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Departments of Medicine (J.L., R.N., S.S.P., M.C.O., B.D.G., M.O.T.), Chemistry (C.E.P., H.M.G.), and Pharmacology (M.L.J., P.V.), University of Virginia, Charlottesville, Virginia 22908; Bristol-Myers Squibb Pharmaceutical Research Institute (D.A.G.), Princeton, New Jersey 08543; Merck Research Laboratories (A.D.H.), Rahway, New Jersey 07065; and Lilly Research Laboratories (D.R.W.), Indianapolis, Indiana 46285
Address all correspondence and requests for reprints to: Michael O. Thorner, University of Virginia Health System, Endocrinology and Metabolism, Box 801411, Charlottesville, Virginia 22908. E-mail: MOT{at}virginia.edu.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionNote Added in ProofReferences |
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Objective: Our objective was to examine changes in circulating ghrelin and des-acyl ghrelin in response to meals and fasting using newly developed two-site sandwich assays and sample preservation protocols to allow specific detection of full-length forms.
Design: Ten-minute sampling was done for 26.5 h during a fed admission with standardized meals and on a separate admission during the final 24 h of a 61.5-h fast and continuing for 2.5 h after terminating the fast.
Setting: The study was conducted at the University Hospital General Clinical Research Center.
Participants: Eight male volunteers participated, mean ± SD age 24.5 ± 3.7 yr and body mass index 24 ± 2.1 kg/m2.
Main Outcome Measures: Ten-minute sampling profiles were assessed for ghrelin and des-acyl ghrelin, fed and fasting.
Results: In the fed state, ghrelin and des-acyl ghrelin showed similar dynamics; both were sharply inhibited by meals and increased at night. During fasting, ghrelin decreased to nadir levels seen postprandially, and des-acyl ghrelin remained near peak levels seen preprandially. Total full-length ghrelin (acyl plus des-acyl) levels remained unchanged.
Conclusions: Meals inhibited secretion of both ghrelin and des-acyl ghrelin, yet long-term fasting inhibited acylation but not total secretion. Acylation may be regulated independently of secretion by nutrient availability in the gut or by esterases that cleave the acyl group. These studies highlight the importance of stringent conditions for sample collection and evaluation of full-length ghrelin and des-acyl ghrelin using specific two-site assays.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionNote Added in ProofReferences |
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During its synthesis, ghrelin is acylated on serine (3) with an ester-linked fatty acid group (9, 10). This unusual acylation is essential for ghrelin's activity at the GH secretagogue receptor (1, 11) but is readily cleaved by endogenous esterase activity (12, 13, 14). Both acyl- and des-acyl ghrelin are found in the circulation (1), with des-acyl being the more abundant form (15). Des-acyl ghrelin was first thought to be inactive (1), however, recent studies suggest that des-acyl ghrelin has multiple biological activities (16, 17, 18, 19, 20, 21, 22). Some of these actions of des-acyl ghrelin oppose those of acyl-ghrelin (18, 23), suggesting that the ratio of acyl- to des-acyl ghrelin may determine the overall physiological response (18).
Most published studies have used single-antibody ghrelin assays that recognize an epitope unique to ghrelin (active ghrelin) or common to both ghrelin and des-acyl ghrelin (total ghrelin). Such assays also detect ghrelin fragments. Two-site sandwich assays, as used in this study, have greater specificity and can avoid cross-reactivity with peptide fragments (24).
Likely because of ghrelin's strong basic charge and hydrophobic acylation, it can stick to surfaces and be difficult to quantitate. In the circulation, ghrelin, but not des-acyl ghrelin, may be predominantly bound to carrier proteins (25), with ghrelin having specific lipoprotein interactions not seen with des-acyl ghrelin (26), and ghrelin antisera differ in their ability to detect these bound forms (25). Together these factors can affect ghrelin measurement as reported by Groschl et al. (27), who found that when two popular commercial RIA kits were compared on the same sample set, a 10-fold difference in the measured total ghrelin levels was seen.
