'.e;.Copyright 1986by Tile Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/86/1004--0293502.80

Uptake of Selenate and Selenite by Isolated Intestinal Brush Border Membrane Vesicles from Pig, Sheep, and Rat S. WOLFFR,M~,* E. ANLIKER, AND E. SCHARRER lnstitut fEir Veterin&r-Physiologie, Universit&t Zfirich, Winterthurerstr. 260, 80:57 ZElrich, Switzerland Received March 10, 1986; Accepted April 15, ] 986

ABSTRACT Selenate and selenite uptakes by isolated intestinal brush border membrane vesicles (BBMV) from pig, sheep, and rat were investigated. Selenate uptake into jejunal and ileal, but not duodenal, BBMV from pig was stimulated by an inwardly directed transmembrane N a ' gradient (Na *out > Na 'in). Selenate transport into rat ileal and sheep jejunal BBMV was also enhanced in the presence of a Na ~ gradient. Unlike selenate uptake, selenite uptake was not Na ' dependent, neither in pig small intestine nor in sheep jejunum and rat ileum. Uptake of selenate represented real uptake into the vesicular lumen, whereas selenite uptake was a result of an extensive binding of 7SSe to the membranes. Thiosulfate at a 250-fold concentration of selenate completely inhibited Na ~-dependent selenate uptake into pig jejunal BBMV. Furthermore, Na '-dependent sulfate uptake was totally inhibited in the presence of a 250-fold selenate concentration. The results clearly show that selenate transport across the BBM of pig jejunum and ileum, sheep jejunum, and rat ileum is partially energized by a transmembrane N a ' gradient. Moreover, it is concluded from the results that there exists a common transport mechanism for sulfate and selenate in the BBM. The extensive binding of 75Se from 75Se-labeled selenite to the membranes could be from a spontaneous reaction of selenite with membrane-associated SH groups. *Author to whom all correspondence and reprint requests should be addressed.

BiologicalTraceElementResearch

293

VoL1O,1986

Wolffram, Anliker, and Scharrer

294

Index Entries: Selenium salts, and uptake into intestinal brush border membrane vesicles; selenium salts, and transport at various intestinal sites; selenium salts, and intestinal transport in pig, sheep, and rat; selenate, and competitive inhibition of sulfate transport.

INTRODUCTION It is well established that selenium (Se) is an essential trace element in h u m a n and animal nutrition. Selenium is a component of glutathione peroxidase (GSH-Px, EC 1.11.1.9), and a low activity of this e n z y m e is related to Se deficiency (1,2). Van Vleet has summarized specific disorders associated with Se/vitamin E deficiency in domestic animals (3). Although the most important forms of Se in naturally occurring foodstuff are the organic, protein-associated forms (4), selenate is also present in certain foodstuff (5), and the addition of inorganic Se salts (selenite, selenate) to mixed feed is the most common and economical means of supplying Se to animals in Se-deficient areas (3). A recent study (6) in an extremely low-Se area in China showed selenite to be less efficient than Se-methionine in increasing plasma and red blood cell Se concentrations w h e n orally applied to humans, whereas selenite was as efficient as Se-methionine in increasing GSH-Px activity. Beilstein and Whanger (7) concluded from the distribution of Se and GSH-Px in blood fractions from humans, rhesus and squirrel monkeys, rats, and sheep that GSH-Px activity may not be a good measure of Se status in higher primates. Nevertheless, knowledge about the mechanisms of enteric Se absorption from selenate and selenite is of interest, at least in animal nutrition. McConnell and Cho (8) concluded from their experiments with everted sacs of hamster small intestine that intestinal absorption of selenite occurs by simple diffusion. Former investigations in our laboratory using an in-vivo-perfusion technique have shown that selenate is absorbed faster than selenite by the rat lower small intestine (9). Experiments with everted gut sacs revealed an active Na * -dependent transport of selenate in the rat ileum, whereas selenite seemed to be absorbed by simple diffusion (10). In a recent study on selenate and selenite uptake by sheep and rat intestinal mucosa (11), selenate as well as selenite uptake occurred faster in rat than in sheep small intestine. With the exception of sheep d u o d e n u m , selenate uptake by rat and sheep intestinal mucosa was Na § d e p e n d e n t (11). Sulfate, thiosulfate (10,ll), and the anions chromate and molybdate (11) inhibited mucosal selenate uptake, probably by sharing a common transport mechanism in the brush border membrane (BBM). The present study was performed to investigate the role of the Na gradient in selenate and selenite transport across the brush border membrane using isolated intestinal brush border membrane vesicles (BBMV) from the pig. Some experiments with sheep and rat intestinal BBMV Biological Trace Element Research

