Studies of Coproporphyrin. Ii. an Investigation of The

Home , Urobilin

STUDIES OF COPROPORPHYRIN. II. AN INVESTIGATION OF THE CONTRIBUTION TO THE URINARY COPROPORPHYRIN OF HEMOGLOBIN AND OF BACTERIAL METABOLISM IN THE INTESTINAL TRACT 1, 2 By EVREL A. LARSON AND CECIL JAMES WATSON (From the Department of Medicine, University of Minnesota Hospital, Minneapolis) (Received for publication July 9, 1948) Stokvis (1) advanced the idea that blood in had previously shown that hemoglobin is converted the intestinal tract was the source of the urinary in small part to protoporphyrin in the colon, and porphyrin. Schumm (2) also held this belief. Sub- Kammerer (16) ascribed its formation to a sym- sequent studies, notably those of Fischer and Hil- biotic activity of the bacterial flora. Hill and mer (3), Brugsch (4, 5), and Kaunitz (6) indi- Holden (17) showed, however, that pure cul- cated that a variable fraction of the urinary copro- tures of E. coli liberated free protoporphyrin from porphyrin was thus derived. The presence of a hemoglobin; this was confirmed by van den Bergh porphyrin in meconium (2, 7, 8), later identified and co-workers (18). No direct evidence has by Waldenstr6m as coproporphyrin I (9), to- been described, however, for the intestinal forma- gether with a number of other evidences of an en- tion of coproporphyrin III from hemoglobin, al- dogenous formation, which are discussed in de- though the above-mentioned work of Jakob's sug- tail in recent reviews- (10, 11), have made it rea- gested that this might take place. In a previous sonably clear that at most not more than a certain study (19), it was shown that in the presence of fraction of the, urinary coproporphyrin (UCP) complete biliary obstruction due to cancer, copro- could be regarded as originating in the intestinal porphyrin could not be demonstrated in the acholic tract. The purpose of the present investigation feces even when there was bleeding into the upper was to obtain more definite information as to the gastrointestinal tract. This observation does not size of this fraction, under various experimental favor an origin from hemoglobin in the bowel, but and clinical conditions. It was also determined the possibility could not be excluded that copro- to study the relative percentage of coproporphyrin porphyrin thus formed had been entirely reab- isomers composing any increases which might be sorbed. observed, since previous studies bearing on these (2) A formation of coproporphyrin, either I questions had dealt only with the total copro- or III, as an independent bacterial synthesis in porphyrin. It was conceivable on a priori reason- the absence of hemoglobin. Fischer and co-work- ing, that any one or a combination of the following ers (3, 20) demonstrated that yeast cells under- might alter the total coproporphyrin concentration going autolysis, synthesize coproporphyrin I. or the isomer distribution in the urine: Kench and Wilkinson (21) have recently shown (1) A formation of coproporphyrin III from that yeast cells elaborate both isomers, the pre- hemoglobin, by the bacterial flora of the colon, ponderance depending on the conditions of the with reabsorption and excretion in the urine. culture. Coulter and Stone (22) found that diph- showed that pure cultures of E. coli theria bacilli in broth cultures form coproporphyrin Jakob (12) in an amount roughly proportional to that of the produce small amounts of coproporphyrin III in Snapper toxin produced. Gray and Holt (23) have re- the presence of hemoglobin. (13-15) cently demonstrated that this is the type III iso- 'Aided by grants from the John and Mary R. Markle mer and that it is formed in relatively large amount Foundation, New York City, and the Medical Research under suitable conditions of growth. All of these Fund of the Graduate School, University of Minnesota, observations suggested that the bacterial flora of Minneapolis. 2A portion of this study was included in a thesis sub- the human colon might, at least under certain cir- mitted (by E. A. L.) in partial fulfillment of the require- cumstances, produce either coproporphyrin I or ments for the Ph.D. degree, University of Minnesota. III, and, if it were absorbed, might thus bring 452 STUDIES OF COPROPORPHYRIN. II. 453 about an increased urinary excretion of the corre- the increases which he noted were composed of sponding isomer. von Mallinkrodt-Haupt (24) has, type III isomer, derived from the fed hemoglobin. in fact, postulated that the urinary coproporphyrin Kaunitz (6) and Boas (25, 26) described increases is wholly related to the bacterial flora in the colon, of urinary coproporphyrin following gastrointes- and that the increases described in pernicious ane- tinal bleeding due to peptic ulcer or malignancy. mia are related to the effect of achylia in altering This increase was likewise assumed to be composed the flora. It will be seen subsequently that the evi- of the type III isomer, although it was not iso- dence in favor of this theory is indeed very meager. lated. There is also the possibility that some de- (3) The further possibility had to be considered gree of liver functional impairment had occurred that liver function plays an important role with re- with resultant increase of type I rather than type spect to coproporphyrin I or III, which might orig- III, an association which has been discussed pre- inate in the intestinal tract whether from hemo- viously (11), and in favor of which there is abun- globin or bacterial metabolism. Kaunitz' study dant evidence in the literature (5, 10, 27-29). It (6) suggested that coproporphyrin is absorbed seems less probable, however, that this factor from the intestine but that it does not appear in the would be important after the simple ingestion of urine in appreciable amount unless there is as- blood by normal individuals, which was reported sociated liver functional impairment. Thus, after by Schumm (2), and Fischer (3) to be followed oral administration of 20 Gm. doses of crystal- by an increased urinary coproporphyrin of un- line hemoglobin, significant increases of urinary determined type. This was not confirmed by other coproporphyrin were observed only in cases of investigators (14, 15, 30, 31), nor were Borst and liver disease, or in instances where liver injury K6nigsd6rffer (32) able to detect the appearance was postulated as a result of severe anemia or of any porphyrin in the blood plasma after in- toxemia. Kaunitz assumed but did not prove that gestion of coproporphyrin.

