Simplified Rapid Procedure for Determinationof Agmatine and Other Guanidino-Containing Compounds Millicent C. Goldschmidtl and Betty M. Lockhart Department of’ Clinical Pathology, The University of Texas M . D. Anderson Hospital and Tumor Institute at Houston, and The University of Texas Graduate School of’Biomedica1 Sciences, Houston, Texas 77025 This paper presents an adaptation of two methods into a single rapid and sensitive micro-quantitative and/or -qualitative procedure for use in the routine laboratory analyses of guanidino compounds. Agmatine can be quantitatively or qualitatively detected in the presence of arginine or creatine following a differential extraction with an alkaline n-butanol solution. 8045% of the agmatine is removed from an aqueous solution by this procedure. The a matine is quickly visualized (within 3 min) by the a d i t i o n of a modified diacetyl reagent to the butanol layer. As little as 5-10 pg/ml of agmatine can be detected. Differential extraction of several other guanidino and related compounds is discussed. In addition, the same reagent can be used to quantitate agmatine, arginine, creatine, and other guanidino-containing compounds over a range of 2-90 pg/ml. Time concentration curves are also presented.
AGMATINE, the amine formed by decarboxylation of arginine, is usually determined in biological systems either by the production of COS ( I ) , a color change in an indicator (2), or fluorometric analysis (3). Sakaguchi’s reagent (4, 5), diacetyl (6-8), and phenanthrequinone (9) have also been used to detect guanidino-containing compounds (IO). This paper presents a rapid and sensitive micromethod for the qualitative and quantitative determination of agmatine, arginine, and several other guanidino-containing compounds. In combination with a modified differential procedure, agmatine can be determined in the presence of arginine. A simplified diacetyl reagent quickly allows the visualization of these compounds on either a qualitative or quantitative basis. EXPERIMENTAL
Reagents and Equipment. All chemicals were reagent grade unless otherwise specified and the solvents were spectro quality. Glass distilled water was used. Solutions of 1naphthol and diacetyl (2,3-butanedione) were stored in dark (amber) glass bottles in the cold. Pre-coated Silica Gel 1 Present address, The University of Texas Graduate School of Biomedical Sciences at Houston, 109 Herman Professional Building Garage, 6410 Fannin, Houston, Texas 77025
( 1 ) E. F. Gale in “Methods of Biochemical Analysis,” D. Glick, Ed., Vol. 4, Interscience, New York, N.Y., 1957, p 285. (2) V. Mpller, Acta. Parhol. Microbiol. Scand., 34, 102 (1954). (3) V. H. Cohn and P. A. Shore, Anal. Biochem., 2,237 (1961). (4 S. Sakaguchi, J . Biochem., 37, 231 (1950).
(5) J . F. Van Pilsum, R. P. Martin, E. Kito, and J. Hess, J . Biol. Chem.,222, 225 (1956). (6) A. H . Ennor and L. A. Stocken, Biochem. J., 55, 310 (1953). (7) H. Rosenberg, A. H. Ennor, and J. F. Morrison, ibid., 63, 153 ( 1956). (8) L. Meites, “Handbook of Analytical Chemistry,” McGrawHill, New York, N.Y., 1963, pp 10-83. (9) B. E. Magun and J. W. Kelly, J . Histochem. Cytochem. 17, 821 (1969). (10) J. E. Bonas, B. D. Cohen, and S. Natelson, Microchem. J . , 7,63 ( 1963).