There are also concerns about the specificity of available acyl-ghrelin assays. In one study examining patients with anorexia nervosa (28) the acyl-ghrelin levels measured in the same sample set were either increased, decreased, or unchanged in subjects with anorexia relative to normal controls, depending on which of three different active ghrelin assays was used.
Human plasma ghrelin levels increase preprandially and drop rapidly after meals, suggesting that ghrelin plays a role in short-term regulation of food intake (29, 30). However, changes in plasma ghrelin levels accompanying a fast in excess of the typical inter-meal interval are not well understood. Previous studies have not examined whether ghrelin and des-acyl ghrelin respond differently to meals or long-term fasting.
Here we report the development of sensitive and specific two-site assays to measure both ghrelin and des-acyl ghrelin and validated protocols for sample preservation. Using these assays, we examined ghrelin and des-acyl ghrelin profiles in normal young men sampled every 10 min for 26.5 h in fed and fasting protocols.
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Subjects and Methods |
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The C-terminal region of human ghrelin [cys-ghrelin (21, 22, 23, 24, 25, 26, 27)] was synthesized using an Fmoc strategy on Rink amide resin. The product was then HPLC purified and verified by mass spectrometry.
Sources of other peptides
Peptides were obtained as follows (all human sequence unless noted): ghrelin (human and rat), des-acyl ghrelin (human and rat), ghrelin(17–28), and acyl-ghrelin(3–28) (rat) from Phoenix Pharmaceuticals (Burlingame, CA); ghrelin 1–5, 1–10, 1–14, and 1–18, from Peptides International (Louisville, KY); and GH-releasing peptide 6, motilin, galanin, somatostatin, cortistatin-14, cortistatin-17, GHRH, pituitary adenylate cyclase-activating peptide(1–38), secretin, gastric inhibitory polypeptide, PTH, and calcitonin, from Bachem (King of Prussia, PA).
Ghrelin antisera
Three different ghrelin antisera were used in these ghrelin assays: 1) C-terminal-specific antiserum, a rabbit polyclonal antiserum generated using full-length human acyl-ghrelin as antigen (gift from Bristol-Myers Squibb) and then affinity purified against ghrelin(21–27); 2) acyl-specific antiserum, a protein A purified polyclonal rabbit IgG generated using the N-terminal fragment of acyl-ghrelin(1–11) as antigen (gift from Merck Research Laboratories); and 3) des-acyl-specific antiserum, a mouse monoclonal antibody generated using full-length human ghrelin as antigen. This antibody recognizes the Gly1 through Pro7 region of the N terminus of des-acyl ghrelin (gift from Eli Lilly & Co.).
Affinity purification and biotinylation of antisera
A monospecific polyclonal antiserum for the ghrelin C terminus was obtained by affinity purification against cys-ghrelin(21–27) linked to iodoacetyl activated resin (Sulfo-Link; Pierce, Rockford, IL) following the manufacturer's protocol. Yield was 300–500 µg purified IgG/ml crude serum.
For biotinylation, purified antisera were bound to a nickel-chelated column (Pierce no. 21440) and reacted with N-hydrosuccinimide-biotin containing a polyethylene oxide spacer arm (Pierce no. 21329). Note that in our hands, it was crucial that the antiserum be free of Tris (introduced during affinity purification) to obtain satisfactory binding to the nickel column (not documented by Pierce).
Dot blotting
Ghrelin and its fragments were dotted on nitrocellulose membrane (Hybond-ECL; GE Healthcare, Piscataway, NJ), blocked, and then detected by standard Western blot techniques (West Pico; Pierce) and x-ray film (X-Omat RP; Kodak, Rochester, NY).
