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.

     

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 USE OF PHOTO-ELECTRIC TURBIDOMETRY IN THE DETERMINATION OF RED CELL COUNTS, HEMATOCRITS, AND HEMOGLOBIN

1. Blood turbidometry is recommended as a screening technic for distinguishingbetween anemic and nonanemic individuals.

2. Blood turbidometry must be supplemented by other technics for an exactdiagnosis of the type of anemia. However, with such help it makes its own contribution to the accuracy of the diagnosis.

3. Blood turbidometry alone would seem to be capable of following an anemicindividual’s response to therapy once the proper diagnosis is established.

4. Determination of hemoglobin concentration by turbidometry appears as adistinct possibility. However, further investigation is necessary to validate itsutility.

5. Evidence is accumulated that the shadow-volume relationship is a constantin several species.

Note: The author is particularly indebted to the following for advice in connection with this work: Dr.Peter Olafson of the Department of Veterinary Pathology, Dr. C. E. Hayden of the Department of Veterinary Physiology, Dr. L. I. Barnes of the Department of Physics, and Dr. W. B. Carver of the Department of Mathematics, all of Cornell University, and Dr. Charles P. Winsor of Johns Hopkins University.Dr. Robert N. Ericson conducted some preliminary explorations of the problem with the authorwhen both were connected with Kansas State College.

     

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.

     

FOLIC ACID IN THE TREATMENT OF PERNICIOUS ANEMIA

1. Folic acid in daily doses of 15 to 50 mg., orally, or 20 mg. intramuscularly,usually produced a submaximal reticulocytosis in patients with pernicious anemia.

2. In 3 patients the hemoglobin and red cells rose to a level of about 12.0 Gm.and 4.3 million respectively without further rise after 3 months of therapy.

3. Folic acid in the above doses failed to prevent the development or progressionof neurological symptoms indicative of subacute combined sclerosis.

4. In 5 patients folic acid in doses of 5 or 10 mg. orally daily combined with unit of liver extract injected intramuscularly daily produced a reticulocytosisgreater than that anticipated from adequate liver extract therapy alone.

5. With combined liver extract and folic acid therapy there was evidence ofimprovement in the symptoms and signs of subacute combined sclerosis in 3patients.

6. Folic acid, combined with unit of liver extract, was found to produce acomplete hematological remission.

7. Folic acid, alone or in combination with small doses of liver extract, producedan improvement in appetite and general well-being in patients with perniciousanemia.

8. The possible enhancing effect of liver extract when combined with folic acidcannot be due to the folic acid content of the former since 1 unit of liver extractcontains only 0.38 micrograms of folic acid.³¹

9. Folic acid administered to a patient with macrocytic anemia due to faultypostoperative intestinal digestion and absorption, produced a complete remissionin the blood picture and a marked improvement in signs and symptoms.

     

EXPERIMENTAL LEUKOCYTOSIS. THE INEFFICACY OF P-CHLOROXYLENOL AND METHYL ACETAMIDE AS BONE MARROW STIMULANTS

1. Single and multiple injections of CXM (methyl acetamide and p-chloroxylenol) have a similar action in increasing the total number of polymorphonuclearleukocytes in the peripheral blood of a group of 32 rats. No "shift to the left"takes place.

2. There is no stimulation of the myeloid elements of the bone marrow to suggest that this leukocytosis is due to hyperplasia.

3. The evidence suggests that the mechanism for the leukocytosis produced bythe CXM consists of releasing blood cells from depots in the body. This action isselective for the granulocytes as the red cells and platelets are not affected.

4. There is progressive degeneration of the parenchymal cells of the liver aftersingle and multiple injections of these substances.

5. If less toxic combinations of methyl acetamide and para-chloro-xylenol couldbe obtained, they might be of value in the treatment of certain patients withleukopenia or agranulocytosis more particularly when the bone marrow is hyperplastic but the granulocytes are not released.

Note: The author expresses his appreciation to Dr. Oscar Riddle for the generous provision of animal andlaboratory facilities.

