9 1992 by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/92/3502~105 $02.80
Selenium Metabolism and Glutathione Peroxidase Activity in Cultured Human Lyrnphoblasts Effects of Transsulfuration Defects and Pyridoxai Phosphate M. A. BEILSTEIN AND P. D. WHANGER Department of Agricultural Chemistry, Oregon State University, Corvallis, OR 97331 Received September 16, 1991; Accepted January 10, 1992
ABSTRACT The metabolism of selenite, selenocysteine (SeCys), and selenomethionine (SeMet) was studied in three human lymphoblast cell lines with defects in the transsulfuration pathway and in control cells without this defect. There were very little differences in the induction of glutathione peroxidase (GPX) activity by selenite and SeCys among these cells. However, markedly higher levels of SeMet were required to induce GPX activity in transsulfuration defective cells than in control cells. Surprisingly, the addition of pyridoxal phosphate (PLP) to the media resulted in elevated GPX activity in all cells regardless of the chemical form of Se used. There is no explanation for this effect of PLP, but it is not through direct reaction with GPX or on the alteration of sulfhydryl groups. Index Entries- Transsulfuration; human lymphoblasts; pyridoxal phosphate; selenite; selenocysteine; selenomethione.
INTRODUCTION S e l e n o m e t h i o n i n e (SeMet) is biologically available as a source of selenium (Se) for incorporation as selenocysteine (fieCys) in glutathione
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Beilstein and Whanger
106 Methionine
"CH3
~
.CH 3
Homoc~'steine Cystathionine Synthase
~
_
Serine
Cystathionine Cystathionine
Lyase
1
Cysteine Fig. 1. Transsulfurationpathway for the conversion of methionine to cysteine.
peroxidase (GPX) (1). However, the intermediates of SeMet metabolism are not completely known. The chemical similarity of methionine and its Se analog, SeMet, may suggest involvement of the transsulfuration pathway for SeMet metabolism. SeMet and its metabolites are substrates for all steps of the transsulfuration pathway (Fig. 1) (2,3). Synthesis of selenocysteine (SeCys) by this route or degradation of a transsulfuration intermediate, such as selenohomocysteine, could produce a precursor form of Se for incorporation in GPX. Despite the in vitro demonstration of SeMet metabolism through the transsulfuration route, the in vivo importance of this pathway for SeMet utilization is uncertain. Two indirect approaches have been used to examine the possible role of the transsulfuration pathway in SeMet utilization. The effects of vitamin B 6 deficiency have been investigated (4,5), since two enzymes of this pathway, cystathionine synthetase and cystathionine lyase, require pyridoxal 5'-phosphate (PLP) as a cofactor (6). Accumulation of homocystine (7) and/or cystathionine (8) occurs in B 6 deficiency. Results from our laboratory (9) indicated a definite inhibition of SeMet utilization in B6-deficient rats. A second approach has been to examine Se status of individuals with genetic deficiencies of transsulfuration enzymes (10). Studies in our laboratory suggested that SeMet utilization for GPX synthesis was enhanced by transsulfuration deficiency and reduced by a Biological Trace Element Research
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Se Metabolism by Lymphoblasts
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defect (of homocysteine remethylation) that tends to force methionine through the transsulfuration route. The results of such studies are less than conclusive because of incomplete knowledge of dietary history, and especially dietary forms of Se and possible effects of therapy on Se utilization. Use of cultured mutant cells allowed direct assessment of the role of transsulfuration in Se utilization, since most environmental and nutritional factors can be controlled and differences in Se metabolism could be attributed to the presence or absence of the enzymes of concern. Studies of the effect of PLP on Se metabolism in cultured lymphoblasts were conducted because of a possible effect of vitamin B 6 status on transsulfuration activity of the mutant cells. The unexpected results of those studies led to further studies of a possible direct chemical interaction between selenite and PLP.
METHODS Mutant Cell Idnes All cell lines were obtained from the NIGMS Human Genetic Mutant Cell Repository, Coriell Institute for Medical Research, Camden, NJ. Genetically deficient cell lines used were GM 1781 (cystathionine lyase deficient, responsive to vitamin B6 therapy), GM 1566 (cystathionine lyase deficient, B6 response unknown), and GM 1532 (cystathionine synthetase deficient, B 6 response unknown). The apparently normal cell line, GM 3299, was used as a control.
Cell Growth and Maintenance The cells were maintained in RPMI 1640 media (11) supplemented with 10% fetal bovine serum (Flow Laboratories, Inc., McLean, VA) and a commercial serum extender (SerXtend, NEN Research Products, Wilmington, DE). All fetal bovine serum used was from a single lot selected for low-Se concentration as determined in a previous survey of Se in commercial fetal bovine serum (12). The SerXtend was specifically formulated with deletion of sodium selenite. Final concentration of Se in the supplemented media was 1.3 ppb (16 nM) as determined by fluorometric analysis (13) after nitric and perchloric acid digestion. The vitamin B 6 of RPMI 1640 media is 1 ng/L as pyridoxine 9 Hcl. Final vitamin B 6 content of the complete media with 10% serum was not determined, but typical serum vitamin B 6 concentration is 30 ~g/L. Gentamicin sulfate at 0.10 mg/ mL, penicillin at 100 U/mL, streptomycin at 0.1 mg/mL, and Fungizone at 0.25 p~g/mL (Whittaker M.A. Bioproducts, Walkersville, MD) were added to the media. Cells were grown in suspension in unstirred culture at 37~ in a 95: 5 air:CO2 atmosphere saturated with water. Cell densities were maintained between 0.2 and 1.5 x 106/mL as determined by Coulter counter Biological Trace Element Research
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Beilstein and Whanger
(Coulter Electronics, Inc., Hialeah, FL). Doubling times of cells varied from 24 to 48 h depending on cell density.
