EPA-600/1-76-016
March 1976
Environmental Health Effects Research Series

        COMPARATIVE METHYLATION CHEMISTRY  OF
   PLATINUM,  PALLADIUM, LEAD,  AND  MANGANESE
                                       Health Effects Research Laboratory
                                       Office of Research and Development
                                      U.S. Environmental Protection Agency
                                 Research Triangle Park, North Carolina 27711

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                 RESEARCH  REPORTING SERIES

Research reports  of the Office of Research and Development,  U.S. Environ-
mental Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application
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    2.    Environmental Protection Technology
    3.    Ecological Research
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                                                EPA-600/1-76-016
                                                March 1976
    COMPARATIVE METHYLATION CHEMISTRY OF PLATINUM,

            PALLADIUM, LEAD, AND MANGANESE
                          By

                   Robert T, Taylor
  Biomedical Division, Lawrence Livermore Laboratory
University of California, Livermore, California  94550
      Interagency Agreement No. EPA-IAG-D4-0439
                    Project Officer

                    Paul E. Brubaker
          Criteria and Special Studies Office
          Health Effects Research Laboratory
          Research Triangle Park, N,C.  27711
         U.S. ENVIRONMENTAL PROTECTION AGENCY
          OFFICE OF RESEARCH AND DEVELOPMENT
          HEALTH EFFECTS RESEARCH LABORATORY
          RESEARCH TRIANGLE PARK, N.C. 27711

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                              DISCLAIMER
     This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication.  Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
                                 11

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                               ABSTRACT

A study was carried out to evaluate the potential for platinum,
palladium, lead, and manganese salts and oxides to be biochemically
methylated.  Methylation is an important, well recognized determinant
of metal toxicity, the striking example being the extreme health hazard
of methylated mercury.  The possible biological methylation of the
metals which are associated with emissions arising from the use of
automotive fuels, fuel additives, and catalytic control devices is of
special concern to the Environmental Protection Agency Fuel and Fuel
Additives Program.

Salts of platinum, palladium, and lead and oxides of lead all containing
the metal in a 4* valence were observed to demethylate methylcobalamin,
a biologically active form of vitamin B-12.  Inorganic salts and oxides
of manganese were unreactive.  No evidence for a stable monomethyl-meta]
derivative was found using palladium and lead compounds as reactants.
However, salts of platinum 4+ do result in the formation of stable
methylation products.  The reaction product formed from methylcobalamin
and hexachloroplatinate was shown definitively to be a monomethyl-
platinum compound.  It is sufficiently stable in aqueous solutions under
a variety of conditions to exist in freshwater ecosystems and to exhibit
toxic effects on mammalian cells.

This report was submitted in fulfillment of EPA-IAG-D4-0439 by the
Lawrence Livermore Laboratory (Energy Research and Development
Administration) under the sponsorship of the Environmental Protection
Agency.  Work was completed as of June 1975.

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                               CONTENTS
Abstract




List of Figures                                                      v



List of Tables                                .                      vi



Acknowledgments                                                    vii



Sections



I      Conclusions                                                   1



II     Recommendations                                               2



III    Introduction                                                  3



IV     Materials and Methods                                         5



V      Results and Discussion                                        6



VI     References                                                   24
                                  IV

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                               FIGURES

No.

1     Demethylation of methyl cobalamin  (MeB-12) with
      potassium hexachloroplatinate  (K2PtCle)                        7

2     pH Dependence of the demethylation with K2PtClg
      and the catalytic effect of K2PtCljt                             8

3     Stoichiometry of demethylation with the amount
      of K2PtClg in the presence of K2PtClt                           9

4     Quantitative demethylation upon the addition of
      K2PtCl6 alone                                                  10

5     Demethylation of MeB-12 by platinic sulfate
                                                                     12
6     Paper chromatography of the reaction products
      from [Me-ll+C]MeB-12 and K2PtCl6                                14

7     Paper electrophoresis of the reaction products
      from mixed labeled [Me-^CJMeB-^ + [Me-3H]MeB-12
      and K2PtCl6                                                    15

8     Absorption spectrum of the [Me-1!*C]Pt reaction
      product from [Me-ll+C]MeB-12 and K2PtCl6                        17

9     Proton nuclear magnetic resonance (NMR) spectrum
      of the [Me-11+C]Pt reaction product                             18

10    Effect: of temperature and light on the UV
      absorption of the [Me-ll*C]Pt product                           19

11    Partial demethylation of MeB-12 with lead
      tetraacetate (Pb(Ac)^)                                         22

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                                TABLES

No.

