THE HIGH SPECIFICITY OF THE MANGANESEPATHWAYTHROUGHTHE BODY1, 2

By GEORGEC. COTZIAS AND JAMES J. GREENOUGH

(From the Medical Department, Brookhaven National Laboratory, Upton, N. Y.)

(Submitted for publication March 13, 1958; accepted May 2, 1958)

The accepted ideas about the physiological roleof manganese have been derived predominantlyfrom in vitro experiments. However, these arecharacterized by lack of specificity: Only a limiteddiscrimination between manganese and some othermetals is shown, for instance, in metal transportsystems (1, 2), in enzyme activation (2-4) andin the preservation of mitochondria (5). Theconclusions from these experiments were of in-terest because they threw new doubt on the func-tional specificity and even on the essentiality ofmanganese in the living organism. Thus it be-came necessary to ascertain whether manganesemay be replaced in the intact animal as well. Toinvestigate this, we have used a variation of theclassical technique of flooding the organism withmetals which we were led to believe would sub-stitute for manganese. Surprisingly, these kindredmetals were ineffective in eluting radiomanganesefrom the body: Only manganese compoundsproved effective in that regard.

The results suggest that there exists a segmentin the pathway of manganese through the body,the properties of which permit the passage of thatmetal only. This paper deals with the implicationsof this finding both in reference to manganese aswell as to the elements which have been thoughtnot to possess such an in vivo specificity.

MATERIALS AND METHODS

Experiments with intact animals

Animals. These were seven week old Swiss albinomale mice, weighing 16 to 18 grams. They were housedin cages, the bedding of which consisted of shreddedcorn cobs. When dietary restrictions were imposed,

'This work was supported by the United States AtomicEnergy Commission. It was accorded an Honorable Men-tion in the A. Cressy Morrison Award in Natural Sciencein 1957, by the New York Academy of Sciences.

2 preliminary report of this paper was presented be-for the Federation of American Societies for Experi-mental Biology in Chicago, March, 1957.

metal screening was used to prevent the animals fromeating the bedding. Regardless of the type of bedding,the furs remained radiologically clean, as shown by thefailure of VerseneQ and detergent baths to lower the totalbody radioactivity.

Diets. Since the manganese turnover depends upondietary intake, an effort was made to keep the manganeseintake constant. In the bulk of the experiments, the samelot of Purina Fox chow was used (MnSO, reported bymanufacturer as 0.02 per cent). As a "manganese free"diet, a vitamin B. deficient rat diet was used.3 To thisa salt-vitamin fortification mixture' was added withoutsupplementary manganese, unless otherwise stated.

Stable metals and their salts. Rhenium dioxide wasprepared by dissolving the metal in nitric acid and heat-ing to dryness with anhydrous hydrazine HCl. The pro-cedure was repeated twice with the precipitate. This wasfinally washed with water, ground into a fine suspensionin normal saline and injected into the animals intra-peritoneally. All the metals, oxides and salts 5 used inthese experiments to challenge the isotope were injectedwith normal saline as the vehicle. In some experiments(see Table I) the sulfates or chlorides of the metals weredissolved with added equimolar amounts of sodium citrate(hereafter referred to simply as "citrate"). In thoselatter experiments the controls received equimoler sodiumcitrate instead of the routinely used saline injections. Thedoses of the challenging materials ranged from 1 X 10'to 1 X 10' mole of metal. These were given either assingle or as daily injections for one week.

3 Nutritional Biochemicals Corporation. The completediet contained 2.5-y of manganese per 100 gram diet (orly of manganese per gram ash).

4 Nutritional Biochemicals Corporation, "Vitamin DietFortification Mixture."

5 Spectrographic tests of the MnSO4powder showed itto be free from contaminating metals, while both theFeSO4 and the CrCl, powders contained 0.01 per centmanganese. The utilized chemical agents and theirsources of purchase are as follows: cobaltous sulfate,ferrous chloride, manganese dioxide, nickelous sulfate andsodium citrate from Baker and Adams Co.; copper sul-fate, magnesium sulfate, potassium permanganate andzinc sulfate from Baker's; ferric chloride from Merck &Co.; manganous chloride from Mallinkrodt; manganesemetal from A. D. Mackay; vanadyl sulfate from Fisher;and vanadium metal from Amend Drug. The rheniumcompounds were obtained from the University of Tennes-see except for the rhenium pentachloride (K & K Labs.).

