Metabolism of heptadecapeptide gastrin in humans studied by region specific antisera

Metabolism of heptadecapeptide gastrin in

humans studied by region-specific antisera.

S Pauwels, … , R Walker, S Marcus

J Clin Invest.

1985;

75(6)

:2006-2013.

https://doi.org/10.1172/JCI111919

.

The metabolism of synthetic human heptadecapeptide gastrin (G17) in vivo, and in serum in

vitro, was studied by radioimmunoassay using region specific antisera, gel filtration, ion

exchange chromatography, and high performance liquid chromatography. After infusion of

G17 intravenously in normal human volunteers, COOH-terminal and NH2-terminal

immunoreactive G17 fragments were generated. At a steady state, approximately 15% of

COOH-terminal immunoreactivity was attributable to G14-like material and up to 25% of

total NH2-terminal immunoreactivity was attributable to two NH2-terminal fragments; one

had the chromatographic properties of 1-13 G17, and the other was less acidic and less

hydrophobic. After stopping the infusion of G17, the latter fragments accounted for

progressively greater proportions of total gastrin activity. When G17 was incubated in serum

in vitro, there was time-dependent and temperature-dependent loss of immunoreactivity,

and again COOH-terminal and NH2-terminal immunoreactive fragments were formed.

Removal of the NH2-terminal pyroglutamic acid was probably the rate limiting step because

synthetic 2-17 G17 was degraded more rapidly in serum (t1/2, 2-3 h) than G17 (t1/2, 3-5 h).

EDTA blocked degradation at the COOH-terminus of both 2-17 G17 and G17 but cleavage

at the NH2-terminus still occurred, giving rise to a G14-like peptide. The rate of conversion

of G17 in serum was not enough to account for the production of fragments in vivo, and it is

proposed that these are […]

Research Article

Metabolism

of

Heptadecapeptide

Gastrin

in

Humans

Studied by Region-specific

Antisera

S. Pauwels, G. J. Dockray, R. Walker, and S. Marcus

Medical Research CouncilSecretoryControl Research Group, Physiological Laboratory, UniversityofLiverpool,

Liverpool, United Kingdom, andGastrointestinal Unit, Walton Hospital, Liverpool, UnitedKingdom

Abstract

The metabolism of synthetic human heptadecapeptide gastrin (G17) in vivo, and in serum in vitro, was studied by radjoim-munoassay using region specific antisera, gel filtration, ion

exchangechromatography, and high performance liquid

chro-matography. After infusion of G17 intravenously in normal

human volunteers, COOH-terminal and NH2-terminal immu-noreactive G17 fragments were generated. At a steady state, -15% ofCOOH-terminal immunoreactivity was attributable

to G14-like material and up to 25% of total

NH2-terminal

immunoreactivity was attributable to two NH2-terminal frag-ments; onehadthe chromatographic properties of1-13 G17, and the other was less acidic and less hydrophobic. After

stopping the infusion of G17, the latter fragments accounted

for progressively greater proportions oftotal gastrin activity. When G17 was incubated in serum in vitro, there was time-dependent andtemperature-dependent loss ofimmunoreactivity, andagain COOH-terminal and NH2-terminalimmunoreactive fragments were formed. Removal of the

NH2-terminal

pyroglu-tamicacid was probably the rate limiting step because synthetic 2-17 G17 was degraded more rapidly in serum

(tl/2,

2-3 h) than G17

(t1/2,

3-5 h). EDTA blocked degradation at the COOH-terminus of both 2-17 G17 and G17 but cleavage at

the NH2-terminus still occurred, giving rise to a G14-like peptide. The rate of conversion of G17 in serum was not enoughtoaccountfor the production of fragments in vivo, and it is proposed that these are formed when G17 encounters enzymesoncell surfaces, perhaps during passage through the capillarycirculation. The production of these fragments needs

tobeconsidered in interpreting studies of the identity, metab-olism, andrelease of gastrin in health and disease.

Introduction

Thepredominant form of theacid-stimulatinghormonegastrin that occurs in antral mucosa is the heptadecapeptide, G17'

Dr. Pauwel's present address is Department of Nuclear Medicine, University of Louvain MedicalSchool, B-1200 Brussels, Belgium.

Address correspondence to Professor Dockray, The Physiology Laboratory, University of Liverpool, P.O. Box 147, Brownlow Hill,

Liverpool, L69 3BX, U.K.

Receivedforpublication23September 1983 and in revised form 6 February 1985.

1.Abbreviations used in thispaper:G14, COOH-terminal tetradeca-peptide fragment; G17, heptadecatetradeca-peptide gastrin; 2-17 G17,

hexa-decapeptide;HPLC, high performance liquid chromatography.

