Iron Enzymes in Iron Deficiency. Vi. Aconitase Activity and Citrate Metabolism

IRON ENZYMES IN IRON DEFICIENCY. VI. ACONITASE ACTIVITY AND CITRATE METABOLISM

Ernest Beutler

J Clin Invest. 1959;38(9):1605-1616. https://doi.org/10.1172/JCI103939.

Research Article

Find the latest version: https://jci.me/103939/pdf IRON ENZYMES IN IRON DEFICIENCY. VI. ACONITASE ACTIVITY AND CITRATE METABOLISM By ERNEST BEUTLER (From the Argonne Cancer Research Hospital, operated by The University of Chicago for the United States Atomic Energy Commission, and the Department of Medicine, The University of Chicago, Chicago, Ill.) (Submitted for publication April 7, 1959; accepted June 1, 1959)

Iron serves vital functions in the economy of marked citric acid diuresis occurred in these ani- the body in a variety of ways. Its role as an inte- mals, especially after loading with citrate and gral part of biologically important substances is butyrate. While we cannot accept Takeda's im- well known. In heme, it forms a part of the oxy- plication that a-a'-dipyridyl-poisoned animals are gen-carrying compounds, hemoglobin and myo- iron-deficient in a sense in any way comparable to globin, and of the cytochromes, cytochrome oxi- iron deficiency produced by bleeding or by a low dase, catalase and perioxidase. Another enzyme, iron diet, it seemed worthwhile to explore aconi- succinic dehydrogenase, appears to contain iron tase activity and the metabolism of exogenously (1), but not bound as heme. Physiologic vaso- administered citrate in truly iron-deficient ani- depressor activity has also been ascribed to the mals and humans. iron storage compound, ferritin (2). Less well known, perhaps, is the relatively recently described MATERIALS AND METHODS function of iron as a cofactor in several important Unless otherwise indicated, female Holtzman strain enzymatic reactions (3-5). Knowledge of this rats have been used throughout these experiments. All function of iron served as the basis for the current animals were fed the iron-poor diet that we have de- investigation. scribed previously (7), and were given water rendered In previous studies of the pathologic physiology iron free by passing it over an ion exchange resin. Un- of the iron-deficiency state, we have examined the less otherwise indicated, each control rat was given 25 mg. of iron, as iron-dextran (Imferon®),' intramuscu- fate of the heme compounds: hemoglobin, cyto- larly into the hamstring muscles in two divided doses, chrome c (6, 7), catalase (7, 8), cytochrome oxi- one at the beginning of the experiment, the other, two dase (9), and succinic dehydrogenase (10). Con- weeks later. The animals were not fasted prior to sac- trary to the earlier conclusions of Hahn and rifice. Each animal was exsanguinated from the heart but in with those of under ether anesthesia. All of the organs that were re- Whipple (11), agreement moved were placed into chilled vessels. Gubler, Cartwright and Wintrobe (12), we found The method of aconitase assay was based on that de- that varying degrees of depletion in several iron scribed by Anfinsen (15). Tissues were homogenized in enzymes occurred in the iron-deficiency state. In ice cold 0.2 M phosphate buffer, pH 7.40, with a Potter- the current study, we have undertaken measure- Elvejhem type homogenizer, and the diluted homogenate ment of aconitase. an enzyme that is not known was filtered through Schleicher and Schuell No. 604 fil- ter paper. Filtrates were kept at 0° C. and assays were to contain iron, but that appears to require iron carried out within an hour. Unless otherwise stated, 0.1 as a specific cofactor (5). Dialyzed solutions of and 0.2 ml. aliquots of filtered tissue homogenate were aconitase lose their activity. Dickman and Clou- added to 4.4 or 4.3 ml. of 0.2 M phosphate buffer, pH 7.4, tier (5) demonstrated that the addition of iron in a 370 C. water bath. The reaction was started by the and a reducing agent reactivated the enzyme. addition of substrate, 0.5 ml. of cis-aconitate solution. The substrate was freshly prepared for each assay by al- None of the many other ions tested had the ca- lowing an aqueous solution of cis-aconitic acid anhydride pacity to reactivate aconitase (5, 13). (Sigma Chemical Corporation) to remain at room tem- Aconitase catalyzes the biologically important perature for one hour, then neutralizing with 0.1 ml. 2 N equilibrium between citrate, isocitrate and cis- NaOH per 2.76 mg. anhydride. The volume was then aconitase. Takeda and Hara (14) have reported adjusted with buffer to give a concentration of 15.6 mg. that activity of aconitase is diminished in livers of 1 Supplied through the courtesy of Lakeside Labora- guinea pigs poisoned with a-a'-dipyridyl, and that tories, Milwaukee, Wisconsin. 1605 1606 ERNEST BEUTLER

