CONCERNING THE NATURALLY OCCURRINGPORPHYRINS. IV. THE URINARY PORPHYRIN INLEAD POISONING AS CONTRASTED WITHTHAT EXCRETED NORMALLY AND IN OTHERDISEASES

Cecil James Watson

J Clin Invest. 1936;15(3):327-334. https://doi.org/10.1172/JCI100783.

Research Article

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CONCERNINGTHE NATURALLYOCCURRINGPORPHYRINS. IV.THEURINARYPORPHYRININ LEAD POISONINGAS CON-

TRASTEDWITH THAT EXCRETEDNORMALLYAND IN OTHERDISEASES'

By CECIL JAMESWATSON

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

(Received for publication January 22, 1936)

In previous communications (1, 2, 3), the lit-erature relating to H. Fischer's "dualism'" ofthe natural porphyrins was discussed. The isola-tion of coproporphyrin I from the urine of a pa-tient with cincophen cirrhosis of the liver, alsofrom the feces in hemolytic jaundice and perni-cious anemia, was reported. Of particular in-terest was the consideration of these findings withrelation to the clinical and experimental observa-tions of Van den Bergh and coworkers (4, 5).These investigators pointed out that human eryth-rocytes regularly contain protoporphyrin which isincreased in amount in the same individuals whoexhibit increases of coproporphyrin in the urine.By perfusion experiments they further demon-strated that the surviving liver of the dog is ableto convert protoporphyrin into coproporphyrin.Correlation.of these studies made it appear likelythat the coproporphyrin of the urine, as well as ofthe bile, may originate from the protoporphyrinof the erythrocytes. Since hemoglobin, followingloss of its protein and iron, yields a protoporphy-rin of the aetioporphyrin III type (6), it wasnaturally assumed that the porphyrins of the urineand bile are directly related to hemoglobin metab-olism. Schreus (7) in particular has pointed toVan den Bergh's results as supporting this con-ception. The idea that the porphyrin of the urineis derived from hemoglobin is by no means a re-cent one. In fact, a considerable literature deal-ing with this question was collected by Garrod in1900 (8). Although Garrod subscribed to thebelief that the urinary porphyrin, then spoken ofas hematoporphyrin, was related to hemoglobindestruction, he observed that increases in amountwere frequently associated with clinical or ana-tomical evidence of liver disease.

1Aided by a grant from research funds of the GraduateSchool of the University of Minnesota.

The concept that the coproporphyrin of theurine and bile is derived from hemoglobin wasnot supported by the findings described in Parts Ito III of this investigation (1, 2, 3). The occur-rence of coproporphyrin I rather than III consti-tuted evidence that at least in the instances stud-ied, porphyrin formation was a process distinctlyseparate from the synthesis or destruction ofhemoglobin. The isolation of coproporphyrin IIIfrom the urine in lead poisoning was described byGrotepass (9), later by H. Fischer and Duesberg(10). Since ordinary hemin and coproporphyrinIII both correspond in configuration to aetiopor-phyrin III, the elaboration of coproporphyrin IIIin lead poisoning could be associated quite readilywith a peculiar disturbance in hemoglobin metabo-lism.

The function of the porphyrins present in theerythrocytes, urine, bile and feces is quite un-known and can hardly be determined until exactknowledge of their origin and isomeric type isobtained. The amount of porphyrin in normalurine is so small that it has not yet been isolated,and the important question as to whether it iscoproporphyrin I or III remains unanswered.The finding of coproporphyrin I in the urine inan instance of advanced liver disease (1), sug-gested that it was the normal urine porphyrin, in-creased in amount for the same reason that uro-bilinogen was increased, i.e., diminished excretoryfunction of the liver.

The purpose of the present investigation was toisolate and identify the coproporphyrin of a nor-mal urine, and of the urine in some of the patho-logical states accompanied by a heightened por-phyrin excretion; further, to determine whethercoproporphyrin III is regularly excreted in theurine in lead poisoning (as yet it has been isolatedbut from one instance).

