the association of the urobilin early erythropoiesis man · the term "urobilin" is used...

Josrnal of Clinical InvestigationVol. 45, No. 11, 1966

The Association of the Urobilin "Early Peak" andErythropoiesis in Man *

PETERV. D. BARRETT,t MARTINJ. CLINE, ANDNATHANIEL I. BERLIN(From the Metabolism Branch, National Cancer Institute, National Institutes of Health,

Bethesda, Md.)

In a normal individual the administration ofisotopically labeled glycine (1, 2) results in twopeaks of radioactivity in the excreted fecal bilepigment. The first or "early" labeled peak of fe-cal bile pigment excretion occurs within the firstfew days and cannot be explained on the basis ofthe breakdown of normal circulating red cells.The second peak occurs 100 to 130 days after ad-ministration of the isotopic precursor and coincideswith the termination of the life-span of the groupof red cells that contains the labeled precursor.When the conversion of intravenously adminis-tered glycine-2-14C to serum bilirubin-14C wasstudied, two peaks of bilirubin specific activitywere found, the first at 24 hours and the secondat the third to fourth day (3). It must be recog-nized that the single observed fecal urobilin earlypeak is a result of fusion of the two early labeledpeaks in the gastrointestinal tract and that thechanges measured in these patients could, intheory, be due to a quantitative change in eithercomponent.

Several theories have been proposed to explainthe first or early fecal peak. The most widely ac-cepted is that this pigment is derived from red cellsthat are destroyed before or shortly after their re-lease from the bone marrow. For this reason theearly peak has been thought of as representing"ineffective" erythropoiesis. With this hypothe-sis, London, West, Shemin, and Rittenberg (1)and Gray, Neuberger, and Sneath (2) calculatedthat in a normal individual 11 to 20%o of erythro-poiesis is represented in this peak. However, re-cent studies have been interpreted as showing thatthe early peak might arise by a direct syntheticpathway from a common precursor pool that does

* Submitted for publication August 9, 1965; acceptedJuly 18, 1966.

t Address requests for reprints to Dr. Peter V. D. Bar-rett, National Cancer Institute, National Institutes ofHealth, Bethesda, Md. 20014.

not require heme as an intermediate and, thus, isnot necessarily related to erythropoiesis (4).

James and Abbott (5) reported the presence ofan elevated early peak in two patients who eventu-ally developed acute granulocytic leukemia. Oneof these patients had erythroid hyperplasia of thebone marrow, and the other had erythroid aplasia.From these observations it was concluded that themajor portion of the early peak could not be as-sociated with erythropoiesis. However, this con-clusion is at variance with studies in which theamount of labeled urobilin in the early peak didincrease after an experimental hemorrhage or wasfound to be increased in disease states associatedwith increased rates of erythropoiesis (6).

The present study was undertaken in an at-tempt to define the relationship between erythro-poiesis and the early labeled peak of urobilin bystudying a given individual at two different ratesof red cell production. With the exception of twopatients who were studied before and after an ex-perimental hemorrhage (7, 8), this has not beendone previously. Four patients with hematologicdiseases that might respond favorably to therapywere selected for study. Before and after treat-ment these patients were given glycine-2-14C, andthe specific activity of urobilin-14C was determined.In addition, erythrokinetic function was evaluatedby using either 59Fe or glycine-2-14C as a precursorof heme. Wefeel that the results of these studiessupport the concept that in man most of the earlylabeled peak is associated with erythropoiesis.

Methods

Fecal urobilin isolation. For each study the patientswere given intravenous injections of 100 ,uc glycine-2-1'C(SA 2.6 to 4.0 ,uc per umole) in a volume of 1 to 5 mlover a 10-second period. Stool samples were collecteddaily and stored at -20° C until analysis. Early in thestudy Watson's original method for the isolation of fecalurobilin was used in a modified form (7, 9), but the most

