Determination of total serum insulin (IRI) in insulin-treated diabetic patients
Determination of Total Serum Insulin (IRI) in Insulin-treated Diabetic Patients
LISE G. I-IEDING 0
0 Novo Research Institute , Copenhagen , Denmark
Summary. A routine method is described for the determination of total II~I (immunoreaetive insulin) in insulintreated diabetics. The method involves an easy acid ethanol extraction, whereby antibody-bound I R I is dissociated and separated, together with the "free" I R I from the serum proteins and the antibodies. The recovery of I R I in this procedure is about 80%. After the separation, the isolated total I R I is measured in an immunoassay, using ethanol for the separation of free and antibody bound l~sI-insulin. I n 169 diabetic patients treated with insulinin doses of from 6 to 120 units/day, the fasting serum total I g I was between 6 and 4374 ~U/ml, with a mean of 392 ~U/ml. During treatment with insulin, the level of total I R I increased from normal values, registered during the first two months, to a higher level which became stable after about 5 months of treatment. The increase in I R I occurred simultaneously with the formation of antibodies. Insulin-resistant patients showed very high I R I levels.
Insulin; radioimmunoassay; total IRI in insulin-treated diabetics; acid ethanol extraction of insulin
Determination de l'insuline totals chez lea diabgtiques
traitgs a l'insuline
t~dsumg. On dgcrit une m6thode de routine pour le
dosage de l ' I R I (insuline immunorgactive) totale chez les
diab6tiques trait6s par Pinsuline. La mdthode comprend
une extraction ~ l'acide-6thanol, tr6s simple, pendant
laquelle I ' I R I lide aux antieorps est dissocige ot s6parge
ainsi qua l'It~I ~>libre<~des prot6ines s6riques, anticorps
eompris. La rgcup6ration de I ' I R I par carte m6thode est
aux environs de 800/0. Aprgs la s6paration, l ' I R I totale
isol6o est mesur6e par u n dosage immunologique q u i s e
sert de l'6thanol afin de s6parer I'I125-insulinelibra de calla
li6e aux antieorps. Chez 169 malades diab6tiques trait6s
par l'insuline k des doses allant de 6 k 120 unit6s par jour,
I ' I R I totMe s6rique ~ jeun 6tait de 6 h 4374 ~U/ml, avee
une moyenne de 392 ~xU/ml. P e n d a n t le traitement par
l'insuline le t a u x de I ' I R I totale est pass6 des niveaux
normaux, enregistrgs pendant les deux premiers mois,
des niveaux plus 61egvs qui se stabilisent 5 mois environ
apres le d6but du traitement. L ' a u g m e n t a t i o n de l'It~I
coincide avee la formation d'anticorps. Les roulades
insulino-rgsistants prgsentent des valeurs trgs hautes
d ' I g I .
