The antigenicity of pig insulin
The Antigenicity of Pig Insulin*
TORSTt~N DECKERT a n d ELSE GRUNDAHL 0 1
0 Die Antigen-
1 Medical Department , Blegdamshospital, Copenhagen , Niels Steensen's Hospital, Gentofte and State Hospital , Nykobing Sjaelland
Summary. Treatment of h u m a n subjects with a neutral solution of pure pig insulin crystals does not lead to the formation of insulin antibodies. Thirty-six non-diabetics with psychiatric diseases were treated with a neutral solution of crystalline pig insulin for a m a x i m u m period of 104 days, using a 24-h dosage of up to 208 i.u. I n patients who had previously not received insulin t r e a t m e n t (24 patients), insulin antibodies could not be demonstrated after the termination of the insulin treatment. However, in patients who had previously received t r e a t m e n t with acid solutions of insulin consisting of pig and ox insulin (12 patients), it was possible in almost all cases to demonstrate insulin antibodies after the termination of the insulin treatment. I n addition, 10 pigs received t r e a t m e n t with solutions of pure pig insulin. Insulin antibodies could be demonstrated in only 2 pigs, both treated with acid solutions of recrystallized pig insulin, whereas antibodies could not be demonstrated in pigs treated with neutral solutions of recrystallized pig insulin. The reason why pure preparations of pig insulin are in most cases also antigenic to man, is presumably t h a t the pig insulin preparations are injected as suspensions (zinc insulins, N P H insulin) or as is the case with acid solutions of insulin, converted to suspensions after injection, since insulin precipitates when the acid insulin solution is neutralized by tissue fluid.
Rdsumd. Le t r a i t e m e n t de sujets humains avec tme
solution neutre de cristaux d'insuline pure de pore ne
provoque pus la formation d'anticorps insuliniques.
Trente-six sujets non-diab6tiques, atteints de maladies
psychiatriques, ont dt6 traitds avec une solution neutre
d'insuline cristallisde de pore, p e n d a n t 104 jours au
maxim m n , en utilisant une dose quotidierme allant jusqu'~
208 U. Chez les patients qui n ' a v a i e n t pus dt6 traitds
a u p a r a v a n t par l'insuline (24 patients), on ne pouvait pas
ddmontrer la prdsenee d'anticorps insuliniques apr~s la fin
du traitement ~ l'insuline. Par contre, chez les patients
(12) qui avaient dt6 traitds a u p a r a v a n t par des solutions
aeides d'insuline (insuline de pore et insuline de boeuf), il
dtait possible duns presque t o u s l e s eas de ddmontrer la
prdsence d'anticorps insuliniques apr6s la fin du
traitem e n t par l'insuline. - - E n outre, 10 pores ont re~u u n
raitement avee des solutions d'insuline pure de pore. On
a p u trouver des anticorps insuliniques seulement chez
2 pores, traitds avec des solutions aeides d'insuline de pore
recristallisde, tandis qu'on n ' a pus pu trouver d'anticorps
ehez les pores traitds avec des solutions neutres d'insuline
de pore recristallisge. La raison pour laquelle les
prdparations pures d'insuline de pore sont duns la plupart des cas
dgalement antigdniques chez l'homme, est probablement
que les prdparations d'insuline de pore sont injeetdes en
suspensions (insuline-zinc,NPH-insuline), ou que, eomme
e'est le cas pour les solutions acides d'insuline, elles se
transforment en suspensions apr~s l'injeetion, puisque
l'insuline pr4cipite quand la solution acide d'insuline est
neutralistic par les liquides tissulaires.
Zusammenfassung. Behandlung von 3~Iensehen mit
neutralen LSsungen yon reinen Schweineinsulinkristallen
fiihrte nicht zur Bildung yon Insulinantik6rpern. 36
Patienten mit psychiatrisehen K r a n k h e i t e n ohne Diabetes
wurden maximal 104 Tage lung t/iglich mit einer neutralen
LSsung yon reinen Schweineinsulinkristallen gespritzt.