1657

P. V. D. BARRETT, M. J. CLINE, AND N. I. BERLIN

recent method of Watson and his co-workers (10) was

used in later isolations.The term "urobilin" is used generically in this report

to include the closely related pigments urobilin IXa,d-urobilin, and l-urobilin (stercobilin), which are identi-cal from a physiologic point of view and which all may

be isolated with the above method (10). However, inany given individual only one species of urobilin is usu-

ally present. Except for patient W.G., the specific opti-cal rotation of the urobilin crystals of each sample was

determined in chloroform,' and the values obtained were

consistently within the accepted limits of purity for i (in-active)-, d-, or l-urobilin (11). In addition, thespecific activity of urobilin crystals obtained by thesemethods was found to be constant after crystallizationfrom ethyl acetate. To determine the specific activity ofthe urobilin crystals, we plated 1 to 2 mg on a preweighed5-cm planchet with the aid of a turntable and counted ina low background thin window 8-counter2 with an effi-ciency of 22% and a background of 2 cpm. All sampleswere counted for at least a half-hour or until a minimumof 1,000 counts had been observed.

Hemin isolation. The method of Fischer (12) was

used for most of the sample--. The crystalline materialwas plated and counted for SC content as with urobilin.However, for patients S.S. and J.F., hemoglobin was ex-

1 Standard polarimeter, Hilger and Watts, Ltd., Lon-don, England.

2 Sharp Laboratories, Inc., La Jolla, Calif.

tracted, and calculations were made to convert the datato the specific activity of hemin (13). The values givenfor each patient represent maximal specific activities at 10to 20 days.

Ferrokinetics. Twenty juc of 'Fe in the form of fer-rous citrate with a specific activity of 2 to 40 mc per mgwas used in each study. The method of Huff and co-workers (14) was used to determine the plasma radioirondisappearance half-time and the red cell radioiron uptake.

Blood volume. The 'Cchromium method of Sterling andGray (15), as modified by Read (16), was used.

DFP red cell life-span. Tritiated diisopropylfluoro-phosphate (DFP-'H) was used as a red cell label as

previously described (17).Isotopic measurements. Samples containing 'Cr and

'Fe were counted in a well-type scintillation counterwith a sodium iodide (thallium-activated) crystal and a

single channel spectrometer. When both "Cr and "Fewere in the same sample, a correction was made for "Feactivity in the "Cr counting window.

Crystalline urobilin-'C or hemin-"C was counted byplating the material infinitely thin on a 5-cm diameterplanchet and counting in a thin window low backgroundcounter. Planchets plated with hemin that contained both"Fe and "C were counted twice, with sufficient time be-tween the two countings to allow at least half of the "Feto decay. Assuming that the radioactivity due to 14C re-

mained constant during this period, one may then calcu-late the radioactivity due to 14C alone.

TABLE I

Erythrokinetic data for four patients with varying rates of erythropoiesisand for two hematologically normal patients*

PlasmaTotal radio-

Plasma RBC iron- iron RBC Hemin- Urobilin-iron iron binding clear- radio- Fecal 14C 14C

turn- turn- Serum capac- ance iron Red cell urobi- day maximalPatient Study over over iron ity tj uptake life-span linogen 9-20 value

mg/kg/day pg/100 ml min % days mg/day dpm/mgR.P. Normal 0.53 0.43 90 290 68 83 110 142

W.G. Normal 0.40 0.37 62 284 52 91 153 223S.S. September 1961 0 210 210 293 0 800t 48

November 1961 800t 461 459

A.H. January 1963 0.31 0.005 271 271 390 1.6 19 400t 0 22(Hb t4)

March 1963 0.74 0.610 80 146 57 82 44 400t 404 1,291(DFP-3H)

July 1964 0.63 0.384 213 213 131 61 86 293 196 596(Hemin-14C)

J.F. April 1962 800t 9 15

L.F. November 1961 1.06 0.15 202 202 95 14 72 400t 30 1,650(DFP-'H)

November 1962 0.72 0.43 60 140 93 60 131 220t 70 125(DFP-SH)

May 1963 0.45 0.18 181 181 154 40 33 81 110 343:(lCr)

January 1964 0.76 0.11 185 207 120 15 14 419 52 502(Hb ti)

* Abbreviations: RBC = red blood cell, Hb = hemoglobin, and DFP-8H = tritiated diisopropylfluorophosphate.t Calculated from transfusion requirement, red cell life-span, or both.- Two-day pool.

