E. Werle

Fluorometric assay of histamine in tissues and body fluids: Choice of the purification procedure and identification in the nanogram range

ZusammenfassungVerschiedene Isolierungsverfahren für Histamin zur fluorimetrischen Bestimmung wurden geprüft. Für Histamingehalte von mehr als 1 μg/g Gewebe oder Milliliter Vollblut eignet sich sowohl die Butanolextraktion als auch die Ionenaustausch-Chromatographie an Dowex 50W-X8, für Vollblut des Hundes nur das letztere Verfahren, für Plasma und Magensaft des Menschen nur die Kombination der beiden Isolierungsmethoden. Histamin wurde identifiziert durch Dünnschicht-Chromatographie auf Cellulose, Umsatz durch angereicherte Diaminoxydase und Histaminmethyltransferase, durch die Fluorescenzspektren nach Kondensation mit o-Phthaldialdehyd und durch seine biologische Aktivität. Die Histamingehalte in Geweben und Körperflüssigkeiten des Menschen und einiger Versuchstiere werden mitgeteilt.SummarySeveral procedures for the isolation of histamine from tissues and body fluids were examined. For the fluorometric assay of histamine concentrations higher than 1 μg/g tissue or ml blood the extraction into n-butanol as well as the cation-exchange chromatography on Dowex 50W-X8 proved suitable. The determination of histamine in whole blood of dogs gave valid results only after chromatography on Dowex 50, in human plasma and gastric juice only after the combined application of both purification methods. Histamine was identified by thin-layer chromatography on cellulose, by inactivation in the presence of purified diamine oxidase from pig kidney and purified histamine methyltransferase from guinea-pig brain, by the biological activities and by the fluorescence spectra of the condensation product between histamine and o-phthaldialdehyde. Histamine concentrations in tissues and body fluids of man and some laboratory animals are reported.

Biochemical and histochemical studies on the distribution of histamine in the digestive tract of man, dog and other mammals.

Summary1.The distribution of histamine was determined in tissues of the digestive tract of man, dog, pig, cow, and sheep, especially in the oral mucosa, stomach, gallbladder, and pancreas.2.After treatment with compound 48/80, histamine was released from the frenulum linguae, soft palate, tongue, and thyroid gland of dogs, but not from the vestibulum oris, hard palate, pharynx, oesophagus, stomach, and pancreas (experiments in the dog). The release of histamine from the tongue showed regional differences and was lowest in the root and highest in the tip.3.A parallelism could be shown between the histamine content and the mast cell density in different parts of the tongue, stomach and in the soft palate of untreated dogs and dogs treated with 48/80. The mast cells in the gastric mucosa could be characterized by their staining properties as “atypical” mast cells, whereas those in the musculature of the tongue were “typical” mast cells.4.The histamine content of the single mast cell was similar in all tissues (3.2 pg/ cell in the tongue, 3.3 in the stomach, 4.8 in the soft palate and 3.4 in the submaxillary gland). Only the mast cells in the fundic mucosa showed a significantly lower histamine content (1.9 pg/cell). The mast cells of the fundus and body of the stomach of the dog seemed to store histamine which was released by 48/80.5.A new classification of histamine stores is proposed: “unspecific mast cell stores” and “tissue specific stores”.

Zur Physiologie der Mastzellen als Träger des Heparins und Histamins

ZusammenfassungAn Hand von Fermentstudien, pharmakologischen Histaminbestimmungen, Papierchromatographie, Dialysierversuchen und Fällungsreaktionen wurde gezeigt:1.Mastzellenreiche Gewebe besitzen keine oder keine nennenswerte Fähigkeit zur Bildung von Histamin aus Histidin.2.Heparin vermag Histamin und andere Di- und Polyamine zu binden. Das in vitro ermittelte Bindungsverhältnis zwischen Heparin und Histamin entspricht größenordnungsmäßig dem Mengenverhältnis, in welchem Heparin und Histamin aus Geweben extrahierbar sind.3.Di- und Polyamine lösen am frisch isolierten Meerschweinchendarm eine histaminartige Kontraktion aus, die auf eine Verdrängung des Histamins vom Heparin der Mastzellen der Darmmucosa zurückgeführt wird. Es wird der Schluß gezogen, daß das Histamin der Mastzellen und so der weitaus überwiegende Teil des Gewebshistamins als Heparinat vorliegt.Zusammenhänge unserer Befunde mit der Histamin-und Heparinausschüttung in vivo werden diskutiert.

