Br. J. Anaesth. (1975), 47, 739
CORRESPONDENCE INTERPRETATION OF DRUG BINDING CHANGES IN RELATION TO PROTEIN CONCENTRATION
JOHN M.
THOMPSON
Birmingham REFERENCES
Crawford, J. S. (1969). Drug binding by serum albumin: implications of some recent observations. Br. J. Anaesth., 41, 543. Davies P., and Davies, P. (1971). Further studies of the binding of bromosulphthalein by serum albumin. Br. J. Anaesth., 43, 344. Hooi, H. W. Y. (1968a). Binding of bromosulphthalein by serum albumin from pregnant women, neonates and subjects on oral contraceptives Br. J. Anaesth., 40, 723. (1968b). Binding of salicylic acid and sulphanilamide in serum from pregnant patients, cord blood, and subjects taking oral contraceptives. Br. J. Anaesth., 40, 825. • Jones, R. L., Thompson, J. M., and Wells, W. D. E. (1972). The binding of bromosulphthalein sodium by human serum albumin using a continuous diafiltration technique. Br. J. Pharmacol, 44, 80. Gretch, M., and Jusko, W. J. (1974). Interpretation of drug binding changes in relation to protein concentration. Br. J. Anaesth., 46, 545. Klotz, I. M. (1974). Protein interactions with small molecules. Accounts Chent. Res., 1, 162. Nichol, L. W., Jackson, W. J. H., and Winzor, D. J. (1967). A theoretical study of the binding of small molecules to a polymerizing protein system: a model for allosteric effects. Biochemistry, 6, 2449. Winzor, D. J. (1972). Migration of interacting systems. Oxford: Clarendon Press. Thompson, J. M. (1973). Further studies of binding of bromosulphthalein sodium by human serum albumin: effects of protein concentration and buffer composition. Br. J. Pharmacol, 47, 133. (1974). Binding of pancuronium by human serum proteins in Krebs-Henseleit buffer. (In preparation.) HALOTHANE AND NITROPRUSSIDE HYPOTENSION
Sir,—Dr Prys-Roberts and his colleagues (1974) may be correct when they suggest that halothane is not a drug with vasodilator properties. It might be described more accurately as a sympathetic blocking agent with actions mainly at ganglionic level (Raventos, 1961, 1962). The response of the peripheral blood vessels to sympathetic blockade at ganglionic or preganglionic level is dilatation of the a- adrenoceptive vessels and constriction of the /3 adrenoceptive vessels. The dilatation of the a-vessels and the constriction of the /3-vessels have been confirmed by plethysmography in patients anaesthetized with halothane, and it has been shown that the effects persist;throughout the period of surgery and anaesthesia (Johnstone, 1972, 1974).
Downloaded from http://bja.oxfordjournals.org/ at Carleton University on May 10, 2015
Sir,—I read with great interest the recent letter by Gretch and Jusko (1974) on the papers of Crawford and Hooi (1968a,b) and Crawford (1969) on the binding of bromosulphthalein, sulphanilamide and salicylic acid by serum albumin. Although the authors invoke simple mass action law behaviour as an explanation of the decrease in binding of the drugs as the protein concentration increases, they have overlooked several points of experimental detail and subsequent publications by Crawford, Davies and Davies (1971), Crawford and others (1972) and Thompson (1973) on bromosulphthalein binding by serum albumin. In the work of Crawford and Hooi (1968a,b) and Crawford, Davies and Davies (1971), the binding was measured at only one constant concentration of unbound drug in the dialysate or in the gel chromatographic eluant, thus the interpretation of the data on binding was open to a number of very different interpretations, including that of Gretch and Jusko (1974) based on a simple mass action equation. However, the method of analysis used by Gretch and Jusko (1974) and also the Scatchard (1949) method (which they proclaim) are generally used for binding data from experiments in which the protein concentration is very small. In such cases, the protein-protein interactions are also likely to be small and the methods discussed above may be valid. However, even under these circumstances Klotz (1974) has shown the great disadvantages of these methods of data interpretation. The experiments described by Crawford and coworkers (1968a,b; 1971) were