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Department of Metabolic Medicine, Imperial College Faculty of Medicine, London Hammersmith Hospital, London W12 ONN, United Kingdom
Address all correspondence and requests for reprints to: Professor Stephen R. Bloom, Department of Metabolic Medicine, Imperial College, London Hammersmith Hospital, Du Cane Road, London W12 ONN, United Kingdom. E-mail: s.bloom{at}imperial.ac.uk.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Interestingly, fasting levels of ghrelin varied dramatically in these studies, suggesting that the assays may be measuring different forms of Ghr-LI. Only Ariyasu et al. (11, 12) used chromatography to confirm the molecular form of Ghr-LI detected by their assays. They used two assays; one specific for acylated ghrelin and the other cross-reacting with both acylated and des-acylated ghrelin (11). In their study, peptide was extracted from plasma using Sep-Pak cartridges (Waters, Milford, CT). The extract was fractionated by reverse-phase HPLC. Two Ghr-LI peaks were detected by the nonspecific assay: a large peak corresponding to synthetic des-acylated ghrelin and a second smaller peak corresponding to synthetic acylated ghrelin. The specific assay detected only the second peak. Using these same assays, Yoshimoto et al. (13) showed that less than 10% of Ghr-LI detected after Sep-Pak extraction was acylated ghrelin. In 2003 Beaumont et al. (14) showed that acylated ghrelin will bind to several plasma proteins in vitro, particularly high-density lipoprotein (HDL)-associated protein apolipoprotein A1, but also another HDL-associated protein, paraoxonase-1. Paraoxonase-1 has esterase activity and might therefore be responsible for the conversion of ghrelin to des-acylated ghrelin. However, the relative amounts of free and protein bound ghrelin in circulation were not established.
The most widely used assay is the human ghrelin RIA (Phoenix Pharmaceuticals, Inc., Belmont, CA). No published data are available characterizing the molecular form of Ghr-LI measured in plasma by this assay. It is therefore unknown how much of the Ghr-LI measured is acylated ghrelin and whether it circulates as free peptide or is protein bound.
In this study we used the Phoenix Pharmaceuticals assay plus two other ghrelin RIAs with different specificities to characterize Ghr-LI in human pre- and postprandial plasma samples.
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Subjects and Methods |
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All studies were performed according to the principles of the Declaration of Helsinki. The Local Research and Ethics Committee at the Hammersmith Hospital approved the study (03/6499). Written informed consent was obtained. Exclusion criteria included chronic medical or psychiatric illness, pregnancy, substance abuse, more than two alcoholic drinks per day, and aerobic exercise for more than 30 min three times per week.
Test meal protocol
Five male and three female healthy subjects aged 20–33 yr (BMI, 23.4 ± 1.2 kg/m2) were fasted for 12 h before receiving a standard breakfast. Males received a 750-kcal breakfast and females a 550-kcal breakfast of the same macronutrient composition to allow for the difference in energy requirements. An iv cannula was inserted into the distal forearm 30 min before the meal. Blood samples (10 ml) were taken at baseline and 15, 30, 60, 120, and 180 min after the meal. Blood samples were collected in lithium heparin tubes containing 200 µl Trasylol aprotinin (Bayer PLC, Berkshire, UK), immediately centrifuged at 4 C for 15 min, and the plasma separated and stored at –20 C before being assayed for Ghr-LI.
RIAs
Samples for all assays were thawed immediately before assay and were assayed within 1 h to minimize any degradation of ghrelin. Our pilot studies had shown that if samples were thawed and remained at 4 C for longer periods of time (2 h or longer), there was a significant loss of Ghr-LI detected by the assay specific for only the active, acylated ghrelin (antibody G0–1) (data not shown).