Gamma/Day

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Copro 20 I 1. -I I

100 -

Proto so0 60 Feces Day Periods 60 Deu tecro so

20I -L Urobil inogen 15 Mgm/day I

4 8 12 16 20 Days FIG. 1. NORMAL EXCRETION OF PORPHYRIN IN DoGs Dog No. 1: Solid lines; diet-milk. Dog No. 2: Broken lines; diet-"kibbled" dog food and milk. 454 EVREL A. LARSON AND CECIL JAMES WATSON

MATERIAL AND METHODS The method used for determining coproporphyrin in the In view of the above considerations, the present study urine, both total and isomer distribution, was that of in Schwartz and co-workers (33). The method for the was planned as outlined the following: feces was that described by Watson and Layne (34). I. Studies on dogs. This is not an exact procedure but permits a rough A. Feeding of blood-two dogs. approximation of the three main types of porphyrins B. Feeding of raw meat-two dogs. found in the feces: 1) coproporphyrin, 2) protoporphyrin, II. Studies on human subjects. 3) deutero- and the pseudo-deuteroporphyrins (19). The A. Cases hating blood in the gastrointestinal tract animals used were shepherd dogs, weighing 20-30 pounds, due either to hemorrhage or to feeding of housed in standard metabolism cages which were so con- blood: structed that the urine could be collected quantitatively Hemorrhagic gastritis, one case; malnutrition, under a small amount of toluene preservative. During one case; hepatic gumma, one case; bacterial the control period, one dog was maintained on a strict endocarditis, one case; cholecystitis and chole- milk diet, productive of marked constipation, the other on lithiasis, three cases; infectious hepatitis (re- a diet of "kibbled dog food" and milk, with which the covery phase), one case; carcinoma of head of bowel movements were normal. The urine coproporphy- pancreas, one case; cirrhosis and chronic al- rin determination was carried out every two days. As coholism, two cases; idiopathic cirrhosis, one mentioned previously, coproporphyrin does not deteriorate case. significantly in this period of time. In the human experi- B. Feeding of crystalline coproporphyrins: ments determinations were made on 24 hour samples of 1) Type III isomer-3-5 mgm. orally. urine. The feces porphyrins in both dogs and humans Bacterial endocarditis, one case; cholecystitis were determined on four-day collections. Urobilinogen and cholelithiasis, one case; acute alcohol- was determined by Watson's modification (35) of Ter- ism, one case; active duodenal ulcer with- wens method (36). Porphyrin isolations and melting out hemorrhage, one case; cirrhosis and point determinations were carried out according to pre- chronic alcoholism, one case. viously described methods (19, 37, 38, 39). The total 2) Type I isomer-3 mgm. orally. heme pigments of the feces were determined by the Cirrhosis and chronic alcoholism, one case. method of Flink and Watson (40). The crystalline

