STUDIES ON URIC ACID AND RELATED COMPOUNDS* II. PAPER CHROMATOGRAPHY OF SUBSTITUTED XANTHINES AND URIC ACIDS BY SHABTAY DIKSTEIN, FELIX BERGMANN, AND MOSHE CHAIMOVITZ (From the Department of Pharmacology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel) (Received for publication, June 23, 1955) Methylated xanthines are converted in the animal organism into a mix- ture of xanthines and uric acids by two simultaneous processes : demethyla- tion and oxidation (l-3). However, the sequence of these two reactions and the exact metabolic pathways are still unknown. A quantitative study of the metabolism of substituted xanthines has been attempted by many investigators. Thus, for instance, Buchanan, Christman, and Block (4) measured the increase, after ingestion of theophylline, in phosphotung- stic acid-reducing material that was not destroyed by the action of uricase. Brodie, Axelrod, and Reichenthal (5) purified the metabolites of theophyl- line, which are excreted in an 18 hour sample of human urine, by counter- current distribution and ion exchange chromatography. The procedure, although lengthy and requiring large volumes of urine, permits the identifi- cation and approximate estimation of the methylated uric acids, which appear as metabolites of theophylline. Weinfeld and Christman (6) identified the uric acids, excreted after application of caffeine or theophyl- line to various species, by paper chromatographic separation, but again took recourse to the method of Buchanan and coworkers (4) for quantita- tive determination. None of these procedures can be applied to the analy- sis of plasma, since the volume of blood required is too large. Paper chromatography represents the most sensitive method for quanti- tative separation of members of a homologous series, but requires a reliable test for spot detection. In the xanthine group, fluorescence in ultraviolet light has been used by Markham and Smith (7) and Weisman et al. (8). The xanthines can also be made visible by complex formation with platinum chloride and potassium iodide (9). Michl (10) obtained fluorescent de- rivatives of xanthines by treatment with chlorine. For uric acid and some of its alkyl derivatives spraying with an arseno- phosphomolybdic reagent has been used by Johnson (11). Vischer and Chargaff (12) located uric acid on paper by applying mercuric nitrate first, * Presented before the Fourteenth International Congress of Pure and Applied Chemistry, Zurich, Switzerland, July, 1955. 239 by guest on May 1, 2020 http://www.jbc.org/ Downloaded from
Transcript
STUDIES ON URIC ACID AND RELATED COMPOUNDS*
II. PAPER CHROMATOGRAPHY OF SUBSTITUTED XANTHINES AND URIC ACIDS
BY SHABTAY DIKSTEIN, FELIX BERGMANN, AND MOSHE CHAIMOVITZ
(From the Department of Pharmacology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel)
(Received for publication, June 23, 1955)
Methylated xanthines are converted in the animal organism into a mix- ture of xanthines and uric acids by two simultaneous processes : demethyla- tion and oxidation (l-3). However, the sequence of these two reactions and the exact metabolic pathways are still unknown. A quantitative study of the metabolism of substituted xanthines has been attempted by many investigators. Thus, for instance, Buchanan, Christman, and Block (4) measured the increase, after ingestion of theophylline, in phosphotung- stic acid-reducing material that was not destroyed by the action of uricase. Brodie, Axelrod, and Reichenthal (5) purified the metabolites of theophyl- line, which are excreted in an 18 hour sample of human urine, by counter- current distribution and ion exchange chromatography. The procedure, although lengthy and requiring large volumes of urine, permits the identifi- cation and approximate estimation of the methylated uric acids, which appear as metabolites of theophylline. Weinfeld and Christman (6) identified the uric acids, excreted after application of caffeine or theophyl- line to various species, by paper chromatographic separation, but again took recourse to the method of Buchanan and coworkers (4) for quantita- tive determination. None of these procedures can be applied to the analy- sis of plasma, since the volume of blood required is too large.
Paper chromatography represents the most sensitive method for quanti- tative separation of members of a homologous series, but requires a reliable test for spot detection. In the xanthine group, fluorescence in ultraviolet light has been used by Markham and Smith (7) and Weisman et al. (8). The xanthines can also be made visible by complex formation with platinum chloride and potassium iodide (9). Michl (10) obtained fluorescent de- rivatives of xanthines by treatment with chlorine.
