The Determination of Iron Absorption and Loss by Whole Body Counting

A technic for the study of radioiron absorption and loss is described employing an NaI (T1) crystal-detector whole body counter and 1-10 µc. Fe⁵⁹in 250 µg. elemental iron. Changes in whole body Fe⁵⁹ activity during thefirst few hours and the next 90-100 days after oral ingestion are describedand their significance discussed. Normal absorption with this technic rangesfrom 5.7-24.7 per cent of the administered tracer. In 14 patients with polycythemia vera, 12 previously phlebotomized and 2 with a recent history ofgastrointestinal hemorrhage, iron deficiency as evidenced by increased ironabsorption (20.6 per cent-96.9 per cent) correlates well with the extent ofpreceding phlebotomy, and relatively well with the plasma iron at the timeof study. Although other parameters reflect iron deficiency, none correlatewell with the absorption of radioiron. Next to increased iron absorption, depletion of iron stores in the marrow seems to be the earliest evidence of irondeficiency.

Iron absorption and erythrocyte incorporation of radioiron was also studiedin several other hematologic disorders, including four heavily menstruatingwomen, three cases of aplastic anemia, and a small number of other conditions.The findings are described and discussed.

Radioiron loss in three normal patients was 0.110 per cent, 0.110 per cent,and 0.182 per cent daily, and in two patients with aplastic anemia 0.103 percent and 0.173 per cent daily, defining the normal range of tracer loss overdays 20-100. Radioiron loss in the polycythemics ranged from 0-0.044 per centdaily. An unusual case of pyridoxine-responsive anemia with increased absorption of radioiron (69.1 per cent), but no red cell incorporation, lost only0.026 per cent/day. Some problems in the interpretation of such data arediscussed.

The results demonstrate the effectiveness of the technic of whole bodycounting in the study of various aspects of iron metabolism.

Submitted on December 26, 1961 Accepted on July 21, 1962     

Polycythemia in primary carcinoma of the liver

In a series of 176 consecutive patients with hepatocarcinoma an increasein red cell count and in hemoglobin significantly above normal levels wereencountered in 17 (10 per cent).

An investigation of 28 patients with hepatocarcinoma developing in acirrhotic liver is reported. In three of the patients (10 per cent), the redcell counts and hemoglobin levels were significantly above those encounteredin healthy Chinese.

In these 28 patients it has been shown that the plasma volume is increased,and this increase does not differ significantly from that encountered in uncomplicated cirrhosis of the liver. The total red cell volume, on the otherhand, is significantly greater than in uncomplicated cirrhosis of the liver. Whilethe mean total red cell volume in hepatocarcinoma is not significantly different from that in healthy controls, consideration of this finding in individualpatients shows that it was above normal in 17, normal in six, and in the remaining five it was below normal.

It is concluded that the polycythemia encountered is a true polycythemiasecondary to the development of the hepatocarcinoma. The expanded plasmavolume is considered probably attributable to the pre-existing cirrhosis ofthe liver. Unfortunately, in the course of this investigation we did not encounter a patient in whom the carcinoma had developed in a liver whichwas not cirrhotic.

Submitted on April 8, 1957 Accepted on November 18, 1957     

The Measure of Erythropoiesis in Anemias. II. The Immediate and Subsequent Loss of Transfused Erythrocytes in Healthy Subjects

1. Fresh and stored red cells, transfused in healthy subjects, disappear fromthe circulation during the first 24 hours at a rate in excess of that prevailing insubsequent 24 hour periods. When fresh red cells are transfused the excess firstday loss is approximately 3 per cent.

2. Comparative survival studies using the nonagglutinable red cell methodof Ashby have indicated that this excess immediate loss is not due to elutionof Cr⁵¹, but to actual loss of red cells.

3. When red cells are stored improperly or for an extended period of time,the excess loss occurs very rapidly, and at one hour is approximately 50 percent of the total loss at 24 hours. The limitations to the use of the dilutiondata of transfused stored cells to establish the 100 per cent value for themeasure of the survival of the transfused red cells are implicit.

4. When the first 24 hour loss of transfused red cells is approximately 30per cent or more, paradoxically the rate of loss in subsequent days is lessthan when fresh, undamaged red cells are transfused. This observation raisesthe question of interpretation of the measure of the red cell survival expressedas T .

