Experimental anemias in the rat; I. Macrocytic anemia in chronic pteroylglutamic acid deficiency and after splenectomy in Bartonella muris infection

1. In agreement with findings by other workers, rats in acute pteroylglutamicacid deficiency showed leukopenia and growth depression followed by death, without any significant change in the red cell picture.

2. In chronic deficiency, however, produced by the addition of small pteroylglutamic acid doses given intermittently, a severe anemia was obtained afterseventy days.

3. The anemia was macrocytic and "normochromic." Price-Jones curves showeda preponderance of macrocytes with anisocytosis. This agreed with findings byother workers for other species.

4. The anemia could be cured by single doses of 40 µg. or more of pteroylglutamicacid.

5. There was no significant difference between sexes to pteroylglutamic aciddeficiency. Reduction in the protein content of the diets, containing 1 per centsulfasuxidine, from 18 per cent to 10.5 per cent, produced no significant differencein the time of onset and severity of the blood symptoms.

6. These results were not due to infection with Bartonella muris. This infectionproduced a macrocytic anemia of a different type, and was curable by treatmentwith neoarsphenamine.

Note: ACKNOWLEDGMENTSWe are grateful to Dr. T. H. Jukes of the Lederle Laboratories for generous supplies of aldehyde-freePGA; and to Dr. K. Folkers of Merck Laboratories for the biotin used in these experiments. We wish tothank Dr. W. Jacobson for his advice during the course of this investigation. Valuable technical helpwas provided by Mr. D. R. Ashby, Mr. S. G. Impey, Miss M. J. Kemp and Mr. P. W. Wilson, to whomthe authors are indebted.

     

Experimental production of a nutritional macrocytic anemia in swine

1. A deficiency of pteroylglutamic acid has been produced in 32 swine fed apurified diet containing casein and supplemented with seven B vitamins, sulfasuxidine and a folic acid antagonist. The casein was fed at two levels, 10 and 26per cent. Two types of casein were used: a crude preparation possessing significant"extrinsic factor" activity and a purified casein with little activity.

2. The hematologic manifestations observed were (a) severe macrocytic anemia,(b) leukopenia, due to a proportionately greater reduction in polymorphonuclearthan in mononuclear cells, (3) slight thrombocytopenia, and (4) hyperplastic bonemarrow with an increase in immature nucleated red cells which resemble themegaloblasts seen in the bone marrow of patients with pernicious anemia.

3. The feeding of a 26 per cent rather than a 10 per cent crude casein diet did notprevent but did delay the onset of the blood changes. Anemia developed mostrapidly in the animals receiving 10 per cent purified casein.

4. The group receiving 26 per cent casein developed a greater degree of macrocytosis in the same period of time than did the group receiving 10 per cent casein.In all groups the degree of macrocytosis increased as the duration of the anemiaincreased.

5. The hematologic manifestations were not delayed nor was their developmentprevented by the intramuscular administration of 15 U.S.P. units of liver extractevery 15 days.

6. The blood and bone marrow returned rapidly to normal following the administration of pteroylglutamic acid, pteroyldiglutamic acid, pteroyltriglutamicand pteroylheptaglutamic acid. Thymine and xanthopterin had little or no activity. Tyrosine, adenine and uracil were inactive.

7. Purified liver extracts and crystalline vitamin B₁₂ were found to possess somehemopoietic activity in several animals but the activity was considerably less thanthat of the pteroylglutamic acid compounds.

8. The urinary excretion of "tyrosyl" (hydroxphenyl compounds) was notabnormal in the pteroylglutamic acid deficient pigs and was not altered by eitherpteroylglutamic acid or liver extract therapy.

9. The urinary excretion of allantoin and uric acid did not differ significantlyfrom the normal. Immediately following therapy with pteroylglutamic acid,however, in association with the reticulocytosis and lasting for the same period,there was a marked increase in the excretion of allantoin.

