Anaesth Intens Care (1991),19,378-381

N-Trifluoroacetyl-Ethanolamine: A Proposed Urinary Metabolite of Halothane: Validation and Measurement in Children H. WARK,* J. EARL,t D. CHAu:J: AND J. OVERTON§ Anaesthetic and Biochemistry Departments of the Royal Alexandra Hospital for Children and the Pharmacy Department of the University of Sydney SUMMARY

It has been postulated that trifluoroacetyl chloride, a halothane metabolite, can bind covalently with the phosphatidylethanolamine component of the hepatic cell membrane and cause cell necrosis. Breakdown of the necrotic hepatocyte would release N-trifluoroacetyl-ethanolamine (TFAE) into the serum with subsequent urinary excretion. An original High Performance Liquid Chromatography (HPLC) method for the measurement ofTFAE is described. In six children 1 % halothane was administered for one hour and the halothane uptake measured. Urinary excretion ofTFAE was measuredfor up to eight days andfound to be 0.09 ± 0.07% or less of the absorbed halothane. In children TFAE is not a major urinary metabolite of halothane. Key Words: ANAESTHETICS: volatile, halothane, metabolism; ANAESTHESIA: paediatric

Halothane-associated hepatitis in children occurs with an incidence of less than 1 in 82,000. I In adults the incidence is 1 in 10,000. 2 This difference in incidence of halothane-associated hepatitis between children and adults has never been adequately explained although it has been postulated that it may be due to either a quantitative or qualitative difference in halothane metabolism. In 1975, Cohen 3 studied eight adult patients receiving halothane and identified TFAE as a major urinary metabolite. The authors speculated that there was covalent binding of the oxidative metabolite of halothane, trifluoroacetyl chloride, with the phosphatidylethanolamine moiety of cephalin, a normal constituent of all cell membranes. This reacton may have a direct toxic effect on the hepatocyte. Enzymatic cleavage of the necrotic hepatocyte would release TFAE into the plasma with subsequent urinary excretion. In adults irreversibly bound halothane metabolites have been demonstrated in the liver both chemically and autoradiographically following halothane anaesthesia. 4,5 "F.e. Anaesth., Staff Specialist. tB.Sc., Ph.D., Development Biochemist. iPh.D., Scientific Officer. §F.e. Anaesth., Director of Anaesthesia. Address for Reprints: Dr. H. Wark, Department of Anaesthesia, Royal Alexandra Hospital for Children, Pyrmont Bridge Road, Camperdown, N.S. W. 2050, Australia. Accepted for publication March IS, 1991

It is possible that children do not metabolise halothane via this mechanism and this may explain why children are less susceptible to halothaneassociated hepatitis than are adults. Our study was designed to measure TFAE in the urine of children following halothane anaesthesia and to assess whether it is a major urinary metabolite of halothane.

PATIENTS AND METHODS N- Trifluoroacetyl-ethanolamine identification TFAE was prepared from ethyl-trifluoroacetate and ethanolamine in chloroform solution (V.S. Patent Specification 1, 123, 672). The compound was analysed by nuclear magnetic resonance spectroscopy NMR,13 C NMR) and mass spectrometry. Spectral data were consistent with the titled structure CF3CONHCH 2CH 20H (Tables 1 and 2). TFAE measurement Anion exchange columns were prepared by packing a 2 m1 syringe barrel with a plug of glass wool and pouring in a slurry of AGI-X2 (Bio-rad, Richmond California) in water to obtain 1 cc of the resin. The resin column was washed with 3 X 1 ml water. Urine was centrifuged, 0.1 mllayered onto the column and washed through with 1.9 ml water. The eluate was collected and stored at - 20°C until analysis.

eH

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A URINARY OXIDATIVE METABOLlTE OF HALOTHANE TABLE I

IH NMR andproton-decoupled 13C NMR data ofTFAE. Chemical shift values 0 and expressed relative to Si (CH}4' and with coupling constants J in Hz given in parenthesis. Symbols d, t, q and m refer to doublet, triplet, quartet and multiplet respectively. Assignments are based on the data of aminoethanol. Assignment

'H NMR data in CDCI 3 3.54 t (J HH 5.0) 3.81 d of t (JH.H 5.0; JH.Fl.O)

OCH 2

3.29 m

58.9 42.3 116.2 q (JcF288) 156.7 q (J ccF 36)

