BIOLOGICAL TRACE ELEMENT RESEARCH 4, 245-258 (1982)

Kinetics of Selenite Uptake by Mononuclear Cells from Peripheral Human Blood J ~ R G E N C L A U S E N * AND JENS T R A N U M

The Laboratory of Biochemistry and Toxicology, Institute of Biology and Chemistry, Roskilde University, DK 4000 Roskilde, Denmark; and The Neurochemical Institute, 58, Ritdmandsgade, DK 2200 Copenhagen N. Received January 27, 1982; Accepted April 9, 1982

Abstract The present study deals with the kinetics and thermodynamics of the uptake of 75Selabeled SeO 2- from incubation media to lymphocytes cultivated from eight normal individuals (14-55 years of age, two females). The uptake of SeO~- was evaluated on the assumption of pseudo-first-order kinetics with regard to a reacting cellular receptor pool, On the basis of the experimental o b s e r v a t i o n s , it was a s s u m e d that the s u g g e s t e d p o o l o f r e c e p t o r molecules-symbolically represented by "s with SeO 2- in the hypothetical reaction: kl

s

4 + SeO 2- +

2H + < > s

+ 3H20

k i

The mean value of the change in standard free energy at 25~ was calculated to be AG ~ = - 141.6 + 1.3 kJ/mol, while the corresponding mean value of the free energy of activation at 25~ was calculated to be AG e+ = - 7 . 8 -+ 0.9 kJ/mol for the forward reaction. The calculated values of the corresponding individual changes in the respective standard enthalpies and entropies were mutually interdependent for all eight donors. 2if/~ = - 152 + 315AS~ corresponding to the common value AG ~ ~ - 152 kJ/mol at 315 ~ K. These mutual interdependencies are possibly the effect of variable conformational states (e. g., the macromolecular compactness) of the cellular receptor pools. This suggestion may furthermore be supported by the correlation traced between M-/~ vs the biological age in years of the donors: ~-/~ --= 76.7-1.0(age)kJ/mol (r = -0.92).

9 1982 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/82/1200-0245 $03.00

245

246

CLAUSEN AND TRANUM

The calculated values of activation enthalpy AH2§ kJ/mol and activation entropy AS2+(kJ/mol K) also mutually correlated linearly (r = 0.998); the regression line was: AHe+ = -8.9 + 305AS2+ (kJ/mol) corresponding to the common value A G 2 + --~

-8.9 (kJ/mol) at 305~

Similarly the activation enthalpy AH2+ vs the biological age in years correlated linearly: AH2+ = 67.4 - 0.73(age) (kJ/mol) (r = -0.76) The range of AH2+ studied was from 13,8 to 53.9 kJ/mol with a linearly corresponding range in AS2+ from 73 to 205 J/mol K. The thermodynamic data reveal the selenite uptake during the hypothetical standard reaction to be exergonic and endothermic. Critical pH dependencies of the selenite uptake were explained. Index Entries: Cellular selenite uptake; thermodynamics of selenite uptake; kinetics of cellular selenite uptake; model for selenite cellular uptake; selenite uptake by in vitro cultivation of lymphocytes; lymphocytes, kinetics of selenite uptake.

Introduction Since Schwarz and Foltz (1) found that selenium is an essential trace element in mammals, this element has been demonstrated to be an integral part of glutathione peroxidase (EC. 1.8.4.2) (2, 3). Selenium may however also be an integral part of other proteins (4-7). Thus in bacteria glycine reductase (8) contains Se and in plants Se may be incorporated both in amino acids and carbohydrates (9). Painter (10) suggested the reaction scheme [Eq. (1)] for selenotrisulfide formation in organic molecules (e.g., GSH) containing SH-groups: 4GSH + SeO~- + 2H+< > G S - - S e - - S G + G S - - S G + 3H20

(1)

wherein GSH may stand for reduced glutathione. The formation of seleno-trisulfides is thus sensitive to changes in pH; alkaline conditions may lead to hydrolytic cleavage of the seleno-trisulfides formed at lower pH values (11). The present study was made in order to investigate the physiological kinetics in vitro of the uptake of selenite by normal human lymphocytes (monocellular cells). Our experimental data support the idea that a reaction scheme such as Eq. (1), or a similar mechanism, may explain the chemical nature of the uptake.