thin-layer chromatography (TLC) plates without fluorescent indicator (E. Merck AG, Darmstadt, Germany) were obtained from Brinkmann Instruments, Inc. A DK-2A Ratio Recording Spectrophotometer (Beckman Instruments, Inc.) and a Zeiss Spectrophotometer PMQlI (Brinkmann Instruments, Inc.) were used. Procedures. DIACETYL REAGENT.The diacetyl reagent consisted of a combination of two solutions which were stored separately and mixed together before use. Solution A : 60 pl of diacetyl were diluted to 100 ml with distilled water and refrigerated in a dark glass bottle for periods not exceeding 2 weeks. Solution B: 1 gram of 1-naphthol, 6 grams of NaOH, and 20 grams of NaCl were dissolved in distilled water, diluted to 100 ml and stored in a dark glass bottle at -5 “C for periods not exceeding 3-4 weeks. This solution remains a liquid at this temperature. The solutions were freshly mixed daily in the proportions of 2 parts A 3 parts B. It was also possible to add each solution individually to the sample in these proportions. Three milliliters of the diacetyl reagent were added to each 6-ml sample. The appearance of a red-purple color within 3-5 min indicated the presence of a guanidino containing compound. DIFFERENTIAL EXTRACTION PROCEDURE. The procedure used by Cohn and Shore (3) to extract agmatine from a mixture of arginine and agmatine has been greatly simplified and modified. A salt-saturated KOH solution was prepared by adding sufficient solid NaCl to 10% KOH so that an excess of NaCl remained in the flask. Equal proportions of the sample and this solution (2 ml of each) were mixed in 125- X 16-mm screw cap test tubes. The differential extraction was completed by adding 2 ml of n-butanol to each tube. The tubes were then agitated for 1-2 min and centrifuged or allowed to settle. Then, 0.5 ml of the top butanol layer was removed and mixed with the diacetyl reagent to detect the appearance of colored derivatives in this layer. If only a qualitative estimation was desired, it was not necessary to remove the butanol layer from the reaction tube. The diacetyl reagent was added directly to the butanol layer, the tube agitated slightly, and this layer observed for color formation. THIN-LAYER CHROMATOGRAPHY. Ascending one-dimensional chromatograms were obtained using Silica Gel thinlayer chromatography plates. These were preactivated by heating for 30 min at 100-110 “ C and could be stored for several months in a desiccator chamber without further reactivation. The solvent system consisted of a mixture of phenol, acetic acid, and water in a ratio of 6:1 :6. The plates were dried at 100-110 “C before developing. Several spray reagents were used to observe the positions of the compounds on the plates. These were prepared as follows: Ninhydrin Spray Reagent Ninhydrin, 0.1 gram, was dissolved in a small volume of 95% ethanol and brought to a final volume of 100 ml with chloroform. The plates were then heated to 100-110 “ C just until the purple-orange color developed. Diacetyl Spray R e a g a . Soution A was sprayed on the plates. After air drying, solution B was sprayed on the plates. The plates were then heated to 100-110 “C just until the red-orange color developed. Sakaguchi Spray Reagent. The thymine, 1-naphthol, and thiosulfate solutions were combined as indicated in the pro-
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Table I. Qualitative Determination of Guanidine-Containing Compounds before and after Differential Butanol Extraction Before extraction In tube On chromatogram Da Sak Da Sak Nin
In tube Da Sak
After extraction On chromatograz Da Sak Nin
Compound Agmatine L-Arginine L-Homoarginine Creatine Creatinine* Spermine Spermidine L-Citrulline L-Canavaninec 4-Guanidinobutyl-1-amine 1,4-Diguanidobutanesulfate = red-purple or orange color indicating presence of guanidino compounds. - = no color or absence of these compounds. Extraction = treatment with NaCl saturated 10% KOH followed by agitation with n-butanol. Sak = Sakaguchi reagent. Da = Diacetyl reagent. Nin = Ninhydrin reagent. a = n-Butanol layer. * = Very very weak diacetyl reactions. c = Very weak Sakaguchi reaction on chromatogram. 20 to 40 micrograms were used to spot the chromatograms; 80-100 micrograms/ml were added to the tubes in these reactions.
+
++ ++ +
++ +
++ +
++ +
++ +
++ + ++
cedure of Bonas et al. (10) except that the NaOCl was not added. The plates were sprayed with this solution and heated to 100-110 "C for 1-3 min. While still hot, they were sprayed with NaOCl (undiluted commercial Clorox) to develop the orange color associated with guanidino groups. SPECTROPHOTOMETRIC DETERMINATIONS. Samples for spectrophotometric determinations were dissolved in distilled water and reacted with the diacetyl reagent. Similar samples were differentially extracted and the butanol layer reacted with the diacetyl reagent. The samples were then pipetted into cuvettes and placed in the Beckman DK-2A Ratio Recording Spectrophotometer t o obtain the characteristic absorption curves. Quantitative measurements were also made on these solutions using a Zeiss Spectrophotometer model PMQII. The wavelength used depended on the absorption characteristics of the compounds and is so indicated in the data. RESULTS AND DISCUSSION
Guanidino compounds are important metabolic precursors of many substances including ornithine, urea, and creatinine. Agmatine has been mentioned as one of the classical substrates of diamine oxidase (3). In addition, one test used in the identification of Gram-negative bacteria involves a lengthy monitoring of arginine decarboxylase and dihydrolase activity. Thus, a rapid method of detecting and quantitating several important guanidino compounds, especially agmatine, would be of value to the microbiologist and the biochemist. Attempts to develop a rapid routine method for detection of arginine decarboxylase activity in microbial cultures resulted in the adaptation and modification of a differential extraction procedure and a diacetyl reagent into this single method for the rapid detection of agmatine in the presence of arginine. The use of this procedure in the routine detection of agmatine produced by various bacteria (media, pH, and other growth parameters) will be published in a separate paper (11). (1 1) M. C.