GPX Response to Se Supplementation Cells were seeded at 3 x 100 (approx 170 ~g cell protein) per 10 mL of media. Se supplements were added in buffered saline in vol of 0-100 p,L to duplicate flasks of cells at Se concentrations of 0, 0.01, 0.05, 0.10, 0.50, 1.0, 5.0, 10.0, and 50.0 ~M. Se compounds tested were sodium selenite, dl-SeCys, and dl-SeMet (Sigma Chemical Co., St. Louis, MO). Because of a difference in GPX response and toxicity to SeMet, the SeMet trials were repeated with triplicate flasks supplemented at 0, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, and 100 p~M. The cells were incubated in unstirred culture for 5 d. Final cell numbers were not determined in all experiments, but the typical cell number was between 11 and 14 x 106 (600 to 800 b~g cell protein) per flask when cells were harvested after 5 d. Cells were harvested by centrifugation, washed in buffered saline, and suspended in approx 1.0 mL ice-cold water. Cell suspensions were kept on ice until GPX assays were performed within 2 h of harvesting. Duplicate or triplicate assays were performed on each harvested flask of cells. Assays were performed by the method of Paglia and Valentine (14) at 30~ with initial concentrations of 5 mM reduced glutathione and 0.17 mM hydrogen peroxide. Several assays were performed with sonicated cell suspensions to test whether suspension in cold water was adequate to lyse the cells completely. No increase in GPX activity was detectable in sonicated cells. Cell protein was determined by the Lowry assay (15) on duplicate aliquots from each flask of cells. GPX activities expressed as nmol NADPH oxidized/min were normalized to cell protein. Concentrations required to induce one-half maximum GPX activity (EC 50) were calculated for the different Se compounds using a linear regression of the logit function (16) of the increase in enzyme activity vs the log Se concentration. Logit is defined as log[Amax - A)/A], where A equals GPX activity. The intercept of the log Se concentration vs logit least squares regression line is equal to log EC50. Effect of PLP on Se Induction of GPX Each ceil type was seeded in triplicate at 3 x 106/10 mL complete media with or without Se compounds added at the calculated EC 50 and with (60 ~g/mL) or without PLP. Additional triplicate flasks of each cell type were supplemented with 0.5 bLM sodium selenite representing a positive control of 100% of inducible GPX activity, without Se toxicity. After 5 d incubation, cells were harvested and assayed for GPX activity as described for the GPX response curves. Enzyme activities normalized to protein were converted to percent of maximum increase over basal (no added Se or PLP) activities. Biological Trace Element Research
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Se Metabolism by Lymphoblasts
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Uptake of 75Se Compounds Each cell type was seeded at 4 x 106/10 mL complete media with 755e as either SeMet or selenite (Amersham Corp., Arlington Heights, IL) plus carrier Se c o m p o u n d at a final concentration of 0.1 p,M Se (0.5 ~Ci/10 mL). Duplicate flasks were harvested at 4, 12, 24, and 48 h after addition of 75Se compounds. Harvested cells were washed once with buffered saline. Radioactivity was determined for each flask of harvested cells using a gamma scintillation counter (Gamma 8000, Beckman Instruments, Inc., Palo Alto, CA). Se uptake in pmoles was calculated by comparison with radioactivity of aliquots of the stock-labeling solutions. Molar uptake of Se at each time-point was normalized to cell protein. Initial rates of uptake were calculated from linear regression of molar Se uptake vs time for the 4- and 12-hour cell samples, including a theoretical 0 S e a t t = 0 ( n = 5).
Effect of PLP on Se Uptake Control lymphoblasts (GM 3299) were seeded at 107/10 mL complete media in triplicate flasks with or without added 60 b~g/mL PLP and with radiolabeled sodium selenite or SeMet plus carrier at 0.1 b~M Se. Cells were harvested after 24 h, and Se uptake determined and normalized to protein as described above.
Chemical Reaction of Selenite and Pyridoxal 5'-Phosphate Visible UV spectral scans from 450 to 200 n m were performed on 0.2 mM PLP solutions in pH 7.0, 0.125M phosphate buffer after addition of sodium selenite at 0, 0.02, 0.04, 0.05, 0.06, 0.10, 0.20, and 0.40 mM final concentration. Control spectra of sodium selenite solutions without PLP were obtained for comparison. The scans were performed on a Beckman DU 64 (Palo Alto, CA) spectrophotometer. Changes in the spectra would have indicated a change in oxidation state of the PLP. Radiolabeled sodium selenite with carrier at 0.20 mM was combined with PLP at 0, 0.1, 0.2, 1.0, and 2.0 mM in 2.0 mL unbuffered aqueous solution. After incubation for 15 min at room temperature, 1.0 mL of 5 mg/mL 2,3-diaminonaphthalene (DAN) in 0.1 N HC1 was added to each trial solution of sodium selenite. After a further incubation for 15 min, each trial was extracted three times with 1.0 mL of cyclohexane. Radioactivity of both the aqueous solutions and the organic extracts was determined. 75Se extracted into the organic phase was considered to be as selenite (/7), whereas radioactivity in the aqueous phase was assumed to be in some other oxidation state. To verify the method, identical assays were conducted with reduced glutathione substituted for PLP at the same concentrations. Biological Trace Element Research
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Beilstein and Whanger
Pjm'doxal 5'-Phosphate Effect on Activity of Purified Glutathione Peroxidase A stock solution of purified bovine erythrocyte GPX obtained from Albrecht Wendel (University of Konstanz, Konstanz, Germany) was prepared at 100 ~g/mL in 0.125 M phosphate buffer, pH 7.0. PLP was added to duplicate aliquots at 0, 50, and 100 ~M. The highest PLP concentration represents an approx 90-fold molar excess of PLP over enzyme. GPX enzyme activity of the stock solutions was determined immediately after addition of PLP, and subsequently after 15 and 30 min and after 2, 3, and 20 h of incubation at 37~
Data Analysis The data were subjected to statistical analyses using linear regression analyses, estimations of variance, determination of confidence intervals, and comparisons of means by the Student's t-test (18).