Submitted on July 25, 1961 Accepted on October 17, 1961     

Hookworm Anemia: Iron Metabolism and Erythrokinetics

Iron metabolism, balance of red cell production and destruction and ironabsorption from hemoglobin were determined in 11 patients with heavy hookworm infection and severe anemia.

The plasma iron, total iron binding capacity, bone marrow hemosiderinand plasma Fe⁵⁹ clearance are in agreement with the idea that the anemia associated with hookworm infection is of the iron deficiency type.

The rate of red cell production measured by the E/M ratio, absolute reticulocyte count and plasma iron turnover showed an increase to about twicenormal, while the rate of destruction estimated by the T erythrocytesurvival showed a destruction about 5 times normal. This unbalance betweenproduction and destruction could explain the severity of the anemia.

The increase of fecal urobilinogen output to twice normal was interpretedas due to the metabolism of the hemoglobin lost into the intestine rather than toan increase of hemolysis.

The estimation of fecal blood loss in the patients whose red cells weretagged with Cr⁵¹ and Fe⁵⁹, showed that the radioactivity counted with Fe⁵⁹was only about 63 per cent of the radioactivity counted with Cr⁵¹. This difference was interpreted as due to iron absorption from the hemoglobin lostinto the intestine.

The mean daily fecal excretion of iron reaches 4.7 mg. Since the ironmetabolism in these patients is in equilibrium, we have concluded that theiron loss is replaced by the iron from food; this is in addition to the 3 mg.hemoglobin iron which is reabsorbed from the blood lost into the gut.

Submitted on January 9, 1961 Accepted on April 2, 1961     

A Study of Histamine in Myeloproliferative Disease

1. Whole blood histamine content was measured in 80 patients with myeloproliferative disease. Increased levels were found in 60 per cent of patientswith uncontrolled polycythemia vera, in 7 per cent of patients with polycythemia vera being controlled by myelosuppressive therapy, and in 71 percent of a group with "spent" polycythemia, myeloid metaplasia and myelofibrosis.

2. The excretion of histamine in the urine was measured in 60 patients,30 with elevated blood histamine and 30 with normal blood histamine. Theurine findings paralleled the blood findings in 90 per cent of the cases.

3. Measurements of cell-poor and cell-rich fractions of blood showed thatthe histamine is contained in the white cell fraction. Elevated basophil countswere present in 50 per cent of the patients and occurred with the greatestfrequency in the groups with elevated blood and urine histamine. A roughcorrelation between the basophil count and the histamine content of bloodand white cell fractions was observed in normal subjects and most cases withmyeloproliferative disease. Data obtained in some cases of myeloproliferativedisease suggest that the histamine content of the basophil may be abnormaland that other granulocytes may contribute to the total leukocyte histamine.

4. Myelosuppressive agents produced a reduction in histamine (expressedper 10⁹ myeloid cells) and a decrease in urine histamine as control of themyeloproliferative process was achieved. Treatment with phlebotomy aloneproduced no change in histamine levels.

5. The incidence of pruritus, upper gastrointestinal distress and urticarialmanifestations was increased 7-fold, 4-fold and 12-fold, respectively, in patients with elevated histamine levels as compared with those who had normalhistamine levels.

6. Cyproheptadine, a potent antihistaminic, successfully controlled pruritus,relieved pyrosis and suppressed urticarial eruptions in patients with elevatedhistamine levels. Suppression of the reaction to subcutaneously administeredcodeine (a histamine-releaser) afforded objective evidence that cyproheptadine blocked the effects of histamine release in vivo.

7. The metabolism of histamine and the role of elevated histamine levelsin the clinical manifestations and pathophysiology of myeloproliferative diseaseare discussed.

Submitted on September 23, 1965 Accepted on May 24, 1966     

Co60 Vitamin B12 Binding Capacity of Human Leukocytes

1. Mature neutrophilic leukocytes show the highest Co⁶⁰B₁₂ binding capacity.

2. Less mature granulocytes, "blast" forms and eosinophils have little or noCo⁶⁰B₁₂ binding capacity.

3. Disintegrated mature leukocytes from chronic myelocytic leukemia andpolycythemia vera show higher B₁₂ binding capacity than intact cells.