For uric acid and some of its alkyl derivatives spraying with an arseno- phosphomolybdic reagent has been used by Johnson (11). Vischer and Chargaff (12) located uric acid on paper by applying mercuric nitrate first,
* Presented before the Fourteenth International Congress of Pure and Applied Chemistry, Zurich, Switzerland, July, 1955.
followed by hydrogen sulfide treatment. Reguera and Asimov (13) used for the same purpose the combination silver nitrate-potassium chromate. The reduction of ferric to ferrous ion and the formation of the red o-phe- nanthroline complex of the latter have enabled Bode and Htibener (14) to detect 0.5 y of uric acid. We have found this method applicable to all methylated uric acids, which contain at least one free NH- group. How- ever, the background also reddens slowly and thus makes quantitative determination difficult. The reaction is negative with xanthines.
We have tried the methods mentioned above, but none of them proved useful for all derivatives under consideration. Color intensity varies in most cases from one homologue to the other and thus not only would require individual standardization, but also would set a different limit to the quan- titative determination of each member of a series. The multitude of reagents, described in the literature, merely indicates the difficulties en- countered in the identification of xanthines and uric acids in chromato- grams. None of the color reactions can unambiguously detect quantities below about 20 to 50 y per sq. cm.l It is, therefore, apparent that the most accurate and reliable method is ultraviolet spectrophotometry, whereas any calorimetric reaction may serve only for qualitative spot de- tection.
The present investigation has led to a simple procedure for the quantita- tive separation of all components of mixtures of xanthines and uric acids, such as occur in plasma and urine. A new method has been found for spot staining, which permits easy and unambiguous determination of Rr values. The ultimate analysis, however, is carried out with the unstained materials, which are extracted from the paper and measured spectrophoto- metrically. Only a few micrograms of each component are required; hence the method is suitable for metabolic studies.
Materials and Methods
‘i-Methyl- and 1,7-dimethylxanthines were obtained through the cour- tesy of Dr. J. J. Fox of the Sloan-Kettering Institute for Cancer Research. 3-Methyl- and 1,3-diethylxanthines were a gift of Dr. V. Papesch of G. D. Searle and Company, Chicago. l-Methyl- and 1,7-dimethyluric acids were kindly contributed by Professor D. Keilin, Molten0 Institute, Cambridge, England, and 1,3,7,9-tetramethyluric acid by Professor E. Boyland. The other derivatives were synthesized, as described in earlier papers (16, 17). 1-Methylxanthine is difficult to obtain in pure form (18). The following synthesis is based on the method of Bredereck et al. (19):
1 With special ultraviolet filters, it is possible to detect purines in quantities of 1 7 per ml. or even less (15). However, the method is much less sensitive in the case of substituted uric acids.
I-Methyluric acid (25 mg.) was added to 0.4 ml. of boiling formamide. After 2 hour the suspension suddenly became clear. Boiling was continued for another 10 minutes; the mixture was cooled and diluted with 10 ml. of water. The solution is acidified with 1 ml. of 10 per cent HCl and passed through Amberlite IR-112-H (column length 15 cm., diameter 6 mm.). After washing the column with 5 ml. of 1 per cent HCl, the ad- sorbed material was eluted with 60 ml. of 6 per cent ammonia and the eluate was evaporated to dryness. The residue was dissolved in 5 ml. of hot 1 per cent ammonia and decolorized with charcoal and the filtrate was again evaporated to dryness. In this way 17.5 mg. of colorless crystals were obtained. The purity of this preparation was checked by chromatographic and spectrophotometric analyses.
The reagents used for spraying are as follows: (a) mercuric acetate, 0.25 per cent in 95 per cent alcohol, with addition of a few drops of glacial acetic acid to prevent precipitation of mercuric oxide, and (b) diphenyl- carbazone, 0.05 per cent in 95 per cent ethanol. Whatman paper n-0. 1 was used in all experiments. All solvent mixtures were prepared from commercial products. RF values were determined on descending chro- matograms. For spot detection under ultraviolet8 light a Magnaflux “black light” lamp was used.
