A practical guide for the stabilization of acylghrelin in human blood collections

Blatnik, Matthew; Soderstrom, Catherine

doi:10.1111/j.1365-2265.2010.03916.x

Cited by 51 publications

(42 citation statements)

References 31 publications

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“…Immediately after withdrawal, AEBSF (dilution 1:100) was directly added to all blood samples to prevent des-acylation of AG (3, 35). Whole blood was carefully mixed by inversion and stored on water ice (4°C) until centrifugation at 2500 g at 4°C for 5min.…”

Section: Methodsmentioning

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: Methodsmentioning

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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“…Hydrochorlic acid was not added to collection containers for AG samples, as recent work demonstrates that it is not required. 20 GLP-1 (active) was assayed using an enzyme-linked immunosorbent assay (ALPCO Diagnostics, Salem, NH, USA). To minimize inter-assay variability, all pre-measurement and post measurements for each subject were analyzed on the same plate.…”

Section: Methodsmentioning

confidence: 99%

Improved acylated ghrelin suppression at 2 years in obese patients with type 2 diabetes: effects of bariatric surgery vs standard medical therapy

et al. 2013

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OBJECTIVE Roux-en-Y gastric bypass (RYGB) produces more durable glycemic control than sleeve gastrectomy (SG) or intensive medical therapy (IMT). However, the contribution of acylated ghrelin (AG), a gluco-regulatory/appetite hormone, to improve glucose metabolism and body composition in patients with type 2 diabetes (T2D) following RYGB is unknown. DESIGN STAMPEDE (Surgical Treatment and Medication Potentially Eradicate Diabetes Efficiently) was a prospective, randomized controlled trial. SUBJECTS Fifty-three (body mass index: 36 ± 3 kg m−2, age: 49 ± 9 years) poorly controlled patients with T2D (HbA1c (glycated hemoglobin): 9.7 ± 2%) were randomized to IMT, IMT + RYGB or IMT + SG and underwent a mixed-meal tolerance test at baseline, 12, and 24 months for evaluation of AG suppression (postprandial minus fasting) and beta-cell function (oral disposition index; glucose-stimulated insulin secretion × Matsuda index). Total/android body fat (dual-energy X-ray absorptiometry) was also assessed. RESULTS RYGB and SG reduced body fat comparably (15–23 kg) at 12 and 24 months, whereas IMT had no effect. Beta-cell function increased 5.8-fold in RYGB and was greater than IMT at 24 months (P < 0.001). However, there was no difference in insulin secretion between SG vs IMT at 24 months (P = 0.32). Fasting AG was reduced fourfold following SG (P < 0.01) and did not change with RYGB or IMT at 24 months. AG suppression improved more following RYGB than SG or IMT at 24 months (P = 0.01 vs SG, P = 0.07 vs IMT). At 24 months, AG suppression was associated with increased postprandial glucagon-like peptide-1 (r = −0.32, P < 0.02) and decreased android fat (r = 0.38; P < 0.006). CONCLUSIONS Enhanced AG suppression persists for up to 2 years after RYGB, and this effect is associated with decreased android obesity and improved insulin secretion. Together, these findings suggest that AG suppression is partly responsible for the improved glucose control after RYGB surgery.

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“…Furthermore, given the post-hoc nature of this study, samples were not pretreated with protease inhibitor. While results were expressed as %Δ, on the other hand future prospective studies with plasma pretreated with protease inhibitor [for details, see: (Blatnik and Soderstrom, 2011)] are needed to validate our preliminary findings. Finally, human studies in the context of alcohol use have shown a possible role of other feeding peptides such as leptin, adiponectin and resistin in alcoholism (Hillemacher et al, 2007; Hillemacher et al, 2009).…”

Section: Discussionmentioning

confidence: 89%

Fasting-induced increase in plasma ghrelin is blunted by intravenous alcohol administration: A within-subject placebo-controlled study

Leggio

Schwandt

Oot

et al. 2013

Psychoneuroendocrinology

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Summary Ghrelin is a 28-amino acid peptide produced mainly by mucosal neuroendocrine cells lining the fundus of the stomach. Preclinical and clinical studies suggest that ghrelin plays a role in alcoholism. Furthermore, human laboratory studies indicate that acute oral administration of alcohol results in reduced circulating ghrelin. As ghrelin is primarily produced in the stomach, one question never previously explored is whether alcohol administered intravenously (IV) results in similar decrease in ghrelin levels. Thus, this study analyzed the potential effects of IV alcohol administration on plasma ghrelin levels in healthy nonsmoking social drinkers (n = 44) who received either a 180-minute IV infusion of 6% v / v alcohol or 0.9% normal saline in two separate counterbalanced sessions. At each session, participants arrived having fasted for ~7 hours and received a light breakfast 60 minutes before the infusion. The percent change (%Δ) in ghrelin levels was 4.5-fold less in the alcohol condition than the saline condition. In fact, there was only a modest change in ghrelin levels from baseline in the IV alcohol condition (9.6%Δghrelin) while in the IV saline condition there was a robust change (43.4%Δghrelin). There was a trend toward significance in %Δghrelin in the alcohol condition compared to the placebo condition (F[1,33] = 3.3, p = 0.07). While the exact mechanisms by which alcohol influences ghrelin levels are unclear, alcohol may act directly in the stomach by inhibiting ghrelin secretion and/or release, and may also attenuate ghrelin levels systemically. Although IV alcohol did not reduce circulating ghrelin levels, as seen in previous studies with oral alcohol administration, the present findings suggest that, despite bypassing the stomach, alcohol still attenuated circulating ghrelin levels, i.e. the fasting-induced increase in circulating ghrelin was blunted by IV alcohol administration. These findings lead us to hypothesize that alcohol might affect ghrelin signaling not only via a local effect on the stomach mucosa, but also via a systemic effect.

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A practical guide for the stabilization of acylghrelin in human blood collections

Abstract: Our data suggest that AEBSF addition to K(2) EDTA blood immediately after collection without plasma acidification, processing on ice and 14-day 70 °C storage is the best treatment for accurately quantifying acylghrelin in human plasma.

Cited by 51 publications

References 31 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?

Improved acylated ghrelin suppression at 2 years in obese patients with type 2 diabetes: effects of bariatric surgery vs standard medical therapy

Fasting-induced increase in plasma ghrelin is blunted by intravenous alcohol administration: A within-subject placebo-controlled study

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