9 1992 by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/92/3301-3--0103 $02.00

Effects of Glutathione and of Cysteine on Intestinal Absorption of Selenium from Selenite E S T H E R SENN, ERWlN SCHARRER,* AND SIEGFRIED WOLFFRAM

Institute of Veterinary Physiology, University of ZiJrich, Winterthurerstrasse 260, CH-8057 ZiJrich, Switzerland Received July 10, 1991; Accepted August 4, 1991

ABSTRACT The influence of gtutathione (1 mmol/L) (GSH) on in vitro mucosal uptake and in vivo absorption of 75Se-labeled selenite (10 ~mol/ L) was investigated in rat jejunum. For comparison, the effect of L-cysteine (1 mmol/L) on in vivo absorption of 75Se-labeled selenite was also studied. In the in vitro uptake experiments, only the mucosal surface was exposed to the incubation medium for 3 min. For the in vivo experiments, a luminal perfusion technique was employed. GSH inhibited in vitro mucosal Se uptake, whereas absorption in vivo was stimulated by GSH. L-Cysteine also stimulated in vivo Se absorption, confirming former in vitro mucosal uptake experiments. Thus, unlike L-cysteine, GSH affected in vitro and in vivo absorption of Se from selenite differently. Enzymatic cleavage of products of the reaction of selenite with GSH occurring more efficiently under in vivo than in vitro conditions may be a prerequisite for the stimulatory effect of GSH on Se absorption. This apparently does not apply to the stimulatory effect of cysteine. Since GSH occurs in the intestinal lumen under physiological conditions, it may contribute to the high bioavailability of Se from selenite. Index Entries: Absorption of selenite in rat jejunum; selenium salts and influence of glutathione on intestinal absorption of selenium; effect of L-cysteine on intestinal selenium absorption; in vivo and in vitro absorption of selenite. *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research

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INTRODUCTION In former work, we have shown that cysteine stimulates mucosal uptake of Se from selenite across the brush border in rat and sheep jejunum (1,2). Since glutathione is present in the lumen of the small intestine under physiological conditions (3), we now have studied whether glutathione affects absorption of Se from selenite in rat jejunum. Both an in vitro mucosal uptake method and an in vivo luminal perfusion technique were employed. The effect of L-cysteine on in vivo absorption of Se from selenite was investigated for comparison.

MATERIALS AND METHODS

Animals Adult male SIVZ 50 rats (Institut ffir Labortierkunde, Universit/it Zfirich) with a mean body wt of 370 g were used for the experiments. The animals were fed a commercial rat diet ad libitum (diet No. 890, NAFAG, Gossau, Switzerland).

#lucosal Uptake Technique (1) The jejunum was removed under ether anesthesia. It was transferred immediately into cold oxygenated Krebs-Henseleit bicarbonate buffer, opened along the longitudinal axis, and cleaned. Twenty centimeters of the proximal, mid~ and distal jejunum were cut into small pieces. For measuring mucosal uptake of 75Se across the brush border, the pieces of proximal, mid-, and distal jejunum were mounted on plastic stoppers so that only the mucosal surface was exposed to the incubation medium (1). After preincubation of the preparations in oxygenated (O2/CO2 = 95/5) Krebs-Henseleit bicarbonate buffer for 10-15 rnin, the preparations were incubated in 100-mL centrifuge tubes containing 25 mL Krebs-Henseleit bicarbonate buffer with 10 i~mol/L 75Se-labeled SeO 2- (Arnersham Int. Plc., England), and with or without 1 mmol/L glutathione. For part of the experiments, Na-free Krebs-Henseleit buffer was used (NaC1 and NaHCO 3 were replaced by choline chloride and KHCO3, respectively). The tubes were gassed with O2/CO 2 (95/5), closed with rubber stoppers, and agitated in an incubator (150 oscillations/min, 37~ 3 min). In order to correct for radioactivity adhering outside the intestinal cells, some preparations were incubated without labeled SeO 2in the presence of 0.2 rnmol/L 3H-labeled polyethyleneglycol 4000 (NEN, Boston). 7SSe activity of the samples was measured in a ~/-counter. 3H activity was determined by liquid scintillation counting following solubil~ ization of samples with a tissue solubilizer (Soluene, Canberra-Packard S. A., Z~irich, Switzerland). Biological Trace Element Research

