TOXICOLOGY AND APPLIED PHARMACOLOGY Im,96- 106 (1989)

Determination and Metabolism of Dithiol Chelating Agents

VIII. Metal Complexes of meso-Dimercaptosuccinic Acid’

MARIO RIVERA,* WEI ZHENG,~ H. VASKEN APOSHIAN,$ AND QUINTUS FERNANDO*

*Department of Chemistry, VDepartment of Pharmacology and Toxicology, and *Department of Molecular and
Cellular Biology, University ofArizona, Tucson, Arizona 85721

Received December 21,1988; accepted April 22, 1989

Determination and Metabolism of Dithiol Chelating Agents. VIII. Metal Complexes of meso-
Dimercaptosuccinic Acid. RIVERA, M., ZHENG, W., APOSHIAN, H. V., AND FERNANDO, Q.
( 1989). Toxicol. Appl. Pharmacol. 100,96- 106. Metal complexes of meso-dimercaptosuccinic
acid (DMSA) with Pb2+, Cd2+, and Hg2+ were studied by potentiometric and infrared methods.
This dimercapto metal-binding agent was found to form complexes whose structures are depen-
dent on the metal ion to be complexed. In the cases of Pb2+ and Cd’+, one oxygen and one sulfur
act as the donor atoms; in the case of Hg*+, two sulfur atoms act as the donors. The solubilities
of all metal chelates were found to be pH dependent. Complexes of cadmium and lead are
insoluble in the pH range 1.O to 7.1, but are solubilized when the noncoordinated sulfhydryl
and carboxylic acid groups are ionized. The mercury complex is insoluble in the pH range 1.O
to 3.0. It dissolves when one of the noncoordinated carboxylic acid groups is ionized. The di-
methyl ester of meso-DMSA (DiMe-meso-DMSA) was synthesized and its acid dissociation
constants were determined (pK, = 6.38 and pK2 = 8.00). Esterification of the carboxyl groups
of meso-DMSA changes its coordination properties in that the two sulfur atoms of DiMe-meso-
DMSA are used to coordinate with Hg 2+, Cd2+ , or Pb2+. Esterification of meso-DMSA also
changes its biological properties. DiMe-meso-DMSA, when given to rats 3 days after Cd admin-
istration, greatly increased the excretion of Cd via bile. In contrast, meso-DMSA was devoid of
such activity. 0 1989 Academic Press, Inc.

meso-Dimercaptosuccinic acid (DMSA) is an ceived increasing attention (see reviews by

effective metal-binding agent that has been
used successfully to increase the urinary ex-
cretion of lead in children (Graziano et al.,
1988) and adults (Friedheim et al., 1978;
Graziano et al., 1985). Because of its poten-
tial for becoming the drug of choice for de-
leading children with elevated levels of lead
in their blood, it has been given orphan drug
classification by the Food and Drug Adminis-
tration.
Although the biological properties of this
dimercapto metal-binding agent have re-
’ This research was supported in part by NIEHS Grant
ES-03356 and NC1 Grant 49252.
Aposhian, 1983; Aaseth, 1983) very little is
known about its physicochemical properties.
Lenz and Martell ( 1965) determined equilib-
rium constants for soluble metal chelates of
meso-DMSA and interpreted them in terms
of the probable structures of the complexes.
Whether metal ions such as Pb*+, Hg*+, or
Cd*+ coordinate with the two sulfur atoms or
with a sulfur atom and an oxygen atom of
DMSA has not been clarified.
This paper presents infrared and potentio-
metric evidence for the following: First, the
coordination sites in meso-DMSA for Pb*+,
Cd*+, or Hg*+ are oxygen and/or sulfur
atoms. Second, the coordination sites depend

