THE INDOLE ACIDS OF HUMAN URINE. PAPER CHROMATOGRAPHY OF INDOLE ACIDS* BY MARVIN D. ARMSTRONG,t KENNETH N. F. SHAW, MELVIN J. GORTATOWSKI, AND HERBERT SINGERt (From the Laboratory for the Study of Hereditary and Metabolic Disorders, and the Departments of Biological Chemistry and Medicine, University of Utah College of Medicine, Salt Lake City, Utah) (Received for publication, October 31, 1957) Nencki and Sieber (1) first reported the formation of a red pigment in urine which had been made strongly acid by the addition of mineral acids, and they named the pigment “urorosein.” At one time considerable im- port was attached to the appearance of a strongly positive urorosein reac- tion (2, 3). Herter (2), however, established that this reaction frequently could be associated with gastrointestinal disturbances, succeeded in iso- lating indoleacetic acid as the urinary chromogen in one such case (4), and inferred that indoleacetic acid of intestinal origin was responsible for the urorosein reaction. This view was widely accepted, and for many years little further work was done on the nature and significance of this reaction. The observation that a strongly positive urorosein reaction always can be demonstrated in the urine of patients with phenylketonuria led to the iden- tification of indoleacetic and indolelactic acids as the major indole com- pounds in their urine (5). In addition, paper chromatograms of extracts revealed the presence of 5-hydroxyindoleacetic acid and many other indole acids. Recently, the excretion of greatly increased amounts of indoleacetic acid, N-(indole-3-acetyl)glutamine, and tryptophan by patients with Hartnup disease was reported (6). Hartnup diseaseis a newly described hereditary abnormality in the metabolism of tryptophan and is character- ized by pellagra-like skin rashes accompanied by intermittent cerebellar ataxia and mental deterioration (7). In addition, it may be recalled that Ross (8) observed that a greatly increased urorosein reaction could be ob- served with the urine of insane patients, as compared with that of normal individuals, and that the occurrence of this strongly increased reaction in two patients probably did not occur as the result of differences in the food intake or in intestinal putrefaction in the case of the patients (9). * This research was supported by research grants from the National Institutes of Health, United States Public Health Service. A preliminary report was presented at the 128th national meeting of the American Chemical Society, Minneapolis, Sept. 11-16, 1955. t Present address, The Fels Research Institute, Antioch College, Yellow Springs, Ohio. 17 by guest on May 16, 2018 http://www.jbc.org/ Downloaded from
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THE INDOLE ACIDS OF HUMAN URINE. PAPER CHROMATOGRAPHY OF INDOLE ACIDS*
BY MARVIN D. ARMSTRONG,t KENNETH N. F. SHAW, MELVIN J. GORTATOWSKI, AND HERBERT SINGERt
(From the Laboratory for the Study of Hereditary and Metabolic Disorders, and the Departments of Biological Chemistry and Medicine, University of
Utah College of Medicine, Salt Lake City, Utah)
(Received for publication, October 31, 1957)
Nencki and Sieber (1) first reported the formation of a red pigment in urine which had been made strongly acid by the addition of mineral acids, and they named the pigment “urorosein.” At one time considerable im- port was attached to the appearance of a strongly positive urorosein reac- tion (2, 3). Herter (2), however, established that this reaction frequently could be associated with gastrointestinal disturbances, succeeded in iso- lating indoleacetic acid as the urinary chromogen in one such case (4), and inferred that indoleacetic acid of intestinal origin was responsible for the urorosein reaction. This view was widely accepted, and for many years little further work was done on the nature and significance of this reaction. The observation that a strongly positive urorosein reaction always can be demonstrated in the urine of patients with phenylketonuria led to the iden- tification of indoleacetic and indolelactic acids as the major indole com- pounds in their urine (5). In addition, paper chromatograms of extracts revealed the presence of 5-hydroxyindoleacetic acid and many other indole acids. Recently, the excretion of greatly increased amounts of indoleacetic acid, N-(indole-3-acetyl)glutamine, and tryptophan by patients with Hartnup disease was reported (6). Hartnup disease is a newly described hereditary abnormality in the metabolism of tryptophan and is character- ized by pellagra-like skin rashes accompanied by intermittent cerebellar ataxia and mental deterioration (7). In addition, it may be recalled that Ross (8) observed that a greatly increased urorosein reaction could be ob- served with the urine of insane patients, as compared with that of normal individuals, and that the occurrence of this strongly increased reaction in two patients probably did not occur as the result of differences in the food intake or in intestinal putrefaction in the case of the patients (9).
