A proton nuclear magnetic resonance study of the interaction of cadmium with human erythrocytes

Biochimica et Biophysica Acta, 762 (1983) 531-541 Elsevier

531

BBA 11171

A P R O T O N NUCLEAR MAGNETIC RESONANCE STUDY OF T H E INTERACTION OF CADMIUM WITH HUMAN ERYTHROCYTES DALLAS L. RABENSTEIN, ANVARHUSEIN A. ISAB, WEBE KADIMA and P. MOHANAKRISHNAN

Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2 (Canada) (Received November 10th, 1982) (Revised manuscript received March 18th, 1983)

Key words: IH- NMR; Cd 2 +; Ion-membrane interaction," Glutathione; (Human erythrocvte)

The binding of C d 2+ by molecules in the intracellular region of human erythrocytes has been studied by I H - N M R spectroscopy. From changes in spin-echo Fourier transform N M R spectra for both intact and hemolyzed erythrocytes to which C d C l 2 w a s added, direct evidence was obtained for the binding of C d 2+ by intracellular glutathione and hemoglobin. Time-courses were measured by IH-NMR for the uptake of C d 2+ by intact erythrocytes in saline/glucose solution and in whole blood. In both cases, the uptake, as indicated by changes in the 1H-NMR spectrum for intracellular glutathione, plateaus after about 30 rain. The effectiveness of the disodium salt of EDTA and of various thiol-chelating agents for releasing glutathione from its C d 2+ complexes in hemolyzed erythrocytes was also studied. EDTA was found to be more effective than thiols, and dithiols more effective than monothiols.

Introduction Although blood is not the target site in cadmium poisoning [1-4], the interaction of cadmium with the components of blood is of considerable interest, since the blood serves to transport cadmium to other tissues of the body [4]. It is well established that the cadmium in blood is located mainly in the erythrocytes [5-9], e.g., more than 90% of the cadmium in the blood of rabbits following repeated exposure was found in the erythrocytes [6]. However, the identity of those components of erythrocytes which bind the cadmium is less well established. It has been suggested that cadmium is bound mainly by hemoglobin [6], by metallothionein [7], and by a high molecular weight fraction, not hemoglobin, and a second fraction which has a molecular weight similar to, but properties different from, metallothionein [9]. In these studies, the identity of the binding agents has been inferred from zone electrophoresis [6] and gel 0166-3178/83/$0.300 © 1983 Elsevier Science Publishers B.V.

filtration [7-9] experiments. In recent studies, we have shown that metal binding in intact erythrocytes can be characterized by ~H - N M R spectroscopy [ 10-13]. From changes in high-resolution ~H-NMR spectra of intact erythrocytes, zinc [ 10], trimethyllead [ 11 ], methylmercury [12] and inorganic mercury [13] were all found to be complexed by intracellular glutathione (GSH) and hemoglobin. Information was obtained about the nature of the complexes formed and, in the case of methylmercury(II), the relative stabilities of the glutathione and hemoglobin complexes were determined. The ~H-NMR method has the advantage that separations are not required, and the measurements on intact erythrocytes are noninvasive. In a continuation of our research on metal ions in erythrocytes, we have studied by IH-NMR the binding of Cd(II) in human erythrocytes. In this report, we present results concerned with the rate of uptake of Cd(II) by intact erythrocytes, the

532

identity of intracellular molecules which bind Cd(II), and the relative effectiveness of a series of ligands for removing Cd(II) from its complexes with the intracellular ligands. Materials and Methods

