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Original Studies |
Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, 2100 Copenhagen, Denmark
Address all correspondence and requests for reprints to: Dr. J. F. Rehfeld, Department Clinical Biochemistry (KB 3011), Rigshospitalet, DK-2100 Copenhagen, Denmark. E-mail: rehfeld{at}rh.dk
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Abstract |
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 Top Abstract IntroductionCCK nomenclatureMaterials and MethodsResultsDiscussionReferences |
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51%) and postprandially (57%), whereas CCK-22 is
the second most abundant (34% and 30%, respectively). In contrast,
CCK-58 is less abundant in human intestines (18%) and plasma
(11%). Its predominance in feline intestines, however, was
confirmed. Hence, the results show a significant species variation and
emphasize the necessity of highly specific and well characterized
assays in molecular studies of CCK. |
Introduction |
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TopAbstractIntroductionCCK nomenclatureMaterials and MethodsResultsDiscussionReferences |
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CCK was first identified in extracts of porcine small intestine as a carboxyamidated and tyrosyl O-sulfated peptide containing 33 amino acid residues (CCK-33) (7). Subsequent studies have shown that pro-CCK is processed at several mono- and dibasic sites to release bioactive forms of different lengths. To date these include CCK-83, CCK-58, CCK-39, CCK-33, CCK-22, and CCK-8, that are all carboxyamidated and O-sulfated (5, 6, 8, 9, 10, 11, 12, 13) and, hence, ligands for the CCK-A receptor. There is general agreement that the predominant form of CCK in central and peripheral neurons is CCK-8 (for reviews, see Refs. 14 and 15). In contrast, there have been remarkably diverging opinions about the main form in plasma and intestinal tissue. The suggestions have varied from CCK-8 to CCK-58 (16, 17, 18, 19, 20, 21, 22, 23).
To understand the function of a hormone, it is essential to know the molecular form in which it circulates. For heterogeneous hormones at least the predominant form in plasma should be known. In this regard it is amazing that consensus still remains to be reached about the molecular nature of CCK in plasma. Much of the problem appears to be due to variations in technology and assay quality (for review, see Ref. 24). Two laboratories have repeatedly reported that CCK-58 is the major hormonal form of CCK in several mammals, including man (22, 25, 26). Their claim has gained widespread acceptance even though it is at odds with observations in other laboratories (18, 20, 21, 27). To settle the discrepancy, we have now studied the molecular pattern of CCK in biopsies of human small intestine and in plasma sampled in fasting and fed normal human subjects using a new assay that measures the bioactive forms of CCK with equimolar potency. Moreover, this assay does not react with the homologous hormone, gastrin (28).
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CCK nomenclature |
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TopAbstractIntroductionCCK nomenclatureMaterials and MethodsResultsDiscussionReferences |
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). The largest bioactive
form is CCK-83, which corresponds to the amidated sequence 1–83 of
pro-CCK (13). This sequence is cleaved at five or more
monobasic sites to release CCK-58, -39, -33, -22, and -8, all of which
have the same C-terminal heptapeptide amide sequence
(-Tyr(SO3-)-Met-Gly-Trp-Met-Asp-PheNH2),
that also is the minimal epitope necessary for receptor binding.
Binding to CCK-A receptors requires that the tyrosyl residue of the
heptapeptide amide is O-sulfated, whereas CCK-B receptors do
not discriminate sulfated from nonsulfated CCK peptides or CCK from
gastrin peptides (for review, see Ref. 29).
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Materials and Methods |
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TopAbstractIntroductionCCK nomenclatureMaterials and MethodsResultsDiscussionReferences |
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Biopsies of human jejunal or ileal mucosa were obtained from the Department of Surgical Gastroenterology, and feline jejunal mucosa from anesthetized cats was obtained from the Department of Experimental Pathology, Rigshospitalet (Copenhagen, Denmark). The biopsies of human small intestine were histologically normal jejunal or ileal mucosa from resections for midgut carcinoid tumors. The tissue samples were immediately frozen in liquid nitrogen. The biopsy collection was approved by the local ethics committee. The frozen tissue was cut into pieces of a few milligrams, boiled in water (10 mL/g tissue) for 20 min, homogenized, and centrifuged at 10,000 x g for 30 min at 4 C. The supernatant was decanted (neutral extract), and the pellet was then redissolved in ice-cold acetic acid (10 mL/g), left at room temperature for 20 min, and centrifuged as described above (acid extract). The extracts were stored at -40 C until analysis.
