Blood carbonic anhydrase activity in anemia,with a note on polycythemia vera

Measurements of blood carbonic anhydrase activity were made in patients witha variety of blood dyscrasias, using a new method. In patients with anemia dueto loss of blood, infection and uremia, and in most of those with anemia associatedwith liver disease and leukemia, a constant relation was found between bloodcarbonic anhydrase activity and the hematocrit; the same holds in polycythemiavera. In patients with pernicious anemia, and in some with "refractory" anemia,and anemias associated with hepatic disease and with myelogenous leukemia,blood carbonic anhydrase activity was in or near the normal range in spite oflowered hematocrit values. The possible relation between these differencesamong anemias and the tolerance of patients with various anemias to exercise isdiscussed.

     

Cyanocobalamin, Ascorbic Acid and Pteroylglutamates in Normal and Megaloblastic Bone Marrow

The B₁₂ activity as estimated by Lactobacillus leichmannii, the folic-acid-like activity by Streptococcus faecalis (F.A.A.) and the ascorbic acid concentration have been determined in the blood and buffy coat of bone marrowof normal subjects, 10 patients with pernicious anemia in relapse, a group ofpatients with non-Addisonian megaloblastic anemia and some patients withiron deficiency.

A correlation between the serum B₁₂ and the plasma ascorbic acid andtheir respective levels in bone marrow was observed. The marrow and serumB₁₂ levels in prenicious anemia were abnormally low, but they did not differfrom a group of 5 patients with hypochromic normoblastic anemia who hadboth low serum and marrow levels. The concentration of F.A.A. in the marrowof patients with pernicious anemia was reduced, but it was felt that this wasmore likely a manifestation of the megaloblastic anemia rather than a causativefactor.

One of six patients with megaloblastic anemia of pregnancy had no detectabledeficiency, while the other five had reduced B₁₂, folic acid and ascorbic acidconcentrations. The possible therapeutic implications are discussed.

There was a significant reduction in the bone marrow concentration ofascorbic acid in all patients with megaloblastic anemia.

Submitted on March 26, 1959 Accepted on October 9, 1959     

Carbonic Anhydrase and Fetal Hemoglobin in Thyrotoxicosis

An adult patient with thyrotoxicosis had erythrocyte carbonic anhydrasedeficiency and an abnormally high level of fetal hemoglobin. After treatmentof the hyperthyroidism, the erythrocyte carbonic anhydrase level became normaland the level of Hb F dropped. Twelve other patients with hyperthyroidismand not related to this index case were studied. Most of them were alreadyunder treatment. Nine had erythrocyte carbonic anhydrase deficiency; two hadan increase of fetal hemoglobin. The possible relationship of increase of fetalhemoglobin, erythrocyte carbonic anhydrase deficiency and thyrotoxicosis isdiscussed.

Submitted on March 1, 1967 Accepted on April 7, 1967     

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.

     

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.

     

Erythrocyte Vitamin B12 Activity in Health, Polycythemia, and in Deficiency of Vitamin B12 and Folate

A method for the estimation of vitamin B₁₂ in erythrocytes using E. gracilisis described. In 29 normal subjects the mean value was 158 pg./ml. (S.D. 31pg.) packed cells.

Five of 14 patients with untreated primary polycythemia had very lowerythrocyte vitamin B₁₂ levels, and in the group as a whole the value wassignificantly less than normal. In eight patients with secondary polycythemiathe erythrocyte vitamin B₁₂ did not differ from normal, though two patients hadvalues below the normal range.

In 19 cases of pernicious anemia the mean erythrocyte vitamin B₁₂ level was53 pg./ml. (S.D. 32 pg.) packed cells which was significantly reduced. Therewas no constant relation between the erythrocyte and plasma levels. In eightcases of folate deficiency the erythrocyte vitamin B₁₂ level was significantlyreduced, with a mean value of 95 pg./ml. (S.D. 57 pg.) packed cells.

Submitted on January 2, 1969 Accepted on July 3, 1969     

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 effect of transfusions of erythrocytes on untreated pernicious anemia

1. Three patients with untreated pernicious anemia were transfused withpacked erythrocytes to produce a normal hematocrit before they were treatedwith vitamin B₁₂. One patient with untreated pernicious anemia was transfusedwith 250 cc. plasma prior to therapy with vitamin B₁₂. No significant changeswere noted in this patient who received plasma.

