METABOLISM OF SODIUM SELENATE AND SELENITE BY THE TISSUES*

Transcription

1 METABOLISM OF SODIUM SELENATE AND SELENITE BY THE TISSUES* BY IRENE ROSENFELD AND. A. BEATH (From the Department of Research Chemistry, University of Wyoming, LaranGe) (Received for publication, October 21, 1947) Various workers investigating the toxic action of selenium postulated that changes in the forms of selenium occur in t,he animal. The mechanism whereby the selenium is changed by the tissues has been the subject of speculation, but no experimental evidence has been reported up to the presect time. Hofmeister (1) has reported that the salts of seler,ium and tellurium are reduced to metallic form in the body; afterwards methyl compounds are formed and these volatile compounds are eliminated by the lung. Challenger (2) pointed out that this conclusion lacks experimental verification. Schultz and Lewis (3) studied the elimination of volatile selenium by the respiratory system when sodium selenite was injected subcutaneously. They reported that withih 8 hours from 17 to 52 per cent of the injected selei-;ium was eliminated as a volatile compomd which could be absorbed by concentrated sulfuric acid. The respiratory excretion of the volatile compounds was not increased by increasing the potential source of methyl groups by methionine or choline chloride. McConnell (4), in time-excretion studies of exhaled selenium after the subcutaneous injection of radioactive selenium in the form of selenate, found that 3 to 1 per cent of the original dose was exhaled during the first 6 hours. Potter (5) investigating the action of various compounds, among them sodium selenate and selenite, on yeast respiration in a glucose substrate observed that with selenite the inhibition began at once, while with selenate the oxygen uptake was reduced only at the end of 2 hours. Potter and Elvehjem (6) studying the succinoxidase as it occurs in chick tissues found that the enzyme system was inhibited by cyanide, sodium selenite, and sodium arsenite, while sodium fluoride, sodium selenate, and arsenate were relatively non-toxic at the end of 1 hour. They suggested that the selenate must be reduced to a.lenite by tissues before any toxicity results. A survey of the literature reveals no experimental evidence on the site of conversion of selez;ate to selecite, formation of volatile selenium, or reduction to elemental selenium. These changes have been postulated, but up to the present time no experiments have been made with organs taking part in these trars?ormations. In our studies in investigating the physiological action of selenate and * Approved for publication by the Director of the Experiment Station. 333

2 334 METAROLISM OF SELENATE AND SELENITE selenite on isolated guinea pig intestine, we observed that 75 parts per million of selenate did not interfere with the normal function of this tissue, while 8 parts per million of selenite completely inhibited the intestinal motility. These experiments indicated that selenate was an atoxic form, at least to the intestine, and that selenite was the toxic form of selenium which could inhibit the normal function of the intestine. If the selenate is converted into selenite before it can exert its toxic effect the site of conversion is not isolated intestine. Details of these experiments will be published in another report. In order to get some evidence on the metabolism of sodium selenate and selenite, we attempted to study the effect of various fresh tissues and autoclaved liver tissue on these compounds. In this report we present the results of these experiments. EXPERIMENTAL Fresh beef liver, spleen, and blood were used in the experiments, since these organs in animal poisoning have a high concentration of selenium (7). Each experiment was repeated three times, and the results given are the closest agreement of two experiments. The tissues were used 3 minutes after the animals were slaughtered. If the tissues were kept longer, there was considerable variation in their action on selenate and selenite. Tissues of other animals, such as sheep, guinea pigs, and rats, showed considerable difference in their action on selenate and selenite. It is known that different species of animals show variations in susceptibility to the toxic action of selenium. In order to investigate the observed differences in the physiological action of selenate and selenite on guinea pig intestine, we have included this organ also in our study. We have observed some variations in the results by changing the temperature, but our report deals only with the results obtained at room temperature. In each experiment we used 2 gm. of tissues and 5 cc. of saline solution containing 5 mg. of sodium selenate and selenite selenium. The method of procedure and scheme of fractionation are presented in the accompanying diagram. Macerated tissue; 5 mg. of selenate or selenite selenium, 25 cc. saline, mixed frequently; room temperature for 2 hours Supernatant liquid decanted Tissues washed repeatedly with 1 cc. saline --tissue washed free of selenium, Total volume of 1 cc. collected and divided into 2 parts I Washing centrifuged Part 1, dried at 7, analyzed I- 1 hr. at 15 R.P.M. for elemental selenium, total I -l selenium Sediment dried at 7 Supernatant liquid Part 2, fresh, analyzed for total Analyzed for elemen- 2 cc. analyzed for selenium tal selenium, total selenate selenium, selenium eelenite selenium, total selenium

