Joiirnol of

Neuruciirniirtri. 1976. Vol 27. pp. 1379- 13x7. Pprpamon Press. Printed in Great Britain

SELENIUM AND GLUTATHIONE PEROXIDASE I N DEVELOPING RAT BRAIN' J. R. PROHASKA and H. E. GANTHER Department of Nutritional Sciences, University of Wisconsin. Madison. WI 53706. U.S.A. (Receiiwd 29 December 1975. Accepted 11 M a y 1976)

Abstract-Week-old rats were given a subcutaneous injection of carrier-free Na27sSe0, and brain 7'Se distribution was studied after 30 days, with special reference to the selenoprotein, glutathione peroxidase (GSH-Px). Chemical fractionation studies showed the 75Se was associated mainly with protein and not extracted by hot trichloroacetic acid or chloroform-methanol, Subcellular fractions also revealed a parallel distribution of 75Se and protein with the notable exception that 75Se was concentrated in the mitochondria and reduced in the cytosol. GSH-Px activity was demonstrated in the isolated mitochondria1 fraction. The estimated biological half-life of brain 75Se was 45 days. Gel filtration (Sephadex G-150) of brain cytosol resulted in four 7'Se peaks: peak I was associated with the void volume, and had the greatest 7sSe content; peak 2 ( V J V , = 1.4) contained nearly as much '%e and had an apparent molecular weight of 94,000; peak 3 (V,,/V, = 2.4) had an apparent molecular weight of 13,500 and was markedly increased when brain was homogenized in the presence of Triton X-100; peak 4 consisted of low molecular weight compounds. When fresh cytosol (with or without Triton X-100) was chromatographed on Sephadex G-150, GSH-Px was detectable only in the void volume; however, storage of cytosol prepared in the presence of Triton X-100 shifted most of the activity to peak 2 (94,000).The GSH-Px activity in the void volume resembled the purified enzyme with regard to pH dependence, K , for cumene hydroperoxide at fixed [GSH]. and first-order kinetic behavior with respect to GSH. A minor peak of GSH-Px activity showing zero-order kinetics with respect to GSH concentration and an apparent molecular weight of 46,000 was detected when larger amounts of protein were chromatographed. The concentration of rat brain Se measured by chemical analysis reached adult levels by 2 weeks after birth. an age when the level of GSH-Px had just begun to rise. It was estimated that only lj5 of the total brain Se may be accounted for by its presence in GSH-Pa, suggesting that the function of the majority of brain Se remains to be determined.

tion against peroxidation, but may have underestimated GSH-Px activity. Our initial studies showed that less than half the total GSH-Px activity was expressed unless detergents were used to disrupt brain tissue. It seemed necessary to investigate this point further. The brain is the target organ in methylmercury toxicity. and dietary selenium has been shown to decrease methylmercury toxicity (GANTHER et al., 1972). Although the mechanism of selenium protection is not known, Se does not reduce methylmercury content in brain. nor is the additional selenium present in brain sufficient to complex with and inactivate more than a small fraction of the mercury (ElBegearmi M., Sunde M. & Ganther H. E.. unpublished). Since little is known about brain selenium metabolism, further studies on selenium in brain seemed warranted to aid in understanding its relationships to methylmercury toxicity. In this study measurements of total Se and GSH-Px distribution in brain were conducted. Animals were also injected with a carrier-free dose of Na,75Se0, during an ' Research supported by the College of Agricultural and active period of brain growth in order to label comLife Sciences, University of Wisconsin, Madison, and the ponents for further investigations of 75Se-seleniurn US. Public Health Service ( A M 14184). Abbrtwiations used: GSH-Px, glutathione peroxidase (EC and GSH-Px distribution in various subcellular fractions and following gel chromatography. In addition, 1.1 1.1.9); TCA, trichloroacetic acid. 1379

THEDISCOVERY of glutathione peroxidase (glutathione: hydrogen peroxide oxidoreductase, EC 1.11.1.9) by MILLS(1957) and the establishment of the essentiality of selenium as a nutrient (SCHWARZ & FOLTZ.1957; PATTERSON e t al.. 1957) were coincident in time. However, it was L6yr later before these two findings merged when it was established (ROTRUCKet ul., 1973; FLOHErt al., 1973; OH et a!., 1974) that selenium was an integral part of glutathione peroxidase. The presence of selenium in GSH-Px, which decomposes lipid peroxides, helped to explain many of the nutritional effects of selenium as an apparent antioxidant. The brain has a high content of unsaturated fatty acids. but is apparently devoid of catalase (HARTZet al., 1973), and has low GSH-Px (DEMARCHENA et al., 1974). It would therefore appear to be vulnerable to peroxide-induced lipid peroxidation. DEMARCHENA et al. (1974) concluded that activity of GSH-Px in brain was insufficient to provide protec~~

~

~

1380

J. R.

PROHASRA

and H. E. GANTHER

Se concentration and GSH-Pt activit) %ere also studied as functions of ape and dietary selenium.

