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Background: Over the last few decades, new synthetic insulin analogues have been developed. Their measurement is of prime importance in the investigation of hypoglycaemia, but their quantification is hampered by variable cross-reactivity with many insulin assays. For clinical analysis, it has now become essential to know the potential cross-reactivity of analogues of interest., Methods: In this work, we performed an extensive study of insulin analogue cross-reactivity using numerous human insulin immunoassays. We investigated the cross-reactivity of five analogues (lispro, aspart, glulisine, glargine, detemir) and two glargine metabolites (M1 and M2) with 16 commercial human insulin immunoassays as a function of concentration., Results: The cross-reactivity values for insulin analogues or glargine metabolites ranged from 0% to 264%. Four assays were more specific to human insulin, resulting in negligible cross-reactivity with the analogues. However, none of the 16 assays was completely free of cross-reactivity with analogues or metabolites. The results show that analogue cross-reactivity, which varies to a large degree, is far from negligible, and should not be overlooked in clinical investigations., Conclusions: This study has established the cross-reactivity of five insulin analogues and two glargine metabolites using 16 immunoassays to facilitate the choice of the immunoassay(s) and to provide sensitive and specific analyses in clinical routine or investigation.

Aim: Glargine, a long-acting insulin analogue, is metabolized in the bloodstream and in subcutaneous tissue. Glargine metabolism and its implications for diabetes therapy remain poorly understood. The aim of our study was to assess in vitro the glargine blood biotransformation and its inter-individual variability., Methods: Formation of M1 glargine metabolite in vitro was studied with Elecsys Insulin immunoassay in pools of sera and sera from patients spiked with glargine. Elecsys Insulin assay is specific of human insulin, does not recognize glargine and its M2 metabolite but does recognize its M1 metabolite., Results: Glargine incubation with serum resulted in M1 metabolite formation which was detected and characterized as an enzymatic process: metabolite kinetics were dependant on temperature, substrate concentration and serum proportion. Carboxypeptidase inhibitors and chelating agents partially inhibited the activity of the enzyme(s). Glargine biotransformation was decreased when blood was collected on EDTA tubes. After 30 min incubation of glargine (100 mU/l) in 69 sera at 37 degrees C, percentage of glargine converted into M1 ranged from 46% to 98% (mean 72%; S.D. 11%)., Conclusion: Glargine blood biotransformation is an enzymatic process probably involving serum carboxypeptidase(s). Metabolite formation is rapid and non negligible. Inter-individual variability of glargine biotransformation is noteworthy and should be confronted to M1 metabolite bioactivity which has not been fully documented yet.

Background: In clinical studies involving rapid-acting analogues (RAAs), insulin immunoreactivity is frequently measured, including endogenous, regular insulin (RI) and RAA immunoreactivities. Such a procedure implies equivalent cross-reactivities of all insulins present in serum. Commercially available human insulin immunoassays have been widely used, but their limitations (including hemolysis and anti-insulin antibodies) were not fully investigated. The aims of our study were to compare cross-reactivities of RI and RAAs in buffer and in serum and to investigate insulin immunoassay pitfalls., Methods: Cross-reactivities were assessed using Bi-insulin IRMA (Schering Cis-Bio International) in phosphate-buffered saline (PBS)-1% bovine serum albumin (BSA) and in pools of sera spiked with RI and RAAs (lispro and aspart). To investigate the influence of hemolysis, a pool of sera spiked with RAA was mixed with a concentrated hemolysate (final hemoglobin concentration 10 g/L) and incubated for 3 h at room temperature. To determine interference by anti-insulin antibodies, insulin was removed using charcoal from 18 sera with anti-insulin antibodies and from 17 sera without detectable anti-insulin antibodies. These insulin-free samples were then spiked with RI and RAAs and the immunoreactivity was determined., Results: Compared with buffer, cross-reactivity in serum for RI, lispro and aspart was lower (35%, 29% and 26% lower, respectively). Hemolysis degraded almost all RI and RAAs contained in the serum (>or=95%). Anti-insulin antibody interference was significant for RI and RAAs (p

