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Endocrinology and Metabolism |
Dept. of Clinical Biochemistry, Rigshospitalet, DK-2100 Copenhagen, Denmark.
a Author for correspondence. Fax 45 3545 4640; e-mail rehfeld{at}rh.dk.
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Abstract |
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 Top Abstract Introductiondefinition of CCK peptidesMaterials and MethodsResultsDiscussionReferences |
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500 000)
with sufficient avidity (K
eff°
1012 mol-1) and the desired specificity. The
antiserum binds the bioactive forms of CCK with equimolar potency and
displays no reactivity with gastrin. CCK concentrations in plasma from
healthy humans rose from 1.13 ± 0.10 pmol/L (mean ± SE,
n = 26) to 4.92 ± 0.34 pmol/L after a mixed meal.
Chromatography of human plasma revealed traces of CCK-58, a
predominance of CCK-33 and CCK-22, and moderate amounts of CCK-8. The
results show that it is possible to produce specific CCK-antisera using
a sulfated CCK-12 analog. |
Introduction |
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TopAbstractIntroductiondefinition of CCK peptidesMaterials and MethodsResultsDiscussionReferences |
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A sensitive and specific bioassay has been described
(5)(6), but it is labor-intensive and has seen
limited use for plasma measurements. Several laboratories have tried to
develop RIAs. The first difficulty in this endeavor was a shortage of
peptides for immunizations. Mutt and Jorpes reported in 1971 the
structure of porcine CCK-33 (7), but synthesis of sulfated
CCK-33 became possible only recently, and only limited amounts of
natural CCK were available. A batch of sulfated CCK-8 was synthesized
(8), but antibodies against it also reacted with gastrin.
The next difficulty was the isotopic labeling, because methionyl
residues are oxidized easily, and CCK-8 contain two such residues. The
problem was solved by nonoxidative labeling (9). The third
difficulty is the low concentrations of CCK in plasma, which requires
antibodies of high affinity and tracers of high specific activity. The
largest obstacle, however, is specificity. Hence, the antibodies should
bind the "active site" of CCK without binding the homologous
gastrin. Alignment of the active site sequences of CCK and gastrin
(Fig. 1
) illustrates the problem, which seems unsurmountable because
antibody binding sites are assumed to harbor epitopes of no more than
four to six amino acid residues (10). The specificity
problem is accentuated by the higher gastrin concentrations in plasma.
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In spite of the somber prognosis, we have tried to raise useful antibodies. Insufficient specificity of the first led to design of haptens to yield antibodies that might bind both the C-terminal phenylalanyl amide and the CCK-specific tyrosyl sulfate in position 7 (as counted from the C-terminus). Using a directionally coupled CCK-12-analog, we have raised such an antiserum.
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definition of CCK peptides |
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TopAbstractIntroductiondefinition of CCK peptidesMaterials and MethodsResultsDiscussionReferences |
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), which is necessary for receptor binding. Binding to CCK-A
receptors requires that the tyrosyl residue of the heptapeptide amide
is O-sulfated (Fig. 1
), whereas CCK-B receptors do not discriminate
between sulfated and nonsulfated CCK-peptides or between CCK and
gastrin peptides (for review, see references 1–3). |
Materials and Methods |
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TopAbstractIntroductiondefinition of CCK peptidesMaterials and MethodsResultsDiscussionReferences |
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The first batch of natural porcine CCK-33 (30 mg, 20% purity) was dissolved in 12 mL of 0.05 mol/L sodium phosphate, pH 7.5, and divided into four portions, which were stored at -20 °C. The next batches of natural porcine CCK-33 (30 mg, 20% purity and 8 mg, 75% purity), synthetic nonsulfated CCK-33 and CCK-29 (6 and 5 mg, 85% purity) were each dissolved in 1.0 mL of N,N-dimethylformamide and conjugated to 25 or 30 mg of bovine serum albumin (the Serum Institute) and dissolved in 2.5 mL of 0.05 mol/L sodium phosphate, pH 7.5, by the addition of 135 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (Sigma Chemical) to give molar ratios between the CCK-peptide, albumin, and ethyl-carbodiimide of 1:0.1:40. The reagents were mixed for 20 h at 20 °C, and each conjugate was then divided into six portions and stored at -20 °C until immunization.
