STUDIES ON COPPER METABOLISM*

XX. ENZYME ACTIVITIES AND IRON METABOLISM IN COPPER AND IRON DEFICIENCIES

BY C. J. GUBLER, G. E. CARTWRIGHT, AND M. M. WINTROBE

(From the Department of Medicine, University of Utah College of Medicine, Salt Lake City, Utah)

(Received for publication, March 22, 1956)

The results of studies on certain enzymes, hemin chromoproteins, and iron and copper in the tissues of normal, copper-deficient, and iron-defi- cient swine are reported in this paper. It has been suggested that the enzymes cytochrome oxidase (l-3), uricase (4), tyrosinase (5), plasma polyphenol oxidase (coeruloplasmin) (6), glutathione oxidase (7), and butyryl coenzyme A dehydrogenase (8) either contain copper or are asso- ciated with copper in their activity. Accordingly a study of the activity of these enzymes in the tissues has been included. The tissue hemin chromoproteins, cytochrome oxidase, cytochrome c, catalase, and myo- globin, were studied because of evidence from earlier work (9, 10) that hemoglobin synthesis is impaired and that copper influences the metabo- lism of iron. Inconsistencies and scarcity of reported data on the hemin chromoprotein content of the tissues in copper and iron deficiencies pro- vided additional reasons for this study.

Methods

The twenty-three swine used in this study were part of a larger group used in studies of the hematological manifestations of copper and iron de- ficiencies (11). The details of the care and feeding of these animals have been described (9). The diet consisted of a commercial brand of canned evaporated milk diluted 1: 1 with water. Six pigs received both iron and copper as supplements (30 mg. and 0.5 mg., respectively, per kilo per day), and served as the control group. Fifteen received only iron in the above amount (copper-deficient) and three received only copper (iron-deficient). Five of the copper-deficient pigs received a total of 1000 mg. of iron intra- venously as saccharated oxide of iron’ prior to the development of anemia. 100 mg. of iron were administered three times a week to these animals.

Uric acid and allantoin determinations were made on 24 hour samples

* This investigation was supported by research grant No. C-2231, from the Na- tional Institutes of Health, Public Health Service.

1 Proferrin, Sharp and Dohme, Philadelphia, Pennsylvania.

533

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534 COPPER AND ENZYMES

of urine from each pig on two or more separate occasions after the develop- ment of the deficiency. The urine was collected without preservative for 24 hour periods. Uric acid was determined by the method of Kalckar (12) as modified by Praetorius (13), and allantoin by the method of Young and Conway (14).

Immediately after each animal was killed by bleeding, the liver, kidneys, heart, and spleen, and a sample of leg muscle (approximately 100 gm.) were removed, blotted free from blood, weighed, wrapped in Parafilm,2 and stored at -37”.

Reduced glutathione was determined by the method of Thompson and Watson (15), cytochrome c by the direct spectrophotometric method of Rosenthal and Drabkin (16), cytochrome oxidase activity by that of Cooperstein and Lazarow (17), butyryl coenzyme A dehydrogenase ac- tivity by that of Mii and Green (18), catalase activity by that of Richard- son et al. (19), and myoglobin by the method of Ginger et al. (20). The hamstring muscles from the same leg area were used in all pigs to minimize any variations in the proportions of light to dark muscle.

The determination of iron in the tissues has been described in an earlier publication (21). Tissue copper was measured by the method of Peter- son and Bollier (22) as modified below for use with tissue digests, the preparation of which has been described previously (23). 1 ml. aliquots of the digests were placed in 4.0 ml. volumetric flasks, to each of which were added 1.0 ml. of a saturated solution of Na2HP04 in copper-free water and 2 drops of a 0.25 per cent solution of phenolphthalein in 95 per cent ethanol. Concentrated NHrOH was added dropwise until a definite pink color developed, after which 2.0 N WC1 was added until the pink color was faint but still readily discernible. Then 0.2 ml. of a saturated solution of Cuprizone3 in 50 per cent ethanol was added, and copper-free water to volume. After mixing the reagents, the samples were allowed to stand for 45 minutes, were centrifuged, and the optical density was determined with a Beckman DU spectrophotometer in a 1.0 cm. cell and at a wave length of 600 rnp. A standard copper solution and a blank were examined with each set of determinations by the same procedure includ- ing the digestion.

