383 — Trace metal requirements in total parenteral nutrition

Bioelectrocizemistry and Bioenergetics, 8 (1981) 49-62 A section of b. Electroanat. Chem., and constituting Vol. 128 (1981) Risevier Sequoia S.A. Lauaanne - Printed in The Netherhands

383 --TRACE METAL NU’I’R?iTXON

REQ tJIREMEmmmTlpLPARENTERAL

Il. POTENTIOMETRIC STUDY OF THE MEX’AL-ION EQUILIBRIA ZINC-HXSTIDINE, ZINC-GLYC-lNEl, ZINC-CYSTEINE-~m, Zmc4.x4YcmE-HIS~~ AND miNc-GLYCmE-CYsrEINEi SYSFEMS UNDER PEYSIOLOGICAL CONDlT’IONS * T@LAY

ALEMDAROGLU

49

and GUY BERTHON

IN THE

**

Laboratoire de Chimie I. Electrochimie et Intemcfions. 40. auenue du Recteur Pineau, 86022 Poitiers (France) (Manuscript received January 24th 1980)

In order to abow a better simulation of the distribution of the zinc complexes that pertain to a nutritive mixture used in total parenterai nutrition (TPN), the equilibrium constants for the ternary systems zincr-cystein~histidine, zincglycine-histidine and zinqlgcinecysteine, and the binary systems zinwhistidine and zineycine have been investigated - or reinv&igateci when already studied - under experimental conditions corresponding to blood plasma (37%, 0.15 M NaCIO4). The incidence of the results on the previousIy predicted dose of zinc for TPN is briefly discussed. INTRODUCTION

It is now well documented that an active competition exists for zinc between certain proteins and amino-acids in plasma [I,21 and that, among the latter, cysteine and histidine are responsible for the largest binding of the low-molecular-weight complexed fraction of zinc in that biofluid [2-63. In addition, these two ammo-acids have also been proved to induce excessive urinary excretions of zinc, when administered intravenously to animals or humans [‘7-lo]. The implications and conseqtienks of such observations for the clinical treat-1 ment known as total parenteral nutrition (TPN) have thus been recently mvestigated on the basis of computer simulation models [II]. The increase of the cysteine and histidine plasma concentrations during TPN have heen recognized as responsible for the mobilization of zinc from loosely bound proteins, inducing the excessive zinc urinary excretions observed, whereas glycine had practically no effect. In fact, all these detailed simulation predictions have confirmed earlier clinical observations [l.O] . Moreover, an attempt has also been made to predict the necessary amounts

l l

PaJitI,seeref_ 11. * To whom correspondence should be addressed

0 3024598/81/0000-0000/$02.50 0 EIsevier secluoia Sk

-

60

7

-_

of metals, particularly zinc, to be included in the nutritive mixtun% so that such extraordinary losses could be balanced [ll]. The calculation of these amounts clearly depends on the metal complex composition of the mixture used. thus on the exactitude of the pertaining stabilim constants available in the literature. Among the distribution of the numerous species corresponding to such direct or statistically combined literature data, the zinc binary and ternary complexes of cysteine and histidine were found to be predominant, together with their related ternary species with glycine, in the most commonly used nutritive mixture in the University Medical Center of Poitiers ]12]. Accordingly, the most urgent task before going further into metal dose predictions was to determin e (or to ascertain when existing) the stability consfzmts of the zinc-cysteine-histidine, zinc-glycine-histidine and zinc-glycinecysteine systems. Indeed, some of the already available ternary constants of the first two systems 131 are somewhat inaccurate or even need confirmation, the more so as the data of the present binary systems used for the previous determinations are also liable for improvement in the experimental conditions required [S]. Thus, together with the study of the three ternary systems quoted above, we also had to reinvestigate the zinc-hi&dine and zineycine binary systems. EXPERIMENTAL