Diet Diet Gamma/Day "Kibbled" Dog Food Mi lk and Milk 75 Copro 50 F Urine 25 Imimiru 2 Day Periods 100 - Copro 75 CHICI 50 25- -I 120 Proto 90 60 - 30 - t i i i -4 - feces 80- 4 Day Periods Deutero 60- 40- .. I 20 -

Hemochromogenf I _ _ 15 a --. Urobilinogen 10 - Mgm/day

4 8 12 20 4 8 12 Days FIG. 2. EFFECT OF DIETARY CHANGE UPON THE PORPHYRIN EXCRETION Dog No. 1. STUDIES OF COPROPORPHYRIN. II. 455

Gamma/Day 100 c c Beef Blood /day orally (12 days) 30 25 20 Copro Urine 151 Periods 10*' 2 Day

70 . I . 60 - 50 - Copro 40 30 20 10

200 175 150 Proto 125 Feces 4 Day Periods 100 75 50 25

Oeutero 20 10 -

Hemochromog en I _. _ _ + 4. 4. - - LJo FI-m Urobilinogen 15 - /A -. in- Mgm/darys-_ Lu ] i . .

4 8 12 16 20 24 28 32 36 Days FIG. 3. EFFECT OF INGESTED BLOOD UPON THE PORPHYRIN ExCRETION IN DOGS Dog No. 2; diet-"kibbled" dog food and milk. coproporphyrins were fed as the hydrochlorides in a "kibbled" dog food and milk, dog No. 1 showed a weakly acid solution. The diet was meat-free only in the significant alteration in porphyrin excretion, i.e., cases indicated in Table I. a decrease in urine and an increase in the feces. E. coli, Aerobacter aerogenes, Pseudomonas aeruginosa, and, in one instance, an inoculum of feces were utilized in The total amount excreted, however, remained studies of the in vitro synthesis of porphyrin by bacteria. approximately the same (Figure 2). This inter- The media employed were nutrient broth and tryptose esting change will be considered subsequently. broth, the same media with 5%0 citrated human blood The porphyrin excretion by dogs No. 1 and No. 2 added, and a synthetic medium of the Gladstone type. the raw, red meat The periods of growth are indicated in Table II. The following feeding of blood and of method of porphyrin extraction and separation in these is shown in Figures 3 and 4. No significant in- experiments was similar to that used for the feces, save creases in the UCP were observed after feeding for the preliminary acetic ether extract of the liquid blood (Figure 3). media. A spectrophotometric method was used in at- of feeding tempting to demonstrate heme pigments in plain broth Figure 4, which shows the effect (40). washed, raw, red meat to dog No. 2, is also be- lieved to be representative of experience gained in RESULTS another similar study. At first glance, it might Dog No. 1 (strict milk diet) excreted more appear that the dog excreted increased amounts urinary and less fecal coproporphyrin than dog of urinary porphyrin following addition of meat No. 2 ("kibbled" dog food and milk) (Figure 1). to the diet. However, this increase was indeed On being changed from a milk diet to a diet of slight and was maintained long after the meat was 456 EVREL A. LARSON AND CECIL JAMES WATSON

Gamma/Day 75 6m Washed Red Meat /day X 10 days 30 25 Copro 20' 15 Urine 10 2 Day Periods 5o **TOt* w w* .* v. . . 9 [ 105 90 Copro 75 60 45 30 15 FFTI ri 125 100 Proto 75 - Feces 4 Day Periods 50 25 I iz IU 15 Deutero 10

