An anaphylactic reaction to highly purified pork insulin. Confirmation by RAST and RAST inhibition

Diabetologia, May 1982

Summary We describe a patient who had an anaphylactic reaction to highly purified pork insulin; he was not allergic however to beef insulin. The sensitivity to pork but not to beef insulin was confirmed by skin testing, radioallergosorbent test (RAST) and RAST inhibition. A scheme is suggested for the investigation of such patients.

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An anaphylactic reaction to highly purified pork insulin. Confirmation by RAST and RAST inhibition

An Anaphylactic Reaction to Highly Purified Pork Insulin. Confirmation by RAST and RAST Inhibition C. C a r i n i 0 1 J. Brostoff ~ a n d A. B. K u r t z 0 1 0 tDepartment of Immunologyand 2TheCobbold Laboratories, Middlesex Hospital Medical School , London , UK 1 Dr. J. Brostoff Department of Immunology Arthur Stanley House Middlesex Hospital Medical School 40-50 Tottenham Street London W1P 9PG , UK Summary. W e describe a p a t i e n t w h o h a d a n a n a p h y - lactic r e a c t i o n to h i g h l y p u r i f i e d p o r k i n s u l i n ; he was n o t allergic h o w e v e r to b e e f i n s u l i n . T h e sensitivity to p o r k b u t n o t to b e e f i n s u l i n was c o n f i r m e d b y s k i n T h e r e h a v e b e e n reports o f a n a p h y l a c t i c r e a c t i o n s to i n s u l i n , the first b e i n g b y W i l l i a m s [1], w h o d e s c r i b e d a p a t i e n t w h o was allergic to i n s u l i n extracted f r o m p o r k p a n c r e a s b u t n o t to a b e e f extract. At t h a t t i m e i n s u l i n was very i m p u r e a n d it is likely t h a t the allergen was n o t i n s u l i n . It has a l w a y s b e e n difficult to p r o v e the precise n a t u r e o f the a l l e r g e n c a u s i n g s u c h r e a c t i o n s a n d with i n c r e a s i n g p u r i t y o f i n s u l i n p r e p a - r a t i o n s allergic r e a c t i o n s h a v e b e c o m e i n c r e a s i n g l y rare. Patients h a v e b e e n d e s c r i b e d w h o h a d allergic A n a p h y l a x i s; r a d i o a l l e r g o s o r b e n t test - 9 Springer-Verlag 1982 testing, r a d i o a l l e r g o s o r b e n t test ( R A S T ) a n d R A S T i n h i b i t i o n . A s c h e m e is s u g g e s t e d for the i n v e s t i g a t i o n o f s u c h patients. r e a c t i o n s to c o n v e n t i o n a l i n s u l i n s [ 2, 4 ]. T h e r e h a v e also b e e n i n s t a n c e s o f r e a c t i o n s to h i g h l y p u r i f i e d i n s u lins [5, 121, b u t with o n e d o u b t f u l e x c e p t i o n all patients h a d received c o n v e n t i o n a l i n s u l i n s p r e v i o u s l y [12]. W e d e s c r i b e a p a t i e n t w h o b e c a m e allergic to h i g h l y p u r i f i e d pork, b u t n o t to b e e f i n s u l i n , in w h o m we h a v e d e m o n s t r a t e d b y s k i n testing a n d R A S T t h a t the i n s u l i n is the a l l e r g e n a n d t h a t the i m m u n o p a t h o logical m e c h a n i s m was I g E m e d i a t e d . Patient and Methods The patient is an Australian male born in 1953.During childhood, he experienced episodes of allergic rhinitis, there being no family history of atopy. Diabetes mellitus was diagnosed in 1968and treatment with insulinstarted. Initially he received beef protamine zinc and soluble insulins. After 2 years of treatment, his insulinrequirement had increased to more than 200 units daily. Treatment was then changed to Actrapid (pork) and Rapitard (pork 25%/beef 75%)insulins(Novo Industri, Denmark) with a reduction in his insulin requirement. He has been seen regularly in our diabetic clinic since 1977and his diabetes has been well controlled on an average dose of 90 U insulin/day (weight: 80 kg) and there have been no complications of the disease. In May 1980,a change was made in his insulin regimen. Mixtard insulin (52 U), a highly purified pork insulin (30% neutral / 70% NPH; Nordisk Denmark), was substituted for the Rapitard insulin, of which he had been taking 72 U daily. After I week, wheals started to appear at injection sites and 2 weeks later a generalised reaction occurred. This reaction started within seconds of an insulin injection. The patient experienced difficulty in breathing with chest