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MILLILITERS OF HOMOGENATE

FIG. 1. THE RELATIONSHIP BETWEEN THE AMOUNT OF CITRATE PRODUCED FROM CIs-ACONITATE AND THE VOLUME OF HOMOGENATE ADDED Assay system: cis-aconitate, 7.8 mg. (as anhydride); tissue homogenate, as indicated; final volume 5 ml. in 0.2' M phosphate buffer, pH 7.4; temperature 370 C.

(as anhydride) per ml. Fifteen minutes after addition of parture from linearity that was sometimes observed when substrate, the reaction was stopped. In the case of the amount of citric acid formed was compared with the heart, kidney and liver, this was done by adding 1 ml. of amount of tissue extract added (Figure 1), two dilu- the reaction mixture to 0.5 ml. of 50 per cent (V/V) tions of tissue-were assayed. These dilutions were chosen sulfuric acid in a tube marked at the 10 ml. mark. The so that one sample usually contained less than one unit total volume was then brought to approximately 5 ml. and the other contained more than one unit of enzyme with distilled water. In the case of brain homogenate, activity. The average of these two determinations has the reaction was stopped by adding 5 ml. of 20 per cent been reported. One-tenth and 0.2 ml. of the following trichloracetic acid to the entire 5 ml. reaction mixture; tissue dilutions were found to be appropriate: kidney, citric acid determinations were done on 5 ml. of the 1 to 25 or 1 to 40; heart, 1 to 50; brain, 1 to 20; liver, filtrate. Citric acid determinations were carried out ac- 1 to 20. cording to our modification of the pentabromacetone The method used for assaying the aconitase activity method (16). In this assay system, the quantity of citric of human white cells has been described in detail else- acid produced is approximately linear with respect to time where (17). of reaction and to volume of tissue extract added (Figure 1). One unit of enzyme activity has been defined arbi- EXPERIMENTAL AND RESULTS trarily as that amount of enzyme that will, in the assay A. The reactivation of aconitase by iron system described, convert 10' Moles of cis-aconitic acid to citric acid per minute.2 Because of the slight de- A one to 800 homogenate was prepared from the kidney of a normal rat in 0.2 M phosphate 2 In two experiments, an assay system containing 3.9 buffer, pH 7.4 The aconitase activity of the mg. cis-aconitic acid as an anhydride was used. It had homogenate was assayed immediately and at in- been determined empirically that one unit of enzyme in tervals after at 370 C. Solutions con- this system converts 0.85 X 10' Moles of cis-aconitic acid incubation to citrate per minute. taining 0.03 M ferrous sulfate, 0.03 M ascorbic ACONITASE ACTIVITY AND CITRATE METABOLISM IN IRON DEFICIENCY 1607 acid, and both were prepared and their pH ad- and aconitase activity of kidneys, hearts and justed to 7.4. After one hour, one volume of one livers were determined. Eighteen 23-day-old rats of each of these solutions was added to each of were given an iron-poor diet, nine of these three nine-volume aliquots of the incubating mix- being injected with iron-dextran. After 10 days, ture. As a control, one volume of buffer was four iron-deficient and four iron-treated (control) added to a fourth aliquot. Aconitase activity was rats were sacrificed, and after 26 days, the re- determined at intervals. The results of this ex- maining five iron-deficient and five iron-treated periment, showing the time course of partial re- (control) animals were sacrified. The hemoglo- activation of aconitase by iron and ascorbate, are bin, hematocrit and plasma iron values and organ shown in Figure 2. No reactivation was achieved aconitase assays of these animals are presented in by the addition of either iron or ascorbate alone. Table I. A well-marked decrease in aconitase The effect of different concentrations of iron activity of the kidneys of the iron-deficient animals, and ascorbic acid has been investigated in a simi- compared with that of the control animals, was lar fashion. In three experiments, one to 800 found. In sharp contrast, the aconitase activity of kidney homogenate, 75 to 80 per cent inactivated the other organs studied was normal, with the by incubating for one hour at 370 C., was reacti- possible exception of a slight reduction of heart vated for 10 minutes with iron and ascorbate. enzymatic activity. The enzymatic activity of Aconitate was then added and enzyme activity mixtures of equal volumes of homogenates from measured in the usual manner. It is apparent iron-deficient and normal animals was the arith- reactivation of incubated enzyme that complete metic mean of the two: no inhibition of normal could be achieved regularly when sufficiently high concentrations of iron and ascorbic acid were used. kidney enzyme or activation of iron-deficient kid- The results of these studies are given in Figure 3. ney enzyme could be demonstrated. Rats that had always been fed stock diet had kidney aconi- B. Aconitase activity of organs of- normal and tase activity comparable with that of the iron- iron-deficient rats dextran controls. Administration of iron-dextran Four 23-day-old rats were sacrificed and the to such rats did not cause an elevation of kidney hemoglobin, hematocrit and plasma iron values aconitase activity.

