Relation Between Severity of Anemia and Erythropoietin Titer in Human Beings

Patients with severe hematologic disorders may have elevated erythropoietintiters in plasma or urine at higher hemoglobin concentrations than those associated with elevated titers in experimental animals or patients anemic as aresult of simple blood loss. Patients with "primary" hematologic disease mayhave a measurable titer of erythropoietin in the plasma and urine at hemoglobinconcentrations up to 8 Gm./100 ml., but patients with iron-deficiency anemiashow elevated titers in the urine only with hemoglobin concentrations at orbelow 4 Gm./100 ml. and in the plasma below 5 Gm./100 ml. The abruptnesswith which the titer rises and the severity of the anemia required beforemeasurable titers appear are similar in man and in rabbits, sheep and dogs.The fact that no measurable erythropoietin titer can be demonstrated whenthe hemoglobin concentration is well below that required for intense stimulation of erythropoiesis and the abruptness of the rise at hemoglobin concentration of 4 Gm./100 ml. or less suggest that erythropoietin may not bean important factor in the control of erythropoiesis except in extreme circumstances. On the other hand, these facts may be explained more simply by theinsensitivity of the assay methods used.

Submitted on March 13, 1961 Accepted on May 6, 1961     

Sickle Cell--Hemoglobin C Disease: Quantitative Determination of Iron Kinetics and Hemoglobin Synthesis

Quantitative determinations of iron kinetics and hemoglobin synthesis weremade on five patients with sickle cell-hemoglobin C disease. The anemia wasmild in all patients but one who had the hemoglobin of 10.1 Gm. per cent.All patients were in a steady state during the period of this study.

The ferrokinetic determinations demonstrated a hemolytic process in allcases. The mean erythrocyte life span in these patients was 18, 20, 29, 46and 56 days respectively (normal range, 110-130 days). The hemoglobinsynthesis was increased in all. Reduced to terms of daily hemoglobin production per liter of blood, the values were 2.2 Gm., 2.9 Gm., 3.6 Gm., 5.8 Gm.and 6.6 Gm. The latter figure represents a five fold increase over the normalmean value of daily hemoglobin synthesis of 1.3 Gm. (normal range, 1.0 to1.6 Gm. of hemoglobin per liter of blood per day).

The results of the in vivo organ counts demonstrated a significant degreeof sequestration of red blood cells in the spleen of two patients. The questionof advisability of splenectomy in such patients was discussed.

Accepted on December 3, 1962     

OBSERVATIONS ON THE EFFECT OF MASSIVE DOSES OF IRON GIVEN INTRAVENOUSLY TO PATIENTS WITH HYPOCHROMIC ANEMIA

Massive doses of iron (from 0.608 to 1.32 Gm. as colloidal terric hydroxide orcolloidal ferric oxide) were given intravenously in single infusions to 8 differentpatients with hypochromic microcytic anemia. One patient was given a secondinjection after an interval of four months, so that nine administrations were made.The following observations were made:

1. The reticulocyte response was higher in each instance than would be expectedin oral therapy. In 3 additional patients in whom injection had to be discontinuedafter 0.070, 0.180, and 0.123 Gm. of elemental iron had respectively been given, thereticulocyte rises were higher than were the average responses reported by Heath¹⁸after optimal oral therapy. This at least suggests that "optimal" oral therapy doesnot provide a maximal stimulus to outpouring of reticulocytes from the bone marrow. Comparable doses of iron given to 3 control subjects with normal hemoglobinlevels did not cause a reticulocytosis.

2. The average rate of hemoglobin regeneration per 100 cc. of blood per day was0.224 Gm.; the lowest value was 0.16 Gm. and the highest 0.27 Gm. These figureswere calculated for the rise that occurred from the day of iron administration to thetime at which the rate of hemoglobin increase was obviously becoming slower.Since correction was not made for blood loss in 3 of the patients during the periodof regeneration, the figures for the rate of hemoglobin formation are lower thanthey otherwise would have been. Even so they are distinctly greater than thoseusually obtained following oral therapy (table 2), but no greater than is found in anoccasional patient given iron by mouth. The data suggest that the fastest rate ofhemoglobin regeneration that can be stimulated by iron in subjects with hypochromic anemia approximates 0.3 Gm. per 100 cc. per day.