327

CECIL JAMES WATSON

MATERIAL ANDMETHODS

The entire amount of urine for a period of 33days was collected from a healthy male student 218 years of age. As soon as 2 to 3 liters hadaccumulated the specimen was subjected to theprocedure previously described (1), with the fol-lowing exceptions. In each instance the primaryether extract after washing with water was re-peatedly extracted with small amounts of 10 percent HCl. The 10 per cent HC1 solutions wereunited and kept in the refrigerator during theperiod of further collection and primary extrac-tion of the urine. All of the hydrochloric acidextract was then covered with a large amount ofether, an excess of sodium acetate was added, andthe mixture at once vigorously shaken. Havingmade certain that the aqueous fraction was nega-tive to Congo paper, ether extraction was re-peated until the porphyrins were removed com-pletely. The further treatment of this solutionof ether was the same as described previously(1). The final coproporphyrin solution in etherwas not dried over sodium sulphate as was donepreviously, since it has been found to be true thata moderate loss of porphyrin occurs by adsorptionon this salt. This was first noted by H. Fischerand Zerweck (11). The ether was simply de-canted into a dry flask and was then removed com-pletely upon the water bath. The porphyrin resi-due after drying in the air was esterified withHCl in methyl alcohol, and the ester was crystal-lized from chloroform-methyl alcohol in -the usualway (1).

Employing the samne method, porphyrin esterswere isolated from the urines of the followingcases.

Case 1. Fever. Male, 37 years of age with lungabscess and empyema. The daily elevation of tempera-ture ranged from 101 to 1030 F. Urobilinuria was 8.3mgm. urobilinogen per day (23). Therewasno jaundice.Moderate anemia was present, and hemoglobin was 55 percent (Sahli method, 17 grams per 100 cc. equivalent to100 per cent). Urine was collected for a period of 12days.

Case 2. Acquired hemolytic jaundice. Female, 18years of age with slight icterus, without anemia, of 3years' duration, increasing icterus and anemia, 4 months.

2 The writer is indebted to Mr. Samuel Schwartz forthe collection and preliminary extraction of this urine.

Hemoglobin was 32 per cent (Sahli), erythrocytes 1,150,-'000 per cu. mm. Stained smears revealed marked macro-anisocytosis, polychromatophilia. Reticulocytes variedbetween 15 and 32 per cent. Resistance of erythrocytesto hypotonic saline was as follows: H1 .38, H, .32, controlH, .38, H2 .30.

There was marked autoagglutination of erythrocytes invitro (below 300 C.), the icterus index was 30, and theVan den Bergh reaction was of the indirect type. Fecesurobilinogen: 1106 mgm. per day. (Normal range 100to 250.) Urine urobilinogen 2.5 mgm. per day. (Nor-mal range 0.5 to 3.0.) (23)

Physical examination revealed nothing except a smoothcystic mass in the left side of the pelvis. The spleen andliver were not palpable. It was considered possible thatchronic hemorrhage into an ovarian cyst with local for-mation of bilirubin might be responsible for the findingsdescribed above. Consequently the cyst was removed atoperation. It contained 800 cc. of a dark brown fluidrich in hematin (identified by hemochromogen spectrumafter treatment with ammonium sulphide). Bilirubin andporphyrins were not identified, although searched for.

There was no improvement following this operation.On the contrary, icterus and anemia increased, and therewas moderate fever. The urobilinogen in the urine in-creased markedly for a few days after operation, thendecreased rapidly again. The highest value noted was380 mgm. per day. During this period the urine wasexamined for porphyrins and a considerable increase ofcoproporphyrin was observed.

After an interval of three weeks during which the pa-tient's condition remained precariously stationary, sple-nectomy was carried out. The spleen weighed but 460grams, exhibiting microscopically a marked pulp conges-tion. The liver appeared to be entirely normal. Thisoperation was followed by prompt improvement. Twomonths after operation the hemoglobin was 70 per cent(Sahli), the icterus index 10, and the feces urobilinogen206 mgm. per day. Eight months later the patient re-ported that she felt entirely well and that there was nojaundice.

