Br. J. Pharmac. (1976), 56, 39-41BIOASSAY OF HISTAMINEIN THE PRESENCE OF PROSTAGLANDINSR.J. GRYGLEWSKI & R. KORBUTDepartment of Pharmacology, Copernicus Academy of Medicine, 16 Grzegorzecka, 31-531 Cracow, Poland I An Amberlite XAD-2 column in a heated (37 0C) jacket was incorporated in between two banks of bioassay organs superfused with Krebs bicarbonate solution in cascade. The column removed pro- staglandins El, E2 and F2a and also rabbit aorta contracting substance (RCS) and possibly slow reacting substance of anaphylaxis (SRS-A). 2 The column gave free passage to histamine in concentrations up to 3000 ng/ml. The method described improves the accuracy of histamine bioassay in the presence of prostaglandins.IntroductionThe technique of Vane (1964) has been used for a gassed with 95% 02 and 5% CO2. Krebs solutionsimultaneous bioassay of histamine, slow reacting (37°C) was dripped over the organs at a rate ofsubstance of anaphylaxis (SRS-A), prostaglandins and 2.5 ml/min, or perfused through the isolated lungs ofrabbit aorta contracting substance (RCS) in the sensitized guinea-pigs before reaching the organseffluent from the shocked perfused lungs of sensitized (Piper & Vane, 1969). All substances were infusedguinea-pigs (Piper & Vane, 1969, 1971; Palmer, Piper directly over the organs at a rate of 0.2 ml/min except& Vane, 1973; Piper, 1974). The cat terminal ileum for ovalbumin (1O mg), which was injected into the(Ferreira, Ng & Vane, 1973) and the mepyramine- pulmonary artery. The combined antagoniststreated guinea-pig trachea were used to differentiate (Gilmore, Vane & Wyllie, 1968), minus mepyramine,between histamine and SRS-A, and prostaglandins were infused directly over the tissues. The movementsand RCS were bioassayed by standard procedures of the assay organs (initial load 2-3 g) were recorded(Ferreira & Vane, 1967; Piper & Vane, 1969). with Patons auxotonic levers connected to Harvard We have observed that the peak concentration of transducers (type 386) and a Watanabe multirecorder.histamine released during anaphylactic shock from Amberlite XAD-2 (Keirse & Turnbull, 1973) orperfused guinea-pig lungs, as indicated by the glass wool (in control experiments) were used to fill thedifference between the responses of the antazoline-free column (2 x 5 cm). The column was maintained atand antazoline-treated guinea-pig ileum (1.08 jtg/ml, 37°C by a water jacket and placed between the twos.e. + 0.21, n= 7) is much higher than that determined banks of the assay tissues (Figure 1). Before beingspectrofluorimetrically by the method of Hakanson & used the Amberlite column was washed twice withR6nnberg (1974) (0.23 ,g/ml, s.e. ± 0.04, n= 7). One 100 ml portions of distilled water and then perfusedof the reasons for this discrepancy may be the with Krebs (2.5 ml/min) for 15 minutes.presence of prostaglandins and RCS in the effluent. The following substances were used: prostaglandins Here we describe a technique, which enables E1, E2 and F2a (Upjohn Co.), histaminehistamine to be bioassayed by the guinea-pig ileum in dihydrochloride (Polfa), antazoline methane-the presence of relatively high concentrations of sulphonate (Polfa), mepyramine maleate (May andprostaglandins. Baker) and Amberlite XAD-2 (BDH Chemicals Ltd).Methods ResultsTwo banks of the assay organs, each consisting of a In our experiments the threshold concentrations ofrabbit aortic strip, a rat stomach strip and a guinea-pig prostaglandins E1 and E2 which contracted the ratileum, were superfused with Krebs bicarbonate stomach strip were 0.5-2 ng/ml, and those of pros-solution of the following composition (mM): NaCl taglandin F2a were 2-10 ng/ml. The guinea-pig ileum118.0, KCI 4.7, KH2PO4 1.2, MgSO4 7H2O 1.17, was usually contracted by histamine in concentrationsCaCl2 6H20 2.5, NaHCO3 25.0 and glucose 8.4, of 5-10 ng/ml, and also by prostaglandins El and E2