tightness and laryngeal obstruction and he developed a generalised urticarial eruption i.e. anaphylaxis. While on the Actrapid and Mixtard insulins diabetic control had been good. After the anaphylactic reaction, the patient resumed his previous insulin regimen and has subsequently been entirely free of allergic symptoms. Prick testing was performed using normal saline, insulinvehicle, and pork and beef insulins (both highly purified and conventional preparations) at a concentration of 40 U/ml. Pork and beef extracts (Bencards, Brentford, UK), histamine (1%) as a positive control and saline were also used. The tests were performed on the volar aspect of the forearm and were read at 20 rain. A positive test was a wheal at least twice the size of the saline control. Intradermal tests were also performed by injecting 0.05 ml of various solutions includinga diluent control. The insulintest dose was initially 0.002 U with subsequent doses increasing tenfold to 2 U. A positive reaction was judged in the same way as with the prick testing and the area of the wheal was measured byplanimetry. Goat anti-humanIgE was supplied by the Rheumatic Diseases Laboratory, Maine Medical Center, Portland, Oregon, USA. Iodinationofthe antiserum was performed by the chloramine T method of Hunter and Greenwood [ 13 ]using 125Iodine.The iodihated antiglobuinwas diluted in phosphate buffer (0.04mol/l), pH 7.4, containingsodium choride 0.15mol/l (PBS) and 1%Tween 20, to give 100,000counts. 100 s-~ 9100 g1-1,specific activity 1.49 108 Ci/mol. Discs of Whatman 54 paper 3 mm in diameter were prepared with a paper punch and activated using cyanogen bromide [ 14 ].The discs were sensitized with purified pork or purified beef insulin (10 ug/ml), diluted in sodium bicarbonate (0.I mol/l) and incubated for 4 h at 4 ~C on a rocking platform. The discs were then washed once in 200 ml of sodium bicarbonate (0.1 mol/1) and unreacted sites were blocked by incubationin 50 mmol/1 ethanolamine 200 ml diluted in sodium bicarbonate 0.1 tool/1 for 1h at 4 ~C. The discs were then washed in sodium bicarbonate 0.l mol/1 and then in PBS and stored at - 2 0 ~ The bindingof 1251labelled insulinby sera was determined using a 15% polyethylene glycol separation [ 15 ].This method determines the bindingof insulinby IgG. The Radioallergosorbent Test (RAST) This test was performed according to the method of Wide et al. [ 14 ], using discs sensitized with highly purified beef or pork insulin. Control sera were obtained from insulintreated diabetic subjects. Two control groups were studied, one with IgG insulin bindingof C. Carini et al.: Anaphylaxis to Highly Purified Pork Insulin Pork zo/ t ! I Patient Normal High Low Patient Normal High Low 0.04 I 0.08 I 0,4 I 0.8 Insulin (mU/I) less than 3% and the other with binding of 20%-40%, the first group having very- low or undetectable insulin binding and the second having moderately high binding without insulin resistance. RAS T Inhibition To test the specificity of the IgE anti-pork insulin antibody, an inhibition RAST was set up as follows: pork or beef insulin at concentrations between 0-4 m U / m l was added to the patient's serum, which was then assayed on a pork insulin sensitized disc to show inhibition of the binding of 125Ianti-human IgE to the discs. Results Skin tests were first performed 4 weeks after the anaphylactic reaction. Positive skin prick tests were obtained with the pork insulins (Actrapid, Leo Neutral and Mixtard insulins) but no responses were seen with beef insulins (Actrapid beef, Wellcome soluble and Wellcome N P H insulins), other allergens or diluent control. Rapitard insulin, which contains both b e e f and pork insulins, also gave a positive reaction. After a further 7 months, skin prick tests were repeated and were negative for both pork and beef insulins. Beef I 4 EAST Pork Beef However, following intradermal challenge, a positive reaction was seen with 2 U pork insulin (Actrapid) with negative reactions to lower doses and to beef insulin (Actrapid beet). IgG Antibodies lg E Antibodies The patient's serum showed only a modest level of insulin binding; 18% of labelled beef insulin and 12% of labelled pork insulin were bound. When the patient's serum was tested by RAST using pork insulin sensitized discs, a positive response was