* This research was supported by research grants from the National Institutes of Health, United States Public Health Service.
A preliminary report was presented at the 128th national meeting of the American Chemical Society, Minneapolis, Sept. 11-16, 1955.
t Present address, The Fels Research Institute, Antioch College, Yellow Springs, Ohio.
In order to understand better the significance of the presence of the indole acids in phenylketonuric urine and in other pathological conditions, it became necessary to study the indole acids of normal urine. A similar study of the phenolic acids of human urine already has been described (10). Recently there have been several reports on the paper chromatography of indole acids (11-21). The purpose of the present paper is to report the indole acids which have been detected by two-dimensional paper chroma- tography of extracts of a large number of normal and pathological urine samples, to present the chromatographic properties of a number of authen- tic indole acids which conceivably could be present in urine, and, where possible, to accord tentative identification of the urinary compounds. It has been found that many samples of urine which give a strongly positive urorosein reaction may contain only minor amounts of indoleacetic acid, but larger amounts of other substances. These findings reopen for study the possible significance of the urorosein reaction in some of the patholog- ical conditions in which it has been observed.
EXPERIMENTAL
Indole, skatole, indoleacetic and indolepropionic acids, and tryptophan were obtained from commercial sources. N-(Indole3-acetyl)glutamine was obtained from Dr. J. B. Jepson and pyrrole-2-carboxylic acid from Dr. C. E. Dalgliesh. The syntheses of P-(5-methoxyindole-3)lactic acid (22) and ,&(5-hydroxyindole-3)lactic acid (22) and P-(indole-3)lactic acid,l /3-(indole-3)pyruvic acid,l /?-(indole-3)acrylic acid,’ sodium indole-3- glycolate,l p-(indole-3)+oximinopropionic acid,r and indole-3-glyoxyl- amide,’ have been described. N-(Indole-3-acetyl)glutamine and N-(in- dole-3-acetyl)asparagine were prepared by coupling indoleacetic acid with the parent amino acids by the mixed anhydride procedure, as described by Wieland and Hijrlein (23). N-(Indole3-acetyl)-ol-alanine, -p-alanine, -glu- tamic acid, and -aspartic acid were synthesized by coupling indoleacetic acid with the appropriate amino acid ester by the mixed anhydride pro- cedure of Vaughan and Eichler (24). N-(Indole-3-acetyl)-y-aminobutyric acid, -P-aminoisobutyric acid, and -glycine were made by coupling indole- acetyl chloride with the proper amino acid by use of Schotten-Baumann conditions. Complete details on the preparation of these derivatives of indoleacetic acid will be presented elsewhere. The remainder of the indole acids was prepared by methods described in the literature.
Chromatography was carried out according to the methods reported previously for the study of phenolic acids (10). Data obtained with the n-butanol-pyridine-dioxane-Hz0 solvent are not recorded here because of
1 Shaw, K. N. F., McMillan, A., Gudmundson, A., and Armstrong, M. D., unpub- lished work.
the absence of any marked advantage of this system for distinguishing the indole acids. Extracts of urine were prepared in the same manner as re- ported for the phenolic acids (10). When possible, urine collected for study was frozen immediately upon being voided and was stored in the deep freeze until it was extracted; when this was unfeasible, it was preserved by the addition of a crystal of thymol and was stored in the cold.