VoL I0, 1986

Selenate and Selenite Uptake into BB34V

295

were also performed for comparison. The use of isolated BBMV allows measurement of substrate transport unaffected by cellular metabolism or efflux processes across the basolateral membrane of the epithelial cell. In particular, unlike whole tissue preparations, isolated BBMV are suitable for investigating the influence of ion gradients on substrate transport under well controlled conditions.

MATERIALS AND METHODS Membrane Isolation Duodenum, midjejunum, and ileum of pigs (about 80 kg body weight) were obtained from freshly killed animals at the local slaughterhouse. Sheep midjejunum was removed from five 3-4-mo-old sheep (mean body weight: 25.5 kg), anesthetized with Xylazin-hydrochloride (Bayer, Leverkusen, FRG, 1 mg/kg body weight, im) and Ketamine-hydrochloride (Parke and Davis Company, Munich, FRG, 2.5 mg/kg body weight, ira). The BBMV from rat ileum (mean body weight: 280 g) were isolated from pooled ileum of eight animals anesthetized with ether. The BBMV were prepared from mucosal scrapings, as described by Christiansen and Carlson (12). The vesicles were preloaded with buffers, as indicated in the legends of the figures and tables. Aliquots of 400 ILL from pig and sheep BBMV preparations were stored under liquid nitrogen (13) until use, whereas the rat ileal BBMV were fresh when used. Purification of BBMV was routinely checked by determination of the activity of the BBM enzyme alkaline phosphatase (AP) (test kit, Boehringer Mannheim GmbH). Final BBM fractions of pig, sheep, and rat intestines showed a 12-18-fold enrichment in AP activity with respect to the original homogenates. Protein was determined by using the Bio-Rad protein assay kit with bovine albumine as standard (Bio-Rad Laboratories AG, Glattbrugg, Switzerland).

Uptake Measurements Uptake of 7SSe-labeled SeO 2 and SeO 2- (7~Se-Na2SeO4, specific activity 190 MBe/mg Se and 75Se-Na2SeO3, specific activity 125 MBe/mg Se, Amersham, International plc. England), 3SS-labeled SO,~ (35SNa2SO4, specific activity 680 MBe/mg S, NEN, Boston), and 3H-labeled D-glucose (3H-D-glucose, NEN, Boston) was determined by a rapid filtration technique described previously (14). Uptake was started by adding a 10-1~L membrane suspension to 20 IxL of incubation medium and stopped by the transfer of 20 IJ,L of this mixture into 1 mL of ice-cold stop solution (mmol/L: 150 NaCl, 10 HEPESFFris, pH 7.4). The composition of the incubation media is indicated in the legends of the figures and tables. The dilution of the sample in chilled stop solution was immediately followed by rapid filtration (nitrocellulose filters, 0.6 p,m pore size, Biological Trace Element Research

Vol. 1O, 1986

Wolffram, Anliker, and Scharrer

296

Schleicher and Schhll, Switzerland). 1he :~!-]and ~~Sactivity remaining on the filters was measured by liquid scintillation counting, whereas the 75Se activity was determined in a Gamma counter. Uptake was related to 1 mg of membrane protein.

Statistical Anatysis The results are presented as means with the standard error of the mean (J? _+ SEM). Generally, group differences were statistically evaluated using the Mann-Whitney u-test (1,5).