Bestimmung des Gesamtserum-Insulins ( I R [ ) bei
Zusammenfassung. Fiir die Bestimmung des
GesamtI R I (immunoreaktiven Insulins) bei Diabetikern, die mit
Insulin behandelt wurden, wird eine l%outinemethode
beschrieben. Die Methode schliel3t eine einfache
S//uroXthanol-Extraktion ein, wobei das antik6rpergebundene
It~I dissoziiert u n d zusammen mit dam ,,freien" I R I yon
den Serumproteinon, einschliel31ieh den Antik6rpern,
get r e n n t wird. Boi diesem Verfahren warden etwa 80% des
I g I wiedergefunden. Naeh der T r e n n u n g wird das isolierte
Gesamt-IRI immunologisch gemessen. Ffir die T r e n n u n g
des freien yon dam an AntikSrper gebundenen l ~ I - I n s u l i n
wird Jkthanol verwendet. Bei 169 Diabetikern, die mit
6 bis 120 E Insulin/Tag behandelt wurden, lag das
Niichternserum-Gesamt-IRI zwischen 6 u n d 4374 ?E/ml
(Mittelwert 392 ~xE/ml). I m Laura der I n s u l i n b e h a n d l u n g
stieg das Gesamt-IRI yon Normalwerten, die w/~hrend
dot ersten 2 Monate registriert wurden, ant ein h6heres
Niveau an, das sich naeh etwa 5 Monarch
Behandlungsdauer stabilisierte. Der Anstieg des It~I erfolgte
gleichzoitig mit der Bildung yon Antik6rpern. Bei
insulinresistenten Patienten ergaben sich sehr hohe I R I - K o n z e n
Yalow a n d Berson (1960) were the first to develop
a n d describe the i n s u l i n r a d i o i m m u n o a s s a y , a n d since
t h e n this assay has been widely used, either i n its
original form or in a form modified, e.g., with respect
to the tracer a n d / o r the s e p a r a t i o n technique, in order
to m e e t the r e q u i r e m e n t s of a r o u t i n e method. As a
result of the a v a i l a b i l i t y of these r o u t i n e methods, an
enormous a m o u n t of i n f o r m a t i o n has been collected
over the past l l years a b o u t the c o n c e n t r a t i o n of
i m m u n o r e a c t i v e i n s u l i n (IRI) in serum from n o r m a l
persons a n d u n t r e a t e d diabetic patients. However,
i n f o r m a t i o n a b o u t I R I levels i n i n s u l i n - t r e a t e d p a t i e n t s
is w a n t i n g .
W h e n diabetic p a t i e n t s are t r e a t e d w i t h
commercial i n s u l i n preparations, n e a r l y all of t h e m develop
i n s u l i n antibodies
(Berson a n d Yalow, 1964)
m o n t h s of t r e a t m e n t . The serum
p a t i e n t s t h e n contains a m i x t u r e of ,,free" insulin,
a n t i b o d y - b o u n d i n s u l i n a n d free antibodies. Due to
the presence of antibodies, it is n o t possible to
determ i n e the t o t a l a m o u n t of I R I b y a direct i m m u n o
assay such as those used for the sera from normals a n d
diabetics n o t t r e a t e d w i t h insulin. The antibodies m u s t
be r e m o v e d from the diabetic serum before a n I i ~ I
d e t e r m i n a t i o n can be performed.
e x t r a c t e d i n s u l i n from the sara of
two r e s i s t a n t p a t i e n t s a n d f o u n d values as high as
12 m U of t o t a l i n s u l i n per m l of serum, t I e d i n g a n d
V M u n d (1967) a n d H e d i n g (1969) described a r o u t i n e
m e t h o d of acid e x t r a c t i o n for the d e t e r m i n a t i o n of
t o t a l i n s u l i n i n i n s u l i n - t r e a t e d patients. I n a series of
i n s u l i n - t r e a t e d diabetic p a t i e n t s (10 to 120 i . U . a day)
the I R I values were found to range between 0 and
Ohneda et al. (1970)
described a plasma
extraction method which abolished a non-specific
inhibitor of the double-antibody method. Furthermore,
the extraction removed the antibodies present in the
serum, making it possible to determine IlgI in sara
from insulin-treated diabetics. Determination of I R I
performed in eight sara gave values of from l0 to
Pearson and Martin (1970)
filtration, on Sephadex G-50, of plasma from diabetic
patients after dissociation of the insulin-antibody
complex at low pH. Using this method, the authors
found between 700 and 6000 ~zU I g I per ml of plasma
of six fasting, insulin-treated diabetic patients.
This paper describes a routine method for the
determination of total I R I in insulin-treated patients,
and consists of two parts: 1. insulin
radioimmunoassay and 2. determination of total I g I in serum from
Isolation of total IR[ from serum
I "IF" + IB-Ab I
+ HCI---~.pH -:~
+ e t h a n o [
+ NaOH----~pH- 7 - 8
1. Insulin radioimmunoassay. The general
principles of radioimmunoassay are well known. One of the
most critical steps in this assay is the separation of
free and antibody-bound l~I-insulin. A number of
techniques are available t o d a y
(Kirkham and Hunter,
In the ethanol method, the free and the
antibodybound insulin are separated b y addition of 96%
ethanol to give a final concentration of 79%. This
method was developed (Heding, 1966) as a substitute
for the paper chromatographic method and the
doubleantibody method, to avoid the time consumed b y the
former and the sources of error of the latter.
