Determination of antipituitary antibody in patients with endocrine disorders by enzyme-linked immunosorbent assay and Western blot analysis.

The identification of pituitary antigens recognized by human antipituitary antibodies (APAs) is important in evaluating the pathophysiology of multiendocrine disorders linked to autoimmune factors. However, there is no convenient method for the quantitative analysis of circulating APAs. This study reports the development of an enzyme-linked immunosorbent assay (ELISA) for the detection of APAs. APAs were measured by ELISA and confirmed by Western blot analysis in sera from patients with endocrine disorders. APAs were detected frequently in patients with autoimmune thyroiditis, insulin-dependent diabetes mellitus (IDDM), or pituitary dwarfism. Circulating APAs were detected in 18% of patients with autoimmune thyroiditis. Confirmation by Western blot revealed positivity for APAs in the serum of 36% of patients with Hashimoto disease and in 29% of patients with Graves disease. Notably, 39% of patients with IDDM were also positive for APAs by ELISA. The identification of APAs by ELISA may be useful in evaluating autoimmune mechanisms involved in patients with multiendocrine disorders.

Enhancement of nasal salmon calcitonin absorption by lauroylcarnitine chloride in rats.

PURPOSE: We investigated optimum formulation characteristics in the nasal absorption of salmon calcitonin (sCT) by incorporation of acylcarnitines. METHODS: Nasal sCT formulations were administered to anesthetized rats. Plasma calcium level was measured and pharmacological bioavailability (P.bioav) was calculated. RESULTS: Nasal sCT absorption was significantly enhanced by carnitines with acyl groups of 12 or more carbon atoms. Enhancement by lauroylcarnitine chloride (LCC) was observed at its critical micelle concentration and reached a plateau at the concentration of 0.1 percent. Optimal absorption was achieved at a molar ratio of LCC to sCT of 5:1. Enhancement was not influenced by osmolarity and maximum enhancement was obtained at pHs 3.1 and 4.0. CONCLUSIONS: The 12-carbon LCC was the strongest enhancer among acylcarnitines. Micelle formation played a key role in this enhancement effect.

Association of sick sinus syndrome with hyperinsulinemia and insulin resistance in patients with non-insulin-dependent diabetes mellitus: report of four cases.

We report four non-insulin-dependent diabetic (NIDDM) patients accompanied by a unique combination of sick sinus syndrome (SSS) and hyperinsulinemia of unknown etiology. SSS of all four cases was due to sinus arrest in association with paroxysmal atrial fibrillation (Rubenstein-III). Of special interest is that one patient showed a high prevalence of SSS and NIDDM among her close relatives. Hyperinsulinemia of moderate degree was seen at fasting state or after carbohydrate ingestion in the absence of obesity. The resistance to the action of insulin on glucose metabolism which was evaluated in three patients by the euglycemic hyperinsulinemic clamp study was found to be comparable to the lowest quartile level for common NIDDM patients. Because insulin is a physiological regulator of cell-membrane Na+/K+-ATPase, we speculate that malfunction of the sinus node automaticity may be caused by chronic exposure to hyperinsulinemia secondary to insulin resistance in these NIDDM patients.

Highly increased insulin secretion in a patient with postprandial hypoglycemia: role of glucagon-like peptide-1 (7-36) amide.

The mechanism(s) of an inappropriate secretion of insulin is poorly understood. We report a case of reactive hypoglycemia associated with an unusually exaggerated insulin secretion. The patient, a 32-year-old man, developed frequent episodes of postprandial hypoglycemia after interferon treatment was begun for chronic type C hepatitis. Oral glucose challenge test confirmed the patient's extremely high plasma IRI response, i.e., more than 1000 microU/ml, and that of plasma C-peptide 56.9 ng/ml at 90 min, followed by symptomatic hypoglycemia (plasma glucose 34 mg/dl) at 240 min. The plasma proinsulin level also was high, but the molar ratio of immuno reactive insulin (IRI)/plasma C-peptide and IRI/proinsulin was within the normal range. Antibodies to insulin or insulin-receptor were negative. Plasma IRI response was apparently greater when the glucose was given orally than when given intravenously. The response of plasma glucagon-like-peptide (GLP)-1 to oral glucose was quite high (from baseline of 45.5 to 303.2 pmol/L) and showed a close parallel with the change in the plasma IRI concentration. The greatly enhanced insulin secretion leading to reactive hypoglycemia in this patient may therefore be attributed to the increased secretion of GLP-1.

