Correlation between HbA1c, purified insulins, diabetic control, and insulin antibodies in diabetic children

Insulin antibodies were determined in sera from 38 children diagnosed as having juvenile diabetes for a duration of 0.7-15.2 years (median = 4.9 years). 8 children were treated with purified porcine insulins from the beginning of their disease, 16 children with bovine insulin NPH alone, and 14 children with non-purified, of whom 9 were later transferred to purified insulins. Serum insulin antibodies were measured by non-specific and specific methods using beef (B) and pork (P) antigens as described by Welborne and Sebriakova, respectively. 12/38 children had insulin binding levels similar to those of normal children, irrespective of the type of insulin used. The concentration of antibodies using radiolabelled B or P insulins as antigens were strongly correlated, by both the non-specific (p less than 0.01) and the specific (p less than 0.01) methods. Children with better score for diabetic control had significantly lower levels of insulin antibodies against B (p less than 0.05) and P (p less than 0.05) than those with poor diabetic control. There was also a significant positive correlation between mean HbA1c concentration and both B and P mean insulin antibody concentration (p less than 0.01). Finally, patients treated with purified porcine insulin had significantly lower levels of antibodies than patients with non-purified bovine insulin (p less than 0.05).     

Heterogeneity and specificity of human anti-insulin antibodies determined by isoelectric focusing

Anti-insulin antibodies are present in the majority of insulin treated diabetics, and in some cases these antibodies have been found to be highly specific for limited epitopes on the molecule. To determine how the human response differs from that seen in inbred animals, we have examined the heterogeneity and specificity of human anti-insulin antibodies by isoelectric focusing (IEF). In addition, we have used human insulin to examine the extent of autoreactivity in the serum of subjects treated with animal insulins. The majority of diabetic sera exhibited complex IEF spectra that were composed of discrete bands and unresolved smears. Autoradiography using 125I-beef, pork, and human insulin revealed some affinity differences; however, the predominant antibodies were capable of binding all insulins, including human. These specificity studies were extended by comparing competitive inhibition with excess cold insulins, and sera with highly specific binding of the A chain loop of beef insulin were identified. The spectra by IEF of these highly specific sera were found to be variable. Our results indicate that the majority of insulin-treated diabetics develop a heterogeneous antibody response that is more complex than the response of inbred animals and includes reactivity with autologous insulin. Although infrequent, individuals having antibodies directed at limited regions of the molecule can be identified and will provide valuable tools for dissecting this complex response.     

Structural and morphological characterization of ultralente insulin crystals by atomic force microscopy: evidence of hydrophobically driven assembly.

Although x-ray crystal structures exist for many forms of insulin, the hormone involved in glucose metabolism and used in the treatment of diabetes, x-ray structural characterization of therapeutically important long-acting crystalline ultralente insulin forms has been elusive because of small crystal size and poor diffraction characteristics. We describe tapping-mode atomic force microscopy (TMAFM) studies, performed directly in crystallization liquor, of ultralente crystals prepared from bovine, human, and porcine insulins. Lattice images obtained from direct imaging of crystal planes are consistent with R3 space group symmetry for each insulin type, but the morphology of the human and porcine crystals observed by AFM differs substantially from that of the bovine insulin crystals. Human and porcine ultralente crystals exhibited large, molecularly flat (001) faces consisting of hexagonal arrays of close packed hexamers. In contrast, bovine ultralente crystals predominantly exhibited faces with cylindrical features assignable to close-packed stacks of insulin hexamers laying in-plane, consistent with the packing motif of the (010) and (011) planes. This behavior is attributed to a twofold increase in the hydrophobic character of the upper and lower surfaces of the donut-shaped insulin hexamer in bovine insulin compared to its human and porcine counterparts that results from minor sequence differences between these insulins. The increased hydrophobicity of these surfaces can promote hexamer-hexamer stacking in precrystalline aggregates or enhance attachment of single hexamers along the c axis at the crystal surface during crystal growth. Both events lead to enhanced growth of ¿hk0¿ planes instead of (001). The insulin hexamers on the (010) and (110) faces are exposed "edge-on" to the aqueous medium, such that solvent access to the center of the hexamer and to solvent channels is reduced compared to the (001) surface, consistent with the slower dissolution and reputed unique basal activity of bovine ultralente insulin. These observations demonstrate that subtle variations in amino acid sequence can dramatically affect the interfacial structure of crystalline proteins.     