Zur Kenntnis der blutdrucksenkenden Wirkung des Trypsins

ZusammenfassungKristallisiertes Trypsin legt aus Blutserum auf Grund seiner proteolytischen Wirkung außer dem Bradykinin noch eine weitere blutdrucksenkende Substanz frei. Nach ihren chemischen Eigenschaften, ihren pharmakologischen Wirkungen und ihrem Verhalten gegenüber Inaktivatoren des Kallikreins und des Trypsins ist die Substanz identisch mit Kallikrein, wie es mit Hilfe früher beschriebener Verfahren aus Blutserum gewonnen werden kann. Ein Verfahren zur quantitativen Bestimmung des Serum-Kallikreinogens wird angegeben. Das Vorkommen des Kallikreinogens im Blutserum des Menschen und verschiedener Tiere sowie in Organen wurde untersucht. Nur im Pankreas wurde das Kallikreinogen in größeren Mengen angetroffen.

Specific histidine decarboxylases in the gastric mucosa of man and other mammals: Determination, location and properties

Abstract Relatively high activities of the specific histidine decarboxylases were found in the gastric mucosa of men, monkeys, pigs, cows, dogs, cats, guinea-pigs, rabbits and rats. Some improvements of the assay of specific histidine decarboxylase were necessary, before these enzymes could be demonstrated in all stomachs investigated. The specific histidine decarboxylase could also be shown in the human gastric carcinomas. By pH-optima, substrate optima, K m , inhibition by α-methylhistidine but not by α-methyldopa, inhibition by benzene and activation by pyridoxal-5′-phosphate the histidine decarboxylases in the gastric mucosa could be characterized as specific histidine decarboxylases. The enzyme in the stomach of guinea-pigs has been purified 22-fold by ultracentrifugation and gelfiltration on Sephadex G 100. The demonstration of the specific histidine decarboxylases (isoenzymes) in the gastric mucosa of numerous mammals has some importance for the hypothesis of a physiological function of histamine as a chemostimulator of gastric secretion. Problems of the nomenclature of histidine decarboxylases are discussed. The terms “acid” and “alkaline” histidine decarboxylases are proposed.

KALLIKREIN, KALLIDIN, KALLIKREIN INHIBITORS

For a long time we have been convinced that kallikrein owes its pharmacological activities to its capacity to form kallidin from kallidinogen. Other enzymes like trypsin have been found in the meantime which also form polypeptides which until recently could not be distinguished from kallidin. If the kinin-forming capacity of the kallikreins depends to some degree on the presence of one of these enzymes in the kallikrein preparations, the ability to develop kallidin would decrease during purification. I would like to review first the procedures for the purification of the different kallikreins.

Histamine and Histamine Methyltransferase in the Gastric Mucosa of Man, Pig, Dog and Cow

SummaryHigh histamine concentrations and histamine methyl transferase activity were demonstrated in the gastric mucosa of man, dog, pig and cow. Modified methods for the determination of histamine and histamine methyltransferase were developed. Histamine was identified by its fluorescence spectrum, by thin-layer chromatography in 8 different solvent systems and by bioassay. Histamine methyltransferase from pig antral mucosa was purified 6-fold by ultra-centrifugation and fractional precipitation by ammonium sulfate. ItsKm for histamine as substrate was 2.3×10−5 M, for S-adenosylmethionine 4.3×10−5 M, the pH-optimum was found to be pH 7.4. Nicotinamide in concentrations up to 1×10−2 M had no effect on the activity of the enzyme.

Über ein blutdrucksteigerndes Prinzip in Extrakten aus der Glandula submaxillaris der weißen Maus

ZusammenfassungExtrakte aus der Submaxillaris der weißen Maus verursachen nach i.v. Injektion bei Hund, Ratte und Maus eine sehr starke, lang anhaltende Blutdrucksteigerung. Wiederholte Injektionen führen rasch zur Tachyphylaxie. Bei dem wirksamen Prinzip dürfte es sich um eine bisher unbekannte Substanz von Eiweißcharakter und sehr hoher spezifischer Wirkung handeln.