0041-008X/89 $3.00 96
Copyright 0 1989 by Academic Press, Inc.
All rights of reproduction in any form reserved.
 meso-DIMERCAPTOSUCCINIC
on the type of metal ion that is to be coordi-
nated. Third, when the carboxylic groups of
meso-DMSA are esterified with methanol,
the two sulfur atoms become the coordina-
tion sites, and the coordination sites are inde-
pendent of the coordinated metal ion. Fi-
nally, esterification not only changes the co-
ordination sites, but the resulting dimethyl
ester, DiMe-meso-DMSA, increases the bil-
iary excretion of Cd, a property consistent
with intracellular distribution and markedly
different from that of meso-DMSA.
METHODS
Potenliometrv
Potentiometric measurements of hydrogen ion con-
centration were performed in a 150-m] titration vessel
provided with a magnetic stirrer and a tightly fitting rub-
ber stopper. The latter was equipped with inlet and outlet
tubes for nitrogen. a buret for delivery of NaOH, and a
combination glass-saturated calomel electrode. To an
aqueous suspension of meso-DMSA. sufficient NaOH
was added to form the species HZD’- (where H4D repre-
sents meso-DMSA). A solution of the metal nitrate or
chloride of known concentration was added slowly to
give a 1:1 mole ratio of metal to H2D’-. The liberated
hydrogen ions were monitored potentiometrically by the
addition of a standard solution of NaOH. The hydrogen
ion:DMSA mole ratio was plotted against the measured
pH. All potentiometric studies were carried out in a ni-
trogen atmosphere to prevent oxidation of the thiol
groups to disulfides.
Synthesis oj’the DimethJ,l Ester of meso-DMSA
A suspension of meso-DMSA in I M methanolic HCl
was refluxed for 1 hr and slowly cooled under nitrogen
to ambient temperature. The methanol was evaporated
to one-third of its original volume and the resulting solu-
tion chilled to 0°C. A white precipitate formed almost
immediately. The solution was maintained at O’C for 2
hr. The precipitate was filtered, dried, and recrystallized
from chloroform. The NMR spectrum of the ester was
determined with a Bruker WM-250 spectrometer using
CDCll as solvent. The infrared spectrum of the com-
pound in KBr was determined with a Perkin-Elmer 987
spectrophotometer.
ACID-METAL COMPLEXES 97
Determination of Acid Dissociation Constants of DiMe-
meso-DMSA
The pK, values of the two sulfiydryl groups were de-
termined potentiometrically with the aid of a glass-satu-
rated calomel electrode. Since DiMe-meso-DMSA is not
water soluble, a 50% (v/v) solution of water-methanol
was used to minimize the hydrolysis of the ester. The
glass electrode was calibrated in the 50% water-methanol
mixture since the dielectric constant of a mixture of 50%
water-methanol is different from that of water. This was
accomplished by the potentiometric titration of a stan-
dard solution of perchloric acid with a standard solution
of NaOH in a 50% (v/v) water-methanol mixture at a
constant ionic strength of 0.1. The measured pH of the
solvent mixture was lower by 0.30 than the pH calculated
from the hydrogen ion concentration at every point in
the titration.
A weighed amount of DiMe-meso-DMSA was dis-
solved in methanol in the vessel that was used for the
potentiometric titrations. When the ester was dissolved,
an equal volume of water was added together with suffi-
cient sodium perchlorate to maintain the ionic strength
at 0.1. The titration vessel was then placed at 25°C in a
constant-temperature water bath and the potentiometric
titration performed with a solution of carbon dioxide-
free NaOH. For every volume increment of NaOH solu-
tion added. the same volume of methanol was added to
maintain a 50% (v/v) aqueous methanol solution
throughout the titration.
Syntheses ql‘Meta1 Compkxes sf meso-DMSA
Syntheses were carried out in a Schlenck line under
nitrogen to prevent oxidation of the mercapto groups to
disulfides. All solvents were freed from oxygen by freeze-
thaw cycles in which the solvent was solidified by cooling
with liquid nitrogen followed by evacuation of the flask:
the flask was then filled with nitrogen gas. The cycle was
repeated three times. All solvents were transferred from
one flask to another with the aid ofa cannula to minimize
contact with oxygen in the air.
C4H40,S2Pb.2H,0. To a solution of 5.28 mmol ot
the disodium salt of meso-DMSA in 30 ml of water, a
stoichiometric amount of lead nitrate in 25 ml of water
was added dropwise from an addition funnel. A yellou
precipitate formed almost immediately. Once the addi-
tion of lead nitrate was completed, the suspension was