(1-4). This molecule is rapidly cleared from the circulation witha half-life of5-11 min in man, but littleisknown of the

metabolic pathways involved and in particular whether or not it might be converted to shorter fragments (5-7). The latter would be biologically active if they included the

COOH-terminal tetrapeptide amide sequence, which is the minimal active fragment. We recently obtained preliminary evidence

to indicate thatGI7mightbedegraded by plasmaenzymesto

fragments with COOH-terminal immunoreactivity (8). We have now used radioimmunoassay

(RIA)

employing a series of region specific antisera together with chromatographic

sep-aration by gel filtration, ion exchange chromatography, and

high performance liquid chromatography (HPLC), to study the forms producedin vivo whenG17is infusedintravenously and in vitro when G17 is incubated in serum. The results provide evidencefortheproductionof both COOH- and

NH2-terminal fragments of G17. One of theNH2-terminal immu-noreactivefragmentshas arelatively longhalf-life and becomes

the predominant molecular species after stopping infusions

of G17.

Methods

Peptides. Synthetic hG17, 2-17hG17, and 1-13 hG17werepurchased from UCB Bioproducts (Brussels, Belgium).

Subjects. Studies were made on five normal male subjects (aged

22-40 yr) whowere members of the laboratoryorclinicalstaff, and

werewithout gastrointestinal disease.All gaveinformedconsent tothe

experiments, whichwereapproved bythelocalethical committee, Experimentalprocedures. Two types of experiments were carried

out. First, metabolism in vivo was studied during and after an

intravenousinfusion of G17. Second, the metabolism of G17and 2-17G17 invitrowasstudiedafter their addition toserumor

EDTA-treated plasma.

In vivometabolism. Subjects fasted from midnight before the test.

An intravenous infusion of 0.15M NaCl was given for a 45-min

control period at 42 ml*h-, followed by synthetic hG17 (400

pmol .kg-'-h') foranother 60_{min.After the infusion, the contents}

of the syringe were removed and stored at -20°C before further

analysis. In control experiments, it was established that there was

negligible adsorption of G17 to the infusion lines in the present

experimental conditions.

Duringeach test, venousbloodwas drawnfromthe arm opposite

that used for infusion. Degradation of peptides by plasma enzymes was inhibited by mixingthe blood immediately in tubescontaining

EDTA and 1:10o-phenanthroline,togive final concentrations of2.7 and 5 mM, respectively. After centrifugation, plasma wasstored at

-20°C beforeRIAorfractionation procedures. Blood samples of5 ml weretaken every 15 minthroughout the test and at 5-minintervals

duringthefinal 15minof infusion.Concentrations of immunoreactive

gastrin in the final three samples were not significantly different,

indicatingthat asteady statehad been attained; these samples were

pooled for chromatography. After the infusion, samples were taken

everyminutefor5 min,andthenevery 5 minfor 30min.

In vitro metabolism. After an overnight fast, venous blood was J.Clin. Invest.

©TheAmericanSociety for Clinical Investigation, Inc.

hG 17 InfusIon (400 pmol-kg-'-

h-1)

STOP INFUSION Ab LW1

0-0 Ab LS

a-- _{Ab LW 11}

TIME (min)

Figure 1. Meanplasma

concentrationof intact

G17 measured by

anti-body L6 (-o-), COOH-terminal

(-*-),and

NH2-termi-nal (--A--) G17 im-munoreactivity measured byLW1 andLW11,

re-spectively, duringand

af-100 terinfusion ofhG17(400

pmol-kg-'-h-')infive normal subjects.Each

point is mean±SE.

collected in aseptic conditions into sterile dry tubes or in tubes containing 2.7 mM EDTA. The bloodwascentrifugedat40C(2,000

g, 10min)andserum orplasmawasseparated. Synthetic hG17,orits

COOH-terminal hexadecapeptide (2-17 G17),wasaddedtothe samples and incubatedat4,20,or370C for 0,6, 12, and 24 h. After incubation,

thesampleswereimmediately frozen in liquid nitrogen and storedat -80'C beforeRIA orfractionation by gel filtration orion-exchange

chromatography.

Columnchromatography. Serumorplasma sampleswere

fraction-ated bygel filtration, ion-exchange chromatography, and HPLC. To obtainenough material for characterization, plasma samples collected during the final 15 min of the infusion of hG 17wereseparately pooled for each subjecttogive volumes of 15-20 ml. In someexperiments, plasma samples from the five subjects were pooled together for chromatography.Foursuchsamplesof 20 mlwereseparatelyprocessed:

(a) plasma collected just before the end of the infusion; (b) plasma collected2and 3 min afterstopping infusion; (c) plasma collected 5, 10, and 15 min after infusion; and (d) plasma collected 20, 25, 30, and 35min after infusion.