A radioreceptor assay for pharmaceutical preparations of insulin

A radioreceptor assay has been developed that is suitable for the measurement of the potency of crystalline insulin and pharmaceutical insulin formulations. It utilizes the well characterized and widely available IM-9 human lymphocyte cell line as the source of receptor. Bovine, porcine and human crystalline and formulated insulins have been assayed against the 4th International and European Standards for Insulin and the potencies compared with those obtained by the mouse blood glucose method. Results with bovine insulin were in full correspondence with the in vivo results. Porcine and human insulins were 15-20% more potent by the radioreceptor assay than by the in vivo method when the mixed bovine and porcine insulin 4th International and European Standards were used, but were equivalent when compared with like materials. Average 95% confidence limits for formulated insulins in two assays were +/- 6% of the mean. The coefficient of variation on repeated assay of the same sample was 3.8%. The three dose parallel line radioreceptor assay with appropriate species species standards is a candidate biological test capable of international adoption as an alternative to in vivo animal testing of insulin.     

A radioreceptor assay for insulin: direct measurement of dog pancreatic vein serum insulin.

A simple radioreceptor assay for insulin rat liver membranes as receptor sites, with sufficient specificity precision, and sensitivity to detect 10 ng or 276 muU/ml of serum insulin, has been developed. In the presence of standard porcine insulin at the concentration of 1.0 ng/tube, approximately 8% of 125I-porcine insulin was bound to the plasma membranes and ninety-five per cent of this binding was inhibited by 1.0 microgram of standard insulin per tube. Four animal insulins inhibited the binding of 125I-insulin while ACTH, glucagon, human growth hormone, and oxytocin were inert. Insulin values in dog pancreatic vein sera obtained during and after glucose loading and measured by the present radioreceptor assay agreed well with immunoreactive insulin. The ratio of IRI to the measurement by radioreceptor assay was 1.09 +/- 0.18 for the same sera.     

World Health Organization International Standards for highly purified human, porcine and bovine insulins

The 4th International Standard (IS) for Insulin, established in 1958, consists of a mixture of relatively impure bovine and porcine insulins and is not suitable as a standard for the assay of highly purified single-species insulins presently used in the treatment of diabetes. Preparations of human, bovine and porcine crystalline insulins, representative of current highly purified therapeutic insulins, have now been studied in an international collaborative study carried out by twenty-three laboratories in fifteen countries. In the collaborative study described here, each of the three preparations was found to be suitable for use as a standard for insulin for bioassay and each was established by WHO in 1986 as an international standard. The 4th IS of Insulin bovine/porcine (code numbered 58/6) has been discontinued. Insulin preparations should now be calibrated in terms of International Units defined by the standard for the appropriate species: the International Standard for Insulin, Human, the International Standard for Insulin, Bovine, or the International Standard for Insulin, Porcine.     

Analytical biotechnology of recombinant peptides and proteins. I. Analysis of the purity, composition and structure of human, porcine and bovine insulins

A method for analysis of the type, purity, and possible structural modifications of insulins of bovine, porcine, and human origin was proposed. It is based on a combination of narrow-bore reversed-phase HPLC and mass spectrometry. The hydrolysis of insulins with highly specific Glu-protease V8 from Staphylococcus aureus followed by peptide mapping of the hydrolysis products and mass spectrometry of the isolated fragments helps rapidly and reliably localize and identify substitutions of amino acid residues in insulin structure by using insulin samples of less than 1 nmol.     

Can insulin antibodies of diabetic patients distinguish human insulin from porcine insulin?

We examined antisera from patients treated with bovine-porcine mixture (hereafter referred to as bovine/porcine), porcine or human insulin, and compared their binding affinities to human insulin with those to porcine insulin. Patients treated with bovine/porcine insulin developed antisera with a higher affinity to porcine insulin compared with that to human insulin in five of nineteen cases. Furthermore, three of these five antisera had a comparable affinity to bovine and porcine insulin and appeared to recognize the amino acid residue at B-30. Treatment with porcine or human insulin, on the other hand, did not result in any significant difference in the affinity to porcine and human insulin in twenty-three patients. These results indicate the significant role of B-30 amino acid residue as an antigenic determinant, and suggest that the amino acid sequence of the A chain of bovine insulin may contribute to the development of antibody recognizing B-30 amino acid residue.     

