More than 130 years ago, in 1858. a Czech physician Bohumil (Theophil) Eiselt (1831-1908)

described s.c. melanogenuria, i.e. the excretion of s.c. melanogen, colorless precursor of the dark pigment melanin, in the urine of three patients with generalized melanoma (23) (fig. 1). He wrote:

"Der Urin, frisch gelassen, war hoch gelb, volkommen klar; bei längeren Stehen wurde er so dunkelbraun wie Porterbier". So, already at that time, Eiselt recognized correctly that colorless precursor and not the dark pigment melanin itself was excreted. Unfortunate!y, this fact has not been respected by many later authors and the incorrect terms melaninuria or vague melanuria instead of correct melanogenuria have been used for many years (21). Thirty years later, in 1887, a German physician Johannes Thormählen (1860-1892) discovered, when he performed the Legal's test for ketone bodies in the urine of melanoma patients, that after the addition of acetic acid no decolorization and also no violet-red, but beautiful blue colour was formed. ("... trat sofort eine prachtvoll blaue Farbe auf.") (65) (Fig. 2). This specific test for indole compounds with an unsubstituted pyrrole ring of the indole nucleus - as Böhm (10) and Leonhardi (34) confirmed much later - carries Thormählen's name up to the present time.

The first man who believed that he had solved the chemical structure of the Thormählen positive melanogen was Hans Eppinger, already in 1910 (24). However, his finding of the sulphate of N-methylhydroxypyrrolidine carboxylic acid has never been confirmed.

In 1920ties, the question of the chemical nature of the melanogen became even more complicated. Thannhauser and Weiss found a compound of phenolic nature - probably the 3.4-dihydro- xyphenylacetic acid - in the melanotic urine (64). However, this compound didn't give a positive result in Thormählen test. The papers of Fürth and Kaunitz (25) and of Barrenscheen and Prinz (8)

___________________

'' Presented under the titel Melamogenuria: History - Present- Perspective as a "special lecture" (together with 50 slides) at the 3rd Annual Meeting of the European Society for Pigment Cell Research held in Amsterdam, September 8-11, 1991; partly up-dated for this 1994 edition.

Mailing address: Prof. MUDr. J. Duchon, DrSc., 2nd Dept. of Medical Chemistry and Biochemistry, 1st Medical Faculty, Charles University, U nemocnice 5, 128 53 Prague 2 (Czech Republic)

Ø Univerzita Karlova, vydrvatelství Karolinum, Praha 1994

published in 1930ties brought a partial solution of this problem: These Vienna authors described the existence of two kinds of melanogen :

1 - compound containing nitrogen, soluble in water and giving positive Thormählen test, probably an indole derivative, and

2 - nitrogen-free compound, soluble in ether and giving negative result of the Thormählen test, probably of phenolic nature.

At the same time. the well-known British biochemist Henry Stanley Raper, together with Louis Linnell, suggested that the Thormählen positive melanogen, i.e. the "chromogen of melanuria" as they called it, might be a simple derivative of 5,6-dihydroxyindole, possibly an ethereal sulphate or glycuronate (38). Linnell and Raper refused the presence of 5,6-dihydroxyindole-2-carboxylic acid in melanoma urine and also were doubtful about the existence of the nitrogen-free, Thormählen negative melanogen.

A substantial progress in this area came only in 1950ties. At that time, a German biochemist and dermatologist Gottfried Leonhardi was able to separate 3 Thormählen positive substances from melanoma urine, using paper chromatography (35, 36). He designated them as compounds A, B, C and

he announced that the main compound A was 5,6-dihydroxyindole bound with a dipeptide formed from glutamic and pyrrolidone carboxylic acid (37). Unfortunately, his opinion concerning this peptide moiety was wrong. The conjugation of glucuronic acid and sulphate to the indole part of Thormählen positive compounds was reported by A. B. Anderson in England in 1962 (2) and one year later, in 1963, M. R. Atkinson from Adelaide, Australia revealed the presence of two isomeric compounds in the melanoma urine (4). He determined the structure of the major isomer as 5-hydroxy-6-methoxy-indoI-5-glucosiduronate and of the minor one as 6-hydroxy-5-methoxy-indol-6- glucosiduronate.