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F IG. 3. THE REACTIVATIOIN OF ACONITASE BY VARIOUS CONCENTRATIONS OF IRON AND ASCORBATE In each of the three experiments, normal rat kidney homogenate diluted one to 800 was incubated for one hour at 370 C. The concentrations of iron and ascorbate indicated were added and an equal volume of buffer was added to a control tube. Aconitase activity was determined after an additional 10 minutes' incubation. Seventy-five to 80 per cent of the enzyme was inactivated in the control tube. The effect of iron and ascorbate is expressed as the percentage of inactivated enzyme which was reactivated.

C. An attempt to reactivate aconitase of kidneys kidney homogenate. In point of fact, approxi- front iron-deficient rats with iron and ascorbic mately 33 per cent inhibition of the enzymatic acid activity of kidney homogenate from iron-deficient rats was observed. Tissue homogenates from iron-deficient and control rats were incubated with ascorbate and CONTROL ferrous sulfate, both in final concentration of | IRON DEFICIENr

0.003 M, for one-half hour. The aconitase ac- Fe tivity of these homogenates was compared with that of control homogenates that had not been incubated 400 with iron and ascorbate acid. No "reactiva- l 100 tion" of enzyme in homogenates prepared from F.~~~~~~~~~~~~~~~F iron-deficient animals could be demonstrated (Fig- ure 4). The use of ferrous sulfate and ascorbate in a higher concentration, 0.005 M, also resulted in no "reactivation." As indicated under B above, normal kidney homogenate also failed to "acti- vate" iron-deficient kidney homogenate. Simi- FIG. 4. THE FAILURE OF IRON AND ASCORBATE TO REACTIVATE' ACONITASE FROM NORMAL AND IRON- larly, 3 ml. of one to 400 normal kidney homog- DEFICIENT RAT KIDNEYS enate completely inactivated by heating to 1000 Bars under arrows represent aconitase activity of ho- C. for five minutes or to 600 C. for 30 minutes, mogenates that had been incubated for one-half hour or partially inactivated by incubating at 370 C. with ascorbate and ferrous sulfate, both in final concen- for two hours, failed to "reactivate" iron-deficient tration of 0.003 M. 1610 ERNEST BEUTLER

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1 2 3 4 5 6 7 8 9 10 1 12 13 14 AGE (WEEKS) FIG. 5. THE EFFECT OF INTRAGASTRIC ADMINISTRATION OF IRON ON THE HEMOGLOBIN AND PLASMA IRON VALUES, AND ON KIDNEY ACONITASE ACTIVITY OF IRON-DEFICIENT RATS Each point represents the value obtained from a single rat. The lines connect the means of each group. Intragastric iron therapy, 2 mg. Fe++ per day, was initiated when the rats were 76 days old.