1658

ASSOCIATION OF THE UROBILIN "EARLY PEAK" ANDERYTHROPOIESISIN MAN

0

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FIG. 1. ERYTHROKINETIC STUDIES OF TWOHEMATOLOGICALLYNORMALPATIENTS, R.P. AND W.G. The radioironplasma disappearance and red cell (RBC) uptake, used here as an index of the rate of erythropoiesis, are shown as

lines connecting the circles; the bar graphs of urobilin-Y4C specific activity represent the "early peak."

HOURS2 3 4 5 6 0

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C14

0Fcm

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2Jn. 1963 Munch 1963 / July 1964 .12^-

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FIG. 2. ERYTHROKINETIC STUDIES AND CLINICAL COURSEOF PATIENT A.H. The three studies illustrated above, from

left to right, indicate the radioiron and urobilin-14C data obtained before treatment of erythroid hypoplasia, after suc-

cessful treatment, and 18 months later. The three small arrows at the top margin of the clinical data indicate the

times when the glycine-2-1'C and radioiron studies were performed. The specific activity of the early peak of uro-

bilin-14C, represented by the bar graphs, varied with changes in the rates of plasma clearance and red cell incorpora-tion of radioiron.

1659

0

200 3

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r8 10 12 14

C4

HEMOGLOBINgm%

RETICULOCYTES 10.Ci% 5.C

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(09/day) 2!

TESTOSTERONE1201tmq/wk)

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I .


Results

Clinical course and erythrokinetic data for eachpatient may be found in Figures 1-6. Case his-tories are in the Appendix. The data have alsobeen tabulated in Table I.

Three of the four patients were studied duringtwo or more phases of their illnesses. For com-parison, two hematologically normal individuals,R.P. and W.G., were also studied (Figure 1).R.P. had a localized carcinoma of the tongue, andW.G. had psoriasis. Their respective ferrokineticstudies were as follows: plasma radioiron disap-pearance half-time, 68 and 52 minutes; and maxi-mal red cell 59Fe incorporation, 83 and 91%o.Plasma iron turnover (PIT) values were 0.53

100ri 90

480

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4 0oi 406 8

2 420

and 0.40 mg per kg per day, and red cell ironturnover (RCIT) -was 0.43 and 0.37 mg per kgper day. Two weeks after injection of 100 ,uc ofglycine-2-1'C, the hemin-"4C specific activitieswere, respectively, 110 and 153 dpm per mg, andin two other hematologically normal patients thevalues were 115 and 152 dpm per mg. The peakspecific activities of the urobilin-14C were 142 and223 dpm per mg. These maximal values for theurobilin-14C specific activity in hematologicallynormal individuals compare favorably with themaximal specific activity of 91 dpm per mg ob-tained by White (18) in a normal adult after theintravenous injection of 100 pc glycine-2-14C.Two additional hematologically normal subjectshave been studied by Gray and Scott (7) and

NOV 19F6I

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12

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453015

SEPT 1961 NOV. 1961 -

- t t t I

0 00 50 100

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FIG. 3. ERYTHROKINETIC STUDIES AND CLINICAL COURSEOF PATIENT S.S. At thetime of the first study, when the patient had profound erythroid hypoplasia, there wasvery little incorporation of isotopic precursors into red cells or the early peak. How-ever, after treatment, red cell production returned, and the amount of isotopic glycineincorporated into hemin and into the urobilin-14C early peak was greater than normal.

1660

ASSOCIATION OF THE UROBILIN "EARLY PEAK" AND ERYTHROPOIESISIN MAN

Israels and Zipursky (19); one was given 25 uLcglycine-2-l4C orally and the other, 50 ,.c intrave-nously. The maximal observed fecal urobilinspecific activities can be extrapolated to a dose of100 lic, assuming factors of 4 and 2, respectively,to yield calculated peak fecal urobilin specific ac-tivities of approximately 75 and 286 dpm per mg,respectively.