RESULTS Maximum inducible GPX activity on Se supplementation was approx 90 EU/mg cell protein for all cell types examined. The concentrations of selenite or SeCys required to achieve half-maximum GPX varied between the four cell types (Table I and Fig. 2). This variation was less for selenite than for SeCys. The EC 50 for the GM 1566 cell type was over fourfold that for the GM 1781 of the control. Concentrations of SeMet required to induce half-maximum enzyme activity were significantly higher compared to selenite or SeCys, and much higher for the mutant cells as compared to controls. Addition of PLP to media significantly increased GPX activity for all cell types and for all forms of Se added (Table 2). There was even stimulation of activity in control cells with no added Se. This stimulation of GPX activity by PLP was not influenced by the form of Se added to the media. Initial rates of cellular incorporation of 75Selabel as SeMet or selenite were not apparently different between cell types (Table 3). However, rates of Se uptake and 48-h Se incorporation were from four to eight times higher for SeMet compared to selenite. Addition of PLP to media approximately doubled the 24-h cellular incorporation of Se from selenite for control lymphoblasts (Table 4). In contrast, SeMet uptake was reduced by supplemental PLP. No evidence was obtained of a direct interaction of selenite with PLP (Fig. 3). Ultraviolet-visible spectra of mixtures of selenite and PLP were equivalent to the sum of the spectra of the two separate compounds. After incubation with PLP, selenite was still extracted with DAN (Table 5). However, this extraction of selenite was reduced in proportion Biological Trace Element Research
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Se Metabolism by Lymphoblasts
111
Table 1 EC 50* (nM) for Se Induction of Glutathione Peroxidase Activity Cell line GM 3299 (Control) GM 1532 (Synthase def.) GM 1781 (Lyase def.) GM 1566 (Lyase def.)
Selenium source SeCys 18 (9-36) 36 (25-52) 18 (14-23) 87 (55-104)
Na2SeO 3
32 (24 42) 21 (16-27) 25 (18-35) 20 (12-33)
SeMet 1580 (1340-1870) 6750 (5920-7700) 11,690 (9510-14,380) 11,980 (10,610-13,520)
*See Methods section for definition of EC 50. (This is calculated on the amount of selenium added and does not include the 16 nM selenium in the basal media.) Values in parentheses represent the 90% confidence limits.
80 -GM 3 2 9 9
801- GM 1789
(Control)
9
(- Lyose)
|
:
f ~ 40
!
" !
I
I
I0 -4 I0 "7
10 - 5
:~ ~.~ X EL (_9
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!
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9
40
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I
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Selenium
=
L
I
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concentration,
I
I0 -6
I0 "5
I
10-4
M
Fig. 2. Response of glutathione peroxidase activity to SeMet supplementation. Arrows indicate EC 50 calculated from logit function. (See Methods Section.) to the concentration of added reduced glutathione. The incubation of selenite with reduced glutathione apparently produces selenodiglutathione, in which the 75Se cannot be extracted by DAN. Incubation of purified bovine erythrocyte GPX with PLP had no marked effect on activity (Fig. 4). PLP slightly accelerated the slow loss of enzyme activity during incubation at 37~ Biological Trace Element Research
Vol. 35, 1992
112
Beilstein and Whanger
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Vol. 35, 1992
Se /Vletabolism by Lyrnphoblasts
113
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VoL 39, 1992
Be#stein and Whanger
114
Table 4 Effect of Pyridoxal Phosphate on Selenium Uptake* by Control Lymphoblasts (GM3299) 0 PLP 60 p~g/mL PLP
Selenite
SeMet
11.6 + 0.6 26.8 ___ 2.5
185 + 8 146 _+ 7
*pmol/mg cell protein after 24-h i n c u b a t i o n + SD, n = 3. Effect of PLP o n Se u p t a k e was significant (p < 0.01) for both forms of Se.
A
B
PLP+
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1.2
).8 U) r
w 2.(} D
-6 .o
1.0
0.4
0
I..p 0
IC2oo
ZOO
24!
;)20
' 240
200
220
240
Wavelength (nm)
Fig. 3. Ultraviolet spectra of Pyridoxal Phosphate (PLP) and sodium selenite as determined in 0.125M phosphate buffer, pH 7.0. Panel A shows superimposed spectra of 0.2 mM PLP and 0.2 mM PLP plus 1.0 mM sodium selenite. Inset in panel A shows difference between these two spectra. Panel B shows spectrum of 2.0 mM sodium selenite. The PLP absorbance peak at 388 nm (not shown) was unchanged by selenite addition.
DISCUSSION The similarity of r e s p o n s e in GPX activity to s u p p l e m e n t a t i o n with selenite or SeCys (Table 1) indicates that the four l y m p h o b l a s t lines examined h a d similar ability to utilize these forms of Se. In contrast, utilization of SeMET for GPX i n d u c t i o n was m a r k e d l y lower for the transsulfuration-deficient cells in c o m p a r i s o n to the controls (Fig. 2 a n d Table 1). The variance in utilization of SeMet was not d u e to differences Biological Trace Element Research
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Se Metabolism by Lymphoblasts
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Table 5 Complexation of Selenite--Pyridoxal Phosphate or Reduced Glutathione Reactions Products by 2,3-Diaminonaphthalene PLP added, mM
% 75Se complexed
0 0.1 0.2 1.0 2.0
95 96 95 94 94
Glutathione added, mM
+ 2 + 1 ___ 2 _+ 2 + 3
0 0.1 0.2 1.0 2.0
% 7SSe complexed 96 95 89 25 1
+ 1 + 2 _+ 3 + 5 + 0.5
Each value represents mean + SE of three samples. A level of 0.2 nM selenite was used in all complexation studies.
120
80
%.
.--.%
\
~_ 4 0 (_9
0
I
I
I!