5 1~~~~~v-I I I rm~

Hemochrom ogen + +4 - _ Urobilinogen 10 Mgm/day 5 4 8 12 16 20 24 28 32 DaYs FIG. 4. EFFECT OF INGESTED RAW MEAT UPON THE PORPHYRIN EXCRETION IN DoGs Dog No. 2; diet-"kibbled" dog food and milk. discontinued; this may represent nothing more liquor following crystallization of the type I isomer, than a normal variation in excretion by the ani- but further crystallization could not be effected. mal, a finding repeatedly demonstrated during the Figures 5, 6, and 7 show the results of studies course of the dog experiments. Furthermore, the on several human subjects. following the feeding coproporphyrin methyl ester isolated from the of hemoglobin, coproporphyrin III, and in one in- urine during the control period and during meat stance, coproporphyrin I (Figure 5). Except feeding melted at 2460 C. Since this is the type I where otherwise indicated, the studies were con- isomer, it appears unlikely that any appreciable in- ducted in consecutive four-day periods. The crease had been derived from the heme pigments results of studies on a 59 year old patient having of the fed meat. The findings in the feces of the outspoken evidence of cirrhosis and severe hepatic two dogs following the feeding of both blood and functional impairment associated with chronic al- raw meat, of which Figures 3 and 4 are repre- coholism and dietary deficiency, are shown in sentative, were quite similar. Increases in all Figure 5. Although the average excretion of uri- three fractions were observed. The copropor- nary porphyrin is slightly greater after the feeding phyrin of the feces after meat feeding was identi- of hemoglobin, the occurrence of gross variations fied as the type I isomer in dog No. 2. The pres- during the control period and in previous untabu- ence of small amounts of coproporphyrin III was lated determinations, minimizes the significance of strongly suggested by a highly colored mother this increase. Moreover, the unexplained decrease STUDIES OF COPROPORPHYRIN. II. 457 in the next four-day period, following the feeding struction to bile flow. There was no increase in of 5 mgm. of coproporphyrin III, is probably a urinary porphyrin excretion following the feeding further coincidental variation in excretion. The of coproporphyrin III, most of which was ac- low values after 3 mgm. of coproporphyrin I were counted for in the feces. Further data on jaun- obtained during a later period, when the average diced subjects are shown in Figure 7: Case 12 urinary excretion was appreciably lower than that (cirrhosis), Case 13 (calculus obstruction), and observed early in the patient's hospital course. Case 14 (carcinomatous obstruction). The re- Of principal importance is the fact that during sults of additional case studies are listed in Table both feeding experiments, approximately all of the I. fed coproporphyrin appeared in the feces and there Case 2 (Table I) requires further explanation. was no increase in the urine. In one instance This individual had taken alcohol rather continu- after the feeding of coroporphyrin III, the same ously until the hour of admission in spite of epi- isomer was easily crystallized and identified from gastric pain and moderate hematemesis. The diet the feces. Figure 6 shows the results of studies had been deficient for a matter of months prior to on a 62 year old woman with a high-grade biliary this time. The results of the study appear to indi- obstruction due to an impacted common duct stone. cate that significantly larger amounts of copro- An increase in the urinary porphyrin excretion porphyrin were excreted in the urine while blood was observed on the third day following hemo- was present in the bowel. However, the "control" globin feeding, but during the corresponding four- study, following cessation of bleeding, was con- day period, there was a diminished fecal copro- ducted eight days later, when the patient was in a porphyrin excretion, suggesting an increased ob- far better nutritional state and had not been drink-

Gamma/day Control 21 Gm. Hgb.5 mgm. CoprO j3 mgm. Copro- Per i od orally m orally I orally ..--I Copro. 300 200

100 Urine Isomere** 0 24 hr. periods %of Total 25

75 100 1000 Consecutive interval Itra 800 periods Copro- 600

2000 Feces 4 day periods 700 Proto- I I

Oeutero- trace _ traceeneg. neg. Quain c _neg. ne. neg. Quant. Hgb.0 0 18.2 GM.

