THE SIGNIFICANCE OF PORPHYRINURIAIN LEAD POISONING1

By ROBERTKARK2 AND ARNOLDP. MEIKLEJOHN3(From the Thorndike Memorial Laboratory, Second and Fourth Medical Sertices (Harvard),

Boston City Hospital, and the Department of Medicine, Harvard Medical School, Boston)

(Received for publication September 24, 1941)

A pathologic increase in the amount of por-phyrin excreted in the urine by patients with leadpoisoning was first reported by Garrod (1) in1892. Since then it has been generally assumedthat this porphyrinuria is related in some way tothe destruction of hemoglobin. Until recentlythere has been no means of testing the validityof this belief. Lately, however, the advance inknowledge concerning the chemistry of the por-phyrins, and the development of methods for dis-tinguishing one type from another, have provideda new approach towards an understanding of theunderlying metabolic disorder responsible for theporphyrinuria of plumbism.

The isomeric identity of the Type III copro-porphyrin found in the urine in lead poisoningwith the Type III protoporphyrin of hemoglobinhas been demonstrated by Grotepass (2) and byWatson (3). The porphyrin in the urine in leadpoisoning thus fundamentally differs from theType I isomer which is the predominant type ex-creted in several other conditions associated withporphyrinuria. These facts justify the assump-tion that the porphyrinuria in lead poisoning is aconsequence of some alteration in the metabolismof hemoglobin itself. In theory, it might originatefrom an altered synthesis of hemoglobin just asprobably as from hemoglobin destruction; butowing to the traditional belief that the anemia inlead poisoning is hemolytic in nature, only thelatter possibility has received any general attentionuntil recently. The hypothesis that the copro-porphyrin excreted in lead poisoning is a productof hemoglobin destruction requires, however, anadditional corollary. In pathologic conditions as-

1The expenses of this investigation were defrayed inpart by the J. K. Lilly gift to the Harvard Medical School.

2 Rockefeller Travelling Fellow; Research Fellow inMedicine, Harvard Medical School; Research Fellow,Thorndike Memorial Laboratory, Boston City Hospital.

8 Instructor and Francis Weld Peabody Fellow inMedicine, Harvard Medical School; Research Fellow,Thorndike Memorial Laboratory, Boston City Hospital.

sociated with excessive hemoglobin destruction,the breakdown products are eliminated in the bilein the normal manner without any large increasein the amount of Type III porphyrin excreted inthe urine. Therefore, if the increased output ofcoproporphyrin in lead poisoning is conceived tobe on the basis of increased destruction of hemo-globin, it is necessary to postulate that one effectof lead is to cause a partial interference in thepathway by which hemoglobin is normally brokendown to bilirubin. Thus, it must be assumed thatpart of the destroyed blood pigment is divertedfrom the bile and appears in the urine with itsoriginal ring structure still intact.

In view of these considerations, it seemed ofinterest to study the mechanism of hemoglobindestruction in patients with lead poisoning. Evi-dence of some defect in this process would lendsupport to the hypothesis that the porphyrin ap-pearing in the urine in this condition is derivedfrom the breakdown of hemoglobin. On the otherhand, failure to obtain such evidence would fur-nish additional support for the more recent theorythat this porphyrinuria is a consequence of defec-tive hemoglobin synthesis.

The experiments described in this paper werecarried out on two patients suffering from leadpoisoning with anemia and porphyrinuria. An at-tempt was made to trace the path of hemoglobindestruction in these patients by observing the effectof injected hemoglobin on the production of bili-rubin and on the excretion of coproporphyrin andurobilinogen in the urine and feces.

METHODSAND MATERIAL.

During the period of investigation the patients wereconfined to bed and, in order to reduce the possibility ofdietary porphyrins contributing to the estimated porphyrinexcretion, partook of a meat, fish, and egg-free diet. Allurine and feces were collected in dark glass containersand kept in an icebox with toluol as a preservative. Insuccessive 24-hour specimens of urine the coproporphyrinwas measured by a modification of the method of Brugschand Keys (4) previously described by us (5). The fecal

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coproporphyrin excretion was measured on aliquots fromsuccessive 3-day accumulations of the stools by a modi-fication of the same method which is described in detailbelow. The urobilinogen excreted in the urine and feceswas estimated by Watson's (6) method. Plasma andurinary hemoglobin were estimated by Bing and Baker's(7) modification of Wu's method; and plasma bilirubinby Barron's (9) method. Hemoglobin solutions wereprepared for intravenous injection by the method ofOttenberg and Fox (8).