Die hbchste Tagesdosis war 208 E. Bei Patienten, die
friiher niemals eine Insulinkur durchgemacht h a t t e n
(24 Patienten), k o n n t e n naeh der K u r mit neutralem
Sehweineinsulin keine Insulinantik6rper naehgewiesen
werden. Bei Patienten, die friiher einmal I n s u l i n k u r e n
mit Sehweine-Rinderinsulinkristallen,gel6st in
verdiinnter HC1, durchgemacht h a t t e n (12 Patienten), k o n n t e n
jedoch in beinahe allen F~llen naeh der K u r mit neutralem
Sehweineinsulin, Insulinantik6rper nachgewiesen werden.
- - Von 10 Sehweinen, die mit Sehweineinsulin gespritzt
wurden, k o n n t e n Insulinantik6rper nur bei Schweinen
nachgewiesen werden, die mit Sehweineinsulin in saurer
L6sung gespritzt waren. Es wird angenommen, dal3 die
Ursache zur Antigenizit/it vieler
Schweineinsulinpr/iparate Mensehen gegeniiber daran liegt, dab sic als
Suspensionen gespritzt werden (Zink-Insuline, N P H - I n s u l i n )
oder -- wie es mit den sauren Insulin-LSsungen der Fall
ist -- naeh der Injektion zu Suspensionen umgewandelt
werden, indem Insulin pr/~eipitiert, wenn die sauren
L6sungen yon der Gewebefliissigkeit neutralisiert werden.
Key-words: Insulin antibodies, pig insulin,
isoimmunization, insulin dose.
H u m o r a l i n s u l i n a n t i b o d i e s can be d e m o n s t r a t e d
i n a l m o s t all subjects who h a v e b e e n t r e a t e d w i t h insu.
O ' D o n o v a n , 1966). This is p r e s u m a b l y because ox
i n s u l i n differs more i n s t r u c t u r e from h u m a n i n s u l i n
lin (Deckert, 1967). I n m a n , however, the a n t i g e n i c i t y
t h a n pig i n s u l i n does.
of p r e p a r a t i o n s of pig i n s u l i n seems to be less pro.
n o u n c e d t h a n the a n t i g e n i c i t y of p r e p a r a t i o n s of pure
ox i n s u l i n or m i x t u r e s of pig a n d ox i n s u l i n (Devlin a n d
The sole s t r u c t u r a l difference b e t w e e n pig i n s u l i n
a n d h u m a n i n s u l i n is the C - t e r m i n a l a m i n o acid i n the
B - c h a i n , which is a l a n i n e i n pig i n s u l i n a n d t h r e o n i n e
i n h u m a n i n s u l i n (Table 1). I t has, nevertheless , b e e n
f o u n d t h a t a n t i b o d y f o r m a t i o n i n diabetic subjects
c a n n o t be avoided, e v e n t h o u g h these p a t i e n t s are
p a r e d e x c l u s i v e l y from pig insulin
(Berson a n d Yalow,
1963; D e c k e r t , 1964; L o c k w o o d a n d P r o u t , 1965)
T h e r e a s o n for t h e a n t i g e n i c i t y of pig insulin in m a n
has n o t been clarified. I t can h a r d l y be due to t h e
s t r u c t u r a l difference existing b e t w e e n pig insulin a n d
h u m a n insulin, since i t has n o t been possible to d e m o n
s t r a f e a n y i m m u n o l o g i c a l difference b e t w e e n p i g
insulin a n d h u m a n insulin
( D e c k e r t a n d J c r g e n s e n , 1966)
n o r b e t w e e n pig insulin a n d d e s a l a n i n e - p i g insulin
(Berson a n d Y&low, 1963), a p r e p a r a t i o n of p i g insulin
from which t h e C - t e r m i n a l a m i n o acid, alanine, h a s
b e e n split off.