MATERIALS A N D METHODS Glutathione reductase (EC I .6.4.2). GSH. NADPH. NADH. sodium pyruvate. r-nialic acid rind Triton X-100 were purchased from Sigma Chemical Co. (St. Louis. MO). Cumene hydroperoxide was obtained from ICN K & K Chemicals (Cleveland. O H ) and found to be 81"" pure based on the amount of GSSG formed in the presence of excess GSH-Px as estimated b! enzymatic assay with glutathione reductase and NADPH. Ne\v England Nuclear (Boston, MA) supplied H2"Se0,. Selenite content was estimated to be 91"" based o n the amount of -$Se cornplexing with 3.?'-diaminohen7idine ICHENG. 19561. Rats used in these studies were purchased from Holtzman Co. (Madison. WI). Sephadex (3-150 \vas obtained from Pharmacia (Piscataway. NJ). ,411imdcare tirid diets. Pregnant rats isere fed a commercial pellet diet (Lab Blox. Allied Mills Inc.. Chicago. IL) containing 0.42 p g Setg. Within I day of parturition offspring were randomly distributed into litters of 8. Some of the pups were allowed to develop without treatment while others were subcutancousl) injected at 7 dabs of age (w v ) sodium acetate with 25 pCi of Naz""SeO, in (0.02mg Se/Kg body a-t). A t 21 d a y of age all offspring were weaned and fed the pellet diet and distilled \vater rid lib. At various intervals animals wcre killed for assay of GSH-Px and Se and to estimate the biological turnover of "Se. In one experiment (Table 4) wennling rats were fed a diet low in selenium ivhich n a s based on Torula yeast (HAFEMASt't c i l . . 1974) for 13 weeks prior to enzyme and Se analysis. A similar group nas fed the same Torula yeast diet supplemented with 0.5 p g Se g as Na2Se0,. Tissue prcprarioii. In some experiments animals were anesthetized with ether and perfused intracardiallj with l 0 m l of chilled 0.9"" (w'v) NaCl before decapitation to reduce red blood cell contamination in brain. Brain was homogenized in 9 vol of 0.32 M-SueroSe and subcellular fractions were prepared on ii discontinuous sucrose density gradient according to WHITTAKER (1969). isolating the crude mitochondrial fraction at 17.000g for 55 min. If enzymatic analyses were carried out the tissue was homogenized in 9 vol of 0.1 w-KCI. 0.02 ht-K,HPO,. 0.001 M-EDTA. 0.5", ( v v) Triton X-100. adjusted to pH 7 with

HCI. Erizyrw assu~.s.Glutathione perouidase was measured b) a coupled enzyme procedure with glutathione reductase & VALLSTINE. 1967) utilizing cuniene hydroperox(PAGLIA ide as substrate (LITTLEt't ul.. 1970). The assa) contained 0.1 M-potassium phosphate pH 7.0. 3 mwEDTA. 1 mM-KCN, 1 mM-GSH. 0.1 1 niwNADPH. 4 p g glutathione reductase. and a sample (0.05 ml) of the supernatant fluid from detergent-treated homogenate centrifuged for 15 min at 15.000y. .After ;I 5-niin preincubation (25 C) the reaction mixture was hrought t o I.0ml with 0.01 ml of 10 m w cumene hydroperoxide dissolved in ethanol. The reaction was followed at 340 nm. 25 C in a Gilford Model 2ooO spectrophotometer. A small non-enzymatic rate (NADPH w a s subtracted from the overoxidation. 0.0015 AA,4,1),, all rate. An enzyme unit is one pnol GSH oxidized min. Fumarase (EC 4.2.1.2) was assayed by the method of R ~ C K I R(1950) as described previously (PROHASKA &

W ~ L L S 1975). . Lactic dehydrogenase (EC 1.1.1.27) was measured according to the procedure of STOLZENBACH (1966) at 25 C. Chetnicul mialyses. Protein was measured by the method of LOWRYet al. (1951) using bovine serum albumin as a standard. Total selenium was determined fluorometrically as the diaminonaphthalene complex following wetashing of the samples (OH er rd., 1974). Radioactivity was determined by counting the "Se in a well-type gamma counter (Nuclear-Chicago) over the range 0.05-0.5 MeV. Consideration was made for influences of sample volume on the counting efficiency and for decay of the '%e (halflife 120 days). Other details can be found in the legends to the figures o r in footnotes to the tables.