Since these animals were not perfused, tissue hemoglobin determina- tions were performed by the method of Greenberg and Erickson (24), and appropriate corrections were made for the iron and catalase activity con- tributed by the blood trapped in the tissues.

2 Marathon Corporation, Menasha, Wisconsin. 3 Biscylclohexanone oxalyldihydraeone, G. Frederick Smith Chemical Company,

Columbus, Ohio.

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GUBLER, CARTWRIGHT, AND WINTROBE 535

RESULTS AND DISCUSSION

In all the determinations in which no significant difference was found between the copper-deficient pigs which received no parenteral iron and those which received 1000 mg. of iron intravenously, the data have been combined into one copper-deficient group.

Hemin Chromoproteins

Cytochrome c--The cytochrome c content of the heart and kidneys is presented in Table I. Liver cytochrome c levels were not measured be- cause of the technical difficulties associated with cytochrome c determina- tions in the liver of non-fasted animals. Although there was a definite tendency for the cytochrome c content of the heart and kidneys to be higher in pigs which received iron intravenously, these differences were not significant when analyzed statistically. There was a significant elevation of both the concentration (t = 2.7, P = 0.016) and the total cytochrome c per organ (t = 4.5, P = 0.001) in the hearts of copper-deficient animals as compared with the controls. The cytochrome c content of the kidneys of copper-deficient pigs was normal.

In iron-deficient animals, a significant reduction in the cytochrome c concentration of the heart (t = 8.9, P = <O.OOl) and the kidneys (t = 3.1, P = 0.02) was found, but there was no significant change in the total per organ. In contrast to the copper-deficient pigs, iron-deficient pigs were unable to maintain a normal cytochrome c concentration in the heart, even though the heart weight was only moderately increased.

Cytochrome Oxtiase-Copper deficiency resulted in an g-fold reduction in the cytochrome oxidase activity of the heart and a 3-fold reduction in the liver activity (Table I). These changes were highly significant (P = <O.OOl). The activity in the kidneys was not altered, and no significant changes were found in the cytochrome oxidase activity in iron-deficient animals.

These results are in agreement with those of Cohen and Elvehjem (25) and of Schultze (1).

Although the cytochrome oxidase complex contains hemin in its pros- thetic group, the reduction in cytochrome oxidase activity in copper defi- ciency cannot be explained on this basis. There was no reduction in cytochrome oxidase activity in iron deficiency and none in cytochrome c activity in copper deficiency. The present studies lend support to the suggestion that cytochrome oxidase is dependent on copper for its ac- tivity (l-3, 26, 27), but definite clarification of this point must await further purification of cytochrome oxidase.

Cutalase-The mean catalase activity in the liver tissue of copper-de- ficient animals was significantly lower (t = 6.8, P = <O.OOl) than that

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GUBLER, CARTWRIGHT, AND WINTROBE 537

of the control animals (Table I). In the one iron-deficient liver available, the catalase act,ivity was below the lowest value in the control group. Neither copper nor iron deficiency had any effect on the catalase activity of the kidneys. The findings in copper-deficient pigs are in agreement with the results in rats reported by Schultze and Kuiken (28), but not with those reported in an earlier paper (23) from this laboratory, nor with those in mice reported by Adams (29). The reduction in liver catalase activity found in the irondeficient swine is consistent with the results of our earlier study (23) and with those of Schultze and Kuiken (28), but not with those of Adams (29). Although a difference in liver catalase activity between females and males was found in rats by Schultze and Kuiken (28) and in mice by Adams (29), no such difference has been found in swine.