     

THE EXCRETION OF UROBILINOGEN IN THE STOOLS AND URINE DURING MALARIAL INFECTION

In 10 cases of malaria (6 benign tertian, 4 malignant tertian), the excretion ofurobilinogen in the stools and in the urine was studied. In all 10 cases the amountof urobilinogen excreted in the stools was found to be increased. After defervescence and disappearance of parasites from the blood the excretion gradually declined. The increased excretion of urobilinogen in the stools was the constant andsometimes the only evidence of increased blood destruction occurring at times in the complete absence of jaundice and reticulocytosis. Increased excretion of urobilinogenin the urine was not a constant feature.

It is suggested that the development of jaundice and of urobiligenuria is duenot only to the liberation of pigments by the hemolysis, but to a disturbance in theliver function.

This study lends further confirmation to the concept that the only unequivocalevidence of increased blood destruction is shown in an increased output of urobilinogen in the feces.

Note: The author is indebted to Dr. M. Rachmilewitz for his suggestions and criticisms.

     

Experimental anemias in the rat; II. The effect of various sulfonamides in producing a pteroylglutamic acid deficiency and the pteroylglutamic acid activity of test substances

1. Different sulfonamides were tested to ascertain their effect in producing thecharacteristic symptoms of acute PGA deficiency in rats fed on synthetic diets.Sulfasuxidine (1 per cent) and the less soluble phthalylsulfathiazole (1 per cent)were equally effective. Sulfathiazole in 1 per cent concentration produced a hemolytic anemia not reversible by PGA or whole liver powder. In a 0.5 per cent concentration it was also effective, but in view of its toxicity, the less soluble sulfonamides were to be preferred. A mixture of 0.5 per cent sulfathiazole and 0.5 percent sulfadiazine was extremely toxic and produced a hemolytic anemia. Sulfaguanidine was toxic at 1 per cent concentration.

2. When intermittent small doses of PGA were given to PGA-deficient rats toprolong their life from 45 up to 155 days, 1 per cent sulfasuxidine or phthalylsulfathiazole, or 0.5 per cent sulfathiazole were equally efficient in producing regularlya macrocytic normochromic anemia.

3. The response of PGA-deficient rats to single doses of PGA has been studiedand an assay procedure has been suggested which uses the weight increase andduration of cure as the measure of the response. The W.B.C. and reticulocyte response can also be used as a qualitative indication of PGA activity.

4. Of the substances tested by this procedure, vitamin B₁₂, purified perniciousanemia preparations, ascorbic acid and xanthopterin showed no PGA activity. Acommercial yeast preparation and Teropterin were found to possess biologic activity comparable with that found by other workers in assays on chicks.

Note: ACKNOWLEDGMENTSWe wish to thank Dr. E. Lester-Smith, of the Glaxo Laboratories, for the generous supply of vitaminB₁₂, Dr. T. H. Jukes, of the Lederle Laboratories, for the gift of PGA and Teropterin, and Dr. W. Jacobson for the samples of purified P. A. liver preparations and xanthopterin.

     

Refractory megaloblastic anemia

1. Fifty-nine cases of megaloblastic anemia refractory partially or completelyto potent liver extracts given parenterally have been investigated in Edinburghduring the past six years. Thirty-four of these cases were associated with pregnancy, the puerperium or the sprue syndrome. No explanation of the cause of themegaloblastic anemia was discovered in the remaining 25 cases.

2. The etiology, clinical features and treatment of 25 cases of idiopathic refractory megaloblastic anemia are described. Attention is directed to the excellenttherapeutic effects produced by proteolysed liver or folic acid.

3. The mechanisms involved in refractoriness to potent parenteral liver extractsare discussed.

4. In certain cases of refractory megaloblastic anemia it is suggested that an unknown hematinic principle, in addition to the liberating factor in purified parenteral liver extract and folic acid, is required for the complete restoration of normoblastic blood formation.