3 I. ROSENFELD AND. A. BEATH 335 The first results with the liver indicated that there was a considerable loss of selenium when selenite salt was in contact with the tissues and less with selenate; Since each fraction of the tissue and was analyzed, we concluded that this loss was due to volatile selenium. Therefore, we inserted several absorption flasks, using procedures similar to that of McConnell (4) or Schultz and Lewis (3) but with some variation in the absorbents used. We used the following absorbents: hydrobromic acid followed by water, sulfuric and nitric acids and sodium hydroxide, or only sulfuric and nitric acids. These modifications were instituted when we became aware that the recovery of volatile selenium was not quantitative. Analysis for selenate, selenite, and elemental selenium was carried out according to Byers et al. (8). Determinations of total selenium and of volatile selenium, determined as total selenium, were carried out according to the method of Kline (9). Results E$ects of Liver Tissue on Selenate and Selenite-Changes in the form of selenium occurred when sodium selenate and selenite were in contact with fresh liver tissue. Table I gives the results obtained. 26 per cent of the original selenate was converted to selenite, indicating that the liver is a site where reduction from selenate to selenite takes place. Calculated on the basis of the selenium added, 17 per cent of selenate and 31 per cent of selenite were changed to volatile selenium. In the selenate experiments we have recovered an average of 5 per cent and in the selenite an average of 15 per cent of volatile selenium. In experiments in tivo, with the same absorbents, other workers were able to recover larger amounts of volatile selenium. The only explanation we have for the lower values in our experiments is that the forms of volatile selenium in vitro may be different from those formed in wivo. Selenite was reduced to elemental selenium by the liver; part of this was present in the cent,rifuged sediment and part in the tissues. No reduction of selenate to elemental selenium took place under our present experimental conditions with liver. In addition to the reduction of selenate to selenite, and volatile selenium, in the, combined selenium was present, as can be seen from the results. Evidence for a combined selenium is the fact that the total selenium content of the was always greater than the sum of the selenate and selenite selenium contained therein. This difference in selenium values is called combined selenium in our tables. Whether this selenium was in colloidal or organic form combined with the water-soluble proteins, we cannot state at the present time. In order to investigate the factors responsible for the changes in the forms

4 336 METABOLISM OF SELENATE.4ND SELENITE of selenium by the fresh tissues, we have studied the effect of autoclaved liver on selenite and selenate. The results of the experiments, given in Table II, indicate that only fresh tissue has the property of decomposing the selenate to selenite and converting the selenite into elemental and vola- TABLE Chemical Decomposition of Selenate and Selenite in Contact with Fresh Beef Liver for % Hours at Room Tempedure I Se recovered from elenitc Form of Se recovered from various fractions Selenate Elemental er cm wr cc?21 per cen1 Supernatant liquid selenate Centrifuged sediment from Macerated liver Supernatant liquid 4. selenite Centrifuged sediment from 3.6 Macerated liver 4.9 *Combined selenium = total Se - (selenate + selenite). - I combined SC* Total - - CI cm 1 i IW cent TABLE II Effect of Autoclaved Liver on Selenate and Selenite per ccn1 Form of Se recovered* fractions Form of Se added Se recovered from 3; from various Selenate per cent Total per cmt selenate Supernatant liquid Centrifuged sediment from 11.5 selenite Macerated liver Supernatant liquid Centrifuged sediment from the Macerated liver * Elemental, none. tile selenium. We have found that selenite has a greater afhnity for tissues than selenate whether it is in contact with fresh or autoclaved tissue. This afhnity of selenite for the tissues may account for its instability, rapid volatilization, and reduction to elemental selenium when it is in contact with fresh tissue. The factor or factors which accomplish the decomposition of

5 I. ROSENFELD AND. A. BEATH 337 selenate and selenite are heat-labile, since the forms of selenium added were not altered by the autoclaved tissue. Perhaps the factors responsible for the changes are enzymatic in nature. E$ect of Blood on Selenate and Selenite-Since blood contains large amounts of selenium during selenium poisoning, we studied the effect of freshly drawn beef blood on selenite and selenate selenium. The method of procedure was the same as that outlined previously. TABLE XII Chemical Changes in Selenate and Selenite in Contact with Bet Blood at Room Tern rature for 2 Hours selenate selenite Tissue used Se recovered from Form of Se recovered from various fractions cpm- Sele- Sele- Ek- Gd nite Kite Total In len tal m w w ted 6.M Cenl Whole bloos Supernatant liquid Centrifuged red blood cells Red blood Supernatant liquid 8.8 cells Red blood cells Plasma Supernatant liquid Whole bloo Supernatant liquid 9.1 Red blood cells.9 Red blood Supernatant liquid 56. cells Red blood cells.7 Plasma Supernatant liquid 5.3 * Combined selenium = total selenium - (selenate + selenite) olatile Se reovered After incubation of the whole blood and blood cells with selenite, considerable hemolysis of the cells occurred, while selenate did not affect the cells. Results of the experiments are given in Table III. The whole blood reduced the selenate to selenite, but the amount of selenite formed was considerably less than with liver. However, combined selenium was higher than that observed in the liver experiments. In order to study the part of the blood responsible for the reduction of selenate, we tested the red blood cells and plasma separately with this compound. The results indicate that the plasma has the ability to reduce the

