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SCREENING OF CERTAIN ANAESTHETIC AGENTS FOR THEIR ABILITY TO ELICIT ACUTE PORPHYRIC PHASES IN SUSCEPTIBLE PATIENTS
Br.J. Anaesth. (1980), 52, 759
SCREENING OF CERTAIN ANAESTHETIC AGENTS FOR THEIR ABILITY TO ELICIT ACUTE PORPHYRIC PHASES IN SUSCEPTIBLE PATIENTS G. H. BLEKKENHORST, G. G. HARRISON, E. S. COOK AND L. EALES SUMMARY
The porphyrias are a group of disorders of haem metabolism characterized by specific patterns of haem precursor overproduction, accumulation and excretion, each pattern defining a particular form of porphyria. Variegate, or South African genetic porphyria, is one of the forms particularly common in southern Africa. It has been estimated that there are more than 8000 susceptible individuals (Dean, 1971) with a frequency of 1 in 250 in one region. The acute phase of this condition and of two other forms of the hereditary "hepatic" porphyrias, acute intermittent porphyria and hereditary coproporphyria, may be life-threatening. It is usually caused by exposure of the susceptible individual to drugs, particularly barbiturates (Eales, 1971). The frequency of acute porphyric attacks in South Africa has been reduced dramatically by effective family surveys, when asymptomatic but biochemically positive members of families have been warned to avoid precipitating agents (Eales, 1971). Identification of drugs which are porphyrogenic is also necessary to prevent lifethreatening acute porphyric crises. Previously this has been largely a process of trial and error or anecdote, but it is now possible to identify the porphyrogenicity of drugs in the laboratory. Rats given a relatively low dose of 3,5-diethoxycarbonyl- 1,4-dihydrocolhdine (DDC) exhibit a condition which resembles latent human variegate porG. H. BLEKKENHORST, PH.D.; E. S. COOK, B.SC.(HONS.);
L. EALES, M.D., F.RX.P.; UCT/MRC Porphyria Research Unit. G. G. HARRISON, M.D., F.F.A.R.C.S., Department of
Anaesthetics. University of Cape Town, Medical School, Observatory 7925, South Africa. 0007-0912/80/080759-O4 $01.00
phyria. The animals become sensitive to drugs which can precipitate the metabolic disorder such that, biochemically, the reaction is typical of the human attack (De Matteis, 1973). We have used the DDCprimed rat to test the porphyrogenicity of phenobarbitone and the i.v. anaesthetic agents Althesin, etomidate, flunitrazepam, propanidid and minaxolone by determination of the activity of hepatic 8-aminolaevulinic acid synthetase (E.C. 2.3.1.37) (ALA-S). This is the initial and rate-limiting enzyme of haem biosynthesis (Granick and Urata, 1963) and is increased in the three hereditary "hepatic" porphyrias (Elder, Gray and Nicholson, 1972; Meyer and Schmid, 1973). MATERIALS AND METHODS
Male Wistar rats (weight 180-220 g) were starved for 24 h but water was freely allowed. In each experiment the five animals in the control group each received arachis oil 1 ml; the second group offivereceived the drug under study; a third group offivereceived DDC 100 mg kg" 1 suspended in arachis oil and the fourth group received DDC lOOmgkg" 1 simultaneously with the drug under study. The drugs were administered i.p. in the doses: phenobarbitone (Gardenal, May and Baker, Port Elizabeth, South Africa) 5 0 m g k g - 1 ; propanidid (Fabantol, Bayer, Leverkusen, Germany) lOOmgkg" 1 ; etomidate (Ethnor, Halfway House, South Africa) Smgkg" 1 ; flunitrazepam (Rohypnol, Roche, Isando, South Africa) 0.5 mg kg" 1 ; Althesin (Glaxo-Allenbury, Wadeville, South Africa) ^ m g k g - 1 and minaxolone (Glaxo Group Research, Greenford, Middlesex, England) © Macmillan Publishers Ltd 1980
Downloaded from http://bja.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
The activity of 8-aminolaevulinic acid synthetase (E.C. 2.3.1.37) (ALA-S) was measured in rat liver after the simultaneous administration of various anaesthetic agents and 3,5-diethoxycarbonyl1,4-dihydrocollidine (DDC) in vivo. Flunitrazepam, Althesin and phenobarbitone caused a significant increase in the activity of the enzyme which was not observed with propanidid, etomidate and minaxolone. It is suggested that DDC-treated rat, which resembles latent human variegate porphyria, may be a more valid method of testing drugs for their ability to elicit acute porphyric phases in susceptible individuals. The anaesthetic agents which induced the activity of hepatic ALA-S in this model are not recommended in patients with genetic hepatic porphyria.