Reagents The three amino-acids employed were supplied by Merck as biochemical grade products and their purity was checked by potentiometric titration before use. Stock solutions of zinc perchlorate in acid were prepared from cLystals supplied by Pierce Inorganics B.V., and the metal and strong acid contents determined by complexometric titration against EDTA 1131 and from direct potentiometric measurements using the equipment described below. Sodium perchlorate used as the background salt was Merck reagent grade. Sodium hydroxide solutions were prepared from BDK concentrated volumetric solutions with freshly boiled deionized water; their titre was systematically checked by volumetric titrations against potassium hydrogenophthalate Prolabo R.P_ p-a. Perchloric acid was also supplied by Prolabo RP., as a Normatom grade product, and was titrated against standardized sodium hydroxide before use_ Method

axd equipment

Ah measurements were carried out as potentiometric titrations in an Ingold reaction cell system, thermostatted at 37 + 0.02”C by circulating water and maintained under an atmosphere of thermostatted, scrubbed, oxygen-free nitrogen supplied as “U” by 1’Air Liquide. The ionic medium to hold the activity coefficients constant was I = 0.15 mol dmm3, this being isotonic with blood plasma. The potentials were measured with a digital mV meter Beckman Model 4500, equipped with a Beckman glass electrode S 39301 and a saturated sodium chlo-

51

ride caIomeI

G.E. I Iigand,

eIecWade

from Beckman,

Zn**, NaCIOi

0.15

arranged

M II NaCI

as below:

I Hg#I,

(1)

- Hg

(=W proved to be carbonate-&e by Gran titration 1143, was stored i_nand delivered from a Radiometer autoThe

standardized

hydroxide solutions,

sodium

TABLE1 Initialtotalconcentrationsofzinc,ligandsandstrongacid used forthe titrationsofthesystemunderinvestigation System

=ZZl (-w

Roton-histidine

CL (=m

cx hw

20.00

24.?5

Titrant

NaOH(mM) 100.0

20.00

24.75

100.0

10.00 10.00

24.75 39.60

100.0 100.0

24.95 25.27 25.79 25.79 25.27 25.27

100.0 100.0 100.0 100.0 100.0 100.0

25.79

25.79 25.79 10.42 25.27

100.0 100.0 100.0 100.0 100.0

10.00 10.00 20.00

24.70 39.60 24.70

99.6 99.6 99.6

25.76 25.24 26.80 25.24 25.45 25.76 24.93

99.6 99.6 99.6 99.6 99.6 99.6 101.5

ZinAistidine

7.12 5.07 10.15 10.15 5.07 5.07

19.58 10.00 10.00 20.00 10.00 20.00

Zincrcyatein&istidine

10.15 10.15 10.15 5.07 5.07

10.00 10.00 20.00 5.00 10.00

Proton-glycine

=H (-1

LO.00 20.00 10.00 5.00 10.00

Zindycine

10.15 5.07 20.30 5.07 7.10 10.15 2.00

10.00 10.00 10.00 20.00 20.00 20.00 20.00

Zixz~lycine-histidine

10.15 10.15 10.15 20.30 5.01 5.01

10.00 20.00 10.00 10.00 5.00 10.00

10.00 10.00 20.00 10.00 5.00 10.00

30.71 25.76 25.76 37.18 25.24 30.19

100.0 100.0 100.0 100.0 100.0 100.0

Znwlycine-cysteine

10.15 10.15 5.01 5.01 5.01 3.00

10.00 20.00 5.00 5.00 10.00 6.00

10.00 10.00 5.00 10.00 5.00 3.00

41.70 35.76 50.02 35.24 30.24 23.09

100.0 100.0 100.0 100.0 100.0 100.0

52

burette ABU 12. The electrode system was cslibrated by determination‘of formal potentials using readings from solutions of known concentrations of hydrogen ion; pK, was used as found (13.38) in an earlier work of one of us

