CONCERNING THE NATURALLY OCCURRING . IV. THE URINARY IN LEAD POISONING AS CON- TRASTED WITH THAT EXCRETED NORMALLY AND IN OTHER DISEASES' By CECIL JAMES WATSON (From the Department of Medicine, University of Minnesota Hospital, Minneapolis) (Received for publication January 22, 1936) In previous communications (1, 2, 3), the lit- The concept that the coproporphyrin of the erature relating to H. Fischer's "dualism'" of urine and is derived from hemoglobin was the natural porphyrins was discussed. The isola- not supported by the findings described in Parts I tion of coproporphyrin I from the urine of a pa- to III of this investigation (1, 2, 3). The occur- tient with cincophen cirrhosis of the , also rence of coproporphyrin I rather than III consti- from the feces in hemolytic and perni- tuted evidence that at least in the instances stud- cious anemia, was reported. Of particular in- ied, porphyrin formation was a process distinctly terest was the consideration of these findings with separate from the synthesis or destruction of relation to the clinical and experimental observa- hemoglobin. The isolation of coproporphyrin III tions of Van den Bergh and coworkers (4, 5). from the urine in lead poisoning was described by These investigators pointed out that human eryth- Grotepass (9), later by H. Fischer and Duesberg rocytes regularly contain protoporphyrin which is (10). Since ordinary hemin and coproporphyrin increased in amount in the same individuals who III both correspond in configuration to aetiopor- exhibit increases of coproporphyrin in the urine. phyrin III, the elaboration of coproporphyrin III By perfusion experiments they further demon- in lead poisoning could be associated quite readily strated that the surviving liver of the dog is able a disturbance in hemoglobin metabo- to convert protoporphyrin into coproporphyrin. with peculiar Correlation.of these studies made it appear likely lism. that the coproporphyrin of the urine, as well as of The function of the porphyrins present in the the bile, may originate from the protoporphyrin erythrocytes, urine, bile and feces is quite un- of the erythrocytes. Since hemoglobin, following known and can hardly be determined until exact loss of its protein and iron, yields a protoporphy- knowledge of their origin and isomeric type is rin of the aetioporphyrin III type (6), it was obtained. The amount of porphyrin in normal naturally assumed that the porphyrins of the urine urine is so small that it has not yet been isolated, and bile are directly related to hemoglobin metab- and the important question as to whether it is olism. Schreus (7) in particular has pointed to coproporphyrin I or III remains unanswered. Van den Bergh's results as supporting this con- The finding of coproporphyrin I in the urine in ception. The idea that the porphyrin of the urine an instance of advanced liver disease (1), sug- is derived from hemoglobin is by no means a re- gested that it was the normal urine porphyrin, in- cent one. In fact, a considerable literature deal- creased in amount for the same reason that uro- ing with this question was collected by Garrod in bilinogen was increased, i.e., diminished excretory 1900 (8). Although Garrod subscribed to the function of the liver. belief that the urinary porphyrin, then spoken of The purpose of the present investigation was to as hematoporphyrin, was related to hemoglobin isolate and identify the coproporphyrin of a nor- destruction, he observed that increases in amount mal urine, and of the urine in some of the patho- were frequently associated with clinical or ana- logical states accompanied by a heightened por- tomical evidence of liver disease. phyrin excretion; further, to determine whether coproporphyrin III is regularly excreted in the 1Aided by a grant from research funds of the Graduate urine in lead poisoning (as yet it has been isolated School of the University of Minnesota. but from one instance). 327 328 CECIL JAMES WATSON