3. Calculations indicated that from 71.8 to 99.7 per cent of the injected iron wasapparently used for the synthesis of hemoglobin. These figures are likewise lowerthan they would have been if several of the patients had not lost blood during therecovery period. The observation of other workers that parenterally administerediron is almost completely retained by the body and converted into hemoglobin wastherefore confirmed.

4. Toxic reactions to the injected iron are described in detail. They were severein all but two instances, and in 3 patients were so alarming that injection of ironhad to be discontinued. There can be no doubt that the reactions to iron parenterally administered in large doses are great enough to contra-indicate use of thismeasure as a therapeutic procedure.

     

Urethane-caused blood and bone marrow changes in agranulocytosis and panmyelopathy of the cat

The investigations reported in the present paper deal with the behavior of theperipheral blood and the bone marrow in urethane-produced agranulocytosis andpanmyelopathy in the cat. The principal conclusions follow:

1. A severe panmyelopathy can be produced regularly in cats by the administration of relatively small doses of urethane: viz., 0.05 to 0.1 Gm. per Kg.per day. The panmyelopathy is still reversible if the urethane is stopped earlyenough and if the animal is maintained with antibacterial therapy.

2. The time required for recovery of the bone marrow after cessation of urethane administration depends on the total dosage of the drug. After small dosesrecovery occurs rapidly; after large doses much more slowly. Individual sensitivity of the experimental animal also plays a role, however.

3. The effect of smaller doses of urethane (0.05 Gm./Kg./day) on the bonemarrow is to produce inhibition of maturation of granulocytopoiesis, and, to alesser degree, of thrombocytopoiesis. To a still lesser extent, there is also inhibition of maturation of the red cell series.

4. The effect of larger doses of urethane (0.1 Gm./Kg./day) on the bonemarrow, the effect on maturation is overshadowed by a generalized depletionof all marrow cells. Part of this effect is due to a decrease in the number of mitoses,with reduction of the mitotic index; as well as qualitative alterations in the mitotic patterns. It is probable that the effect on mitoses results in the formation ofcells which are incapable of life as well as in an overall decrease in the formationof cells.

5. In the cat, the neutrophilic granulocytes are most sensitive to the actionof urethane. The lymphocytes are next most sensitive; then the megakaryocytes,finally the erythroblasts. The eosinophils were remarkably insensitive.

6. The normal plasmacellular reticulum cells are relatively insensitive to urethane. This is in contrast to the neoplastic myeloma cells in human beings whichhave been reported to be sensitive to urethane.

7. The administration of folic acid or of vitamin B₆ to the experimental animals had no effect on the course of urethane agranulocytosis.

8. The normal granulocytopoiesis of the cat is about as sensitive towardsurethane as the pathologic granulocytopoiesis of chronic myeloid leukoses ofhuman beings. This is in contrast to the findings in other animals, and to normal granulocytopoiesis of man. The variation in behavior possibly indicatesvariations in enzyme systems in the various types of granulocytopoiesis, withonly certain enzymes being affected by urethane.

9. Further experiments are in progress to determine if the effect of urethaneon the bone marrow can be counteracted by means of other growth substances.It is hoped that better insight may thus be gained into the mechanism of actionof urethane.

     

A Comparison of the Percentage of Fetal Hemoglobin in Human Umbilical Cord Blood as Determined by Chromatography and by Alkali Denaturation

A comparison has been made of the determination of fetal hemoglobin inhuman umbilical cord blood by column chromatography and alkali denaturation. A careful study has also been made of the variables that control theaccuracy and precision of the methods. Minor modification has led to muchimproved control of the 1-minute alkali denaturation procedure.

The percentage of fetal hemoglobin in the umiblical cord blood of full terminfants has been found to cover a far narrower range than is commonly reported. By chromatography, the average is 85.5 per cent with a range from79 to 91 per cent that includes more than 95 per cent of normal full terminfants. By alkali denaturation, the average is 74.0 per cent with a range from63 to 87 per cent.

Possible correlations with several clinical parameters have been examined.The highest correlation by both methods of determination occurred in thegroup of 12 samples from infants with a duration of gestation less than 37weeks. In this group the linear correlation with weight was greater than 0.6.

