Scandinavian Journal of Clinical and Laboratory Investigation
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Prevalence of anti-insulin antibodies and its relation to severe hypoglycaemia in insulin-treated diabetic patients R. Wredling, P. E. Lins & U. Adamson To cite this article: R. Wredling, P. E. Lins & U. Adamson (1990) Prevalence of antiinsulin antibodies and its relation to severe hypoglycaemia in insulin-treated diabetic patients, Scandinavian Journal of Clinical and Laboratory Investigation, 50:5, 551-557, DOI: 10.1080/00365519009089170 To link to this article: http://dx.doi.org/10.1080/00365519009089170
Published online: 29 Mar 2011.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iclb20 Download by: [RMIT University Library]
Date: 10 May 2016, At: 13:54
Scand J Clin Lab Invest 1990; 50: 551-557
Prevalence of anti-insulin antibodies and its relation to severe hypoglycaemia in insulin-treated diabetic patients R . W R E D L I N G , P. E . L I N S & U. ADAMSON
Scandinavian Journal of Clinical and Laboratory Investigation 1990.50:551-557.
Department of Medicine, Danderyd Hospital, Danderyd, Sweden
Wredling R, Lins PE, Adamson U. Prevalence of anti-insulin antibodies and its relation to severe hypoglycaemia in insulin-treated diabetic patients. Scand J Clin Lab Invest 1990; 50: 551-557. The prevalence of anti-insulin antibodies (AIABs) and their association with clinical parameters, metabolic control and severe hypoglycaemia were investigated in a geographically defined population of insulin-treated diabetic patients. Eighty per cent of the patients (479) delivered venous blood samples and answered a questionnaire on severe hypoglycaemic problems during a 12-month period. Circulating AIABs were demonstrable in 78% of the patients, being more common among those with type 1 diabetes and in long-duration patients. High levels of AIABs were also more frequent in patients in whom insulin treatment had been initiated prior to the era of highly purified insulins. The AIABs did not correlate to metabolic control, insulin dose or severe hypoglycaemia. It is concluded that AIABs is not a risk factor for severe hypoglycaemia in insulin-treated diabetic patients.
Key words: duration of insulin treatment; highly purified insulins; insulin dose; metabolic control; type 1 diabetes; severe hypoglycaemia Regina Wredling, Department of Medicine, Danderyd Hospital, S- 18288 Danderyd, Sweden
Anti-insulin antibodies (AIABs) have been demonstrated to moderately increase the insulin requirement in patients with type 1 (insulindependent) diabetes in some studies [ 1-51, while other investigators have failed to demonstrate a relationship between AIABs and insulin requirement (6-121. In some rare cases of insulin resistance, defined as patients requiring more than 200 U/day of regular insulin to maintain metabolic homeostasis, extremely high levels of AIABs have been demonstrated ~31. It is disputed whether AIABs exert a stabilizing effect on glycaemic control, assuming that
dissociating insulin-antibody complexes would help to mimic basal insulin secretion [14-161, or whether they may cause a hyperlabile state of glycaemic control [17- 191. Recently, hypoglycaemia has been the focus of attention, as it has been demonstrated that patients with AIABs exhibit an impaired restoration of blood glucose following insulin-induced hypoglycaemia due to a low clearance rate of insulin from the circulation [20, 211. The existence of AIABs as a risk factor for hypoglycaemia during insulin treatment has been considered [22-251. However, this has been supported by only a few case reports to date [26-281 and 551
552
R . Wredling et al
there is little epidemiological data on this issue. The present study of a large group of diabetic patients aimed at analysing whether severe hypoglycaemia is related to AIABs. METHODS
Scandinavian Journal of Clinical and Laboratory Investigation 1990.50:551-557.