10. The results suggest that both pteroylglutamic acid and a factor in liverextract similar to or identical with vitamin B₁₂ are required for normal hemopoiesisin the pig.

Note: ACKNOWLEDGEMENTSThe crude methylfolic acid antagonist, xanthopterin, and the pteroylglutamic acid compounds, withthe exception of pteroylheptaglutamic acid, were kindly furnished by the Lederle Laboratories, PearlRiver, New York, through the courtesy of Dr. T. H. Jukes and Dr. S. M. Hardy.Sulfasuxidine was generously furnished by Sharp & Dohme, Inc., Philadelphia, Pa., through thecourtesy of Dr. W. A. Feirer.Pteroylheptaglutamic acid and Natola were supplied by Parke, Davis & Company, Detroit, Mich.,through the courtesy of Dr. A. E. Sharp and Dr. J. J. Pfiffner.Biotin was obtained from Hoffmann-LaRoche, Inc., Nutley, N. J., through the courtesy of Dr. E. L.Sevringhaus.The vitamins, with the exception of pteroylglutamic acid and biotin and including vitamin B₁₂ werekindly furnished by Merck and Company, Inc., Rahway, N. J., through the courtesy of Dr. A. Gibsonand the late Dr. D. F. Robertson.Experimental liver extracts (No. 1124, 1063, 1066 and 1067) were generously furnished by Armour andCompany, Chicago, Illinois through the courtesy of Dr. E. E. Hays.We are indebted to Mrs. Darlene Kehl, Mr. George Trappett, and Mr. Ocie Hadley for technicalassistance.

     

THE ACTION OF PTEROYL GLUTAMIC ACID AND NATURAL SOURCES OF FOLIC ACID ON BLOOD DYSCRASIAS INDUCED BY SULFONAMIDE DRUGS

Sulfanilamide, sulfathiazole, and sulfadiazine have been fed at 1 per cent levelsin highly purified diets and their effect on growth, mortality, and blood dyscrasiascompared with that of sulfasuxidine.

The soluble drugs produce conditions which are similar to those produced bysulfasuxidine. The growth depression is alleviated in large measure in the case ofsulfanilamide and to a lesser extent for sulfathiazole and sulfadiazine by folic acid.liver extract powder, and dried yeast extract as well as by para-aminobenzoic acid,

The blood dyscrasias due to sulfanilamide, sulfathiazole, and sulfadiazine aresevere leukopenia, granulocytopenia, and mild-to-severe anemia. These are uniformly prevented or the severity greatly curtailed by feeding folic acid, liver extract powder, or dried yeast extract. PABA has a lesser effect in the amounts fed.

Liver extract powder seems to have a beneficial effect on growth and mortalitywhich is not shown by the other supplements. Both free and conjugated folic acid(as yeast extract and in liver extract powder 1:20) are active in combating thedyscrasias.

Evidence from in vitro experiments with Str. haemolyticus (B Lancefield) indicates that neither folic acid, liver extract powder, nor dried yeast extract in ratiosto sulfonamide which are effective in preventing the blood dyscrasias will inhibitor block the bacteriostatic action of the sulfonamide drugs in vitro.

It is suggested that the action of folic acid, liver powder, and yeast extract is notwholly explained by alleviating a folic acid deficiency caused by intestinal bacteriostasis due to the drugs, but by an increased demand of the animals for folicacid in the presence of certain sulfonamides.

Note: We are indebted to Harold Buskirk, Alice Bergdahl, Hallie Ferguson, E. M. Stapert, F. La Plante,and Evon Saggio for valuable assistance in carrying out the experimental work reported here.

     

Dangerous universal donors; II. Further observations on in vivo and in vitro behavior of isoantibodies of immune type present in group O blood

1. Severe hemolytic reactions were observed in 3 group A (subgroup A₁) recipients transfused with group O whole blood or plasma. In one case, 10 ml. of acommerical preparation of soluble A and B factors had been added to 500 ml. ofwhole blood prior to the transfusion and it is believed that the reaction mighthave been even more serious had not this material been added.

