The metabolism of gastrin-52 and gastrin-6 in pigs
C. Palnæs
Hansen,
J. P.
Goetze,
F.
Stadil, and
J. F.
Rehfeld
Departments of Gastrointestinal Surgery and Clinical Biochemistry,
Rigshospitalet, University of Copenhagen, DK-2100 Copenhagen,
Denmark
 |
ABSTRACT |
The
kinetics and metabolism in various organs of three bioactive products
of progastrin, the small sulfated and nonsulfated gastrin-6 and the
large nonsulfated gastrin-52, were examined during intravenous
administration in anesthetized pigs. The kidney, hindlimb, liver, head,
and gut eliminated the hexapeptides efficiently, with a fractional
extraction ranging from 0.50 to 0.28 (P < 0.001-0.05). No metabolism was recorded in the lungs, and
sulfation was without influence on the extraction of gastrin-6.
Gastrin-52 was eliminated only in the kidney and the head, with a
fractional extraction between 0.23 and 0.11 (P < 0.01-0.05). The half-life of sulfated and nonsulfated gastrin-6
was 1.5 ± 0.4 and 1.4 ± 0.3 min, the metabolic clearance
rate (MCR) was 80.8 ± 7.6 and 116.0 ± 13.5 ml · kg
1 · min
1
(P < 0.05), and the apparent volume of distribution
(Vdss) was 199.3 ± 70.1 and 231.4 ± 37.3 ml/kg,
respectively. The decay of gastrin-52 in plasma was biexponential. The
half-lives of this biexponential after a bolus injection were 3.9 ± 0.5 (T1/2
) and 25.7 ± 1.4 (T1/2
) min, and the MCR and Vdss
were 4.2 ± 0.4 ml · kg
1 · min
1 and
116.2 ± 16.2 ml/kg1. We conclude that there is a
differential elimination of progastrin products in splanchnic and
nonsplanchnic tissue, which depends on the chain length of the
peptides. Sulfation of gastrin-6 had no influence on the organ-specific
extraction but reduced the MCR. Our results are in keeping with
previous studies of nonsulfated gastrin-17, which is extracted in the
kidney, head, limb, and gut but not in the liver.
gastrointestinal hormones
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INTRODUCTION |
GASTRIN IS AN
IMPORTANT gastrointestinal hormone that regulates gastric acid
secretion and the growth of gastric mucosal cells (for review, see Ref.
6). Gastrin is synthesized mainly in antroduodenal G cells. The
cellular synthesis is a complex process, during which progastrin is
processed to a number of bioactive gastrins of different length (Fig.
1). The bioactive gastrins all share the
same
-amidated COOH terminus
(Tyr-Gly-Trp-Met-Asp-Phe-NH2), of which the tetrapeptide
amide constitutes the active site (19), and the length of
the NH2 terminal extensions governs the metabolism and
clearance of circulating gastrins. Today gastrin-71, -34, -17, -14, and
-6 have been structurally identified as antral progastrin products, all in Tyr-sulfated and nonsulfated forms, and gastrin-52 has
been identified by immunochemical methods (9-12, 27,
29). In antral venous blood, gastrin-17 accounts for
>80% of postprandially released
-amidated gastrin. The remainder
is mainly gastrin-34 and -6, which are released in approximately equal
amounts, and gastrin-71 and -52 each account for <1% (13,
27).

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Fig. 1.
Major processing products of human progastrin in antral G
cells. The -amino group of COOH terminal
Gly72 of glycine-extended gastrins constitutes amide donor
for bioactive end products. All bioactive gastrins are carboxyamidated
with COOH terminal active site Trp-Met-Asp-Phe-NH2,
corresponding to sequence 68-71 of progastrin. Biosynthetic end
products exist in Tyr6-sulfated and nonsulfated forms.
