiol VS. Metrizamide: Study of efficacy and morbidity in cervical myelography AJNR 1985; 6: 931-33.

remain unknown.4,5 Medical evidence and legal decisions that have incriminated halothane, mainly by temporal association of anaesthesia with jaundice, have caused a dramatic decline in halothane use for adults in the USA. Elsewhere, the rarity of unexplained hepatitis after halothane and the failure to find irrefutable evidence against halothane have ensured this agent’s continued popularity and sustained the controversy. 1,7 Diagnosis of mild halothane-induced hepatitis is difficult whether made on clinical, biochemical, or histological criteria. Plasma or serum aminotransferase activity8 is generally regarded as a sensitive measure of acute hepatic damage.9 However, aminotransferase activity is not specific to the liver, because these enzymes are released in other conditions,8 and may correlate poorly with hepatic histology.lO Alanine and aspartate aminotransferase (ALT and AST) are commonly used to assess hepatic injury after general anaesthesia but have yielded conflicting results and, if used alone, may be misleading.4 The measurement of hepatic glutathione S-transferase liver

damage

(GST) by radioimmunoassay (RIA)11 offers potential advantages over the aminotransferases in the investigation of hepatic damage. GST has a molecular mass of 45 000 to 50 000 daltons and is readily and rapidly released into blood after hepatic damage. In contrast to the periportal location of aminotransferases, GST is primarily distributed in centrilobular hepatocytes.12 This location may be more relevant to the study of unexplained hepatitis after halothane, which classically causes centrilobular necrosis.13 Plasma GST concentrations provide a more sensitive index of acute hepatocellular damage than aminotransferase activity. 14,15 Also, when the active phase of hepatic damage is over, plasma GST concentrations rapidly revert to normal, a feature of the short plasma half-life (under 90 min), whereas aminotransferase activity may be abnormal for much longer. 14 Plasma GST concentrations correlate better than aminotransferases with hepatic histology, 10 an important factor when inferring hepatotoxicity by suspected agents.9 These features suggest that GST measurement would have specific advantages over conventional hepatic enzymes in the investigation of acute hepatic damage.

772 In a pilot study to test the value of GST measurement after volatile agent anaesthesia in 28 selected patients, significant increases in GST were observed postoperatively in 23 patients.16 We have now examined the concentrations of plasma GST after anaesthesia in 71 patients with halothane or isoflurane.

Patients and Methods Patients Patients who

were to

have minor elective

gave informed consent for

a

urological procedures study approved by our ethical

committee. Those who had received halothane anaesthesia in the previous 3 months or who had clinical or biochemical evidence of liver disease were excluded. On entry to this open trial patients were randomly allocated by drawing envelopes to one of three groups for anaesthetic maintenance--group I received halothane in 30 % oxygen/70% nitrous oxide; group II received halothane in 100% oxygen; and group III received isoflurane in 30% oxygen/70% nitrous oxide. The randomisation plan allowed half the patients to be allocated to group I while the remainder were equally divided between groups II and III. No premedication was given and induction was achieved with thiopentone 4-6 mg/kg. All patients breathed spontaneously from a non-rebreathing system. The inhaled concentration of halothane varied from 0-5 to 3% in group I and from 1.5to 4% in group II; and that of isoflurane varied from 1 to 3% in group III. Blood was sampled from an indwelling cannula inserted in a forearm vein with local anaesthesia before induction of general anaesthesia. Further samples were taken at the end of anaesthesia (time zero) and 1, 3, 6, and 24 h later. Samples were stored in lithium-heparin tubes at 4°C until the plasma was separated for analysis. Plasma was stored at -20°C before GST measurement.

Assay Procedures The activity of ALT, y-glutamyltransferase (y-GT), and alkaline phosphatase (AP), and the level of bilirubin in plasma were measured with a multichannel analyser (’SMAC II’, Technicon). TABLE I-DEMOGRAPHIC DATA

NumDer or

Postoperative changes

from

patents

pre-induction level in GST.

Group I received halothane in 30% 02/70% N2O; group II received halothane in 100% O2; and group III received isoflurane in 30% O2/70% ’

N2O. Individual changes ce) shown as increased to more than 4 Ilg/l at any time.

for patients whose GST level

The concentration of the GST Bl-subunit in plasma was measured by RIA," with a reference range for GST of 0-7 to 40 µg/1. The interassay coefficient of variation was less than 10% over the range 0-2-12 )ig/l.

