Departments of Gastrointestinal Surgery C and Clinical Biochemistry, Rigshospitalet, University of Copenhagen, DK-2100 Copenhagen, Denmark
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ABSTRACT |
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The antral hormone gastrin is
synthesized by processing progastrin into different peptides that
stimulate gastric secretion. The effect on acid secretion depends
mainly on the metabolic clearance rate of the peptides, but some of
them may differ in potency and maximum acid output at similar
concentrations in plasma. Sulfated and nonsulfated gastrin-6 are the
smallest circulating bioactive gastrins in humans. Their effect and
metabolism have now been investigated in nine normal subjects and
compared with nonsulfated gastrin-17, a main product of progastrin.
Maximum acid output after stimulation with gastrin-17, sulfated
gastrin-6, and nonsulfated gastrin-6 were 28.3 ± 2.0, 24.5 ± 2.0 (P < 0.02), and 19.3 ± 2.3 (P < 0.05) mmol H+/50 min, respectively,
and the corresponding EC50 values were 43 ± 6, 24 ± 2 (P < 0.01), and 25 ± 2 (not
significant) pmol/l. The half-life of gastrin-17 was 5.3 ± 0.3 min, the metabolic clearance rate (MCR) was 16.5 ± 1.3 ml · kg1 · min
1, and the
apparent volume of distribution (Vd) was 124.3 ± 9.6 ml/kg. The half-lives of sulfated and nonsulfated gastrin-6 were 2.1 ± 0.3 and 1.9 ± 0.3 min, the MCRs were 42.8 ± 3.7 and 139.4 ± 9.6 ml kg
1 min
1
(P < 0.01), and the Vd were 139.0 ± 30.5 and 392.0 ± 81.6 (P < 0.01) ml
kg
1. All pharmacokinetic parameters differed
significantly from gastrin-17 (P < 0.01). We conclude
that gastrin 6 has a higher potency but a lower efficacy than
gastrin-17. The efficacy of gastrin-6 is increased by tyrosine
O-sulfation, which also enhances the protection against elimination.
gastric acid; pharmacodynamics; pharmacokinetics
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INTRODUCTION |
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GASTRIN WAS
THE FIRST GASTROINTESTINAL hormone to have its structure
determined (9). Gastrin is a major regulator of gastric acid secretion and growth of gastric mucosa cells (for review, see Ref.
30). The active site of gastrin is the COOH-terminal tetrapeptide amide
Trp-Met-Asp-Phe-NH2 (17). Progastrin
is synthesized in antral G cells and processed into a number of
bioactive peptides, of which the heptadecapeptide gastrin-17 is
the main product (9, 11, 23) (Fig.
1). All bioactive gastrin peptides are
carboxyamidated and exist in nonsulfated and sulfated forms, due to
O-sulfation of the tyrosyl residue (Tyr6), as
counted from the COOH terminus (1, 9, 11). A minor fraction of gastrin-17 is cleaved in G cells and released as short COOH-terminal peptides (24). These peptides have been
identified in porcine and human antral tissue as a mixture of
gastrin-7, -6, and -5, of which sulfated gastrin-6 is the predominant
form released to antral venous blood (10, 22).
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The kinetics and pharmacodynamics of gastrin-52, -34, -17, and -14 have been well described (6, 8, 18, 31). Gastrin-17 is equipotent with gastrin-34 at similar plasma concentrations, and sulfation influences neither gastric acid secretion nor metabolic clearance rate (MCR) (4, 5, 8). In cats, however, short gastrin peptides have a lower potency than gastrin-17, and sulfation reduces their MCR (3, 12).
So far, human studies of gastrin-6 have not been reported. Therefore, we have now investigated the pharmacodynamics and pharmacokinetics of sulfated and nonsulfated gastrin-6 in humans. The peptides were compared with nonsulfated gastrin-17, which has been used in most studies of gastrin physiology.
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MATERIALS AND METHODS |
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Peptides
Sulfated and nonsulfated human gastrin-6 were custom synthesized by Cambridge Research Biochemicals (Zeneca; Alderley Park, UK). The content and purity of the peptides were controlled by amino acid analysis (LKB amino acid analyzer 4000 with fluorescence detection; LKB Biochrom, Cambridge, UK) and reverse-phase HPLC (Hewlett-Packard 1084 B; Hewlett-Packard, Palo Alto, CA). Synthetic human nonsulfated gastrin-17 was purchased from Sigma Chemical (St. Louis, MO).Subjects
The studies were carried out in nine healthy volunteers (6 men and 3 women, ages 22-36 yr; Table 1) without a history of medical or surgical illness. Peak acid output was determined in all subjects before the studies by intramuscular injection of 6 µg pentagastrin (Peptavlon; Zeneca). Informed consent was obtained, and the study was approved by the Ethics Committee for Medical Research in Copenhagen in accordance with the Helsinki II declaration.