*CCitrated Human Blood *Plain--Copro-X Stippled--Copro- m 0-As total heme pigment 40) FIG. 5. EFFECT OF THE INGESTION OF HEMOGLOBIN AND OF COPROPORPHYRIN III AND I UPON THE PORPHYRIN EXCRETION IN THE PRESENCE OF SEVERE LIvER DAMAGE ' 458 EVREL A. LARSON AND CECIL JAMES WATSON

Control 12.5 Gm Hgb Copro m Gamma /dav period I orally 3 mgm orally I 500 X 400 Copro- 300 200 Urine 100 24 hr periods

Isomers Z of Total 25 Copro- m Stippled 50 Copro-1 75 Plain 100

600 Copr0- 400

500 300 200

100

300 Feces 4 Proto- 200L day periods

100

Deutero- Neg. Neg. Trace Guwac Neg. Neg. 8. 6 GM Quant. Hgb /specimen Citrated human blood FIG. 6. EFFECT OF ORAL HEMOGLOBIN AND COPROPORPHYRIN III UPON THE PORPHYRIN ExcmRIoN IN HUMANS

ing. The effect of acute alcoholism upon the uri- provide, we are inclined to favor the second of nary porphyrin excretion is shown in Case 7, these two interpretations. It is quite possible that Table I, and will be discussed in detail in a subse- the bile flow is a major factor in the difference quent paper of this series. between the two diets; there is reason to believe The results of studies of the synthesis of por- that less bile would be formed on the milk diet phyrins by bacteria are shown in Table II. alone because of its relatively higher carbohydrate, and lower protein content (3.5% as against DISCUSSION 22.5%), and hence that less coproporphyrin might The results of the studies on dogs maintained on be excreted by the liver. different diets appear to permit either of two No conclusive evidence was obtained in the dogs interpretations: (1) That the constipating effect of a process of reabsorption and increased excre- of a milk diet promotes absorption of coproporphy- tion of coproporphyrin in the urine following the rin from the bowel and consequent increase in feeding of blood or meat. The increase of fecal urinary excretion; or (2) That the excretory func- coproprophyrin excretion observed in each ani- tion of the liver is relatively sluggish on a milk diet mal in these experiments is probably related to an with the result that a larger fraction of the total increased flow of bile, although a bacterial syn- appears in the urine. Since it would appear that thesis cannot be excluded. The finding that this a normal liver might be expected to handle the in- increase is represented by the type I rather than crement of coproporphyrin which an enterohepatic the type III isomer favors the former rather than circulation due to constipation might conceivably the latter possibility, since the coproporphyrin of STUDIES OF COPROPORPHYRIN. II. 459 the bile has been shown to be type I, while the pre- investigation by the observation that the relative existing evidence and that furnished by the present percentages of isomer types did not change. This study indicate that bacteria elaborate type III. finding practically excludes any appreciable con- The more detailed studies in human subjects tribution to the UCP of fed protoporphyrin type also fail to furnish any conclusive evidence for a III (hemoglobin) or coproporphyrin III and I. reabsorption of porphyrin from the bowel follow- Any increase in urinary coproporphyrin excretion ing the feeding of blood, regardless of the state of following the feeding of either porphyrin would liver function. Moreover, the excretion of urinary necessarily be of the same type. This assumption porphyrin after the feeding of coproporphyrin III is based on chemical evidence that a transforma- in five instances and of the type I isomer in one in- tion of one type of isomer to another would entail stance actually provides significant evidence to the a disruption and complete resynthesis of the mole- contrary. Although these findings are at variance cule. Although quantitative recoveries were sel- with those of Kaunitz and others after the feeding dom attained, it was obvious that the bulk of the of hemoglobin or blood (3, 6) and coproporphyrin fed porphyrin could be found in the following four- I (3), they are strongly supported in the present day collection of feces.

Case Xi I Case X II Urine Copro Control 18 Gm.. 1gb Control 19.5 Gm. 11gb 6amma /day period oral1ly " * Period orally - 400 I I 300-

200- 100- I~~~~~~~~~~~~~ Isomers- 0 - % of Total 25- 50 r--~j 75. 100

Case XIV Control 15 Gm. Hgb Urine Copro ora 6amma /day Per i od llys 600 -I-_

500 -

400 -

300 -

200 - 100

1somers I" 0- % of Total 25- 50 - 74-, 100- lIWlJ *Citrated Human Blood -Plain-Copro L Stippled-Copro 33I Consecutive hr. urine collections in each case.