G0–1 RIA
The G0–1 RIA was performed as previously described by English et al. (10). Briefly G0–1 antibody was raised in a rabbit immunized with synthetic human ghrelin (Bachem UK Ltd., Merseyside, UK) conjugated to BSA (Sigma-Aldrich, St. Louis, MO) by glutaraldehyde (Sigma-Aldrich) and used at a final dilution of 1:70,000. The antibody cross-reacted 100% with human and rat acylated ghrelin and rat acylated ghrelin (octnoylated and decanoylated) but did not cross-react with human or rat des-acylated ghrelin or any other known gastrointestinal or pancreatic peptide or hormone. The 125I ghrelin was prepared by Bolton & Hunter reagent (Amersham International UK, Aylesbury, UK) and purified by reverse-phase HPLC using a linear gradient from 10 to 40% acetonitrile (AcN), 0.05% trifluroacetic acid (TFA) over 90 min. The specific activity of ghrelin label was 48 Bq/fmol. The assays were performed in a total volume of 0.7 ml of 0.06 M phosphate buffer (pH 7.2) containing 0.3% BSA and incubated for 3 d at 4 C before separation of free and bound label by sheep antirabbit antibody (Pharmacia & Upjohn, Stockholm, Sweden). The assay detected changes of 15 pmol/liter of plasma ghrelin with 95% confidence limit. The intra- and interassay coefficients of variation were 6.2 and 9.5%, respectively.
SC-10368 (SC) RIA
The SC RIA was developed in-house using a commercial antibody (SC-10368) purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). SC-10368 is a goat polyclonal antibody raised against an internal region of human ghrelin. This antibody fully cross-reacts with human and rat acylated ghrelin (octnoylated and decanoylated) and des-acylated ghrelin and was used at a final dilution of 1:50,000. The assay was performed as detailed for G0–1 using the same 125I ghrelin label. After 3 d incubation-free and antibody-bound labels were separated by charcoal adsorption of the free 125I ghrelin fraction. The assay detected changes of 25 pmol/liter plasma ghrelin with 95% confidence limit, with an intra- and interassay coefficient of variation of 5.5 and 10.1%, respectively.
Commercial RIA
The commercial RIA used was the human ghrelin RIA (Phoenix Pharmaceuticals). This assay used an antibody specific for total human ghrelin (acylated and des-acylated). To allow comparison with G0–1 and SC, in-house standards were used in addition to those provided with the kit.
Peptide extraction procedure
Peptide was extracted from plasma using Sep-Pak C18 cartridges (Waters). Sep-Pak C-18 cartridges were activated using 10 ml of 100% methanol and then 20 ml water. A 1-ml volume of plasma was mixed with 1 ml of 0.1 M HCl loaded onto the cartridge. The plasma eluant was collected and assayed for G0–1 Ghr-LI by RIA. The cartridge was then washed with 10 ml of 4% acetic acid (vol/vol). The Sep-Pak bound sample was then eluted in 1.5 ml methanol. The eluate was dried in a Savant vacuum centrifuge and reconstituted in assay buffer for direct RIA or in water plus 0.05% TFA (vol/vol) for fast protein liquid chromatography (FPLC). As a control 1 pmol synthetic ghrelin dissolved in phosphate buffer (pH 7.2) and the peptide extracted. Recovery was calculated as Ghr-LI recovered from each sample, compared with Ghr-LI recovered from control, and was expressed as a percentage.
Chromatography
Sephadex G-100 gel permeation chromatography. Ghr-LI was fractionated by eluting 0.06 M phosphate buffer containing 0.3% BSA (vol/vol) at 3.2 ml/h through a Sephadex G100-Superfine (Pharmacia, Uppsala Sweden) gel column (60 x 0.9 cm). Before loading 30 mg/ml Dextran Blue (molecular weight > 2 million), 30 mg/ml horse heart cytochrome C (molecular weight, 12,384) and 5 Bq Na125I was added to 1 ml plasma, giving a final volume of 1.1 ml. Of this volume, 0.8 ml was loaded on the column, and the remaining 0.3 ml was used to calculate the recovery. Fractions of 0.75 ml were collected, and the three RIAs were used to determine the elution profile of Ghr-LI and calculate a recovery. Elution positions of Dextran Blue and 125 I were used to calculate the relative elution coefficent (Kav) as previously described (15).