2. Determination of total I R I in serum of
insulintreated persons. The principle of this method (Heding,
1967, 1969) is shown in Fig. 1. The serum sample
contains free I R I , antibody-bound I R I and a surplus of
antibodies. The pI-I is adjusted to approximately 2
with HC1; at this low p H the insulin-antibody complex
dissociates immediately into free insulin and
antibody. Ethanol is then added, and due to the low p H
no precipitation of the serum proteins will take place
until NaOI-I is added to bring the p H close to 7. The
ethanol concentration of this neutral mixture is
approximately 75%, at which level insulin does not
react with antibody. The insulin remains in the
supern a t a n t and is separated from the precipitated
antibodies and other serum proteins by eentrifugation.
The supernatant is evaporated in an exsiecator in
vacuum, and the residue is dissolved in buffer and
Materials and Methods
hionocomponent-insulins of h u m a n , porcine and
b o v i n e origin, w i t h biological activities of 25.4, 27.2 a n d
25.7 i. U . / m g , respectively, (supplied b y t h e N O V O Insulin
L a b o r a t o r y ) , were used as standards.
12~I-pork insulin was used as tracer. I t was p r e p a r e d
b y iodinating m o n o c o m p o n e n t - p o r k insulin w i t h 1251
according to J o r g e n s e n a n d B i n d e r (1966). I t s specific
a c t i v i t y was a p p r o x i m a t e l y 30 mCi/mg, l ~ I - i n s u l i n and
anti-insulin sera were diluted in p h o s p h a t e buffer (0.04 M,
p t I 7.4) c o n t a i n i n g h u m a n a l b u m i n (Behringwerke) (1 g/l)
a n d t h i o m e r s a l (0.2 g/l) (subsequently referred to as
FAM). All s t a n d a r d s and samples were dissolved a n d
diluted in p h o s p h a t e buffer (0.04 hi, p H 7.4) c o n t a i n i n g
NaC1 (6 g/l), h u m a n a l b u m i n (60 g/l) a n d t h i o m e r s a l
(1 g/l) (subsequently referred to as N a F A M ) .
Insulin antibodies were raised b y i n j e c t i n g guinea pigs
(weighing 300--400 g) w i t h 0.5 m l of an emulsion of
3.75 m l sterile w a t e r and 6.25 m l of Zinc P r o t a m i n e Insulin
(NOVO, p o r k insulin, 80 i . U . / m l ) and i0 m l of F r e u n d ' s
a d j u v a n t , corresponding to a dose of 12.5 units of insulin;
this dose was s u b s e q u e n t l y increased to 25 units. The
guinea pigs were allowed 10% glucose w a t e r ad libitum
after t h e injections.
The r a d i o i m m u n o a s s a y procedure was as follows:
to triplicates of 100 ~1 of s t a n d a r d solutions (containing
f r o m 10 to 100 ~lY of insu]in/ml) or samples was a d d e d
100 fxl of anti-insulin guinea-pig s e r u m diluted 1 : 35000.
A f t e r 20 h of i n c u b a t i o n at 4~ 100 ~l of 125I-insulin
(200 ixU/ml) was a d d e d at 4~ a n d after a n o t h e r
incubation period at 4~ (4--20 h), 1.6 m l of e t h a n o l was a d d e d
in order to separate the free and t h e a n t i b o d y - b o u n d
insulin. A f t e r m i x i n g a n d centrifugation for 10 m i n at
2500 rpm, t h e s u p e r n a t a n t c o n t a i n i n g the free 125I-insulin
was d e c a n t e d into disposable plastic tubes (diameter:
10 ram, h e i g h t : 75 ram, N U N C , I~oskilde, D e n m a r k ) , t h e
tubes were stoppered w i t h plastic stoppers, and t h e
radioa c t i v i t y counted.
