THE ORIGIN OF NATURAL CRYSTALLINE UROBILIN (STERCOBILIN) *

BY CECIL JAMES WATSON

(From the Department of Medicine, University of Minnesota Hospitals, Minneapolis)

(Received for publication, October 14, 1935)

Previous communications have dealt with the isolation and nature of crystalline urobilin and stercobilin (l-9). These sub- stances, as obtained from human urine and feces respectively, were proved identical. It was further shown that crystalline mesobilirubmogen (urobilinogen) does not yield this type of uro- bilin upon exposure to air and light (8,9). Instead of this a uro- bilin was isolated possessing a different crystal form and failing to yield the characteristic crystalline compounds of the natural substance; i.e., the iron chloride molecular compound and the hydrobromide. This work has been confirmed and extended by Fischer, Halbach, and Stern (10). These investigators found that natural crystalline stercobilin is strongly levorotatory, while meso- bilirubinogen and the urobilm prepared from it, previously spoken of as K-urobilin (9),1 are optically inactive. Natural urobiim further differed from K-urobilin in that the melting point of the crystalline, free substance from acetone was 236”, while the free K-urobilm was found to melt at 190”.

From the foregoing it would appear that natural crystalline urobilin cannot be derived in vitro from mesobilirubinogen. Since the latter substance is identical with urobilinogen (11,12), it re- mained to be determined whether a different urobilinogen is the parent substance of natural crystalline urobilin or whether the latter is derived from some other substance not having urobilino- gen characteristics. The following studies were believed to be important to the solution of this question.

* Aided by a grant from the research fund of the Graduate School, Uni- versity of Minnesota.

~Ktin-stliches urobilin.

47

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48 Origin of Crystalline Urobilin

1. Repetition of the isolation of a crystalline urobilin in vitro from mesobilirubinogen. This was desirable for the sake of fur- ther confirmation of results previously described (8, 9).

2. (a) Isolation of crystalline urobilinogen from urine in order to compare it again with mesobilirubinogen. (b) Isolation of crystalline urobilin from the mother liquor of the crystalline urine urobilinogen, after allowing it to develop urobilin characteristics by exposure to light and air.

3. Isolation of a urobilinogen from the feces in order to de- termine whether crystalline urobilin might be obtained from it after exposure to light and air.

4. Comparison of normal feces with feces during hemolytic jaundice as to total urobilmogen content estimated colorimetri- tally, and actual yield of crystalline stercobilin. This was desir- able in order to decide whether stercobilin formation was more than proportionately increased in hemolytic jaundice.

EXPERIMENTAL

Preparation of K-Urobilin from Mesobilirubinogen-The method employed has been described previously (8, 9). The yield of K-urobilin from 75 mg. of crystalline mesobilirubinogen was but 2 mg. The crystals were identical in appearance with those al- ready described. The substance again failed to yield a crystalline iron chloride molecular compound (7,9) although this was readily obtained from a smaller amount of natural crystalline stercobilm. The absorption spectrum of solutions of these crystals does not differ from that of natural crystalline urobilin (9). The intensify- ing effect of the addition of water to an alcoholic solution, previ- ously described for the natural substance (9), was also observed with K-urobilin.

Isolation of Crystalline Urobilinogen from Urine-The urine was obtained from a patient with hemolytic jaundice. The amount employed measured approximately 4 liters, requiring a period of 6 days to collect. The urine was collected in a brown glass bottle beneath a considerable amount of petroleum ether. At the time the isolation procedure was begun the urine exhibited a very in- tense Ehrlich reaction. The entire amount together with the petroleum ether was placed in a separatory funnel of 6 liters capac- ity. More petroleum ether was added until about 1 liter covered

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C. J. Watson 49

the urine. The urine was next acidified with 100 cc. of glacial acetic acid and very gently mixed. After the carbon dioxide had entirely formed and disappeared, the urine and petroleum ether were mixed by repeated inversion for several minutes. The mix- ture was not shaken vigorously. Nevertheless a partial emulsion formed, most of which separated after a short period of standing; addition of a few cc. of 95 per cent alcohol furthered separation of the emulsion. The remainder of the emulsified portion was filtered with suction on a large Buchner funnel; this accomplished complete separation. The further treatment of the petroleum ether solution was as follows:

Petroleum ether

Washed with water 2 times

5% N$&03 (5 times) Acidified with 50% HtSOl until just blue to Congo paper

I

CH&13 (8 times)

After concentration in vucuo the CHCl, solution was subjected to Fischer’s procedure (11) for crystallizing mesobilirubinogen. A considerable amount of a very light yellow, almost white residue resulted when the final petroleum ether solution, obtained by this procedure, was concentrated to dryness in vacua. This resi- due was dissolved in about 8 cc. of warm acetic ester, which was then concentrated to about 3 cc. After 5 hours in the ice box a considerable separation of light yellow, almost colorless crystals had occurred. The yield was 44 mg., 16 mg. of this amount being obtained by further concentration of the mother liquor. The substance was readily recrystallized from warm acetic ester. The crystals as first obtained are shown in Fig. 1. Recrystallization yielded small but perfect crystals such as are illustrated in Fig. 2.

The latter were examined by Professor Gruner of the Depart- ment of Geology, University of Minnesota, to whom the writer is indebted for the following report: “Small, probably monoclinic crystals. So far as I can determine, they appear to correspond entirely with the descriptions of hemibilirubin and mesobilirubino- gen as given by Steinmetz in the reports of Fischer (ll), Fischer and Meyer-Betz (12), and Fischer and Hess (13). Because of their small size, measurements could not be made.” The first

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Origin of Crystalline Urobilin

FIG. 1. Crystalline urine urobilinogen as first obtained

3”*#,-,* i* x a I *1 Iri i

FIG. 2. Urine urobilinogen after recrystallization

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C. J. Watson

crystals obtained melted at 193-196” with some shrinking above 190”; after recrystallization the melting point was 200-202’ with slight shrinking above 192”. The melting point for mesobiliru- binogen and urobilinogen was given by Fischer (14) as 201”. The crystals gave an intense Ehrlich reaction; the solution showed absorption at 568.3 to 542.2 mp, maximum 558.9 mp. A small amount of the crystalline substance was sent to Professor H. Fischer in Munich, for whose report, which follows, the writer acknowledges his gratitude.

“The absorption spectrum after carrying out the Ehrlich reaction is identi- cal with that of mesobilirubinogen treated in the same manner. Crystals were submitted to Professor Steinmetr who stated that they were probably identical with those of urobilinogen as isolated by Fischer and Meyer-Betz (12), but that measurement could not be carried out because of the small size. After recrystallization from acetic ester-petroleum ether somewhat larger crystals were obtained; these were also believed by Professor Stein- metz to be probably identical with the urobilinogen of Fischer and Meyer- Bets, but were still too small for measurement.”

Isolation of Crystalline Urobilin from Mother Liquor of the Above Crystalline Urobilinogen-After removal of the 44 mg. of crystal- line urobilinogen mentioned above, the acetic ester mother liquor was allowed to dry. The residue remained exposed for several days to light and air and was then dissolved in 8 cc. of glacial acetic acid. This solution was allowed to stand in a lightly corked tube in the light for 2 weeks. At the end of this period its color was dark brown and the Ehrlich reaction negative. Green fluores- cence with alcoholic zinc acetate solution was intense. The solu- tion was poured into 4 volumes of chloroform and this was further treated in exactly the same manner as the first chloroform extract in the method previously described for the isolation of urobilin from the urine or stercobilin from the feces (3, 4). 17 mg. of crystals having the typical appearance of urobilin hydrochloride were obtained. From these the crystalline, free substance, hy- drobromide, and iron chloride molecular compound were readily prepared according to the procedures previously described (2,6,7). The characteristic form of the crystals, particularly of the last two compounds, served to identify the substance with natural crystalline urobilin.

Methods Employed in Attempt to Isolate Crystalline Urobilinogen from Feces, and in Isolating Crystalline Stercobilin from Various

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52 Origin of Crystalline Urobilin

Fractions Resulting during This Attempt-202 gm. of feces were obtained over a 4 day period from a male patient, aged 20, who had suffered an injury to the left kidney with a resulting hema- toma. The urobilinogen in this sample of feces was greatly in- creased. The average amount excreted per day during the 4 day period was found to be 1010 mg. A modification of a previously described method (15) was used for estimation of thii amount. This modified procedure will be described in a separate communi- cation.