Four main groups devoted to the study of melanogens and melanogenuria have been formed during the last three decades: The Czech, the Swedish, the Dutch and the Japanese goups.

l At the beginning of 1960ties my friend Zdenek Pechan from Brno and I (J. D.) in Prague fell in this melanin, i.e. "black" bussines or "black magic". as we called it, and later, at that time very young and innocent boys - especially Bohuslav Matous and Stanislav Pavel (and also. in another field, Jan Borovanský), joined us. Zdenek Pechan in Brno elaborated a new and exact quantitative method for the sum of Thormählen positive melanogens estimation (52), he was also able to confirm, independently, Anderson' s and Atkinson's findings and he also discovered two other Thormählen positive compounds, designated as D and E (22, 53). We in Prague could demonstrate the existence of the nearly forgotten Thormählen negative melanogen in the urine of melanoma patients (12). We found that it consisted of at least 4 compounds which we originally separated by two-dimensional paper chromatography (21). Later we identified them as homovanillic (compound Nr. 1)(15), vanillactic (Nr. 2) (20). vanilpyruvic (Nr. 3) (20) and especially the compound Nr.4(19) as a mixture of isomeric 5-hydroxy-6-methoxy-and 6-hydroxy-5-methoxy-indole-2 carboxylic acids (16). The structure of these two isomers was immediately confirmed by French authors Badinand and Vallon (6, 67) and later by a lot of others (27, 31, 32, 42, 43, 44, 47. 48, 51, 56, 57. 58, 68, 69, 70, 71. 72, 73. 74).

Of course. the time didn't stop and in Sweden, in Lund, another very productive group was formed. The "spiritus movens" of it was Hans Rorsman. His destiny was firmly connected with a new, key compound of melanogenessis - 5-S-cysteinyldopa - which was for the first time described by Italien chemist Giuseppe Prota and co-workers who identified it as a key precursor of pheomelanins (55).

Hans Rorsman and his closest-co-workers Gunar Agrup, Christer Hansson and Evald Rosengren found 5-S-cysteinyldopa (and also some of its derivatives) in melanoma tissue, in the blood and urine of melanoma patients, as well as in the body fluids of healthy persons, as an important marker of melanogenesis. They have done really a tremendeous work in this field. Besides many original papers, their work is summarized also in several reviews, e.g. (1, 27, 56, 57, 58). However, Christer Hansson later became unfaithfull to 5-S-cysteinyldopa and turned his attention to indole melanogens (27). He declared them more specific markers of melanogenesis than 5-S-cysteinyldopa was supposed to be.

At the beginning of 1980ties, another group started its activity in the Netherlands. However, the founder of this group was not of Dutch but of Czech origin. In 1981 he illegaly left Prague for the Netherlands and in this free and friendly country he developed and finished, with a great success the work which he had started already in Prague. His name is Stan(islav) Pavel. Stan Pavel. together with his co-workers. elaborated a modem, specific and very sensitive gas-chromatography mass-spectrometry and later even high-performance liquid chromatography methods for melanogens estimation (49, 50). He safely and definitely determined the structure of Thormählen positive melanogens A, B, C (45, 46. 49) and he discovered independently on Hansson's finding. the presence of 5,6-dihydroxyindole-2-carboxylic acid and its derivatives also in the urine of healthy people (43, 44). He applied his findings to the study of melanoma tissue cultures on the one hand (48) and to the following of the course of malignant melanoma on the other (47).

Finally, at the end of 1980ties another very active group was formed in this area in Japan. Shosuke Ito. well-known chemist in the area of melanin chemistry, together with his co-worker Kazumasa Wakamatsu, elaborated very useful and simplifted procedure for preparing subgrams quantities of 5,ó-dihydroxyindole 5,6-dihydroxyindole-2-carboxylic acid and their O-methyl derivatives (68). Further, Wakamatsu and Ito found that even Thormählen negative, i.e. carboxylated indolic melanogens occur also as conjugates in meIanoma urine (69). They also studied in detail the correlation between meIanogenuria and the growth of the tumour on the animal model of B 16 mouse melanoma (32, 70) and also estimated normal values of 5-cysteinyldopa and 6-hydroxy-5-methoxy indole-2-carboxylic acid in the urine and serum of healthy persons (31, 71).

Of course. during the past decades they were not only these 4 groups, i.e. the Czech, Swedish, Dutch and Japanese, but also many other scientists who contributed to the elucidation of the enigma of melanogens and melanogenuria. So, e.g. J. A. Scott was the first who found dopa in melanoma urine only in 1962 (59), Hana Hiinterberger (ovà) and co-workers in Australia estimated the s.c. "free catechols" which included not only dopa but also dopac and dopamine (29, 30), Gruhn and co-workers described a new compound in melanoma urine which was probably dopa-tetramer (26), M. S. Blois and P. W. Banda detected 3-O-methyldopa using their diphenylpicrylhydrazyl method (9), G. Prota and co-workers described the occurrence of trichochromes in the urine of some melanoma patients (54), B. Kagedahl and A. Pettersson elaborated authomated HPLC method for 5-S-cysteinyldopa estimation (33). etc. Also e. g. Coward and co-workers (75). Crawhall and co-workers (76), Haberman and co-workers (77), Morgan and co-workers (78), Schwartze and Grüneberg (79), Trapeznikov and co-workers (80) and also some other authors (e. g. 81, 82) contributed to the elucidation of the biochemical and clinical significance of melanogenuria.