Patients Five hundred and ninety-three patients with insulin-treated diabetes, who visited our outpatient department during 1984 were invited to participate in the study. The patients belonged t o the catchment area of Danderyd Hospital and included the majority of type 1 diabetic patients in this area. All patients were asked in a questionnaire to report severe hypoglycaemic reactions over a 12-month period and to deliver a blood sample for the analysis of anti-insulin antibodies (AIABs). Severe hypoglycaemia was defined as an event with unconsciousness and a need for help from another person. Informed consent was obtained from all the patients and the study was approved by the local Ethics Committee. Clinical data were collected from each patient file. Analytical procedures Glucosylated haemoglobin (HbAlc) was determined by a cation-exchange chromatographic method with fast protein liquid chromatography (291. Usually H b A l c analysis was made at 3-month intervals. The values given below represent the mean of H b A l c determinations during the actual 12-month period. The reference interval in non-diabetics was 4.0- 5.6'Yo. The AIAB level was analysed by a combined polyethylene glycol and anti-IgG precipitation method [30] as follows: to 100 yl of sodium barbital buffer (pH 8.6) 100 yl of serum sample diluted 1 : 5 in buffer was added, followed by 100 yl of '"I-bovine insulin at a concentration of 0.5 mU/ml buffer with ii specific activity of approximately 30 mCi/mg. The mixture was incubated for 20-24 h at 4 "C. Insulin-antibody complexes were precipitated by adding 300 yl of 25% polyethylene glycol in H 2 0 followed by 100 yl undiluted rabbit anti-human IgG (Dakopatts, Glostrup, Denmark). The mixture was vortexed and centrifuged at 2 500 g for 30 min. The precipitate was washed with 1 ml of 12.5'X
polyethylene glycol and finally dissolved in 0.5 ml of 0.05 mol/l NaOH and coupted. All precipitation and washing steps were carried out at 4 "C. Results were calculated from the percentage of total tracer-insulin hound, corrected for the average nonspecific binding to IgG from three normal sera. Sera with binding values >2.8% (99% upper limit of normal range) were judged antibody positive. All samples were assayed in duplicate and the intraand interassay of variation was 3.8Y0 and 5.2'y0, respectively. In the present material, we performed two sub-analyses. In the first, 58 type-I patients with severe hypoglycaemia were compared with a group without severe hypoglycaemia matched for sex, disease duration and age. In the second, 17 type-1 patients who had recurrent severe hypoglycaemia during the last 3 years were compared with a control group without severe reactions that was selected from the study population and matched for sex, disease duration and age. Statistics Linear regression analysis was used to evaluate the correlate between the AIABs on the one hand, and the duration of insulin treatment, age, age at inception, H b A l c , insulin dose, body weight and amount of insulin per kilogram of body weight on the other. Correlation of AIABs to sex, type of diabetes and hypoglycaemia was analysed by Wilcoxon's non-parametric, signed-rank test. In the stepwise regression procedure, the AIABs were used as the dependent variable. Values of p
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Prevalence of anti-insulin antibodies and its relation to severe hypoglycaemia in insulin-treated diabetic patients R. Wredling, P. E. Lins & U. Adamson To cite this article: R. Wredling, P. E. Lins & U. Adamson (1990) Prevalence of antiinsulin antibodies and its relation to severe hypoglycaemia in insulin-treated diabetic patients, Scandinavian Journal of Clinical and Laboratory Investigation, 50:5, 551-557, DOI: 10.1080/00365519009089170 To link to this article: http://dx.doi.org/10.1080/00365519009089170
Published online: 29 Mar 2011.
Submit your article to this journal
Article views: 16
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iclb20 Download by: [RMIT University Library]
Date: 10 May 2016, At: 13:54
Scand J Clin Lab Invest 1990; 50: 551-557
Prevalence of anti-insulin antibodies and its relation to severe hypoglycaemia in insulin-treated diabetic patients R . W R E D L I N G , P. E . L I N S & U. ADAMSON
Scandinavian Journal of Clinical and Laboratory Investigation 1990.50:551-557.
Department of Medicine, Danderyd Hospital, Danderyd, Sweden
Wredling R, Lins PE, Adamson U. Prevalence of anti-insulin antibodies and its relation to severe hypoglycaemia in insulin-treated diabetic patients. Scand J Clin Lab Invest 1990; 50: 551-557. The prevalence of anti-insulin antibodies (AIABs) and their association with clinical parameters, metabolic control and severe hypoglycaemia were investigated in a geographically defined population of insulin-treated diabetic patients. Eighty per cent of the patients (479) delivered venous blood samples and answered a questionnaire on severe hypoglycaemic problems during a 12-month period. Circulating AIABs were demonstrable in 78% of the patients, being more common among those with type 1 diabetes and in long-duration patients. High levels of AIABs were also more frequent in patients in whom insulin treatment had been initiated prior to the era of highly purified insulins. The AIABs did not correlate to metabolic control, insulin dose or severe hypoglycaemia. It is concluded that AIABs is not a risk factor for severe hypoglycaemia in insulin-treated diabetic patients.
