Nutrient Requirements

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1 Nutrient Requirements Selenium from Selenium-Rich Spirulina Is Less Bioavailable than Selenium from Sodium Selenite and Selenomethionine in Selenium-Deficient Rats 1 Julien Cases, Véronique Vacchina,* Anthony Napolitano, Bertrand Caporiccio, Pierre Besançon, Ryszard Lobinski* and Jean-Max Rouanet 2 Unité Nutrition, Laboratoire Génie Biologique et Sciences des Aliments, Université Montpellier II, Montpellier, France and *Group of Bio-inorganic Analytical Chemistry, CNRS-UMR 5034, Hélioparc, Pau, France ABSTRACT The bioavailabilty of selenium (Se) from selenium-rich Spirulina (SeSp) was assessed in Se-deficient rats by measuring tissue Se accumulation and glutathione peroxidase (GSH-Px) activity. For 42 d, rats were subjected to dietary Se depletion by consumption of a Torula yeast (TY)-based diet with no Se; controls were fed the same diet supplemented with 75 g Se/kg diet as sodium selenite. Se-deficient rats were then repleted with Se (75 g/kg) by the addition of sodium selenite, selenomethionine (SeMet) or SeSp to the TY basal diet. Selenium speciation in SeSp emphasized the quasi-absence of selenite (2% of total Se); organic Se comprised SeMet ( 18%), with the majority present in the form of two selenoproteins (20 30 kda and 80 kda). Gross absorption of Se from SeSp was significantly lower than from free SeMet and sodium selenite. SeMet was less effective than sodium selenite in restoring Se concentration in the liver but not in kidney. SeSp was always much less effective. Similarly, Se from SeSp was less effective than the other forms of Se in restoring GSH-Px activity, except in plasma and red blood cells where no differences were noted among the three sources. This was confirmed by measuring the bioavailability of Se by slope-ratio analysis using selenite as the reference form of Se. Although Se from SeSp did not replenish Se concentration and GSH-Px activity in most tissues to the same degree as the other forms of Se, we conclude that it is biologically useful and differently metabolized due to its chemical form. J. Nutr. 131: , KEY WORDS: selenium Spirulina rats glutathione peroxidase Selenium (Se), an essential trace element for animals (1,2) and humans (3), is obtained mainly from cereal products, fish, poultry and meat, almost exclusively in organic compounds. At least 13 different selenoproteins have now been identified (4,5), but only glutathione peroxidase ( GSH-Px), 3 selenoprotein P and Type I iodothyronine 5 -deiodinase have been well characterized in animals. Thus, selenium plays a vital part in many metabolic functions as a key component of GSH-Px, which is involved in the removal of hydrogen peroxide and lipid peroxides generated in cells (6) during oxidative processes. The Se responsiveness of an endemic cardiomyopathy called Keshan disease, which is found in areas of China with low soil Se, emphasized the human essentiality of this element as recently as 1979 (7,8). A correlation between the lack of Se in food and different diseases such as cardiovascular disease (9), cancer (10,11), rheumatoid arthritis (12) and cataract (13) has been proposed. Although human Se deficiency has also been reported in patients subjected to long-term parenteral nutrition (14,15), Se deficiency in humans is relatively 1 Supported in part by the Région Languedoc Roussillon. 