Plasma for CCK quantitations
Blood samples were collected into chilled tubes containing 3.9 µmol ethylenediamine tetraacetic acid (EDTA)/mL blood. Within 30 min, the samples were centrifuged at 3000 x g 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 2 vol 960 mL/L ethanol on a whirlmixer for 10 s. The mixture was then centrifuged for 30 min at 1200 x g, and the supernatant was decanted and evaporated at 37 C in a Speed-Vac concentrator (SVC 200 H, Savant Instruments). 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 five healthy females and males (mean age, 36 yr) after an overnight fast. The meal consisted of an omelet (two eggs mixed with 10 g flour, 25 mL cream, salt, and pepper) with two slices of bacon, 250 mL orange juice, 250 mL milk, 250 mL 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; mean age, 31 yr) 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 (30) 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)/5 mL blood (acid sample). Immediately after centrifugation, 50 mL of the neutral plasma samples were extracted directly on Sep-Pak (Waters Corp., Milford, MA) cartridges, and 50 mL of the acidified plasma were poured slowly into 150 mL 20 g/L trifluoroacetic acid (TFA) under constant stirring (23, 28). This mixture was extracted on Sep-Pak C18 cartridges prewashed with 10 mL 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 at a flow rate of 1 mL/min. After the cartridge was washed with 10 mL 13 mmol TFA/L, the CCK peptides were eluted by 2 mL 800 mL/L ethanol containing 13 mmol TFA/L. Evaporation of the eluates was performed as previously 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 BSA 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 [125I]albumin and 22NaCl to indicate void (Vo) and total (Vt) volumes.
Enzyme analysis
To measure the immunoreactivity of the larger endogenous forms of CCK, chromatographic fractions of the jejunal tissue extracts were also measured after tryptic cleavage. Each fraction was incubated with trypsin (100 mL/L trypsin-L-p-tosylamino-2-phenylethyl chloromethyl ketone; Worthington Biochemical Corp., Freehold, NJ) for 30 min at 20 C. The cleavage was terminated by boiling the fraction for 10 min. Similarly, tissue extracts were cleaved with trypsin before chromatography to ensure their molecular nature. The principles and performance of the tryptic analysis were reported in detail previously (31).
CCK measurements
The measurements of CCK in tissue extracts, plasma, and
chromatographic fractions were performed with RIA using a panel of
eight high titer CCK antisera specific for the C-terminal 
-amidated
sequence of CCK as well as an antiserum specific for the N-terminus of
human CCK-22 and an antiserum specific for CCK glycine-extended at the
C-terminus. Characteristics of these antisera are presented in Tables 1 and 2.
O-Sulfated CCK-8 and CCK-22 were used as standards and in
monoiodinated form as tracer labeled by the Bolton-Hunter technique
(32). Details of antibody production and assay
characteristics were previously reported (28, 32, 33, 34, 35). It
should be noted that the antiserum library includes Ab 92128. As
previously demonstrated (28), Ab 92128 displays a unique
specificity, comprising equimolar binding of all CCK-A receptor ligand
forms of CCK regardless of size and length, and at the same time it
does not bind any gastrins. The CCK assay using Ab 92128 consequently
quantitates the different molecular forms of CCK accurately. Also the
use of Ab 89009 (directed against the N-terminus of human CCK-22) to
measure CCK precursors (34) and Ab 3208 to measure
glycine-extended CCKs should be noted (35).
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Control measurements of related gastrin peptides were performed by RIA using antiserum 2604, which binds all carboxyamidated gastrins (gastrin-71, -34, -17, and -14) with equimolar potency regardless of their degree of sulfation. Antiserum 2604 does not bind any CCK peptide (33).