Insulin-receptor kinase is enhanced in placentas from non-insulin-dependent diabetic women with large-for-gestational-age babies.

The function of insulin receptor and IGF-1 receptor was investigated in placentas from 10 healthy control mothers, 8 diabetic mothers with appropriate-for-gestational-age babies (AGA group) and 9 diabetic mothers with large-for-gestational-age babies (LGA group). None of the diabetic mothers were obese before pregnancy; their blood glucose was well controlled during pregnancy and glycosylated HbA1c was 6.52 +/- 0.71% (M +/- S.E.). Insulin and IGF-1 receptors were partially purified from placentas using wheat germ agglutinin chromatography. The insulin-binding capacity was significantly increased in both the AGA and the LGA groups compared to the control, whereas the IGF-1 binding capacity was similar in the three groups. Autophosphorylation studies were performed with partially purified receptors equalized for similar binding capacity, then immunoprecipitated with anti-insulin receptor antibody or anti-IGF-1 receptor antibody. Insulin-stimulated 32P-incorporation into the insulin receptor beta-subunit was increased by 133% in the LGA group versus the control, whereas incorporation in the AGA group was equivalent to the control. Insulin-stimulated tyrosine kinase activity of the receptor preparation for histone H2B phosphorylation was also significantly increased in the LGA group compared to the control. 32P-incorporation into beta-subunit IGF-1 receptor and IGF-1-stimulated tyrosine kinase activity did not show any significant differences among the three groups. The data in the present study suggest that elevated insulin receptor kinase might be involved in fetal overgrowth in diabetic mothers.

Insulin autoimmune syndrome (Hirata disease): clinical features and epidemiology in Japan.

Since Hirata et al. first reported a patient with insulin autoimmune syndrome in 1970, 197 cases have been reported in Japan as of December, 1992. The clinical profiles of these 197 cases were as follows; the peak age at onset was 60-69 years and peak duration of hypoglycemic attacks was more than 1 and less than 3 months. There was no gender difference in the peak age of onset or duration of hypoglycemic attacks. Approximately 82% of the IAS patients had spontaneous remission without any positive treatment. Before diagnosis of IAS, 43% of the patients with IAS had been taking medication; methimazole (MTZ) for Graves' disease, alpha-mercaptopropionyl glycine (MPG) for cataracts, liver disease or rheumatoid arthritis, or glutathione for liver disease, all of which are sulfhydryl compounds. After such sulfhydryl compounds were discontinued, the hypoglycemic attacks subsided. Three patients with IAS experienced recurrence of the hypoglycemic attacks after re-administration of MTZ and MPG, although 6 patients who developed IAS without exposure to any drug had recurrent attacks without exposure to any drug around 1 year after the first hypoglycemic attacks had stopped. Thus, hypoglycemia in IAS is mainly transient and the development of IAS may be related to sulfhydryl compounds.

Transient extreme insulin resistance in shock during diabetic ketoacidosis.

Transient extreme insulin resistance was encountered during an episode of diabetic ketoacidosis (DKA) in an insulin-treated diabetic patient. On admission, the plasma glucose level was 1241 mg dl-1 and arterial blood pH 6.895 with HCO3- 4.7 mEql-1. An intravenous bolus injection of 20 units, followed by continuous infusion of 20 units h-1 of short-acting regular human insulin, was instituted. Ischemic myocardial changes were noted on the initial electrocardiogram, therefore fluid replacement was limited to 1,000 ml of 0.9% saline solution in the first hour. As the plasma glucose level declined by only 203 mg dl-1 (41 mg dl-1 h-1) in the first 5 h, the insulin dose was doubled every 2 h. At hour 4, the patient developed circulatory shock which required vasopressor support and respiratory assistance. A plasma glucose level of 300 mg dl-1 was not achieved until the total dosage of insulin amounted to 91,580 units at hour 25. Insulin resistance was not observed from that point on. The patient had neither insulin antibodies nor anti-insulin receptor antibodies in serologic testing. The insulin binding characteristics of the patient's erythrocytes were similar to those from healthy controls both with and without experimental acidosis and with a high level of beta-hydroxybutyrate. Among multiple potential factors, the severe shock associated with DKA has been considered as a primary cause of the transient severe insulin resistance in this case.