press. 1476
Qualitative Determinations. Diacetyl and Sakaguchi reagents have been used to detect certain guanidino compounds (4, 7, 8). However, the Sakaguchi reagent is only specific for monosubstituted guanidino derivatives (IO). In addition, the control blank is colored, thus making the detection of small amounts very difficult. We have modified the diacetyl reagent so that amounts as low as 2-10 pg of guanidino compounds such as agmatine and arginine can be detected. Related compounds such as citrulline, spermine, and spermidine were also tested to determine the specificity of the reaction with the modified reagent. These data are presented in Table I. Chromatograms were run on pre-coated thin-layer silica gel plates to observe the position of the different compounds. Although DiJeso (12) reported the use of hand-prepared cellulose TLC plates using several different solvent systems followed by many different spray reagents, ours was a simple procedure for these compounds. Duplicate samples were differentially extracted and the butanol layer was tested for compounds which would react with diacetyl and Sakaguchi reagents. Chromatograms similarly monitored the differential extraction process and indicated the presence of only a few guanidino compounds in the butanol layer. These were agmatine, 1,4-diguanidobutane sulfate and 4-guanidinobutyl-1-amine. Chromatograms were also sprayed with ninhydrin to detect the compounds with NH2 groups and observe their presence before and after differential extraction. A summary of these experiments is presented in Table I. As can be seen, the extraction procedure also removed spermine and spermidine from the mixture. Meites (8) indicated that creatinine gave a positive reaction with diacetyl reagent. However, we observed only a weak reaction with our modified reagent (Table I) and could not detect a reaction with the concentrations used in the recording spectrophotometer (Figure 1). Therefore, this reagent is not recommended for the determination of small amounts of
Goldschrnidt and B. M. Lockhart, Appl. Microbiol., in (12) F. DiJeso, J . Chromatogr., 32,269 (1968). ANALYTICAL CHEMISTRY, VOL. 43, NO. 1 1 , SEPTEMBER 1971
1.00.
1.4-Disuanidobutonc-sulfate
0.80
4-
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Gu.nidinobutrl-l-amine
0.60
z Figure 1. Absorption spectra of various compounds after reacting with the modified diacetyl reagent in water (-) and in the n-butanol layer following the differential extraction procedure (- -)
-
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4 - Guanidinobutyl-I-amine FAgmlrtine
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creatinine (20-50 pg). Addition of 0.1 ml of dimethylsulfoxide per 3 ml of diacetyl reagent slightly intensified the color reaction. The absorption spectra of several guanidino derivatives produced during the reaction with the diacetyl reagent were recorded and reproduced in a composite figure (Figure 1). Several other compounds, such as citrulline, spermine, and spermidine were included in order to observe possible interference and areas of maximal absorbance. The spectral curves for the guanidino derivatives again provided supportive data since they indicated that only agmatine, 1,Cdiguanidobutane sulfate, and 4-guanidinobutyl-1-amine were present after the differential extraction procedure with butanol. Since different initial amounts were used in these experiments, the relative proportions before and after extraction cannot be determined from the data. Actually, about 8085 % agmatine was extracted by this method. Quantitative Determinations. There are several methods reported in the literature for quantitative determinations of various guanidino compounds. Cohn and Shore (3) presented a sensitive fluorometric assay for agmatine. Unfortunately, the derivative lost its stability 15 min after the reaction was completed. Several modifications of Sakaguchi's procedure have been published (4, 5, IO). Hutzler, Odievre, and Dancis (13) reported a n indirect method for detecting agmatine using dinitrofluorobenzene. However, many of these procedures have high color control blanks or are too lengthy and complicated for routine laboratory procedures. Figures 2-5 show the time-concentration curves for arginine, creatine, agmatine, and 1,4-diguanidobutane sulfate in water, respectively. No one has reported the use of diacetyl in the quantitative determination of agmatine. Ennor and Stocken (6) used a diacetyl reagent to assay creatine in the urine. Rosenberg, Ennor, and Mor(13) J . Hutzler, M. Odievre and J. Dancis, Anal. Biochem., 19, 529 ( 1966).