6 338 METABOLISM OF SELENATE AND SELENITE selenate to selenite and volatile selenium, and to change the form of selenium, since 55.6 per cent of selenium recovered was combined selenium. The red blood cells did not convert the selenate to selenite and only 1.5 per cent of combined selenium was recovered. Similar experiments with the selenite were carried out, the results of which are also given in Table III. The selenite was reduced to elemental selenium by whole blood and by the red blood cells, while the plasma caused no reduction. The distribution of selenite in the with whole blood, plasma, and red blood cells showed considerable variation. Small amounts of selenite remained in the with whole selenate eelenite TABLE Chemical Changes in Selenate and Selenite in Contact with Beef Spleen at Room Temperature for d Hours Supernatant Serecoveredfrom liquid Centrifuged sediment from Macerated spleen Supernatant liquid Centrifuged sediment from Macerated spleen - I IV Wenite Form of Se recovered from various fractions wenate C cm< 42.4 * Combined selenium = total Se - (selenate f selenite).. El* nental CI cm Total.- CI Cenl 1 per CM com- i5tf blood and plasma, while with the red blood cells 56 per cent of the selenite was unchanged. The of the whole blood, red blood cells, and plasma contained combined selenium. The amounts present were as follows: whole blood, 75. per cent; red blood cells, 31.1 per cent; plasma, 87.7 per cent. E$ect of Spleen on Selenate and Se&&-The sequence of changes when selenate and selenite were in contact with beef spleen was similar to that observed with the liver, as can be seen from Table IV. This was the only tissue in which we have obtained elemental selenium when we started with selenate. Although the amount was small, it is an indication that after selenite is formed from selenate reduction to elemental selenium can take place

7 I. ROSENFELD AND. A. HEATH 339 Treating the spleen with selenite selenium, we obtained 19.5 per cent of elemental selenium. Reduction of selenite to the elemental form with this tissue was greater than with any of the other tissues tested. Only 5 per cent of selenite, the form added, was present; the remainder was either in combination with tissue proteins or was converted into volatile selenium. E$ect of Guinea Pig Intestine on Selenate and Selenite-According to our observation, selenite and selenate showed remarkable differences in their physiological action on guinea pig intestines. Using the same method as in the previous experiments, we have investigated the effect of this organ on sodium selenate and selenite. The results of these experiments are given in Table V. It is interesting to note that the guinea pig intestine did not produce any changes in the form of selenium added, except the attachment of selenium to the tissue. TABLE V Effect of Fresh Guinea Pig Intestine on Selenate and Selenite Form of Se added Se recovered from aelenate selenite * Elemental, none. Supernatant liquid Guinea pig intestine Supernatant liquid Guinea pig intestine _-- I Form of Se recovered* from various fractions Selenite Selenate per csni gcr Ccnl 89.5 Total )W ccn With this tissue as in the previous experiments the selenite showed a greater aflinity than the selenate for the tissue. These results indicate that the difference in the physiological action of selenate on guinea pig intestine was due to the failure of this organ to change the selenate to selenite. DISCUSSION It is apparent from the data presented here that fresh beef liver, spleen, whole blood, and plasma change the forms of selenium in vitro, while autoclaved liver and fresh guinea pig intestine do not produce similar changes. The red blood cells produce changes by forming combined selenium when selenate or selenite is added. These results suggest that the fresh guinea pig intestine does not contain the necessary factor or factors (enzymes) to decompose the selenium in vitro. The factor or factors (enzymes) in the liver which decompose the selenite are heat-labile, since autoclaving the tissue destroys the substances which brought on the decomposition of the