RESULTS Figure 1 depicts the time course of SeO~ uptake into BBMV from pig d u o d e n u m , jejunum, and ileum in the presence of an inwardly directed Na ' or K * gradient (100 mmol/L). Selenate uptake at 15 s-3 rain incubation into jejunal and ileal, but not duodenal, BBMV was significantly (p < 0.05) higher under Na *-gradient conditions compared to K *-gradient conditions, whereas equilibrium values were similar under both conditions (Fig. 1, A-C). Thereby, N a ' - d e p e n d e n t SeO~ uptake into jejunal BBMV was somewhat greater than into ileal BBMV. The N a ' - i n d e p e n d e n t uptake (K' gradient) of SeO 2 was quite similar in all segments (Fig 1, A-C). In additional experiments, SeO]uptake into pig jejunal BBMV in the presence of a transmembrane Na ' t.

m

"" o 150-

A

o.. (3)

E -~ 1 0 0 -

E

~

1 50-

! I

O4

d ~

0

0 1A. Biological Trace Element Research

I

!

l

1

2 time, min

3

30 Vol. I O, 1986

r ~

0 Q.

150

C

O}

E 0

E

I

100-

Q.

*/

d se r'~

50-

I I cq

cY 0

O9

0 lB.

i

I

I

1

2 t i m e , min

3

,'//~"

I

30

c~

150-

B

0 Q.

E

~0 I 0 0 -E

d -

g3--

*-" 5 0 ! I

d

CD

O9

0

w

0 1C.

1

I

2 t i m e , min

I

3

~

I

3O

Fig. 1A-C. Time course of SeO~ uptake into pig duodenal (A), jejunal (B), and ileal (C) BBMV; vesicles were preequilibrated in 300 mmol/L D-mannitol and 20 mmol/L HEPESFFris, pH 7.4; incubation media contained (mmol/L): 100 D-mannitol, 100 NaCI (O) or KCI (O), 20 HEPESFFris, pH 7.4, and 0.04 7SSelabeled Na2SeO4; values are means _+ SEM from five pigs; * significant difference (p < 0.05). Biological Trace Elernent Research

297

Vol. I0, 1986

Wolffram, Anfiker, and Scharrer

298 200-

_Z 150c-

O D. 0')

E "5 E c~

/: -t

100-

O. iI

~ 0

50-

0 0

I

I

I

1

2

3

~'//

I

3O

time, min

Fig. 2. Time course of SeO42- uptake into pig jejunal BBMV in the presence of an inwardly directed Na' gradient (O) and under Na ' -equilibrium conditions (9 vesicles were preequilibrated in 300 mmol/L D-mannitol and 20 mmol/L HEPESFFris, pH 7.4 (Na + gradient) or 100 mmol/L D-mannitol, 100 mmol/L NaCI, and 20 mmol/L }]EPESFI'ris, pH 7.4 (Na' equilibrium) and incubated in a medium containing7~(mmol/L): 100 D-manmtol," 100 NaC1, 20 HEPES/ Tris, pH 7.4, and 0.04 Se-labeled Na2SeO4; values are means + SEM of three animals. gradient was c o m p a r e d with u p t a k e u n d e r Na*-equilibrium (Nao,,t = Nai,,) conditions. Uptake u n d e r Na+-gradient conditions clearly exceeded short-term u p t a k e u n d e r Na +-equilibrium conditions, w h e r e a s equilibrium values (30-min incubation) did not differ (Fig. 2). Unlike SeO 2- uptake, SeO 2 u p t a k e by pig intestinal BBMV in the presence of a Na ' or K + gradient did not differ in any of the investigated intestinal s e g m e n t s (Table 1).