The estimation of ether-soluble porphyrin in the fecesThe 3-day collection of feces is weighed as collected

and transferred to the beaker of an electric mixer. Thelarge lumps are broken down with a glass stirring rod,and water is gradually stirred in to bring the volume toa given amount (usually 1000 ml.). The feces andwater are thoroughly mixed into a paste by means ofan electric stirrer and a measured volume, equivalent to10 grams of the original weight of the stool, is taken outwith a measuring cylinder and transferred to a mortar.The paste is then repeatedly ground up with 1 to 2 ml.glacial acetic acid and 20 to 40 ml. of ether. The aceticadd-ether extracts are decanted into a brown half-literbottle and, finally, when no more color can be extractedfrom the stool by the acetic acid-ether mixture, the stoolis also added to the contents of the bottle. The bottle isthen shaken on a machine for 2 hours. The acetic acid-ether and feces mixture is filtered. The residue on thefilter paper is mixed with more acetic acid, furtherether is added and the filtration repeated. The two fil-trates are then combined.

The total acetic acid-ether extract is washed six timeswith water in a separatory funnel, drained and extractedthree times on the rotary machine with 10 ml. lots of5 per cent HC1 solution. A final extraction is madewith 10 ml. of 10 per cent HC1. The HC1 extracts arecombined and neutralized to Congo red by the additionof sodium acetate crystals. The neutral solution is re-peatedly extracted with 10 ml. amounts of acetic add and100 ml. amounts of ether until there is no red fluorescencevisible in the acetic acid-ether layer when examined byultraviolet light.

The acetic acid-ether extracts are combined, washed sixtimes with water, drained and reextracted with 5 per centand 10 per cent HC1 as previously. This whole pro-cedure is repeated three times but during the final HCIextraction only 5 per cent HCI is used.

The combined 5 per cent Ha extracts are now shakenrepeatedly with 5 ml. chloroform until the chloroformlayer shows no red fluorescence when held in front ofthe ultra-violet lamp. The acid fraction is now dilutedto 0.05 per cent HC1 by the addition of distilled waterand again repeatedly extracted with 5 ml. amounts -ofchloroform. These steps are introduced to remove anyprotoporphyrin which may be present. The 0.05 per centHC1 extract is then made neutral to Congo red by theaddition of sodium acetate crystals and the neutral solu-tion repeatedly extracted with acetic acid and ether as

previously. The combined acetic acid-ether extracts arewashed, drained and reextracted with 5 ml. amounts of5 per cent Ha. The combined Ha extracts are madeup to a suitable volume and read in the same manner asthe final Ha extracts obtained from the urine. The perdiem excretion of porphyrin in the feces can be calcu-lated by using the following equation:

Total weight of 3-day Final volume of The reading incollection of feces Haextract micrograms

x x10 Volume of HCI 3

extract used forreading against

standard- daily excretion of porphyrin in micrograms.

By this method the excretion of porphyrin in normalindividuals varied from 80 to 280 micrograms per diem.

The two patients selected for study each pos-sessed the characteristic features of lead poisoning.

Case 1. Case 1 was a 24-year-old white male whohad been employed in polishing soldered metal surfaces.He was admitted to the hospital following an attack ofcolic and complained of weakness, nervousness and con-stipation. His gums showed a well-marked lead line, butthere was no evidence of neuritis. His hemoglobin was54 per cent (Sahli), red blood cells 2.73 million percubic millimeter, hematocrit 25 per cent, reticulocytes 9.6per cent with 1 or 2 stippled cells visible in each highpowered field. The blood lead was 0.007 mgm. per 10grams of blood (normal 0.002 mgm.).4

During his stay in the hospital, the urine was con-sistently normal in color, yet contained about 1000 micro-grams of coproporphyrin in each 24-hour collection (Fig-ure 1). The normal urinary excretion of coproporphyrin,as measured by the method employed, usually ranges be-tween 20 and 40 micrograms, and rarely exceeds 9¢micrograms in 24 hours. The daily fecal coproporphyrinexcretion of Case 1 averaged about 200 micrograms.The urine never contained more than a trace of urobili-nogen, and the fecal excretion of urobilinogen was alwayswithin normal limits.

