Bruno Hansen

Impaired meal stimulated glucagon-like peptide 2 response in ileal resected short bowel patients with intestinal failure

BACKGROUND Glucagon-like peptide 2 (GLP-2) is a growth factor for the intestinal epithelium in rodents and may affect intestinal transit. AIMS To study the GLP-2 response to nutrient ingestion in seven short bowel patients with intestinal failure and seven controls. METHODS The patients and controls were admitted twice for two test meals after a night of fasting. Meal A was liquid (300 ml, 1.88 MJ); meal B was a regular breakfast (755 g, 3.92 MJ). Plasma samples were collected for 180 minutes; GLP-2 immunoreactivity was measured with an NH2 terminal specific radioimmunoassay. RESULTS Both meals elicited significant increases in plasma GLP-2 in controls. The magnitude and duration of the responses were dependent on the meal size: the maximum median (25–75%) increases after meal A and B were 24 (3–28) and 48 (33–56) pmol/l. Plasma GLP-2 returned to basal concentrations 180 minutes after meal A, but remained at 50% of peak values after meal B. In the patients neither meal significantly changed the GLP-2 concentration; the maximum median elevation after meal B was 5 (2–8) pmol/l. There were significant differences between patients and controls with respect to the GLP-2 responses to meals A and B. CONCLUSION Identification of GLP-2 as a tissue specific intestinal growth factor and demonstration of an impaired meal stimulated GLP-2 response in short bowel patients raises the possibility that GLP-2 administration may constitute a new therapeutic strategy, enhancing jejunal adaptation in ileum resected short bowel patients with intestinal failure.

Short-term Administration of Glucagon-like Peptide-2. Effects on Bone Mineral Density and Markers of Bone Turnover in Short-Bowel Patients with No Colon

Background: Glucagon-like peptide 2 (GLP-2) is a newly discovered intestinotrophic hormone. We have recently reported that a 5-week GLP-2 treatment improved the intestinal absorptive capacity of shortbowel patients with no colon. Additionally, GLP-2 treatment was associated with changes in body composition that included a significant increase in total body bone mass. This article describes the effect of GLP-2 on spinal and hip bone mineral density (BMD) and biochemical markers of bone turnover in these patients. Methods: In an open-labelled pilot study, eight short-bowel patients (3M, 5F; mean age 49 years) with small-bowel resection and no colon received 400 μg s.c. of GLP-2 twice daily for 5 weeks. Four received home parenteral nutrition (mean length of residual jejunum 83 cm) and 4 did not (mean length of ileum resected 106 cm). The outcome measures were the mean percent change from baseline in spinal and hip BMD measured by dual-energy X-ray absorptiometry, changes in four biochemical markers of bone-turnover, PTH, 25-hydroxy vitamin-D, and the intestinal absorption of calcium. Results: Mean ± s x (SEM) percent changes in spinal and hip BMD were 1.1 ± 0.4% ( P < 0.05) and 1.9 ± 0.8% ( P = 0.06), respectively. The intestinal calcium absorption increased by 2.7% ( P = 0.87). Serum ionized calcium increased in 5/8 patients with a concomitant decrease in serum PTH values. Three of the four markers of bone turnover decreased. Conclusion: A 5-week GLP-2 administration significantly increased spinal BMD in short-bowel patients with no colon. The mechanism by which GLP-2 affects bone metabolism remains unclear, but may be related to an increased mineralization of bone resulting from an improved intestinal calcium absorption.

Effects of soy supplementation on blood lipids and arterial function in hypercholesterolaemic subjects.

Background:Studies on soy supplementation suggest a cardioprotective potential.Objective:To examine the effects on LDL cholesterol and arterial function as a result of dietary enrichment with soy supplementation.Design:A Randomized, double blind, parallel intervention trial.Setting:Department of Endocrinology and Metabolism C, Aarhus University Hospital, and Department of Human Nutrition, The Royal Veterinary and Agricultural University, Denmark.Subjects:In all, 100 hypercholesterolaemic but otherwise healthy subjects were included in the study of which 89 completed it.Interventions:Subjects were randomly assigned to 24 weeks of daily intake of either a soy supplement, Abalon® (30 g soy protein, 9 g cotyledon fibre and 100 mg isoflavones) or placebo (30 g of casein). The soy supplement and placebo were provided in two sachets daily that were stirred in water. Fasting plasma lipids, TNF-α, homocysteine, insulin sensitivity, homeostasis model assessment (HOMA-IR), serum insulin, serum glucose, blood pressure as well as Glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP) and plasma lipids to a fat-rich meal were recorded before and after the intervention. In a sub study in 32 subjects, arterial dilatory capacity, compliance, and distensibility were recorded before and after the intervention.Results:In the main study, no difference in fasting plasma lipid levels or insulin sensitivity was found between soy-based supplement and placebo. A significant postprandial increase in GIP to the meal test was observed in the soy group (P<0.05). In a substudy, no difference between the groups in changes in flow-mediated vasodilatation (P=0.84) was detected, while the soy supplementation caused a reduction in LDL and total cholesterol.Conclusions:No significant effects on blood lipids were observed in the main study to a soy supplementation in hypercholesterolaemic subjects after 24 weeks. In the substudy, the soy supplementation, however, reduced LDL and total cholesterol but did not influence markers of arterial function.Sponsorship:Nutri Pharma ASA, Oslo, Norway.

Biological potency and binding affinity of monoiodoinsulin with iodine in tyrosine A14 or tyrosine A19.