Patient A.H. (Figure 2), a 57-year-old manwith chronic lymphocytic leukemia, was found tohave almost complete erythroid aplasia of the bonemarrow at the time of the initial study (January1963). The plasma radioiron disappearance half-time was 390 minutes, and there was virtually noincorporation of radioiron into red cells and of gly-cine-14C into urobilin and hemin. The specificactivity of the urobilin was 22 dpm per mg, andthat of the hemin was 0 dpm per mg. PIT was0.31 and RCIT 0.005, mg per kg per day. How-ever, after treatment with prednisone and testos-terone a reticulocytosis occurred, and a second studywas performed (March 1963). Plasma radioirondisappearance half-time was 57 minutes, with amaximal radioiron red cell uptake of 82%. PITwas 0.74 and RCIT 0.61, mgper kg per day. Thespecific activity of hemin-14C after 2 weeks was404 dpm per mg, and the maximal specific activityof urobilin-14C was 1,291 dpm per mg, which isgreatly elevated. The patient was studied again inJuly 1964. Plasma radioiron disappearance half-time was 131 minutes, and maximal radioiron redcell uptake was 61%o. PIT was 0.63 and RCIT0.38, mg per kg per day. The specific activity ofthe hemin-14C was 196 dpm per mg, and hemin-14C red cell life-span was 86 days. The specificactivity of urobilin-14C reached a maximum of596 dpm per mg.

Patient S.S. was a 45-year-old woman withchronic lymphocytic leukemia. In the first study(Figure 3) her bone marrow showed almost com-plete erythroid aplasia. Ferrokinetic studiesshowed a prolonged plasma radioiron disappear-ance half-time of 293 minutes, with no radioironincorporation into circulating red cells. The maxi-mal specific activity of urobilin-"4C was 48 dpmper mg. The second study was done shortly afterthe peak of reticulocytosis, when the bone marrowcontained 10%o erythroid precursors, many ofwhich showed megaloblastoid changes. A radio-iron study was not performed, but the maximal

J. F

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TRANSFUSIONS 3units of blood 2

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40PREDNISONE 30

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-10aC)C M

toz

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[. ..:..... ...

10 20 30 40DAYS

FIG. 4. ERYTHROKINETICSTUDIES ANDCLINICAL COURSEOF PATIENT J.F. This patient with aplastic anemia wasstudied on only one occasion. The maximal specific ac-tivities of the hemin-j'C and urobilin-14C were both ap-proximately 10% of the values found in normal indi-viduals after a similar amount of glycine-2-"C.

specific activity of hemin-14C was 461 dpm per mg,which is greater than normal (20). The specificactivity of the urobilin-'4C early peak was alsogreater than normal, with a maximal value of 459dpm per mg.

J.F., a patient with aplastic anemia (Figure 4),was found to have hypoplastic bone marrow.Glycine-'4C incorporation into hemin decreasedmarkedly (9 dpm per mg). Maximal urobilin-'4Cspecific activity was very low (15 dpm per mg).

Patient L.F. had refractory anemia with eryth-roid hyperplasia of the bone marrow, but at au-topsy a diagnosis of multiple myeloma was made.Over 21 years, four studies were performed onthis patient. In the first (Figure 5, November

1661


L. FHOURS

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TESTOSTERONE(mg /wk)

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50 150 200 400DAYS

FIG. 5. ERYTHROKINETIC STUDIES AND CLINICAL COURSEOF PATIENT L.F. Four studies were performed on L.F., apatient with refractory anemia. At the time of the first study, there was a large transfusion requirement and an ex-tremely high specific activity of the urobilin-G'C early peak. The second study, performed after treatment, showedthat the red cell production had returned towards normal and the specific activity of the urobilin-J"C early peak waswithin the normal range. The third and fourth studies are shown in Figure 6.

1961), plasma radioiron disappearance half-timewas normal (87 minutes), but red cell radioironuptake was low (14%o). PIT was 1.06 and RCIT0.15, mg per kg per day. The maximal specificactivity of urobilin-14C was 1,650 dpm per mg,which is markedly elevated. A hemin- 4C red celllife-span was 52 days, with a maximal specificactivity of 30 dpm per mg. In November 1962(Figure 5), after treatment with testosterone, thepatient's plasma radioiron uptake was 60%o.PIT at this time was 0.72 and RCIT 0.43, mgperkg per day. Red cell life-span (DFP-5H) was131 days. The specific activity of urobilin-14C was125 dpm per mg, which is within the normal range,

and the maximal specific activity of hemin-l4C wasslightly low (70 dpm per mg) (20). In May1963 (Figure 6), when the patient's rate of eryth-ropoiesis had decreased, plasma radioiron disap-pearance half-time was 154 minutes, and red celluptake was 40%o. PIT was 0.40, and RCIT 0.18,mgper kg per day. The 51chromium red cell sur-vival half-time was 33 days. Maximal specific ac-tivity of urobilin-14C was 343 dpm per mg, andthat of hemin-14C was 110 dpm per mg. In Janu-ary 1964 (Figure 6), when the patient had a largetransfusion requirement, plasma radioiron disap-pearance half-time was 120 minutes, with a redcell radioiron uptake of 15%o. PIT was 0.76 and