RESULTS

Distrihtiori

Preliminary chemical fractionations were carried out to learn something about the form of "Se in brain I month after injection of carrier-free 75Seselenite. Trichloroacetic acid (TCA) or HC104 treatments were used to precipitate proteins. hot TCA was used to extract nucleic acids, and ethanol or chloroform-methanol was used to extract lipids. Less than 5"" of the "Se in brain homogenates was soluble in these solvents (data not shown). Similarly, more than 95"., of the "Se in brain cytosol was precipitated by cold TCA. Selenium was determined by chemical analysis in various anatomical regions of brain (Table 1). Small variations were present, the cerebellum having the highest concentration and the medulla oblongata the lowest. The relative distribution of "Se in these brain regions (not shown) was comparable to that of total Se. Subcellular distribution of 75Se was determined 15 weeks after injection of "Se-selenite (Table 2). The distribution of protein is also presented for comparison. Selenium and protein show a close parallel except that selenium is conc. in the mitochondria1 fraction (relative to protein) and diluted in the cytoSol.

TABLE1. CONCENTRATION OF SELENIUM IN OF RAT BRAIN* Region Whole brain Cortex Cerebellum Medulla oblongata Remaindert

VARIOUS REGIONS

Selenium

Tissue weight

( m / g wet wt)

(8)

0.192 f 0.010 0.186 f 0.005 0.221 I O.(K)9 0.166 f 0.01 1 0.201 f 0.033

1.94 k 0.11 0.785 0.073 0.276 f 0.011 0.29-1 t 0.050 0.555 k 0.065

+

*Values are means f S.D. of four 3-month-old male rats fed a commercial pellet diet from weaning. lntracdrdiac perfusion \vith saline was performed to reduce blood contamination. Dissection was performed according to the procedure of GLOWINSKI & I v m s m . 1966. tRemainder includes midbrain. hypothalamus, striatum and hippocampus.

Brain selenium and glutathione peroxidase TARLE2. SLIRCELLULAR

DISTRIBUTION

OF 75Se IN

RAT

BRAIN* 0,;

Total

__

Fraction Nuclear Mitochondria1 Cytosolic Microsomal Myelin Synaptosomal

75

Selenium

16.3 f 1.34 20.9 f 1.46 9.68 f 1.38 4.13 0.29 16.7 f 1.54 22.4 f 0.67 ~

~~

Protein 17.6 0.47 10.7 _+ 0.68 20.7 f 0.17 6.20 f 0.16 16.2 f 0.65 21.1 0.78 ~

~~

*Values represent means f S.D. of 3 female rats (age 16 weeks). Intracardiac perfusion with saline was performed prior to decapitation. Fractions were isolated as detailed in Methods and measured for total radioactivity and protein.

1381

An increase (up to 1600,:) in GSH-Px activity resulted when brain homogenates were treated with Triton X-100 prior to assay (Table 3). A maximal effect was obtained with 0.5oi;, (v/v) Triton X-100. The increase in latent activity with Triton treatment was not so evident in the case of liver or kidney samples (Table 3). Other studies (not shown) revealed that Triton X-100was wither inhibitory or stimulatory when tested with GSH-Px isolated from ovine erythrocytes. Note (Table 3) that the large sex difference in GSH-Px activity found in liver was not apparent in brain.