The most useful reagent for the detection of the indole acids is an acidic solutionof p-dimethylaminobenzaldehyde. The reagent described by Berry et al. (25) is somewhat unsatisfactory, since the slow evaporation of water from sprayed chromatograms prolongs the time period during which maxi- mal color development is attained with many of the compounds. In the present study the following spray reagent (PDAB) was used: 2 gm. of p-dimethylaminobenzaldehyde dissolved in a mixture of 80 ml. of 95 per cent ethanol and 20 ml. of 6 N HCl. It is advisable to mix the ethanol and acid before dissolving the reagent; otherwise, local high acid concentration may cause the development of a strong yellow color in the reagent and lead to a yellow background which makes difficult the detection of faint spots. PDAB causes the appearance of spots ranging in color from blue-green through blue to bright pink with the authentic indole compounds and with acids present in urine. In addition, many substances in urine respond with the formation of yellow colors. None of the substances which give rise to yellow colors have been identified with any degree of certainty and are not shown on the chart of urinary compounds (Fig. 1). Compounds giving purple or blue colors with the reagent most probably contain the indole nucleus; although other substances give purple colors, the extraction pro- cedure used in processing urine excludes them from the compounds studied here. Since some indole compounds give pink colors with the reagent, the unknown substances which give rise to pink colors may be indoles, but with less probability than the ones which give purple colors.
Despite the improvement in the rate of color development with the reagent described above, a considerable difference in the time for maximal color formation, ranging from 5 minutes to over 24 hours, still exists. Be- cause of variations in humidity, temperature, ventilation, and amount of solvent sprayed onto chromatograms, it is impossible to ascribe a definite time of maximal color development. In Table I the rate is listed as being fast, medium, or slow, with reference to the time required for maximal color formation with indoleacetic acid. In some cases, however, differences in the rate of development of color may be observed on chromatograms of urine extracts as compared with pure compounds. This is particularly true of N-acetyltryptophan and indoleacetylglutamine. On chromato- grams containing pure compounds, both of these develop color at the same rate as indoleacetic acid; in urine extracts, their color development is
somewhat slower. Also, despite the uncertainties caused by the differing rates of appearance and the different colors given by different compounds, an estimate of the intensity of maximal color is also referred to indoleacetic acid; i.e., the amount of each compound which forms a color comparable in intensity to that given by 5 y of indoleacetic acid. Almost all the indole compounds develop a maximal color within 5 to 30 minutes and fade. There are notable exceptions to this behavior. Indole-2- and 3-carboxylic acids develop much stronger colors after 24 hours or longer standing. 2-Pyrrolecarboxylic acid develops an early strong pink color, which after 24 hours is an equally strong purple color which persists. Indoleacrylic acid develops an early strong pale green color which fades; then a stronger blue-green color develops during the next 24 hours and remains. In this case the initial color is usually not observed in urine extracts because of proximity of indoleacetic acid on the chromatograms.
Distinguishing characteristics of the authentic indole compounds other than their behavior when treated with PDAB are not given, except in cases in which a particular reaction is especially useful. The papers cited above from other laboratories list many of the other qualitative reactions which have been applied to many of the compounds reported. In practice, how- ever, PDAB is the most useful reagent for detecting and characterizing indole acids. This is particularly true when the additional information included here is available; i.e., rate of development of maximal color, color at time of maximal intensity and after 24 hours, and the intensity of the color as compared with that given by indoleacetic acid.
Most of the indole acids show some type of fluorescence when examined under ultraviolet light. The fluorescence varies in color and intensity, however, depending upon the solvent system used, the length of time the developed chromatograms are allowed to stand before examination, and the amount of compound present. In a few cases, the ability of compounds to quench the background fluorescence of the paper has been consistent and is useful, and a few of the compounds show a characteristic fluorescence. These cases are noted in Table I.
Variations in RR values are to be expected since factors such as tempera- ture, humidity, and minor variations in solvent composition have a pro- nounced effect. Temperature has appeared to be the major factor which influences these variations. It has been observed that, the greater the volatility of the solvent system used, the greater will be the temperature effect; tix., in the case of 20 per cent KCI, differences in temperature have little effect, whereas in the isopropyl alcohol-ammonia-water system tem- perature effects are marked. However, relative RF values under given conditions on the same chromatogram remain the same, despite consider- able variation in absolute RF values. In order to overcome these differ-
ences, exceptional precautions were taken to establish precise values for indoleacetic acid in each solvent system; the RF values listed in Table I for the other compounds were adjusted to correspond with the standard value for indoleacetic acid.