2. The anti-A antibodies in the serum of the dangerous universal donors causingthe hemolytic reactions fixed complement, acted as hemolysins, were difficult toneutralize with soluble A and B factors, were capable of giving positive Coombstests and their ability to agglutinate A cells was enhanced by the presence of normal human serum. These characteristics were similar to those observed in serumfrom donors known to be actively immunized against the A factor, but the stimulusfor development of "immune" anti-A antibodies in the dangerous group O donorswas not apparent.

3. Small amounts of immune A antibody were consistently demonstrated in 12of 100 random group O sera which, after neutralization, produced indirect Coombstests with A₁ cells and agglutinated A₁ cells suspended in compatible normal humanserum.

4. Screening procedures for elimination of dangerous group O donors are discussed.

Note: ACKNOWLEDGMENTSIt is a pleasure to thank Mrs. Jane Peters, Mrs. Nieves Dole and Miss Jean Dorothy for technicalassistance and Dr. R. Wendell Davis for collaboration in the study of Case 1.

     

Autohemagglutinins and hemolysins with hemoglobinuria and acute hemolytic anemia,in an illness resembling infectious mononucleosis

A case is reported of a young man with acute hemolytic anemia and hemoglobinuria who presented: an initial blood picture consistent with infectious mononucleosis, associated with a heterophile agglutination test positive in high dilution;auto-hemagglutinins, active in the cold, at room temperature and at 37 Centigrade;a hemolysin active at 37 C. after chilling, requiring the presence of a thermolabilecomponent of serum for hemolysis; a positive Donath-Landsteiner test but no evidence of syphilis. In addition there was clubbing of the digits with certain otherroentgenologic changes in the bones; absence of any other etiologic factors knownto be concerned with such anemia; uneventful improvement under massive transfusion therapy, with apparent recovery from his hematologic disorder whenstudied two years later.

Note: ACKNOWLEDGMENTOur thanks are due to Dr. Maxwell Finland and Dr. Philip F. Wagley for suggestions in preparingthis report. We are especially indebted to Dr. T. Hale Ham for his patient and critical advice and encouragement.

     

Experimental production of nutritional macrocytic anemia in swine. V. Hematologic manifestations of a combined deficiency of vitamin B12 and pteroylglutamic acid

A total of 20 swine were fed a diet adequate in all known respects except thatsoybean protein was substituted for casein, succinylsulfathiazole and a folic acidantagonist were added, and vitamin B₁₂ and pteroylglutamic acid were withheldfrom the vitamin supplement.

The animals developed macrocytic anemia, leukopenia and neutropenia, accompanied by erythroid hyperplasia of the bone marrow. Tue erythroblasts consisted mainly of immature macronormoblasts but a few atypical megaloblastswere also observed.

The anemia responded rapidly and completely to the administration of bothvitamin B₁₂ and pteroylglutamic acid. The administration of pteroylglutamicacid alone resulted in an immediate return of the blood and bone marrow towithin normal limits but after several months there was a partial hematologicrelapse in spite of continued therapy with this vitamin. The administration ofvitamin B₁₂ alone resulted in only partial remission of the anemia and the bonemarrow remained macronormoblastic although the megaloblasts tended to disappear.

Growth of the animals was stimulated by the administration of either vitaminbut the administration of both vitamins simultanseously resulted in the greatestrate of growth.

No manifestations of neurologic disturbances or of inscreased pigment excretionwere observed in the deficient swine.

Submitted on May 15, 1952 Accepted on July 22, 1952     

Experimental megaloblastic anemia in young guinea pigs

Megaloblastosis has been produced in young guinea pigs by feeding thempurified diets deficient its pteroylglutamic acid (PGA) or vitamin B₁₂ or both.The withholding of ascorbic acid from the diet did not produce megaloblastosisbut it greatly enhanced its development when PGA was also withheld. Spontaneous remission took place in several instances.