Statistical Analysis For comparisons between the groups, we used one-way analysis of variance (age, height, and weight) or, for all the other data (which were not normally distributed), the Kruskal-Wallis one-way analysis of variance. Levels of GST and ALT within each group were compared over time with the Friedman two-way analysis of variance (excluding 24 h because of 11missing values at this time). Changes in GST from pre-induction levels were analysed with the Kruskal-Wallis analysis of variance. The frequency of abnormal values in each group was compared with the chi-squared test. Correlation analysis was done with the Spearman rank test.

Results in

TABLE II-GST CONCENTRATION

(MEDIAN

[1st, 3rd QUARTILE],

µg/1)

Therewere no significant differences between the groups demographic or other background data (table 1),

including consumption of alcohol and cigarettes. No patient showed clinical evidence of hepatic dysfunction at any time and no patient was taking a concurrent medication associated with hepatotoxicity except for 1 patient who was taking phenytoin. There was no significant increase in ALT, y-GT, or AP activity, or in bilirubin concentration. GST concentrations did not change significantly in patients given isoflurane but they increased in most patients in both halothane groups (table II, and figure). GST concentrations usually reached a peak in the 3-6 h samples. TABLE III-INCIDENCE OF ABNORMAL VALUES OF GST AT ANY TIME AFTER END OF ANAESTHESIA**

II, p < 0-001; and III, Analysis of variance within groups-I, p<0.0001; p < 0-7. Analysis of variance between groups-not significant except for 3 h; p = 0-038.

*Table shows number of patients.

773

in group I and 1 patient in group II, GST concentrations had returned to normal by 24 h. At 3 h the changes in GST in groups I and II were significantly different to those in group III. There was no significant correlation between the changes in GST concentration and the total dose of halothane received. The occurrence of abnormal GST values after anaesthesia (table III) was greater in the patients who received halothane than in the group who received isoflurane, in which no patient showed an abnormal GST level (p < 0-02).

Except for 4 patients

Discussion

Unexplained hepatitis after halothane probably consists of two distinct entities, one mild (type I) and one severe (type II).17 Type II leads to hepatic failure with features of hypersensitivity 17 and familial constitutional susceptibility.18 Type I, transient and subclinical, may be more common. However, conventional biochemical tests of hepatic function give conflicting results in postoperative halothane-induced hepatic damage.4°6 Even complex animal models which enhance the unexplained hepatitis after halothane have not provided conclusive evidence for the mechanism.4,6 We have found, in plasma GST levels, some evidence for mild hepatic dysfunction post-halothane. Although the changes in median GST levels were small, some individual increases were large. This impaired hepatocellular integrity resulted from a single, brief halothane administration. Isoflurane did not impair hepatocellular integrity. Enzymes other than GST are claimed to have advantages in detecting hepatic damage after anaesthesia, but in practice these enzymes have little advantage over aminotransferases.19 The timing of the changes observed may be important in the mechanism of GST release, with an increase in GST between 3 and 6 h and resolution by 24 h in most patients. However, a few patients had a rise in GST at 24 h. In our pilot study, 16 we also saw two distinct phases of increased GST-the first within 3 h in most patients and the second at 24 h in a smaller number of patients. These discrete responses suggest two mechanisms. In animal models, hepatic damage has been attributed to both metabolic and hypoxic causes. Halothane is usually metabolised by hepatic mixed-function oxidases.4 In hypoxic conditions halothane can be reduced to unstable electrophilic intermediates which may bind covalently to liver tissue macromolecules to cause necrosis .20 Reduction of halothane has been demonstrated in man and explained by a theoretical pathway involving reduced glutathione.21 In animals, hepatic damage is related to halothane concentration and can be ameliorated by cimetidine which inhibits the reductive pathway.6 This evidence supports a metabolic theory with toxic intermediates that are formed during halothane reduction. Whether cimetidine could obviate the changes in GST after halothane anaesthesia in man is unknown. In the guineapig, only halothane produces hepatic necrosis.22 The guineapig model may therefore be more relevant to halothane hepatotoxicity in man since neither hypoxia nor enzyme induction are required.6 Nitrous oxide potentiates halothane hepatotoxicity in rats23 but we found no difference between our group I patients who received nitrous oxide and group II patients who did not. The finding that hepatic damage resulted from hypoxia in association with other agents has led to the theory that hypoxia alone is the cause.6 Hepatic hypoxia results from either hypoxaemia or decreased hepatic blood-flow; both would cause centrilobular necrosis. Halothane anaesthesia is

