Diabetes and hypoglycemia due to insulin antibodies

Diabetes and Hypoglycemia Due to Insulin Antibodies

JAMES H ANDERSON, Jr , MD WILLIAM Richmond,

G BLACKARD,

MD

Virginia

JOSE GOLDMAN,

MD.,

Ph D

ARTHUR H. RUBENSTEIN, M.D Chicago, Illinois

From the Departments of Medtcine, Medrcal College of Virgrnia, Richmond, Vrrginra and the Unrversity of Chrcago, Prrtzker School of Medicine, Chicago, Illinois This work was supported In part by U.S. Public Health Servrce Grants 5M09RR65, AM 18093 and AM 05227 Requests for reprints should be addressed to Dr W G. Blackard, Box 155, MCV Station, Richmond, Vtrginra 23298. Manuscript accepted July 26, 1977

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A patient with “autoimmune diabetes” with reactive hypoglycemia due to anti-insulin antibodies is described. The pathophysiology of the impaired carbohydrate tolerance and reactive hypoglycemia is illustrated by measurements of C-peptide, and free and antibody-bound insulin. Detailed analysis of her antibodies did not provide any evidence in favor of exogenous insulin immunization. Nevertheless, a possible cause of unsuspected insulin immunization was present in her history which should be considered in other reported cases of “autoimmune diabetes” due to insulin antibodies in which exposure to insulin cannot be documented. Autoimmunity has been considered a possible cause of some cases of diabetes. The increased association of autoimmune disorders, such as pernicious anemia and thyroiditis with diabetes [l-3] and islet cell antibodies in recent onset juvenile diabetes mellitus [4], suggests the possibility of an autoimmune destruction of the endocrine pancreas by a cell-mediated or delayed hypersensitivity reaction in a manner similar to the “insulitis” produced by injection of pancreatic islet preparations [5] The concept that diabetes might be caused by circulating antibodies to insulin has not been emphasized; in fact, it was specifically rejected as an important pathogenetic mechanism by Yalow and Berson [6]. Although the presence of anti-insulin antibodies is as common as the use of insulin itself, the presence of antibodies in the serums of insulin-treated diabetic patients is a result of therapy and is not usually considered important in the pathogenesis of diabetes. Anti-insulin antibodies are present in all persons who receive commercial insulin for a period of time, usually exceeding six weeks [7-91 Although nonspecific insulin-binding proteins have been described in both diabetic and nondiabetic subjects [lo], rarely have specific insulin antibodies been described in diabetic subjects not treated with insulin [ 111 Hirata et al. [ 121 were the first to describe insulrn antibodies in conjunction with hypoglycemic attacks and decreased glucose tolerance in a patient who had not previously received insulin injections Since that time there have been additional reports in the Japanese literature [ 13-151; more recently, Folling and Norman [ 161, and also Ohneda and associates [ 171 have presented extensive immunologic studies in patients suspected of having anti-insulin antibodies without known exposure to insulin. In this report we describe a similar patient with anti-insulin antibodies without evidence of previous insulin administration. C-peptide studies as well as measurements of free and antibodybound insulin (indices of pancreatic beta cell secretion) illuminate the pathophysiology of the reactive hypoglycemia and carbohydrate intolerance in this patient. In addition, special circumstances in this