stirred for 30 min, filtered under nitrogen, and washed
with water (15 ml) and with methanol (15 ml). The com-
pound was filtered and dried in vacua. The dried com-
pound is stable in air. The yellow compound obtained
was suspended in dimethylsulfoxide (DMSO) to dissolve
any unreacted DMSA, filtered, and washed with water
and then with methanol: the last traces of solvent were
98 RIVERA
removed with a vacuum pump. Pb found, 49.06%; Pb
calculated, 48.95%.
C,H,O&Cd. 2H2 0. The synthesis was carried out in
a manner similar to that described for the Pb2+ analog.
Cadmium nitrate was used as a source of Cd*+. The cad-
mium-DMSA complex was obtained as a white solid.
The dried white compound is not air sensitive. Cd found,
34.05%; Cd calculated, 34.22%.
C4H404S2Hg.2H,0. meso-DMSA (10 mmol) was
placed in a Schlenk flask filled with nitrogen. A solution
of 20 mmol of triethylamine in 35 ml of methanol was
added with the aid of a cannula into the flask containing
meso-DMSA to solubilize it as an ion pair with triethy-
lammonium ion. A solution of HgC12 (10 mmol in 30 ml
of methanol) was then added dropwise. The white precip-
itate that formed initially completely dissolved in about
15 min. Addition of about 60 ml of water to the solution
resulted in precipitation of a white solid, which was fil-
tered, washed with water, and dried in vacua. Hg found,
48.29%; Hg calculated, 48.13%.
Infrared Spectra
Infrared spectra of all compounds were obtained with
a Perkin-Elmer 983 spectrophotometer. All compounds
were suspended in KBr and pressed to form pellets.
Animals
Male Sprague-Dawley rats (150 g) were purchased
from Harlan Sprague-Dawley Inc. (Indianapolis, IN).
They were kept in a temperature-controlled facility, with
a 12-hr light/dark cycle, and fed ad libitum Teklad rat
diet purchased from Teklad (Madison, WI). Rats were
allowed to acclimate for 1 week after arrival.
Administration of Chelating Agents
The compounds that were injected intravenously were
dissolved in 80% ethanol and administered via the jugu-
lar vein at a rate of 0.1 ml/min.
Biliary Excretion of Cadmium
Rats (200-220 g) were injected intraperitoneally with
1 mg Cd (3.08 X 10’ cpm of ‘09Cd)/kg. Three days later,
the rats were anesthetized with urethane 1.Og/kg, ip. The
bile duct and jugular vein were cannulated with PE-50
polyethylene tubing (Clay Adams). The tubing was in-
serted into the bile duct, close to the side where the duct
joins the duodenum and deeply enough (about 1- 1.5 cm)
to avoid contamination with any exocrine excretion
ET AL.
from the pancreas. During the experiment, rats were in-
fused with saline via the jugular vein at a rate of 1.5 ml/
hr. After a steady bile flow rate was established, bile sam-
ples were collected at 30-min intervals for 6.5 hr. Rats
were kept on an electric heating pad so that their core
temperature was maintained at 36 + 0.5”C during bile
collection. The radioactivity of the bile samples was
counted in a LKB type- 1282 CompuGamma Counter.
Assay for Biliary GSH and Dithiols
Bile samples were collected at 10-min intervals directly
into a vial containing 0.1 ml of 80 mrvt monobromobi-
mane (mBBr) and 1.8 ml of 0.1 M ammonium bicarbon-
ate buffer (pH 8.4) to derivatize the thiol groups and to
prevent the spontaneous oxidation of GSH to GSSG.
Vials were wrapped with aluminum foil to avoid expo-
sure to light. After collection, the volume of each bile
sample was measured and the samples were extracted
with 4 ml of methylene chloride to remove excess mBBr.
The aqueous phase was then diluted lo-fold and exam-
ined by HPLC as described below.
HPLC Analysis
HPLC analysis of dithiol-BB derivatives was per-
formed using the method of Maiorino et al. (1986). Sam-
ples were fractioned on a 250 X 4.6-mm Ultrasphere IP
C- I8 reversed-phase column, using a Beckman Model
157 fluorescence detector with 356-nm excitation and
450 f 20-nm emission filters. Separations were per-
formed at room temperature and at a rate of 1 ml/min.
The mobile-phase gradient system was described in the
procedure reported by Maiorino and Aposhian (1989).
RESULTS
Coordination of meso-DMSA to HP via
Two Sulfur Atoms
When an aqueous suspension of meso-
DMSA (H,D) is titrated with NaOH, com-
plete solubilization of the ligand occurs after
1 mol of NaOH has been added. The ionic
species H3D- is formed. When a second mole
of NaOH is added to the solution of the am-
pholyte H3D-, the species H@- is formed.
The pH of the resulting solution is in the rap-
 meso-DIMERCAPTOSUCCINIC ACID-METAL COMPLEXES 99