MATERIAL AND METHODS Hemoglobin was 32 per cent (Sahli), erythrocytes 1,150,-' 000 per cu. mm. Stained smears revealed marked macro- The entire amount of urine for a period of 33 anisocytosis, polychromatophilia. Reticulocytes varied days was collected from a healthy male student 2 between 15 and 32 per cent. Resistance of erythrocytes 18 years of age. As soon as 2 to 3 liters had to hypotonic saline was as follows: H1 .38, H, .32, control accumulated the specimen was subjected to the H, .38, H2 .30. procedure previously described (1), with the fol- There was marked autoagglutination of erythrocytes in vitro (below 300 C.), the icterus index was 30, and the lowing exceptions. In each instance the primary Van den Bergh reaction was of the indirect type. Feces ether extract after washing with water was re- : 1106 mgm. per day. (Normal range 100 peatedly extracted with small amounts of 10 per to 250.) Urine urobilinogen 2.5 mgm. per day. (Nor- cent HCl. The 10 per cent HC1 solutions were mal range 0.5 to 3.0.) (23) united and kept in the refrigerator during the Physical examination revealed nothing except a smooth cystic mass in the left side of the pelvis. The spleen and period of further collection and primary extrac- liver were not palpable. It was considered possible that tion of the urine. All of the hydrochloric acid chronic hemorrhage into an ovarian cyst with local for- extract was then covered with a large amount of mation of might be responsible for the findings ether, an excess of sodium acetate was added, and described above. Consequently the cyst was removed at the mixture at once vigorously shaken. Having operation. It contained 800 cc. of a dark brown fluid rich in hematin (identified by hemochromogen spectrum made certain that the aqueous fraction was nega- after treatment with ammonium sulphide). Bilirubin and tive to Congo paper, ether extraction was re- porphyrins were not identified, although searched for. peated until the porphyrins were removed com- There was no improvement following this operation. pletely. The further treatment of this solution On the contrary, icterus and anemia increased, and there of ether was the same as described previously was moderate fever. The urobilinogen in the urine in- (1). The final coproporphyrin solution in ether creased markedly for a few days after operation, then decreased rapidly again. The highest value noted was was not dried over sodium sulphate as was done 380 mgm. per day. During this period the urine was previously, since it has been found to be true that examined for porphyrins and a considerable increase of a moderate loss of porphyrin occurs by adsorption coproporphyrin was observed. on this salt. This was first noted by H. Fischer After an interval of three weeks during which the pa- and Zerweck (11). The ether was simply de- tient's condition remained precariously stationary, sple- nectomy was carried out. The spleen weighed but 460 canted into a dry flask and was then removed com- grams, exhibiting microscopically a marked pulp conges- pletely upon the water bath. The porphyrin resi- tion. The liver appeared to be entirely normal. This due after drying in the air was esterified with operation was followed by prompt improvement. Two HCl in methyl alcohol, and the ester was crystal- months after operation the hemoglobin was 70 per cent lized from chloroform-methyl alcohol in -the usual (Sahli), the icterus index 10, and the feces urobilinogen 206 mgm. per day. Eight months later the patient re- way (1). ported that she felt entirely well and that there was no Employing the samne method, porphyrin esters jaundice. were isolated from the urines of the following The urine employed for the isolation of coproporphyrin cases. consisted of the entire amount for a period of four days immediately following the first operation (coincident with Case 1. Fever. Male, 37 years of age with lung the highest urobilinogen excretion). This urine was first abscess and empyema. The daily elevation of tempera- employed for the isolation of crystalline urobilinogen, a ture ranged from 101 to 1030 F. Urobilinuria was 8.3 procedure which will be described elsewhere (24). Suf- mgm. urobilinogen per day (23). Therewasno jaundice. fice it to say that this entailed a preliminary extraction Moderate anemia was present, and hemoglobin was 55 per with petroleum ether in which coproporphyrin is insolu- cent (Sahli method, 17 grams per 100 cc. equivalent to ble. Following this, the coproporphyrin was extracted 100 per cent). Urine was collected for a period of 12 with ether in the manner already referred to (1). It is days. worthy of note that this urine contained another pigment resembling bilirubin, but differing from it in that it failed to a test as well as the more delicate reaction Case 2. Acquired hemolytic jaundice. Female, 18 give Gmelin years of age with slight icterus, without anemia, of 3 for bilirubin devised by Harrison, and described by God- years' duration, increasing icterus and anemia, 4 months. fried (12). Also differing from bilirubin the substance could not be extracted with chloroform from the urine. 