Chemicals. Cadmium chloride was obtained from J.T. Baker Chemical Co. Mercaptoacetic acid (Eastman Kodak Co.), cysteine, penicillamine, 2mercaptosuccinic acid, 2,3-mercapto-l-propanesulfonic acid, sodium salt (Aldrich Chemical Co.), glutathione, N-acetylpenicillamine, meso-2,3-dimercaptosuccinic acid and dithioerythritol (Sigma Chemical Co.) were used as received. Their purities had been determined previously [17,18]. Erythrocytes. Erythrocytes were isolated from venous blood as described previously [12]. The blood was collected in Vacutainers (Becton, Dickinson and Co.) containing E D T A solution or, for the cadmium uptake experiments, heparin. Packed erythrocytes were hemolyzed by the freeze-thaw technique or by sonication. NMR meaJurements. 1H-NMR spectra were measured at 360 M H z on a Bruker WM-360 spectrometer or at 400 MHz on a Bruker W H - 4 0 0 / D S spectrometer. Both spectrometers were operated in the pulsed Fourier transform mode. Spectra were measured at 25°C on 0.4-0.5 ml samples of packed cells, cell suspensions, hemolyzed cells or glutathione solutions in 5 m m o.d. N M R tubes. The free induction decay was collected in 8K or 16K of data points with a spectral width of 4000 Hz (at 360 MHz) or 5000 Hz (at 400 MHz). Quadrature detection was used, and generally 100 transients were collected for each spectrum. Chemical shifts are reported relative to the methyl resonance of sodium 2,2-dimethyl-2-silapentane-5-sulfonic acid, based on the resonance for the methylene protons of glycine having a chemical shift of 3.540 ppm. The resonances of interest are those from the small molecules of the erythrocyte. JH - N M R spectra were measured by techniques with which the interfering hemoglobin resonances can be either partially or completely eliminated. In one technique, a selective presaturation pulse is applied at 8.1 p p m for 1-2 s prior to the 90 ° observation pulse [14]. Even though the presaturation pulse is selective, the entire hemoglobin envelope of reso-

nances is suppressed to some extent due to transfer of saturation by cross-relaxation throughout the hemoglobin spin system. In the second technique, spectra were measured by the spin-echo Fourier transform method, which is based on the pulse sequence 90 °-~-2-180°-~'2-acquisition. Interfering h e m o g l o b i n resonances are selectively eliminated on the basis of their short T2 values [ 15,16,19]. Spectra were measured with delay times, ~2, of 0.015, 0.060 or 0.150 s. A ~2 of 0.015 s was used to observe resonances from some of the imidazole side-chains of the hemoglobin histidine residue, and a r 2 of 0.060 s was used to observe resonances from the small molecules of erythrocytes, except in the experiments to follow the time-course for the uptake of CdCI 2 by intact cells, where the longer delay time of 0.15 s was used to eliminate the water resonance [20]. Results and Discussion

The binding of cadmium by erythrocytes The 400 MHz spin-echo 1H-NMR spectrum of packed intact human erythrocytes which had been washed twice with isotonic saline/0.005 M glucose in 2H20 is shown in the upper half of Fig. I. The intense resonance at 4.77 p p m is due to residual H 2 H O in the cells, the other resonances are due to imidazole groups of some of the hemoglobin histidine residues (6.85-8.3 ppm) and to small molecules in the cells. Many of the resonances from small molecules have been assigned [15,21], those of interest in this study are identified in Fig. 1. The lower spectrum is for packed erythrocytes from the same sample after incubation for 1 h in an equal volume of isotonic 2HzO/saline/glucose solution containing 0.002 M CdC12. The most significant change is the disappearance of the glutathione resonances (gl-g5), indicating a decrease in their T2 values. Except for the resonance at 1.28 ppm, there are no significant changes in the other small molecule resonances, indicating little if any interaction of Cd 2+ with these molecules, which include several of the amino acids [21]. The resonance at 1.28 p p m is due to the methyl protons of lactic acid; the change in this resonance is most likely due to the metabolic activity of the cells and not to Cd 2+ binding.

533

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Fig. 1. The 400 MHz IH spin-echo N M R spectrum of 0.5 ml of packed h u m a n erythrocytes (top) and 0.5 ml of packed erythrocytes which had been incubated for 1 h in an equal volume of isotonic 2 H 2 0 / s a l i n e / g l u c o s e solution containing 0.002 M CdC12 (bonom). A r value of 0.060 s was used in the spin-echo pulse sequence. See text for details of cell preparation.