Key words: duration of insulin treatment; highly purified insulins; insulin dose; metabolic control; type 1 diabetes; severe hypoglycaemia Regina Wredling, Department of Medicine, Danderyd Hospital, S- 18288 Danderyd, Sweden
Anti-insulin antibodies (AIABs) have been demonstrated to moderately increase the insulin requirement in patients with type 1 (insulindependent) diabetes in some studies [ 1-51, while other investigators have failed to demonstrate a relationship between AIABs and insulin requirement (6-121. In some rare cases of insulin resistance, defined as patients requiring more than 200 U/day of regular insulin to maintain metabolic homeostasis, extremely high levels of AIABs have been demonstrated ~31. It is disputed whether AIABs exert a stabilizing effect on glycaemic control, assuming that
dissociating insulin-antibody complexes would help to mimic basal insulin secretion [14-161, or whether they may cause a hyperlabile state of glycaemic control [17- 191. Recently, hypoglycaemia has been the focus of attention, as it has been demonstrated that patients with AIABs exhibit an impaired restoration of blood glucose following insulin-induced hypoglycaemia due to a low clearance rate of insulin from the circulation [20, 211. The existence of AIABs as a risk factor for hypoglycaemia during insulin treatment has been considered [22-251. However, this has been supported by only a few case reports to date [26-281 and 551
552
R . Wredling et al
there is little epidemiological data on this issue. The present study of a large group of diabetic patients aimed at analysing whether severe hypoglycaemia is related to AIABs. METHODS
Scandinavian Journal of Clinical and Laboratory Investigation 1990.50:551-557.
Patients Five hundred and ninety-three patients with insulin-treated diabetes, who visited our outpatient department during 1984 were invited to participate in the study. The patients belonged t o the catchment area of Danderyd Hospital and included the majority of type 1 diabetic patients in this area. All patients were asked in a questionnaire to report severe hypoglycaemic reactions over a 12-month period and to deliver a blood sample for the analysis of anti-insulin antibodies (AIABs). Severe hypoglycaemia was defined as an event with unconsciousness and a need for help from another person. Informed consent was obtained from all the patients and the study was approved by the local Ethics Committee. Clinical data were collected from each patient file. Analytical procedures Glucosylated haemoglobin (HbAlc) was determined by a cation-exchange chromatographic method with fast protein liquid chromatography (291. Usually H b A l c analysis was made at 3-month intervals. The values given below represent the mean of H b A l c determinations during the actual 12-month period. The reference interval in non-diabetics was 4.0- 5.6'Yo. The AIAB level was analysed by a combined polyethylene glycol and anti-IgG precipitation method [30] as follows: to 100 yl of sodium barbital buffer (pH 8.6) 100 yl of serum sample diluted 1 : 5 in buffer was added, followed by 100 yl of '"I-bovine insulin at a concentration of 0.5 mU/ml buffer with ii specific activity of approximately 30 mCi/mg. The mixture was incubated for 20-24 h at 4 "C. Insulin-antibody complexes were precipitated by adding 300 yl of 25% polyethylene glycol in H 2 0 followed by 100 yl undiluted rabbit anti-human IgG (Dakopatts, Glostrup, Denmark). The mixture was vortexed and centrifuged at 2 500 g for 30 min. The precipitate was washed with 1 ml of 12.5'X
polyethylene glycol and finally dissolved in 0.5 ml of 0.05 mol/l NaOH and coupted. All precipitation and washing steps were carried out at 4 "C. Results were calculated from the percentage of total tracer-insulin hound, corrected for the average nonspecific binding to IgG from three normal sera. Sera with binding values >2.8% (99% upper limit of normal range) were judged antibody positive. All samples were assayed in duplicate and the intraand interassay of variation was 3.8Y0 and 5.2'y0, respectively. In the present material, we performed two sub-analyses. In the first, 58 type-I patients with severe hypoglycaemia were compared with a group without severe hypoglycaemia matched for sex, disease duration and age. In the second, 17 type-1 patients who had recurrent severe hypoglycaemia during the last 3 years were compared with a control group without severe reactions that was selected from the study population and matched for sex, disease duration and age. Statistics Linear regression analysis was used to evaluate the correlate between the AIABs on the one hand, and the duration of insulin treatment, age, age at inception, H b A l c , insulin dose, body weight and amount of insulin per kilogram of body weight on the other. Correlation of AIABs to sex, type of diabetes and hypoglycaemia was analysed by Wilcoxon's non-parametric, signed-rank test. In the stepwise regression procedure, the AIABs were used as the dependent variable. Values of p