760
BRITISH JOURNAL OF ANAESTHESIA
2001
150-
3 o 100-
T
I T
I
50-
RESULTS
A preliminary experiment established that a dose of DDC 100 mg kg" 1 gave a maximal increase in ALA-S activity when administered with phenobarbitone, a known porphyrogen (Eales, 1971). When administered alone, this dose of DDC gave only a modest increase in ALA-S activity. The activity of hepatic ALA-S in rats treated with the anaesthetic agents together with DDC is shown in figure 1. Phenobarbitone and DDC caused an increase in the activity of the enzyme but no change was observed in the activity when propanidid and DDC were administered compared with the activity of ALA-S when DDC alone was given. No significant difference in hepatic ALA-S activity was observed when etomidate and minaxolone were given together with DDC, but flunitrazepam and Althesin given with DDC caused an increase in the activity of hepatic ALA-S (P< 0.05). The activities of the enzyme in rats treated with only the drug under test were in all cases found to be not significantly different from those in rats receiving oil only. DISCUSSION
The results of this study demonstrate that phenobarbitone and the anaesthetic agents flunitrazepam and Althesin, when given simultaneously with DDC to rats cause a significant increase in the activity of hepatic ALA-S compared with the activity of the enzyme when DDC is given alone. On this basis, these drugs must be regarded as porphyrogenic and contraindicated in patients with genetic porphyria.
Phenobarbitone Etomidate Althesin Propanidid Flunitrazepam Minaxolone
FIG. 1. Hepatic 8-aminokevuIinic add synthetase (ALA-S) activity in male rats treated simultaneously with 3,5diethoxycarbonyl-l,4-dihydrocollidine (DDC) and various drugs. Each result represents the mean for five animals; bars indicate SD. One hundred per cent of DDC control= ALA 479 ± SD 58 (n= 10) nmol formed per hour per gram of liver. Hatched bars indicate a significant difference (P < 0.05) when compared with DDC controls.
A similar investigation (Parikh and Moore, 1978) showed that repeated doses of various anaesthetic agents, including Althesin and etomidate, increased hepatic ALA-S activity. The experimental approach adopted by these workers may not be correct to screen drugs for their porphyrogenicity, since it appears that increased activity of hepatic ALA-S results from the coexistence of two requirements, each of which alone may have little or no effect on the enzyme. These are: first, an effect on haem synthesis and, second, exposure to lipid-soluble inducers of haemoprotein synthesis (Maxwell and Meyer, 1978). Thus when rats are primed with small doses of DDC which cause a partial block at the ferrochelatase level (De Matteis, 1973), and a lipidsoluble inducer of haemoprotein synthesis such as phenobarbitone is administered, a potentiation of ALA-S activity is observed. However, single doses of
Downloaded from http://bja.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
. The volume of the drug injected was usually 1 ml and drugs were diluted if necessary in sodium chloride solution (0.15 mmol litre; saline) After 17 h, the animals were sacrificed by decapitation, exsanguinated and the livers removed, blotted free of blood and washed with ice-cold saline. For measurement of ALA-S, the liver was weighed, chopped into small pieces, washed twice with icecold saline and homogenized in Tris : HC1 buffer 0.01 mol litre" 1 , pH 7.4 containing sodium chloride 0.15 mmol litre" 1 and EDTA 0.5 mmol litre" 1 using a motor-driven Teflon pestle in a glass mortar to give a 10% (w/v) homogenate. The assays for ALA-S were performed as described previously (Pimstone, Blekkenhorst and Eales, 1973) using a modification of the method of Strand and others (1972b). The significance of difference between mean values was assessed by Student's t test.
EXPERIMENTAL STUDY OF PORPHYROGENICITY
761
Chem., 238, 821.
