British Journal of Anaesthesia 1992; 69: 508-510
SHORT COMMUNICATIONS
IN VITRO DISPLACEMENT OF VASOACTIVE MEDIATORS FROM PLASMA PROTEINS: A POSSIBLE MECHANISM FOR PSEUDO-ALLERGIC REACTIONS TO NEUROMUSCULAR BLOCKING DRUGS W. R. WILLIAMS AND D. J. SHALE
SUMMARY
Anaesth. 1992; 69: 508-510) KEY WORDS Complications: histamine release, prostaglandin release. Neuromuscular relaxants. Protein: binding.
Adverse reactions to neuromuscular blocking drugs are sometimes categorized as pseudo-allergic because they appear to produce their effect by mechanisms similar to those of the immediate immurfe response [1]. Histamine has been identified as an important instigator of adverse reactions to neuromuscular blocking drugs and its release has been attributed to the direct action of these drugs on histaminecontaining cells [2]. Release of vasoactive mediators from plasma protein in response to neuromuscular blocking drugs has not been considered as an alternative mechanism. This in vitro study has investigated the effects of neuromuscular blocking drugs on prostaglandin F 2o (PGF 2 J and histamine binding in serum. METHODS AND RESULTS
Binding of PGF,O and histamine was measured by equilibrium dialysis [3] in serum from healthy volunteers, who were not taking any medication. Tritiated PGF^ 20 ul (0.74 KBq) (6.55 TBq mmol"1; Amersham pic) was added to serum 1.5 ml diluted 1:1 in phosphate buffered saline (PBS).
W. R. WILLIAMS, PH.D.; D. J. SHALE, B.SC, M.D., F.R.C.P.; Uni-
versity of Wales College of Medicine, Section of Respiratory Medicine, Llandough Hospital, Penarth, South Glamorgan CF6 1XX. Accepted for Publication: June 4, 1992.
Downloaded from http://bja.oxfordjournals.org/ by guest on August 20, 2015
We have studied the re/ease of prostaglandin F2x (PGF2J and histamine from serum proteins by neuromuscular blocking drugs using equilibrium dialysis, with tracer quantities of radio-labelled mediators as probes. Small concentrations (0.050.25 mmol litre'1) of competitive neuromuscular blocking drugs displaced 16-67% of bound histamine. Greater concentrations of suxamethonium (2 mmol litre'1) were required for histamine displacement (19%). There was a significant release of PGF2* by atracurium 1 mmol litre'1 and pancuronium 0.69 mmol litre'1. These findings suggest an alternative mechanism of histamine release by neuromuscular blocking drugs which may be relevant to adverse reactions during use. (Br. J.
Serum samples in visking tubing were dialysed against an equal volume of PBS containing 0.1% sodium azide for 36 h before the unbound PGF2ri was measured in a scintillation counter. To measure histamine binding, tritiated histamine dihydrochloride 20 ul (0.74 KBq) (1.4 TBq mmol-1; New England Nuclear) was added to undiluted serum and dialysed against PBS containing 1 % phenol. Neuromuscular blocking drugs were added (in 30 ul of distilled water) to the serum samples before dialysis, although larger volumes of injectable drug preparations were also used. Injectable drug preparations consisted of alcuronium chloride (Roche), pancuronium and vecuronium bromides (Organon Teknika), atracurium besylate and tubocurarine chloride (Calmic). Atracurium besylate, suxamethonium chloride and tubocurarine chloride were also provided as pure agents by Burroughs Wellcome. Pancuronium bromide, histamine dihydrochloride and sodium salicylate were purchased from Sigma Chemical Co. Dorset, England. Under these experimental conditions, 44.0 (SD 7.3)% (n = 20) of the added PGF !a and 13.4 (4.1)% {n = 20) of histamine became associated with serum proteins. Atracurium and pancuronium, in final concentrations of 1 and 0.69 mmol litre"1, respectively, inhibited PGF 2a binding by > 20 % (table I). At this concentration, atracurium had an inhibitory potency similar to that of salicylate. The binding of PGF2o was increased significantly by alcuronium. The neuromuscular blocking drugs were more potent at displacing histamine from plasma protein than at displacing PGF, a . With the exception of suxamethonium, at 0.05 mmol litre"1 all inhibited histamine binding by > 25 %; greater drug concentrations (0.25 mmol litre"1) were more inhibitory. In a concentration of 0.05 mmol litre"1, the histamine releasing effects of both atracurium and tubocurarine were similar to that of "cold" histamine 0.01 mmol litre"1. Greater concentrations of suxamethonium (2 mmol litre"1) displaced 19% of the histamine associated with serum protein, although this effect
ADVERSE REACTIONS TO NEUROMUSCULAR BLOCKERS
509