1     Alkyl corrinoid specificity of the dem0thylation
      by K2PtCl6                                                    11

2     Relative rates of MeB-12 demethylation by
      K2PtCl6 versus Pt(SOl|)2                                       13

3     Purification summary of the [Me-ll*C]Pt product
      from [Me-^C]MeB-12 and K2PtCl6                               16

4     Stability of the [Me-^CJPt product under
      various conditions                                            20

5     Extents of [Me-ll*C]MeB-12 demethylation and the
      recovery of ll*C after prolonged incubation with
      various lead salts and oxides                                 2,3

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                           ACKNOWLEDGMENTS

The technical assistance of Mr. Richard Ryon and Mr. James A. Happe of
the Lawrence Livermore Laboratory General Chemistry Division were
invaluable aids in this study.  Mr. Ryon performed the analytical
determinations for platinum using X-ray fluorescence, while Mr. Happe
obtained the NMR spectrum of our  [Me-ll*C]Pt compound.  I also wish to
acknowledge that Mr. Robert E. Elson  (Lawrence Livermore Laboratory
Organic Materials Division) demonstrated the release of methyl groups
from MeB-12 as MeCl under certain conditions.

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                              SECTION I

                             CONCLUSIONS

Methylcobalamin (MeB-12), a biologically active form of vitamin B-12 is
readily demethylated at 22C under slightly acid conditions by halogen
platinate salts and platinic sulfate.  At pH 1.0 in the presence of
1.0 M sodium chloride (NaCl) and potassium hexachloroplatinate (F^PtClg)
almost half of the methyl groups appear as MeCl.  This is indicative of
an overall transfer from MeB-12 to ionic platinum and a subsequent
transfer to chloride ions.  At pH 2.0 in the absence of high concen-
trations of NaCl a mono-MePt compound is formed from MeB-12 and ^PtClg.
This methylated Pt compound has characteristic UV light absorption and
NMR spectra, it is quite soluble in water, and it carries a net negative
charge at pH 7.0.  Time-storage experiments indicate that our Me-Pt
product is sufficiently stable with respect to temperature, salt, and
pH to exist in freshwater systems and to exhibit biological activity.

Salts containing ionic palladium (Pd) can also demethylate MeB-12 at
pH 2.0 but the rates are much slower than are observed with Pt1** salts.
The three Pd compounds shown to be reactive were K2PdCl6>PdSOi+>K2PdCltt.

No significant demethylation was detected when MeB-12 was incubated at
22C for as long as 24-48 hrs with various lead salts containing Pb*-*
ions.  However, salts and insoluble oxides containing lead in the 4+
valence state slowly demethylated MeB-12 to extents of 40-100% after
24 hrs at 22C.  Demethylation by Pb compounds was correlated with a
parallel volatilization of the MeB-12 methyl group.

No reactivity was detected between MeB-12 and several manganous salts at
any pH for periods up to 24-48 hrs at 22.  Manganese dioxide suspensions
were also unreactive.

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                              SECTION II

                           RECOMMENDATIONS

We have demonstrated that MeB-12 can be demethylated by three metals of
interest to the EPA Fuel and Fuel Additives Program.  Also, we have
definitively shown that one platinum salt (K^PtClg) reacts to form a
rather stable mono-MePt compound that has never been described
previously in the literature.  As a consequence of these findings many
new questions have been raised about the biochemical methylation of
platinum and palladium in particular.  It is recommended that additional
research be carried out to characterize adequately the new MePt compound
that we have purified as well as any stable methylation products that
may be formed from platinic sulfate under neutral as opposed to acidic
conditions,  It should also be carefully determined whether the halogen
palladate salts react with MeB-12 to generate small amounts of stable
MePd.  Furthermore, if it is determined in the near future that catalyst
attrition products contain ionic forms of Pt and Pd, then the reactivity
of small emission particles with MeB-12 should be examined.  It wottld
lend considerable credence to our in vitro studies on the methylation of
Pt-salts if emission particles from catalyst equipped cars were found
to demethylate MeB-12.  However, even if such particles contain only
unreactive, non-ionic Pt and Pd, this does not preclude their subsequent
oxidation to reactive, ionic forms in the environment.

As soon as methylated forms of Pt and Pd have been characterized
chemically, it will be imperative to carry out research on their
biological effects.  A logical approach would be to compare the cellular
and molecular toxicity of the methylated compound with the parent metal
salt used as a reactant with MeB-12.  Some comparative studies should
be feasible in the near future using our newly isolated mono-MePt
material and l^PtClg.  Since the amounts of MePt available will be small,
the biological test systems would be restricted to small animals
(rodents) and cultured mammalian cells.  The types of studies that I
would recommend are as follows:  1) chronic low dose administration to
rodents with subsequent monitoring of organ and subcellular organelle
distribution; this could be coupled with assays of key enzymes known
to be sensitive to SH reagents; 2) acute effects on cultured mammalian
cells such as cell killing, growth inhibition, depression of macromolec-
ular synthesis, and induction of DNA strand scissions; 3) induction of
chromosome breakage and aberrations in cultured mammalian cells or
human lymphocytes; and 4) mutagenicity in mammalian test systems using
several established phenotypic markers.