Downloaded from http://bja.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
In this investigation of the porphyrogenidty of the inducing drugs administered to animals with an intact haem biosynthetic pathway have little efiFect on newer i.v. anaesthetic agents, we conclude that hepatic ALA-S activity (De Mattds, 1978; Maxwell etomidate and minaxolone are probably nonand Meyer, 1978). porphyrogenic in susceptible patients, whereas Similarly, inducing drugs can superimpose their Althesin and flunitrazepam are likely to be dangerous. effects on ALA-S in the hereditary hepatic porphyrias. Nevertheless, the ultimate test of porphyrogenidty of The basis genetic defects in the haem biosynthetic these anaesthetics can only be the response of pathway have been shown to be decreased uro- individuals who suffer genetic porphyria to adminisporphyrinogen synthetase in acute intermittent tration of these agents. porphyria (Strand et al., 1972a), decreased coproporphyrinogen oxidase in hereditary coproporphyria ACKNOWLEDGEMENT (Elder et al., 1977) and a decreased ferrochelatase We thank the South African Medical Research Council for activity (Becker et al., 1977) and decreased protofinancial support. porphyrinogen oxidase, or both (Brenner and Bloomer, 1979)t, in variegate porphyria. When these drugs are REFERENCES administered to patients with the metabolic defect, the acute porphyric phase becomes manifest, with an Anderson, K. E. (1978). Effects of antihypertensive drugs on hepatic heme biosynthesis, and evaluation of ferroinappropriate increase in hepatic ALA-S activity chelatase inhibitors to simplify testing of drugs for heme (Meyer and Schmid, 1973). pathway induction. Biockim. Biophys. Acta, 543, 313. The determination of hepatic ALA-S in the DDC- Becker, D. M., Viljoen, J. D., Katz, J. and Kramer, S. (1977). Reduced ferrochelatase activity: a defect common primed rat, which biochemically resembles latent to porphyria variegata and protoporphyria. Br. J. variegate porphyria, appears to be a reliable technique Haematol., 36, 171. in screening drugs for their potential porphyrogenidty. Brenner, D. A., and Bloomer, J. R. (1979). The enzymatic Phenobarbitone is the classic porphyrogenic agent, defect in variegate porphyria. Clin. Res., 27, 274A. while propanidid is known from clinical use to be Brodie, B. B., and Reid, W. D. (1971). The value of determining the plasma concentration of drugs in animals and free of this disadvantage. The inclusion of these man; in Fundamentals of Drug Metabolism and Drug drugs among those tested serves to an extent as a Disposition (eds B. N. La Du, H. G. Mandel and E. L. control of the method. This method appears more Way), p. 328. Baltimore: Williams and Wilkins. sensitive than determination of hepatic haem pre- Dean, G. (1971). The Porphyrias. A Story of Inheritance cursors after administration of DDC and drugs and Environment, 2nd edn. London: Pitman Medical. (Eales and Blekkenhorst, 1978), when large variations De Matteis, F. (1973). Drug interactions in experimental hepatic porphyria. Enzyme, 16, 266. in the individual responses of rats to the drugs were (1978). Hepatic porphyrias caused by 2-allyl-2observed. It should be pointed out, however, that isopropyl-acetamide, 3,5-diethoxycarbonyl-1,4-dihydroinduction of ALA-S by drugs is a dose-related collidine, griseofulvin and related compounds; in phenomenon, and a large spedes variation in the Handbook of Experimental Pharmacology 44, Heme and Hemoproteins (eds F. de Matteis and W. N. Aldridge). therapeutic or toxic effects of chemical substances New York: Springer Verlag. exists. Furthermore, there is an interspedes variation in drug metabolism and pharmacological response Eales, L. (1971). The acute porphyria attack. Ill: Acute porphyria: the precipitating and aggravating factors. espedally to liposoluble drugs (Brodie and Reid, S. Afr.J. Lab. Clin. Med., 17,120. 1971), all of which must be considered when extraBlekkenhorst, G. H. (1978). The use of the rat in the experimental investigation of the porphyrias. J. S. Afr. polating these findings to man. In the present Vet. Assoc., 49, 249. experiments, large doses were used, but less than the Elder, G., Evans, J. O., Thomas, N., Cox, R., Brodie, M. J., known lethal doses of the anaesthetic agents. The Goldberg, A., and Nicholson, D. C. (1977). The primary in vivo screening technique presented here is more defect in hereditary coproporphyria. Lancet, 2, 1217. valid than the exquisitely sensitive chick embryo liver Gray, C. H., and Nicholson, D. C. (1972). The cell culture (Granick, 1966) and chick embryo in porphyrias: a review. J. Clin. Pathol., 25, 1013. vivo (Anderson, 1978) screening systems, in which Granick, S. (1966). The induction in vitro of the synthesis of 8-aminolaevulinic acid synthetase in chemical pormetabolites which may augment synthesis of ALA-S phyria. A response to certain drugs, sex hormones, and cannot be readily excreted. It is conceivable that foreign chemicals. J. Biol. Chem., 241,1359. some drugs dassed as porphyrogenic using these Urata, G. (1963). Increase in the activity of S-aminomethods might be safe to use in the hereditary hepatic laevulinic acid synthase in liver mitochondria induced by • feeding of 3,5-dicarbethoxy-l,4-dihydrocollidine. J. Biol. porphyrias.