performed at albumin concentrations at which protein-protein interactions are very probable, although they do not discuss the possibilities explicitly. Their work on bromosulphthalein binding by serum albumin was continued by Crawford and colleagues (1972) and Thompson (1973) in a manner designed to remove some of the possible ambiguities of interpretation. In the latter two studies complete binding isotherms were measured at different albumin concentrations. From that work it has now become obvious that the interpretation suggested by Gretch and Jusko (1974) is not correct but that protein-protein interactions are a dominant factor. If Gretch and Jusko (1974) were correct, then a series of plots of moles of drug bound per mole of protein versus free drug concentration for several different protein concentrations would overlie each other. The work of Crawford and colleagues (1972) and Thompson (1973) shows quite clearly that the complete isotherm moves down as the protein concentration increases. Nichol, Jackson and Winzor (1967) and Nichol and Winzor (1972) have suggested an elegant way of analysing binding data in order to distinguish between the presence or absence of competitive or non-competitive isomerization or polymerization of the binding protein along with the binding of a ligand (such as a drug). These processes cannot be distinguished unambiguously without a detailed examination of the binding process as a function of both protein concentration and free drug concentration. The processes discussed by Nichol, Jackson and Winzor (1967) and Nichol and Winzor (1972) are more general in nature than the simple allosteric processes cited in Settle, Hegeman and Featherstone (1971) referred to by Gretch and Jusko (1974). The variation of protein binding
capacity with protein concentration described by Crawford and co-workers (1968a,b, 1969, 1971, 1972) and Thompson (1973) appears to occur quite commonly. Steroid binding is influenced by protein concentration variation (see references cited in Thompson, 1973). Pancuronium binding by gamma globulin and possibly by serum albumin appears also to be influenced in this way (Thompson, 1974). We are at present investigating the behaviour of tubocurarine and alcuronium binding by serum proteins in this respect.
740
BRITISH JOURNAL OF ANAESTHESIA
MICHAEL JOHNSTONE
Manchester
diagnostic procedures. Lately, we have used it in a similar manner for laparoscopic tubal ligations. J. ANTONIO ALDRETE
Louisville, Kentucky REFERENCES
Aldrete, J. A., Clapp, H. W., Fishman, J., and O'Higgins, J. W. (1971). Pentazepam, a supplementary agent. Anesth. Analg. (Cleve.% 50, 498. Schoenfeld, A., Goldman, J. A., and Levy, E. (1974). Pentazocine and diazepam analgesia for minor gynaecological operations. Br. J. Anaesth., 46, 385. NEUROLEPTANAESTHESIA FOR MAJOR SURGERY
Sir,—Is it not a sad comment on our specialty that a valuable clinical study like that of Morgan, Lumley and Gillies (1974) on neuroleptanalgesia can be published without any reference of any kind to what the patients thought about it afterwards? One real disadvantage of the technique has often been described. The authors refer, for instance, to the paper by Edmonds-Seal and Prys-Roberts (1970), who wrote: "Although these drugs are generally potent tranquillizers, it must be emphasized that in some subjects acute administration of clinically effective doses may induce a state of mental restlessness and agitation. This may be completely masked to the observer by the overt appearance of tranquillity and dissociation". In my experience, about 10% of patients find the period after surgery most unpleasant because of this. Pharmacological detachment is quite different from philosophical detachment: it is a physical cutting-off of a patient's feelings so that they no longer find expression in his face nor outlet in his speech. His ordinary means of communication are detached from him and he is shut up in the private world of his own fear and pain. Where cardiovascular stability and postoperative tranquillity are essential this is an acceptable price to pay. How often are they essential in ordinary anaesthetic practice?