About 2 gm. of the feces were employed in carrying out a test for occult blood with the benzidine method, which proved nega- tive. The remaining 200 gm. were first ground in a mortar and washed repeatedly with petroleum ether, which was simply de- canted in each instance. These washings gave only a weak Ehrlich reaction. The entire amount was next thoroughly ground with 1 liter of 5 per cent sodium carbonate in distilled HzO. This mixture was at once poured into 1 liter of 8 per cent ferrous sulfate (FeSOd. 7HzO) solution with constant shaking. Filtration was begun immediately and was complete at the end of 1 hour. The ferrous carbonate was employed at this stage to facilitate ,extraction of the urobilinogen. The character of the precipitate permits quicker titration, and the urobilinogen is protected dur- ing its extraction by the mildly reducing action of the ferrous carbonate.

The alkaline filtrate was washed three times with petroleum ether without vigorous shaking. It was next acidified with glacial acetic acid and extracted three times with petroleum ether. A partial emulsion formed which was quickly separated by filtration with suction through a Buchner funnel. The combined petroleum ether solution gave a strong Ehrlich reaction. This solution was now subjected to the same procedure as was applied to the primary petroleum ether extract of the urine, mentioned above. Then with Fischer’s method (11) the final CHCL solution was treated according to the following plan. It should be noted that precipi- tates and solutions left behind in this fractionation which gave either a relatively weak, or no Ehrlich reaction, but which ob- viously contained substances having stercobilin characteristics, were saved in order to determine whether crystalline stercobilin might be isolated from them. In the following plan these frac- tions are designated by Roman numerals.

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C. J. Watson 53

CHC18 concentrated in vacua to 20 cc. -1

1 liter petroleum ether

L Orange ppt. (Fraction I) Concentrated in vacua to dryness Light yellow residue dissolved in 15 cc. warm ethyl acetate Concentrated to 5 cc. (Ehrlich’s reaction intense) No crystallization after standing overnight at 4”

4, 600 cc. petroleum ether

1 I Light yellow!preci itate

R* Filtrate concentrated in vacua to 10 cc.

Attempt to crysta ize out Separation of 130 mg. of almost white sub- of eth 1 acetate unsuc- bessf UT

stance giving intense Ehrlich’s reaction

Strong Ehrlich’s reacy CoFd not be crystallized out of acetrc ester

\ /

Combined i< CHCh

Combined 5 CHCls solutions L

5yo NaHCOz

5 Washed with CHClr 3 times Acidi6ed with 50yo HSO,

-1 Aqueous Fraction II CHCh concentrated in vacua to 8 cc.

-1 500 cc. petroleum ether

I Ppt. (Fraction III) Concentrated in vacua to dryness Attempt to crystallize residue out of ethyl acetate un-

successful Dissolved in 25yo HCI 2 cc. of 5% FeCh in 2% HCl added _

-1 Filtrate Voluminous brown granular

iron chloride double salt o P pt. similar in appearance to mesobilirubinogen (Fischer

and Niemann (16))

-1 Washed with 25yo HC13 times 1% NaOH

-1 Ppt. of Fe(OH), Filtrate acidified to Congo paper with 50% Has01

-i Aqueous Fraction IV CHCh (concentrated in vacua to 5 cc.)

I 200 co. petroleum ether (concentrated in vacua to dryness) Almost white residue (intense Ehrlich’s reaction)

1 3 cc. ethyl acetate (concentrated in vacua to 1 cc.) No crystallization even after seeding with crystalline

urobilinogen (from urine)

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54 Origin of Crystalline Urobilin

The dried residue was now subjected to the procedure described above by which crystalline urobilin was obtained from the mother liquor of the urine urobilinogen. In this way 13 mg. of crystals having the typical appearance of natural stercobilin hydrochloride were obtained. This material was identified by preparation of the crystalline hydrobromide and the iron chloride molecular compound.