BRITISH JOURNAL OF ANAESTHESIA
762
DEPISTAGE DE CERTAINS AGENTS ANESTHESIANTS SUSCEPTIBLES DE PROVOQUER DES PHASES PORPHYRIQUES AIGUES SUR DES MALADES SUJETS A CE GENRE DE REACTION
PRUFUNG GEWISSER NARKOSEMITTEL AUF IHRE EIGENSCHAFT, AKUTE PORPHYRIEPHASEN IN ANFALLIGEN PATIENTEN HERVORZURUFEN ZUSAMMBNFASSUNG
Die Aktivitit von 5-aminolavulinische S3ure-Synthetase (E.C. 2.3.1.37) (ALA-S) wurde in der Rattenleber gemessen nach gleichzeitiger Verabreichung verschiedener Narkosemittel und von 3,5-Diethoxycarbonyl-l,4-dihydrocollidin (DDC) in vivo. Flunitrazepam, Althesin und Phenobarbiton bewirkten einen wesentiichen Aktivitfitsanstieg des Enzyms, der bei Propanidid, Etomidat und Minazolon nicht beobachtet wurde. Man nimmr an, dass cine mit DDC behandelte Ratte, die eine menschenahnliche Porphyrie aufweist, eine gultigere Methode zur Uberprufung von Drogen auf ihre Eigenschaft hin darstellt, akute Porphyriephasen bei anfUligen Individuen hervorzurufen. Die Narkosemittel, die in diesem Modell die Aktivitfit von hepatischer ALA-S hervorriefen, werden fur Patienten mit genetischer hepatischer Porphyrie nicht empfohlen.
SELECCION DE CIERTOS AGENTES ANESTESICOS CON ARREGLO A SU HABILIDAD PARA ELUCIDAR LAS FASES PORFIRICAS EN PACEENTES SUSCEPTIBLES
RESUME
SUMARIO
On a mesure l'activite de l'acide 8-aminolevulinique synthetase (E.C. 2.3.1.37) (ALA-S) dans le foie d'uu rat apres l'administration simultanee de divers agents anesthesiants et de 3,5-diethoxycarbonyle-l,4-dihydrocollidine (DDC) in vivo. Le flunitrazepam, l'Althesine et le phenobarbitone ont provoque une augmentation significative de l'activite de l'enzyme, ce que Ton n'a pas observe avec le propanidide, l'6tomidate et le minaxolone. Ceci laisse penser que le rat traite au DDC, lequel ressemble a la porphyrie humaine diversicolore latente, peut constiruer une methode plus valable d'essayer les medicaments susceptibles de provoquer des phases porphyriques aigues sur les individus sujets a ce type de reaction. Les agents anesthesiants qui ont provoque l'activite de l'ALA-S hepatique dans ce modele ne sont pas recommandes pour les malades souffrant de porphyrie hepatique genetique.
Se midi6 la actividad de la sintetasa de acido S-aminolevulinico (B.C. 2.3.1.37) (ALA-S) en el hlgado de rata, despues de la administraci6n simultinea de varios agentes anestesicos y de 3,5-dietoxicarbonil-l,4-dihidrocollidina (DDC) in vivo. El flunirrazepam, la Altesina y la fenobarbitona ocasionaron un incremento significativo en la actividad de la enzima, que no se observ6 con la propanidida, etomidate ni con la minaxolona. Parece ser que el tratamiento de la rata con DDC, que se asemeja a la variedad de porfiria latente en el ser humano, es un metodo de mayor validez para la verificaci6n de dxogas respecto a su habilidad para elucidar las fases porfiricas agudas en individuos susceptibles. Los agentes anestesicos que indujeron la actividad del ALA-S hepatico en este modelo, no se recomiendan para pacientes con porfiria hepatica de tipo genetico.
