17
JULY' 9 5 4 5 0 9 METABOLISM OF HEMOGLOBIN AND OF BILE PIGMENT* IRVING M. LONDON Associate Professor of Medicine, College of Physicians and Surgeons, Columbia University MOGLOBIN IS One of a class of compounds which occupy a central role in metabolic processes. Hemoglobin is com- posed of a pigment, heme, or ferrous protoporphyrin (Fig. I), and of a protein, globin. Heme, the pigment portion of hemoglobin, is also a constituent of myoglobin, or muscle hemoglobin, and is the prosthetic group of the respiratory enzymes, catalase, peroxidase, and the cyto- chromes. The essential function of the heme compounds is to make oxygen available to the cell. Hemoglobin serves to transport oxygen from the lungs to the tissues. This transport is based on the capacity of hemo- globin to bind oxygen reversibly. The hemoglobin molecule is com- posed of four heme molecules bound to one globin molecule with a molecular weight of about 68,ooo. The porphyrin is bound to the protein via the carboxyl groups of the propionic acid substituents. The iron which is in the ferrous state is bound in coordinate linkage to the four nitrogen atoms of the porphyrin, the fifth link is believed to be bound to the imidazole nitrogen of the histidine of globin, and the sixth link binds oxygen reversibly. The same capacity to bind oxygen is shared by myoglobin, a com- pound of one heme molecule bound to a protein with a molecular weight of about 17,000. Myoglobin serves as an oxygen store. It can load and unload oxygen rapidly and its function seems to be that of providing a supply of oxygen when the muscular need is greatest, as well as smooth- ing out the fluctuations in oxygen content during intermittent muscular activity. Oxygen, which is carried from the lungs by hemoglobin, is -passed Presented at the 26th Graduate Fortnight of The New York Academy of Medicine, October 20, 1953. From the Department of Medicine, College of Physicians and Surgeons, Columbia University, and the Presbyterian Hospital, New York. The work from the author's laboratory was aided by grants from the American Cancer Society on the recommendation of the Committee on Growth of the National Research Council and by a contract between the Office of Naval Research and Columbia University, NR 26616.

Haemoglobin and Bile Pigment Metabolism

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Page 1: Haemoglobin and Bile Pigment Metabolism

JULY' 9 5 4 5 0 9

METABOLISM OF HEMOGLOBINAND OF BILE PIGMENT*

IRVING M. LONDONAssociate Professor of Medicine, College of Physicians and Surgeons, Columbia University

MOGLOBIN IS One of a class of compounds which occupya central role in metabolic processes. Hemoglobin is com-posed of a pigment, heme, or ferrous protoporphyrin(Fig. I), and of a protein, globin.

Heme, the pigment portion of hemoglobin, is also a

constituent of myoglobin, or muscle hemoglobin, and is the prostheticgroup of the respiratory enzymes, catalase, peroxidase, and the cyto-chromes.

The essential function of the heme compounds is to make oxygenavailable to the cell. Hemoglobin serves to transport oxygen from thelungs to the tissues. This transport is based on the capacity of hemo-globin to bind oxygen reversibly. The hemoglobin molecule is com-

posed of four heme molecules bound to one globin molecule with a

molecular weight of about 68,ooo. The porphyrin is bound to theprotein via the carboxyl groups of the propionic acid substituents. Theiron which is in the ferrous state is bound in coordinate linkage to thefour nitrogen atoms of the porphyrin, the fifth link is believed to bebound to the imidazole nitrogen of the histidine of globin, and thesixth link binds oxygen reversibly.

The same capacity to bind oxygen is shared by myoglobin, a com-pound of one heme molecule bound to a protein with a molecular weightof about 17,000. Myoglobin serves as an oxygen store. It can load andunload oxygen rapidly and its function seems to be that of providinga supply of oxygen when the muscular need is greatest, as well as smooth-ing out the fluctuations in oxygen content during intermittent muscularactivity.

Oxygen, which is carried from the lungs by hemoglobin, is -passedPresented at the 26th Graduate Fortnight of The New York Academy of Medicine, October 20, 1953.From the Department of Medicine, College of Physicians and Surgeons, Columbia University, andthe Presbyterian Hospital, New York.The work from the author's laboratory was aided by grants from the American Cancer Societyon the recommendation of the Committee on Growth of the National Research Council and by acontract between the Office of Naval Research and Columbia University, NR 26616.