Sulfated and nonsulfated synthetic CCK-4, CCK-8, CCK-12, and CCK-13 (2.0, 4.0, and 6.0 mg of each) were dissolved in 10 mL of 0.05 mol/L sodium phosphate, pH 7.5, and conjugated to 15 mg of bovine serum albumin by dropwise addition of 100 µL of 500 g/L glutaraldehyde. The solution was mixed for 4 h at 20 °C, applied to a calibrated Sephadex G-10 column (10 x 60 mm), and eluted at 20 °C with 0.05 mol/L sodium phosphate, pH 7.5, in fractions of 1.0 mL. The void volume fractions containing the conjugate were pooled, divided into six portions, and stored at -20 °C until immunization.
tracers
Two types of tracers were used: Sera from rabbits immunized with
CCK-33 and CCK-29 were examined with CCK-33 labeled by nonoxidative
conjugation of
[I]hydroxyphenylpropionic-succinimide ester to
either the 
- or 
- NH2 groups of the N-terminal
lysyl residue as detailed elsewhere
(9)(11)(12). Sera from rabbits
immunized with CCK-13, CCK-12, CCK-8, CCK-4, and the
corresponding analogs were examined with Bolton-Hunter-labeled CCK-8
(Amersham). The tracers displayed specific radioactivities of
1500–1700 kCi/mol. Thus, the 1000-cpm tracer used for the RIA
incubations corresponds to ~0.5 fmol of peptide.
immunizations
Randomly bred white Danish rabbits (n = 78) were used for
immunizations in nine series of 6–16 rabbits each. In addition, one
series of 29 guinea pigs and one of 8 mice were immunized (Table 1
). On the basis of our experience with the production of
antibodies towards other peptides (13)(14)(15)(16)(17)(18)(19)(20)(21), we chose a
specific immunization procedure for all of the animals: The first
portion of the antigen was suspended in 8.5 g/L saline to a volume of 5
mL and emulsified with an equal volume of Freund's complete adjuvant
(the Serum Institute). Two subcutaneous injections of the mixture were
given over the hips in amounts corresponding to 100 µg of CCK-33
(~40 µg of CCK-12 or 25 µg of CCK-8) per animal. Five or more
booster injections using Freund's incomplete adjuvant were
administered simultaneously at 8-week intervals, using one-half of the
initial dose of antigen per immunization. The rabbits were bled from an
ear vein 10 days after immunization. Guinea pigs and mice were bled by
cardiac puncture 10 days after immunization. Sera from the bleedings
were separated and stored at -20 °C.
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antiserum evaluation
Four characteristics of sera from the immunized animals were
examined: (a) Titer was defined as the serum
dilution that binds 33% of the 0.5-fmol tracer at equilibrium.
(b) Affinity (expressed by the "effective"
equilibrium constant (K
eff°) was
determined as the slope of the curve at zero peptide concentration in a
Scatchard plot (22)(23). (c)
Specificity was determined in percentage as the molar
ratio of the concentrations of the CCK standard and the related peptide
that produced a 50% inhibition of the binding of the tracer.