Downloaded from http://bja.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
Maxwell, J. D., and Meyer, U. A. (1978). Pharmacogenetics in the field of heme metabolism. Drug sensitivity in hereditary hepatic porphyria: in Handbook of Experimental Pharmacology 44. Heme and Hemoproteins (eds F. de Matteis and W. N. Aldridge). New York: Springer Verlag. Meyer, U. A., and Schmid, R. (1973). Hereditary hepatic porphyrias. Fed. Proc, 32, 1649. Parikh, R. K., and Moore, M. R. (1978). Effect of certain anaesthetic agents on the activity of rat hepatic 8aminolaevulinate synthase. Br. J. Anaesth., 50, 1099. Pimstone, N. R., Blekkenhorst, G. H., and Eales, L. (1973). Enzymatic defects in hepatic porphyria. Preliminary observations in patients with porphyria cutanea tarda and variegate porphyria. Enzyme, 16, 354. Strand, L. J., Meyer, U. A., Felsher, B. F., Redeker, A. G., and Marver, H. S. (1972a). Decreased red cell uroporphyrinogen I synthetase activity in intermittent acute porphyria. J. Clin. Invest., 51, 2530. Strand, L. J., Swanson, A. L., Manning, J., Branch, S., and Marver, H. S. (1972b). Radiochemical microassay of 8-aminolevulinic acid synthetase in hepatic and erythroid tissues. Analyt. Biochem., 47, 457.
SCREENING OF CERTAIN ANAESTHETIC AGENTS FOR THEIR ABILITY TO ELICIT ACUTE PORPHYRIC PHASES IN SUSCEPTIBLE PATIENTS G. H. BLEKKENHORST, G. G. HARRISON, E. S. COOK AND L. EALES SUMMARY
The porphyrias are a group of disorders of haem metabolism characterized by specific patterns of haem precursor overproduction, accumulation and excretion, each pattern defining a particular form of porphyria. Variegate, or South African genetic porphyria, is one of the forms particularly common in southern Africa. It has been estimated that there are more than 8000 susceptible individuals (Dean, 1971) with a frequency of 1 in 250 in one region. The acute phase of this condition and of two other forms of the hereditary "hepatic" porphyrias, acute intermittent porphyria and hereditary coproporphyria, may be life-threatening. It is usually caused by exposure of the susceptible individual to drugs, particularly barbiturates (Eales, 1971). The frequency of acute porphyric attacks in South Africa has been reduced dramatically by effective family surveys, when asymptomatic but biochemically positive members of families have been warned to avoid precipitating agents (Eales, 1971). Identification of drugs which are porphyrogenic is also necessary to prevent lifethreatening acute porphyric crises. Previously this has been largely a process of trial and error or anecdote, but it is now possible to identify the porphyrogenicity of drugs in the laboratory. Rats given a relatively low dose of 3,5-diethoxycarbonyl- 1,4-dihydrocolhdine (DDC) exhibit a condition which resembles latent human variegate porG. H. BLEKKENHORST, PH.D.; E. S. COOK, B.SC.(HONS.);
L. EALES, M.D., F.RX.P.; UCT/MRC Porphyria Research Unit. G. G. HARRISON, M.D., F.F.A.R.C.S., Department of
Anaesthetics. University of Cape Town, Medical School, Observatory 7925, South Africa. 0007-0912/80/080759-O4 $01.00
phyria. The animals become sensitive to drugs which can precipitate the metabolic disorder such that, biochemically, the reaction is typical of the human attack (De Matteis, 1973). We have used the DDCprimed rat to test the porphyrogenicity of phenobarbitone and the i.v. anaesthetic agents Althesin, etomidate, flunitrazepam, propanidid and minaxolone by determination of the activity of hepatic 8-aminolaevulinic acid synthetase (E.C. 2.3.1.37) (ALA-S). This is the initial and rate-limiting enzyme of haem biosynthesis (Granick and Urata, 1963) and is increased in the three hereditary "hepatic" porphyrias (Elder, Gray and Nicholson, 1972; Meyer and Schmid, 1973). MATERIALS AND METHODS
Male Wistar rats (weight 180-220 g) were starved for 24 h but water was freely allowed. In each experiment the five animals in the control group each received arachis oil 1 ml; the second group offivereceived the drug under study; a third group offivereceived DDC 100 mg kg" 1 suspended in arachis oil and the fourth group received DDC lOOmgkg" 1 simultaneously with the drug under study. The drugs were administered i.p. in the doses: phenobarbitone (Gardenal, May and Baker, Port Elizabeth, South Africa) 5 0 m g k g - 1 ; propanidid (Fabantol, Bayer, Leverkusen, Germany) lOOmgkg" 1 ; etomidate (Ethnor, Halfway House, South Africa) Smgkg" 1 ; flunitrazepam (Rohypnol, Roche, Isando, South Africa) 0.5 mg kg" 1 ; Althesin (Glaxo-Allenbury, Wadeville, South Africa) ^ m g k g - 1 and minaxolone (Glaxo Group Research, Greenford, Middlesex, England) © Macmillan Publishers Ltd 1980
Downloaded from http://bja.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
The activity of 8-aminolaevulinic acid synthetase (E.C. 2.3.1.37) (ALA-S) was measured in rat liver after the simultaneous administration of various anaesthetic agents and 3,5-diethoxycarbonyl1,4-dihydrocollidine (DDC) in vivo. Flunitrazepam, Althesin and phenobarbitone caused a significant increase in the activity of the enzyme which was not observed with propanidid, etomidate and minaxolone. It is suggested that DDC-treated rat, which resembles latent human variegate porphyria, may be a more valid method of testing drugs for their ability to elicit acute porphyric phases in susceptible individuals. The anaesthetic agents which induced the activity of hepatic ALA-S in this model are not recommended in patients with genetic hepatic porphyria.