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5 I 0 THE BULLETIN

MPC" G" G"3 3

CN N

C + C

N N

CH3 ."g C"2 C"3GOO" COO"

H EMEFigure I

on in tissue cells, to cytochrome oxidase, a heme enzyme, which activatesthe oxygen to accept electrons. The activated oxygen accepts electronsfrom the cytochromes which are in the final position of the oxidativechain. Their iron atoms are reduced from the ferric to the ferrous state

by accepting electrons from the substrates of the dehydrogenatingsystems, and they are oxidized by molecular oxygen to which they, inturn, transfer electrons. By repeated alternate oxidation and reduction,small amounts of these cytochromes participate in the oxidation ofenormous quantities of material in the cell.

The heme enzymes, catalase and peroxidase, are also involved inoxidative processes. Peroxidase forms a complex with hydrogen peroxideas a result of which it is capable of acting as a hydrogen acceptor forthe oxidation of other substances. Catalase splits hydrogen peroxide to

water and oxygen and probably serves to "mop up" hydrogen peroxidewhich is produced by the reaction of molecular oxygen with a varietyof agents and which might be injurious to the cell if it were not rapidlyremoved. It has been suggested that catalase may serve as a peroxi-dase also.

In brief, the heme compoun& are concerned with transporting andstoring oxygen, with making oxygen available to the cell, and with theoxidation of cellular constituents. These are the basic energy-yieldingreactions of living matter.A related compound is concerned with the basic energy-storing

reaction. This is chlorophyll, a magnesium-porphyrin complex, which is

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Metabolism of Hemoglobin and of Bile Pigment 5 1 1

an essential agent in the mechanism of photosynthesis, the mechanism bywhich energy derived from the sun is stored in organic chemical'consti-tuents of plants. I

The heme compounds reflect the development of a specialization infunction. The reversible binding of oxygen, and the splitting of hydro-gen peroxide and its activation are in each case best performed by theheme-protein molecule specific for that function. Heme itself is capableof carrying out these functions but at a markedly lower level, and,indeed, inorganic iron has a primitive or even lower level of function..It is the protein moiety which markedly enhances and differentiatesthese functions of heme.

In the case of hemoglobin, the protein portion has received muchrenewed interest in the past few years because of the -finding that theglobin portion is abnormal in some disease states.' At the present time,in addition to normal adult human hemoglobin, there are fetal hemo-

2globin, sickle cell hemoglobin, hemoglobin c and hemoglobin d. Sicklecell hemoglobin exists in the homozygous, state as sickle cell anemia, inthe heterozygous state as sickle cell trait. Hemoglobin c has also beenfound in both homozygous and heterozygous states.3--l' These hemo-

globins, whose protein portions vary in ways not yet thoroughly defined,can be distinguished by methods designed to separate proteins, namely,electrophoresis, alkali denaturation, solubility properties, and chromato-graphy. The heme portion of these hemoglobins is, as expected, normal.A number of exciting possibilities for further investigation are

afforded by these findings, but discussion of them is beyond the scopeof this paper.

Let us proceed then to the biologic synthesis of hemoglobin, or, more

specifically, to the question of how the heme molecule is formed in

biologic systems. During the past several years considerable progresshas been made in the elucidation of the mechanism of synthesis of heme.

Isotopic techniques have played a crucial role in advancing our knowl-

edge in this field. It was first shown that acetate and glycine are utilized11-12in the biologic formation of protoporphyrin. Glycine has been shown

to be a specific precursor, not only of protoporphyrin which is of theetioporphyrin III configuration but also of uroporphyrin 1, copropor-phyrin I and coproporphyrin III.11.3-15 The nitrogen atom of glycineprovides all four nitrogen atoms and the methylene carbon of glycineprovides eight of the 34 carbon atoms of protoporphyrin.11 Acetate is