I
'
4f5 550 WAVELENGTH (nrn )
P
700
I
67 8 3 3 0 167 2133 500 L.AR GININE HCL CONCENTRATION ( In y l m l l
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Figure 2. Time course of development, intensity, and stability of the colored derivative formed from the reaction of arginine with the modified diacetyl reagent
rison (7) used a modification of their method for arginine and other guanidino compounds (but not agmatine) and added propanol to increase the sensitivity. The former authors reported that a tenfold increase in the concentration of arginine was needed to produce any results comparable to those obtained with creatine. They further stated that the arginine reaction showed no signs of reaching completion even after 50 min. Our modification lowered the value for arginine to a 2.6-fold equivalency compared to creatine and
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
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o I" I
3
66
133
200
,
266 0
,
,
66
,
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200
CREATINE CONCENTRATION Ipqfmll
Figure 3. Time course of development, intensity, and stability of the colored derivative formed from the reaction of creatine with the modified diacetyl reagent the time curves straightened out after 30 min (Figure 3). The results with agmatine (Figure 4) were similar. 1,4Diguanidobutane sulfate was the least sensitive of the four compounds (Figure 5). As can be seen, these curves had not straightened out by 2 hours. Under our conditions, propanol did not enhance the time or intensity of the color reaction as might possibly be expected from the report of Rosen ef a / . (7). Of all the compounds tested here, creatine was the most sensitive to this reagent as much lower amounts (2 pg/ml) were detected compared to the other compounds. In addition, all time curves were straight even from the earliest assay period of 15 min (Figure 3). The last four figures showed that the reaction with diacetyl first intensified and then slowly faded with time. However,
0
r
,
167
,
333
,
,
I
r
,
,
,
I
500 667 833 0 I67 333 500 667 1,4-DIGUANIDOBUTANE SULFATE CONCENTRATION lygimll
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833
Figure 5. Time course of development, intensity, and stability of the colored derivative formed from the reaction of 1,4diguanidobutane sulfate with the modified diacetyl reagent
as long as concurrent standards are run, any straight portion of the curves or any standard time could be used for the assay procedure. Probably 35-40 min would be the most convenient. We next investigated the possibility of diluting the derivatives after a 30- to 40-min color development so that those samples with high values could be determined more accurately on the spectrophotometer. If this were possible, it would save time needed to rerun unknowns with an initial concentration higher than 80 pg/ml. If the samples of agmatine or arginine were diluted with the color control "blank" (Extra color control blanks were prepared for this purpose at the beginning of these experiments.), an average error of 3-6z lower than the theoretical value occurred. When the samples were diluted with water and read against an undiluted color control, the error was 35 above the theoretical value. If both sample and color control were diluted with water, an average error of 7-12 higher than the theoretical value was noted. Errors observed with creatine were very high (25-35 with any of these procedures. 1,4-Diguanidobutane sulfate was not run. Therefore, only an approximation of the value can be obtained by diluting samples of arginine or agmatine after color development regardless of the diluent employed. The concentration of agmatine in the butanol layer could also be determined. Since only 80-85 was removed in the first extraction, a second extraction with 2 ml of butanol was necessary to remove the remainder. The two extractions When colored derivatives obtained removed 98-99 from reacting diacetyl with the guanidino compounds extracted into the butanol layer were run, the samples eventually became cloudy. Results were more accurate when the agmatine was first removed from the butanol layer with water before forming the colored derivatives. Tests showed that all of the agmatine was removed by this method.
z
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i
90mn
z
z)
z
z.