It is interesting to note that the urinary excre-tion of coproporphyrin was unaffected by the ad-ministration of 500 mgm. of nicotinic acid or of5 U.S.P. units of liver extract daily for 12 days(Figure 1). This therapy was administered inview of a published report (10) which claims thatnicotinic acid reduces the porphyrinuria in leadpoisoning.

4The blood lead determination was very kindly per-formed by Dr. A. J. Plummer of the Evans MemorialHospital.

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FIG. 1. CASE 1. EXCRETIONOF COPROPORPHYRININ URINE AID FECES, ANDFECAL EXCRETION OF UROBILINOGEN BEFORE AND AFTER Two INJECTIONS OFABOUT7.5 AND 19 GRAMSOF HEMOGLOBIN, RESPECTIVELY

Case 2. Case 2 was a 40-year-old colored male leadsmelter. He had been suffering from colic prior toadmission and was constipated, nervous and "shaky." Adefinite lead line was present in the gums. No evidenceof neuritis was noted. The hemoglobin was 60 per cent(Sahli), red blood cells 3.9 million per cubic millimeter,hematocrit 38 per cent, reticulocytes 6.7 per cent, stippledcells 1.8 per cent.

The urine was normal in color5 throughout his stayin the hospital but contained an average of 1200 micro-grams of coproporphyrin in each 24-hour collection (Fig-ure 2). The urinary excretion of urobilinogen variedfrom a trace to 4 mgm. a day. The amount of copro-

5Wewish to emphasize that in both the cases reportedhere the urine was entirely normal in appearance despiteits abnormal content of porphyrin. It is evident that theport wine coloration, traditionally associated with por-phyrinuria, is not found in lead poisoning owing to theabsence of other associated pigments which are usuallypresent where the urine contains excessive amounts ofporphyrin. This question will be discussed more fullyin a later communication.

porphyrin in the feces was at first somewhat increased,averaging 450 micrograms a day for the first 9 days, butlater fell to within normal limits. The excretion ofurobilinogen in the feces was always within the limitsof normal.

RESULTS

After a suitable control period in each case, solu-tions of hemoglobin were injected intravenouslyduring a period of 30 minutes.

Case 1. In the first test, about 7.5 grams ofhemoglobin derived from 50 ml. of normal bloodwere injected. This amount of hemoglobin, iftotally destroyed, would yield theoretically about250 mgm. of protoporphyrin. Following the in-jection, hemoglobin was detectable in the plasmaand reached a peak of 55 mgm. per cent after onehour. The plasma bilirubin rose from a controllevel of 0.38 to a peak of 0.70 per cent in 4 hours.There was, therefore, a maximum increase of 0.32

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FIG. 2. CASE 2. EXCRETIONOF COPROPORPHYRININ URINE AND FECES, AND FECALEXCRETION OF UROBILINOGEN BEFOREANDAFrER AN INJECTION OF ABOUT7.9 GRAMSOF HEMOGLOBIN

mgm. of bilirubin per 100 ml. of plasma. Assum-ing the blood volume to have been approximately5 liters, with the observed cell volume 25 per cent,about 12 mgm. of bilirubin derived from the in-jected hemoglobin must have been present in thecirculating blood stream during the height of itsproduction. It is fair to assume, therefore, thatat least this amount of the 250 mgm. of proto-porphyrin present in the injected hemoglobin wasconverted to bilirubin; yet none appeared as addi-tional coproporphyrin in the urine. The urinaryexcretion of coproporphyrin was not increasedeven by 100 micrograms, an amount that wouldcertainly have been detected by the method of es-timation employed.

The fecal excretion of urobilinogen and copro-porphyrin remained unchanged during the test(Figure 1) as did the urinary excretion of uro-bilinogen.

The second test on Case 1 was even more de-finitive. About 19 grams of hemoglobin derived

from 125 ml. of normal blood and equivalent to630 mgm. of protoporphyrin were injected intra-venously. The maximum increase in plasma bili-rubin following the injection was 2.8 mgm. per100 ml. of plasma (Figure 3), from which thecalculated maximum quantity of circulating bili-rubin derived from the injected hemoglobin wasabout 100 mgm. However, as will be seen fromFigures 1 and 3, the urinary excretion of copro-porphyrin remained at a level of approximately500 micrograms per 24 hours.