D. The effect of treatment on kidney aconitase of kidney aconitase activity toward normal values of iron-deficient animals was observed in the iron-treated group. The individual values were too scattered to warrant A group of weanling rats was placed on an conclusions about the rate of return of aconitase iron-poor diet. The "control" group was given to normal activity, as compared with hemoglobin mg. of iron in the form of iron-dextran 37.5 and plasma iron values (Figure 5). intramuscularly in three divided doses. Four animals were sacrificed at the beginning of the E. The effect of anemia on aconitase activity of experiment and the hemoglobin, plasma iron and non-iron-deficient animals kidney aconitase were determined. Thereafter, groups of two or three animals were sacrificed at Holtzman strain, retired male breeder rats, intervals, and hemoglobin, plasma iron and kid- weighing 450 to 510 Gm., were placed on the ney aconitase assays were carried out. Ap- iron-poor diet and were divided into four groups. proximately seven weeks after initiation of the A control group of four rats received no treat- experiment, one-half of the iron-deficient group ment. Another group of four animals was in- was treated by the daily intragastric administra- jected subcutaneously with 10 or 20 mg. of tion of 2 mg. of iron in the form of iron sulfate phenylhydrazine at two to four day intervals in 0.01 N hydrochloric acid. A prompt return (Figure 6). The other two groups of six rats ACONITASE ACTIVITY AND CITRATE METABOLISM IN IRON DEFICIENCY 1611 each were bled 5 ml. from the heart three times F. Aconitase activity of human white cells weekly for a total of six bleedings. Six of these Semiquantitative estimation of the aconitase animals were intramuscularly injected weekly with activity of human white cells, reported elsewhere 12.5 mg. of iron as iron-dextran. Only six bled (16), revealed a mean value of approximately animals, three iron-treated and three untreated, 98 units per 1010 leukocytes. Aconitase determi- survived. The average hemoglobin levels and nations of the white cells of four iron-deficient treatment schedules of the entire surviving ex- subjects have given an average aconitase value of perimental group are presented in Figure 6. At 88. All of the individual values (106, 94, 77 the end of the 27 days, all animals were sacrificed and 77 units per 1010 leukocytes) were within and the hemoglobin, plasma iron and kidney the normal range, and the difference in means is aconitase activity were measured. The results, not statistically significant. presented in Figure 7, demonstrate that iron- deficient rats (bled, but not treated with iron) G. Metabolism and excretion of a citrate load in iron-deficient and normal had diminished kidney aconitase activity, even subjects after relatively short-term iron deprivation. Fasting subjects were given disodium hydro- Anemia, induced by phenylhydrazine injections gen citrate, 130 mg. per Kg. of body weight, in or by bleeding when iron supplements were the form of a commercial preparations Citrate given, failed to affect kidney aconitase activity. 3 Citralka®, Parke, Davis and Co.

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levels in plasma and urine were measured be- lower cumulative urinary citrate excretion of the fore ingestion of the loading dose and at subse- iron-deficient subjects are deemed to be of little quent intervals. A light breakfast consisting of importance. In one mildly iron-deficient sub- a roll and coffee was given one hour after ad- ject with high urinary citrate excretion, repeated ministration of citrate. The results of studies measurements were made of six hour urinary carried out in three normal and six iron-deficient citrate excretion following standard loading, both subjects are shown in Figure 8. Pertinent clinical before and after iron therapy. No change in and hematologic data are presented in Table II. citrate excretion could be demonstrated (Fig- The slightly higher plasma average levels and ure 9). ACONITASE ACTIVITY AND CITRATE METABOLISM IN IRON DEFICIENCY 1613

DISCUSSION rats did not appear to be due merely to a lack of iron acting as a cofactor for aconitase been dem- activity, A decrease in aconitase activity has since addition of iron failed to "reactivate" aconi- onstrated in the kidneys of iron-deficient rats. tase from iron-deficient animals. Normal kid- Weanling rats deprived of iron failed to develop ney tissue also failed to reactivate enzyme from increase in kidney aconitase activity the rapid and it may therefore be that occurs when iron is administered to young iron-deficient kidneys, rats. The activity of this enzyme decreased in presumed that the decrease of aconitase activity mature animals rendered iron-deficient by re- was due to actual depletion of the protein moiety peated bleeding while on an iron-poor diet. It of the enzyme. In this respect, aconitase may be is of particular interest that the decrease in en- regarded to be similar to apoferritin, which is zymatic activity in kidneys from iron-deficient synthesized in response to iron (18), and to the