Analytical biotechnology of recombinant peptides and proteins: I. determination of the purity, composition, and structure of human, porcine, and bovine insulins

A method for analysis of the type, purity, and possible structural modifications of insulins of bovine, porcine, and human origin was proposed. It is based on a combination of narrow-bore reversed-phase HPLC and mass spectrometry. The hydrolysis of insulins with highly specific Glu-protease V8 fromStaphylococcus aureus followed by peptide mapping of the hydrolysis products and mass spectrometry of the isolated fragments helps rapidly and reliably localize and identify substitutions of amino acid residues in insulin structure by using insulin samples of less than 1 nmol.     

Very pure porcine insulin in clinical practice.

In a one-year follow-up study the insulin dose in diabetic patients using very pure porcine insulin was compared with that in patients using conventional preparations. The dose of insulin used to obtain diabetic control was reduced by 7% in 108 patients treated solely with very pure porcine insulin from the start of insulin treatment when compared with 108 matched patients who had received conventional insulins. In 117 patients whose treatment had been changed from conventional bovine or bovine-porcine insulin to very pure porcine insulin the dose was reduced by 9%. A further 511 patients receiving conventional insulins were examined for local cutaneous or subcutaneous abnormalities at insulin injection sites. Lipoatrophy was found in 49 of these patients (10%), but not in patients using very pure porcine insulin. The results confirm that very pure porcine insulin reduces the insulin dose needed to maintain diabetic control and may resolve or prevent local reactions such as lipoatrophy. Long-term advantages in reduced antigenicity to insulin and contaminating peptides remain to be established.     

Immunocytochemical localization and immunochemical characterization of an insulin-related peptide in the pancreas of the urodele amphibian,Ambystoma mexicanum

Summary The pancreas of the axolotl, Ambystoma mexicanum, was investigated by immunocytochemical methods for the presence of immunoreactivity to a number of antisera raised against mammalian insulins. All anti-insulin antisera tested revealed substantial amounts of reaction products confined solely to the aldehyde-fuchsinophilic B cells of the endocrine pancreas. The reactive cell population was detected by use of one polyclonal antiserum against bovine insulin and eight different monoclonal antibodies against insulins from various mammalian species. Six of these antibody clones have known specificity to sub-regions of the insulin molecule. Additionally, fractions of an ethanol-HCl extract of pancreatic tissue from Ambystoma was studied in both conventional dot-blot tests by means of the same panel of antibodies and a two-site sandwich time-resolved immunofluorometric assay for human insulin involving two of the monoclonal antibodies. These experiments support the immunocytochemical observations by demonstrating the existence of an insulin-related peptide with a great deal of structural resemblance to mammalian insulins and displaying antigenic determinants in common at least with the amino acid residues A_8–10 and B_26–30. In conclusion, we interpret the findings as indicating that the immunocytochemically revealed tissue bound antigen in the Ambystoma pancreatic B-cells may be a peptide related to human insulin.Supported in part by SNF grant 11-5082 (G.N.H.). The authors are indebted to Dr. P. Rosenkilde for the gift of the Ambystoma material     

Radioimmunoassay for zearalenone and zearalanol in human serum: production, properties, and use of porcine antibodies

To produce antigens susceptible to raise antibodies for resorcylic acid lactones, the 6'-carboxymethyloxime derivatives of zearalenone and zearalanone were bound to bovine serum albumin. Pigs could be immunized by using these antigens, the best titer in antibodies being obtained with the zearalenone antigen. The porcine antibodies were specific for the resorcylic acid lactones of structural resemblance with zearalenone. This specificity made the antibodies usable for a radioimmunoassay of zearalenone and zearalanol, which may be found in human and animal sera. The range of the assay was between 0.25 and 10 ng. The limit of detection was 5 ppb (5 ng/ml) in human serum.     