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Luminal Perfusion Technique The distal jejunum (length of ligated segments: about 5 cm) of anesthetized rats was perfused with Krebs-Henseleit bicarbonate buffer (0.5 mL/min) containing 10 ~mol/L 75Se-labeled selenite, or in addition, 1 mmol/L glutathione or L-cysteine. The animals were anesthetized by intramuscular injection of xylazin-hydrochloride (11.7 mg/kg, Bayer AG, Leverkusen) and ketamine-hydrochloride (50 mg/kg, Parke Davis & Co., Berlin). The perfused solution contained phenolred as an unabsorbable marker (4). Absorption of Se was determined during a 30-min period following a 45-rain equilibration period from the luminal disappearance of 75Se. Se absorption was related to 100 mg intestinal dry wt. In one series of experiments, the distal jejunum was simultaneously perfused with 0.5 mL/min fluid and 0.5 mL/min air (= segmental perfusion). This procedure has been shown to stimulate absorption of several substrates owing to the reduction of the unstirred layer adjacent to the mucosal surface by intensive stirring of the fluid (5). Statistical Analysis The results are presented as means with the standard error of the mean (2 + SEM). Group differences were statistically evaluated with the Mann-Whitney U-test (n < 10) or the t-test (n > 10).

RESULTS As shown in Fig. 1, glutathione (1 mmol/L) inhibited in vitro mucosal uptake of Se from selenite (10 ~mol/L) in all three jejunal segments tested. Furthermore, in the distal jejunum, the inhibitory effect of glutathione was similar in the presence (inhibition: 46%, n = 28, p < 0.001) and absence of Na (inhibition: 53%, n = 12, p < 0.01). In the respective series of experiments, the absence of Na did not significantly affect rnucosal Se uptake. Figure 2 shows that, unlike in the in vitro experiments, glutathione clearly stimulated in vivo absorption of Se from selenite in the luminally perfused distal jejunum. L-Cysteine also enhanced in vivo Se absorption (Fig. 2), confirming previous in vitro observations (2). The in vivo effect of c-cysteine appeared to be greater than that of glutathione, but the difference was not statistically significant (Fig. 2). Segmental perfusion (0.5 mL/min fluid and 0.5 mL/min air) did not influence Se absorption in the absence or presence of c-cysteine (Fig. 2). Therefore, an unstirred layer effect apparently did not occur.

DISCOSSION The results show that glutathione (GSH) stimulates in vivo absorption of Se from selenite, whereas in vitro GSH inhibited mucosal uptake Biological Trace Element Research