F u r t h e r , t h e s u p p o s i t i o n t h a t t h e s t r u c t u r e of
circulating endogenous h u m a n insulin is different from
t h a t of c r y s t a l l i z e d pig insulin or h u m a n insulin a f t e r
p r e p a r a t o r y purification, m u s t be r e g a r d e d as unlikely,
since i t can be shown t h a t p l a s m a insulin from n o r m a l
s u b j e c t s as well as o v e r w e i g h t s u b j e c t s a n d d i a b e t i c s
does n o t differ i m m u n o l o g i c a l l y from c r y s t a l l i n e p i g
insulin or h u m a n insulin
(Deekert a n d J r
The following i n v e s t i g a t i o n s were m a d e in a n
attempt to determine the reason for the antigenicity of
l o n g - a c t i n g pig i n s u l i n p r e p a r a t i o n s .
Material and Methods
T w e n t y - f o u r p a t i e n t s w i t h p s y c h i a t r i c diseases who
were n o n - d i a b e t i c s a n d who h a d n o t p r e v i o u s l y
received t r e a t m e n t w i t h insulin, were g i v e n a n i n t r a
m u s c u l a r i n j e c t i o n of a n e u t r a l solution of r e c r y s t a l l i z e d
p i g insulin n e a r l y e v e r y d a y for p e r i o d s u p to 104 d a y s .
The size of t h e insulin dose v a r i e d from p a t i e n t t o
p a t i e n t a n d from d a y t o d a y , d e p e n d i n g on t h e a i m
a n d effect of t h e t r e a t m e n t . T a b l e 2 shows t h e d u r a t i o n
of t h e t r e a t m e n t a n d t h e m a x i m u m dose of insulin
p e r 24 h. A similar t r e a t m e n t was given t o 12 p a t i e n t s
w i t h p s y c h i a t r i c diseases who were n o n - d i a b e t i c , b u t
who h a d p r e v i o u s l y , some m o n t h s t o several y e a r s
before, been t r e a t e d w i t h acid solutions of r e c r y s t a l l i z e d
p i g - o x insulin for a s h o r t e r p e r i o d (Table 3). B l o o d
samples were t a k e n from t h e c u b i t a l vein on t h e d a y prior to
t h e c o m m e n c e m e n t of t h e insulin t r e a t m e n t , a n d 2 d a y s
a f t e r t h e t e r m i n a t i o n of t h e insulin t r e a t m e n t . S e r u m
for insulin a n t i b o d y i n v e s t i g a t i o n was s t o r e d a t - - 20 ~C.
A . B . H .
L . P .
J . J . H .
G . L . J .
E . P .
L . R .
K . B . J .
B . P .
I . B . M .
A . H . B .
I . N .
E . R . J .
E . T .
B . H .
J . O .
J . B .
V . J .
K . I . N .
E . L . H .
E . N .
N . E . M .
E . T .
A . H .
F o r i s o i m m u n i z a t i o n tests, 12 pigs were used
weighing b e t w e e n 22 a n d 28 kg, 11 of t h e m f r o m one
l i t t e r
( B r u n f e l d t a n d D e c k e r t , 1964a)
. Six pigs were
t r e a t e d w i t h r e c r y s t a l l i z e d p i g insulin dissolved in
p h o s p h a t e buffer ( p t I 7.3), free from antiseptics. F o u r
pigs were t r e a t e d w i t h r e c r y s t a l l i z e d pig insulin
dissolved in d i l u t e d h y d r o c h l o r i c acid ( p H 3.2) t o which
g l y c e r o l a n d a n a n t i s e p t i c h a d been a d d e d . Two pigs
were u s e d as controls (Table 4). The biological s t r e n g t h
of t h e insulin p r e p a r a t i o n s was 2 4 - - 2 6 i . u . / m g . I n s u l i n
w i t h o u t t h e a d d i t i o n of a d j u v a n t s was i n j e c t e d
subc u t a n e o u s l y daffy for 8 7 - - 9 0 days. Nine of t h e a n i m a l s
received a total of 2250 i.u. insulin each, beginning
with 15 i.u. daily, later with increasing doses. One
animal (Brunfeldt and Deekert, 1964 a) received a total
of 1050 i. u. insulin. The d a y prior to a n d two d a y s after
the t e r m i n a t i o n of the insulin t r e a t m e n t , blood samples
were taken and serum for antibody investigation was
stored at -- 20 ~ C.