Reverse-phase FPLC
Peptide extracts from plasma were dissolved in 1 ml distilled water plus TFA 0.05% (vol/vol) and then filtered through 0.2 µm hydrophilic membranes (Satorius, Gottingen, Germany). Of this volume, 0.5 ml was fractionated by FPLC on a high-resolution reverse-phase (Pep RPC 1 ml HR) C-18 column (Pharmacia, Uppsala Sweden). The column was eluted with a 10–40% gradient of AcN/water 0.05% (vol/vol) TFA over 60 min. Fractions from all runs were dried in a Savant vacuum centrifuge, reconstituted in assay buffer, and Ghr-LI content was determined by RIA. The remaining 0.2 ml was used to calculate the percentage recovery.
Incubating plasma with ghrelin
A volume of 100 µl phosphate buffer containing 1 pmol synthetic human ghrelin, 1 pmol synthetic human des-acylated ghrelin, or control (no ghrelin) was added to a 0.9-ml aliquot of human plasma, which was then incubated for 10 min at 37 C. Samples were frozen at –20 C until they were fractionated on a Sephadex G100-Superfine column and assayed using the SC and G0–1 RIAs as detailed above.
Treatment of plasma with chaotropic agents and detergents
Human plasma (1 ml) was incubated for 1 h at 37 C with 8 M urea, 8 M guanindine hyperchloride, 0.5 or 1% Triton X-100. This concentration of urea was chosen because it was used by Orth and Mount (16) to separate CRH from CRH binding protein. Guanidine hyperchloride was used at the same concentration as an alternative chaotropic agent. Triton X-100 was not used at concentrations above 1% because this caused interference with the G0–1 assay. After incubation the plasma was immediately assayed using both the G0–1 assay and SC assay. Samples treated with 0.5 and 1% Triton X-100 were run on a Sephadex G100 column as described previously.
Statistical analysis
Hormone levels are expressed as means ± SEM. Fasting and postprandial levels of Ghr-LI were compared by two-tailed, paired Student’s t test.
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Results |
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We initially used only results obtained using the in-house standard to allow direct comparison of Ghr-LI levels detected by each assay. A postprandial fall in circulating Ghr-LI level was observed with all assays (Fig. 1
, A and B). However, both pre- and postprandial G0–1 Ghr-LI levels were 500 and 460% greater than levels detected by SC and the commercial assay, respectively. Furthermore, the timing of the postprandial fall in Ghr-LI differed. G0–1 Ghr-LI levels fell significantly 30 min post meal (P < 0.05) and had returned to basal by 90 min (Fig. 1A). In contrast, SC Ghr-LI levels did not fall significantly until 90 and 120 min (P < 0.01) (Fig. 1B). Ghr-LI levels detected by the commercial assay were similar to levels detected by the SC assay and showed a similar pattern, falling significantly only at 90 and 120 min (P < 0.01) (Fig. 1B). Ghr-LI measured by G0–1 therefore differs from that measured by the SC and commercial assays.
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Ghr-LI detected by the commercial and SC assays correlated strongly (Fig. 1
, B and C). However, when using the standard supplied with the commercial kit, Ghr-LI levels detected by the commercial assay were approximately 3-fold lower than those detected by the SC assay (Fig. 1C). To allow comparison, all the data shown except Fig. 1C were calculated with the in-house standards.
Ghr-LI after peptide extraction
After peptide extraction by Sep-Pak cartridge, more than 90% of Ghr-LI detected by the SC and commercial assays was recovered, whereas less than 10% of G0–1 Ghr-LI was recovered. The plasma eluant did not contain any G0–1 Ghr-LI.
Chromatography
G100 Sephadex gel fractionation.
Pre- and postprandial samples were fractionated using G100 Sephadex gel columns. All columns had a recovery of more than 80%. Figure 2A shows the elution profile of G0–1 Ghr-LI. Two G0–1 Ghr-LI peaks were identified: one smaller peak (13%) eluting at Kav of 0.1 and a larger peak (83%) eluting at Kav of 0.5. The two peaks identified by G0–1 both have a molecular weight greater than 12,384. The SC and commercial assays identified only one major Ghr-LI peak at Kav of 0.75, eluting in the same position as acylated and des-acylated synthetic ghrelin (Fig. 2, B and C). This peak was not detected by the G0–1 assay and is therefore likely to be nonacylated ghrelin.
