Editorial
`
`On the Measurement of Cholecystokinin
`
`A hormone is a chemical transmitter that is secreted from
`one part of the body and circulates in the bloodstream to
`reach a distant target in another part of the body where it
`exerts its biologic effect. At its inception, this was a
`revolutionary concept that gave birth to an entire physi-
`ological and, later, medical discipline. Early on, hormones
`were discovered by their biologic actions. However, the
`physiology of hormones could not be assessed without
`quantification. Fundamental to the study of hormones
`was the ability to measure concentrations in the blood.
`Although biological assays have been the cornerstone of
`endocrinologic measurements, the development of the
`radioimmunoassay (RIA) completely changed the field of
`endocrinology (1). The attractive features of RIA include
`(a) applicability to most hormones, (b) ease of perfor-
`mance, and (c) relatively low cost, as well as high degrees
`of (d) accuracy, (e) sensitivity, and (f) specificity.
`Cholecystokinin (CCK) was discovered in 1928 on the
`basis of the ability of intestinal extracts to stimulate
`gallbladder contraction in dogs (2). Later it was recog-
`nized that CCK was a potent stimulant of pancreatic
`enzyme secretion (3). However, it was not until 1966,
`when CCK was purified, that the primary sequence was
`determined (4, 5). Early estimates of CCK-like activity in
`blood were based on biological assays such as pancreatic
`secretion or gallbladder contraction. However, these esti-
`mates were fraught with confounding problems that
`existed in whole animals, such as the effects of other
`hormones or neural influences. To circumvent these prob-
`lems, a sensitive and specific in vitro bioassay was devel-
`oped (6), but this method is labor-intensive and cumber-
`some and is not readily available to clinical laboratories.
`Attempts to develop an RIA for CCK had to overcome a
`number of unique challenges. Among these were (a) the
`multiple molecular forms of CCK, (b) amino acid se-
`quence similarity between CCK and gastrin, (c) low blood
`concentrations of CCK, (d) limited peptide availability,
`and difficulties with (e) isotope labeling and (f) peptide
`synthesis. It should not be surprising then, that solutions
`to these many problems were slow in coming.
`CCK was originally identified as a 33-amino acid pep-
`tide (CCK-33); however, since its discovery, multiple
`molecular forms have been described. In several species,
`biologically active forms ranging in size from CCK-83 to
`CCK-8 have been found to exist in intestine, brain, and
`blood (7, 8). All forms are derived from a single CCK gene
`by posttranslational or extracellular processing (9). The
`biologically active portion of the molecule is its amidated
`carboxyl terminus. All forms of CCK larger than CCK-8
`are biologically active. Therefore, to measure physiologi-
`cally relevant CCKs, assays must detect the carboxyl
`terminus of all molecular forms. A major difficulty in
`developing CCK assays has been its structural similarity
`to gastrin. CCK and gastrin comprise a family of gastro-
`intestinal peptides that share an identical carboxyl-termi-
`nal pentapeptide sequence. To develop a specific CCK
`
`RIA that does not cross-react with gastrin, the antisera
`should recognize the tripeptide sequence at the amino
`terminus of CCK-8, which is common to all forms of CCK
`but is dissimilar to gastrin.
`Over the last 20 years, a number of RIAs and a bioassay
`for CCK have been developed (6, 10 –24). Although most
`have not undergone the extensive validation described in
`the current study by Rehfeld (25) in this issue, there is
`general agreement that CCK concentrations in the circu-
`lation are relatively low (in the picomolar range). In
`contrast, plasma concentrations of gastrin are 20–100
`times higher. Thus, even slight antibody cross-reactivity
`with gastrin poses a substantial problem for accurately
`measuring blood concentrations of CCK. Accordingly, the
`sensitivity and specificity of an accurate CCK assay must
`be extremely high.
`Even after the discovery of CCK-33, only limited
`amounts of material were available for raising antibodies.
`The largest bioactive form of CCK that has been described
`is CCK-83 (26). Abundant forms of CCK in tissue and
`blood include CCK-58, CCK-33, CCK-22, and CCK-8.
`Unfortunately, large forms of CCK are difficult to purify
`and still are not readily available. As such, CCK-83 and
`CCK-58 have not been used as standards for most assays,
`and the cross-reactivity with many antibodies is un-
`known. It has even been suggested that larger forms of
`CCK are less immunoreactive than smaller forms, per-
`haps because of tertiary structure (27).
`Sulfation of the tyrosine residue at position seven from
`the carboxyl terminus of CCK is critical for biological
`activity. Because of this, synthesis of moderately large
`forms of CCK had not been possible until recently, and
`these peptides are still not commercially available.
`The final problem in the development of a CCK RIA has
`been difficulty with isotopic labeling of the peptide.