60
120
180
I
I
1200
Time (rain) Fig. 4. Changes in activity of purified bovine glutathione peroxidase during incubation with and without pyridoxal phosphate at 37~
in rates of SeMet incorporation into cells, since similar rates of uptake were found for all cell lines (Table 3). Deficiency of cystathionine synthetase activity (homocystinuria) results in elevated physiological levels of both homocystine and methionine through remethylation of accumulated homocysteine (19). SeMet and methionine compete for the same transport mechanism in intestine (20), and competition for uptake has been demonstrated in other cell culture systems (21). Competition with methionine for transport w o u l d be expected to have a greater effect on SeMet uptake in the cystathionine-synthetase-deficient cells than in either the controls or the cystathionine-lyase-deficient cells, because cysBiological Trace Element Research
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Be#stein and Whanger
tathionine synthetase deficiency does not result in accumulation of methionine. However, no such effect was observed in either initial rates of SeMet uptake or in SeMet accumulation after 48 h incubation (Table 3). In humans with transsulfuration defects (10), the major effect on Se utilization is possibly through competition of SeMet with methionine for protein synthesis. Defects that inhibit transsulfuration, thus elevating physiological methionine levels, result in lower levels of Se in the nonSe-requiring protein, hemoglobin, and result in increased plasma and erythrocyte GPX activity (10). Deficiency of either cystathionine synthetase or lyase resulted in reduced induction of GPX by SeMet, and had no effect on SeMet accumulation in cells. The effects of PLP supplementation were examined because transsulfuration defects are often the result of reduced affinity of the enzyme for this cofactor. In such cases, supplementation with pyridoxine at 50 to 100 times the vitamin B6 RDA is effective in alleviating the deficiency (19). Vitamin B6 was provided to the lymphoblasts as PEP rather than as pyridoxine because of uncertainty concerning the capacity of lymphoblasts to generate the cofactor. The vitamin Bo response of the patientsource of the lymphoblasts was known only for the cystathionine-lyasedeficient cell line, GM 1781. An increased GPX response to SeMet in the presence of elevated PLP was sought in this cell line as verification that correcting the enzyme deficiency also normalized SeMet utilization. Such a response was obtained, but a similar response was also observed for the control cell line, GM 3299, and the other mutant cell lines (Table 2). We have no explanation for the increase in GPX activity with selenite and SeCys supplementation with added PLP. Assays of reduced and oxidized glutathione in both media and cells revealed no effect of incubation with PEP (data not shown). The possibility of a direct chemical reaction of PLP with selenite was examined through both spectroscopy (Fig. 3) and by assay for Se as selenite with 2,3-diaminonaphthalene (Table 5). However, no evidence of a direct reaction was obtained. The effect of PLP on selenite incorporation was similar to the effect of sulfhydryl-reducing agents (22). The increase in cellular Se was principally in the loosely protein-bound fraction that was removed from protein by exposure to 2-mercaptoethanol, and similarly, PLP increased Se binding to media proteins (data not shown). Although GPX is not a PLP-dependent enzyme, effects of exposure to PEP on enzyme activity were examined because PLP is known to stabilize some enzymes for which it is not a cofactor (23). One possible mechanism of PEP effects on cellular GPX activity is through activation of an enzyme involved in intermediary Se metabolism. Selenocysteine lyase is such an enzyme (24), although it is debatable whether SeCys is the physiological substrate for this enzyme (25). These studies provide some supportive evidence for a role of the transsulfuration enzymes in utilization of SeMet, as evidenced by the higher EC 50 of SeMet for GPX induction in the transsulfuration-deficient Biological Trace Element Research
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Se Metabolism by Lymphoblasts
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cells. The effects of PLP on selenite utilization and on GPX induction are intriguing, but not readily explained on the basis of a direct reaction with Se c o m p o u n d s or by interaction with the GPX.
ACKNOWLEDGMENTS We wish to thank A. Wendel (Faculty of Biology, Biochemical Pharmacology, P.O. Box 5560, D-7750, Konstanz, Germany) for the gift of purified bovine erythrocyte glutathione peroxidase. This work was published with the approval at Oregon State University Experiment Station as technical paper n u m b e r 9703. This study was supported by Public Health Service Research Grant DK 38306 from The National Institute of Diabetes and Digestive and Kidney Diseases.
REFERENCES 1. S. T. Omaye and A. L. Tappel, J. Nutr. 104, 747 (1974). 2. F. Pan and H. Tarver, Arch. Biochem. Biophys. 119, 429 (1967). 3. E. Nobuyoshi, T. Nakamura, H. Tanaka, T. Suzuki, Y. Morino, and K. Soda, Biochemistry 20, 4492 (1981). 4. K. Yasumoto, K. Iwami, and M. Yoshida, J. Nutr. 109, 760 (1979). 5. R. A. Sunde, W. K. Sonenburg, G. E. Gutzke, and W. G. Hoekstra, TEMA-4, (J. McHowell, J. W. Gawthorne, and C. L. White, eds., Aust. Acad. Sci., Canberra. 4, 165 (1981). 6. D. M. Greenberg, Metabolic Pathways, 3rd ed., vol. 7 Academic Press, New York, 1975, p. 505. 7. L. A. Smolin and N. J. Benevenga, J. Nutr. 112, 1264 (1982). 8. D. B. Hope, Biochem. J. 66, 486 (1957). 9. M. A. Beilstein and P. D. Whanger, J. Nutr. 119, 1962 (1989). 10. M. A. Beilstein, W. A. Gahl, and P. D. Whanger, TEMA-6, L. S. Hurley, C. L. Keen, B. Lonnerdal, and R. B. Rucker, eds., 6, 323 (1989). 11. G. F. Moore, R. E. Gerner, and H. A. Franklin, Am. Med. Assoc. 199, 519 (1967). 12. M. A. Beilstein, P. D. Whanger, and L. Wong, in Selenium in Biology and Medicine, Part A, Van Nostrand Reinholt Co., New York, 1987, p. 197. 13. M. W. Brown and J. M. Watkinson, Analyt. Chim. Acta 89, 29 (1977). 14. D. E. Paglia and W. N. Valentine, J. Lab. Clin. Med. 70, 158 (1967). 15. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 16. D. J. Finney, in Statistical Method in Biological Assay, Hafner Publishing Co., New York, 1952, p. 454. 17. W. H. Allaway and E. E. Cary, Anlyt. Chem. 36, 1359 (1964). 18. J. Neter, W. Wasserman, and W. H. Hunter. Richard D. Irwin, Inc., Homewood, IL, 1983. 19. S. H. Mudd and H. L. Levy, in The Metabolic Basis of Inherited Disease. McGraw-Hill, New York, 1983, p. 522. 20. K. P. McConnell and G. J. Cho, Am. J. Physiol. 208, 1191 (1965). 21. M. A. Beilstein and P. D. Whanger, J. Inorg. Biochem. 29, 137 (1987). Biological Trace Element Research
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Beilstein a n d W h a n g e r
22. P. D. Whanger and M. A. Beilstein, FASEB J. 2, A1439 (1988). 23. M. A. Grillo, Enzymologia 34, 7 (1967). 24. N. Esaki, N. Karai, T. Nakamura, H. Tanaka, and K. Soda, Arch. Biochem. Biophys. 238, 418 (1985). 25. J. T. Deagen, J. A. Butler, M. A. Bei|stein, and P. D. Whanger, ]. Nutr. 117, 91 (1987).