(d) Homogeneity of the antibodies with respect
to binding kinetics as expressed by the Sips index (24). An
index of 1.0 indicates homogeneity of both the tracer and the antiserum
in the binding, otherwise seen only for monoclonal antibodies
(25). The ability of the peptides to displace tracers from
the antisera was tested in peptide concentrations of 0, 3, 10, 30, 100,
1000, 10 000, and 100 000 pmol/L.
ria procedure
The RIA was carried out as an equilibrium system at pH 8.4 in
disposable plastic tubes, using 0.02 mol/L barbital buffer, pH 8.4,
containing 1 g/L bovine serum albumin (Ortho). Incubation mixtures of
2.4 mL contained 2.0 mL of antiserum dilution, 250 µL of tracer
solution (giving 1000 cpm, corresponding to 0.5 fmol of freshly
prepared I-labeled CCK-peptide), and 150 µL
of standard solution or sample. The assays were set up at room
temperature and incubated at 4 °C for 48 h to reach
equilibrium. Antibody-bound (B) and free (F) tracers were separated by
the addition of 0.5 mL of a suspension of 20 mg of activated charcoal
(Merck) and blood plasma (equivolume mixture of buffer and outdated
human plasma from the Blood Bank of Rigshospitalet, Denmark) in 0.02
mol/L sodium phosphate, pH 7.4, to each tube. The tubes were
centrifuged for 10 min at 2000 rpm, and the supernatant (B) and
sedimented charcoal (F) were counted in automatic 
-scintillation
counters for 5 min. The binding percentage was calculated as
B - [(B F) x D]/(B F) - [(B F) x D] x
100, where the "damage" (D) is defined as B x
100/(B F) in the absence of antiserum. The damage was usually 2–3%,
and the antisera were used at dilutions giving a binding percentage of
~33%. All samples were assayed in duplicate.
assay reliability
The assays were evaluated with respect to detection limit,
specificity, between- and within-assay reproducibility, and accuracy.
tissue extracts
Biopsies of human jejunal mucosa were obtained from the Department
of Surgical Gastroenterology and porcine jejunal mucosa from
anesthetized pigs at the Department of Experimental Pathology,
Rigshospitalet. The tissue samples were immediately frozen in liquid
nitrogen. The frozen tissue was cut in pieces of a few milligrams,
boiled in water (10 mL/g tissue) for 20 min, homogenized, and
centrifuged at 10 000g for 30 min at 4 °C. The
supernatant was decanted (neutral extract), and the pellet was
redissolved in ice-cold acetic acid (10 mL/g), left at room temperature
for 20 min, and centrifuged as described above (acid extract).
plasma extracts
Blood samples were collected into chilled tubes containing 3.9
µmol of EDTA per mL of blood. Within 30 min, the samples were
centrifuged at 3000g at 4 °C for 10 min. The plasma was
stored at -20 °C until extraction, which was performed as follows.
One volume of plasma (usually 1.0 mL) was mixed with two volumes of 960
mL/L ethanol on a whirlmixer for 10 s. The mixture was then
centrifuged in 30 min at 1200g; the supernatant was decanted
and evaporated at 37 °C in a speed-vac concentrator (SVC 200 H,
Savant). The dried extracts were then reconstituted to the original
volume with assay buffer and assayed. The basal and postprandial
concentrations in plasma were measured in 15 healthy females and 11
healthy males (mean age, 36 years) after an overnight fast. The meal
consisted of an omelet (two eggs mixed with 10 g of flour, 25 mL
of cream, salt, and pepper) with two slices of bacon, 250 mL of orange
juice, 250 mL of milk, 250 mL of yogurt, and two slices of toasted
bread with butter and cheese, i.e., 1470 calories of which 45% was
fat, 37% was carbohydrates, and 18% was protein. Blood samples were
taken from each of the subjects from 60 min before to 125 min after
ingestion of the meal.
plasma for chromatography
Four healthy persons (two of each sex, 23- to 40-years-old)
ingested a meal as described above. Blood samples (200 mL) were drawn
from an arm vein immediately before the meal as well as 30, 90, and 150
min postprandially. Because it has been suggested (26) that
acidification is necessary to prevent in vitro degradation, one-half of
each blood sample was drawn into EDTA tubes as described above (neutral
sample), and the other half was drawn into EDTA tubes containing 1 mL
0.5 mol/L sodium acetate buffer (pH 3.6) per 5 mL of blood (acid sample
(26)). Immediately after centrifugation, 50 mL of the
neutral plasma samples was extracted directly on Sep-Pak cartridges,
and 50 mL of the acidified plasma was poured slowly into 150 mL of 20
g/L trifluoroacetic acid (TFA) under constant stirring (26).
