Immunological and biological properties of iodoinsulin labeled with one or less atoms of iodine per molecule

Experiments were designed to compare the distribution of free and antibody-bound unlabeled insulin to the distribution of free and antibody-bound insulin-(125)I. The insulin antibody was incorporated in a specific immune precipitate similar to the one used by Hales and Randle for the radioimmune assay of insulin. Insulin which was not bound by the specific immune precipitate was measured by the immune hemolysis inhibition assay. This report contains evidence that the addition of the unlabeled insulin in the radioimmune assay results in relatively more insulin-(125)I which remains free and less bound by antibodies than is the case with the unlabeled insulin. Methods are described for the separation of an electrophoretically homogeneous iodoinsulin from samples of crude iodoinsulin with average incorporations of less than 0.2 atoms iodine per molecule. These purified iodoinsulin fractions have a markedly attenuated biological activity. Evidence is presented which supports the postulate that only a portion of the antibodies in guinea pig insulin antiserum are capable of effectively binding with purified iodoinsulin.     

Dextran-charcoal and dioxane phase separation methods in the radioimmunoassays for parathyroid hormone and calcitonin

Dioxane precipitation and dextran-charcoal adsorption were used to separate free from bound ¹²⁵I-labelled hormone in the radioimmunoassays of calcitonin and parathyroid hormone. Changes in the fraction of bound radioactivity, independent of hormone concentrations, were seen with changes in temperature, plasma concentration, time of standing and amount of dioxane or dextran-charcoal added. Characterization of these variables affecting bound radioactivity, and careful standardization of the conditions of phase separation, indicate that dioxane precipitation is a satisfactory method for the calcitonin immunoassay while dextran-charcoal adsorption provides the most reliable results in the parathyroid hormone immunoassay.     

Free and total insulin as determined after precipitation with polyethylene glycol: analytical characteristics and effects of sample handling and storage

We evaluated results of radioimmunoassays of free and total insulin after precipitation of endogenous antibodies with polyethylene glycol (PEG), and we investigated the influence of collection time, temperature, and storage in heparin- cr EDTA-treated plasma or serum on results for free insulin. Analytical recovery of free insulin was 99.3%, of total insulin 96.4%. For free insulin, assay precision (CV) was 4.0-13.0% (intra-assay) and 7.8-10.7% (inter-assay); for total insulin, 3.6-9.5% and 6.6-11.7%, respectively. Free insulin decreased in plasma (p less than 0.05) and serum (p less than 0.01) at room temperature after 3 h and in promptly analyzed serum (p less than 0.01). Storage of samples at -20 degrees C increased the concentration of free insulin in plasma (p less than 0.025) and serum (p less than 0.005), whereas the free insulin content of supernates after PEG precipitation was stable, except for a slight decrease in serum samples (p less than 0.02). We conclude that, for radioimmunoassay of free and total insulin, plasma should be used, treated with PEG without delay; supernates then are analytically stable for as long as 26 weeks at -20 degrees C.     

Double-antibody method and the protein-A-bearing Staphylococcus aureus cells method compared for separating bound and free antigen in radioimmunoassay.

We compared the protein-A-bearing Staphylococcus aureus immunoadsorbent to the double-antibody precipitation method for separating bound and free radiolabeled antigen in a radioimmunoassay. With human albumin (antigen) and rabbit anti-human albumin (antibody) as a model, our results indicate that formalin-fixed, heat-killed S. aureus cells could be substituted for the double-antibody precipitation method. Ease of preparation and high adsorption capacity of protein-A-bearing S. aureus for most mammalian IgG make this method economical and time saving.     

Demonstration of the insulin receptor in vivo in rabbits and its possible role as a reservoir for the plasma hormone.

