Measurement of free insulin concentrations: the influence of the timing of extraction of insulin antibodies

Summary Plasma insulin concentrations of insulin-treated diabetic patients must be measured after removal of insulin antibodies, usually by precipitation with polyethylene glycol (PEG). Details of the procedure vary between laboratories; commonly, frozen plasma is thawed and incubated at 37 °C to restore a presumed equilibrium between free and antibody bound insulin before extraction. The present study was designed to investigate methodological factors that could affect the measured free insulin concentration. In normal subjects PEG extraction of globulins did not disturb measurement of insulin concentrations, whether carried out after incubation for 2 h at 37 °C, or storage at -20 °C, in either order. Freezing or incubation of PEG extracts of plasma from insulin-treated patients also failed to disturb the measured concentrations of free insulin. When plasma from patients was incubated for 2 h after storage, a marked scatter (51–272%) of measured results occurred when compared to bedside extraction. This problem was not overcome by buffering with HEPES or storage at a lower temperature (-40 °C). Incubation at 0 °C also severely disturbed the apparent concentrations. Incubation of plasma before extraction and freezing also disturbed the measured result, a problem not corrected by maintaining near physiological pH. Total insulin concentrations measured on acid extracts were not disturbed by any of these manoeuvres. The temperature of centrifugation of blood at the time of venepuncture did not influence the result. Furthermore, when insulin concentrations were rising or falling similar percentage changes were seen over a 30-min incubation of plasma before extraction on the day of venepuncture, suggesting that equilibrium between free and bound insulin is maintained in vivo. We suggest that, for accurate estimation of free insulin concentrations in insulin-treated diabetic patients, immediate centrifugation of blood and extraction of insulin antibodies is mandatory.     

Serum insulin,insulin antibodies and insulin requirement in the first period of insulin treatment in non-insulin resistant diabetics

Summary Twelve insulin-sensitive diabetics were studied for 200 days after the initiation of mixed beef-pork NPH insulin. Normalization of the fasting blood glucose was not accompanied by any elevation in the pre-treatment fasting immunoreactive insulin level. Insulin antibodies appeared in 2 patients on the second week of insulin treatment, in 6 others within 87 days. In 4 patients no antibodies were found 200 days after the start of insulin. The appearance of antibodies was accompanied in two patients by a decrease in insulin requirement, in others there was no change. When antibodies were present, the total maximum insulin binding capacity was 4 to 12 U/1, but the total insulin constituted only 3 to 36% of the binding capacity. Insulin wastage caused by the destruction of the immune complexes was calculated to be 0.35 to 5.6 U/die only, and this explains the negligible effect of insulin antibodies on insulin requirement in non-resistant patients. Presented at the 10th Annual Meeting of the European Association for the Study of Diabetes in Jerusalem, September 11–13, 1974.     

Controlled study comparing treatment with monocomponent insulin and conventional insulin in patients with lipoatrophy

Summary Twenty-one patients with evident lipoatrophy treated with conventional (Conv.) insulin were either allocated to continuation of treatment with previously used insulin (Conv. group, n=10) or were transferred to Lente? MC (monocomponent) insulin with or without supplementary Actrapid? MC insulin (MC group, n=11). On entry and after 3, 6 and 12 months of follow-up, serum insulin-, pancreatic polypeptide- and proinsulin-binding IgGs were determined by radioimmunoelectrophoresis according to the method of Christiansen. Prior to determination of proinsulin-binding IgG, the insulin-binding IgG was removed by means of sepharose-bound insulin according to the method of Heding. In both groups a slight decrease in the titer of insulinbinding IgG was observed: in the Conv. group from 5.33±0.92 (SEM) to 4.66±1.17 mU/ml after 12 months, and in the MC group from 3.22±0.64 to 2.66±0.46 mU/ml, respectively. Due to the small number of patients with pancreatic polypeptide antibody titers above the detection limit no statistical evaluation was carried out. The level of serum proinsulin-binding IgG decreased in the MC group only (from 9.3±2.2 to 1.9±0.6 ng/ml after 12 months), and even showed a slight increase in the Conv. group (the respective titers were: 14.0±4.6 and 14.9±4.6 ng/ml). In the MC group 10 patients (91%) showed improvement and 7 (64%) complete regression of their lipoatrophy corresponding to 6 (60%) and 2 (20%) in the Conv. group. This finding suggests a possible role of proinsulin-binding antibodies in the pathogenesis of insulin lipoatrophy.     