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5 I 2 THE BULLETIN

H00C

H? COOH

H2 CHp

C C

11 IHC C-CH2NH2

NH

Porph obi I inogenFigure 2

utilized via a succinyl intermediate which is formed from the condensa-tion of succinate and glycine.16 This intermediate is delta-amino levulinicacid (COOHCH2CH2CH2CH2NH2) formed from the condensationof succinate and glycine and the decarboxylation of the glycine moiety.Two molecules of delta-amino-levulinic acid combine to form a pyrrolewhose structure is most likely similar to, or identical with, that of por-phobilinogen. Porphobilinogen, which is excreted in the urine of patientswith acute intermittent porphyria, has recently been isolated17 and itsstructure has been determined (Fig. 2) .18-20 This structure is similar to

that of the projected parent pyrrole. 10, 21

Porphobilinogen can be converted to uroporphyrin III spontaneouslyin vitro. 17, 22 In addition, evidence has recently been presented 'Whichindicates that po'rphobilinogen is a precursor of protoporphyrin, ofuroporphyrin III, and of coproporphyrin III.2-3Evidence has also beenproposed for the biologic conversion of uroporphyrin III to protopor-Phyrin24 but similar conversion of coproporphyrin III to protoporphyrinis not observed.

Our present knowledge and prevailing concepts may be summarizedin the scheme shown in Figure 3.

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Metabolism of Hemoglobin and of Bile, Pipment 5 1 3

Scheme of blosynthesisof protoporohyrin

HOOC CH9 CHp COOH + CH2 NH2 COOHSUCCINATE GLYCINE

HOOC CH2 CH2 CO CH2 NH2delta AMINOLEVULINIC ACID

2 delta AMINOLEVULINIC ACID-----41-1 MONOPYRROLE (porphobilinogen)

PORPHOBILINOGEN

UROPORPHYRIN GOPROPORPHYRIN PROTOPORPHYRIN

Figure 3

Whether uroporphyrin III and coproporphyrin III are by-productsor are on the main route of synthesis of protoporphyrin remains to beestablished.

Many of these studies were made feasible by the demonstration thatsynthesis of heme occurs in immature mammalian2' and avian erythro-cytes" and in hernolyzed preparations of these ceJIS..27-29These systemsprovide an in vitro model of the formation of heme in vivo in the imma-ture cells of the erythrocyte series in the bone marrow.

Let us next see what happens to heme newly formed in the marrow:

If glycine labeled with N15 is administered to a normal man, andhemin is subsequently isolated from the erythrocytes in the peripheralblood and the N15 concentration in the hemin is plotted against time,the.type of curve shown in Figure 4 is obtained.

The isotope concentration rises to a maximum between the 2oth and3oth days, maintains a plateau for several weeks, and then declines more

or less abruptly. The upward slope of the curve represents the introduc-tion into the circulation of newly formed erythrocytes containinglabeled hemoglobin which are replacing cells that were formed priorto the administration of glycine and that are therefore without labeledhemoglobin. The plateau is reached and maintained when the-erythro-cytes entering the circulation and those leaving the circulation containlittle or no labeled hemoglobin. Eventually, the erythrocytes which were

formed during and shortly after the administration of isotopic glycine

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THE BULLETIN5 1 4

00.50ui0x0.4.w

onz0.3.10-z1-1 0.20ccwa- 0.1a0I.-9 0-

TIME IN DAYS

Figure 4-N15 concentration in hemin after feeding N14-labeledglycine for 2 days.

and which contain the bulk of the labeled hemoglobin undergo' destruc-tion and are replaced by cells containing little or no labeled hemoglobin.The result is a decline in the isotope concentration in the hemin. Thiscurve reflects the introduction into the circulation of a population oflabeled erythrocytes, their survival and their disappearance from thecirculation, in brief, the life span and pattern of destruction of thenormal human erythrocyte. The average life span of the human erythro-cyte is approximately I 2o days. The erythrocyte is destroyed not in a

random fashion but rather as a function of its age. There appears to beno significant destruction of cells before the first month of age. Themajor portion of the cell population is destroyed between about thegoth and I 5oth days.

Once the cell is destroyed, what is the fate of its hemoglobin? Balancestudies in dogs have indicated that 8o to i oo per cent of the heme portionof hemoglobin is excreted as bile pigment.-" There appears to be no

significant reutilization of the heme for new hemoglobin formationalWhere and how is hemoglobin converted to bile pigment? The

available evidence indicates that hemoglobin is converted to bile pigment32not only in the liver but throughout the reticulo-endothelial system.