TABLE I. Changes (%) in the binding of iritiated PGFa and histamine in the presence of neuromuscular blocking (NMB) drugs, histamine and salicylate in concentrations of 0.05-1.0 mmol litre''. Pancuronium 0.69 mmol litre'', suxamethonium 2 mrnpl litre'' and histamine 0.01 mmol litre'1 were also tested. Mean (SD) from five to 10 experiments. Significant differences from normal (Student's t test): * P < 0.05; **P < 0.01
Histamine binding (% change)
PGF M binding_(% change) 1
NMB concn (mmol 1itre"1)
NMB concn (mmol litre" ) 0.2
Alcuronium Atracurium Pancuronium (0.69 mmol litre"1) Suxamethonium (2 mmol litre"1) Tubocurarine Vecuronium Histamine (0.01 mmol litre"1) Salicylate
1.0
122.1 (7.6)** 105.9(14.1) 96.4 (6.9) 60.8 (3.0)* — 97.0 (5.3) — 79.0 (5.2)** 106.3 (9.7) 95.8 (3.6) — 92.6(13.5) 108.9(6.1) 108.7(11.4) 102.3 (8.5) 83.0 (5.7) 105.0(12.2) 105.7 (7.9)
0.05
0.25
73.2(15.0) 45.0(12.1)* 72.1 (25.2)
70.9 (20.0)* 33.0(25.6) 67.1 (28.4)**
— —
— — —
53.0(12.1)* 73.4(21.2)
40.4(17.5) 56.8(17.0)**
— —
—
— — —
11.2(6.4)*
114.5(30.2)
—
—
52.1 (16.2)*
86.0(5.6)**
58 2(10.8)*
—
COMMENT
It is difficult to accept that immediate hypersensitivity is the sole cause of adverse reactions to neuromuscular blocking drugs, because of the small concentrations of specific antibody in reactive patients, the observed cross-reaction to different drugs and the apparent development of immunity with no previous exposure [4]. While there is apparent consensus on the'important role of histamine in these adverse reactions, the mechanism of its release remains unknown. The distinction between allergic and pseudo-allergic reactions is more clear with other drugs. Aspirin sensitivity is a more typical pseudo-allergic reaction because of the absence of IgE antibodies in reactive patients. Aspirin and other non-steroidal anti-inflammatory drugs induce adverse reactions through inhibition of cyclo-oxygenase or by their protein binding characteristics, which displace PGF2[1 from plasma protein [3]. The release of this bronchoconstricting prostaglandin may be relevant to the development of aspirininduced asthma. As this displacement mechanism may be applicable to pseudo-allergic reactions in general, it was of interest to investigate the potential of neuromuscular blocking drugs for the displacement of PGF 2a and histamine from plasma protein. We demonstrated that small concentrations of neuromuscular blocking drugs can displace radiolabelled histamine from serum protein tn_vitro/T\\§_ "inhibitory effects of these drugs on histamine binding increased with concentration. Furthermore, salicylate and cold histamine showed specificity in inhibiting the binding of radio-labelled PGF t e and of histamine, respectively. Although suxamethonium displaced histamine only at greater concentrations,
— — —
97.1 (15.3) 80.6 (24.3)
—
134.3(17.7)
the displacement mechanism is still relevant to this drug, which is used in clinical practice in concentrations greater than those used for competitive neuromuscular blocking drugs. The concentration of suxamethonium in preparations for injections is 126 mmol litre"1 (50 mg ml"1). In contrast, the concentrations of the other tested drugs ranged from 2.7 mmol litre"1 (pancuronium: 2 mg litre"1) to 13.0 mmol litre"1 (tubocurarine: 10 mg ml"1). PGF 2a binds to plasma albumin, a protein with many low affinity binding sites for drugs and endogenous substances, including histamine. The large plasma concentration of albumin gives this protein a binding capacity that is not readily saturated. Almost all (approximately 99 %) PGF 2a in plasma, of the order of 100 pg ml"1, is protein-bound [3]. Knowledge about histamine binding to plasma proteins in normal subjects is limited. A peptide responsible for binding histamine dissociates from albumin during coagulation or enzymic digestion [5]. Although the basic quarternary amine structure of neuromuscular blocking drugs should promote their binding to alpha^acid glycoprotein (AAG), both alcuronium and gallamine are bound predominantly by plasma albumin [6]. However, increased resistance to the action of atracurium in patients may be associated with an increase in AAG concentration. The binding characteristics of albumin and AAG may form a natural protective mechanism which reduces the effects of released vasoactive mediators. The presence of these proteinbound mediators may not become evident unless they are displaced by increased drug concentrations associated with i.v. injection. Our results provide some support for this hypothesis and suggest that the displacement mechanism may be relevant in a proportion of adverse reactions to neuromuscular blocking-drugs — - --
ACKNOWLEDGEMENTS The authors are grateful to Dr E. B. Williams and the Wellcome Foundation for interest in and financial support of this work.