2 The writer is indebted to Mr. Samuel Schwartz for However, it gave a delayed and an indirect Van den the collection and preliminary extraction of this urine. Bergh reaction, and was precipitated by a barium chlo- CONCERNING THE NATURALLY OCCURRING PORPHYRINS. IV 329 ride solutioni. In dilute NaOH ill defined absorption recalilbrated, these are believed attributable to between 536 to 477 mny was observed. Attempts to crys- very sliglht changes in the relation of the wave tallize the substance were unsuccessful, probably because length scale of the to its of its obvious lability, and it cani only be stated that its instrtumiient grating. Of characteristics as nioted above appeared to be those of the chief importance is the identificationi 1y miieanis of " himorubin " of Jenike (13), which, it is noteworthy, superimposition of spectra, employing a pure was likewise obtainied from the uriine of an individual with coproporphyrin to compare with that un(ler in- hemiiolytic jaunldice. vestigation. With the apparatus use(l (1), this permlits detection of the slightest (lifferenices in Case 3. Lead poisoning. Male, 67 years of age. 'Mild of anemia, hemoglobini 69 per cent. Considerable basophilic positionl absorption band(ls. stippling of ervthrocytes. Urine collection for a perio(d of 5 days. RESULTS (SEE TABLE I)

Case 4. Lead poisoning. Male, 46 years of age. Colic, ANrorimial irile aniemia, hemoglobin 66 per cent, marked basophilic stip- After four recrvstallizations 0.13 Ilmgiml. of pling of erytlhrocytes. Urine collection for a period of 4 days. porphyrin ester were obtained fromn 33 (lays nor- mal urine. This will be spoken of as Fraction a. Case 5. Lead poisoning. Male, 38 years of age. The ester composing this fraction crystallized rap- Weakniess of extensor muscles, mild anemia, hemoglobini idly in the form of fluffy imiasses consisting of 67 per cenit, conisiderable basophilic stippling of the fine curving and branclhing needles. This mlaln- erythrocytes. Urine collection over a period of 6 days. ner of crystallization is vTery characteristic of coproporphyrin I ester wlhile coproporphyrin III Cases 3, 4 and 5 were members of a large group ester crystallizes muclh mlore slowly in smiialler of foundry emiployees suffering from lead poison- clumiips of crystals often adhering to the walls of ing.3 The bearings in this foundry contained lead the tube. These have the form of nee(dle-like and were believed responsible for the numerous prisms whiicl are usually broader than those of instances of lead poisoning which were encoun- coproporphyrin I, and do( not show the curving tered. and branching of the latter. The crystals of The total amuount of urine collected in each of coproporphyrin III ester rather frequently ar- the above instances was subjected to the method range themselves in concentrically uniited grotups, of isolation already referred to. It is imlportant or rosettes (10, 14, 15. 16). During crystalliza- to note that the fractionation of porphyrins em- tion and first recrystallization of Fraction a, it bodied in this method depends upon character- was observed that the miotlher liquior obviously istic solubilities (1, 2). Consequently, the copro- contained more porphyrin thani is usually true isolated porphyrins and described in the following when coproporphyrin I ester is crystallized from in instance the paragraphs possessed each typical chloroformii methyl alcohol. These two miotlher solubilities of a coproporphyrin, particularly in liquors were combined and allowed to not leaving 0.2 per cent HCI for chloroform. concentrate The sodiumn salt in each instance was entirely sol- slowly at roomii temperature. After stan(ling sev- uble in 10 per cent NaOH, a further character- eral days a further se)aration of crystals ap- istic of coproporphyrins. peared. These were in the formii of smnall aggre- Spectroscopic stu(lies were carried out by miieans gates part of which adlhered to the walls of the of apparatus ancd miiethods dlescribed previously smiiall test tube in wlhich the solution was conl- (1, 2). Sliglht variations in the miiaximiia for the taine(l. This material will be sI)oken of as Frac- absorption bands of the coproporphyrins isolated tion b; its amiiount was evidently considerably less will be notel, if comiipared with those observed thani that of Fraction a, nevertheless it was pos- hefore (1, 2, 3). Since the spectrometer was not sible to recrystallize it twice in or(ler to further observe its miianner of crystallization; the amiiount The writer is inidebted to Dr. E. C. Emerson of St. did not suffice for a miielting point (letermiiniationi. Paul, Minnesota, for the opportunlity to inivestigate the The characteristics of these fractionis were as fol- urinie of these cases. lows. ...4

