A Quantitative Study of the Complexation of Cadmium in Hemolyzed Human Erythrocytes by ‘H NMR Spectroscopy Webe Kadima and Dallas L. Rabenstein Department of Chemistry, University of Alberta, Edmonton,
Alberta, Canada
ABSTRACT The stability of complexes formed by Cd 2f in hemolyzed human erythrocytes was studied by spin-echo ‘H NMR spectroscopy. Changes in resonances for the carbon-bonded protons of glutathione (GSH) upon addition of the ethylenediaminetetraacetic acid complex of Cd2+ (Cd(EDTA)‘-) and the appearance of resonances for Mg(EDTA)*indicate that the Cd(EDTA)*- complex dissociates in hemolyzed erythrocytes with the formation of Cd(GSH), and Mg(EDTA)2complexes. A semiquantitative estimate of the overall stability constant for the complexation of Cd*+ in hemolysed erythrocytes was obtained from spin-echo ‘H NMR data. The stability constant is consistent with the majority of the Cd*+ in erythrocytes present as Cd(SG)T*A conditional equilibrium constant was also determined for the complexation of Mg *+ by ligands in hemolyzed human erythrocytes.
INTRODUCTION Blood participates actively in the toxicology of cadmium through its role as the transport medium. Regardless of the source of cadmium (injection, inhalation, or ingestion), it is transported by blood to other tissues in the body where it is ultimately accumulated [ 11. As a consequence, studies on the toxicology of cadmium have been in part concerned with the distribution of cadmium in the different compartments of blood and the interactions of cadmium with blood components [l-4]. Cadmium in blood is mainly localized in the erthrocytes. Its removal from the erythrocytes is much slower than its removal from the plasma [3, 41, which suggests that cadmium rdight be bound tightly by molecules in the erythrocytes. The interactions of Cd’+ in red blood cells have been investigated by electrophoresis and gel filtration [3-41. It was suggested that cadmium is bound to hemoglobin [2], to metallothionein
Address reprint requests and correspondence to: Dr. Dallas L. Rabenstein, University of California, Riverside, CA 92521. Journal of Inorganic Biochemistry, @ 1990 Elsevier Science Publishing
40, 141-149 (1990) Co., Inc., 655 Avenue of the Americas,
Department
of Chemistry,
141 NY, NY 10010
0162-0134/90/$3.50
CADMIUM
I
I
4.5
4.0
IN HEMOLYZED
3.0
2.0
2.5
143
ERYTHROCYTES
Y-
I
I
I
3.5
HUMAN
I
I
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pm
FIGURE
1.
A portion of the 360 MHz spin echo ’ H NMR spectrum of hemolyzed erythrocytes, measured with Q = 0.060 sec. The resonances for the carbon-bonded protons of glutathione and glycine are identified.
RESULTS
AND DISCUSSION
A portion of the ‘H NMR spectrum of hemolyzed erythrocytes is shown in Figure 1. The spectrum was measured by the spin echo pulse sequence with 7-2 = 0.06 sec. Resonances of interest to the present study are those of glutathione (gl-g5) and glycine (g6), and are identified in Figure 1. When Cd(EDTA)2is added to hemolyzed red blood cells, resonances for GSH decrease in intensity or disappear and a new resonance is observed at 2.69 ppm for the ethylenic protons of Mg(EDTA)2(Fig. 2). This indicates that, even though the Cd(EDTA)2complex is very stable (log Kcd(nnr~) = 16.6) [8], it dissociates in hemolyzed erythrocytes due to competitive complexation reactions. Free Mg2+ FIGURE
2. The spin echo ‘H NMR spectrum of hemolyzed erythrocytes to which 2.62 x lop3 M Cd(EDTA)*and Mg *+ have been added to give a total Mg2+ concentration of 4.61 x 10F3 M. The resonance for the ethylenic protons of Mg(EDTA)*is identified.
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W. Kadima and D. L. Rabenstein
alone cannot displace EDTA from Cd(EDTA)*-- (log KM~(EDTA~= 8.7 [9]), however, the necessary additional driving force for dissociation of Cd(EDTA)‘-~ is provided by complexation of the released Cd’ ’ by ligands in the hemolyzed erythrocytes. as described by the reaction: Cd(EDTA)‘-m +- XL + Mg + CdL, 4 Mg(EDTA)‘~
.