ISI. The initial overall concentrations of the reactants used for the titrations of

the proton-@and, Table 1. CWcuhtion

zinc binary and ternary systems investigated are detailed in

of formutixz

constants

The potentiometric titration data for the proton-hgand and zinc binary systems were treated with the Miniquad program 1151. As in our experience the SCOGS least-squares program [ 161 is able to accommodate estimations of stability constants more widely removed &om the limit values than Miniquad, both SCOGS and Miniquad computations were employed for the zinc ternary systems, in such a way that the SCOGS outputs were finally refined by Miniquad. The principle of the two-stepped (optimization/simulation) computational approach used has already been thoroughly developed in an earlier paper [S] . Nevertheless, particular emphasis should be given on the fact that the different sets of constants corresponding to all the possible combinations for each system were not only tested on the basis of numerical criteria (sum of squared residuals), but also on graphical comparisons between experimental and simulated data. The latter were obtained from the proton concentrations iteratively calculated from the set of constants under consideration and the analytically known total concentrations of the reagents. For the binary systems, the calculation was carried out by means of the Pseudoplot program 1171, making use of the formation function: T=

CL-

([L] f [HL] + [H*L] +- _.__) Chf

_

(2)

in which cL and cbl represent the total ligand and total metal concentrations respectively, and which is established independently of the metal complex species existing in the solution. For the ternary systems, the original Pseudoplot pmgram was modified to allow the iterative calculation of the pseudo-averaged degree of protonation of the two ligands L and X: s= c~+NDP~X~~+NDP~X~~-~~~+[OH]-[H] CL+

(3)

cx

where cB cL cxl con respectively stand for total concentrations of strong acid, first ligand L, second figand X and sodium hydroxide in the solution, and NDP for the number of Wiable protons of each ligand. RESULTS

The final results chosen as the be& sets of constants for each system are shown in Table 2. The other possible combinations which gave rise to numerical

53 TABLE2

.

FormationconstantsasobtainedfromMiniquadcalculations. Thegeneralformda of acompk~i~Z~XJr?s whereLandXrepresenttheligandsintheorderofthedeftitionofthe -systems. H=protonwhenpositive,hydrosidewhennegative;n = numberofexperimentalobservations;S=sumof.squeredresidu& System

P

q

p

s

log8

c_

S

n

1 2 3

8.770 14.643 16.400

0.002 0.003 0.007

O.lllE-05

180

0 0

6.385 11.558

0.003 0.006

0.148E-O5

230

Sameset asinRefs_ 3.19-24

0 0 1

6.388 11.592 10.525

0.003 0.007 0.063

0.1223-05

230

Sameset asinRefs. 19and22

0 0 1

6.351 11.550 16.713

0.006 0.006 0.058

0.120E-05

230

0 0 1 1

6.336 11.599 10.718 16.919

0.004 0.004 0.031 0.030

0.6093-06

230

1 2

9.239 11.654

0.005 0.007

O-2423-05

115

0 0 0 -1

4.792 8.795 11.181 -3.365

0.006 0.020 0.050 0.081

O-2323-05

272

0.006 0.008 0.155 0.047

272

0 0 -1

0.006 0.008 0.020

0.2083-05

272

8 0

4.784 8.897 10.707 -0.602 4.783 8.900 -0.467

0.1893-05

0

0 0 0 -1

4.832 8.931 10.767 10.073 -0.572

0.005 0.008 0.137 0.079 0.045

0.167E-05

272

0 0

0 0 0 1 -1 0

4.832 a.919

0.005 0.012

0.166E-05

272

0.2143-05

272

1

0

0

1 1

0 0

II-AZinc1 histidine 1

1 2

0

II-BZinc1 histidine 1 1

1 2 1

0 0

DIGZinc1 bistidine 1 1

1 2 2

0

II-DZinchktidine

1 1 1 1

1 2 1 2

0 0

JIIRotop glycine

0 0

1 1

IV-AZincr glycine

1 1 1 1

1 2 3 1

0

1 1 1 1 1 1 1

1 2 3 2 1 2 2

0 0

1 1 1 1 1

1 2 3 1 2

1

1

0

: 1 1 1

: 1 1 2

ii : 0 1 0 -1 0 -1

1 1 1 -a

1 2 3 *

0

IRoto~ 0 histidine 0

IV-BZincr glycine

Iv-cZin~ glycine IV-DZincr glycine

TV-EZinr glycine

IV-FZincglycine

0

0

0 0

0

0 0

0

0 0 0

0

0

0 0

0

0 0 0

n

1

0

10.762

0.126

10.065 -4.230 -0.591

0.081 0.561 0.053

4.835 8.834 11.234 Inn.29

0.006 0.020 0.047 nna7

Notes

Bestset, sameas in Ref. 24

Bestset

Sameset asinreh.

