Antibody-bound Insulin Research Articles

Assessment and Management of Anti-Insulin Autoantibodies in Varying Presentations of Insulin Autoimmune Syndrome.

ContextInsulin autoimmune syndrome (IAS), spontaneous hyperinsulinemic hypoglycemia due to insulin-binding autoantibodies, may be difficult to distinguish from tumoral or other forms of hyperinsulinemic hypoglycemia, including surreptitious insulin administration. No standardized treatment regimen exists.ObjectivesTo evaluate an analytic approach to IAS and responses to different treatments.Design and SettingObservational study in the UK Severe Insulin Resistance Service.PatientsSix patients with hyperinsulinemic hypoglycemia and detectable circulating anti–insulin antibody (IA).Main Outcome MeasuresGlycemia, plasma insulin, and C-peptide concentrations by immunoassay or mass spectrometry (MS). Immunoreactive insulin was determined in the context of polyethylene glycol (PEG) precipitation and gel filtration chromatography (GFC). IA quantification using ELISA and RIA, and IA were further characterized using radioligand binding studies.ResultsAll patients were diagnosed with IAS (five IgG, one IgA) based on a high insulin/C-peptide ratio, low insulin recovery after PEG precipitation, and GFC evidence of antibody-bound insulin. Neither ELISA nor RIA result proved diagnostic for every case. MS provided a more robust quantification of insulin in the context of IA. One patient was managed conservatively, four were treated with diazoxide without sustained benefit, and four were treated with immunosuppression with highly variable responses. IA affinity did not appear to influence presentation or prognosis.ConclusionsIAS should be considered in patients with hyperinsulinemic hypoglycemia and a high insulin/C-peptide ratio. Low insulin recovery on PEG precipitation supports the presence of insulin-binding antibodies, with GFC providing definitive confirmation. Immunomodulatory therapy should be customized according to individual needs and clinical response.

Measurement of insulin in human sera using a new RIA kit. 2. Determination of free and total insulin--correlations to insulin antibody levels.

Using the micro-scale modification of a newly developed RIA kit for insulin, we established methods for the determination of free and total insulin in serum of insulin-treated diabetics. Precipitation with polyethylene glycol 6000 or acid alcohol extraction of sera was carried out to remove or to dissociate antibody-bound insulin. Both assays permit precise and accurate measurement of either serum insulin fraction. In 50 diabetic sera with tracer insulin binding of 0-97%, free (after equilibration of the sera at 37 degrees C) and total insulin levels as well as insulin antibody binding parameters were determined. There was a good correlation of free to total insulin levels with maximally 10-fold higher values of total insulin. Both free and total insulin were found to be correlated with the ability of the serum to bind insulin. In detail, binding affinities (i.e. the reciprocal of equilibrium dissociation constants) and binding site concentrations were evaluated which were shown to be positively correlated with free and total insulin levels as well. From these data we conclude that insulin antibodies in the serum may accumulate therapeutic insulin and function as a depot for delivering insulin in insulinopenic episodes (Keilacker et al., 1982 and 1986).

Treatment With Insulin and Its Analogs in Pregnancies Complicated by Diabetes

Before the advent of insulin in 1922, 90% infant mortality rate and a 30% maternal mortality rate. As late as 1980, physicians were still counseling diabetic women to avoid pregnancy. This philosophy was justified because of the poor obstetric history in 30–50% of diabetic women. Improved infant mortality rates finally occurred after 1980, when treatment strategies stressed better control of maternal plasma glucose levels, once self-monitoring of blood glucose and A1C became available. As the pathophysiology of pregnancy complicated by diabetes has been elucidated and as management programs have achieved and maintained near-normoglycemia throughout pregnancy complicated by diabetes, perinatal mortality rates have decreased to levels seen in the general population (2–5). This review reports the literature on the safety and efficacy of insulin analogs in pregnancy and thereby enables the clinician to choose the optimal insulin treatment protocol to achieve and maintain normoglycemia throughout pregnancies complicated by diabetes. Maternal glucose freely crosses the placenta. Maternal insulin does not cross the placenta unless it is bound to IgG antibody, which carries it through the placenta or insulin is forced through the placenta by high perfusion (6,7). Diabetic fetopathy is thought to be the result of fetal hyperinsulinemia (1–9). Thus, our treatment must be designed to normalize maternal blood glucose concentrations without the use of exogenous insulins that cross the placenta. Placental transfer of insulin complexed with immunoglobulin has also been associated with fetal macrosomia in mothers with near-normal glycemic control during gestation. Menon et al. (8) reported that antibody-bound insulin transferred to the fetus was proportional to the concentration of antibody-bound …

Development and validation of radioligand binding assays to measure total, IgA, IgE, IgG, and IgM insulin antibodies in human serum.