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Reverse-phase FPLC chromatography was used to further analyze Ghr-LI extracted from plasma by Sep-Pak cartridge. Reverse-phase C-18 FPLC had the resolution to separate the more hydrophobic acylated ghrelin from des-acylated ghrelin. All columns had a recovery of more than 78%. In extract from 0.5 ml plasma, the SC assay detected an early small peak (17 ± 3.9%) (mean ± SEM) and a larger peak (83 ± 3.9%) corresponding to the des-acylated ghrelin (n = 4). A representative profile is shown in Fig. 3A. The commercial assay detected a similar profile, with 15.8 ± 3.5% Ghr-LI eluting in the early peak and a further 84.2 ± 3.5% corresponding to des-acylated ghrelin (n = 4). A representative profile is shown in Fig. 3B. There was insufficient G0–1 Ghr-LI in 0.5 ml of extract to detect a peak. The G0–1 assay detected a clear peak corresponding to acylated ghrelin when extract from 3 ml plasma was pooled and fractionated. A representative profile is shown in Fig. 3C. Acylated ghrelin accounted for 37.5 ± 2.5% of Ghr-LI. An early peak represented 5 ± 1%, but the majority of Ghr-LI 58 ± 4% eluted at 40% AcN (n = 2). The second peak may represent more hydrophobic fragments of ghrelin peptide. In extract from 3 ml plasma, the SC assay detected a Ghr-LI peak corresponding to acylated ghrelin. This accounted for 8 ± 1% of the total Ghr-LI loaded on the column. An early peak represented 3 ± 2%, and des-acylated ghrelin the remaining 89 ± 3 (n = 2). A representative profile is shown in Fig. 3D. Both octanoylated ghrelin (the major form of acylated ghrelin) and the less common decanoylated ghrelin eluted at the same position marked acylated ghrelin in Fig. 3. We were therefore unable to determine the relative amounts of each form of acylated ghrelin detected in Fig. 3, C and D.
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After incubation of 1 pmol synthetic acylated ghrelin with 1 ml plasma for 10 min at 37 C, only 40% of added peptide was recovered by the SC and 20% by the G0–1 assay, respectively. This relatively low recovery may be due to degradation of the ghrelin and/or neither antibody detecting certain forms of circulating ghrelin. G100 Sephadex gel permeation chromatography detected two peaks of G0–1 Ghr-LI in identical positions for both the plasma incubated with synthetic ghrelin and the control plasma (Fig. 4A). No peak correlated with free synthetic ghrelin, suggesting the synthetic ghrelin had bound to larger plasma proteins or been degraded. SC detected one Ghr-LI peak for both the control plasma and plasma incubated with synthetic ghrelin, which eluted at the position of free synthetic ghrelin (Fig. 4B). When the experiment was repeated with synthetic des-acylated ghrelin, only the SC assay detected a rise in Ghr-LI (110% recovery). Chromatography showed that this Ghr-LI also eluted in a single peak at the same position as free synthetic ghrelin (data not shown).
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Samples were treated with chaotropic agents or detergents in an attempt to separate ghrelin species detected by the G0–1 RIA from binding proteins. After treatment with 0.5 and 1% Triton X-100, there was no change in Ghr-LI detected by the G0–1 or SC assays. After G100 Sephadex gel fractionation of plasma treated with 0.5 or 1% Triton X-100, an identical elution profile was detected in treated and untreated samples.