So when we at the end of 1980ties tried to summarize the contemporary knowledge of the chemical structure of melanogens (14, 18), we could say that more than 20 urinary melanogens- newly also called "eumelanin and/or pheomelanin - related metabolites" - had been identified so far in the urine - and also in the blood - of melanoma patients, as well as some of them even in the tumours themselves. They were also described in the body fluids of healthy persons, however in much lower concentrations. In accordance with their chemical structure, these compounds could be divided into two principal groups: phenolic melanogens (PMG) and indolic melanogens (IMG). On the basis of the Thormählen test, it was possible to distinguish Thormählen negative (TNM) and Thormählen positive (TPM) melanogens (14, 17, 18).

It is generally believed that urinary melanogens are precursors (and/or metabolites of these precursors) on the tyrosine-to-melanins biosynthetic pathway. Our present idea of their biosynthesis is shown on the Fig. 3 (14, 18). To date, however, the detailed mechanism of their formation has not been exactly elucidated in all types of them. The most valuable results in this respect were obtained in the case of cysteinyldopas, from the work of the already mentioned Swedish group in Lund (27, 56, 57, 58). As far as the IMG, i.e. indolic melanogens, are concerned, our previous discovery of the structure of the TNM-IMG as 5-hydroxy-6-methoxy-and 6-hydroxy-5-methoxy-indole-2-carboxylic acids (16) clearly proved that these compounds originated from dopa - there is a preserved carboxyl group in the position 2 of the indole nucleus (14).

Returning to the general scheme of the biosynthesis of urinary melanogens (Fig. 3), we see that it can be divided into 2 main sections: The first is the section of metabolites of dopa itself: it includes all PMG with the exception of cysteinyldopas. The second is the section of metabolites of dopaquinone: it indudes cysteinyldopas and all IMG. Only these latter metabolites are strictly specific for melanogenesis, since - as it is well known - not only tbe "cresolase", i.e. tyrosine hydroxylase, but also the "catecholase", i.e. dopa oxidase activity of tyrosinase is necessary for the formation of dopaquinone which is one of the key compounds in melanogenesis (66). In the second group, i.e. the group of - we can say - "real melanogens" we can distinguish the following "subgroups":

I - for melanogenesis the only usualy specific PMG - i.e. cysteinyldopas (in the dotted frame on the Fig. 3);

2 - for melanogenesis specific IMG-TNM - indole-2-carboxylic acids (in the dashed frame on the Fig. 3);

3 - for melanogenesis specific IMG-TPM - conjugates of 5,6-dihydroxyindole or (5,ó)-hydroxymethoxyindole (in dot-dashed frame on the Fig. 3).

This classification, i.e. for melanogenesis specific melanogens on the one hand and for melanogenesis non- or less specific melanogens on the other, is also in agreement with clinical observations (39, 42). Each of these metabolites is, of course, the product of additional activities of further enzymes which have to be involved in some manner in the regulation of melanogenesis in vivo. We mean e.g. the participation of "dopachrome conversion factor" (51), "dopachrome oxidoreductase" (7), "dopachrome isomerase" (51) or better "dopachrome tautomerase" (3, 63), further of catechol-O-methyltransferase (44, 62), etc.

As far as the clinical significance of urinary melanogens. i.e. their practical importance for detection, differential diagnosis and prognosis of malignant melanoma is concerned, it depends on: I - their specificity, 2 - the frequency of their occurrence, and 3 - the mutual relationship between the stage and type of the disease and the corresponding analytical data (13, 17. 18).

1 - The elevated excretion of the IMG and partly also of 5-S-cysteinyldopa is highly specific for melanoma, whereas the PMG excretion is less specific. The PMG may also occur in elevated levels in other tumours of neural crest origin, e.g. neuroblastomas, pheochromocytomas, etc., generally in all cases which are characterized by dopa overproduction.

2 - Already several years ago (13), we compared results obtained by various authors in the past and we were able to conclude that the elevated level of urinary melanogens occurred, on the average, in only 20 - 30 % of all melanoma patients. Since that time two further reports evaluating the clinical significance of melanogenuria in sufficiently large groups of melanoma patients were published: One by the already mentioned Swedish group and concerning 5-S-cysteinyldopa determination (1), and the other by us concerning the sum of TPM determination (41). When we compare these two groups studied so far, we can conclude that the results are very similar, in fact nearly the same: In 571 melanoma patients the level of 5-S-cysteinyldopa in the urine was elevated in 182 cases, i.e. in 31,8 % (1), and in 690 melanoma patients the level of the sum of TPM in the urine was elevated in 216 patients, i.e. in 31,3 % (41).

