Hiroshi Hosoda1, Kenji Kangawa. 1. Department of Regenerative Medicine and Tissue Engineering, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan. hosoda.hiroshi.ri@mail.ncvc.go.jp
Abstract
BACKGROUND: Octanoyl modification of ghrelin is rapidly hydrolyzed to des-acyl ghrelin in blood samples. Owing to the increased interest in ghrelin measurement, a standardized method of sample collection is required. METHODS: This chapter investigates the effect of a variety of anticoagulants and storage conditions on ghrelin stability. Experiment 1 evaluates the effects of anticoagulants on ghrelin measurements. Experiment 2 evaluates the effect of plasma pH on ghrelin stability. Experiment 3 evaluates the mechanisms of degradation of the active form of ghrelin in plasma. Experiment 4 investigates the kinetics of ghrelin following intravenous injection of rat ghrelin. RESULTS: In whole blood and plasma, octanoylated ghrelin is highly unstable. The collection of blood samples with EDTA-aprotinin under cooled conditions was appropriate to maintain ghrelin stability. Acidification of plasma to pH 3-4 and storage at 4°C maintained ghrelin stability. The degradation of ghrelin was shown to be due to the hydrolysis to des-acyl ghrelin. After intravenous administration to rats, plasma ghrelin levels rapidly decreased with a half-life of 8 min. CONCLUSION: The results showed that the ghrelin values measured in human blood samples were markedly affected by the conditions of collection and storage, the pH, and the RIA method in measurement. Measuring the ghrelin values of the active form is useful for studying plasma ghrelin changes over short time periods. As ghrelin is highly unstable, it is necessary to standardize the preparation of samples to ensure reliable ghrelin measurements.
BACKGROUND:Octanoyl modification of ghrelin is rapidly hydrolyzed to des-acyl ghrelin in blood samples. Owing to the increased interest in ghrelin measurement, a standardized method of sample collection is required. METHODS: This chapter investigates the effect of a variety of anticoagulants and storage conditions on ghrelin stability. Experiment 1 evaluates the effects of anticoagulants on ghrelin measurements. Experiment 2 evaluates the effect of plasma pH on ghrelin stability. Experiment 3 evaluates the mechanisms of degradation of the active form of ghrelin in plasma. Experiment 4 investigates the kinetics of ghrelin following intravenous injection of ratghrelin. RESULTS: In whole blood and plasma, octanoylated ghrelin is highly unstable. The collection of blood samples with EDTA-aprotinin under cooled conditions was appropriate to maintain ghrelin stability. Acidification of plasma to pH 3-4 and storage at 4°C maintained ghrelin stability. The degradation of ghrelin was shown to be due to the hydrolysis to des-acyl ghrelin. After intravenous administration to rats, plasma ghrelin levels rapidly decreased with a half-life of 8 min. CONCLUSION: The results showed that the ghrelin values measured in human blood samples were markedly affected by the conditions of collection and storage, the pH, and the RIA method in measurement. Measuring the ghrelin values of the active form is useful for studying plasma ghrelin changes over short time periods. As ghrelin is highly unstable, it is necessary to standardize the preparation of samples to ensure reliable ghrelin measurements.
Authors: Fabio Bernardoni; Nadine Bernhardt; Shakoor Pooseh; Joseph A. King; Daniel Geisler; Franziska Ritschel; Ilka Boehm; Maria Seidel; Veit Roessner; Michael N. Smolka; Stefan Ehrlich Journal: J Psychiatry Neurosci Date: 2020-07-01 Impact factor: 6.186