B l o o d was d r a w n f r o m the a n t e c u b i t a l v e i n into glass
tubes and allowed to clot for one h o u r at r o o m t e m p e r a t u r e
before eentrifugation. The s e r u m was p i p e t t e d into plastic
tubes a n d stored at - - 1 8 ~ until used.
D e t e r m i n a t i o n of t h e t o t a l a m o u n t of I R I was done
as follows: to duplicates of 500 ~zl of s e r u m was a d d e d
100 ~l of 1 N tIC1 to give a pI-t of a p p r o x i m a t e l y 2.5. The
tubes were shaken and i n c u b a t e d at r o o m t e m p e r a t u r e
for I0 min, w h e r e u p o n 2.5 m l of 95% e t h a n o l was a d d e d
and t h e c o n t e n t s m i x e d b y v i g o r o u s l y i n v e r t i n g the tubes.
D u e to the low pI-I, t h e p l a s m a proteins did n o t preeipitate.
100 ~zl of 1 N NaOlaI was t h e n added, and again, t h e
m i x t u r e was shaken vigorously. A h e a v y protein
prec i p i t a t e was formed: The p r e c i p i t a t e was s e p a r a t e d b y
e e n t r i f u g a t i o n for 10 rain at a b o u t 2000 G, and t h e
s u p e r n a t a n t , containing t h e insulin, was transferred to a
small b o t t l e a n d e v a p o r a t e d to dryness in a dessiccator
under a pressure of 10 mm Hg overnight. One hundred
samples could easily be handled simultaneously. The dry
residue was dissolved in 1 ml of NaFAM buffer and
1. Insulin radioimmunoassay
Concentration of ethanol, salt, albumin
Two requirements should be fulfilled in connection
with the use of ethanol for the separation of free and
antibody-bound ~2~I-insulin, viz.: 1. complete
precipitation of all the ~sI-insulin in an incubation medium
containing insulin antibodies in surplus, and 2. no
precipitation of ~sI-insulin in solutions containing no
antibodies. As previously shown (Healing, 1966), these
requirements are fulfilled b y adding 1.6 ml of 96%
ethanol to 0.3 ml of an incubation mixture to give a
final ethanol concentration of approximately 79%.
Complete immunoassays (including standard curves
and serum samples) were run simultaneously using
double-antibody, paper chromatographic or ethanol
separation techniques. No significant differences could
be found in the per cent ~sI-insutin bound to
antibodies either in the standards or in the samples.
A minimum concentration of 0.02 M C1- (or other
monovalent anions) was found necessary to ensure
complete precipitation o~ the insulin-antibody
complex; furthermore, the insulin-antibody complex
was shown to be stable in 79% ethanol for several
The protein concentration influences the
copreeipitation of the ~I-insulin. Copreeipitation of free
~ I - i n s u l i n in the presence of different serum samples
and 6% albumin containing buffer was between 4.6%
and 5.2%. I t is important t h a t the protein
concentration should be the same in all incubation mixtures, and
a final concentration of 2% has been used throughout.
At temperatures below 10~ the coprecipitation
increases, and the temperature in the final mixture
should therefore be above 10~ (e. g., ethanol at room
Adsorption of ~ I - i n s u l i n to different types of tubes,
concentration of ~ I - i n s u l i n in foam. I t is a well-known
phenomenon t h a t insulin in low concentration (ng
range) is adsorbed to the surface of glass-ware unless
other substances are present -- preferably proteins.