12;00-

9.00 -

‘ I
O.OOobo! 1.00 2.00 3.00
mole H*/ mole Lipand mole H’ i mole Ligond

FIG. I. Potentiometric titration of (a) I mmol DiMe- FIG. 3. Potentiometric titration of (a) 1 mmol DiMe-
meso-DMSA + 1 mmol HgCL, (b) 1 mmol meso- meso-DMSA + 1 mmol Cd(NO+, (b) I mmol meso-
DMSA, and (c) I mmol meso-DMSA + 1 mmol HgCIZ. DMSA. and (c) I mmol mes+DMSA + I mmol
Cd(NO,)z

idly rising portion of the titration curve

(curves b. Figs. l-3).
the suspension is 3.2. If this were the case,
When an equimolar amount of Hg’+ is
continued addition of two additional moles
added to this solution, the pH drops to 3.2
of NaOH should give a result similar to the
(curve c, Fig. 1). This pH drop occurs because
result obtained when a suspension of the li-
Hg”- is complexed, and as a result, protons
gand alone is titrated (curve b, Fig. 1). This is
are released. An insoluble white precipitate is
indeed what occurs, and is strong evidence
formed. If the two sulfur atoms are coordi-
that the two sulfur atoms are coordinated to
nated to the Hg2+ ion, the liberated protons
the Hg2+ ion.
would be expected to protonate the carbox-
ylic acid groups since the equilibrium pH of
Coordination of mesa-DMSA to Pb’+ or Cd’+
via One Oxygen Atom and One Su&
Atom

Addition of an equimolar concentration of

Pb2+ or Cd2+ to a solution of the species
HzD2- results in completely different behav-
ior upon titration with NaOH. When 1 mole
of NaOH is added to a solution containing a
suspension of 1 mole of the solid yellow com-
plex of Pb”+ with DMSA, the pH of the solu-
tion, which is in equilibrium with the solid,
continues to increase from 2.4 to 6.2 (curve
mole W’ l mole Ligwid c, Fig. 2). In this pH region, one carboxylic
acid group in the ligand is neutralized, identi-
FIG. 2. Potentiometric titration of (a) 1 mmol DiMe-
mes+DMSA + 1 mmol Pb(NO&, (b) I mmol meso- cal behavior is observed with the Pb-DMSA
DMSA, and (c) I mmol meso-DMSA + 1 mmol complex. Upon addition of a second mole of
Pb(NO&. NaOH, the pH of the solution increases from
100 RIVERA ET AL.