Insulin assays play a central role in the investigation of glucose metabolism disorders (investigation of the causes of hypoglycemia, assessment of beta-cell function and determination of pathogenesis of type 1 and type 2 diabetes), and in studies on the pharmacology of insulin itself. Greater convenience and improved reproducibility, sensitivity and specificity have been achieved with human insulin immunometric assays. Besides human insulin, insulin analogues (lispro, aspart and glargine) have been introduced to therapeutic use. The specificity of human insulin assays to these analogues should be assessed. Anti-insulin antibodies are present in a significant proportion of sera to be analyzed for insulin. The sensitivity of insulin assays to interference from these antibodies should also be assessed. Interferences from anti-insulin antibodies and hemolysis, which degrades insulin molecules, remain the main pitfalls of insulin assays. Immunometric assays have also improved the sensitivity and reproducibility of free insulin measurements. Standardization of insulin immunoassays is still called for.

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.

Objective: To assess the antigenicity of the insulin Hoechst 21PH (Hoe21PH) using continuous subcutaneous insulin infusion (CSII) and to compare the antigenicity of this insulin when administered intraperitoneally or subcutaneously. RESEARCH DESIGN AND METHODS; Peritoneal administration of Hoe21PH (Hoechst-Roussel, Somerville, NJ) insulin using implantable devices (continuous peritoneal insulin infusion [CPII]) increases anti-insulin antibody (AIA) levels in type 1 diabetic patients. Intraperitoneal administration, addition of a stabilizer (polyethylene polypropylene glycol), or insulin modifications due to storage in the pump may be involved in this antigenicity. In this nonrandomized study, 24 type 1 diabetic patients were treated with either CSII (n = 11, group 1) or CPII (n = 13, group 2). AIA levels were measured by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) before starting patients on Hoe21PH and again after 3 and 6 months., Results: Patients were comparable in the two groups. AIA levels (RIA) remained stable (24.3 +/- 8.5% [month 0] to 24.9 +/- 8.5.5% [month 6]) in group 1 and increased (21.8 +/- 6.7% [month 0] to 41.8 +/- 6.9% [month 6]) in group 2 (P = 0.005, Wilcoxon's rank-sum test). Using ELISA, AIA remained stable in the patients in group 1 (n = 9; 3.8 +/- 0.8 units/ml [month 0] and 4.1 +/- 1.0 units/ml [month 6]) and tended to increase in the patients in group 2 (n = 12; 4.1 +/- 0.7 units/ml [month 0] to 17.5 +/- 4.6 units/ml [month 6]) (P = 0.07). Comparison of the evolution of AIA formation between the two groups, using RIA at months 0, 3, and 6 showed a significant difference (analysis of variance, P = 0.009)., Conclusions: No increase in AIA levels was demonstrated when Hoe21PH insulin was administered subcutaneously as assessed by two different assays. CPII is proven to be more antigenic than CSII, and this is not related to a specific antigenicity of Hoe21PH insulin. The intraperitoneal route of administration or insulin modifications due to insulin storage in implantable devices might explain this antigenicity.

Intraperitoneal insulin infusion of Genapol stabilized insulin via implantable devices significantly improves diabetes control and hypoglycemia frequency in type 1 diabetes while it increases insulin antibody levels. Causes for this particular antigenicity remain unknown. The role of insulin modifications occurring in the reservoir on the antigenicity observed was assessed by comparing the antigenicities of the insulin coming from the vial or from the pump reservoir. Rats were injected intraperitoneally with insulin sampled either from a vial (group 1) or from a pump reservoir during a refill of a clinical trial (group 2). Two control groups, one without insulin, the second one receiving a mixture of silicone and insulin were also studied. Human insulin antibody levels were assessed by RIA 10 days after 4 weekly immunizations. AIA levels were higher in group 1 compared to group 2 (P = 0.003 for the first experiment, P = 0.04 in the second experiment). The increased antigenicity of the insulin sampled from the implanted pump might be due to the insulin modifications occurring during the storage in the device. Insulin aggregates could be involved in this antigenicity since they are known to be antigenic and their concentration was shown to be related to the amplitude of the antigenic response.