.ws .. X..i ..., R

13 .: ,.),(5Ch xioo~~~~~~~~~J

I

5r 5b X 75C0r K/50

6 ~~~~7 X28_0 X300m FIG. 1. COPROPORPHYRIN I ESTER FROM NORMAL URINE. FIG. 2. COPROPORPHYRIN ESTER FROM N'ORMAL URINE, CRYS- TAL FORM SUGGESTIVE OF COPROPORPHYRIN III. FIG. 3. COPROPORPHYRIN I ESTER FROM URINE OF PATIENT WITH FEVER DUE TO LUNG ABSCESS AND EMPYEMA. FIG. 4. COPROPORPHYRIN I ESTER FROM URINE OF PATIENT WITH ACQUIRED HEMOLYTIC JAIJNDICE. FIG. 5. COPROPORPHYRIN III ESTER FROM URINE OF FIRST CASE OF LEAD POISONING. FIG. 6. COPROPORPHYRIN III ESTER FROM URINE OF SECOND CASE OF LEAD POISONING. FIG. 7. COPROPORPHYRIN III ESTER FROM URINE OF THIRD CASE OF LEAD POISONING. CONCERNING THE NATURALLY OCCURRING PORPHYRINS. IV 331 Fraction a. The crystals had the form of long Pathological urines curving needles (Figure 1). On recrystallization Case 1. Fever due to lung abscess and empy- these appeared rather quickly in the light fluffy ema. After three recrystallizations 1.5 mgm. of clumps characteristic of coproporphyrin I ester. porphyrin ester were obtained. This crystallized After four recrystallizations the amount (0.13 rapidly in the manner of coproporphyrin I, and mgm.) sufficed for one melting point determina- the individual crystals were fine curving needles tion. The substance did not melt sharply; shrink- (Figure 3). The melting point was 204 to 2080 ing was observed above 1900, melting from 218 C. The remaining substance was recrystallized to 2230. (Coproporphyrin I methyl ester, for twice more, following which the melting point which the writer is indebted to Professor H. was 215 to 218° C. All that remained was now Fischer in Munich, melted at 245 to 2460 C.) mixed with coproporphyrin I methyl ester of No change whatever was exhibited at the lower melting point 245 to 2460 C., in an approximate temperatures critical for coproporphyrin III ester ratio of one part of the former to four of the lat- (1420, 1720). The absorption spectrum in acetic ter. The melting point of this mixture was 240 to 2430 C. From this it is evident that while the and ether solution was: I. 624.86 ; IL porphyrin isolated was still slightly impure, it 582.7- melted far above the melting point of copropor- faint maximum 597.5; III 577.4568.3 ; IV. phyrin III ester, and did not depress the melting 533.5 -526.0 507.0- 487.0 point of known coproporphyrin I ester suffi- 530.0 ; V. 496.6 * Order of in- ciently to suggest that it was another porphyrin. tensity: V, IV, I, III, II. This was identical The absorption spectrum was that of a copropor- 626.7- 622.2 583.3 -567.6 with the superimposed absorption spectrum of phyri, i.e., I. ,; IL known coproporphyrin I in similar solution. 624.8 577.8 534.9-525.5 506.4-490.3 Fraction b. The crystals of this fraction ap- II. IV. Order peared very slowly in the manner already re- 530.9 497.5 ferred to. The majority of the crystals were of intensity: IV, III, I, II. This absorption was long narrow prisms similar to those of copropor- identical with that of coproporphyrin I when the phyrin III ester; a few curved and branching spectra were superimposed in the comparison spectrometer. needles of the coproporphyrin I type were also During the isolation of the above coproporphy- noted. After two recrystallizations the appear- rin it was noted that another porphyrin remained ance of crystals was still delayed and not prompt in the ether and was not extracted by 1 per cent as is usual with even very small amounts of copro- HCI, but was removed quantitatively by 10 per porphyrin I ester. The crystals now appeared to cent HCI. This porphyrin was chloroform solu- be uniform in type, namely, needle-like prisms ble, and was readily removed from HCI solutions frequently in groups (Figure 2), strongly sug- by chloroform. The amount was too small to gestive of coproporphyrin III methyl ester. be obtained in a crystalline form; it was pos- Spectroscopically this material was a copropor- sible, however, to examine the substance spectro- phyrin, having the following absorption spec- scopically. The acetic and ether solution was trum: bluish pink and exhibited the following absorp- I. 627.1-9II6 faint maximum 634.7-625.6. 