760
BRITISH JOURNAL OF ANAESTHESIA
2001
150-
3 o 100-
T
I T
I
50-
RESULTS
A preliminary experiment established that a dose of DDC 100 mg kg" 1 gave a maximal increase in ALA-S activity when administered with phenobarbitone, a known porphyrogen (Eales, 1971). When administered alone, this dose of DDC gave only a modest increase in ALA-S activity. The activity of hepatic ALA-S in rats treated with the anaesthetic agents together with DDC is shown in figure 1. Phenobarbitone and DDC caused an increase in the activity of the enzyme but no change was observed in the activity when propanidid and DDC were administered compared with the activity of ALA-S when DDC alone was given. No significant difference in hepatic ALA-S activity was observed when etomidate and minaxolone were given together with DDC, but flunitrazepam and Althesin given with DDC caused an increase in the activity of hepatic ALA-S (P< 0.05). The activities of the enzyme in rats treated with only the drug under test were in all cases found to be not significantly different from those in rats receiving oil only. DISCUSSION
The results of this study demonstrate that phenobarbitone and the anaesthetic agents flunitrazepam and Althesin, when given simultaneously with DDC to rats cause a significant increase in the activity of hepatic ALA-S compared with the activity of the enzyme when DDC is given alone. On this basis, these drugs must be regarded as porphyrogenic and contraindicated in patients with genetic porphyria.
Phenobarbitone Etomidate Althesin Propanidid Flunitrazepam Minaxolone
FIG. 1. Hepatic 8-aminokevuIinic add synthetase (ALA-S) activity in male rats treated simultaneously with 3,5diethoxycarbonyl-l,4-dihydrocollidine (DDC) and various drugs. Each result represents the mean for five animals; bars indicate SD. One hundred per cent of DDC control= ALA 479 ± SD 58 (n= 10) nmol formed per hour per gram of liver. Hatched bars indicate a significant difference (P < 0.05) when compared with DDC controls.
A similar investigation (Parikh and Moore, 1978) showed that repeated doses of various anaesthetic agents, including Althesin and etomidate, increased hepatic ALA-S activity. The experimental approach adopted by these workers may not be correct to screen drugs for their porphyrogenicity, since it appears that increased activity of hepatic ALA-S results from the coexistence of two requirements, each of which alone may have little or no effect on the enzyme. These are: first, an effect on haem synthesis and, second, exposure to lipid-soluble inducers of haemoprotein synthesis (Maxwell and Meyer, 1978). Thus when rats are primed with small doses of DDC which cause a partial block at the ferrochelatase level (De Matteis, 1973), and a lipidsoluble inducer of haemoprotein synthesis such as phenobarbitone is administered, a potentiation of ALA-S activity is observed. However, single doses of
Downloaded from http://bja.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
. The volume of the drug injected was usually 1 ml and drugs were diluted if necessary in sodium chloride solution (0.15 mmol litre; saline) After 17 h, the animals were sacrificed by decapitation, exsanguinated and the livers removed, blotted free of blood and washed with ice-cold saline. For measurement of ALA-S, the liver was weighed, chopped into small pieces, washed twice with icecold saline and homogenized in Tris : HC1 buffer 0.01 mol litre" 1 , pH 7.4 containing sodium chloride 0.15 mmol litre" 1 and EDTA 0.5 mmol litre" 1 using a motor-driven Teflon pestle in a glass mortar to give a 10% (w/v) homogenate. The assays for ALA-S were performed as described previously (Pimstone, Blekkenhorst and Eales, 1973) using a modification of the method of Strand and others (1972b). The significance of difference between mean values was assessed by Student's t test.
EXPERIMENTAL STUDY OF PORPHYROGENICITY
761
Chem., 238, 821.