The possible role of growth of the animal and changes in the bacterial flora ofthe gastrointestinal tract has been discussed. Bacteriologic studies appeared toindicate a possible interrelationship between the occurrence of Escherichia coliin the stools of guinea pigs consuming deficient diets and the development ofmegaloblastosis. Diarrhea and infections are considered important in the pathogenesis of megaloblastic anemia but infections were not observed in animals ofthe present study. However, diarrhea was a prominent feature and appeared tobe a factor associated with megaloblastosis.

Anemia was usually present in association with megaloblastosis; however,megaloblastosis of a mild degree appearing in some of the animals fed the dietsdeficient in PGA or vitamin B₁₂ was not accompanied by anemia. When ascorbicacid was withheld in addition, then anemia always developed.

A dimorphic picture of macrocytosis and microcytosis was found most commonly in the peripheral blood of animals with megaloblastosis. This has beenexplained on the basis of regenerative macrocytosis or as a disturbance of thematuration of erythrocytes in such deficiencies.

Data obtained from peripheral blood are not a reliable indicator of megaloblastosis, nor is the clinical condition of the animal indicative of the pathologicstate.

One animal which had been fed a diet deficient in vitamin B₁₂ and PGA andwhich showed megaloblasts in its bone marrow was given injections of vitaminB₁₂. The megaloblastosis was corrected but the animal died on the sixteenth dayof treatment.

Two animals fed diets deficient in PGA and ascorbic acid were subsequentlygiven injections of PGA and ascorbic acid. The results demonstrated that administration of ascorbic acid alone did not interrupt the development of severemegaloblastosis in the continuing absence of PGA. When PGA was given, reticuocytosis then ensued and there was a reversal of megaloblastic erythropoiesis.

Submitted on March 31, 1955 Accepted on August 27, 1955     

Hemolytic disease of the newborn due to anti-A1

1. The authors report clinical, hematological and serological data in a case ofA₁ iso-sensitization of pregnancy.

2. The mother’s serum displayed immune characters specific to A₁ cells immediately after delivery. On the 24th day post partum the specificity extendedto A₂ cells.

3. The disease exhibited by the infant was very mildly hemolytic. It wasmarked by a deep jaundice, repeated alimentary vomiting and a progressive stateof drowsiness. There was no anemia. The direct anti-globulin test was negative.

4. It is shown that the mildness of the hemolytic process in cases of placentaltransfer of immune anti-A or anti-B into the incompatible A or B fetus is probablydependent upon a peculiar "resistance" of the fetal cells. This may be demonstrated in vivo and in vitro. In the present case replacement transfusion withA₁ adult blood resulted in its in vivo sensitization, detectable by the antiglobulintest and eventually leading to hemolytic anemia.

Submitted on September 28, 1953 Accepted on August 15, 1954     

Free Serum Vitamin B12 Level in Certain Hematologic Disorders

The variation of the free fraction of serum vitamin B₁₂ level was studied incertain hematologic disorders. In normals, the mean ratio of free to totalvitamin B₁₂ was 10.2 per cent. In leukemias—chronic myeloid, acute myeloidand acute lymphatic—the mean value of free vitamin B₁₂ was higher thannormal. In chronic myeloid and acute lymphatic leukemias, the free vitaminB₁₂ level did not on an average exceed 20 per cent of the total. In acute myeloid leukemia, the free fraction was relatively more increased, constituting31.1 per cent of the total on an average. In aplastic anemia, the mean valuesof free vitamin B₁₂ and the ratio (31.2 per cent) of free to total vitamin B₁₂were higher than normal. The patterns presented by acute myeloid leukemiaand aplastic anemia appeared to be more or less similar.