598.0-571.9 tion:tlon:I. ;I. 580.9 597.9; III. 581.8- 568.0 IV 534.0- 525.2 V 631.5 597.9;III.. 577.2 IV* 531.0 V 554.8 -530.5 514.8-492.0 ; IV 505 Order of intensity: V, IV, I, 536.6 502 . Order of in- -49687.7 tensity: IV, III, I, II. This absorption spectrum III, II. This was identical with the superim- is quite characteristic of protoporphyrin (15), posed spectrum of a similar solution of copro- and in fact complete identity with protoporphyrin porphyrin I. was observed when the spectra were superim- 332 CECIL JAMES WATSON posed. This confirms the report of Boas (17) there was almost, if not entire identity of the regarding the occasional finding of protoporphy- spectra. (Questionable difference in position of rin in the urine. Band I.) It should be noted that the absorption Case 2. Acquired hemolytic jaundice. The spectra of all four isomeric coproporphyrins have yield of porphyrin ester was 0.6 mgm. This been shown to be identical (16). crystallized rapidly in the fluffy masses of long Case 4. Lead poisoning. There were 4.1 curving needles characteristic of coproporphyrin mgm. porphyrin ester after 3 recrystallizations. I ester (Figure 4). The melting point after five Crystallized slowly in the manner characteristic recrystallizations was 235 to 2380 C. with con- of coproporphyrin III (Figure 6). After an- siderable shrinking above 2200. A mixture with other recrystallization the substance melted at approximately equal parts of coproporphyrin I. 1560, with marked shrinking above 1460. After ester (melting point 245 to 2460), also melted a fifth recrystallization, the melting point was at 236 to 2380 C., but with less preceding shrink- 147 to 150°, and after a sixth, 144 to 1490. A ing. small amount of the latter material was submitted The absorption spectrum of this porphyrin in to Prof. Hans Fischer in Munich for whose re- acetic and ether solution was as follows: I. port the writer wishes to acknowledge his grati- 62624-6227 faint maximum 596.1; tude. "The mixed melting point (with copro- IL. porphyrin III ester) gave no depression. The III. 581.2 -567.5 IV 535.2 524.9 V crystal form of the concentrically united needles 577.5 531.3 is characteristic of coproporphyrin III." 506.8-486.2 Order of The material exhibited the spectroscopic 496.3 . intensity: V, IV, I, absorption of a coproporphyrin, as follows: was III, II. This identical with the superim- 626.7-622.6 I 583.5 - 567.5 . posed absorption spectrum of coproporphyrin I 624.8 577.0 in a similar solution. 534.6- 525.3 507.1-489.3 Case 3. Lead poisoning. One milligram por- 531.3 ; IV. 496.9 Order of phyrin ester after 3 recrystallizations. This crys- intensity: IV, III, I, II. With the absorption tallized slowly in the characteristic manner of spectra superimposed in the usual way, complete coproporphyrin III (Figures Sa and 5b). The identity with coproporphyrin I was noted. melting point was 161 to 1640 C. The remaining material was recrystallized once after which the TABLE I melting point was 164 to 1660. The amount of Summary of results substance did not suffice for another recrystalliza- Num- Amount that copro- Sourceof ~. ber of ry Melting tion. H. Fischer (6, 16) observed Source of urine oftaioe Crystal type collec-edays ester oint porphyrin III methyl ester exhibits a double melt- tion obtained upon tautomerism. ing point probably dependent M1M. a C. At times the ester melts at 1420, again at 1720. 1. Normal...... 33 0.13+ CoproporptyrinI 218to223 2. Fever ...... 12 1.5 Coproporphyrin I 215 to 218 (H. Fischer, Platz and Morgenroth (16) in 3. Hemolytlo jaundice. 4 0.6 Coproporphyrn I 236to 238 4. Cinoophen oirrhosi *. 8 1.2 Coproporphyrin 241to 243 studying synthetic coproporphyrin III observed 5. LIad poisoning 5 1.0 Coproporpyrin III 164to 166 6. Lead poisoing . 6 1.4 Coproporphyrin fII 143to 144 melting points of the first tautomer between 144 7. Lead poiBonng . 4 4.1 CoproporpHyrin III 141to 149 and 1480 even after several recrystallizations.) The absorption spectrum in acetic and ether solu- * This instance, described previously (1), is included here for purposes of comparison. tion was as follows: I. 624.7-621.0; I weak 623.6 Case 5. Lead poisoning. There were 1.4 mgm.