The precision and accuracy of the chromatographic method recommend itin the study of such subjects as prematurity, twinning, dysmaturity, intrauterine growth retardation, and infants of diabetic mothers.

Submitted on November 16, 1962 Accepted on June 3, 1963     

A simplified technic of spectrography and its application to the study of intracellular hemoglobin

1. A simplified microspectrograph for use in the visible range is described.

2. A nonrecording microdensitometer for use with the above is discussed.

3. The Soret band of hemoglobin is demonstrable in individual erythrocytesand is not affected by fixing or staining.

4. The ratio of the hemoglobin concentration to cosin staining is constant inerythrocytes and the cytoplasm of immature red cells but not in the nucleus.

5. There is no detectable hemoglobin or verdoperoxidase in the granules present in the eosinophile granulocytes of the camel.

6. Both dried and fixed and stained erythrocytes can take up carbon monoxidefrom the atmosphere.

Submitted on November 7, 1950 Accepted on May 21, 1951     

Protein Synthesis in Rat Lymphocytes: Radioautographic Studies of Availability and Utilization of Labeled Amino Acids in Vivo

Injections of H³-methionine and H³-leucine were combined with radiochemical and radioautographic technics to study the availability time ofH³-methionine and the protein synthetic ability of rat lymphocytes in vivo.

Although 98.5 per cent of H³-methionine was removed from the serum5 minutes after injection, sufficient quantities persisted and/or re-entered theserum from tissues to cause increasing grain counts in radioautographs oflarge lymphocytes for 1 hour after isotope administration. A small amount ofadditional labeling occurred during the 2nd hour, but it is calculated thatlabeling is 97-98 per cent complete by 1 hour.

All of the large and medium lymphocytes were labeled in the thymus, lymphnode, and thoracic duct lymph at short intervals after injection of 4 µc./Gm.body weight of H³-methionine. Evidence is presented that protein synthesisoccurs in the nucleus as well as in the cytoplasm and that newly formed protein is equally distributed between daughter cells following mitosis. Previousimmunochemical studies are combined with information on generation timeand disappearance rates of radioactivity to suggest that large and mediumlymphocytes are constantly producing and releasing proteins. Large andmedium cells in lymph and lymph node are more active in this than aresimilar cells in the thymus. Evidence of reutilization of labeled metabolitesin the lymph node and especially in the thymus is discussed.

Although not all small lymphocytes were labeled by 4 µc./Gm. body weightof H³-methionine, it was shown that larger doses of isotope would label 100per cent of them. Small lymphocytes in thoracic duct lymph evidenced significant turnover of labeled protein during the 1st day after isotope administration.

Submitted on August 21, 1963 Accepted on November 9, 1963     

Hematologic observations on patients with sickle cell anemia sustained at normal hemoglobin levels by multiple transfusions

(1) Three sickle cell patients were sustained at normal hemoglobin levels for3-4 months by means of repeated transfusions of fresh blood.

(2) In response to transfusions, there was a decline in reticulocytes to normallevels during the first 5-7 days of observation. During periods in which the hemoglobin was maintained at high-normal or super-normal levels, the reticulocytevalues were depressed below the normal range. A distinct reticulocyte responsewas observed when the hemoglobin declined to approximately 11.0 Gm. per centfollowing cessation of transfusions.

(3) Employing anti-M differential agglutination, a simple exponential declinewas observed in the number of sickle cells in each patient’s circulation duringthe period of sustained normal hemoglobin concentration.

(4) The continued production of new sickle cells during the first week ofobservation complicated the interpretation of the differential agglutinationdata, but provided indirect evidence for the presence of an especially short-livedproportion of the patient’s cells. Support for this concept was derived from theradioactive iron utilization studies performed in Case 1.

Submitted on November 14, 1955 Accepted on March 9, 1956     

Prophylaxis of hookworm anemia-deficiency disease

1. In individuals severely infested with Ancylostoma or Necator, it is possible tomaintain the normality of blood value by the administration of a sufficient dose ofan iron salt.

2. The minimum dose necessary to maintain normality of the blood in an individual weighing 45 kilograms, with 1051 helminths, was 0.2. Gm. daily of ferrous sulfate, administered in mixture with manioc flour.