17and25

54 TABLE2(contiimed) P

9

r

s

1-B

r

s

n

Notes

V-AZine 1 cysteine 1 histidine

1 1

1 1

0 1

15.090

0.032 0.028

0.862345

288

Bestset

21.233

V-BZincr 1 cysteine 1 histidine 1

1 1 1

1 1 2

0 1 1

15.041 21.228 25.536

0.034 0.026 0.131

0.691E-05

288

Sameset asin Ref.3

V-CZincrcystcine histidine V-DZinc~ cysteinhistidine

1 1 1 1 1 1 1

1 1 1 1 1 1 1

1 1 1 1 1 2 1

0 1 2 0 1 1 2

15.136 21.256 25.952

0.028 0.027 0.141

0.6763-05

288

15.073 21.272 25.555 25.928

0.030 0.025 0.124 0.143

0.621E-05

288

Vi-AZinr1 glycinehistidine

1

1

0

10.590

0.023

0.2213-05

212

VI-BZinc~ 1 glycine- 1 histidine

1 1

1 1

0 1

10.623 16.576

0.021 0.066

0.181E-05

212

VI-CZine giycinehktidine

1 1 1 2

1 1 2 1

0 1 1 1

0.173E-05 212 10.602 0.022 16.518 0.077 21.890 0.143 constantmadenegativeduringrefmement)

1 2 1

1 1 2

1 1 1

15.738 21.780 21.820

0.713 0.060 0.311

0.4833-05

212

1

1

1

1

19.892

0.091

0.5603-06

155

VII-BZincr- 1 glyciIl@F 1 cysteine

1 1

1 1

0 1

11-987 19.895

t-109 0.091

0.5593-06

155

M-CZincrglycille cysteine

1 2 2 1

1 1 1 2

1 0 1 0

19.922 16.166 24.752 19.747

0.069 0.051 0.148 0.148

O-3563-06

155

System

1 1 1 (1 VI-DZincr1 glycine 1 histidine 1 VII-AZincglycine cysteine

1 1 1 1

Bestset

Sameset asinRef. 3

Bestset

fits of about the same order, but which have been finally discarded for definite graphical reasons, are also given. The technicaI observations tbrcughout the calculations and the details of the numerical and graphical comparisons between the different sets of constants are successively reported below for each system investigated.

55

Zinc-his

fidine

Un@e that previously found for the zinW& system [18], it appears from the protonation curve of histidine in the presence of various concentrations of zinc (Fig. 1) that this l&and may bind the metal under its protonated fOEStl_ This observation is supported by the variation of the sum of squared residuals S from one set of con&auk to another, as can be seen in Table 2. The addition of the MLH or MLH species separately to the initial set of the main complexes ML and ML does not notably improve the sum S, but when they are introduced together the sum S is decreased almost to half of its value in the initial set. Moreover, the existence of MLH and ME&I is definitely contied by the plots of the formation function of the system for the two cases under examination. It can indeed be seen &om Fig. 2 that the existence of MLH and M&H is required to interpret properly the experimental splitting observed on the curve aroundF = 1.