The urine employed for the isolation of coproporphyrinconsisted of the entire amount for a period of four daysimmediately following the first operation (coincident withthe highest urobilinogen excretion). This urine was firstemployed for the isolation of crystalline urobilinogen, aprocedure which will be described elsewhere (24). Suf-fice it to say that this entailed a preliminary extractionwith petroleum ether in which coproporphyrin is insolu-ble. Following this, the coproporphyrin was extractedwith ether in the manner already referred to (1). It isworthy of note that this urine contained another pigmentresembling bilirubin, but differing from it in that it failedto give a Gmelin test as well as the more delicate reactionfor bilirubin devised by Harrison, and described by God-fried (12). Also differing from bilirubin the substancecould not be extracted with chloroform from the urine.However, it gave a delayed and an indirect Van denBergh reaction, and was precipitated by a barium chlo-

328

CONCERNINGTHE NATURALLY OCCURRINGPORPHYRINS. IV

ride solutioni. In dilute NaOH ill defined absorptionbetween 536 to 477 mny was observed. Attempts to crys-tallize the substance were unsuccessful, probably becauseof its obvious lability, and it cani only be stated that itscharacteristics as nioted above appeared to be those of the" himorubin " of Jenike (13), which, it is noteworthy,was likewise obtainied from the uriine of an individual withhemiiolytic jaunldice.

Case 3. Lead poisoning. Male, 67 years of age. 'Mildanemia, hemoglobini 69 per cent. Considerable basophilicstippling of ervthrocytes. Urine collection for a perio(dof 5 days.

Case 4. Lead poisoning. Male, 46 years of age. Colic,aniemia, hemoglobin 66 per cent, marked basophilic stip-pling of erytlhrocytes. Urine collection for a period of4 days.

Case 5. Lead poisoning. Male, 38 years of age.Weakniess of extensor muscles, mild anemia, hemoglobini67 per cenit, conisiderable basophilic stippling of theerythrocytes. Urine collection over a period of 6 days.

Cases 3, 4 and 5 were members of a large groupof foundry emiployees suffering from lead poison-ing.3 The bearings in this foundry contained leadand were believed responsible for the numerousinstances of lead poisoning which were encoun-tered.

The total amuount of urine collected in each ofthe above instances was subjected to the methodof isolation already referred to. It is imlportantto note that the fractionation of porphyrins em-bodied in this method depends upon character-istic solubilities (1, 2). Consequently, the copro-porphyrins isolated and described in the followingparagraphs possessed in each instance the typicalsolubilities of a coproporphyrin, particularly innot leaving 0.2 per cent HCI for chloroform.The sodiumn salt in each instance was entirely sol-uble in 10 per cent NaOH, a further character-istic of coproporphyrins.

Spectroscopic stu(lies were carried out by miieansof apparatus ancd miiethods dlescribed previously(1, 2). Sliglht variations in the miiaximiia for theabsorption bands of the coproporphyrins isolatedwill be notel, if comiipared with those observedhefore (1, 2, 3). Since the spectrometer was not

The writer is inidebted to Dr. E. C. Emerson of St.Paul, Minnesota, for the opportunlity to inivestigate theurinie of these cases.

recalilbrated, these are believed attributable tovery sliglht changes in the relation of the wavelength scale of the instrtumiient to its grating. Ofchief importance is the identificationi 1y miieanis ofsuperimposition of spectra, employing a purecoproporphyrin to compare with that un(ler in-vestigation. With the apparatus use(l (1), thispermlits detection of the slightest (lifferenices inpositionl of absorption band(ls.

RESULTS (SEE TABLE I)

