Study of paper electrophoresis as a potential method for the separation of histamine from spermidine

Paper electrophoresis is described as a potential method for quantitative separation of histamine from spermidine. When 0.2 M sodium phosphate buffer pH 9.5 and 1000 V for four hours were used, amounts of histamine less than 120 ng migrate behind spermidine. With the came conditions for electrophoresis, amounts of histamine greater than 120 ng migrate a head of spermidine. The migration distance of spermidine was constant, irrespective of quantity. In contrast, when 0.2 M potassium phosphate buffer pH 9.5 and 1000 V for four hours were used, the migration distance of histamine was constant, irrespective of quantity, and the distance was such that no separation from spermidine could be achieved. Therefore, the finding that only in a narrow range of quantity can histamine be separated from spermidine render the method prohibitive for quantitative evaluation of biological material. The result of this study show that spermidine is a potent quenching agent of the fluorescence elicited by the histamine-OPT complex. If 95% of the spermidine within a brain extract were removed, the residual spermidine would still be capable of quenching the fluorescence of the histamine-OPT complex by 25%. In addition, the present finding are in disagreement with those of other investigators with regard to the fluorescence spectra of the orthophthaldialdehyde condensates of histamine and spermidine.

A STUDY OF PAPER ELECTROPHORESIS AS A POTENTIAL METHOD FOR THE SEPARATION OF HISTAMINE FROM SPERMIDINE by Ernst Herbert Behr A thesis submitted to the faculty of the University of Utah in partial fulfillment of the requirements for the degree of Master of Science Department of Pharmacology University of Utah June 1971 This Thesis for the Master of Science Degree by Ernst Herbert Behr has been approved September 1970 Supervis ory Committee / J .supervisory Committee Head, Major Department ACKNOWLEDGMENTS I wish to thank the members of my committee for making this opportunity possible: To Dr. s. C. Harvey, I am indebted for the many helpful suggestions pertaining to my research and for his generous assistance in the editing of the numerous drafts that went into writing this thesis; to Dr. J. W. Kemp I am indebted for his direction, as well as his interest in my educational and professional development; and to Dr. D. M. Woodbury, I am indebted for his patience and counsel during the course of my training. A special note of thanks must be extended to my mother, whose example has continually inspired me to strive toward excellence. Lastly, I wish to express incessant gratitude to my wife, without whose constant encouragement and faith in me this thesis would not have been possible. This work was supported by U.S.P.H.S. Pharmacology Training Grant No. GM 00153 iii TABLE OF CONTENTS ACKNOWLEDGMENTS iii ABSTRACT vi I. INTRODUCTION II. METI-IODS A. Solutions 1 15 15 1. Amine Standards 15 2. Reagents 16 B. Electrophoresis Buffer Solutions 17 C. Paper Electrophoretic Separation 18 D. Elution of Color- Developed Standards and 21 Quantification of Test Components E. Radiochemical Methods III • RESULTS A. Conditions for Optimal Electrophoretic 23 26 26 Separation of Histamine B. Separability Determined by Visual and Optical 28 Means 1 . Visual Spotting 29 2. Photographic Recording 29 C. Elution of Histamine 30 D. Investigations of the Photofluorometric 31 Characteristics of Histamine iv E. Photofluorimetry of Spermidine and Interference 33 with Histamine 34 F. Radioactivity Studies 38 IV. DISCUSSION 49 V. TABLES AND FIGURES 94 VI. REFERENCES 99 VII. VITA v ABSTRACT Paper electrophores is is described as a potential method for the quantitative separation of histamine from spermidine. When 0.2. M sodium phosphate buffer at pH 9.5 and 1000 V for four hours were used, amounts of histamine les s than 120 ng migrate behind spermidine. With the same conditions for electrophoresis, amounts of histamine greater than 120 ng migrate ahead of spermidine. The migration distance of spermidine was constant, irrespective of quantity. In contrast, when 0.2 M potassium phosphate buffer at pH 9.5 and 1000 V for four hours were used, the migration distance of histamine was constant, irrespective of quantity, and this distance was such that no separation from spermidine could be achieved. Therefore, the finding that only in a narrow range of quantity can histamine be separated from spermidine render the method prohibitive for quantitative evaluation of biological material. The results of this study show that spermidine is a potent quenching agent of the fluorescence elicited by the histamine-OPT complex. If 95% of the spermidine within a brain extract were removed, the residual spermidine would still be capable of quenching the fluorescence of the histamine-OPT complex by 25%. In addition, the present findings are in disagreement with those of other investigators with regard to the fluorescence spectra of the orthophthaldialdehyde condensates of histamine and spermidine. vi I. INTRODUCTION In a study of histamine and its behavior in peripheral nerve, it became evident that there were problems associated with available methods used for the quantitative determination of histamine. With extracts of nerve used in this study, the method of Shore, Burkhalter and Cohn (1959) did not appear to be precise; variation was encountered among aliquots of anyone sample. Furthermore, there was some question as to the specificity of this method (see below). Anton and Sayre (1969) modified the method of Shore, Burkhalter and Cohn. Although their procedure purported to improve specificity, the method of Anton and Sayre did not afford a constant degree of recovery. Consequently, a study of paper electrophoretic separation of histamine from interfering substances was undertaken with hopes of achieving a method with better specificity and reproducibility than the aforementioned. The earliest determination of histamine was made by bioassay. These methods were founded on the observation of Dale and Laidlaw (1910), who characterized the pharmacodynamics of what they called /3- iminazolylethyl . amine, now known as histamine. Various organ systems of dog, cat, rat, and guinea pig were tested in the presence of atropine. In the pithed animal, intravenous infusion caused a biphasic vasodepressin: a small transitory fall followed by a larger and more pronounced drop in blood pressure. This effect on smooth musculature of blood vessels was contrasted 2 to the response of bronchiolar smooth musculature. A minimal amount caused acute bronchiolar spasm, which was manifested by a striking obstruction of the pulmonary airways. When its influence on isolated uterine smooth muscle was appraised, it was discovered that histamine was capable of producing maximum contraction at a concentration of 1:3, 50~ 000 histamine in Tyrode's solution, and a state of tonus continued until the solution was changed. The last organ tested for pharmacological activity by Dale and Laidlaw was the loop of jejunum of the cat. Not only did histamine cause increased rate and strength of contraction of jejunum with intact circulation, but it also appeared to enhance the force of contraction when the organ was isolated in a smooth muscle bath. In the next two decades, a great amount of interest was generated in histamine and its physiological function. At the same time there arose the need for a method capable of estimating tissue concentrations. Koessler and Hanke (1919) developed the first chemical method for the determination of histamine. 'Trey. utilized the reaction between imidazole derivatives and 1-phenyldiazonium sulfonate, which form a colored complex that could be optically quantified. This method was later improved by Hanke and Koessler (1920). However, much more refinement of the extraction process and the actual s:eparation of histamine was required before the method would gain widespread acceptance. 3 During the early period of development of chemical methods, Burn and Dale (1926) applied Dale and Laidlaw's earlier pharmacodynamic findings to the bioassay of histamine. They found that the vasodilator effect in the cat could be used as a quantitative index. Shortly thereafter, Macgregor and Peat (1933) used the Burn and Dale method to evaluate the histamine- histaminase system in an isolated perfused kidney-lung preparation. In general, they concluded that the Dale and Laidlaw method was not preCise, since the experimental error is about 20%. When compared to the precision of the Koessler- Hanke chemical method, the biological system, nevertheless, appeared to be more suitable. Early attempts to quantitate histamine in blood with bioassay were hindered by the presence of active substances which added to the pharmacological activity of histamine. The first widely accepted quantitative method for histamine which obviated contamination was that of Barsoum and Gaddum (1935). Although the procedure was designed specifically for blood, extracts from other tissue could be prepared in a similar manner. That the procedure is complex is shown by the following: The protein is precipitated with trichloroacetic acid (TeA), and the supernatant is filtered. The TeA is then removed from the filtrate with ether, after which the residue is treated with Hel. The excess acid is then removed by drying in vacuo and distillation with alcohol. After further extraction with 4 hot alcohol saturated with NaCl, the dried residue is taken up in water and neutralized to litmus. The lower portion of guinea pig ileum was used for bioassay. Barsoum and Gaddum undertook exhaustive studies to compare bioas say on the guinea pig ileum, rectal caecum of fowl, and blood pressure in the cat. By these three methods they obtained mean values of 0.11, 0.10 and 0.14 micrograms per m!, respectively. The high value obtained on the blood pressure of the cat was reduced to approximately the companion values when the extract was assayed 'with the atropinized cat. The procedure of Barsoum and Gaddum represented the first major attempt to refine the separation of histamine from a tissue extract; nevertheless, Code (1937) contended that the method of Barsoum and Gaddum was much too time consuming for the assay of numerous samples. Furthermore, he demonstrated differences of as much as 30% between duplicates of the same sample. He concluded that there was a distinct risk of unreliability associated with the method. The chief modification proposed by Code was the omission of the removal of TCA, which he claimed was the cause for differences and losses encountered in recovery. He also substituted bromothymol blue for litmus, in order to fix more accurately the pH of the final extract. Anrep (1939) defended the method of Barsoum and Gaddum with the results of an intensive study, which repeated the original work. He found no such deviation among sample