Results
Detection of Xanthines and Uric Acids on Paper Chromatograms
In the method of Vischer and Chargaff (12) the paper is first sprayed with mercuric nitrate, the excess reagents are removed by washing, and the spots are then made visible by blackening with hydrogen sulfide. Since the mercuric complexes of the methylated derivatives are soluble in acid media (Bergmann and Dikstein (20)), they are easily removed by the washing step, especially if present in microgram quantities only. However, the mercuric complexes of all xanthines and uric acids are practically in- soluble in organic liquids. We, therefore, turned to the use of alcoholic solutions for spraying; e.g., spraying with mercuric acetate in acidulated ethanol, without subsequent washing, permits location of the complexes. Under ultraviolet light they show light blue fluorescence on a dark violet background. However, the amounts necessary for unambiguous identi- fication of a spot of about 1 sq. cm. are in the order of 20 to 50 y. Thus this procedure, like the “direct” method of Markham and Smith (7), is not sufficiently sensitive. In addition, it was found that ultraviolet ir- radiation decomposes the mercuric complexes slowly.
Because of the limited detectability in ultraviolet light of the mercuric complexes, we tried to make them visible with some of the color reagents used in inorganic analysis. Ionic mercuric derivatives, e.g., give a blue-
violet color with diphenylcarbazone (DPC), whereas covalent compounds, such as mercuric chloride, do not stain with the reagent. When solid mercuric urate is immersed in an alcoholic solution of DPC, the surface of
* The RF values were measured in descending chromatograms, after the solvent front had advanced for about 30 cm. The chromatography chambers were kept at about 27”.
t Solvent A, 95 per cent ethanol 85 ml., acetic acid 5 ml., water 10 ml. $ Solvent B, 95 per cent ethanol 70 ml., pyridine 20 ml., water 10 ml. $ Solvent C, pyridine 94 ml., 25 per cent NHB- 6 ml.; final ammonia concentra-
tion, 1.5 per cent. 11 Since caffeine and tetramethyluric acid do not stain with the Hg++-DPC reagent,
their positions were determined spectroscopically.
the particles stains blue-violet, indicating the ionic nature of this derivative. This method proved to be generally applicable: Upon spraying the paper first with mercuric acetate and then with DPC, the whole sheet turned red-violet, but the spots could be recognized by their different and more intense shade. Upon heating the paper over a hot-plate, the color of the background gradually faded, and the spots of the xanthines and uric acids
FIG. 1. The relationship between log (l/R F - 1) and n, the number of carbon atoms in the alkyl groups attached to the heterocyclic nucleus. X, methylated uric acids (U) ; 0, methylated xanthines (X). 1,3-E indicates the diethyl derivative of the homologous series. A, Solvent A (see Table II), note the relatively small devi- ations from the straight lines both in the U and X series. B, Solvent B, more pro- nounced deviations appear in the U series. In the X series the experimental points can be represented only by two lines with a different slope. C, Solvent C, the xan- thines are again represented here by two straight lines, but in this case with an identical slope.
stood out as well defined areas. Upon continued heating, these also dis- appeared. It was, therefore, necessary to determine the heat stability of the various mercuric derivatives. This was achieved in the following way: 10 y of each material were put on spots of about 1 sq. cm. area, stained by the above procedure, and the time required to bleach the color at various temperatures was determined by putting the paper into a constant temper- ature oven. The results of these experiments are summarized in Table I. In the final procedure the paper is always heated to 90” for about 15 min- utes. The exact time is not critical under these conditions. Direct heat- ing over a hot-plate, under eye control, is a simplified modification of this procedure. The stained spots preserve their color for several months.
We now were in a position to study the lower limit of detectability for each individual derivative. The results, which are included in Table I, indicate that the procedure is suitable for the identification of microgram
quantities, such as are to be expected in biological experiments. The fully methylated derivatives, caffeine and 1,3,7,9-tetramethyluric acid, do not form mercury complexes, apparently because of the lack of a free NH- group. These two compounds were therefore located in chromatograms by extracting each cm. of the paper strip separately and measuring the amount present by ultraviolet spectrophotometry.
The only calorimetric reaction, which proved applicable to all xanthines and uric acids, is the method of Reindel and Hoppe for the detection of amino acids, peptides, and related compounds (21). These authors exposed the paper to the gases, produced from permanganate and hydrochloric acid, and then treated it with a mixture of o-tolidine and potassium iodide to obtain a blue color. In this procedure a positive reaction is obtained even with caffeine and tetramethyluric acid (5 y per spot). However, with such small quantities of material, the color disappears within 1 to 2 hours. In addition, this method is very sensitive to impurities of the paper and is inapplicable after the use of basic solvents (e.g. pyridine), which cause the whole paper to react. For the purposes of the present investigation, therefore, the “mercury” method proved to be more useful.