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The metabolism and clearance from circulation of gastrin-34, -17, and
-14 have been well studied in humans and other mammals (2, 4, 24,
31, 32, 34-36). Moreover, the metabolism of
glycine-extended gastrin-17 has been thoroughly investigated (3,
24). Glycine-extended gastrins are the immediate precursors of
the carboxyamidated active gastrins, and they are also released from G
cells into circulation (13, 14). Under normal conditions, gastrin-17 and -34 are the predominant plasma gastrins in most mammals
(25). However, as the molecular forms in plasma change toward longer or smaller, less processed forms in diseases with increased gastrin synthesis (16), it is relevant to
examine the metabolism and kinetics of all circulating gastrin forms.
Gastrin-71, -52, and -6 are the most recently identified gastrins
(12, 27, 29). So far, gastrin-71 has not been synthesized or purified in amounts sufficient for metabolic studies. In this study
we have, therefore, examined only gastrin-52 and -6. The study was
undertaken in anesthetized pigs during separate administration of each
peptide, and the metabolic parameters were compared with those of
gastrin-17 (24).
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MATERIALS AND METHODS |
Peptides
Sulfated gastrin-6 (mol wt 897 Da), nonsulfated gastrin-6 (mol
wt 817 Da), and nonsulfated gastrin-52 (mol wt 6,018 Da) were custom
synthesized by Cambridge Research Biochemicals (Zeneca, Cheshire, UK).
Peptide content and purity were controlled in our laboratory by amino
acid analysis (LKB amino acid analyzer with fluorescence detection; LKB
Biochrom, Cambridge, UK) and reverse-phase HPLC (model 10843;
Hewlett-Packard, Palo Alto, CA). Before infusion, the peptides were
diluted in isotonic saline with 1 g/l of human albumin.
Animal Preparations
The investigations were carried out in four groups of eight pigs
(Danish Landrace-Yorkshire breed, 30-40 kg) under general anesthesia. The animals were fasted overnight but had free access to
water. The study conformed with the legal requirements for animal experiments.
Anesthesia was introduced with ketamine (8 mg/kg Ketalar;
Warner-Lambert/Parke Davis, Morris Plains, NJ) and a gas mixture of 1%
halothane (Halocarbon Laboratories) and nitrous oxide in oxygen (2:1).
After intubation, halothane was withdrawn and anesthesia was continued
with repeated doses of pentobarbital sodium (2.5 mg/kg Mebumal; Nycomed
DAK, Copenhagen, Denmark) and pancuronium (0.1 mg/kg Pavulon; Organon,
Oss, The Netherlands) as a supplement to nitrous oxide under
intermittent positive-pressure ventilation. Isotonic saline was infused
at 10 ml · kg
1 · h
1 in all
pigs during the whole experiment. In addition, donor blood from
siblings was constantly infused with a pump in volumes equal to that
removed during blood sampling. Arterial blood pressure and
electrocardiograms were continuously monitored, and cardiac output was
measured regularly with a Swan-Ganz thermodilution catheter (Swan-Ganz
pediatric 5F; Baxter Health Care, Santa Ana, CA). Carbon dioxide
tension, oxygen tension and saturation, pH, and standard bicarbonate
were measured in all vascular beds used for blood sampling (ABL 2;
Radiometer, Copenhagen, Denmark).
Catheters for blood sampling were placed as follows. Polyethylene
catheters were positioned in the thoracic aorta via the left carotid
artery and cephalically in the left internal jugular vein. Via the
external jugular veins, two angiography catheters were introduced into
a major hepatic vein and the left renal vein. Blood from a
peripheral lung artery was drawn from the Swan-Ganz catheter. The
positions of all catheters were controlled fluoroscopically. Neither
the hepatic nor the pulmonary catheters were wedged, to avoid mixing
with portal and pulmonary venous blood when samples were drawn. After a
midline laparotomy, a catheter was introduced into the entrance of the
portal vein via the splenic vein and another catheter was positioned in
one of the major tributaries of the mesenteric vein draining the small
intestine. Blood from the femoral vein was drawn from an inserted
catheter after exposure of the vessels in the groin. Peptides and
indicator substance were infused into catheters with their tips placed
in the right atria. After completion of surgery, a stabilization period
of 1 h was allowed before start of the experiments.