Note: ACKNOWLEDGMENTSMy thanks are due to many members of my staff who have helped in theseinvestigations, particularly to Professor L. J. Davis formerly lecturer in Medicinein the University of Edinburgh, and to Dr. Girdwood. Grateful acknowledgmentmust also be made to Doctor Riding, Medical Director of Evans Medical SuppliesLtd. and his research chemists who were responsible for the preparation of proteolysed liver and the other fractions of liver mentioned above.

     

SERUM PROTEIN CHANGES IN MYELOGENOUS AND LYMPHOCYTIC LEUKEMIAS AND HODGKIN'S DISEASE

1. Using a methyl alcohol fractionation technic, the albumin, globulin, andtotal protein levels were determined in a series of normal adults and compared withcases of myelogenous and lymphocytic leukemias and Hodgkin’s disease.

2. Statistically significant decreases in albumin and increases in globulin werefound in the cases of Hodgkin’s disease and myelogenous leukemia, but withoutsignificant changes in total protein. Globulin levels above the highest normal valuewere found in 23 per cent of the former and 33 per cent of the latter group.

3. No apparent relationship was noted between the levels of the serum proteinfractions and (1) the hemoglobin level, (2) the erythrocyte count, (3) the peripheral white blood cell picture, and (4) the bone marrow smears.

Note: The authors wish to express their appreciation to Professor Ovid O. Meyer, Department of Medicine,for making available the clinical material in this study and for valuable suggestions. The authors arealso indebted to Professor J. A. E. Eyster, Department of Physiology, for suggestions as to statisticaltreatment of the data.

     

THE ERYTHROPOIETIC ACTIVITY OF CHOLINE CHLORIDE IN MEGALOBLASTIC ANEMIAS

1. The effect of the administration of choline chloride has been observed in 10cases of megaloblastic anemia of various types.

2. Choline was without effect in a case of untreated Addisonian perniciousanemia which subsequently responded to parenteral liver therapy.

3. Choline was also without effect in a case of nutritional megaloblastic anemia,in a case of megaloblastic anemia of pregnancy, and in two cases of megaloblasticanemia associated with the sprue syndrome. All these cases had proved refractoryto injections of potent liver extract before the choline was given, and all respondedto subsequent oral liver or folic acid therapy.

4. A significant erythropoietic response to choline occurred in two cases resembling Addisonian pernicious anemia which were refractory to parenteral liverextracts.

Secondary responses followed the administration of choline in two other casesof Addisonian pernicious anemia and in a case of megaloblastic anemia of pregnancy, all of which had already responded to injections of liver extract.

5. The significance of these observations is discussed. It is concluded that choline possesses no direct erythropoietic activity, but that under certain circumstances it may potentiate the effect of liver extracts.

It is suggested that refractory megaloblastic anemias may be divided into twogroups. In one, represented by well known syndromes associated with defectiveabsorption or pregnancy, the lack of response to parenteral liver extracts is notcorrected by choline. In the other, represented by two cases simulating Addisonianpernicious anemia, choline is effective in overcoming, partially or completely, therefractoriness to parenteral liver therapy. Consideration is given to the view thatthe refractoriness of this group results from hepatic dysfunction.

6. The most satisfactory method of administering choline probably consists ofintravenous injections in daily doses of 1 gram. Larger doses given intravenouslyare frequently accompanied by unpleasant side effects, while oral administrationappears to be relatively less effective.

7. It seems unlikely that choline will be of practical value in the treatment ofrefractory megaloblastic anemias, for which oral liver preparations provide themost certain and effective treatment. It is possible, however, that choline may beof use in cases complicated by severe hepatic disease.

Note: Acknowledgment: We wish to thank Dr. L. D. W. Scott for permission to include his patient (case10) in our series.

     

THE TREATMENT OF LYMPHOBLASTIC LEUKEMIA WITH CRUDE MYELOKENTRIC ACID

Eight cases of blastic lymphoid leukemia have been treated with myelokentricacid in crude form, because hypothetically in blastic lymphoid leukemia there isa deficiency of this material. The crude myelokentric acid was used because it wasmore easily obtained than partially purified material. Purification of biologicallyactive materials by methods of extraction and precipitation necessarily results in aconsiderable loss of material. Thirteen partial remissions occurred following theadministration of crude myelokentric acid. Seven of the 8 patients have died, and5 necropsies were performed.