The human sera were examined for insulin
antibodies by a modification of the double antibody
technique described b y S k o m a n d Talmage (1958). 100 ~1
of serum to which 2.5 ~1 heparin L E O was a d d e d
(5000 units/ml) and 100 ~1 of blind (0.04 M p h o s p h a t e
buffer, pI-I 7.4 with 5o/o h u m a n albumin) were b o t h
i n c u b a t e d for 4 days at 4~ with 100 ~1 of an 0.04 M
p h o s p h a t e buffer, p H 7.4, containing 0.5% h u m a n
alb u m i n a n d 0.9% NaC1 t o g e t h e r with n o t quite 0.2 ixg
l~SI-pig insulin/ml. The specific r a d i o a c t i v i t y of the
iodinated insulin was a b o u t 60 mCi/mg. After s t a n d i n g
for 4 days, 50 F1 of the i n c u b a t i o n m i x t u r e was diluted
with 5000 ~zl 0.04 M p h o s p h a t e buffer, p i t 7.4,
containing 0.25% h u m a n albumin. Two h u n d r e d F1 of this
dilution was i n c u b a t e d for 24 h with 100 ~l of a dilution
of serum f r o m rabbits i m m u n i z e d with h u m a n g a m m a
globulin. The dilution of the r a b b i t a n t i h u m a n g a m m a
globulin was m a d e with 0.04 M p h o s p h a t e buffer,
pI-I 7.4, a n d was so adjusted t h a t 100 ~1 of the dilution
could completely precipitate the a m o u n t of g a m m a
globulin present in the reaction mixture. After the
incubation, the precipitate formed was filtered off on
oxoid filters. The precipitate was rinsed twice with
0.04 M p h o s p h a t e buffer containing 0.5% h u m a n
albumin, a n d the r a d i o a c t i v i t y t h e n m e a s u r e d in a
The "insulin-antibody c o n c e n t r a t i o n " was
expressed as the a m o u n t of radioactive insulin b o u n d to the
immunoglobulins, as a percentage of the t o t a l
radioactivity, i.e. the r a d i o a c t i v i t y of the sample on the
filter minus the r a d i o a c t i v i t y of the blind sample on
the filter, as a percentage of the total activity. The
total activity corresponded to the overall radioactivity
in the second incubation mixture (corresponding to
approximately 2 mFg 125I-insulin). All determinations
were performed as double tests. SEM (double
determinations) = 0.19%.
The 1~'sI-piginsulin was supplied by Nordisk
Insulinlaboratorinm. The rabbit anti-human gamma-globulin
was supplied by Dansk Svovlsyre and Supcrphosphat
Fabrik A/S, and the freeze-dried human albumin was
supplied by Statens Seruminstitut.
Using the same method, the insulin antibodies were
examined for their ability to discriminate between pig
insulin and ox insulin, after varying amounts of
recrystallized pig insulin and recrystallized ox insulin,
respectively, had previously been added to the serum.
The pig and ox insulin crystalline powders were
supplied by Nordisk Insulinlaboratorinm.
After the addition of 13~I-pig insulin, the sera from
the pigs were examined by means of
immunoelectrophoresis and autoradiography as previously described
(Brunfeldt and Deckert, 1964a)
. The specific activity
of the isotope-labelled insulin was 140--180 mCi/mg.
It was used in a concentration of approximately 0.04 Fg
Patients who h a d n o t previously been t r e a t e d with
insulin h a d an "insulin-antibody c o n c e n t r a t i o n " of <
1.2%, n a m e l y 0.46 ~: 0.34.