Blood flow in the renal and femoral artery and the portal vein was
measured with an electromagnetic flowmeter (Nycotron, Oslo, Norway),
and pulmonary flow was regarded as equivalent to the cardiac output.
Blood flow was converted to plasma flow from the hematocrit. Hepatic
plasma flow was measured by continuous infusion of indocyanine green
(Cardiogreen; Becton Dickinson, Cockeysville, MD) at 0.130 µmol/min.
After a calibration period of 90 min, simultaneous samples from the
aorta and the hepatic vein were drawn at 10-min intervals. The
concentration of indocyanine in plasma was measured spectrophotometrically, and hepatic plasma flow was calculated according to Fick's principle as
where Q is dose rate, BW is body weight, Ca and
Cv are plasma concentrations in the aorta and the hepatic
vein, and dCa/dt and
dCv/dt are the linear regressions of the
arterial and venous concentrations with time (t).
Experiments
Elimination of sulfated and nonsulfated gastrin-6.
The elimination of the peptides was measured from the concentration
gradient across the vascular beds during separate infusion of sulfated
gastrin-6 (460 pmol · kg
1 · h
1) and
nonsulfated gastrin-6 (600 pmol · kg
1 · h
1) in two
groups of eight pigs. The infusion rates were determined from previous
pilot studies. Arterial samples were taken from the aorta every 15 min
during infusion. After 90 min, venous samples were drawn every 15 min
for 1 h from a lung artery and the femoral, renal, portal,
hepatic, internal jugular, and cranial mesenteric veins. The cranial
mesenteric vein is almost equivalent to the superior mesenteric vein in
humans and drains the major part of the blood from the small intestine
and the first part of the large intestine. When the infusion of the
peptides was terminated after 170 min, aortic blood was sampled at
regular intervals for 90 min to determine the half-life of the peptides.
Elimination of gastrin-52.
Gastrin-52 was infused at 12 pmol · kg
1 · h
1 in eight
pigs; the experimental design was otherwise similar to the study of
gastrin-6. Because the disappearance of gastrin-52 from plasma follows
a two-compartment model, the kinetic parameters were also calculated after a bolus injection (5 pmol · kg
1 · h
1) in another
group of eight pigs. Arterial blood samples were drawn at regular
intervals until 90 min from the time of the injected bolus.
In vitro metabolism and recovery of gastrin-6 and -52.
The metabolism of peptides in plasma was evaluated by separate
incubation of sulfated and nonsulfated gastrin-6 as well as gastrin-52
in porcine plasma at room temperature for 0, 1, 2, 4, 8, and 24 h.
After incubation, the samples were immediately frozen in liquid
nitrogen and stored at
20°C until analysis.
Laboratory Analyses
Radioimmunoassay.
Blood samples were collected in ice-chilled tubes containing 50 IU
heparin and 250 µl aprotinin (5,000 KIU) and placed on ice. After
centrifugation, plasma was stored at
20°C until
radioimmunoanalysis. All radioimmunoassay measurements were performed
with previously described assays developed in our laboratory.
All samples of each experiment were performed in a single assay.
Antiserum 2604 measures total concentration of carboxyamidated gastrins
larger than the hexapeptides (30). The antiserum was
raised against the
-amidated 2-17 fragment of human gastrin-17 and is directed against the COOH terminal part of the peptide. The
antiserum binds gastrin-71, -52, -34, and -17 with equimolar potency;
the reactivity with CCK is <0.5%. Synthetic human gastrin-17 was used
as standard, and monoiodinated 125I-gastrin-17 was used as
a tracer (26). The accuracy of the assay was evaluated by
the recovery of known amounts of peptide added to plasma. The precision
(intra-assay variation) was expressed by the coefficient of variation
of repeated measurements of peptide in plasma at three different
concentrations. The accuracy ranged from 84 to 102%, and the precision
was 5%.