In Fig. 2 are shown portions of 1H spin-echo spectra for the small molecules in hemolyzed erythrocytes when titrated with CdC12. Spectrum A is for the lysate before any CdC12 was added. Spectra B and C are for lysate containing 0.29 mM and 0.85 m M CdC12, respectively. As in the incubation experiment with intact cells, CdC12 causes the G S H resonances (indicated by the arrows) to decrease in intensity and disappear at sufficiently high concentrations. The absence of resonance g2 from spectrum B indicates this resonance to be particularly sensitive to Cd 2+. To determine whether this is the behavior expected when G S H is

complexed by Cd 2+, N M R spectra were measured by the single-pulse method and by the spin-echo method for G S H in solutions to which varying amounts of CdC12 were added. In one experiment, CdC12 was added to a solution containing 2 m M G S H and 0.16 M NaC1, in a second experiment CdC12 was added to a solution containing 2 mM G S H , 0.16 M NaC1 and alanine, creatine, ergothionein and glycine at concentrations similar to those in erythrocytes. In both experiments, CdC12 caused a broadening of the G S H resonances in the single-pulse spectrum, which corresponds to shorter T2 values and thus decreased

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Fig. 2. Portions of the 400 MHz JH spin-echo N M R spectra of hemolyzed erythrocytes to which CdCI 2 had been added. The Cd 2+ concentrations are (A) zero, (B) 0.29 mM or (C) 0.85 mM. The arrows indicate the positions of GSH resonances gl, g2, g3 and g4 (left to right).

intensities in the spin-echo spectra. Results from the first experiment are shown in Fig. 3. Of particular interest is resonance g2, which is quite broad in spectra B - D and either very much reduced in intensity or completely eliminated from spectra B' D'. These spectra indicate that resonance g2 is most sensitive to binding of Cd 2+ because its chemical shift changes more than that of the other resonances upon complexation. Thus, exchange of G S H between its free and complexed forms on the N M R time-scale causes more broadening of resonance g2, and a shorter effective T2. This is consistent with previous studies which have shown Cd 2+ to bind preferentially to the sulfhydryl group of G S H [22]. The complete disappearance of resonance g2 when only 0.29 mM CdC12 has been added (spectrum B in Fig. 2) indicates exchange of G S H between its free and complexed forms on the N M R time-scale. If exchange were very slow, resonance g2 would decrease in intensity in a stepwise fashion until all the G S H was complexed. Although the stoichiometry of the GSH complex(es) which form in erythrocytes is not known, it is unlikely that all of the G S H is complexed in spectrum B, since the normal G S H level in erythrocytes is 2.1 m M [23]. This is supported by the additional decrease in resonances gl, g3 and g4 when more CdC12 is added (spectrum C). Thus, for resonance g2 to disappear completely in spectrum B, the G S H must be exchanging between its free and complexed forms. The behavior of resonance g2 in the spectra measured by the singlepulse method in Fig. 3 indicates this to be the case. From these results, the average lifetime of free G S H in the lysate containing 0.28 mM Cd 2+ is estimated to be less than 0.6 s. Resonance gl is also broadened and, in addition, it is split into an AB pattern from the binding of C d 2+. The AB pattern is most clearly evident in spectrum C' in Fig. 3. This causes a reduction in the intensity of gl in the spin-echo spectrum; however, it never completely disappears as happens when sufficient Cd 2+ is added to cell lysate. (The residual signal at the gl position in spectrum C in Fig. 2 is due to another resonance which is nearly coincident with resonance gl, as can be seen more clearly in Fig. 7.) It is important to note that, although resonances gl, g3 and g4 in spectra B' D' are decreased in intensity by complexation

535

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of Cd 2÷, they have not completely disappeared, even at a concentration twice that at which they have disappeared from spectra of lysate to which CdC12 has been added. To determine if the binding of C d 2+ by GSH in erythrocytes is reversible, lysate containing CdC12 was titrated with EDTA. The spectra in Fig. 4 are for lysate containing 0.85 mM CdC12 and 0.56, 1.6, 2.6 or 3.5 mM EDTA (spectra A - D , respectively). As compared to spectrum C in Fig. 2, resonance gl is increased in intensity in spectrum

A in fig. 4, indicating the release of some of the GSH by the EDTA. In spectrum B, resonance g2 is clearly present, and resonances gl, g3 and g4 are increased in intensity, indicating further release of GSH from its Cd 2÷ complex. Comparison of spectra C and D in Fig. 4 with spectrum A in Fig. 2 indicates complete release of the GSH due to complexation of the Cd 2÷ by EDTA. The additional resonances at 2.68 and 3.64 ppm in spectra B - D are due to the ethylenic protons of EDTA in its Mg 2* complex and to the methylenic protons

536

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Fig. 5. Portions of the 400 MHz I H spin-echo NMR spectra of hemolyzed erythrocytes to which EDTA was added. The EDTA concentrations were (A) zero, (B) 1.2 mM or (C) 2.7 mM. The arrow in spectrum B indicates the resonance for the ethylenic protons of the Mg2+-EDTA complex. The arrow in spectrum C indicates the resonance for the methylenic protons of the acetate arms of free EDTA. Spectrum D is for a 2H20 solution (pH* 7.0) containing 3 mM EDTA and 2 mM MgCI 2.