Zinqlycine

For the different ligand/metal ratios shown in Table 1, the formation curves of this system are superimposable within the range P = 0 to ?; = 2, except a few points around7 = 1. The deviation of these points is probably attributable to the formation of MLOH as a minor species (Fig. 3). Beyond T; = 2, the split curves rise to F = 3 without reachirig a limit and would pass overF = 3 if no precipitate had appeared in the solution. This particular shape can stem from the formation of ML3 and/or hydroxylated species of ML*. From the set of constants shown in Table 2 IV-A. it seems that ML, ML, and

zinc 0 A Q 2

7 5 10

or020 .5 D520

HIS (mM) 20 10 T-0 lo

56

Fig. 2 Foxoation ewe of the zinehistidine system. The solid line is given for the &st set of constants (Table 2 II-D). Only the experimental points are materialized.

Zmc 0

A v 0

f b x

70 5 20

Gly (m&f) 10 70 10

52c 7 20 x3 20 2 20

Fig. 3. Formation curve of the zineycine system. The solid line is given for the ksf set of constants (Table 2 IV-D). The broken Line is given for the remIts in Table 2 IVC. The experimental points are materialized.

57

MLa, are the main species, but the corresponding pseudo-formation function does not fit with the experimental one. If MLOH is replaced by MLOH in this set, the latter becomes the major species at the end of the formation curve in place of ML3 (Table 2 IV-B), which greatly improves the coincidence beCween the pseudo and experimenti formation curves (Fig. 3). Finally, ML, ML, and M&OH are recognized as the main complexes of this system (Table 2, W-C), ML, and MLH appearing as minor species (Table 2 IV-B and IV-D) and MLOH as negligible (Table 2 IV-E). Zinc--qwteinetrisfine

As indicated above, both the SCOGS and the Miniquad programs were successively used for the treatment of the da+& As their result.swere very close to one another, only those obfained by Miniquad have been introduced in Table 2. As it can be seen from the lanker,the main species are MUC and MLXH (Table 2 V-A). The complexes MI&H and MLXH, were also possibly present iu the solution as minor species, but they did not noably improve either the sum S or tie graphical fiti (Fig. 4). On addition, their maximum percentage in the solutions investigati were only 11% and 5% respectively (Table 2 V-D).

MLX was formed as the major species of this system (Table 2 VI-A). Indeed, the presence of MLXH together with MLX in the set VI-B in Table 2 brought Little improvement to the SW of squared residuals and, even though it improved the standard error observed on MLX refined by itself, its maximum percentage throughout all the experiments reached only about 6%. Furthermore, when the constant.sof the MLX, MLXH and ML&H species

l-

Fig.4. Roto~tionc~forthe~~~e-irist~~~~(c~,=10mM, ~,~=lOmM,~=ZO~~esolidLinerepresentsthesystem tithoutanyteernarpspecies. The broken lime is giGen for the best set of constants (Table2 V-A) and the dotted linefor the resulbin Table 2 V-R The experiatenhl points are materialized.

58

I

2

3

1

1

5 -Log

I

A

7

e

[H-]

Fig 5. Protonation curve for the zinc-glycine-histidine ternary system (ca = 10 mM. = 10 m&f, cIris = 10 m&f)_ The solid line represents the system without any ternary species. The broken line is given for the best set of constants (Table 2 VI-B). The experimentai points are materSized. %lY

were refined together, almost no improvement was noted in the sum S with respect to that of the set MLX, MLXH (Table 2 VI-C). The fact that the maximum percentage reached by MLX*Ei is only 4.5% in the 1 : 1 : 2 experiment inclines to the consideration of this complex as a minor species. As on the other hand its introduction into VI-C worsens the standard errors on MLX and MLXH, MI&H can definitely be regarded as negligible, the more so as: (i) it is less likely from the chemical point of view than ML2XH which was put out during Miniquad refinement, and (ii) it induces no improvement in the graphical simulations (Fig. 5).

The first observation that can be made on this system is the low importance of the ternary species to be expected from the fairly good coincidence between the experimental protonation curves in the presence of zinc and their simulations taking account of the binary species only (Fig. 6). Accordingly, the constant for MIX was made negative during refinement by Miniquad MLXH was then tried and gave rise to a satisfactory fitting from the numerical viewpoint (VII-A), but its maximum percentage did not exceed 4%, MLX remaining quite negligible (<1.5%) when refined together with it (VII-B). Among the successive combinations of M&X, MI&H, MIX&I and MI&H,, together with MLX and MLXH which did not improve the preceding result, we

59

Fig. 6. Rotonation curve for the zindycineTysteine ternary system (c% = 3 mM)_ The solid line represents the system without any temary 6df,c, the experimental points are materialized.