ANrorimial irile

After four recrvstallizations 0.13 Ilmgiml. ofporphyrin ester were obtained fromn 33 (lays nor-mal urine. This will be spoken of as Fraction a.The ester composing this fraction crystallized rap-idly in the form of fluffy imiasses consisting offine curving and branclhing needles. This mlaln-ner of crystallization is vTery characteristic ofcoproporphyrin I ester wlhile coproporphyrin IIIester crystallizes muclh mlore slowly in smiiallerclumiips of crystals often adhering to the walls ofthe tube. These have the form of nee(dle-likeprisms whiicl are usually broader than those ofcoproporphyrin I, and do( not show the curvingand branching of the latter. The crystals ofcoproporphyrin III ester rather frequently ar-range themselves in concentrically uniited grotups,or rosettes (10, 14, 15. 16). During crystalliza-tion and first recrystallization of Fraction a, itwas observed that the miotlher liquior obviouslycontained more porphyrin thani is usually truewhen coproporphyrin I ester is crystallized fromchloroformii methyl alcohol. These two miotlherliquors were combined and allowed to concentrateslowly at roomii temperature. After stan(ling sev-eral days a further se)aration of crystals ap-peared. These were in the formii of smnall aggre-gates part of which adlhered to the walls of thesmiiall test tube in wlhich the solution was conl-taine(l. This material will be sI)oken of as Frac-tion b; its amiiount was evidently considerably lessthani that of Fraction a, nevertheless it was pos-sible to recrystallize it twice in or(ler to furtherobserve its miianner of crystallization; the amiiountdid not suffice for a miielting point (letermiiniationi.The characteristics of these fractionis were as fol-lows.

329

...4

..

R...,

13,.),(5Ch

.ws

X..i

.:xioo~~~~~~~~~J

I

5rX 75C0r

5bK/50

6 ~~~~7X28_0 X300m

FIG. 1. COPROPORPHYRINI ESTER FROMNORMALURINE.FIG. 2. COPROPORPHYRINESTER FROMN'ORMAL URINE, CRYS-

TAL FORMSUGGESTIVEOF COPROPORPHYRINIII.FIG. 3. COPROPORPHYRINI ESTER FROMURINE OF PATIENT

WITH FEVER DUE TO LUNG ABSCESSAND EMPYEMA.FIG. 4. COPROPORPHYRINI ESTER FROMURINE OF PATIENT

WITH ACQUIRED HEMOLYTIC JAIJNDICE.FIG. 5. COPROPORPHYRINIII ESTER FROM URINE OF FIRST

CASE OF LEAD POISONING.FIG. 6. COPROPORPHYRINIII ESTER FROMURINE OF SECOND

CASE OF LEAD POISONING.FIG. 7. COPROPORPHYRINIII ESTER FROM URINE OF THIRD

CASE OF LEAD POISONING.

CONCERNINGTHE NATURALLYOCCURRINGPORPHYRINS. IV

Fraction a. The crystals had the form of longcurving needles (Figure 1). On recrystallizationthese appeared rather quickly in the light fluffyclumps characteristic of coproporphyrin I ester.After four recrystallizations the amount (0.13mgm.) sufficed for one melting point determina-tion. The substance did not melt sharply; shrink-ing was observed above 1900, melting from 218to 2230. (Coproporphyrin I methyl ester, forwhich the writer is indebted to Professor H.Fischer in Munich, melted at 245 to 2460 C.)No change whatever was exhibited at the lowertemperatures critical for coproporphyrin III ester(1420, 1720). The absorption spectrum in acetic

and ether solution was: I. 624.86 ; IL

faint maximum 597.5; III 582.7- 568.3 ; IV.577.4533.5 -526.0 507.0- 487.0

530.0 ; V. 496.6 * Order of in-tensity: V, IV, I, III, II. This was identicalwith the superimposed absorption spectrum ofknown coproporphyrin I in similar solution.

Fraction b. The crystals of this fraction ap-peared very slowly in the manner already re-ferred to. The majority of the crystals werelong narrow prisms similar to those of copropor-phyrin III ester; a few curved and branchingneedles of the coproporphyrin I type were alsonoted. After two recrystallizations the appear-ance of crystals was still delayed and not promptas is usual with even very small amounts of copro-porphyrin I ester. The crystals now appeared tobe uniform in type, namely, needle-like prismsfrequently in groups (Figure 2), strongly sug-gestive of coproporphyrin III methyl ester.Spectroscopically this material was a copropor-phyrin, having the following absorption spec-

trum: I. 627.1-9 II6 faint maximum597.9; III. 581.8- 568.0 IV 534.0- 525.2 V597.9;III.. 577.2 IV* 531.0 V

505 -49687.7 Order of intensity: V, IV, I,

III, II. This was identical with the superim-posed spectrum of a similar solution of copro-porphyrin I.