6.2 to 10.9 (curve c, Fig. 2). It is unlikely that

a carboxylic hydrogen is neutralized in this
pH region; a sullhydryl hydrogen in the Pb-
DMSA complex is neutralized between pH
6.2 and 9.2. This is strong evidence that
meso-DMSA is coordinated to Pb2+ via one
sulfur atom and one oxygen atom.
When 1 mol of NaOH is added to a solu-
tion containing a suspension of 1 mol of the b/
solid white complex of Cd2+ with DMSA
(curve c, Fig. 3), the pH increases from 2.3 to
5.5, as in the case of its lead analog. Addition
of a second mole of NaOH causes the pH to
increase from 5.5 to 10.8. Hence, it may be /
concluded that mesu-DMSA is coordinated C

to Cd2+ via one sulfur atom and one oxygen

atom, as in the lead complex.

Coordination of DiMe-meso-DMSA to H$+,

Pb2+, or Cd2+ via Two Surfur Atoms

When a solution of Hg2+, Pb2+, or Cd2+ in

a 50% water-methanol mixture is added in
equimolar amounts to a solution of DiMe-
meso-DMSA, the pH drops to 1.6 (curves a,
Figs. l-3). In the cases of Hg2+ and Cd*+ a
white precipitate is formed; a yellow precipi-
tate is formed in the case of Pb2+. The DiMe-
meso-DMSA complexes have limited solubil-
ity over the pH range 2- 12. When a solution
containing a suspension of the Hg2+, Cd2+, or
Pb*+ complex of DiMe-meso-DMSA is ti-
trated potentiometrically, 2 mol of NaOH are
necessary per mole of metal complex to raise
the pH from 1.6 to 10.7. This behavior dem-
onstrates that DiMe-meso-DMSA coordi-
nates to Hg2+, Pb2+, and Cd*+ ions by using
two sulfur atoms as the donors.
In the infrared spectrum of meso-DMSA
(Fig. 4a). The peak at 17 15 cm-’ corresponds
to the C=O stretching of a protonated car-
boxylic group. In the infrared spectra of the
Pb*+ and Cd2+ complexes of meso-DMSA
(Figs. 4b and 4c), the peak at 1700 cm-’ cor-
responds to the C=O stretching frequency of
a protonated carboxylic group. In addition to
4ooo 3ooo zoo0 mm lM0 800 400
FIG.4. Infrared spectra of (a) meso-DMSA, (b) meso-
DMSA-PbZf complex, (c) meso-DMSA-Cd*+ complex,
and (d) meso-DMSA-Hg’+ complex.
this peak, two bands are found that corre-
spond to asymmetric and symmetric stretch-
ing frequencies at 1537 and 1366 cm-r, re-
spectively. These correspond to a coordi-
nated carboxylate group.
In contrast, the infrared spectrum of the
Hg*+ complex (Fig. 4d) shows only the band
that corresponds to the protonated carboxylic
acid at 1692 cm-‘. These results corroborate
the results obtained in the potentiometric
studies.
 ~WW-DIMERCAPTOSUCCINIC ACID-METAL COMPLEXES 101

12.00
1
. . . l

9.00 -
J

,,H 6.00-
/**
- .

4000 3000 2000 1600 1200 600 400

Volume of N-OH Id)
FIG. 5. Infrared spectrum of DiMe-meso-DMSA.
FIG. 7. Potentiometric titration of 0.01089 M DiMe-
meso-DMSA with 0.1463 M NaOH.