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patient provide a logical hypothesis to explain the formation of anti-insulin antibodies in patients without history of insulin injections. CASE REPORT A 43 year old, married, white, woman of Honduran ancestory was admitted to the hospital for evaluation of hypoglycemic attacks. The patient had been in good health until one year prior to admission when she noted the onset of attacks characterized by light-headedness, dizziness, clammy sensations and tremulousness which were relieved by drinking or eating “something sweet.” These attacks would typically occur once or twice a day, usually 3’& to 4 hours after meals. The patient had noted similar, but milder, symptoms in the morning on awakening, but she reported that during the preceding two month period these morning episodes had become particularly severe, associated with headaches, dizziness and sweating. Two weeks prior to admission, the patient had an oral glucose tolerance test which revealed the following plasma glucose levels: fastng, 84 mg/dl; 1 hour, 296 mg/dl; 2 hours, 305 mg/dl; 3 hours, 186 mg/dl; 4 hours, 30 mg/dl; and 5 hours, 40 mg/dl She had become symptomatic at 4 and 5 hours with complaints identical to those with which she presented Following this test, an 1,800 calorie diabetic diet and chlorpropamide therapy (Diabenese@ 250 mg orally, each morning were instituted. The patient continued the medication for several days but noted accentuation of her hypoglycemic symptoms. One early morning attack was particularly severe resulting in confusion and disorientation. After this episode, the ingestion of chlorpropamide was discontinued The patient’s weight had been stable at 62 kg. Polyuna, polydipsia or changes in appetite were denied. Systemic and endocrine reviews of systems were essentially nc contributory except for frequent bladder infections. The patient had always had irregular periods and was PARA 2-2-O. Although no previous psychiatric problems were recalled, the patient stated she had always been high strung and nervous Past history revealed an ovarian biopsy in 1953 (results unknown), an abdominal operation with an appendectomy and resection of a segment of small intestine in 1958, and a total abdominal hysterectomy and bilateral salpingooophorectomy in 1964 for uterine fibroids and “abnormal” ovaries. The patient had been receiving cyclic estrogen (Premarin@) therapy since the oophorectomy. She was questioned in detail about possible exposure to insulin and any previous parenteral therapy Although she denied any insulin injections and was considered incapable of giving herself injections, her mother, an insulin-requiring diabetic, had given her vitamin Bj2 and estrogen injections with glass syringes (boiled between usage) at approximately monthly intervals over several years until her hospitalization A strong family history of diabetes mellltus existed, with her mother, maternal grandmother and maternal uncle having diabetes. The patient weighed 9 pounds, 8 ounces at birth. She had six siblings, two of whom died, one at birth and one of a lung abscess. The other four siblings are living and well, without known diabetes.

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physical examination revealed a cooperative and alert Honduran woman. Blood pressure was 150/100 mm Hg and pulse rate 84/min. Body weight was 62 kg. The skin was normal in texture and moisture, and demonstrated no evidence of old or recent needle marks. Funduscopic examination revealed only slight arteriolar constriction. Ear, nose and throat examination disclosed no abnormalities. The thyroid was of normal size. Heart, lungs, abdomen and extremities were within normal limits as was the neurologic examination An intravenous tolbutamide (Orinase@)test was performed because of the history compatible with early morning hypoglycemia. The plasma glucose decreased from 77 mg/dl to its lowest value of 41 mg/dl at 150 minutes. Plasma immunoreactive insulin values at all times during the intravenous tolbutamide test including the basal level were “greater than 1,000 PUlml.” The extremely high immunoreactive insulin values suggested an artifact so qualitative aspects of her plasma insulin were investigated. One milliliter samples of both fasting plasma and plasma obtained 5 minutes after tolbutamide administration were enriched with 13’1and placed on a G50 Sephadexm column for gel filtration. One milliliter fractions of the eluted material were collected and assayed for immunoreactive insulin. The results from the fasting sample are demonstrated in Figure 1. The material eluted in the first third of the distance from the albumen peak to the salt peak is, by convention, “big insulin” or pro-insulin and the material following that is “little insulin” or the usual physiologic moeity of 6,000 molecular weight immunoreactive insulin. The immunoreactive material in the patient’s samples eluted prior to and with the albumen peak indicating that the immunoreactive material being measured had a

120-

IOO-

, d

5

lo

I5 20 1 ml fractions

-

25

30

Figure 7. Gel filtration on G50 Sephadex of fasting plasma with “immunoreactive insulin”determined in each fraction. Most of the immunoreactive material eluted in the void volume indicating a molecular weight greater than 50,000.