aliquots as claimed 5 by Code and suggested that successive alcohol extractions might be introduced to improve recovery. During the time that these assay procedures were under intensive study, Feldberg and O'Connor (1937) extended the earlier investigation of Feldberg and Kellaway (1937) on the physiological function of histamine. They performed a series of experiments utilizing an isolated lung preparation, infusing into the lung agents that were known to cause release of histamine: peptone and snake venom. To assay the lung tissue at various times after onset of perfusion, they employed both the procedures of Barsoum and Gaddum (1935) and Burn and Dale (1926). They reported that available methods of assay were unreliable and agreed with the criticisms of Macgregor and Peat. Since the possibility of interfering substances present along with histamine in the final extract was not excluded by the use of available methods at the time, McIntire, Roth and Shaw (1947) undertook further studies in an attempt to resolve this problem. In their procedure, tissue homogenate was extracted with butanol. The histamine was recovered from the solvent by means of adsorption onto a column of acid succinate cation exchange cotton. Subsequently, the histamine was eluted from the acid succinate cotton with acid and then neutralized to give an isotonic solution suitable for bioassay on the blood pressure of the cat. The significant 6 contribution in this work came from the reported findings that described the aforementioned extraction procedure. The authors observed the effect of pH and salt extraction on the distribution of histamine between the aqueous and organic phases, finding that the use of high pH and a highly soluble salt such as ~a)2S0 4 expelled the histamine into the organic phase. Also, n- butanol was superior to isobutanol, tertiary butanol, n-amyl alcohol and isoamyl alcohol in their extraction of histamine from water. The optimum condition that could be created within the aqueous phase to drive 90% of all histamine into the n-butanol phase was a pH of 12.5 and a concentration of 25% (Na)2S04 (w Iv). In the late 1940's, there was a reactivation of interest in chemical methods for the quantitative estimation of histamine. Rosenthal and Tabor (1948) sought further to exploit the reaction between diazonium salts and imidazole compounds. In general, there appeared to be a lac\< of specifiCity, an instability of the color complex formed, and interference by numerous substances present in biological extracts. Of the reagents tested, the diazonium salt of 4-nitroaniline provide to be the most promising. The colored azo compound was formed by reacting the imidazole derivative and 4nitrobenzene in an alkaline medium, and then isolated and concentrated by extraction into a small amount of organic solvent, which also served to stabilize the color produced. At the appropriate pH, and 7 by use of the optimum organic solvents, the azo compounds of most interfering substances would remain in the aqueous phase. The histamine-azo compound was extracted into the organic layer, creating a rose color, whereas the azo compounds of those interfering substances which entered into the organic layer caused a yellow hue. The color of the organic phase thus provided an index of purity. Among the substances which inhibited the reaction, uric acid was the greatest concern. However, by briefly heating the raw histamine extract, or mixing it with nitrous acid, the effect of uric acid could be overcome. Because of continued dissatisfaction with the use of a coupling reaction between histamine and diazotized aromatic amines, McIntire, White, and Sproull (1950) investigated dinitrofluorobenzene (Sanger's Reagent, DNFB). At that time it was recognized not only that the reagent could be used to introduce a color tag on the amino end of a peptide but also that the reagent formed a yellow complex with amines. McIntire et al. (1950) adopted the reaction for the determination of histamine. After a separation procedure, histamine was coupled with DNFB. The histamine derivatives were then purified and measured spectrophotometrically. Two dinitrobenzyl derivatives of histamine are formed: one containing a single dinitrobenzyl group attached to the primary amino nitrogen; and the second having two dinitrobenzyl groups, one attached as just mentioned, the other attached to the 8 nitrogen at the one position of the imidazole ring. Since measurement depends on the presence of the former only, any histamine that proceeds to the latter derivative lowers the actual amount of histamine recorded and thus poses a distinct disadvantage. The method contains an additional inherent drawback with respect to numerous substances capable of inhibiting the reaction between histamine and DNFB. Heavy metals such as Cu+, Au+, Ni* in minute quantities are capable of inhibiting the reaction up to 98%, and the inhibition follows closely the ratio of metal ions to histamine concentration. Aldehydes also inhibit the reaction. The inhibition imposed by metal ions can be overcome with the use of chelating agents, but the problem with aldehydes has not been satisfactorily resolved. Lowry, Graham, Harris, Priebat, Marks and Bregman (1954) improved upon the method of McIntire, perfecting the method such that it permits measurement of quantities less than 0.1 }lg histamine with a precision of 25% and of quantities greater than 0.1 IJg with a precision of 5 -10%. A solution the histamine content of which is. to. be measured colorimetrically is prepared by an initial extraction from biological material with TCA, followed by separation of histamine from other amines on a Decalso column. After elution, the colored derivative is formed by reaction with DNFB, then concentrated by extraction into methyl-n-hexyl ketone, and finally suspended in HCl. The authors claimed that with Decalso, specificity no longer 9 posed a problem, since most amines having the potential for interference are not adsorbed onto Decalso and in most circumstances are excluded from the methyl- n -hexyl ketone. With respect to precision, Lowry's results were comparable to those obtained by McIntire. Although all previous attempts to enhance the accuracy of quantitative methods for histamine were regarded as distinct contributions' Shore et al. (1959) were of the opinion that current methodology was yet overly laborious and that specificity was not assured. In this method of isolating histamine, tissues are first homogenized in perchloric acid and centrifuged. The supernatant is saturated with NaCl, made alkaline with NaOH, and extracted into n-butanol. Following removal of the aqueous layer, the butanol phase is extracted with a salt solution of lower concentration, which serves to remove residual amounts of histidine. Heptane and a small volume of HCl are then added to butanol, and the mixture is shaken in order to extract the ·histamine back into the aqueous phase. The histamine in this final extract is condensed with orthophthaldialdehyde in a strongly alkaline solution. The resulting labile fluorescent product is stabilized upon the addition of acid. A spectrophotofluorometer is used to determine the intensity of fluorescence. The authors claimed that interference from most amines had virtually been eliminated in this method, since spermidine and spermine failed to produce significant fluorescence at the spectral peaks for histamine. 10 The claim that the method was specific soon was challenged. The question of the validity of this claim arose when Carlini and Green (1963) became involved with the measurement and distribution of histamine in the brain of rats. Their extracts were prepared according to Shore et al., but they were assayed on guinea pig ileum. Their results indicated varying amounts of histamine throughout the brain, the highest content being in the midbrain. These findings contrasted with those of Michaelson and Dowe (1963), who demonstrated uniform distribution of histamine in the guinea pig brain. They had used the entire method of Shore, Burkhalter and Cohn. Carlini and Green (1963) could not attribute these conflicting data to species differences and hypothesized that they were more likely due to the fact that two different quantitative indices had been used. They proceeded to demonstrate that brain extract contained at least five substances which were capbale of contributing to the apparent values of histamine obtained by the fluorometric method. In addition, they were able to show that when analyzetl by bioassay, most extracts of brain also give erroneously high values of histamine. The latter was demonstrated by comparing the apparent histamine content of an extract in the presence of atropine alone and in the presence of atropine and mepyramine. The difference was a figure very close to the amount of histamine introduced as an internal standard. The authors called the non-histaminergtc, active substance 11 in brain extract slow releasing substance, or SRSI. A brief attempt to characterize this unidentified component showed its activity could not be decreas ed by vigorous boiling, thus diminishing the probability that SRS is a protein. They also found their observations of the action of this compound to agree with those of Andrews, Roberts, and Sanders (I960~ who originally showed that the active substance has a latency of about 45 seconds and continues to evoke an increase in tone over the next five minutes. This is in contrast to the effect of histamine, which is immediate. Kremzner and Pfeiffer (1966) were the first to undertake the task of attempting to identify the unknown active substance(s) previously encountered in brain extract. They isolated the so-called SRS from the final extract by exposing it to a phosphorylated cellulose column. Mter various elutions, they observed chromatographic behavior and chemical properties, which led these investigators to the conclusion that the active substance is spermidine. Michaelson (1967) had a more direct way of proving the identity of the contaminant in a n-butanol extract of brain, as prepared by the method of Shore, Burkhalter, and Cohn. He extracted the histamine from brain and from a standard in an identical manner. To obtain the activation spectra, he set the fluorescent monochromater at a wavelength of INot to be confused With the aut:aCdd, slow reacting substance, thought to be partly responsible for mediating hypersensitivity reactions. 