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Metabolism of Hemoglobin and of Bile Pigment 5 I 5

TABLE I-CONVERSION OF HEMATIN TO BILE PIGMENTIN NORMAL DOG

Amount of Hematin N15 Atom Per Cent N5 Atom Per CentInjected Excess in Hematin Excess in Stercobilin800 mg. 1.425 0.325

An early school of thought held that the conversion of hemoglobinto bile pigment entails the splitting of hematin from globin and theconversion of hematin to bile pigment with loss of iron and rupture ofthe porphyrin ring. Much conflicting evidence was presented concerningthe role of hematin in the formation of bile pigment. It is now establishedthat hematin can be converted to bile pigment in the mammalianorganism.

If hematin labeled with N'5 is administered to a normal dog, sterco-bilin, a bile pigment isolated from the feces of the dog, is found to behighly labeled (Table I).33 In this experiment, at least i 8 per cent ofthe administered hematin was converted to bile pigment within the firstnine days after the administration of the hematin.

Although it is clear that hematin can be converted to bile pigmentin the mammalian organism, there is no information concerning theextent to which this pathway of bile pigment formation is normallyoperative.

An alternative route for the formation of bile pigment has beenproposed,34 which consists of oxidation of the a-methene carbon atomof the heme and the formation of at least one green compound, chole-globin. Then the porphyrin ring is opened, the a-methene carbon atomis lost, and the iron is split out. Biliverdin, a green bile pigment, is formed.Precisely when the globin is split from the pigment is unknown; but inman this step plus the reduction of biliverdin to bilirubin most likelyoccur in the reticulo-endothelial system.

In disease states in which red cells are destroyed intravascularlythe hematin which is formed becomes bound to albumin with the formna-tion of a compound called methemalbumin.35 As we have seen, thiscompound can be converted to bile pigment.33

In normal man, however, the breakdown of hemoglobin appears tooccur principally in the reticulo-endothelial tissue. Whatever the route

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51I6 THE BULLETIN

BILIRUBIN AND RELATED COMPOUNDS

Orange BILIRUBINRed C33l3N406

Reddish DIHYDROBILIRUBINYellow C33H38N406

#2HLight MESOBILIRUBINYellow C33H4N4O6

DIHYDROMESOBILIRUBIN d-UROBILINOGENFaintly C33H42N406 C33H42N406Yellow

+ 2HS *+2H1

Colorless MESOBILIRUBINOGENC35H44N406

~ #4fill -2H

Colorless STERCOBILINOGENC33H48N406

Orange i-UROBILIN STERGOBILIN d-UROBILINYellow C33H42N406 C33H46N406 C33H4N406

Figure 5

Lowry, P. T., Ziegler, N. R. and Watson, C. J.3' in Bull. Univ.Minn. Hosp. 24:166-80, 1952. Reprinted by permission.

of catabolism of hemoglobin may be, the bile pigment which is releasedfrom the reticulo-endothelial cells is bilirubin.

The bilirubin is transported in the blood bound to protein. In theliver the bilirubin is excreted into the bile apparently no longer inbound form.

In the intestine, the bilirubin undergoes a series of reductions byreducing systems of intestinal bacteria. A scheme for this sequence ofreactions has been proposed recently (Fig. 5).36 Bilirubin, an orange-redpigment is converted to mesobilirubin, a light yellow pigment viadihydrobilirubin, which is reddish yellow. Under normal conditions itappears that dihydromesobilirubin is formed and that this is reducedto the colorless mesobilirubinogen. But if Terramycin or Aureomycin isgiven by mouth, a bile pigment with the same empirical formula asdihydromesobilirubin can be isolated. This is d-urobilinogen. Whetherthis compound is a normal intermediate in the formation of mesobili-

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Metabolism of Hemoglobin and of Bile Pigment

rubinogen, or whether it is an abnormal metabolite is not known. Onoxidation of d-urobilinogen, an orange yellow pigment, d-urobilin,which is very strongly dextro-rotatory, is formed.36'37

The colorless mesobilirubinogen can be further reduced to formstercobilinogen. Together, mesobilirubinogen and stercobilinogen consti-tute what is clinically known as "urobilinogen." This urobilinogen isto a large extent reabsorbed from the intestine and returned to the livervia the portal circulation. In the liver it may be reexcreted into the bileand then into the intestine. This constitutes the entero-hepatic circulationof urobilinogen. When mesobilirubinogen is oxidized, an orange-yellowpigment which is optically inactive is formed. This is i-urobilin. Andwhen stercobilinogen is oxidized an orange-yellow pigment which isstrongly levo-rotatory is formed. This is called stercobilin.