Biological Trace Element Research

Vol. I0. 1986

Selenate and Selenite Uptake into BBh,IV

299

+1 +1 +1 +1 +1

~

d d d d d ~ +1 +1 §

~

m

+1 +1

m

N

d d d d d ~ +1 +1 +1 +1 +1

e

2

ddddd'~ +1 +1 +1 +1 +1

+1 +1 +1 +1 +1

i

~

0

2E §

§

+, §

§

~

~

0 e" 9

Biological Trace Element Research

~.~ m~

Vol. 10, 1986

Wolffram, Anliker, and Scharrer

300

TABLE 2 Uptake of SeO 2 and SeO 2- by Sheep Jejunal and Rat Ileal BBMV"

Uptake, pmol/mg protein Substrate, mmol/L

SeO (0.o4)

Incubation time 15 s 30 s 1 rain 3 min 30 min

Sheep jejunum Na ' K' 19.4 25.1 35.0 54.1 78.5

+_ 1.7 _+ 0.6 +_ 1.5 _+ 5.1 +_ 2.4

13.0 16.6 23.3 34.2 74.3

_+ 1.5 _+ 0.2 _+ 1.9 +_ 1.9 _+ 2.5

Rat ileum Na ' K' 43.2 54.8 64.0 68.8 90.8

24.4 28.0 46.4 73.2 94.8

Uptake, nmol/mg protein SeO 2 (0.05)

15 s 30 s 1 min 3 min 30 rain

2.38 3.32 4.65 6.65 7.34

_+ 0.42 _+ 0.31 _+ 0.21 _+ 0.24 + 0.13

2.52 3.54 4.73 6.59 7.85

:+_ 0.38 _+ 0.14 + 0.23 _+ 0.44 _+ 0.11

2.13 2.20 3.59 3.77 5.72 5.93 9.98 10.17 1 5 . 7 3 16.10

"Values are means +_ SEM ot three sheep and of one membrane preparation of the pooled ileum of eight rats; vesicles were preequilibrated and incubated as described for Fig. 1 and "['able 1.

In both sheep jejunal and rat ileal BBMV, SeO42- uptake in the presence of a N a ' gradient occurred faster than u n d e r K+-gradient conditions, whereas SeO 2 uptake was not different u n d e r the two conditions (Table 2). Because the equilibrium values (30-min incubation) of SeO 2 uptake exceeded those of SeO 2 uptake about 60- (pig), 100- (sheep), and 170(rat) fold, further experiments were performed to examine binding of SeO 2 and SeO 2 to the membranes. For these experiments, pig and sheep jejunal and rat ileal BBMV were incubated for 30 min at varying osmolarities of the incubation m e d i u m . Equilibrium uptake of SeO4s h o w e d inverse relation of uptake and osmolarity (Fig. 3A). These findings indicated that SeO42- was transferred into an osmotic reactive space (vesicular lumen). Since extrapolation of SeO 2 uptake to infinite osmolarity resulted in a very small SeO 2 uptake, it can be a s s u m e d that only negligible binding of SeO 2 to the m e m b r a n e s occurred (Fig. 3A). Unlike SeO 2 uptake, SeO 2- uptake was not influenced by increasing m e d i u m osmolarity (Fig. 3B). Extrapolation of SeO,~- uptake revealed that the SeO 2- uptake was totally the result of binding to the membranes, at least at a 30-min incubation (Fig. 3B). Furthermore, unlike SeO 2- uptake, SeO 2 uptake was not related to the protein concentration of the m e m b r a n e suspension (Fig. 4, A and B). This explains the difference in the equilibrium values of SeO 2- uptake between rat BBMV (protein: 8 mg/mL) and pig or sheep BBMV (protein: about 15 mg/mL), expressed as nmol/mg protein (Tables 1 and 2 and Fig. 3B). Biological Trace Element Research

1/ol. 10, 1986

Selenate and Selenite Uptake into BB/~ V 9

301

@

U9

...t

I ~

~

0

E CO

Ol

-

o

Ckl

I (

I

I

I

I I

I

~

0~, .- ~- =~

!