3. The patient observed became clinically normal two weeks after the beginningof blood regeneration up to the end of the trial period one year later. In this period,with the various doses of iron tried, hemoglobin varied from 8.0 to 11.0 per 100ml. of blood.

Note: ACKNOWLEDGMENTWe owe thanks to the kindness of our colleague, Dr. Genard Nobrega, for the case report and electrocardiographic study of the patient.

     

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     

Erythrokinetics in Cooley''s anemia

Blood production and destructions have been measured in four patients withCooley’s anemia. Methods employed included the determination of erythroid/myeloid ratio of the marrow, reticulocyte count, plasma iron turnover and redcell utilization, Cr⁵¹ survival and fecal urobilinogen. Rates of production obtainedby these measurements have been compared to normal.

Patients with Cooley’s anemia have been shown to have an increased turnover of hemoglobin constituents comparable to the maximal response seen inother hemolytic anemias. There is, howvever, a marked decrease in maximaldelivery of erythrocytes to the peripheral blood amounting to about 50 per centin the mildly anemic patients and 85 per cent in severely anemic patients. Therate of destruction of circulating erythrocytes was similar in the three patientsstudied. The severity of anemia was therefore largely related to the productiondefect.

It was concluded that the defect in Cooley’s anemia is not in total hemoglobin synthesis, but in the fabrications of circulating erythrocytes, which inturn have the associated manifestations of hypochromia, increased percentageof fetal hemoglobin and shortened survival time.

Submitted on May 23, 1956 Accepted on June 25, 1956     

Studies on abnormal hemoglobins. VIII. The gelling phenomenon of sickle cell hemoglobin: its biologic and diagnostic significance

1. When sufficiently concentrated sickle cell hemoglobin containing solutionsare exposed to a constant stream of CO₂ gas, the hemolysates gel. This gellingphenomenon is indicative of the presence of S hemoglobin and cannot be obtainedwith any other type of human hemoglobin in the absence of S pigment. Thelowest S hemoglobin concentration (Gm. per cent) of a hemolysate at which thegelling phenomenon can still be elicited is designated as its lowest gelling point.

2. A simple apparatus was developed to analyze the gelling phenomenon understandardized conditions. It could be shown that the lowest gelling points ofhemolysates prepared from erythrocytes of the sickle cell trait (containing A +S hemoglobins), of the "C variant" (containing C + S hemoglobins), and fromsickle cell anemia cells (containing S + F hemoglobins) differ distinctly. Furtherexperiments suggest that the presence of A hemoglobin decreases the minimalamount of S pigment required for gel formation, and that type C hemoglobinreduces this amount even further. F hemoglobin seems to exert no significantinfluence on the gelling phenomenon. Serum albumin is also capable of decreasingthe amount of S hemoglobin required for gelation.

3. A sickled erythrocyte is visualized as an S hemoglobin tactoid or gel, specifically influenced by the companion pigment which interacts with the S compound.Thus, in the sickle cell trait, a positive sickling test is not only caused by thepresence of S hemoglobin, but also by its interaction with A hemoglobin. Onlyin the sickle cell anemia cells does sickling seem to depend solely upon the interaction of the S hemoglobin molecules.

4. The readily demonstrable differences of the lowest gelling points of hemolysates prepared from the various types of sickling red cells form the basis of thediagnostic gelling test which distinguishes sharply between sickle cell anemia andsickle cell trait erythrocytes. By this procedure atypical cases of sickle cell disease,for example, those whose erythrocytes contain C hemoglobin, may also bedetected.

Submitted on April 21, 1953 Accepted on May 25, 1953     

Porphyria Cutanea Tarda Associated with Chronic Granulocytic Leukemia Treated with Busulfan (Myleran)

A patient with chronic granulocytic leukemia terminating in "blast crisis"and marked porphyria is presented. Quantitative porphyrin studies revealed:urinary uroporphyrin 8894 to 11,453 µg./24 hours, urinary coproporphyrin 107-486 µg./24 hours, fecal coproporphyrin 55-116 µg./Gm. dry weight and fecalprotoporphyrin 22-38 µg./Gm. dry weight. A review of the clinical aspects ofporphyria is briefly presented, together with some speculation as to the etiologyof the porphyria in the present case. The possibility is present that the busulfantherapy either initiated the disorder or triggered its development from a previously occult state. If so, this would be another manifestation of busulfantoxicity, of which pigmentation is the most common.