581.3 - ester after 3 at maximum 596.6; III 567.1; IV porphyrin recrystallizations. Crys- 577.1 tallized slowly in small aggregations of long, nar- 532.5 -524.3 505.8 -483.8 row, well defined prisms (Figure 7). Melting 529.5 ; V. 495S5 Order of in point 143 to 144°. The following absorption tensity: V, IV, I, III, II. Superimposed with 627.5 62. coproporphyrin I in the comparison spectrometer spectrum was observed: I. 625.3-623. ; II. CONCERNING THE NATURALLY OCCURRING PORPHYRINS. IV 333 faint maximum 607.7; III. 583.0- IV. of urine were necessary for the isolation of the 577.6568.2; coproporphyrins. 535.6- 525.4 507.7-485.5 In instances 2, 3 and 4, above, all of the por- 530.9 ; V. 487.1 Order of in- phyrin ester obtained had the crystal form char- tensity: V, IV, I, III, II. There was complete acteristic of coproporphyrin I, and crystals of the identity of absorption spectra when superimposed coproporphyrin III type were not obtained by with coproporphyrin I in acetic and ether solution. further concentration of the mother liquors. The opposite was true in instances 5, 6 and 7. COMMENT In this investigation no attempt to isolate a Although most of the porphyrin obtained from uroporphyrin was made. This porphyrin is in- the normal urine was evidently coproporphyrin I, soluble in ether and according to H. Fischer (6) a very small fraction crystallized in the manner is formed secondarily from coproporphyrin. of coproporphyrin III ester, and it is believed It is noteworthy that in each of the above cases, probable that coproporphyrin III was actually 2 to 4, urobilinogen was considerably increased in present in an extremely small proportion. In this the urine, this lending some support to the state- connection the earlier observations of Schumm ment made previously (1) that coproporphyrin I (18) and H. Fischer (16, 19) must be men- may increase in the urine because of diminished tioned. These' concerned the increase of urinary hepatic excretory function. coproporphyrin after ingestion of blood or blood The above findings reveal that coproporphyrin containing foods. It is en'tirely conceivable that I is of rather frequent occurrence in the urine, coproporphyrin III might originate from proto- while coproporphyrin III has so far been identi- porphyrin (16) derived from hemoglobin in the fied with certainty only in lead poisoning and rare bowel, and it is equally conceivable that the copro- instances of congenital porphyrinuria (1).' The porphyrin content of the urine might be increased significance of the occurrence of 'coproporphyrin in this way (16). The normal individual whose I in the urine is not yet clear; the possibility that urine was used in the above investigation was not it is exogenous has already been mentioned, and restricted as to diet, and ate considerable meat some evidence against this view was presented (1, throughout the period of collection of the urine, 2, 3). The question of the origin and signifi- so that small amounts of hemoglobin may have cance of the coproporphyrin in the urine, bile and been supplied. The fact remains, however, that feces will be discussed in more detail in a later the great majority of the porphyrin isolated had communication. the characteristics of coproporphyrin I, and co- proporphyrin III, if present, was in much smaller SUMMARY amount. It is quite possible that the melting Coproporphyrin I has been isolated from the point of Fraction a, i.e., of the crystals of copro- urine of a normal individual. It is probable that porphyrin I type, was lowered because of the relatively very small amounts of coproporphyrin presence of traces of coproporphyrin III. This III were likewise present. Coproporphyrin I possibility may also account for the lowering in was obtained in much larger amount from the other of the instances noted above where the crys- urine of an individual with fever due to pul- tal form was that of coproporphyrin I. H. monary suppuration, and from a patient with Fischer, Platz, and Morgenroth (16) noted in- hemolytic jaundice during a postoperative " hemo- stances where natural coproporphyrin I ester clastic " crisis. In both of the latter instances could not be brought to a melting point of over considerable increases in the urine urobilinogen 240° C. even after repeated recrystallization; they observed that this was also true in the case of an 4Hoerburger and Fink (20) have recently isolated a artificial mixture