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Fig. 1. Influence of glutathione (1 mmol/L) on in vitro mucosal Se uptake from selenite (10 ~mol/L) in the proximal, mid-, and distal jejunum. The number of preparations is in parentheses. Values are means + SEM. * ***Significantly different from controls (*p < 0.05, ***p < 0.001). [] Selenite ( = control); ~ selenite + GSH. of Se from selenite across the jejunal brush border. Unlike GSH, cysteine enhanced in vivo absorption and in vitro mucosal uptake of 75Se from selenite similarly. The pertinent in vitro effect of cysteine has already been reported (2). Since selenodicysteine, cysteine selenopersulfide, selenide, and elemental selenium are produced by the spontaneous reaction of selenite with cysteine (6,7), the stimulatory effect of cysteine on in vivo and in vitro absorption of Se from selenite appears to be the result of rapid absorption of some of these compounds. In particular, absorption of the selenoamino acids, deriving from this reaction, by the amino acid carriers of the brush border membrane seems to contribute to the stimulatory effect of L-cysteine, because some nonthiol L-amino acids competitively diminished the in vitro effect of L-cysteine (1,2). There is some evidence that rapid absorption of selenide might also contribute to cysteine's stimulatory action on selenite absorption (2,8). At first glance, the inhibitory effect of GSH on in vitro mucosal uptake of Se from selenite (Fig. 1) is surprising because, in analogy to the reaction of cysteine with selenite, selenodiglutathione (GSSeSG), glutathione selenopersulfide (GSSeH), elemental selenium, and selenide are produced by the reaction of GSH with selenite (7). However, the inhibition could be easily explained if the Se derivatives of GSH (GSSeSG, GSSeH) could not pass the jejunal brush border membrane. During luminal perfusion of the jejunum, GSSeSG and GSSeH might be hydroBiological Trace Element Research

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Fig. 2. Influence of glutathione (1 mmol/L) and L-cysteine (1 retool/L) on in vivo absorption of Se from selenite (10 ~mol/L) by luminally perfused segments of distal jejunum. The number of experiments is in parentheses. Values are means + SEM. * ** Significantly different from controls (* p < 0.05, ** p < 0.01) Continuous perfusion: 0.5 mL fluid/rain; segmental perfusion: 0.5 mL fluid and 0.5 mL air/min (segmental perfusion has been shown to reduce the unstirred layer adjacent to the epithelium). [] Selenite (= control); ~ selenite + GSH; '~:" ~-~ selemte + cysteine. lyzed to a great extent by the mucosal enzymes catalyzing cleavage of the peptide bonds of GSSG and GSH (9) because of a relatively long exposure to a large intestinal surface compared to the in vitro mucosal uptake experiments. Selenodicysteine and cysteine selenopersulfide released in this way might then be rapidly absorbed by the amino acid carriers of the jejunal epithelium (1,2). This could be an explanation for the stimulatory effect of GSH on in vivo absorption of Se from selenite. It is also possible that GSSeSG formed from GSH and selenite is transformed in the intestinal lumen via GSSeH and GSH to selenide (3,7), which seems to be efficiently absorbed from the gastrointestinal tract (10). Since elemental Se being formed in the course of this reaction (7) is poorly absorbed (10), it cannot be involved in the stimulatory effect of GSH on in vivo selenite absorption. In the in vitro mucosal uptake experiments, small pieces of jejunum (0.45 cm 2) were incubated for a short time (3 min) in a relatively large volume of oxygenated incubation medium (25 mL). Therefore, the ratio between tissue mass and the amount of substrate present in the incubation was certainly unfavorable for cleavage of GSSeSG and GSSeH by mucosal enzymes, which is probably a prerequisite for effective absorpBiological Trace Element Research

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tion. This can explain the inhibitory effect of GSH on in vitro mucosal uptake of Se from selenite, which is not in accord with experiments of others (11) employing isolated enterocytes. In this study, GSH slightly stimulated intracellular uptake of Se from selenite. The discrepancy between these and the present in vitro findings may be owing to a more efficient enzymatic cleavage of GSSeSG by the isolated enterocytes because of a more favorable relationship between membrane surface and amount of substrates present in the incubation medium. Since the results obtained under in vivo conditions are closer to the physiological situation than those obtained in vitro, the in vivo findings may reflect physiological conditions at the GSH concentration (1 mmol/L) tested. GSH concentrations of up to 1 mmol/L have been reported for the liquid phase of the intestinal contents (3). Thus, in addition to cysteine released by protein digestion, glutathione ingested with food or entering the small intestine through bile (3,12,13) might increase bioavailability of Se from selenite and, thus, may contribute to the relatively high bioavailability of selenite (14).

ACKNOWLED

NT

This work was supported by the Swiss National Foundation (Grant No. 3.931-0.88).

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