Pitfalls in the determination of human acylated ghrelin plasma concentrations using a double antibody enzyme immunometric assay

Trivedi, Arjun; Babic, Sandra; Chanoine, Jean‐Pierre

doi:10.1016/j.clinbiochem.2011.10.023

Cited by 9 publications

(8 citation statements)

References 7 publications

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“…Trivedi and coworkers (36) showed that AEBSF may inhibit acetylcholinesterase (AChE) activity from the kits and thus may change AG levels. However, this problem of AEBSF suppressing AChE activity is circumvented using the applied processing method and Bertin Pharma EIA kits described by Delhanty and coworkers (3).…”

Section: Discussionmentioning

confidence: 99%

Plasma acylated and plasma unacylated ghrelin: useful new biomarkers in patients with neuroendocrine tumors?

Adrichem

Lely

Huisman

et al. 2016

Endocrine Connections

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To date, the value of fasting plasma acylated ghrelin (AG) and unacylated ghrelin (UAG) as potential novel biomarkers in patients with neuroendocrine tumors (NETs) is unknown. The aims of this study are to (i) compare fasting AG and UAG levels between nonobese, nondiabetic NET patients (N=28) and age- (±3 years) and sex-matched nonobese, nondiabetic controls (N=28); and (ii) study the relationship between AG, UAG, and AG/UAG ratios and biochemical (chromogranin-A (CgA) and neuron-specific enolase (NSE) levels) and clinical parameters (age at diagnosis, sex, primary tumor location, carcinoid syndrome, ENETS TNM classification, Ki-67 proliferation index, grading, prior incomplete surgery) in NET patients. Fasting venous blood samples (N=56) were collected and directly stabilized with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride after withdrawal. Plasma AG and UAG levels were determined by ELISA. Expression of ghrelin was examined in tumor tissue by immunohistochemistry. There were no significant differences between NET patients and controls in AG (median: 62.5 pg/mL, IQR: 33.1–112.8 vs median: 57.2pg/mL, IQR: 26.7–128.3, P=0.66) and UAG in levels (median: 76.6pg/mL, IQR: 35.23–121.7 vs median: 64.9, IQR: 27.5–93.1, P=0.44). No significant correlations were found between AG, UAG, and AG/UAG ratios versus biochemical and clinical parameters in NET patients with the exception of age at diagnosis (AG: ρ= −0.47, P=0.012; AG/UAG ratio: ρ= −0.50, P=0.007) and baseline chromogranin-A levels (AG/UAG ratio: ρ= −0.44, P=0.019). In our view, fasting plasma acylated and unacylated ghrelin appear to have no value as diagnostic biomarkers in the clinical follow-up of patients with NETs.

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Section: Discussionmentioning

confidence: 99%

Plasma acylated and plasma unacylated ghrelin: useful new biomarkers in patients with neuroendocrine tumors?

Adrichem

Lely

Huisman

et al. 2016

Endocrine Connections

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“…The assays differ in that the capture antibody in the Bertin Pharma ELISA plate will bind both forms of ghrelin, and it is the tracer (detection antibody) that provides specificity for the assay. The tracer is an AChE‐conjugated Fab, requiring a stringent wash step following sample incubation in the plate as AChE is inhibited by AEBSF . The SCETI assay is essentially the reverse, as it uses capture antibodies that are specific for each form of ghrelin with a common, nonspecific (for ghrelin), tracer (HRP‐conjugated) antibody.…”

Section: Discussionmentioning

confidence: 99%

The acylated (AG) to unacylated (UAG) ghrelin ratio in esterase inhibitor‐treated blood is higher than previously described

Delhanty

Huisman

Julien³

et al. 2014

Clinical Endocrinology

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AG and UAG levels measured in AEBSF-stabilized plasma indicate that the AG/UAG ratio is markedly higher than previously described and that UAG is a physiological component of the circulation. This highlights the importance of immediately stabilizing blood samples on collection for determination of both AG and UAG concentrations and provides a valuable tool for their measurement in physiological and interventional studies.