3 - This low percentage of positive results in all melanoma patients is caused by the fact that it depends first of all on the stage or progress of the disease. Since our first report on this (21, 22), this fact has been repeatedly proved not only by us (13, 40, 4l)but also by many authors using different methods for different urinary melanogens determination. E.g. (1, 13, 56).

An important conclusion resulting from these findings is that melanogenuria, i.e. the eIevated excretion of urinary melanogens has - in general - a low value for very early diagnosis of malignant melanoma. In the first stage (primary tumour) the values in melanoma patients mostly overlap or interfer with the values found in other diseases or in healthy persons (1, 13, 17, 18, 41). However, the detection of melanogenuria may be very useful for differential diagnosis and prognosis of the disease (18, 41). It can in many cases come before the clinical symptomas of dissemination, i.e. it can predict them (18). It also can be very useful for the following of the course of the therapy of melanoma (40, 47). However, there are many other factors which may influence the excretion of urinary melanogens, e.g. the tumour type (melanotic or amelanotic), tumour origin (skin or eye), localization of the metastases (liver or other organs), the metabolic activity of the tumour, the response of the host organism, the effect of UV-irradiation, etc. (13, 18, 42, 47).

There is no agreement, at present time, which of the urinary melanogens is the best marker of elevated melanogenesis and, consequently, of malignant melanoma. Some authors prefer 5-S-cysteinyldopa (1, 56), the other indolic metabolites (27, 47) and in that case again some authors recommend 5-hydroxy-6-methoxy-indols (27) and the other 6-hydroxy-5-methoxy-indols (31, 71), usually the Thormählen negative, i.e. carrying carboxyl group at the position 2. However, e. g. Stan Pavel (47) prefers to follow 5-hydroxy-6-methoxy-indol itself which seems to be the most sensitive indicator of increased eumelanin production in patients with pigmented malignant melanoma. On the other hand, 5-hydroxy-6-methoxy-indole-2-carboxylic acid - and no 5-hydroxy-6-methoxy-indole itself - was recommended as the best urinary marker of melanin formation in the skin pigmentary system by Westerhof and co-workers (72). It seems that it is also important whether the level of melanogens is followed in the urine or in the blood (71).

At the end of this paper we should like to emphasize that irnportance of melanogens consists not only in their diagnostic and prognostic value, but also in their biochemical, i.e. metabolic significance (44). Of course, it is necessary to introduce new approaches for further elucidation of their biosynthesis and biodegradation. Such approaches are e.g. the biochemical study of melanoma tissue cultures (48), the biochemical research of subcellular compartments of meIanocytes (28), the following of the localization of different enzymes activities in melanoma cells (60), etc. Jan Borovanský, Pavel Mirejovský and Patrick Riley suggested a hypothesis that the elevated levels of melanogens in melanoma may be in connection with the destruction of tumour melanosomes which are more desintegrated than the melanosomes of normal melanocytes (11). Therefore, in this connection, such studies as e.g. by Hatta and co-workers (28) who found 5,6-dihydroxyindole and 2-carboxylic acid in coated vesicles and premelanosomes of pigment cells, are very promising. As well as e.g. the recent paper of Wilczek and Mishima (73). Also the papers of Shibata, Smit, Pavel and co-workers concerning the determination of catechol-O-methyltransferase (60, 62) are very important. This enzyme whose activity was found in melanoma tissue by Julius Axelrod and Aaron Bunsen Lerner already in 1963 (5), may play an important role in melanogenesis. The methylation of dihydroxyindole precursors by catechol-O-methyltransferase may serve as a protective mechanism of the melanocyte against its own toxic products (44). The presence of specific catechol-O-methyltransferase isoenzymes in the melanocyte couId be of great importance in order to find selective inhibitors of the enzyme causing the self-destruction of the malignant melanocyte (43, 61 ), This might offer new insight into strategies for melanoma therapy (61).

As far as the further research in the field of melanogens and melanogenuria in maligant melanoma at our Department is concerned, the work of Bohuslav Matous and his group seems to be promising (39).

1. Agrup. G., Agrup, P., Andersson, T., Hafstrom, L., Hansson, C., Jacobsson, S., Jonsson, P. E., Rorsman, H., Rosengren, A. M., Rosengren, E.: 5 years experience of 5-S-cysteinyldopa in melanoma diagnosis. Acta Dermatovener. (Stockholm) 59: 381-388, 1979.

2. Anderson, A. B.: Urinary melanogens. Biochem. J. 83: 10P, 1962.

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15. Duchon, J., Gregora, V.: Homovanillic acid and its relation to tyrosine metabolism in melanoma. Clin. Chim. Acta 7: 443-446, 1962.

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