This mixture was extracted on Sep-Pak C-18 cartridges (Waters
Associates) prewashed with 10 mL of 960 mL/L ethanol followed by 10 mL
of a 13 mmol/L solution of TFA. Ten milliliters of ice-cooled plasma
were then loaded on each cartridge with a flow rate of 1 mL/min. After
the cartridge was washed with 10 mL of 13 mmol TFA/L, the CCK-peptides
were eluted by 2 mL of 800 mL/L ethanol containing 13 mmol TFA/L.
Evaporation of the eluates was performed as described. All steps were
performed consecutively without freezing the plasma or extracts.
chromatography
One milliliter of tissue extract or plasma concentrate was applied
to a Sephadex G-50 superfine column (10 x 1000 mm) and eluted
with either 125 mmol/L NH4HCO3, pH 8.2, or
20 mmol/L sodium veronal, pH 8.4, containing 0.6 mmol/L thiomersal and
1 g/L bovine serum albumin at 4 °C with a flow rate of 4 mL/h.
Fractions of 1.0 mL were collected. The columns were calibrated with
human CCK-33, CCK-22, and CCK-8, as well as with
I-albumin and NaCl to indicate void
(Vo) and total (Vt) volumes.
enzyme analysis
To measure the immunoreactivity of the larger endogenous forms of
CCK using antiserum 92128, chromatographic fractions of the jejunal
tissue extracts were also measured after tryptic cleavage. Each
fraction was incubated with trypsin (100 mg/L Trypsin-TPCK
,Worthington) for 30 min at 20 °C. Tryptic cleavage was terminated
by boiling the fraction for 10 min. Similarly, tissue extracts were
also cleaved with trypsin before chromatography to ensure their CCK
nature. Principles and details of the tryptic analysis have been
described elsewhere (27).
gastrin measurements
Control measurements of gastrin in plasma were performed with a
RIA using antiserum 2604, which binds all bioactive, carboxyamidated
gastrins (gastrin-71, -34, -17, and -14) with equimolar potency
irrespective of their degree of sulfation
(14)(15). The assay does not measure
CCK-peptides.
gastrin infusions
After an overnight fast, six healthy male volunteers (24- to
29-years-old) were intubated with a nasogastric tube (AN 10, Anderson
Amplers). The position of the tube was controlled by fluoroscopy. The
gastric contents were aspirated by intermittent pump suction. The
infusions were preceded by an equilibrium period of at least 30 min,
during which saline was infused. Subsequently, increasing doses of
synthetic human nonsulfated gastrin-17 (0–60 pmol · kg · h;
Sigma Chemical) were administered continuously into a cubital vein.
Each dose (0, 10, 30, and 60 pmol · kg · h) was administered
for 45 min. Venous blood samples were drawn from the opposite cubital
vein every 15 min into EDTA tubes as described above. The tubes were
immediately placed in crushed ice and centrifuged at 4 °C within 30
min. The plasma samples were stored at -20 °C until the measurement
of gastrin and CCK.
ethics
The studies reported here were approved by the local committee for
ethics in studies of human subjects.
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Results |
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TopAbstractIntroductiondefinition of CCK peptidesMaterials and MethodsResultsDiscussionReferences |
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). The modest or low antibody titers in the early series
(I-VII) may be due to inadequate methods for measuring hapten in the
antigen-conjugate, a low degree of hapten purity, degraded peptides,
and less efficient coupling procedures. Nevertheless, a few antisera in
series I and III had titers and binding affinities of some usefulness
(Table 1
). Two of these (4478 and 4698) were also specific for CCK and
have proved useful for the characterization of CCK-peptides in tissue
(9)(28)(29)(30). However, they were not useful for
plasma measurements because there was too little antiserum (small
volume of heartblood from guinea pigs) in titers that were too modest
(<50 000) and because the animals died before the quality of their
antibodies was known. In addition, only antiserum 4698 had the desired
specificity, but it lacked sufficient affinity to measure plasma
concentrations in fasting subjects.