Sulfated and nonsulfated gastrin-6 were measured with antiserum 2609 after extraction of the peptides from plasma. Antiserum 2609 was also
raised against the 2-17 fragment of human nonsulfated gastrin-17
(28). In contrast to antiserum 2604, which requires an
epitope of seven residues or more for binding, antiserum 2609 requires
only the COOH terminal tetrapeptide amide sequence for binding. The
reactivity is 63% with gastrin-34 and 20% with CCK-8. One milliliter
of plasma was extracted with 2 ml of 96% ethanol, and the supernatant
was evaporated under a constant air flow. The dry extracts were then
reconstituted in assay buffer. Sulfated and nonsulfated gastrin-6 were
used as standards, and monoiodinated 125I-gastrin-17 was
used as a tracer. Measurements of sulfated gastrin-6 had an accuracy of
91-98% and a precision of 1-8%. For nonsulfated gastrin-6,
the accuracy ranged from 86 to 95% and the precision ranged from 4 to
6%.
Gastrin-52 was measured using antiserum 88235, which was raised against
fragment 20-33 of human progastrin that corresponds to the
NH2 terminus of gastrin-52 (29). The antiserum
is specific for the sequence 20-25 of progastrin. The antiserum
does not bind gastrin-34, -17, or smaller carboxyamidated gastrins, nor
does it react with CCK. Synthetic human gastrin-52 was used as
standard, and monoiodinated 125I-gastrin-52 was used as a
tracer (26). The accuracy of measurements ranged from 80 to 94%, and the precision was 20%.
Chromatography.
The elution profile of each peptide was determined by means of gel
chromatography. Plasma from eight pigs of each group was pooled, and
1-ml samples were applied to Sephadex G-50 superfine columns (10 × 1,000 mm and 12 × 2,000 mm; Pharmacia, Uppsala, Sweden). The
short columns were eluted with 0.125 M NH4HCO3
(pH 8.2) with a flow rate of 4 ml/h and calibrated with sulfated and nonsulfated gastrin-6. The long columns were eluted like the short columns but with a flow rate of 3 ml/h and calibrated with gastrin-52. Void volume and total volume were determined by elution of
125I-albumin and 22NaCl. Fractions of 1 ml were
obtained and analyzed with antisera 2604, 2609, and 88235. The elution
position (Kd) was calculated as
where Ve is the elution volume of the peptides and
Vo and Vt are elution volumes of
125I-albumin and 22NaCl, respectively.
Calculations
The fractional extraction of the peptides in the vascular beds
was calculated as
where Ci and Co are inflow and outflow
concentrations in plasma. The fractional extraction in the liver was
grossly estimated from the difference between the inflow concentrations
in arterial (Ca) and portal (Cp) plasma and the
outflow concentration in hepatic venous plasma (Ch) and the
respective flows (F)
Arterial flow was estimated by subtraction of portal flow from
total liver flow. Clearance of the peptides at the metabolic sites was
calculated by multiplying extraction ratio with plasma flow.
The kinetics of sulfated and nonsulfated gastrin-6 were calculated
according to a one-compartment open model
where Co and Ct are plasma
concentrations at zero time and t. The half-life was
determined from the elimination in arterial plasma after termination of
the infusion. Plasma concentrations were plotted on semilogarithmic
graph paper after subtraction of basal values. Linear regression of the
logarithm of plasma concentration vs. time was computed to yield the
slope (ke), from which the half-life was
calculated by dividing into 0.693. The metabolic clearance rate (MCR),
which is total body clearance, was calculated from dose rate divided by
the plateau increment of peptide in plasma. The apparent volume of
distribution (Vd) was determined by dividing MCR with
ke of the regression line for disappearance.
The kinetics of gastrin-52 were analyzed according to a two-compartment
open model
where A and B are the zero intercepts with
the ordinate of the individual exponential terms and
and
are
the slopes of the curves. When a substance displays two- or
multicompartment kinetics, prolonged infusion may load the deep
compartment with the substance, so that the postinfusional plasma curve
shows little distributional activity and is of limited analytic value.