I

I

~

l

0

"1 oO

~6~

o 9"u @ ,-.t ~ ."=- L,.) ~ l (D

I ~1"

9 ~ Z~

0

I 04

O

U!UJOE 9Ll!O.],OJd 6w/IOWU 'o~leidn -_sEOOS

~-I.O

0 .,,,u 8 ~

CO

C~

"6 < = o =~ E ~u -- 0,~=~ o~ ~'c-

E

9 .+~

g .,..,, > ::z: ~ ~,'c,,

Or,

Z

~

X \ \\\ I

O O w--

I

O cO

I'

O CO

I

O ,~-

1

O Ckl

0

O

o~:Zo

U!U,J OE: "U!O],oJd 5LU/ IOLUd 'O)lei.dn-_E~'OOS Biological Trace Element Research

Vol. 1O, 1986

Wolffram, Anliker, a n d Scharrer

302

0 "04

t

o

_0 .~

. c" -

~

!~

8~ ~~8 ~ ~EE~ = o.~.~

~

6

rn m

t

d

I

o

I

I

d

d

o

"=

".20

"5" >-S~ Z .~ . ~ ~ .~_-~

ultu OC / lOUJU 'a~e~.dn -_i~s

_o

~ . e N d '~o

\ ""o -lid

I

0

I

I

140

0

ulw OC / I o w d Biological Trace Element Research

I ~0

'sEfi~.'S ~.

"--

~0 ,~

0

._~

~ 6 _.~

~

~

0 0

9

'e~le~,dn- _at'oe9 Vol. 10, 1986

Selenate and Selenite Uptake into BB/vIv

303

140-

control

120G r 0

"{3..

100-

t

_L

E "~

E O. d ,-

80-

4-

+ 1.0 mmol / L $2032~

+

60-

0.

~

4-

!

'

40-

9

~ ~ 0

m m o l / L $2032-

20-

01 r

,

0

1

,

,

2

3

/,"

,

30

t i m e (min)

Fig. 5. Influence of 1.0 (~) or 10.0 (0) mmol/L 520 2 on Na +-dependent SeO 2 uptake into pig jejunal BBMV; incubation conditions were identical to those described for Fig. 4, with the exception of the addition of 1.0 or 10.0 mmol/L Na2S203; values are means _+ SEM of 7-9 pigs; significant differences from the control values are indicated by + (p < 0.05) or * (p < 0.002).

T h e influence of $ 2 0 2- (1.0 a n d 10.0 mmol/L) on SeO 2 u p t a k e into pig jejunal BBMV is s h o w n in Fig. 4. Thiosulfate at both c o n c e n t r a t i o n s significantly (p < 0.05 at 1.0 m m o l / L a n d p < 0.01 at 10.0 m m o l / L $20~ ) d e c r e a s e d SeO 2 u p t a k e in the p r e s e n c e of a 100-mmol/L Na + g r a d i e n t (Fig2, 5). T h e inhibitory effect was m o r e p r o n o u n c e d at 10.0 m m o l / L $ 2 0 ~ - t h a n at 1.0 m m o l / L $ 2 0 2 in the incubation m e d i u m . Furtherm o r e , Na ' - d e p e n d e n t SO 2 u p t a k e into pig jejunal BBMV was strongly inhibited by 10.0 m m o l / L SeO 2 , w h e r e a s D-glucose u p t a k e w a s not i n f l u e n c e d by the p r e s e n c e of 10.0 m m o l / L S e O ~ (Table 3). Sulfate u p t a k e u n d e r Na ' - g r a d i e n t c o n d i t i o n s in the p r e s e n c e of 10.0 m m o l / L SeO 2 w a s similar to SO 2 u p t a k e u n d e r K ' - g r a d i e n t c o n d i t i o n s .