=

3 idf,

species.

cgly

=

Only

can select as the best possibility of existence for ternary species the set shown in Table 2 VII-C. In that case, the percentage of MUCH, ML& MLIXH and MLX, respectively reached 4%, 20%, 12% and 11.5% for the experiments pertinent to the corresponding M/L/X ratios. Nevertheless, in our opinion, due to the graphical reasons mentioned above, the observation of these species might only arise fiorn the mathematical requirements of the matrix minimization. DISCUSSION

Let us now examine our results comparatively with those obtained by earlier authors and discuss the particular tendency of each ternary system to form mixed-l&and species. Then, since the zinc ternary complexes v&h cysteme, histidine and glycine, as taken from previous studies, conditioned to a large extent the dose of zinc to be incorporated in a nutritive mixture used in TPN [12], we shah briefly investigate the incidence of the present results on its determination. Binmy

systems

Among the various studies avai!abIe in the Iiterature on the xiuc-histidine system [3,19-241, most of them mention the &stence of the ML and ML, complexes exclusively. only Perrin et al. [19,22] cdcuiated the MLH constant, and very recently Cergely et al. obtained both MLH and M&H [24]_ Our results as shown in Table 2 IT-D confirm the latter. The zindycine system has been studied by several authors, some of them

60

mentioning ML and ML, [24], others ML, ML, and ML3 126,271 or even ML, Mb, ML3, MLH and MLOH [17,25]. Our final results in Table 2 IV-D differ slightly from those of the latter which had been obtained in the same experimental conditions as our results. However, none of these authors had considered M&OH as a possibly existing species in their final choice, whereas we have demonstrated that MLOH is more concentrated in solution than ML+ MLOH was also found negligible by us. For the sake of comparison, the set of conskuks proposed by these authors was tried with our data, but it did not give satisfactory fits (Table 2 IV-F). Ternary systems

The zinc-cysteine-histidine system had already been studied by Perrin et al. [ 31 who had characterized the species (with the corresponding constants) MLX

(l&23), MLXH (21.60) and MLX*H (26.50). Our findings (Table 2) are in good agreement with the previous ones as faras the first two constants are concerned, but the present results suggest that MLX,H is almost negligible, since its constant * is found ten times lower than Perrin’s. The ziuc?glycine--histidine system had also been studied earlier by Perrin et al. [3]. They had mentioned the existence of the three species MLXH (17.72), M&XII (23.10) and ML&H (22.79). We do not confirm these results as MLX, which was not mentioned by the previous authors, has been found to be the main species in this system (Table 2 VI-A). Its stability, as well as that of the complex of the same stoichiometry of the zinc-cysteine-histidine system, is a litiJe higher than expected from the following relation [28] : 1% PMIX

= :(log

Parr, f log Pbfx* * log 4)

(4)

which yields 10.56. Moreover, in the recent study cited above [24], Gergely

et al. stated MIX to be the oniy ternary species in this system, which can be considered as a confirmation of our results. As for the zin~ycine-cysteine system, no study on this system was found in the literature to which our results could have been compared. Our main finding is the poor ability of this system to give rise to the formation of tern= species. This is well illustrated by the comparison between the experimental and (respectively 11.987 and 13.718) the statistkelly deduced 1281 values of &= which confirm the destabilization of the mixed-l&and complex.