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TABLE I

5 Hour Oral Glucose Tolerance PfaslM6iucoss (Wdf)

TIW (hr) Fasting l/2 1 2 3 4 5

70 165 209 222 125 61 41

Test (40 g/m*) TotalIB Wmi)

FreeIRI Nmi)

1,297 1,759 2,846 2,982 2,809 1,979 1.414

54 16 3 43 7 74.3 37 7 14 3 70

TotalCPA (WV 16 20 25 28 25 25 22

2 2 1 9 7 3 6

IRG (Wml) 100 100 100 50 120 100 100

NOTE. IRI = immunoreactive rnsultn;CPR = C-peptide immunoreactivrty, IRG = immunoreactive glucagon

molecular weight greater than 50,000. This high molecular weight immunoreactive material is compatible with insulin bound to large proteins or an artifact in the radioimmunoassay produced by insulin antibodies migrating in this position Additional proof of the presence of an insulin-binding protein in the serum was obtained by incubating 1251-labelled insulin with the patient’s serum and subjecting the incubation mixture to gel filtration. The majority of radioactivity was eluted from the G50 Sephadex column in the void volume indicating a molecular weight greater than 50,000 To further characterize the nature of the insulin-binding proteins, the patient’s serum was incubated for 24 hours with labelled porcine insulin. Antihuman globulin, produced In rabbits specific for either IgG, IgM, IgA or IgD, was then added to the incubation as a second antibody. After an additional 24 hours’ incubation the tubes were centrifuged and the precipitate counted in the usual manner for a double antibody radioimmunoassay Significant binding of the tracer by IgG was demonstrated. Small, but significant binding of tracer by IgM was also observed. Having demonstrated the antibody nature of the insultnbinding protein, we performed additional tests to determine if the antibody production occurred de novo or in response to inadvertent, unsuspected prior insulin administration Skin tests to beef and pork protein antigens (insulin) were negative. Serums were tested for binding with bovine and porcine C-

TABLE II

Intravenous

Time (min) 0 5 10 15 20 30 45 60 90 120 150 180

Tolbutamide

peptide with no binding demonstrated. In addition, serums were studied for binding to both 1251porcine and 1251bovine proinsulin. These tracers were completely displaced by cold insulin indicating that specific C-peptide directed antibodies were not present. Also, the same technic (using 1251-labelled glucagon) failed to demonstrate any antibodies against glucagon. After extraction of antibodies specific for insulin by a coupled insulin-sepharose technic, the serum was again tested for binding of beef and pork proinsulin tracer. Although significant binding of tracer has been demonstrated by serums from 90 per cent of insulin-treated diabetic subjects by this technic, the extracted serum from our patient failed to bind beef or pork protnsulin tracers C-peptide, as well as free and antibody-bound insulin, (total Insulin minus free insulin) was determined to assess beta cell activity during a 5 hour glucose tolerance test (Table I) and a repeat intravenous tolbutamide test (Table II). During the latter test, both free C-peptide, plus total C-peptide immunoreactivity (free C-peptide plus proinsulin bound to antibody) were determined. (In normal fasting man, C-peptide levels are 1.5 to 2.8 ng/ml risrng to 4 to 8 ng/ml after a glucose load ) Insulin was determined by radioimmunoassay using a double-antibody method [ 181 C-peptide reactivity was measured by a similar technic using 1251-labelled synthetic tyrosylated C-peptide as the tracer, and a rabbit antiserum

Test (2120175)

Plasma6iucoss (mg/dl)

Total IRI WJ/mi)

Free Ml (Nmi)

Total CPA Wmi)

Fraa CPA Wmi)

71 68 65 63 61 58 50 46 48 45 43 45

795 1,092 1,103 1,271 1,800 1,414 1,344 1,250 1,218 1,085 984 861

86 11 6 104 13 9 17.9 165 14 0 143 11.8 10 0 94 11 1

6.45 8.65 9 70 10 45 9 30 9.25 8 80 a 00 7 20 6.90 7 10 6 65

1 22 3 85 4 57 5 15 4 02 3 69 2 92 2 75 2 44 191 2 04 1 62

NOTE: IRI = rmmunoreactwity insulin; CPR = C-peptrde immunoreactivity.