Figure 4. Time course of development, intensity, and stability of the colored derivative formed from the reaction of agmatine with the modified diacetyl reagent 1478
CONCLUSIONS
The combination of a differential extraction procedure with a modified diacetyl reagent allowed the rapid detection
ANALYTICAL CHEMISTRY, VOL. 43, NO. 1 1 , SEPTEMBER 1971
of agmatine in the presence of arginine. The method is simple and convenient to perform and would be easily adaptable for routine automated procedures employing autoanalyzers or flow-through cuvette systems now available with many spectrophotometers. In addition, the procedure can be used to quantitate guanidino-containing compounds in general. It should be of value both in routine monitoring of arginine decarboxylase activity or other systems in which
agmatine is present in the presence of arginine as well as the detection and assay of other guanidino-containing compounds in general.
RECEIVED for review April 19, 1971. Accepted June 14, 1971. This research was supported, in part, by a research grant from the National Institutes of Health (NCI) No. CA-06939.
On-Line Interactive Data Processing I. As Applied to Mass Spectrometry and Gas Chromatography J. W. Frazer, L. R. Carlson, A. M. Kray, and M. R. Bertoglio' Lawrence Radiation Laboratory, University of California, Livermore, Gal$ 94550
S. P. Perone Chemistry Department, Purdue University, Lafayette, Ind. 47907
An interactive computer system used for simple instrument control, data acquisition, and interactive data reduction is described. Emphasis is placed on the interactive data reduction aspects. The approach taken establishes convenient and effective communications links between the processing digital computer and the experimenter-operator such that the computer can execute a variety of tedious processing operations under the continuous guidance of the operator. Thus, the operator can impose his ex erienced judgment on the net processing procedqtre by interacting with the computer during data reduction. The operator can oversee the processing functions by graphical communications with the computer through an oscilloscopic display terminal. Applications to spark-source mass spectrometry and gas chromatography are illustrated.
THEWORK PRESENTED here and in the following paper ( I ) provides a description of our interactive computer system used for simple instrument control, data acquisition, and interactive data reduction. The emphasis here will be placed on the interactive data-reduction aspects of this automation. The work describes data reduction of nonroutine data by means of operator interaction with digitized waveforms generated as outputs from chemical instrumentation. Eventually, the work reported here will help support the development of a real-time interactive system for experimentation. For routine data reduction applications, where the boundary conditions are well defined, it is usually preferable to provide computerization via predetermined algorithms. However, it is difficult to incorporate completely into a computer program the complex interpretive processes required to analyze the nonroutine data obtained from research and development projects. These waveforms, obtained as transducer outputs or from the correlation of the data, are often very complex and noisy; the information obtained is often unpredictable in form at the onset of the work; and the waveforms often Present address, Chemistry Department, University of Illinois, Urbana, Ill. (1) S. P. Perone, J. W. Frazer, and A. M. Kray, ANAL.CHEM., 43, 1485 (1971).
have drifting or abruptly changing backgrounds. Under those conditions the ability to have dynamic interactive capabilities for the retrieval of information is advantageous. The data processing approach developed here involves establishing a convenient and effective communications link between the processing digital computer and the experimenteroperator such that the computer can execute a variety of tedious processing operations under the continuous guidance of the operator. Thus, the operator can impose his experienced judgment on the net processing procedure by interacting with the computer during data reduction. The interactive system developed here incorporates the experimenter-operator into the data processing system in a most efficient manner. The computer is programmed to carry out control functions, data acquisition, and the tedious and difficult computational handling of the data, whereas the operator is required t o make judgments regarding the selection of regions of the data to be analyzed and how the analysis should proceed for maximum benefit. These functions are readily selectable by the operator through an oscilloscopic display system which allows graphical communication with the computer. Because of this dynamic computer-displayoperator interaction, it is also possible to obtain unique visual perspectives on experimental data. The operator quickly makes a selection, by visual methods, and by mathematical tests, of the best approach for data reduction. The principal interaction capability is provided by an oscilloscope, a Graf-Pen (Science Accessories Corporation, 65 Station Street, Southport, Conn.), and a special function panel. The Graf-Pen permits the input of information in a manner similar to that provided by a light pen. The special function panel consists of several switches and push buttons which are used by the operator to communicate with the software program to call special calculational, control, and display functions. Data-processing software will vary with the requirements of various analytical tasks to be performed. Therefore, software was developed such that all programming for data processing was done in the FOCAL language (FOCAL is a trademark of the Digital Equipment Corporation, Maynard,
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