of coproporphyrin I and copro- coproporphyrin from the urine of a patient treated with porphyrin III esters. In this connection it is salvarsan (no mention of jaundice). Because of the type of crystal and the pH fluorescence curve they believed believed significant that the melting points were this to be coproporphyrin III. The significance of this higher in the present instances (such as 3 and 4 finding will not be clear until further studies of porphyrin in the above table) where much smaller amounts excretion following salvarsan treatment have been made. 334 CECIL JAMES WATSON were observed simultaneously with the heightened 7. Schreus, H. T., Ergebnisse und Probleme der Por- excretion of coproporphyrin. A porphyrin was phyrinforschung. Klin. Wchnschr., 1934, 13, 121. isolated from the urine in three cases of lead 8. Garrod, A. E., The urinary pigments in their patho- logical aspects. Lancet, 1900, 2, 1323. poisoning. The crystal form and melting point 9. Grotepass, W., Zur Kenntnis des im Harn auftre- of the ester indicated clearly that this was, in each tenden Porphyrins bei Bleivergiftung. Ztschr. f. instance, coproporphyrin III. physiol. Chem., 1932, 205, 193. 10. Fischer, H., and D-uesberg, R., tber Porphyrine bei Since this communication was submitted for klinischer und experimenteller Porphyrie. Arch. f. exper. Path. u. Pharmakol., 1932, 166, 95. publication, an important finding of Hoerburger's, 11. Fischer, H., and Zerweck, W., Zur Kenntnis der mentioned in his inaugural dissertation (21), has natiirlichen Porphyrine. V. Uber Koproporphyrin come to the writer's attention. Hoerburger iso- im Ham und Serum unter normalen und patho- lated a crystalline coproporphyrin from normal logischen Bedingungen. Ztschr. f. physiol. Chem., urine; the crystal form and pH fluorescence curve 1924, 132, 12. (22) were characteristic of coproporphyrin I. 12. Godfried, E. G., Clinical tests for bilirubin in urine. Biochem. J., 1934, 28, 2056. The amount obtained was presumably too small 13. Jenke, M., Vber die Natur des indirekten Bilirubins for determination of the ester melting point. (Hamorubin). Verhandl. d. deutsch. Gesellsch. f. An extensive study of the urinary porphyrins inn. Med., 1931, 43, 387. has recently been reported by Dobriner (25). 14. Fischer, H., Hilmer, H., Lindner, F., and Piutzer, B., Coproporphyrin I was obtained in a variety of Zur Kenntnis der naturlichen Porphyrine. XVIII. Chemische Befunde bei einem Fall von Porphyrin- diseases, including hemolytic jaundice and pul- urie (Petry). Ztschr. f. physiol. Chem., 1925, 150, monary infection; this finding is confirmed in the 44. present report. Dobriner found coproporphyrin 15. Kirstahler, A., Blutfarbstoff und Derivate. Tabulae I in the urine in a number of instances of liver Biologicae Periodicae, 1931, Bd. VII, 48. disease, which is in keeping with the writer's pre- 16. Fischer, H., Platz, K., and Morgenroth, K., Synthese von Koproporphyrin III und IV, ein Beitrag zur vious report concerning cincophen cirrhosis. In Kenntnis der Porphyrie. Ztschr. f. physiol. Chem., this connection it is of considerable interest that 1929, 182, 265. Dobriner found coproporphyrin III in one in- 17. Boas, I., tJber das Vorkommen von Protoporphyrin stance of atrophic cirrhosis, and in one of pigment im Ham. Klin. Wchnschr., 1933, 12, 589. cirrhosis. 18. Schumm, O., tYber die natiirlichen Porphyrine. Ztschr. f. physiol. Chem., 1923, 126, 169. BIBLIOGRAPHY 19. Fischer, H., Farbstoffe mit Pyrrolkernen. Oppen- 1. W-atson, C. J., Concerning the naturally occurring heimer's Handb. der Biochem, des Menschen u. d. porphyrins. I. The isolation of coproporphyrin I Tiere, Zweite Auflage, Band I, Baustoffe der tier- from the urine in a case of cincophen cirrhosis. J. ischen Substanz. G. Fischer, Jena, 1924, p. 351. Clin. Invest., 1935, 14, 106. 20. Hoerburger, W., and Fink, H., Ueber Porphyrine bei 2. Watson, C. J., Idem, II. The isolation of a hitherto klinischer Porphyrie. Ztschr. f. physiol. Chem., undescribed porphyrin occurring with an increased 1935, 236, 136. amount of coproporphyrin I in the feces of a case 21. Hoerburger, W., Zur Kenntnis der Porphyrinfluor- of familial hemolytic jaundice. J. Clin. Invest., eszenz und deren Anwendung bei physiologischen 1935, 14, 110. Untersuchungen. Inaug. Diss. Erlangen, 1933. 3. Watson, C. J., Idem, III. The isolation of copropor- 22. 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