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“…Of note, it is customary when preparing plasma for subsequent assay of acyl ghrelin to add a protease inhibitor such as PHMB (p-hydroxymercuribenzoate) prior to the HCl stabilization step as another step to stabilize the acyl group (26,35,58). To test the requirement for the protease inhibitor with the current cell culturebased system, pilot secretion studies were performed as described above in serum-free DMEM medium (containing penicillin, streptomycin, and octanoate-BSA) plus 1, 5, or 10 mM D-glucose (as described above) with or without 0.05% DMSO.…”

Section: Methodsmentioning

confidence: 99%

Glucose-mediated control of ghrelin release from primary cultures of gastric mucosal cells

Sakata

Park

Walker

et al. 2012

American Journal of Physiology-Endocrinology and Metabolism

108

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-The peptide hormone ghrelin is released from a distinct group of gastrointestinal cells in response to caloric restriction, whereas its levels fall after eating. The mechanisms by which ghrelin secretion is regulated remain largely unknown. Here, we have used primary cultures of mouse gastric mucosal cells to investigate ghrelin secretion, with an emphasis on the role of glucose. Ghrelin secretion from these cells upon exposure to different D-glucose concentrations, the glucose antimetabolite 2-deoxy-D-glucose, and other potential secretagogues was assessed. The expression profile of proteins involved in glucose transport, metabolism, and utilization within highly enriched pools of mouse ghrelin cells and within cultured ghrelinoma cells was also determined. Ghrelin release negatively correlated with D-glucose concentration. Insulin blocked ghrelin release, but only in a low D-glucose environment. 2-Deoxy-D-glucose prevented the inhibitory effect of high D-glucose exposure on ghrelin release. mRNAs encoding several facilitative glucose transporters, hexokinases, the ATPsensitive potassium channel subunit Kir6.2, and sulfonylurea type 1 receptor were expressed highly within ghrelin cells, although neither tolbutamide nor diazoxide exerted direct effects on ghrelin secretion. These findings suggest that direct exposure of ghrelin cells to low ambient D-glucose stimulates ghrelin release, whereas high D-glucose and glucose metabolism within ghrelin cells block ghrelin release. Also, low D-glucose sensitizes ghrelin cells to insulin. Various glucose transporters, channels, and enzymes that mediate glucose responsiveness in other cell types may contribute to the ghrelin cell machinery involved in regulating ghrelin secretion under these different glucose environments, although their exact roles in ghrelin release remain uncertain. secretion THE PEPTIDE HORMONE GHRELIN is the endogenous ligand of the growth hormone secretagogue receptor (GHSR) and is named for its ability to stimulate growth hormone release (32, 59). Ghrelin also regulates gastrointestinal motility, chronic stressinduced mood-related behaviors, and alcohol-seeking behaviors, among many other actions (1,13,15,18,31,32,34,37). Perhaps best studied are ghrelin's actions in signaling and responding to states of energy insufficiency. Regarding its role in signaling energy-insufficient states, ghrelin levels are known to rise prior to set meals, following food deprivation, and after weight loss linked to exercise, cachexia, and anorexia nervosa (9,10,33,39,42,45,55,57,60). Several lines of evidence suggest that the rise in plasma ghrelin upon caloric restriction is likely related, at least in part, to binding of norepinephrine released from the sympathetic nervous system to ␤ 1 -adrenergic receptors embedded in the plasma membranes of ghrelin cells (14,28,41,63). Regarding ghrelin's role in responding to energy-insufficient states, infusions of ghrelin or GHSR agonists increase body weight via proorexigenic actions and/or decreases in energy expenditure (1...

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Pitfalls in the determination of human acylated ghrelin plasma concentrations using a double antibody enzyme immunometric assay

Abstract: Hemolysis and AEBSF may affect AG determination.

Cited by 9 publications

References 7 publications

Plasma acylated and plasma unacylated ghrelin: useful new biomarkers in patients with neuroendocrine tumors?

Plasma acylated and plasma unacylated ghrelin: useful new biomarkers in patients with neuroendocrine tumors?

The acylated (AG) to unacylated (UAG) ghrelin ratio in esterase inhibitor‐treated blood is higher than previously described

Glucose-mediated control of ghrelin release from primary cultures of gastric mucosal cells

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