Therefore, the parameters were calculated from postinfusional data and
from the decay in plasma after a bolus injection. When model parameters were calculated from postinfusional data, the values of A
and B, which would have followed a single intravenous dose
of the same magnitude, were calculated from the zero intercepts
R and S at the start of the postinfusional period
where T is the period of infusion (18).
Area under the curve (AUC) was determined as
During constant-rate infusion, the MCR was calculated the same
way as for gastrin-6. After bolus injection, MCR was calculated from
injected dose divided by AUC. The apparent volume of distribution at
steady state (Vdss) was calculated as the sum of the
central and peripheral compartment volumes. The rate constants of the model and the volumes of the central and peripheral compartments (V1 and V2) were calculated as
where k12 and k21
are the first-order rate constants of distribution between the central
and peripheral compartment and k10 is the sum of
the simultaneous processes of metabolism and excretion.
All results are expressed as means ± SE. Statistical analysis was
performed by Wilcoxon's test for paired samples and the Mann-Whitney
test for unpaired samples. P values <0.05 were considered significant.
 |
RESULTS |
Elimination of Sulfated and Nonsulfated Gastrin-6
The concentrations of the peptides during the studies are shown in
Fig. 2. The corresponding dose rates of
sulfated and nonsulfated gastrin-6 estimated from the concentrations in
the infusion lines were 462.0 ± 20.4 and 597.9 ± 53.5 pmol · kg
1 · h
1,
respectively.

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Fig. 2.
Concentration (means ± SE) in arterial plasma
during and after infusion of sulfated gastrin-6 (460 pmol · kg 1 · h 1) and
nonsulfated gastrin-6 (600 pmol · kg 1 · h 1) in 2 groups of 8 pigs.
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Extraction of sulfated gastrin-6 was recorded over the kidney
(P < 0.01), liver (P < 0.01), gut
(P < 0.01), hindlimb (P < 0.01), and
head (P < 0.01), whereas pulmonary extraction was not significant (Fig. 3; Table
1). Extraction of nonsulfated gastrin-6 was observed over the kidney (P < 0.01), liver
(P < 0.05), gut (P < 0.01), hindlimb
(P < 0.01), and head (P < 0.01) but
not over the lungs (Fig. 4; Table 1). The
fractional extraction of sulfated and nonsulfated gastrin-6 was not
significantly different.

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Fig. 3.
Plasma concentrations (means ± SE) of sulfated
gastrin-6 during infusion of the peptide (460 pmol · kg 1 · h 1) in 8 pigs.
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Fig. 4.
Plasma concentrations (means ± SE) of nonsulfated
gastrin-6 during infusion of the peptide (600 pmol
kg 1h 1) in 8 pigs.
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The kinetics of sulfated and nonsulfated gastrin-6 followed a
one-compartment open model (Fig. 5). The
MCR, half-life, and Vd of sulfated gastrin-6 were 80.8 ± 7.6 ml · kg
1 · h
1,
1.5 ± 0.4 min, and 199.3 ± 70.1 ml/kg, respectively. For
nonsulfated gastrin-6, the values amounted to 116.0 ± 13.5 ml · kg
1 · h
1
(P < 0.05), 1.4 ± 0.3 min (not significant), and
231.4 ± 37.3 ml/kg (not significant), respectively. The
hemodynamic values measured during the experiments and the calculated
clearance of the peptides at the metabolic sites are presented in
Tables 2 and
3.

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Fig. 5.
Elimination of sulfated (A) and nonsulfated
(B) gastrin-6 in arterial plasma after termination of
infusion of the peptides in 2 groups of 8 pigs.
ke, Slope.
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Elimination of Gastrin-52
Plasma concentrations of gastrin-52 during the study are shown in
Fig. 6. The corresponding dose rate of
the peptides was 11.7 ± 1.7 pmol · kg
1 · h
1.