Biological Trace Element Research

VoL I0. 1986

304

Wolffram, Anliker, a n d Scharrer

TABLE 3 Influence of 10 mmol/L SeO42 on SO 2 and D-Glucose Uptake into Pig Jejunal BBMV'

Incubation, condition

Incubation, time

SO~ uptake, l prnol/mg protein

Glucose uptake, 2 nmol/mg protein

Na* gradient

15 30 1 3 30

s s rain rain min

57 91 112 120 83

_+ 12 _+ 18 _+ 19 _+ 9 _+ 3

0.80 1.03 1.13 0.64 0.20

Na- gradient + 10 mmol/L SeO~

15 s 30 s 1 rain 3 rain 30 rain

14 21 32 44 68

_+ 1 _+_ 5 _+ 5 _+ 11 __ 7

0.98 1.27 1.18 0.67 0.20

K

15 s 30 s 1 rain 3 rain 30 min

13 25 34 45 74

_+_ _+ _+ _+ _+

0.09 (I.10 0.12 0.16 0.20

gradient

2 5 6 8 7

"Values are means _+ SEM of one (glucose uptake, triplicate determination) or three (SO~ uptake, duplicate determination) experiments; vesicles were preequilibrated with 300 mmol/L D-mannitol and 20 mmol/L HEPES/Tris, pt] 7.4; inct,bation media contained

(mmol/l,): 100 l)-mannitol; 100 NaCI or KCI; 20 [IEPES/Tris, pH 7.4; 0.04 >'S-labeled Na2SO4 or 0.1 ~Hqabeled D-glucose; and 10.0 Na2SeOa, as indicated.

DISCUSSION The results presented here clearly d e m o n s t r a t e that selenate uptake into BBMV from pig small intestine, with the exception of the duodenum, is stimulated by a t r a n s m e m b r a n e Na" gradient. The N a + - d e p e n d e n t selenate uptake into jejunal BBMV seemed to be s o m e w h a t greater than into ileal BBMV, whereas N a + - i n d e p e n d e n t uptake was quite similar in the d u o d e n u m , jejunum, and ileum. Selenate uptake into sheep jejunal and rat ileal BBMV could also be divided into Na ~d e p e n d e n t and - i n d e p e n d e n t components. The results obtained with intestinal BBMV are in keeping with our former results derived from experiments employing perfusion of intestinal segments in vivo (9), everted gut sacs (10), and a mucosal uptake preparation (11). Those experiments d e m o n s t r a t e d Na ' - d e p e n d e n t , active selenate uptake in the rat (lO, ll) and sheep (ll) small intestines, with the exception of sheep d u o d e n u m . The greatest uptake rates were found in rat ileum and sheep jejunum. Thus, the middle and lower small intestines seem to be the major site of selenate absorption in rat, sheep, and pig. Biological Trace Element Research

VoL 10. 1986

Selenate and Selenite Uptake into BB~,IV

305

In our previous work concerning intestinal selenate absorption, we could not, however, distinguish between the influence of Na + and a Na ' gradient. In the present experiments it was therefore shown for the first time that a Na ' gradient across the BBM stimulates selenate transport. Unlike selenate uptake, selenite uptake was not enhanced in the presence of a transmembrane Na ' gradient in pig, sheep, or rat intestinal BBMV. This agrees in general with our previous investigations, which apart from sheep midjejunum (11), did not yield a Na ~ dependence of intestinal selenite transport (I0,1I). To prove whether selenate and selenite are taken up into the vesicular lumen or are only bound to the membranes, equilibrium uptakes of selenate and selenite were measured at varying osmolarities of the incubation medium (16). Increasing the osmolarity resulted in a decrease of selenate but not of selenite uptake. Furthermore, extrapolation to infinite osmolarity (16) indicated only a negligible binding for selenate but an extensive binding of 7~Se from selenite to the membranes. The binding of Se from selenite might be the result of a spontaneous reaction of selenite with membrane-associated SH groups, because selenite undergoes reductive metabolism without enzymatic catalysis (17). Thus, Anundi and coworkers (18) found a rapid initial loss of selenite from the incubation medium, probably resulting from cellular binding, when hepatocytes were incubated in selenite-containing media. In previous work, an inhibitory effect of sulfate and thiosulfate on selenate uptake by the rat ileum and sheep jejunum was demonstrated (lO,1l). Therefore, a common transport mechanism for sulfate and selenate was suggested. This is also supported by the findings of Cardin and Mason (19), who found an inhibition of active sulfate uptake into everted sacs of rat ileum in the presence of selenate. The results of the present study clearly demonstrated that selenate, thiosulfate, and sulfate are transported across the BBM of pig jejunum by a common, Na '-dependent mechanism because 10.0 mmol/L of thiosulfate completely inhibited Na ~-dependent selenate uptake, and 10.0 mmol/L of selenate in the incubation medium resulted in a complete inhibition of Na+-dependent sulfate uptake. Although Se salts are not necessarily the optimal sources of supplemental Se for man and animals, our findings on intestinal selenate and selenite transport are of some practical interest for animal nutrition because oral administration of Se salts in live stock production is a common practice in Se-deficient areas (20-22).