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FIG. 8. PLASMA CITRATE AND CUMULATIVE URINARY CITRATE EXCRETION IN THREE NORMAL AND SIX IRON-DEFICIENT SUBJECTS At time = 0, 130 mg. of disodium hydrogen citrate was given per Kg. of body weight. Pertinent clinical and hematological data for each patient are given in Table II. 1614 ERNEST BEUTLER

TABLE II Clinical and laboratory findings of nine subjects studied after citrate loading

Serum unsaturated iron- Patient Plasma binding Marrow No. Sex Age Clinical diagnosis Hb iron capacity iron Remarks

Gm. % pg.f100 ml. "g.f 100 ml. 1 F 28 Cardiac arrhythmia 13.3 ++ 2 M 22 Normal male 3 M 25 Normal male 4 F 21 Pseudohemophilia 10.9 5 540 (no recent patho- logical bleeding) 5 F 22 Menorrhagia 4.8 5 450 6 F 25 Menorrhagia 10.8 5 540 (inactive rheumatic heart disease) 7 F 35 Menorrhagia 12.7 50 140 Patient had been treated 50 160 for iron deficiency a year previously and her normal Hb was known to be higher than the present level. Hb rose to 14.5 Gm. % following IV iron therapy (see Figure 9). 8 F 43 Menorrhagia 13.6 46 180 0 44 270 9 F 62 Gastrointestinal 6.7 5 400 bleeding

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p 100 LU 0 Menses I I I a I " I I I 0 10 20 30 40 50 60 ( 100 110 TIME (-DAYS) FIG 9. CUMULATIVE Six HOUR URINARY CITRATE EXCRETION OF SUBJECT No. 7 Measurements of citrate excretion were made following oral citrate loading on repeated occasions, before and after intravenous administration of iron in the form of saccharated iron oxide. ACONITASE ACTIVITY AND CITRATE METABOLISM IN IRON DEFICIENCY 1615 globin moiety of hemoglobin, synthesis of which of iron and ascorbate, homogenate of normal appears to be inhibited in iron deficiency (19). rat kidney, or heat-inactivated normal rat kid- Takeda and Hara (14) have reported that ney homogenate. This finding is interpreted as poisoning with a-a'-dipyridyl results in greatly. indicating an actual depletion of the protein increased urinary excretion of citric acid by moiety of aconitase in iron-deficient animals. guinea pigs, especially after they had been fed 4. Treatment of iron-deficient rats with iron large quantities of citrate and butyrate. Using restored their kidney aconitase activity to normal. rats as the test animal, we have been unable to 5. Anemia, induced by the administration of duplicate these results, using our modification of phenylhydrazine or by repeated bleeding of rats the pentabroacetone method for citrate analysis given supplemental iron, failed to reduce kidney (16). The less specific sodium sulfide (20) aconitase activity; blood loss anemia in rats method used by Takeda and Hara gives a false not given iron supplement resulted in a decrease positive reaction forcitricacid with a-a'-dipyridyl in kidney aconitase activity. (21). It is possible that Takeda and Hara meas- 6. Aconitase activity of human white blood ured a-a'-dipyridyl, or a metabolic product as cells from iron-deficient subjects was found to be citric acid. However, even true citric aciduria, normal. if it occurs under these experimental conditions, 7. Plasma citrate levels and urinary citrate does not necessarily indicate profound changes excretion after oral loading with citrate has been in intracellular citric acid metabolism: citric studied. No difference could be demonstrated acid excretion is extremely sensitive to urine between normal and iron-deficient subjects. pH changes (22). In spite of our failure to confirm the results REFERENCES of Takeda and Hara in rats, we studied citric 1. Kearney, E. B., and Singer, T. P. On the prosthetic acid excretion in iron-deficient and normal sub- group of succinic dehydrogenase. Biochim. biophys. jects following loading with citric acid, hoping Acta 1955, 17, 596. 2. Shorr, E. Intermediate metabolism and biological ac- that a clinically useful means for diagnosis of tivities of ferritin. Harvey Lectures, 1954-55, 50, iron deficiency might emerge. However, no 112. significant difference was observed either in 3. Patwardhan, M. V. Role of ferrous iron in enzymatic blood or urine citrate levels of iron-deficient and transamination. Nature (Lond.) 1958, 181, 187. normal subjects. Obviously, this does not prove 4. Suda, M., and Takeda, Y. Metabolism of tyrosine. nor- II. Homogentisicase. J. Biochem. (Tokyo) 1950, that the operation of the citric acid cycle is 37, 381. mal in iron-deficient subjects: the metabolism 5. Dickman, S. R., and Cloutier, A. A. Activation and of an exogenously administered dose of citrate stabilization of aconitase by ferrous ions. Arch. may be quite different from that of citrate pro- Biochem. 1950, 25, 229. duced within the cell by condensation of acetate 6. Beutler, E. Iron enzymes in iron deficiency. I. and oxalacetic acid. Cytochrome C. Amer. J. med. Sci. 1957, 234, 517. 7. Beutler, E., and Blaisdell, R. K. Iron enzymes in iron deficiency. III. Catalase in rat red cells and SUM MARY liver with some further observations on Cyto- chrome C. J. Lab. clin. Med. 1958, 52, 694. 1. The observation of Dickman and Cloutier 8. Beutler, E., and Blaisdell, R. K. Iron enzymes in (5, 13), that inactivated solutions of aconitase iron deficiency. II. Catalase in human erythrocytes. may be reactivated by iron and a reducing agent, J. clin. Invest. 1958, 37, 833. has been confirmed. 9. Beutler, E. Iron enzymes in iron deficiency. IV. 2. The aconitase activity of kidneys from iron- Cytochrome oxidase in rat kidney and heart. Acta Haemat. (Basel) 1959, 21, 371. deficient rats was found to be reduced consist- 10. Beutler, E. Iron enzymtes in iron deficiency. V. Suc- ently, but the aconitase activity of the hearts, cinic dehydrogenase in rat liver, kidney, and heart. livers and brains of iron-deficient rats was Blood. In press. normal. 11. Hahn, P. F., and Whipple, G. H. Iron metabolism. II. Its absorption, storage and utilization in ex- 3. Aconitase from the kidneys of iron-deficient perimental anemia. Amer. J. med. Sci. 1936, 191, rats could not be "reactivated" by the addition 24. 1616 ERNEST BEUTLER