`Oxidative methods to label CCK tended to destroy bio-
`logical activity of the molecule through oxidation of the
`methionine residue in position three from the carboxyl
`terminus. Oxidation of CCK reduces its biological activity
`100- to 1000-fold.
`Identifying the problems in developing a CCK assay is
`one thing, successfully overcoming those problems is
`another. In the current issue of Clinical Chemistry, Rehfield
`has undertaken the ambitious task of developing an
`accurate RIA for measuring blood concentrations of CCK.
`He has tackled each of the problems listed above. Selec-
`tion of a proper antigen for raising specific CCK antisera
`was a critical initial step. A variety of antigens, including
`natural porcine CCK-33 and synthetic nonsulfated
`CCK-33 and -29, as well as synthetic sulfated CCK-4, -12,
`and -13, were all used as immunogens. Two types of
`tracers were used, including CCK-33 labeled by nonoxi-
`dative conjugation and Bolton-Hunter-labeled CCK-8.
`Characteristics of the antisera that were critically evalu-
`ated included the titer, affinity, specificity, and homoge-
`neity of binding kinetics. Seventy-eight rabbits, 29 guinea
`
`Clinical Chemistry 44, No. 5, 1998
`
`903
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`-903-
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`MAIA Exhibit 1012
`MAIA V. BRACCO
`IPR PETITION
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`

`

`904
`
`Editorial
`
`pigs, and 8 mice were immunized. The most successful
`immunization strategy used an immunogen consisting of
`a CCK-12 analog corresponding to O-sulfated CCK-10
`extended at the N-terminus with a diglycine bridge for
`carrier coupling. The resulting rabbit antiserum (Ab.
`92128) was of high titer (.500 000) and bound sulfated
`CCK-8, CCK-22, CCK-33, and CCK-58 with nearly
`equimolar potency and with essentially no cross-reactiv-
`ity with gastrin. This latter point was demonstrated in
`three ways. First, Ab. 92128 did not detect gastrin in
`chromatographically purified plasma extracts. Second,
`there was no correlation between CCK and gastrin plasma
`concentrations in humans. And third, infusion of gas-
`trin-17 into human subjects did not increase plasma CCK
`immunoreactivity. With this RIA, plasma CCK concentra-
`tions averaged ;1 pmol/L under basal conditions and
`increased to ;5 pmol/L after ingestion of a meal. These
`estimates are in the range of other accepted assays.
`The discovery of CCK-58 in tissue and the circulation
`required special treatment to preserve forms larger than
`CCK-33 (28). It was suggested that immediate acidifica-
`tion of plasma was necessary to prevent the in vitro
`degradation of CCK. The current study confirmed that
`larger forms of CCK were detectable only after plasma
`was acidified and that only CCK-22 and smaller forms
`were present in neutral extracts, thus confirming that
`CCK-58 is a major component of CCK-like immunoreac-
`tivity in plasma. Unfortunately, large forms of CCK are
`not available as standards for RIAs, and it still remains to
`be determined whether the immunoreactivity and biolog-
`ical activity of CCK-58 are equivalent to other molecular
`forms. In future studies, these determinations and the
`ability to compare CCK values by various RIAs will
`depend on the manner in which plasma is collected and
`extracted, the antibody that is used, and the epitope to
`which it is directed. These challenges and the lack of CCK
`RIAs for general use are hurdles that remain to be
`overcome. Nevertheless, this report by Rehfeld represents
`an exhaustive and careful attempt to develop the best
`characterized CCK RIA to date.
`
`References
`1. Yalow RS, Berson SA. Assay of plasma insulin in human subjects by
`immunologic methods. Nature 1959;184:1648.
`2. Ivy AC, Oldberg E. A hormone mechanism for gallbladder contraction and
`evacuation. Am J Physiol 1928;65:599 – 613.
`3. Harper AA, Raper HS. Pancreozymin, a stimulant of secretion of pancreatic
`enzymes in extracts of the small intestine. J Physiol 1943;102:115–25.
`4. Jorpes E, Mutt V. Cholecystokinin and pancreozymin, one single hormone?
`Acta Physiol Scand 1966;66:196 –202.
`5. Mutt V, Jorpes J. Structure of porcine cholecystokinin-pancreozymin. Eur
`J Biochem 1968;6:156 – 62.
`6. Liddle RA, Goldfine ID, Rosen MS, Taplitz RA, Williams JA. Cholecystokinin
`bioactivity in human plasma: molecular forms, responses to feeding, and
`relationship to gallbladder contraction. J Clin Invest 1985;75:1144 –52.
`
`7. Eysselein VE, Eberlein GA, Schaeffer M, Grandt D, Goebell H, Niebel W, et al.
`Characterization of the major form of cholecystokinin in human intestine:
`CCK-58. Am J Physiol 1990;258:G253– 60.