1662


L F* 2 3 4

100 ! -- t- --- I-Z \ MAY 1963L USA PLASMA

L801 \TiTV,' 154 minW BO\-a.0

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TESTOSTERONE(mg/wk)

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2 0-

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FIG. 6. ERYTHROKINETICSTUDIES AND CLINICAL COURSEOF PATIENT L.F. The third and fourth studies in L.F., whohad previously responded to treatment, were performed when red cell production decreased and the patient becamerefractory to treatment. Concomitantly, the specific activity of the urobilin-A'C early peak increased to levels greaterthan normal.

RCOT0.10, mg per kg per day. Maximal spe-

cific activity of urobilin-14C was 502 dpm per mg,

and that of hemin-14C was 52 dpm per mg.

Discussion

The early labeled peak was first observed byGray and co-workers (2) and London and co-

workers (1) in 1950. Both groups felt that theearly peak was probably related to erythropoiesis.Subsequent studies in disease states with increasederythropoiesis and after an experimental hemor-rhage indicated that the maximal specific activitywas increased when the rate of erythropoiesis was

increased (6). However, James and Abbott (5)reported studies performed on a patient in whichan increased early labeled peak was found at a

time when the bone marrow showed erythroidaplasia.

The present study was designed to determinewhether the early peak in man is in fact relatedto erythropoiesis by measuring the incorporationof glycine-2-14C into the urobilin-14C early peakat a time when erythropoiesis was markedly re-

duced or absent as judged by bone marrow exami-nation, reticulocyte count, radioiron plasma clear-ance and red cell uptake, and glycine-2-"4C in-corporation into red cell heme.

-J00- 90 _

zw 5080a-w 70a-o 306z'r 50-L*J-Ju

or 40-t-

u' 30

a-

1663

a


Before treatment three patients (A.H., S.S.,and J.F.) were found to have a very small earlylabeled peak when erythropoiesis was markedlyreduced and erythroid hypoplasia of the bone mar-

row was present. After treatment had restorederythropoiesis in two of these patients (A.H. andS.S.), the early labeled peak of urobilin was foundto be increased above normal and markedly in-creased in comparison with the studies performedwhen erythropoiesis was absent. The third pa-

tient (J.F.) did not respond to therapy.Patient L.F., in contrast to the preceding three

patients, initially had erythroid hyperplasia of thebone marrow. In his first study (Figure 5, No-vember 1961), the presence of anemia and the de-creased incorporation of radioiron into circulatingred cells, together with a normal plasma radioirondisappearance half-time, a markedly elevated earlypeak, and erythroid hyperplasia, suggest that a

large amount of ineffective erythropoiesis was

present. Maximal specific activity of the earlypeak was 1,650 dpm per mg, which is much higherthan the normal range of 75 to 286. A year later(November 1962), the second study was per-

formed. The patient did not require transfusions,and the ferrokinetic studies were normal, suggest-ing that the synthesis of red cells had returnedtoward normal. The early peak had also fallento normal levels. In the ensuing months the pa-

tient's effective red cell production slowly de-creased, as evidenced by anemia and an increas-ing transfusion requirement. Concomitantly, thestudies in May 1963 and January 1964 (Figure6) showed an increasing urobilin-14C specific ac-

tivity, suggesting that the bone marrow was syn-

thesizing red cells in adequate numbers but thatthere were defective cells that were catabolizedeither in the marrow or shortly after their re-

lease into the peripheral blood. The hemoglobin-14C contained in the defective red cells was pre-

sumably converted to urobilin-14C, and thus an

increased isotope content of the early peak was ob-served. This combination of findings can be desig-nated as ineffective erythropoiesis.