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(final dilution 1:15,000) to synthetic human C-peptide (both kindly furnished by Dr. N. Yanaihara) [ 191. The assay was calculated using a human C-peptide standard extracted from the pancreas removed at autopsy. Serum free C-peptide was determined after precipitating the proinsulin bound to circulating insulin antibodies with polyethylene glycol. Serum “free” insulin was estimated after precipitating the insulin bound to circulating insulin antibodies with polyethylene glycol, and the total insulin content of plasma was measured by first separating the antibody-bound insulin with 0.1 N hydrochloric acid and then removing the antibodies by polyethylene glycol precipitation [ 201. Since the time of the original testing, the patient has been maintained on a frequent feeding, low carbohydrate diet which she tolerates well with only occasional episodes of hypoglycemic symptoms. COMMENTS

The presence of anti-insulin antibodies in the serum of patients treated with insulin is anticipated and can be demonstrated to occur in diabetic subjects who have received insulin injections for over six weeks. Usually the antibody titers are low, resulting in only a modest increase in insulin requirements. Few cases have been reported of the presence of insulin antibodies in patients not known to have received an insulin injection. The majority of cases (seven of eight) have been Japanese [ 12- 15,171 with one case being reported from Norway [ 161. In all of these cases, the patients presented with either diabetes or reactive hypoglycemia or both. The subject of this report also exhibited abnormal glucose tolerance and reactive hypoglycemia (Table I) due to anti-insulin antibodies. C-peptide determinations were performed in this patient in hopes that they would be a more reliable indication of beta-cell secretion than immunoreactive insulin measurements in the presence of insulin antibodies. Previous studies have shown that insulin and C-peptide are secreted in equimolar quantities and that C-peptide measurements correlate well with insulin secretion in normal subjects [ 191. However, in patients with anti-insulin antibodies, significant amounts of proinsulin (containing the Cpeptide within its structure) may bind to insulin antibodies. Proinsulin-antibody complexes have a prolonged half-life and, therefore, distort the precise relationship between circulating C-peptide levels and pancreatic beta-cell secretory response. Thus the markedly increased total C-peptide immunoreactivity observed during the oral glucose tolerance test (Table I) reflects the sum of both free C-peptide and antibody-bound prornsulin secreted over a prolonged period of time. That most of the basal immunoreactive C-peptide represents antibody bound proinsulin can be observed from the data obtained during the tolbutamide test (Table II). Only a small amount of the total C-peptide immunoreactivity in the basal state is free C-peptide. How-