The fractional extraction of gastrin-52 was significant in kidney (P < 0.01) and head (P < 0.05) but
nonsignificant in the liver, gut, hindlimb, and lung (Fig.
7; Table 1). The extraction in kidney and
head was lower than of sulfated and nonsulfated gastrin-6 (P < 0.01 and 0.05, respectively).

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Fig. 6.
Concentration (means ± SE) of gastrin-52 in
arterial plasma during and after infusion of the peptide (12 pmol · kg 1 · h 1) in 8 pigs
with the use of antiserum 88235.
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Fig. 7.
Plasma concentrations (means ± SE) of gastrin-52
during infusion of the peptide (12 pmol · kg 1 · h 1) in 8 pigs
with the use of antiserum 88235.
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The kinetics of gastrin-52 followed a two-compartment open model (Fig.
8). When the pharmacokinetic parameters
were calculated from postinfusional data, the half-lives of the
biexponential, T1/2
and
T1/2
, were 4.3 ± 0.8 and 29.3 ± 3.1 min, and the MCR and Vdss were 3.1 ± 0.2 ml · kg
1 · min
1 and
98.5 ± 9.4 ml/kg, respectively. When the pharmacokinetics were
calculated from plasma concentrations after a bolus injection (5.3 ± 0.2 pmol/kg), the half-lives were 3.9 ± 0.5 (T1/2
) and 25.7 ± 1.4 min
(T1/2
), and MCR and Vdss
amounted to 4.2 ± 0.4 ml · kg
1 · min
1
and 116.2 ± 16.2 ml/kg, respectively. The kinetics of the two peptides were not significantly different. The respective fractions of
elimination during the
-phase,
[(B/
)/AUC], were 0.75 and 0.71, which implies that the
major part of the elimination took place during the
-phase.
Hemodynamic values measured during the investigations and the
calculated clearance of the peptides at the metabolic sites are
shown in Tables 1 and 2.

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Fig. 8.
Elimination of gastrin-52 in arterial plasma after
termination of constant-rate infusion (12 pmol · kg 1 · h 1;
A) and after bolus injection (5 pmol/kg; B) in 2 groups of 8 pigs. The 2-compartment model calculated from
postinfusional data had volumes of central (V1) and
peripheral (V2) compartments, respectively, of 46.6 ± 6.6 and 51.9 ± 12.1 ml/kg, and and rate constants of
k12 = 0.098 ± 0.034, k21 = 0.068 ± 0.006, and
k10 = 0.073 ± 0.012 pmol/min. The
respective values after a bolus injection were V1 = 57.2 ± 6.0 and V2 = 59.0 ± 12.5 ml/kg and
k12 = 0.076 ± 0.012, k21 = 0.073 ± 0.009, and
k10 = 0.075 ± 0.005 pmol/min. In this
instance, k12 and k21 are
the first-order rate constants of distribution between the central and
peripheral compartments, and k10 is the sum of
the simultaneous processes of metabolism and excretion.
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In Vitro Degradation of Progastrin Products
The recovery of sulfated and nonsulfated gastrin-6 during the
first 8 h of incubation in plasma ranged from 90 to 97%.
Gastrin-52 was stable for 4 h when immunoreactivity was measured
with the NH2 antiserum. After 8 and 24 h, the
immunoreactivity in plasma had decreased to 85 and 68%, respectively.
There was no reduction in COOH terminal immunoreactivity during 24 h of incubation.
Chromatography
Gel chromatography of arterial plasma during infusion of sulfated
and nonsulfated gastrin-6 revealed a peak at Kd
of 1.26 and 1.12, which corresponds to the elution positions of the
standard calibration peptides (Fig. 9).
Chromatography of gastrin-52 in arterial plasma revealed one major peak
at a Kd of 0.29, which is the elution position
of the intact peptide. This peak was visible with the NH2
terminal (antiserum 88235) and the COOH terminal antibody
(antiserum 2604) (Fig. 9). Smaller NH2 or COOH terminal fragments were measured neither in arterial nor in venous plasma from
the metabolically active sites in head and kidney.