Based on studies of the interaction of insulin with its receptors in vitro, we calculated that a receptor compartment should be measurable directly in vivo. For this purpose, rabbits were injected intravenously with a labeled insulin that has low affinity for receptors in combination with a radioiodinated insulin that has high affinity for receptors. Plasma concentrations of labeled insulins were measured at selected intervals after injection. Apparent volumes of distribution were calculated by extrapolation of plasma distribution were calculated by extrapolation of plasma disappearance curves; high affinity insulins consistently distributed into spaces that were two-three times greater than those of the low affinity insulins. Injections of unlabeled pork insulin before tracer insulins decreased the distribution space of the high affinity insulin in a dose-dependent manner while having little or no effect on the distribution space of the low affinity labeled insulin. When unlabeled insulin was injected after the tracer insulins, there was an immediate rise in the plasma concentration of the high affinity insulin with only a slight change in the plasma concentration of the low affinity insulin. These results demonstrate that high affinity insulins distribute into a body compartment which has many properties of the insulin receptor previously studied in vitro. This receptor compartment: (a) recognizes insulins based on their biological potencies; (b) is saturated by elevated concentrations of insulin; and (c) insulin bound to receptors is in equilibrium with free hormone in plasma. Further, the bound to free ratios for hormone, calculated from these data, suggest that in vivo greater than 50% of the extrapancreatic insulin is bound to receptors during normal physiological states.     

Hypoglycemia due to serum-complexed insulin in a patient with diabetes mellitus

A 28-year-old woman with insulin-dependent diabetes mellitus presented with a "hyperlabile" state of hyperglycemia and ketoacidosis alternating with hypoglycemia. Measurements of total and free insulin levels suggested that the clinical syndrome may have been due to antibody binding of insulin. Equilibrium analysis of insulin binding to the patient's serum demonstrated two classes of anti-insulin activities. The first class was of high affinity (dissociation constant approximately equal to 10(-9) M) and low capacity (150 microU/ml). At low total serum insulin concentrations, most of the circulating insulin was bound to the high-affinity binding activity, and the patient presented with hyperglycemia or ketosis. The second class of insulin binding activity had a lower affinity (dissociation constant approximately equal to 5 X 10(-7) M). The insulin that was bound to this low-affinity serum substance still maintained biologic activity in vivo. Isophane insulin (NPH) had a markedly prolonged serum half-life, which resulted in delayed hypoglycemia. Serum insulin complexes--that is, bound insulin--may not be "inactive" but may contribute to total insulin action. A determination of insulin activity, not only free insulin levels, may help explain hypoglycemia in selected patients with diabetes mellitus.     

Free insulin, bound insulin, C-peptide and the metabolic control in juvenile onset diabetics: comparison of C-peptide secretors and non-secretors during 24 hours conventional insulin therapy

In two groups of juvenile onset diabetics similar in age, weight, diet and daily insulin dosage (eight without C-peptide, group I; eight with C-peptide, group II) the serum levels of free and antibody bound insulin, C-peptide, glucose, lactate, alanine and FFA were determined over 24 h. In addition the affinity and binding capacity of the insulin antibodies were determined in vitro. No correlation was found between free or bound insulin and glucose. This holds true for the individual profiles as well as for the averaged profiles of the two groups. Free insulin and lactate or alanine were positively correlated in the C-peptide secreting group. C-peptide secretion followed the flucturations of the glucose level during 24 h in each individual patient. As a group, C-peptide secretors were better controlled than non-secretors with respect to mean blood glucose, M-value and the lability index and showed higher free insulin levels despite a similar daily insulin dosage. The possible reasons for this fact are discussed. No correlation was found between the affinity characteristics of the insulin antibodies and the degree of metabolic control or the daily insulin dosage.     

The interference of insulin antibodies in insulin immunometric assays.

We investigated the interference of insulin antibodies in two insulin immunometric assays (Bio-Rad and Elecsys) by measuring direct and free insulin in plasma from 30 patients without insulin antibodies (group 1), as screened by a sensitive radio-binding assay, and in plasma from 80 patients with insulin antibodies (group 2). In group 1, the direct/free insulin ratio did not differ from 1, showing the equivalence of free and direct insulin results in theses samples. In group 2, this ratio was markedly increased (mean: Bio-Rad 2.63, Elecsys 5.02) and correlated positively with the insulin antibody radio-binding assay result (r=0.92 for the correlation between Bio-Rad and Elecsys assays after log-transformation of the ratios). In samples containing insulin antibodies, direct insulin concentration was frequently lower than total (bound and unbound) insulin measured with the Bio-Rad and Elecsys assays. This study underlines the interference of insulin antibodies in insulin immunometric assays and the importance of assessing an insulin immunometric assay for sensitivity towards the presence of these antibodies.     