Dimethyl Ester of meso-DMSA

quartet at 6 = 3.65, and S-H protons show
The compound obtained by the esterifica- the same pattern at 6 = 2.24. The multiplicity
tion of methanol with meso-DMSA was ana- of these signals arises from the coupling of
lyzed by NMR and by IR spectroscopy. The methine and sullhydryl protons.
absence of bands in the IR spectra of the ester
(Fig. 5) that correspond to the O-H stretching Acid Dissociation Constants (11DiMe-meso-
frequency of the carboxylic acid group indi- DMSA
cates that the ester is not contaminated with
unreacted DMSA. The NMR spectrum of the The titration curve for DiMe-meso-DMSA
ester is shown in Fig. 6. Methyl protons show was obtained in a 50% (v/v) water-methanol
a singlet at d = 3.80, methine protons show a mixture (Fig. 7). Since titration of two sulfhy-
dry1 protons results in two distinct buffer re-
gions, the acid dissociation constants of the
1 sulfhydryl groups were calculated indepen-
dently of each other: pK, was found to be 6.38
and pK;, 8.00.
The basis for these calculations follows.
The dissociation of DiMe-meso-DMSA
can be represented as
A-
H2Y 2 HY- + H+
ii.
HY- 2 y’- + H+
where H?Y represents DiMe-meso-DMSA.
K = [H+I[HY-1
aI (‘1
6 5 4 3 2 1
J WhYI
FIG. 6. NMR spectrum of DiMe-meso-DMSA in deu- k’ = [H+lW’l (2)
terochloroform, referenced to TMS. a? [HY-]
102 RIVERA ET AL.

O-O.5 0.5-l.Al.-l.5 1.5-2. 2.-3. 3.-4.*4.-4.5 4.5-5.55.5-6.5

HOUR

FIG. 8. DiMe-mesc-DMSA increases biliary excretion of cadmium-109. Rats were injected ip with 1 mg
Cd (4.2 X 10’ cpm of “‘Cd)/kg. Three days later, the bile duct and jugular vein were cannulated. DiMe-
meso-DMSA and meso-DMSA were dissolved in 80% ethanol. Chelating agents (0.10 mmol/kg) or 80%
ethanol (control) was administered via the jugular vein as indicated by the arrows.

The following equation was used to deter- Within 30 min of DiMe-DMSA administra-
mine K,, and K,, from the potentiometric ti- tion, biliary excretion of Cd increased 54-
tration data: fold. A second injection of DiMe-DMSA 3 hr
after the first elicited a 49-fold increase in bil-
4, or K,, iary excretion within 30 min. DMSA did not
influence the biliary excretion of Cd, con-
“’ + [H+] - [OH-]
IHfl i (V, + v) I firming previous results (Zheng et al., unpub-
lished). The action of DiMe-meso-DMSA in
= (hca - vcb) _ [Hf] + [OH-] (3)
increasing the biliary excretion of Cd was not
(Vo + v
due to an increase in bile flow rate (Table 1).
where all concentration terms are expressed
in moles per liter, C, is the analytical concen-
Biliary GSH Is Not Increased by DiMe-meso-
tration of the acid (DiMe-meso-DMSA), V.
DMSA
is the initial concentration of acid, V is the
volume of base added, and Cb is the concen-
Bile samples from a rat given DiMe-meso-
tration of base. The acid dissociation con-
DMSA (0.20 mmol/kg iv) were analyzed by
stants were obtained from Eq. (3) by a linear
least-squares method. HPLC and the results compared with the
HPLC profile of bile from a control rat that
had received only alcohol. No increase in bil-
DiMe-meso-DMSA Increases Biliary Excre- iary GSH was found (Fig. 9).
tion of Cadmium

DiMe-meso-DMSA, when given iv to rats Is DiMe-meso-DMSA Excreted in the Bile?

that had received ‘09Cd 3 days previously, in-
creased the biliary excretion of lo9Cd com- Dithiols are labile compounds. They are
pared with DMSA and the controls (Fig. 8). easily oxidized to form disulfides with them-
 meso-DIMERCAPTOSUCCINIC ACID-METAL COMPLEXES 103

TABLE 1
INCREASEIN THE BILIARY EXCRETION OF Cd BY DiMe-meso-DMSA
Is NOT DUE TO INCREASEIN THE RATE OF BILE FLOW’

Response

From 30 min before From time of injection

to time of injection to 30 min after % Increase

Bile flow rate (ml/30 min) 0.314 '_ 0.067 0.347 kO.076 10
Biliarv Cd (cpmlml/30 mitt) 324kSl 8788+1054 2612

a Data represent F + SE. Experimental conditions as described in the legend to Fig. 8, except that 1 mg Cd (3.08
X 10’ cpm of ‘@‘Cd)/kg was injected ip.