Gelfiltration. Samples were appliedto Sephadex G-50 superfine

columns(1 X 100cm) elutedwith0.05 Mphosphate, pH7.5 at4VC

withaconstantflowrateof 6 ml * h-'. Traceamountsof '251-Nawere

added as a calibration marker; in addition, the void volume was

routinely identified by the elution position of plasma proteins detected byODat280nm.Columnswerealso calibratedonseparate occasions

withsynthetic standard hG 17 and 1-13 hG 17.

Ion-exchange chromatography. Samples were applied to DEAE

columns (1 X 15 cm) with trace amountsof 125I labeled hG17 as a

marker. After equilibration with 0.6% ammonium carbonate at4VC, thecolumnswere eluted withagradient from 0.6to6% ammonium

carbonate using a 50-ml mixing vessel and a constant flow rate of 10 ml/h.

Reversephase HPLC. Material from DEAE column eluates was

separated by reversed-phase HPLC on a Techsil C18 5Am (5 X 250 mm) column using an Altex system. The samples were reduced in

volume in a Speed-vac (Savant Instruments, Inc., Hicksville, NY)to

3-6 ml and pH was brought below 7.0 with trifluoroacetic acid.

Elutionwasmade withagradient of acetonitrile in 0.05%trifluoroacetic

acid.Inseparaterunsthecolumnswerecalibrated with 1-13 G 17 and

synthetic hG 17 in total quantities comparable to those in the test

samples. Column eluates (I ml) were collected into 0.1 ml of 1 M

ammonium bicarbonate;recovery was90-100%.

Gastrin RIA. Gastrin wasestimated by RIA using three typesof region specific antisera: (a) antiserum L6, which is in practice absolutely specific forG17 (9), (b), antiserum LW1, which was raisedto hG17

conjugated to bovine serum albumin (BSA) and is COOH-terminal specific (10), and (c) antiserumLWl1,whichwasraisedto 1-13hG17 conjugatedtoBSA,andisNH2-terminal specific (10).

251I-labeledsynthetic hG17wasusedfor RIA(11, 12).Thepeptide wasiodinated by thechloramine-T method and the reaction mixture was purified on AEcellulose. Incubation and separation conditions were as previously described (8, 10). With all antisera, assays were

carried out using synthetic hG171 asstandard. The concentration of

hGl71 for halfmaximal inhibition of binding was 2 fmol ml-' for

Table I. MetabolismofhGI7ImmunoreactivityinFive NormalSubjects

(Mean±SE)

InfusedwithhGJ7Measuredwith Antibodies L6, LWI, andLWIJ

Antisera hG17Infusionrate Plasmaplateau level Half-life Metabolicclearancerate Volume ofdistribution pmol-kg-]'*h- pmol-1` min ml-kg-' min-' ml-kg]

L6 396±6 617±47 3.23±0.17 10.97±0.91 51.39±5.80

LW1 389±11 691±34 4.88±0.08 9.52±0.68 67.08±4.90

LW1 406±10 815±49 11.71±0.60 8.44+0.57 143.30±14.17

1.

1.0.

0.8-' 0.6

0.4

o

I-4 w

0

z

n r

n

z

I--4c

to 4c -1

LW1, 1 fmol-_{mlP' for LWI 1, and 2.5 fmol *}ml-' for L6. Inassaysin

which >20,lof plasma was added to the tube (up toamaximum of 100Ml),the standard curves wereprepared withanappropriate volume of plasma stripped of gastrin. Thedetection limit ofgastrin immuno-reactivity in plasma ranged from 3 to 10 pmol I_{depending onthe}

antiserum.

Analysis of the data.Allresultsareexpressedasmean±SEM,and

theinfusiondatais analyzed here accordingtothe plateauprinciple.

Plasma steady-stategastrin concentrations were taken as the mean of values obtained during the final 15 min of infusion ofhGl7. The plasma gastrinconcentrations after stoppingtheinfusionwereconverted to the naturallogarithm, and linearregression ofInplasmagastrinvs.

time was computed toyield the slope, from which the half-life (t1/2)

was determined bydividing into 0.693. The metabolic clearance rate, expressed in ml kg-' min-', was calculated bydividingthe infused

dosein pmol *kg-'*min-',by theplateau increment inplasma gastrin in pmol * 1-'. The apparent volume of distribution, expressedas ml*kg-',

was obtained bydividing the infusionratebythe product of plateau concentration and the slope ofregressionfordisappearance.

terminal specific antibody,LW11,was- 30%higherthanwith

antibody L6,which measures intactG17.Theplateau

concen-tration with theCOOH-terminal specific antibody, LWl, was

- 15% higher than withL6(Fig. 1 and TableI).Themetabolic clearance rate,volume ofdistribution,and half-life determined

with antibodies L6, LW1, and LWl1 aregiven in Table I. Of

particular interest is the difference in half-lives measured by different antibodies; thus, intact circulating G17 (read by L6) disappeared withacalculatedmeanhalf-life of 3.23±0.17min, whereas total COOH-terminal and NH2-terminal GI7 immu-noreactivity disappeared slower with half-lives of 4.88±0.08 min and 11.71±0.60min, respectively (Fig. 2and TableI).