A highly sensitive enzyme immunoassay of anti-insulin antibodies in human serum

A highly sensitive enzyme immunoassay of anti-insulin antibodies in human serum is described. Serum samples were subjected to successive processes of the incubation with insulin, the dextran-charcoal treatment to remove free insulin, the precipitation of insulin anti-insulin antibodies by polyethylene glycol, the acid treatment of the precipitates to inactivate anti-insulin antibodies, and the measurement of insulin by sandwich enzyme immunoassay technique. By this enzyme immunoassay, anti-insulin antibodies were demonstrated in most of serum samples from patients who had been treated with insulin for 0.6–24 months. The detection limit of anti-insulin IgG in human serum was 1,000 to 3,000-fold less than that obtained by the previously reported enzyme immunoassay, in which an insulin-coated polystyrene ball was incubated with diluted serum and subsequently with (antihuman IgG γ-chain) Fab'-horseradish peroxidase conjugate. The present enzyme immunoassay may be useful for the measurement of antibodies for not only insulin but also other antigens that are not precipitated by polyethylene glycol.     

An evaluation of serum high density lipoproteins-phospholipids

Phospholipids in high density lipoproteins (HDL) is being used as a negative risk indicator of atherosclerosis. Phospholipids in HDL may not demonstrate the actual level of HDL-phospholipids when determined by the precipitation or ultracentrifugal methods, because HDL fractions contain very high density lipoproteins (VHDL) and albumin. In the present study, the true level of phospholipids in HDL was estimated using high performance liquid chromatography (HPLC), and it was compared with the level of phospholipids in HDL determined by the precipitation method. Sera from 18 healthy subjects were used as materials. In the HPLC method, the HDL fraction was extracted making sure that it contained no free albumin, which is albumin not bound to phospholipids. The HDL fraction was separated into subfractions. It was found that phospholipids in the VHDL fraction make a 20.2 +/- 7.3% (mean +/- S.D.) part of the total HDL-phospholipids. A large part of the VHDL fraction was constituted of albumin-bound phospholipids. A significant correlation was observed between HDL-phospholipids determined by the precipitation method, which contain albumin, and the actual HDL fraction phospholipids determined by HPLC, which do not contain VHDL (r = 0.903, p less than 0.01). These results suggest that HDL-phospholipids values determined by the precipitation method give useful clinical data.     

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.     

Characteristics of Membrane-Bound and Free Hepatic Ribosomes from Insulin-Deficient Rats I. ACUTE EXPERIMENTAL DIABETES MELLITUS

Membrane-bound and free ribosomes were prepared by discontinuous density gradient centrifugation from livers of rats 2-3 days after receiving alloxan (75 mg/kg) or streptozotocin (100 mg/kg). Hepatocytes from these animals were also examined by electron microscopy and subjected to quantitative morphometric analysis. The results indicated that the two populations of hepatic ribosomes respond differently to acute insulin deficiency. There was an overall reduction (P < 0.001) in total number of bound ribosomes per volume cytoplasm: the remaining bound ribosomes underwent a shift to smaller-sized ribosomal messenger RNA (mRNA) aggregates (P < 0.02); and the proteinsynthetic activity of these bound ribosomes was less than normal (P < 0.02) when protein synthesis was directed by endogenous mRNA. However, there was no difference between bound ribosomes from livers of normal and diabetic rats when protein synthesis was directed by polyuridylic acid. In contrast, free ribosomes were unchanged in number and degree of ribosomal mRNA aggregation, but displayed a significantly increased rate of in vitro protein synthesis (P < 0.01) as compared to normal controls. This increased protein-synthetic activity occurred when amino acid incorporation was directed by endogenous mRNA or polyuridylic acid. These changes in structure and function of bound and free hepatic ribosomes were prevented by the concomitant administration of insulin. The decrease in protein-synthetic activity of bound hepatic ribosomes from acutely diabetic rats seems to be secondary to marked disruption and disaggregation of the rough endoplasmic reticulum (RER) with production of smaller ribosomal mRNA aggregates which incorporate less amino acids into protein. Increased protein synthetic activity of free ribosome appears to be related to the ability of these ribosomes to copy mRNA more efficiently.     