CON~H2

3.48 m

CF 3 CO

TF AE was analysed by high pressure liquid chromatography (HPLC). The mobile phase was 25 mM aqueous sodium phosphate pH 7.0 at a flow rate of 1.0 ml.min - 1. The equipment consisted of a Waters M6000 pump, Rheodyne injector and Waters 480 variable wavelength UV detector. The compound was measured by absorption of the amide group in the compound at 205 nm. To achieve resolution of this very polar compound, several Brownlee reverse phase columns were used in series, first a Newguard 1.5 cm X 3.2 mm RP-18 guard column, second a 25 cm X 4.6 mm RP-18 Spheri-5 column and third a 10 cm X 4.6 mm RP-18 Spheri-5 cartridge. These were linked together with 5 cm pieces of Alltech 1/16" X 0.01" pre-cut stainless steel tubing using Activon/ Upchurch fingertight fittings and equivalent to a 35 cm reverse-phase column. 20 ~l of sample or standard was injected giving a retention time of7.8 minutes. The limit of detection was 0.3 ~M in the extracted samples, equivalent to 6.0 ~M in the original urine. Peak height was linear with concentrations from 0.3 ~M to 1.0 mM. Standards of 640 ~M and 64 ~M TFAE extracted through the system gave recoveries of 99% and 96%. The coefficient of variation for five replicate determinations of an extracted 640 ~ standard was 2.8%. Separation of TFAE from major UV-absorbing constituents of urine was confirmed by comparing the chromatograms of unspiked urine and urine spiked with TFAE (Figure 1).

children having elective surgery. Patient six, a tenyear-old boy had cystic fibrosis, chronic lung disease and biopsy-proven liver disease. Full patient details are given in Table 3. Halothane 1% v/v was administered from a Mark III fluotec vapouriser for one hour in an inspiratory gas mixture of oxygen 2 litre.min - 1 and nitrous oxide 4 litre.min - 1. After the cessation of halothane, anaesthesia was mainained with nitrous oxide and oxygen with bolus doses of 2 mg.kg - 1 thiopentone given if required. Inspiratory gas samples from the fresh gas line were collected every fifteen minutes into gas-tight glass syringes (SGE Ringwood, Victoria, Australia) and halothane concentration measured by gas chromatography. Expiratory minute ventilation was measured with a Wright respirometer (BOC Healthcare, Surrey, England). Residual halothane in expired gas was measured by gas chromatography. Halothane uptake was calculated from the patient's inspiratory and expiratory halothane concentration and minute ventilation. The experimental method has been described in detail elsewhere. 5 Urine was collected continuously until the patient left hospital and stored at - 4°C until analysed for TFAE. Length of urine collections varied from three to eight days.

I

0.02 AU

Patient details Six children were included in the study with one child being studied twice. Five patients were fit TABLE 2 Methane chemical ionization mass spectral data of TFAE

mle

Assignment

158 140

MH+ MH+ -HP

Relative Abundance (%) lOO 63

Anaesthesia and Intensive Care. Vol. 19. No. 3, August. 1991

I

o Time (minutes)

I

10

I

10

Time (minutes)

FIGURE I.-HPLC measurement of urinary TFAE with an arrow indicating the position of the TFAE peak. (a) Sample taken from a patient's first 24-hour collection following halothane anaesthesia. (b) The same sample spiked with 64 I1M TFAE.

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TABLE 3 Patient details.

Patients

1 2 3 4 5 5* 6t Mean ± SD

Age at operation (months)

Weight (kg)

Surgical procedure

Duration of anaesthesia (min)

14 39 47 88 101 110 119

9.7 15.0 14.5 28.5 32.5 35.7 35.8

Hypospadias repair Pelvic/femoral osteotomy Pelvic/femoral osteomony Pelvic/femoral osteotomy Pelvic/femoral osteotomy Pelvic/femoral osteotomy Choledochoduodenostomy

90 200 190 115 190 225 300

74 ± 37

24.5 ± 10.3

187

± 64

*This patient had bilateral hip surgery for Perthe's disease. tThis patient had cystic fibrosis with a stenosis of the distal end of the common bile duct. A liver biopsy showed moderate fatty change. His medications were ticarcillin, tobramycin, Vitamin E, pancrease and multivitamins. necrosis is shown in Figure 2. In 1975 Cohen and RESULTS

Details ofTFAE excretion are shown in Table 4. On four of the seven occasions that halothane metabolism was investigated a HPLC peak with the same retention time as TFAE was identified in preoperative urine samples. This peak was due to an unidentified interfering substance. In calculating TFAE excretion the assumption was made that all UV absorbing material with a retention time at 7.8 minutes was TFAE and no correction was made for interfering substances in the control urine. The results obtained represent the upper possible limit of TFAE excretion. Total urinary excretion varied from 8 to 40 ~ moles and 0.09 ± 0.07% of the absorbed halothane was metabolised to TFAE. DISCUSSION

A proposed oxidative metabolic pathway of halothane which has the potential to cause liver cell

colleagues 4 reported a study of eight adult patients following intravenous radiolabelled halothane administration and found by counting urinary radioactivity that TFAE constituted 20% of urinary halothane metabolites. In this paper a quantitative technique for measuring TFAE was not described. These authors postulated that the halothane metabolite trifluoroacetyl chloride combined by a nucleophilic substitution reaction with the phosphatidylethanolamine component of the hepatic cell membrane causing cell necrosis. Enzymatic breakdown of the necrotic cell membrane resulting in TFAE release into the serum with subsequent urinary excretion. Our study was designed to develop a measurement technique for TFAE, and then to confirm or deny whether TFAE is a major urinary metabolite of halothane in normal children. An initial difficulty was that TFAE was not

TABLE

4

Details of urinary excretion of TFAE after halothane anaesthesia in children.