Simulation

of the distribution

of the species in the three systems

ln order to appreciate the respective importance of each complex in the three systems considered as a whole, we have run COMICS [ZS] calculations for different concentration ratios of the components of these systems. As our principal aim was to define the influence of the preceding results on the determination of the daily dose of zinc to be included in a nutritive mix-

*Thisconstanthasalreadybeentakenaccount 12.

ofinthe cakulationsreportedinRef.lland

61

6

1

2

3

4

-Tog

6

7

0

9

lo

[H-]

Fig. 7. Distribution of the various complexes of zinc with cysteine, glycine and histidine in the concentration conditions of the most commonly used TPN mixture in the University Medical Center of Poitiers M = zinc, L = cysteine, X = histidine, Y = glycine; 1 = M&, 2 = MX,3=MX~,4=MXH,5=MX2H,6=MY,7=MYH,8=MLX,9=MLXH,l0=MLX~H, 11 = MXY, 12 = MXYH, 13 = MXzYH, 14 = MLY*, 15 = MLY,H, 16 = M&Y_ c, = concentration of a given complex; c~ = 0.19 miW, c~ = 0.34 tmIf, c x = 6.24 m&f, cy = 23.22 DIM.

ture, we have, as far as possible, chosen for these calculations concentration ratios similar to those actually present in this mixture. Figure 7 shows the simulation of this distribution. As appears from examination of Fig. 7, the complexes zinc-(cysteine)-(histidine~+3, zindglycine)(hi&idine~-ES, zin~glycine),-(histidine)-I-i which were previously taken as analyfkally important in the mixture on the basis of literature data turn out to be almost negligible. We intend to examine in more detail the simulation of the real system in a following paper, but depending upon the results obtained in the present study, we can already estimate that the above species w-ill be of decreasing importance in the nutritive mixture under consideration [11,12]_ This finally implies that the corresponding dose of zinc sbolrld also be diminkbed.

62 REFERENCES 1 2 3 4

AS_ RasadaadD.

ObezIeas.J.Iab.Qinh?&76

(1970>416.

ELGiroux andRLHenkin.Biochim BiophpzActa.273~l-S72~64. P.S. Hallman. D.D. Pemiu end kE Watt, Biochem. J, 121(1871) 54% D.D. Penin and R_P. Agawal. in H. SigeI (Editor).Metal IOnS in BiOlo&d New York. 1973.p.167. May. P.W. LindaaadD~R.W;tri.m..J.Chem.Soc.~on~.
6 7 8 9 10 11 12 13 14 15 16 17 16 19 20 21 22 23 24 25 26 27 28 29

SYS@~S.

~

me&

G B.er?.&on.P.?&May andD.RWmJ. Cbem 6oc.DelfonRam,~l978~1483RL H-RR Keiser aodD. Bronzert.J. -Invest.. 51<1972)44a. RL Henki~Adv.Exp.Med Biot48<1974)299. RF.% Freeman and P-R Tayloz Am. J. Cliu. Nut?.. 30 (1977) 623. A_A_YUUX, RW. King.Jr..s Kra&itpauitch,C.C.HaygoodandRD. Lindemaa. Am. J-Pb~oL 235<1978)F40. GButhoa.P.M~~andC.~tu~y.Ex:perientia.submitted forpublication G -on. c_ rwatuchauskYand PAL May. J.Inorg. Biocbem.13<1980)63. CompIerometdcTitratonz.Methuen.London.1957. G.8eboparzenhch. KRorrottiThestud~ofIopic~~ri4Longmao. London.1978. k.Sabatki.kVacc_endP. Gens.Telaz1ta.21<1974)53. LG. 8ayce. TaIanta.15(1968)1397. A_bz.CorrIe. GEC.Rm ~.LD.ToucheandD.RW~J.~emSoc.~tOnTranr. <1975)105. kKaya.IiaudG Ber&bon,J.chim. Pbyss..77<1980)333. mD_ Perrim andV-8. F~J_CbemSoc,A(1967)724RP_ ~~dbL~c.BuIlsoe-(3him,6<1969~1866. D_RWilIkc&J_ ChemSoc. A<1970)1550. RP_AgarwaIandD.D. P~J.(=hem~DsltonTrarm.<1975)1045. P-G DanieleaadG OstacoIi.AnnChixn