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SPECIFICITY OF THE MANGANESEPATHWAY

Isotopes. Mn' (T/2 = 5.7 days) was prepared by theCr" (d, 2n) Mn' reaction in a 60 inch cyclotron by oneof us (J. J. G.). Separation of the tracer Mn' from thechromium necessitated a precipitation with manganesecarrier. When the latter was found to have a markedinfluence on the phenomena under study, this preparationwas abandoned in favor of Mn". The latter isotope wasprepared carrier free by the Nuclear Science and Engi-neering Corporation. It was received as Mn"Cl, inhydrochloric acid (200 fc. per ml.). Neutral dilutions(0.8 and 1.6 uc.) were given intraperitoneally to theanimals in 0.1 or 0.2 ml. of saline. While with the Mn"variations even from 0.1 to 0.2 uc. brought upon con-cordant variations of the turnover rate, the Mn" prepara-tion was free from such complicating effects. Accidentalinjections into a hollow viscus were soon followed byalmost complete loss of the animal's radioactivity.

Measurements of the radioactivity. Each tagged an-imal was placed in a 50 ml. plastic centrifuge tube withits mouth opposite to the outlet of an oxygen line. Afterplugging with cotton, the tube was inserted into thehorizontal well of a Texaco® counter. The same pro-cedure (minus the ventilation) was used for pooled or-gans, to permit the direct comparison between animalsand their organs. As a rule counting was accurate towithin 1 per cent in both cases, and its geometry wasconstant between 3 and 30 ml.

Calculations of turnover rates. The animal's total bodyradioactivity was counted within one hour after isotopeinjection. This was repeated daily, in some cases for aslong as 20 to 30 days. The subsequent counts were ex-pressed as percentages of the first, after being correctedfor background and, if needed, for isotopic decay. Thedata were then plotted on semilogarithmic paper as afunction of time and the curves were drawn (Figure 1).Whennecessary, these were analyzed graphically for theirconstituent components (Figure 2) by subtracting thestraight end from the remainder of each curve (6). Inthe experiments in which challenging injections of carriermetals were given, the following controls were used: 1)saline- or sodium citrate-challenged animals; 2) eachanimal's own prechallenge turnover rate; 3) animals thathad received MnCl, in amounts often less than equimolarto that of the challenging metal. At the end of someexperiments, in which the challenge did not effect theturnover rate, the isotope's continued availability forexchange was demonstrated by injecting MnCl, solutions.

Summary of typical protocol. Thirteen mice were in-jected intraperitoneally with 1.0 ,uc. of Mn"Cl, in 0.2 ml.saline, and their total body radioactivity determined.They were then divided into four groups. The retainedradioactivity was measured at daily intervals. The cor-rected counts were plotted semilogarithmically againsttime as per cents of the first count. On the third day,Group I received 1X 10' mole of CrCl,; Group II, 0.2 ml.of saline; Group III, 2 X 10" MnCl; Group IV, 1 X 10'FeCl,. The day following the injection the phenomenonillustrated in Figure 4 was seen. It was followed forfive days and then the 2 X 10' mole of MnCl were givento the animals that had received CrCl2 and FeCl,. This

was done in order to test whether their tracer had re-mained exchangeable. The animals were then sacrificed,and the peritoneal cavities were found to be free ofmetallic deposits.

OBSERVATIONS

Turnover of body manganese. The resultsshown in Figure 1 were obtained with a group ofnine Mn54 tagged animals. These animals weremaintained on Purina Fox chow. The individualdata fell into exceedingly smooth curves so that.subsequently, breaks in the curves which were ex-perimentally induced could be used as additionalcontrols. When these curves were graphicallyanalyzed, at least two components were found(T/2 = 48 to 68 hours and T/2 = 230 to 300hours).