The necropsy material adds further weight to the belief that the remissions wereinduced by the myelokentric acid in that in all 5 necropsies there was a definitealteration in the histologic morphology as contrasted with the findings in thenecropsies of the controls.

It seems inadvisable, however, to treat a large number of patients with this material because it is crude, it is relatively unavailable, and no standard dose has yetbeen devised.

Note: We wish to t hank Dr. D. L. Turner and Dr. W. A. Hause for valuable assistance in this work.

     

The effect of quantitative and qualitative protein deficiency on blood regeneration; hemoglobin

1. The effect of diets, varying in quantity or quality of protein, on hemopoiesiswas studied in protein depleted and anemic adult rats.

2. In experiments with diets containing different amounts of casein (0, 3, 9 and18 per cent, respectively), and fed ad libitum, it was found that with a protein-freediet a further decrease of hemoglobin occurred, whereas the other three dietsinitiated a regeneration of hemoglobin; its degree being more or less proportionalto the casein content.

3. In experiments, in which diets with 9 and 18 per cent of casein, respectively,were given in restricted amounts, it was found that the degree of hemoglobinformation was similar to that with the same diets when given ad libitum, whereasthe weight gain was considerably less. It is concluded, therefore, that in caloricdeficiency hemoglobin formation has a priority over weight recovery.

4. A diet containing 30 per cent casein and given in restricted amounts induceda further weight loss, whereas the concentration of hemoglobin showed a markedincrease. Comparing the results obtained by this diet with those observed with 18per cent casein diets, given either ad libitum or in controlled amounts, it was evident that restriction of the quantity of food protein interferes more seriously withhemopoiesis than restriction of calories.

5. Diets containing nutritionally inferior proteins fed at 9 per cent level, alsoimpaired normal hemopoiesis. Hemoglobin regeneration induced by the proteinsinvestigated was found to decrease in the following order: eggs, meat, processedsoya, casein, peanut, maize, wheat, gelatin.

6. Comparing the nutritive value of various proteins for regeneration of hemoglobin and of granulocytes it was found, that casein and soya and maize proteinsare considerably more efficient for hemoglobin formation than for production ofgranulocytes, whereas wheat protein and gelatin have a higher granulocytopoieticcapacity.

     

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.

     

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.

     

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.

     

The relationship between carbonic anhydrase activity and zinc content of erythrocytes in normal,in anemic and other pathologic conditions

A good correlation exists between zinc content and carbonic anhydrase activityof the red blood cells under all conditions studied, including anemia and polycythemia. In almost all patients with anemias other than pernicious anemia, bothzinc and carbonic anhydrase levels were lowered in parallel fashion. These changeswere proportional to decreases in hematocrit and hemoglobin levels and erythrocyte counts so that both zinc and carbonic anhydrase values per unit of RBC werein the normal range. In a few instances of anemia associated with leukemia andin one of sickle cell anemia, neither zinc content nor carbonic anhydrase activitywas decreased in proportion to the anemia; in these cases the zinc and carbonicanhydrase levels per unit of blood were both elevated to the same degree.

Patients with pernicious anemia showed no decrease in absolute values for zincand carbonic anhydrase activity in spite of marked lowering of hematocrit andhemoglobin levels and of erythrocyte count. Accordingly, both zinc concentrationand carbonic anhydrase activity per unit of blood were elevated, often to a markeddegree. These increases were parallel, varying inversely with the degree of anemia;when they regressed under treatment, both did so at the same rate.

There are no methods available for estimating carbonic anhydrase concentration;all methods now in use measure only the activity of the enzyme. It is suggestedthat zinc concentration could be used as an indicator of carbonic anhydrase contentof the red blood cells.

Note: ACKNOWLEDGMENTSDrs. Joseph C. Aub and Ira T. Nathanson were kind enough to refer several patients for study. Dr.Byrl J. Kennedy was most helpful in regard to obtaining samples of blood. The technical work wasperformed by Miss Mary Lou Roney, Betty Hickey and Marion Taylor.