It is of interest that, in this test, the injectionwas followed by the excretion of 0.47 mgm. ofhemoglobin in the urine, and in the 24 hours fol-lowing the injection 7 mgm. of urobilinogen ap-peared in the urine.

As a control to these experiments, the levels ofplasma bilirubin and urinary coproporphyrin ex-cretion were measured over a previous 24-hour pe-riod. As may be seen in Figure 4, their levels didnot fluctuate markedly.

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FIG. 3. CASE 1. PLASMA BILIRUBIN AND HEMOGLOBINAND URINARYCOPROPORPHYRINAND HEMOGLOBINFOLLOWINGTHE INTRAVENOUSADMINlIS-TRATION OF ABOUT 19 GRAMSOF HEMOGLOBIN

Case 2. About 7.9 grams of hemoglobin de-rived from 100 ml. of the patient's own blood wereinjected. The amount of protoporphyrin whichwould be liberated if all this hemoglobin werebroken down is about 260 mgm. Following theinjection, the plasma bilirubin showed a maximumincrease of 1.1 mgm. per cent (Figure 5). Thered blood cell volume was 38 per cent and, assum-ing again that the blood volume was approximately5 liters, 33 mgm. of bilirubin derived from theinjected hemoglobin may be assumed to have beencirculating in the blood stream at this time. Theurinary excretion of coproporphyrin during thetest is recorded in Figures 2 and 5. It will be seenthat no variations, even of the order of 100 micro-grams, were observable in the 24-hour amountexcreted.

There was no significant change in the fecal ex-cretion of urobilinogen or coproporphyrin (Figure4), though 0.8 mgm. of hemoglobin appeared in

the urine shortly after the injection and 14 mgm.of urobilinogen was excreted in the urine duringthe succeeding 24 hours.

DISCUSSION

In both these patients the introduction of freehemoglobin into the blood stream was followedby a rapid rise in plasma bilirubin. This rise inbilirubin resembles very closely both in degree andtime of onset the bilirubinemia observed by Gilli-gan et al. (11) in normal subjects under the sameconditions. It is evident, therefore, that thesetwo patients were able to convert hemoglobin tobilirubin without evidence of impairment of thisprocess. Nevertheless, the transient increase inhemoglobin destruction produced by this experi-mental means resulted in no appreciable alterationin the amount of coproporphyrin excreted in theurine or feces, although the amount of hemoglobininjected was presumably sufficient to have pro-

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FIG. 4. CASE 1. PLASMA BILIRUBIN AND URINARY COPROPORPHYRINDURING A 24-HOUR CONTROLPERIOD

duced a definite rise in porphyrin excretion if any

significant part of it had been eliminated in thisform. These results suggest that there is no ob-vious defect in the mechanism of hemoglobin de-struction in lead poisoning, and consequently thatthe porphyrinuria characteristic of this conditioncannot be explained on the basis of such a defectoccurring as a result of increased destruction ofred blood cells.

A possible objection to this conclusion is the factthat these experiments did not succeed in demon-strating the ultimate fate of the injected hemo-globin. Though it was not excreted as porphy-rin, it did not appear as an increase in fecalurobilinogen as might have been expected. How-ever, it seems certain that at least some part of thehemoglobin was broken down, as judged by theconsistent appearance of bilirubin in the plasmaafter each injection of hemoglobin, and the tran-sient increase in urobilinogen in the urine in twoinstances. If a defect in hemoglobin breakdownwas responsible for the increased porphyrin ex-

cretion before the injection of hemoglobin, one

would have expected that at least a few additionalmilligrams of porphyrin would have appeared inthe urine or feces as a result of the hemoglobininjections. However, no detectable alteration inporphyrin excretion was observed. The lack ofincrease in the fecal excretion of urobilinogenfollowing the injection of hemoglobin was prob-ably due to the conservation of bilirubin withinthe body. A similar discrepancy between theamount of hemoglobin destroyed and the excretionof its waste products in the bile has been notedby other authors (12).