12 450 nm and scanned for peak activation. To obtain the fluorescent spectrum, he used an activation wavelength of 360 nm. The results indicated that the peaks of activation for tissue extract and histamine standard were the same (i. e., 360 nm), but that the intensity with histamine was greater. However, the peaks of fluorescence were different (400 and 450 nm, brain and histamine.,respectively). Further investigation showed that the spectral peaks for spermidine matched those of the raw extract. In view of this difference in spectral characteristics, the contribution of spermidine to the fluorescence of histamine may be explained by the fact that the former is present at levels sometimes in excess of 500 times that of histamine in various parts of the brain, although on a mole··to - mole basis the histamine fluorogen appears to be about 30 times more fluorescent than that of spermidine. Until 1966, the only defect of the method of Shore, Burkhalter and Cohn was the interference from substances other than histamine, leading to the limitation that it could not be used for the assay of brain tissue. But Beall (1966) found other disadvantages. Despite careful attention to water, reagents, glassware, and technique, there were large variations in the results of the fluor metric determination when the same tissue extract was assayed repeatedly. He believed that this problem is inherent in the chemical method and represents a significant drawback. In his opinion, it is probable that substances 13 in tissues may be sometimes present which can either produce interfering fluorescence, or perhaps quench the fluorescence resulting from the histamine fluorogen. He concluded that there is in the method a definite element of unreliability, which severely limits the decisiveness of the information obtained with it. However, he did concede that the extremes of variability were not encountered in blood, plasma, or histamine solutions, as in other tissues. In order to obviate the several undesirable traits of the methods of Shore, Burkhalter, and Cohn, Anton and Sayre (1969) imposed some important modifications on the original procedure. In this method, the authors incorporated the use of isoamyl alcohol in lieu of nbutanol for the extraction of histamine. As a substitute for NaCI to effect salting out, they employed K2HPO4' which also served to fix the pH of the aqueous layer at an optimum. They also replaced HCI with citric acid to terminate the condensation between histamine and orthophthaldialdehyde, which replacement resulted in a greater degree of stabilization. By improving the method of Shore, Burkhalter, and Cohn in this manner, they claimed to attain 85% recovery and nearly' absolute specificity. However, in the present author's experience, the best recovery that could be obtained was 55%, and recoveries were sometimes as low as 28%; but, more important, it was never constant from one extraction to the next. In addition, for the evaluation of numerous samples, the method of Anton and 14 Sa yre proved to be more laborious than the method of Shore, Burkhalter, and Cohn, because of additional steps included into the former. The present author holds the view that much of the emphasis of prior attempts to quantitate histamine have been channeled in the wrong direction. Most of the effort in this area has been spent trying to refine the extraction procedure for the separation of histamine from tissue components. In every instance, one phase of the process is modified by the use of different reagents, or simply by the more complex procedure. Specifically, no method to date has been able to achieve significant specificity without sacrificing recovery. This has been especially true in the separation of histamine from spermidine, two amines with very similar chemical and physical properties. As long as investigators continue to pursue this problem by striving to isolate histamine with chemical extraction procedures, the prospects for finding the ideal method seem to be dim. It is hoped that the following work will stimulate its readers to study the potential of other physicochemical media, heretofore, unexplored for the purpose of isolating and quantitating this elusive component in biological material. II. METHODS A. Solutions 1. Amine Standards. Standard histamine solutions were prepared from histamine hydrochloride (Nutritional Biochemicals). The salt was weighed out in 16 mg (10 mg histamine base) portions to the nearest 0.1 mg and diluted to volume with water. Solutions of lower concentration were prepared by serial dilution. Standard solutions of spermidine, spermine, putrescine, and histidine were similarly prepared from spermidine trihydrochloride (Cal Biochem), spermine tetrahydrochloride (Cal Biochem), putrescine dihydrochloride (Cal Biochem), and 1- histidine free base (Nutritional Biochemicals l respectively. Fresh solutions were made up monthly and stored in polyethylene tubes at 4 oC. A standard mixture of the aforementioned components was also prepared. In this solution, all the standards were present in equal proportions. Solutions of 2-C I4-histamine (Amersham Searle, Inc.) were 5 prepared from a 5. O-ml stock solution containing 10.2 x 10- g histamine with an activity of 1 ~c per ml. Aliquots of this solution were added to stock solutions containing histamine in various concentrations, such that each of a serial dilution of histamine had equivalent amounts of radioactivity. For the quantitative assay of histamine in nerve tissue, sections of cat sciatic nerve trunk were homogenized in 5 ml 0.4 N perchloric 16 acid (HClO4). These sections weighed from 0.1 g to 0.5 g. The tissue was homogenized in 2.0 ml HClO 4 and transferred to a collecting tube. The pestle and homogenizing tube were rinsed with 3.0 ml 0.4 N HClO4' and this rinse was added to the homogenate in the collecting tube. The homogenate was then centrifuged. The supernatant was used for subsequent paper electrophoresis. Standards which were exposed to electrophoresis concomitantly with aliquots from the supernatant of biological material were also made up in 0.4 N HCl04 • 2. Reagents. Four color reagents were used to locate amines and amino acids on the electropherograms. A ninhydrin solution contained 85 ml acetone, 15 ml water and 0.4 g ninhydrin (Dougherty Chemicals). Empirical observations indicated that when Ba ++ and collidine solution in the presence of glacial acetic acid were added to ninhydrin, the resulting reagent appeared able to detect more minute amounts of histamine. Therefore, modifications of the ninhydrin reagent solution were made by adding approximately 0.05 g barium acetate, 0.1 ml collidine solution, and 0.5 ml glacial acetic acid. Also, it was of interest to compare the detection limit of ninhydrin as determined by decreasing amounts of histamine with two other color reagents. An iodoplatinic acid reagent was prepared after the method of Mannering, Dixon, Carroll and Cope (1954) • Pauly's reagent, or diazotized sulfanilic acid, was prepared by the procedure of Berry (see Hais and Macek, 1963). Thus, 4.5 g sulfanilic 17 acid was mixed with 45 ml 12 N Hel; the mixture was gently heated and diluted to 500 ml. This mixture constituted solution A. In another flask, equal volumes of 4.5% NaN02 and 10% NaC03 were mixed to constitute solution B. Both solutions were stored under refrigeration. When the reagent was needed, equal volumes of solutions A and B were mixed and kept chilled in ice for 15 minutes, at the end of which time the mixture could be used for spraying an electropherogram containing various test compounds. The fluorescence reagent for histamine and spermidine usually contained 10% orthophthaldialdehyde (OPT) in anhydrous methanol. This reagent was used both for developing the zones of test compounds and for fluorometric quantification of histamine. In several experiments' the OPT was dissolved in either xylene or diethyl ether, and used for a spraying reagent. Other reagents used in the fluorescence assay were 0.4 N NaOH and 6 N citric acid. These two latter solutions were prepared freshly, biweekly, although not stored under refrigeration. B. Electrophores is Buffer Solutions The formulae for these solutions are taken directly from Gortner and Gortner (1949). Before use, the pH of each of these buffers was checked with a Beckman Model G pH meter. The buffer solutions were prepared as follows: 18 pH 1.6: pH 4.8: 14.6 g. KCI, 131.5 ml 0.2M HCI, dilute to 1000 mI. 40.8 g. potassium acid phthalate, 88.5 ml 0.2 M NaOH, dilute to 1000 ml. pH 8.0: 34.8 g. KH2PO4' 234 ml 0.2 M NaOH, dilute to 1000 mI. pH9.0: 12.3 g. H3B03 , 14.6g. KCI, 8.0 g. NaOH, dilute to 1000 ml. pH 9. 1: 24.0 g. NaH2 P04, dilute to 1000 mI. pH 9.3: 24.0 g. NaH2 P04 , 0.1 g. NaOH, dilute to 1000 ml. pH 9.5: 24.0 g. NaH 2PO4' 0.2 g. NaOH, dilute to 1000 ml. pH 10.0: 24.0 g. NaH2 P04 , 0.3 g. NaOH, dilute to 1000 mI. C • Paper Electrophoretic Separation Electrophoretic separation was effected with an apparatus designed by J. W. Kemp and fabricated in this department. The dimensions of the glass plate were such that it could accommodate chromatography paper 45 cm x 56 cm. Along each edge of the plate, a trough for buffer was positioned so that wicks could pass from the buffer to the electropherogram. In each of the troughs, a platinum wire was extended the entire length. These wires were connected to the terminals of the power supply such that anyone of a pair of troughs represented the opposite pole of the trough positioned across from it. The water supply of two tanks was arranged so that it could be pumped continuously through a network of vented pipes, positioned directly beneath the glass plate. One tank was kept cooled by means of a cooling unit, while the second was heated to 800 C with a heating coil. By selecting water from the hot or the cold tank to run through the system, one could cool or heat the plate as desired. Whatman No. 54 chromatography grade filter paper (Reeve-Angel) 19 was used routinely, but Whatman No. 1 and Whatman 3 MM papers were also employed in initial experiments. Also, in several early trials the paper was prewashed with 0.2 M acetic acid, followed by a rinse with buffer or 0.4 N NaOH. Prior to electrophoresis, the dry paper was marked off in 1 1/2" channels along an arbitrary line of origin 5 1/4" from and parallel to the longer edge. After being placed in position on the glass plate, the paper was saturated with buffer. Excess buffer and air bubbles were eliminated by gentle rolling of the paper with a rubber roller, which was rolled from the center toward the edge of the paper. This procedure was followed by filling each of the troughs with 250 ml buffer and placing the wicks in position. One edge was immersed in the buffer, and the other was folded on the electropherogram with an overlap of about 1/2". The wicks, with dimensions of 2 1/4" x 22 1/2", were made from Whatman No. 1 chromatography grade filter paper. The purpose of the wicks was to allow for the conduction of current from the electrode trough of buffer to the paper. After the paper and wicks were so positioned, the electropherogram was covered with a plexiglass safety shield, and the pump was engaged to distribute water from the cold tank along the lower surface of the glass plate. Through a slit in the safety shield, each test solution was then applied to the line or origin at the center of a respective channel. Voltage was then