Radioligand binding assays for total and Ig classes of insulin antibodies (IAB) were developed and validated. For each assay, insulin-extracted serum samples were incubated with radiolabeled insulin in the presence and absence of high levels of unlabeled insulin to determine nonspecific binding and total binding, respectively. To measure total IAB, antibody-bound insulin was precipitated with a polyethylene glycol solution, washed, and counted in a gamma-counter. To measure IgG IAB, samples were treated with protein G-Sepharose beads, centrifuged, washed, and counted. For the measurement of IgA, IgE, and IgM IAB, IgG was removed from the samples and treated with anti-IgA, -IgE, or -IgM conjugated to Sepharose beads, centrifuged, washed, and counted. The acid/charcoal extraction of bound and unbound insulin from serum samples was optimized. Specificity and binding capacity of the protein G and antibody-bound beads were evaluated and optimized. The linear region of the total and IgG IAB assays was determined using serum samples containing high levels of insulin antibodies. The limit of quantitation, limit of detection, and precision for all the assays were also determined.

Anti-insulin antibodies and birth weight in pregnancies complicated by diabetes.

Free insulin cannot cross the placenta but insulin complexed to anti-insulin antibodies has been demonstrated in cord blood. We studied whether antibody-bound insulin in diabetic patients can evoke fetal macrosomia independently of maternal metabolic control. In 457 non insulin-treated controls and 173 insulin-treated diabetic patients we measured 1187 anti-insulin antibody levels and maternal blood glucose, maternal fructosamine, cord blood insulin, cord blood C-peptide, cord blood fructosamine and amniotic fluid insulin. Mean anti-insulin antibody levels in maternal blood and cord blood were significantly higher in insulin treated diabetic patients (4.6 and 5.4 U/ml) than in controls (1.8 and 1.7 U/ml) with maxima of 89.2 in maternal and 120.0 U/ml in cord blood, respectively. In insulin treated diabetic patients 16.6% (maternal blood) and 22% (cord blood) anti-insulin antibody levels were above the 97th percentile. There was a high significant correlation between maternal and cord blood anti-insulin antibodies (R = 0.987, P = < 0.0001), but no correlation of anti-insulin antibodies with maternal (glucose, fructosamine) or fetal (insulin, C-peptide, and fructosamine in cord blood, amniotic fluid insulin) metabolic parameters. While maternal and fetal metabolic parameters correlated with birth weight neither maternal nor cord blood anti-insulin antibody levels correlated with birth weight. These findings do not support the hypothesis that maternal anti-insulin antibodies independently influence fetal weight.

Transplacental Passage of Insulin in Pregnant Women with Insulin-Dependent Diabetes Mellitus

Fetal macrosomia occurs despite nearly normal maternal blood glucose levels in women with diabetes treated with insulin. We examined the hypothesis that it may be caused by insulin transferred as an insulin-antibody complex from the mother to her fetus. We adapted and validated a method based on high-performance liquid chromatography and used it to quantitate insulin in small volumes (0.5 to 1.0 ml) of cord serum from 51 infants born to mothers with insulin-dependent diabetes mellitus. In mothers receiving only human insulin (n = 6), only human insulin was detected in cord serum. Of the remaining 45 infants, whose mothers received animal insulin during pregnancy, 28 (group 1) had levels of animal (bovine or porcine) insulin (mean [+/- SE], 707 +/- 163 pmol per liter) that constituted 27.4 +/- 2.5 percent of the total insulin concentration (2393 +/- 500 pmol per liter) measured in the cord serum. The cord-serum insulin concentration in the remaining 17 infants (group 2), in whom only human insulin was detected (381 +/- 56 pmol per liter), was only 15 percent of that in group 1 (P less than 0.001). There was a significant correlation between the maternal and the cord-serum concentrations of anti-insulin antibody and the concentration of animal insulin in the baby (r = 0.77, P less than 0.01, and r = 0.76, P less than 0.001, respectively), suggesting that the animal insulin was transferred as an insulin-antibody complex. In group 1 the mean concentration of animal insulin in cord serum was higher in the 12 infants with macrosomia than in the 16 infants without the condition (1113 +/- 321 vs. 402 +/- 110 pmol per liter; P less than 0.05), and the concentration of animal insulin in cord serum correlated with birth weight (r = 0.39, P less than 0.05). The maternal glycosylated hemoglobin values and the incidence of respiratory distress syndrome were similar in groups 1 and 2. Considerable amounts of antibody-bound insulin are transferred from mother to fetus during pregnancy in some women with insulin-dependent diabetes mellitus; the extent of transfer correlates with the maternal concentration of anti-insulin antibody. The correlation between macrosomia and the concentrations of animal insulin in cord serum indicates that the transferred insulin has biologic activity and suggests that the formation of antibody to insulin in the mother is a determinant of fetal outcome independent of maternal blood glucose levels.