TABLE II Hemoglobin Content of Blood and Myoglobin Content of Leg and Heart Muscle of Swine _

Group

No.of pigs.................. Blood hemoglobin, gm. per

100 ml.................... No. of pigs.................. Leg muscle myoglobin, mg.

per gm. . . . . . No.of pigs.................. Heart myoglobin, mg. per

gm . . . . . . . . Heart myoglobin, mg. per

heart......................

-

-

Control

6

13.2 f 0.20 6

0.44 f 0.056 6

2.99 f 0.093

322 f 32.5

-

_-

-

Cu-deficient

13

4.2 f 0.32 13

0.43 f 0.027 13

2.25 f 0.091

417 f 30.8

-

- - Fe-dgicimt

3

3.2 i 0.42 3

0.12 f 0.017 3

1.38 f 0.085

194 f 41.3

Myoglobin--The results of the myoglobin determinations are summar- ized in Table II, blood hemoglobin concentrations being included for com- parison. The copper-deficient animals were severely anemic, their mean blood hemoglobin concentration being only 32 per cent of that in the control group. In contrast, the concentration of myoglobin in the leg muscles remained normal. There was a significant reduction (t = 4.9, P = 0.001) in the concentration of myoglobin in the heart muscle, whereas the total myoglobin per heart was normal or slightly increased. This can be attributed t.o the marked cardiac hypertrophy.

The iron-deficient animals were likewise severely anemic, with a mean blood hemoglobin concentration only 24 per cent of that in the control group, However, in iron deficiency in contrast to copper deficiency, a marked reduction was found in the myoglobin concentration in both the leg muscles (t = 15.9, P = <O.OOl) and the heart muscle (t = 10.9, P =

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538 COPPER AND ENZYMES

<O.OOl). These reductions in myoglobin concentration were of the same order of magnitude as the reduction in blood hemoglobin concentration. The total myoglobin per heart was also reduced (t = 2.3, P = 0.05) in spite of a moderate degree of hypertrophy. In rats deficient in both iron and copper, Cowan and Bauguess (30) found a slight decrease in myo- globin concentration and a marked increase in the total myoglobin of the heart.

The present data suggest that an impairment in hemin chromoprotein synthesis becomes manifest in copper deficiency only when the require- ment is increased as a result of an increased turnover rate of the com- ponent or of an increase in the mass of the tissue requiring it. The low liver catalase may be the result of a rapid turnover rate, a relative increase in the size of the liver, and other factors such as inanition. Since the turnover rate of myoglobin is slower than that of hemoglobin (31-33), impaired synthesis of myoglobin in copper deficiency becomes evident only in a tissue such as the heart, which increased 3-fold in size relative to body weight. The reason for the increase in cytochrome c of the heart in copper-deficient animals is not apparent. It may be an attempt to compensate for the reduced oxidative capacity resulting from the low cytochrome oxidase activity. If this represents an increase in the rate of cytochrome c synthesis, it would suggest a priority over the synthesis of other hemin chromoproteins.

In iron deficiency, on the other hand, an impairment in the synthesis of all of the hemin chromoproteins except cytochrome oxidase has been demonstrated. This is presumably due to a lack of iron. This finding is especially noteworthy in that it does not substantiate Hahn and Whip- ple’s suggestion (34, 35) that severe iron deficiency has no effect on the levels of myoglobin and the “parenchymal” iron components of the tis- sues. In the present study the diet was very low in iron, and young, rapidly growing swine were used which were expanding their tissue mass at a rapid rate. In the studies of Hahn and Whipple on adult dogs, anemia and a relative iron deficiency were produced and maintained by repeated bleedings. The diet used was not strictly iron-deficient and hence may have supplied enough iron to meet the small requirements for tissue hemin chromoprotein synthesis under these conditions, but not enough to meet the needs for new hemoglobin synthesis to replace that lost through repeated phlebotomy. Under these conditions, blood loss depleted the body of hemoglobin but not necessarily of myoglobin and cytochrome c. The results of Cowan and Bauguess (30) have been cited to support the view that myoglobin synthesis is not impaired in iron de- ficiency. However, their results are complicated by the fact that their rats were deficient in both iron and copper. The present study has shown

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GUBLER, CARTWRIGHT, AND WINTROBE 539

that, in uncomplicated iron deficiency in growing animals, the lack of iron prevents the synthesis of tissue hemin chromoproteins in a manner quan- titatively similar to its effect on hemoglobin synthesis.