Submitted on September 13, 1963 Accepted on November 17, 1963     

An Attempt to Produce Hypersplenism in the Dog, Using Methylcellulose

(1) Hematological and histopathologic changes were studied in dogs andrats after infusions of solutions of methylcellulose.

(2) After 4 to 8 weeks of daily intravenous infusions of 0.6 Gm. of 400 centipoise methylcellulose, the dogs developed moderate anemia with a reductionof the mean red cell volume to 27 ± 4 cc./Kg., compared with 38.6 ± 3 cc./Kg.in the controls, and a reduction in the apparent red cell life span studied withCr⁵¹ from 24 ± 3 days in the controls to 18 ± 3 in the treated animals.

(3) Following the infusions in the dog there was an increase of the meanblood urea nitrogen from 15 mg. per cent to 96 mg. per cent, with only a smallincrease in the rat.

(4) At necropsy there was foamy cytoplasmic vacuolization throughout thereticuloendothelial systems of both rat and dog. In the dog, the spleens werepale and moderately enlarged. Large cells with vacuolized cytoplasm largelyreplaced the normal structures of this organ. In the markedly enlarged ratspleen there were islands of methylcellulose-filled cells, surrounded by a rimof lymphocytes and strikingly engorged red pulp.

In the rat there was vacuolization of the renal glomerular cells but no significant change in the tubules or interstitial tissue. In the dogs sacrificed oneweek after the termination of the methylcellulose infusions, foamy cytoplasmicdistention of the glomeruli and interstitial cells and tubular dilatation and cystformation were noted. Progressive fibrosis and hyalinization of the glomerulusand fibrosis and calcification and lymphocytic infiltration of the interstitialtissue were seen in the kidneys of dogs sacrificed from 3 months to 3 years aftertermination of the methylcellulose infusions.

(5) Although methylcellulose infusions produce splenomegaly and anemiain the dog, the associated uremia precludes this preparation as a model forthe study of hypersplenism.

Accepted on February 23, 1961     

Hb MANAWATU [α37(C2)Pro→Leu]: A NEW MILDLY UNSTABLE MUTATION AT AN INVARIANT PROLINE RESIDUE

α-Thalassemia is associated with a defect in the production of the α-globin chains of hemoglobin. There are four common varieties in decreasing order of severity: the hemoglobin (Hb) Bart's (γ₄) hydrops fetalis syndrome, hemoglobin H disease, “severe” heterozygous α-thalassemia or α-thal-1 trait and “mild” heterozygous α-thalassemia or α-thal-2 trait (1) and there is now evidence that these may be the result of deletion of 4, 3, 2, and 1 respectively of the α-globin structural genes (2, 3, 4). As a result of defective α-globin chain production, the umbilical cord blood of infants with Hb Bart's hydrops fetalis contains from 80 to 90 per cent Hb Bart's (1) and those with Hb H disease, about 26 per cent (5). In newborn infants with α-thalassemia trait, lesser amounts of Hb Bart's have been found (5, 6, 7, 8). Observations in Thailand suggest that they fall into two groups, those with about 5 per cent Hb Bart's and others with from 1-2 per cent (9). The former were designated to have α-thal-1 trait and the latter the α-thal-2 trait. However, there is uncertainty as to whether a clear-cut division between these two groups of infants can be made on this basis (1) and in the present study we have been unable to find a bi-modal distribution of Hb Bart's levels in the cord blood of Chinese infants with α-thalassemia trait.     

Brief Note: Tritiated Thymidine as a Cytocidal Agent in Human Leukemia

1. Two patients with acute leukemia had considerable decreases in leukemiccells in the peripheral blood as well as reduction in size of spleen and leukemicmasses after 10 injections of ³H-TDR given over a 5-day period. Each injectionwas 0.25 µc./Gm. body weight.