Varying smaller amounts of crystalline stercobilm were likewise obtained from each of the Fractions I to IV in the accompanying diagram. To accomplish this, those of the fractions which were aqueous were acidified with hydrochloric acid, and the stercobilin hydrochloride was extracted with chloroform. Fractions con- sisting of precipitates were dissolved in 25 per cent HCl; after dilution with water the hydrochloride was extracted with chldro- form. From this point the chloroform solutions were handled according to the method described previously (4). The crystal- line iron chloride molecular compound was prepared in each in- stance for the sake of identification.

Comparison of Normal Feces and Feces during Hemolytic Jaun- dice As to Total Urobilinogen Content and Yield of Crystalline Stercobilin-The total amount of feces for 4 days was collected from a healthy male student 25 years old. This amount weighed 223 gm. (moist). The urobilinogen content was found to be 359.2 mg. The amount of crystalline stercobilin isolated was 73 mg. or 20.3 per cent of the estimated urobilinogen content.

The total amount of feces from a male patient, 18 years old, suffering from familial hemolytic jaundice, was collected for a period of 8 days. This weighed 920 gm. During the first 4 days the urobilinogen content was 4560 mg., and during the second 4 days 4880 mg., a total of 9440 mg. The amount of stercobilm isolated from the feces for 8 days was 191U mg., or 20.1 per cent of the estimated urobilinogen content.

DISCUSSION

The results of the above experiments confirm those previously reported (S-lo), which indicated that mesobilirubinogen does not yield natural crystalline urobilin or stercobilin. They further prove that the latter substances, already shown to be identical, may be obtained from a chromogen having urobilinogen charac-

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C. J. Watson 55

teristics, occurring in both urine and feces. This chromogen un- doubtedly differs from mesobilirubinogen or crystalline urobilino- gen as isolated from urine by Fischer and Meyer-Beta (12), and again in the present investigation. Of distinct importance was the isolation of natural crystalline urobilin from a mother liquor which had already yielded crystalline mesobilirubinogen. These findings point to but one conclusion ; i.e., the existence in urine and feces of two urobilinogens. The following scheme may be used in further considering their relationship.

Bilirubin in intestine

L Urobilinogen A

(mesobilirubinogen)

I

1 Urobilinogen B

I Crystalline urobilin

K-urobilin or ster cobilin

Urobilinogen B has not been obtained in crystalline form, prob- ably because of its poor ability to crystallize. It was previously noted (4) that the leuco compound obtained by amalgam reduc- tion of crystalline stercobilin could not be crystallized. This has been confirmed by Fischer, Halbach, and Stern (10). Probably for the same reason, K-urobilin has not been isolated from urine or feces, and can be crystallized only with difficulty after formation in vitro. There is every reason to believe that it accompanies the other, easily crystallized urobilin in nature. The failure to ob- tain mesobilirubinogen, or urobilinogen A, from the feces in con- trast to the ease with which it may be isolated when present in comparable amount in the urine, suggests that this substance is more readily and completely reabsorbed from the bowel.

The above scheme assumes that urobilinogen B is derived from bilirubin. Direct proof of this is lacking, but the close similarity of the two urobilinogens as well as of their respective urobilins makes this most probable. The assumption is further supportedby the recent observation of Fischer and associates (10) that crystal- line stercobilin yields a porphyrin very similar spectroscopically to that obtained from bilirubin or mesobilirubinogen by the same treatment; i.e., heating with glacial acetic and hydrobromic acids in a sealed tube at 180”.

Another possibility must be mentioned; i.e., that urobilinogen B is a derivative of mesobilirubinogen by virtue of some process

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Origin of Crystalline Urobilin

peculiar to the chemical activity in the bowel. Two facts appear to be opposed to this theory: (1) the failure to obtain methyl- ethylmaleinimide by the nitric acid oxidation of stercobilin (1, 2, 10) ; (2) the failure by Fischer and coworkers (10) to obtain hema- tinic acid from stercobilin following oxidation with chromic acid, instead of which succinic acid was isolated. Mesobilirubinogen yields both of these substances when oxidized by the same re- agents (17).