     

Congenital Nonspherocytic Hemolytic Anemia, Associated with Glutathione Deficiency of the Erythrocytes: Hematologic, Biochemical and Genetic Studies

1. A new biochemical defect of erythrocytes is described: glutathionedeficiency (reduced glutathione less than 10 per cent of the amount of reducedglutathione in normal erythrocytes).

2. The defect is associated with a clinical picture of congenital nonspherocytic hemolytic anemia which is fairly well compensated.

3. The results of a family study are consistent with an autosomal recessivepattern of inheritance.

4. Labeling with Na₂Cr⁵¹O₄ has a damaging effect on glutathione-deficienterythrocytes. The erythrocyte life span, as estimated by a serological method(Ashby), was markedly shortened (30 days instead of 100-120 days).

5. Red cell destruction could be increased by the administration of primaquine.

6. Secondary to the glutathione deficiency, low glyoxalase activity wasobserved. The glutathione-reducing capacity, glycolytic activity, and the ATPlevel of the abnormal red cells were found to be within the normal range.

7. On incubation of the glutathione-deficient erythrocytes in vitro withglycine-C¹⁴ and glutamine-C¹⁴, no formation of labeled glutathione could bedemonstrated.

Submitted on October 7, 1964 Accepted on June 29, 1965     

Total Body Counting in the Assessment of Vitamin B12 Absorption in Patients with Pernicious Anemia, Achlorhydria Without Pernicious Anemia and in Acid Secretors

The absorption of ⁵⁸Co-vitamin B₁₂ was assessed by the method of total bodycounting in 25 patients with frank P.A., five with latent P.A., 47 withachlorhydria but without malabsorption of vitamin B₁₂ and 47 acid-secretingpatients. A total of 192 tests was done. With or without prior stimulation ofintrinsic factor secretion by pentagastrin the upper level of absorption ofvitamin B₁₂ in patients with frank or latent P.A. was 25 per cent. Taking anequivocal result to be 21-25 per cent inclusive, the total body counting methodwas able to distinguish P.A. patients from those with achlorhydria withoutP.A. with 87 per cent accuracy and one per cent error and from non-P.A.achlorhydric patients and acid secretors combined with 91 per cent accuracyand 0.5 per cent error.

Pentagastrin stimulation did not improve the separation between achlorhydric patients with and without P.A. but raised the lower limit of retentionof vitamin B₁₂ from 24 to 44 per cent in the acid secreting patients.

The method of total body counting is recommended as a method of assessingvitamin B₁₂ absorption.

Submitted on September 26, 1969 Revised on January 26, 1970 Accepted on February 20, 1970     

Surface scintillation measurements in humans of the uptake of parenterally administered radioactive vitamin B12

1. The distribution of radioactive vitamin B₁₂ in humans was studied by scintillation counting of radioactivity over various skin projections of underlyingorgans in five individuals, following parenteral administration of radioactivevitamin B₁₂. The results of these investigations showed that the scintillation surface measurements of the radioactivity following parenteral administration ofradioactive vitamin B₁₂ may be profitably applied to the study of the metabolicturnover of vitamin B₁₂ under normal and pathological conditions in humans.

2. In two young normal control subjects over 96 per cent of the parenterallyadministered radioactive vitamin B₁₂ (5 and 10 µg. containing 0.925 µc. of Co⁶⁰)disappeared from the site of the intramuscular injection within three to fourhours, and during that time radioactivity rose to its peak value over the areascorresponding to kidney, spleen and iliac crest, and somewhat later over the muscles of the extremities. Subsequently, a gradual decline of radioactivity wasobserved over the spleen, kidney and extremities, so that at the end of threemonths in the normal subject from to of radioactivity as compared to peakvalues was observed over the spleen and left kidney and about over the calfmuscle.

The projection areas of the liver differed from all the other areas of the bodyin requiring about five to six days to build up the peaks of radioactivity. Theseexceeded counts over the kidney and spleen about 2 times, counts over theiliac crest approximately 7 times, and those over the calves about 17 times. Thedecline of radioactivity over the liver and iliac crest was very slow and small.