FIG. 7. EFFECT OF THE INGESTION OF HEMOGLOBIN UPON THE URINARY PORPHYRIN EXCRETION IN THE PRESENCE OF SEVERE LIVER DAMAGE (CASE 12) AND OF OBSTRUCTIVE JAUNDICE (CASES 13 AND 14) 460 EVREL A. LARSON AND CECIL JAMES WATSON

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TABLE II variably observed following the feeding of blood to Observations on the bacterial synthesis of porphyrins humans. One notable exception occurred in the case of high-grade biliary obstruction (Figure 6) Organism mediumCulture Dayscul- Typepor- Amountin Isomers which suggests that tured phyrin gamma the elevations in the other instances were on Copro- 35/500 cc. I -15% the basis of increased flow of bile Plain Proto- Trace III =85% Broth 15 Deutero- Neg. into the intestine. However, an alteration of the B. coli 1 Copro- 15/500 cc.* I- 9% bacterial flora and consequently of bacterial syn- Blood Proto- Trace III =91% Broth 15 Deutero- Neg. thesis of porphyrin, due to the exclusion of bile cannot Copro- 38/500 cc. I = 14% from the intestine, be excluded. In this Plain Proto- 7/500 cc. III =86% Broth 14 Deutero- Neg. connection, it is of interest that preliminary stud- Aerobacter any aerogenes t Copro- 34/500 cc. I = 11% ies have failed to reveal effect of aureomycin, Blood Proto- 16/500 cc. III =89% Broth 14 Deutero- Neg. given orally, on the UCP. It is known that Copro- 31/100 cc. 1 =27% aureomycin markedly inhibits the bacterial flora of Tryptose Proto- 28/100 cc. III =73% Broth 12 Deutero- Neg. the colon, especially E. coli. Pseudomonas aeruginosa t Tryptose Copro- 42/100 cc. I =17% In the studies on bacteria, pure cultures of sev- Blood Proto- 38/100 cc. III -83% Broth 12 Deutero- Neg. eral organisms in broth media were found capable Feces Synthetic 11 None None/500 cc. of synthesizing both coproporphyrin and proto- Pseudomonas Synthetic 11 None None/500 cc. porphyrin. In contrast to the results of previous aeruginosa t studies (17, 18), relatively little protoporphyrin Pseudomonas Synthetic 11 None None/500 cc. aeruginosa t was recovered from the culture of E. coli in a medium containing blood. The explanation may lie Heme compounds not demonstrable in 200 cc. of plain in the much larger amount of hemoglobin which broth after extraction and eight-fold concentration (40). * An estimated half of the total porphyrin lost during was exposed to bacterial action in the previous extraction. studies; it is also possible that the longer period of t Inoculated organism recovered in pure culture at end of experiment. culture in the present study resulted in degradation and disappearance of an undetermined fraction of The increases in UCP observed in the cases of the protoporphyrin. The synthesis progressed alcoholism included in the present study, are con- despite the absence of demonstrable heme com- sistent with the usual finding in acute alcoholism pounds, lending support to recent tracer studies of distinct increases of coproporphyrin III, suffi- which indicate that this process proceeds from cient as a rule to elevate the UCP value into the simple precursor molecules such as acetate and range of 120-500y/day. This will be considered glycine (41, 42). in detail in a subsequent paper of this series. The regular increases in alcoholics not having blood in SUMMARY AND CONCLUSIONS the intestinal tract, obviate the need of ascribing to 1. Studies in dogs have shown that a milk diet such a source the increase observed in the cases of is associated with a higher urinary and lower fecal hemorrhagic (alcoholic) gastritis included in the coproporphyrin excretion than a diet composed of present study. milk and prepared (kibbled) dog food. The total Previous investigators (3, 6) have utilized a amount of coproporphyrin excreted is approxi- diet low in protoporphyrin content as a