REFERENCES
Johnstone, M. (1961). Halothane: the first five years. Anesthesiology, 22, 591. (1972). The cardiovascular effects of oxytocic drugs. Br. J. Anaesth., 44, 826. (1974). Facial vasomotor behaviour. Br. J. Anaesth., 46, 765. Prys-Roberts, C , Lloyd, J. W., Fisher, A., Kerr, J. H., and Patterson, T. J. S. (1974). Deliberate profound hypotension induced with halothane: studies of haemodynamics and pulmonary gas exchange. Br. J. Anaesth., 46, 105. Ravent6s, J. (1961). The action of Fluothane on the autonomic nervous system. Helv. Chir. Acta, 28, 358. (1962). Action du Fluothane sur mechanisms regulateurs de la tension arterielle. Anesth. Analg. {Paris), 19, 27. ANALGESIA FOR MINOR GYNAECOLOGICAL OPERATIONS
Sir,—I write in relation to the paper on the combination of pentazocine and diazepam by Drs A. Schoenfeld, J. A. Goldman and E. Levy (1974). The combination of these two drugs is indeed a form of neuroleptanalgesia and I was pleased to find that the authors, using exactly the same doses as we did, found the same results (Aldrete et al., 1971). In our publication, the mixture given i.v. was found to be a satisfactory supplementary agent for a variety of minor surgical and
T. H. SPREADBURY
Warwick REFERENCES
Edmonds-Seal, J., and Prys-Roberts, C. (1970). Pharmacology of drugs used in neuroleptanalgesia. Br. J. Anaesth., 42, 207. Morgan, M., Lumley, J., and Gillies, I. D. S. (1974). Neuroleptanaesthesia for major surgery. Br. J. Anaesth, 46, 288. PREDICTION OF Pa C O 2 IN A CIRCLE SYSTEM
Sir,—I was interested to read the paper by Drs Scholfield and Williams reporting Pacoa values in patients ventilated using large minute volumes and a circle system without carbon dioxide absorption (Scholfield and Williams, 1974). I would like to suggest two additional explanations for the discrepancy between the predicted and observed Pacoa values that they found. First, it is possible that their assumption that elimination of carbon dioxide from the system is directly related to the fresh gas flow, is not correct. In the circuit shown in their figure 1, the spill valve is immediately adjacent to the reservoir bag. During the inspiratory phase of the ventilator, fresh gas will flow into the reservoir bag. It is possible that the composition of the gas spilt from the valve during the inspiratory phase will alter with the composition of gas in the bag,
Downloaded from http://bja.oxfordjournals.org/ at Carleton University on May 10, 2015
A feature of the arterial hypotension produced by halothane is that it is associated with bradycardia. This suggests that the baroreceptor system is inactivated by the drug. Atropine prevents the bradycardia and may modify the hypotension to some extent (Johnstone, 1961). The overall cardiovascular effects of halothane indicate that at least two factors are involved in the causation of hypotension: one is the blockade of the sympathetic efferents to the heart and the peripheral blood vessels; the other is increased vagal tone with its negative chronotropic and inotropic effects. It is not improbable that the "resistance" of some patients to the hypotensive effect of certain sympathetic blocking agents may be related to the constrictive effect of sympathetic blockade on the /3-adrenoceptive blood vessels. This is most obvious in young adults with good muscle perfusion. For obvious reasons, the alterations in peripheral vascular tone caused by variations in sympathetic activity may not be reflected by changes in the total peripheral resistance in normovolaemic subjects. Sodium nitroprusside is a vasodilator substance which acts directly on the smooth muscle of both the a- and the y3-adrenoceptive vessels. It is not a sympathetic blocking drug. In patients with intact sympathetic reflexes, the hypotensive action of nitroprusside, like that of oxytocin (Johnstone, 1972), is antagonized by brisk baroreceptor activity with reflex adrenergic stimulation of the heart producing tachycardia and an increased cardiac contractility and output, provided that the venous return is not impeded. Undoubtedly nitroprusside potentiates the hypotensive effect of halothane by dilating the constricted /8-adrenoceptive vessels. It remains to be seen whether or not this is a desirable procedure in anaesthetic practice, especially in view of the depression of the baroreceptor system by halothane. Unless considerable care is taken in their use, the combined effects of the two drugs may precipitate a serious failure of venous return in patients who are anaesthetized in the head-up position.