The recent development of specific radioimmunoassays of insulin (Ins) and proinsulin (PI) led some authors to question the classical data on insulinosecretion in patients with abnormal glucose tolerance. The aim of this work was to determine the participation of intact proinsulin (iPI) and its split fragments to total insulinsecretion in obese subjects and in various stages of glucose intolerance determined by an oral glucose load according to the WHO recommendations. Five groups were constituted: non obese controls (C), obese subjects with normal glucose tolerance (O), non obese subjects with impaired glucose tolerance (I), obese subjects with impaired glucose tolerance (OI) and diabetic patients (D). The basal level of total Proinsulin (tPI) of D and OI was significantly higher than that of C, but the tPI/Ins ratio did not differ between the five groups. After glucose load, this ratio tended to be higher in D, but not significantly. No statistical difference between groups was observed for the iPI/Ins ratio. These results indicate that the determination of tPI is at least as informative or more than that of iPI. Furthermore, the proportionally constant participation of PI to insulin secretion observed in various stages of glucose intolerance suggests that the results obtained in the past with non specific insulin radioimmunoassays remain valid.

Insulin was assayed directly using radioimmunoassay and immunometric assay in 31 sera containing anti-insulin antibodies. Anti-insulin antibodies were determined by radio-binding-assay. Insulin measurements were compared with those of free (unbound to antibodies, polyethylene glycol precipitated) insulin measurements. Compared with free insulin concentrations, radioimmunoassay and immunometric assay yielded falsely increased insulin results. The degree of overestimation by radioimmunoassay and by immunometric assay correlated with the anti-insulin antibody value. Anti-insulin antibodies still remain a possible pitfall in the insulin-specific immunometric assays which are now being widely used.

Intraperitoneal insulin infusion using implantable devices in insulin-dependent diabetic (IDDM) patients is promising since it improves diabetic control and decreases frequency of hypoglycaemia. However, preliminary data show a striking increase in plasma levels of anti-insulin antibodies with this therapy. In order to more precisely evaluate the immunogenicity and its consequences, anti-insulin antibody levels in 62 IDDM patients were assessed every 3 months during a 2-year period following pump implantation. At the same time, diabetes control was evaluated with HbA1c, mean blood glucose levels, standard deviation of the daily blood glucose levels and the frequency of low blood glucose (< 3.58 mmol/l). Factors involved in antibody formation such as age, gender, HLA typing, and complement C4 alleles were also studied. After implantation, anti-insulin antibody levels increased significantly from 3.14% (range 0-26%) to 8.34% (0-49%) after 1 year and remained elevated. Patients were divided into two groups: responders able to show at least one antiinsulin antibody titre higher than 15% and non-responders whose titres were always lower than 6%. None of the factors studied was shown to statistically influence the anti-insulin antibody titres. Non-responders had significantly better metabolic results than the responders. Severe hypoglycaemic episodes decreased dramatically in both groups. Insulin requirements were comparable at time 0 and decreased initially in both groups. They remained low for the non-responders but returned to pre-implantation values for responders. Intraperitoneal insulin infusion led to a high immunogenetic response towards insulin in about half of the patients, leading to only moderately deleterious effects on metabolic control.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin secretion is assessed by simultaneous radioimmunological assay of insulin (R.I.I.) and C-peptide (R.I.C.P.) levels under basal conditions, and after stimulation by oral or intravenous glucose administration. Subjects with abnormal glucose tolerance demonstrate an increase in the ratios R.I.C.P. divided by glucose and R.I.C.P. divided by R.I.I. after fasting, increase in R.I.I. and R.I.C.P. occurring later and lasting longer after glucose loading. These anomalies are observed in both obese subjects and those with normal body weights. Simultaneous determination of R.I.I. and R.I.C.P. levels appears of value in a limited number of cases where glucose loading tests do not supply precise information on the quality of glucose tolerance. Obese subjects, in whom abnormal hyperglycemia levels provoked by oral glucose are observed, but in whom the peripheral glucose assimilation coefficient is normal, can be considered to be non-diabetic as shown by the levels of R.I.I. and R.I.C.P. and more particularly by the molar ratio R.I.C.P. divided by R.I.I.