2 To whom correspondence should be addressed. rouanet@arpb. univ-montp2.fr. 3 Abbreviations used: GSH-Px, glutathione peroxidase; GSH-Px ec, extracellular GSH-Px; ICP-MS, inductively coupled plasma mass spectrometry; SeSp, Se-enriched Spirulina; TY, Torula yeast. rare and many populations with low Se intakes show no apparent ill-effects. The major forms of selenium occurring in foodstuffs are the organic, protein-associated forms, selenomethionine (SeMet, plant and animal sources) and selenocysteine (SeCys, animal sources). Selenate is also present in some foodstuffs (16), and in selenium-deficient areas, inorganic selenium salts (selenite, selenate) are added to the food (17). The activity of selenoproteins depends on an adequate selenium supply from the diet. Because Se enters the food chain through plants, its availability may vary with climatic and soil fertilization conditions. Moreover, Se in foodstuffs is not always available for intestinal absorption, and there are differences both between and within species (18 21). The metabolism of selenium varies according to the form of selenium ingested. It was demonstrated (22) that SeMet and selenate are more diffusible than selenocysteine and selenite under simulated gastrointestinal conditions, contributing to their high absorption in vivo. Moreover, reutilization of organic selenium is one of the most important differences in the metabolism of SeMet and selenite (23). It is very important to know the bioavailability of selenium present in the diet to establish nutritional Se status in relation to dietary intake and to intervene promptly in cases of Se deficiency. In the present study, we examined the bioavailabil /01 $ American Society for Nutritional Sciences. Manuscript received 29 January Initial review completed 20 February Revision accepted 23 May

2 2344 CASES ET AL. ity of Se from SeMet, sodium selenite (Na 2 SeO 3 ) and from enriched Spirulina platensis, a blue-green alga which is commercially available for human consumption, and now used as a health food source of Se for humans (24). We reported previously that Spirulina grown in the presence of selenium showed health benefits in rats fed high cholesterol diets (25). The ability to control its chemical composition by varying cultivation conditions makes Spirulina a vegetable that can easily be enriched with Se by way of the aquatic medium. This work investigated the ability of Se-enriched Spirulina (SeSp) to provide biologically available Se to rats. Experiments using Se-deficient rats were conducted to measure tissue uptake of the element and GSH-Px activity. MATERIALS AND METHODS Spirulina selenium fortification. This step was performed at Aquamer S.A. (Mèze, France). Algae (Spirulina platensis) were grown in a 130-L photobioreactor under continuous lighting on Zarouk s medium at 22 C and ph 10.5 in the presence of SeO 2. This medium contained NaHCO 3, 16.8 g/l; K 2 HPO 4, 0.5 g/l; NaNO 3, 2.5 g/l; K 2 SO 4, 1.0 g/l; NaCl, 1.0 g/l; MgSO 4 7H 2 O, 0.2 g/l; CaCl 2, 0.04 g/l; FeSO 4 7H 2 O, 0.01 g/l; EDTA, 0.08 g/l; H 3 BO 3, 2.86 mg/l; MnCl 2 4H 2 O, 1.81 mg/l; ZnSO 4 7H 2 O, 220 g/l; CuSO 4 5H 2 O, 79 g/l; MoO 3,15 g/l; and Na 2 MoO 4,21 g/l and was supplied with light aeration (30 L/min) and the addition of 0.03% CO 2. At the end of the growth, the biomass was recovered and filtered through a 20- m membrane, thoroughly washed with distilled water, frozen and lyophilized. Characterization of selenium speciation in fortified Spirulina. Selenium speciation was characterized by a sequential extraction with reagent solutions designed to selectively leach different classes of selenium species into an aqueous phase. The latter was subsequently characterized by size exclusion HPLC with Se-specific detection by inductively coupled plasma mass spectrometry (ICP-MS) (26). A 0.2-g sample was taken for analysis. It was extracted sequentially as follows: 1) 5 ml of hot water (85 90 C) by agitation for 1hfor TABLE 1 Composition of experimental diets Diet component Control diet Se-deficient diet g/kg Torula yeast2 (N 6.25) DL-Methionine 1 1 Cornstarch Sucrose Cellulose Corn oil Mineral mix3 40 Mineral mix4 40 Vitamin mix Total selenium, g/kg Metabolizable energy, kj/g All diets were formulated to contain 15% protein. 