Comparison of liquid phase radiobinding assay and solid phase ELISA for the measurement of insulin antibodies and insulin autoantibodies in serum

Sera containing insulin antibodies from 20 insulin-treated diabetic patients, sera containing insulin autoantibodies from 20 insulin-naive non-diabetic patients, and from 10 normal controls, were tested at 1:20 dilution in three different radioimmunoassays (RIA) and an enzyme linked immunosorbent assay (ELISA), using a highly purified human insulin ligand. The RIA using insulin radiolabelled at multiple sites detected insulin antibodies in 17/20 and insulin autoantibodies in 13/20 sera. The same RIA using A-14-monoiodinated insulin was sensitive to antibodies and autoantibodies in all the sera. The same RIA using sera after insulin extraction detected only 13/20 diabetic sera and 9/20 autoimmune sera as positive, owing to a substantial rise in non-specific binding of the control sera. ELISA was sensitive to insulin antibodies and autoantibodies in every case. When binding curves for ELISA and the most sensitive RIA were compared using serial dilutions of four insulin antibody containing sera and four insulin autoantibody containing sera, antibody titres varied from 1.1 to 3.8 times higher in ELISA, and autoantibody titres from 10.6 to 28.6 times higher in ELISA. These studies indicate that ELISA is more sensitive than RIA to insulin antibodies, and in particular to insulin autoantibodies.     

胰岛细胞抗体亚型与胰岛素、胰高血糖素分泌的关联

目的　通过研究胰岛细胞抗体 (ICA)不同亚型阳性患者胰岛素、胰高血糖素水平的差异 ,探讨成人隐匿性自体免疫性糖尿病 (LADA)的发病机制。方法　免疫组化法测定的ICA边缘型阳性LADA患者 2 9例、弥漫型阳性LADA患者 2 8例、ICA阴性正常人 17例分为 3组 ,测定空腹、餐后 3 0、60、12 0min血糖、胰岛素、胰高血糖素。结果　 ( 1)与对照组比较 ,ICA阳性两组的胰高血糖素曲线下面积 (AUC)均高于对照组 (均P <0 .0 5 ) ;餐后 3 0、60min胰岛素 :对照 >边缘型阳性 >弥漫型阳性 ;( 2 )多元逐步回归显示胰岛素、胰岛素 /胰高血糖素 (AUC ,60、12 0min)均与ICA亚型、胰岛素敏感性显著负相关。结论　ICA边缘型及弥漫型阳性LADA患者中 :( 1)胰高血糖素均高于正常人 ,且两组间差异无显著性 ;( 2 )胰岛素水平均低于正常人 ,但弥漫型阳性者更为明显 ,显示其 β细胞胰岛素分泌功能受损更明显 ;( 3 )对于胰岛素水平的影响 ,胰岛素敏感性的作用比ICA更强     

胰岛素抵抗机制的探研——阻断型胰岛素抗体是导致胰岛素抵抗的原因之一

目的　探讨胰岛素抗体导致胰岛素抵抗发生的机制。方法　利用单抗制备技术和酶联免疫方法获得１７ 株抗人胰岛素单克隆抗体（ＭＡｂｓ） ,测定这些抗体阻断胰岛素与其受体结合、抑制ＣＨＯ 细胞（Ｃｈｉｎｅｓｅｈａｍｓｔｅｒｏｖａｒｙｃｅｌｌｓ）上胰岛素受体自身磷酸化、以及免疫结合共价交联的胰岛素胰岛素受体的能力。结果　在这组ＭＡｂｓ 中,多数ＭＡｂｓ（１６／１７）阻断胰岛素与其受体结合或抑制ＣＨＯ细胞上胰岛素受体自身磷酸化,其识别胰岛素上的位点与胰岛素的受体结合区域相重叠。有１ 株ＭＡｂ 具有免疫结合已与受体交联的胰岛素的能力,但不阻断胰岛素的作用。结论　在胰岛素抗体阳性的糖尿病患者中,其胰岛素抗体的识别位点对胰岛素抵抗的发生有重大作用。     