4. Mature leukocytes from patients with chronic myelocytic leukemia andpolycythemia vera show a two-phase B₁₂ curve suggesting specific and nonspecificbinding, similar to that observed in human serum.

5. Disintegration products from mature neutrophilic leukocytes probablycontribute largely to increased B₁₂ binding capacity of serum in chronic myelocyticleukemia and polycythemia vera.

Submitted on August 30, 1961 Accepted on October 17, 1961     

The Red Cell Chromium Elution Rate in Patients with Some Hematologic Diseases

1. The erythrocyte Cr⁵¹ elution rate was determined in 38 patients withhematologic diseases.

2. In four patients with finite red cell life spans, two exponential Cr⁵¹elution rate constants could be calculated. In the remaining 34 patients, thedata were consistent with a single exponential elution rate constant from day1 to day 30-40 following Cr⁵¹ administration.

3. The single elution rate varied from 0.62 to 2.27 per cent per day.

4. In two patients, the chromium elution rates determined on two separateoccasions were not significantly different. In a third individual, the chromiumelution rate constant was 0.75 per cent per day when the red cell life span was66 days and 1.07 per cent per day when red cell life span was 79 days.

Submitted on April 23, 1962     

Studies on Erythropoiesis. XI. Reticulocyte response of transfusion-induced polycythemic mice to anemic plasma from nephrectomized mice to and plasma from nephrectomized rats exposed to low oxygen

Mice with transfusion-induced polycythemia have been used to assayerythropoietic activity in plasma derived from mice and rats subjected tovarious stimuli and experimental procedures.

1. Anemic, anoxic, or cobalt mouse plasma increased erythropoiesis fromthe zero baseline to about the normal range after four 0.5 cc. injections.No significant effect is observed from the same number of injections of normalrat or mouse plasma or normal saline. Similarly, plasma from rats starvedfor 4 days had no erythropoietic activity, but erythropoietic activity waspresent in the plasma of pregnant mice during the last trimester of pregnancy.

2. Plasma from mice made anemic by phenylhydrazine and then subjectedto bilateral nephrectomy contained slight erythropoietic activity (less than0.2 per cent reticulocytes). The plasma of sham-operated anemic animals hadconsiderably more erythropoietic activity (2.0 per cent). The plasma wascollected for assay 10 hours after the operative procedures.

3. Plasma from nephrectomized rats exposed to hypoxic anoxia for 8, 16,or 24 hours, had slight erythropoietic activity when measured by the peripheralreticulocyte response of the recipients (0.18, 0.11, and 0.00 per cent reticulocutes, respectively). The same plasma had no erythropoietic activity whenjudged by the Fe⁵⁹ red cell incorporation response in starved rats.

Plasma from rats subjected to bilateral ureter ligation and similarly exposedhad an erythropoietic activity (0.7, 1.38, and 1.26 per cent reticulocytes, respectively), comparable with that of the plasma from normal rats exposed tohypoxic anoxia (0.9, 1.71, and 1.78 per cent, respectively). The low and inconstant erythropoietic activity in the plasma of nephrectomized mice andrats subjected to anemic or hypoxic anoxia is more or less comparable withthe response occasionally produced by normal plasma. We are, therefore,disinclined to consider the data as evidence against the renal origin oferythropoietins, but the alternative possibilities are discussed.

Submitted on July 28, 1958 Accepted on December 9, 1958     

Studies on the pathogenesis of splenic anemia

Splenomegaly was produced in rats by repeated intraperitoneal injections ofmethylcellulose. Anemia and marked reticulocytosis resulted. Coombs tests werenegative.

Comparative measurements of red cell life with Cr⁵¹ in normal, methyl-cellulose treated, and splenectomized animals showed a marked decrease of red cellsurvival in the methylcellulose rats. Slight but statistically insignificant increasedsurvival could be demonstrated in the splenectomized animals.

Excessive red cell destruction occurred in the spleen as evidenced by rapidlocalization of Cr⁵¹-tagged cells in this organ. Direct correlation between severityof hemolysis and splenic size could be demonstrated. The hemolytic processdecreased significantly after splenectomy.

