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.

     

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.

     

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     

DIETARY FACTORS CONCERNED IN ERYTHROPOIESIS--Continued

Riboflavin is essential for normal erythropoiesis in rats, dogs, pigs, and monkeys.There is no evidence that this vitamin is required for normal erythropoiesis in man.The anemia in swine is normocytic.

Nicotinic acid deficiency is accompanied by a severe anemia in dogs. The type ofanemia produced is normochromic and may be either macrocytic or normocytic andis associated with a mild reticulocytosis. Limited observations indicate that thebone marrow is hypoplastic and that erythropoiesis stops at the erythroblasticlevel. An anemia due to a deficiency of this vitamin has not been demonstrated inother species nor in man.

Pyridoxine is essential for normal erythropoiesis in chicks, rats, dogs, and pigs.The anemia is microcytic and slightly hypochromic in type. Anisocytosis, microcytosis, polychromatophilia, and normoblasts can be seen in the blood smear.An irregular reticulocytosis is present. The bone marrow is hyperplastic and thereis an increase in the nucleated red blood cells. The anemia is accompanied byhemosiderosis of the tissues, an elevated serum iron level, and degeneration in thenervous system. There is no evidence of an increased rate of hemolysis. No relationship between pyridoxine and erythropoiesis has been demonstrated in man.

The "Lactobacillus casei group" includes the norite eluate factor, the L. caseifactor from liver, folic acid, the Streptococcus lactis R factor of Keesztesy et al., theyeast factor of Stokstad, the factor of Hutchings et al., vitamin M₁₁, xanthopterin,vitamin B_c, vitamin B_c conjugate, vitamins B₁₀ and B₁₁, and pyracin.

The L. casei factor from liver has been identified as pteroylglutamic acid. Theavailable evidence indicates that the norite eluate factor, folic acid, vitamin M,vitamin B_c, vitamin B₁₀, and vitamin B₁₁ are identical with pteroylglutamic acid.The Streptococcus lactic R factor of Keesztesy et al. may be pteroic acid. The yeastfactor of Stokstad is unidentified. The fermentation factor of Hutchings et al. hasbeen identified as pteroyltriglutamic acid. Vitamin B_c conjugate is now known tobe pteroylheptaglutamic acid. Thus the various members of this group are closelyrelated chemically and represent minor alterations of a basic structure. The corresponding deficiency syndromes are probably identical. In the rat the deficiency ismanifested by severe normocytic anemia, severe granulocytopenia, leukopenia,and thrombocytopenia. Nucleated red cells appear in the peripheral blood. Bonemarrow studies suggest a maturation arrest in the early stage of development of allthree of the cellular elements of the blood. The manifestations of the deficiency inthe chick are macrocytic anemia, leukopenia, and thrombocytopenia. Again immature red cells are present in the peripheral blood. In the monkey the manifestations of the deficiency are normocytic anemia, leukopenia, and thrombocytopenia.In human beings the synthetic L. casei factor from liver (pteroylglutamic acid) hasbeen shown to be effective in the treatment of various types of macrocytic anemiaincluding pernicious anemia and sprue. The relation of this substance to the antipernicious anemia substance in liver remains to be determined.

The extrinsic factor of Castle is still unidentified. It now seems reasonable thatit is related in some way to pteroylglutamic acid. It is unlikely that it is identicalsince the synthetic L. casei factor is effective even in the absence of normal gastricjuice. A deficiency of the extrinsic factor in man results in an anemia which isidentical with pernicious anemia and the bone marrow is cytologically indistinguishable. An accompanying neutropenia and thrombocytopenia are also frequently seen. The anemia responds rapidly to the parenteral administration ofhighly purified antipernicious anemia liver extracts and to pteroylglutamic (folic)acid. Achlorhydria is generally not present. Macrocytic anemia of nutritional originoccurring in the tropics varies from this anemia in one important aspect. It fails torespond to highly purified liver extracts. This strongly suggests that the factorresponsible for the deficiency is distinct from that of the extrinsic factor of Castle.A deficiency of this factor has been produced in monkeys and the deficiency syndrome consists of a macrocytic anemia with a megaloblastic bone marrow. Theanemia fails to respond to highly purified liver extracts which are effective in thetreatment of pernicious anemia but does respond to crude liver extracts and tomarmite, an autolyzed yeast extract. The relation between this factor and the L.casei factor has not been investigated.