When patients A.H., S.S., and J.F. had eryth-roid hypoplasia of the bone marrow, essentiallyno erythropoiesis, and a low early peak, one may

speculate that the metabolic defect was in the syn-

thesis of hemoglobin. However, in patient L.F.with bone marrow erythroid hyperplasia, an ele-

vated early peak, and a normal radioiron plasmaclearance, it is likely that the defect occurred at alater stage and could involve the mechanism ofnuclear extrusion. Electron microscopic studiesby Bessis, Breton-Gorius, and Thiery (21) sug-gested that 5 to 10%o of the reticulocyte cyto-plasmic hemoglobin is lost at the time of nuclearextrusion, and Hammel and Bessman (22) haveindicated that the nucleus itself may be the siteof a significant amount of hemoglobin. It is pos-sible that if the nuclear extrusion mechanism wentawry, large amounts of labeled hemoglobin wouldbe found. It is also possible that in the normalindividual the hemoglobin contained in the peri-nuclear cytoplasm and in the nucleus itself couldrepresent a major source of the early peak of la-beled urobilin, a possibility that would be consist-ent with our studies in which changes in theamounts of labeled precursor in the early peakwere associated with changes in the rate of ef-fective erythropoiesis.

After the injection of glycine-2-14C, Yamamoto,Skanderbeg, Zipursky, and Israels (23) found twopeaks of isotopic incorporation in plasma bilirubin,one occurring within the first day and the secondduring the fourth day. The occurrence of the sec-ond peak corresponds to the time of nuclear ex-trusion of the group of red cells that would con-tain the largest amount of an isotopic precursor(24). Evidence that labeled bilirubin appears indog fistula bile before labeled heme can be detectedin the bone marrow has also been presented (4),suggesting that a nonheme pathway for labeled bilepigment might exist. However, it is more likelythat various heme-containing enzymes with rapidturnover times, such as those in the liver, may con-tribute to the early labeled peak, and it may be thissource that accounts for the first of the two plasmabilirubin peaks. This concept is supported by re-cent studies in bile-fistula dogs (25, 26) in whichthe early peak of bile bilirubin was separated intoerythropoietic and nonerythropoietic fractions bystimulating or suppressing erythropoiesis. It wasfound that in the normal state one-half to two-thirds of the early peak was of erythropoietic ori-gin, and that the remainder could be accounted forby the radioactivity present as hepatic nonhemo-globin heme. The presence of a hepatic contri-bution to the early peak of labeled urobilin in manis suggested by studies in which an early peak ap-

1664


peared in a normal individual after the ingestionof delta-aminolevulinic acid- 14 C, a poor in vivoprecursor of red cell heme (27), and also by theinitial studies in patients A.H. and S.S., in whomurobilin specific activity was found to be very lowbut never absent, whereas virtually no radioironwas incorporated into the red cells (Figures 2 and3). In man, however, pigment production asso-ciated with erythropoiesis certainly constitutes thebulk of the fecal urobilin early peak, as shown bythe extremely low urobilin specific activity in theabsence of erythropoiesis in patients A.H., S.S.,and J.F.

Theoretically, once the specific activity of theurobilin is known, it should be possible to calcu-late the total amount of precursor in the early peakif the total urobilin excretion is also known. How-ever, the quantitative fecal urobilinogen determina-tion is inexact, and meaningful calculations cannotbe made (6, 8). Alternatively, the amount of la-beled precursor in the early and late peaks could becompared, but this would require steady state con-ditions for more than 120 days and would be im-possible in patients without a "late" peak, such asthose with rapid rates of hemolysis or those inwhom the amount of labeled precursor incorpo-rated into circulating red cells is very small.These difficulties make it necessary for one toevaluate the early peak of labeled urobilin excre-tion in terms of the maximal specific activity.This is a valid concept if the precursor is givenintravenously over a short period of time and iffecal samples are analyzed in 1-day blocks as wasdone in this study. This method of comparisonrequires that the area under the curve be propor-tional to the maximal value and that the urobilinproduction be constant, thus making the maximalvalue the principal variable reflecting the totalamount of isotope in the peak. With these as-sumptions, the contribution to the early peak inman that is not due to erythropoiesis may be esti-mated as approximately 7 to 10%o (J.F.), 10 to15%o (A.H.), and 22 to 34%o (S.S.).Y

The possibility that the differences observed inthe urobilin specific activities before and aftertreatment could be the result of changes in the

3 These estimates represent a comparison of the maxi-mal urobilin-"C specific activities in these patients whenerythropoiesis was virtually absent to those observed incontrol patients R.P. and W.G.