Downloaded from http://bja.oxfordjournals.org/ by guest on August 20, 2015
was not statistically significant. Salicylate • 1 mmol litre"1 increased the quantity of histamine associated with serum protein. In contrast, the same concentration of cold histamine displaced nearly 90 % of the radio-labelled histamine.
1.0
510 REFERENCES 1. Schlumberger HD. The pseudo-allergic reactions to drugs and chemicals. Annals of Allergy 1983; 51: 317-324. 2. Vervolet D, Nizankowska E, Arnaud A, Senft M, Alazia M, Charpin J. Adverse reactions to suxamethonium and other muscle relaxants under general anesthesia. Journal of Allergy and Clinical Immunology 1983; 71: 552-559. 3. Williams WK, Pawlowicz A, Davies BH. Aspirin sensitive asthma: significance of the cyclooxygenase-inhibiting and protein-binding properties of analgesic drugs. International Archives of Allergy and Applied Immunology 1991; 95: 303-308.
BRITISH JOURNAL OF ANAESTHESIA 4. Watkins J. Anaphylactoid response to neuromuscular blockers: atracurium in perspective. In: Jones RM, Payne JP, eds. Recent Developments in Muscle Relaxation: Atracurium in Perspective. London: Royal Society of Medicine Services International Congress and Symposium Series, 1988; 131: 13-20. 5. Geese A, Lonovics J, Szekeres L, Zsilinsky E, West GB. On the histamine-binding property of human serum. Journal of Pharmacy and Pharmacology 1972; 24: 70-71. 6. Hunter JM. Resistance to non-depolarizing neuromuscular blocking agents. British Journal of Anaesthesia 1991; 67: 511-514.
Downloaded from http://bja.oxfordjournals.org/ by guest on August 20, 2015
SHORT COMMUNICATIONS
IN VITRO DISPLACEMENT OF VASOACTIVE MEDIATORS FROM PLASMA PROTEINS: A POSSIBLE MECHANISM FOR PSEUDO-ALLERGIC REACTIONS TO NEUROMUSCULAR BLOCKING DRUGS W. R. WILLIAMS AND D. J. SHALE
SUMMARY
Anaesth. 1992; 69: 508-510) KEY WORDS Complications: histamine release, prostaglandin release. Neuromuscular relaxants. Protein: binding.
Adverse reactions to neuromuscular blocking drugs are sometimes categorized as pseudo-allergic because they appear to produce their effect by mechanisms similar to those of the immediate immurfe response [1]. Histamine has been identified as an important instigator of adverse reactions to neuromuscular blocking drugs and its release has been attributed to the direct action of these drugs on histaminecontaining cells [2]. Release of vasoactive mediators from plasma protein in response to neuromuscular blocking drugs has not been considered as an alternative mechanism. This in vitro study has investigated the effects of neuromuscular blocking drugs on prostaglandin F 2o (PGF 2 J and histamine binding in serum. METHODS AND RESULTS
Binding of PGF,O and histamine was measured by equilibrium dialysis [3] in serum from healthy volunteers, who were not taking any medication. Tritiated PGF^ 20 ul (0.74 KBq) (6.55 TBq mmol"1; Amersham pic) was added to serum 1.5 ml diluted 1:1 in phosphate buffered saline (PBS).
W. R. WILLIAMS, PH.D.; D. J. SHALE, B.SC, M.D., F.R.C.P.; Uni-
versity of Wales College of Medicine, Section of Respiratory Medicine, Llandough Hospital, Penarth, South Glamorgan CF6 1XX. Accepted for Publication: June 4, 1992.
Downloaded from http://bja.oxfordjournals.org/ by guest on August 20, 2015
We have studied the re/ease of prostaglandin F2x (PGF2J and histamine from serum proteins by neuromuscular blocking drugs using equilibrium dialysis, with tracer quantities of radio-labelled mediators as probes. Small concentrations (0.050.25 mmol litre'1) of competitive neuromuscular blocking drugs displaced 16-67% of bound histamine. Greater concentrations of suxamethonium (2 mmol litre'1) were required for histamine displacement (19%). There was a significant release of PGF2* by atracurium 1 mmol litre'1 and pancuronium 0.69 mmol litre'1. These findings suggest an alternative mechanism of histamine release by neuromuscular blocking drugs which may be relevant to adverse reactions during use. (Br. J.