2 Torula yeast contained 7.42% N. 3 Mineral mixture contained (mg/kg diet): CaHPO 4, 17,200; KCl, 4000; NaCl, 4000; MgO, 420; MgSO 4, 2000; Fe 2 O 3, 120; FeSO 4 7H 2 O, 200; trace elements, 400 (Na 2 SeO 3, 0.165; MnSO 4 H 2 O, 98; CuSO 4 5H 2 O, 20; ZnSO 4 7H 2 O, 80; CoSO 4 7H 2 O, 0.16; KI, 0.32; sufficient starch to equal 40 g (per kg diet). 4 Mineral mix as described above and free from Na 2 SeO 3. 5 Vitamin mixture containing (mg/kg diet): retinol, 12; cholecalciferol, 0.125; thiamin, 40; riboflavin, 30; pantothenic acid, 140; pyridoxine, 20; inositol, 300; cyanocobalamin, 0.1; all-rac- -tocopherol, 340; menadione, 80; nicotinic acid, 200; choline, 2720; folic acid, 10; p-aminobenzoic acid, 100; biotin, 0.6; sufficient starch to equal 20 g (per kg diet). FIGURE 1 Speciation of selenium in Spirulina by size-exclusion HPLC-inductively coupled plasma mass spectrometry (ICP-MS) after sequential extraction. Extractions were with (A) hot water, (B) Driselase solution (mixture of laminarinase, xylanase and cellulase), (C) SDS solution and (D) lipase-pronase solution. Dotted lines mark the elution volumes of Se(VI), Se(IV) and selenomethionine standards. water-soluble selenospecies; 2) 5 ml of 4% Driselase in 30 mmol/l Tris-HCl buffer (ph 7.0) in the presence of 1 mmol/l phenylmethylsulfonyl fluoride by agitation for1hat25 C for selenium associated with cell wall; 3) 5 ml of 30 mmol/l Tris-HCl buffer (ph 7.0) containing 4% SDS by agitation for 1hat25 C for water-insoluble selenoproteins; and 4) 5 ml of phosphate buffer (ph 7.5) by incubation for 16 h at 37 C with a mixture containing 20 mg of Pronase and 10 mg of lipase to break the residual selenoproteins. Selenium was determined by ICP-MS in the supernatant remaining after the centrifugation. The final residue was dissolved completely in 2 ml of 250 g/l tetramethylammonium hydroxide in water to determine the residual Se. A 100- L aliquot of extract (10-fold diluted) was injected on a Superdex Peptide HR 10/30 column (Pharmacia, Uppsala, Sweden) and eluted at 0.75 ml/min with 30 mmol/l Tris-HCl buffer (ph 7.5). 78 Se, 80 Se and 82 Se were monitored on-line using an ELAN 6000 ICP MS spectrometer (PE-SCIEX, Thornhill, Canada). Animals and diets. Male Sprague-Dawley rats (Iffa Credo, L Arbresle, France) weighing 68 g were used. Rats were housed individually in stainless steel metabolic cages and had free access to deionized water. They were kept under light from 0700 to 1900 h, and the room temperature was 23 1 C, with constant humidity. Rats were fed a Torula yeast (TY)-based selenium-deficient diet through-

3 BIOAVAILABILITY OF SE FROM SPIRULINA 2345 TABLE 2 Body weight and food intake of rats during Se depletion and Se repletion with sodium selenite, Group Body weight, g Control a Se-deficient Selenite 306 4b SeMet 336 6a Spirulina 328 7a Food intake, g/d Control Se-deficient Selenite SeMet Spirulina out the 98-d experiment (Table 1). They were maintained by pairfeeding to the food intake of the group with the lowest intake over the experimental periods, i.e., each group received daily a quantity of food equal to that eaten by this group the day before. Rats were weighed weekly. On arrival and just before the beginning of the experimental period, five rats were selected at random and killed to establish baseline values for tissue GSH-Px activity and Se concentration. At the start of the experiment, rats (n 70) were divided into two dietary groups on the basis of average body weight. The control group (n 20) received the TY diet to which was added 75 g Se/kg as Na 2 