Probably, these proteins, e.g. albumin, compete with
the insulin for the binding site on the glass-ware, and
if albumin is present in great excess, the loss of insulin
due to adsorption will be minimal. However, the
degree of insulin adsorption depends not only on the
amount oi protein present but also on the type of glass
or plastic-ware used. The adsorption of ~2~I-labelled
ox proinsulin to four different~types of glass and
plastic tubes was examined. I t was found t h a t the
plastic tube absorbed 2% of the ~ I - l a b e l l e d ox
proinsulin, whereas the glass tubes adsorbed between 13
and 37% of the tracer at 0.1% albumin concentration,
The adsorption was diminished b y addition of, e.g.,
serum or by increasing the concentration of albumin.
The adsorption to glass-ware was also demonstrated
using ~25I-insulin. I t is obvious t h a t the type of tube
used for immunoassay should be carefully selected.
The same holds true for the glass-ware t h a t is used to
store and prepare the ~25I-insulin solutions.
Solutions of 125[-insulin in FAM containing 0.1%
albumin foam readily upon shaking. The concentration
of 125I-insulin is higher in the foam t h a n in the solution.
This circumstance m a y induce serious errors. A frozen
solution of ~25I-insulin was shaken during thawing and
100 ~1 volumes were pipetted into tubes and counted.
The first 90 tubes contained the same amount of
~25I-insulin. In the subsequent tubes the radioactivity
increased, with the final 4 - - 5 tubes exhibiting v e r y
high concentrations of ~25I-insulin. Obviously, the
foam with its higher concentration of ~25I-insulin had
gradually settled thereby increasing the concentration
of 12~I-insulin in the solution.
Degradation of int',utin by different type's of albumin
cpm in precipitate
5000 . ~ .
(a--a 2 days at 4~
Armour .Jo.,...o fresh
albumin|x-,-~x 2 days at 4~
/ 50 KIE trasylollml
~ i ~ B e h r i n g
~.. ~ albuWmienrke e~.4 .z .na.ys. . a.t 4
Quality of albumin and 125I-insulin; degradation of
insulin by serum. As mentioned before, albumin is
added to all buffer solutions in order to prevent
adsorption of insulin to glass-ware and to attain the same
protein concentration in serum and standards. Many
publications mention using bovine albumin fraction V
(Armour) for this purpose. I t was found, however, t h a t
this albumin contained enzymes capable of degrading
insulin. The problem is illustrated in Fig. 2. A series of
insulin standard solutions was prepared with bovine
albumin made by Armour, and another one with
human albumin made b y Behringwerke. I t was found
t h a t the insulin solution prepared with bovine albumin
retained only about 65~/o of its original content of
L.G. tIeding: Determination of Total Serum Insulin (IRI)
insulin after 2 days of storage at 4 ~C. The reason was,
no doubt, enzymatic degradation (and not the binding
of insulin to albumin) because this degradation could
be prevented with trasylol (a proteinase inhibitor).
A common " m u s t " in all immunoassays is the use
of a high-quality tracer. The significance of this
requirement varies with the different methods of
separation. Table 1 shows some results obtained with two
a the concentration of 125I-insulin in both preparations
was approx. 220 ~zU/ml.
different ~sI-insulin preparations: one of t h e m was
labelled b y the method of K. Jorgensen
, the other one was a commercially
available preparation. I t is obvious t h a t a p a r t of the
radioactivity in the commercial preparation was not
bound to insulin since only 84% could be " b o u n d " to
insulin antibodies in surplus, and since more t h a n 20%
was precipitated in the absence of antibody.
The degradation of insulin in plasma and serum
has been discussed in numerous papers. No detectable
degradation occurs in serum stored at --18~ the
problem first arises when serum is handled at
temperatures above 0~ No difference could be shown in
the I R I content of six serum samples obtained from a
normal person during an oral glucose tolerance test
after 4 h of storage at --18~ 4~ a n d 30~ (IRI
values between 17 a n d 68 ~U/ml).