applied. During initial experiments, voltage and time were 20 varied to determine the best separation. However, for most of the studies, 1000 volts for 4 hours was routinely employed. At the end of electrophoresis, the current was shut off, and the ionogram was dried directly on the glass plate. This was accomplished by now allowing hot water to come into contact with the glass plate. Simultaneously, the hot air jet of an electric hair dryer was passed rapidly and systematically over the entire surface area. After the electropherogram was dried, it was suspended by clips on a glass rod and sprayed with the appropriate coloring reagent. In a few cases the chromatogram was dipped in a large shallow pan containing the color-producing reagent. In order to locate the histamine zone when it was undesirable to use a coloring reagent (as, for example, when fluorimetric determination was to be subsequently employed, or when the quantity of histamine was below the detection limit), the zone was assumed to lie at the same distance from the origin as that of a detectable reference in an isolated channel from the same ionogram. When fluorescence photography was employed, the sprayed electropherogram was positioned against the emulsion face of a piece of photographic paper (Kodak Ad, type A2) in the darkroom. It o was then exposed to ultraviolet light at 2537 A , and a contact print of the electropherogram was made. In addition to photography, direct visualization of the fluorescent zone was employed. The fluorescenceproducing reagents are described under Reagents. However, it 21 should be noted that in several experiments the OPT- spray was preceded by a spray of 0.4 N NaOH, in order to promote condensation between OPT and histamine, which condensation is enhanced in an alkaline medium. D. Elution of Color- Developed Standards and Quantification of Test Components Sections of channel containing a violet ninhydrin-histamine zone were isolated and immersed in 3.5 ml of 0.4 N NaOH, then allowed to steep for about one hour. The eluate was then assayed by spectrophotometry, using the Beckman DU Spectrophotometer. The optimal wavelength was determined from a spectrum obtained both from eluate and from chromogens formed in vitro. In the latter instance, a solution containing 2.0 ml 0.4 N NaOH, 0.5 ml 1.0% histamine, and 0.5 ml ninhydrin reagent was used. Similar attempts were made us ing Pauly's reagent. The photofluorimetric method used for the determination of histamine was the method of Shore, Burkhalter and Cohn, (1959) with the modifications outlined by Anton and Sayre (1969). A one-inch square section of a electropherogram channel was removed for the determination of histamine, the location of the appropriate section having been determined from the reference channel (see page 20 ). The histamine was eluted from the section in the same manner as the ninhydrinhistamine spot mentioned above. In some instances, the eluate was 22 filtered after extraction in order to remove light-scattering fibers. At time zero, 0.2 ml OPT was added to 3.0 ml of eluate. After the reaction between OPT and histamine had been allowed to proceed for four minutes, it was terminated by the addition of 0.5 ml 6 N citric acid. At five minutes (i.e., one minute after the addition of citric acid), the fluorescence intensity of the final solution was determined in an Aminco-Bowman Spectrophotofluorometer, using an activating wavelength of 360 nm and a fluorescence wavelength of 440 nm. The appropriate wavelength was determined by spectral anal ys is. In this procedure, the spectrogram was recorded by means of a Mosely-Autograf X- Y recorder, which was activated from the output of the spectrophotofluorometer. After each addition of a reagent to a tube, the contents were agitated for several seconds with a vortex mixer. Electropherograms containing spermidine alone or spermidine plus histamine were eluted and assayed in the same manner. In order to ascertain the degree of recovery by elution, controls were established by loading sections of paper with amounts of histamine comparable to those used at the line of origin on an electropherogram. The paper from which the control spots were isolated was previously saturated with the same buffer used in electrophoresis and subsequently dried. The load zones were then eluted, and the histamine content quantified by the above method. In a number of experiments, the influence of spermidine on the 23 fluorescence of histamine was determined directly from the standard mixtures, without their having been subjected to electrophoresis and elution. The method of determination and the relative quantities of solution are the same as those described beforehand. It was of interest to examine the stability of the fluorogen result- ing from condensation between OPT and histamine. Accordingly, the progress of the OPT-histamine reaction was not arrested by adding citric acid, but rather fluorescence was simply measured at predetermined intervals following the addition of OPT. This stability was also examined at different temperatures. Three groups of samples containing equal amounts of histamine in equal volumes of 0.4 N NaOH were maintained at a respective temperature for the duration of the study: the first group was maintained at SoC in an ice bath, the second o at 2S oC, and the third at 8S C in a hot water bath. E. Radiochemical Methods In a number of experiments, the histamine solutions contained C l4-labelled histamine (see page IS, Solutions). Mter electrophoresis and drying was completed, the electropherogram were sectioned into 1 1/2" parallel channel strips. The distribution of the radioactivity in the paper channel was ascertained and quantified by passing the dried channel strip through a 2'1f windowless counter fitted with a S mm x 38 mm aperture to a proportional counter head, as described by Kemp and Woodbury (196S). Respective channels were joined with 24 tape forming one continuous strip. At the junction of two adjoining channels, heavy pencil lines were drawn across the tape with a SensaMark 2100 pencil, which caused a small excursion of the recorder pen when coming into contact with an electro-conducting sensing device arranged in the path of the advancing strip. In this way, it was possible to make an accurate determination of the distance of migration of the labelled histamine from the line of origin. The movement of C 14 -labelled spermidine, was also studied in the same manner. The movement of labelled histamine-spermidine combinations were similarly examined. By scintillation counting, attention was also given to the distribution of tracers in a channel. The tracer-containing channel was divided into numerous sections. Begirming at the line of origin, twenty-five 3/8" segments were cut from a channel and their sequence numbered in the direction of ionic movement toward the cathode. Each numbered segment was shredded, immersed in 1.0 ml of distilled water and left overnight, in order to effect elution. A 0.5-ml aliquot was removed from each eluate and pipetted with a 500-ml Eppendorf pipette into a series of scintillation vials, each containing 10 ml 2', 5'-diphenyloxozoltoluene (18 g ppot/31 toluene) and 6.0 ml cellosolve (ethoxyethanol). The radioactivity within the vials was determined with a Chicago Nuclear liquid scintillation counter. In some experiments, the 3/8" segments were isolated as described 25 above, cut in half, and the pieces placed directly into planchets for counting in a Tracer Lab low background flow counter. III • RESULTS A. Conditions for Optimal Electrophoretic Separation of Histamine The electrophoretic mobilities of putrescine, histamine, spermidine, spermine, and histidine were studied at several pH's. In each trial, every test compound was ecposed to electrophoresis in a series of channels, in order to allow for identification of the respective standards in channels where a mixture had been applied. The distance of migration1 of each test compound was determined by locating the substance with ninhydrin spray treatment (see Methods, page 20). At pH 1.6 all compounds with the exception of histidine migrated completely to the cathodal edge of the paper, so that separation was interderminant (fig. 1). With 1.S watt-hours of exposure, the migration of all compounds could be kept within the limits of the paper; however, putrescine, histamine, and spermidine migrated identical distances, with spermine lagging slightly behind them (fig. 2). Histidine was found at a distance of 9.3 cm behind the leading compounds. At pH 4.8, some separation was achieved, the migration distance of putrescine being slightly greater and that of spermidine and spermine slightly less than that of histamine (fig. 3). Again, histidine remained a considerable distance behind histamine. At a pH of 8.0, the migration distance for histamine was 1 The distance of migration was arbitrarily taken to be the distance from the line of origin to the center of density of the spot, the position of which was determined by eye. 27 1.0 em greater than that for spermidine (fig. 4), but the leading edge of spermidine zone significantly overlapped the trailing edge of histamine. The other test substances appeared to be adequately separated from histamine. A pH of 9.1 appeared to increase the degree of separation between test compounds (fig. 5). The spots on the electropherogram exhibited a sequence of putrescine, histamine, spermidine, spermine, and histamine; respectively, putrescine having migrated the farthest toward the cathode. There was a 2. 7-cm separation between points of maximum color intensity and spermidine. However, the histamine spot was elongated at the trailing edge; thu~ the "tail" again over- lapped the following spermidine spot. The "tailing" was measureably diminished at pH 9.5 (fig. 6), and there was a difference of 3.4 cm in the distances of migration for histamine and spermidine. At pH 10.0, the distance of separation between histamine and spermidine decreased to 2.5 cm, and, at pH 10.5, the two spots were essentially fused together. Extens ive tailing reappeared, which made a determination of distance between center of zones meaningless. The molarity of the buffer was important for discrete separations. A sodium phosphate buffer with a molarity of less than 0.2 produced diffuse zonal patterns with a considerable degree of tailing; a concentration greater than 0 .3 M appeared to restrict the movement of histamine, and this concentration was the limit above which phosphate 0 salts preCipitated on the paper at the 4 C operating temperature. 