412 [Vol. 32,

96. Studies on Bile Pigments. IV. Mechanism of Ehrlich's Aldehyde Reaction o f . 1 Studies on Reactive Position of Urobilinogen towards Ehrlich's Aldehyde Reagent By Kenji YAMAOKA,Kiyowo KOSAKA,Jiro NAKAGAWA, Nobuo YADA, and Motomu HOSOKAWA First Departmentof InternalMedicine, Okayama University Medical School (Comm. by T. SHIMIZU,M.J.A., June 12, 1956)

P. Ehrlich'' found, in 1901, a pathological urine from which there appeared a strong red tint by the addition of p-dimethylamino- benzaldehyde in an acid medium by hydrochloric acid, and the sub- stance to offer the phenomenon was proved to owe to urobilinogen by 0. Neubauer2' in 1903. The Ehrlich's aldehyde reaction is, accord- ing to H. Fischer, 3' a specific reaction to pyrrole and its derivatives of which a or R position has methenyl group, especially in the former. But urobilinogen has no methenyl, and then, in 1931, H. Fischer and R. Hess4' suggested that urobilinogen will be split into neo(isoneo)- bilirubinic acid at the central methylene bridge by the Ehrlich's aldehyde reagent and that p-dimethylaminobenzaldehyde will be more reactive to the free end methenyl. But H. Fischer and E. Adler, 5' in 1932, denied the Hess's suggestion judging from the fact that a condensation product of 2 moles of neo(isoneo)-xanthobilirubinic acid and 1 mole of aldehyde or aliphatic ketone strongly reacts with the Ehrlich's aldehyde reagent, and they pointed out that the Ehrlich's aldehyde reagent perhaps acts upon three methylene bridges of urobilinogen respectively. According to H. Fischer,s' the condensation product of neobilirubinic acid and p-dimethylaminobenzaldehyde was light yellow needle-like crystal and not identical with the Ehrlich's aldehyde reactant, and its colour remained yellow even when it was dissolved into hydrochloric acid, and its oxidation product by ferric chloride was light pink. And then, the mechanism of the Ehrlich's aldehyde reaction has not been cleared up till now.7' From various experiments in our Department,8' on the other hand, it was pointed out that the central methylene bridge of grows to be active by a combination of atoms, atom groups, or molec- ules with hydroxypyrromethenes at both sides, and then the Ehrlich's diazo reaction becomes positive although bilirubin itself denotes no diazo reaction. So it may be suggested from the above facts that the central methylene bridge of urobilinogen, such as mesobilinogen of which hydroxypyrromethenes have 4 hydrogen atoms respectively, No. 6] Studies on Bile Pigments. IV-1 413 and as stercobilinogen of which hydroxypyrromethenes have 6 hydro- gen atoms respectively, is active. In fact, urobilinogen gave the positive direct diazo reaction, and after an addition of such a micro- quantitatively active methylene-detecting reagent as sodium nitro- prusside or sodium 1, 2-naphthoquinone-4-sulf ovate with or without a subsequent use of N/2 sodium hydroxide to aqueous solutions of uro- bilinogen of various concentrations and pH values, no specific colouring towards the Ehrlich's aldehyde reagent occurred even by adding the reagent till the optimal pH should be introduced. When a mixture of the active methylene-detecting reagent and the Ehrlich's aldehyde reagent in equal volume was poured out dropwise upon the above- mentioned aqueous solutions of urobilinogen, the typical Ehrlich's aldehyde reaction occurred only when the medium of the initial urobilinogen solution had been acid. This means that both reagents react upon urobilinogen independently, that the active methyl ene- detecting reagent reacts in basic medium contrary to the Ehrlich's aldehyde reagent reacting in acid one, and further that both reagents react at the same position. Then, urobilinogen has active methylene radical, to which the Ehrlich's aldehyde reagent is condensed. When a minute volume of formaldehyde is preliminarily added to an uro- bilinogen solution, no Ehrlich's aldehyde reaction occurs and no urobilinogen is transformed into . Although the reductive power of formaldehyde must be considered, it will not concern to an inhibition of the Ehrlich's aldehyde reaction by the reason why l-ascorbic acid, of which reductive power is stronger than that of formaldehyde, does not inhibit the reaction. And then the reactive positions of p-dimethylamino- benzaldehyde and formaldehyde are the same, and the reaction between urobilinogen and formaldehyde is suggested to proceed as shown in Fig. 1, giving no space of an oxida- tion of urobilinogen into uro- .