total daily excretion of bile pigment was also ex-amined. Although fecal urobilinogen determina-tions were not obtained in every case, it was pos-sible to estimate the rate of pigment excretion fromthe transfusion requirement and the rate of changeof venous hemoglobin concentration. When wemade these calculations, we found that the maxi-mal change in total bile pigment excretion wastwofold, whereas the smallest change in the pre-and post-therapy urobilin maximal specific activi-ties was ninefold. Thus-despite some variationin the total pigment excretion-because of thelarge change in the specific activity before and af-ter treatment, it is clear that the maximal urobilinspecific activities are a presently acceptable reflec-tion of the rate of isotope excretion.

SummaryAn "early peak" of labeled fecal urobilin excre-

tion occurs during the first few days after the in-travenous administration of glycine-2-14C to man.To determine if this early peak is associated witherythropoiesis, we gave four patients with hemato-logic diseases and two hematologically normal pa-tients glycine-2-14C, a precursor of bile pigment,and thereafter made determinations of the amountof radioactivity in the fecal urobilin. Three pa-tients were studied when erythropoiesis was vir-tually absent, and in all three instances the specificactivity of the fecal urobilin-14C was markedly re-duced when compared to hematologically normalsubjects. Two patients (S.S. and A.H.) werestudied a second time during a period of reticulo-cytosis and rising hemoglobin concentration. Themaximal specific activity in the urobilin-14C in-creased approximately tenfold in patient S.S. and60-fold in A.H. Thus, the maximal urobilin-14Cspecific activity was shown to be low initially whencompared with normal and to increase markedlywhen erythropoiesis was active. This suggeststhat a small early labeled peak is present in manin the absence of erythropoiesis but that the bulkof the peak is associated with red cell formationin the marrow in these patients. A fourth patientwith anemia and normoblastic hyperplasia of themarrow was studied four times. In this patientthe maximal urobilin-14C specific activity wasshown to decrease by approximately 13-fold con-comitant with return of the bone marrow towardnormal and return of effective erythropoiesis.

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AcknowledgmentsWe thank Drs. Cecil J. Watson and Samuel Schwartz

and their laboratory staff, Irene Bossenmaier, Ruth Car-dinal, and Mary Weimer, for generous advice and en-couragement. In addition, we thank Dr. John J. Scottfor help in the initial phases of the study and JeanneWaggoner and Donald Van Houten for technical as-sistance.

Appendix

Case histories of patientsA.H. (NIH no. 02-63-51), a 57-year-old white man,

was found in 1959 to have a leukocyte count of 290,000per mm', with 90% lymphocytes, and a bone marrowcharacteristic of chronic lymphocytic leukemia. He wastreated in 1960 and 1961 with a total of 160 roentgens ofwhole body X-irradiation. In July 1962, the hemoglobin(Hb) was 12.0 g per 100 ml, but by November it haddropped to 5.0, and in January 1963, at the time of ini-tial study, it was found to be 2.9 g per 100 ml. His bonemarrow showed 90% lymphocytes and no erythroid cells.He was treated with prednisone, but because there wasno apparent response after 3 weeks, intramuscular in-jections of testosterone 4were begun. After 2 weeks ofthis treatment, an increase in reticulocyte count wasnoted, and a second study was performed. The bonemarrow was hypercellular with 35% lymphocytes and45% erythroid precursors. One and one-half years later,at the time of the third study, the Hb was 12.6 g per100 ml, reticulocyte count was 1.5%, and bone marrowaspiration revealed a hyperplastic marrow packed withlymphocytes and containing abundant reticulin and col-lagen fibrosis. Occasional nortnoblasts were seen.