Serum samples in visking tubing were dialysed against an equal volume of PBS containing 0.1% sodium azide for 36 h before the unbound PGF2ri was measured in a scintillation counter. To measure histamine binding, tritiated histamine dihydrochloride 20 ul (0.74 KBq) (1.4 TBq mmol-1; New England Nuclear) was added to undiluted serum and dialysed against PBS containing 1 % phenol. Neuromuscular blocking drugs were added (in 30 ul of distilled water) to the serum samples before dialysis, although larger volumes of injectable drug preparations were also used. Injectable drug preparations consisted of alcuronium chloride (Roche), pancuronium and vecuronium bromides (Organon Teknika), atracurium besylate and tubocurarine chloride (Calmic). Atracurium besylate, suxamethonium chloride and tubocurarine chloride were also provided as pure agents by Burroughs Wellcome. Pancuronium bromide, histamine dihydrochloride and sodium salicylate were purchased from Sigma Chemical Co. Dorset, England. Under these experimental conditions, 44.0 (SD 7.3)% (n = 20) of the added PGF !a and 13.4 (4.1)% {n = 20) of histamine became associated with serum proteins. Atracurium and pancuronium, in final concentrations of 1 and 0.69 mmol litre"1, respectively, inhibited PGF 2a binding by > 20 % (table I). At this concentration, atracurium had an inhibitory potency similar to that of salicylate. The binding of PGF2o was increased significantly by alcuronium. The neuromuscular blocking drugs were more potent at displacing histamine from plasma protein than at displacing PGF, a . With the exception of suxamethonium, at 0.05 mmol litre"1 all inhibited histamine binding by > 25 %; greater drug concentrations (0.25 mmol litre"1) were more inhibitory. In a concentration of 0.05 mmol litre"1, the histamine releasing effects of both atracurium and tubocurarine were similar to that of "cold" histamine 0.01 mmol litre"1. Greater concentrations of suxamethonium (2 mmol litre"1) displaced 19% of the histamine associated with serum protein, although this effect
ADVERSE REACTIONS TO NEUROMUSCULAR BLOCKERS
509
TABLE I. Changes (%) in the binding of iritiated PGFa and histamine in the presence of neuromuscular blocking (NMB) drugs, histamine and salicylate in concentrations of 0.05-1.0 mmol litre''. Pancuronium 0.69 mmol litre'', suxamethonium 2 mrnpl litre'' and histamine 0.01 mmol litre'1 were also tested. Mean (SD) from five to 10 experiments. Significant differences from normal (Student's t test): * P < 0.05; **P < 0.01
Histamine binding (% change)
PGF M binding_(% change) 1
NMB concn (mmol 1itre"1)
NMB concn (mmol litre" ) 0.2
Alcuronium Atracurium Pancuronium (0.69 mmol litre"1) Suxamethonium (2 mmol litre"1) Tubocurarine Vecuronium Histamine (0.01 mmol litre"1) Salicylate
1.0
122.1 (7.6)** 105.9(14.1) 96.4 (6.9) 60.8 (3.0)* — 97.0 (5.3) — 79.0 (5.2)** 106.3 (9.7) 95.8 (3.6) — 92.6(13.5) 108.9(6.1) 108.7(11.4) 102.3 (8.5) 83.0 (5.7) 105.0(12.2) 105.7 (7.9)
0.05
0.25
73.2(15.0) 45.0(12.1)* 72.1 (25.2)
70.9 (20.0)* 33.0(25.6) 67.1 (28.4)**
— —
— — —
53.0(12.1)* 73.4(21.2)
40.4(17.5) 56.8(17.0)**
— —
—
— — —
11.2(6.4)*
114.5(30.2)
—
—
52.1 (16.2)*
86.0(5.6)**
58 2(10.8)*
—
COMMENT
It is difficult to accept that immediate hypersensitivity is the sole cause of adverse reactions to neuromuscular blocking drugs, because of the small concentrations of specific antibody in reactive patients, the observed cross-reaction to different drugs and the apparent development of immunity with no previous exposure [4]. While there is apparent consensus on the'important role of histamine in these adverse reactions, the mechanism of its release remains unknown. The distinction between allergic and pseudo-allergic reactions is more clear with other drugs. Aspirin sensitivity is a more typical pseudo-allergic reaction because of the absence of IgE antibodies in reactive patients. Aspirin and other non-steroidal anti-inflammatory drugs induce adverse reactions through inhibition of cyclo-oxygenase or by their protein binding characteristics, which displace PGF2[1 from plasma protein [3]. The release of this bronchoconstricting prostaglandin may be relevant to the development of aspirininduced asthma. As this displacement mechanism may be applicable