Two groups of three mice each were given 1 percent ammonium chloride and 1 per cent sodiumbicarbonate instead of drinking water. After noeffect on the turnover was seen, their bottles wereswitched. This again failed to produce any changein the turnover rates.

Effect of diet on manganese turnover. Thefeeding of the "manganese-free" diet to a group ofthree mice a few days prior to and during the ex-periment did not effect the rate of the first com-ponent (T/2 = 54 hours), while that of the sec-ond increased to T/2 = 860 hours. The diet wasenriched with carrier manganese sulfate (0.01 percent of Mn++ w/w), and the procedure was re-peated in three other mice. This showed turnover

Ia

'I

FIG. 1. TOTAL BODYTURNOVEROF MN" IN MICE(SEE TEXT)

The dashed lines define the envelope of all the data.

1299

GEORGEC. COTZIAS AND JAMES J. GREENOUGH

a

0

at

l-:

0 60 120 ISO 240 300 31iELAPSED TIME IN HOURS

FIG. 2. TOTAL BODYTuRNovER OF MN' AS INnUENcEDBY DITARY MANGANESE

rates of T/2 = 38 and T/2 = 238 hours for eachof the two components. Increase of the manganesesupplement to 0.05 per cent caused further de-creases of these numbers to T/2 = 31 and T/2= 208 hours in another group of three mice.Starvation of three animals markedly deceleratedthe turnover. Obstruction of the intestine at theanus in another three mice stopped the turnoverentirely.

These experiments show that the rate of radio-manganese loss from the body is sensitive to thelevel of dietary manganese and to the state ofgastrointestinal function. They are compatiblewith intestinal excretion of this metal (7). Con-cordantly, the acceleration of the observed turn-over by added dietary manganese might have hadthe following implications: 1) that more carriermanganese is absorbed from the gut than hithertosuspected and elutes the radioisotope from thetissues; or 2) that on elimination from the gut,the stable metal carries with it a significant frac-tion of the bile-bound tracer (6, 7) which nor-mally might have been reabsorbed (8). Regard-less of which explanation might be correct, thisuncertainty would throw doubt on experimentswith any stable metal, if these were fed. Thus theoral route was abandoned in favor of the intra-peritoneal.

Accelerated elimination of radiomanganese fol-lowing challenge with manganese carrier. BothMn54 and Mn52 injected animals were used inthese experiments.

As can be seen in Table I, various forms ofthe stable metal were given at various times. Thevalence state of the carrier ranged from 0 in the

metal powder to 7 in the permanganate. Injec-tions of all of these compounds were followed byacceleration of the rate of turnover, which re-sulted in losses ranging from 15 per cent to 80 percent within twenty-four hours. Figure 3 illus-trates two characteristic experiments at two dif-ferent postinjection times with two differentisotopes. By varying the time at which the

I-z0 208-J49

ILZUi.0

at

40

-

0U

-J.4I-

z 20UI.00

315 335 355 375 395TIME IN HOURS

415 435 455

FIG. 3. EFFECT OF ADDING CARRIER MANGANESEONRADIOACTIVE MANGANESETURNOVER

A = Total body turnover of Mn in mice receiving 8 X10 mole of carrier manganese as MnSO4 at the 86thhour of the experiment

B = Averages of total body turnovers of Mn" in threemice compared with those of three mice receiving 4 X 10Omole of carrier manganese as MnSO, at the arrow.

--

Mn54 IN MICE

MnSO4

x-"

B

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SPECIFICITY OF THE MANGANESEPATHWAY

challenge was made, it was found that an ap-preciable fraction of the tracer had remained ex-changeable, even three weeks after tagging theanimals. The exchange has not been quantitatedas yet. The impression was gained, however, thatelution of the tracer manganese was more obviouswhen the challenge was made early or with highdoses of carrier. An easily observed accelerationof the turnover was evident following injections of9 x 10-8 mole of MnSO4as late as the sixth day.The extreme valence states (Mn0 and Mn7+)seemed less effective than Mn.2+.

These experiments proved that the radioisotoperemained largely and continuously available forelution by carrier. The elution was obvious afterchallenges with MnSO4amounting to 30 per centof the estimated manganese content of the body.