3. The patterns of the uptake of injected radioactive vitamin B₁₂ by the totallygastrectomized patient were grossly similar to those observed in normal controls,except for possibly slightly faster absorption of the injected material from the siteof injection, and somewhat faster and larger accumulation, but also faster discharge of radioactivity from the liver area.

4. In a patient with pernicious anemia in partial remission who received threeparenteral doses of 2 respectively 10 µg. radioactive vitamin B₁₂ (0.185-0.925µc. Co⁶⁰), the uptake of the radioactive vitamin B₁₂ by the kidney and spleen areawas slightly higher than that of the normal controls, that of the liver similar tothe normal, and that over the iliac crest showing a significantly faster declinethan in normals during the course of study. The uptake of radioactive vitamin B₁₂in the same patient following intravenous injection of the same dose of radioactivevitamin B₁₂ was grossly similar to that following intramuscular injection of thesame dose of radioactive material, except for the faster accumulation of radioactivity over the kidney area.

5. From 65 to 86 per cent of the peak radioactivity persisted over the liver 2-3months after parenteral administration of radioactive vitamin B₁₂ to two controlsubjects. After five months 60 per cent of the initial peak of radioactivity wasstill observed over the liver in the patient with anemia of sprue in remission, 85per cent in a patient with pernicious anemia in partial remission, and at the endof eight months still 35 per cent of the peak liver count in a patient with total gastrectomy without anemia. This does not take into account the normal decay ofCo⁶⁰ which is about 1 per cent per month.

6. If the presence of Co⁶⁰ in the liver indicates the deposition of Co⁶⁰-B₁₂ in thisorgan, which is most probable, then the long storage of vitamin B₁₂ in the livermay explain the long time needed in humans for depletion of hepatic stores ofvitamin B₁₂, as well as for long remissions observed in pernicious anemia followingparenteral treatment with liver extracts or vitamin B₁₂.

Submitted on May 10, 1954 Accepted on July 21, 1954     

Rhisosensitization in the American Negro

1. Studies of the Rh factor in 11,486 pregnant female American Negroes revealed 1,302 who were Rh₀ negative (8.4 per cent). Sixty-four cases of isosensitization were encountered which gave an incidence of 4.9 per cent in the Rh-negativepatients. In comparison, among 8,889 Rh₀-negative Caucasians, 460 cases ofisosensitization (5.2 per cent) were encountered. The difference was found to bestatistically insignificant.

2. Two typical examples of erythroblastosis fetalis in American Negro infantsare presented.

     

Fast Hemoglobin in Lead Poisoning

1. An electrophoretically fast hemoglobin was found in approximately 40per cent of preschool children with elevated blood lead levels.

2. Fast hemoglobin was found more often in lead-poisoned patients withhypochromic anemia than in patients with normochromic red cells.

3. Fast hemoglobin differed from hemoglobins produced in vitro by incubation with chromate or oxidized glutathione. It had electrophoretic propertiessimilar to that found in a few patients receiving tolbutamide.

4. Fast hemoglobin could not be differentiated from normal hemoglobin A₃by any technic utilized.

5. Both lead and A₃ hemoglobins were heterogeneous molecular species.

6. The mechanisms leading to the production of hemoglobin A₃ and leadhemoglobin remain unknown.

Submitted on October 7, 1965 Accepted on December 10, 1965     

Reticulocytopenia in autoimmune hemolytic anemia

1. In a series of 57 cases of autoimmune hemolytic anemia it was found that25 or 44 per cent had a relative reticulocytopenia at times of hemolytic crisis.The mortality rate in this group was significantly higher than in those whoshowed a reticulocyte response consistent with the severity of anemia. Specialsignificance is given to the high mortality rate among the patients with idiopathic AHA.