Biological TraceElementResearch
Vol. 35, 1992
Selenium Metabolism and Glutathione Peroxidase Activity in Cultured Human Lyrnphoblasts Effects of Transsulfuration Defects and Pyridoxai Phosphate M. A. BEILSTEIN AND P. D. WHANGER Department of Agricultural Chemistry, Oregon State University, Corvallis, OR 97331 Received September 16, 1991; Accepted January 10, 1992
ABSTRACT The metabolism of selenite, selenocysteine (SeCys), and selenomethionine (SeMet) was studied in three human lymphoblast cell lines with defects in the transsulfuration pathway and in control cells without this defect. There were very little differences in the induction of glutathione peroxidase (GPX) activity by selenite and SeCys among these cells. However, markedly higher levels of SeMet were required to induce GPX activity in transsulfuration defective cells than in control cells. Surprisingly, the addition of pyridoxal phosphate (PLP) to the media resulted in elevated GPX activity in all cells regardless of the chemical form of Se used. There is no explanation for this effect of PLP, but it is not through direct reaction with GPX or on the alteration of sulfhydryl groups. Index Entries- Transsulfuration; human lymphoblasts; pyridoxal phosphate; selenite; selenocysteine; selenomethione.
INTRODUCTION S e l e n o m e t h i o n i n e (SeMet) is biologically available as a source of selenium (Se) for incorporation as selenocysteine (fieCys) in glutathione
Biological Trace Element Research
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Beilstein and Whanger
106 Methionine
"CH3
~
.CH 3
Homoc~'steine Cystathionine Synthase
~
_
Serine
Cystathionine Cystathionine
Lyase
1
Cysteine Fig. 1. Transsulfurationpathway for the conversion of methionine to cysteine.
peroxidase (GPX) (1). However, the intermediates of SeMet metabolism are not completely known. The chemical similarity of methionine and its Se analog, SeMet, may suggest involvement of the transsulfuration pathway for SeMet metabolism. SeMet and its metabolites are substrates for all steps of the transsulfuration pathway (Fig. 1) (2,3). Synthesis of selenocysteine (SeCys) by this route or degradation of a transsulfuration intermediate, such as selenohomocysteine, could produce a precursor form of Se for incorporation in GPX. Despite the in vitro demonstration of SeMet metabolism through the transsulfuration route, the in vivo importance of this pathway for SeMet utilization is uncertain. Two indirect approaches have been used to examine the possible role of the transsulfuration pathway in SeMet utilization. The effects of vitamin B 6 deficiency have been investigated (4,5), since two enzymes of this pathway, cystathionine synthetase and cystathionine lyase, require pyridoxal 5'-phosphate (PLP) as a cofactor (6). Accumulation of homocystine (7) and/or cystathionine (8) occurs in B 6 deficiency. Results from our laboratory (9) indicated a definite inhibition of SeMet utilization in B6-deficient rats. A second approach has been to examine Se status of individuals with genetic deficiencies of transsulfuration enzymes (10). Studies in our laboratory suggested that SeMet utilization for GPX synthesis was enhanced by transsulfuration deficiency and reduced by a Biological Trace Element Research
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Se Metabolism by Lymphoblasts
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defect (of homocysteine remethylation) that tends to force methionine through the transsulfuration route. The results of such studies are less than conclusive because of incomplete knowledge of dietary history, and especially dietary forms of Se and possible effects of therapy on Se utilization. Use of cultured mutant cells allowed direct assessment of the role of transsulfuration in Se utilization, since most environmental and nutritional factors can be controlled and differences in Se metabolism could be attributed to the presence or absence of the enzymes of concern. Studies of the effect of PLP on Se metabolism in cultured lymphoblasts were conducted because of a possible effect of vitamin B 6 status on transsulfuration activity of the mutant cells. The unexpected results of those studies led to further studies of a possible direct chemical interaction between selenite and PLP.
METHODS Mutant Cell Idnes All cell lines were obtained from the NIGMS Human Genetic Mutant Cell Repository, Coriell Institute for Medical Research, Camden, NJ. Genetically deficient cell lines used were GM 1781 (cystathionine lyase deficient, responsive to vitamin B6 therapy), GM 1566 (cystathionine lyase deficient, B6 response unknown), and GM 1532 (cystathionine synthetase deficient, B 6 response unknown). The apparently normal cell line, GM 3299, was used as a control.
Cell Growth and Maintenance The cells were maintained in RPMI 1640 media (11) supplemented with 10% fetal bovine serum (Flow Laboratories, Inc., McLean, VA) and a commercial serum extender (SerXtend, NEN Research Products, Wilmington, DE). All fetal bovine serum used was from a single lot selected for low-Se concentration as determined in a previous survey of Se in commercial fetal bovine serum (12). The SerXtend was specifically formulated with deletion of sodium selenite. Final concentration of Se in the supplemented media was 1.3 ppb (16 nM) as determined by fluorometric analysis (13) after nitric and perchloric acid digestion. The vitamin B 6 of RPMI 1640 media is 1 ng/L as pyridoxine 9 Hcl. Final vitamin B 6 content of the complete media with 10% serum was not determined, but typical serum vitamin B 6 concentration is 30 ~g/L. Gentamicin sulfate at 0.10 mg/ mL, penicillin at 100 U/mL, streptomycin at 0.1 mg/mL, and Fungizone at 0.25 p~g/mL (Whittaker M.A. Bioproducts, Walkersville, MD) were added to the media. Cells were grown in suspension in unstirred culture at 37~ in a 95: 5 air:CO2 atmosphere saturated with water. Cell densities were maintained between 0.2 and 1.5 x 106/mL as determined by Coulter counter Biological Trace Element Research
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Beilstein and Whanger
(Coulter Electronics, Inc., Hialeah, FL). Doubling times of cells varied from 24 to 48 h depending on cell density.