High titers and affinities were obtained in series VIII and X (Table 1
), but none of the antisera had the specificity necessary for plasma
measurement. Although several high-titer and high-affinity antisera
were C-terminal directed (1559, 1560, 1564, 8007, and 8011), they all
cross-reacted with gastrin peptides to a degree making them useless for
plasma measurement. One high-titer antiserum in series VIII (1561) was
monospecific for sequence 16–20 of porcine CCK-33. This antiserum has
proved valuable for plasma measurement and affinity purification of
porcine N-terminal desocta- and desnona-CCK-58, CCK-39 and CCK-33
fragments (31)(32). Unfortunately, the epitope
was specific for a porcine CCK sequence. Hence, species variations of
the sequence N-terminal for the bioactive C-terminal heptapeptide amide
make antisera like 1561 useless for plasma measurements in man and
other nonporcine species.
The last series (XI) was composed of 16 rabbits that all responded;
some with high titers and affinities (Table 1
). Among the five antisera
containing C-terminal-directed antibodies with binding affinities
sufficient for measurement of basal CCK concentrations in plasma and
with titers sufficient for long-term measurements (Table 2
), only one antiserum (92128) displayed a specificity acceptable
for the plasma measurement of CCK. In addition, it also binds the
nonmammalian CCK homologs, cionin and caerulein, with full potency
(Table 2
).
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specificity characteristics of antiserum 92128
The epitope for antiserum 92128 was delineated on the basis of the
reactivity with related peptides (Table 2
). The specificity was then
corroborated by measurements of tissue and plasma extracts (Figs. 2
and
3). The results confirmed that antiserum 92128 binds O-sulfated
CCK-8, CCK-22, CCK-33, and CCK-58 (as shown by tryptic cleavage) with
equimolar potency (Table 2
, Figs. 2
and 3
).
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The single most crucial specificity problem for plasma CCK assays is
interference from circulating gastrins. This point was examined in
different ways: (a) Chromatography of plasma extracts
revealed that antiserum 92128 bound no gastrin (data not shown);
(b) no correlation existed between CCK and gastrin
concentrations in human plasma samples covering a wide range of
concentrations (Fig. 4
); and (c) infusion of gastrin-17 into human subjects
did not increase CCK concentrations in plasma. On the contrary, gastrin
induced a decrease in plasma CCK (Fig. 5
). Stripping of the antiserum showed that CCK circulates mainly
as CCK-22 and CCK-8 in rabbit 92128 (data not shown).
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Plasma CCK concentrations in healthy young human subjects in the basal
state were 1.13 ± 0.10 pmol/L (mean ± SE, n = 26),
whereas the peak concentration at 60 min (or later) after onset of the
meal was 4.92 ± 0.34 pmol/L (mean ± SE, n = 26; Fig. 6
).
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ria reliability
The CCK concentration two SD below the binding percentage at
zero binding, (i.e., mean ± SD, 33.5 ± 0.4%, n = 10),
was 0.1 pmol/L, which was defined as the detection limit of the assay.
According to Ekins and Newman (23) this detection limit
corresponds to 50 amol CCK in the assay tube.
The intra- and interassay variation at different concentrations within
the working range of the assay ranged between 5% and 15% (Table 3
).
|
Accuracy evaluations by the addition of CCK-peptides to plasma, the dilution of plasma samples with high concentrations of endogenous CCK, and the mixing plasma samples with high and low concentrations of CCK showed a high degree of correlation with deviations <15% from the expected concentrations.