The further fate of stercobilin, of i-urobilin, or d-urobilin is notknown but it is likely that at least a portion of these pigments is degradedto other substances. In any case there is normally a smaller daily produc-tion of urobilinogen, which represents mesobilirubinogen, stercobilino-gen and the reduction products of i-urobilin and stercobilin, than theamount of bilirubin calculated from the daily destruction of hemoglobin.The possibility that smaller pyrrole units may be derived from bilirubinand so account for part of this discrepancy requires further exploration.

Let us return to the question of the transport bf bilirubin in theblood. The bilirubin in the serum can be measured by the van den Berghreaction in which bilirubin reacts with diazotized sulfanilic acid. Innormal sera and in the sera of many patients with hemolytic jaundicethe addition of alcohol is essential for a rapid reaction to occur. In casesof obstructive jaundice, however, the reaction occurs rapidly in theabsence of alcohol. This is a "direct" van den Bergh reaction. Thereaction in the presence of added alcohol is an "indirect" reaction.

The basis of the difference between the direct and indirect reactionsis still in dispute. Recently, evidence has been presented which indicatesthat indirect reacting bilirubin is precipitated with the globulin fractionon ammonium sulfate fractionation while the direct reacting bilirubinis precipitated with the albumin fraction.38 It was suggested also thatthe direct reacting bilirubin might be in the form of a metal complexwith albumin.

Other workers39 have extracted bile pigments from the sera ofjaundiced patients and have separated them into two types. One type,

5 I 7

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THE BULLETIN

Gross metabolic changes of bilirubin

BILIRIUBIN BILIRUBIN

/Reexcre ion ad

INTESTI NEKIDNEY LIVER AnUrobilinoqen"

| \ ~~~~~Reabscurption A

V~~~~URI NARY joUrobilinogenU nogen

FECALUrobil inogen

Figure 6

which is more soluble in organic solvents, manifests an indirect van denBergh reaction and is believed to be bilirubin. The other type, whichis more soluble in water, gives a direct van den Bergh reaction. Neitherpigment appears to contain protein and the nature of the reaction withdiazotized sulfanilic acid appears to be unrelated to linkage of the pig-ment with protein. It is clear that the mechanism of the van den Berghreaction remains the subject of controversy, and these various findingsrequire confirmation and further exploration.

It may be well at this point to summarize the gross metabolic changesof bilirubin (Fig. 6).

In normal man about 250 to 300 mg. of bilirubin should be produceddaily from the degradation of the hemoglobin of newly destroyederythrocytes. This bilirubin, presumably of the indirect reacting type,is converted in the liver to the direct type. In the intestine the bilirubinis reduced to the "urobilinogens," a large portion of which is reabsorbedand reexcreted into the intestine. Normally, the urobilinogen is excretedalmost completely in the feces, with up to 240 mg. daily as the normaladult value.40 Little or no urobilinogen (up to 4 mg.) is normallyexcreted in the urine.

This pattern may be altered in various disease processes. In hemolyticprocesses the capacity of the liver to handle bilirubin may be exceededso that bilirubinemia of the indirect reacting type occurs; in additionthe large amounts of urobilinogen which are formed and reabsorbedfrom the intestine may exceed the capacity of the liver for reexcretion

5 I 8 THE BULLETIN

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Metabolism of Hemoglobin' and of Bile Pigment

with the result that increased amounts of urobilinogen appear in theurine.

Complete extrahepatic obstruction, as by a carcinoma of the headof the pancreas, will result in jaundice with bilirubin of the directreacting type, with no fecal or urinary urobilinogen but with bili-rubinuria.

Parenchymatous diseases of the liver may produce varying patterns.There may be increased amounts of indirect reacting bilirubin if thecapacity of the liver to convert the indirect to the direct type isexceeded. If the process is primarily one of intrahepatic obstruction,there may be direct reacting bilirubin in the blood and in the urinewith diminished amounts of fecal urobilinogen. If adequate flow ofbilirubin to the intestine is restored, hepatic damage may be reflectedin a diminished capacity of the liver to reexcrete urobilinogen with theresult that urinary urobilinogen will increase.

An evaluation of gross bile pigment metabolism in a patient mustinclude these considerations: i) The amount of hemoglobin which isbeing destroyed daily; 2) the functional capacity of the liver to excretebilirubin into the bile; 3) the patency of the biliary tree; 4) the func-tional capacity of intestinal bacteria to reduce bilirubin to the urobilino-gens; 5) the functional capacity of the liver to reexcrete urobilinogen;6) the threshold of the kidney for the excretion of direct bilirubin.