Molecular forms. Gel filtration ofplasma samples taken during the plateau phase of the infusion revealed in each

subject a major peak of activity elutingin thepositionof the standard (Fig. 3) and read equally by the three antisera. In

addition, there was a peak of late-eluting COOH-terminal

Results

G17

In vivo metabolism. The effective infusion rate of hG17

measuredwith L6,LWl,andLW11 was396±6,

389+11,

and 406±10 pmol-kg-' h-', respectively, asestimated byRIAof the solutions remaining in the syringe after infusion. Gel filtration and ion-exchange chromatography of this material showed a single peak with the chromatographic and immu-nochemical propertiesofstandard hG17.

During the initial saline infusion, plasma gastrin immu-noreactivity was undetectable with L6 (<10 pmol -'), was

10.1±2.6 pmol l1-' with antiserum LW1, and was 10.0±0.7 pmol

I`

with antiserum LW 11. 30 min after beginning infusion ofhG 17, plasmagastrin immunoreactivity reached a

plateau level. The meanplateau concentration withthe

NH2-

2.0- I-->

1.6*

0

-0

*-o _ 1.2

z =

0 2Z_E

0.8-z

E-

0.4

m

-C

0-- 0.5

C)

E0. 101~~~~~~b1w I

0 z

0.05\

eZ A~b LW1

I-C)

a ~~~~~AbLO \

0.01;

10 15 20 2S 30 35

TIMsE(min)

Figure2.Disappearanceof intactGI17(o),COOH-terminal(-),and

NH2-terminal (A)immunoreactivityafterstoppinginfusion of hG17. SeeFig. I for further details.

100->

80-C.) wI

E _ 23

D

._

a ! E

40-z

IC

20.

I-0

co

4c

a

0~j

1-13 G17 G17 G14

11

1

0 20 40 80 80 100 120 PERCENT ELUTION VOLUME PROTEIN TO

1251

Figure 3. Sephadex G50 elution profile of standardG17 (top) and plasma taken during the plateau phase of intravenousinfusion of G17 (bottom)in a normalhumansubject. The columns were assayed for intact G17 (-o-), COOH-terminal (- * -), and

immunoreactive material measured by LW1, and a peak of NH2-terminal gastrin immunoreactivity eluting just before G17 revealed by LW I 1.

Ion-exchange chromatography of plasma samples taken during the plateau phase also revealed ineach subjectamajor peak coeluting with standard G17 and reacting with all three antibodies (Fig. 4). Inaddition, antibodyLWl revealed a peak of COOH-terminal activity (18% total) that eluted later and progressively declined after stopping the infusion. Moreover, antiserum LW 1 revealed two further peaks of NH2-terminal Gl7 immunoreactivity. These eluted close to each other and

just in front of hG 17; they probably coelutedongel filtration. PeakIIcorresponded in elution position to 1-13 G17standard. Tubes corresponding to peaks I and II of NH2-terminal immunoreactivity were also fractionated by HPLC. Peak 1I emergedasasingle peak that again had the retention time of 1-13 G17, and Peak I emerged 2-3 min earlier, indicating a less basic and less hydrophobic peptide (Fig. 5). In plasma samples collected after the infusion, the same peaks of NH2-terminal activity were found. However, the two earlier eluting peaksincreased from 26% of total NH2-terminal immunoreac-tivity during the infusion to 75% in the 20-35-min samples

800

600

-F.

400-

200-z 1,000

0

Z 800 -400

-200

PEAK II

PEAK I

_-1-13G17

h

w

l1

60 I

40 Z

[₂₀0I-_wu

C) o 10 20 30 40 50 60

RETENTION TIME (min)

Figure 5. Separation by reversed phaseHPLC onTechsilC18of tubes inDEAEcolumneluatescorrespondingtopeaksIandII of NH2-terminal immunoreactivity. Sampleswereconcentrated ina

Speed-vacand pHwastakentobelow7beforeinjection. Gradient elution

wascarriedoutwith acetonitrile(---).Arrowattopindicates the retention time of standard 1-13G17; in thissystemintact G17 hasa

retention time of55 min.Assayswith LW11.