Pathological urinesCase 1. Fever due to lung abscess and empy-

ema. After three recrystallizations 1.5 mgm. ofporphyrin ester were obtained. This crystallizedrapidly in the manner of coproporphyrin I, andthe individual crystals were fine curving needles(Figure 3). The melting point was 204 to 2080C. The remaining substance was recrystallizedtwice more, following which the melting pointwas 215 to 218° C. All that remained was nowmixed with coproporphyrin I methyl ester ofmelting point 245 to 2460 C., in an approximateratio of one part of the former to four of the lat-ter. The melting point of this mixture was 240to 2430 C. From this it is evident that while theporphyrin isolated was still slightly impure, itmelted far above the melting point of copropor-phyrin III ester, and did not depress the meltingpoint of known coproporphyrin I ester suffi-ciently to suggest that it was another porphyrin.The absorption spectrum was that of a copropor-

626.7- 622.2 583.3 -567.6phyri, i.e., I. 624.8 ,; IL 577.8534.9-525.5 506.4-490.3II. 530.9 IV. 497.5 Order

of intensity: IV, III, I, II. This absorption wasidentical with that of coproporphyrin I when thespectra were superimposed in the comparisonspectrometer.

During the isolation of the above coproporphy-rin it was noted that another porphyrin remainedin the ether and was not extracted by 1 per centHCI, but was removed quantitatively by 10 percent HCI. This porphyrin was chloroform solu-ble, and was readily removed from HCI solutionsby chloroform. The amount was too small tobe obtained in a crystalline form; it was pos-sible, however, to examine the substance spectro-scopically. The acetic and ether solution wasbluish pink and exhibited the following absorp-

tion: I. 634.7-625.6 . 598.0-571.9tlon: I. 631.5 ;I. 580.9554.8 -530.5 514.8-492.0

536.6 ; IV 502 . Order of in-tensity: IV, III, I, II. This absorption spectrumis quite characteristic of protoporphyrin (15),and in fact complete identity with protoporphyrinwas observed when the spectra were superim-

331

CECIL JAMES WATSON

posed. This confirms the report of Boas (17)regarding the occasional finding of protoporphy-rin in the urine.

Case 2. Acquired hemolytic jaundice. Theyield of porphyrin ester was 0.6 mgm. Thiscrystallized rapidly in the fluffy masses of longcurving needles characteristic of coproporphyrinI ester (Figure 4). The melting point after fiverecrystallizations was 235 to 2380 C. with con-

siderable shrinking above 2200. A mixture withapproximately equal parts of coproporphyrin I.ester (melting point 245 to 2460), also meltedat 236 to 2380 C., but with less preceding shrink-ing.

The absorption spectrum of this porphyrin inacetic and ether solution was as follows: I.62624-6227 IL. faint maximum 596.1;

III. 581.2 -567.5 IV 535.2 524.9 V577.5 531.3

506.8-486.2

496.3 . Order of intensity: V, IV, I,

III, II. This was identical with the superim-posed absorption spectrum of coproporphyrin Iin a similar solution.

Case 3. Lead poisoning. One milligram por-phyrin ester after 3 recrystallizations. This crys-

tallized slowly in the characteristic manner ofcoproporphyrin III (Figures Sa and 5b). Themelting point was 161 to 1640 C. The remainingmaterial was recrystallized once after which themelting point was 164 to 1660. The amount ofsubstance did not suffice for another recrystalliza-tion. H. Fischer (6, 16) observed that copro-

porphyrin III methyl ester exhibits a double melt-ing point probably dependent upon tautomerism.At times the ester melts at 1420, again at 1720.(H. Fischer, Platz and Morgenroth (16) instudying synthetic coproporphyrin III observedmelting points of the first tautomer between 144and 1480 even after several recrystallizations.)The absorption spectrum in acetic and ether solu-

tion was as follows: I. 624.7-621.0; I weak623.6

at maximum 596.6; III 581.3 - 567.1; IV577.1

532.5 -524.3 505.8 -483.8529.5 ; V. 495S5 Order of in

tensity: V, IV, I, III, II. Superimposed withcoproporphyrin I in the comparison spectrometer

there was almost, if not entire identity of thespectra. (Questionable difference in position ofBand I.) It should be noted that the absorptionspectra of all four isomeric coproporphyrins havebeen shown to be identical (16).

