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 , 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 [
]. 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
. 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 [
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
Discs of Whatman 54 paper 3 mm in diameter were prepared
with a paper punch and activated using cyanogen bromide [
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 [
determines the bindingof insulinby IgG.
The Radioallergosorbent Test (RAST)
This test was performed according to the method of Wide et al. [
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
Patient Normal High
Low Patient Normal High Low 0.04
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.
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.
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.
Insulin preparations can be immunogenic and
immunoglobulins of all classes can be induced in patients
during treatment [
]. The immunogenicity appears to
be related more to the physical characteristics of the
insulin than to the species of origin [
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 [
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Received: 5 June 1981
and in revised form: 18 January 1982