PHYSIOLOGIC, PHARMACOLOGIC, AND CLINICAL ASPECTS OF PROTEINASE INHIBITORS

It is generally assumed that the pharmacological action of a biologically active substance, which is synthesized by the body, is, in some way, paralleled by its physiological function. Provided that selective inhibition of distinct proteolyticesterolytic enzymes is the sole function of naturally occurring proteinase-inhibitors and that synthesis of these inhibitors is not merely a rudimental and now meaningless genetic cell-function, the physiological function of these inhibitors is selective inhibition of enzymatic-proteolytic processes. They probably also protect tissues against undue activation of proteolytic enzymes. Inhibitors, which only occur in certain species and, even in these, only in certain organs, and which do not occur in interstitial and other body fluids (TABLE l ) , probably inhibit and regulate enzymatic reactions at an intracellular level. This applies to the polyvalent trypsin-kallikrein-inhibitor from bovine organs, which occurs also in secretory organs like salivary glands and pancreas, but is not found in secretions of these organs nor in other body fluids (Frey et al., 1950). Stimulation-e.g., by pilocarpin-which usually results in an unphysiological high secretion rate, does not bring about any secretion of inhibitor. Following i.v. injection of pure preparations of trypsin-kallikrein-inhibitor from bovine organs up to 10,000 KIU/kg in cattle, dogs, and hogs, we could not demonstrate the inhibitor in saliva nor in pancreatic juice. Furthermore, the inhibitor reaching salivary glands and pancreas via the bloodstream did not accumulate in these organs. On isolated perfusion of dog pancreas, addition of Trasylol@ to the perfusion fluid does not. impair secretion of zymogens or insulin. From our former experiments, we know that intravenous Trasylol is distributed to the various organs according to their blood supply (FIGURE 1 ) . Following injection of 12,000 KIU/kg in rats and 3500 KIU/kg in dogs, inhibitor concentration in the liver attains a temporary maximum after 30 min. After a latent interval of about 10 min, inhibitor concentrations begins to increase in the kidneys. One hour after injection, the inhibitor content in renal tissue is higher than in the liver (50% and 36% respectively of the total amount). The blood still contains less than 10% of the injected inhibitor amount. About five hours after injection, almost the entire amount of inhibitor administered is to be found in an active form in the kidneys. The results suggest that the inhibitor undergoes structural change in the liver which condition it for fixation in the kidney. Using tritiumlabeled Trasylol, we could show that, before excretion, the bulk of inhibitor is degraded and is demonstrable in urine as tritium activity only (FIGURE 2) (Trautschold et ul., 1964). As the trypsin-kallikrein-inhibitor occurs selectively in cattle and perhaps more generally in ruminants, a physiological function can be discussed for this species only. From the information available to us, we believe this inhibitor is identical with the inhibitor found by Kunitz (Kunitz et al., 1936). This evidence was neglected by many authors in discussing the meaning of this inhibitor in acute pancreatitis. It may be that the inhibitor has something to do with the more complicated digestion system of ruminants and the growth of bacteria and protozoa in their gastrointestinal tract. (Vogel et al., 1966). Since the inhibitor also

CHEMISTRY AND BIOCHEMISTRY OF PROTEINASE INHIBITORS FROM MAMMALIAN TISSUES

Since uses have been found for naturally occurring proteinase inhibitors in medical therapy, it is essential to find new inhibitors and to simplify their isolation. The previous methods for isolating proteinase inhibitors (Reviews: Laskowski ei al., 1954, 1955; VogeI et al., 1966) of low molecular weight include precipitation of ballast protein and additional chromatographic purification with principally an acid ion exchanger. For the high molecular inhibitors, conventional methods for fractionation of proteins are used, as for example, fractional salting out, distribution, adsorption chromatography, and electrophoresis. All of these methods are nonspecific and, because their repetition is necessary, the yield will be low. Both the biologically active and inactive protein material have similar chemical properties and the active molecules are distinguished only by their ability to combine with enzymes. In order to further distinguish each inhibitor, their selective reaction with distinct enzymes may be used (TABLE 1 ) . Recently, it has been possible to transform enzymes and still retain their biological activity in an insoluble form, as for example, a carboxymethyl cellulose derivative of trypsin (Mitz and Summaria, 1961) or by coupling polytyrosyl trypsin with a insoluble polydiazonium salt derived from a copolymer of an amino acid derivative (Bali-Eli and Katchalski, 1963). Especially suitable for our purpose are copolymers of maleic anhydride and ethylene cross-linking with hexamethylene diamine as prepared by Levin et al. (1964) for trypsin. The €-amino groups of the lysyl residues of the enzyme surface react with the maleic anhydride residues of the copolymer. The cross-linking is affected by the enzyme molecule as well as by the hexamethylene diamine (FIGURE 1 ) . It was demonstrated that the Kunitz-inhibitor is still capable of inhibiting esterolytic activity of the trypsin resin (Levin et al., 1964). We succeeded in using the trypsin method for producing insoluble resins with kallikrein and chymotrypsin. After having proven that the known as well as the newly isolated inhibitors would react with these enzyme resins and that this reaction is reversible, a simple method for isolating inhibitors resulted (FIGURE 2) . In the simplest case, the organ containing the inhibitor is first homogenized in a buffer solution, separated from the insoluble portion by centrifugation, and the supernatant filtered through a trypsin resin column. The protein material may also previously be removed by deproteinization. Remaining impurities not bound to the resin are then removed by a neutral salt solution. The salt serves to limit the colloidal solubility of the resin and to suppress any ion exchanger effect by the carboxylic groups. Elution is done with acid salt solutions. There is no advantage to be gained in adjusting the pH to a value at which the complete dissociation of the inhibitor complex results, as the continual elution produces a constant dissociation of the complex-bound inhibitor. In a single procedure, it is possible to isolate an inhibitor free from impurities which will not react with the trypsin. Important in the production of the enzyme resin is the existing weight ratio between the enzyme and the copolymer ( 5 : 1 ) . This must be regulated so that no