2. Four illustrative cases are presented.

3. There is a discussion of mechanisms that may be responsible for reticulocytopenia in the presence of severe anemia and erythroid hyperplasia of the marrow.

Submitted on January 23, 1956 Accepted on March 7, 1956     

Granulomatous lesions in the bone marrow in infectious mononucleosis; a comparison of the changes in the bone marrow in infectious mononucleosis with those in brucellosis,tuberculosis, sarcoidosis and lymphatic leukemia

1. The findings in the blood and in aspirated bone marrow in 23 cases of infectious mononucleosis have been described.

2. Unequivocal evidence of involvement of the bone marrow has been found in70 per cent of the cases.

3. Evidence of granulomatous inflammation of the marrow was found in 48 percent of the cases.

4. Epithelioid cells were found in the films of bone marrow in 48 per cent of thecases. These cells appear morphologically identical with those seen in imprints oflymph nodes from infectious mononucleosis and sarcoidosis and with the epithelioid cells seen in films of the marrow in brucellosis, sarcoidosis and tuberculosis.

5. The granulomatous lesions of infectious mononucleosis seem most similar tothose of brucellosis, but they also resemble the small granulomatous lesions ofsarcoidosis and tuberculosis.

6. Lymphocytosis of the marrow as well as of the blood was demonstrated in allcases. Evidence of formation of lymphocytes in the marrow was presented, and thealtered lymphocytes of infectious mononucleosis were found in films of the marrow.The degree of lymphocytosis of the marrow in infectious mononucleosis was shownto be less than that in lymphatic leukemia. Lymphocytosis of the marrow was notfound in brucellosis, sarcoidosis or tuberculosis. The lymphocytic reaction demonstrable in the marrow in infectious mononucleosis is believed to be of value in differential diagnosis.

Note: ACKNOWLEDGMENTSWe wish to acknowledge the generous cooperation of Dr. Ruth E. Boynton and the Staff of theStudent’s Health Service of the University of Minnesota throughout the course of this year long studyof infectious mononucleosis. We are indebted to Dr. T. Edward Bell and Dr. James Cardy for performingthe sternal aspirations. The photomicrographs were made by Mr. Henry Morris.

     

STUDIES ON THE PHYSIOLOGY OF THE WHITE BLOOD CELL: THE GLYCOGEN CONTENT OF LEUKOCYTES IN LEUKEMIA AND POLYCYTHEMIA

The technic of determining glycogen in isolated white blood cells was appliedto the study of the different types of leukemia and of polycythemia, in order toobtain information on the physiology of the white blood cell. From this study itis concluded that the granulated leukocyte is the only carrier of glycogen in wholeblood. The "reducing substances" in lymphocytes and blast cells are not consideredas true glycogen.

The glycogen content of wet white blood cells in the rabbit amounts to about1 per cent. In the human being a range of from 0.17 to 0.67 per cent was calculated.In disease higher percentages occur, in polycythemia up to 1.64 per cent and inglycogen storage disease up to 3.05 per cent.

The glycogen concentration of normal white blood cells is within the same rangeas that of the striated muscle.

Note: I acknowledge with gratitude my indebtedness to Dr. William Dameshek for giving me the opportunity of analyzing the blood of some of the patients studied. Miss M. H. Campbell, Miss H. A. Clark,and Miss L. M. Garofalo have aided in carrying out many of the blood counts.

     

The Rh chromosome frequencies in England

The results are reported of testing 1073 English bloods with the Rh antibodiesanti-C, anti-C^w, anti-c, anti-D, anti-E and anti-e. The results of another series of927 bloods, already published, are here reproduced. The total of 2000 bloods hasbeen used by Fisher to estimate, by his method of maximum likelihood, the Rhchromosome frequencies in England. The estimates are: CDe 40.75 per cent, cde38.86 per cent, cDE 14.11 per cent, cDe 2.57 percent, C^wDe 1.29 per cent, cdE 1.19per cent, Cde 0.98 per cent, and CDE 0.24 per cent.

