Determination of total serum insulin (IRI) in insulin-treated diabetic patients

Diabetologia, Aug 1972

A routine method is described for the determination of total IRI (imraunoreactive insulin) in insulintreated diabetics. The method involves an easy acid ethanol extraction, whereby antibody-bound IRI is dissociated and separated, together with the “free” IRI from the serum proteins and the antibodies. The recovery of IRI in this procedure is about 80%. After the separation, the isolated total IRI is measured in an immunoassay, using ethanol for the separation of free and antibody bound125I-insulin. In 169 diabetic patients treated with insulin in doses of from 6 to 120 units/day, the fasting serum total IRI was between 6 and 4374 μU/ml, with a mean of 392 μU/ml. During treatment with insulin, the level of total IRI 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 IRI occurred simultaneously with the formation of antibodies. Insulin-resistant patients showed very high IRI levels.

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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 insulinbehandelten Diabetikern 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 trationen. Introduction 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) after a few m o n t h s of t r e a t m e n t . The serum from these 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. Grodsky (1965) 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 3000 ~U/ml. 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 390 ~zU/ml. Pearson and Martin (1970) developed gel 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 insulin-treated persons. Isolation of total IR[ from serum I "IF" + IB-Ab I [ + HCI---~.pH -:~ I ''+ I + e t h a n o [ 1 + NaOH----~pH- 7 - 8 precipitate supernatant Fig. I 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, 1971) . 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 Diabetologia,Vo!. 8 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 immunoassayed. 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 immunoassayed. Results 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 hours. 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 temperature). 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 . ~ . 9 \ (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 3ooc 2000 1000 "~:. 5t0 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 (Jorgensen and Binder, 1966) , 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 Volund (1972) . 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 69.6 ~U/ml). 90 "% free 1251-insulin 8(1 70 60 50 -* , , M. 40' 0, 1,2,0 5 , , , 50 75 100 pU/rnl standard 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 concentra 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 8O 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 results. 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 test samples. IRI Glucose ~U/ml mg~ 600 500 o--o total IRI glucose 100 ~ 0 I r I 8 a.m. 12 4 ~' r 8 meals l 12 t 4 a.m. 8 i.u. Actrapid 12 i.u. Retard 8 LU. Retard Fig. 6 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 separation techniques. showed t h a t chickens treated with insulin for 19 weeks developed vascular lesions faster t h a n untreated chickens. Thus, the high total serum I R I found in diabetics could be contributory to the development of vascular complications in diabetes. for immunoassay. following: treated persons. The presence of antibodies and antibody-bound insulin in serum from insulin-treated 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 monocomponent-insulin (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 little from 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. 4. Insulin-resistant patients (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. Stout and 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. Berson , S.A. , Yalow , R. S. : The present status of insulin antagonists in plasma . Diabetes 13 , 247 -- 259 ( 1964 ). Grodsky , G.M. : Production of auto-antibodies to insulin in man and rabbits . Diabetes 14 , 396 -- 403 ( 1965 ). tIeding , L.G.: ~ simplified insulin radioimmunoassay method, in "Labelled Proteins in Tracer Studies" , Ed. L. Donate et M. Euratom , Brussels 345-- 350 ( 1966 ). -- Nielsen , A. Volund: Determination of free and antibody-bound I R I in serum from insulin-treated diabetic patients . Abst . in Exerpta Medics international congress series No. 140 , Sixth Congress of the International Diabetes Federation , Stockholm, 1967 . -- Determination of free and antibody-bound insulin in insulin-treated diabetic patients . Horm. Metab. l~es. 1 , i ~ 5 -- 146 ( 1969 ). Jorgensen , K. , Binder , C. : 12~I-insulinas a tracer of insulin in different chemical processes, in "Labelled Proteins in Tracer Studies" , Ed. L. Donate et M. Euratom , Brussels, 329 -- 333 ( 1966 ). Kirkham , K.E. , Hunter , W.M.: t~adioimmunoassay Methods, Churchill Livingstone, Edinburgh and London ( 1971 ). Ohneda , A. , Toyota , T. , Sate , S. , Yamagata , S. : Extraction of plasma insulin in radioimmunoassay for removal of nonspecific inhibitor and of circulating insulin antibody . T6hoku J. exp. Med . 109 , 75 -- 84 ( 1970 ). Pearson , M.J. , Martin , F . I . ~ . : The separation of total plasma insulin, from binding proteins using gel filtration : its application to the measurement of rate of insulin disappearance . Diabetologia 6 , 581 -- 585 ( 1970 ). Schlichtkrull , J., tteding, L.G., Christiansen , Aa.H., VMund , An. : Immunological aspects of insulin therapy. Paper presented at the V I I t h Acts endocrinologiea Congress , Copenhagen, Denmark ( 1971 ). Stout , I~.W. , Vallance-Owen , J :: Insulin and atheroma . Lancet 1969 I , 1078 -- 1080 . -- Development of vascular lesions in insulin-treated animals fed a normaI diet . Brit. med. J. 1970 III , 685 -- 687 . Volund , A. : Computerized calculation and control of radioimmunoassay or bioassays illustrated for the insulin assay . Paper to be published ( 1972 ). YMow , R.S. , Berson , S.A. : Immunoassay of endogenous plasma insulin in man . J. clin. Invest . 39 , 1157 -- 1175 ( 1960 ).


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Lise G. Heding. Determination of total serum insulin (IRI) in insulin-treated diabetic patients, Diabetologia, 1972, 260-266, DOI: 10.1007/BF01225569