StdhG17 Std 1-13 hG17 1251-hG17

I p

iateauG17Infusion

2-3 minafter infusion

5-15 minaftrinfusion

20-35 minafter Infusion

100 120 140 160

FRACTION NUMBER

Figure 4. Ion exchange chromatographyonDEAE cellulose of

plasma taken during the plateau phase of intravenousinfusion of

G17 (top), andatintervals (2-3, 5-15, and 20-35 min) after stopping infusion ofG17.Arrowsatthetopindicate elution positionof standards. 251I-hGl7wasaddedroutinelytosamples,and 1-13 hG17

orG17 were runinseparateexperiments. Note the increase in relativeconcentrations of the early eluting NOOH-terminal fragment

afterstopping infusion. Symbolsare asinFig. 1.

after stopping the infusion (Fig. 4). The peak corresponding

to hG17wasproportionally reduced(Table II).

In vitrometabolism.Allantisera revealed a time-dependent and temperature-dependent decrease of immunoreactivity when G17 was incubated in serum (Fig. 6). The COOH-terminal and NH2-terminal G17 immunoreactivities were slower to

disappear than intact G17-immunoreactivity measured by L6. This suggests that both NH2- and COOH-terminal fragments of G17 were produced during the incubation, and that the degradation of these was slower than that of intact G17. In

support of this, gel filtration studies revealed a major peak with the properties of the intact heptadecapeptide together with an additional peak of COOH-terminal immunoreactivity elutingafter G17andapeakofNH2-terminal immunoreactivity eluting justbefore, andimperfectly separated from, G17 (Fig. 7). The COOH-terminal and NH2-terminal immunoreactive components were also separated on ion exchange chromatog-raphy(Fig. 8).

Addition ofEDTAprevented the loss ofimmunoreactivity with theCOOH-terminal antibody LW1, but not with LW 1I and L6(Fig. 6). Gel filtration (not shown) and ion exchange chromatography(Fig. 9)suggested that in these samples there was material with the properties of G17 together with a COOH-terminalfragmentthatincreased inconcentration dur-ingincubation and that had similarchromatographic properties

totheCOOH-terminal fragmentgenerated in vivo. Therewas

alsoaminor peak ofNH2-terminal immunoreactive material.

A third peak ofCOOH-terminal immunoreactivity was read by antiserum LW I in samples after prolonged incubation. This material eluted just in front of G17 and was not well

resolved from it. TotalCOOH-terminal

immunoreactivity

was

relatively constant, sothat thetwoCOOH-terminal

fragments

generated during incubation became

progressively

more

im-portant and

finally

accounted for close to 100% ofthe total COOH-terminal activity.

When 2-17 G17 was incubated in serum, there was a

time-dependent and

temperature-dependent

loss of immuno-reactivity, whichwasabouttwice thatofG17(Fig. 10).EDTA

3.0-

2.0-IA I

a 1.61

-C)

IC

0.6

-0.3

-0 _

0 20 40 60 80

a---I I "no%

1-0

./.2L.

Al&&

-TableII.COOH-terminal and NHrterminal Gastrin ImmunoreactiveFormsSeparated byIonExchange Chromatography in Plasma Pools of FireSubjectsDuringandAfterInfusion of hGI7(400pmol*kg'*h-'*

NT-G17measuredby antibodyLWI I

G17 CT-G17measured by antibodyLWI NT-G17

measured by

antibody L6 Total G17 CT-G17 Total G17 PeakI PeakII

Plasmapoolduring infusion 532 708 580(82%) 128(18%) 860 636(74%) 139(16%) 85(10%)

Plasmapoolsafter infusion

2-3 min 386 563 490(87%) 73(13%) 824 560(68%) 172(21%) 92(11%)

5-15 min 180 262 231 (88%) 31 (12%) 515 237(46%) 231 (45%) 99(19%)

20-35 min 5 30 30(100%) -_(0%) 190 46(24%) 118(62%) 26 (14%)

*Values are presented aspmol lI-'equivalent of plasma. Values inparenthesisgivethe percentagesofeach componentof total immunoreactiv-ity measured by antibody LWl or LWl 1.CT-G17, COOH-terminal G17;NT-G17, NH2-terminalG17.

completelyinhibited the loss ofimmunoreactivity. Separation byion-exchangechromatographyindicated that in bothserum

and EDTA-treated plasma there was conversion to a more

SERUM PLASMA-EDTA

acidic COOH-terminal fragment (Fig. 11), and withprolonged incubation there was further accumulation of a molecular species thatwasless acidic than 2-17 GI 7. Presumably EDTA

stopsthefurther degradation of theCOOH-terminal fragments.

Discussion

100-801

3

601

40 201

01

so

1 00,

601

40

20-101

a-0.