2. The pertinent aspects of cytotoxic effects of ³H-TDR are reviewed.

3. The radiation doses delivered to the nucleus are estimated from autoradiographic data.

4. Evidence is presented for the observed effects being due to ³H-TDR.

Submitted on July 19, 1965 Accepted on May 10, 1966     

Platelet determination by turbidimetry

A method to determine the amount of circulating platelets without the aid ofany special apparatus is described. The method consists in separating the platelets from the other blood elements by fractional centrifugation and in measuringthe light absorption of the platelet suspension in a photoelectric apparatus. Lightabsorption is standardized against the platelet volume found by the Van Allenthrombocytocrit, and results presented as platelet volume in 100 ml. of bood.

The error of the method was found to be 6 per cent. To test the consistencyof the method, normal dogs and patients of both sexes with different pathologicconditions were studied, the results agreeing with the data found in the literature. In dogs of both sexes, twenty males showed 0.50 ml. per 100 ml. blood witha standard deviation of ±0.15, and ten females 0.53 ml./100 ml. ±0.17; fiftytwo male patients showed 0.45 ml. per 100 ml. of blood with a standard deviationof ±0.20, and thirty-one females 0.44 ml./100 ml. ±0.23.

Still further to test the reliability of the method, a confirmation was undertaken of the fact of platelet increase in the postoperative period in major surgicalinterventions. Twenty-seven patients with 0.38 ml./100 ml. (±0.16 as standarddeviation) platelets showed soon after the operation 0.44 ml./100 ml. ±0.19 adifference not significant according to statistical tests; eleven days after theoperation, average platelets were 0.60 ± 0.21 ml./100 ml. a highly significantincrease.

Submitted on November 23, 1953 Accepted on December 29, 1953     

Studies on abnormal hemoglobins. III. The interrelationship of type S (sickle cell) hemoglobin and type F (alkali resistant) hemoglobin in sickle cell anemia

1. It could be demonstrated that of the three tested types of human hemoglobin—N (normal adult), S (sickle cell) and F (fetal)—only the reduced S compound shows tactoid and gel formation in sufficiently concentrated solutions.These physico-chemical phenomena may be used for the qualitative identificationof S hemoglobin.

2. The alkali resistant hemoglobin fraction, present in sickle cell anemiaerythrocytes (but not in trait red cells), was concentrated in purified form. Notactoid or gel formation could be elicited. Therefore, this alkali resistant pigment does not appear to be a variant of S hemoglobin. It seems probable thatsickle cell anemia erythrocytes contain two separate types of pathologic hemoglobin (S and F) which are not directly related to each other.

Submitted on June 21, 1951 Accepted on July 23, 1951     

An Isotopic Method for the Determination of Vitamin B12 Levels in Blood

Vitamin B₁₂ is normally present in plasma mainly bound to a specific binding protein. Addition of exogenous vitamin ultimately results in saturation ofprotein binding sites and excess vitamin remains in free form. The ratio offree to bound fractions thus quantitatively depends upon the concentration ofexogenous compound. This observation has been utilized to determine amountsof vitamin B₁₂ extracted from the blood of normal subjects and of patientswith certain pathologic conditions.

The method is simple and reproducible. The sensitivity of the method issuch that vitamin levels down to roughly 20 µµg./ml. may be evaluated usinglabeled vitamin B₁₂ of a specific activity of about 1 µc./µg. Repeated assays onidentical specimens of normal plasma have shown a reproducibility of about5-6 per cent. Results on 39 normal subjects gave a range of 330-1070 µµg./ml.with an average of 611 ± 167. Values observed in plasma taken from patientssuffering from pernicious anemia were around 100 µµg./ml. or less. Resultson subjects with other pathologic conditions are also presented and thelimitations of the method are discussed.

Submitted on April 11, 1962     

Conversion of cyanocobalamin to a physiologically occurring form

Results are presented which indicate that in guinea pigs cyanocobalaminundergoes conversion to hydroxocobalamin, in vivo and in vitro, at the approximate rate of 0.1-0.4 mµg./day/Gm. of liver. In boiled liver, no conversion wasfound.

The radioactivity excreted in human urine the first 12 hours after a therapeutic cyanocobalamin dose, on the other, is still cyanocobalamin. It issuggested that demonstrated metabolic differences between therapeuticallyused cyanocobalamin and hydroxocobalamin are explained, in part, by thetime needed to convert cyanocobalamin to hydroxocobalamin.

Submitted on June 11, 1965 Accepted on July 8, 1966