Srphades G-150 chronzcitogrnphy

Fresh brain cytosol prepared in 0.32 M-sucrose was chromatographed on Sephadex (3-150 and the collected fractions were assayed for protein (AZRO),7’Se The subcellular distribution of GSH-Px is shown content and GSH-Px activity (Fig. 2). Four main in Fig. 1 along with fumarase (a marker for the mito- radioactive peaks were observed: the first was associchondrial matrix) and lactic dehydrogenase (a marker ated with the void volume (V,) and main ALSOpeak; for the cytosol). GSH-Px was highest in the cytosol the second peak ( VJV0 = 1.4), comparable in arca to (37.1%) followed by mitochondria1 (24.9) and synapto- the first, and the third peak ( Ve,’K, = 2.4) had elution soma1 (1 6.1) fractions: lactic dehydrogenase was also volumes corresponding to apparent molecular highest in the cytosol(38.0) followed by synaptosomal weights of 94,000 and 13.500, respectively when com(23.9) and microsomal (16.9); fumarase was highest pared with proteins of known molecular weight (gluin the mitochondria1 (48.0) and synaptosomal (25.3) cose oxidase, glucose 6-phosphate dehydrogenase. fractions. While approx 40% of the total GSH-Px and bovine serum albumin. ovalbumin. myoglobin); the lactic dehydrogenase were found in the cytosol, some fourth peak was eluted with low molecular weight cytosolic enzymes may be present in synaptosomes compounds associated with the other major A z s o as a result of occlusion (see Discussion). In compari- peak. Only the void volume fractions (up to r.:,iV, son, 65”G of the total GSH-Px and 92:d of the total of 1.25) contained measurable GSH-Px. Kinetic lactic dehydrogenase were found in the cytosol of rat analysis suggested that this activity was similar to purified GSH-Px from bovine erythrocytes ( F L O H&~ liver. GUNZLER,1974) since the enzyme reaction showed the same pH optimum (approx 8.5), the reaction rate was first order with respect to GSH up to 7 mM-GSH and the apparent K , for cuinene hydroperoxide was 2.8 x M when GSH was fixed at 1 mM. For ’%e. TABLE 3. EFFECTOF TRITON X-100 ON GLUTATHIONE PEROXIDASE ACTIVITY*

Triton X-100

[%(v/v)l 0

0.1 0.25 0.5 1.0 2.0

Glutathione peroxidase (units/g tissue) Brain Liver Kidney F M F M F M 1.14 1.33 110 55.2 1.79 - - 2.10 - - 2.43 2.00 130 60.4 2.28 - - -. 2.23 ~

-

25 3

~-

-

-

-

-

32.0

-

-.

-

*Fresh tissue homogenates [?09, ( w ~ ) ]weru prepared using a Potter-Elvehjem type homogenizer fitted with a Teflon pestle using 10 full strokes and buffered KCI as medium (see Methods). The homogenates were thcn diluted to IOS, (w/v) in the same medium with varying FIG. 1. Subcellular distribution of lactic dehydrogenase R, amounts of buffer or Triton X-100 then centrifuged and glutathione peroxidase E l and fumarase E4 in rat brain. the supernatant fluids assayed for glutathione peroxidase Values represent means +_ S.D. for three adult male rats. as described in Methods. Values shown are representative Fractions were treated with Triton X-100 and assayed as of results obtained from several experiments and are the described in Methods. means of duplicate assays.

I382

.I. R. P K ~ H A S and K A H. E. GANTHCR

the peak height ratio of peak 2,peak 3 was consistently 5 when cytosol was prepared from brains homogenized in sucrose. When cytosol was prepared from brain homogenized in 0.5:#0 ( v i v ) Triton X-100 and frozen (- 15°C)the Sephadex G-150profiles changed (Fig. 3). Four radioactive peaks with the same 1.:.'I,b ratios as before were observed. but two notable changes were evident. First, the GSH-Px was split into two peaks with the main one coinciding with the second "Se peak (Ve/V, = 1.4). Secondly. "Se peak 3 (LL.,! V , = 2.4) was markedly increased so that now the peak height ratio of peak ?:'peak 3 was 2 instead of 5. The A280eluting with peak 3 was shown from U.V. spectra to be due to the presence of Triton X-100. Mixing experiments using purified enzyme from ovine erythrocytes and samples from both peaks 1 and 2 in Figs. 2 and 3 (i.e. with and without measurable GSH-Px) failed to reveal the presence of inhibitors or activators of enzyme activity. If fresh detergent-treated supernatant fluid was chromatographed on Sephadex G- I50 (Fig. 3A) the major GSH-Px peak was associated with the void volume. rather than peak 2, as seen with frozen detergent-treated cytosol (Fig. 3). A common feature of either fresh or frozen detergent-treated samples following chromatography was the relative increase in the "Se peak 3 (Lily,= 2.4) (Figs. 3 and 4A). Besides the four "Se peaks described previously. Fig. 4A also shows the presence of a new "Se peak