28 Optimum mobility of the several amines tested in a sodium phosphate buffer occurred at a molarity of 0.2. In some experiments, the effect of washing the paper with 0.2 N acetic acid (see Methods, page 19) was observed. Prewashing tended to make the results erratic, such that the distancES of separation of the histamine and spermidine spots were often diminished, and tailing was increased. If the time allowed for exposure to the buffer rinse was sufficiently extended, this diminution in the distance of separation and increase in tailingwere not seen. In no instance was interzone distance increased or the tailing decreased by prewashing. Three grades of chromatography paper (What man No.1, Whatman No. 54, Whatman 3 MM) were evaluated with respect to the following qualities: 1) The ability to support cohesive migration with a minimum of "tailing, " 2) ease of handling when saturated with buffer, and 3) the presence of lint in eluates from the paper. It was observed that the least amount of tailing occurred on Whatman No. 54 paper. Although ease of handling was not determined by objective criteria, subjectively, Whatman No. 54 again appeared to be the most suitable paper. The elution of lint was determined by the extent of light scattering measured in the photofluorometer; Whatman No. 54 produced the least amount of lint. Therefore, Whatman No. 54 was used throughout this work, except where specifically stated to the contrary. B. Separability Determined by Visual and Optical Means 29 1 . Visual Spotting. A ninhydrin spray containing traces of Ba+t was capable of rendering visible a minimum quantity of 2.0 jJg histamine. This finding, which was observed on an electropherogram, maybe contrasted to the sensitivity of the color test when the amine was applied to a section of dry paper and sprayed immediately. In the latter instance, ninhydrin produced a colored zone with as little as 0.1 jJg histamine; however, that latter zone covered a much smaller area than the spot developed on the electropherogram. Paul y' s reagent was evaluated in a similar manner. It exhibited a limit of sensitivity on the electropherogram of about 5.0 jJg. The sensitivity limit of Pauly's reagent for samples applied to paper but not exposed to electrophoresis was also approximately 0.1 jJg. 2. Photographic Recording. Ultraviolet photography of undeveloped electropherograms (i .e., no spray treatment with color reagent) failed to record any zones of putrescine, histamine, spermidine, spermine, or histidine, when 10-30 jJg of compound was exposed to electrophoresis. Likewise, fluorescent photography of the OPT-treated electropherograms failed to reveal any discrete zones that correlated with ninhydrin-identified loci. Substitution of xylene or diethyl ether for methanol as the solvent medium for OPT did not improve detection. Because 30 jJg was well in excess of the expected quantity of any of the test compounds in one·· gram samples of biological material, no follow-up trials with greater amounts were undertaken. The attempts 30 made to observe the various spots under the influence of U. V. light and to encircle them directly on the electropherogram for subsequent elution likewise proved fruitless, because of a lack of definition of the boundaries of a respective zone. C . Elution of Histamine Histamine-ninhydrin spots were eluted according to the procedure outlined under Methods (page 21). The wavelength of maximal absorp- tion for the histamine-ninhydrin complex, generated by adding ninhydrin reagent directly to a solution of histamine (see Methods, page 21) was 430 nm, and it was at this wavelength that spectrophotometric assay of the eluates from Whatman No. 54 paper was made. The recovery of histamine by elution was low and erratic. Even when the paper which contained the developed zone was immersed in water (see Methods, page 21) for as long as 16 hr, violet color could be observed on the paper. Based upon the amount of histamine applied at the origin of an electropherogram, recovery by elution from sections of electropherogram ranged between 0.0% and 35 .0% . Since recovery of standards was not linear (fig. 7), no further investigation was pursued with this method. Elution of zones rendered visible with Pauly's reagent was similarly of low and erratic yield. Likewise, color was visibly retained on the paper after allowing several hours for elution. When samples of histamine not exposed to electrophoresis were eluted, the recovery was quantitative (fig. 8). In this work, the 31 recovery of histamine from an electropherogram determined photofluorometrically with orthophthaldialdehyde was low ranging from about 10% when the amount of histamine applied to the origin was 1.0 ~g to about 68% when 10 ~g was applied. These data are plotted in figure 8. The values plotted in this figure represent the recovery from a one-inch-square segment of the electropherogram; the location of the segment was detennined from a ninhydrin-developed spot in an adjacent and isolated channel to which 10 ~g of histamine had been applied. D. Investigations of the Photofluorometric Characteristics of Histamine Fluorescent spectral studies of histamine were carried out using 1.0 ~g histamine. The histamine-OPT complex exhibited an activation peak at 360 nm when the fluorescence monochrometer was set at 440 nm (fig. 9A). The larger peak in figure 9A is a scatter peak. Conversely, a, scan of fluorescence with an activation wavelength of 360 nm produced a peak at 440 nm (fig. 9B). The larger peak in figure 9B is also a scatter peak. All subsequent photofluorometric analyses of histamine were performed under these conditions of activation and fluorescence. These wavelengths were also used to assess the fluorescence spectrum of reagent blanks; this study revealed that there was minimal activation or fluorescence contributed by the reagent blanks at the wavelength found to produce maximum activation and fluorescence for histamine. In the modification of Anton and Sayre (1969) of the Shore, 32 Burkhalter and Cohn method (1959) for the determination of histamine, citric acid is added to the alkaline histamine-OPT mixture, in order to terminate the reaction and stabilize the fluorogen. Accidental observation indicated that the fluorescence of the reagent blank was higher when citric acid was added than when it was omitted. This finding suggested the need for further investigation on the sensitivity, stability, and preCision of the method. In separate experiments, the fluorescence of six reagent blanks was determined in solutions to which no citric acid was added. The addition of OPT was recorded as time zero and fluorescence was determined five minutes later. The mean deviation of fluorescence among a group of blanks so determined was 73 fluorescence units, with a standard deviation of 96 fluorescence units. When citric acid was added at four minutes and fluorescence determined at five minutes, the mean deviation of fluorescence was 42 fluorescence units and the standard deviation was. 50 fluorescence units. The time course of the development of fluroescence was followed by determining the fluorescence of a given sample at various times without the addition of citric acid. The same experiment was also 0 conducted with groups of blanks maintained at SoC and at 85 C • Table 1 is a composite of these data. It may be seen that fluorescence intensity varied with temperature. However, it was also evident that as the temperature was increased to 85 0 C, precision among samples 33 became considerably less, so that no advantage accrued to increasing the temperature of the reaction. Since maximum fluorescence of blanks at room temperature was for the most part attained within five minutes in those samples of the group to which citric acid was added at four minutes following addition of OPT (table 1), this schedule of reagent additions was adapted for all subsequent photofluorometric determinations. At one stage of the investigation, consideration was given to the advisability of including an antioxidant in the buffer solution. However, it was first necessary to examine the effects of antioxidants on the histamine-OPT complex. It was observed that the inclusion of NaHS0 3 or ascorbic acid caused a prominent depression of the fluorescence of histamine. However, the intensity of the glutathione-OPT complex was so great that the presence of increasing amounts of histamine could not be detected. With respect to the fluorescence of the glutathione reagent blank, the fluorescence intensity value obtained was equal to that recorded for 15 ~g histamine. These data are summarized in figure 10. E. Photofluorimetry of Spermidine and Interference with Determination c of Histamine Spectral analysis of 10 ~g spermidine demonstrated an activation peak at 360 nm when fluorescence was recorded at 440 nm (fig. 11A). A scan of fluorescence at an activation wavelength of 360 nm produced 34 nothing more than a scatter peak between 365 nm and 380 nm (fig. lIB). Even with 100 Ilg of spermidine, no definite peak of fluorescence was demonstrable. These studies were repeated with OPT which had been recrystallized in petroleum ether. The peak of activation remained at 360 nm, but the scatter peak was diminished (figs 12A, 12B). In a scan of fluorescence, the scatter peak was sharp at 370 nm, but no fluorescence peak was observable. Experiments in which spermidine standards were assayed spectrophotofluorometrically demonstrated that no amount of spermidine could evoke the intensity of fluorescence equal to that of 1.0 Ilg histamine. Maximum fluorescence was attained with 200 Ilg; with quantities greater than 200 Ilg, the fluorescence diminished. Determination of the fluorescence of a solution containing both histamine and spermidine gave rise to values that were not additive; indeed, spermidine quenches the fluorescence of histamine. Even in quantities as low as 10 Ilg, spermidine depresses the fluorescence of 1 Ilg histamine by more than 25%, although such a quantity of spermidine does not by itself evoke fluorescence. These data are summarized in figure 13. F. Radioactivity Studies The method employed to establish the zone of undeveloped electropherogram to which histamine was assumed to have migrated (see Methods, page 20), indicated the practicability of electrophoretic separation of histamine from spermidine. However, when the 35 reference distance for 10 llg histamine was used to locate a zone within a channel spotted with 20 ng of C 14 -labelled histamine, the area of electropherogram isolated for counting exhibited only background radioactivity. In channels that were loaded with 5, 000 ng and 10, 000 ng radiolabelled histamine, the percent of total applied radioactivity found in the zone at reference distance increased to 65% and 67%,respectively (see table 2 and fig. 14). The explanation for this finding proved to be that varying quantities of histamine migrate different distances. The 217" strip counter was employed to produce a recorded profile of radiolabelled histamine. For example,. 