likely to result in hepatic hypoxia than isoflurane because halothane specifically decreases the ability of the hepatic artery to alter its flow in relation to portal blood-flow.25 In hypoxic conditions, halothane but not isoflurane reduces portal blood-flow.25 The hypoxia theory is supported by animal work which demonstrates that factors which cause increased oxygen consumption enhance the degree of hepatic necrosis;6 these factors include enzyme induction25 and thyroid hormone administration.6 In hyperthyroid patients and patients receiving excess thyroxine, hepatic damage has been demonstrated with GST measurement.26 Although enzyme induction was not thought to be a primary factor in unexplained hepatitis after halothane,4 single halothane anaesthesia for minor surgery has intrinsic more

enzyme-inducing properties.27 Types I and II hepatic damage after halothane anaesthesia are demonstrated only in the first and second postoperative week by conventional enzyme analysis. Using GST levels, we could demonstrate hepatic damage much earlier. The early phase of damage up to 6 h after halothane may well be due to the direct effect of halothane on hepatic blood-flow, producing a relative hepatic hypoxia. The later phase, less common, but with larger changes in GST at 24 h, may result from the production of toxic metabolites from halothane.

Hepatic damage has always been associated with general anaesthesia and the hope that halothane would be free from hepatotoxicity has not heen realised. Halothane was investigated for hepatotoxicity during development and there was no reason to suspect that hepatic damage would result from its use.4,6 It is now generally accepted that halothane can induce unexplained hepatitis, particularly when used repeatedly at short intervals in middle-aged obese women.6,17 Our study, however, implies that even a single halothane exposure for anaesthesia in minor surgery can produce hepatic damage, albeit transiently. Isoflurane may be free of this potential. The measurement of GST by RIA may help to make clearer scientific decisions about the future of halothane. GST assay may also help to define the occurrence of hepatic dysfunction after halothane and other volatile agents, and may even shed some light on the mechanism of halothane hepatitis. This work was supported by a grant from the Scottish Home and Health Department to Dr G. J. Beckett and Dr J. D. Hayes.

Correspondence should be addressed to L. G. A., Department of Anaesthetics, Royal Infirmary, Edinburgh EH3 9YW; from June 1, Department of Anaesthesia, Northwick Park Hospital and Clinical Research Centre, Harrow, Middlesex HA1 3UJ. REFERENCES 1. Editorial. Halothane-associated liver damage. Lancet 1986; i: 1251-52. 2. Touloukian J, Kaplowitz N. Halothane-induced hepatitic disease. Semin Liver Dis 1981; 1: 134-42. 3. Virtue RW, Payne KW. Postoperative death after Fluothane. Anesthesiology 1958; 19: 562-63. 4. Strunin L. Postoperative hepatic dysfunction. In: Strunin L, ed. The liver and

anaesthesia. Major problems in anaesthesia, vol III. London: WB Saunders, 1977: 144-81. 5. Cousins MJ. Halothane hepatitis: What’s new? Drugs 1980, 19: 1-6. 6. Stock JGL, Strunin L. Unexplained hepatitis following halothane. 1985; 63: 424-39.

Anesthesiology

Blogg CE. Halothane and the liver: The problem revisited and made obsolete. Br Med J 1986; 292: 1691-92. 8. Zimmerman HJ, Seeff LB. Enzymes in hepatic disease. In: Coodley EL, ed Diagnostic enzymology Philadelphia: Lea and Febiger, 1970: 1-38. 9. Zimmerman HJ. Experimental hepatotoxicity. In: Zimmerman HJ, ed. 7.

10.

Hepatotoxicity. The adverse effects of drugs and other chemicals on the liver. New York: Appleton-Century-Crofts, 1978: 167-97. Sherman M, Bass NM, Campbell JAH, Kirsch RE. Radioimmunoassay of human ligandin. Hepatology 1983; 3: 162-69.