CORRESPONDENCE INTERPRETATION OF DRUG BINDING CHANGES IN RELATION TO PROTEIN CONCENTRATION
JOHN M.
THOMPSON
Birmingham REFERENCES
Crawford, J. S. (1969). Drug binding by serum albumin: implications of some recent observations. Br. J. Anaesth., 41, 543. Davies P., and Davies, P. (1971). Further studies of the binding of bromosulphthalein by serum albumin. Br. J. Anaesth., 43, 344. Hooi, H. W. Y. (1968a). Binding of bromosulphthalein by serum albumin from pregnant women, neonates and subjects on oral contraceptives Br. J. Anaesth., 40, 723. (1968b). Binding of salicylic acid and sulphanilamide in serum from pregnant patients, cord blood, and subjects taking oral contraceptives. Br. J. Anaesth., 40, 825. • Jones, R. L., Thompson, J. M., and Wells, W. D. E. (1972). The binding of bromosulphthalein sodium by human serum albumin using a continuous diafiltration technique. Br. J. Pharmacol, 44, 80. Gretch, M., and Jusko, W. J. (1974). Interpretation of drug binding changes in relation to protein concentration. Br. J. Anaesth., 46, 545. Klotz, I. M. (1974). Protein interactions with small molecules. Accounts Chent. Res., 1, 162. Nichol, L. W., Jackson, W. J. H., and Winzor, D. J. (1967). A theoretical study of the binding of small molecules to a polymerizing protein system: a model for allosteric effects. Biochemistry, 6, 2449. Winzor, D. J. (1972). Migration of interacting systems. Oxford: Clarendon Press. Thompson, J. M. (1973). Further studies of binding of bromosulphthalein sodium by human serum albumin: effects of protein concentration and buffer composition. Br. J. Pharmacol, 47, 133. (1974). Binding of pancuronium by human serum proteins in Krebs-Henseleit buffer. (In preparation.) HALOTHANE AND NITROPRUSSIDE HYPOTENSION
Sir,—Dr Prys-Roberts and his colleagues (1974) may be correct when they suggest that halothane is not a drug with vasodilator properties. It might be described more accurately as a sympathetic blocking agent with actions mainly at ganglionic level (Raventos, 1961, 1962). The response of the peripheral blood vessels to sympathetic blockade at ganglionic or preganglionic level is dilatation of the a- adrenoceptive vessels and constriction of the /3 adrenoceptive vessels. The dilatation of the a-vessels and the constriction of the /3-vessels have been confirmed by plethysmography in patients anaesthetized with halothane, and it has been shown that the effects persist;throughout the period of surgery and anaesthesia (Johnstone, 1972, 1974).
Downloaded from http://bja.oxfordjournals.org/ at Carleton University on May 10, 2015
Sir,—I read with great interest the recent letter by Gretch and Jusko (1974) on the papers of Crawford and Hooi (1968a,b) and Crawford (1969) on the binding of bromosulphthalein, sulphanilamide and salicylic acid by serum albumin. Although the authors invoke simple mass action law behaviour as an explanation of the decrease in binding of the drugs as the protein concentration increases, they have overlooked several points of experimental detail and subsequent publications by Crawford, Davies and Davies (1971), Crawford and others (1972) and Thompson (1973) on bromosulphthalein binding by serum albumin. In the work of Crawford and Hooi (1968a,b) and Crawford, Davies and Davies (1971), the binding was measured at only one constant concentration of unbound drug in the dialysate or in the gel chromatographic eluant, thus the interpretation of the data on binding was open to a number of very different interpretations, including that of Gretch and Jusko (1974) based on a simple mass action equation. However, the method of analysis used by Gretch and Jusko (1974) and also the Scatchard (1949) method (which they proclaim) are generally used for binding data from experiments in which the protein concentration is very small. In such cases, the protein-protein interactions are also likely to be small and the methods discussed above may be valid. However, even under these circumstances Klotz (1974) has shown the great disadvantages of these methods of data interpretation. The experiments described by Crawford and coworkers (1968a,b; 1971) were performed at