Reproducibility of standard curves, standard
deviation of triplicates, day-to-day variation in serum I R I
determinations, normal I R I values. Fig. 3 shows the
reproducibility of 7 different standard curves obtained
over a period of three weeks. The d a y - t o - d a y variation
is small, but it is advisable always to set up a standard
curve together with the samples to be assayed.
The counts from the i m m u n o a s s a y were recorded
direct on a punch tape, which was transferred to an
IBIV[ 1130 computer and processed according to a
program developed b y
. The results
were written as mean values with the 95% interval.
I n the range of 0 - - 8 0 ~xU/ml, the 95% interval of
59 determinations (2 experiments) was 2.83 ~U/ml
4-0.82 (mean ~ s.d.). The 95 % interval was the same
throughout the entire range, meaning t h a t the most
accurate determinations are obtained using the upper
p a r t of the standard curve. Two normal sera were
assayed in 10 different immunoassays, giving the
following results: 3 9 . 6 j : 3 . 0 and 8 1 . 1 ~U/ml
(mean :L s. d.). Dilution of the serum samples gave the
expected values, and recovery of added insulin was
practically 100%. Twenty-four normal fasting persons
showed I R I values between 0 and 16 ~U/ml (mean
7.2 ~zU/ml). One hour after 1.75 g of glueose/kg, their
I R I values were between 30 and 150 fxU/ml (mean
90 "% free 1251-insulin
-* , , M.
40' 0, 1,2,0 5
, , ,
50 75 100
L.G. Heding: Determination of Total Serum Insulin (I1~I)
Recovery; serial dilutions of the extracted insulin.
The r e c o v e r y of insulin in t h e e x t r a c t i o n p r o c e d u r e
has been d e t e r m i n e d using t h e following t h r e e
app r o a c h e s :
1. a d d i t i o n of 125I-insulin to n o r m a l a n d d i a b e t i c sera
2. use of n o r m a l sera w i t h a k n o w n insulin
t i o n
3. use of n o r m a l sera w i t h a k n o w n insulin c o n c e n t r a
t i o n a d d e d to g u i n e a - p i g a n t i - i n s u l i n s e r u m (AIS).
The r e c o v e r y of 125I-pork insulin a d d e d to d i a b e t i c
sera v a r i e d from 79 to 84% in 12 sera. T h e r e c o v e r y of
insulin from s t a n d a r d solutions c o n t a i n i n g from 25 to
100-% bound 1251-insulin
Total I R I in insulin-treated patients. T a b l e 2
shows t h e results of t o t a l - I R I d e t e r m i n a t i o n s in sera
from 169 i n s u l i n - t r e a t e d fasting d i a b e t i c p a t i e n t s . T h e
s e r u m was d r a w n 1 4 - - 2 4 h after t h e l a s t insulin
injection, All p a t i e n t s h a d been t r e a t e d w i t h insulin for
more than six months. The majority, b y far, had
elevated total I R I levels as compared to normals and
untreated patients. Two examples of the variation in
fasting total 1RI in the course of treatment are shown
in Table 3. A marked increase in total I R I was
observed after 2 - - 3 months of treatment.
1. I n s u l i n radioimmunoassay. Each
radioimmunoassay method has a number of sources of error, some
of which are common to most methods, others being
specific for a particular method.