774 IMMUNE DONORS CAN PROTECT MARROW-TRANSPLANT RECIPIENTS FROM SEVERE CYTOMEGALOVIRUS INFECTIONS

J. P. GROB1 H. G. PRENTICE1 A. V. HOFFBRAND1 T. TATE4

J. E. GRUNDY2 P. D. GRIFFITHS2 M.D. HUGHES3 J. Z. WIMPERIS1 M. K. BRENNER1

Departments of Haematology,1 Virology,2 Clinical Epidemiology,3 and Radiotherapy,4 Royal Free Hospital, London

study the importance of transferred immunity against cytomegalovirus (CMV) in allogeneic, HLA-matched, T-cell-depleted bonemarrow transplantation, the incidence, severity, and

Summary

We have shown that immunisation of the marrow donor with tetanus toxoid or hepatitis-B vaccine allows B-cell immunity to be adoptively transferred from the donor to the recipient. This transfer produces protective levels of antibody in the recipient,4,5even when the donor marrow is first depleted of T-cells to prevent graft-versus-host disease (GvHD).6 To study the relevance of this observation to the outcome of infections in vivo, we analysed retrospectively the effects of marrow-donor immunity against CMV on the incidence and severity of CMV infections in CMV-

seropositive recipients. Patients and Methods

To

of CMV infections were studied in 40 CMVseropositive recipients in relation to the donors’ immunity against CMV. There was no significant difference in the incidence of CMV infections between recipients of seropositive (n = 27) and seronegative (n = 13) marrow. However, the incidence of CMV pneumonitis (8/13 compared with 4/27; p < 0·001) and the mortality attributable to CMV infection (6/13 compared with 1/27, p < 0·01) were significantly greater in the group with seronegative donors than in those with seropositive donors. Multivariate regression analysis showed that recipients of seronegative marrow had a fifteen-fold greater risk of CMV pneumonitis and a fifty-fold increase in risk of a fatal CMV infection than recipients of seropositive marrow. Thus, after T-cell depletion CMV-seropositive marrow protects seropositive recipients against severe CMV infections; whenever possible, therefore, such recipients should be given marrow from seropositive donors. Ultimately, active immunisation of CMV-seronegative donors might help to protect seropositive recipients of T-cell-depleted marrow transplants against severe CMV infections. outcome

Introduction ALLOGENEIC bone-marrow transplantation (BMT) is followed by a period of profound immunosuppression often complicated by serious, life-threatening, infections.! Cytomegalovirus infections (CMV) are of special concern since the most serious manifestation, CMV pneumonitis, is the commonest infectious cause of death after BMT.2 CMV infections are probably due to reactivation of endogenous virus3 and so are commonest among CMV-seropositive recipients, in whom the frequency approaches 70%.2 CMV pneumonitis occurs in 25% of seropositive patients and has a mortality of 85%.

All 42 CMV-seropositive patients who had received an allogeneic T-depleted BMT since February, 1983, in our T-depletion study and who survived at least 30 days after the procedure were included in the initial analysis. 2 patients in whom marrow engraftment did not occur were subsequently excluded. Of the 40 remaining patients, 37 received HLA-matched, mixed-lymphocyte-culture non-reactive, sibling grafts, and 3 had a single-locus-mismatched sibling donor. The standard conditioning for BMT consisted of cyclophosphamide, (two doses of 60 mg/kg) and total body irradiation (maximum prescribed dose to the lung 7-5 Gy in 27 or 8-0 Gy in 6); 7 patients received an intensified conditioning regimen of cyclophosphamide (two doses of 45 mg/kg), cytarabine (6 doses of 3 g/m2), and total body irradiation (7-5 Gy in 6,8-0 Gy in 1). Two monoclonal anti-T antibodies (anti-CD8 and anti-CD6) and rabbit serum (as a complement source) were used to deplete the donor marrow of mature T-lymphocytes.6 No immunosuppression for GvHD prophylaxis was used after BMT for HLA-matched donor-recipient pairs. The 3 patients with minor HLA mismatches received oral cyclosporin (5 mg/kg daily) and oral prednisolone (1 mg/kg/daily) in an attempt to prevent graft rejection. All patients received blood products unscreened for CMV. 3 patients were included in a randomised study investigating the value of CMV immune globulin for the prevention of CMV pneumonitis (unpublished). 2 of them (1in each group) received the control, non-hyperimmune immunoglobulin. The third patient, with a CMV-seropositive donor, received CMV hyperimmune globulin and no CMV infection developed. Serum samples taken from donor and recipient before BMT were analysed for IgG anti-CMV antibodies by means of a solid-phase radioimmunoassay7 The amount of antibody in each serum sample was expressed as a binding ratio at a serum dilution of 1/100. This figure indicates how many multiples of the background radioactivity were bound at this dilution and is directly proportional to antibody titre as determined by end-point dilution.7 After BMT serum was taken from the recipient once a week; samples were stored at - 20°C and analysed in a single assay for each recipient at 3 months.