40 R.J. GRYGLEWSKI & R. KORBUT Perfused Lungs Lungs II at 10-150 ng/ml and also RCS from shocked guinea- lungs 10 min pig lungs are reduced to undetectable levels during one b 750OmV passage through XAD-2. Amberlite absorbs neither histamine (10-3000 ng/ml) nor catecholamines RbALJ (10-100 ng/ml). The guinea-pig ileum pretreated with antazoline (0.5 jig/ml) or with mepyramine (1.0 jg/ml) and situated beneath the XAD-2 column RSS -J* was contracted neither by histamine nor by the effluent from shocked lungs (5 experiments). It seems likely therefore that SRS-A is also absorbed by GPI XAD-2. Using the above method we have found that the Off On effluent from shocked lungs of sensitized guinea-pigsXAD-2 contains histamine in the range 225-700 ng/ml (470 ± 54 ng/ml, mean ± s.e., n = 10) together with a RbA L _J prostaglandin-like substance (43.1 + 3.7 ng/ml pros- taglandin E2 equivalents). RSS Discussion b * *4 U * 4 * E2 H E2 H E2H An Amberlite XAD-2 column removes pros- mgmlr1minr1 30200 50 400 40400 taglandins, RCS and possibly SRS-A, but not OVA OVA histamine from the effluent from the shocked lungs of 10mg 10mg sensitized guinea-pigs, and therefore the guinea-pig ileum situated distal to the column registers the actual Figure 1 Isolated lungs of two sensitized guinea- amount of histamine in the effluent. A similar pigs (lungs I and lungs 11) were perfused with Krebs absorbent column, but filled with aluminium oxide, bicarbonate solution (2.5 ml/minute). The effluent has been proposed by us for the bioassay of pro- was used to superfuse two banks of the assay tissues. staglandins in the presence of high concentrations of Each consisted of a rabbit aortic strip (RbA), a rat stomach strip (RSS), and a piece of guinea-pig ileum catecholamines (Korbut, 1975; Grodzifiska, (GPI). All tissues were blocked with combined Panczenko & Gryglewski, 1975). antagonists minus mepyramine (Gilmore et al., Differential assay of biologically active substances 1968). Calibration amounts (U) of prostaglandin E2 in a mixture is possible with organ cascades because (E2) and histamine (H) were infused directly into the of the selective sensitivity of the assay organs towards effluent at the top of the cascade. Antigen (ovalbumin individual hormones (Vane, 1964). This may be 10 mg) was injected into the pulmonary artery (OVA). further improved by administration of combined During the perfusion of lungs 11 the XAD-2 column antagonists (Gilmore et al., 1968). The procedure was placed between the two banks of tissues. The described here provides another way of facilitating the difference in the responses of the guinea-pig ilea above and below the column shows the influence of specific bioassay of hormones and autacoids in the interfering substances on histamine bioassay by mixtures. the upper guinea-pig ileum. We thank the Trustees of The Welicome Trust (London) for the generous grant of equipment, and Dr J. Pike, Upjohnin concentrations of 10-20 ng/ml. As partially Co., Kalamazoo, U.SA. for kindly supplying us with pros-exemplified in Figure 1, prostaglandins E1, E2 and F2a taglandins.ReferencesFERREIRA, S.H., NG, K.K. & VANE, J.R. (1973). The GILMORE, N., VANE, J.R. & WYLLIE, J.H. (1968). continuous bioassay of the release and disappearance of Prostaglandins released by the spleen. Nature, Lond., histamine in the circulation. Br. J. Pharmac., 49, 218, 1135-1137. 543-553. GRODZINSKA, L., PANCZENKO, B. & GRYGLEWSKI, RJ.FERREIRA, S.H. & VANE, J.R. (1967). Prostaglandins: their (1975). Generation of prostaglandin E-like material by disappearance from and release into the circulation. the guinea pig trachea contracted by histamine. J. Nature, Lond., 216, 868-873. Pharm. Pharmac., 27, 88-9 1.
HISTAMINE ASSAY IN THE PRESENCE OF PGs 41HAKANSON, R. & RONNBERG, A.L. (1974). Improved factors in anaphylaxis and its antagonism by anti- fluorimetric assay of histamine: condensation with o- inflammatory drugs. Nature, Lond., 223, 29-35. phthalaldehyde at -20°C. Anal. Biochem., 60, 560-567. PIPER, P.J. & VANE, J.R. (1971). The release ofKEIRSE, M.J.N.C. & TURNBULL, A.C. (1973). Extraction of prostaglandins from lung and other tissues. Ann. N.Y. prostaglandins from human blood. Prostaglandins, 4, Acad. Sci., 180, 363-385. 607-617. PIPER, P.J. (1974). Release and metabolism ofKORBUT, R. (1975). Bioassay of prostaglandins in the prostaglandins in lung tissue. Pol. J. Pharmac. Pharm., presence of high concentrations of catecholamines. Pol. 26, 61-72. J. Pharmac. Pharm. (in press). VANE, J.R. (1964). The use of isolated organs detectingPALMER, M.A., PIPER, PJ. & VANE, J.R. (1973). Release of active substances in the circulating blood. Br. J. rabbit aorta contracting substance (RCS) and pros- Pharmac., 23, 360-373. taglandins induced by chemical or mechanical stimulation of guinea pig lungs. Br. J. Pharmac., 49, 226-242. (Received April 25, 1975.PIPER, P.J. & VANE, J.R. (1969). Release of additional Revised July 1, 1975)