The isolation of crystalline stercobilin from the various Frac- tions I to IV mentioned above is of importance, since it proves that stercobilin formation is not dependent upon any particular pro- cedure employed, such as that previously used (4) in its isolation.

The amounts of stercobilin which may be isolated from the feces in hemolytic jaundice are relatively so large that the ques- tion arose as to whether they were not actually out of proportion to the increased blood destruction in this disease. That this is not the case is shown by the close agreement of yields of ster- cobilin in terms of per cent of total urobilmogen contents of feces in normal cases and in patients with hemolytic jaundice.

SUMMARY

1. Mesobilirubinogen, although identical with a urobilinogen occurring in the urine, is not the parent substance of crystalline urobilin or stercobilin as obtained from urine or feces. A differ- ent crystalline urobilin may be obtained in vitro from mesobiliru- binogen. This urobilin probably occurs in nature but has not yet been isolated because of greater lability and poorer ability to crystallize.

2. The existence of a second urobilinogen in the feces and urine is indicated by the following: (a) The isolation from the feces of a non-crystalline chromogen possessing urobilinogen charac- teristics, which, after exposure to light and air, yielded natural crystalline stercobilin. (b) Th e isolation of natural crystalline urobilin from a mother liquor which had been allowed to develop urobilin characteristics by exposure to light and air. This mother liquor originally contained only chromogen, a part of which was isolated in the form of crystalline mesobilirubinogen.

3. The yield of crystalline stercobilin from feces in cases of

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C. J. Watson

hemolytic jaundice is in the same proportion to the initial urobili- nogen content as in normal feces.

Addendum-Recent studies, which have been reported since this paper was submitted for publication, suggest that natural crystalline urobilin may be derived from mesobilirubin. A bilirubinoid substance similar in constitution to mesobilirubin, but more closely resembling crystalline urobilin in physical properties, was synthesized by Siedel (18). Fischer and Halbach (19) were able to convert natural crystalline urobilin into glaucobilin. This substance, a derivative of mesobilirubin first described by Fischer, Baumgartner, and Hess (ZO), has not been found in nature. Fischer and Halbach’s work is proof of the bile pigment nature of crystal- line urobilin.

BIBLIOGRAPHY

1. Watson, C. J., 2. physiol. Chem., 204,57 (1932). 2. Watson, C. J., Z. physiol. Chem., 203,101 (1932). 3. Watson, C. J., 2. physiol. Chem., 221,145 (1933). 4. Watson, C. J., J. Biol. Chem., 106, 469 (1934). 5. Watson, C. J., Proc. Sot. Ezp. Biol. and Med., 30,121O (1933). 6. Watson, C. J., Proc. Sot. Exp. BioZ. and Med., 32, 534 (1934). 7. Watson, C. J., Proc. Sot. Exp. BioZ. and Med., 32, 1506 (1935). 8. Watson, C. J., Proc. Soe. Exp. BioZ. and Med., 32,1508 (1935). 9. Watson, C. J., Z. physiol. Chem., 233, 39 (1935).

10. Fischer, H., Halbach, H., and Stern,.A., Ann. Chem., 619,254 (1935). 11. Fischer, H., 2. physiol. Chem., 73, 204 (1911). 12. Fischer, H., and Meyer-Beta, F., Z. physiol. Chem., 76, 232 (1911). 13. Fischer, H., and Hess, R., 2. physiol. Chem., 194, 193 (1931). 14. Fischer, H., 2. Biol., 66, 163 (1914). 15. Watson, C. J., Arch. Znt. Med., 47,698 (1931). 16. Fischer, H., and Niemann, G., 2. physiol. Chem., 137, 292 (1924). .17. Fischer, H., and R&e, H., Z. physiol. Chem., 89,255 (1914). 18. Siedel, W., Z. physiol. Chem., 237, 8 (1935). 19. Fischer, H., and Halbach, H., Z. physiol. Chem., 238, 59 (1936). 26. Fischer, H., Baumgartner, H., and Hess, R., Z.physioZ. Chem.,206,201

(1932).

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Cecil James Watson(STERCOBILIN)

CRYSTALLINE UROBILIN THE ORIGIN OF NATURAL

1936, 114:47-57.J. Biol. Chem. 

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