2. Following erythrocyte transfusions the bone marrows of these patientswere considered to show the megaloblastic type of maturation with a reductionin percentage of the early megaloblasts (Stages I and II). The ME ratio of themarrows increased. A decrease in the absolute reticulocyte count occurred. Nochange occurred in the peripheral leukocyte and platelet counts and there wasno significant change in the clinical condition of the patients.

3. After therapy with vitamin B₁₂, at a time when the hematocrits were normal, the bone marrows became normoblastic, a small but definite reticulocytosisoccurred, the leukocyte and platelet counts returned to normal, and there wassubjective improvement in the condition of the patient.

4. It is concluded that in these three patients with untreated pernicious anemiamultiple transfusions of packed erythrocytes decreased erythropoiesis but didnot cause any basic change in the type of erythrocyte maturation.

Submitted on August 1, 1955 Accepted on November 17, 1955     

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.

     

The coincidence of Mediterranean anemia and pernicious anemia in a young Sicilian

1. Coincidental Mediterranean anemia and pernicious anemia were found in a26 year old soldier of Sicilian parentage.

2. The diagnosis of pernicious anemia was made on the finding of achlorhydriaafter histamine, glossitis, megaloblastic bone marrow and macrocytic anemiawhich responded to liver extract on two occasions.

3. The diagnosis of mild Mediterranean anemia was made by finding the target-oval-cell trait in the patient and in five members of his family.

4. It is of interest that target cells were not found in the peripheral blood untiltreatment with liver was begun. While pernicious anemia dominated, the characterof the peripheral blood picture was macrocytic. Liver therapy corrected this,whereupon "hypochromic polycythemia" characteristic of mild Mediterraneananemia was found.

     

The Activity of Carbonic Anhydrases B and C from Human Erythrocytes and the Inhibition of the Enzymes by Copper

The catalytic activity of two isolated forms of human erythrocytic carbonic anhydrase, HCAB and HCAC, was studied at 1.6° C by means of a pH-stat method. 80 per cent of the activity of the carbonic anhydrase with high specific activity (HCAC) was inhibited in the presence of 1.6 μM CuSO₄, whilst the activity of the form with low specific activity (HCAB) was the same as that in a reaction medium which contained no inhibiting metallic ions. A chelating agent (EDTA), which eliminated the effect of CuSO₄, had no effect on the enzyme activity in reaction media in which the concentration of the ions of heavy metals was below a critical level. The fact that HCAC is inhibited more effectively by copper than HCAB, was utilized in a procedure by means of which the activity of HCAC and HCAB was determined separately in solutions which contained both enzymes. The same procedure was used to determine the enzyme content of the erythrocytes, on the assumption that HCAC and HCAB in haemolysates had the same specific catalytic activity and were inhibited by CuSO₄ to the same extent as in preparations of the isolated forms of carbonic anhydrase which were used in the investigations. On the basis of results from investigations of the blood from 24 donors it was calculated that HCAC comprises 80 per cent of the activity and about 20 per cent of the carbonic anhydrase concentration in red cells. The total carbonic anhydrase activity in erythrocytes from 5 new-born infants was about one tenth of that in the erythrocytes from adults. A high erythrocytic carbonic anhydrase activity in 2 patients with untreated pernicious anaemia was found to be the result of a parallel increase in the activity of HCAB and HCAC.     

Comparison of vitamin B12 from liver and from Streptomyces griseus in the treatment of pernicious anemia

1. A clinical remission in one case of pernicious anemia in relapse treated with50 µg. of vitamin B₁₂ from liver is reported; the patient was followed for 114 daysafter two doses of 25 µg. were given on successive days and a peak of 3,900,000erythrocytes occurred on the eighty-sixth day.

2. Preliminary observations are reported in 2 patients with pernicious anemiain relapse treated with a vitamin B₁₂ derived from Streptomyces griseus; the firstpatient who had no neurologic complaints received 32 µg. while the second wasgiven two doses of 32 µg. each because of severe, subacute, combined degeneration.A good hematologic response and a satisfactory clinical remission occurred inboth cases. There was definite improvement neurologically in the second case.

3. The administration of 32 µg. of vitamin B₁₂ derived from Streptomyces griseusto each of 5 cases of pernicious anemia in remission resulted in minor subjectiveand objective improvement.