Failure of some other stable metals to affectMn54 turnover. Manganese is a member of boththe first transition group and of group VIIB ofthe elements. Many of the members of the transi-tion group (in periods four and five) have beenknown to substitute for manganese in vitro (1-5,9). Manganese and rhenium are the only stablemembers of group VIIB. Rhenium is Men-deleef's celebrated dvi-manganese (10). His eka-manganese (technetium), which would have beenvery crucial in these studies, has only radioactiveisotopes.

These metals were used as eluting agents in ex-periments identical with those described in thecase of carrier manganese. Magnesium, the ele-ment most widely used in vitro instead of man-ganese, was also included. In another experi-ment, a member of group VIIA was tested becauseof its possible interchange with manganese in thethyroid ( 11 ).

The upper limit of the concentration in thechallenging injections was set by the toxicity ofthe individual compounds. Safe doses werechosen after preliminary experimentation withgroups of mice. Care had to be taken to avoidinterference with the animals' eating and bowelhabits which were known to affect the end point.

The lower limit was set at 30 per cent of themouse's total body content of each of the stablemetals used as eluting agents. This value waschosen because of the effect of carrier manganesedescribed above. Furthermore, 30 per cent ofanything appeared to be a large amount. Table I

shows both the metals' estimated body content andthe challenging doses given. High values wereintentionally chosen in making these estimates.A mouse weighing 20 grams was chosen as aprototype in spite of the fact that the experimentalanimals weighed 16 to 18 grams. Even so, thedesired value of 30 per cent was reached or ex-ceeded by the individual injections. It was mark-edly surpassed in the experiments in which theinjections were given daily for one week.

Every effort was made to compare similarvalence states with each other. Some animalswhich had received toxic doses of ferrous andchromous chlorides had small peritoneal metallicdesposits at autopsy. The mouse peritoneum hadshown a high absorptive capacity in the experi-ments with manganese metal and dioxide powders.In spite of this the precaution was taken of includ-ing citrate in many of these injections, since thismarkedly facilitates absorption by forming che-lates with these metals (12). The metals' releasein the body was expected because citrate is rapidlymetabolized.

These stable metals were incapable of affectingin any way the rate of elimination of radioman-ganese from the body, in spite of all the specifica-tions listed above. Figure 4 illustrates the failureof ferrous and chromous citrates to bring uponany change of the radiomanganese turnover inconcentrations 50 times higher than an effectiveamount of manganous sulfate. That the isotopewas available for exchange in these animals alsowas shown by the effect of the manganous salt atthe end of that observation.

Experiments uwith organs

Following the observation that challenges withcarrier manganese had caused elution of radio-manganese readily, reproducibly and regardless ofthe state of the carrier, the failure of any of theother stable metals to do so seemed incongruous.The following explanation for the apparent in-congruity was considered and tested: It wasthought that the nonmanganese metals might havecaused elution of the tracer from one organ intoanother instead of elimination out of the body.This would not have been detected by our method,since it was designed to quantitate the radioactiv-ity of the entire body and not to differentiate be-

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GEORGEC. COTZIAS AND JAMES J. GREENOUGH

TABLE I

Summary of metals administered to manganese" injected mice

No. of Metal Atomic Moles per At pmice or salt number 20 Gm. mouse* Moles per challenge isotope

7 MgSO4 12 4.2 X 104t 5 X 10--5 X 10-4 146

3 VOSO4 23 1.3 X 107t 1.0 X 10-' 310-505§

9 Cr Cit 24 3.1 X 10-7$ 2 X 10-6 - 1 X 10-6 (-)6-505§13 CrCl2 24 4 X 10-6 - 1 X 10-' 50-650

3 Mn° 25 2.8 X 10-7 9 X 10-6-4.5 X 104 6-14611 Mn Cit 25 1.8 X 106 - 9 X 10-6 (-)6-674§28 MnSO4 25 9 X 10-8 -1 X 10-' 50-650