Experimental 1. Materials. Urobilinogen was extracted from the sound human feces by C. J. Watson's method,9' and also prepared by a reduction of crystalline bilirubin, supplied from Kodak Co., with sodium amal- gam by C. J. Watson's method.9' Extraction of urobilinogen from urine was undertaken as follows: Fresh urine containing urobilinogenn and no bilirubin was acidified till pH 3 to 4 with glacial acetic acids 414 K. YAMAOKA et al. [Vol. 32,

After extraction of urobilinogen into an equal volume of diethyl ether, the ether was washed with water, and the urobilinogen was trans- ferred into a sodium hydroxide solution by mixing a third or an equal volume of N/10 sodium hydroxide to the ether solution. The sodium hydroxide solution of the urobilinogen was adjusted to pH 7.0 to 7.2 with N/10 hydrochloric acid. Feces offer stercobilinogen, bilirubin does mesobilinogen, and the prepared from urine contains the two. No indol could be detected in the prepared urobilinogen solution. 2. Ehrlich's Aldehyde Reagent. To 50 cc of concentrated hydro- chloric acid, 2 g of p-dimethylaminobenzaldehyde was dissolved, and then 50 cc of distilled water was further added. 3. Surveys on Activity of Central Methylene of Urobilinogen. Two drops of 5% sodium nitroprusside aqueous solution or 50 alcohol saturated with sodium 1,2-naphthoquinone-4-sulfonate were added to 1 cc of an aqueous or alcoholic solution of urobilinogen of various concentrations and pH values, and after adding 2 drops of N/2 sodium hydroxide or not, 2 to 10 drops of the Ehrlich's aldehyde reagent were added until the pH value of the medium grew to 1

Table I. Influences of active methylene group-detecting reagent on the Ehrlich's aldehyde reaction

4. Influences o f Formaldehyde on Ehrlich's Aldehyde Reaction of Urobilinogen. When the Ehrlich's aldehyde reagent was added to a mixture of 5 cc of an urobilinogen solution of 1.03 mgr/ and a minute volume of f ormalin, a red tone discoloured paralleling with an increase of the added volume of f ormalin (Fig. 2). An aqueous urobilinogen solution of 3.81 mgr/ and a mixture of 5cc of the solution No. 6] Studies on Bile Pigments. Iv-1 415Y and 0.05 cc of formalin were partitionated by ascending one-dimen- sional paper chromatography under development with methanol-water (2:1) under the aeration of coal gas. A yellow zone of urobilin was separated in both cases at Rf 0.77. By spraying the Ehrlich's aldehyde reagent to the papers, a red zone appeared at Rf 0.83 in the former, while no coloured zone appeared anywhere in the latter (Fig. 3), and further no yellow zone appeared by letting the raw developed papers in a room. And although, after 50 cc of a mixture of an aqueous urobilinogen solution of 10 mg/ and formalin, which was introduced in an amount of 3 / to the urobilinogen solu- tion, was aerated with oxygenn for 1.5 hours at a rate of 200 bubbles per minute, it was left standing in a room, no in- crease of urobilin could be recognized (Table II).

Fig. 2. Influences of formalin on the Ehrlich's reaction Fig. 3. Paper chromatograms of then aqueous solution of urobilinogen with or without the addition of formalin Materials; I • • •Aqueous solution of uro- bilinogen lI Aqueous solution of uro- bilinogen with the additionn of formalin in 1%

5. Reductive Power of Formaldehyde. After 10 minutes of the addition of 5 cc of formalin or a 5% aqueous solution of l-ascorbic acid to 5 cc of a 1 % aqueous solution of methylene blue, extinction coefficients of the solution by filter S51 of Pulfrich's photometer was 1.6 or 0.04. 416 K. YAMAOKA et al. [Vol. 32,

Table II. Influence of 02-aeration on the formaldehyde-urobilinogen reaction

Conclusion

1. The Ehrlich's aldehyde reaction may be due to a reaction between the reagent and the active methylene bridge, perhaps the central, of urobilinogen. 2. The Ehrlich's aldehyde reaction does not occur by a prelimi- nary addition of formaldehyde. 3. Formaldehyde condensates itself perhaps to the central active methylene bridge, and the condensation is so stable as not to be transformed into urobilin type under a mild oxidation as clearly anticipated from the chemical structure.

References

1) Ehrlich, P.,: Med. Wschr., 1, 151 (1901). 2) Neubauer, 0.,: Munch, med. Wschr., 50, 1846 (1903). 3) Fischer, H., and Orth, H.,: Die Chemie des Pyrrols, I, Akadem. Verlagsgesellschaft, Leipzig, 66 (1934). 4) Fischer, H., and Hess, R.,: Z. physiol. Chem., 194, 193 (1931). 5) Fischer, H., and Adler, E.,: Z. physiol. Chem., 206, 187 (1932). 6) Fischer, H., and Orth, H.,: Die Chemie des Pyrrols, II, Erste Halfte, Akadem. Verlagsgesellschaft, Leipzig, 716 (1937). 7) Lemberg, R., and Legge, J. W.,: Hematin Compounds and Bile Pigments, Intersci. Publ., Inc., New York, 137 (1949). 8) Yamaoka, K.,: J. Jap. Soc. Inter. Med., 42, 531 (1953). 9) Watson, C. J.,: J. Biol. Chern., 200, 691, 697 (1953).