Rapid acyclovir radioimmunoassay, using charcoal adsorption.

Charcoal adsorption of unbound acyclovir rather than ammonium sulfate precipitation of bound acyclovir to facilitate the separation of bound antigen from free antigen gave rise to a radioimmunoassay which was quicker yet still as sensitive and accurate as that previously used.     

Nonspecific blinding of insulin to gamma globulin in the serum of black patients with hepatocellular carcinoma and other forms of liver disease

The study was prompted by the apparent detection of insulin antibodies in a black patient with HCC and recurrent hypoglycemia who had never received insulin. It consisted of two parts. Initially the sera of 30 individuals (six normoglycemic HCC patients, three with HCC and recurrent hypoglycemia, 11 patients with noncancerous liver diseases, and 10 healthy black controls) were analyzed for the presence of insulin (and glucagon) antibodies by precipitating the bound, labeled hormone with ethanol and also by the technique of radioimmunoelectrophoresis. In the nine HCC patients, binding of 125I-insulin averaged 13% by ethanol separation and 0.018 mU/ml with radioimmunoelectrophoresis, levels that were similar to those of patients with noncancerous liver disease and significantly higher than those of the healthy controls. Mean binding of 125I-glucagon was 11% in HCC sera. Serum binding of labeled hormones correlated significantly with IgG concentrations in the patients. The second part of the study attempted to define the nature of insulin binding in HCC and other forms of liver disease. After confirmation of the increased serum binding of labeled insulin by another method of precipitation, PEG, an attempt was made to compete with the labeled insulin for its serum binding sites by adding a large amount of unlabeled insulin. This binding was not displaceable, however, and was therefore considered nonspecific. When the same procedures were repeated using normal serum to which increasing amounts of gamma globulin were added, the nonspecific binding of insulin increased in a linear fashion. Furthermore, a similar degree of high nonspecific insulin binding occurred in six patients with multiple myeloma and raised serum IgG concentrations. We therefore conclude that in the many clinical situations where hypergammaglobulinemia exists, false positive tests for the detection of antibodies against insulin (and probably other peptide hormones) will emerge unless appropriate methods are used to check for nonspecific peptide binding.     

Radioimmunoassay of oestriol in pregnancy plasma

A method for the estimation of oestriol in pregnancy plasma is described. The principal steps of the method are addition of internal standard, precipitation of proteins, acid hydrolysis, solvent partition and estimation by radioimmunoassay using an oestriol antiserum. The radioimmunoassay is based on the precipitation of the “bound” tritiated oestriol with ammonium sulphate. Details of the precision, accuracy, sensitivity and specificity of the method are presented.     

Salivary insulin in normal and type I diabetic subjects

We studied the relationship of salivary insulin to serum insulin concentrations in normal subjects and type I (insulin-dependent) diabetic patients to test the hypothesis that salivary insulin might be a simple measure of insulinemia in diabetes. In 8 nondiabetic subjects, salivary insulin levels increased after an oral glucose load but with a delay in peak concentrations of approximately 45 min in comparison with serum insulin levels. There was a significant correlation (r = .810, P less than .01) between mean serum insulin and the salivary insulin 30 min later. In 12 type I diabetic patients, day profiles of saliva and serum insulin were obtained during usual insulin treatment, diet, and physical activity. In serum, the mean (+/- SE) percentage of bound insulin was 58.8 +/- 5.2%, and in saliva it was 45 +/- 3.5%. The mean ratio of salivary to serum free insulin throughout the day was 1:1.6. Although there was a significant correlation (r = .913, P less than .001) between mean serum free insulin for all patients and the corresponding mean free salivary insulin, several individual profiles showed marked discrepancies between the timing and magnitude of insulin changes in the two compartments. We would not, therefore, recommend salivary insulin concentrations as a reliable index of insulinemia in individuals with type I diabetes.     