selves or mixed disulfides with other thiol previous experiments, neither unaltered nor
compounds such as GSH or proteins. For ex- altered mesu-DMSA was detected in the bile
ample, meso-DMSA, when given po to hu- after administration of meso-DMSA. Un-
mans, is biotransformed to a mixed disulfide altered meso-DMSA in extremely small
in which two molecules of cysteine are indi- amounts, however, appeared in the bile
vidually linked, by disulfide bonding, to the within 30 min of administration of DiMe-
sulfur atoms of meso-DMSA (Maiorino et al., meso-DMSA (Fig. 10).
1989). After injection of DiMe-meso-DMSA
iv, the bile was examined using bro- DISCUSSION
mobimane derivatization, HPLC, and fluo-
rescence detection. Unchanged or unaltered Valuable qualitative information can be
DiMe-meso-DMSA was found in the bile obtained if potentiometric titrations are per-
within 30 min of injection (Fig. 10). The peak formed on a system containing a suspended
concentration occurred within 15-30 min. In precipitate in equilibrium with the solution.
The results of the experiments presented in
this paper demonstrate the following: When
solutions containing equimolar amounts of
H2D-* and Hg(I1) are mixed, the compound
that precipitates is the neutral species
HgH2D:
0 0
II II
-0-C-CH-CH-C-O-+Hg*+ +
I I
SH SH
0 0
. IIIN
II II
HO-C-,CH-CJ-C-OH (4)
FIG. 9. DiMe-meso-DMSA does not increase biliary
GSH. Rats (200-220 g, n = 3) were anesthetized ip with S S
I .O g urethane/kg. DiMe-mesuDMSA (0.20 mmol/kg) ’ Hg ’
or 80% ethanol (equal volume) was injected via the jugu-
lar vein at zero time. The bile sample was collected di- In this neutral complex, HgH2D, the carbox-
rectly into a bromobimane-containing solution. ylate groups are protonated in the presence of
104 RIVERA ET AL.

protons that are released upon complex for- Addition of a solution of NaOH to an aque-
mation [Eq. (4)]. Addition of a solution of ous suspension of the species PbHzD or
NaOH to an aqueous suspension of HgH2D CdH2D neutralizes a carboxylate group in the
neutralizes the carboxylic acid groups in the pH region 2.4 to 6.2 (curves c, Figs. 2 and 3)
pH region 3.2-l 1.5 (curve c, Fig. 1): with generation of the insoluble species Pb-
HD- or CdHD-:
0 0 0
II II 0
HO-C-CH-CH-C-OH+OH- e B II
C-CH-CH-C-OH+OH- )_
/ \ / \ I
S S 0 SH
’ Hg ’ \M/’
0 0 0
0
II II \ II
HO-C-,$H-C\H-C-0-+H,O (5) C-CC\H-y-C-0-+H,O (8)
/
S S 0 S SH
’ Hg ’ ‘M’
0 0 Continued addition of NaOH to the aqueous
II II suspension of PbHD- or CdHD- neutralizes
HO-C-,CH-C\H-C-0-+OH- __
a sulfhydryl group in the pH region 6.2 to
s s 11.5 (curves c, Figs. 2 and 3), with formation
’ Hg / of the soluble compound PbDv2 or CdD’-:
0 0 0 0
II II \ II
-O-C-,y--C\H-C-0-+H,O (6) C-CH-CH-C-0-+OH- __
S S 1 \ I
0 S SH
’ Hg ’ ‘M’
0 0
When solutions containing equimolar \
amounts of H2D2- and Pb(I1) or [Cd(II)] are C-CF-YH-!-0-+H,O (9)
/
mixed, the compound that precipitates is the 0 s-
neutral species PbHzD [or CdH2D]. In the lMIS
presence of protons released upon complex
formation, one carboxylic group and one When solutions containing equimolar
sullhydryl group are protonated: amounts of DiMe-meso-DMSA (H,Es) and
Hg(II), Pb(II), or Cd(I1) are mixed, the com-
0
II pound that precipitates is MEs, where M rep
-O-i-,, -CH-C-0-+M*+ -3 resents Hg(II), Pb(II), and Cd(U). The pro-
I I tons released upon complex formation are
SH SH neutralized by the addition of a solution of
0
0 NaOH to the aqueous suspension of MEs
\
C-Ct-+-OH (7) (Eq. 10). This occurs between pH 1.5 and
/ 11.5 (curves a, Figs. l-3). Thus, it is not un-
0 SH reasonable to predict that the chelate struc-
\M/’
tures of meso-DMSA are those shown in
M = Pb” or Cd*+ Fig. 11.
 meso-DIMERCAPTOSUCCINIC ACID-METAL COMPLEXES 105