(1)
where L represents potential ligands in hemolyzed erythrocytes. The disappearance of resonances for GSH indicates that at least some of the Cd’ ’ is complexed by GSH [S]. It is interesting to note that resonances are not observed for the Cd(EDT,4)“in hemolyzed erythrocytes to which Cd(EDTA)‘-- has been added [51. in hemolyzed crythroThe equilibrium constant for dissociation of Cd(EDTA)’ cytes was determined from the intensity of the Mg( EDTA!‘. resonance in spin echo ‘H NMR spectra. The equilibrium constant for the reaction in Eq. i 1) is:
K
~~~~~~-----_~.-.---..-
.__.
[Cd(EDTA) Assuming negligible
]
[CdL,][Mg(EDTA)“z:
~_
.
][Mg][Ll‘
that changes in the concentrations of the ligands represented by L are when some reacts with Cd’ * , the equilibrium constant can be rewritten as:
K’
z=
K
[L]”
x
r
[CdL,] [Mg( EDTA )’ f __--------l..---.-. _. [CdCEDTA)- j[Mg]
(3)
To determine K’, the total concentration of magnesium in hemolyzed erythrocytes was first determined by titration with EDTA, as described in the next section. while monitoring the titration by NMR. The concentration of Mg( EDTA)‘.. in hemolyzed erythrocytes to which Cd(EDTA)’ was added was then determined from the intensity of the resonance for the ethylenic protons of Mg( EDTA)‘~ by using a calibration curve which was prepared as described below. The other concentrations in Eq. 13) were obtained by difference. assuming that [M~rlEDTAI’ i ICtii., i.
Determination
of Total Magnesium
in Hemolyzed
Erythrocytes
The total magnesium concentration of hemolyzed erythrocytes was determined by titration of 0.5 ml of hemolyzed cells with five ~1 increments of a 0.04 M EDTA solution in D20 at pH ~7 7.4. Shown in Figure 3 are spectra of hemolyzed erythrocytes to which 0, 15. 25, and 35 ~1 of the EDTA solution were added. The resonances for and free EDTA appear at 2.69 and 3.61 ppm. the ethylenic protons of Mg(EDTA)’ respectively. The end point of the titration was taken as the intersection of the rwo linear portions of a titration curve prepared by plotting the ratio of the intensiq of the resonance for the cthylenic protons of free EDTA to that of the gl resonance of GSH as a function of the volume of EDTA solution added (Fig. 4). The gi rcsonancc of GSH and the other GSH resonances are not affected by the presence of EDTA. The blood used for ali the experiments was from one (Jonor to keep the concentration of magnesium constant. The average magnesium concentration from seven determinations over a period of 40 days was 2.6 i -t 0.2’~ IO ’ M. For comparison.
CADMIUM
I
I
4.0
3.0
IN HEMOLYZED
2.0
1.0
FIGURE 4. NMR titration curve for the determination of magnesium in hemolyzed erythrocytes. The ratio of the intensity of the resonance for the ethylenit protons of free EDTA to the intensity of the resonance for the gl protons of glutathione is plotted as a function of the volume of EDTA added. The end point in the titration is the volume at the intersection of the two straight line portions.
6-
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. E
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e 2
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FIGURE 3. Spin echo ’ H NMR spectra measured after the addition of (A), 0 pL; (B), 15 pL; (C), 25 pL; and (D), 35 FL of 0.040 M EDTA solution to 0.50 mL of hemolyzed erythrocytes. The resonances for the ethylenic protons of Mg(EDTA)2and free EDTA are identified.