     

Red-Cell Carbonic Anhydrase Isoenzymes in Megaloblastic Anaemia

Erythrocyte carbonic anhydrase isoenzymes B and C have been measured by means of electrophoresis on cellulose acetate membrane strips followed by staining, elution and spectrophotometry. The results from 30 normal subjects showed for carbonic anhydrase B a mean and standard deviation of 12.1 ± 1.4 mg/g Hb, and for carbonic anhydrase C, 3.9 ± 0.9 mg/g Hb. Both isoenzymes were markedly increased in all 10 cases of untreated megaloblastic anaemia, the mean ± SD for carbonic anhydrase B being 24.8 ± 7.6 mglg Hb and for the isoenzyme C, 7.7±k 1.2 mg/g Hb. These results are both highly significantly greater than the normal means (P < 0.001). The elevated levels of both isoenzymes returned to the normal range within 2–4 weeks of starting vitamin B₁₂ therapy in 3 patients with pernicious anaemia in whom serial enzyme measurements were made. In a group of 13 patients with pernicious anaemia, who had been adequately treated with vitamin BIZ for 1–15 years, the mean levels of isoenzymes B and C were 10.3 ± 0.6 and 2.9 ± 0.7 mg/g Hb respectively; both these mean results are significantly less than the corresponding normal means (P < 0.001).     

Urinary excretion of vitamin B12

1. Twelve patients, 6 with pernicious anemia and 6 without pernicious anemiaas controls, were studied as to urinary excretion of vitamin B₁₂.^*

2. Microbiologic tube assay using Lactobacillus leichmanii A.T.C.C. # 4797was the method of B₁₂ assay.

3. Dosages used were 42.2, 63.3, 84.4, 211.0 µg given both in ascending anddescending order at daily intervals.

4. Amounts of 84.4 to 211.0 µg of vitamin B₁₂ parenterally resulted invariablyin the excretion of 53 to 68 per cent of the injected vitamin B₁₂ within 18 hours.The percentage of excretion rose remarkably as the dosage increased.

5. An amount of 42.2 µg of vitamin B₁₂ parenterally resulted in relativelylittle excretion when this was the initial dose, but the excretion was appreciablewhen these doses followed the injection of the larger doses. "Saturation" of thebody apparently occurs. In general more vitamimi B₁₂ is excreted in the smallerdoses if these follow the larger doses.

6. No significant differences could be detected between the pernicious anemiaand nonpernicious anemia patients.

7. A possible explanation of these results is proposed.

Submitted on July 18, 1951 Accepted on October 1, 1951     

Pernicious anemia and related anemias treated with vitamin B12

Eleven cases treated with vitamin B₁₂ have been presented. Eight patients withpernicious anemia in relapse responded hematologically. Two patients with mildneurologic involvement were relieved by therapy with B₁₂ alone.

Consideration of the quantities of the crystalline vitamin required to promotemaximal erythropoiesis in pernicious anemia indicates that less than about 0.75µg. daily in doses at intervals of several days will not suffice to establish and maintain blood values as high as does adequate treatment with liver extract. Parenteraldaily doses of 1.0 µg. promoted good erythropoiesis in one patient, although itappears that the maximum rate of hemopoiesis may require the initial averagedaily dose of approximately 3.0 µg.

The reticulocyte count is an unreliable quantitative criterion of activity or adequacy of therapy.

It is suggested that hemopoietic factors in addition to PGA and B₁₂ may berequired by some patients to obtain maximal erythrocyte levels.

Vitamin B₁₂, as well as PGA, effects a reduction in the fecal urobilinogen output of patients with pernicious anemia. The significance of this finding is discussed.

No change in urinary excretion of pteroylglutamate or of porphyrin was detected in patients treated with vitamin B₁₂.

     

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

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

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

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

The Maturation Rate of Reticulocytes

An in vitro culture technic for the study of reticulocyte maturation wasdescribed. The method gave reproducible results and proved to be of valuein the comparative study of reticulocyte maturation in blood disorders. By thismethod it was shown that variations in the reticulocyte maturation in vitroparalleled similar variations present in vivo.

The maturation of reticulocytes from patients with different types ofanemia was investigated. In some anemias the in vitro maturation of reticulocytes was prolonged, not only because younger reticulocytes were present inthe blood, but also because the rate at which the reticulum substance disappeared was delayed. This was particularly evident in the anemia of chronicuremia, in Cooley’s anemia and in pernicious anemia in relapse. In only occasional cases of hereditary spherocytosis and of autoimmune hemolyticanemia was the rate of reticulocyte maturation found to be moderatelydelayed. In patients with iron deficiency anemia or bleeding anemia it wasalways normal.