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Experimental Procedures
Gastric acid secretion during infusion of nonsulfated gastrin-17,
sulfated gastrin-6, and nonsulfated gastrin-6.
Three experiments were carried out randomly in each subject on separate
days. After an overnight fast, a nasogastric tube was inserted with the
tip in the antrum under fluoroscopic control. After residual gastric
content was evacuated, gastric juice was aspirated continuously by
intermittent pump suction (Egnell) in periods of 10 min with the
subject in the supine position. The recovery of gastric juice was
determined by continuous infusion of a marker in the lateral lumen of
the tube (230 kBq 57Co-labeled cobalamin, 1.25 mg
cobalamin, and 1 g human albumin diluted in 1,000 ml isotonic
saline; 60 ml/h). The lateral lumen ended 10 cm proximal to the
openings of the central canal. The volume of gastric juice was measured
for each 10-min period, and the concentration of H+ was
determined by titration to pH 7.0 with an autotitrator (PHM 26;
Radiometer, Copenhagen, Denmark). Sterile filtrated gastrin peptides
were diluted in isotonic saline containing 1 g/l of human albumin. The
syringes were weighed before and after the infusions, and remaining
peptide was stored at 20°C until radioimmunoassay.
Metabolism of nonsulfated gastrin-17, sulfated gastrin-6, and nonsulfated gastrin-6. After termination of the infusion, the intravenous catheter was rapidly removed and blood samples were taken at 1, 2, 3, 4, 5, 10, 15, 20, 30, 45, and 60 min to determine the half-life of the peptides in plasma.
In vitro degradation of nonsulfated gastrin-17, sulfated
gastrin-6, and nonsulfated gastrin-6.
Venous blood from normal subjects who had fasted overnight was
collected in dry heparinized tubes. Known amounts of the peptides were
added to plasma and incubated at 37°C for 0, 1, 2, 4, 8, and 24 h. Then the incubation samples were immediately frozen in liquid
nitrogen and stored at 20°C until radioimmunoassay.
Laboratory Analyses
Radioimmunoassay.
Blood was collected in chilled tubes containing 50 IU of heparin and
250 µl of aprotinin (5,000 kallikrein inhibitor units) and
immediately placed on ice. After centrifugation, plasma was stored at
20°C until radioimmunoassay. All radioimmunoassay measurements were performed by previously described assays developed in our laboratory. The concentration of gastrin-17 was measured using antiserum 2604 (25, 28). This antiserum was raised against the 2-17 sequence of human nonsulfated gastrin-17 and is specific for the bioactive COOH-terminal heptapeptide. It binds both
sulfated and nonsulfated gastrin-71, -52, -34, and -17 with equimolar
potency, whereas the reactivity with the homologous hormone
cholecystokinin is <0.5% (25). Synthetic human
gastrin-17 was used as standard, and monoiodinated
125I-gastrin-17 was used as tracer (27).
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Chromatography.
Blood samples drawn at the termination of the last dose interval during
infusion of nonsulfated gastrin-17, sulfated gastrin-6, and nonsulfated
gastrin-6 were studied by gel chromatography. Plasma from all subjects
was pooled, and 1-ml samples were applied to Sephadex G-50 superfine
columns (10 × 1,000 mm; Pharmacia, Uppsala, Sweden) and eluted
with 0.125 mol/l NH4HCO3, pH 8.2, at room
temperature with a flow rate of 3 ml/h. Void volume and total volume
were determined by elution of 125I-labeled albumin and
22NaCl, and fractions of 1 ml were collected and assayed
with antisera 2604 and 2609. The elution positions,
Kd, were calculated as
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Calculations and Statistical Analysis
Correction for nonrecovered gastric juice was made for every sampling period as
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The pharmacodynamics of the peptides were evaluated by three
parameters: EC50, efficacy (maximum response), and potency.
EC50 was estimated from a Hill plot (14),
which is the logit of the secretory response vs. log plasma
concentration. The theoretical maximum acid output (Em) was
estimated by linear transformation of the Michaelis-Menten kinetics
according to Hofstee (7). This transformation has the
equation
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The pharmacokinetic analysis was made according to a one-compartment,
open model
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Results are expressed as means ± SE. Curve fitting was made from linear and nonlinear regression using GraphPad Prism (GraphPad Software, San Diego, CA). Data were analyzed using Wilcoxon's test for paired samples and Friedman's test for analysis of variance. P values <0.05 were considered significant.