containing GSH-Px which was eluted at Ve/yl= 1.8, corresponding to an apparent MW of 46,000. This GSH-Px peak was observed only when rather large amounts of enzyme were applied to the column, since it represented only I/5 of the activity found in the GSH-Px peak eluted at the void volume. Since AWASTHI c't al. (1975)had isolated a GSH-Px (form B) from human erythrocytes with an apparent MW of 47,000, another experiment was run to verify the existence of a similar activity in brain tissue. Detergent-treated supernatant fluid was prepared from frozen brains of animals fed the Se-deficient Torula yeast diet for 8 weeks. which is known to deplete GSH-Px in blood and other tissues but not in brain (HOEKSTRA, 1974). Se deficiency was indicated by liver GSH-Px activities less than lo",, of that from animals fed the Se supplemented diet. The level of GSH-Px in blood should also be lower in these animals (Table 4); intracardial perfusion was used to further reduce the red blood cell GSH-Px contamination i n brain samples. The Sephadex G-I50 profile shown in Fig. 4B verifies the presence of the 46.000 M W GSH-Px peak (VJ C:, = 1.8). in addition to a GSH-Px peak eluting at the void volume. The 46,000 M W peak from this Sephadex G-150run and a replicate run were pooled, concentrated by ultrafiltration (Amicon Model 202, PM 10 membrane) and samples were used for a kinetic study. This glutathione peroxidase activity showed zero-order kinetics with respect to GSH and

FIG.2. Elution profile of fresh rat brain cqtosol fractionated on Sephadex G-150. One month following injection of Na2"Se0, three rats \*ere perfused intracardially H ith NaCl 0.9", ( y / v j and brains were removed and homogenized in 9 vol of 0.32br-sucrose and the cytosol was prepared. Equal samples of each cytosol were pooled and a 10-ml portion (18 mg protein) was applied to a 2.5 x 95 cm column of Sephadex G-150 equilibrated with 0.05 wpotassium phosphate ( p H 7.01 at 4'C. Fractions of 5.95 ml glutathione peroxidase ( M j and '$Se were collected (23.8mlVh) and analyzed for A Z R OI-) ).-.I as described in Methods. The void \olume was determined wing blue devtran as a inarkcr in order t o calculate ratios.

Brain selenium and glutathione peroxidase

1383

VQ/VO

FIG.3. Elution profile of frozen-thawed Triton X-IM) treated rat brain supernatant fluid fractionated on Sephadex G-150. Three weeks after injection of Na2”Se0, four rats were perfused and brains were homogenized in the presence of Triton X-100(see Methods). Supernatant fluid following centrifugation at 100,OOOy. 1 h was pooled and frozen ( - IS’C).Ten ml of the thawed sample was layered on to Sephadex G-150 as described in the legend to Fig. 2. Fractions were measured for A,,, (-). glutathione peroxidase (-0) and ”Se (.---a).

an apparent K , of 0.4 mM was obtained when cumene hydroperoxide was employed at 0.5 m ~This . enzyme activity was also observed to have a slightly altered pH dependence and slightly higher apparent K, (4.6 x 1 0 - 5 ~ )for cumene hydroperoxide at fixed GSH (1 mM) when compared to the activity in the void volume. Freezing cytosol prepared in 0.32 M-sucrose for an appropriate time prior to Sephadex G-150 chromatography did not itself shift the GSH-Px to the second pcak ( V J V , = 1.4), as shown in Fig. 4C. As is the case in brain, the shifting of GSH-Px to peak 2 following freezing of detergent-treated supernatant fluid was observed when liver and testes samples were chromatographed on Sephadex G- 150 (not shown). Posriici tu 1 development Several litters of noninjected rats were used to investigate the postnatal changes in brain selenium and GSH-Px (Fig. SA); corresponding values for blood are also presented in Fig. 5B for comparison. Following an initial drop, the adult concentration of brain Se was achieved 14 days after birth whereas brain GSH-Px was still increasing at 40 days of age (approx 3/4 the adult level). The mean adult values for brain were 0.205 pg/Se/g and 2.07 Units GSH-Px/g. Blood selenium and GSH-Px levels fell during the first week, and rose steeply during the last 2 weeks of lactation to approx 314 the adult levels. During the first 40 days of age the blood/brain ratio for GSH-Px (units per ml blood/units per g