Haemolysis affects insulin but not C-peptide immunoassay

Summary Venous blood was taken at the end of a glucose infusion test in 19 individuals and divided into four aliquots, 3 of which were variably haemolysed by repeated passage through a 23-gauge needle to simulate traumatic venepuncture. Plasma insulin (measured by both a charcoal separation and a double-antibody method), C-peptide, and haemoglobin were measured in each aliquot, and haemolysis was also assessed visibly. A significant loss of immuno-assayable plasma insulin was found in samples with only a trace of visible haemolysis, with up to 90% lost in severely haemolysed samples. Plasma C-peptide was unaffected by haemolysis. This represents an additional advantage for the use of plasma C-peptide in assessing insulin secretion.     

Cyclophosphamide inhibition of insulin antibody production,insulin resistance and experimental immunodiabetes

Summary Hyperglycemia, increase of insulin tolerance and striking changes in B- and A-cells of the islets of Langerhans occurred in guinea pigs producing precipitating insulin antibodies after immunization with insulin in adjuvant. Treatment with cyclophosphamide inhibited the production of insulin antibodies while the blood sugar and insulin tolerance remained on the level of nonimmunized control animals. Also the islets of Langerhans were less severely injured than those of animals not treated with cyclophosphamide.

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Zusammenfassung Hyperglykämie, erhöhte Insulintoleranz und auffällige Veränderungen der A- und B-Zellen der Langerhansschen Inseln wurden bei Meerschweinchen beobachtet, welche infolge Immunisierung mit Insulin in Adjuvans Insulin-präzipitierende Antikörper bilden. Cyclophosphamid-Behandlung verhindert die Bildung von Insulin-Antikörpern, während Blutzucker und Insulintoleranz auf dem gleichen Niveau bleiben wie bei nicht immunisierten Kontrolltieren. Auch die Langerhansschen Inseln sind weniger schwer geschädigt als bei nicht mit Cyclophosphamid behandelten immunisierten Tieren.

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Resumen En covayos productores de anticuerpos antinsulina precipitantes, tras inmunización con insulina y adyuvante, se ha podido observar: hiperglicemia, aumento de la tolerancia a la insulina y conspícuas modificaciones a cargo de las células A y B de las islas de Langerhans. El tratamiento con ciclofosfamida inhibía la producción de anticuerpos antinsulina, mientras la glucemia y la tolerancia insulínica permanecían en los mismos niveles que podían observarse en los animales de control no inmunizados. Las islas de Langerhans aparecían menos dañadas respecto a las de los animales no tratados con ciclofosfamida.

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Résumé Hyperglycémie, amélioration de la tolérance à l'insuline et modifications considérables à niveau des cellules B et A des ilots de Langerhans ont fait l'objet d'une observation, après immunisation par insuline et adjuvant, en cobayes produisant des anticorps anti-insuline précipitants. Le traitement par le cyclophosphamide inhibait la production d'anticorps anti-insuline, alors que la glycémie et la tolérance à l'insuline restaient aux mêmes niveaux qu'on pouvait vérifier chez les animaux de contrôle non immunisés. Même les îlots de Langerhans semblaient avoir subi des dommages moins importants par rapport à ceux des animaux qui n'avaient pas été traités par le cyclophosphamide.