Paper Chromatography of Xanthines and Uric Acids
In view of the limited solubility of these substances in organic solvents, aqueous mixtures appeared most promising for the development. The butanol-formic acid mixture, used previously by Markham and Smith (7), was tried first. It suffers from the disadvantage that its components react slowly with each other and that the increasing percentage of butyl formate changes the results from 1 day to the other. When the final equilibrium mixture was used, separation of the homologues under investigation was unsatisfactory; therefore, various mixtures with acetic acid or pyridine were studied. The RF values of three selected solvents are summarized in Table II.
Fig. 1, A to C, demonstrates the varying separating power of three sol- vent mixtures. For xanthines two-dimensional chromatography is re- quired for the analysis of an unknown mixture. In general, the RF values of isomers are closely related and well spaced from the next higher and lower homologue. Therefore, a plot of log (l/R, - 1) versus n, the num- ber of carbons in the side chains attached to the heterocyclic nucleus, gives in most cases the linear relationship postulated by Martin (22).2
Attempts were made to modify the chromatographic procedure in such a way as to be able to observe the migration of the various components with the naked eye. For this purpose, mercuric acetate was applied di-
2 It should be noted that the RF values change with the distance of travel. The values in Table II were all obtained with a solvent front about 30 cm. from the origin.
rectly to the original spot and DPC dissolved in the developing mixture. The Rp values mere identical with those obtained in the absence of mercury, indicating that the solvent extracts the organic molecules and the metal ions separately and independently. It was accordingly observed that during “mercuric chromatography” the color of the advancing spots faded gradually.
I SOLVENT c (~.DIMENsI~N)
5 I ,” so=A STARTING POINT
(i?.DIMENSION) OF ORIGINAL MIXTURE
FIG. 2. Two-dimensional paper chromatogram of methylated xanthines. Loca- tion of the homologous xanthines after application of Solvent C is shown on the right side. Although a-methyl- and 1,7-dimethylxanthines are closely connected, the two spots are distinguishable by the different color of their complexes with mercury and diphenylcarbazone (see Table I). Since these two derivatives are extracted together from the paper, it is convenient to run the chromatogram in a second dimension with Solvent A. However, it is also possible to determine a mixture of these two homo- logues spectrophotometrically, since their absorption maxima are entirely different (to be published).
Separation Procedure
Caffeine, the most highly substituted xanthine, ingested with normal food, may give a mixture of fourteen methylated xanthines and uric acids. Successful separation and determination of all possible metabolites require, therefore, first separation of the two main groups, A suitable procedure can be based on the observation that only xanthines form cations in strongly acid solutions (16). They are therefore adsorbed by a cation exchanger, such as Amberlite IR-112-H, and can be subsequently eluted by ammonia with recovery of at least 95 per cent. Similar procedures have been used
previously by Johnson (11) and Weisman, Bromberg, and Gutman (8). The separation of the members of either the xanthine or uric acid series is shown in Figs. 2 and 3. For the former group we use first Solvent C (pyridine-ammonia) and in the second direction, Solvent A (ethanol-acetic acid) .3 For the separation of uric acids Solvent B (ethanol-pyridine) has been applied in both directions. Since the ratio of the distances, covered
I SOLVENT B (~.DIMENSION)
STAkG POINT
(2 DIMENSION) OF ORIGINAL MIXTURE
FIG. 3. Two-dimensional paper chromatogram of methylated uric acids. In both directions Solvent B was used. Therefore the spots, obtained in the first direction (at the right-hand side), as well as those in the second dimension, are situated along a straight line. The spots of l- and 3-methyluric acids lie close together, but can be clearly distinguished because of the different shade of their Hg++-DPC complexes (see Table I). For quantitative analysis these two isomers are extracted together from the paper, but do not interfere with each other in the spectrophotometric de- termination, since the pH dependence of their maximal extinction is entirely differ- ent (to be published).
by any individual component in the two perpendicular directions, is con- stant, it is to be expected that all spots are situated along a straight line. This is borne out by the experiment shown in Fig. 3. It should be noted that the solvent combinations were selected after a large number of trials and do not represent the only possible solution of the present problem.
3 If adenine, guanine, and hypoxanthine are also present, they are adsorbed as cations and thus accompany the xanthines. In this case, the above procedure has to be modified, as will be described in the application of the method to biological fluids.
Thus, xanthines have been separated by Weisman et al. (8), using butanol- ammonia first and subsequently butanol-formic acid.