Affected spleens showed red cell congestion of the pulp microscopically. Hemochromogen determinations revealed approximately twice the normal amount ofhemoglobin per unit splenic mass.

Quantitative measurements of splenic phagocytosis showed a marked increasesof total phagocytic mass with no significant increase of phagocytes per unit ofspleen mass.

The mechanism of splenic anemia in these animals is thought to be a combination of an enlarged pulp compartment with resultant stasis and cell destruction.

Methylcellulose-induced splenomegaly may be considered as an experimentalmodel for the study of the hyperfunctional spleen in human disease.

Submitted on March 28, 1956 Accepted on June 17, 1956     

Studies of the production and life span of erythrocytes in myeloid metaplasia

1. Four patients with agnogenic myeloid metaplasia and one patient withpolycythemia vera and myeloid metaplasia were studied with Fe⁵⁹, Cr⁵¹ andglycine-2-C¹⁴.

2. Three of the patients with agnogenic myeloid metaplasia had activesplenic, hepatic or renal erythropoiesis. One had deficient erythropoiesis.

3. The red cell life span was short in all of the patients with agnogenicmyeloid metaplasia, definite splenic sequestration occurring in two patients.The red cell life span was normal in the patient with polycythemia vera andmyeloid metaplasia.

4. The possible indications for splenectomy were discussed.

Submitted on July 25, 1958 Accepted on October 12, 1958     

Haptoglobin Metabolism in Polycythemia Vera

1. Turnover studies with ¹²⁵I-labeled haptoglobin (Hp) were performed in7 patients with polycythemia vera, 2 patients with erythrocytosis of unknownetiology, and 2 control subjects.

2. The T of plasma radioactivity was shortened in 6 of the 7 patients withpolycythemia vera; 3 of these had diminished plasma Hp levels but lackedother evidence of hemolysis.

3. The fractional catabolic rate exceeded 40 per cent/day in all subjectswith a shortened half-time of plasma radioactivity.

4. Increases in the fractional catabolic rate were not accompanied by increases in Hp turnover (mg./kg./day), suggesting that accelerated Hp catabolism per se does not provide a stimulus to Hp production.

5. It is concluded that patients with polycythemia vera catabolize Hp morerapidly than nonpolycythemic subjects, possibly because of increased formation and removal of the haptoglobin-hemoglobin complex.

Submitted on November 5, 1968 Accepted on January 21, 1969     

The life span of the red blood cell and the red blood cell volume in the chicken,pigeon and duck as estimated by the use of Na2Cr51O4, with observations on red cell turnover rate in the mammal,bird and reptile

Eight to 25 per cent of intravenously injected Na₂Cr⁵¹O₄ binds firmly witherythrocytes of the chicken, pigeon and duck. Calculation of the maximum lifespan of these avian red cells was made from the disappearance time of circulatingradioactivity. The maximum life span of the chicken erythrocyte was found to be35 days, of the pigeon erythrocyte 35-45 days, and the duck erythrocyte 42 days.Comparing the life span of avian erythrocytes with those of other animal species,the rate of red cell turnover in the mammals, birds, and reptile (turtle) was foundto correlate directly with basal heat production per kilogram body weight.

Using erythrocytes tagged with Na₂Cr⁵¹O₄ in vitro, the total red blood cellvolume was found to be 17-24 ml. per Kg. body weight in the rooster, 9-12 ml.per Kg. in the hen, 25-31 ml. per Kg. in the duck, and 31-34 ml. per Kg. in thepigeon. These values proved somewhat lower than those obtained from theindirect estimates of red cell volume, using plasma volume figures and peripherablood hematocrit.

Submitted on June 25, 1956 Accepted on August 18, 1956     

Radioactive Iron Studies in Patients with Iron Deficiency Anemia with Concurrent Abnormal Hemolysis

A report is presented on two women suffering from severe iron deficiencyanemia, concomitant with abnormal hemolysis. The life span of the patients’red cells was shortened; transfused normal red cells were more rapidlybroken down. Extracorpuscular factors seem to be responsible for the pathologic hemolysis. There was no pathologic sequestration of red cells labeledwith Cr⁵¹ in the spleen.