Comparative study of binding of Scorpaena porcus scorpion fish insulin and pig insulin by specific muscle plasma membrane receptors

Insulin from the scorpion-fish and porcine insulin have been compared with respect to their capacity to interact with the specific receptors in plasma membrane of skeletal muscle fibers of rats. Both insulins inhibited membrane binding of insulin-125J; the dose of porcine insulin which inhibited binding by 50% was equal to 3.7 nM, that of fish insulin -- to 100 nM. These figures indicated lower affinity of fish insulin to mammalian receptors (4% of the porcine affinity), being in accordance with lower biological activity of fish insulin in diaphragmal test. Other peculiarity of fish insulin was revealed in studies on dissociation of insulin-125J, which was previously bound to isolated diaphragm muscle of the rat. In contrast to mammalian insulin, it was not capable of increasing the dissociation when being added to a solution in doses from 7.10(-11) to 7.10(-9) M. This fact is analysed in term of the "site--site" interactions concept.     

Human insulin: study of safety and efficacy in man.

The safety and efficacy of a new highly purified neutral soluble human insulin produced by conversion of porcine insulin was compared with a highly purified neutral soluble porcine insulin in six normal men. The insulins were administered by subcutaneous injection at a dose of 0.075 U/kg body weight. Somatostatin was infused during the experiment to suppress endogenous insulin secretin. No difference was found in the plasma glucose, insulin, or metabolite responses. Thus the potency, onset, and duration of effect were identical with the two insulins. No short-term side effects to either insulin were observed. Highly purified, semi-synthetic human insulin offers a safe and effective means to explore the possible advantages of homologous human insulin in the management of diabetes mellitus.     

Transfer from porcine insulin to human insulin in insulin-dependent diabetes mellitus: effects on insulin binding to IgG and glycemic control

Thirty type I diabetic patients who were treated for at least 2 years with a combination of regular and lente monocomponent porcine insulins were allocated in a double-blind study to either continued porcine insulin treatment or a transfer to the corresponding semi-synthetic human insulins. Insulin binding to IgG measured by an immunoelectrophoretic method, was followed at 3-month intervals for 1 year, and did not change after the transfer. The glycemic control, as assessed by hemoglobin A1 levels, tended to deteriorate in the human insulin group during the first 3 months of the trial and then return to the baseline level. It is concluded that a transfer from highly purified porcine insulin to human insulin apparently does not change the insulin binding to IgG in already sensitized patients.     

Radioimmunoassay for Zearalenone and Zearalanol in Human Serum: Production, Properties, and Use of Porcine Antibodies

To produce antigens susceptible to raise antibodies for resorcylic acid lactones, the 6′-carboxymethyloxime derivatives of zearalenone and zearalanone were bound to bovine serum albumin. Pigs could be immunized by using these antigens, the best titer in antibodies being obtained with the zearalenone antigen. The porcine antibodies were specific for the resorcylic acid lactones of structural resemblance with zearalenone. This specificity made the antibodies usable for a radioimmunoassay of zearalenone and zearalanol, which may be found in human and animal sera. The range of the assay was between 0.25 and 10 ng. The limit of detection was 5 ppb (5 ng/ml) in human serum.     

Comparison of the effect of semisynthetic human insulin and porcine insulin on glucose kinetics, plasma free fatty acid and amino acid levels in man

The effect of semisynthetic human insulin on hepatic glucose output, peripheral glucose clearance, plasma levels of C-Peptide, free fatty acids and amino acids was compared with purified pork insulin using the glucose clamp technique. 8 normal overnight-fasted subjects received intravenous infusions of either human or porcine insulin at 20 mU/m2.min(-1) during 120 min achieving plasma insulin levels of approximately equal to 50 mU/l. Plasma glucose levels were maintained at euglycaemia by variable rates of glucose infusion. Hepatic glucose production measured by continuous infusion of 3-(3) H-glucose was similarly suppressed by both insulins to rates near zero. The metabolic clearance rate of glucose increased during infusion of human insulin by 120%, C-peptide levels decreased by 41% and plasma FFA concentrations fell by 74%. The respective changes during infusion of pork insulin were similar, 118%, 48% and 72%. Both insulins decreased the plasma levels of branched-chain amino acids, tyrosine, phenylalanine, methionine, serine and histidine similarly. Thus, the results demonstrate that semisynthetic human and porcine insulin are aequipotent with respect to suppression of hepatic glucose output, stimulation of glucose clearance, inhibition of insulin secretion, lipolysis and proteolysis.     