GPX Response to Se Supplementation Cells were seeded at 3 x 100 (approx 170 ~g cell protein) per 10 mL of media. Se supplements were added in buffered saline in vol of 0-100 p,L to duplicate flasks of cells at Se concentrations of 0, 0.01, 0.05, 0.10, 0.50, 1.0, 5.0, 10.0, and 50.0 ~M. Se compounds tested were sodium selenite, dl-SeCys, and dl-SeMet (Sigma Chemical Co., St. Louis, MO). Because of a difference in GPX response and toxicity to SeMet, the SeMet trials were repeated with triplicate flasks supplemented at 0, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, and 100 p~M. The cells were incubated in unstirred culture for 5 d. Final cell numbers were not determined in all experiments, but the typical cell number was between 11 and 14 x 106 (600 to 800 b~g cell protein) per flask when cells were harvested after 5 d. Cells were harvested by centrifugation, washed in buffered saline, and suspended in approx 1.0 mL ice-cold water. Cell suspensions were kept on ice until GPX assays were performed within 2 h of harvesting. Duplicate or triplicate assays were performed on each harvested flask of cells. Assays were performed by the method of Paglia and Valentine (14) at 30~ with initial concentrations of 5 mM reduced glutathione and 0.17 mM hydrogen peroxide. Several assays were performed with sonicated cell suspensions to test whether suspension in cold water was adequate to lyse the cells completely. No increase in GPX activity was detectable in sonicated cells. Cell protein was determined by the Lowry assay (15) on duplicate aliquots from each flask of cells. GPX activities expressed as nmol NADPH oxidized/min were normalized to cell protein. Concentrations required to induce one-half maximum GPX activity (EC 50) were calculated for the different Se compounds using a linear regression of the logit function (16) of the increase in enzyme activity vs the log Se concentration. Logit is defined as log[Amax - A)/A], where A equals GPX activity. The intercept of the log Se concentration vs logit least squares regression line is equal to log EC50. Effect of PLP on Se Induction of GPX Each ceil type was seeded in triplicate at 3 x 106/10 mL complete media with or without Se compounds added at the calculated EC 50 and with (60 ~g/mL) or without PLP. Additional triplicate flasks of each cell type were supplemented with 0.5 bLM sodium selenite representing a positive control of 100% of inducible GPX activity, without Se toxicity. After 5 d incubation, cells were harvested and assayed for GPX activity as described for the GPX response curves. Enzyme activities normalized to protein were converted to percent of maximum increase over basal (no added Se or PLP) activities. Biological Trace Element Research
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Se Metabolism by Lymphoblasts
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Uptake of 75Se Compounds Each cell type was seeded at 4 x 106/10 mL complete media with 755e as either SeMet or selenite (Amersham Corp., Arlington Heights, IL) plus carrier Se c o m p o u n d at a final concentration of 0.1 p,M Se (0.5 ~Ci/10 mL). Duplicate flasks were harvested at 4, 12, 24, and 48 h after addition of 75Se compounds. Harvested cells were washed once with buffered saline. Radioactivity was determined for each flask of harvested cells using a gamma scintillation counter (Gamma 8000, Beckman Instruments, Inc., Palo Alto, CA). Se uptake in pmoles was calculated by comparison with radioactivity of aliquots of the stock-labeling solutions. Molar uptake of Se at each time-point was normalized to cell protein. Initial rates of uptake were calculated from linear regression of molar Se uptake vs time for the 4- and 12-hour cell samples, including a theoretical 0 S e a t t = 0 ( n = 5).
Effect of PLP on Se Uptake Control lymphoblasts (GM 3299) were seeded at 107/10 mL complete media in triplicate flasks with or without added 60 b~g/mL PLP and with radiolabeled sodium selenite or SeMet plus carrier at 0.1 b~M Se. Cells were harvested after 24 h, and Se uptake determined and normalized to protein as described above.
Chemical Reaction of Selenite and Pyridoxal 5'-Phosphate Visible UV spectral scans from 450 to 200 n m were performed on 0.2 mM PLP solutions in pH 7.0, 0.125M phosphate buffer after addition of sodium selenite at 0, 0.02, 0.04, 0.05, 0.06, 0.10, 0.20, and 0.40 mM final concentration. Control spectra of sodium selenite solutions without PLP were obtained for comparison. The scans were performed on a Beckman DU 64 (Palo Alto, CA) spectrophotometer. Changes in the spectra would have indicated a change in oxidation state of the PLP. Radiolabeled sodium selenite with carrier at 0.20 mM was combined with PLP at 0, 0.1, 0.2, 1.0, and 2.0 mM in 2.0 mL unbuffered aqueous solution. After incubation for 15 min at room temperature, 1.0 mL of 5 mg/mL 2,3-diaminonaphthalene (DAN) in 0.1 N HC1 was added to each trial solution of sodium selenite. After a further incubation for 15 min, each trial was extracted three times with 1.0 mL of cyclohexane. Radioactivity of both the aqueous solutions and the organic extracts was determined. 75Se extracted into the organic phase was considered to be as selenite (/7), whereas radioactivity in the aqueous phase was assumed to be in some other oxidation state. To verify the method, identical assays were conducted with reduced glutathione substituted for PLP at the same concentrations. Biological Trace Element Research
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Beilstein and Whanger
Pjm'doxal 5'-Phosphate Effect on Activity of Purified Glutathione Peroxidase A stock solution of purified bovine erythrocyte GPX obtained from Albrecht Wendel (University of Konstanz, Konstanz, Germany) was prepared at 100 ~g/mL in 0.125 M phosphate buffer, pH 7.0. PLP was added to duplicate aliquots at 0, 50, and 100 ~M. The highest PLP concentration represents an approx 90-fold molar excess of PLP over enzyme. GPX enzyme activity of the stock solutions was determined immediately after addition of PLP, and subsequently after 15 and 30 min and after 2, 3, and 20 h of incubation at 37~
Data Analysis The data were subjected to statistical analyses using linear regression analyses, estimations of variance, determination of confidence intervals, and comparisons of means by the Student's t-test (18).