After incorporation of radioactive iron into the red cells, the utilizationpercentage of Fe⁵⁹ under iron therapy fell to about 15 per cent within a fewweeks. This, too, indicates that the pathologic hemolysis was to be ascribedto extracorpuscular factors. The Fe⁵⁹ was apparently not sufficiently reutilized.The constantly decreased serum iron concentration might also indicate adisturbance in the reutilization of iron liberated during red cell breakdown.

Six other patients with less severe iron deficiency anemia and an insufficientresponse to iron therapy were examined in addition. In 5 of these patients,the life span of red cells labeled with radioactive chromium was found shorterthan normal. An insufficient response to iron therapy in patients with chroniciron deficiency anemia may be ascribable, in some instances, to concomitantpathologic hemolysis.

     

Erythrokinetic Studies in Severe Bone Marrow Failure of Diverse Etiology

1. Erythropoiesis was studied in seven patients with refractory anemia.

2. In all seven patients the total red cell volume was low and the plasmavolume elevated.

3. The serum iron was elevated and iron binding protein saturated in sixof seven patients.

4. The T for clearance plasma Fe⁵⁹ was decreased in one, normal in two,and increased in four patients.

5. The red cell iron turnover was decreased in six of seven patients.

6. Radioiron accumulated in liver and spleen.

7. Red cell life span was difficult to measure but probably shortened.

8. Androgen therapy in two cases was ineffective, and was associated witha slight effect in one patient.

Submitted on April 18, 1961 Accepted on September 14, 1961     

The binding of Cr 51 to hemoglobin. II. In vivo elution rates of CR 51 from Hb CC, Hb CS, and placental red cells

In vivo elution rates of Cr⁵¹ from red cells containing Hb C and fromplacental red cells containing large amounts of Hb F have been determined.These were found to be 1.8 and 0.85 per cent per day, respectively, and do notdiffer greatly from those of normal adult red cells. Therefore, no special correction factors for elution need be used in evaluating Cr⁵¹ survival curves inthese clinical situations. The in vivo elution rate of 3.5 per cent/day observedfor Hb C-S is significantly greater than that of normal red cells.

Submitted on December 1, 1965 Accepted on January 16, 1966     

Studies of the Fate of Platelets in Rats and Man

The tissue distribution of ⁵¹Cr-labeled platelets and erythrocytes was determined in normal and asplenic rats. Two hours after injection, the distributions in tissues of RBC and platelet ⁵¹Cr were not significantly differentexcept in the spleen which contained platelet ⁵¹Cr (12 per cent), significantlyin excess of red cell ⁵¹Cr (1.4 per cent). As labeled platelets were removedfrom the bloodstream, 80.2 per cent of the injected ⁵¹Cr was deposited in thespleen, liver and marrow of normal animals. During the same interval, radioactivity in all other sites examined diminished to less than 10 per cent ofinitial values. In splenectomized animals, hepatic uptake of radioactivity wastwice that observed in normal animals.

Studies in normal human subjects using external scintillation scanningsuggest that similar quantitative considerations apply to man in that, normally, one-third of total platelets are sequestered in the liver and one-thirdin the spleen while in asplenic individuals two-thirds are destroyed in the liver.

These observations suggest that the majority of platelets are cleared fromthe circulation by the reticuloendothelial system after becoming "senescent"but they do not elucidate the nature of "senescence" or the circumstancesleading to it.

Submitted on September 6, 1968 Accepted on March 27, 1969     

The Urinary Excretion of Orally Administered Tritium-labeled Folic Acid as a Test of Folic Acid Absorption

The urinary excretion of radioactivity following an oral test dose of tritium-labeled folio acid was determined in a group of 40 subjects which includedcontrols and patients with a variety of malabsorptive disorders. The resultswere compared with the occurrence of folic acid deficiency, as detected bysubnormal serum L. casei folate levels, and with a variety of other testsavailable for the evaluation of intestinal absorption, including the determination of folio acid absorption based on the microbiological assay of peakserum Str. faecalis folate concentrations following the oral test dose.

