Endogenous Gastric Mediators: Patho‐Physiological Role and Measurements

Stanislaw J. Konturek1, Wladysław Bielanski1, Peter C. Konturek2, Thomas Brzozowski1

1 Jagiellonian University, Cracow, Poland, 2 Thueringen‐Kliniken, Saalfeld, Germany
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 21.6
DOI:  10.1002/0471140856.tx2106s45
Online Posting Date:  August, 2010
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Abstract

The protocols described in this unit are designed to present the major endogenous gastric mediators involved in the control of gastric acid secretion, namely gastrin and histamine, and in the regulation of gastric motility, which include motilin and ghrelin, under physiological and pathological conditions. The measurement of these mediators in plasma or serum of humans and animals by radioimmunoassay are described and their pathophysiological role is discussed. Curr. Protoc. Toxicol. 45:21.6.1‐21.6.24. © 2010 by John Wiley & Sons, Inc.

Keywords: gastrin; histamine; motilin; ghrelin; stomach

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Gastrin
  • Basic Protocol 2: Measurement of Histamine
  • Basic Protocol 3: Measurement of Motilin
  • Basic Protocol 4: Measurement of Ghrelin
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Gastrin

  Materials
  • Fasting (≥10 hr) serum or plasma samples without heparin
  • GASK‐PR radioimmunoassy kit (CIS Bio International) containing:
    • Lyophilized gastrin‐125I tracer; i.e., synthetic gastrin I (1 through 17), containing <37 kBq 125I
    • Lyophilized anti‐gastrin rabbit antiserum
    • Lyophilized synthetic gastrin I (1 through 17) standards
    • Ready‐for‐use immunoprecipitating reagent (insoluble complex of anti‐rabbit IgG sheep serum and non‐immunized rabbit IgG)
  • 100‐, 300‐, and 1000‐µl precision micropipets (±1%)
  • 3‐ml disposable polystyrene plastic tubes
  • Vortex‐type mixer
  • Refrigerated centrifuge
  • Invertable tube racks
  • Gamma scintillation counter calibrated for 125I measurements

Basic Protocol 2: Measurement of Histamine

  Materials
  • Plasma (from blood collected in EDTA/heparin) samples
  • 125I‐histamine radioimmunoassay kit (BioSource Europe S.A, cat. no. KIPL 1000) containing:
    • 125I‐histamine tracer, activity <150 kBq
    • Acylation buffer
    • Histamine antiserum: goat anti‐N‐acyl‐histamine
    • Precipitating antiserum containing donkey anti‐goat‐IgG in PEG/phosphate buffer
    • Histamine standards: A through F (0, 0.4‐, 1.2‐, 4‐, 12‐, and 40‐ng/liter)
    • Controls
    • Acylation reagent
  • 10‐, 25‐, 50‐, 100‐, and 1000‐µl automatic micropipets
  • 3‐ml polypropylene or polystyrene tubes and suitable rack
  • Refrigerated swinging‐bucket centrifuge
  • Suitable device for aspirating or decanting tubes
  • Vortex mixer
  • Gamma scintillation counter calibrated for 125I measurements
NOTE: All above reagents are ready to use except the acylation reagent, which is supplied as a concentrated solution and equalizing reagent is supplied lyophilized, to be reconstituted with distilled water. The histamine RIA kit contains materials for 100 quantitative determinations (including the standards) of histamine.NOTE: For safety measures and basic radioprotection rules, see protocol 1.CAUTION: The reagents should be stored at 2° to 8°C. Avoid microbiological contamination of the reagents. Do not use components beyond the expiration date shown on the kit labels. Do not mix various lots of any kit component within an individual assay. Inappropriate handling of the test samples or deviations from the test instructions can affect the result.

Basic Protocol 3: Measurement of Motilin

  Materials
  • Serum or EDTA‐plasma samples without heparin or citrate
  • Aprotinin
  • Motilin RIA kit (Phoenix Pharmaceuticals, cat. no. RK‐045‐03) containing:
    • RIA concentrated buffer
    • Lyophilized motilin‐125I tracer (radioactivity 55.5 kBq)
    • Lyophilized motilin standard
    • Rabbit antiserum specific for the motilin (lyophilized powder)
    • Goat anti‐rabbit IgG serum (GAR; lyophilized powder)
    • Normal rabbit serum (NRS; lyophilized powder)
    • Positive control (lyophilized powder)
  • Lavender vacutainer tubes
  • Platform rocker
  • 7‐ml centrifuge tubes
  • Precision pipettors: 10‐, 25‐, 50‐, 100‐, and 1000‐µl (±1%)
  • 3‐ml disposable polypropylene or polystyrene tubes and suitable rack
  • Refrigerated centrifuge
  • Aspirator
  • Vortexer
  • Gamma scintillation counter calibrated for 125I measurements
NOTE: The standard size of the RIA kit is sufficient for 125 RIA tubes. The 125I‐motilin will expire in ∼6 weeks. Store at −20°C upon receipt.

Basic Protocol 4: Measurement of Ghrelin

  Materials
  • Serum or EDTA‐plasma samples without heparin or citrate
  • Aprotinin
  • Ghrelin (human) RIA kit (Phoenix Pharmaceuticals, cat. no. RK‐31‐30) containing:
    • RIA buffer
    • Lyophilized ghrelin‐125I tracer (radioactivity 55.5 kBq)
    • Lyophilized ghrelin standard
    • Rabbit antiserum specific for the ghrelin (lyophilized powder)
    • Goat anti‐rabbit IgG serum (GAR; lyophilized powder)
    • Normal rabbit serum (NRS; lyophilized powder)
    • Positive control (lyophilized powder)
  • Lavender Vacutainer tubes containing EDTA
  • Platform rocker
  • 7‐ml centrifuge tubes
  • Precision pipettors: 10‐, 25‐, 50‐, 100‐, and 1000‐µl (±1%)
  • 3‐ml disposable polypropylene or polystyrene tubes and suitable rack
  • Refrigerated centrifuge
  • Aspirator
  • Vortexer
  • Gamma scintillation counter calibrated for 125I measurements
NOTE: The standard size of the kit is sufficient for 125 RIA tubes. The 125I‐ghrelin will expire in ∼6 weeks. Store at −20°C upon receipt.NOTE: See protocol 1 for instructions for possession, handling, and use of radioactive material.CAUTION: Due to the short half‐life of iodine‐125 (the tracer used with this kit), it is highly recommended to use the kit as soon as possible after receiving. All solutions should be used on the same day of rehydration. Never store reconstituted kit components as they are unstable. Use of stored reconstituted kit components to perform RIA may yield poor and unacceptable results. When using the ghrelin (human) RIA kit (Phoenix Pharmaceuticals), carefully follow the procedure for utilization of the RIA as described in “General protocol for radio immunoassay kit of peptides”.
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Figures

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Literature Cited

Literature Cited
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