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RESULTS |
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Effective Dose Rates and Gastrin Concentration in Plasma
The effective dose rates were calculated from the concentration of peptide in the infusion lines as shown in Fig. 2. During infusion, a concentration plateau was reached for all peptides within each of the four dose intervals.
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Acid Output
The acid output before infusion of nonsulfated gastrin-17 was 5.2 ± 0.7 mmol H+/50 min and increased stepwise to 15.7 ± 2.2 (P < 0.01), 26.2 ± 3.1 (P < 0.001), 25.6 ± 2.8, and 26.4 ± 2.4 [not significant (NS)] mmol H+/50 min during the four consecutive dose intervals (Fig. 2). During infusion of sulfated gastrin-6, acid output increased from 5.7 ± 0.7 to 12.5 ± 1.2 (P < 0.05), 20.1 ± 2.5 (P < 0.005), 22.3 ± 2.2, and 23.6 ± 1.7 (NS) mmol H+/50 min, and the output of nonsulfated gastrin-6 increased from 4.5 ± 0.9 to 10.6 ± 1.6 (P < 0.005), 17.3 ± 1.7 (P < 0.001), 19.6 ± 2.0 (P < 0.001), and 18.9 ± 1.9 (NS) mmol H+/50 min. Maximum and peak acid output of nonsulfated gastrin-17, sulfated gastrin-6, and nonsulfated gastrin-6 differed significantly, as shown in Table 3. The theoretical Em of nonsulfated gastrin-17 and sulfated gastrin-6 were similar, both being significantly above the Em of nonsulfated gastrin-6 (Table 3). The EC50 of nonsulfated gastrin-17 exceeded the EC50 of sulfated and nonsulfated gastrin-6 (P < 0.01); the respective concentrations in plasma estimated from the regression lines were 43 ± 6 (r = 0.91), 24 ± 2 (r = 0.92), and 25 ± 2 (r = 0.90) pmol/l (Fig. 3). LCR curves computed from EC50 and the respective Hill coefficients are shown in Fig. 4.
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Metabolism
The elimination of COOH-terminal immunoreactivity in plasma after infusion of the three peptides was monoexponential (Fig. 5). The half-life, MCR, and Vd of nonsulfated gastrin-17 were 5.3 ± 0.3 min, 16.5 ± 1.3 ml · kg
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Chromatography
Gel chromatography of plasma sampled during infusion of sulfated and nonsulfated gastrin-6 revealed peaks that eluted at the positions of the standard calibration peptides (Fig. 6).
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In Vitro Degradation of Peptides
There was a time-dependent loss of nonsulfated gastrin-17 as well as of sulfated and nonsulfated gastrin-6 after incubation in plasma. The concentrations of nonsulfated gastrin-17 declined to 85, 73, 56, 30, and 18% after 1, 2, 4, 8, and 24 h of incubation, respectively. The concentrations of sulfated gastrin-6 declined to 47, 28, 20, 12, and 4%, and the concentrations of nonsulfated gastrin-6 declined to 55, 15, 4, 2, and 1% at the same intervals (Fig. 7).
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DISCUSSION |
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This study showed that gastrin-6 has a higher potency but a lower efficacy than nonsulfated gastrin-17. The efficacy of gastrin-6, however, increased by tyrosine sulfation, which also decreased the MCR. When the theoretical Em was calculated from Michaelis-Menten kinetics of the concentration-response data, the efficacy of sulfated gastrin-6 and nonsulfated gastrin-17 was not significantly different but exceeded the Em of nonsulfated gastrin-6. Comparison between potencies were more complex because of the different slope of the LCR curve. Thus different values would emerge depending on the intensity of effect at which comparisons were made. However, the EC50 of the hexapeptides was lower than the EC50 of nonsulfated gastrin-17.
Due to the different rates of metabolism, dose rates of the peptides had to be different. Although plasma concentrations of the hexapeptides did not reach the same level as nonsulfated gastrin-17, no further increase in acid output was recorded after the third dose interval of either sulfated nor nonsulfated gastrin-6. Fading, i.e., decreased response with time, may distort dose-response studies when increasing doses are administrated consecutively without intervening rest periods. Our secretory data did not suggest fading, and peak acid output during infusion of nonsulfated gastrin-17 reached the same level as observed during the preceding pentagastrin test. Calculation of the theoretical value Em remains controversial. The method requires that concentration and response follow Michaelis-Menten kinetics, a precondition that was met in our study. But the reliability of the calculations also depends on the mathematical transformation of the Michaelis-Menten equation and the error of the data. However, it has been shown experimentally that the Hofstee transformation is acceptable even when response is subject to a considerable error (7).