Isolation, primary structure, and biological and immunological properties of pink and chum salmon insulins

1. Insulins have been isolated from islet tissue of pink (Oncorhynchus gorbuscha) and chum (Oncorhynchus keta) salmon. The primary structure of chum and pink salmon insulins was found to be identical. Compared to the amino acid sequence of human insulin, the salmon insulins under study differed at 14 positions. 2. Biological activity of pink salmon insulin was 83% of that of standard porcine insulin. 3. The immunological properties of fish insulins were investigated in specific radioimmunoassay (RIA) systems, based on porcine and pink salmon insulins. 4. A significant difference in the antigenic determinants of these fish and mammalian hormones was revealed.     

Double blind clinical and laboratory study of hypoglycaemia with human and porcine insulin in diabetic patients reporting hypoglycaemia unawareness after transferring to human insulin.

OBJECTIVES--To compare awareness of hypoglycaemia and physiological responses to hypoglycaemia with human and porcine insulin in diabetic patients who reported loss of hypoglycaemia awareness after transferring to human insulin. DESIGN--Double blind randomised crossover study of clinical experience and physiological responses during slow fall hypoglycaemic clamping with porcine and human insulin. SETTING--Clinical investigation unit of teaching hospital recruiting from diabetes clinics of five teaching hospitals and one district general hospital. SUBJECTS--17 patients with insulin dependent diabetes mellitus of more than five years'' duration who had reported altered hypoglycaemia awareness within three months of transferring to human insulin. MAIN OUTCOME MEASURES--Glycaemic control and frequency of hypoglycaemic episodes during two months'' treatment with each insulin. Glucose thresholds for physiological and symptomatic responses during clamping. RESULTS--Glycaemic control did not change with either insulin. 136 hypoglycaemic episodes (eight severe) were reported with human insulin and 149 (nine severe) with porcine insulin (95% confidence interval -4 to 2.5, p = 0.63). 20 episodes of biochemical hypoglycaemia occurred with human insulin versus 18 with porcine insulin (-0.8 to 1, p = 0.78). During controlled hypoglycaemia the mean adrenaline response was 138 nmol/l/240 min for both insulins; neurohormonal responses were triggered at 3.0 (SE 0.2) versus 3.1 (0.2) mmol/l of glucose for adrenaline and 2.5 (0.1) versus 2.5 (0.1) mmol/l for subjective awareness. CONCLUSIONS--These data suggest that human insulin per se does not affect the presentation of hypoglycaemia or the neurohumoral, symptomatic, and cognitive function responses to hypoglycaemia in insulin dependent diabetic patients with a history of hypoglycaemia unawareness.     

Human insulin

The two human insulins of clinical importance are (a) semisynthetic human insulin prepared from pork pancreas by enzymatically substituting threonine for alanine-the last amino acid in the beta chain-thereby transforming pork insulin in vitro to human insulin; and (b) biosynthetic human insulin synthesized biotechnologically in Escherichia coli-K12. Using this latter technique, it is possible to produce mass quantities of highly purified insulin for the treatment of insulin-dependent diabetics, avoiding the problems inherent in supplies of insulin produced from animal pancreas. It has been suggested that to avoid confusion the two human insulins should be called semisynthetic human insulin of pork origin and biosynthetic human insulin of E. coli origin, respectively. These insulins have four advantages over highly purified animal insulins: (a) they induce lower titers of circulating insulin antibodies; (b) their subcutaneous injection is associated with fewer skin reactions; (c) they are absorbed more rapidly from the injection site; and (d) less degradation occurs at the site of injection. These data indicate that newly diagnosed insulin-dependent diabetes, particularly in children, should be treated with either of the two human insulins. The warranty against inadequate supplies of insulin offered by biosynthetic human insulin makes the use of pork insulins unnecessary and beef insulins totally useless.