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ever, under conditions of acute secretion, the increase in total C-peptide immunoreactivity can be mainly attributed to free C-peptide. Although basal free insulin values are relatively normal (Tables I and II), total insulin values are greatly increased indicating a large amount of insulin bound to antibody. The results demonstrated in Table I illustrate the pathophysiology of the carbohydrate intolerance and reactive hypoglycemia in our patient. Plasma glucose and free insulin are normal in the fasting state, but a large amount of insulin is present in the plasma bound to antibody. Although the fasting free insulin and free C-peptide reactivity levels were normal at the time of the oral glucose tolerance test and tolbutamide tests, the fasting hypoglycemic symptoms which occurred at other times suggest dissociation of insulin bound to antibody complexes as a possible pathogenic mechanism. As glucose is administered, the free and antibody bound insulin (reflected by total immunoreactive insulin) increases. Even though the increased insulin secretion would ordinarily result in a rapid return of plasma glucose to normal, most of the insulin is bound to antibody and is biologically ineffective resulting in a diabetic glucose pattern. Because of the persistent hyperglycemia, insulin secretion continues and results in reactive hypoglycemia at 5 hours. Hypoglycemia may be attributed to prolonged beta-cell stimulation by hyperglycemia as well as to dissociation of insulin from the greatly increased antibody-bound insulin pool. The increase in total C-peptide immunoreactivity after the administration of glucose reflects the heightened beta-cell secretory activity. Plasma samples during the tolbutamide test (Table II) were analyzed for free C-peptide as well as total C-peptide immunoreactivity (C-peptide plus proinsulin bound to antibody). As can be seen from Table II, most of the C-peptide immunoreactivity represents material bound to larger proteins (i.e., proinsulin bound to insulin-binding antibodies). Free C-peptides levels were not excessively elevated either before or after tolbutamide stimulation. Free C-peptide levels may rise to 5 to 7 ng/ml during tolbutamide testing in control subjects, and the contribution of proinsulin to the total C-peptide immunoreactivity levels is less than 3 per cent in these persons [21]. The presence of insulin antibodies suggests previous immunization, but thorough questioning failed to elicit such a history. Although insulin injections were specifically denied, inadvertent insulin administration should be considered a possibility in that the patient lived next door to her diabetic mother who gave her injections of estrogens and vitamin B12. It is extremely difficult to distinguish anti-insulin antibodies produced as a result of previous immunization from those produced to a patient’s own insulin without

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exogenous stimulation. A review of the literature indicates that one cannot clearly differentiate the source of antigenic stimulation on the basis of monoclonal versus polyclonal immunoglobulin patterns or specific immunoglobulin class. In a report by Faulk et al. [22] serums from 50 insulin-treated diabetic subjects were studied; the most common type of anti-insulin immunoglobulin found was IgG (the level of which correlated well with anti-insulin titers). Antibodies of the IgM class were rarest with only one patient in Faulk’s series having detectable quantities [22]. IgM class antibodies were also least common in a study by Yagi et al. [ 231 In addition, the presence of IgM class anti-insulin antibodies were detected before but not after the 19th day of insulin therapy in a diabetic subject studied by Dolovich and co-workers [24]. Although Nakagama and Ohneda argue that the presence of only IgG is evidence for the endogenous stimulation of antibody production [ 17,251, other investigators have shown that exogenous insulin administration may also produce antibodies of only one class [26]. A marker for identifying previous exogenous immunization would be the presence of antibodies to beef or pork C-peptide or proinsulin since proinsulin and related proteins have been shown to be contaminants of commercial insulin [27]. Several workers have demonstrated that C-peptide and proinsulin have common antigenic determinants located in the C-peptide region and that C-peptide and proinsulin compete in specific immunosystems. Although only minor immunologic differences exist among human, bovine and porcine insulin, connecting peptide segments display greater immunologic differences. Previous studies have shown only slight cross reactivity among C-peptides and proinsulin of different species [28] Failure to demonstrate antibodies to beef or pork C-peptide or the C-peptide region of proinsulin may not, however, conclusively prove an autoimmune nature. Although most diabetic subjects treated persistently with insulin exhibit specific antibodies to these foreign antigenic determinants [29-321, intermittent or infrequent injection of commercial insulin might not result in significant titers of such antibodies, One might postulate that prior injection of commercial insulin may have given rise to insulin antibodies which could possibly be sustained by antigenically similar endogenous insulin, whereas antibodies to the C-peptide region of the original immunogenic material may not have been stimulated or may have decreased to nondetectable levels. Evidence in our case for de novo productron of antiinsulin antibodies would rnclude the absence of antibodies to both bovine and porcine C-peptide and proinsulin, the absence of skin reaction to beef and pork antigens, and the persistence of an immunoglobulin