The role of ascorbic acid in erythropoiesis is not clear. Although the scorbuticstate in both guinea pigs and human beings is frequently accompanied by anemiait is questionable whether the anemia is due specifically to a deficiency of ascorbicacid. Much of the animal experimentation is inconclusive because pure ascorbicacid supplements were not used. Further work in animals is needed. In man it hasbeen both asserted and denied that synthetic ascorbic acid is effective in relievingthe anemia. It would seem, however, that there are some scorbutic patients whorespond specifically to pure ascorbic acid. The anemia accompanying scurvy hasbeen reported as macrocytic, normocytic, and microcytic. An induced, uncomplicated ascorbic acid deficiency in a human being did not result in anemia.

Pantothenic acid deficiency results in a normocytic anemia of moderate degree inpigs in about two-thirds of the animals. There is evidence which suggests that adeficiency of this vitamin in rats may result in anemia, granulocytopenia, and bonemarrow hypoplasia. Not all animals show these changes and pantothenic acid,although completely preventive, does not exert a curative action in all animals.There seems to be a relation between pantothenic acid deficiency and a deficiencyof the L. casei factor in the rat.

Choline deficiency in dogs results in a severe anemia. In many animals this changeis irreversible. This may be explained by the irreversible liver damage which ispresent.

Biotin is necessary for the production of hemoglobin values greater than 14grams per cent in dogs maintained on a highly purified ration. There is no evidencethat biotin has an effect on erythropoiesis in other species.

In addition to the factors described above it has been shown that monkeys,pigeons, and guinea pigs require at least one more additional factor for normalerythropoiesis.

There is no evidence that thiamine, p-aminobenzoic acid, and inositol are concerned in erythropoiesis in any species.

Considering the relative size of the globin fraction of the hemoglobin moleculeit is understandable that a deficiency of protein results in anemia. This has beendemonstrated in rats and dogs. It has been pointed out that because of a markedreduction in the total blood volume only when the total circulating hemoglobinis determined and adjusted to a unit of surface can the true severity of the anemiabe appreciated. Equine globin contains all ten of the "essential" amino acids andat least nine "nonessential" amino acids. Human globin has not been so extensivelystudied. It would be expected that a deficiency of any one of the "essential" aminoacids would give rise to anemia. Actually, specific deficiencies of tryptophan,lysine, phenylalanine, and isoleucine have been produced in the rat and anemiadeveloped in each instance. The morphological characteristics of these anemiashave not been carefully investigated. The anemia due to tryptophan deficiency inthe rat has been stated to be normocytic and normochromic. An anemia probablydue to a lack of tryptophan has been produced in pigs. This anemia is normocytic,normochromic, and accompanied by a hypoplastic or normoplastic bone marrowand a normal level of iron in the serum. No increase of hemosiderin in the tissueshas been noted. Whether the anemia produced in rats by feeding deaminized caseinis due to a toxic substance rather than a deficiency of lysine is unsettled althoughlarge amounts of lysine prevent its development. Evidence that glycine is utilizedin the synthesis of the pyrrole rings of protoporphyrin has been obtained by labeling this amino acid with N¹⁵ and feeding the labeled compound to rats. Pyrroleshave also been synthesized in vitro from glycine. Similar evidence is available toindicate that acetic acid, or a derivative of it, is utilized for porphyrin synthesis.

Three mineral elements, iron, copper, and cobalt, have been shown to be essentialfor normal erythropoiesis in at least one species each. Iron is probably required forerythropoiesis in all mammals. A deficiency results, at least in the chronic stages,in a microcytic hypochromic anemia and is accompanied by a normoblastic, hyperplastic bone marrow and a low serum iron level, an increased amount of protoporphyrin in the erythrocytes, and an elevated serum copper level. Nucleated red bloodcells are occasionally seen in the peripheral blood and the reticulocytes are increased.