Methods Chemicals Commercial chemicals used were of highest analytical purity from BDH, Poole, Dorset, England.

SELENITE UPTAKE BY LYMPHOCYTES

247

Cell Separation (12) All operations were performed under sterile conditions. Human lymphocytes were isolated from anticoagulated whole blood [to each 12 mL of blood was added 200 ~L 10% (w/v) of disodium ethylenediaminetetraacetate, NazEDTA, pH 7.3]. Similarly, 48 mL of anticoagulated venous blood from each of eight normal individuals from 14 to 55 years of age, and including two female donors of 26 and 41 years age, were collected. The blood was then diluted 1 : 1 (v/v) with 0.9% (w/w) aqueous NaCI (saline). A 4 mL sample of the diluted blood was layered on 3 mL of Lymphoprep (Nyegaard & Co. A/S Norway). The tubes were centrifuged for 30 min at 400g (Heraeus Christ Labofuge 1) and the plasma layer removed, whereafter the intermediate layer of lymphocytes was isolated and suspended three times in 10 mL saline, and each time sedimentated at 400g (5 rain). The yield of lymphocytes was 10 6 cells/mL blood. Differential counting revealed 96% of the cells to be lymphocytes. Hereafter, the rinsed cells were ready for subsequent incubations in tissue culture media.

Cell Cultures Initial studies were made in order to study the effects of changes in pH on the selenite uptake. These studies showed that selenite taken up by the cells at pH 7.3 was released again from the cells at pH > 7.3 and it was obvious that a precise pH control maintaining the incubation medium at pH 7.3 is essential for investigation of the kinetics of the uptake. Thus, by increasing the pH to - 8 . 0 because of degasification of C Q from the cell cultures [cf. the Henderson-Hasselbach buffer equation (13)], the selenite taken up at the physiological normal value, pH 7.3, desorbed from the cells by a factor of 3 within 40 h. Thus, in order to fix the pH in the standard assay procedure, a commercial (HEPES + CO2/HCO7 buffer system, Flow Laboratories) with a pH of 7.3 was used. The cell suspension was adjusted to 0.5 • 10 6 cells/mL tissue culture medium (total, 80 mL) with the following composition of the aqueous solution (Flow laboratories): 14.128 g H199/L (with L-glutamine) + 20 mM HEPES buffer, pK~ 7.3 at 37~ (Flow Labs, Biological Cat. No. 10-235) and 20% heat-inactivated fetal calf serum (v/v) + 0.35 g/L NaHCO3 + penicillin: 100 ixg/mL + streptomycin: 100 Ixg/mL. The 80 mL cell suspension was split into four parts of 20 mL each and an aqueous NazSeO3 solution, labeled with 3,-active 75Se (0.56 mCi/mL, Amersham Radioactive Centre, England) was added at four selenite concentrations: 0.9, 1.8, 3.5, and 7.1 ~xM.

Apparatus It was possible by means of "Titertek Microplates" [Flow Labs ISMRC-96 Sterile (MRG)-U-shaped wells] to cultivate 344 samples each with 200 ixL containing 105 cells/well. The cultures with four selenite concentrations were incubated at each of four temperature levels indicated below.