DISCUSSION
The curves in Fig. 1, A to C, reveal certain interesting relationships. While in Solvents A and C the straight lines for xanthines and uric acids are approximately parallel, this is not the case for Solvent B. Therefore, in both Solvents A and C, the rule of Martin (22) is obeyed; i.e., for each additional CHZ- group the same increment in AF is obtained within a homologous series. However, in Solvent B, MF (increment in AF) de- creases sharply for xanthines, while remaining at the same level for uric
TABLE III Free Energy of Transfer of Methylene Groups Between
Stationary and Mobile Phases
The figures were calculated from the slopes of the straight lines in Fig. 1, A to C, by using the equation AAF = 2.3 RT log (l/R pn - l)/(l/Rp,el - 1). T = 300” absolute.
* It should be noted that the MF values are of the same magnitude as those ob- tained for the transfer of a methylene group in cholinesterase inhibitors from the free solution to the enzyme surface (23).
acids. The values of AAF, derived from Fig. 1, A to C, are given in Table III. It is concluded that in Solvent B the distribution of xanthines be- tween the stationary and mobile phases involves an additional factor, which is of no importance for uric acids. A discussion of this problem will be given later.
All compounds included in the present investigation combine with mer- curic ions, with the exception of the fully methylated derivatives, caffeine, and 1,3,7,9-tetramethyluric acid. However, the nuances of the color, formed by staining the mercuric complexes with DPC, vary from one substance to the other (Table I). It is thus indicated that the position of NH- groups, participating in complex formation, determines the specific structure and stability of the coordinative compound. Our results suggest that conjugation of an NH- group with another unsaturated structure,
e.g. C=O, is the minimal requirement for the formation of stable mercuric complexes. However, in testing this assumption, it was found that simple amides, amidines, or guanidines do not give a positive reaction. Only biuret can be stained with Hg++ and DPC. It thus appears that an NH- group has to be cross-conjugated with at least two unsaturated groups in order to give a positive test. All heterocyclic compounds studied so far follow this general rule. The procedure, developed in this investigation, should, therefore, be applicable to a large number of compounds, including pyrimidines, barbiturates, purine derivatives, and imidazoles. The ex- tension of the method to these heterocycles is now under study, and the results obtained will be reported later.
The thermal stability of the complexes with Hgff and DPC shows an interesting relation to structure. In the xanthine series all dimethyl deriv- atives fade at about the same high rate, then follows 3-methylxanthine, thereafter xanthine itself, while 7-methylxanthine is the most stable one. Evidently, when positions 1 and 3 are free, the complex is more heat-stable than when positions 1 and 7 are unsubstituted.
In the uric acid series the most stable complexes are obtained when only positions 7 and 9 are free, since both 1,3-dimethyl- and diethyluric acids excel the mother substance in their heat stability. On the other hand, if all nitrogens are occupied, besides N-l or N-9, very unstable complexes result. This may serve as an indication that the heavy metal atom at- taches itself to at least 2 heteroatoms in the uric acid structure. There- fore, the intramolecular distance between the free nitrogens determines the heat stability of the complex. However, this does not explain why the mercuric complex of 1,3-dialkyluric acids should be more stable than that of uric acid itself. Probably, free NH- groups in positions 1 and 3 com- pete with N-7 and N-9 for the metal and thus decrease the over-all stability of the complex. Therefore, the observed order of heat stability is 1,3- dimethyl > l- or 3-methyluric acid or uric acid itself.
The application of the chromatographic procedure, described in this paper, to biological fluids will be reported in a future communication.
SUMMARY
Microgram quantities of xanthines and uric acids can be detected on paper by staining their mercuric complexes with diphenylcarbazone. This method is also applicable to related heterocyclic systems.
A two-dimensional paper chromatographic procedure has been developed for the separation of homologues within each series. Xanthines are first quantitatively adsorbed from a strongly acid solution onto a cation ex- changer, Amberlite IR-112-H, and quantitatively eluted with ammonia before spotting. The uric acids, which pass through the column, are chromatographed separately.
A plot of log (l/RF - 1) versus n, the number of CHZ- groups attached to the ring system of uric acid, gives straight lines with almost identical slopes for various solvents. However, for xanthines the slope varies from one solvent to the other, and in some cases two straight lines, rather than one, represent the above function for various methylated derivatives.
This work was supported by a grant from the Hadassah Medical Organi- zation.
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