Collection of human plasma samples for ghrelin assay validation
Volunteers gave written informed consent, and all procedures followed a protocol approved by the Institutional Review Boards of the University of Virginia and the General Clinical Research Center (GCRC). Overnight fasting blood samples (collected before breakfast) were drawn into a cold syringe and added to chilled EDTA Vacutainer tubes preloaded with 4-[2-aminoethyl benzene] sulfonyl fluoride (AEBSF; Alexis Biochemicals, San Diego, CA) (4 mM final concentration) on ice. The blood was promptly centrifuged and the plasma separated and acidified with 200 µl 1 N HCl/ml. Aliquots were stored frozen at –20 C.
Stripping of ghrelin from plasma by C-18 extraction
Human plasma from the University of Virginia Hospital Blood Bank was dosed with AEBSF and HCl as above, and the plasma was then sequentially extracted three times on a C-18 column (3M Bioanalytical Technologies, St. Paul, MN). Previous experiments with [3H]ghrelin (Phoenix Pharmaceuticals) had established that under these acid conditions, both acyl- and des-acyl ghrelin are dissociated from carrier proteins and can be quantitatively retained on this resin (31).
Ghrelin sandwich assay
Plates (384-well Maxisorb; Nunc, Roskilde, Denmark) were coated with acyl-specific antiserum at 1 µg/ml overnight. The plate was then blocked, washed, and loaded with 25 µl/well wetting/neutralization buffer (0.5 M phosphate buffer with 1% BSA, pH 7.4) and 25 µl/well ghrelin standards or unknown samples and incubated overnight at 4C. The washed plate was then incubated 1 h with the biotinylated C-terminal ghrelin antiserum in blocking buffer and then for 30 min with streptavidin-poly-HRP80 (RDI Fitzgerald, Concord, MA). Finally, the plate was detected with the fluorescent substrate Amplex Red (Molecular Probes, Eugene, OR). Fluorescence was read using excitation/emission wavelengths of 535/590 nm (Tecan Genios plate reader; Phenix Research, Hayward, CA). All unknowns were run in duplicate, and all samples for each admission of each subject were run on the same plate. Standards were made up in acid/AEBSF-treated stripped plasma.
Des-acyl ghrelin sandwich assay
The protocol for des-acyl ghrelin assay follows that used for the ghrelin sandwich assay with the substitution of affinity-purified C-terminal ghrelin antiserum for the capture step and biotinylated N-terminal des-acyl ghrelin-specific monoclonal antiserum as the reporter. All other steps are unchanged.
Butyrylcholinesterase (BuChE) assay
BuChE was assayed in 96-well plates following established methods (32). Hydrolysis of butyrylthiocholine iodide substrate was detected with DTNB and absorbance read at 415 nm in a plate reader at 1-min intervals for 10 min (Tecan Genios plate reader; Phenix Research). The assay showed an intraassay coefficient of variation (CV) of 3.6% and an interassay CV of 7.2%. For the 10-min sampling studies, BuChE was assayed in serum samples without preservative. All unknowns were run in duplicate. Assay results are reported as percent inhibition relative to controls or as enzyme units relative to the activity units of purified bovine BuChE (no. C1057; Sigma-Aldrich, St. Louis, MO) run as a control on each plate.
Subjects and study design for 10-min sampling protocols (Fig. 1
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The study protocol was approved by the Institutional Review Boards noted above. Eight healthy men were recruited by advertisement and gave written informed consent before participating in the study. Mean age ± SD was 24.5 ± 3.7 yr (range 18–28) and body mass index (BMI) ± SD was 24 ± 2.1 kg/m2 (range 20.6–26.2). Each subject was admitted for both a fed and a fasting admission in random order.
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During the fed admission, subjects were admitted to the GCRC, served a standardized dinner at 1800 h and then fasted overnight except for water ad libitum. The following morning, a venous cannula was inserted into a forearm vein of each arm. Blood sampling started at 0800 h and continued every 10 min for 26.5 h. During the sampling period, four standardized meals were served. Subjects were asked to consume each meal in its entirety within 30 min. The total calories given each subject were calculated using the Harris-Benedict equation and supplied as 20% protein, 30% fat, and 50% carbohydrate. Each sample was assayed for ghrelin, des-acyl ghrelin, and BuChE.