-

Samples of urine and saliva (in viral transport medium) and heparinised venous blood were collected from the recipients before

________________________

GJ, Hayes JD. Development of specific radioimmunoassays for the of human hepatic basic and NA/2b glutathione S-transferase. Clin Chim Acta 1984; 141: 267-73. Redick JA, Jakoby WB, Baron J. Immunohistochemical localisation of glutathione

11.Beckett

measurement

12.

S-transferases m livers of untreated rats. J

Biol Chem 1982; 257: 15200-03. 13. Peters RL, Edmondson HA, Reynolds TB, Meister JC, Curphey TJ. Hepatic necrosis associated with halothane anesthesia. Am J Med 1969; 47: 748-64. 14. Beckett GJ, Chapman BJ, Dyson EH, Hayes JD. Plasma glutathione S-transferase measurements following paracetamol overdose: Evidence for early hepatocellular damage. Gut 1985; 26: 26-31. 15 Bass NM, Kirsch RE, Tuff SA, Saunders

16.

17. 18.

19.

SJ Radioimmunoassay of plasma ligandin: A sensitive index of experimental hepatocellular necrosis. Gastroenterology 1978; 75: 589-94. Hussey AJ, Howie J, Allan LG, Drummond GB, Hayes JD, Beckett GJ. Impaired hepatocelluir integrity during general anaesthesia, as assessed by measurement of plasma glutathione S-transferase. Clin Chim Acta 1986; 161: 19-28. Neuberger J, Williams R. Halothane anaesthesia and liver damage Br Med J 1984; 289: 1136-39. Farrell G, Prendergast D, Murray M. Halothane hepatitis. Detection of a constitutional susceptibility factor N Engl J Med 1985; 313: 1310-14. Strunin L. Liver function tests. In: Strunin L, ed. The liver and anaesthesia: Major

problems in anaesthesia, vol III. London. WB Saunders, 1977: 24-34 20. Gandolfi AJ, White RD, Sipes IG, Pohl LR. Bioactivation and covalent binding of halothane in vitro: Studies with [3H] and [14C) halothane. J Pharmacol Exp Ther 1980; 214: 721-25. 21. Cohen EN, Trudell JR, Edmunds HN, Watson E. Urinary metabolites of halothane in man. Anesthesiology 1975; 43: 392-401. 22. Lunam CA, Hall P de la M, Cousins MJ. Cadiovascular and hepatic effects of halothane and isoflurane in a guinea pig model of ‘halothane hepatitis’. Clin Exp Pharmacol Physiol 1983; 10: 726 (abstr). 23. Ross JAS, Monk SJ, Duffy SW. Effect of nitrous oxide on halothane-induced hepatotoxicity in hypoxic enzyme-induced rats. Br J Anaesth 1984, 56: 527-33 24. Vina JR, Davis DW, Hawkins RA. The influence of nitrous oxide on methionine, S-adenosylmethionine and other amino acids. Anesthesiology 1986; 64: 490-95. 25. Gelman S, Rimerman V, Fowler KC, Bishop SP, Bradley EL. The effect of halothane, isoflurane and blood loss on hepatotoxicity and hepatic oxygen availability in phenobarbital-pretreated hypoxic rats. Anesth Analg 1984; 63: 965-72 26. Beckett GJ, Kellett HA, Gow SM, Hussey AJ, Hayes JD, Toft AD. Elevated plasma glutathione S-transferase concentrations in hypothyroid patients receiving thyroxine replacement: evidence for hepatic damage. Br Med J 1985; 291: 427-31 27. Haxholdt O St, Loft S, Clemmensen A, Hjortso E. Increased hepatic microsomal activity after halothane anaesthesia in children Aneasthesia 1986; 41: 579-81

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