Case 4. Lead poisoning. There were 4.1mgm. porphyrin ester after 3 recrystallizations.Crystallized slowly in the manner characteristicof coproporphyrin III (Figure 6). After an-other recrystallization the substance melted at1560, with marked shrinking above 1460. Aftera fifth recrystallization, the melting point was147 to 150°, and after a sixth, 144 to 1490. Asmall amount of the latter material was submittedto Prof. Hans Fischer in Munich for whose re-port the writer wishes to acknowledge his grati-tude. "The mixed melting point (with copro-porphyrin III ester) gave no depression. Thecrystal form of the concentrically united needlesis characteristic of coproporphyrin III."

The material exhibited the spectroscopicabsorption of a coproporphyrin, as follows:

626.7-622.6 I 583.5 - 567.5 .

624.8 577.0534.6- 525.3 507.1-489.3

531.3 ; IV. 496.9 Order ofintensity: IV, III, I, II. With the absorptionspectra superimposed in the usual way, completeidentity with coproporphyrin I was noted.

TABLE I

Summary of results

Num- AmountSourceof ~. ber of of ry MeltingSource of urine edays taioe Crystal type ointcollec- ester

tion obtained

M1M. a C.1. Normal .................. 33 0.13+ CoproporptyrinI 218to2232. Fever .................. 12 1.5 Coproporphyrin I 215 to 2183. Hemolytlo jaundice . 4 0.6 Coproporphyrn I 236to 2384. Cinoophen oirrhosi .* 8 1.2 Coproporphyrin 241to 2435. LIad poisoning 5 1.0 Coproporpyrin III 164to 1666. Lead poisoing . 6 1.4 Coproporphyrin fII 143to 1447. Lead poiBonng . 4 4.1 CoproporpHyrin III 141to 149

* This instance, described previously (1), is includedhere for purposes of comparison.

Case 5. Lead poisoning. There were 1.4 mgm.porphyrin ester after 3 recrystallizations. Crys-tallized slowly in small aggregations of long, nar-row, well defined prisms (Figure 7). Meltingpoint 143 to 144°. The following absorption

627.5 62.spectrum was observed: I. -623. ; II.625.3

332

CONCERNINGTHE NATURALLYOCCURRINGPORPHYRINS. IV

faint maximum 607.7; III. 583.0- 568.2; IV.577.6535.6- 525.4 507.7-485.5

530.9 ; V. 487.1 Order of in-tensity: V, IV, I, III, II. There was completeidentity of absorption spectra when superimposedwith coproporphyrin I in acetic and ether solution.

COMMENT

Although most of the porphyrin obtained fromthe normal urine was evidently coproporphyrin I,a very small fraction crystallized in the mannerof coproporphyrin III ester, and it is believedprobable that coproporphyrin III was actuallypresent in an extremely small proportion. In thisconnection the earlier observations of Schumm(18) and H. Fischer (16, 19) must be men-tioned. These' concerned the increase of urinarycoproporphyrin after ingestion of blood or bloodcontaining foods. It is en'tirely conceivable thatcoproporphyrin III might originate from proto-porphyrin (16) derived from hemoglobin in thebowel, and it is equally conceivable that the copro-porphyrin content of the urine might be increasedin this way (16). The normal individual whoseurine was used in the above investigation was notrestricted as to diet, and ate considerable meatthroughout the period of collection of the urine,so that small amounts of hemoglobin may havebeen supplied. The fact remains, however, thatthe great majority of the porphyrin isolated hadthe characteristics of coproporphyrin I, and co-proporphyrin III, if present, was in much smalleramount. It is quite possible that the meltingpoint of Fraction a, i.e., of the crystals of copro-porphyrin I type, was lowered because of thepresence of traces of coproporphyrin III. Thispossibility may also account for the lowering inother of the instances noted above where the crys-tal form was that of coproporphyrin I. H.Fischer, Platz, and Morgenroth (16) noted in-stances where natural coproporphyrin I estercould not be brought to a melting point of over240° C. even after repeated recrystallization; theyobserved that this was also true in the case of anartificial mixture of coproporphyrin I and copro-porphyrin III esters. In this connection it isbelieved significant that the melting points werehigher in the present instances (such as 3 and 4in the above table) where much smaller amounts

of urine were necessary for the isolation of thecoproporphyrins.