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Results |
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TopAbstractIntroductionCCK nomenclatureMaterials and MethodsResultsDiscussionReferences |
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-amidated C-terminus
CCK-58-like peptides in chromatographic fractions of the
neutral and acid extracts of the four biopsies (i.e. eight
individual extract pools) were assayed in five different dilutions. As
shown in Table 2
, the assays using C-terminal specific antisera
measured the content of CCK-58-like peptides differently. The assay
using Ab 92128 have been shown to measure CCK-58, CCK-33, CCK-22, and
the standard peptide (CCK-8) with equimolar potency (28).
Therefore, the results obtained using this antiserum are assumed to be
accurate. Table 2 then shows that the assays employing antisera 92127
and 92138 overestimate the amount of CCK-58, whereas the assays
employing antisera 8007 and 92132 measures CCK-58 with almost the same
potency as Ab 92128. Finally, the assays using antisera 2609, 2717 and
92136 underestimate the amount of CCK-58 in the jejunal mucosa.
CCK in extracts of jejunal mucosa
When measured with the specific CCK assay that binds the four main
forms of CCK with equimolar potency (using Ab 92128), the human jejunal
mucosa contained, as shown in Table 3, a
total of 37.7 ± 11.9 pmol bioactive CCK/g tissue (mean ±
SEM; n = 5); bioactive CCK were products of pro-CCK
that are both carboxyamidated and O-sulfated. In addition,
the mucosal extracts contained, on the average, 1.9 pmol/g tissue of
the immediate precursor, the glycine-extended CCK, and 56.1 pmol/g
tissue of even less mature processing-intermediates and pro-CCK. Tiny
amounts of amidated gastrin (<1 pmol/g) were also detected in the
control measurements (Table 3). Chromatography revealed a molecular
pattern as shown in Fig. 2. Of the
amidated and O-sulfated bioactive forms, the longer CCK-58-
and CCK-33-like peptides were extracted under acidic conditions,
whereas the neutral extracts contained mainly CCK-22- and CCK-8-like
forms (Fig. 2, left). CCK-33-like peptides were predominant
and more abundant than CCK-58- and CCK-22-like peptides (Fig. 2 and
Table 4). Nonamidated pro-CCK and/or
processing intermediates occurred mainly in the neutral intestinal
extract (Fig. 2, right). As they were measured using an
assay against glycine-extended CCK after in vitro cleavage
of the chromatographic fractions with trypsin and carboxypeptidase B,
the chromatography suggests that pro-CCK is cleaved at four basic sites
(or more) in its N-terminal sequence.
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The concentrations of carboxyamidated and O-sulfated
CCK in plasma rose 5-fold after a protein-rich meal, i.e.
from 0.9 to 4.6 pmol/L (Table 5). As
shown in Table 6 and Fig. 4 (left), bioactive CCK eluted
by chromatography in peaks corresponding to CCK-58, CCK-33, CCK-22, and
CCK-8. Of these, the CCK-33-like peptides predominated, and the
CCK-22-like form was the second most abundant. CCK-58 and CCK-8
constituted minor fractions of the plasma CCK regardless of whether
neutral or acid extractions were examined chromatographically (Table 6).
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, right). As in the intestinal mucosa, the nonamidated
processing intermediates of pro-CCK in plasma eluted in four major
peaks. The exact molecular identities of these peaks were not examined
in this study. |
Discussion |
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TopAbstractIntroductionCCK nomenclatureMaterials and MethodsResultsDiscussionReferences |
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35%; Table 4 and Fig. 2)
and plasma (53%; Table 6 and Fig. 4) in man. As mentioned
previously, it is possible that a small fraction of what here has been
characterized as CCK-33 is, in fact, CCK-39 (11).
The tissue extracts also contain substantial amounts of CCK-8 (31%),
which contrasts with the lower percentage of CCK-8 in plasma (10%).
We attribute this discrepancy to a difference in the clearance rates of
circulating CCKs. Hence, the short CCK-8 peptide is cleared
considerably faster from plasma than the longer CCK-33
(36). The low fraction of CCK-58-like peptides in human
plasma (10%) cannot be explained by a rapid metabolism of CCK-58.