`8. Paloheimo LI, Rehfeld JF. A processing-independent assay for human
`procholecystokinin and its products. Clin Chim Acta 1994;229:49 – 65.
`9. Deschenes RJ, Haun RS, Funckes CL, Dixon JE. A gene encoding rat
`cholecystokinin. J Biol Chem 1985;260:1280 – 6.
`10. Schlegel W, Raptis S, Grube D, Pfeiffer EF. Estimation of cholecystokinin-
`pancreozymin (CCK) in human plasma and tissue by a specific radioimmu-
`noassay and the immunohistochemical
`identification of pancreozymin-
`producing cells in the duodenum of humans. Clin Chim Acta 1977;80:305–
`16.
`11. Marshall CE, Egberts EH, Johnson AG. An improved method for estimating
`cholecystokinin in human serum. J Endocr 1978;79:17–27.
`12. Byrnes DJ, Henderson L, Borody T, Rehfeld JF. Radioimmunoassay of
`cholecystokinin in human plasma. Clin Chim Acta 1981;111:81–9.
`13. Calam J, Ellis A, Dockray GJ. Identification and measurement of molecular
`variants of cholecystokinin in duodenal mucosa and plasma. J Clin Invest
`1982;69:218 –25.
`14. Maton PN, Selden AC, Chadwick VS. Large and small forms of cholecysto-
`kinin in human plasma: measurement using high pressure liquid chroma-
`tography and radioimmunoassay. Regul Pept 1982;4:251– 60.
`15. Chang TM, Chey WY. Radioimmunoassay of cholecystokinin. Dig Dis Sci
`1983;28:456 – 68.
`16. Jansen JBMJ, Lamers CBHW. Radioimmunoassay of cholecystokinin in
`human tissue and plasma. Clin Chim Acta 1983;131:305–16.
`17. Himeno S, Tarui S, Kanayama S, Kuroshima T, Shinomura Y, Hayashi C, et
`al. Plasma cholecystokinin responses after ingestion of liquid meal and
`intraduodenal
`infusion of fat, amino acids, or hydrochloric acid in man:
`analysis with region specific radioimmunoassay. Am J Gastro 1983;78:
`703–7.
`18. Izzo RS, Brugge WR, Praissman M. Immunoreactive cholecystokinin in
`human and rat plasma: correlation of pancreatic secretion in response to
`CCK. Regul Pept 1984;9:21–34.
`19. Becker HD, Werner M, Schafmayer A. Release of radioimmunologic chole-
`cystokinin in human subjects. Am J Surg 1984;147:124 –9.
`20. Ohgo S, Takemura J, Oki Y, Nishizono F, Ishikawa E, Yoshimi T, et al.
`Radioimmunoassay of cholecystokinin in plasma. Clin Chem 1988;34:
`1579 – 84.
`21. Hocker M, Schmidt WE, Creutzfeldt W, et al. Determination of plasma
`cholecystokinin (CCK) concentrations by bioassay and radioimmunoassay in
`man. A critical evaluation. Regul Pept 1992;37:255– 69.
`22. Ballinger AB, Clark ML. L-Phenylalanine releases cholecystokinin (CCK) and
`is associated with reduced food intake in humans: evidence for a physio-
`logical role of CCK in control of eating. Metabolism 1994;43:735– 8.
`23. Paloheimo LI, Rehfeld JF. Quantitation of procholecystokinin and its prod-
`ucts in plasma by processing-independent analysis. Clin Chim Acta 1995;
`238:21–33.
`24. Rehfeld JF. The molecular nature of cholecystokinin in plasma. An in vivo
`immunosorption study in rabbits. Scand J Gastroenterol 1994;29:110 –21.
`25. Rehfeld JF. Accurate measurement of cholecystokinin in plasma. Clin Chem
`1998;44:991–1001.
`26. Eberlein GA, Eysselein VE, Davis MT, Lee TD, Shively JE, Grandt D, et al.
`Patterns of prohormone processing: order revealed by a new procholecys-
`tokinin-derived peptide. J Biol Chem 1992;267:1517–21.
`27. Reeve JR, Jr, Eysselein VE, Rosenquist G, Zeeh J, Regner U, Ho FJ, et al.
`Evidence that CCK-58 has structure that influences its biological activity.
`Am J Physiol 1996;270:G860 – 8.
`28. Eberlein GA, Eysselein VE, Hesse WH, Goebell H, Schaefer M, Reeve JR Jr.
`Detection of cholecystokinin-58 in human blood by inhibition of degradation.
`Am J Physiol 1987;253:G477– 82
`
`Rodger A. Liddle
`Department of Medicine
`Box 3913
`Duke University Medical Center
`Durham, NC 27710
`Fax 919-684-8857
`
`-904-
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