3 MnO2 25 1.1 X 10-4 (-)243 KMnO4 25 2.5 X 10-6 354

6 FeCl2 26 2.1 X 10-6' 9 X 10-6 -1 X 10-6 50-65011 Fe++ Cit 26 1.8 X 10-6 (-)6-480§

3 Fe+++Cit 26 1 X 10-6 13 FeCI3 26 3 X 10-6 48

6 Co Cit 27 9 X 10-,$ 1.7 X 10-6 - 8.5 X 10-6 311-480§3 CoSO4 27 3.5 X 10-7 266

3 Ni Cit 28 6.1 X 10-7t 1.7 X 10-6 - 8.5 X 10-6 311-480§

7 Cu Cit 29 6.3 X 10-7 1 X 10-7 -3 X 10-7 (-)6-364§19 CUS04 29 1 X 10-7 - 6 X 10-7 50-318§

3 Zn Cit 30 1.6 X 10-'t 1.5 X 10-6 - 7.6 X 10-6 311-480§3 ZnSO4 30 3.5 X 10-7 266

3 KI03 53 7.1 X 10-8t 4.6 X 10-6 354

7 Re° 75 5.5 X 10-6 6-507 ReO2 75 4.6 X 10-6 (-)24-270§3 Re2O7 75 9.7 X 10-6 13 NaReO4 75 2.7 X 10-6 354

* These are estimates of the mouse's total body content prior to challenge. All estimates pertain to the individualmetal involved in the test and not to the salt listed.

t Estimated from Hawk, Oser and Summerson (14).* Estimated from Tipton and co-workers (18).§ Include daily injections for one week.1I Estimated from Underwood (9).

80-

z

40

a MnC12

o Fe C1210, 24 48 72 96 120 144 168 192 216 240

HOURS AFTER ISOTOPE INJECTION

FIG. 4. TOTAL BODYTURNOVEROF MN' IN MICE AS

INFLUENCED BY SOMECARRIER METALSAt first arrow 0.2 ml. of saline, 1 X 10' mole of either

FeCl, or of CrCl,, or 2 x 10' mole of MnCl, were injectedin each of the groups. The 2 X 10 mole of MnCl, were

injected, at the second arrow, to the FeCl, and CrCl,treated animals.

tween its parts. Thus we investigated the effectsof some of these stable metals on the partition ofthe tracer amongst the organs of the body. Theintracellular organelles could be also tested fromthat standpoint, particularly since it was observedearlier (13) that the mitochondria concentratemanganese isotopes. For this, one might havechosen an organ on the basis of some gross ob-servation: As will be seen below, no such lead wasforthcoming.

In view of the size of the enterprise involvedin using all of the previously tested metals in thisstudy as well, we had to arrive at a compromise:Wechose elements 24, 25 and 26 (Cr++, Mn++ andFe++) .

For brevity, only Experiments A and B (Fig-

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SPECIFICITY OF THE MANGANESEPATHWAY

ure 5) are described out of four concordant ones(the others differed in several experimental de-tails). In both of these the animals were dividedinto groups of three and eight, respectively, in-jected with radiomanganese tracer and tested forradioactivity on the first day. In Experiment Athese groups were injected with 1 X 10-5 mole ofsodium citrate or manganous, ferrous or chromouscitrates per animals, respectively. The injectionswere made both in the morning and the eveningof the second day. On the third, they weresacrificed within the hour following the test oftheir radioactivity. The ferrous citrate animalshad given the impression of toxicity by displayinglesser physical activity and rougher furs than thecontrols. Thus, in Experiment B, the number ofmice per group was increased, the manganouscitrate group was omitted, the challenging dose offerrous citrate was reduced to 2 x 10-8 mole andall the carriers were injected on the second andthird days. The animals were sacrificed on thefourth day and were handled as in Experiment A.

The bodies of the animals were dissected andtheir organs divided as shown in Figure 5. Theorgans of each group were pooled and tested for

radioactivity. Careful inspection of the peritonealsurfaces showed no metallic deposits.

As is shown in Figure 5, the only appreciableeffect on the distribution of the tracer amongst theorgans tested was again brought about by themanganese preparation. The others yielded a dis-tribution comparable to that of the controls.