TABLE III Content of Iron in Blood and Several Tissues* of Swine

Group

No. of pigs Blood$.

Liver.

Kidney.. _.

Spleen......

Heart

Total mg. Fe......

Body weight kg..

Mg. Fe, per kg..

Control

6 468 If, 111.1 f51.f

172 87 f14.8 f9.5

51 7 f4.7 f0.4

127 6 zklO.5 f0.3

35 4 f2.2 f0.2

1083 f 93.4

27.0 f 2.70

40.0 f 0.95

Copper-deficient

7 50 189

f30.4 f22.f 5 27

zklO.2 f6.1 2 3

f2.6 lto.3 91 12

f37.1 f3.1 4 ,9

f2.2 f0.8

239 xk 35.9

15.4 f 1.50

15.5 f 1.09

-

--

3 1

D 1

6

6,

‘6

-

Copper-deficient + iron intravenouslyj

Y ger 6-m. mg. gcr

LWgWI

‘3 20 178 f32.9 f62.!

117 631 3t87.8 19.2

1 17 f5.3 ’ f0.7

81 35 X234.8 f17.:

2 13 It8.3 f2.9

866 f 53.6

17.7 f 2.43

49.0 f 3.33

-

I - _

7

3 II

5 9

11

3’ 1

21

_-

-

Iron-deficient

, per gm. mg. )CI

organ

3 08 185 f14.5 53.9

5 f0.7 10.9

D 1 zko.9 fO.l

9 3 f3.8 f0.7

D 3 f0.9 10.4

196 i 2.6

22.1 i 2.50

8.9 f 1.00

* All irons were corrected for blood iron entrapped in the tissues. t These pigs received a total of 1000 mg. of iron intravenously as Proferrin. $ The blood iron is expressed as micrograms of Fe per ml. and as mg. per total

red cell mass. The total red cell iron was calculated from the concentration and the total red cell mass obtained as shown in the text.

Total Iron of Blood and Tissues

The mean values for the content of iron in the blood, liver, kidneys, spleen, and heart of control, copper-deficient, and iron-deficient swine are presented in Table III. The total amount of iron in the blood hemoglobin compartment was calculated by the formula: blood Fe in mg. = Hb in gm. per cent X ((total blood volume in ml.)/lOO) X 3.4. An approxi- mation of the total blood volume was obtained from data reported by Bush et al. (36) for normal and anemic swine. In both the copper-defi- cient and the iron-deficient groups, the blood iron was markedly reduced

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540 COPPER AND ENZYMES

as a result of the anemia. In the copper-deficient group the storage iron compartment was significantly reduced as compared to the control group, but not to the same degree as in the iron-deficient group. In the animals which received 1000 mg. of iron intravenously, most of this iron could be accounted for in the storage compartment (673 mg.). The amount of iron in the spleens of the copper-deficient animals was increased, presum- ably owing to the hemolytic process which has been shown to be a part of the copper deficiency syndrome (10).

The total iron contributed by the sum of the tissues analyzed and the blood was approximately the same per kilo of body weight in the copper- deficient (15 mg.) as in the iron-deficient (9 mg.) animals, and was much less than that found in the control animals (40 mg.). The marked reduc- tion in tissue and total body iron found in copper-deficient animals con- firms the results of an earlier study from this laboratory (21) and indicates an iron deficit nearly as great in degree as that found in iron-deficient ani- mals. This is in spite of the large doses of iron (30 mg. per kilo per day) received by the copper-deficient swine. This lends further support to the suggestion (21, 37) that copper deficiency results in a reduced absorption of iron. The increase in the iron content of the heart in copper-deficient animals is not surprising in view of the increase in cytochrome c content.