In instances 2, 3 and 4, above, all of the por-phyrin ester obtained had the crystal form char-acteristic of coproporphyrin I, and crystals of thecoproporphyrin III type were not obtained byfurther concentration of the mother liquors. Theopposite was true in instances 5, 6 and 7.

In this investigation no attempt to isolate auroporphyrin was made. This porphyrin is in-soluble in ether and according to H. Fischer (6)is formed secondarily from coproporphyrin.

It is noteworthy that in each of the above cases,2 to 4, urobilinogen was considerably increased inthe urine, this lending some support to the state-ment made previously (1) that coproporphyrin Imay increase in the urine because of diminishedhepatic excretory function.

The above findings reveal that coproporphyrinI is of rather frequent occurrence in the urine,while coproporphyrin III has so far been identi-fied with certainty only in lead poisoning and rareinstances of congenital porphyrinuria (1).' Thesignificance of the occurrence of 'coproporphyrinI in the urine is not yet clear; the possibility thatit is exogenous has already been mentioned, andsome evidence against this view was presented (1,2, 3). The question of the origin and signifi-cance of the coproporphyrin in the urine, bile andfeces will be discussed in more detail in a latercommunication.

SUMMARY

Coproporphyrin I has been isolated from theurine of a normal individual. It is probable thatrelatively very small amounts of coproporphyrinIII were likewise present. Coproporphyrin Iwas obtained in much larger amount from theurine of an individual with fever due to pul-monary suppuration, and from a patient withhemolytic jaundice during a postoperative " hemo-clastic " crisis. In both of the latter instancesconsiderable increases in the urine urobilinogen

4Hoerburger and Fink (20) have recently isolated acoproporphyrin from the urine of a patient treated withsalvarsan (no mention of jaundice). Because of the typeof crystal and the pH fluorescence curve they believedthis to be coproporphyrin III. The significance of thisfinding will not be clear until further studies of porphyrinexcretion following salvarsan treatment have been made.

333

CECIL JAMES WATSON

were observed simultaneously with the heightenedexcretion of coproporphyrin. A porphyrin wasisolated from the urine in three cases of leadpoisoning. The crystal form and melting pointof the ester indicated clearly that this was, in eachinstance, coproporphyrin III.

Since this communication was submitted forpublication, an important finding of Hoerburger's,mentioned in his inaugural dissertation (21), hascome to the writer's attention. Hoerburger iso-lated a crystalline coproporphyrin from normalurine; the crystal form and pH fluorescence curve(22) were characteristic of coproporphyrin I.The amount obtained was presumably too smallfor determination of the ester melting point.

An extensive study of the urinary porphyrinshas recently been reported by Dobriner (25).Coproporphyrin I was obtained in a variety ofdiseases, including hemolytic jaundice and pul-monary infection; this finding is confirmed in thepresent report. Dobriner found coproporphyrinI in the urine in a number of instances of liverdisease, which is in keeping with the writer's pre-vious report concerning cincophen cirrhosis. Inthis connection it is of considerable interest thatDobriner found coproporphyrin III in one in-stance of atrophic cirrhosis, and in one of pigmentcirrhosis.

BIBLIOGRAPHY

1. W-atson, C. J., Concerning the naturally occurringporphyrins. I. The isolation of coproporphyrin Ifrom the urine in a case of cincophen cirrhosis. J.Clin. Invest., 1935, 14, 106.

2. Watson, C. J., Idem, II. The isolation of a hithertoundescribed porphyrin occurring with an increasedamount of coproporphyrin I in the feces of a caseof familial hemolytic jaundice. J. Clin. Invest.,1935, 14, 110.

3. Watson, C. J., Idem, III. The isolation of copropor-phyrin I from the feces of untreated cases of per-nicious anemia. J. Clin. Invest., 1935, 14, 116.