Downloaded from http://bja.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
In this investigation of the porphyrogenidty of the inducing drugs administered to animals with an intact haem biosynthetic pathway have little efiFect on newer i.v. anaesthetic agents, we conclude that hepatic ALA-S activity (De Mattds, 1978; Maxwell etomidate and minaxolone are probably nonand Meyer, 1978). porphyrogenic in susceptible patients, whereas Similarly, inducing drugs can superimpose their Althesin and flunitrazepam are likely to be dangerous. effects on ALA-S in the hereditary hepatic porphyrias. Nevertheless, the ultimate test of porphyrogenidty of The basis genetic defects in the haem biosynthetic these anaesthetics can only be the response of pathway have been shown to be decreased uro- individuals who suffer genetic porphyria to adminisporphyrinogen synthetase in acute intermittent tration of these agents. porphyria (Strand et al., 1972a), decreased coproporphyrinogen oxidase in hereditary coproporphyria ACKNOWLEDGEMENT (Elder et al., 1977) and a decreased ferrochelatase We thank the South African Medical Research Council for activity (Becker et al., 1977) and decreased protofinancial support. porphyrinogen oxidase, or both (Brenner and Bloomer, 1979)t, in variegate porphyria. When these drugs are REFERENCES administered to patients with the metabolic defect, the acute porphyric phase becomes manifest, with an Anderson, K. E. (1978). Effects of antihypertensive drugs on hepatic heme biosynthesis, and evaluation of ferroinappropriate increase in hepatic ALA-S activity chelatase inhibitors to simplify testing of drugs for heme (Meyer and Schmid, 1973). pathway induction. Biockim. Biophys. Acta, 543, 313. The determination of hepatic ALA-S in the DDC- Becker, D. M., Viljoen, J. D., Katz, J. and Kramer, S. (1977). Reduced ferrochelatase activity: a defect common primed rat, which biochemically resembles latent to porphyria variegata and protoporphyria. Br. J. variegate porphyria, appears to be a reliable technique Haematol., 36, 171. in screening drugs for their potential porphyrogenidty. Brenner, D. A., and Bloomer, J. R. (1979). The enzymatic Phenobarbitone is the classic porphyrogenic agent, defect in variegate porphyria. Clin. Res., 27, 274A. while propanidid is known from clinical use to be Brodie, B. B., and Reid, W. D. (1971). The value of determining the plasma concentration of drugs in animals and free of this disadvantage. The inclusion of these man; in Fundamentals of Drug Metabolism and Drug drugs among those tested serves to an extent as a Disposition (eds B. N. La Du, H. G. Mandel and E. L. control of the method. This method appears more Way), p. 328. Baltimore: Williams and Wilkins. sensitive than determination of hepatic haem pre- Dean, G. (1971). The Porphyrias. A Story of Inheritance cursors after administration of DDC and drugs and Environment, 2nd edn. London: Pitman Medical. (Eales and Blekkenhorst, 1978), when large variations De Matteis, F. (1973). Drug interactions in experimental hepatic porphyria. Enzyme, 16, 266. in the individual responses of rats to the drugs were (1978). Hepatic porphyrias caused by 2-allyl-2observed. It should be pointed out, however, that isopropyl-acetamide, 3,5-diethoxycarbonyl-1,4-dihydroinduction of ALA-S by drugs is a dose-related collidine, griseofulvin and related compounds; in phenomenon, and a large spedes variation in the Handbook of Experimental Pharmacology 44, Heme and Hemoproteins (eds F. de Matteis and W. N. Aldridge). therapeutic or toxic effects of chemical substances New York: Springer Verlag. exists. Furthermore, there is an interspedes variation in drug metabolism and pharmacological response Eales, L. (1971). The acute porphyria attack. Ill: Acute porphyria: the precipitating and aggravating factors. espedally to liposoluble drugs (Brodie and Reid, S. Afr.J. Lab. Clin. Med., 17,120. 1971), all of which must be considered when extraBlekkenhorst, G. H. (1978). The use of the rat in the experimental investigation of the porphyrias. J. S. Afr. polating these findings to man. In the present Vet. Assoc., 49, 249. experiments, large doses were used, but less than the Elder, G., Evans, J. O., Thomas, N., Cox, R., Brodie, M. J., known lethal doses of the anaesthetic agents. The Goldberg, A., and Nicholson, D. C. (1977). The primary in vivo screening technique presented here is more defect in hereditary coproporphyria. Lancet, 2, 1217. valid than the exquisitely sensitive chick embryo liver Gray, C. H., and Nicholson, D. C. (1972). The cell culture (Granick, 1966) and chick embryo in porphyrias: a review. J. Clin. Pathol., 25, 1013. vivo (Anderson, 1978) screening systems, in which Granick, S. (1966). The induction in vitro of the synthesis of 8-aminolaevulinic acid synthetase in chemical pormetabolites which may augment synthesis of ALA-S phyria. A response to certain drugs, sex hormones, and cannot be readily excreted. It is conceivable that foreign chemicals. J. Biol. Chem., 241,1359. some drugs dassed as porphyrogenic using these Urata, G. (1963). Increase in the activity of S-aminomethods might be safe to use in the hereditary hepatic laevulinic acid synthase in liver mitochondria induced by • feeding of 3,5-dicarbethoxy-l,4-dihydrocollidine. J. Biol. porphyrias.