537 of the acetate arms of free EDTA, respectively. This is substantiated by the spectra in Fig. 5 for hemolyzed erythrocytes to which no, 1.2 m M or 2.7 m M E D T A was added ( A - C , respectively). Spectrum D is for a pH* 7.0 2 H 2 0 solution (pH* indicates p H meter readings which have not been corrected for deuterium isotope effects) containing 3 m M E D T A and 2 m M MgC12. The resonance for the methylenic protons of the acetate arms of E D T A in the Mg(EDTA) 2 complex is an AB pattern which is phase modulated in the spin-echo spectrum [16]. From a plot of the ratio of the intensity of the Mg(EDTA) 2- resonance to the intensity of creatine resonance c2 (Fig. 1) versus the concentration of added EDTA, a concentration of 2.5 m M is obtained for the Mg 2÷ in the hemolyzed erythrocytes, as compared to an average value of 2.35 mM [24]. Resonances are not observed for the protons of the Cd(EDTA) 2- complex. The ethylenic protons of the complex in 2H20 solution, pH* = 7.4, give a resonance at 2.69 ppm, while the methylenic protons of the acetate arms give an AB pattern centered at 3.146 ppm. Comparison of Fig. 4D with Fig. 2A indicates no additional resonances in these regions after the E D T A has apparently complexed the Cd 2÷. The spectra in Fig. 6 suggest that Cd 2+ is also being complexed by hemoglobin. These spectra were obtained on the same samples as in Fig. 2, but with a shorter ~'2 value in the spin-echo pulse sequence so as to observe more resonances from the imidazole side-chains of hemoglobin. Resonances in the 6.85-7.5 and 7.5-8.5 p p m regions are due to the C-4 and C-2 hydrogens, respectively, of some of the imidazole side-chains. The stepwise decrease in intensity of the resonances at 6.98 and 7.87 p p m as the lysate is titrated with CdC12 indicates that Cd 2+ is binding to at least one histidine residue of hemoglobin. These two resonances have not been assigned to specific histidine residues, but their parallel decrease in intensity suggests that they are from C2-H and C4-H, respectively, of the same residue. The decrease in intensity indicates a considerable decrease in local mobility of the particular imidazole side-chain upon Cd 2+ binding, which taken with the stepwise decrease in intensity suggests a strong binding. It is of interest to note that this pair of

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hemoglobin histidine resonances is affected in the same way by Z n 2+ [10], which suggests the Zn 2+and CdZ+-binding sites of hemoglobin to be the same. The increased sensitivity of G S H resonances to Cd 2÷ in red cells (Figs. 2 and 3) might indicate that the resonances of the Cd2+-GSH complexes are naturally broader in this medium, due, for example, to a different viscosity, or it might indicate that the Cd2+-GSH complexes which form in erythrocytes are different from those which form

538 in simpler a q u e o u s solutions c o n t a i n i n g G S H and C d 2+. F o r example, t e r n a r y m i x e d ligand complexes might form in erythrocytes. The spectra in Fig. 6 indicate b i n d i n g b y h e m o g l o b i n , which, t a k e n with the d i s a p p e a r a n c e of the G S H resonances, suggests the f o r m a t i o n of t e r n a r y G S H C d 2 + - h e m o g l o b i n complexes. T h e G S H in such a c o m p l e x w o u l d p r e s u m a b l y have m o t i o n a l p r o p e r ties similar to those of the h e m o g l o b i n molecule, which w o u l d cause the spin-spin r e l a x a t i o n times for its resonances to be similar to those of h e m o g l o b i n resonances and the resonances would not be o b s e r v e d with a spin-echo d e l a y time of 0.060 s [15,161.

that, i m m e d i a t e l y after addition, sufficient C d C l 2 has been taken up b y the cells to cause the d i s a p p e a r a n c e of r e s o n a n c e g2 and a significant decrease in r e s o n a n c e g l . Then, as time passes, the intensity of r e s o n a n c e gl decreases further, reaching a m i n i m u m intensity at a b o u t 30 min. Identical b e h a v i o r was observed in the spectra