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Riassunto Iperglicemia, aumento della tolleranza all'insulina e cospicue modificazioni a carico delle cellule B ed A delle isole di Langerhans sono stati osservati, dopo immunizzazione con insulina in adiuvante, in cavie produttrici di anticorpi anti-insulina precipitanti. Il trattamento con ciclofosfamide inibiva la produzione di anticorpi anti-insulina, mentre la glicemia e la tolleranza insulinica rimanevano agli stessi livelli riscontrabili negli animali di controllo non immunizzati. Anche le isole di Langerhans apparivano meno danneggiate rispetto a quelle degli animali non trattati con ciclofosfamide.

     

Dextran-coated charcoal immunoassay of insulin

Summary 1. The use of dextran-coated charcoal allows a rapid, simple and relatively precise radioimmunoassay of insulin. — 2. The factors affecting the selectivity of coated charcoal for insulin and antibody-bound insulin have been examined and optimum operating conditions selected. — 3. The original method has been modified to allow the use of a commercially available antiserum. — 4. The accuracy and precision of the method have been compared with those of a double antibody technique.

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Immunologische Insulinbestimmung mit dextranvorbehandelter Tierkohle

Zusammenfassung 1. Die Verwendung von dextranvorbehandelter Tierkohle erlaubt eine schnelle, einfache und relativ präzise radio-immunologische Insulinbestimmung. — 2. Die Faktoren, die das unterschiedliche Ansprechen von dextranvorbehandelter Tierkohle auf freies und antikörper-gebundenes Insulin beeinflussen, wurden untersucht und die günstigsten Bedingungen für die Durchführung der Untersuchung herausgearbeitet. — 3. Diese Modifikation der Originalmethode erlaubt auch die Verwendung des handelsüblichen Anti-Insulin-Serums. — 4. Die Richtigkeit und die Genauigkeit der beschriebenen Methode wurden mit der eines Doppel-Antikörper-Verfahrens verglichen.

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Dosage immunologique de l'insuline avec le charbon recouvert de dextran

Résumé 1. L'utilisation de charbon recouvert de dextran permet un dosage radioimmunologique de l'insuline rapide, simple et relativement précis. — 2. Les facteurs affectant la sélectivité du dextran-charbon pour l'insuline et les complexes insuline-anticorps ont été examinés et les meilleures conditions d'opération ont été sélectionnées. — 3. La méthode originale a été modifiée afin de permettre l'usage d'un antisérum commercial. — 4. La justesse et la précision de la méthode ont été examinées et comparées avec celles de la méthode au double anticorps.

     

Failure of appearance of insulin antibodies in dogs adapted to bovine-porcine insulin

Summary 1. After a slowly increased adaptation to insulin (commercial normal insulin, i.e. a mixture of bovine-porcine insulin) two dogs showed no circulating insulin antibodies, despite daily subcutaneous application of 4 IU/kg body weight for more than 2 years. — 2. The non-appearance of circulating insulin antibodies was confirmed by several experimentsin vitro andin vivo especially by autoradiography — immunoelectrophoresis and determination of IBC before as well as after the removal of insulin from the serum. — 3. The biological activity of unlabelled bovine insulin after I.V. injection was not diminished in the dogs adapted to insulin.

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Fehlen von Insulinantikörpern bei an Binder- und Schweininsulin angepaten Hunden

Zusammenfassung 1. Nach einer langsam ansteigenden Anpassung an Insulin (kommerzielles Alt-Insulin, eine Mischung von Rinder- und Schweineinsulin) zeigten 2 Hunde keine zirkulierenden Insulinantikörper, obwohl 4 IE/kg Körpergewicht über zwei Jahre täglich subcutan injiziert wurden. — 2. Das Fehlen von zirkulierenden Insulinantikörpern wurde durch verschiedene Experimentein vitro undin vivo, insbesondere durch Autoradiographie — Immunelektrophorese und Bestimmung der IBC vor und nach Entfernung des Insulins aus dem Serum bekräftigt. — 3. Die biologische Aktivität unmarkierten Rinderinsulins war nach i.v.-Injektion bei den adaptierten Hunden nicht vermindert.