Fasting admission
During the fasting admission, subjects were admitted to the GCRC in the early evening. Upon completion of dinner at 1830 h, no food was allowed for 61.5 h except for free access to water. Sampling at 10-min intervals was started at 0800 h, 37.5 h into the fast. Breakfast was served at 0800 h the following day ending the fast, but sampling continued for another 2.5 h (total of 26.5 h of sampling). Ketones were measured three times per day to verify compliance with fasting.
Statistical analysis
All results are expressed as mean ± SEM unless otherwise noted. Statistical comparisons were performed using two-tailed paired t tests. P < 0.05 was considered statistically significant. In all cases, nonparametric Wilcoxon paired rank tests gave the same conclusions as t tests. Statistical, regression, curve fitting, and IC50 calculations were made using GraphPad Prism version 4.03 for Windows (GraphPad Software, San Diego, CA).
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Results |
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Figure 2 demonstrates that each antisera recognized the peptides and fragments containing its expected epitope.
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The dose-response curve for the ghrelin sandwich assay and lack of cross-reactivity with des-acyl ghrelin, ghrelin fragments, or other peptides is shown in Fig. 3A. The assay sensitivity was 6.7 pg/ml with an intraassay CV of 9.1% at 30 pg/ml, 12.6% at 100 pg/ml, and 16.8% at 300 pg/ml. The interassay CV was 17.8% at 50 pg/ml. Figure 3B shows the dose response of the des-acyl sandwich assay. The assay sensitivity was 4.6 pg/ml with an intraassay CV of 12.5% at 50 pg/ml, 10.7% at 150 pg/ml, and 18.0% at 500 pg/ml. The interassay CV was 20.8% at 30 pg/ml. There was no significant cross-reactivity with ghrelin fragments or nonspecific peptides, but the assay does show a cross-reactivity of less than 3% with acyl-ghrelin. Because acyl-ghrelin was generally less abundant in clinical samples, this is not a significant error in this study and was not corrected for.
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Studies were performed to evaluate methods to prevent degradation of ghrelin after blood samples are drawn. Figure 4A shows average percent acylation over time (time was defined as the delay in hours between blood draw and plasma acidification). The initial overnight fasting ghrelin levels in six subjects (three men and three women, ages 26–60 yr) varied from 26–234 pg/ml, but in the samples without AEBSF, all were degraded to below the level of detection within 24 h. In the samples incubated at room temperature with and without AEBSF, levels of acyl-ghrelin decreased over time, whereas des-acyl increased. Those incubated with AEBSF on ice showed only 6.2% loss of acylation in 24 h (not statistically different from zero loss). Samples on ice without AEBSF were tested in other runs (data not shown) and gave a deacylation half-life of about 6.4 h. All samples for additional studies were collected with AEBSF on ice and acidified within 1 h of collection.
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Recovery of acyl-ghrelin added to plasma samples
Spiking experiments were used to confirm the specificity of the assays and the efficacy of the protective agents. Two doses of ghrelin were spiked into plasma samples from four volunteers and into stripped blood bank plasma with and without AEBSF and acid. Fasting ghrelin levels (overnight, before breakfast) varied from 43–366 pg/ml in four volunteers. The increase due to the added exogenous ghrelin could be assayed quantitatively in all subjects and in the stripped plasma with recoveries of 102.9 ± 4.2% (n = 5 at 50 pg/ml) and 108.2 ± 8.9% (n = 5 at 200 pg/ml). Without AEBSF and acid, 100% of the 50 pg/ml dose and 53% of the 200 pg/ml dose of spiked acyl-ghrelin was lost during assay.