From the above findings the following conclusions were derived:

1. The reticulocyte number in the circulating blood is the resultant of threevariables: (a) the rate of output of new reticulocytes from the bone marrow;(b) the stage of maturation at which reticulocytes are delivered into theperipheral circulation; (c) the rate of disappearance of the reticulum substance.

2. The number of reticulocytes in the circulating blood cannot be indiscriminately used as a precise index of red cell production in erythrokinetics.

3. There is good reason to believe that a defect in the rate at which thereticulocytes mature in the circulating blood is an index of a similar defectin the process of erythroblastic differentiation in the bone marrow.

     

The chemistry and functioning of the mammalian erythrocyte

The erythrocyte is a unit of protoplasm highly specialized for the functions ofO₂ and CO₂ transport but still containing sufficient repair systems for maintainingitself for about 120 days.

The erythrocyte develops through a complex series of changes, arising from areticular cell of the, bone marrow and differentiating into an actively synthesizingand dividing nucleated cell. After a time the cell stops dividing, the nucleus beginsto degenerate and a differentiation takes place in the cytoplasm, the complex mixture of cytoplasmic proteins including mitochondria being replaced almost butnot completely by a single kind of protein, namely, hemoglobin.

The functions of several of the anatomic features of the hemoglobin molecule areconsidered. The hemoglobin molecule has a molecular weight of 68,000 with 4planar heme units which lie parallel to each other, two being on the proximal andtwo on the distal surface of the globin. The globin appears to be made up of 4 polypeptide layers with the planar heme units lying perpendicular to the polypeptidelayers.

The heme units possess a resonating ring structure which stabilizes the unit.The two vinyl groups on the periphery of the heme appear to be necessary ifan iron atom is to be inserted into the newly formed protoporphyrin ring. Thetwo ionized propionic acid groups also at the periphery of the heme, appear to berequired for orienting and attaching the heme unit to two strongly basic groups ofthe globin, possibly guanidine groups of arginine. The attachment of the iron ofheme is postulated to be to an imidazole nitrogen of a histidine residue of theglobin. This latter attachment is by itself a weak bonding but is stabilized not onlyby the coulombic attraction of the ionized propionic acid groups but also by theVan der Waals forces between the globin and the planar resonating porphyrin.

The attachment of the iron to the imidazole group endows the iron of the hemewith the property of combining with O₂ reversibly, the addition of O₂ being connected with a pairing of all the unpaired electrons in the complex. Probably, as aconsequence of this pairing of electrons, the O₂ does not act as an oxidant as itwould if the special iron link to globin were destroyed. The iron of the heme isbound to 6 atoms or atom groups. It binds 4 nitrogens of the protoporphyrin in theplane of the ring. Below the plane of the ring it binds one nitrogen of the imidazolegroup, and above the plane of the ring it may then bind O₂ reversibly. The secondnitrogen of the imidazole group is postulated to change its ionization with achange of oxygenation of the hemoglobin; this change in ionization makes possible the conversion of some 50 per cent of the CO₂ transported in the blood tobicarbonate ion, without appreciably changing the pH of the blood. A zinc protein, carbonic anhydrase, is present in the erythrocytes to catalyze the normallyslow hydration-dehydration of the CO₂-H₂CO₃ system.

How this non-nucleated erythrocyte is maintained in a functional state for a lifespan of 120 days is poorly understood. The erythrocyte has a very low O₂ utilization which is compatible with the fact that the mitochondnia, which are believedto be the seat of cytochrome oxidase activity, are absent. However, there is presenta rather complete glycolytic system which appears to play a major role in themetabolic life of the mature erythrocyte. Hemoglobin is slowly converted in theintact erythrocyte to ferric hemoglobin, i.e., methemoglobin. The methemoglobinis reduced back to the functional ferrous form by reduced diphosphopyridine nucleotide arising during glycolysis. Riboflavin enzymes appear to act as the intermediators between reduced DPN and methemoglobin. The pyridine and flavineenzymes which are slowly undergoing hydrolysis are regenerated by adenosinetriphosphate produced in glycolysis. Catalase is present, probably to protect theheme units of hemoglobin against H₂O₂, the hemes being especially vulnerable toperoxidative attack at the methene links.

Note: ACKNOWLEDGMENTSThe author wishes to acknowledge his gratefulness to L. Michaelis, E. Racker, and A. Rothen fortheir suggestions and criticisms.

     

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.

     

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.