This phenomenon had the characteristics of theeffect we had planned to show with the otherstable metals, namely elution from one to anotherpart of the body. It occurred above and beyondthe accelerated elimination induced by manganese:In the experiments with the dissected organs, wehave reported only the radioactivity retained inthe body.

DISCUSSION

The uncertainty of projecting facts from thetest tube into the realities of integrated life isclearly illustrated by this study. At the outsetone was led to expect that several metals mightelute radiomanganese from the body. Such ex-pectation was based on the following facts: 1)Many of the in vitro substitutes for manganese

530 U:

o 0

CARCASS LIVER G. I. KIDNEYS BRAIN SPLEEN LUNGSB CARCAS LIVER DLI KIDNEYS BRAIN SPLEEN HEART BTRACT HEART TRACT LUNGS

FIG. 5. PARTITION OF RADIOMANGANESEAMONGTISSUES OF MICE FOLLOWING INJECTION OF STABLEMETAL COMPOUNDS

A and B summarize the results of Experiments A and B, respectively, described in the text.

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I.

F

cLcL

LII

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GEORGEC. COTZIAS AND JAMES J. GREENOUGH

bind more strongly with tissue constituents (2).2) Some are abundant, like magnesium, while thetrace concentrations of manganese might not suf-fice to satisfy all its postulated reactions (14).3) The known in vitro transport mechanism doesnot guard against competitors (1).

A review of the coordination chemistry ofmanganese (2, 12) did not reveal any one singleproperty which alone could explain these results.If all the known coordination properties are con-sidered, however, this specificity can indeed beeasily explained. Therefore, one is justified to as-sume that there exist in the body micro-com-partments possessing many properties, the sumtotal of which determine the entrance of manga-nese and manganese only. These might include:a fairly resilient octahedral arrangement of sixcharges; oxygen as the predominating donoratom; a space with a radius of 0.5 to 0.9 A.Such an environment would discriminate effec-tively against the entrance of other metals. If onealso assumes that a given redox potential prevailsat such a site, then specific reactions would befavored. A redox potential of minus 1.33 volts,for instance, would favor the reaction Mn+ +H20 = MnO2+ 4H+ + 2E-, while it would dis-criminate against other reactions.

The sum of such factors would constitute aspecific segment in the anatomical pathway ofmanganese through the tissues of the body. Theexistence of specific atom configurations wouldnot preclude binding of manganese by other lessspecific sites (2, 12). If this hypothesis weregranted, one could ascribe many of the in vitroresults to oversaturation of the few specific man-ganese sites with excess of metal. Such over-saturation would bring into evidence the prop-erties of the numerous nonspecific sites. However,regardless of whether one favors this explanation,the observed irreplaceability of manganese in thebody supports the concept of its performing spe-cific tasks, rather than functions which might betaken over by other metals whenever the random-ness inherent in the mass-law so dictates.

There is another extrapolation which one mighthave made a priori which also was shown by thiswork to be incorrect. One might have expectedthat manganese would be displaced by some metal(other than manganese) because of such well-known precedents as the displacement in the body

of bromide by chloride (15), of molybdenum bytungsten (16) and of strontium by calcium (17).There exists, however, at least one metabolic dif-ference between manganese and the elementslisted above: the route of excretion. Manganeseis excreted in the feces, while the others (Br, Mo,Sr, and so forth) are excreted primarily in theurine. This suggests that a search for other com-mon differences might be rewarding. Further-more it is obviously doubtful that manganese isthe only substance whose pathway is strewn withspecific segments, since one would expect similarbehavior from iron also. With this in mind, aninvestigation of the pathways of other elements isnow in progress.

SUMMARY

1. A procedure is described which permits ob-servation of the elution of radiomanganese by in-jected stable metal compounds in intact mice andtheir tissues. This delineates an area for directcorrelations between in vivo and in vitro phe-nomena.

2. Stable manganese compounds ranging frompowdered metal to permanganate were effectiveeluting agents of the body's radiomanganese.

3. Injections of stable manganous citrate causeda pronounced change in the isotope's partitionamongst the organs of these mice.