The adsorption of nsI-insulin to plastic and
glassware is probably of much greater consequence than
one usually realizes. I t has been generally accepted
t h a t the presence of 0.1% albumin effectively prevents
this adsorption of nsI-insulin. However, this is not the
ease in every type of tube. The ability of nsI-insulin
to concentrate in foam m a y completely invalidate the
Variation in the protein concentration will influence
most of the separation techniques used in
radioimmunoassay. Thus, the binding of free nsI-insulin
to cellulose, ion-exchangers, charcoal and similar
substances is diminished at higher albumin
concentrations. When ethanol is used for the separation of free
and bound nsI-insulin, the eopreeipitation of h'ee
nsI-insulin will increase at higher protein
concentrations. I t is therefore obvious t h a t the protein
concentration in the standards must be the same as in the
o--o total IRI
0 I r I
8 a.m. 12 4
8 i.u. Actrapid
12 i.u. Retard
8 LU. Retard
Enzymatic degradation of insulin during
incubation will, of course, give false values. The degree of
degradation in human sera was found to be very low,
but sera from other species exhibited a much more
pronounced degradation of l~sI-insulin. Some types of
albumin contain enzymes t h a t degrade the standard
insulin (Fig. 4) and thereby reduce the stability of the
standard solutions. When use was made of a pure
albumin, solutions containing from 10 to 5000 3U
insulin/ml were found to remain stable for more than
a year at --18~ Anti-insulin sera stored at --18~
have been used since 1966 without our detecting a n y
changes in their binding capacity.
The immunoassay method here described makes
use of 96% ethanol for the separation of free and
antibody-bound 125I-insulin. This method has several
advantages : it is accurate, quick and easy to perform,
and the 96% ethanol is an ordinary, inexpensive
standard chemical. For optimal results a good l~hI- the development of vascular disease. Stout (1970)
insulin preparation is required, as shown in Table 1.
Ethanol separation has yielded results identical with
those of the chromatographic and double-antibody
showed t h a t chickens treated with insulin for 19 weeks
vascular lesions faster t h a n
chickens. Thus, the high total serum I R I found in
diabetics could be contributory to the development
of vascular complications in diabetes.
treated persons. The presence of antibodies and
antibody-bound insulin in serum
persons necessitates carrying out an acid ethanol
extraction to obtain an estimate of the total It~I. The
method involves a complete dissociation of the
insulinantibody complex, followed b y a precipitation of the
antibodies in ethanol at neutral pH. This method
offers several advantages as compared to separation
by gel filtration described b y Pearson and
Martin (1970) -- it is a routine method in which more than 50 samples can easily be handled simultaneously; the dry residue can be dissolved direct in the buffer used
The results obtained with this method showed the
1. Total fasting I R I in a randomly selected diabetic
population treated with insulin in doses of between
6 and 120 units/day varied from 6 to 4347 ~U/ml
(mean 392 ~xU/ml), and b y far the majority of the
patients had substantially elevated I R I values when
compared with normal subjects and diabetics not
treated with insulin.
2. The total I R I increased during the period of
treatment from the normal values of the first month or
two to a higher level, which then became fairly
stable after about 5 months of treatment (two
examples are shown in Table 3). The increase in
I g I was observed simultaneously with the
formation of antibodies. If no antibodies were produced,
for instance due to treatment with
(Schlichtkrull et al., 1971)
, the serum
I R I stayed within normal limits.
3. The fasting total I g I in diabetic patients treated
with insulin for over five months varied only a
week to week. Even in those treated
several months longer, the changes observed were
minor (Tables 3 and 4). That is to say, each patient
has his own total I R I level.
(defined as diabetics
whose daily dose of insulin exceeds 100 units) had
so far proved to have extraordinarily high levels of
I R I (one such case is demonstrated in Table 4).
5. The daily insulin injection(s) caused variations in
the I ~ I level (Fig. 6).
The composition of the total I g I is of great interest.
Most of the I R I is, naturally, bound to antibodies and
has hardly any biological effect in vivo or in vitro.
Vallance-Owen (1969) put forward the hypothesis t h a t hyperinsulinism plays a major role in
Acknowledgements. I wish to thank Jergen
Schlichtkrull, D. So., for his interest, constructive criticism and
valuable advice; Mrs. Ulla Dalai Larsen for preparing the
excellent 12hi-insulin; the staff of Hvidore Diabetes
Hospital for drawing most of the serum samples used in
this study; Mrs. Majken Petri Petersen, Mrs. Connie
Eriksen and Miss Lene I-Iansen for their excellent technical
assistance; and Mrs. Birgit Jensen for drawing the curves.
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