12. Gubler, C. J., Cartwright, G. E., and Wintrobe, M. M. 17. Beutler, E., and Yeh, M. K. Y. Aconitase in human Studies on copper metabolism. XX. Enzyme ac- blood. J. Lab. clin. Med. In press. tivities and iron metabolism in copper and iron de- 18. Granick, S. Ferritin. IX. Increase of the protein ficiencies. J. biol. Chem. 1957, 224, 533. apoferritin in the gastrointestinal mucosa as a 13. Dickman, S. R., and Cloutier, A. A. Factors affect- direct response to iron feeding. The function of ing the activity of aconitase. J. biol. Chem. 1951, ferritin in the regulation of iron absorption. J. 188, 379. biol. Chem. 1946, 164, 737. 14. Takeda, Y., and Hara, M. Significance of ferrous 19. Beutler, E. Iron content of haemoglobin in iron ion and ascorbic acid in the operation of the tri- deficiency. Nature (Lond.) 1958, 181, 837. 20. Lardy, H. A. Method for citric acid in Manometric carboxylic acid cycle. J. biol. Chem. 1955, 214, 657. Techniques, W. W. Umbreit, R. H. Burris and 15. Anfinsen, C. B. Aconitase from pig heart muscle in J. F. Stauffer, Eds. Minneapolis, Burgess Pub- Methods in Enzymology, S. P. Colowick and N. 0. lishing Co., 1957, p. 240. Kaplan, Eds. New York, Academic Press Inc., 21. Beutler, E. Unpublished observations. 1955, vol. I, p. 695. 22. Ostberg, V. 0. Studien uber die zitronensiureaus- 16. Beutler, E., and Yeh, M. K. Y. A simplified method scheidung der menschenniere in normalen und for the determination of citric acid. J. Lab. clin. pathologischen zustinden. Skand. Arch. Physiol. Med. 1959, 54, 125. 1931, 62, 81.