SeO 3. This suboptimal level was chosen to generate nonplateauing GSH-Px activities (27). In fact, the use of the optimal Se level (100 g/kg) most likely would prevent detection of any metabolic differences that might have been detected with a level lower than the dietary Se requirement. The remaining rats (n 50) were fed the TY Se-deficient basal diet (Se-deficient dietary group) which contained 7 g Se/kg. After 42 d, five rats from each group were anesthetized by an intraperitoneal injection of pentobarbital, and tissues (liver, kidney and blood) were removed for Se bioassays and GSH-Px activity to ascertain that the Se-deficient diet had depleted Se. The repletion period lasted from d 43 to 98. The rats from the Se-deficient group were randomly divided into three groups of 15 and fed diets containing 75 g Se/kg as Na 2 SeO 3, SeMet or SeSp. Five rats of each group were killed at d 7, 28 and 56 of the repletion period. Five animals from the control group were also killed at the same times. Analytical procedures. Rats were handled according to NIH guidelines (28). They were deprived of food overnight and anesthetized with pentobarbital (60 g/l pentobarbital, 60 mg/kg body) before tissues were excised. The liver was perfused with 0.15 mol/l KCl to remove residual blood, rapidly excised, rinsed in ice-cold saline, blotted dry, weighed, sectioned for analyses and stored in liquid nitrogen. Kidneys were also removed, washed in ice-cold saline, blotted, weighed and frozen immediately in liquid nitrogen. Liver and kidney were homogenized in 5 volumes of ice-cold 0.1 mol/l potassium phosphate buffer (ph 7.4), and the homogenate was centrifuged at g for 15 min at 4 C. The supernatant was then centrifuged at g for 60 min at 4 C and the cytosol was stored at 80 C for subsequent assay of GSH-Px activity. Glutathione peroxidase activity was measured by the method of Wendel (29) using 0.2 mmol/l hydrogen peroxide as the substrate and including 1.0 mmol/l sodium azide to inhibit catalase; thus, only Se-dependent GSH-Px activity was measured. The cytosolic protein content was determined by a commercial protein assay (Sigma, Saint Quentin Fallavier, France) according to Smith et al. (30) and using bovine serum albumin as the standard. Blood was withdrawn by cardiac puncture and aliquots were transferred to heparinized tubes, then centrifuged at 1000 g for 10 min to separate plasma and erythrocytes. The erythrocytes obtained were washed with saline and hemolyzed in 9 volumes of hypotonic buffer (5 mmol/l sodium phosphate buffer, ph 7.0). Feces were collected from each rat during the last 5dofthe repletion period for intestinal absorption measurements. Selenium was analyzed by ICP-MS after samples were digested in nitric acid and hydrogen peroxide. Assessment of Se bioavailability. The bioavailability of Se from sodium selenite, SeMet and SeSp was assessed by using sodium selenite fed control rats as the reference. The deposition of Se and the increase in GSH-Px activity in different tissues were used as the responses to time of repletion (T). Because this response R can be described by the equation, R mt k, the relative bioavailability of Se from the three sources was estimated by the slope-ratio technique, which compares the slope of the time-response plots observed for SeMet and SeSp to that observed for sodium selenite. Statistical analyses. Data are shown as the means SEM, n 5. Data were subjected to logarithmic transformation where necessary to FIGURE 2 Percentage of dietary selenium absorbed by Se-deficient rats after the 56-d period of repletion with DL-selenomethionine (SeMet), sodium selenite (selenite) or Se-rich Spirulina. Values are means SEM, n 5. Bars with different index letters differ, P 0.05.