E l e v a t e d insulin-antibody concentration could n o t
be d e m o n s t r a t e d in a n y of the patients or the animals,
prior to the c o m m e n c e m e n t of the insulin t r e a t m e n t .
1816 o-----_oy~__.~__~x o
o ox insulin
x pig insulin
0.01 0.05 0.1 0.5 1
.u,g insulin/m[ serum
I n o n l y one (O.J.) of the 24 patients who had n o t
previously been t r e a t e d with insulin, could insulin
antibodies be d e m o n s t r a t e d after the t e r m i n a t i o n of
the insulin t r e a t m e n t (Table 2). However, the
eoncenafter the termination of the insulin treatment.
However, 2 of the 4 pigs t h a t had been treated with
recrystallized pig insulin in acid solution showed insulin
antibodies after the termination of the insulin t r e a t m e n t
(Fig. 2, Table 4).
tration of insulin antibody was very slight, being only
significantly higher t h a n in non-insulin-treated patients
at the 5 ~ level, but not at the 1% level.
I n contrast to the absence of antibody formation in
the patients not previously treated with insulin, 10 out
of the 12 patients who had previously received insulin
t r e a t m e n t with mixed pig-ox insulin preparations
showed insulin antibodies following the renewed insulin
treatment (Table 4).
I n one case, the insulin antibodies formed were
examined for their ability to discriminate between pig
and ox insulin. No discrimination was found (Fig. 1).
None of the 6 pigs treated with recrystMlized pig
insulin in neutral solution showed insulin antibodies
D i s c u s s i o n
The absence of insulin antibody formation after
t r e a t m e n t with solutions of neutral pig insulin in
patients not previously treated with insulin, is not due to
the t r e a t m e n t being too low in intensity, since a
corresponding intensity of t r e a t m e n t with acid pig-ox
insulin solutions resulted in the formation of insulin
antibodies (Table 5) in almost all cases - - as previously
I t is remarkable t h a t the t r e a t m e n t of h u m a n
subjects with large doses of pig insulin in neutral somtion
did not lead to a demonstrable formation of antibodies.
This is in contrast to the findings in patients treated
with longacting pig insulin preparations, since it had
already been reported
t h a t circulating
insulin antibodies could be demonstrated in 11 out of
12 patients after t r e a t m e n t with N P H - p i g insulin
(Table 5). I t thus appears as if the v e r y slight
antigenicity of pig insulin in h u m a n subjects is potentiated
when the insulin is injected subcutaneously as a
suspension of crystals with a long-acting effect. There are
a n u m b e r of factors which suggest t h a t a suspension of
insulin particles constitutes a greater stimulus to the
reticuloendotheliM system of the organism around the
site of injection t h a n a neutral solution, which is rapidly
reabsorbed. Thus, experiments with pigs show t h a t the
injection of acid insulin preparations, which following
injection are transformed into a suspension as a result
of isoelectric precipitation at the site of injection,can
likewise lead to the formation of insulin antibodies,
whereas this is not the case when the insulin is injected
as a solution whose p t I corresponds to the p H of the
Frankhauser and Morell (1968)
rarely able to demonstrate insulin antibody formation
in patients treated with neutral solutions of pig insulin,
whereas in patients treated exclusively with semilente
suspensions, which are almost pure pig insulin, Devlin
and Duggan (1968) found insulin antibodies in all eases.