248

CLAUSEN AND TRANUM

The cultures were exposed to sterile air. The sterile microplates were covered with gas-tight, self-adhesive foil (Flow Labs) and incubated in four thermostats at 5, 15, 25, and 37~ +- 0.1~ respectively. The CO2 evolved by the cell cultures is present under the gas-tight plastic foil cover. However, if the foil is punctured, e.g., to compare it with the gas-tightness of the intact foil, the pH in the incubation medium will rise to alkaline values above 7.3 (in spite of the HEPES buffer) and the cells will die. Thus the maintenance of constant pH is obtained by use of a gas-tight foil cover (for details concerning effects of pH on Se uptake, cf. later). Cell viabilities were intermittently tested by Trypan-blue in saline, taking 10 ~L aliquots immediately before each harvesting of the respective cell cultures. The viabilities found were all near 100% of the cells during the incubations. The phenol red indicator in the H 199 tissue culture medium (Flow Labs) showed that the pH was 7.3 during the incubation periods because of the (HEPES + CO2/HCO3) buffer system.

Cell Harvesting of Incubated Cells The respective cell cultures were successively transferred to fiber filter discs (Flow Labs, Scotland) using a semiautomatic "Titertek" cell harvester (Sharron, Lier, Norway). After the filtration, the cells were washed free of incubation medium by a 20 s passage of distilled water through glass fiber filters. Thereafter the filters with the rinsed cells were dried in air for 30 rain at 50~

Radioactivity Counting The counting of the ",/-active 75Se taken up by the incubated cells was made with a Beckman LS-230 liquid scintillation counter assayed as "Bremsstrahlung" when lead ions absorbed the ~/-rays (of Beckman's manual). The dried filters with ",/-active cells were applied in 7-mL polyethylene vials at 15 mm (Lumac Systems AG, Basel, Switzerland). To each vial was added 10 mL Ready Solv HP scintillation liquid saturated with lead acetate and the polyethylene vial with the filter were applied herein, sealed 5 mm above the bottom of the Beckmann glass vial (to obtain a sufficient layer of ~/-scintillation liquid below the filters). Measurements with Beckmann's LS-230 were performed also on the incubation media, permitting calculation of the specific ratio of radioactivity between the lymphocytes and the medium (cf. below). The reproducibilities of the CPM measurements on the triplets were all within 5% relative deviations.

Theoretical Bases for the Calculation of Thermodynamic Data As will be demonstrated under fixed experimental conditions, the uptake of selenite asymptotically approaches an equilibrium state in which the uptake is equal to the cellular loss of selenite. The specific CPM ratio between 75Setaken up by the cells and that of the media was used to calculate the molar concentration ratio (2tR) between the total number

249

SELENITE UPTAKE BY LYMPHOCYTES

of selenium atoms take up by the cells and the corresponding molar concentration of selenite added to the incubation media. At equilibrium this ratio is expressed as Although lymphocytes of the peripheral blood contain subpopulations of different functions and size, we have used the known mean diameter of lymphocytes for the present calculations (13). Thus with 0.5 x 106 cells/mL medium, the volume ratio (0.12 -+ 0.02) x 10 -3 VOl cells/vol medium was calculated, the cells roughly being considered as spherical with a radius of 3.8 i~m (13). The SeO 2- concentrations in the media were constant during the incubation, since less than 1% of the SeO 2- pool in the media actually was taken up by the cells. The progress of the specific selenite uptake during the incubations, 2ff~ vs time, shows the typical course, by which an uptake asymptotically approaches a saturation level as the equilibirum successively is established. Considering observed responses of the experimentally accessible values of to time, temperature, selenite concentrations, and pH, we may suggest the validity of the hypothetical reaction scheme: kl