BRITISH JOURNAL OF ANAESTHESIA
762
DEPISTAGE DE CERTAINS AGENTS ANESTHESIANTS SUSCEPTIBLES DE PROVOQUER DES PHASES PORPHYRIQUES AIGUES SUR DES MALADES SUJETS A CE GENRE DE REACTION
PRUFUNG GEWISSER NARKOSEMITTEL AUF IHRE EIGENSCHAFT, AKUTE PORPHYRIEPHASEN IN ANFALLIGEN PATIENTEN HERVORZURUFEN ZUSAMMBNFASSUNG
Die Aktivitit von 5-aminolavulinische S3ure-Synthetase (E.C. 2.3.1.37) (ALA-S) wurde in der Rattenleber gemessen nach gleichzeitiger Verabreichung verschiedener Narkosemittel und von 3,5-Diethoxycarbonyl-l,4-dihydrocollidin (DDC) in vivo. Flunitrazepam, Althesin und Phenobarbiton bewirkten einen wesentiichen Aktivitfitsanstieg des Enzyms, der bei Propanidid, Etomidat und Minazolon nicht beobachtet wurde. Man nimmr an, dass cine mit DDC behandelte Ratte, die eine menschenahnliche Porphyrie aufweist, eine gultigere Methode zur Uberprufung von Drogen auf ihre Eigenschaft hin darstellt, akute Porphyriephasen bei anfUligen Individuen hervorzurufen. Die Narkosemittel, die in diesem Modell die Aktivitfit von hepatischer ALA-S hervorriefen, werden fur Patienten mit genetischer hepatischer Porphyrie nicht empfohlen.
SELECCION DE CIERTOS AGENTES ANESTESICOS CON ARREGLO A SU HABILIDAD PARA ELUCIDAR LAS FASES PORFIRICAS EN PACEENTES SUSCEPTIBLES
RESUME
SUMARIO
On a mesure l'activite de l'acide 8-aminolevulinique synthetase (E.C. 2.3.1.37) (ALA-S) dans le foie d'uu rat apres l'administration simultanee de divers agents anesthesiants et de 3,5-diethoxycarbonyle-l,4-dihydrocollidine (DDC) in vivo. Le flunitrazepam, l'Althesine et le phenobarbitone ont provoque une augmentation significative de l'activite de l'enzyme, ce que Ton n'a pas observe avec le propanidide, l'6tomidate et le minaxolone. Ceci laisse penser que le rat traite au DDC, lequel ressemble a la porphyrie humaine diversicolore latente, peut constiruer une methode plus valable d'essayer les medicaments susceptibles de provoquer des phases porphyriques aigues sur les individus sujets a ce type de reaction. Les agents anesthesiants qui ont provoque l'activite de l'ALA-S hepatique dans ce modele ne sont pas recommandes pour les malades souffrant de porphyrie hepatique genetique.
Se midi6 la actividad de la sintetasa de acido S-aminolevulinico (B.C. 2.3.1.37) (ALA-S) en el hlgado de rata, despues de la administraci6n simultinea de varios agentes anestesicos y de 3,5-dietoxicarbonil-l,4-dihidrocollidina (DDC) in vivo. El flunirrazepam, la Altesina y la fenobarbitona ocasionaron un incremento significativo en la actividad de la enzima, que no se observ6 con la propanidida, etomidate ni con la minaxolona. Parece ser que el tratamiento de la rata con DDC, que se asemeja a la variedad de porfiria latente en el ser humano, es un metodo de mayor validez para la verificaci6n de dxogas respecto a su habilidad para elucidar las fases porfiricas agudas en individuos susceptibles. Los agentes anestesicos que indujeron la actividad del ALA-S hepatico en este modelo, no se recomiendan para pacientes con porfiria hepatica de tipo genetico.
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