to pseudo-allergic reactions in general, it was of interest to investigate the potential of neuromuscular blocking drugs for the displacement of PGF 2a and histamine from plasma protein. We demonstrated that small concentrations of neuromuscular blocking drugs can displace radiolabelled histamine from serum protein tn_vitro/T\\§_ "inhibitory effects of these drugs on histamine binding increased with concentration. Furthermore, salicylate and cold histamine showed specificity in inhibiting the binding of radio-labelled PGF t e and of histamine, respectively. Although suxamethonium displaced histamine only at greater concentrations,
— — —
97.1 (15.3) 80.6 (24.3)
—
134.3(17.7)
the displacement mechanism is still relevant to this drug, which is used in clinical practice in concentrations greater than those used for competitive neuromuscular blocking drugs. The concentration of suxamethonium in preparations for injections is 126 mmol litre"1 (50 mg ml"1). In contrast, the concentrations of the other tested drugs ranged from 2.7 mmol litre"1 (pancuronium: 2 mg litre"1) to 13.0 mmol litre"1 (tubocurarine: 10 mg ml"1). PGF 2a binds to plasma albumin, a protein with many low affinity binding sites for drugs and endogenous substances, including histamine. The large plasma concentration of albumin gives this protein a binding capacity that is not readily saturated. Almost all (approximately 99 %) PGF 2a in plasma, of the order of 100 pg ml"1, is protein-bound [3]. Knowledge about histamine binding to plasma proteins in normal subjects is limited. A peptide responsible for binding histamine dissociates from albumin during coagulation or enzymic digestion [5]. Although the basic quarternary amine structure of neuromuscular blocking drugs should promote their binding to alpha^acid glycoprotein (AAG), both alcuronium and gallamine are bound predominantly by plasma albumin [6]. However, increased resistance to the action of atracurium in patients may be associated with an increase in AAG concentration. The binding characteristics of albumin and AAG may form a natural protective mechanism which reduces the effects of released vasoactive mediators. The presence of these proteinbound mediators may not become evident unless they are displaced by increased drug concentrations associated with i.v. injection. Our results provide some support for this hypothesis and suggest that the displacement mechanism may be relevant in a proportion of adverse reactions to neuromuscular blocking-drugs — - --
ACKNOWLEDGEMENTS The authors are grateful to Dr E. B. Williams and the Wellcome Foundation for interest in and financial support of this work.
Downloaded from http://bja.oxfordjournals.org/ by guest on August 20, 2015
was not statistically significant. Salicylate • 1 mmol litre"1 increased the quantity of histamine associated with serum protein. In contrast, the same concentration of cold histamine displaced nearly 90 % of the radio-labelled histamine.
1.0
510 REFERENCES 1. Schlumberger HD. The pseudo-allergic reactions to drugs and chemicals. Annals of Allergy 1983; 51: 317-324. 2. Vervolet D, Nizankowska E, Arnaud A, Senft M, Alazia M, Charpin J. Adverse reactions to suxamethonium and other muscle relaxants under general anesthesia. Journal of Allergy and Clinical Immunology 1983; 71: 552-559. 3. Williams WK, Pawlowicz A, Davies BH. Aspirin sensitive asthma: significance of the cyclooxygenase-inhibiting and protein-binding properties of analgesic drugs. International Archives of Allergy and Applied Immunology 1991; 95: 303-308.
BRITISH JOURNAL OF ANAESTHESIA 4. Watkins J. Anaphylactoid response to neuromuscular blockers: atracurium in perspective. In: Jones RM, Payne JP, eds. Recent Developments in Muscle Relaxation: Atracurium in Perspective. London: Royal Society of Medicine Services International Congress and Symposium Series, 1988; 131: 13-20. 5. Geese A, Lonovics J, Szekeres L, Zsilinsky E, West GB. On the histamine-binding property of human serum. Journal of Pharmacy and Pharmacology 1972; 24: 70-71. 6. Hunter JM. Resistance to non-depolarizing neuromuscular blocking agents. British Journal of Anaesthesia 1991; 67: 511-514.
Downloaded from http://bja.oxfordjournals.org/ by guest on August 20, 2015