Strong association of insulin autoimmune syndrome with HLA-DR4.

Insulin autoimmune syndrome is characterised by spontaneous hypoglycaemia without evidence of exogenous insulin administration, a high serum concentration of total immunoreactive insulin, and the presence of insulin autoantibodies in high titre. HLA typing of 27 patients with insulin autoimmune syndrome showed that all had DR4, which was present in only 43% of 51 healthy controls (odds ratio 72.1, p less than 2 x 10(-6), and 19 (70%) of the patients were positive for the allelic combination, Cw4, Bw62, and DR4. Analysis of the nucleotide sequences of the DRB1, DQA1, and DQB1 genes showed that all the patients had DRB1*0406, DQA1*0301, and DQB1*0302, compared with only 14% of the controls (odds ratio 281, p less than 1 x 10(-10). We conclude that the development of insulin autoimmune syndrome is associated with a strong genetic predisposition.

Inducement of antibody that mimics insulin action on insulin receptor by insulin autoantibody directed at determinant at asparagine site on human insulin B chain.

We previously showed that purified human IgG1 insulin autoantibody (IAA) from the serum of male patient T.H. with insulin autoimmune syndrome is directed at a determinant at the asparagine site on the human insulin B chain. An anti-idiotypic antibody (anti-TH) that inhibited TH-IAA binding to human insulin was obtained by immunizing BALB/c mice with TH-IAA. Anti-TH bound to viable IM-9 cells and the purified insulin receptor from IM-9 cells. Anti-TH binding to IM-9 cells and the insulin receptor was inhibited by TH-IAA but not by human IgG. Moreover, incubation of HepG2 cells with anti-TH had an inhibitory effect on insulin binding to HepG2 cells. Anti-TH, like insulin, stimulated amino acid uptake in HepG2 cells. These findings indicate that the conformation of TH-IAA idiotope is a mirror image of the determinant on the insulin B chain, the binding site for TH-IAA on anti-TH is also related to the insulin binding site on the insulin receptor, and anti-TH mimics insulin action on the insulin receptor.

Insulin autoimmune syndrome is the third leading cause of spontaneous hypoglycemic attacks in Japan.

From 1979 to 1981, questionnaires were sent to 2094 hospitals throughout Japan to investigate the causes of severe hypoglycemic attacks other than the administration of oral hypoglycemic agents or insulin preparations. The survey revealed three main causes for the attacks, of which the first was insulinoma, the second extrapancreatic neoplasms, and the third was insulin autoimmune syndrome (IAS), in descending order. Seven years later, a second survey was carried out, which showed the order of the three disorders as the cause of the hypoglycemic attacks to be the same as in the first survey. In both studies it was suggested that the IAS was frequently induced by thiol compounds.

Significant myocardial pathology and increase of alpha 1-adrenergic receptor number affecting the arrhythmogenic condition in cases with ventricular tachycardia.

To study the pathogenesis of idiopathic ventricular tachycardia with left bundle branch block morphology (LBBB-VT), histopathological findings in 34 patients and adrenergic receptor (AR) density obtained by micro-autoradiography in 11 patients were analyzed, using endomyocardial biopsy samples. According to the ventricular volume, we divided the patients into 3 groups. Group A patients (n = 8) were diagnosed as having arrhythmogenic right ventricular dysplasia (ARVD) showing right ventricular enlargement, group B patients (n = 7) showed decreased left ventricular function and/or enlarged left ventricle, and group C (n = 19) included patients with idiopathic ventricular tachycardia (VT) without enlargement or dysfunction of either ventricle. Histologically, there was a high incidence of significant pathology showing myocardial hypertrophy, degeneration, abnormal branching, and interstitial fibrosis in all groups (group A, 100%; group B, 86%; group C, 56%). There was a higher incidence of fatty tissue infiltration in groups A (100%), B (71%), C (48%) than in the control groups. As for AR, specific grains of alpha 1- and beta-AR were 23.0, and 18.3 (grains/25 x 25 mm square) respectively, in patients with LBBB-VT. The number of alpha 1 grains in patients with LBBB-VT was apparently higher than in the control group, sick sinus syndrome (SSS; 11.2), and the beta-AR density in the LBBB-VT group was the same as in the control group (SSS; 15.4). We concluded that the significant pathology in cases with VT might affect the arrhythmogenic condition. Moreover, these results suggested that the increase in the number of alpha 1-AR might have a great influence in idiopathic VT.