8 34 METABOLISM OF SELENATE AND SELENITE compounds. Selenate is converted into selenite by various fresh tissues. These tissues show a great deal of variation in their ability to change the selenate to selenite. The highest activity is shown by the liver, followed by spleen, whole blood, and plasma. The red blood cells are unable to change selenate to selenite. The attachment of selenite to tissue proteins is evidenced from the results. Only a small amount of selenite was recovered from the supernatant fluid as selenite; the larger amounts were in the centrifuged sediment or tissues, or they combined with the soluble proteirs and were recovered as combined selenium. The distribution of the selenate and selenite after contact with the various tissues shows some variations, but the trend appears to be similar. These experiments indicate that the metabolism of selenate and selenite is similar, but the amount of volatile selenium formed from the selenite is much greater than from selenate. This may explain the differences in the results obtained by Schultz and Lewis (3) and McConnell (4) in their studies on the respiratory elimination of volatile selenium. The former used selecite and obtained 17 to 52 per cent volatile selenium and the latter used selenate and obtained 3 to 1 per cent of volatile selenium. The amount of volatile selenium formed by the addition of selenite and selenate to the liver tissue approximates the amounts reported in viva. However, the total volatile selenium in all the organs studied was higher than reported by these workers. We must take into consideration that in in viva experiments the volatile substances are transported by the blood and that considerable changes may occur in the volatile substarices. Wright (1) in studying the effect of sodium selenate and selenite on oxygen consumption of mammalian tissues observed that a higher concentration of selenate than selenite was necessary for equivalent depression. He observed that brain slices were little affected by selenate, while the oxygen uptake of liver slices was altered by a lower concentration. Kidney and muscle were slightly more sensitive than brain but less so than liver. Interpreting his results on the basis of our present observations would indicate that the addition of selenate to liver tissue, which in our experiment was able to convert the selenate into selerite, would interfere with the oxygen consumption. After conversion occurred, the effect would be the same as that of equivalent amounts of selenite. Selenite depressed the rate of oxygen consumption in all of the tissues studied. This fact suggests that the toxicity of selenate to the tissue may depend upon the ability of the tissues to convert the selenate into selenite. The sequence of transformation by the various tissues indicates that the decomposition of selenium follows a definite pattern, from selenate to selenite, combined selenium, and volatile and elemental selenium. There is a possibility that in viva the reactions are reversible. Observations in ani-

9 I. ROSENFELD AND. A. BEATH 341 ma1 poisoning seem to indicate this fact. Beath (11) observed that symptoms of poisoning, due to the delayed action of selenium, occurred several months after the animals were removed from seleniferous areas. SUMMARY 1. In in vitro experiments we demonstrated that sodium selenate is converted into selenite and volatile selenium by beef liver, spleen, whole blood, and plasma. These tissues also convert the selenite to volatile selenium and reduce selenium to the elemental form. There are variations in the ability of the tissues to bring about these changes. 2. Autoclaved beef liver and fresh guinea pig intestine are unable to effect any changes in selenate or selenite. The factor or factors present in liver which decompose selenium are heat-labile since they are destroyed by autoclaving. The fresh guinea pig intestine does not contain the factors necessary for the decomposition of the compounds studied. 3. Decomposition of selenate and selenite follows a definite sequence of reactions. Some of the changes in the forms of selenium were determined by chemical means and others by the differences in the results obtained, since no methods at the present time are available for their determination. 4. Recovery of volatile selenium was not quantitative with the absorbents used. 5. On the basis of these experiments we have proved the postulation that selenate and selenite are changed by the tissues and the toxicity of selenate is due to its conversion to selenite by the tissues. BIBLIOGRAPHY 1. Hofmeister, F., Arch. ezp. Path. u. Pharmakol., 33,198 ( ). 2. Challenger, F., Chcm. and Ind., 657 (1935). 3. Schultz, J., and Lewis, H. B., J. Biol. Chem., 133, 199 (194). 4. McConnell, K. P., J. Biol. Chem., 146,55 (1942). 5. Potter, V. R., Thesis, University of Wisconsin (1936). 6. Potter, V. R., and Elvehjem, C. A., J. Biol. Chem., 117,341 (1937). 7. Rosenfeld, I., and Beath,. A., J. Nutr., 3,443 (1946). 8. Byers, H. G., Miller, J. T., Williams, K. T., and Lakin, H. W., U. S. Dept. Agr., Tech. Bull. 61 (1938). 9. Kline, A. K., J. Assn. s. Agr. Chem., 24,363 (1941). 1. Wright, C. I., Pub. Health Rep., U. S. P. H. S., 63,1825 (1938). 11. Beath,. A., Science, 81, 617 (1935).

10 METABOLISM OF SODIUM SELENATE AND SELENITE BY THE TISSUES Irene Rosenfeld and O. A. Beath J. Biol. Chem. 1948, 172: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites references, of which can be accessed free at tml#ref-list-1