4 2346 CASES ET AL. TABLE 3 Liver total selenium concentration of rats during Se depletion and Se repletion with sodium selenite, nmol/g wet liver Control a a a b Se-deficient b Selenite b b a SeMet c c c Spirulina c d d achieve homogeneity of variances. Statistical analyses of data were performed by one-way ANOVA followed by Fisher s Protected Least Significant Difference post-hoc procedure using a StatView 512 microcomputer program (Brain Power, Calabasas, CA). Differences were considered significant when P RESULTS The concentration of Se in the fortified algae was g/g. The speciation of this selenium is characterized in Figure 1. Water-soluble selenium accounted for 23% of the total selenium. Selenite corresponded to only 10% of the watersoluble Se (2% of total Se), indicating that algae had metabolized the SeO 2 from the culture medium. Most of the watersoluble selenium was present as a macromolecule of kda. The attack with Driselase (an enzyme preparation containing laminarinase, xylanase and cellulase) liberated an additional 18% of Se (bound to cell walls) that eluted at the elution volume of selenomethionine. The majority of Se in Spirulina (40%) was present in the form of selenoproteins, which can be solubilized by a solution of SDS. Figure 1c shows that these proteins represent two molecular mass fractions, one close to the excluded volume of 80 kda and the other in the kda range. Leaching with SDS did not allow all of the selenoproteins present in the sample to solubilize. A proteolytic attack of the residue allowed recovery in the aqueous phase of an additional 12% present in the form of a macromolecular compound but also Se(IV) and selenomethionine. The remaining 7% of selenium was resistant to the above reagents. As a consequence of the feeding protocol, there was no difference in food intake among the dietary groups at 42 d of the experiment or at 7, 28 and 56 d of Se repletion (Table 2). Therefore, daily Se intakes were not different among groups; Se intake averaged 1.40 g/d. Body weights generally were not affected by dietary Se source during the repletion period (Table 2). Apparent absorption of Se was significantly less from SeSp (82%) than from sodium selenite (89%) and SeMet (90%), which did not differ (Fig. 2). Liver selenium concentration in Se-depleted rats was 6.3% of controls (Table 3). The repletion of liver selenium was greatest in rats fed sodium selenite, lower in rats fed SeMet and lowest in rats fed SeSp. Normal hepatic Se concentration was found only in the selenite group at the end of the experimental period (110% of controls). SeMet and SeSp did not result in normal hepatic Se concentration (73 and 53% of controls, respectively). Sodium selenite and SeMet did not differ in their ability to normalize kidney Se concentrations after 56 d (100 and 99% of controls, respectively; Table 4). SeSp did not restore kidney selenium (83% of controls). Kidney selenium concentration in depleted rats was only 47% of controls. Repletion of GSH-Px activity in liver cytosol was consistent with repletion of selenium concentration in this tissue (Table 5), i.e.,103% of the control value for sodium selenite, TABLE 4 Kidney total selenium concentration of rats during Se depletion and Se repletion with sodium selenite, nmol/g wet kidney Control a a a a Se-deficient b Selenite b b a SeMet c c a Spirulina c c b

5 BIOAVAILABILITY OF SE FROM SPIRULINA 2347 TABLE 5 Liver glutathione peroxidase activity of rats during Se depletion and Se repletion with sodium selenite, mol/(min mg protein) Control a a a a Se-deficient b Selenite b b b SeMet b c c Spirulina b d d 71% for SeMet and 50% for SeSp. Tissue Se repletion and GSH-Px activity recovery were correlated at d 56 (r 0.95, 0.97 and 0.96 for selenite, SeMet and SeSp, respectively). Correlation coefficients were lower in kidney than in liver (r 0.86, 0.67 and 0.67 for selenite, SeMet and SeSp, respectively); no Se form led to a full recovery of kidney cytosol GSH-Px activity (Table 6) and the repletions ranged from 87% of control for sodium selenite to 57% for SeSp. Erythrocyte enzyme activity (Table 7) was not restored at 56 d in any of the groups, which did not differ (they represented, on average, 79% of the controls). Similarly, extracellular GSH-Px activity (GSH-Px ec ) in plasma (Table 8) was not normalized (77 84% of controls) in any of the groups. Table 9 summarizes the results of regression analyses between the repletion time and either the tissue Se concentration or GSH-Px activities. Table 10 compares relative bioavailabilities of Se in SeMet and SeSp as assessed by comparing the regression slopes with that of Se in selenite. Both tissue Se concentration and GSH-Px activity data gave relative