seen while a weakly positive response was shown using beef insulin sensitized discs. None of the diabetic control subjects had allergic symptoms and none showed any IgE antibodies directed to either pork or b e e f insulin (Fig. 1). RAST inhibition showed that the binding of the IgE to the pork insulin sensitized discs could be inhibited by pork insulin but not by beef insulin in equivalent concentrations (Fig.2), thus confirming the clinical history, skin tests and RAST. Discussion Insulin preparations can be immunogenic and immunoglobulins of all classes can be induced in patients during treatment [ 16 ]. The immunogenicity appears to be related more to the physical characteristics of the insulin than to the species of origin [ 15 ]. Anaphylaxis is the most dangerous and dramatic immunlogical side effect of insulin treatment and is now very rare, most probably because currently used insulin preparations are less impure than they used to be. In patients treated with highly purified pork insulin, the induction of insulin binding IgG is much less than in tion o f b o u n d insulin specific I g E with radiolabelled insulin. H o w e v e r , using this m e t h o d , insulin specific I g E was detected in m a n y subjects w h o h a d not experienced allergic reactions. R A S T inhibition seems a satisfactory way o f investigating species specificity, a l t h o u g h it m a y not be an especially sensitive m e t h o d in this respect [ 8 ]. 1. WilliamsJR ( 1922 ) A clinical study ofthe effectsofinsulin in severe diabetes . J Metab Res 2 : 729 - 731 2. Patterson R , Mellies CJ , Roberts M ( 1973 ) Immunologic reactions against insulin II. IgE anti insulin, insulin allergy and combined IgE and IgG immunologic insulin resistance . J Immunol 110 : 1135 - 1145 3. Kumar D ( 1977 ) Anti-insulin IgE in diabetics . J Clin Endocrinol Metab 45 : 1159 - 1164 4. Kumar D , Rosenquist RJ , Parameswaran V ( 1979 ) Insulin allergy . Reaginic antibodies to insulin and proinsulin . J Clin Metab 49 : 252 - 254 5. Leslie D ( 1977 ) Generalised allergic reaction to monocomponent insulin . Br Med J 2:736 737 6. Goldman RA , Lewis AE , Rose LI ( 1976 ) Anaphylactoid reaction to single component pork insulin . J Am Med Assoc 236 : 1148 - 1149 7. Reisner C , Moul DJ , Cudworth AG ( 1978 ) Generalised urticaria precipitated by change to highly purified porcine insulin . Br Med J 2 : 56 - 60 8. Nakagawa S , Nakayama H ( 1979 ) Immunogenicity of insulin with special reference to IgE antibody . In: Baba S, Kameko T , Yamaihara N (eds) Proinsulin, insulin, C peptide. Excerpta Medica, Amsterdam ICS 468:170 "173 9. Borsey DQ , Malone DNS ( 1979 ) Local cutaneous allergy to monocomponent insulin . Postgrad Med J 55 : 199 - 200 10. Goldman JM , Brynildsen HC ( 1980 ) Generalised allergy to porcine and bovine monocomponent insulins . Br Med J 2 : 1494 - 1497 11. Simmonds JP , Russell GI , Cowley AT , Sewell HF , Hearnshaw JR ( 1980 ) Generalised allergyto porcine and bovine monocomponent insulin . Br Med J 281 : 355 - 356 12. Schernthaner G , Ludwig H , Jarisb R , Bruneder H ( 1981 ) Immediate-type allergy against insulin itself: clinical and immunological studies on a diabetic patient with insulin intolerance . Diabetes Care 4 : 196 - 201 13. Hunter WM , Greenwood FC ( 1962 ) Preparation of iodine 131 labelled human growth hormone of high specific activity . Nature 194 : 495 - 497 14. Wide L , Bennich H , Johansson SGO ( 1967 ) Diagnoses of allergyby an in vitro test for allergicantibodies . Lancet 2 : 1105 - 1107 15. Kurtz AB , Nabarro JDN ( 1980 ) Circulating insulin binding antibodies . Diabetologia 19 : 329 - 334 16. Hamilton RG , Rendell M , Adkinson NF ( 1980 ) Serologicalanalysis of human IgG and IgE anti-insulin antibodies by solid phase radio-immuno assays . J Lab Clin Med 96 : 1022 - 1024 17. Shore RN , ShelleyWB, Kyle GC ( 1975 ) Chronic urticaria from isophane insulin therapy: sensitivityassociated with non-insulin components in commercial preparations . Arch Dermatol 111 : 94 97 Received: 5 June 1981 and in revised form: 18 January 1982


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C. Carini, J. Brostoff, A. B. Kurtz. An anaphylactic reaction to highly purified pork insulin. Confirmation by RAST and RAST inhibition, Diabetologia, 1982, 324-326, DOI: 10.1007/BF00253575