After treatment with 8 M urea or 8 M guanidine hyperchloride, no Ghr-LI was detected by the G0–1 assay.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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After peptide extraction from plasma by Sep-Pak cartridge, recovery of G0–1 Ghr-LI was less than 10%, whereas more than 90% Ghr-LI was recovered by the SC and commercial assays. This suggested that Ghr-LI detected by the G0–1 assay was not free peptide. G0–1 Ghr-LI was not present in plasma eluant, suggesting that it had bound to the cartridge. It is possible that G0–1 Ghr-LI is denatured during the Sep-Pak process or does not elute from the cartridge. Reverse-phase chromatography (FPLC) demonstrated that less than 10% of Ghr-LI in the extract recovered by the SC assay was eluting in the position of acylated ghrelin. These results are similar to those of Yoshimoto et al. (13), who have previously shown that acylated ghrelin accounts for less than 10% of Ghr-LI extracted from human plasma by Sep-Pak cartridge. However, we did see an additional early peak. This early peak consists of Ghr-LI that has not bound to the column. However, it is difficult to speculate what this immunoreactivity may represent. Our results demonstrate that Sep-Pak extraction leads to the loss of large forms of Ghr-LI that may represent ghrelin bound to larger proteins. Researchers should be aware that ghrelin bound to larger plasma proteins may be lost when treating with Sep-Pak cartridges. Similar observations were found during the development of assays for CRH in plasma. Immunoreactivity lost after extraction was originally thought to be interference but was later demonstrated to be CRH bound to a specific binding protein (16).
We attempted to separate Ghr-LI detected by G0–1 from possible binding proteins using detergents and chaotropic agents. After treatment with detergent (Triton X-100), there was no change in the elution position of the Ghr-LI detected by G0–1. This indicated that Ghr-LI had not been separated from binding proteins. Although some binding protein interactions can be reversed by detergents (18), other strong protein-protein interactions are not (19). In certain cases detergents can increase a protein affinity for its binding proteins (20).
Treatment with urea at concentrations previously used to separate CRH from its binding protein (16) resulted in a loss of G0–1 Ghr-LI. The chaotropic agents may have disrupted the structure of the relevant protein to the extent it was no longer recognized by G0–1. Guanidine hyperchloride was used as an alternative chaotropic agent. However, again, no G0–1 Ghr-LI was detected after treatment.
Ghr-LI levels detected by the commercial assay were 3-fold greater when calculated with our in-house standard than when calculated with the standard supplied with the kit. Groschl et al. (21) previously demonstrated that the 10-fold difference in Ghr-LI levels detected by the Phoenix Pharmaceuticals assay used in this study and another commercial RIA produced by Linco Research were solely due to differences in the concentration of the standards supplied. These differences make it difficult to compare studies using different assays. It would be useful were standards for all ghrelin assays obtained from a common source.
The timing of the postprandial fall in Ghr-LI measured by the three assays differed. The fall in plasma G0–1 Ghr-LI was rapid, with a significant reduction in Ghr-LI seen by 30 min. G0–1 Ghr-LI had returned to basal levels after 90 min. This is similar to the postprandial profile of Ghr-LI we previously published using this assay (10). In contrast, there is no significant reduction in Ghr-LI measured by SC and the commercial assay until 90 min, and levels remain suppressed at 120 min. It is interesting that postprandial acylated ghrelin levels fall more quickly than total ghrelin. This may reflect changes in secretion of acylated ghrelin and/or the des-acylation of acylated ghrelin. Hosoda et al. (22) have shown that after an oral glucose tolerance test, acylated ghrelin levels return to baseline values more quickly than total ghrelin levels. We have now shown that this is also the case with a mixed macronutrient meal using our acylated ghrelin-specific RIA. Assays measuring specific forms of ghrelin may therefore be more useful in determining its physiological role than those that detect both acylated and des-acylated forms.
Our study characterizes circulating Ghr-LI detected by three antibodies. The results suggest that des-acylated ghrelin circulates as free peptide, whereas the majority of acylated ghrelin circulates bound to larger molecules in plasma. This could be important in the transport of ghrelin to centers of appetite control.
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Footnotes |
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First Published Online January 18, 2005
Abbreviations: AcN, Acetonitrile; BMI, body mass index; FPLC, fast protein liquid chromatography; G0–1, RIA specific for only the active, acylated ghrelin antibody; Ghr-LI, ghrelin-like immunoreactivity; HDL, high-density lipoprotein; Kav, relative elution coefficient; SC, RIA (SC-10368) developed in-house using a commercial antibody from Santa Cruz Biotechnology; TFA, trifluroacetic acid.
Received August 17, 2004.
Accepted January 10, 2005.
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