Fig. 1 is a schematic diagram which indicates the compounds which have been definitely characterized in the course of an examination of over 1000 samples of urine from healthy and sick individuals. As with the phenolic acids (lo), the compounds are assigned numbers, with an increasing number indicating a decreasing order in the frequency with which the compound has been observed. Considerable variation has been noted in the amounts of the compounds present in samples of urine from various persons. In most cases, however, indoleacetic acid has been observed in amounts rang- ing from less than 1 y to approximately 10 y per mg. of creatinine, indole- lactic acid from less than 0.2 to 2 y per mg. of creatinine, 5-hydroxyindole- acetic acid from 1 to 3 y per mg. of creatinine, and indoleacetylglutamine from 1 to 5 y per mg. of creatinine. The other compounds are usually observed in smaller amounts in most samples of urine, but each of the com- pounds has been observed in greatly increased amounts in certain speci- mens. Many materials which give red, purple, or blue colors when treated with PDAB have been observed in the course of this work. The com- pounds shown on Fig. 1 are those which have been observed in several samples of urine, and which might be expected to be encountered frequently.
DISCUSSION
The chromatographic behavior of thirty-eight authentic indole com- pounds is described in Table I. These are mainly acids, with the exception of a few derivatives which might be expected to occur as products formed in the course of chromatography, and one compound, the oxime of indole- pyruvic acid, which might be useful as a derivative of the unstable parent compound. Data for twelve of these substances have not been recorded previously. The chromatographic characteristics of the indole acids which were detected frequently in human urine in the course of screening more than 1000 specimens of normal and pathological samples are described (Table II and Fig. 1). The indole nature of these substances is presumed from the fact that they yield blue, purple, or red colors when treated with a developing spray of acidic p-dimethylaminobenzaldehyde; the extraction procedure used to prepare the acids for chromatography should eliminate most of the non-indole compounds known to respond to thii reagent. Of the thirty-eight substances described, ten have been accorded a tentative identification in view of correspondence of the chromatographic properties of the urinary compound with authentic material. Six of these indole acids have not been reported previously to occur in urine. In each case, an
aliquot of an extract which contained a barely detectable amount of a specific compound was subjected to chromatography after the addition of a small amount of authentic compound in order to assure identity of their Rp values. This procedure is necessary because the RF value of some compounds in the urine extracts might differ appreciably from that ob- served with the pure substances. This behavior was noted particularly in some extracts containing indoleacetamide, in which the actual amount of the same extract used exerted a marked influence upon the relative Rp value of the compound.
Despite the precautions taken to minimize decomposition of indole com- pounds during the extraction procedure, such decomposition most certainly occurs. This is indicated by the appearance of purple or red solutions, particularly upon acidification of the bicarbonate extracts. After acidi- fication of these extracts and preparation of the final ethyl acetate extracts, the spent aqueous phase frequently is colored, and almost always will give a positive urorosein test, either pink or purple in color. Also, the appear- ance of material which runs to the front in both solvent systems and gives a blue or green color with the PDAB is frequent; the material responsible for this reaction undoubtedly is formed by decomposition of unstable sub- stances during the extractions, during storage of the extracts, or in the course of chromatography with the ammoniacal solvent system.
It has been difficult to assign any significance to the appearance of any of the unusual indole acids. In urine from patients with phenylketonuria, indolelactic acid is always present in greatly increased amounts, and indole- acetic acid is usually present in increased amounts. Also, Compounds 14 and 15 usually are apparent in specimens from phenylketonuric inmates of institutions, but not from patients living at home. Compound 14 may be the indole substance which has been reported by other workers (28, 29) to be present consistently in phenylketonuric urine. Urine specimens from three patients with carcinoid tumors have been studied in the course of this work. In confirmation of the reports from many laboratories, 5-hy- droxyindoleacetic acid was found to be present in greatly increased amounts. It has not been possible to examine urine from a patient with Hartnup disease, but the excretion of large amounts of indoleacetic acid and indoleacetylglutamine in this condition seems well documented (6). The excretion of an increased amount of indoleacetylglutamine after the ingestion of indoleacetic acid by humans has been confirmed in this labora- tory. In addition, a greatly increased excretion of Compound 7 and a slightly increased excretion of indoleacetylglutamic acid were observed.2 The results of the screening of many urines have shown quite clearly, how- ever, that there is not a direct relationship between the amount of indoleace-
2 Singer, H., and Armstrong, M. D., unpublished observations.
tic acid ingested or produced and the pattern of these four substances in the urine. Many specimens of urine have been found to contain large amounts of indoleacetic acid but only normal or small amounts of indole- acetylglutamine and Compound 7. On the other hand, many samples
X
SOLVENT FRONT
0 31 0 20
FIQ. 1. Indole acids in human urine. The numbers refer to the compounds listed in Table II. Substances which give red colors are cross-hatched; the open areas represent compounds.which form blue to purple colors with PDAB.
