An Improved Method for the Determination of Islet Amyloid Polypeptide Levels in Plasma

We describe an improved method for the determination of islet amyloid polypeptide (IAPP) levels in plasma. Plasma is first extracted with acid-acetone, followed by a specific and sensitive radioimmunoassay (RIA) for IAPP using rabbit-anti-human-IAPP serum. Recovery of synthetic IAPP from plasma was 82±6% (n = 16). Standard samples, prepared in ‘hormone-free’ serum, were also extracted with acid-acetone. Displacement curves of serially diluted acid-acetone extracted plasma samples were parallel to the standard curve. The lower detection limit of the RIA was 2·3 ± 0·1 fmol/sample (n = 5). Intra-assay variations for IAPP concentrations of 4, 17 and 32 pM were 16·3% (n = 10), 9·2% (n = 10) and 6·2% (n = 10); inter-assay variations were 35·9% (n = 14), 19·9% (n = 15) and 15·4% (n = 15), respectively. Non-stimulated IAPP levels ranged from 2·4 to 12 pM (mean 6±4pM, n = 10) in healthy control subjects. IAPP was not detectable in type 1 (insulin-dependent) diabetic patients before and after glucagon administration. In type 2 (non-insulin-dependent) diabetic patients basal levels ranged from 2·2 to 14·5 pM and glucagon-stimulated levels ranged from 2·2 to 38·9 pM. The increase in IAPP varied from 0 to 24·4 pM. The anti-human-IAPP serum had full cross-reactivity with rat IAPP (= mouse IAPP). Transgenic mice overexpressing the human IAPP gene showed elevated plasma IAPP levels as compared to (non-transgenic) control mice. It is concluded that the method presented for the determination of IAPP in plasma is reliable and easy to perform, yielding reproducible results. The ability to measure plasma IAPP levels will help to elucidate the physiological function of IAPP as well as its pathophysiological role in type 2 diabetes mellitus.

[1]  D. Erkelens,et al.  Plasma Concentrations of Islet Amyloid Polypeptide After Glucagon Administration in Type 2 Diabetic Patients and Non‐diabetic Subjects , 1993, Diabetic medicine : a journal of the British Diabetic Association.

[2]  E. Wilander,et al.  Islet amyloid polypeptide-producing pancreatic islet cell tumor. A clinical and biochemical characterization. , 1992, Scandinavian journal of gastroenterology.

[3]  S. Bloom,et al.  Very high concentrations of islet amyloid polypeptide are necessary to alter the insulin response to intravenous glucose in man. , 1992, The Journal of clinical endocrinology and metabolism.

[4]  A. Kassir,et al.  Lack of Effect of Islet Amyloid Polypeptide in Causing Insulin Resistance in Conscious Dogs During Euglycemic Clamp Studies , 1991, Diabetes.

[5]  G. Greeley,et al.  Inhibitory Action of Islet Amyloid Polypeptide and Calcitonin Gene‐related Peptide on Release of Insulin from the Isolated Perfused Rat Pancreas , 1991, Pancreas.

[6]  G. S. Brooke,et al.  Human and rat amylin have no effects on insulin secretion in isolated rat pancreatic islets. , 1991, Biochemical and biophysical research communications.

[7]  L. Rossetti,et al.  In Vivo Insulin Resistance Induced by Amylin Primarily Through Inhibition of Insulin-Stimulated Glycogen Synthesis in Skeletal Muscle , 1991, Diabetes.

[8]  P. Gluckman,et al.  Radioimmunoassay for insulin-like growth factor-I: solutions to some potential problems and pitfalls. , 1991, The Journal of endocrinology.

[9]  B. Ahrén,et al.  Effects of amidated rat islet amyloid polypeptide on glucose-stimulated insulin secretion in vivo and in vitro in rats. , 1991, European journal of pharmacology.

[10]  R. Silvestre,et al.  Inhibitory effect of rat amylin on the insulin responses to glucose and arginine in the perfused rat pancreas , 1990, Regulatory Peptides.

[11]  P. Westermark,et al.  Islet amyloid polypeptide (IAPP) does not inhibit glucose-stimulated insulin secretion from isolated perfused rat pancreas. , 1990, Biochemical and biophysical research communications.

[12]  D. Matthews,et al.  The effect of islet amyloid polypeptide (amylin) and calcitonin gene-related peptide on glucose removal in the anaesthetized rat and on insulin secretion from rat pancreatic isletsin vitro , 1990, Bioscience reports.

[13]  D. Steiner,et al.  Lack of Islet Amyloid Polypeptide Regulation of Insulin Biosynthesis or Secretion in Normal Rat Islets , 1990, Diabetes.

[14]  R. Rizza,et al.  Effects of Meal Ingestion on Plasma Amylin Concentration in NIDDM and Nondiabetic Humans , 1990, Diabetes.

[15]  Michael W. Schwartz,et al.  Evidence of Cosecretion of Islet Amyloid Polypeptide and Insulin by β-Cells , 1990, Diabetes.

[16]  B. Ahrén,et al.  Failure of islet amyloid polypeptide to inhibit basal and glucose-stimulated insulin secretion in model experiments in mice and rats. , 1990, Acta physiologica Scandinavica.

[17]  H. Datta,et al.  Amylin and amylin-amide lack an acute effect on blood glucose and insulin. , 1990, The Journal of endocrinology.

[18]  J. Olefsky,et al.  Induction of Insulin Resistance In Vivo by Amylin and Calcitonin Gene–Related Peptide , 1990, Diabetes.

[19]  M. Nakazato,et al.  Establishment of radioimmunoassay for human islet amyloid polypeptide and its tissue content and plasma concentration. , 1989, Biochemical and biophysical research communications.

[20]  W. Lems,et al.  Aggravation of hypoglycemia in insulinoma patients by the long-acting somatostatin analogue octreotide (Sandostatin). , 1989, Acta endocrinologica.

[21]  H. Makino,et al.  Islet amyloid polypeptide inhibits glucose-stimulated insulin secretion from isolated rat pancreatic islets. , 1989, Biochemical and biophysical research communications.

[22]  R. Holman,et al.  Islet amyloid, increased A-cells, reduced B-cells and exocrine fibrosis: quantitative changes in the pancreas in type 2 diabetes. , 1988, Diabetes research.

[23]  G. Cooper,et al.  Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro , 1988, Nature.

[24]  J. Rothbard,et al.  Amylin found in amyloid deposits in human type 2 diabetes mellitus may be a hormone that regulates glycogen metabolism in skeletal muscle. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[25]  D. W. Hayden,et al.  Immunolocalization of islet amyloid polypeptide (IAPP) in pancreatic beta cells by means of peroxidase-antiperoxidase (PAP) and protein A-gold techniques. , 1988, The American journal of pathology.

[26]  R. Turner,et al.  Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[27]  D. W. Hayden,et al.  Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[28]  C. Wernstedt,et al.  A novel peptide in the calcitonin gene related peptide family as an amyloid fibril protein in the endocrine pancreas. , 1986, Biochemical and biophysical research communications.

[29]  F. Greenwood,et al.  Preparation of Iodine-131 Labelled Human Growth Hormone of High Specific Activity , 1962, Nature.