The fundamental concepts of iron metabolism have changed greatly in recentyears. These may be summarized. Iron is absorbed chiefly in the duodenum. In manit is absorbed principally as ferrous iron. Dogs absorb both valence forms wellalthough some animals absorb the ferrous form more readily than the ferric form.Rats absorb both forms equally well. The absorption of iron is also dependent uponthe concentration of the iron in the intestine, upon the solubility of the iron salt,and in the human being at least upon the presence of reducing substances in the dietas well as the reducing action of the gastric hydrochloric acid. In addition to thesefactors the need of the body for iron may determine, to a certain degree, the amountabsorbed. This is known as the "selective absorption" theory. Recently it has beensuggested that apoferritin acts as a receptor compound in the mucosal cell. As theconcentration of the plasma iron falls, ferrous iron is removed from the mucosal cellresulting in a diminution of ferritin in the mucosa. When the ferritin has diminishedto a point where the cell is no longer saturated with respect to ferrous iron, moreiron is absorbed into the mucosal cell. Once absorbed the iron is transported in theplasma to the tissues where it is stored to a great extent as ferritin, a protein-ironcomplex. The iron is then used over and over again for hemoglobin synthesis. Ironis excreted from the body in only insignificant quantities. This theory requiressubstantiation.

Copper has been shown to be essential for normal erythropoiesis in chickens,mice, rats, rabbits, dogs, pigs, sheep, cattle, and infants. A deficiency of this mineral in rats is manifested by a microcytic hypochromic anemia and a moderatereticulocytosis. A condition due to a deficiency of copper, known as "enzooticataxia," occurs in sheep in Western Australia. Anemia may be severe. In younglambs it is microcytic and hypochromic and is accompanied by demyelinization ofthe nervous system and hemosiderosis of the tissues. In adult sheep the anemia isslightly macrocytic and hypochromic. Blood smears reveal anisocytosis, poikilocytosis, Howell-Jolly bodies, normoblasts, numerous macrocytes, stippling, andpolychromatophilia. Similar blood changes have been reported in copper-deficientcattle in Western Australia. In nutritional anemia in infants the rate of erythropoiesis is accelerated when copper is given in addition to iron. In adults supplementalcopper therapy may be of value in a few cases. Such cases, if they occur, are rare.Most cases will respond if adequate doses of iron are given. This does not necessarilyindicate that copper is not needed for erythropoiesis or that it is not a dietary essential but rather that the quantities needed are so small that sufficient copper is present in the body stores in adult life, in the diet, or as a contaminant in the iron usedtherapeutically to supply the needs. No case of uncomplicated copper deficiency hasbeen reported in man. The manner in which copper is related to the formation of redcells is not understood.

The role of cobalt in erythropoiesis is unique. A deficiency results in anemia. Theadministration of small amounts to normal animals produces a polycythemia,whereas the administration of large amounts depresses erythropoiesis. The enzooticoccurrence of cobalt deficiency in sheep and cattle has been reported from variousregions of the world. Anemia is present and is oftentimes severe. The anemia iseither normocytic or microcytic and usually hypochromic. Blood smears revealanisocytosis and poikilocytosis. There is a hypoplasia of erythrogenic tissue in thebone marrow, hemosiderosis of the tissues and a reduction in reticulocytes in theblood. An experimental anemia due to cobalt deficiency has not been produced ineither rats or dogs. There is no substantial or convincing evidence that cobalt isneeded by human beings for normal erythropoiesis. The administration of smallamounts of cobalt to normal rats, dogs, guinea pigs, frogs, mice, rabbits, chickens,pigs, and ducks produces a marked polycythemia which is accompanied by areticulocytosis, hyperplasia of the bone marrow, and an increased erythropoieticactivity in the spleen and liver. Larger doses of cobalt inhibit erythropoiesis. Themetabolism of cobalt is unlike that of iron. The excretion of cobalt from the bodyonce it is absorbed is exceedingly rapid and is principally through the kidneys.