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Fig. 9.
Gel chromatography of arterial plasma from pigs during
infusion of sulfated or nonsulfated gastrin-6 (A) and
gastrin-52 (B). Pooled samples of plasma from pigs who had
either sulfated or nonsulfated gastrin-6 were applied to Sephadex G-50
superfine columns (10 × 1,000 mm) and eluted with 0.125 M
NH4HCO3 (pH 8.2). Eluates were assayed with
antiserum 2609. Pooled samples from pigs who had gastrin-52 were
applied to Sephadex G-50 superfine columns (12 × 2,000 mm), and
eluates were assayed with antiserum 88235. Columns were calibrated with
125I-albumin (Vo), 22NaCl
(Vt) as well as sulfated and nonsulfated gastrin-6 and
nonsulfated gastrin-52 (Ve). (Ve Vo)/(Vt Vo) is the elution
constant.
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 |
DISCUSSION |
The present study revealed a differential elimination of bioactive
gastrins in organs as well as in nonsplanchnic tissue. Previous studies
have shown that the major biosynthetic endproduct of progastrin,
gastrin-17, is eliminated at multiple sites in the body (4, 24,
32). These observations led to the conclusion that gastrin is
cleared in all major vascular beds. However, the present results
suggest that this conclusion needs modification, since we found that
the metabolic sites for bioactive gastrin varied according to the
molecular size of the peptides. A differential metabolism of gastrin as
well as other gastrointestinal peptides has so far only been recorded
in the liver (7, 8, 34), but the present study also
demonstrated a differential extraction in the gut and hindlimb. Only
the head and kidneys were capable of eliminating gastrin peptides
independent of their size but with a higher fractional extraction for
small peptides.
An arteriovenous concentration gradient has been measured in the heads
of different animals during infusion of gastrin-17 (4, 20,
32). In pigs, blood from the brain and the musculoskeletal part
of the head have a common drainage into the internal jugular vein.
Although selective venous samples from the brain were not obtained in
this study, two observations are in favor of a cerebral gastrin
metabolism. One is an abundant expression of the
gastrin/CCKB receptor in the brain (17). The
other is the selective extraction of the hexapeptides and gastrin-52 in
the musculoskeletal system (the hindlimb), whereas gastrin-6 and -52 were both extracted in the head.
The kidneys have been regarded as important sites of extraction of
circulating peptides. The glomerular filtration of gastrin is
influenced by the Donnan effect, with an enhanced filtration of
negatively charged peptides (21, 23, 33). After glomerular filtration, the peptides undergo an almost complete tubular absorption and metabolism, since excretion of intact peptides in urine accounts for <1% of the filtered amount. Since most gastrins, including gastrin-34, -17, and -6, are eliminated at several sites in the body
with an integrated clearance exceeding that of the kidneys, renal
contribution to the MCR seems only to be of major importance for large
gastrins like gastrin-52 and, probably, gastrin-71. However,
hypergastrinemia found in some patients with renal failure is only
partially explained by low renal clearance of gastrin. An increased
release of gastrin caused by hypo- or achlorhydria and hypercalcemia in
combination with a decreased metabolism in other vascular beds are more
likely explanations. Moreover, hemodialysis may contribute to the
complexity of gastrin physiology in these patients (20).
The role of the liver in the metabolism of gastrointestinal peptides
has been debated because of the number of conflicting results. Animal
experiments in pig, dog, and rat with infusion of gastrin and CCK
fragments of different chain length have shown that peptides with eight
or fewer amino acid residues are subject to hepatic extraction, whereas
larger peptides are increasingly resistant to elimination in the liver
(5, 7, 8, 34). The present results confirm these
observations, since only the hexapeptides were cleared in the liver.