On the contrary. If carboxyamidated CCK peptides are released from the
I cells to plasma in ratios similar to those found in the extracts of
intestinal tissue, a high fraction of CCK-58 should be expected in
peripheral plasma, because the long CCK-58 peptide, in analogy with
other heterogeneous peptide systems (37, 38), survives
longer in circulation than shorter forms. Other mechanisms, therefore,
have to be considered to explain the discrepancy between the abundance
of CCK-58 in tissue and that in plasma. Such explanation might be that
I cells contain a mixture of immature and mature secretory granules,
and that the immature granules contain a larger fraction of the less
processed CCK-58 than mature granules. Accordingly, the prohormone
convertases (PC 1/3, PC 2, and other endoproteases) have had more time
for truncation of the peptides in mature granules. As a larger fraction
of mature granules is secreted upon stimulation, the percentage of
CCK-58 released from I cells is presumably lower than the percentage in
tissue extracts. Notably, in vitro degradation at neutral pH
cannot explain the low fraction of CCK-58 in plasma, as the fraction of
CCK-58 in blood samples drawn in acidified vials contained an even
lower percentage of CCK-58. Hence, our data cannot support the
contention of rapid in vitro cleavage at neutral pH
(22).
One of the new results of this study is the demonstration of large quantities of biologically inactive processing intermediates in both tissue and plasma. Due to the nature of the analysis used for their demonstration, i.e. measurement of glycine-extended CCK after trypsin and carboxypeptidase B cleavage, these long nonamidated polypeptides are processing intermediates, which are extended beyond Gly84 at the C-terminus of pro-CCK. In other words, they are not simple N-terminal desocta or desnona fragments of longer bioactive forms of CCK. Such inactive fragments are also present in tissue and plasma (23, 39), but they have not been measured in this study. The occurrence of substantial amounts of nonamidated CCK processing intermediates in human plasma has been corroborated by processing-independent analysis (40).
The debate about the molecular nature of CCK in plasma, in particular the nature of CCK in human plasma, during the last decade has to a large extent been a debate about CCK-58. In other words, is CCK-58 indeed the predominant hormonal form of CCK in man (for review, see Ref. 24)? It is by now generally agreed that the molecular pattern of CCK in plasma varies among mammals. Hence, plasma from pigs, rats, and rabbits contain mainly CCK-22 and CCK-8 (23, 24, 41), whereas canine plasma has been claimed to harbor mainly CCK-58 (30). In man, however, the reports have been particular controversial. Eysselein, Reeve and co-workers have in analogy with their canine studies (30) maintained that CCK-58 is the major form in human plasma (22), whereas others have found human plasma to contain a heterogeneous mixture, with CCK-33 and CCK-22 being the major forms (18, 21, 27, 28, 42).
Three explanations have been offered to explain the discrepancy concerning CCK-58 in plasma. First, it was suggested that the amount of CCK-58 in plasma has been underestimated, because of specific in vitro degradation at neutral pH (22). Therefore, a procedure involving acidification during sampling and processing of blood was proposed to prevent degradation (22). Even strict adherence to the proposed procedure, however, did not increase the amount of CCK-58 in human plasma. It only decreased the extraction efficiency for the shorter forms, but still not to a degree making CCK-58 the predominant form. Thus, acid extraction results in false low and unequal extraction of different CCK peptides in circulation. Specific in vitro degradation of plasma CCK-58, therefore, cannot explain the discrepancy.
The second explanation suggests that CCK-58, in contrast to other forms of CCK, is bound less well to CCK receptors and antibodies specific for the common C-terminal epitope (43, 44). Accordingly, Reeve et al. suggested that CCK-58 has a structure in which the N-terminal part shields the C-terminal bioactive sequence from receptor or antibody binding (44, 45). Such shielding should reduce the detection by bioassays and immunoassays unless the CCK-58-like material is exposed by tryptic release of CCK-8 (45). It is difficult to know whether the shielding idea is correct. It is, however, an old empirical observation that some C-terminal-directed antibodies may have a lower affinity for N-terminally extended peptides. This was described 2 decades ago for the longer forms of CCK and its homolog gastrin (31, 46). At that time, trypsination was also proposed to ensure accurate immunochemical quantitation of larger CCKs (31). Some antibodies do, however, bind the longer forms with the same affinity as shorter forms (28, 33, 46). Thus, the new antiserum, Ab 92128, binds CCK-58, CCK-33, and CCK-22 with the same affinity as CCK-8 (28). Shielding, therefore, does not interfere with measurements based on Ab 92128, which, as shown in this study, discloses that CCK-58 is only a minor form of CCK in human plasma and tissue, but is a major form in the feline jejunum. In human plasma CCK-33 and -22 are the two most abundant CCKs.