I

4°C

20°C

The principal finding of this study is that G 17canbe converted

to COOH-terminal and NH2-terminal immunoreactive

frag-mentsinthe human circulation in vivo, and inserumincubated in vitro. There have been many previous studies of the concentrations, identity, and metabolism of G17-like immu-noreactivity in the human circulation, butlittle attention has

beengiventothepossibility of its conversion tosmallerforms.

0

E

c

5

37°C

0 6 12 24 0 12 ₂₄

TIME (h)

Figure 6. Loss ofimmunoreactivity whenhG17wasincubatedin

humanserumat4,20,or37°C forupto24h(left),and the effects ofEDTA (right). Sampleswereassayed withthreeantibodies(seeFig.

1). Note that EDTAstopsthe lossof COOH-terminal

immunoreac-tivitybut notthat of NH2-terminal and intact G17 immunoreactiv-ity. -,antibody LW1;-o-,antibody L6;-A-,antibody

LWI1.

1-13G17 G17 G14

3.0-Oh

2.0-

1.0-OJ MWOM0409

0.8 ui

0 z

Z 0.6-a

Z 0.4-i

I.-'C _0.2.

6 h

0 20 40 60 80 100 120 140 160 PERCENT ELUTION VOLUME PROTEN TO 1251

Figure 7.SeparationonSephadex G50 superfine (1 X 100cm)of

synthetic hG17 before (top)and 6 h after incubation inserumat 370C(bottom).Columnswere_assayedwith thesameantibodiesas

usedinFig. 1.Notethat after 6 h incubation therewas alate

running peak of COOH-terminalimmunoreactivityandan imper-fectly separated peakofNH2-terminal immunoreactivityemerging

beforeG17.

(A)

44

0 w

w 0

z

a

z

w 0

z

4c

z C)

I--z

'U

cc

'U

1251-G17

3 0

0I

0

E

-C 5

oc

-

W-0

z

-0 0

2.0-

1.

0- 0.8-

0.6-

0.4-

0.2-O0

O h

6 h

1251

-G17

N1I

0r20 40a60_80100 120 140

0

2'0

40 60 80 100 120 140

FRACTION NUMBER

Figure8. Separation onDEAECellulose(1 X 15 cm) of human GI 7 before (top) and 6hafter incubation in serum at 370C(bottom). See legend to Fig. I forfurther details about antibodies. Note the late-eluting peak of COOH-terminal immunoreactivity and the early elut-ing peak ofNHrterminalimmunoreactivity, in addition to

G17-immunoreactivity. Columns were calibrated with '25I-G1i 7,indicated by the arrow.

Indeed, for practical purposes it often seems to have been assumed that G17 is stable in human plasma. Our results show that this assumption is unjustified. In species such as dogandcat,it isalready well establishedthat circulatingG17 is converted in the circulation to COOH-terminal immuno-reactive

fragmen4

(13-15). The maindifference between dog, cat, and man liesin the rate oftheconversion. Thus in dog, intact G17 and NH2-terminal immunoreactive fragments are

virtually absent from the systemic circulation (13), whereas in

man COOH-terminal or

NH2-terminal

immunoreactive

frag-ments generated from G17 occur in concentrations up to

-25% of total G17

immunoreactivity.

The main COOH-terminal product of G17 metabolism both in vivo and in vitro has the properties ofminigastrin, which is known now to be theCOOH-terminal tetradecapeptide fragment, G14 (16). One of the two NH2-terminal immuno-reactive forms generated in vivo had the

properties

of 1-13 G17. The otherNH2-terminal product isprobably afragment of 1-13 G 17, sincewefoundcloselysimilar materialproduced when 1-13 G17 was infused intravenously in humans (10). Both G14 and 1-13 G17 have previously been reported to occur in the humanperipheralcirculation (11, 17). Thesame

forms have also been reported inextractsof antral mucosa, or

gastrinoma tissue, and have beenisolated andsequencedfrom these sources (16, 18, 19). It is generally thought that in man

the circulating peptides are derived intact from the tissue

125I-G17

3.0-

2.0-

1.0-

O- 0-10

._

B C

0.5

-< 1.2,

mi

0 .I z 0

0.8-a a Z 0.4

O

I-. 0

1.2-

0.8-

0.4-

0-O h

O_hI

0 20 40 60 80 100 120 140 160

FRACTION NUMBER

Figure9.Separationon DEAE celluloseof samples 6, 12,and 24 h

after incubation of hG17in plasma containingEDTA. Seelegendto

Fig. 1 for furtherdetails.

stores.Thisstudyindicatesthatthisneednotbetrue.Instead,

a proportion ofthe circulating pools of these peptides could beobtained by cleavageofG17 followingsecretion.Wefound that after stopping an infusion of G17 the NH2-terminal fragments accounted for progressively greater proportions of total immunoreactivity until they were the predominant im-munoreactive forms; presumably thesefragmentshavealonger half-lifethan G17.