Other Enzymes Associated with Copper

No glutathione oxidase or tyrosinase activity could be demonstrated in the liver of either control or deficient animals. Butyryl coenzyme A de- hydrogenase activity was measured in crude liver homogenates of control and copper-deficient pigs. The optical density, measured in 6.0 ml. of final volume due to the formation of formazan from 2,3,5-triphenyl-2H- tetrazolium by 0.1 ml. of 1: 10 liver homogenate, was the same in both groups (0.218). Hence, copper deficiency did not exert any influence on the activity of this enzyme in the crude system studied.

Uric Acid and Allantoin-Uric acid determinations were made in the plasma of several control and copper-deficient pigs. In no case was a measurable quantity found. The excretion of uric acid and allantoin in 24 hour collections of urine was measured in six control, fourteen copper- deficient, and three iron-deficient pigs. The results are summarized in Table IV. The mean uric acid excretion (in mg. per kilo per 24 hours) was 460 per cent greater in the copper-deficient than in the control group, while the allantoin excretion was only 175 per cent greater. This resulted in a significant increase (t = 4.3, P = <O.OOl) in the ratio of uric acid to allantoin excreted. Iron deficiency had no effect, either qualitative or quantitative, on the excretion of uric acid and allantoin. The relation- ship between the volume of packed red cells and the uric acid and allantoin

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GUBLER, CARTWRIGHT, AND WINTROBE 541

excretion is shown in Fig. 1. As can be seen, there was a significant nega- tive correlation between the volume of packed red cells and the excretion of uric acid (T = -0.46, P = 0.003), or the ratio of uric acid to allantoin (T = -0.64, P = <O.OOl). Since uricase is claimed (4) to be a copper- containing enzyme, the increase in the ratio of uric acid to allantoin ex- cretion in the copper-deficient animals suggests that the activity of uricase may be impaired by a lack of copper. However, the total purine excretion is likewise increased, presumably as a result of the shortened life span of the red cells. The possibility therefore arises that this relatively greater

TABLE IV

Excretion of Uric Acid and Allantoin in Urine of Control, Cu-Deficient, and Fe-Deficient Swine*

Controls Group Cu-deficient

No. of pigs 6 No. of determinations 12 Volume packed red cells, 41.0 f 1.14

% Body weight, kg.? 18.0 f 1.70 Urine uric acid, mg. per 1.3 f 0.10

kg. per 2’4 hrs. Urine allantoin, mg. per 13.5 f 1.28

kg. per 24 hrs. Total uric acid + allan- 14.8 f 1.37

toin, mg. per kg. per 84

hrs. Ratio, allantoin-uric acid 0.10 f 0.008

* Each value is a mean accompanied by its standard error. t Body weight at the time samples were collected.

14 25 12.9 f 0.69

14.5 f 0.69 6.0 f 2.60

23.5 f 2.30

29.2 f 3.25

0.24 f 0.002

-

-

-

Fedclicient

3 5

14.5 zk 1.17

19.0 f 1.99 2.1 i 0.46

16.2 f 2.29

18.3 zlz 2.72

0.12 i 0.016

output of uric acid is merely a function of the increase in the total purine excretion.

Glututhione-The values obtained for the reduced glutathione content of the red blood cells and the liver are summarized in Table V. The mean concentration of glutathione in microgram per ml. of red cells was sig- nificantly increased in both the copper-deficient (t = 2.31, P = 0.04) and the iron-deficient (t = 7.6, P = <O.OOl) swine. Since the red cells were microcytic (ll), the glutathione content per unit number of red cells was essentially normal in the iron-deficient group and was only moderately increased in the copper-deficient group (t = 2.4, P = 0.03). In contrast, the liver glutathione content was decreased significantly (t = 6.3, P = <O.OOl) in the copper-deficient, animals. There was no significant change in the liver glutathione of iron-deficient animals.