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The uptake of cadmium by erythrocytes Since p l a s m a c o n t a i n s small molecules a n d p r o teins which p o t e n t i a l l y can b i n d Cd 2+, the u p t a k e of Cd 2+ b y e r y t h r o c y t e s in suspension in isotonic s o l u t i o n was c o m p a r e d with u p t a k e b y e r y t h r o cytes in whole b l o o d . This was d o n e b y m e a s u r i n g time courses for the u p t a k e of C d 2+ b y erythrocytes in the two media, using as an i n d i c a t o r of the u p t a k e the intensity of r e s o n a n c e gl of G S H . The p r o c e d u r e involved dividing 50 ml of b l o o d into two portions. One p o r t i o n was washed twice with isotonic 2 H 2 0 / s a l i n e / g l u c o s e solution, and then the p a c k e d cells were r e s u s p e n d e d in an equal v o l u m e of wash solution. To this suspension a n d to the whole b l o o d sufficient CdCI 2 solution was a d d e d to give a final total C d 2+ c o n c e n t r a t i o n of 2 m M . 2 ml samples were taken from the suspension a n d the whole b l o o d at 10-min intervals up to 120 min after the a d d i t i o n of CdCI2. Each was centrifuged a n d the ~ H - N M R s p e c t r u m m e a s u r e d for the p a c k e d cells. Because the p a c k e d cells isolated from the whole b l o o d c o n t a i n e d H20, the reson a n c e of which is m u c h m o r e intense than those from G S H , the spin-echo spectra were m e a s u r e d with a d e l a y time of 0.15 s to e l i m i n a t e the H 2 0 r e s o n a n c e [20]. R e p r e s e n t a t i v e spectra from the time-course s t u d y for cells in suspension are shown in Fig. 7. S p e c t r u m A was o b t a i n e d from cells before any C d C I 2 had been a d d e d , s p e c t r u m B was o b t a i n e d from cells r e m o v e d from the suspension imm e d i a t e l y after the a d d i t i o n of CdC12, a n d spectra C, D and E from cells r e m o v e d from the suspension 10, 20 and 50 min later. S p e c t r u m B indicates

I 4

L

[ 3

L

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I

t 1

pprn Fig. 7. Portions of 360 MHz IH spin-echo NMR spectra of packed human erythrocytes. Spectrum A was obtained after washing with isotonic ZH 20/saline/glucose. Spectra B-E were obtained from cells from the same preparation but had been resuspended in an equal volume of isotonic 2H20/saline / glucose. Portions of the suspension were removed and the cells packed (B) immediately, (C) 10 min, (D) 20 min and (E) 50 min after sufficient CdCI2 had been added to give a concentration of 2 mM. Spectra were measured with a delay time of 0.15 s in the spin-echo pulse sequence.

539

obtained from erythrocytes isolated from whole blood to which CdCI 2 was added, with the exception that resonance gl was slightly more intense, when it levelled off, suggesting that some of the Cd 2+ was complexed by molecules in the plasma. However, the time courses for the uptake of Cd 2+ by erythrocytes in the two media, as represented by a plot of the ratio of the intensity of resonance gl to the intensity of resonance e2 of ergothionein or C2 of creatine (which are not affected by the Cd 2+) as a function of time, are similar in that the intensity of resonance gl reaches its minimum at approx. 30 min in both cases. The results indicate the uptake of Cd 2÷ by intact erythrocytes to be fairly rapid, although somewhat slower than has been observed for trimethyllead [11], methylmercury(II) [12] and mercury(II) [ 13]. This study demonstrates that 1H-NMR is a powerful method for following the dynamics of metal transport into intact erythrocytes. It also is of interest to note that the methyl resonance of lactic acid, identified as 1D in Fig. 7, increases in intensity with time, indicating that there is some metabolism of glucose, even in the presence of