Theconversion ofG17 toCOGH-terminal fragments like G14 is notlikely to make a significant changein circulating gastrin biological

activity

because long COOH-terminal

frag-ments are equipotent with G17 (20). It is generally thought that NH2-terminal fragments of G17 are inactive, so that conversion ofG17to1-13 G17 would reduce

activity.

However, if1-13 G17 wasgenerated by the action ofanendopeptidase, the COOH-terminal tetrapeptide might be produced as a

coproduct and this is the minimal fragment with

biological

activity. The potency of the

tetrapeptide

is about 10 timesless

than that ofG17 for acid secretion. In this

study

it would have escaped detection because it reacts weakly with the COOH-terminal antibody LW1. It should also be noted that

Petersen et al. (21) have recently

reported

that 1-13 G17

-i.^-be-S-- xr -^-^ C__aa~

SERUM

100

80

z 60

I- 40

0 8 4

ol

P 80

0

60

b

z

0 _40X

z

20-w

0100:

z

60-I-.

40-z

w

0.

10.

PLASMA-EDTA

*

4°C

20C

37&C

01 2 4 6

TIME (h)

Figure10. Companrsonof thestabilityof h

I-U

41

w

0

01 2 4 6

0

4

G17(oo~nd2-17

HG17(. 6)inserumandinplasmacollectedin EDTAduring incubationsat4, 20,and 370C forperiodsupto6 h. Allsamples

wereassayed withaCOOH-termninalspecific antibody (LW1).

inhibited acid secretion. Cleavage of G17 to 1-13 G17 might therefore generate inhibitory activity inthe circulation.

How-ever, we have been unable to confirm the observations of

Petersen etal., so that fornow the significance ofcirculating 1-13 G17 for acid secretion remainsindoubt(10).

Todetermine iftheconversionof G 17 toCOOH-terminal and NH2-terminal fragments wasdue to the action ofserum

enzymes, we performed aseriesofincubation experiments in

vitro. We found thatin serum thereareenzyme systemsthat can degrade G17to G14-like and 1-13 G17-like peptides, as

well as to forms not recognized by COOH-terminal or

NH2-terminalspecific antibodies. Thehexadecapeptide (2-17G17)

wasdegraded more rapidly than G17, indicatinga protective

function for the NH2-terminal pyroglutamyl residue in G17. In the presence of EDTA both G17 and 2-17 G17 were

converted to COOH-terminal immunoreactive peptides that

were resistant to further degradation, so that one of the

enzymes acting at the NH2-terminus is presumably a

metal-loenzyme.

The rate of conversion of G17 to COOH-terminal and NH2-terminal fragments in vitro, and the subsequent degra-dation to nonimmunoreactive fragments was relatively slow;

forexample, at370C the half-life measured by L6was3-5 h.

In contrast, the half-life ofintact GI 7 in vivo is <5 min. Evidently the plasma enzyme systems we have identified are

not likely to be ofphysiological importance in the clearance

1.2

0.8-

0.4-

0-CONTROL

20 40 60 S0 100 120 140

FRACTION NUMBER

Figure11.SeparationonDEAE Cellulose of samples taken before

(bottom) and during incubation of 2-17 hG17 inserumfor 30min

and 120 min,andinplasma containing EDTA. Assays with

COOH-terminalspecific antiserum, LW1. Note the progressiveappearancein

serumofaG14-like peptide (elution volume, 110-115 ml) followed byanearlier-eluting peptide (60-70 ml). In EDTA-treated samples

theG14-likecomponentpredominates.

of G 17. We havepreviouslyobservedthat indog,there is also

degradation of G 17 by plasma enzymes to COOH-terminal

fragments,andagaintheratesof conversionarerelativelyslow

(although fasterthaninhumans) (13).Becauseplasmaenzymes

1251lG17

1.6

1.2-

0.8-

0.4-

0-

1.6-

1.2-EDTA-30

SERUM-120

SERUM-30'

0.8-

0.4-

0-1.2

0.8-0.4

areunableto account for therateofconversion in vivo, it has been postulated that enzymes either on or near the capillary endothelial cells mediate the conversion (13). Similar systems

have also been postulated to account for the production of fragments of the NH2-terminal tryptic peptide of G34 that

have been found in the peripheral circulation in man (22). Thepresentfindingsemphasizethe carethatmustbe taken in processing samples for gastrin RIAand in interpretation of the results of studies of gastrin molecular forms. They also

indicate the importance of using several region specific antisera to.gether with chromatographic separation in studying the

physiology and pathophysiology of this hormone.

References

1. Gregory, R. A., and H. J. Tracy. 1964. The constitution and

properties oftwo gastrins extracted from hogantral mucosa. Gut. 5: 103-117.