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COPPER AND ENZYMES

q m Allontoin UricAcid

20-30 ABOVE

VPRC

FIG. 1. The relationship of the excretion of uric acid and allantoin to the volume of packed red cells (VPRC) in copper-deficient swine.

TABLE V Reduced Glutathione and Copper Content of Red Cells

and Some Tissues of Swine*

Tissue

No. of pigs Red cell glutathione, y per ml. red

blood cells Red cell glutathione, -y per 1010 red

blood cells No. of pigs Liver glutathione, y per gm.t

“ “ mg. per liver No. of pigs Liver copper, y per gm.t

‘I “ mg. per liver Kidney copper, y per gm.t

I‘ ‘I mg. per 8 kidwys

-

-- Control

6 465 f 9.6

250 f 3.8

6 2189 i 90.2 1126 f 133.0 6 10.9 f 2.63 5.3 f 1.17 4.7 f 0.42 0.7 f 0.09

Cu-deficient

11 703 f 65.2

374 f 36.2

9 1226 f 108.4 522 f 53.2 8 0.5 f 0.04 0.2 f 0.02 2.1 f 0.14 0.3 f 0.03

- Fe-deficient

3 1080 f 121.8

307 i 31.0

3 2039 f 49.7 999 f 80.1 3 10.8 & 2.47 5.5 f 1.65 9.4 f 1.54 1.4 f 0.33

* All the values represent means f the standard error. t Based on wet weight of the tissue.

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GUBLER, CARTWRIGHT, AND WINTROBE 543

The significance of the marked reduction in liver glutathione in copper deficiency is not clear. The anemia per se had no influence since equally anemic iron-deficient pigs showed no reduction in liver glutathione. The data suggest an association between copper and glutathione metabolism in the liver, but this may not necessarily be a direct relationship.

Tissue Copper

The content of copper in the livers and kidneys is presented in Table V. In confirmation of the results of an earlier study (23), the copper content

TABLE VI

Injluence of Copper and Iron Deficiencies on Weights of Various Organs of Swine

Group

No. of pigs Total body weight,

kg. Heart, weight, gm.

“ % body

weight Liver, weight, gm.

‘I % body

weight Kidney, weight,

cm. Kidney, y. body

weight No. of pigs Spleen, weight, gm.

Spleen, % body weight

Control

6 27.0 f 2.70

108 f 9.7 0.40 f 0.016

508 f 38.0 1.88 f 0.085

150 f 13.0

0.56 f 0.029

6 47 f 5.6 0.18 f 0.006

Cu-deficient

9 15.2 f 1.20

183 zk 21.9 1.22 f 0.089

407 f 32.7 2.71 f 0.181

116 f 10.0

0.78 f 0.014

7 56 i 22.7 0.29 f 0.045

-

--

/

I

-

Cu-deficient + intravenous Fe

5 16.1 f 2.22

195 f 16.4 1.26 f 0.100

625 f 60.0 4.37 f 0.880

114 f 17.8

0.72 f 0.071

3 48 f 8.5 0.28 zk 0.065

Fe-deficient

3 22.1 zk 2.50

140 f 24.7 0.63 i 0.057

492 f 51.8 2.24 f 0.284

140 i 15.2

0.63 f 0.053

3 75 & 27.2 0.33 f 0.094

of these tissues was found to be markedly reduced in all copper-deficient animals. Copper levels in the liver were in the normal range in the iron-deficient pigs, and the copper content of the kidneys was signifi- cantly elevated (P = 0.01).