albumin concentrations at which protein-protein interactions are very probable, although they do not discuss the possibilities explicitly. Their work on bromosulphthalein binding by serum albumin was continued by Crawford and colleagues (1972) and Thompson (1973) in a manner designed to remove some of the possible ambiguities of interpretation. In the latter two studies complete binding isotherms were measured at different albumin concentrations. From that work it has now become obvious that the interpretation suggested by Gretch and Jusko (1974) is not correct but that protein-protein interactions are a dominant factor. If Gretch and Jusko (1974) were correct, then a series of plots of moles of drug bound per mole of protein versus free drug concentration for several different protein concentrations would overlie each other. The work of Crawford and colleagues (1972) and Thompson (1973) shows quite clearly that the complete isotherm moves down as the protein concentration increases. Nichol, Jackson and Winzor (1967) and Nichol and Winzor (1972) have suggested an elegant way of analysing binding data in order to distinguish between the presence or absence of competitive or non-competitive isomerization or polymerization of the binding protein along with the binding of a ligand (such as a drug). These processes cannot be distinguished unambiguously without a detailed examination of the binding process as a function of both protein concentration and free drug concentration. The processes discussed by Nichol, Jackson and Winzor (1967) and Nichol and Winzor (1972) are more general in nature than the simple allosteric processes cited in Settle, Hegeman and Featherstone (1971) referred to by Gretch and Jusko (1974). The variation of protein binding
capacity with protein concentration described by Crawford and co-workers (1968a,b, 1969, 1971, 1972) and Thompson (1973) appears to occur quite commonly. Steroid binding is influenced by protein concentration variation (see references cited in Thompson, 1973). Pancuronium binding by gamma globulin and possibly by serum albumin appears also to be influenced in this way (Thompson, 1974). We are at present investigating the behaviour of tubocurarine and alcuronium binding by serum proteins in this respect.
740
BRITISH JOURNAL OF ANAESTHESIA
MICHAEL JOHNSTONE
Manchester
diagnostic procedures. Lately, we have used it in a similar manner for laparoscopic tubal ligations. J. ANTONIO ALDRETE
Louisville, Kentucky REFERENCES
Aldrete, J. A., Clapp, H. W., Fishman, J., and O'Higgins, J. W. (1971). Pentazepam, a supplementary agent. Anesth. Analg. (Cleve.% 50, 498. Schoenfeld, A., Goldman, J. A., and Levy, E. (1974). Pentazocine and diazepam analgesia for minor gynaecological operations. Br. J. Anaesth., 46, 385. NEUROLEPTANAESTHESIA FOR MAJOR SURGERY
Sir,—Is it not a sad comment on our specialty that a valuable clinical study like that of Morgan, Lumley and Gillies (1974) on neuroleptanalgesia can be published without any reference of any kind to what the patients thought about it afterwards? One real disadvantage of the technique has often been described. The authors refer, for instance, to the paper by Edmonds-Seal and Prys-Roberts (1970), who wrote: "Although these drugs are generally potent tranquillizers, it must be emphasized that in some subjects acute administration of clinically effective doses may induce a state of mental restlessness and agitation. This may be completely masked to the observer by the overt appearance of tranquillity and dissociation". In my experience, about 10% of patients find the period after surgery most unpleasant because of this. Pharmacological detachment is quite different from philosophical detachment: it is a physical cutting-off of a patient's feelings so that they no longer find expression in his face nor outlet in his speech. His ordinary means of communication are detached from him and he is shut up in the private world of his own fear and pain. Where cardiovascular stability and postoperative tranquillity are essential this is an acceptable price to pay. How often are they essential in ordinary anaesthetic practice?