s

+ SeO 2- + 2H 2
0.80. As already stressed, the calculation of the total receptor pool (s was based upon the rough assumption that lymphocytes have mean volume of VL = 2.30 • 10 -~5 L (13). The s pool was then calculated to be: s = NoPVL = 0.14 X P x 10 9 s 4 molecules (No being Avogadro's number, P as mM). The linear regression of the calculated values of In Keq vs T- ~ (cf. also Fig. 2b) gave the thermodynamic constants AG ~ Aft/~ and AS~ shown in Table 2. No significant differences in the AG ~ values among the eight individuals studied were found. (Mean value: AG ~ -- - 141.6 + 1.3 kJ/mol at 25~ However, 2if-/~ and AS~ seem interrelated. AH ~ = - 1 5 2 + 315 x AS~ kJ/mol (r = 0.998) corresponding to the common value AG ~ = - 152 k J/tool at 315 K. Furthermore, a correlation was discovered between the enthalpy 2d4~ kJ/mol and the biological age in years of the eight donors: AH ~ = 76.7 - 1.0 • (age) kJ/mol (r = -0.92). By plotting the actual values of In ki - - v s T -I 1)

~R 100 -

/ J

/ J

/ _

m

f

/ / / /

f

50-

J

t-

"/~/, /

~-~-~1

~_L__L____ 50

,

l

1 O0

I i i I I 150 HOURS

Fig. 1. The experimental relationship of AR (i.e., the molar concentration ratio of intra- to extracellular selenium) vs t in incubation media containing 7.1 M and 0.7 laM selenite, respectively, is shown. Data were taken from Experiment No. 4, and values of 2tR at three experimental temperature levels [15~ ( I ) , 25~ (V), and 37~ (O) are given. The fully drawn curves (--) were calculated from linear regression data by insertion of the experimental values of z ~ in Eq. (10) with succeeding linear regressions of _+AR~ In [2tR~/(2d~ - ~ ) ]

= aot

vs t (of the text). The punctuated curves ( - - - - - - ) were calculated from Eq. (11): AR = 2d~ (1 - e ~~ by insertion of the corresponding values of Oto and zS~R~as taken from the corresponding Arrhenius and van't Hoff linear regression lines: Oto = k l K ~

s- l

and AR~ = (PC~+/K)

with K = (C320/Keq) + C2+

9 Cseo~

and kl and Keq from the corresponding Arrhenius and van't Hoff lines (cf. Figs. 2a and b). The stipulated saturation levels ( - - - - - ) are the respective asymptotic values of ~ = corresponding to the hypothetical progress [Eq. (11)] of ~ vs t.

255

SELENITE UPTAKE BY LYMPHOCYTES

TABLE l Percentage of Cellular Ligand Pool Bound to Selenium at Equilibrium" Selenite concentration, % Temperature, ~ 5 15 25 37

0.9 laM

1.8 IxM

3.5 g M

7.1 g M

4.78 5.55 6.54 8.17

9.12 10.52 12.28 15.10

16.32 18.61 21.40 25.70

28.35 31.67 35.58 41.23

"Calculated as Xeq/P = 2dL~Csc~-/P x 100%, wherein Xeq is the molar concentration of a hypothetical cellular compound s at t = 2, Csco~ is the extracellular selenite concentration in the medium, and ~ is the ratio at equilibrium of cellular to extracellular molar concentration of selenium. Data from experiment No. 1.

(cf. Fig. 2a), the c o r r e s p o n d i n g values o f 2if-/2+ and A S 2+ w e r e o b t a i n e d for the eight individuals studied. A g a i n correlating 2if-/z+ vs c o r r e s p o n d i n g values o f AS 2+ and vs the biological age o f the d o n o r s in years, we get similar correlations for the kinetic constants as for the t h e r m o d y n a m i c constants, n a m e l y 2if-/2+ = - 8 . 9 + 305 x AS 2+ kJ/mol (r = 0 . 9 9 8 ) c o r r e s p o n d i n g to the c o m m o n value AG 2+ = - 8 . 9 kJ/mol at 305 K, and 2if-/2+ -~ 6 7 . 4 - 0.73 x (age) kJ/mol (r = - 0 . 7 6 ) , respectively.

TABLE 2 Calculated Values of Thermodynamic and Kinetic Constants Blood donors Exp't No.