The third explanation addresses some technological problems in the studies, which have proposed CCK-58 to be the major CCK component in human plasma (22, 30). As emphasized previously (24, 28, 47, 48) measurement of CCK in plasma is unusually difficult for several reasons, including extensive C-terminal homology between CCK and gastrin, low plasma concentrations of CCK (>10-fold lower than those of gastrin), and a high degree of molecular heterogeneity for both CCK and gastrin. For practical purposes, plasma CCK assays therefore have to be sensitive RIAs specific for the C-terminus of CCK, but without cross-reactivity with gastrin. The proponents of high levels of CCK-58 in human plasma, however, used subtraction assay systems (22, 30). Subtraction based on combinations of a C-terminal assay, which measures all the carboxyamidated CCKs and gastrins, a gastrin-specific assay, and/or high pressure liquid chromatography separation of the molecular forms of gastrin and CCK may in theory provide accurate measurements, but in practice they never do (see also arguments in Ref. 47). First, both CCK and gastrin circulate in forms of different lengths and degrees of amino acid derivatization. The reactivity of all circulating forms of CCK and gastrin therefore has to be tested with both the cross-reacting and the gastrin-specific assay. None of the laboratories employing subtraction assays has to date reported such specificity evaluations. For instance, neither sulfated human gastrin-34, sulfated human gastrin-14, nor sulfated gastrin-6 has been tested in RIAs or high pressure liquid chromatography systems employed in subtraction assays. Second, since gastrin circulate in concentrations 10–50 times above those of CCK, even a minor gastrin component may easily appear as a major CCK component. Third, identification by reverse phase HPLC of the various molecular forms of CCK and gastrin requires caution. Minute modifications by, for instance, methionyl oxidation or conservative amino acid substitutions among species (i.e. man vs. pig) may change the elution position of sample or calibrator peptide.
Taken together, evaluation of available data indicates that CCK-58 is not the major form of CCK in human plasma. The predominant form is CCK-33, and CCK-22 is the second most abundant. Moreover, the data suggest that subtraction assay technology cannot be used to characterize CCK in plasma. A sensitive and specific bioassay has been shown to provide accurate plasma CCK results (18), but it is too labor-intensive, costly, and inconvenient. Therefore, only sensitive and specific CCK RIAs that recognize all the molecular forms of CCK with similar potency can be used to characterize CCK in human plasma.
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Acknowledgments |
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Footnotes |
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Received May 16, 2000.
Revised September 5, 2000.
Accepted September 15, 2000.
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References |
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TopAbstractIntroductionCCK nomenclatureMaterials and MethodsResultsDiscussionReferences |
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-amidated hormones by radioimmunoassay of
glycine-extended peptides after trypsin-carboxypeptidase B
cleavage. Anal Biochem. 152:119–126.[CrossRef][Medline]
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) and neutral
(•) extracts of human jejunal mucosa. The extracts were applied to
Sephadex G-50 superfine columns (1 x 100 cm) and eluted with 0.02
mol/L sodium veronal, pH 8.4, containing 1 g/l BSA. The chromatographic
elutions were monitored with the CCK-specific RIA using antibody 92128.
The elution positions of known molecular forms of CCK are indicated.
The elution of the large nonamidated processing intermediates
(right) was monitored by a RIA specific for
glycine-extended CCKs (using Ab.3208) after each fraction was
incubated with trypsin and carboxypeptidase B. The specificity was
subsequently corroborated by measurement of the enzyme-treated
fractions with the human CCK-specific RIA (using Ab89009)
(