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Discussion |
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TopAbstractIntroductiondefinition of CCK peptidesMaterials and MethodsResultsDiscussionReferences |
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). Therefore, the poor results
with immunization against small CCK-peptides led to the design of a
CCK-12 analog corresponding to O-sulfated CCK-10 extended at the
N-terminus with a diglycine bridge for carrier coupling–the idea
being that coupling at the N-terminus would inevitably produce
antibodies directed towards the amidated C-terminus (common also for
gastrin) but at the same time expose the CCK-specific sequence 3–5 of
CCK-10 (Asp-Tyr (SO
3-)-Met-; see Fig. 1
). In other words, this hapten should offer a chance for the
production of an antibody specific for the entire bioactive
CCK-site–the sulfated heptapeptide amide sequence. For theoretical
reasons (10), however, such a chance should be small because
antibody combining sites usually have a size corresponding to the
epitopes of five or six small amino acid residues. Therefore, the
second precaution to be taken was to immunize a larger number of
animals to increase the chance of obtaining an antiserum with the
desired specificity. This explains why series XI was composed of 16
rabbits. It may seem odd to describe the development of a RIA for a gut hormone in the 1990s. Such studies were generally reported in the 1970s and early 1980s (for a review, see reference 33). But the problems in developing a reliable CCK-RIA for plasma measurements have by far exceeded those of other gut hormones and of peptide hormones in general (4).
Since 1969, various reports have claimed to measure CCK in plasma
(6)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63). Meaningful measurements, however,
were not reported until the mid-1980s
(6)(47)(52)(53)(57)(58)(59)(60)(61)(62);
most of the antisera used in these assays suffer shortcomings. Some
antisera bind gastrins to such a degree
(51)(54)(59), that apparent
hypercholecystokininemia has been assumed to occur during clinical
hypergastrinemias and during infusion of gastrin. Thus, the phenomenon
of gastrin-suppressed CCK secretion (Fig. 5
) has thus far escaped
detection. Moreover, such antisera cannot be used in the search for
CCK-producing tumors. Another shortcoming has been that too few
bleedings, low titer, and/or suboptimal assay technology have limited
the amounts of antiserum available for distribution so that only a few
laboratories have successfully measured CCK in plasma in a reliable
manner.
Another shortcoming has been the variable extent to which the molecular
forms of CCK in plasma have been measured. This again has contributed
to the confusion about the molecular nature of CCK in plasma
(5)(6)(26)(31)(42)(43)(48)(61)(63)(64)(65)(66)(67)(68)(69)(70)(71).
The molecular pattern reported thus far has varied considerably both in
man
(6)(42)(43)(48)(61)(63)(64)(65)(66)(67)
and between mammalian species. Hence, the evidence in favor of
predominance of either CCK-8 (42)(43) or CCK-58
(26)(64) is declining, and a pattern of mixed
occurrences of CCK-33, CCK-22, and CCK-8 is emerging (Fig. 3
). Even in
acidified human plasma, CCK-58 predominated neither in the basal state
nor after a meal (Fig. 3
). In addition, the use of different analytical
principles in the earlier studies, such as RIAs with different
antisera, subtraction assays, combined RIA and enzyme assays, and
bioassays have contributed to the confusion.
As mentioned in the introduction, a single bioassay has met the reliability demands for accurate plasma CCK measurements (5)(6). However, the complexity, labor-intensiveness, and costs for this bioassay measurements have precluded broader use. Therefore, the present RIA using antiserum 92128 should open avenues for new studies of the old hormone, CCK.
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Acknowledgments |
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References |
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TopAbstractIntroductiondefinition of CCK peptidesMaterials and MethodsResultsDiscussionReferences |
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-amidated hormones by radioimmunoassay of glycine-extended peptides after trypsin-carboxypeptidase-B cleavage. Anal Biochem 1986;152:119-126.
[ISI][Medline]
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) and after (
) incubation of each fraction with trypsin. The
elution positions of known molecular forms of CCK are indicated by
arrows. Similar chromatographic patterns were obtained with
extracts of human jejunal mucosa (data not shown).