2. Berson,S.A., andR.S.Yalow.1971.Natureof immunoreactive gastrin extracted from tissues of gastrointestinaltract.Gastroenterology. 60:215-222.

3. Malmstrom, J., F. Stadil,and J. F. Rehfeld. 1976. Gastrins in tissue: concentration andcomponent pattern ingastric, duodenaland

jejunalmucosaof normal humansubjectsandpatients withduodenal

ulcer.Gastroenterology. 70:697-703.

4. Calam, J., G. J. Dockray, R. Walker, H. J. Tracy, and D.

Owens. 1980. Molecular forms ofgastrin in pepticulcer: comparison ofserum and tissue concentrations ofG17 andG34 in gastric and duodenal ulcersubjects.Eur.J. Clin. Invest. 10:241-247.

5. Schrumpf, E., L. S. Semb, and H. Vold. 1973. Metabolic

clearance anddisappearance ratesofsynthetichumangastrinin man.

Scand. J. Gastroenterol. 8:731-734.

6. Walsh,J.H.,J. I. Isenberg,J.Ansfield, and V. Maxwell. 1976.

Clearanceandacidstimulating action ofhuman big and little gastrin in duodenal ulcer subjects.J. Clin. Invest.57:1125-1131.

7.Boniface, J.,D. Picone, M.Schebaline, A.M.Zfass,and G. M.

Makhlouf. 1976. Clearance rate, half-life and secretory potency of humangastrin-17-I indifferentspecies. Gastroenterology. 71:291-294. 8. Pauwels, S., andG. J. Dockray. 1982. Identification of NH2-terminalfragments of big gastrin in plasma. Gastroenterology.

82:56-61.

9.Dockray,G.J.,andI.L.Taylor. 1976.Heptadecapeptidegastrin:

measurement in blood by specific radioimmunoassay. Gastroenterology. 71:971-976.

10. Pauwels, S., G. J. Dockray, R. Walker, and S. Marcus. 1985. Metabolismof 1- 13 G 17 in man andfailuretoinfluenceacid secretion.

Gastroenterology. Inpress.

11.Dockray,G.J., and J. H. Walsh. 1975.Aminoterminal gastrin fragment inserum of Zollinger-Ellison syndrome patients.

Gastroen-terology. 68:222-230.

12. Dockray, G. J., J. H. Walsh, and M. I. Grossman. 1976. Biological activity of iodinated gastrins. Biochem. Biophys. Comm. 69: 339-345.

13. Dockray, G.J., R. A. Gregory, H. J.Tracy, andW. Y. Zhu. 1982. Post-secretoryprocessing ofheptadecapeptide gastrin: conversion toC-terminal immunoreactive fragmentsin the circulationofthedog.

Gastroenterology. 83:224-232.

14. Uvnas-Wallensten, K., and J. F. Rehfeld. 1976. Molecular forms ofgastrin in antral mucosa, plasma and gastricjuice during vagal stimulation of anesthetized cats. Acta Physiol. Scand. 98:217-226.

15.Blair,E.L.,E. R. Grund, P. K. Lund, and D. J. Sanders. 1977. A possible origin of circulating gastrin component IV in cats. J.

Physiol. 273:561-572.

16. Gregory, R. A., H. J. Tracy, J. I. Harris, M. J.Runswick, S.

Moore,G.W. Kenner, and R. Ramage. 1979. Minigastrin:corrected

structureandsynthesis.Hoppe-Seyler'sZ.Physiol. Chem.360-73-80.

17. Petersen, B., and J. F. Rehfeld. 1980. The NH2-terminal

tridecapeptide fragment of gastrin-17 in serum from duodenal ulcer

patients. Scand.J. Gastroent. 15:29-31.

18.Gregory, R. A. 1974.Thegastrointestinal hormones: areview

of recent advances. J. Physiol. 241:1-32.

19.Gregory, R. A. 1979.Some recentaspects of the structure of gastrin and gastrin-like forms and fragments in gut and brain. In

Gastrins and the Vagus. J. F. Rehfeld and E. Amdrup, editors. AcademicPress, London. 47-55.

20. Carter, D. C., I. L. Taylor, J. Elashoff, and M. I. Grossman. 1979. Reappraisal of thesecretorypotency and disappearance rate of pure humaitminigastrin. Gut. 20:705-708.

21. Petersen, B., J. Christiansen, and J. F.Rehfeld. 1983. The

N-terminal tridecapeptide fragment of gastrin 17 inhibits gastric acid secretion. Reg. Peptides. 7:323-334.

22.Pauwels, S., G. J. Dockray, R. Walker, and S. Marcus. 1984.

N-terminal tryptic fragment of big gastrin: metabolismandfailureto