have been observed which contain much more indoleacetylglutamine than usual along with only trace amounts of indoleacetic acid.
There has been some indication that the excretion of increased amounts of indolelactic acid may be characteristic of poor liver function. Urine from patients known to suffer from faulty liver function invariably has contained an increased amount of this acid. In addition, urine from very young infants usually contains a larger amount of indolelactic acid.
Color with PDAB is listed as follows: color at maximal intensity; rate of color development; color after 24 hours (see Table I for the abbreviations). Compounds marked with an asterisk had not been reported previously to be constituents of hu- man urine.
The most strikingly “abnormal” patterns of indole acid excretion have been observed with urine from institutionalized patients. This has been true in the case of urine specimens from severely mentally retarded patients and also the mentally ill. Compounds 14 and 15 have been observed in the urine of a majority of patients in these categories, and in many cases are quantitatively by far the most important indole compounds present. The possibility of a significant relationship between the excretion of these com- pounds and the illness of the patients is rendered tenuous by several facts: (1) the administration of broad spectrum antibiotics results in a disappear- ance of the substances from urine; (2) the amounts of the compounds ex- creted by an individual patient are not consistent but quite variable; (3) non-institutionalized patients in the same age groups and equally as ill as those in institutions usually do not excrete the compounds, or they are present in only trace amounts; and (4) the very high proportion of patients with different illnesses who excrete these substances makes it seem improb- able that they can be characteristic of a discrete disorder. Further and more detailed data on the occurrence of these substances will be presented elsewhere.
A dietary origin seems possible for some of the compounds given a tenta- tive identification in this work. The occurrence of indoleacetamide in plants has been demonstrated by Good, Andreae, and van Ysselstein (30, 31). It is not clear how indoleacetamide appears in the acidic fraction of the urine extract, since indoleacetamide added to urine is not carried from ethyl acetate into the bicarbonate extract. Indole-3-carboxaldehyde and carboxylic acid have been reported recently to occur in plants (32) ; Power and Sherwin (33) observed that indole3-carboxylic acid is excreted essen- tially unchanged by the human. Indoleacrylic acid might be expected to occur in plants in analogy with the widespread natural occurrence of aromatic acrylic acids. Its instability1 would probably hinder attempts to demonstrate a natural occurrence. Baugess and Berg (34) have reported that it is excreted in large part unchanged by the rat or the rabbit after oral ingestion. The occurrence of indoleglycolic acid in plants has been claimed by Fischer (35). It has also been reported to be a breakdown product formed from indolepyruvic acid during chromatography (27). Its possible natural occurrence has been discussed recently (36).
The valuable technical assistance of Kathryn S. Robinson, Patricia E. Wall, Eleanor Bethsold, and Lurrine Burgess is gratefully acknowledged. We are deeply indebted to Dr. Vernon F. Houston, Director of the Utah State Training School, and to Mrs. Betty Fernelius and Mrs. Avis Thayne of that institution for their help in providing material from mentally defi- cient patients. Dr. J. F. Bosma and Dr. N. L. Low, Department of Pedi-
atrics, University of Utah College of Medicine, provided material from out-patients. Dr. L. 0. Currier, Dr. E. L. Bliss, and Dr. L. D. Clark, Department of Psychiatry, University of Utah College of Medicine, ob- tained material from psychiatric patients.
SUMMARY
The chromatographic behavior of thirty-eight authentic indole com- pounds, mostly acids, is described. The chromatographic properties of thirty-eight compounds which are presumed to contain an indole nucleus and which have been observed in the urine of human subjects are described, and ten of these have been accorded a tentative identification. The pos- sible significance of the appearance of these substances in urine is discussed briefly.
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