bioavailability measures 100% except kidney Se (SeMet) and plasma GSH-Px (SeSp). These data confirm those in Tables 3 8. DISCUSSION The bioavailability of Se in foods can be assessed in three the following different ways: 1) the prevention of Se deficiency, 2) the functional assay of GSH-Px activity and 3) tissue retention of Se (31). Because the second and third approaches are much more sensitive than the first, (32,33), tissue GSH-Px activity and Se concentration were employed as the criteria of bioavailability in this study. Selenium consumed in foods and supplements exists in a number of organic and inorganic forms, including selenomethionine (plant and animal sources and supplements), selenocysteine (SeCys) (mainly animal sources), selenite and selenate (mainly supplements). Bioavailability and tissue distribution depend on the form ingested. For instance, SeMet is more effective in increasing Se status because it is nonspecifically incorporated into proteins in place of methionine (34). It must, however, be catabolized to an inorganic precursor before entering the available Se pool. Selenite is a more available metabolic source of Se than SeMet because it needs only to be reduced to selenide to provide selenophosphate, the precursor of SeCys, which is the active form of Se in selenoproteins (35). Despite this, organic forms are often preferred in interventions, partly because they are less acutely toxic (36). However, the supplemental form of Se should not accumulate excessively in the body. This work shows that SeSp meets this criterion for a good supplemental form of Se because it does not replenish the Se pool to the same extent as sodium selenite, for example, and consequently it does not accumulate in the tissues studied (kidney, liver). It must be pointed out that SeMet as well as selenoproteins from Spirulina must be digested for intestinal absorption and subsequent metabolism; TABLE 6 Kidney glutathione peroxidase activity of rats during Se depletion and Se repletion with sodium selenite, mol/(min mg protein) Control a a a a Se-deficient b Selenite b b b SeMet b,c b c Spirulina c b d

6 2348 CASES ET AL. TABLE 7 Erythrocyte glutathione peroxidase activity of rats during Se depletion and Se repletion with sodium selenite, mol/(min mg protein) Control a a a a Se-deficient b Selenite b b b SeMet c c b Spirulina c c b selenite does not. The blue green algae Spirulina belongs to the cyanobacteria family; in this regard, it is closely related to bacteria, in which the Se metabolism incorporates SeCys into proteins; such a process also occurs in mammals (37). Elsewhere, we could consider that these microalgae belong too to the plant kingdom. This dual-property makes spirulina an ubiquitous material which could contain Se into forms identical to SeMet (plant and animal sources) and SeCys (animal sources) and probably other metabolites. In fact, selenium speciation in Spirulina seems to be distinctly different from that in selenized yeast and allium plants. Moreover, lowmolecular-mass compounds (amino acids and oligopeptides), which account for the majority of Se in yeast and garlic (38), are present only in small amounts in the microalgae investigated. Few previous studies feeding rats Se as high selenium garlic (in the form of seleno-methyl selenocysteine) (39) and high selenium broccoli (40), in the same chemical form as in garlic (41), reported that these organic forms of Se tended to incorporate less into most tissues than Se fed as SeMet. Our results are consistent with such findings, although Isp and Lisk (39) fed 3 mg Se/kg diet and Finley (40) refed rats with 100 g Se/kg diet for 63 d vs. 75 g Se/kg diet for 56 d in our study. Moreover, Finley used L-SeMet, whereas we chose DL-SeMet. The lower dietary Se level (75 g/kg diet) and reduced intestinal absorption of Se from SeSp could in part explain our results. Moreover, several forms of Se were present in these algae; SeMet accounted for a small part of Se, the majority of which was linked to cell walls and therefore not readily available to the body. It is likely that the effects observed were to a great extent due to the proteins detected (20 30 kda and 80 kda). Although kidney is the tissue in which Se is most concentrated (42), GSH-Px activity predominates in the liver (43). This difference would suggest that renal Se is used in activities other than GSH-Px activity. Selenium-dependent glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase have been shown to be very active in renal membranes (44). The selenoprotein type I iodothyronine 5 -nucleotidase is localized on the cytosolic surface of the renal cell basolateral plasma membrane (45); the plasma GSH-Px (i.e., GSH-Px ec ) is synthesized mainly