Patterns of Hyperinsulinaemia in Type 1 Diabetic Patients With and Without Nephropathy

The prevalence and patterns of insulinaemia in five groups of patients with Type 1 diabetes have been reinvestigated using a free insulin assay which minimizes in vitro redistribution of the free and antibody-bound insulin components. In 18 diabetic patients managed by conventional insulin injection treatment and 19 patients treated by continuous subcutaneous insulin infusion (CSII), the mean 24-h serum free insulin level exceeded a non-diabetic reference range in 78% (injections) and 68% (CSII). Twenty-four-hour profiles showed that hyperinsulinaemia occurred in the basal state before meals and at night, but not immediately post-prandially. The serum free insulin concentration at 0800 h, 1200 h, and 1600 h was significantly (p less than 0.001) correlated with mean 24-h free insulin in injection-treated and CSII patients, and samples at one of these time-points may thus provide a simple, single measure of integrated insulinaemia for population studies. There was no significant difference in 24-h mean free insulin levels in 7 patients randomly crossed-over between injection treatment and CSII, but the profiles had a more physiological pattern during CSII, with a mean post-prandial serum free insulin level which was significantly higher on CSII than injections (mean +/- SE = 37.2 +/- 3.1 vs 26.9 +/- 2.5 mU l-1, p = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Major malformations in infants of IDDM women. Vasculopathy and early first-trimester poor glycemic control.

From animal and in vitro studies, it has been suggested that high environmental glucose, ketone, or insulin concentrations and low glucose or insulin concentrations may be etiologic factors for congenital malformations (CMs) in infants of diabetic mothers (IDMs). Transplacental passage of antibody-bound insulin has been demonstrated in humans. Controversy exists regarding the pathophysiology of CMs in human insulin-dependent diabetes mellitus (IDDM) pregnancies. We hypothesized that CMs in IDMs are associated with maternal vasculopathy, poor first-trimester glycemic control (i.e., hyper- and/or hypoglycemia), advanced White class, and high insulin requirements. We studied 165 first pregnancies of women with IDDM from 1978 to 1986. The goals of glucose control were a fasting blood glucose of less than 100 mg/dl and a 90-min postprandial blood glucose of less than 140 mg/dl. Insulin requirements, body weight, and pre- and postprandial blood glucose were recorded at weekly clinic visits. Maternal blood HbA1 was measured on entry and every 4 wk to confirm that adequate glycemic control was achieved. Women who enrolled in the project were interviewed during gestation by a geneticist/dysmorphologist who obtained genetic and environmental histories using a standard questionnaire. All live-born infants and stillbirths were examined. Each live-born infant was assessed systematically by two independent examiners, a neonatologist and a geneticist/dysmorphologist; examination with standardized checklists was performed in the newborn nursery as soon after birth as was practical. In first pregnancies in the study, there were 13 IDMs with major CMs (7.9%).(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous hypoglycemia and insulin autoantibodies in a patient with Graves' disease

A 31-year-old woman with Graves' disease developed fasting hypoglycemia after treatment for 3 weeks with methimazole. Although the patient had not received exogenous insulin, high titers of insulin autoantibodies were found in serum and large amounts of total and free insulin (1550 and 82 microU/ml, respectively) and C-peptide reactivity (CPR, 22 ng/ml) were detected in serum. After glucose loading, blood glucose and total insulin levels increased abnormally. The immunoglobulin class of the autoantibodies was IgG and the light chains were of the kappa type. The titers of insulin autoantibodies, elevated serum total and free insulin, and CPR levels decreased gradually, but insulin autoantibodies and elevated insulin levels were still present in the serum 8 months after the episode of hypoglycemia. These findings suggest that the patient's fasting hypoglycemia was due to excess free insulin released from antibody-bound insulin, and that methimazole might play a role in the initiation of production of insulin autoantibodies.