2 .0 ng migrated an average distance of 10.1 cm, whereas 5020 ng migrated 17 .7 cm. In contrast, a similar study with radiolabelled spermidine demonstrated no such variation. Quantities between 24 ng and 10,024 ng migrated an average distance of 13.8 cm (± 0.3 cm). The data for migration distances of histamine and spermidine are tabulated in table 3 and plotted in figure 15. Figure 16 shows the increasing migration distances with increasing amounts of histamine as revealed by the 2'1f' strip counter. In figure 17, the same strip used to produce the tracings in figure 16 was passed through the 2'11" strip counter with a higher sensitivity. The significant observation from this trial is that recordings of the lower amounts of histamine appear to have a more gentle slope on the anodal side of the peak than the cathodal side. It may be seen that as the amount of histamine on the strip 36 increases, the recordings of the respective peaks become much more symmetrical. Trials in which C l4 -histamine and C 14 -spermidine were exposed to electrophoresis simultaneously in the same channel show that histamine can be separated from spermidine only when the amount of histamine is large enough to promote maximum mobility or small enough to manifest minimum mobility. An experiment displaying the variable distances of migration of varying amounts of histamine applied to respective channels in combination with a constant amount of spermidine is shown in figure 18. The solid tracings are profiles of the combinations, while the dotted tracings represent the superimposed profiles of a control amount of histamine (equal to those amounts of histamine exposed to electrophoresis in combination with spermidine). These histamine controls were electr:ophoresed concurrently with the combination, but in separate channels. Two peaks in the scanogram of the profile of the combination may be seen. One is essentially fixed in distance from the origin; the second varies in its position and this position coincides with the peak of the corresponding histamine control. If it is assumed that the variable peak corresponds to histamine, then it may be said that quantities of histamine less than 20 ng migrated behind spermidine and quantities greater than 120 ng of histamine migrated ahead. However, the recording did not give indication of adequate separation until histamine was present either in excess of 37 1000 ng or in amounts too small to be detected by chemical means. Figure 18 also shows that spermidine does not appear to affect the mobility of histamine. Since it was desirable to investigate further the possible interactions between histamine and spermidine, experiments were performed in which the quantity of spermidine used was higher or lower than that employed in the preceding experiment. With the exception of two anomalous findings, no interactions were noted. In two experiments, spermidine appeared to retard the migration of histamine, in one so much that histamine hardly moved from the origin. In these experiments, the migration of spermidine was normal. Figure 19 shows several of the recordings obtained from one of these experiments. Similar experiments, in which quantities of histamine were varied in the presence of a constant quantity of C 14 -spermidine, were performed in potassium phosphate. Figure 20 shows that in 0.2 M potassium phosphate at pH 9.5 the mobility of histamine did not vary with quantity. However, under these conditions, the mobilities of histamine and spermidine were essentially identical, and no separation was possible. Figure 21 shows an experiment with a combination of C 14 -histamine and C 14-spermidine; the presence of a single peak in each scanogram indicates that the two substances are superimposed. 38 IV. DISCUSSION Under a specific set of conditions, electrophoresis may be considered an adequate method for the separation of histamine from spermidine. The most obvious condition to manipulate in order to effect a separation is that of pH. The electrophoretic mobility of an amine would be expected to decrease with the hydronium ion concentration, inasmuch as at higher pH the proportion of time spent as an ion would diminish. The decrease in mobility of histamine observed in this study and also by Shelley and Juhlin (1966) is consistentwith this prediction. When two or more amines are to be separated, there should be an optimal pH for separation according to the different pKa' s involved. Although the pKa' s of histamine are known, the pKa's of spermidine could not be found by the author, so that no theoretical basis was provided for predicting the optimal pH for the separation of histamine from spermidine. The optimal pH observed was approximately 9. 5. At low pH, closely related amines should be difficult to separate. In this work, amines as different as histamine and spermidine migrated essentially equal distances at pH's below 4. O. Consequently, it is hard to understand on the ~bas is of pKa and per cent ionization alone the findings of Navert, Flock, Tyce and Code (1969). In their study, histamine was separated from its various metabolites in a buffer system of formic acid, acetic acid and, cadmium acetate. Their findings were published too late to be followed up in the present study as affording a system for the poss ible separation of histamine from spermidine. 39 Once separation is achieved, quantification can be accomplished after elution, providing elution is quantitative. In studies in which the degree of recovery is of no concern, the method may be routinely employed as a qualitative tool to detect as little as 2.0 ~g histamine. However, there are several features about the method that render it imprecise for use with quantities found in biological material. The problem of varying mobility of histamine is the greatest drawback to the quantitative separation of histamine from spermidine. Within a critical range of histamine content (0.1- 0 •5 ~g), it is evident that the peaks observed on a radioscano- gram of the two amines appear at almost equal distance from the origin, and this range corresponds closely to those amounts of histamine that might be expected when working with 0 .5-g samples of most tissues. The amounts of histamine that are clearly separable from spermidine are quantities that are beyond the levels found in biological samples. This fact, then, places limitations on the usefulness of electrophoretic separation. Histamine in amounts as low as 0.1 ~g could be detected on chromato- graphy paper by color-developing techniques, when the color reagent was applied to non -electrophoresed spots. However, histamine could be rendered visible on an electropherogram only when the quantity of histamine applied exceeded 2. 0 ~g, a quantity higher than that expected to be present in most biological samples. This lack of chromogenesis consequent to treatment of the electropherogram with ninhydrin or other reagents may be explained by a diffusion phenomenon extant during the course of 40 electrophoresis. Furthermore, even when a discrete spot occurred, it could not be quantitatively eluted from the paper. Therefore, colorimetric determinations of histamine from eluates of chromatogramsv.ere not feasible. The problem of recovery is magnified by the finding that the extent of 14 tailing appeared to vary inversely with the amount of C -labelled histamine applied at the origin. That is to say, in sodium pJ'losphate buffer, the distance along the baseline bounded by the leading edge and the trailing edge of a peak on the scanogram was greater when 20 ng was electrophoresed than when 1020 ng was electrophoresed (fig. IS). The appearance of variation in the extent of tailing is explicable in terms of a tail of constant quantity. Thus, when larger amounts of labelled histamine are applied, the tail becomes a lesser proportion of the total amount of label than when small amounts of labelled histamine are applied. In figure IS, the dilution of the isotope in E is 251 times that in A; therefore, the amount of label in the tail is undetectable, and it would appear that a tail did not exist. ,;A reduction in the proportional counter gain has the same effect as that of dilution of the isotope, in that the amount of histamine in the tail contains too little tracer to be recorded at low gain (compare B of ffgure 17 with B of figure IS). This phenomenon of a tail of constant quantity is unusual. It was neither observed with spermidine nor has it been described in the literature to the knowledge of the author. Since the tail is a lesser pro- portion of a zone produced by a higher quantity of histamine, the definition and recovery are higher with large than with small amounts of histamine. 41 The results from electrophoresis performed with potassium phosphate buffer provide an interesting contrast to the findings obtained when sodium was used as the cation in the buffer. Histamine migrated the same distance regardless of the amount applied to the origin. Furthermore, the scanogram showed smooth, nearly symmetrical peaks and thus no apparent tailing. Spermidine migrated approximately the same distance, so that the distance of separation between histamine and spermidine is reduced to less than O. 5 cm, which is too small to permit quantitative separation. To the extent that in potassium phosphate buffer both the histamine and the spermidine zones are discrete (such that scanograms show distinct peaks and no apparent tailing) and migration distances are constant irrespective of the quantity of amine, potassium phosphate is an ideal buffer for paper electrophoresis. However, its ideal character is defeated by the nearly identical migration distances of spermidine and histamine. It is ironic that it is the anomalous behavior of histamine in the presence of sodium phosphate buffer that allows for any possible separation. Findings such as those reflected by figure 19 were observed only twice. There is uncertainty as to the significance that may be attached to these observations. They may illustrate a potential pitfall in a determination using electrophoresis as a method for separating the two amines in question. Beyond such significance, it is an interesting phenomenon, the chemical nature of which is difficult to explain. Inasmuch as in the two instances in which this phenomenon was apparent, crystallization of buffer occurred in 42 the paper, it is possible that the resulting crystal lattice may have selectively impeded the movement of histamine. The crystals were not identified, but it is probable that they were dis odium orthophosphate dodecao hydrate, which has a low solubility at 4 C. Attempts were made to experiment with the use of higher operating temperatures during electrophoresis, in order to prevent crystallization. However, the problems of electrolytic heat accumulation did not allow for useful observations to be made. The fluorescent spectrum of the histamine-OPT complex is not reported to be the same by all investigators. In the literature, the fluorescent peak ranges between 440 and 460 nm (Michaelson and Coffman, 1967; Kremzner and Wilson, 1961; Shore et- al., 1959; Anton and Sayre, 1969). In the present studies, a fluorescent peak of 440 nm