In conclusion, certain vitamins, namely, riboflavin, nicotinic acid, pyridoxine,"folic acid," and the extrinsic factor, have been shown to be essential for normalerythropoiesis in at least one species each. It has been claimed that ascorbic acid,pantothenic acid, choline, and biotin play a role in erythropoiesis but these claimsneed substantiation. There is no substantial evidence that thiamine or inositol isconcerned in red cell formation. The significance of p-aminobenzoic acid has yet tobe determined. Protein is essential for normal red blood cell formation. The globinfraction of the hemoglobin molecule contains all ten of the "essential" amino acidsas well as many of the "nonessential" ones. The stroma of the red cells also contains amino acids. It is logical, therefore, to assume that in the absence of any oneof the so-called essential amino acids hemoglobin formation cannot take placenormally. Actually specific deficiencies of tryptophan, lysine, phenylalanine, andisoleucine have been produced in the rat and anemia has developed in each instance.There is evidence to show that glycine and acetic acid, or a derivative of it, areutilized in the synthesis of the pyrrole rings of protoporphyrin. Three mineralelements, iron, copper, and cobalt, have been shown to be essential for normalerythropoiesis.

Note: I gratefully acknowledge my indebtedness to Dr. Maxwell M. Wintrobe for his kind advice and aidin the preparation of this review as well as for the liberal use of his extensive reprint file.

     

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.

     

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.

     

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.

     

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.

     

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.

     

RENAL FUNCTION DURING CHRONIC ANEMIA IN MAN

1. Renal function has been studied quantitatively in 15 patients with chronicanemia, 8 of whom were proved to have pernicious anemia. In 7 the anemia wassecondary to chronic blood loss, iron deficiency, paroxysmal nocturnal hemoglobinuria, and leukemia. The effective renal plasma flow and glomerular filtrationrate were measured by clearance technics; and tubular function, by saturationmethods (diodrast Tm and glucose Tm).

2. The effective renal plasma flow, the glomerular filtration rate, and the filtration fraction (percentage of plasma filtered at the glomerulus) were reduced slightlybelow the normal values in most subjects. The effective renal whole blood flowwas always greatly reduced, by 46 per cent on the average in males and by 31.8per cent in females.

3. Since arterial pressure was not significantly depressed it was concluded thatrenal vasoconstriction occurs in chronic anemia, possibly as a homeostatic devicefor the diversion of blood to tissues more sensitive to oxygen lack. The relativelysmall reduction of filtration fraction implies afferent and efferent arteriolar vasoconstriction with dominance by the afferent arterioles. These changes were shownto be reversible, a return to normal values paralleling the return of the blood pictureto normal.

4. Diodrast Tm was reduced significantly in 9 of 10 patients while the values ofglucose Tm were normal in 6 of 7 patients. The normal values for glucose Tm indicated continued operation of all glomeruli and implied the absence of shuntingor of cessation of blood flow in any significant portion of the kidney. The fall indiodrast Tm, which appeared to be reversible in 2 of 4 individuals, was interpretedas evidence of intracellular dysfunction rather than destruction or inactivation ofnephrons.

Note: We wish to express our gratitude to the Misses Elizabeth Nolan and Constance Qua for technicalassistance.

     

Studies on free erythrocyte protoporphyrin,plasma iron and plasma copper in normal and anemic subjects

1. A total of 108 erythrocyte protoporphyrin determinations has been made in 66normal individuals. The geometric mean ± standard error of the mean was 31(26-38).

2. A total of 196 determinations of plasma iron in 92 normal individuals wasmade. The mean ± standard error of the mean was 104.7 ± 3.4 µg per cent.

3. In a total of 150 determinations of plasma copper in 105 normal individuals,the mean ± standard error of the mean was 118.6 ± 1.2. µg per cent.

4. No significant difference in plasma iron was noted between the sexes but infemales the plasma copper was significantly higher and the erythrocyte protoporphyrin slightly higher than in males.

5. Erythrocyte protoporphyrin, plasma iron, and plasma copper determinationshave been made in over 112 patients with a variety of clinical conditions associatedwith anemia. In general, it was found that in pernicious anemia in relapse theerythrocyte protoporphyrin values were normal, the plasma iron normal or highand the plasma copper usually normal. Anemia due to iron deficiency as well as theanemia of infection were accompanied by high values for erythrocyte protoporphyrin, hypoferremia and hypcrcupremia. In nephritis with anemia the erythrocyteprotoporphyrin was generally increased, the plasma iron low or normal and theplasma copper increased. Anemia associated with lymph node disorders or leukemiawas accompanied by a normal or high EP, a low or normal plasma iron and anincrease in plasma copper. Thalassemia major was found to be accompanied byboth hypercupremia and hyperferremia; in thalassemia minor the serum iron valueswere normal although hypercupremia was found. Hyperferremia was noted inaplastic anemia. In cases of plumbism the erythrocyte protoporphyrin wasmarkedly increased. Hypocupremia was noted only twice, in one patient withsevere nephritis and hypoalbuminemia and in one patient with hemochromatosis.