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class (IgM) not usually seen with exogenously produced antibodies. On the other hand, the history of having received vitamin B12 injections from her mother, an insulin-requiring diabetic subject, raises the possibility of inadvertent immunization by contamination of glass syringes used. Reviewing the literature, seven of the eight cases of “autoimmune diabetes” due to insulin antibodies, were reported from Japan, the remaining one from Norway. During the time period of these reports, some Japanese physicians were known to administer hormones, liver extracts, vitamins and other parenteral preparations to their nondiabetic patients, in addition to providing insulin injections for diabetic patients. These reports also occurred prior to the widespread use of disposable syringes in Japan It is conceivable that the production of insulin antibodies in our patient and possibly in some of the others reported on might be due to insulin contamination of glass syringes, which even washed and autoclaved (or boiled in water), retained enough material that was antigenically, but not biologically active, to induce antibody production in a manner identical to the heat killed vaccines in use today. Differentiation of endogenously produced antibody from exogenously stimulated antibody production is virtually impossible, so that this hypothesis cannot be proved in these reported cases. The possibility of producing “autoimmune diabetes” must be considered, however, in patients who are treated with insulin because of transient diabetes (or stress diabetes), the misdiagnosis of diabetes, nursing errors, insulin shock therapy or provocative hormone testing using insulin-induced hypoglycemia. The latter procedure seems less likely to be even a rare cause of immunization since repeated testing is usually not performed and since the polypeptide hormone is given intravenously instead of subcutaneously which would be a more efficient method for immunization. “Autoimmune diabetes” requires a more precise definition of the disease process. In some reports “autoimmune diabetes” has referred only to production of anti-insulin antibodies in the absence of exogenous immunization. Whether the antibody formation in our case was initiated by exogenous administration of antigen or initiated by abnormal immunologic reaction to endogenous insulin, the continued production of antibody might be stimulated by the patient’s endogenous insulin release, thus qualifying as an autoimmune state. The important consideration in our patient, even in the uncertainty of endogenous versus exogenous stimulation of antibody production is the evolution of glucose intolerance from a demonstrable cause, illustrating the heterogenous nature of the carbohydrate tolerance.

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REFERENCES

2

3

10. 11

12

13

14.

15.

16

17

Stauffacher W, Renold AE. Pathophysiology of drabetes mellitus Joslin’s Diabetes Mellitus (Marble A, Whrte P, Bradley RF, Krall LP, eds), Phrladelphia, Lea & Febiger, 1971, p 35 Ungar B, Stocks AE. Martin FIR, et al . Intrinsic-factor Antibody, Parietal-cell Antibody, and Latent Pernrcrous Anemia in Diabetes Mellitus. Lancet 2 415, 1968. Landrng BH, Pettit MD, Wrens RL, et al : Anti-thyrord Antibody and Chronic Thyroiditis in Diabetes J Clin Endocrinol Metab 23: 119, 1963. Lendrum R, Walker G, Gamble DR: Islet-cell antibodres In juvenile diabetes mellrtus of recent onset Lancet 1 880, 1975 Heydrnger DK, Lacy PE: Islet changes In the rat following injection of homogenized islets Diabetes 23: 579, 1974. Berson SA, Yalow RS. Some current controversres in diabetes research. Diabetes 14. 549, 1965 Deckert T Insulin Antibodies, Copenhagen, Munksgarrd, 1964 Berson SA, Yalow RS Quantitative aspects of the reaction between insulin and insulrn-binding antibody J Clan Invest 38: 1996, 1959. Berson SA, Yalow RS, Bauman A, et al . Insulin 13’1 metabolism in human subjects: demonstratron of rnsulrn binding globulin In the circulation of insulin-treated subjects J Clin Invest 35: 170. 1956 Pav J, Jezkova Z, Skrha F Insulin antrbodres Lancet 2 221, 1963. Penchev I, Andrew D, Ditzov S. Insukn-precipitating antibodies in Insulin-treated and untreated diabetic patients Drabetologia 4. 164, 1968 Hirata Y, lshizu H, Ouch1 N, et al. Insulin autoimmunity in a case with spontaneous hypoglycemia. Tonyobyo 13 312, 1970 (Japanese). Sato T, Saito T, Hanma K, et al . A case with considerable hyperrnsulinemia and decreased glucose tolerance. Tonyobyo 15 (suppl). 176, 1972 (Japanese) Shida N, Kawarazakr T, Kudo M, et al.: A Newborn infant with hypoglycemia due to antibodies to endogenous insulin observed in the patient and her mother Tonyobyo 15 (suppl). 177, 1972 (Japanese). Hrrata Y, Nrshrmura H, Tominaga N, et al On Insulin Autoimmune Syndrome Tonyobyo 15 (suppl): 179,1972 (Japanese) Folkng I, Norman N: Hyperglycermia, hypoglycemia attacks and production of anti-insulin antibodies without previous known immunization: immunological and functional studies in a patient Diabetes 21: 814, 1972. Ohneda A, Matsuda K, Sato M, et al . Hypoglycemra due to