It is logical to conclude that in plumbism theporphyrin excreted in the urine is not derivedfrom any abnormal destruction of red blood cells,but is more probably a result of defective synthesisof hemoglobin. This conclusion is in agreementwith the views recently expressed by Watson (13)and by Rimington (14). Watson states "It seems

more likely that the formation of coproporphyrinIII is related to a disturbance in the formation ofhemoglobin rather than to its destruction." Rim-ington believes that the abnormal excretion of

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PORPHYRINURIAIN LEAD POISONING

FIG. 5. CASE 2. PLASMA BILIRUBIN AND URINARY EXCRETION OF Co-PROPORPHYENAND HEMOGLOBINFOLLOWINGTHE INTRAVENOUS ADMINIS-TRATION OF ABOUT7.9 GRAMSOF HEMOGLOBIN

coproporphyrin III results from partial block bytoxic substances of the entrance of iron into theporphyrin ring and consequent defective synthesisof hemoglobin.

In view of this evidence it seems pertinent toreconsider the traditional belief that the anemiaof lead poisoning is hemolytic in nature, and toinquire whether there is a possibility that defectivehemoglobin synthesis may play a part in its pro-duction. The traditional hypothesis is supportedby the following evidence. Aub, Reznikoff andSmith (15) clearly showed that if lead salts areadded to norrnal blood the red cells shrink andseem to become more brittle, with resulting "frac-tionation" and escape of hemoglobin. Key (16)has produced a marked anemia in rabbits followingthe administration of lead salts, resulting in theloss of over one million red blood cells per cubicmillimeter in 24 hours. Furthermore, the anemiaof lead poisoning in man is accompanied by an

apparent reticulocytosis comparable to the reticulo-cytosis occurring in hemolytic jaundice; it is pos-sible, however, that this reticulocytosis may be ofa "spurious" type such as occurs in cases of per-nicious anemia treated with arsenic.

Against the hemolytic hypothesis it may be saidthat, although Aub and his collaborators showedthat hemolysis may be caused by the addition oflead salts to blood in vitro, the amount of leademployed to produce this effect was very muchmore than is usually present in the blood in chroniclead poisoning. Thus, although their results maywell explain the acute anemia produced in rabbitsby Key, it is doubtful whether they furnish anexplanation of the anemia in chronic leadpoisoning.

As Watson (13) has pointed out, the anemia inhuman lead poisoning is usually associated with acolor index below 1.0, and by a reduced mean cor-puscular hemoglobin concentration. This sug-

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ROBERTKARK AND ARNOLDP. MEIKLEJOHN

gests a disturbance or retardation in the formationof hemoglobin. Moreover, as he (17) has shown,the excretion of urobilinogen in the feces is notincreased as in other hemolytic conditions. Thisobservation is confirmed by the results reportedin the present communication. It must be con-cluded that, while the anemia produced by acutelead poisoning in rabbits is apparently hemolyticin nature, the evidence is against the view that theanemia commonly observed in chronic human leadpoisoning can be explained on the same basis.

It is pertinent now to consider the possible originof the coproporphyrin found in the urine in leadpoisoning. Watson and Clarke (18) have latelyshown that reticulocytes contain a considerableamount of protoporphyrin, and have suggestedthat this protoporphyrin is the material responsiblefor the reticulum in these cells. It is tempting tosuggest that the characteristic appearance of thestippled cells found in the blood in lead poisoningis also due to a porphyrin, though of sufficientlydifferent composition to modify its morphologicappearance. As pointed out above, there is evi-dence that the synthesis of hemoglobin is impairedin lead poisoning. The appearance of stipplingin the red cells may therefore be an expression ofan arrest in the maturation of hemoglobin andconsequent accumulation of porphyrin productswithin the cells. It seems likely that, when thestippled cells are ultimately destroyed, this materialwould be liberated and, being an abnormal me-tabolite, might be excreted in an abnormal manner.This, therefore, might well account for at leastsome part of the larger amount of coproporphyrinIII appearing in the urine. In support of thisview, the observations of Thomas (19) may bementioned. He has pointed out that, whereashemoglobin injected into animals is excreted asbile pigments, injected hematin is recovered asporphyrin in the urine.

Whatever the ultimate explanation of porphyrin-uria in lead poisoning, we believe that it is atleast very intimately related to the same metabolicdisturbance that gives rise to the stippling in thered blood corpuscles. This belief is borne out bysome unpublished observations in which we foundthat, in a group of fourteen lead workers withporphyrinuria, the majority also showed the pres-ence of stippled cells in the blood.

SUMMARYANDCONCLUSIONS

Solutions of hemoglobin were injected into twosubjects with lead poisoning, anemia and porphyri-nuria. The injection was followed by a rise inplasma bilirubin resembling closely both in degreeand time of onset the bilirubinemia which has beenobserved (11) in normal subjects under com-parable conditions. It was also accompanied bytransient increase in urinary urobilinogen excre-tion. The injection resulted in no detectable in-crease in the excretion of coproporphyrin eitherin the urine or feces.