Note: ACKNOWLEDGMENTSFor samples of plasma from patients with thalassemia we are indebted to Drs. W. N. Valentine,Rochester, N. Y., P. Sturgeon, Los Angeles, and L. K. Diamond, Boston. The following gave valuabletechnical assistance: Misses Mary Iles, Betty Tatting and Wanda Worth.

     

Refined liver extract in tropical macrocytic anemia

1. A series of 45 cases of T.M.A. treated with "refined" liver extract is reported.

2. "Refined" liver extract was found to be effective in 39 cases.

3. It was found that 2 or 3 ml. of refined liver extract (Examen N.P.) was sufficient to produce an optimum response.

4. As judged from therapeutic observations, it is suggested that in the majorityof cases of T.M.A. the deficiency is similar to that in Addisonian pernicious anemia, though the mode of production of the deficiency may not be the same.

Note: ACKNOWLEDGMENTOur thanks are due to Dr. R. Row, Hon. Director, P.G. Singhance Hindu Hospital, Bombay, whereall the cases were treated, for permission to publish these reports, and the firms concerned for the generousgift of liver extracts.

     

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.

     

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 Mode of Inheritance of PTA Deficiency: Evidence for the Existence of Major PTA Deficiency and Minor PTA Deficiency

Plasma thromboplastin antecedent (PTA) activity was measured with aquantitative assay in the available members of the families of eight propositiwith severe PTA deficiency. PTA deficiency was found to exist in two forms:major PTA deficiency, characterized by PTA levels of up to 20 per cent of ourstandard reference plasma and by the potential for serious surgical bleeding,and minor PTA deficiency, characterized by PTA levels between 30 and 65per cent of our standard reference plasma and by the absence of significantsurgical bleeding. Minor PTA deficiency was found in parents and childrenof subjects with major PTA deficiency.

It would appear that the gene for PTA deficiency is an incompletely recessive or "intermediate" gene which produces major PTA deficiency in the homozygote and minor PTA deficiency in the heterozygote.

Submitted on March 27, 1961 Accepted on May 16, 1961     

The macrocytosis of hepatic disease. I. Thin macrocytosis

A macrocytic blood picture was present in 62 per cent of 222 patients withvarious types of hepatic disease.

Three different types of macrocytes were present in the blood films: a thinmacrocyte, a target macrocyte and a thick macrocyte. Just as leukocytosismight be described as neutrophilic, basophilic or eosinophilic according to thepredominate leukocyte present, so macrocytosis has been defined accordingto the predominate macrocyte present: thin macrocytosis, target macrocytosisand thick macrocytosis. This report deals with the first type, thin macrocytosis.

1. Thin macrocytosis is, by definition, the type of macrocytosis in which allthe macrocytes are thin macrocytes. It is the commonest type of macrocytosis(59 per cent of all types of macrocytosis).

2. The thin macrocyte is a flattened erythrocyte. It has the same volume asthe normal erythrocyte but is broader and thinner. As the diameter of the cellincreases its thickness decreases.

3. Thin macrocytosis is caused by an alteration in erythropoiesis in the bonemarrow and not by a flattening or swelling of normal erythrocytes in the peripheral circulation, as suggested by various workers, because:

(a) Normal size erythrocytes transfused into the circulation of patientswith thin macrocytosis do not become macrocytic.

(b) Anisocytosis is increased.

(c) The nucleated red cells in the marrow are macrocytic.

4.The thin macrocyte is produced by a macronormoblastic (or rarely atypical megaloblastic) type of maturation in the bone marrow.

5. This altered erythrocyte maturation which gives rise to thin macrocytosisis a specific response to nonspecific hepatic parenchymal cell damage. It occursin a significant percentage of patients suffering from all types of parenchymaland obstructive hepatic disease. It does not occur in patients with simple fattyliver or obstructive jaundice of short duration where hepatic cells are notdamaged. A deficiency of any known hematopoietic factor plays no part inthe etiology of this disorder.