0 H H H H
o=c-c-c-c=0 o=c-c-c-CZEO
II
I I I I I I I I
MeO-C-CH-CH C-OMe+M’+ __ ~0 s s o-
I I H H ;/” fl O
SH SH Cd
0 0
H H H H
II II o=c-c-c-c=0 o=c-c-c-c=0
MeO-C-CH-CH-C-OMe+2H’ I I I I I I I !
/ \ .o s s 0
s S v “H O- \ /
‘M’ Pb w

(10) FIG. Il. Structures of meso-DMSA-metal chelates.

2H++20H- + 2H,O
M2+ = Pb Cd or Hg

When given iv to rats, DiMe-meso-DMSA ministration, DiMe-meso-DMSA must enter

greatly increased the elimination of Cd via the hepatocytes. This property of entering
bile from the liver (Fig. S), the organ in which cells may be one reason that it can mobilize
the greatest amount of Cd deposited is found Cd deposited in liver. On the other hand.
(Lucis el al., 1969). This action is not due to meso-DMSA has an extracellular distribu-
DiMe-meso-DMSA’s increasing the rate of tion, does not enter hepatocytes and does not
bile flow (Table 1). Neither is it related to a increase the biliary excretion of cadmium
change in biliary glutathione content (Fig. 9). (Zheng et al., 1989). After administration of
Since it is found in the bile after its iv ad- DiMe-meso-DMSA, a small amount of
meso-DMSA was found in the bile (Fig. 10).
Previous work, however unequivocally dem-
onstrated that meso-DMSA, when given iv.
‘T
is not found in the bile (Zheng et al., 1989),
indicating that DiMe-meso-DMSA under-
goes biotransformation to some extent in he-
3- patocytes or bile canaliculi.
meso-DMSA is emerging as the drug of
choice for treatment of lead intoxication
(Graziano et al., 1985, 1988), because of its
low toxicity (Aposhian et al., 1983). oral use-
fulness, and chelation activity. Its low toxicity
is due in part to its inability to enter the cell.
DiMe-meso-DMSA enters the cell.
The structures proposed for the metal che-
lates of meso-DMSA and supported by the
evidence in this paper are shown in Fig. 11.
It should be noted that a structure for a Hg-
-I DMSA chelate different from that for a Pb-
MIN
DMSA chelate has not been proposed pre-
FIG. 10. DiMe-meso-DMSA and meso-DMSA are viously. It has always been assumed that di-
found in the bile after DiMe-meso-DMSA administra-
tion. DiMe-meso-DMSA (0.20 mmol/kg) was adminis- mercapto chelates were formed by coordina-
tered via the jugular vein at zero time. See legend to Fig. tion with two sulfur atoms. Our groups are in
9 for methods. the midst of collaborative efforts to determine
106 RIVERA
whether the structures of the chemically syn-
thesized chelates (Fig. 11) are the same as
those found in, for example, the urine of rab-
bits given Hg or Pb followed by meso-DMSA
treatment.
ACKNOWLEDGMENT
We thank Johnson and Johnson Baby Products, Inc.,
for providing us with meso-DMSA.
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