REFERENCES
Johnstone, M. (1961). Halothane: the first five years. Anesthesiology, 22, 591. (1972). The cardiovascular effects of oxytocic drugs. Br. J. Anaesth., 44, 826. (1974). Facial vasomotor behaviour. Br. J. Anaesth., 46, 765. Prys-Roberts, C , Lloyd, J. W., Fisher, A., Kerr, J. H., and Patterson, T. J. S. (1974). Deliberate profound hypotension induced with halothane: studies of haemodynamics and pulmonary gas exchange. Br. J. Anaesth., 46, 105. Ravent6s, J. (1961). The action of Fluothane on the autonomic nervous system. Helv. Chir. Acta, 28, 358. (1962). Action du Fluothane sur mechanisms regulateurs de la tension arterielle. Anesth. Analg. {Paris), 19, 27. ANALGESIA FOR MINOR GYNAECOLOGICAL OPERATIONS
Sir,—I write in relation to the paper on the combination of pentazocine and diazepam by Drs A. Schoenfeld, J. A. Goldman and E. Levy (1974). The combination of these two drugs is indeed a form of neuroleptanalgesia and I was pleased to find that the authors, using exactly the same doses as we did, found the same results (Aldrete et al., 1971). In our publication, the mixture given i.v. was found to be a satisfactory supplementary agent for a variety of minor surgical and
T. H. SPREADBURY
Warwick REFERENCES
Edmonds-Seal, J., and Prys-Roberts, C. (1970). Pharmacology of drugs used in neuroleptanalgesia. Br. J. Anaesth., 42, 207. Morgan, M., Lumley, J., and Gillies, I. D. S. (1974). Neuroleptanaesthesia for major surgery. Br. J. Anaesth, 46, 288. PREDICTION OF Pa C O 2 IN A CIRCLE SYSTEM
Sir,—I was interested to read the paper by Drs Scholfield and Williams reporting Pacoa values in patients ventilated using large minute volumes and a circle system without carbon dioxide absorption (Scholfield and Williams, 1974). I would like to suggest two additional explanations for the discrepancy between the predicted and observed Pacoa values that they found. First, it is possible that their assumption that elimination of carbon dioxide from the system is directly related to the fresh gas flow, is not correct. In the circuit shown in their figure 1, the spill valve is immediately adjacent to the reservoir bag. During the inspiratory phase of the ventilator, fresh gas will flow into the reservoir bag. It is possible that the composition of the gas spilt from the valve during the inspiratory phase will alter with the composition of gas in the bag,
Downloaded from http://bja.oxfordjournals.org/ at Carleton University on May 10, 2015
A feature of the arterial hypotension produced by halothane is that it is associated with bradycardia. This suggests that the baroreceptor system is inactivated by the drug. Atropine prevents the bradycardia and may modify the hypotension to some extent (Johnstone, 1961). The overall cardiovascular effects of halothane indicate that at least two factors are involved in the causation of hypotension: one is the blockade of the sympathetic efferents to the heart and the peripheral blood vessels; the other is increased vagal tone with its negative chronotropic and inotropic effects. It is not improbable that the "resistance" of some patients to the hypotensive effect of certain sympathetic blocking agents may be related to the constrictive effect of sympathetic blockade on the /3-adrenoceptive blood vessels. This is most obvious in young adults with good muscle perfusion. For obvious reasons, the alterations in peripheral vascular tone caused by variations in sympathetic activity may not be reflected by changes in the total peripheral resistance in normovolaemic subjects. Sodium nitroprusside is a vasodilator substance which acts directly on the smooth muscle of both the a- and the y3-adrenoceptive vessels. It is not a sympathetic blocking drug. In patients with intact sympathetic reflexes, the hypotensive action of nitroprusside, like that of oxytocin (Johnstone, 1972), is antagonized by brisk baroreceptor activity with reflex adrenergic stimulation of the heart producing tachycardia and an increased cardiac contractility and output, provided that the venous return is not impeded. Undoubtedly nitroprusside potentiates the hypotensive effect of halothane by dilating the constricted /8-adrenoceptive vessels. It remains to be seen whether or not this is a desirable procedure in anaesthetic practice, especially in view of the depression of the baroreceptor system by halothane. Unless considerable care is taken in their use, the combined effects of the two drugs may precipitate a serious failure of venous return in patients who are anaesthetized in the head-up position.