The antigenicity of pig insulin would thus appear to
depend on whether the pig insulin is injected as a
suspension or whether it becomes precipitated at the
site of injection. I t is well known in experimental
immunology t h a t an antigen is more immunogenic as
a suspension t h a n a s a solution, since phagoeytosis of
the particles of antigen b y macrophages potentiates the
antigenieity of protein
(Frei et al., 1965)
I t cannot be overlooked t h a t factors other t h a n the
state of the insulin m a y be of significance for
potentiating the antigenieity of pig insulin. Thus, it is possible
t h a t contamination of pig insulin b y small amounts of
ox insulin, proinsulin
(Steiner st al., 1967)
alien to insulin
(Brunfeldt and Deekert, 1964b)
m a y
be of significance for the formation of insulin antibodies
in patients treated with suspensions of pig insulin. Nor
can the possibility be excluded t h a t a small p a r t of the
pig insulin is so modified during preparation t h a t it
m a y be significant for the antigenicity of the pig
insutin. For example, the splitting-off of amide groups might
well be significant for the antigenic characteristics of
I t is not clear why patients who had previously
produced insulin antibodies following t r e a t m e n t with
mixed pig-ox insulin preparations in acid solution could
be re-immunized following t r e a t m e n t with neutral
solutions of pig insulin. The result m u s t presumably
m e a n t h a t neutral solutions of pig insulin represent
such a weak antigenic stimulus t h a t a n t i b o d y formation
does not occur unless the i m m u n e a p p a r a t u s of the
organism is already sensitized. The fact t h a t the insulin
antibodies formed are unable to discriminate between
pig insulin and ox insulin agrees well with this.
The present investigations suggest t h a t the
formation of insulin antibodies in h u m a n subjects can be
avoided b y using rapidly-acting neutral solutions of pig
insulin crystals. However, the problem of insulin
antibodies has already been reduced to a clinically
insignificant phenomenon even with the use of long-acting
insulin preparations. This is seen from the experience
obtained in the Scandinavian countries, where insulin
resistance is a practically unknown situation. During
the last 15 years, several thousand diabetics with
insulin requirements have undergone t r e a t m e n t at Niels
Steensen's ttospitM, Gentofte. During this period, not
one single ease of insulin resistance has been observed.
Likewise, the insulin requirements of patients in
Scandinavia appear to be lower t h a n elsewhere. At
Niels Steensen's Hospitat, the 24-hour insulin dosage in
diabetics was examined over 2 periods, namely
1942-1952 and 1963--1967. The patients, practically all with
diabetes starting before the age of 30, had all received
insulin suspensions containing mainly pig insulin for a
period of more t h a n 1 year, all patients were over the
age of 18 years, had normal renal function, no infectious
disease, and were not overweight or pregnant. All
patients had recently terminated a hospital s t a y for the
control of their diabetes, were well controlled and
ambulant. The 24-hour insulin dosage the d a y prior to
returning home from hospital was noted. The
distribution of the 24-hour insulin dosage during the 2 periods
mentioned is seen in Fig. 3. This shows t h a t only 1 out
of 1006 patients had an insulin requirement which was
greater t h a n 100 i.u./24 h. For comparison, it m a y be
mentioned t h a t out of 1089 patients examined at the
university diabetes clinic in F r a n k f u r t a m Nain, 36
patients were found with an insulin requirement greater
0 / , %
Fig. 3. The distribution of the size of the dose of insulin
in 1006 diabetic subjects treated with insulin during the
periods 1942--1952 and 1963--1967. n = number of
t h a n 100 i.u. per 24 hours
(Ditschuneit and Federlin,
. Insulin resistance is almost always due to the
presence of large amounts of insulin antibodies, and as
there is in addition a relationship between insulin
requirement and insulin a n t i b o d y titer, the extremely
rare occurrence of insulin resistance and the low insulin
requirement might suggest t h a t the formation of insulin
antibodies is less pronounced using the insulin
preparations employed in Scandinavia.
I t is not clear why there should be a shift in the
distribution curve for 1963--1967 towards a lower
24-h dosage (cf. Fig. 3), b u t this is p r o b a b l y related to
the fact t h a t in 1963--1967 the insulin preparations
had a greater degree of purity.
Acknowledgements. The author wishes to thank
Physician-in-chief Jacob E. Poulsen, 3/[.D., K. Brunfeldt,
~ . Sc., and Physician-in-chief Otto Jacobsen, for their
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