Analysis of ghrelin concentrations in serum samples processed at one, two, and three hours after collection

Obesity is becoming an epidemic of modern society. While the health-related consequences of obesity have been well studied, scientists continue to search for biological risk factors associated with this disease. Since the 1999 discovery of an appetite-stimulating hormone, ghrelin, researchers have further questioned the physiological interactions that lead to obesity. The fundus of the stomach, as well as the intestines and kidneys produce ghrelin before it circulates throughout the body and attaches to its receptor sites located within the section of the brain responsible for appetite-regulation. The hormone increases and decreases in a circadian rhythm that may dictate the routine human eating patterns of breakfast, lunch and dinner. While the field of ghrelin research continues to grow as evidenced by the increasing number of published articles, the majority of the ghrelin research occurs in the laboratory setting. This environment has provided researchers with the preservation and storage techniques necessary to analyze ghrelin. For example, the current Millipore Human Ghrelin (Total) ELISA kit protocol for measuring ghrelin requires the collected blood be centrifuged thirty minutes after collection and immediately placed in a freezer. This limitation alone may impede researchers wishing to conduct clinical research because these requirements are frequently not possible. The objectives of this study were to determine the optimal storage time before processing blood samples collected in the clinical setting to ensure the preservation of ghrelin and to conclude whether a change in the manufacturer\u27s protocol was warranted. By comparing blood samples processed according to the Millipore ELISA kit protocol to samples stored for one, two, and three hours, it was determined that mean ghrelin concentrations at thirty minutes, one hour and two hour were not significantly different, suggesting that the current Millipore ELISA protocol of thirty minutes can be extended for a period of up to two hours. When the crude model was adjusted to include the variables time, waist circumference and exercise, the statistically significant variables in previous ANOVA models, the presence or absence of an exercise routine was found to have the strongest association to mean ghrelin concentrations. These findings will facilitate the experimental design of research aimed at studying ghrelin levels in individuals outside of the laboratory setting, and can also support new research analyzing the effect of exercise on ghrelin levels