npm
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-
20
I
1
I
40
60
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EDTA
(pl)
146
W. Kadima and D. L. Rabenstein
I--J-A____-_ii__-i 4.0
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3c
PP”
/ .o
FIGURE 5. Representative spin echo ‘H NMR spectra used for the calibration curve to determine the concentration c~f’Mg(EDT.4) 2 in hemoly& erythrocytes. The con. centrstion of !vlg(EDTA,i2 was (Aj. 3.65 ‘Y 10 ‘M: (B). 6.09 x Ii! ‘%I: and (
Alberta, Canada
ABSTRACT The stability of complexes formed by Cd 2f in hemolyzed human erythrocytes was studied by spin-echo ‘H NMR spectroscopy. Changes in resonances for the carbon-bonded protons of glutathione (GSH) upon addition of the ethylenediaminetetraacetic acid complex of Cd2+ (Cd(EDTA)‘-) and the appearance of resonances for Mg(EDTA)*indicate that the Cd(EDTA)*- complex dissociates in hemolyzed erythrocytes with the formation of Cd(GSH), and Mg(EDTA)2complexes. A semiquantitative estimate of the overall stability constant for the complexation of Cd*+ in hemolysed erythrocytes was obtained from spin-echo ‘H NMR data. The stability constant is consistent with the majority of the Cd*+ in erythrocytes present as Cd(SG)T*A conditional equilibrium constant was also determined for the complexation of Mg *+ by ligands in hemolyzed human erythrocytes.
INTRODUCTION Blood participates actively in the toxicology of cadmium through its role as the transport medium. Regardless of the source of cadmium (injection, inhalation, or ingestion), it is transported by blood to other tissues in the body where it is ultimately accumulated [ 11. As a consequence, studies on the toxicology of cadmium have been in part concerned with the distribution of cadmium in the different compartments of blood and the interactions of cadmium with blood components [l-4]. Cadmium in blood is mainly localized in the erthrocytes. Its removal from the erythrocytes is much slower than its removal from the plasma [3, 41, which suggests that cadmium rdight be bound tightly by molecules in the erythrocytes. The interactions of Cd’+ in red blood cells have been investigated by electrophoresis and gel filtration [3-41. It was suggested that cadmium is bound to hemoglobin [2], to metallothionein
Address reprint requests and correspondence to: Dr. Dallas L. Rabenstein, University of California, Riverside, CA 92521. Journal of Inorganic Biochemistry, @ 1990 Elsevier Science Publishing
40, 141-149 (1990) Co., Inc., 655 Avenue of the Americas,
Department
of Chemistry,
141 NY, NY 10010
0162-0134/90/$3.50
CADMIUM
I
I
4.5
4.0
IN HEMOLYZED
3.0
2.0
2.5
143
ERYTHROCYTES
Y-
I
I
I
3.5
HUMAN
I
I
I.5
I.0
pm
FIGURE
1.
A portion of the 360 MHz spin echo ’ H NMR spectrum of hemolyzed erythrocytes, measured with Q = 0.060 sec. The resonances for the carbon-bonded protons of glutathione and glycine are identified.
RESULTS
AND DISCUSSION
A portion of the ‘H NMR spectrum of hemolyzed erythrocytes is shown in Figure 1. The spectrum was measured by the spin echo pulse sequence with 7-2 = 0.06 sec. Resonances of interest to the present study are those of glutathione (gl-g5) and glycine (g6), and are identified in Figure 1. When Cd(EDTA)2is added to hemolyzed red blood cells, resonances for GSH decrease in intensity or disappear and a new resonance is observed at 2.69 ppm for the ethylenic protons of Mg(EDTA)2(Fig. 2). This indicates that, even though the Cd(EDTA)2complex is very stable (log Kcd(nnr~) = 16.6) [8], it dissociates in hemolyzed erythrocytes due to competitive complexation reactions. Free Mg2+ FIGURE
2. The spin echo ‘H NMR spectrum of hemolyzed erythrocytes to which 2.62 x lop3 M Cd(EDTA)*and Mg *+ have been added to give a total Mg2+ concentration of 4.61 x 10F3 M. The resonance for the ethylenic protons of Mg(EDTA)*is identified.
DO
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7.0
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4.0
30
ppm
0
2.0
1.0
144
W. Kadima and D. L. Rabenstein
alone cannot displace EDTA from Cd(EDTA)*-- (log KM~(EDTA~= 8.7 [9]), however, the necessary additional driving force for dissociation of Cd(EDTA)‘-~ is provided by complexation of the released Cd’ ’ by ligands in the hemolyzed erythrocytes. as described by the reaction: Cd(EDTA)‘-m +- XL + Mg + CdL, 4 Mg(EDTA)‘~
.