Human monoclonal IgG1 insulin autoantibody from insulin autoimmune syndrome directed at determinant at asparagine site on insulin B-chain.

Purified human insulin autoantibody (IAA) collected from the serum of a man (T.H.) with insulin autoimmune syndrome was characterized. The TH-IAA was found to be of IgG1 (lambda-light-chain) subclass. In addition to the single-binding affinity of TH-IAA to human insulin that we have shown in previous studies, the TH-IAA binding to human insulin was completely inhibited by a mouse monoclonal anti-idiotypic antibody against TH-IAA. A competitive inhibition study with various insulins revealed an epitope of human insulin against TH-IAA. These findings suggest that TH-IAA is monoclonal and is directed at a determinant at B-3 (asparagine) on the human insulin B-chain.

Autoantibodies to the insulin receptor (B-10) can stimulate tyrosine phosphorylation of the beta-subunit of the insulin receptor and a 185,000 molecular weight protein in rat hepatoma cells.

The immunoglobulin G (IgG) fraction obtained from the serum of a patient (B-10) with type B insulin resistance and acanthosis nigricans stimulated both glucose oxidation in rat adipocytes and autophosphorylation of tyrosine residues in the beta-subunit of insulin receptors in H-35 hepatoma cells. Partially purified insulin receptor from H-35 cells, when incubated with B-10 IgG, had increased tyrosine kinase activity for a synthetic peptide sequentially similar to the site of tyrosine phosphorylation in pp60v-arc (the gene product responsible for cellular transformation by the Rous sarcoma virus). In H-35 cells, both B-10 IgG and insulin stimulated tyrosine phosphorylation in an endogenous 185,000 mol wt protein. This phosphoprotein may be similar to the cellular substrate for insulin in hepatoma and other cultured cell lines demonstrated by others. These results suggest that antiinsulin receptor antibodies (B-10) may initiate their insulin-like effects via tyrosine phosphorylation of the insulin receptor, activation of its tyrosine kinase activity, and phosphorylation of a cellular protein substrate of 185,000 mol wt.

The immunoglobulin class, the subclass and the ratio of kappa:lambda light chain of autoantibodies to human insulin in insulin autoimmune syndrome.

The immunoglobulin class, subclass and the k:lambda light chain ratio of insulin autoantibodies were determined in the sera of twenty-four patients with insulin autoimmune syndrome. All sera proved to be of the IgG immunoglobulin class but exhibited various IgG1:IgG2:IgG3:IgG4 ratios. The ratio of k:lambda light chain ranged from 1:0.13 to 1:0.75 with the exceptions of two sera that were characterized as IgG1(k) and IgG1(lambda).

Digoxin degradation in acidic dissolution medium.

The release of digoxin and its simultaneous conversion to digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside, and digoxigenin in a USP dissolution test medium were followed by high-pressure liquid chromatography. Two products, Tablets A and B, were manufactured by solvent deposition and simple blending methods, respectively. Tablet A released digoxin faster than Tablet B in distilled water and in artificial intestinal juice, and no decomposition was observed. In the USP dissolution test medium, the rate of hydrolysis to digoxigenin bisdigitoxoside was almost equal to that of hydrolysis to digoxigenin monodigitoxoside, and a comparatively large formation rate of digoxigenin was observed. Concentrations of digoxin and its decomposition products were described by differential equations that included dissolution rates of digoxin (rapidly dissolving digoxin and digoxin crystals) and an apparent hydrolysis rate. In the earlier stage of dissolution, hydrolysis was rate determining; in the later stage, dissolution became the rate-determining step for overall digoxin degradation. To suppress digoxin hydrolysis in the USP dissolution test medium, a developmental formulation study was performed. The incorporation of magnesium oxide and magnesium hydroxide-aluminum hydroxide in the tablet formulations inhibited digoxin hydrolysis by 15.3 and 14.5%, respectively, after dissolution for 30 min without serious delay of drug release.