in the proximal renal tubules and appears to be secreted through the tubule basolateral membrane (46). Because kidney is the site of synthesis for these selenoproteins, this could explain in part the high uptake of Se by this tissue and its need for a reserve at a site in which Se accumulation is not toxic for cells (47). Thus, kidney could serve as a storage site before the release of seleniated molecules into the bloodstream or as Se reserve for the body after glomerular filtration and reabsorption. Although Se from SeSp was the least effective in restoring kidney Se concentration and GSH-Px activity, the similar plasma GSH-Px ec activities obtained for all of the treatment groups after 56 d would suggest that this Se source is able to supply sufficient Se for this purpose and TABLE 8 Plasma glutathione peroxidase activity of rats during Se depletion and Se repletion with sodium selenite, mol/(min mg protein) Control a a a a Se-deficient b Selenite b b b SeMet c c b Spirulina c c b

7 BIOAVAILABILITY OF SE FROM SPIRULINA 2349 TABLE 9 Regression analyses between tissue selenium or glutathione peroxidase (GSH-Px) activities and time in Se-deficient rats repleted with sodium selenite, selenomethionine (SeMet) or selenium-rich spirulina (SeSp)1 Dependent variable Slope, m Constant, k Correlation coefficient, r Selenite SeMet SeSp Selenite SeMet SeSp Selenite SeMet SeSp Se concentration Liver Kidney GSH-Px activity Liver Kidney Erythrocytes Plasma Regression was fitted to the equation R mt k, where R represented tissue Se concentration or GSH-Px activity. Units of dependent variables were as in Tables 3 8. explain why renal Se is used in other activities than GSH-Px, a protein relatively low in the hierarchy of Se use. In this study we reported that Se from SeSp was less effective than organic and inorganic supplements in restoring GSH-Px activity and Se concentrations in several tissues and organs of Se-depleted rats. Plasma and erythrocyte GSH-Px activities were exceptions because no effect of a particular chemical form was observed, as has been reported (48). However, it must be pointed out that for each tissue studied, GSH-Px activity increased after refeeding with SeSp, although the increase was not as rapid as with other sources. Thus, Se from SeSp is less bioavailable than selenite and SeMet. As clearly stated by Finley et al. (49), bioavailability can be used synonymously with biological usefulness. Therefore, Se-enriched Spirulina represents a useful source of selenium. This work was devoted to Se bioavailability from Spirulina; to our knowledge, it is the first study relative to this microalgae and contributes novel data concerning this potentially beneficial Se supplement. The primary public impetus for Se supplementation is the potential benefit for reduction of cancer. Ip and co-workers (39,50 52) as well as the study by Finley et al. (49) of colon cancer and high selenium broccoli demonstrated that cancer reduction is not a function of tissue selenium concentrations or GSH-Px activities and that the least bioavailable Se compounds may exhibit potent biological activities. Thus, the anticancer effect of a Se-containing compound may be more closely related to its ability to produce TABLE 10 Relative biological availability of selenium contained in selenomethionine (SeMet) and Se-rich spirulina (SeSp) in rats1 Parameter Selenomethionine Spirulina Se concentration Liver Kidney GSH-Px activity Liver Kidney Erythrocytes Plasma Relative biological availability was estimated by using the slope of the regression line described in Table 9; (slope of SeMet or SeSp)/ (slope of selenite) 100. GSH-Px, glutathione peroxidase. antitumorigenic metabolites, such as methyl selenol (52). It is very important, therefore, to define more precisely the forms of Se present in Se-Spirulina to permit identification of the most active anticarcinogenic component(s) and to recommend such a source as a Se supplement. Before such a study can be conducted, due to the several forms of selenium in Spirulina, fractionation of the algae is in progress currently to identify more clearly the chemical forms of Se in the issuing fractions. ACKNOWLEDGMENTS We would like to thank Bruno Baroux (Aquamer S.A.) for the selenium fortification of Spirulina and Jean-Claude Baccou (Plant Physiology and Technology Unit) for his help. LITERATURE CITED 1. Schwarz, K. & Foltz, C. M. (1957) Selenium as an integral part of factor 3 against dietary necrotic liver degeneration. J. Am. Chem. Soc. 79: You-Ping, Z. & Combs, G. F., Jr. (1984) Effect of dietary protein level and level of food intake on the apparent bioavailability of selenium for the chick. Poult. 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