The urinary excretion of H³FA in 15 subjects with normal intestinal absorption ranged from 26.0 to 57.8 per cent of the oral test dose, with a meanof 40.7 per cent. The H³FA excretion in 25 patients with intestinal malabsorption ranged from 1.9 to 38.7 per cent, with a mean of 15.3 per cent.Twenty patients in this group excreted less than 26 per cent. Those five patients who excreted greater than 26 per cent were considered to have normalabsorption of folic acid since absorption studies of peak serum Str. faecalisfolate levels were within the normal range.

The results of the H³FA urinary excretion test correlated well with otherparameters of intestinal absorption, particularly in the instances of severemalabsorption, although three cases with malabsorption were observed inwhich H³FA excretion was subnormal and peak serum Str. faecalis folateconcentrations were normal. Folic acid deficiency was observed in 12 patients and its incidence appeared to be related to the severity of impairmentof folic acid absorption in the majority of cases.

It is concluded that the H³FA urinary excretion test is a simple, rapid andreliable index of folic acid absorption.

Submitted on October 11, 1962 Accepted on December 11, 1962     

Studies on the Erythropoiesis Inhibiting Factor in the Plasma of Animals with Transfusion Polycythemia

Further investigations of the action of polycythemic plasma filtrate were madeusing Fe⁵⁹ and with detailed examination of the blood and bone marrow. Thesestudies confirmed the appearance of an active thermostabile plasma factor(erythropoiesis inhibitor) which depressed erythropoiesis in normal rabbits orrats. The plasma obtained from bilaterally nephrectomized sheep subjectedto transfusion polycythemia also contained the erythropoiesis inhibitor. Blockade of the reticuloendothelial system using trypan blue, in sheep, inducedproduction of the active substance similar or identical with the erythropoiesisinhibitor produced after transfusion polycythemia.

Submitted on November 2, 1960 Accepted on October 10, 1961     

Red cell mass measurement in patients with clinically suspected diagnosis of polycythemia vera or essential thrombocythemia

The cut off for hemoglobin or hematocrit that indicates the need for an isotopic red cell mass study was investigated in 179 patients with a presumptive diagnosis of polycythemia vera or essential thrombocythemia. Hematocrit showed better diagnostic accuracy than hemoglobin. Hemoglobin over 18.5 g/dL in males or over 16.5 g/dL in females showed a high specificity indicating that red cell mass study could be avoided in such cases, but it showed low sensitivity leading to 46% false negatives. The best value of hematocrit to indicate a red cell mass study was 0.50 L/L in males (specificity 75%, sensitivity 87.5%) and 0.48 L/L in females (specificity 73%, sensitivity 94%). Lowering the hematocrit threshold to 0.48 L/L in males increased sensitivity up to 95%. A red cell mass study should be performed in patients with suspected diagnosis of essential thrombocythemia or polycythemia vera and with hematocrit between 0.48 L/L and 0.52 L/L.Key words: polycythemia vera, essential thrombocythemia, red cell mass, hemoglobin, hematocrit     

Blood destruction in the polycythemia induced by hypoxia

The hemoglobin catabolism during the development and during the disappearance of polycythemia induced by hypoxia was studied by measuring the totalcirculating hemoglobin and the daily bile pigment excretion in bile-fistula dogsbefore, during, and after prolonged periods of exposure to 20,000 feet simulatedaltitude.

1. The inscreased erythropoiesis during the first weeks of altitude exposure wasaccompanied by a signiflcant increase in bile pigment output. The possible sourcesof this pigment excretion are discussed.

2. The life spans of the red cells during altitude exposure was found to be about115 days. No differences were observed in the longevity of the cells in animals atground level and at altitude.

3. The normalization of the polycythemic blood levels took place within sixto eight weeks after returns to ground level, and was achieved by the combinedeffect of a depressed erythropoiesis and of an increased blood destruction. Theincrease in red cell destruction observed under these conditions demonstratesthe existence of an "active" mechanism of blood destrunction by which the organism is able to destroy normal blood cells before their life span is exhausted. Thisincreased red cell destruction, however, accounted for only 21 to 39 per cent ofthe hemoglobin which disappeared from circulation after return to ground level.The major part of the normalization of altitude polycythemia was brought aboutby a temporary depression of erythropoiesis which was estimated to amount to30 or 40 per cent of the normal cell production in the six weeks after the discontinuation of the altitude exposure.

Submitted on September 15, 1951 Accepted on October 25, 1951