A brief account is given of the three pairs of alternative antigens shown byFisher to be the basis of the Rh blood groups. Fisher’s interpretation must now beconsidered as established beyond doubt. A possible genetic basis of these relatedantigens is discussed.

Note: ACKNOWLEDGMENTSWe are deeply indebted to Professor Fisher for many reasons, but we should particularly like toacknowledge his kindness in allowing us to publish the results of his calculations of the chromosomefrequencies.For the antisera used in the investigations we are indebted to the following: Doctors E. F. Aubert,Sheila Callender, D. S. Dick, R. J. Drummond, Mr. I. Dunsford, Doctors A. J. McCall, Brenda Morrison,J. Murray, E. Wordley and R. A. Zeitlin.We also thank Dr. H. F. Brewer of the London Red Cross Blood Donor Service, and Dr. J. F. Loutit ofthe National Transfusion Service, for providing us with large numbers of blood samples.We also wish to acknowledge the assistance of Dr. Marjory N. McFarlane in the earlier part of thisinvestigation.

     

Liver extract refractory megaloblastic anemia

1 . The patient described in this report had macrocytic anemia, megaloblasticmaturation arrest in the bone marrow, glossitis, hyper-reflexia and diminished vibration perception in the feet. None of these abnormalities was improved by liverextract or vitamin B₁₂ but all responded rapidly to folic acid except the neurologicsigns.

2. This patient appears to have had a megaloblastic anemia which has been described in European clinics under the names "achrestic anemia" and "refractorymegaloblastic anemia." It appears to be similar to "Wills" factor deficiency anemia" and some cases of pernicious anemia of pregnancy.

3. This patient did not appear to have a primary deficiency of folic acid since theexcretion of this substance in the urine was within normal limits. A deficiency ofan unknown factor probably equivalent to "the Wills’ factor" is suggested.

4. It seems likely that folic acid induced a remission in this case by a "massaction" effect. The possible relationship of folic acid, vitamin B₁₂, the unknownfactor and liver extract to nucleo-protein synthesis is discussed.

Note: ACKNOWLEDGMENTWe wish to thank Doctor Charles Foertmeyer for referring this patient to us for the clinical study usedin this report.

     

Response of tropical sprue to vitamin B12

These findings show that the administration of vitamin B₁₂ to patients with tropical sprue was followed by general clinical and hematologic improvement providedthe dosage was adequate. A single dose of 4 micrograms administered in case 4produced little or no change. The larger dosage of 10-25 micrograms administeredin the other cases was accompanied by striking increase in strength and vigor anda decrease in the diarrhea; however, in no instance was a maximal dose given andthese patients quickly tended to relapse clinically and hematologically. Theycould be relieved promptly again either by another injection of vitamin B₁₂ orby a compound of folic acid. (The conjugated compounds of folic acid used in thesecases were used for experimental purposes, and they produced the same hematologicresponse as that of folic acid per se.) Case 3, who had an excellent hematologicresponse after eating one serving of 400 grams of liver, is regarded as especiallysignificant in that it suggests that, as powerful as vitamin B₁₂ is as a therapeuticagent, it is more effective when given with liver. It is especially noteworthy thatcases 1 and 2, who had three injections of vitamin B₁₂, have had steady clinicaland hematologic improvement. The reader should have in mind that a singleinjection of approximately 100 micrograms of vitamin B₁₂ probably would beneeded to produce a full hematologic response in persons so ill. This tentative appraisal would suggest that this therapeutic compound, per unit of weight, is moreeffective in treating human disease than any compound that yet has been used.

Note: ACKNOWLEDGMENT We are very much indebted to others who have aided us in selecting cases and in observing results.Especially we wish to thank Dr. F. Hernandez-Morales, Dr. Hector Marchand, Miss Clemencia Benitez-Gautier, and Miss Sara Torres.