Solid-phase fluoroimmunoassay of human albumin in biological fluids.

A simple fluoroimmunoassay for the determination of albumin levels in serum, urine and cerebrospinal fluid is described. It employs magnetisable particles to which antibodies to human serum albumin are covalently linked, and albumin labelled with fluorescein. Equilibrium is reached within 30 min, when separation of the bound and free fractions of the labelled albumin is performed by precipitation of the particles either with a magnet or by centrifugation. Measurement of the fluorescence in the supernatant (the free fraction) reflects the albumin concentration of the standards or samples. Correlation studies with an automated immunoprecipitation technique show good agreement.     

A highly sensitive enzyme immunoassay of anti-insulin antibodies in human serum

A highly sensitive enzyme immunoassay of anti-insulin antibodies in human serum is described. Serum samples were subjected to successive processes of incubation with insulin, dextran-charcoal treatment to remove free insulin, precipitation of insulin-anti-insulin antibodies by polyethylene glycol, acid-treatment of the precipitates to inactivate anti-insulin antibodies and measurement of insulin by sandwich enzyme immunoassay technique. The detection limit of anti-insulin IgG in human serum was 50 pg/assay or 450 ng/l of serum. This was 1,000- to 3,000-fold less than that obtained by a conventional enzyme immunoassay, in which an insulin-coated polystyrene ball was incubated with diluted serum and subsequently with (anti-human IgG gamma-chain) Fab'-horseradish peroxidase conjugate. By the present enzyme immunoassay, anti-insulin antibodies were demonstrated in most (89%) of serum samples from diabetic patients who had been treated with porcine insulin and porcine insulin plus bovine insulin for 0.6-10 mth, while only a small proportion (3%) of serum samples from the same patients was positive by the conventional enzyme immunoassay. Similar results were obtained with serum samples from diabetic patients who had been treated with human insulin for 0.5-8.2 mth. The present enzyme immunoassay may be useful for the measurement of antibodies not only for insulin but also other antigens which can be removed by dextran-charcoal treatment and are not precipitated by polyethylene glycol.     

Endocytotic uptake, processing, and retroendocytosis of human biosynthetic proinsulin by rat fibroblasts transfected with the human insulin receptor gene.

The cellular itinerary and processing of insulin and proinsulin were studied to elucidate possible mechanisms for the observed in vivo differences in the biologic half-lives of these two hormones. A rat fibroblast cell line transfected with a normal human insulin receptor gene was used. Due to gene amplification, the cells express large numbers of receptors and are ideal for studying a ligand, such as proinsulin, that has a low affinity for the insulin receptor. Competitive binding at 4 degrees C showed that the concentration of unlabeled insulin and proinsulin that is needed to displace 50% of tracer insulin or proinsulin was 0.85-0.95 nM and 140-150 nM, respectively. Binding to surface receptors and internalization occur at rates that are four to five times faster in cells incubated with insulin compared with proinsulin. Chloroquine led to an increase in cell-associated radioactivity of approximately 1.4-fold in cells incubated with insulin or proinsulin, but inhibited the appearance of degraded insulin by 54% and degraded proinsulin by only 10%. To study the fate of internalized ligand, cells were incubated with insulin and proinsulin until steady state binding occurred. Surface bound ligand was removed by an acid wash and the remaining cell-associated radioactivity represented internalized ligand. Cells were then reincubated in 37 degrees C buffer and the cell-associated radioactivity and radioactivity released into the medium were analyzed by TCA precipitation, Sephadex G-50, and HPLC. The results demonstrated that proinsulin more readily bypasses the intracellular degradative machinery and is therefore released intact from the cell via the retroendocytotic pathway. These results may help to explain the prolonged metabolic clearance rate and biologic responsiveness of proinsulin in vivo.     

Quantitative determination of insulin-binding antibodies in human serum

Methods are described for the assay of insulin antibodies using ¹²⁵I-labeled insulin and for separation of free from antibody-bound hormone, by acrylamide gel electrophoresis. The sensitivity of the method depends upon the specific activity of the labeled hormone; a simple method is described for the calculation of this. Prior to use, the labeled hormone is highly purified by gel electrophoresis.