     

Preparation of stabilized solutions of hemoglobin

It is possible to prepare self-stabilizing solutions of hemoglobin from humanerythrocytes by the use of dextrose, nicotinic acid amide and ammonia during thepreparation and in the final solutions themselves. Co⁺⁺, Mn⁺⁺ and Mg⁺⁺ ions,nile blue and hexose diphosphate contribute to the speed of stabilization of thesesolutions. Stabilization is obtained by the faculty of the solutions, presumablyby enzymic action, to convert the hemoglobin to the reduced form and to maintainit in this form. The hemoglobin solutions described are suitable for intravenousadministration.

Note: ACKNOWLEDGMENTWe wish to acknowledge our deep indebtedness to Miss Lois Priester and to Mr. Edward Smith fortheir technical assistance in this work. We wish also to express our gratitude to the Biological ProductionDivision and to the Biological Control Division of Sharp and Dohme for assistance in many phases of thework.

     

Chronic marrow failure,myelosclerosis and extramedullary hematopoiesis

Thirty cases, including 20 autopsied, of chronic bone marrow failure, myelosclerosis and osteosclerosis have been presented and compared with similar reportsin the literature. The bone marrow histopathologic sequence observed involvedrepeated necrobiosis of maturing hematopoietic cells, followed by overgrowth ofmarrow reticulum and frequent ossification. Immature erythrocytes and leukocyteswere often found in the circulation, and extrameduallary hematopoiesis was characteristic. One case was complicated by leukemia. Etiologic factors implicatedincluded exogenous toxic chemicals, liver dysfunction, endocrine abnormalities,blood loss or destruction and cardiovascular disease. Suspicion was directed towardthe pathogenetic importance of protracted bone marrow exposure to certain substances normally conjugated rapidly in the liver and excreted.

Note: ACKNOWLEDGMENTThe authors acknowledge gratefully the continued assistance of Dr. John Norcross and Dr. DonatP. Cyr of Lahey Clinic, Boston, who permitted use of material from living cases.We are indebted to Dr. Shields Warren, New England Deaconess Hospital, Boston, for permissionto review 10 cases; to Dr. Olive Gates, Harvard Medical School, for advice and assistance with 7 cases,including material from the Collis P. Huntington Hospital; to Professor William Boyd for 5 cases fromBanting Institute, Toronto; to Dr. Paul D. Rosahn, New Britain General Hospital, New Britain, Conn.,and Dr. William Dameshek for one case; to Dr. A. J. Blanchard for several cases from Christie St. Hospital, Toronto; to Drs. Lorne Whittaker and Sabin of St. Catherine’s Hospital for one case; and to Dr.John Brownlee for hematologic studies from one case.

     

The influence of dietary fat and protein on the aging hamster: Hemoglobin,glucose, triglycerides,and serum proteins

Hamsters were fed one of three levels of casein (9, 18, and 36 g/385 Kcal) combined with one of three levels of corn oil (4.5, 9.0, and 18.0 g/385 Kcal) from 8 weeks of age. Blood samples were collected 2, 5, 11, and 17 months after animals began to receive the various diets. Whole blood hemoglobin was highest in animals fed the high-protein diets at the 5-and 11-month measurements, but also high in animals fed the medium-protein diet at the 17-month measurement. The medium-and high-fat diets resulted in the highest hemoglobin values. Serum triglycerides and plasma glucose were elevated in hamsters fed high-fat or high-protein diets at nearly every collection period. Each serum protein fraction was influenced differently by the diets. Increasing dietary fat and/or protein increased serum albumin. Serum alpha one globulin was elevated in hamsters of both sexes fed high-fat diets. In contrast, low-protein diets reduced the concentration of this fraction in males only. Beta globulins were influenced by dietary fat. At 5 and 17 months, high-fat diets elevated this fraction, but at 11 months depressed it. Gamma globulins were elevated by feeding high-fat diets to female hamsters. The age and sex of hamsters are influential in determining their responses to different diets.