prerequi- mately the same, and the interpretation is favored site to the demonstration of reabsorption of por- that the hepatic coproporphyrin excretory function phyrin from the bowel after the feeding of blood or the bile flow, or both, are relatively diminished or porphyrin. Wherever it was feasible clinically, on a milk diet. this plan was followed in the present investigation, 2. No evidence was obtained either in dogs or in spite of which, the results in the studies on hu- humans, with normal or impaired liver function, mans do not indicate any differences in urinary of an absorption of coproporphyrin from the in- porphyrin excretion which might be attributed to testine and excretion in the urine, following the the diet. feeding of blood or raw meat, or spontaneous gas- Just as in the dog experiments, increases of trointestinal hemorrhage. Increases were com- fecal coproporphyrin excretion were almost in- monly encountered in the feces in such experi- STUDIES OF COPROPORPHYRIN. II. 463 ments, but in instances where the porphyrin was 12. Jakob, A., Vber den Abbau von Blutfarbstoff zu identified it was shown to be the type I rather Porphyrinen durch Reinkulturen von Bakterien und uber eine neue biologische Synthese von Kopro- than the type III isomer. porphyrin III. Klin. Wchnschr., 1939, 18, 1024. 3. No evidence was obtained in human experi- 13. Snapper, I., Enterogenes Entstehen von Porphyrinen ments for the absorption from the intestine and ex- aus Blutfarbstoff. Arch. f. Verdauungskr., 1919, cretion in the urine of fed coproporphyrin I or III. 25, 230. 14. Snapper, I., Die Bildung von Porphyrinen in Darm- This was true in individuals with markedly im- kanal. Berlin. klin. Wchnschr., 1921, 58, 800. paired as well as normal liver function. The bulk 15. Snapper, I., and van Creveld, S., Uber okkulte of the fed porphyrin was recovered in the feces. Blutungen. Ergebn. d. inn. Med. u. Kinderh., Any small fractions that may have been absorbed 1927, 32, 1. were either metabolized or reexcreted in the bile. 16. Kammerer, H., Bacterien und Blutfarbstoff. Arch. f. exper. Path. u. Pharmakol., 1920, 88, 247. 4. A variety of bacterial strains, in pure culture, 17. Hill, R., and Holden, H. F., The preparation and were found to synthesize both protoporphyrin 9 some properties of the globin of oxyhaemoglobin. (type III) and coproporphyrin III. This syn- Biochem. J., 1926, 20, 1326. thesis did not proceed on artificial media of the 18. van den Bergh, A. A. H., Grotepass, W., and Revers, Gladstone type, but required presum- F. E., Beitrag uber das Porphyrin in Blut und substances, Galle. Kin. Wchnschr., 1932, 11, 1534. ably of protein character or origin, present in 19. Watson, C. J., Concerning the naturally occurring broth. The synthesis of porphyrins proceeded in porphyrins. V. Porphyrins of the feces. J. Clin. broth media which did not contain demonstrable Invest., 1937, 16, 383. hemoglobin or heme pigments. 20. Fischer, H., and Hilger, J., Zur Kenntnis der natur- lichen Porphyrine. X. tYber Blutfarbstoff im der Hefe; Nachweis von Porphyrin in Pflanzen. BIBLIOGRAPHY Ztschr. f. physiol. Chem., 1924, 138, 289. 1. Stokvis, B. J., Zur Pathogenese der Himatopor- 21. Kench, J. E., and Wilkinson, J. F., Porphyrin for- phyrinurie. Ztschr. f. klin. Med., 1895, 28, 1. mation by yeast. Nature, 1945, 155, 579. 2. Schumm, O., Uber die natfirlichen Porphyrine. I. 22. Coulter, C. B., and Stone, F. M., The occurrence of Das in Ham Gesunder gefundene Porphyrin. porphyrins in cultures of C. diphtheriae. J. Gen. Ztschr. f. physiol. Chem., 1923, 126, 169. Physiol., 1930, 14, 583. 3. Fischer, Hans, and Hilmer, H., 23. Gray, C. H., and Holt, L. B., The porphyrin produced Uber Koproporphyrin- by diphtheria bacillus. J. Biol. 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