Studies of small gastrin peptides have so far been restrained due to a lack of useful radioimmunoassays. Dose-response studies of tetra- and pentagastrin as well as the commercial analog of pentagastrin (Peptavlon) have previously been undertaken to examine their use in gastric function tests (21). Experiments in cats showed that the potencies of tetra- and pentagastrin were lower than the potency of nonsulfated gastrin-17. But since the results were evaluated from the respective dose rates of the peptides, the difference could be explained by the higher MCR of the small peptides (3). In another study, the efficacy of the modified hexapeptide amide fragment of gastrin-17, butyl-oxycarbonyl gastrin-6 (BOC-gastrin-6), was equal to that of nonsulfated gastrin-17, but potency was lower when circulating concentrations were taken into account (12). This observation was explained by a higher binding affinity of nonsulfated gastrin-17 to its receptor (30). Extrapolation of results between species needs caution, especially for peptide analogs. However, other studies suggest that not only the structure but also the size of the peptide may influence the secretory response. Hence, gastrin-17 has a similar efficacy and potency to gastrin-34 and -14 in humans and animals (6, 8), whereas gastrin-52 studied under conditions similar to the present study revealed a lower efficacy than nonsulfated gastrin-17 (18). Gastrin-52 is the largest progastrin product examined so far, and since the half-life is ~50 min, the concentration-response was not studied during steady state concentration in plasma. Therefore, it is possible that differences in equilibration time between plasma and receptor may have influenced the results. In the present study, steady state was achieved for all three peptides during each dose interval. With reference to earlier results, it looks as if medium-sized gastrins like gastrin-34, -17, and -14 may have a higher efficacy than peptides with either a shorter or a longer chain length.
Sulfation of gastrin peptides is another modification that we found had an effect on gastric acid output. Tyrosine O-sulfation is a common posttranslational modification of peptides and proteins, and for some peptides it is necessary for biological activity (5, 15, 26). Gastrin retains its biological activity after desulfation, and studies in both animals and humans have shown that the efficacy and EC50 of sulfated and nonsulfated gastrin-17 are similar (4, 13). However, the higher efficacy of sulfated gastrin-6 compared with its nonsulfated counterpart, as found in the present study, suggests that the biological activity of short gastrin peptides is enhanced by sulfation of tyrosine.
Gastrin-6 had a higher MCR than gastrin-17, which is in keeping with the general experience of an inverse relationship between MCR and peptide length. Whether or not sulfation of gastrin also changes, the MCR has been a subject of discussion. In one study, sulfation of gastrin-17 was found to decrease MCR (19). Other investigations, however, showed that nonsulfated and sulfated gastrin-17 had the same potency evaluated from plasma concentrations of the peptides and, therefore, the same MCR (4, 5). Studies in cats supported the conclusion (13). We found that sulfation of gastrin-6 reduced the MCR, and similar observations were made from sulfation of BOC-gastrin-6 in cats (12). But these findings may apply only to small gastrin peptides. In vitro studies have shown that sulfation of peptides protects against degradation (16, 19), as recorded in the present study with gastrin-6. But since ~50% of the immunoreactivity was still present after incubation for 1 h, enzymatic degradation in plasma does not contribute significantly to the MCR. Nonsulfated gastrin-17 had a lower MCR than gastrin-6 because of a different distribution and extraction in the vascular beds, as recently shown in pigs (unpublished observations). The difference between the MCR of sulfated and nonsulfated gastrin-6 was due to the different Vd. The half-life of the hexapeptides was not significantly different. This secondary pharmacokinetic parameter depends on the two primary parameters MCR and Vd through the expression t1/2 = (0.693 Vd)/MCR. Since the MCR and Vd both differed to the same extent with a factor of ~3, a major difference between the half-lives was not to be expected.
We conclude that gastrin-6 has a higher potency but a lower efficacy than nonsulfated gastrin-17. The efficacy of gastrin-6 was increased by sulfation, which also reduced the MCR. Since preliminary studies have identified a specific G cell enzyme active in the processing of gastrin-6, the peptide should not be regarded merely as a waste product. In spite of its modest contribution to the pool of circulating gastrin, gastrin-6 may still contribute to subtle modifications of gastric acid secretion.
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ACKNOWLEDGEMENTS |
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The expert technical assistance of Inge Mortensen and Winna Stavnstrup is gratefully acknowledged.
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FOOTNOTES |
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The study was supported by grants from the Danish Hospital Foundation, Region of Copenhagen, the Faroe Islands and Greenland, the Danish Foundation for the Advancement of Medical Science, the Danish Biotechnology Program for Signal Peptide Research, and Mogens Andreassen's Foundation.
Address for reprint requests and other correspondence: C. Palnæs Hansen, Dept. of Gastrointestinal Surgery C, Rigshospitalet, DK-2100 Copenhagen, Denmark.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 January 2000; accepted in final form 13 May 2000.
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