Organ Weights

The organ weights and their relationship to total body weight are sum- marized in Table VI. The weights of the liver (t = 3.5, P = 0.004), kidneys (t = 4.2, P = O.OOl), and heart (t = 7.7, P = <O.OOl) were sig- nificantly greater in proportion to body weight in the copper-deficient animals than in the controls. The actual heart weight was significantly

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544 COPPER AND ENZYMES

greater in the copper-deficient group (t = 3.5, P = 0.005), in spite of their smaller total body weight. The intravenous administration of 1000 mg. of iron to copper-deficient animals led to a significantly greater increase (t = 3.5, P = 0.005) in liver weight than that found in the copper-de- ficient pigs receiving no parenteral iron. Iron deficiency was associated with a significant increase (t = 5.2, P = <O.OOl) in relative heart weight. This change was, however, much smaller in degree than that found in the copper-deficient animals, and the weights of the liver and kidneys were normal. The spleen weight was not affected by either deficiency.

Cardiac hypertrophy was a prominent feature in the copper-deficient pigs. Others (1, 30, 38) have found cardiac hypertrophy in rats with anemia owing to a deficiency of both iron and copper. In our pigs, the cardiac hypertrophy cannot be explained entirely by the presence of ane- mia, since the iron-deficient swine were equally anemic and did not have as great a degree of cardiac hypertrophy. It seems possible that the cardiac hypertrophy in copper deficiency is an effort to compensate for the reduction in respiratory activity. The marked reduction in cyto- chrome oxidase activity may well be the explanation of the sudden cardiac failure which we as well as others (39) have observed in copper-deficient animals. It may be significant that, in those copper-deficient pigs which received massive doses of iron parenterally, the relative liver weights were much greater than in those which stored little iron. Earlier studies (23, 40) would suggest that these increases in tissue weight are due to bis- tologically normal tissue and not to fatty infiltration or to necrotic tissue.

SUMMARY

A study has been made of the influence of copper and iron deficiencies on the hemin chromoprotein, copper, iron, and enzyme content of the blood and various tissues of swine, as well as on the size of various organs.

In general, iron deficiency resulted in a decrease in the hemin chromo- protein content of the blood and tissues. Copper deficiency was associ- ated with a decrease in concentration of hemin chromoproteins only when an increased requirement was evident, as indicated by an increased turn- over rate or a disproportionate increase in tissue mass. In the heart, both the concentration and the total cytochrome c as well as the total myoglobin were increased in copper-deficient animals.

Cytochrome oxidase activity was not influenced by iron deficiency but was markedly reduced in copper deficiency. Liver catalase activity was decreased in both iron and copper deficiencies.

The butyryl coenzyme A dehydrogenase activity in liver homogenates was not altered by copper deficiency.

The content of glutathione in the liver was decreased in copper deficiency

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GUBLER, CARTWRIGHT, AND WINTROBE 545

and normal in iron deficiency. The red cell concentration was increased in both iron and copper deficiencies, but the content of the individual red cell was only moderately increased in copper deficiency and was normal in iron deficiency.

The excretion of uric acid and allantoin was normal in iron deficiency. In copper deficiency, an increase in allantoin and a relatively greater in- crease in uric acid excretion were found. This resulted in a 2.5-fold in- crease in the ratio of uric acid to allantoin excreted.

Copper levels were very low in the tissues of copper-deficient swine and normal or high in iron-deficient swine.

Total tissue iron was moderately reduced and hemoglobin iron was markedly reduced in copper deficiency. There was a marked reduction in both iron compartments in iron deficiency. Total iron per kilo of body weight in copper-deficient pigs did not differ greatly from that in iron- deficient pigs.

Iron deficiency was associated with a 50 per cent increase in heart weight relative to body weight. The other organs were not affected.

Copper deficiency resulted in a marked (200 per cent) increase in heart weight and a moderate increase in liver and kidney weight relative to body weight. The other organs were not affected.

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WintrobeC. J. Gubler, G. E. Cartwright and M. M.

DEFICIENCIESMETABOLISM IN COPPER AND IRONXX. ENZYME ACTIVITIES AND IRON STUDIES ON COPPER METABOLISM:

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