Free insulin profiles in insulin-dependent diabetics treated with one or two insulin injections per day.

Twenty-four hour profiles of free insulin and blood glucose were determined in 12 healthy controls and 10 insulin-dependent diabetics treated with insulin regimens based on intermediate-acting insulin injected subcutaneously once or twice a day. The diabetics were ambulatory and in a good glycemic control, i.e. without hyperglycemic symptoms or frequent hypoglycemias and with HbA1 less than 9% (reference value 5.9-7.8%). Body weight was normal and median age (32 years) was the same in both groups. Free insulin was determined after polyethylene glycol precipitation of antibody-bound insulin. The controls had a low basal insulin level (median fasting value 3.9 mU/l) and postprandial peaks with a maximum within 30-60 min. There was no rise in plasma free insulin or blood glucose in the early morning hours. The free insulin profiles in the diabetics were highly unphysiological with hyperinsulinemia between the meals and during the night. The highest plasma free insulin value during the 24 hours was reached before lunch (approximately 5-fold compared to normals, p less than 0.01). Postprandially the free insulin concentrations did not reach the peak levels of the normals. After breakfast, blood glucose rose considerably in the diabetics (p less than 0.02 compared to normals) while the rise after lunch and dinner was not higher than in the healthy controls. The difficulties in glycemic control in the diabetic group, i.e. a blood glucose rise after breakfast and hypoglycemias in some patients, could largely be explained by the unphysiological insulin profiles.

Sample preparation and radioimmunoassay for circulating free and antibody-bound insulin concentrations in insulin-treated diabetics: a re-evaluation of methods.

Methods for blood sample preparation and radioimmunoassay of free and antibody-bound serum or plasma insulin concentrations, using polyethylene glycol (PEG) solution to precipitate bound insulin, have been evaluated and compared. Method A was rapid mixing of whole blood with PEG, followed by separation of bound insulin by centrifugation into Ficoll; method B was rapid PEG addition to whole blood, followed by centrifugation alone; method C was conventional PEG addition to thawed plasma or serum, followed by centrifugation to remove bound insulin. Method A was found to have acceptable performance and reproducibility; serum free insulin levels were not significantly different from those measured by the simpler method B. In samples from insulin-dependent diabetics, serum free insulin was significantly higher and bound insulin significantly lower in method A compared to the conventional method C. However, in samples from non-diabetics there was no significant difference between methods A and C. Spurious results were obtained by addition of PEG and assay of plasma and serum samples after either freezing and thawing or incubation at 37 degrees C for 2h, compared to PEG addition and assay of fresh plasma or serum samples. We conclude that conventional, delayed addition of PEG to plasma or serum may result in redistribution of free and bound insulin and that values more representative of in vivo blood insulin levels are obtained by rapid PEG addition methods.

Separation of free and antibody-bound insulin in plasma using a bench ultracentrifuge (Beckman ‘Airfuge’)

Separation of free and antibody-bound insulin in plasma using a bench ultracentrifuge (Beckman ‘Airfuge’)

The bioavailability of circulating antibody-bound insulin following insulin withdrawal in Type 1 (insulin-dependent) diabetes.

Insulin withdrawal studies were performed in 12 Type 1 (insulin-dependent) C-peptide negative diabetic patients with low to moderate insulin antibody levels, to assess the biological availability of antibody-bound insulin and its clinical significance. There was a highly significant correlation between the extent to which the free insulin concentration was maintained during the period of insulin withdrawal and both the level of insulin-binding by serum and the total insulin concentration at the start of the study. During insulin withdrawal, the patients who best maintained their circulating free insulin levels showed the smallest increases in blood glucose and 3-hydroxybutyrate concentrations. We conclude that antibody-bound insulin is available for physiological action, and that in those individuals with moderate antibody concentrations it is capable, in the fasting state, of maintaining free insulin levels. In these circumstances insulin antibodies are behaving as simple carrier proteins.

Method for rapid quantitation and characterization of insulin antibodies.

