Chemistry, isolation and purification of gastrointestinal hormones.

The isolation in the early 1960s (Gregory & Tracy, 1975; Jorpes & Mutt, 1973) from porcine intestinal and gastric antral tissues of the classical gastrointestinal hormones gastrin, secretin and cholecystokinin, the latter having also the properties ascribed to pancreozymin, confirmed the long-suspected peptide nature of these substances. It then seemed-for a time-that the field of gastrointestinal-hormone research had been revealed in its main features and that future work in it would proceed in obvious directions such as the isolation of the above-mentioned hormones from various different species and the isolation of other assumedly known hormones, such as enterogastrone, incretin, villikinin, enterocrinin and others (Greengard, 1948), which still were in the state of physiological principles, something that those isolated had also been for many decades. Some of those expected developments did actually take place; others did not, but unexpected discoveries have convincingly shown that the field is far more complicated than previously believed and that much more work remains to be done before we will recognize all that is in it. All gastrointestinal hormones are still peptides, but this is partly because of a somewhat arbitrary definition, since substances like prostaglandins and biogenic amines also may exert hormone-like actions on organs of the digestive tract. Other definitions, such as a gastrointestinal hormone being biosynthesized, or stored exclusively in cells of the gastrointestinal tract have proved to be untenable (Pearse, 1976). Enterogastrone was transformed from a unique substance to a concept. since several different intestinal peptides have been found to inhibit gastric acid secretion (Gregory, 1967; Johnson & Grossman, 1971). The possibility still remains, however, that some hitherto-unrecognized peptide, or other type of substance, having enterogastrone activity as its main physiological function may exist. Incretin has similarly converted from substance to concept (Creutzfeldt, 1979). Several peptide hormones, such as insulin (Abel, 1926) and also glucagon (Staub et al., 1953), had been isolated by techniques that were developed at the very beginning of peptide-hormone chemistry, namely fractionations of aqueous hormone solutions with organic solvents or neutral salts, isoelectric precipitations of impurities or of the active substances, etc. Applied to gastrointestinal hormones, such techniques had resulted in varying degrees of purification but not in isolation. A retrospective explanation for this undoubtedly is that all gastrointestinal hormones have been found to occur in their tissues of origin in very low concentrations, much lower than that of insulin and glucagon in the pancreas. Isolation became possible when the older separatory techniques had been replaced by, or complemented with newer, more efficient, methodology. Techniques such as chromatography on cellulose-based ionexchangers, countercurrent distribution, and molecular sieving became available in the 1950s and made the progress of the 1960s possible. These methods have remained a mainstay in preparative work in the field until quite recently, when newer techniques, like high-performance liquid chromatography, affinity chromatography, isoelectric focusing, isotachophoresis and others, seem to be turning a page and introducing a new era of accelerated progress. Isolation of gastrointestinal hormones has been greatly facilitated by their relative thermostability, which has made it possible to remove both enzymes and structural tissue proteins at an early stage of the preparative procedures by heat denaturation. Two complementary extraction procedures have been in use for many years. One devised by Gregory & Tracy (1975) for gastrin entails extraction of the tissue with boiling water, followed by adsorption of the peptides from the extract to aminoethylor diethylaminoethyl-cellulose and elution with a weakly alkaline solution (Gregory, 1973). This technique works well for other acidic peptides also (Dockray et al., 1978). presumably because acidic peptides are not adsorbed to tissue proteins, most of which carry a negative charge at neutral pH values (Jorpes, 1968). It may be mentioned that already Edkins (1906) and later Blair ef a/. (1961) had observed that gastrin could be extracted by boiling water. The other technique, worked out in our laboratory (Jorpes & Mutt, 1973), is adapted for basic and neutral peptides, and excludes the very acidic ones. The tissue is briefly boiled in water and then extracted with cold dilute acetic acid. The rationale being that extraction with cold acid should minimize cleavage of acid-labile peptide bonds. The initial boiling does not extract other than small amounts of secretin, as had been found already by Bayliss & Starling (1902). From the extract the peptides are adsorbed to alginic acid and eluted with 0 . 2 ~ H C i . It may be mentioned that the extraction method for substance P that finally led to its isolation from horse intestine (Studer er al., 1973) entailed boiling in only weakly acidified water (Pernow. 1953). Substance P had, however, then already been isolated from bovine hypothalamic tissue by Chang & Leeman (1 970) by using extraction with acid aqueous acetone, i.e. an extraction method similar to that used in work on insulin. Carraway & Leeman ( 1 973) discovered neurotensin in bovine hypothalamic extracts prepared by the same type of extraction procedure they had used for substance P, and the identical peptide has, with the same technique, been isolated also from bovine intestine (Carraway et al., 1978). Enough of extraction technology! Some 15 peptides have by now been isolated from intestinal and gastric tissues and many