So far we have considered the conversion of hemoglobin to bilepigment. And the usually accepted theory of the origin of bile pigmentis that it is derived exclusively, or very nearly so, from the hemoglobinof mature circulating erythrocytes. The advent of the isotope techniquefor the study of the life span of the erythrocyte afforded an opportunityfor reinvestigation of the origins of bile pigment. If bile pigment isderived exclusively from the hemoglobin of mature erythrocytes incirculation, in experiments in which N15 labeled glycine has been admin-istered, one would expect little or no N15 in the bile pigment duringthe first few weeks of the experiment; and then as the labeled erythro-cytes begin to be destroyed there should be a progressive rise in theN15 of the bile pigment with a peak concentration at the time of maximaldecline in isotope concentration in the hemin.

In a normal man hemin was isolated from the erythrocytes andstercobilin from the feces after the administration of N15 labeled glycine(Fig. 7).4142During the latter period of the experiment, i.e., from about

5 I 9

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520 THE BULLETIN

F-Z .3-,

2 -

0!a4l

TIME IN DAYSFigure 7 N15 concentration in hemin and stercobilin of a normal man

after the start of feeding N15-labeled glycine for 2 days.

the eightieth day onward, there is indeed a rise in the N15 concentrationin the stercobilin which reflects the destruction of the hemoglobin ofcirculating erythrocytes. But during the first week of the experimentwhen the cells which are being destroyed are those formed approxi-mately I20 days earlier and when there is no apparent destruction ofnewly formed labeled cells in the peripheral blood, the N15 concentrationin the stercobilin attains a high value. This finding indicates that aportion of bile pigment is derived from one or more sources other thanthe hemoglobin of mature circulating erythrocytes. In normal man thisfraction is at least io to iS per cent of total bile pigment production.Confirmatory findings have been reported by other workers.43

What are the possible sources for this portion of the bile pigmentin normal man? They may be considered in terms of four categories:i) The hemoglobin of newly formed erythrocytes which are destroyedin the bone marrow or very shortly after entering the peripheral bloodfrom the marrow. There is, however, no evidence as yet to supportthis possibility; 2) myoglobin and the respiratory heme enzymes. Therates of turnover of these heme compounds are unknown and consequentlyan evaluation of their contribution to the production of bile pigmentis only conjectural; 3) heme and porphyrins which are not utilized forhemoglobin formation. Evidence bearing on this possibility will bepresented below; 4) direct formation of bile pigment without prior

5 2 0 THE BULLETIN

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Metabolism of Hemoglobin and of Bile Pigment

30

PERNICIOUS ANEMIA

'2 -]

i 0.8 i pEMINw

Cr064g_ _ , , _ r0-04

0021

2040 60

80oo 120 140 60 180 200 220 240 1

TIME IN DAYS

Figure 8-N15 concentrations in hemin and stercobilinafter the start of feeding Nu5-labeled glycine for 2 days.

formation of a porphyrin ring. There is as yet no evidence to supportthis possibility.

The finding that a portion of bile pigment is normally derived froma source other than the hemoglobin of mature circulating erythrocytesseemed to offer a possible explanation for the discrepancy between thevery high levels of bile pigment produced and the relatively low levelsof hemoglobin and red cells turned over in the peripheral blood inuntreated pernicious anemia. A study with N15 labeled glycine in such apatient reveals an average survival of erythrocytes of about eighty-fivedays and a striking elevation in the isotope concentration in the sterco-bilin (Fig. 8).44 One can calculate that at least 40 per cent of the bilepigment produced in this patient was derived from one or more sourcesother than the hemoglobin of circulating erythrocytes.

Further evidence for multiple sources of bile pigment is providedby a study in a patient with congenital porphyria (Fig. 9).46 Thispatient, producing large amounts of uroporphyrin I and coproporphyrin1, had no anemia and indeed had a normal erythrocyte life span. Andyet from the isotope concentrations in the stercobilin one can concludethat at least 30 per cent of the bile pigment was not derived from thehemoglobin of mature circulating erythrocytes. Similar studies on the

5 2 I

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52 H B LEI

CONGENITAL PORPHYRIA

2 ° - ERoll

- STERCOS.IIN

10

NEMIN

10 20 30 40 50 60 70 80 90 100 110 120 13o10 1OInoISOIO ISOTIME IN DAYS

Figure 9-N'5 concentrations in hemin and stercobilinfollowing the start of feeding labeled glycine for

3 days.