ACKNOWLEDGMENT This work was supported by the Schweizerische Nationalfonds (Grant No. 3.937-0.84) Biological Trace Element Research

VoL I O, 1986

306

Wolffram, Anliker, and Scharrer

REFERENCES 1. J. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. E. Hoekstra, Science 179, 588 (1973). 2. W. G. Hoekstra, Fed. Proc. 34, 2083 (1975). 3. J. F. VanVleet, ]. Am. Vet. Med. Assoc. 176, 321 (1980). 4. D. E. Ullrey, in Selenium in Biology and Medicine (J. E. Spallholz, J. L. Martin, and H. E. Ganther, eds.), Avi Publishing Company Inc., 1981, pp. 176-197. 5. O. E. Olson, E. ]. Novacek, E. I. Whitehead, and I. S. Palmer, Phytochemistry 9, 1181 (1970). 6. X. Luo, H. Wei, C. Yang, J. Xing, X. Liu, C. Qiao, Y. Feng, J. Liu, Y. Liu, Q. Wu, X. Liu, J. Guo, B. J. Stoecker, J. E. Spallholz, and S. P. Yang, Am. ]. Clin. Nutr. 42, 439 (1985). 7. M. A. Beilstein and P. D. Whanger, J. Nutr. 113, 2138 (1983). 8. K. P. McConnell and G. J. Cho, Am. ]. Physiol. 208, 1191 (1965). 9. S. Wolffram, F. Ard/iser, and E. Scharrer, J. Nutr. 115, 454 (1985). 10. F. Ardhser, S. Wolffram, and E. Scharrer, J. Nutr. 115, 1203 (1985). 11. F. Ardhser, S. Wolffram, E. Scharrer, and B. Schneider, Biol. Trace Elem. Res., in press. 12. K. Christiansen and J. Carlsen, Biochim. Biophys. Acta 647, 188 (1981). 13. B. R. Stevens, S. H. Wright, B. S. Hirayama, R. D. Gunther, H. J. Ross, V. Harms, E. Nord, I. Kippen, and E. M. Wright, Membr. Biochem. 4, 271 (1982). 14. S. Wolffram, H. Giering and E. Scharrer, Comp. Biochem. Physiol. 78A, 475 (1984). 15. L. Sachs, Angewandte Statistik, 6th ed., Springer Verlag, New York-Heidelberg-Berlin, 1984, pp. 230-235 and 244-246. 16. U. Hopfer, Am. ]. Physiol. 233, E445 (1977). I7. G. F. Combs and S. B. Combs, Ann. Rev. Nutr. 4, 257 (1984). 18. I. Anundi, J. H6gberg, and A. Stahl, Arch. Toxicol. 50, 113 (1982). 19. C. J. Cardin and J. Mason, Biochim. Biophys. Acta 394, 46 (1975). 20. E. D. Andrews, W. J. Hartley and A. B. Grant, NZ Vet. ]. 16, 3 (1968). 21. J. F. VanVieet, Aln. J. Vet. Res. 48, 1180 (1982). 22. A. Mathis, H. Jucker, and H. Horber, Schweiz. Landwirtsch. Monatsh. 60, 443 (1982).

Biological Trace ElementResearch

Vol. 10, 1986