RESULTS Maximum inducible GPX activity on Se supplementation was approx 90 EU/mg cell protein for all cell types examined. The concentrations of selenite or SeCys required to achieve half-maximum GPX varied between the four cell types (Table I and Fig. 2). This variation was less for selenite than for SeCys. The EC 50 for the GM 1566 cell type was over fourfold that for the GM 1781 of the control. Concentrations of SeMet required to induce half-maximum enzyme activity were significantly higher compared to selenite or SeCys, and much higher for the mutant cells as compared to controls. Addition of PLP to media significantly increased GPX activity for all cell types and for all forms of Se added (Table 2). There was even stimulation of activity in control cells with no added Se. This stimulation of GPX activity by PLP was not influenced by the form of Se added to the media. Initial rates of cellular incorporation of 75Selabel as SeMet or selenite were not apparently different between cell types (Table 3). However, rates of Se uptake and 48-h Se incorporation were from four to eight times higher for SeMet compared to selenite. Addition of PLP to media approximately doubled the 24-h cellular incorporation of Se from selenite for control lymphoblasts (Table 4). In contrast, SeMet uptake was reduced by supplemental PLP. No evidence was obtained of a direct interaction of selenite with PLP (Fig. 3). Ultraviolet-visible spectra of mixtures of selenite and PLP were equivalent to the sum of the spectra of the two separate compounds. After incubation with PLP, selenite was still extracted with DAN (Table 5). However, this extraction of selenite was reduced in proportion Biological Trace Element Research
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Se Metabolism by Lymphoblasts
111
Table 1 EC 50* (nM) for Se Induction of Glutathione Peroxidase Activity Cell line GM 3299 (Control) GM 1532 (Synthase def.) GM 1781 (Lyase def.) GM 1566 (Lyase def.)
Selenium source SeCys 18 (9-36) 36 (25-52) 18 (14-23) 87 (55-104)
Na2SeO 3
32 (24 42) 21 (16-27) 25 (18-35) 20 (12-33)
SeMet 1580 (1340-1870) 6750 (5920-7700) 11,690 (9510-14,380) 11,980 (10,610-13,520)
*See Methods section for definition of EC 50. (This is calculated on the amount of selenium added and does not include the 16 nM selenium in the basal media.) Values in parentheses represent the 90% confidence limits.
80 -GM 3 2 9 9
801- GM 1789
(Control)
9
(- Lyose)
|
:
f ~ 40
!
" !
I
I
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10 - 5
:~ ~.~ X EL (_9
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9
!
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1 10-5
1566
(- Lyose) ~,
9
40
|
9
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o
9
40 !
!
! I
I0 -7
I
10-4"
I0 -6
i.
I
I0 -5
Selenium
=
L
I
I0-7
concentration,
I
I0 -6
I0 "5
I
10-4
M
Fig. 2. Response of glutathione peroxidase activity to SeMet supplementation. Arrows indicate EC 50 calculated from logit function. (See Methods Section.) to the concentration of added reduced glutathione. The incubation of selenite with reduced glutathione apparently produces selenodiglutathione, in which the 75Se cannot be extracted by DAN. Incubation of purified bovine erythrocyte GPX with PLP had no marked effect on activity (Fig. 4). PLP slightly accelerated the slow loss of enzyme activity during incubation at 37~ Biological Trace Element Research
Vol. 35, 1992
112
Beilstein and Whanger
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Vol. 35, 1992
Se /Vletabolism by Lyrnphoblasts
113
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VoL 39, 1992
Be#stein and Whanger
114
Table 4 Effect of Pyridoxal Phosphate on Selenium Uptake* by Control Lymphoblasts (GM3299) 0 PLP 60 p~g/mL PLP
Selenite
SeMet
11.6 + 0.6 26.8 ___ 2.5
185 + 8 146 _+ 7
*pmol/mg cell protein after 24-h i n c u b a t i o n + SD, n = 3. Effect of PLP o n Se u p t a k e was significant (p < 0.01) for both forms of Se.
A
B
PLP+
Na2Se03 2.0
1.2
).8 U) r
w 2.(} D
-6 .o
1.0
0.4
0
I..p 0
IC2oo
ZOO
24!
;)20
' 240
200
220
240
Wavelength (nm)
Fig. 3. Ultraviolet spectra of Pyridoxal Phosphate (PLP) and sodium selenite as determined in 0.125M phosphate buffer, pH 7.0. Panel A shows superimposed spectra of 0.2 mM PLP and 0.2 mM PLP plus 1.0 mM sodium selenite. Inset in panel A shows difference between these two spectra. Panel B shows spectrum of 2.0 mM sodium selenite. The PLP absorbance peak at 388 nm (not shown) was unchanged by selenite addition.
DISCUSSION The similarity of r e s p o n s e in GPX activity to s u p p l e m e n t a t i o n with selenite or SeCys (Table 1) indicates that the four l y m p h o b l a s t lines examined h a d similar ability to utilize these forms of Se. In contrast, utilization of SeMET for GPX i n d u c t i o n was m a r k e d l y lower for the transsulfuration-deficient cells in c o m p a r i s o n to the controls (Fig. 2 a n d Table 1). The variance in utilization of SeMet was not d u e to differences Biological Trace Element Research
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Se Metabolism by Lymphoblasts
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Table 5 Complexation of Selenite--Pyridoxal Phosphate or Reduced Glutathione Reactions Products by 2,3-Diaminonaphthalene PLP added, mM
% 75Se complexed
0 0.1 0.2 1.0 2.0
95 96 95 94 94
Glutathione added, mM
+ 2 + 1 ___ 2 _+ 2 + 3
0 0.1 0.2 1.0 2.0
% 7SSe complexed 96 95 89 25 1
+ 1 + 2 _+ 3 + 5 + 0.5
Each value represents mean + SE of three samples. A level of 0.2 nM selenite was used in all complexation studies.
120
80
%.
.--.%
\
~_ 4 0 (_9
0
I
I
I!