Age, Sex years,

1

~"

49

2

6'

52

3 4

8' 6'

28 48

5

6'

14

6

9

41

7

$

26

8

d

55

s

pool

Thermodynamic constants, 25~

Kinetic constants, 25~

conc'n, P, mM

AG ~, kJ/mol

~M-/~, k J/tool

AS~, J/tool 9 K

AG 2+ ' kJ/mol

z~kH2+ ' kJ/mol

AS2+ ' J/tool 9 K

2.65 -+0.70 (4) 0.77 + 0 . 2 2 (3) -+0.26 (1) 1.22 -+0.12 (3) 1.95 9+ 0 . 0 2 (2) 0.84 -+0.35 (3) 3.64 -+2.50 (3) 1.00 -+0.22 (3)

- 141.7

27.6

568

-8.9

30.4

132

- 143.0

30.7

583

-8.0

13.8

73

-- 141.6

-24.1

-556

-6.1 -7.7

53.5 25.1

200 110

-138.8

57.9

660

-7.2

53.9

205

-143.2

28.7

577

-9.1

44.8

181

-141.1

61.2

679

-7.7

46.8

183

- 141.9

21.1

547

-7.8

41.7

166

256

CLAUSEN AND TRANUM DETERMINATION OF KINETICAL CONSTANTS

i

-'7'

(A) LU r

i

32

:

-

0,87

1,

I

3,3

3.4

........

103 x RECIPROCAL TEMPERATURE(~]"~ (

,,..v.w

I

I

5,5

3,6

K-t)

5

DETERMINATION OF THERMODYNAMICALCONSTANT5

66

(B)

- 0,81

r ~

J

$

.... 3,2

g

Fig. 2.

L

I

I

3.~

L5

].b

I 5.3

lO5 X RECIPROCAL TEMPERATURE (,L~ K-I) I

Values of the equilibrium constant Keq = k t / k

I

and of the kinetic constant kl =

v

e -AG2§

for the forward reaction: (2)s

+ SeO 2- + 2H +

s

+ 3H20

k-I

were calculated from experimental data in experiments No. I to 8. Linear regression of In K~q and

In (kl/v)

respectively vs T - l K - ~ were performed, and the corresponding coefficients of correlation r ( - 1 --< r --< 0) were calculated.

257

S E L E N I T E U P T A K E BY L Y M P H O C Y T E S

As indicated above, the cellular uptake of selenite was sensitive to the pH of the medium. Our studies of the effect of pH on the selenite uptake were found quantitatively in agreement with Eq. (21). Also, at elevated pH the hydrolysis of s was found in close agreement with Eq. 21.

Discussion The present communication revealed the uptake of selenite to be a rather slow process taking more than 3 days to arrive at equilibrium. This is in contrast to the exchange of anions and cations participating in the establishment of membrane potentials (14). Since the shape of the uptake curves was essentially the same at 5 and at 37 ~ it was not possible in the present assay to differentiate between uptake from receptor binding and real cellular uptake. Even the highest selenite levels (7.1 pM) used in the present assay caused only 41% at a maximum of the total receptor pool to be saturated with selenium. This relatively low binding level may be explained by a direct (metabolic) transformation of selenite rather than by a simple ionic transportation of selenite. The uptake of selenite resembles more the uptake of precursors to (glyco)-proteins, [e.g., amino acids or monosaccharides (15)] than a fast ionic uptake process. AG ~ was in all cases negative, which may indicate that the uptake reactions are of the exergonic type. Thus, the reaction may occur spontaneously (16) giving rise to liberation of free energy. Furthermore, the positive AH ~ may indicate that the reaction is associated with an uptake of heat, i.e., it is an endothermic reaction. The finding of the present communication that AG ~ is constant for the uptake in lymphocytes of selenite from different individuals may argue for a rather fixed and constant affinity of the uptake process for selenite. However, the age-dependent variation in AH ~ and AS~ may probably be explained by age-dependent changes in the conformational state of the membrane protein. The present data is based upon the assumption that the uptake of selenite is caused by a reaction with an unknown receptor possibly containing--SH groups. Steam (17) demonstrated that during the denaturation of enzymes the enzymic inactivation is presumably associated with splitting o f - - S - - S - - and hydrogen bonds, giving rise to increased AH and AS values for the enzymic reaction, but constant AG values. These data support our finding of constant AG values, but parallel linear changes in M4 and AS values, for selenite uptake in individuals of different age. Finally, the fast hydrolysis of the s complex at elevated pH may argue for the s complex to be a rather unstable receptor-ligand complex.