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Non-apparition d'anticorps antiinsuline chez des chiens adaptés à de l'insuline bovine et porcine

Résumé 1. Après une adaption lentement progressive à l'insuline (insuline normale du commerce, mélange d'insulines de boeuf et de porc), deux chiens n'ont montré aucun anticorps circulant contre l'insuline, malgré l'application quotidienne sous-cutanée de 4 IU/kg durant plus de deux ans. — 2. La non-apparition d'anticorps circulants contre l'insuline a été confirmée par plusieurs expériencesin vitro etin vivo, en particulier par autoradiographie — immunoélectrophorèse et détermination d'IBC aussi bien avant qu'après avoir retiré l'insuline du sérum. — 3. L'activité biologique de l'insuline de boeuf non marquée, après l'injection intra-veineuse, n'a pas diminué chez les chiens adaptés à l'insuline.

     

动物实验中胰岛素抵抗检测的几个问题

本文讨论了有关动物实验中检测胰岛素抵抗的几个问题.除了葡萄糖钳夹试验外,其他方法在动物实验中都存在问题.     

Comparison of two commonly used insulin injection techniques

In ten patients with insulin-dependent diabetes we compared postprandial blood glucose levels and plasma free insulin concentrations after the administration of insulin mixtures with two commonly used injection techniques. The morning dose of insulin was administered once using a 13 mm needle, inserted perpendicularly, and once with a 20 mm needle, inserted deep subcutaneously just above the muscle fascia at an angle dependent on the skinfold thickness. Plasma free insulin concentrations and postprandial blood glucose levels were virtually identical for either technique. We conclude, therefore, that the simpler perpendicular method is the technique of choice, saving the diabetics much unnecessary anxiety over a daily procedure.     

The antigenicity of pig insulin

Summary Treatment of human subjects with a neutral solution of pure pig insulin crystals does not lead to the formation of insulin antibodies. Thirty-six non-diabetics with psychiatric diseases were treated with a neutral solution of crystalline pig insulin for a maximum period of 104 days, using a 24-h dosage of up to 208 i.u. In patients who had previously not received insulin treatment (24 patients), insulin antibodies could not be demonstrated after the termination of the insulin treatment. However, in patients who had previously received treatment with acid solutions of insulin consisting of pig and ox insulin (12 patients), it was possible in almost all cases to demonstrate insulin antibodies after the termination of the insulin treatment. In addition, 10 pigs received treatment with solutions of pure pig insulin. Insulin antibodies could be demonstrated in only 2 pigs, both treated with acid solutions of recrystallized pig insulin, whereas antibodies could not be demonstrated in pigs treated with neutral solutions of recrystallized pig insulin. The reason why pure preparations of pig insulin are in most cases also antigenic to man, is presumably that the pig insulin preparations are injected as suspensions (zinc insulins, NPH insulin) or as is the case with acid solutions of insulin, converted to suspensions after injection, since insulin precipitates when the acid insulin solution is neutralized by tissue fluid.Delivered as a lecture in a slightly altered form, at the III Congress of the Deutschen Diab. Gesellschaft, Göttingen 1968.     

Chronic insulin resistance

Summary Chronic insulin resistance has been defined and its clinical characteristics summarized. Although increased insulin antibodies can be demonstrated in most patients with chronic insulin resistance, there is not always a good correlation between the severity of the insulin resistance and the titer of insulin antibodies. Several insulin resistant patients have been described who appear to represent examples of peripheral tissue unresponsiveness to insulin. Various forms of treatment of chronic insulin resistance have been reviewed.This work was supported by the United States Public Health Service Grant AM 06865 from the National Institutes of Health.     