S.S. (NIH no. 01-49-65), a 41-year-old white woman,first sought medical attention in 1956 because of fatigue.She was found to have cervical adenopathy, splenomegaly,and profound anemia. The leukocyte count was 1,000 permm', with 80%o lymphocytes, and the reticulocyte countwas 0%. Her bone marrow showed normal cellularitywith infiltration by small lymphocytes; a diagnosis ofchronic lymphocytic leukemia was made. Chemotherapywas given, and subsequently the cellular elements of theblood increased and transfusions were no longer neces-sary. In late 1959 pancytopenia again developed but wassuccessfully treated by splenectomy. In mid-1960, how-ever, transfusions again became necessary. The bonemarrow showed a striking decrease in the number oferythroid precursors, with a myeloid: erythroid ratio of20: 1. During the summer of 1961, the patient wastreated with a testosterone preparation 5 with no ap-parent effect. The first of the patient's two studies wasdone at this time. In October the patient began on tri-amcinolone,6 and 1 week later reticulocytosis began. The

'Depo-testosterone cyclopentylpropionate, Upjohn Co.,Kalamazoo, Mich.

5 Halotestin, Upjohn Co.6 Aristocort, Lederle Laboratories, Pearl River, N. Y.

second study was done during reticulocytosis. The pa-tient developed chronic otitis media and died with asepticemia in February 1962.

J.F. (NIH no. 04-19-59), a 17-year-old Negro malestudent, was admitted in April 1962 with a diagnosis ofidiopathic aplastic anemia. No abdominal organs werepalpable, and an axillary lymph node biopsy was nor-mal. The Hb was 8.7 g per 100 ml, the platelet countwas 5,000 per mm, and the leukocyte count was 800 permm', with 90% lymphocytes and 8% polymorphonuclearleukocytes (PMN). The reticulocyte count was 0, andthe bone marrow was markedly hypocellular, with 20%onormoblasts and 66% lymphocytes. Prednisone andtestosterone4 were given without apparent response.The patient died several weeks after discharge.

L.F. (NIH no. 03-92-55), a 65-year-old white engineer,was first admitted in November 1961, with refractoryanemia. There was no adenopathy or organomegaly.The transfusion requirement was 2 U per month, thereticulocyte count was 1%, the platelet count was 82,000per mm', and the leukocyte count was 3,000 per mm8,with 30% PMNand 70% lymphocytes. There were 75nucleated red cells per 100 leukocytes. Moderate aniso-cytosis and poikilocytosis of the red cells were present,and the mean cell volume was 108 A'. The bone marrowwas moderately hypercellular, with occasional megalo-blastoid cells. Seventy-three per cent of the marrowcells were in the erythroid series, with abundant sidero-blasts. No periodic acid-Schiff positive cytoplasmic in-clusions were seen in the erythroid cells. Injections ofa long acting testosterone preparation 4 were begun, andprednisone was given until May 1962.

The patient was readmitted in November 1962. Notransfusions had been necessary during the preceding 5months. The Hb was 15.2 g per 100 ml, the reticulocytecount was 0.5%, and the leukocyte count was 3,800 permm', with 60% PMNand 28% lymphocytes. The meancell volume was 110 j?. The bone marrow hypercellu-larity was less marked, few sideroblasts were seen, andthe maturation of the granulocytic and erythrocytic se-ries was normal. The testosterone therapy was stopped.

The patient was admitted again in May 1963. In theinterim the Hb had fallen slowly to 12 g per 100 ml.The fecal urobilinogen was 81 mg per day, and serumprotein electrophoresis revealed a total protein of 6.3,albumin of 3.6, and gammaglobulin of 1.2 g per 100 ml.The bone marrow contained many sideroblasts, and therewas a mild shift to the left with 27% myeloblasts. Tes-tosterone injections were reinstituted.

Transfusions became necessary in October 1963, at therate of 3 to 4 U per month. The patient was readmittedin January 1964. The fecal urobilinogen was 419 mg perday. Bone marrow was hypercellular, with 40% of thecells present as abnormal erythroid elements with megalo-blastoid changes. Many sideroblasts were present. Thetotal protein was 6.6 g per 100 ml, and the albumin was3.5 g per 100 ml. Unsuccessful trials of a number ofhematinic agents were given, and the patient was thenbegun on prednisone in addition to testosterone. After 4months no improvement was noted on this regimen. 'Be-

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cause of dysuria the patient was admitted to another hos-pital, where serum protein electrophoresis revealed a

total protein of 7.8 g per 100 ml, with 32% albumin and39% gamma globulin (broad band). The patient diedsuddenly after a prostatic biopsy. Autopsy examinationrevealed a hyperplastic marrow with many plasma cells,and a diagnosis of multiple myeloma was made.

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the association of the urobilin early erythropoiesis man · the term "urobilin" is used...

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