Examining BuChE inhibition in serum samples
BuChE is one of the activities found in human serum that must be inhibited to prevent ghrelin degradation (12). We found that AEBSF potently inhibited BuChE (see supplemental Fig. 1, published as supplemental data on The Endocrine Society's Journals Online web site at http://jcem.endojournals.org). At room temperature, the 4 mM concentration used in our ghrelin samples inhibited 86–96% of the BuChE activity, consistent with the partial loss of ghrelin acylation seen in Fig. 4A
(middle curve).
Different ghrelin assay protocols specify 50, 100, or 200 µl of 1 N HCl/ml plasma to preserve samples. Figure 4B shows the result of adding varying amounts of HCl to plasma (without AEBSF), incubating 1 h, and then diluting the plasma into neutralizing buffer and assaying BuChE activity. This is relevant because samples must be thawed and neutralized before being incubated with antibodies for immunoassay. Figure 4B shows that 200 µl 1 N HCl/ml plasma (pH 
3) inactivates this esterase within 1 h leaving no measurable activity. Using 100 µl of 1 N HCl/ml plasma, 32.5% of the BuChE remained after 1 h (Fig. 4B), but when incubated for 6 h, only 3% of the BuChE activity remained (not shown). With 50 µl acid, 81.6% of the enzyme activity was still present even after 6 h incubation (not shown). Thus, the 50 µl HCl/ml recommended in some protocols seems insufficient to prevent BuChE activity. Acidification is crucial to protect ghrelin against loss of acylation on storage and freeze/thaw (33), but the ester linkage on ghrelin can become unstable if the pH is allowed to drop to less than 2 (33).
Ghrelin profiles during fed and fasting admissions
Figure 5 shows the fed and fasting profiles for acyl- and des-acyl ghrelin over the 26.5 h of the sampling protocol (Fig. 1).
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shows the pulsatile pattern of acyl-ghrelin levels. During the fed admission, preprandial increases and postprandial suppression of acyl-ghrelin were observed. Marked suppression of acyl-ghrelin was seen in the same subjects on the fasting admission. Note that on the fasting admission, ghrelin did not rise before customary mealtimes, and ghrelin levels remained near the nadir seen on the fed admission. Despite this suppression, there was a further suppression of ghrelin after breaking the 61.5-h fast (P = 0.039, paired t tested for 1-h intervals before and after breakfast).
Figure 5B compares the profiles for des-acyl ghrelin on the fed and fasting admissions. During the fed day, the pattern was similar to that observed for acyl-ghrelin, but the levels (picograms per milliliter) were higher. With fasting, des-acyl ghrelin levels remained at preprandial (peak) levels without the postprandial suppression.
Figure 5C compares acyl- and des-acyl ghrelin on the fed admission. However, the patterns, including response to meals and rise at night, are nearly identical (acyl-ghrelin tends to show somewhat larger percent changes with a suggestion of more extreme peaks and valleys).
Figure 5D compares the acyl- and des-acyl ghrelin profiles on the fasting admission and demonstrates the change in the relative abundance of the two forms on fasting.
To quantitate the effects of fasting, we compared the 24-h sampling period while fasting (before breaking the fast at 0800 h) with the same time period on the fed admission. A value for each subject was computed as the average of the 144 time points over these 24 h. Figure 6A shows the average of these values for the eight subjects for both ghrelin and des-acyl ghrelin. On the fasting admission, ghrelin was decreased by 58% (27.9 ± 3.9 vs. 11.8 ± 1.7 pg/ml; P = 0.0025), whereas des-acyl ghrelin was increased by 19% (102.2 ± 13.4 vs. 121.9 ± 11.8 pg/ml; P = 0.040). The sum of acyl- plus des-acyl ghrelin was not different (130.1 ± 13.7 vs. 133.7 ± 12.5 pg/ml; P = 0.66).