Earlier investigations by us (24) failed to demonstrate
elimination of human gastrin-17 in the porcine liver, but hepatic
elimination of postprandially released gastrin-17 has been recorded in
pigs in two independent studies (3, 22). Since human
gastrin-17 has been used in most animal studies of gastrin, the
conflicting results could result from the species difference of the
peptide. Despite the close structural similarity of gastrin-17 among
mammalian species, only gastrin-6 is identical in animals and man,
whereas larger gastrins differ with increasing chain length. Studies of
the CCK octapeptide, which bears a close resemblance to gastrin-6, have shown that hepatic extraction is sensitive to even minor modifications of the molecule (8).
Besides the liver, both the gut and hindlimb displayed a differential
elimination of gastrin with extraction of the hexapeptides, whereas
gastrin-52 passed unhindered. Since clearance of gastrin-17 has also
been recorded in the hindlimb of pigs (24), the
musculoskeletal system, therefore, represents the major metabolic site
of short- and medium-sized gastrin peptides.
The lungs were the only site without a significant gastrin elimination,
and earlier studies in pigs also failed to demonstrate pulmonary
extraction of gastrin-17. However, a small pulmonary extraction of
gastrin-17 of ~10% has been measured in sheep (4). This
means that the lungs in sheep are responsible for the metabolism of as
much as 50% of circulating gastrin and, therefore, represent the major
metabolic organ in gastrin metabolism. However, even small variations
in the measurement of venous and arterial concentrations may lead to a
false high clearance due to the high pulmonary blood flow.
Degradation of the peptides in vitro was slow, since gastrin-52 and -6 retained their immunoreactivity for several hours after incubation in
plasma. Metabolic products of gastrin-52 in plasma were recorded
neither in vitro nor in vivo, probably due to low concentrations of the
intact peptide.
Derivatization of gastrin peptides increases their resistance to
metabolism. Thus amidation of the COOH terminal carboxy group protects
against carboxypeptidases. This was evident from incubation of
gastrin-52 in plasma, which resulted in a decreased NH2
terminal immunoreactivity, whereas the COOH immunoreactivity was
retained after 24 h. Also, sulfation of gastrin enhances
resistance to metabolism, but this may only apply to small peptides. In
humans, the MCRs of sulfated and nonsulfated gastrin-17 were found to be approximately the same (1), whereas MCR of sulfated
hexagastrin in cat was nearly sixfold higher than the nonsulfated form
(15). In the present study, sulfated gastrin-6 had a 30%
lower MCR than the nonsulfated form. Apart from derivatizations, the
MCR also depends on the chain length of the peptides. Thus the higher
MCR of gastrin-6 compared with gastrin-52 is in accordance with the general experience that clearance of peptides varies inversely with
chain length. There was a good agreement between the MCR of gastrin-52
and the integrated organ clearance, but a considerable difference was
recorded for gastrin-6, also when an estimation for the clearance by
the trunk was made. A similar discrepancy was present in metabolic
studies of the octapeptide of CCK in pigs (5). A false
high value of the MCR may result from either intravascular degradation
or from sequestration of the small peptides at extravascular sites.
This would also lead to a high Vd, which for sulfated and
nonsulfated gastrin-6 was twice as high as Vd for
gastrin-52 and -17 (24).
We conclude that the differential elimination of gastrin found in pigs
is related to chain length of the peptide. A selective elimination was
recorded in most vascular beds. Only the kidney and the brain displayed
a nonselective extraction of gastrin.
 |
ACKNOWLEDGEMENTS |
The expert technical assistance of Inge Mortensen, Winna
Stavnstrup, Letty Klarskov, Mette Olesen, and Sørn Haagen Nielsen is
gratefully acknowledged.
 |
FOOTNOTES |
The study was supported by grants from the Danish Hospital Foundation
for Medical Research, Region of Copenhagen, the Faroe Islands, and
Greenland, and the Danish Foundation for the Advancement of Medical Science.
Address for reprint requests and other correspondence: C. Palnaes 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. §1734 solely to indicate this fact.
Received 21 December 1999; accepted in final form 30 March 2000.
 |
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