Some of the possible origins ofbile pigment

MYOGLOBIN SHEME ENZYMES

rProtoporphyrin -*' HEME WHEMOGLOBINPCoproporphyrin _-__ BILE PIGMENT

PYRROLES > Uroporphyrin ~

[MesoporphyrinDeuteroporph yrin

Figure 10

formation of bile pigment in porphyria have been reported from otherlaboratories.13 43, 46, 47A schema representing some of our presently available knowledge

is shown in Figure io. Pyrroles are utilized for the formation of a varietyof porphyrins. Of these protoporphyrin is converted to heme which isused for the various heme compounds. Hemoglobin is in turn convertedto bile pigment. The questions which we have raised involve the conver-sion of some of these intermediates to bile pigment without goingthrough hemoglobin. The first of these which was tested was hematin,and as we have described earlier, this was readily converted to stercobidin.In addition, protoporphyrin labeled with C4 was administered to a dogand the stercobilin isolated from the feces was found to be significantly

5 2 2 THE BULLETIN

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Metabolism of Hemoglobhz and of Bile Pigmnent 5 2 3

labeled, indicating that at least 6 per cent of the protoporphyrin hadbeen converted to bile pigment.4

Studies in progress now are concerned with determining the extentto which other porphyrins may serve as precursors of bile pigment andthe extent to which bile pigment may be formed directly without priorformation of a porphyrin.

SUMMARY

Hemoglobin, myoglobin and the respiratory heme enzymes occupya central role in metabolic processes for they are concerned with theprocesses by which oxygen is made available to the animal cell and withthe processes of cellular oxidation.

In the biologic synthesis of heme, glycine and succinate are utilizedfor the formation of delta-amino-levulinic acid from which a pyrrole,probably identical with porphobilinogen, is formed. UroporphyrinIII can be formed from porphobilinogen, and protoporphyrin IXapparently can be derived, at least in part, from uroporphyrin III.

The conversion of hemoglobin to bile pigment may proceed via theformation of choleglobin. Evidence is presented to indicate that thisconversion might also proceed via hemnatin. A possible scheme for thesequence of reactions by which bilirubin is converted to other bilepigments in the intestine is presented.

Recent studies on the direct and indirect van den Bergh reactionsindicate qualitative differences in the chemical constitution of the pig-ments which give these reactions.

Studies on the origin of bile pigment indicate that in normal manat least i o to i 5 per cent of total bile pigment is derived from one ormore sources other than the hemoglobin of mature circulating erythro-cytes. In disease states such as pernicious anemia and congenital por-phyria, the proportion derived from alternative sources may be increased.Investigation of the nature of these alternative sources has revealed thathematin and protoporphyrin, unbound to globin, can be convertedto bile pigment in the mammalian organism.

REFERENCES

1. Pauling, L., Itano, H. A., Singer, S. .J.and Wells, I. C. Sickle cell anemia, amolecular disease, Science 110:543-48,1949.

2. Itano, H. A. Human hemoglobin, Science

3. Kaplan, E., Zuelzer, W. W. and Neel,J. V. A new inherited abnormality of

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5 24 THE BULLETIN

hemoglobin and its interaction withsickle cell hemoglobin, Blood 6:1240-59,1951.

4. Itano, H. A. and Neel, J. V. A new in-herited abnormality of human hemoglo-bin, Proc. Nat. Acad. Sci. 36:613-17,1950.

5. Ranney, H. M., Larson, D. L. and Mc-Cormack, G. H. Some clinical, biochemi-cal, and genetic observations on hemo-globin C, J. clin. Invest. 32:1277-84,1953.

6. Bloch, K. and Rittenberg, D. Estima-tion of acetic acid formation in rat,J. biol. Chem. 1579:45-58, 1945.

7. Shemin, D. and Rittenberg, D. Utiliza-tion of glycine for synthesis of por-phyrin, J. biol. Chem. 159 :567-68, 1945;and The biological utilization of glycinefor the synthesis of the protoporphyrinof hemoglobin, J. biol. Chem. 166:621-25,1946.

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