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BuChE on fed and fasting admissions
BuChE activity was examined to test the hypothesis that increased circulating activity of this esterase was the cause of the observed decrease in ghrelin acylation seen on fasting. Figure 6B shows that although BuChE activity was present, there was no significant change in activity with fasting (55.5 ± 3.7 fed vs. 57.8 ± 4.1 U/ml fasting; P = 0.13; n = 8; paired t test, 144 samples for each subject over the same time course as in Fig. 6A).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionNote Added in ProofReferences |
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Assays for ghrelin and des-acyl ghrelin
Consistent with improved specificity for the two-site assays, ghrelin profiles show larger relative changes in response to meals than previously seen by single-site assays (29, 30). The extensive literature on ghrelin measurements has generally assumed that single-antibody total ghrelin measurements reflect ghrelin activity; the results presented here from fasting subjects demonstrate that this assumption is not always correct and strongly argues for the specific assay of both forms. This is especially important in the face of a growing literature on the unique activities of des-acyl ghrelin.
Akamizu et al. (34), using their own sandwich assays for acyl- and des-acyl ghrelin, concluded that 40–60% of the signal in a standard total ghrelin RIA in plasma was actually from inactive C-terminal ghrelin fragments. Results with our sandwich assays confirm this, showing that the levels of full-length ghrelin are lower than suggested by single-site assay and that the fraction of acyl-ghrelin is higher. Reported values for ghrelin acylation in normal men were 18.0% in Akamizu's study and 22.0% in our study vs. 8.3–9.4% in similar acid-preserved samples assayed by RIA (35, 36) and 1.8% acylation in plasma assayed by RIA in older studies without appropriate sample preservation (15).
Our results document the importance of measures to protect ghrelin acylation. The half-life of endogenous acyl-ghrelin in blood without AEBSF found here (2.9 h at room temperature) is comparable to that reported by De Vriese et al. (12) (4 h at 37 C), who added synthetic ghrelin(1–23) or ghrelin(1–28) to human serum and analyzed the acylation levels by HPLC.
Changes in ghrelin and des-acyl ghrelin in the fed state and during prolonged fasting
On the fed admission, ghrelin levels varied markedly with rises preprandially and at night. Under these conditions, ghrelin and des-acyl ghrelin levels changed in parallel with sharp declines on feeding, but the percentage of the circulating hormone that was acylated remained fairly constant (22.0 ± 0.3%, mean ± SEM; SD = 3.8%). During the fasting admission, the levels of des-acyl ghrelin were near the peak levels observed during the fed admission, whereas acyl-ghrelin levels were near the nadirs observed on the fed day, and the percent acylation dropped (9.9 ± 0.2%; SD = 2.7%). The sum of acyl- plus des-acyl ghrelin was no different on the fasting than on the fed admission. This is consistent with other studies that found that in contrast to shorter fasts, on long-term fasting, total ghrelin levels were not increased (37, 38, 39, 40). The dissociation between changes in ghrelin and des-acyl ghrelin seen in this study demonstrates the importance of measuring both forms simultaneously.
During fasting, the balance of ghrelin to des-acyl ghrelin was altered. This ratio has been proposed to determine ghrelin's overall adipogenic action (18) and may have other consequences related to the specific actions of des-acyl ghrelin. Our results suggest separate regulation of ghrelin secretion and ghrelin acylation. Consistent with independent mechanisms, the breakfast that was served after 61.5 h of fasting further suppressed ghrelin levels below the maximal suppression caused by fasting and also sharply suppressed des-acyl ghrelin (Fig. 5).
The change in the relative amounts of ghrelin and des-acyl ghrelin could be caused by either an inhibition of the acylation of ghrelin as it is synthesized (a process not well understood, see Note Added in Proof) (10) or an increase in the activity of ghrelin esterases. We have shown that serum BuChE, a significant ghrelin esterase in human serum (12), was not increased during fasting, but this does not rule out the possible contribution of other esterases, especially in tissues such as liver.