was observed. Such discrepancies conceivably might be attributed to errors in calibration of fluorometers; in this laboratory, the fluorometer was calibrated with both a mercury vapor ultra-violet lamp (Aminco No. 4-8}75) and quinine. However, the simple explanation that differences in calibration or lack of calibration account for such discrepancies' is refuted by the fact that all investigators (Anton and Sayre 1969; Shore et al., 1959, Kremzner, 1961; Michaelson and Coffman, 1967) and the present author find a common activation peak, namely, 360 nm. A similar situation exists with spermidine in that the fluorescent wavelength of the complex formed with OPT found by the author did not agree with that reported by others (Anton and Sayre, 1969: 400 nm; 43 Kremzner '.1966: 410 nm or Michaelson, t 19~67: 410 nni). In the present work, I the peak found was between 365 and 380 nm, the exact location of the peak being somewhat indeterminant. It was uncertain whether this peak was a true fluorescent peak or simply a scatter peak with a degree of Raman displacement. as many It was thought advisable to recrystallize the OPT to remove i~purities as possible, hopefully to reduce the amount of extraneous scatter. The peak found in solution of OPT alone was 355 nn.1; with the addition of spermidine, the peak became 370 nm. Thus it is evident that spermidine affects the peak wavelength, and hence the peak cannot be considered to be simply the result of scatter. No explanation is offered as to why the peaks reported by Anton and Sayre, Kremzne:t; and Michaelson were not observed. That the compound employed was actually spermidine is discussed later (see page 45). Because the literature implies that the presence of spermidine causes spuriously high readings for histamine, as observed by Michaelson (1967), it was surprising to find that spermidine quenches or dampens the fluorescence of histamine. Furthermore, the error caused by this quenching was much greater than the ability of spermidine to generate its own fluorescence. If it is assumed that spermidine is indeed capable of forming a fluorescent complex with OPT, the resulting fluorogen must necessarily exhibit a great deal less intenSity of fluorescence than that of the histamine-OPT complex. Michaelson and Coffman (1967) reported that histamine is about thirty times more fluorogenic than spermidine. However, no mention was made as to 44 the amounts of histamine and spermidine used to establish this relationship. There is the possibility that the relative fluorescence varies according to the amount of both components evaluated. Nevertheless, in this study, in which a wide range of amounts of both histamine and spermidine were determined, the relative intensity was never less than 800: 1. Indeed, figure 14 shows that spermidine standards containing less than 10 jJg were essentially indistinguishable from blanks, whereas levels of histamine as low as 0.1 jJg were readily detectible. The high threshhold amount for the fluorescence of spermidine is especially interesting in view of the fact that quantities as low as 10 jJg are capable of significantly reducing the fluorescence of histamine. The interference by spermidine with the fluorescence of histamine places demands on the best established methods for separating histamine from spermidine. Kremzner (1966) states that in brain tissue levels of spermidine may exceed those of histamine by almost 500:1. Therefore, even in the event that 95% of the spermidine present could be removed from the solution assayed for histamine spectrophotofluorometrically, the remaining five percent would be sufficient to cause spurious results. Because of the conspicuous discrepancies between the findings of the author and those of Anton and Sayre (1969), Kremzner, (1966) and Michaelson and Coffman (1967) regarding the fluorometric; behaVior of 45 spermidine, it seems appropriate to ask whether it was indeed spermidine that was being evaluated in this study or perhaps some mixture of breakdown products. However, this implication maybe dispelled by two observations. Firstly, there was only one peak on a radioscanogram of an electropherogram containing an arbitrary amount of spermidine, to which the radio-labelled species was added. Had there been a mixture of breakdown products present in the applied spot, the tracing would have revealed more than one peak. Secondly, a comparison of the distances of migration of labelled spermidine on a scanogram with non-labelled spermidine on a ninhydrin developed electropherogram showed nearly equal mobilities. Thus"it appears highly probable that spermidine was in fact, the substance, employed. Ideally, a reagent blal1k should exhibit a low intensity of fluorescence, so that small quantities of histamine can be readily discriminated from the blank. However, in the Shore, Burkhalter and Cohn and the Anton and Sayre methods, the reagent blanks account for a significant proportion of the total fluorescence when the amount of histamine assayed is equivalent to that contained in most biological samples. The chance observation that the reagent blank exhibited a less intense fluorescence in an alkaline than in an acid medium indicated the desirability of investigating further the factors that modulate the fluorescence of the reagent blanks. Although the reagent blanks were lower and the fluorescence of the histamine-OPT complex uneffected 46 in the absence of acid, the intensity of the blanks varied to such an extent that a disadvantage accrued to the omission of acid. Consequently, in all subsequent fluorometric determinations of histamine, acid was added to the reaction mixture prior to the measurement of the intensity of fluorescence. Since the present findings demonstrated that the fluorescence of a blank continually increases for at least 60 minutes in greater proportion than the fluorescence of the histamineOPT complex, no purpose was served in increasing the time beyond the standard five minutes. The mechanism of acid to enhance and simultaneously stabilize the fluorescence of the blank is unknown; likewise,it is not known how acid terminates the reaction between histamine and OPT. However, one explanation for the termination might be that the acid produces an ammonium salt of the histamine, which then would not condense with carbonyl groups. The manipulation of the temperature at which histamine reacts with OPT does not afford an improvement in the fluorometric method of determination of histamine. The fluorescence of the reagent blank is increased more at high temperature than that of histamine, and the precision was greatly diminished. Although at low temperature the precision was good, the sensitivity of the method was not improved; the slow rate of development of fluorescence, therefore, was a detriment not counterbalanced by an advantage. The investigation of the effects of certain antioxidants on the 47 development of the histamine-OPT fluorophore was the product of an early lack of awareness of the cause of the low recoveries of histamine from eluates from electropherogram. At this time, the very extensive tailing behind the so-called histamine zone had not been discovered, and hypotheses to explain the failure to elute most of the theoretical quantity of histamine from the visible zone were generated. One explanation, admittedl y not in accord with the known properties of his tam ine , was that the substance was oxidized to a non-fluorogenic compound during the course of electrophoresis .. The imidazole ring is subject to oxidation under certain circumstances (Paiva and Paiva, 1961). If oxidation occurred, inclusion of an antioxidant in the buffer solution might improve recovery. If the hypothesis were to be tested in this way, it would also be necessary to determine the effect of antioxidants on the development of fluorescence. The finding that NaHS0 and ascorbic acid diminish the develop,ment of fluorescence 3 was as expected, since bisulfite is a well known reagent of carbonyl groups, and ascorbate would be expected to reduce carbonyl groups to carbinols, thus diminishing the concentration of free OPT .. The reason for the ability of glutathione to form a fluorescent product in the presence of OPT is inexplicable at this time. However, a knowledge of the structure of the OPT-histamine complex might shed some light on this subject, and as yet this has not been determined . In summary, the small range in which histamine could be separated 48 from spermidine renders the paper electrophoretic method generally inapplicable to the separation and determination of histamine in the presence of spermidine. In addition, the amounts of histamine that could be adequately separated electrophoretically could not be eluted from electropherograms without erratic recovery. These findings of variable recovery from elution of histamine as well as of the histamine-OPT complex corroborate the observations of Shelley and Juhlin (1966). The extent of interference by relatively small amounts of spermidine in the fluorometric assay of histamine not only defeats the use of the paper electrophoretic method for the isolation of histamine but should serve as warning to investigators using other methods of supposed separation and determination. 49 TABLE 1 The effect of acid and temperature on the development of fluorescence of OPT - blanks. Initial Mixture Additions 0.4 N NaOH, 3.5 ml 1% OPT, 0.2ml 0.4 N NaOH, 3.0 ml 1% OPT, 0.2 ml 6 N citric acid, 0.5 ml 0.4 N NaOH, 3.0 ml 1% OPT, 0.2 ml 6 N citric acid, 0.5 ml at t = 4* Time after addition of OPT (min)* 0 1 2 3 4 5 10 20 40 60 0 1 2 3 4 5 10 20 40 60 0 1 2 3 4 5 10 20 40 60 Temperature 4°C 126 114 142 117 119 141 162 186 216 300 530 530 530 520 520 510 520 540 530 530 130 138 148 156 430 430 470 500 500 500 1171=1= 117 117 126 135 123 141 156 202 220 530 520 520 520 510 510 500 510 530 520 142 141 137 149 530 540 550 560 560 560 25°C 135 132 144 141 162 156 180 138 195 174 186 180 220 207 213 202 222 207 228 213 470 470 460 490 460 470 470 480 480 480 480 480 480 490 480 500 500 510 480 500 126 118 130 123 147 132 153 141 540 490 540 490 530 500 550 510 560 510 540 500 85°C 300 280 270 270 270 250 195 150 126 340 310 300 290 280 270 210 174 168 141 159 590 560 570 590 580 580 610 640 670 650 284 287 284 309 570 590 620 750 780 840 550 550 550 560 580 580 580 630 530 570 340 308 317 331 810 860 880 1080 1080 1101 *Minutes after addition of OPT to complete initial mixture; t stands for minutes after addition of OPT. tValues of readings expressed in arbitrary fluorescence units. One fluorescent unit is equal to 1 percent of transmission at the highest gain (sensitivity= .001). =f:Samples were studied in duplicate, and both values are shown. 