(1)
where L represents potential ligands in hemolyzed erythrocytes. The disappearance of resonances for GSH indicates that at least some of the Cd’ ’ is complexed by GSH [S]. It is interesting to note that resonances are not observed for the Cd(EDT,4)“in hemolyzed erythrocytes to which Cd(EDTA)‘-- has been added [51. in hemolyzed crythroThe equilibrium constant for dissociation of Cd(EDTA)’ cytes was determined from the intensity of the Mg( EDTA!‘. resonance in spin echo ‘H NMR spectra. The equilibrium constant for the reaction in Eq. i 1) is:
K
~~~~~~-----_~.-.---..-
.__.
[Cd(EDTA) Assuming negligible
]
[CdL,][Mg(EDTA)“z:
~_
.
][Mg][Ll‘
that changes in the concentrations of the ligands represented by L are when some reacts with Cd’ * , the equilibrium constant can be rewritten as:
K’
z=
K
[L]”
x
r
[CdL,] [Mg( EDTA )’ f __--------l..---.-. _. [CdCEDTA)- j[Mg]
(3)
To determine K’, the total concentration of magnesium in hemolyzed erythrocytes was first determined by titration with EDTA, as described in the next section. while monitoring the titration by NMR. The concentration of Mg( EDTA)‘.. in hemolyzed erythrocytes to which Cd(EDTA)’ was added was then determined from the intensity of the resonance for the ethylenic protons of Mg( EDTA)‘~ by using a calibration curve which was prepared as described below. The other concentrations in Eq. 13) were obtained by difference. assuming that [M~rlEDTAI’ i ICtii., i.
Determination
of Total Magnesium
in Hemolyzed
Erythrocytes
The total magnesium concentration of hemolyzed erythrocytes was determined by titration of 0.5 ml of hemolyzed cells with five ~1 increments of a 0.04 M EDTA solution in D20 at pH ~7 7.4. Shown in Figure 3 are spectra of hemolyzed erythrocytes to which 0, 15. 25, and 35 ~1 of the EDTA solution were added. The resonances for and free EDTA appear at 2.69 and 3.61 ppm. the ethylenic protons of Mg(EDTA)’ respectively. The end point of the titration was taken as the intersection of the rwo linear portions of a titration curve prepared by plotting the ratio of the intensiq of the resonance for the cthylenic protons of free EDTA to that of the gl resonance of GSH as a function of the volume of EDTA solution added (Fig. 4). The gi rcsonancc of GSH and the other GSH resonances are not affected by the presence of EDTA. The blood used for ali the experiments was from one (Jonor to keep the concentration of magnesium constant. The average magnesium concentration from seven determinations over a period of 40 days was 2.6 i -t 0.2’~ IO ’ M. For comparison.
CADMIUM
I
I
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3.0
IN HEMOLYZED
2.0
1.0
FIGURE 4. NMR titration curve for the determination of magnesium in hemolyzed erythrocytes. The ratio of the intensity of the resonance for the ethylenit protons of free EDTA to the intensity of the resonance for the gl protons of glutathione is plotted as a function of the volume of EDTA added. The end point in the titration is the volume at the intersection of the two straight line portions.
6-
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. E
‘-
e 2
145
FIGURE 3. Spin echo ’ H NMR spectra measured after the addition of (A), 0 pL; (B), 15 pL; (C), 25 pL; and (D), 35 FL of 0.040 M EDTA solution to 0.50 mL of hemolyzed erythrocytes. The resonances for the ethylenic protons of Mg(EDTA)2and free EDTA are identified.
npm
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-
20
I
1
I
40
60
60
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EDTA
(pl)
146
W. Kadima and D. L. Rabenstein
I--J-A____-_ii__-i 4.0
2.0
3c
PP”
/ .o
FIGURE 5. Representative spin echo ‘H NMR spectra used for the calibration curve to determine the concentration c~f’Mg(EDT.4) 2 in hemoly& erythrocytes. The con. centrstion of !vlg(EDTA,i2 was (Aj. 3.65 ‘Y 10 ‘M: (B). 6.09 x Ii! ‘%I: and (