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i

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The release of GSH by chelating agents The most common treatment for cadmium poisoning is to increase the rate of elimination of cadmium from the body with high-affinity chelating agents [4]. Chelating agents which have been used for this purpose include E D T A and monoand dithiols [4]. The relative effectiveness of various chelating agents for removing Cd 2÷ from its complexes in erythrocytes was determined from their effect on the G S H resonance of Cd2÷-con taining red cell lysates. Spectrum A in Fig. 8 is the spectrum for the lysate before the addition of CdC12; spectrum B after the addition of 1.01 mM CdC12, and spectra C E after the addition of 0.74, 0.98 and 1.46 m M 2,3-dimercaptopropanesulfonic acid to the Cd2+-containing lysate. The increased intensity of the G S H resonances in spectrum E indicates that G S H has been released from its Cd 2+ complex. To determine the relative effectiveness of several thiols for releasing GSH, each was added at concentrations of 1.0 mM and 1.9 m M to lysates containing 1.01 m M CdC12. Spectra obtained when

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Fig. 8. Portions of the 400 MHz XH spin-echo N M R spectra (absolute value display) for (A) hemolyzed erythrocytes and hemolyzed erythrocytes containing 1.01 mM CdC12 and (B) no, (C) 0.74 mM, (D) 0.98 mM or (E) 1.46 mM 2,3-dimercaptopropanesulfonic acid. The resonances for GSH are indicated by the arrows. Spectra were measured with a delay time of 0.060 s in the spin-echo pulse sequence.

mercaptosuccinic acid, N-acetylpenicillamine, Dpenicillamine, dithioerythritol and 2,3-dimercaptosuccinic acid were added to lysate containing 1.01 m M CdC12 are shown in spectra B - F , respectively, of Fig. 9. F r o m the intensity of resonance gl relative to el and C2 in these and other spectra, the effectiveness of the thiols for releasing G S H increases in the order mercaptosuccinic acid = N-

540

particularly effective at complexing Cd 2+, as expected since the Cd(EDTA) z complex has a high thermodynamic stability [25]. Comparison of the data in Fig. 4 with those in Fig. 8 indicates that EDTA is more effective than 2,3-dimercaptopropanesulfonic acid for releasing GSH. This is consistent with the finding that EDTA is more effective than 2,3-dimercaptopropanol at reducing the whole-body retention of C d 2+ by mice [27]. The effectiveness of a series of thiols at reducing whole-body retention was found [27] to increase in the order cysteine < mercaptoacetic acid < penicillamine < 2,3-dimercaptopropanol. The greater effectiveness of the dithiol as compared to the monothiols parallels the finding here that dithiols are more effective for releasing GSH from its Cd z+ complexes, which suggests that the strength of binding is an important factor in the effectiveness of thiol-chelating agents as treatments for Cd 2+ poisoning. Conclusion

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4

i

l

3 ppm

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L

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2

Fig. 9. Portions of the 400 M H z I H spin-echo N M R spectra (absolute value display) for hemolyzed erythrocytes containing 1.01 m M CdCI2 and (A) no added thiol, (B) 1.92 m M mercaptosuccinic acid, (C) 1.92 m M N-acetylpenicillamine, (D) 1.92 m M penicillamine, (E) 0.98 m M dithioerythritol and (F) 0.98 m M 2,3-dimercaptosuccinic acid. The resonances for GSH are indicated by the arrows.

acetylpenicillamine < penicillamine < mercaptoacetic acid < cysteine < 2,3-dimercaptopropanesulfonic acid < dithioerythritol < 2,3-dimercaptosuccinic acid. The spectra in Fig. 4 indicate EDTA to be

This study has provided direct evidence for the binding of Cd 2+ by GSH in human erythrocytes. This is perhaps not surprising, since GSH is known to form strong complexes with Cd 2+ [26] and is present at a rather high concentration [23]; however, it has not been identified previously as a Cd 2+-binding site in erythrocytes [5-9]. This result and the previous finding that GSH also binds zinc [10], trimethyllead [1 l], methylmercury [12], mercury [13], lead (Rabenstein and Isab, unpublished data) and gold [28] in erythrocytes suggest that GSH is probably an important intracellular binding site for all metals that have a high affinity for sulfur. The release of GSH when EDTA is added (Fig. 4) indicates the binding is reversible, and suggests the distribution of Cd 2+ is governed by equilibrium principles. Acknowledgements

This research was supported by the University of Alberta and by a strategic grant from the Natural Sciences and Engineering Research Council of Canada (D.L.R.). A.A.I. was supported by an Alberta Heritage Foundation for Medical Research Postdoctoral Fellowship.

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