brain) seemed rather constant and a pooled valuc of 13.5 1.9 (mean f s.D.) was calculated from the data in Fig. 5. The corresponding pooled value for selenium was 2.08 f. 0.22 (mean f s.D.). These ratios appeared to be higher in adult rats (Table 4). Assuming a 2”/, blood contamination (OWEN,1971) the amount of GSH-Px/g tissue due to blood in brain samples would be approx 0.5 units and for selenium 0.01 pg. These corrections could affect the accuracy of estimates of brain GSH-Px. The changes in brain Se and GSH-Px seen in Fig. 5A were not related to blood contamination. Retention of selenium During the first weeks following the injection of Na,”SeO, the brain was in an active phase of both hyperplasia and hypertrophy and therefore estimations of a biological half-life for 75Se were coniplicated. However, a half-life of 45 days was calculated from data (Fig. 6) taken between 70 and 115 days postinjection. The distribution of ”Se in subcellular fractions also changed with time. For example, about 18% of the total ’%e was found in the cytosol at 5 weeks after injection, 12% at 10 weeks (not shown), and 10% at 15 weeks (Table 2). During the same period the 75Se retention in the myelin fractions (per cent of the total) increased from a value of about 10% at 5 weeks after injection to 170; at 15 weeks (Table 2). These results suggest that turnover times are different for 7sSe in the various subcellular fractions.

J. R . PROHASKA and H. E. GANTHER

1384

rats fed the same diet plus 0.5 p.p.m. Se or to rats fed the stock diet. However, the brain Se level was not significantly reduced nor was brain GSH-Px changed. Rats fed stock diet had values for blood Se and GSH-Px and for brain GSH-Px similar to those obtained from the basal synthetic diet supplemented with selenium, but did have higher brain Se levels (Table 4). This difference in brain Se does not appear to be related to the difference in sex of animals fed synthetic versus stock diets since both male (Table 1) and female rats (Table 4) fed the stock diet had similar Se values.

DISCUSSION

These experiments indicate that 1 month following injection of Na2"Se0, rat brain has retained a substantial portion of the 7sSe, largely associated with protein. Anatomical distribution (Table 1) also suggested an association with protein: the concn. of Se was higher in the cerebellum than in the medulla oblongata, similar to their relative protein content. This pattern of grey matter being higher than white matter in Se levels has been more thoroughly documented (HOCK t't al.. 1975) for human brain. TRAPP & MILLAM (1975) reported that the cerebellum accumulated more '>Se than the cerebral hemispheres when Na,"SeO, was injected into Se-deficient rats. FIG.4. Elution profile of rat brain cytosol fractionated on Our results for total selenium levels support this findSephadex G-150. (A) Ten ml of fresh supernatant fluid ing (Table I). The subcellular distribution also sugfrom a 25"; (wlv) homogenate (treated with Triton X-100) gested that in general "Se follows protein except that of a pool of 3 brains 30 days after injection of Na275SeOj. the "Se was relatively more enriched in the mitoThe column was 75 x 2.5 cm and 8 ml fractions were col- chondria and diluted in the cytosol (Table 2). lected. (B) Supernatant fluid. 7 ml, was prepared as a 25", The amount of 15Se found in the cytosol of brain homogenate with Triton X-100 from 3 frozen brains of was only half that found in the mitochondria and rats fed the Se-deficient diet 8 weeks. The column was 92 x 2.5 cm and 6.8 ml fractions were collected. ( C ) Cyto- only 1/1&1/5 of the total brain "Se (Table 2). sol. 10 ml. which was prepared in 0.32 M-sucrose and frozen BROW & BURK(1973) noted a similar dislribution for 5 days before thawing for chromatography. Conditions of 75Sei n testes, whereas in liver the cytosol had five are described in the legend to Fig. 2. Fractions were ana- times the "Se content of the mitochondria and half (-). GSH-Px (M), and '%e lvzed for the total liver "Se. These facts can be explained by (O---O) as described in Methods. the fact that rat liver has much more GSH-Px (a selenoprotein) than either testes or brain (HOEKSTRA, When rats were fed a diet low in Se for 13 weeks 1974) and 213 of the liver enzyme is found in the ef al. beginning at age 3 weeks. the blood Se and GSH-Px cytosol (GREEN& O'BRIEN. 1970). LEVANDER values were markedly reduced (Table 4) compared to (1974) characterized liver mitochondria1 15Se using TABLt

4 EFFECTOF

DIET OU SELtNILM LEVFLS A h D CLLTATHIOVE PEROXIDASE ACTIVITIES IN RAT B L 0 0 U AND BRAIN*

Blood Dietary selenium (pg'g) Basal Basal + Se Stock