It is well established that glucagon plays an important role in the regulation of fuel supplies as its plasma level increases during the first days of a complete fast. However, it is not certain that glucagon is involved in the adaptation to chronic starvation. In the present study, this problem was investigated by the determination of the changes in the plasma glucagon level elicited by an i.v. glucose tolerance test followed by an i.v. arginine perfusion in 26 self starved patients suffering from anorexia nervosa (AN) and 14 control patients having only minor neurotic disorders. The basal plasma glucagon level tended to be higher in the AN patients than in the controls; but the difference was not statistically significant. Glucagon responses to glucose and arginine observed in the AN patients were not significantly different from those seen in the control patients. In the AN patients, the insulin response to both loads was reduced and the plasma GH level increased paradoxically after the glucose load, whereas it rose normally after the arginine load. It may be concluded that in chronic starvation by AN the regulation of fuel supplies depends mainly on decreased insulin and increased growth hormone secretion. The role of glucagon seems to be of minor importance in this condition.

In order to investigate the controverted effect of ammonia on insulin and glucagon secretion 3 groups of 55 rats were perfused either by Na+ acetate (controls), either by NH4+ acetate: in the first group plasma insulin (IRI) and glucagon (IRG) levels were compared before and after perfusion; the second group was supplemented by glucose perfusion (1 g/100 g/hour); in the last group an arginine perfusion (1 g/kg/min) was started 15 min after the beginning of NH4+ or Na acetate and IRI and IRG levels were determined in the portal blood. Hyperammonemia, which reached about 500 microgram/dl, reduced significantly the IRI portal level and the IRI secretion induced by glucose or arginine, whereas basal or stimulated IRG levels are not modified. The ratio IRI:IRG is diminished in each group perfused with NH4+ acetate. Our results show that ammonia inhibits insulin without modifications in the glucagon secretion; hyperglucagonemia and hyperinsulinism reported in hepatic encephalopathy with chronic hyperammonemia may not be attributed to an effect of ammonia.

The existence of a preabsorptive insulin reflex is well known in animals but remains controversial in humans. Glycemia and insulin variations following olfactive and visual presentation of a standard meal were studied in 25 subjects, 10 of them (5 men and 5 women) of normal weight and 15 overweight (7 men and 8 women), after a 15 hour fast. Blood samples were collected continuously, every minute for 16 minutes after the meal was presented. The presentation produced an early blood insulin increment, variable in magnitude and time course and occurring between the 3rd and 9th minute, in both normal and overweight subjects. Glycemia variations were not significant. Our study demonstrated a positive correlation between the reflex insulin release, body weight and a conscious effort to maintain current body weight. However, the differences between overweight and normal subjects remained small. The physiological and psychological determinants of the cephalic phase of insulin secretion are discussed.

In order to evaluate the relationship between nutritional status and insulin secretion in cirrhosis, the following parameters of caloric (tricipital skin fold, prealbumin) and proteic (arm muscle size, transferrin, 24 h-urinary creatinine excretion) nutritional status were compared in 20 alcoholic cirrhotics and 10 normal subjects. Insulin secretion was evaluated in both groups by insulin and C-peptide response to an intravenous glucose tolerance test and by 24 h urinary excretion of C-peptide. When compared to normals, cirrhotics have lower values for all nutritional status parameters and individually for at least three of those in 14 (70 p. 100) patients. In cirrhotics there is a significant decrease of the 4-min poststimulative response of insulin and C-peptide, contrasting with higher basal and late poststimulative values than in normals. This contrast could be explained by a reduced metabolic clearance rate of insulin (consistent with insulin resistance) and of C-peptide (the urinary clearance of which is 2.5 times lower in cirrhotics than in normals). The 24-h urinary excretion of C-peptide, probably weakly dependent of this reduced clearance, is 50 p. 100 lower in cirrhotics: 12.9 +/- 1.6 nM/24 h than in normals: 26.0 +/- 2.4 nM/24 h (p less than 0.001). In cirrhotics there is a significant linear correlation between 24 h urinary C-peptide excretion and all the nutritional status parameters but one (prealbumin). These results indicate that in cirrhosis: 1) urinary C-peptide excretion rate is a good index of insulin secretion; 2) urinary C-peptide indicates a marked deficit in insulin secretion.(ABSTRACT TRUNCATED AT 250 WORDS)