Interestingly, the data of Kim et al. (41) suggest that the decrease in ghrelin during the fasting admission might be counterbalanced by an increase in central ghrelin receptor expression, showing that in rodents, 48-h fasting resulted in an 8-fold increase in hypothalamic ghrelin receptor expression. This suggests that the changes in circulating ghrelin levels do not necessarily reflect the actual changes in its biological activity.
During fasting, we did not see changes in ghrelin levels around customary mealtimes. Such changes were seen in a study of a 33-h fast (39) but may not have been apparent here because we did not initiate sampling until 37.5 h of fasting.
Regulation of acylation
These new sandwich assays show that with normal feeding, ghrelin and des-acyl ghrelin rise and fall together in response to meals. But on long-term fasting, the proportion of ghrelin that is acylated falls, with no change in the total levels of circulating full-length ghrelin. Thus, under these conditions, the balance between ghrelin and des-acyl ghrelin may be changed by the regulation of ghrelin acylation. Toshinai et al. (42) report that in the stomach of rats fasted for 48 h, "the ratio of des-octanoylated ghrelin to n-octanoylated ghrelin markedly increased after fasting." This result is consistent with our findings, and together these data suggest that long-term fasting regulates ghrelin activity by a mechanism that inhibits the addition of the acyl group as ghrelin is synthesized. It can be speculated that X/A cells require an intraluminal nutrient source to allow acylation. This is consistent with observations that fatty acids from the diet can be directly used for ghrelin acylation (43).
Conclusions
Our results suggest that acyl-ghrelin levels decline with long-term fasting, whereas des-acyl ghrelin levels are increased. It is interesting that increased appetite occurs during short-term fasting but wanes over time; this may relate to the lowering of acyl-ghrelin levels seen here. New assays that specifically measure the full-length forms of ghrelin and des-acyl ghrelin will be crucial to defining ghrelin's physiological role and delineating its regulation.
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Note Added in Proof |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionNote Added in ProofReferences |
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Acknowledgments |
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Footnotes |
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Disclosure Information: The authors have declared that no conflicts of interest exist. D.A.G reports a financial interest in Bristol-Myers Squibb. M.O.T. reports he is a paid consultant for Novo Nordisk and Tercica. Part of this work was supported by an unrestricted grant from Bristol-Myers Squibb.
First Published Online March 18, 2008
1 J.L. and C.E.P. participated equally in this work. 
Abbreviations: AEBSF, 4-[2-Aminoethyl benzene] sulfonyl fluoride; BMI, body mass index; BuChE, butyrylcholinesterase; CV, coefficient of variation; GCRC, General Clinical Research Center.
Received October 4, 2007.
Accepted February 19, 2008.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionNote Added in ProofReferences |
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) were tested in both assays at varying doses shown on a log scale. Controls for nonspecific cross-reactivity were tested in both assays at one dose (all shown as open squares). Ghrelin fragments tested at 20 ng/ml were acyl-ghrelin(1–5), acyl-ghrelin(1–10), acyl-ghrelin(1–14), acyl-ghrelin(1–18), ghrelin(17–28), and ghrelin(3–28) (rat). Other peptides tested at 100 nM (87–506 ng/ml depending on molecular weight) were GH-releasing peptide 6, motilin, galanin, somatostatin, cortistatin-14, cortistatin-17, GHRH, pituitary adenylate cyclase-activating peptide (1–38), secretin, gastric inhibitory polypeptide, PTH, and calcitonin. Although the acyl-ghrelin assay showed no response to des-acyl ghrelin or other tested peptides, the des-acyl ghrelin assay had 3% cross-reactivity to acyl-ghrelin and is an inherent property of the monoclonal antibody's specificity (note faint cross-reactivity seen in Fig. 2
– –), and no AEBSF and no ice (···
···). B, Inhibition of BuChE activity by HCl. Plasma was incubated 1 h with the indicated dose of HCl (microliters per milliliter plasma) and then diluted into a neutralizing buffer for assay. Blank indicates no enzyme negative control. The y-axis is percentage of activity without acid.