Patient

Duration of post-operative urine collection (days)

Total halothane uptaket m moles

Total urinary excretion of TFAEt ~ moles

Absorbed halothane excreted as TFAE (%)

3 8 7 4 7 7 7

13.30 23.65 17.41 25.38 49.14 37.36 45.30

26 8 30 16 8 40 10

0.20 0.03 0.17 0.06 0.02 0.11 0.02

1 2 3 4 5 5* 6 Mean ± standard deviation

0.09 ± 0.07

Halothane 1% v/v from a fiuotec Mark III vapouriser was administered for one hour and the uptake measured (5). tIn patients 1, 2, 3, 5* a substance was identified in control urine with the same HPLC peak as TFAE. In these patients the urinary excretion of TFAE was overestimated. *This patient was studied twice. Anaesthesia and intensive Care, Vo/. 19, No. 3, August, 1991

A URINARY OXIDATIVE METABOLITE OF HALOTHANE HALOTHANE hydroxylation -HBr

1 1 1

TRIFLUOROACETYL CHLORIDE (ACYLATING AGENT) Phosphatidyl ethanolamine (derived from the cephalin of liver cell endoplasmic reticulum)

PHOSPHATIDYL (N-TRIFLUOROACETYL)ETHANOLAMINE (TFA linked to phosopholipid) enzymatic cleavage

N-TRIFLUOROACETYL ETHANOLAMINE (TFAE) (in urine)

FIGURE 2.-Proposed pathway for the oxidative metabolism of halothane.

commercially available. A quantity of TFAE was synthetized from ethyl-trifluoroacetate and ethanolamine and its authenticity validated using nuclear magnetic resonance and mass spectroscopy. A direct measurement technique for TFAE was devised using HPLC with ultraviolet detection. In some patients an interfering substance was identified in preoperative urine samples which had the same UV absorption spectra as TFAE. Despite trying different compositions of mobile phase solutions with varying pH and ionic strength, the UV absorption peak of the trace level of interfering substance could not be separated from that of TFAE. In our study of six children aged from 14-119 months, urinary TFAE constituted O.O? ± 0.07% or less of the absorbed halothane. U nne samples from the six patients included in the present study were analysed for TFA and the results published. 5 In the children studied the urinary TF A: TFAE

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ratio was greater than 100: 1, indicating that TF AE is not a major urinary metabolite. It is postulated that patients who develop hepatic cell necrosis following halothane either metabolise halothane differently from normal patients producing a toxic metabolite or produce an antibody or antibodies to a normal halothane metabolite - cell membrane combination. 6 If covalent binding of trifluoracetyl chloride to the phosphatidyl ethanolamine component of the hepatic cell membrane played a role in halothane hepatitis, then in these patients TFAE excretion should be increased. A further study is planned to investigate urinary excretion of TF AE in patients who develop unexplained hepatitis following halothane. If it was shown that TFAE excretion is not increased in these patients, it would suggest that covalent binding of trifluoroacetyl chloride reactive intermediates to cell membrane phosphatidylethanolamine does not have an etiological role in the production of halothane induced liver injury, ACKNOWLEDGEMENTS This study was supported by research grants from the Children's Medical Research Foundation and the Children's Hospital Fund. Dr. A. Cheong from the Pharmacy Department, University of Sydney, is thanked for his discussion. REFERENCES 1. Cohen E, Metabolism of the volatile anesthetics. Anesthesiology 1971; 35: 193-202, 2. Van Dyke RA, Gandolfi AJ, Studies on irreversible binding of radioactivity from ('4C) halothane to rat hepatic microsomallipids and protein, Drug Metab Dispos 1975; 3:51-57. 3. Cohen EN, Metabolism of halothane-2- '4 C in the mouse. Anesthesiology 1969; 31 :560-565. 4. Cohen EN, Trudell JR, Edmunds RN, Watson E, Urinary metabolites of halothane in man. Anesthesiology 1975; 43:392-401. 5, Wark H, Earl J, Chau D, Overton J. Halothane metabolism in children. Br J Anaesth 1990; 64:474-481. 6. Neuberger J, Williams R. Halothane anaesthesia and liver damage. BMJ 1984; 289: 1136-1139.

N-Trifluoroacetyl-ethanolamine: a proposed urinary metabolite of halothane: validation and measurement in children.

It has been postulated that trifluoroacetyl chloride, a halothane metabolite, can bind covalently with the phosphatidylethanolamine component of the h...
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