These results failed to demonstrate any inter-ruption in the path by which hemoglobin is de-stroyed in the body. It is, therefore, concludedthat the porphyrinuria occurring in lead poisoningcannot be explained on this basis.

The relation of these findings to the problem ofthe causation of anemia in lead poisoning is dis-cussed. It is concluded that they lend support tothe view that this anemia is dyshematopoieticrather than hemolytic in nature.

Wewish to express our grateful thanks to Miss Con-stance Brooks who performed the bilirubin and hemo-globin estimations, and to Frank P. Cohen, M.S., for hisvaluable assistance in carrying out quantitative deter-minations of porphyrin and urobilinogen. Wealso wishto thank Dr. Dorothy Rourke Gilligan for her advice onthe- procedure for injecting hemoglobin.

BIBLIOGRAPHY

1. Garrod, A. E., The occurrence and detection ofhematoporphyrin in the urine. J. Physiol., 1892,13, 598.

2. Grotepass, W., Zur Kenntnis des in Ham auftre-tenden Porphyrins bei Bleivergiftung. Ztschr. f.physiol. Chem., 1932, 205, 193.

3. Watson, C. J., Concerning the naturally occurringporphyrins. IV. The urinary porphyrins in leadpoisoning as contrasted with that associated nor-mally and in other diseases. J. Clin. Invest., 1936,15, 327.

4. Brugsch, J. T., and Keys, A., Quantitative separa-tion and estimation of various porphyrins in bio-logical materials. Proc. Soc. Exp. Biol. and Med.,1938, 38, 557.

5. Kark, R., and Meiklejohn, A. P., Pellagra andporphyrinuria. Am. 3. M. Sc., 1931, 201, 380.

6. Watson, C. J., Studies of urobilinogen. I. An im-proved method for the quantitative estimation ofurobilinogen in urine and feces. Am. J. Clin.Path., 1936, 5, 458.

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7. Bing, F. C., and Baker, R. W., The determination ofhemoglobin in minute amounts of blood by Wu'smethod. J. Biol. Chem., 1931, 92, 589.

8. Ottenberg, R., and Fox, C. L., The rate of removalof hemoglobin from the circulation and its renalthreshold in human beings. Am. J. Physiol., 1938,123, 516.

9. Barron, E. S. G., Bilirubinemia. Medicine, 1931, 10,77.

10. Gross, E. S., Sasaki, Y., and Spies, T. D., Effect ofnicotinic acid on increased porphyrinuria occurringin seven painters. Proc. Soc. Exp. Biol. and Med.,1938, 38, 289.

11. Gilligan, D. R., Altschule, M. D., and Katevsby,E. M., Studies of hemoglobinemia and hemoglo-binuria produced in man by intravenous injectionof hemoglobin solution. J. Clin. Invest., 1941, 20,177.

12. Watson, C. J., in Downey's Handbook of Hematol-ogy. Paul B. Hoeber, Inc., New York, 1938, vol.IV, p. 2490.

13. Watson, C. J., in a symposium on the blood andblood forming organs. University of WisconsinPress, Madison, 1939, p. 25.

14. Rimington, C., Porphyrinuria following sulphanilam-ide: Sulphanilamide dermatitis. Lancet, 1938, 1,770.

15. Aub, J. C., Reznikoff, P., and Smith, D. E., Leadstudies. III. The effect of lead on red blood cells.Part I. Changes in hemolysis. J. Exper. Med.,1924, 40, 151.

16. Key, J. A., Lead studies. IV. Blood changes in leadpoisoning in rabbits, with especial reference tostippled cells. Am. J. Physiol., 1924, 70, 86.

17. Watson, C. J., Concerning the naturally occurringporphyrins. V. Porphyrins of the feces. J. Clin.Invest., 1937, 16, 383.

18. Watson, C. J., and Clarke, W., The occurrence ofprotoporphyrin in the reticulocytes. Proc. Soc.Exp. Biol. and Med., 1937, 36, 65.

19. Thomas, J., Contribution a l'etude des porphyrines enbiologie et en pathologie. Duclume. Lonx-le-Saunier, 1938, p. 73, 121.