18. 19

20

21.

22. 23.

24

25

26.

27

28.

29

30

31 32.

May 1978

apparent autoantibodies to insulin. characterization of insulin-binding protein. Diabetes 23. 41, 1974. Morgan CR, Lazarow A: Immunoassay of insulin: two antibody system. Diabetes 12: 115, 1963 Horwitz DL, Starr JI, Mako ME, et al Proinsulin. insulin and C-peptide concentration in human portal and peripheral blood. J Clin Invest 55. 1278, 1975 Nakagawa S, Nakayama H, Sasaki T, et al . A simple method for the determrnatron of serum free Insulin levels in insulin-treated patients. Diabetes 22: 590, 1973. Sandler R, Horwitz DL, Rubenstern AH Hypoglycemia and endogenous hyperinsulrnism complrcatrng diabetes mellrtus Am J Med 59: 730, 1975. Faulk W, Karam J, Fudenberg H: Human anti-insulin antrbcdres J lmmunol 106_1112, 1971 Yagi Y, Maier P, Pressman D, et al: Multipkcity of insulinbinding antibodies In human sera J lmmunol 90: 760, 1963. Dolovich J, Schnatz J, Rersman R, et al . Insulin allergy and insulin resistance. Case report with immunologic studies J Allergy 46: 127, 1970. Nakagawa S, Shrda N, Kudo M: A study of the insulin antibody of insulin autoimmune syndrome Tonyobyo 15: 409, 1972 (Japanese) Faulk W, Tomsovie E, Fudenberg H lnsukn resistance in JUvenile diabetes mellitus Immunological studies. Am J Med 49: 133.1970 Chance RE: Chemical, physical and immunological studies on porcine and related polypeptides. Proceedings of the 7th Congress International Diabetic Federation, Buenos Aires, 1970 Excerpta Medica International Congress Series No. 231, p 292 * Steiner DF, Oyer P, Cho S, et al Structural and immunological studies on human proinsulin Proceedings 7th Congress International Diabetes Federation IDF, Buenos Aires, 1970, Excerpta Medrca International Congress Series No 231, ~281 Rubenstein A, Steiner D, Cho S, et al.. Immunological properties of bovine proinsulin and related fractions Diabetes 18: 598, 1969 Schlichtkrull J, Brange J, Christiansen A, et al.: Clinrcal aspects of insulin-antigenicity Diabetes 21 (suppl 2) 649, 1972 Kumor D, Miller LV: Proinsulin-specific antibodies in human sera. Diabetes 22. 361, 1973. Kerp L, Steinhilber S, Kasemrr H Besrtzt Die ProinsulinVereinigung Kommerzieller lnsulinpraparate Bedeurung fur die Stimulrerung van Insulin-Antikorpern Klin Wochenschr 48 884, 1970

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