60
120
180
I
I
1200
Time (rain) Fig. 4. Changes in activity of purified bovine glutathione peroxidase during incubation with and without pyridoxal phosphate at 37~
in rates of SeMet incorporation into cells, since similar rates of uptake were found for all cell lines (Table 3). Deficiency of cystathionine synthetase activity (homocystinuria) results in elevated physiological levels of both homocystine and methionine through remethylation of accumulated homocysteine (19). SeMet and methionine compete for the same transport mechanism in intestine (20), and competition for uptake has been demonstrated in other cell culture systems (21). Competition with methionine for transport w o u l d be expected to have a greater effect on SeMet uptake in the cystathionine-synthetase-deficient cells than in either the controls or the cystathionine-lyase-deficient cells, because cysBiological Trace Element Research
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Be#stein and Whanger
tathionine synthetase deficiency does not result in accumulation of methionine. However, no such effect was observed in either initial rates of SeMet uptake or in SeMet accumulation after 48 h incubation (Table 3). In humans with transsulfuration defects (10), the major effect on Se utilization is possibly through competition of SeMet with methionine for protein synthesis. Defects that inhibit transsulfuration, thus elevating physiological methionine levels, result in lower levels of Se in the nonSe-requiring protein, hemoglobin, and result in increased plasma and erythrocyte GPX activity (10). Deficiency of either cystathionine synthetase or lyase resulted in reduced induction of GPX by SeMet, and had no effect on SeMet accumulation in cells. The effects of PLP supplementation were examined because transsulfuration defects are often the result of reduced affinity of the enzyme for this cofactor. In such cases, supplementation with pyridoxine at 50 to 100 times the vitamin B6 RDA is effective in alleviating the deficiency (19). Vitamin B6 was provided to the lymphoblasts as PEP rather than as pyridoxine because of uncertainty concerning the capacity of lymphoblasts to generate the cofactor. The vitamin Bo response of the patientsource of the lymphoblasts was known only for the cystathionine-lyasedeficient cell line, GM 1781. An increased GPX response to SeMet in the presence of elevated PLP was sought in this cell line as verification that correcting the enzyme deficiency also normalized SeMet utilization. Such a response was obtained, but a similar response was also observed for the control cell line, GM 3299, and the other mutant cell lines (Table 2). We have no explanation for the increase in GPX activity with selenite and SeCys supplementation with added PLP. Assays of reduced and oxidized glutathione in both media and cells revealed no effect of incubation with PEP (data not shown). The possibility of a direct chemical reaction of PLP with selenite was examined through both spectroscopy (Fig. 3) and by assay for Se as selenite with 2,3-diaminonaphthalene (Table 5). However, no evidence of a direct reaction was obtained. The effect of PLP on selenite incorporation was similar to the effect of sulfhydryl-reducing agents (22). The increase in cellular Se was principally in the loosely protein-bound fraction that was removed from protein by exposure to 2-mercaptoethanol, and similarly, PLP increased Se binding to media proteins (data not shown). Although GPX is not a PLP-dependent enzyme, effects of exposure to PEP on enzyme activity were examined because PLP is known to stabilize some enzymes for which it is not a cofactor (23). One possible mechanism of PEP effects on cellular GPX activity is through activation of an enzyme involved in intermediary Se metabolism. Selenocysteine lyase is such an enzyme (24), although it is debatable whether SeCys is the physiological substrate for this enzyme (25). These studies provide some supportive evidence for a role of the transsulfuration enzymes in utilization of SeMet, as evidenced by the higher EC 50 of SeMet for GPX induction in the transsulfuration-deficient Biological Trace Element Research
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Se Metabolism by Lymphoblasts
117
cells. The effects of PLP on selenite utilization and on GPX induction are intriguing, but not readily explained on the basis of a direct reaction with Se c o m p o u n d s or by interaction with the GPX.
ACKNOWLEDGMENTS We wish to thank A. Wendel (Faculty of Biology, Biochemical Pharmacology, P.O. Box 5560, D-7750, Konstanz, Germany) for the gift of purified bovine erythrocyte glutathione peroxidase. This work was published with the approval at Oregon State University Experiment Station as technical paper n u m b e r 9703. This study was supported by Public Health Service Research Grant DK 38306 from The National Institute of Diabetes and Digestive and Kidney Diseases.
REFERENCES 1. S. T. Omaye and A. L. Tappel, J. Nutr. 104, 747 (1974). 2. F. Pan and H. Tarver, Arch. Biochem. Biophys. 119, 429 (1967). 3. E. Nobuyoshi, T. Nakamura, H. Tanaka, T. Suzuki, Y. Morino, and K. Soda, Biochemistry 20, 4492 (1981). 4. K. Yasumoto, K. Iwami, and M. Yoshida, J. Nutr. 109, 760 (1979). 5. R. A. Sunde, W. K. Sonenburg, G. E. Gutzke, and W. G. Hoekstra, TEMA-4, (J. McHowell, J. W. Gawthorne, and C. L. White, eds., Aust. Acad. Sci., Canberra. 4, 165 (1981). 6. D. M. Greenberg, Metabolic Pathways, 3rd ed., vol. 7 Academic Press, New York, 1975, p. 505. 7. L. A. Smolin and N. J. Benevenga, J. Nutr. 112, 1264 (1982). 8. D. B. Hope, Biochem. J. 66, 486 (1957). 9. M. A. Beilstein and P. D. Whanger, J. Nutr. 119, 1962 (1989). 10. M. A. Beilstein, W. A. Gahl, and P. D. Whanger, TEMA-6, L. S. Hurley, C. L. Keen, B. Lonnerdal, and R. B. Rucker, eds., 6, 323 (1989). 11. G. F. Moore, R. E. Gerner, and H. A. Franklin, Am. Med. Assoc. 199, 519 (1967). 12. M. A. Beilstein, P. D. Whanger, and L. Wong, in Selenium in Biology and Medicine, Part A, Van Nostrand Reinholt Co., New York, 1987, p. 197. 13. M. W. Brown and J. M. Watkinson, Analyt. Chim. Acta 89, 29 (1977). 14. D. E. Paglia and W. N. Valentine, J. Lab. Clin. Med. 70, 158 (1967). 15. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 16. D. J. Finney, in Statistical Method in Biological Assay, Hafner Publishing Co., New York, 1952, p. 454. 17. W. H. Allaway and E. E. Cary, Anlyt. Chem. 36, 1359 (1964). 18. J. Neter, W. Wasserman, and W. H. Hunter. Richard D. Irwin, Inc., Homewood, IL, 1983. 19. S. H. Mudd and H. L. Levy, in The Metabolic Basis of Inherited Disease. McGraw-Hill, New York, 1983, p. 522. 20. K. P. McConnell and G. J. Cho, Am. J. Physiol. 208, 1191 (1965). 21. M. A. Beilstein and P. D. Whanger, J. Inorg. Biochem. 29, 137 (1987). Biological Trace Element Research
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Beilstein a n d W h a n g e r
22. P. D. Whanger and M. A. Beilstein, FASEB J. 2, A1439 (1988). 23. M. A. Grillo, Enzymologia 34, 7 (1967). 24. N. Esaki, N. Karai, T. Nakamura, H. Tanaka, and K. Soda, Arch. Biochem. Biophys. 238, 418 (1985). 25. J. T. Deagen, J. A. Butler, M. A. Bei|stein, and P. D. Whanger, ]. Nutr. 117, 91 (1987).
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