Summary The present paper demonstrates that selenite is slowly taken up by cultured lymphocytes in vitro. The uptake is associated with a positive heat salvage, i.e., it is an endothermic reaction. The change in free energy during the uptake of selenite is negative; thus this exergonic process occurs spontaneously.

258

CLAUSEN AND TRANUM

The change in free energy is constant for selenite uptake in lymphocytes from different individuals. However, the change in enthalpy and entropy are agedependent. The data suggest that inorganic selenite is directly metabolized during uptake rather than simply transported across the cell membrane. The uptake is agedependent, probably as a result of conformational changes in lymphocyte membranes during aging.

Acknowledgment The present study was made possible by economic support from "KCbmand Sven Hansen og Hustru Ina Hansen's Foundation" and by a gift from Mrs. and Mr. H. and F. Holmberg.

References 1. K. Schwarz and C. M. Foltz, J. Am. Chem. Soc. 79, 3292 (1957). 2. L. Floh6, W. A. Gtinzler, and H. H. Schock, FEBS Left. 32, 132 (1973). 3. J. T. Rostuck, W. C. Hoekstra, A. L. Pope, H. E. Ganther, A. Swanson, and D. Hareman, Fed. Proc. 31,691 (1972), 4. H. E. Ganther, in Selenium, (R. A. Zingaro and W. C. Cooper, eds., Van NostrandReinhold, Princeton, New Jersey, 1974, pp. 546-614. 5. J. T. Rostruck, A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. G. Hoekstra, Science 179, 588 (1973). 6. D. E. Metzler, Biochemistry. The chemical reactions of living cells, Academic Press, New York, 1977, p. 536. 7. I. M. Aria and W. B. Jacoby, eds., Glutathione, Metabolism and Function, Raven Press, New York, 1976, p. 117. 8. H. Tanaka and T. C. Stadtman, J. Biol. Chem. 254, 447 (1979). 9. R. A. Zingaro, J. E. Price, and C. R. Benedict, J. Carbohyd. Nucleosides Nucleotides 4(5), 271 (1977). 10. E. P. Painter, Chem. Rev. 28, 179 (1941). 11. M. Sandholm and P. Sipponen, Arch. Biochem. Biophys. 155, 120 (1973). 12. A. B6yum, Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 77 (1968). 13. K. Diem and C. Lentner, eds., Documenta Geigy, Scientific Tables, Basel, Switzerland, 1975. 14. H. Netter, "Membranpotentiale, Donnan, Gleichgewichte und active Transport," in Biochemisches Taschenbunch, Vol. II, H. M. Rauen, ed., Springer Verlag, Berlin, 1964, p. 192. 15. G. E. Jensen, G. G. Nielsen, and J. Clausen, J . Neurol. Sci. 48, 61 (1980). 16. E. A. Dawes, Quantitative Problems in Biochemistry. Longman, London, 1980, p. 71. 17. E. A. Stearn, Adv. Enzymol. 9, 25 (1949).

Kinetics of selenite uptake by mononuclear cells from peripheral human blood.

The present study deals with the kinetics and thermodynamics of the uptake of(75)Se-labeled SeO 3 (2-) from incubation media to lymphocytes cultivated...
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