4. Van den Bergh, A. A. H., and Hyman, A. J., Studienuber Porphyrin. Deutsche med. Wchnschr., 1928,54, 1492.

5. Van den Bergh, A. A. H., Grotepass, W., and Revers,F. E., Beitrag uber das Porphyrin in Blut undGalle. Klin. Wchnschr., 1932, 11, 1534.

6. Fischer, H., Farbstoffe mit Pyrrolkernen. A. UeberBlut, Blatt und Gallenfarbstoffe. Oppenheimer'sHandb. der Biochem, des Menschen u. der Tiere.Zweite Auflage. Erganzungsband. G. Fischer,Jena, 1930, p. 72.

7. Schreus, H. T., Ergebnisse und Probleme der Por-phyrinforschung. Klin. Wchnschr., 1934, 13, 121.

8. Garrod, A. E., The urinary pigments in their patho-logical aspects. Lancet, 1900, 2, 1323.

9. Grotepass, W., Zur Kenntnis des im Harn auftre-tenden Porphyrins bei Bleivergiftung. Ztschr. f.physiol. Chem., 1932, 205, 193.

10. Fischer, H., and D-uesberg, R., tber Porphyrine beiklinischer und experimenteller Porphyrie. Arch. f.exper. Path. u. Pharmakol., 1932, 166, 95.

11. Fischer, H., and Zerweck, W., Zur Kenntnis dernatiirlichen Porphyrine. V. Uber Koproporphyrinim Ham und Serum unter normalen und patho-logischen Bedingungen. Ztschr. f. physiol. Chem.,1924, 132, 12.

12. Godfried, E. G., Clinical tests for bilirubin in urine.Biochem. J., 1934, 28, 2056.

13. Jenke, M., Vber die Natur des indirekten Bilirubins(Hamorubin). Verhandl. d. deutsch. Gesellsch. f.inn. Med., 1931, 43, 387.

14. Fischer, H., Hilmer, H., Lindner, F., and Piutzer, B.,Zur Kenntnis der naturlichen Porphyrine. XVIII.Chemische Befunde bei einem Fall von Porphyrin-urie (Petry). Ztschr. f. physiol. Chem., 1925, 150,44.

15. Kirstahler, A., Blutfarbstoff und Derivate. TabulaeBiologicae Periodicae, 1931, Bd. VII, 48.

16. Fischer, H., Platz, K., and Morgenroth, K., Synthesevon Koproporphyrin III und IV, ein Beitrag zurKenntnis der Porphyrie. Ztschr. f. physiol. Chem.,1929, 182, 265.

17. Boas, I., tJber das Vorkommen von Protoporphyrinim Ham. Klin. Wchnschr., 1933, 12, 589.

18. Schumm, O., tYber die natiirlichen Porphyrine.Ztschr. f. physiol. Chem., 1923, 126, 169.

19. Fischer, H., Farbstoffe mit Pyrrolkernen. Oppen-heimer's Handb. der Biochem, des Menschen u. d.Tiere, Zweite Auflage, Band I, Baustoffe der tier-ischen Substanz. G. Fischer, Jena, 1924, p. 351.

20. Hoerburger, W., and Fink, H., Ueber Porphyrine beiklinischer Porphyrie. Ztschr. f. physiol. Chem.,1935, 236, 136.

21. Hoerburger, W., Zur Kenntnis der Porphyrinfluor-eszenz und deren Anwendung bei physiologischenUntersuchungen. Inaug. Diss. Erlangen, 1933.

22. Fink, H., and Hoerburger, W., Isolierung von kris-tallisiertem Koproporphyrin I aus normalenmenschlichem Urin. Die Naturwissenschaften,1934, 18, 292.

23. Watson, C. J., Studies of urobilinogen. II. Uro-bilinogen in the urine and feces of individuals with-out evidence of disease of the liver or biliary tract.III. Urobilinogen excretion in the common formsof jaundice and liver disease. Arch. Int. Med. (Inpress).

24. Watson, C. J., The origin of natural crystalline uro-bilin (stercobilin). J. Biol. Chem. (In press).

25. Dobriner, K., Urinary porphyrins in disease. J. Biol.Chem., 1936, 113, 1.

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