Submitted on July 7, 1958 Accepted on October 28, 1958     

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 effects of combined folic acid and liver extract therapy

1. Folic acid, when administered alone, did not prevent the development or progression of subacute combined degeneration in 12 of 22 patients receiving this agent for from twelve to twenty-five months.

2. One patient with total gastrectomy and a macrocytic anemia developed subacute combined degeneration after five months of folic acid therapy.

3. Neurologic disease did not develop in 6 pernicious anemia patients treated with folic acid and liver extract for three and one-half to thirty-nine months.

4. In 10 pernicious anemia patients with good nutrition, neurologic relapses did not progress when liver extract or vitamin B₁₂ therapy was instituted, even though folic acid therapy was continued. In 2 patients with abnormal nutrition and complicating organic abnormalities, nervous system disease progressed after institution of liver extract therapy.

5. Our observations are best explained by the theory that the hematologic and neurologic manifestations of pernicious anemia and other macrocytic anemias associated with gastro-intestinal tract pathology and inadequate nutrition are due to a deficiency of more than one substance. The administration of folic acid may improve the hematologic status but induce a deficiency of another substance or substances, e.g., vitamin B₁₂, which are essential for the maintenance of a normal blood picture and the integrity of the central nervous system. This deficiency will eventually result in the development of a suboptimal blood picture or subacute combined degeneration of the spinal cord, or both.

6. The hematologic status of patients with pernicious anemia is not maintained in a more satisfactory state by supplementation of liver extract or vitamin B₁₂ therapy with folic acid.

7. Folic acid therapy did not produce neurologic disease in patients with iron deficiency anemia who had free gastric hydrochloric acid in their gastric secretions and presumably sufficient intrinsic factor. It did not influence response to ferrous sulfate therapy.

8. Patients with sprue, nutritional macrocytic anemia and other macrocytic anemias associated with gastro-intestinal tract pathology who are treated with folic acid should also be given supplemental liver extract or vitamin B₁₂ to insure against the development of nervous system disease.

Submitted on April 16, 1951 Accepted on July 23, 1951     

Some aspects of the metabolism of antimegaloblastic substances in man

1. When the sera of pernicious anemia patients or controls were heated at100 C. for 30 minutes, they developed the ability to support the growth of L.leichmannii by virtue of some substance other than vitamin B₁₂. It seemedlikely, however, that following the administration of the vitamin, such heatingalso liberated free B₁₂ in the serum from a combined form.

2. The L. leichmannii assay did not appear to be satisfactory for showingpossible difference in the levels of vitamin B₁₂ in the urines or sera of perniciousanemia patients and controls.

3. Parenterally administered vitamin B₁₂ did not cause any measurable risein the serum level of folic acid or citrovorum factor in pernicious anemia patientsor controls.

4. The synthetic folic acid conjugates pteroyldiglutamic acid and pteroyltriglutamic acid appeared in the sera and urines of pernicious anemia patientsas pteroylglutamic acid or some related substance with folic acid activity forS. faecalis.

5. Orally administered citrovorum factor appeared to be largely converted bythe gastric juice to folic acid if free hydrochloric acid was present. When administered parenterally, however, citrovorum factor was excreted in the urinelargely unchanged.

Submitted on October 19, 1952 Accepted on January 6, 1953     

The genetic relation and clinical differentiation of Cooley's anemia and Cooley's trait

1. Four additional families illustrating the clinical and genetic relationshipsof Cooley’s anemia and Cooley’s trait have been presented.

2. Blood findings in an offspring of a patient with Cooley’s anemia are recorded.

3. The asymptomatic nature of Cooley’s trait and its differentiation fromCooley’s anemia has been emphasized.

4. The inheritance of Cooley’s trait and Cooley’s anemia may be best explainedin terms of an incomplete dominant or of the simultaneous appearance of twononallelomorphic genes.

Note: ACKNOWLEDGMENTWe are indebted to Dr. John H. Linner for many of the observations on the Ca family and to MissClara Gillette for observations on the Cr family.