Summary Monoiodoinsulin with 99% of the iodine in the A chain was separated in two bands by disc-electrophoresis using long polyacrylamide gel rods. The bands contained monoiodoinsulin with the iodine in tyrosine A14 and tyrosine A19, respectively. The biological potency and binding affinity of A14 [ 127 I]monoiodoinsulin on rat adipocytes was indistinguishable from that of insulin, whereas the A19 derivative was only half as potent. Similarly, the maximum binding of “tracer” A19 [ 125 I]monoiodoinsulin to adipocyte receptors was only half of that obtained with A14 [ 125 I]monoiodoinsulin.

Stable lodinated polypeptide hormones prepared by polyacrylamide gel electrophoresis

Iodination of several insulin and proinsulin preparations, human growth hormone and bovine pancreatic polypeptide was performed using H2O2 and lactoperoxidase or chloramine T. The iodination mixtures were fractionated by polyacrylamide gel electrophoresis at pH 9.15 in long gel rods followed by simple elution of the iodinated products from thin gel slices. With this method 125I tracers with long shelf life and high specific activity suitable for radioimmunoassays could easily be obtained.

[24] Preparation of stable radioiodinated polypeptide hormones and proteins using polyacrylamide gel electrophoresis

Publisher Summary This chapter discusses the preparation of stable radioiodinated polypeptide hormones and proteins using polyacrylamide gel electrophoresis. Radioactively labeled polypeptide hormones and proteins are widely used as tracers in radioimmunoassays and receptor studies. The peptide or protein is most easily labeled using iodination with 125 I or 131 I. The radioactive iodine is substituted in the tyrosine groups of the peptide as monoiodotyrosine or di-iodotyrosine, resulting in a heterogeneous mixture of iodine-substituted molecules plus native peptide and unreacted iodide. The iodination mixture has to be fractionated to obtain a well-characterized iodinated product to be used as a tracer. An ideal tracer should retain the full biological activity or immunological reactivity of the native peptide hormone or protein and have a high specific activity and a long shelf life. Studies in the laboratory have shown that it is possible to fractionate iodinated insulin preparations into homogeneous monoiodoinsulin derivatives by polyacrylamide gel electrophoresis using long rods. The same fractionation technique has been useful for the preparation of a number of radioiodinated polypeptide hormones and proteins.

Reversed-phase high-performance liquid chromatographic analyses of insulin biosynthesis in isolated rat and mouse islets

Two RP-HPLC systems were developed for the separation of the products of the conversion of proinsulin into insulin in rat and mouse islets, including proinsulin I and II. Peaks were identified by microsequencing and radiosequencing. It was confirmed that mouse C-peptide I has a two amino acid deletion compared to rat C-peptide I. A marked species difference in the ratio between insulin I and II was observed, i.e., 2:1 in the rat and 1:2 in the mouse. Pulse-chase experiments in rat islets have demonstrated that the ratio between insulin I and II in newly synthesized insulin is higher than that of the stored insulin, indicating a slower conversion rate of proinsulin II compared to proinsulin I.

Separation, isolation and characterization of the four monoiodinated insulin tracers using reversed-phase high-performance liquid chromatography

Baseline separation between insulin and insulin monoiodinated in Tyr A14, A19, B16 and B26 can be obtained using isocratic elution from a C18 column with triethylammonium trifluoroacetate-acetonitrile and the iodinated insulin derivatives can be isolated by lyophilization. Compared with similar tracers purified and isolated by disc electrophoresis/ion-exchange chromatography, the reversed-phase high-performance liquid chromatographically purified tracers are more homogeneous but show reduced binding affinity to adipocytes.

Recovery of polypeptides after reversed-phase high-performance liquid chromatography☆

Mass recovery of individual polypeptides may be estimated under various practical conditions. With the purpose of obtaining rapid and reliable standard procedures for recovery measurements, we have compared five individual methods utilizing a silica-based stationary phase [Nucleosil C18 (7 microns)/ammonium sulphate-perchlorate-acetonitrile, pH 3.0] and a resin-based stationary phase (TSK Phenyl 5 PW RP/ammonium phosphate-acetonitrile, pH 7.0). The recoveries of insulin (6 kilodaltons), human growth hormone (22 kilodaltons) and human serum albumin (68 kilodaltons) estimated under five different experimental conditions were found to be concordant. Variations in column load, flow-rate, gradient shape and column dwell time and addition of cyclame did not increase the (reduced) recovery of serum albumin and growth hormone.

Reversed-phase high-performance liquid chromatography of insulin and insulin derivatives : A comparative study☆

Abstract The reversed-phase separation of crystalline insuline (I) and monoiodoinsulins (II) has been investigated, with respect to the effects of buffer, substitution group, pore-size and column support backbone. The separations were performed either isocratically (for II) or by gradient elution with very narrow gradients (for I). Fourteen reversed-phase columns, the majority being silica-based, were investigated, and three main result emerged. (1) Trifluoroacetic acid is unsuitable as a buffer for this type of analysis, whereas trialkylammonium phosphates are very suitable. (2) The separation between the major components in crystalline insulin was comparable in the main for all the columns tested except one. However, the ability to distinguish between the numerous minor components (co-extracted with insulin peptide) varied a great deal between the columns. (3) In an optimized buffer system only three columns were able to separate insulin peptide and the four monoiodoinsulin isomers; all three were 80—100-A silica-based C 18 columns.