Nearly all insulin-treated diabetic patients develop antibodies to insulin. The clinical importance of these antibodies is not exactly known regarding their general influence on the therapeutic control of the diabetic and the development of secondary complications. There is no doubt, however, about the role of the antibody titer in some forms of insulin resistance. Besides cases of hypoglycemia caused by insulin injections and the rare forms of diabetes caused by autoimmune insulin antibodies, insulin resistance is the main indication for measuring insulin antibodies. On the other hand, measurement of free and bound insulin in insulin-treated patients and the characterization of the antibodies is of broad scientific interest. The classical serological methods such as immunoprecipitation, complement binding, agglutination, and hemagglutination have proved not to be applicable to the insulin antibody, or too insensitive (see e.g., 1). The introduction of radiolabeled insulin by Kallee (2) made possible direct determination of antibody-bound insulin. In recent years several such methods have been described. Free and antibody-bound insulin are separated by either electrophoresis on various supporting materials (3-9); ultracentrifugation (1, 10, 11); adsorption by dextran-charcoal (12, 13), ion-exchange resin (14), or cellulose (15); gel filtration (16); or by precipitation methods (17-20). In some of these methods the equilibrium between free and antibody bound insulin is influenced during the procedure; moreover, most of them are too laborious for routine use. In 1971, Desbuquois and Aurbach (21) described how free and antibody-bound peptide hormones could be separated by use of polyethylene glycol (PEG). In the following we describe a method involving PEG for rapidly measuring and characterizing insulin antibodies.

Diabetes and hypoglycemia due to insulin antibodies

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.

Effects of insulin-free plasma on the charcoal-separation method for radioimmunoassay of insulin.

Radioimmunoassay of insulin in rat plasma using a popular method (Albano, Ekins, Maritz and Turner 1972) involving charcoal-separation of free and antibody-bound insulin was found to be unsatisfactory despite inclusion in standard tubes of insulin-free plasma prepared in either of two ways. Insulin-free plasma and untreated plasma had different effects on adsorption of free insulin to the charcoal. It was concluded that separation with charcoal is very sensitive to any prior treatment of the plasma. Particular care must be taken to ensure that hormone-free plasma is identical in all other respects to untreated plasma.

Quantitation of Free, Total, and Antibody-bound Insulin in Insulin-Treated Diabetics

We describe a simplified method for measuring free, total, and antibody-bound insulin in insulin-treated patients in whom antibodies to insulin are present. The free, active insulin is extracted from the serum with a polyethylene glycol solution. Total insulin is extracted from the serum with a polyethylene glycol solution after dissociation of the antibody-antigen complex with dilute HCI. Aliquots of the extracts are used in the radioimmunoassay system. The figure for antibody-bound insulin is the difference between the total and free insulin values and reflects the concentration of insulin antibodies present. A commercially available ("Phadebas") radioimmunoassay for immunoreactive insulin was used to quantitate the insulin present in the two extracts. Recovery of added insulin averaged 85% for the free insulin and 87%for the total insulin.

The Use of Plain Charcoal to Separate Free Insulin from Antibody-Bound Insulin

The Use of Plain Charcoal to Separate Free Insulin from Antibody-Bound Insulin

Sharp temporary drop in insulin requirement after cesarean section in diabetic patients

Twelve consecutive severe diabetics undergoing elective cesarean section were given infusion of isotonic saline without insulin for 48 hours starting on the morning of the operation. In all but one case blood glucose remained relatively stable and insulin was not resumed until 54 to 84 hours after its last injection. Three patients in spite of the temporary withdrawal of insulin, developed hypoglycemia necessitating glucose infusion. Serum free insulin showed no definite changes after the operation, but antibody-bound insulin and insulin-binding capacity showed clear temporary drop by about half their preoperation values. Perhaps the phenomenon was related to corticoid medicated influence of stress on increased clearance of immunoglobulins with resulting partial liberation of the biologically active insulin.

A radioimmunoassay for plasma insulin using zirconyl phosphate gel

A procedure for the detection of immunoreactive insulin in human plasma is described. The procedure is a rapid radioimmunoassay (3 hr) which uses zirconyl phosphate gel (Z-gel) at a pH of 6.25 to adsorb antibody-bound insulin while leaving free insulin in solution. Thirty-two samples of human plasma which had been assayed for immunoreactive insulin using a commercially available assay (Pharmacia Fine Chemicals, Piscataway, NJ) were also assayed on a blind basis for immunoreactive insulin using the Z-gel technique. A statistical comparison of the data obtained by the two methods yielded a highly significant ( P < 0.01) coefficient of correlation (0.998). A known amount of bovine insulin was added to each of these plasma samples, and the Z-gel assay repeated. The mean percentage recovery of added insulin was 100.4 ± 17.0 (SD). In addition, the insulin response of two healthy individuals to the ingestion of 100 g of glucose was determined using the Z-gel assay. The insulin response curves were similar to those described by other investigators.