50 TABLE 2 Recovery of histamine from sections of electroph~erogr~m, the location ot which were established by the reference distance method. * Quantity C 14 histamine appliedt (ng) 2 1000 2000 3000 4000 5000 10,000 Recovery at level of histamine-ninhydrin spot (%) 0 29 39 56 64 65 69 0 31 41 54 69 67 67 0* 39 43 52 63 64 65 Average (%) 0 33 41 54 65 65 67 *Reference distance was taken as the distance of migration in a separate channel containing 10 llg of histamine, the location of which was determined by treatment with ninhydrin reagent. tQuantity of histamine applied to origin, prior to electrophoresis. =t=Experiment was executed in triplicate; a.ll three per cent recoveries are shown. 51 TABLE 3 Distance traversed by various amounts of histamine and spermidine at pH 9.5, with 0.2 M sodium phosphate. Quantity of histamine applied* (ng) 2 20 120 520 1,020 5.p20 Quantity of spermidine applied * (ng) 24 124 240 524 1,024 5,024 10,024 Distance of migration (cm) 10.1 12.4 16.4 16.7 17.1 17.4 10.1 13.9 16.4 17 .1 17.3 17.8 Average (cm) 10.1+ 13.3 16.4 16.9 16.8 17.8 10.1 13.2 16.4 16.9 17.1 17.7 Distance of migration Average (cm) (cm) 13.8 13.9 13.8 13.8 14.0 13.7 13.5 14.0 13.7 13.5 13.6 13.5 13.8 13.8 14.0 13.7 13.7 13.8 13.9 13.8 13.9 13.9 13.7 13.7 13.7 13.8 13.8 13.7 *Quantity of histamine or spermidine applied to origin prior to electrophoresis. +:Experiment was executed in triplicate; all three distances of migration are shown. 52 Figure 1. Electrophoretic separation at pH 1.6. 1000 V was applied for four hours. Each darkened zone in the respective channels is the area produced from ninhydrin spray treatment of a 20 Ilg sample, applied to the origin prior to electrophoresis. •... • -•• •.. -• 38 34 30 26 22 18 em SPERMIDINE SPERMINE PUTRESCINE HISTIDINE HISTAMINE HISTAMINE SPERMIDINE SPERMINE PUTRESCINE HISTIDINE HISTAMINE HISTAMINE 14 10 6 .. 2 -+ ORIGIN 54 Figure 2. Electrophoretic separation at pH 1.6.1000 V was applied for one and one-half hours. Each darkened zone in the respective channels is the area produced from ninhydrin spray treatment of a 20 llg sample, applied to the origin prior to electrophoresis. MIXTURE SPERMIDINE SPERMINE PUTRESCINE HISTAMINE HISTIDINE MIXTURE SPERMIDINE SPERMINE PUTRESCINE HISTAMINE HISTIDINE 22 rB '14 10 em 2 6 ... - + ORIGIN 56 Figure 3. Electrophoretic separation at pH 4.8. 1000 V was applied for four hours. Each darkened zone in the respective channels is the area produced from ninhydrin spray treatment of a 20 llg sample, applied to the origin prior to electrophoresis. MIXTURE SPERMIDINE • • • SPERMINE PUTRESCINE • HISTIDINE HISTAMINE SPERMIDINE SPERMINE PUTREscrNE HISTIDINE HISTAMINE 26 " 22 HISTAMINf 18 14 em 10 2 6 ... -+ ORIGIN 58 Figure 4. Electrophoretic separation at pH 8.0. 1000 V was applied for four hours. Each darkened zone in the respective channels is the area produced from ninhydrin spray treatment of a 20 sample, applied to the origin prior to electrophoresis. ~g - -• MIXTURE SPERMIDINE SPERMINE PUTRESCINE HISTIDINE •• •- HlSTAMINE HISTAMINE SPERMIDINE SPERMINE PUTRESCINE HISTIDINE e 18 • 14 HISTAMINE HISTAMINE 10 em 2 6 ... - + ORIGIN 60 Figure 5. Electrophoretic separation at pH 9.1. 1000 V was applied for four hours. Each darkened zone in the respective channels is the area produced from ninhydrin spray treatment of a 20 Ilg sample, applied to the origin prior to electrophoresis. • • • • MIXTURE SPERMIDINE SPERMINE PUTRESCINE HISTAMINE SPERMIDINE SPERMINE PUTRESCINE HISTAMINE HISTIDINE 22 18 14 em 10 6 -+ ORIGIN 62 Figure 6. Electrophoretic separation at pH 9.5. 1000 V was applied for four hours. Each darkened zone in the respective channels is the area produced from ninhydrin spray treatment of a 20 Ilg sample, applied to the origin prior to electrophoresis. ... MIXTURE SPERMIDINE SPERMINE PUTRESCINE - HISTAMINE HISTIDINE MIXTURE SPERMIDINE SPERMINE PUTRESCINE HISTAMINE HISTIDINE 22 18 10 14 2 6 em .... -+ ORIGIN 64 Figure 7. Attempted recovery of histamine-ninhydrin complex from electropherograms containing varying quantities of histamine. The elucate was assayed spectrophotometrically at nm. A = 430 • - iC' ::t. Q- • C\J UJ z ::e ~ en :::J: >~ t= z<[ :::::> o q • • • • • rt') d C\J d A1ISN30 lV:>lldO 66 Figure 8. Apparent recoveries of histamine from electropherograms. Circles (0) represent fluorescence intensity obtained from test solutions (0.2 ml, 1% OPT in methanol and 6 N citric acid added at the appropriate times to 3 .0 ml histamine standard or eluate). Squares (D) depict fluorescence intensity obtained from eluates prepared by immersing into the test solution 1 in2 sections of Whatman No. 54 chromatography paper previously saturated with pH 9.5 sodium phosphate buffer (0.2 M) to which histamine standards were applied. Triangles (A) exhibit fluorescence intensity units obtained from eluates prepared by immersing into the test solution 1 in2 sections of electropherogram, the location of which sections were determined by the reference method. o ai o o ~ o o v o o o 0 0 CD AlISN31NI3~N3~S3~Onl~ o o N 68 Figure 9A. Spectrophotofluorometric activation scan of 1.0 I-lg histamine recorded at a fluorscence wavelength of 440 nm. B. Spectrophotofluorometric fluorescence scan of 1.0 I-lg histamine when the activation wavelength is set at 360 nm. ® I 200 @ I 300 I 400 WAVELENGTH mp.. I 500 I 200 I 400 WAVE LENGTH mp. I 500 i 600 70 Figure 10. Comparison of fluorescence intensity obtained from histamine standards developed with OPT in the presence (open circles, 0) and absence(filled circles, e) of glutathione. 3~N3~S3HOn1.:t 0 0 o 0 0 q ~~__~~~~~~______~~____~N____~q o • - A1ISN31NI o o 0 0 t.O ..,.q I I I I 01 q- "'5 I I I au z ::E t! en ::z:: I I )0- t- q!z N« =:) f a I I I I 1 q 0 I I I I I 0 0 q 0 ~ 0 0 q 0 ", o - A!ISN3!NI 0 0 0 0 N 0 0 0 cS 3~N3lS3HOn':I 0 72 Figure l1A. Spectrophotofluorometric activation scan of 10 J-lg spermidine when fluorescence wavelength is set at 440 nm. B. Spectrophotofluorometric fluorescence scan of 10 J-lg spermidine, when the activation wavelength is set at 360 nm. ® ® I 200 300 400 WAVELENGTH liP. 500 600 200 I 300 I 400 WAVELENGTH m}l 500 , 600 74 Figure 12A. Spectrophotofluorornetric activation scans of recrystallized OPT blank and 10 Ilg spermidine plus OPT. j The fluorescence wavelength is set at 440 nrn. B. Spectrophotofluorornettic fluorescence scans of recrystallized OPT blank and 10 Ilg spermidine plus OPT. The activation wavelength is set at 360 nrn. ® ® DINE -- -1 0 mg SPERMI DINE ......-10 mg SPERMI K l..-...--t-OPT BLAN I-----~OPT I 200 300 WAVELENGTH mSl SlANK 200 400 300 l mj H GT WAVElEN 500 j j j j j j 76 Figure 13. Effect of increasing amounts of spermidine on the intensity of fluorescence developed by 1.0 IJg histamine. Open squares (0) depict fluorescence intensity generafed by spermidine standards alone, and squares containingcrooses (a) represent fluorescence intensity obtained in the presence of 1.0 IJg histamine. The single cross (x) on the ordinate axis is the fluorescence intensity of 1.0 IJg of histamine in the absence of spermidine. B 8 0 I/') a a ~ LLI 2: - :E <[ l- - V) ::J: -- Ot ::t.. ot. 0. 0- "'w Iii Z -a:: Q :IE LLJ a.. (f) >- 010- 0 NtZ c:r: => 0 B ~ 0 0 N 0 0 a B B Q 8 0 B 0 0 CX) A1ISN31NI 0 0 0 <D 0 0 ~ 3JN3JS3~Onl.:J 0 0 N cg 0 0 78 Figure 14. Per cent recovery as a function of the amount of histamine applied to a channel. The per cent recovery is the per cent of applied radioactivity found in the zone at reference distance, the reference quantity of histamine being 10 jJg. • • -- • C" C • LaJ Z ::E • ~ ~ N • :x: >.... 0 - tZ <t ::l o ~----~------~------~------~--------------~----~O o 0 o CD V A~3AOJ3~ lN3J~3d N 80 Figure 15. Distance of migration as a function of the quantity of amine applied to acharmel. o o o c 10 o o o" , -- 0'1 C LaJ Z :IE « >01:: 0 ..... OZ N~ o o o c Q [J 8 N c c o (W:» NOI1'l~~IW .:10 3JN'I1SIO 82 Figure 16. Radioscanograms of channels of an electropherogram containing varying amounts of radiolabelled histamine as recorded by a 2'11' strip counter. The amount of histamine applied to each channel and its radioactive equivalent are given for each recording. A 2.0", Hil.oO .001 pC B 20", His.c .01pC _A----..-- C 120ng Hil.c .00JlC D 520nll His.c DIJle ~A~ E 1020ng Hil.eIl .00JlC ~ ~ 20 ~ ~ ~ ~ ~ 8 6 4 em -ORIGIN 84 Figure 17. Radioscanograms of the same strips scanned in Figure 16, but recorded at a 3-fold higher gain. The amount of histamine applied to each channel and its radioactive equivalent are given for each recording. I I I I I I I I I I I~-+- 520n9 His,"" ,0 IJlC ~ I A I I I B 1_-t--1020 n9 His,"" Dr"c ..........--+-- 5020 n9 His,·· .0IJlC c I I I I , I 2 em -1+ I -ORIGIN 86 Figure 18. Radioscanograms of channels from an electropherogram to which increasing amounts of histamine were applied simultaneously with a constant amount of spermidine. The dotted tracings depict superimposed responses obtained from channels to which varying amounts of histamine were applied alone. The solid recordings depict the responses elicited by the channels where histamine and spermidine were exposed to electrophoresis in combination. The amounts of histamine and spermidine applied to each channel and their radioactive equivalent are stated for each tracing. ----2.0no Hil.o.OOIJlC -1240", Sp.c.OIJ1C 20no His.C.OIJlC 1240 no $p.o.OIJlC B 12011CJ Hi•. c.OIJ1C 1240 no Sp.o.oIJlC c 520", Hi•. c.olJlC 1240 no $p. C .0IJlC o 1020no His. c .OIj1C 1240 no $p.c.OIj1C E 5020no His.o.olpC 1240 no Sp.o.olj1C F 24 em 10 8 6 4 2 _I + I -ORIGIN 88 Figure 19. Radioscanograms of channels from an electropherogram demonstrating anomalous retention of histamine at the origin. The amounts of histamine and spermidine applied to each channel and their radioactive equivalent are stated for each tracing. 2.0", His. C .oolpC 24n9 Sp. C .001pC 2.0ng His.c .OOIIlC 124", Sp. C .001 pC 2.0", His. c .001 IlC 524", Sp. co .0011lC 2.0ng His. C .001 pC 1024n9 Sp.c.OOIJlC 2.0RQ His.c.OOlpC 5024RQ Sp.o.OOIIlC 102", His.o.oolpC 24nv Sp..... .oOlpC F 26 24 w m ~ em K ~ W 8 6 4 2 90 Figure 20. Radios canograms of channels from an electropherogram containing varying amounts of histamine. The electrophoresis was conducted in 0.2 M potassium phosphate buffer at pH 9.5. The amount of histamine applied to each channel for electrophores is and its radioactive· equivalent are given. in each. recording. 2.0 ng His. c .001 jiC A 20 no His.:::::: .01)l.C 8 120", His. C.OI)1C c 520no Hiao.OIp.C o 1020 ng His.o.OI)1C E 5020ng His. c .oI)1C F 26 24 22 20 18 16 em 14 12 10 8 6 4 2 -1+ -ORIGIN 92 Figure 21. Radioscanograms of channels from an electropherogram containing varying amounts of histamine in combination with a constant amount of spermidine. Electrophores is was conducted in 0.2 M potassium phosphate buffer at pH 9.5 .. The amounts of histamine and spermidine applied to each channel for electrophoresis and their radioactive equivalents are stated for each tracing. 2 ng His.:Q: .001 JLC 1240ng Sp.o.OIJLC 20ng His.:Q: .0IJLC 1240 ng Sp. c .oIJLC 120 ng His. Co DI)LC 1240ng Sp.:Q: .oIJLC 520ng His.o.OIJLC 1240", Sp. 0 .01 JLC 1020 ng His. c .01)LC 1240 ng Sp. 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