胰岛素自身抗体对成人隐匿性自身免疫糖尿病的诊断价值

目的 探讨胰岛素自身抗体(IAA)对成人隐匿性自身免疫糖尿病(LADA)的诊断价值.方法 选取2003年10月至2007年3月连续在中南大学湘雅二医院就诊的1003例初诊2型糖尿病、110例1型糖尿病患者,并选取同期米院体格检杏的317名健康对照者,采用微量平板放射免疫法和放射配体法检测IAA及谷氨酸脱羧酶抗体(GADA)和蛋门酪氨酸磷酸酶抗体(IA-2A)水平,了解IAA阳性率及与其他抗体重叠情况.对4例IAA单独阳性的LADA患者进行了4年随访,观察其临床特征变化.采用卡方榆验比较初诊2型糖尿病组、健康对照组和初诊1型糖尿病组IAA阳性率,采用t检验比较IAA阳性组和阴性匹配组空腹胰岛素(FINS)水平下降速率.结果 (1)初诊2型糖尿病患者IAA阳性率3.39%(34/1003)高于健康埘照组0.95%(3/317)(X2=5.3,P<0.05),但低于1型糖尿病组21.82%(24/110)(x2=68.2,P<0.01).(2)初诊2型糖尿病患者三种抗体联合检测阳性率为10.47%(105/1003),高于GADA 6.58%(66/1003)、IA-2A 2.79%(28/1003)、IAA3.39%(34/1003)单个抗体检测(x2值分别为9.2、37.8和46.2,P值均<0.05).IAA联合检测可提高LADA阳性检出率2.39%.(3)在4年随访期间,IAA阳性者逐年转阴,4例中的2例患者合并GADA阳性;IAA阳性组FINS水平下降速率较阴性匹配组呈增高趋势(分别为15.37%和5.29%;t=1.7,P=0.059).结论 IAA对初诊2型糖尿病患者筛查LADA有一定价值;联合检测IAA、GADA、IA-2A能提高LADA诊断效率.     

血清胰岛素免疫检测及相关问题

血清胰岛素检测对糖代谢疾病的研究有着重要的临床参考价值.胰岛素检测技术经历了近50年的衍变历程,大体上能符合临床研究的要求.但由于胰岛素分子免疫原性和生物活性的多元性、检测体系的复杂性和多重因素的干扰性,目前仍缺乏统一的参考标准,给临床和不同实验室进行胰岛素测定值比较带来一定困难.     

The immunogenicity of insulin preparation. Antibody levels before and after transfer to highly purified porcine insulin

Summary Ninety-two insulin-dependent diabetics (aged 4–20 years, mean±SD: 13±4) with a duration of diabetes from 2 to 17 years (7±3) were transferred from Lente or NPH (5 × crystallised insulin) to Monotard insulin (highly purified insulin). Total serum immunoreactive insulin levels and concentrations of antibodies against insulin, porcine proinsulin, a-component and pancreatic polypeptide were determined prior to [I] and at a mean of 220 [II], 460 [III], 830 [IV], and 1170 [V] days after the change. All but two subjects had insulin antibodies (IgG) at the start, with a mean value of 2864 U/ml. There was a significant fall in the mean insulin antibody level between [I] and [II] to 2165 U/ml (p<10^-7), followed by an increase between [II] and [III] whereafter a slight decrease was observed being significant between [III] and [IV], as well as between [IV] and [V] (p<0.05); some patients showed a constant fall over the entire period, while others showed fluctuations. Total serum insulin showed a similar pattern, with a mean value of 1141 U/ml at [I] declining to 522 U/ml at [V]. The percentage fall between [I] and [V] was greater (54%) than that in the insulin antibodies (30%). Antibodies against acomponent, proinsulin and pancreatic polypeptide were present in 96%, 72% and 41% of the patients respectively before the change in therapy. There was a decline in these antibodies between each sampling (p values between <10^-3 and 10^-8) and, at the end of the investigation antibodies against a-component were above the detection limit in only 4 patients, and none of the patients showed antibodies against proinsulin or pancreatic polypeptide. Thus, removal of the impurities, including the hormonal contaminants of insulin, leads to a slow fall in antibodies to insulin and a much faster disappearance of antibodies against acomponent, proinsulin and pancreatic polypeptide.