4. Stable members of the first transition groupand of group VII of the elements as well asmagnesium were tested for their capacity to elutethe manganese tracer. In spite of injections oflarge doses, none of these affected the animal'snormal rate of elimination of radiomanganese.

5. Chromous and ferrous citrates failed to causedeviations from the normal partition of Mn54 inthe body.

6. The hypothesis is proposed that there exists aspecific segment in the pathway of manganesethrough the body.

7. The specificity displayed by the manganesepathway is compared with the apparent lack ofspecificity illustrated by the in vivo displacementof bromide by chloride, of molybdenum by tung-sten, of strontium by calcium and -others. Onecommon metabolic difference between these ele-ments and manganese is noted: the route of ex-cretion. This is entirely fecal for manganese andprimarily renal for the other elements cited.

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SPECIFICITY OF THE MANGANESEPATHWAY

ACKNOWLEDGMENTS

The spectrographic analyses reported here were doneby courtesy of Morris Slavin, Chemistry Department,Brookhaven National Laboratory. The authors wishto thank Mrs. Helen Hamel for assistance. Dr. LeeE. Farr's continued encouragement is gratefully recog-

nized.

REFERENCES

1. Saltman, P., Fiskin, R. D., Bellinger, S. B., and Alex,T. The metabolism of iron by rat liver slices.The effect of chemical agents. J. biol. Chem. 1956,220, 751.

2. Williams, R. J. P. Metal ions in biological systems.Biol. Rev. 1953, 28, 381.

3. Sumner, J. B., and Somers, G. F. Chemistry andMethods of Enzymes. New York, Academic PressInc., 1953, p. 40.

4. Schroeder, H. A. Trace metals and chronic diseases.Advanc. intern. Med. 1956, 8, 259.

5. Fonnesu, A., and Davies, R. E. The prevention ofswelling of liver mitochondria in vitro. Biochem.J. 1956, 64, 769.

6. Maynard, L. S., and Fink, S. The influence of chela-tion on radiomanganese excretion in man andmouse. J. clin. Invest. 1956, 35, 831.

7. Greenberg, D. M., Copp, D. H., and Cuthbertson,E. M. Studies in mineral metabolism with the aidof artificial radioactive isotopei. VII. The distri-bution and excretion, particularly by way of thebile, of iron, cobalt, and manganese. J. biol.Chem. 1943, 147, 749.

8. Cotzias, G. C., and Curtis, B. A. Unpublished data.9. Underwood, E. J. Trace Elements in Human and

Animal Nutrition. New York, Academic PressInc., 1956.

10. Druce, J. G. F. Rhenium, Dvi-Manganese, theElement of Atomic Number 75. Cambridge, TheUniversity Press, 1948.

11. Shellabarger, C. J. Studies on the thyroidal ac-cumulation of rhenium in the rat. Endocrinology1956, 58, 13.

12. Martell, A. E., and Calvin, M. Chemistry of theMetal Chelate Compounds. New York, Prentice-Hall, 1952.

13. Maynard, L. S., and Cotzias, G. C. The partition ofmanganese among organs and intracellular or-ganelles of the rat. J. biol. Chem. 1955, 214, 489.

14. Hawk, P. B., Oser, B. L., and Summerson, W. H.Practical Physiological Chemistry, 13th ed. NewYork, McGraw-Hill, 1954, p. 1077.

15. Goodman, L. S., and Gilman, A. The Pharmacologi-cal Basis of Therapeutics, 2nd ed. New York,Macmillan, 1955, p. 156.

16. Higgins, E. S., Richert, D. A., and Westerfield,W. W. Molybdenum deficiency and tungstate in-hibition studies. J. Nutr. 1956, 59, 539.

17. Spencer, H., Brothers, M., Berger, E., Hart, H. E.,and Laszlo, D. Strontium-85 metabolism in manand effect of calcium on strontium excretion.Proc. Soc. exp. Biol. (N. Y.) 1956, 91, 155.

18. Tipton, I. H., Cook, M. J., Steiner, R. S., Foland,W. D., Bowman, D. K, and McDaniel, K KSpectrographic analysis of tissues for trace ele-ments. Oak Ridge National Laboratory Report,1956. C. F. 56-3-60.

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