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                             SECTION III

                             INTRODUCTION

GENERAL

Methylation is an important determinant of metal toxicity, particularly
for metals such as Hg, Pb, and Sn (1).  There was little impetus for
studying the possible biological methylation of heavy metals, however,
until several investigators (1967-1970) described the conversion of
HgCl2 to both mono- and di-MeHg by lake bottom sediments, homogenates of
decaying fish, methanogenic (sewage) anaerobes, and aerobic micro-
organisms.  Wood et^ al_. demonstrated that the Me donor in sewage bacteria
was in fact. MeB-12 (2).  Several laboratories have since shown that free
MeB-12 even in the absence of any cellular system will react to form
first mono- and then di-MeHg (3-5).

In addition to acquiring preformed MeHg1* from the environment, evidence
is accumulating that mammalian tissues containing the Hg2+ ion can
convert it to MeHg1+.  This has been demonstrated with fish liver
homogenates and again the methylating agent was shown to be MeB-12 (6).
Based on several biological and chemical studies, MeB-12 seems to be
generally responsible for the environmental methylation of not only Hg
but also selenium and tellurium.  No other known biological Me donor has
yet been shown to alkylate these metals.  These processes are biological
in that they require a ubiquitous, biologically active compound (i.e.
MeB-12) and they are mediated by cells which supply the Me group donor.
However, there is no definitive evidence that any cell contains an
enzyme that will accelerate the chemical reaction between MeB-12 and
Hg2* or any other metal ion.  Consequently, we prefer to term the
formation of MeHg1*, or any other methylated metal, in the presence of
MeB-12 as "biochemical methylation".

Because of the experience with MeHg1*, the EPA Fuel and Fuel Additives
Program continues to be concerned about the possible biochemical
transformation of the oxides and soluble salts of Pt, Pd, Pb, and Mn.
These four metals are associated with emissions arising from the use of
automotive fuels, fuel additives, and hydrocarbon control devices.  Pb
and Mn are present in fuel additives, while Pt and Pd are used as smog
control catalysts.  These latter two metals are of special concern
because they are novel pollutants with which mankind has limited
biological experience (7,8).  Soluble  platinum  compounds (Pt4**) are
more toxic to experimental animals than compounds of other metals of
interest (Mn, Pb) when administered orally; however, palladium salts are
more toxic than these metals when administered intravenously.  Analyses
of the impact of these control devices on the future use and demand for
Pt and Pd indicate that these metals will appear at readily detectable
levels in the environment by the end of the decade.  Yet, there is no
information as to the likely biotransformation (methylation) of Pt and
Pd or the relative hazards to expect from any organo-Pt or Pd compounds.

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OBJECTIVE

The objective of this project is to compare the methylation chemistry of
Pt, Pd, Pb, and Mn using MeB-12 as a biological alkylating agent.  The
ability to demethylate MeB-12 in vitro is being used to indicate whether
ionic forms of these four metals are potentially methylatable either in
the environment or within mammalian tissues.  Any of these metal ions
which demethylate MeB-12 will then be studied in detail to determine
whether such reactions yield stable Me-metal compounds.  Once any newly
discovered Me-metal compounds have been adequately characterized, their
toxic effects on mammalian cells will have to be evaluated.  Estimation
Of their effects at the cellular or macromolecular level is not within
the scope of this methylation chemistry project.

We were prompted to suggest a thorough study of the biochemical methyl-
ation of Pt, in particular, because of an unconfirmed communication in
1971 which merely stated (no data given) that Pt1** can demethylate
MeB-12 (9).  As will become apparent under RESULTS and DISCUSSION
(SECTION V) we have obtained firm data that Pt can be biochemically
methylated, yielding a rather stable organo-Pt compound.  Furthermore,
we have demonstrated chemical reactivity of MeB-12 with Pd and Pb ions
for the first time.  In view of our findings and recent reports  (10,11)
that microorganisms in lake sediments can form tri- and tetra-Me lead,
continued research on the biochemical methylation of selected metals
should be of vital interest to the EPA.

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                              SECTION IV

                         MATERIALS and METHODS

All metal salts and oxides were purchased from either Chemical
Procurement Labs, Research Organic/Inorganic Chem. Co., or the J.T.
Baker Chem. Co. and were of the highest purity commercially available.
Vitamin B-12 (cyanocobalamin) was obtained from Calbiochem; methyl
iodide and other unlabeled alkyl iodides from Eastman Chem. Co.;
[Me-3H]methyl iodide (300 pCi/pmole) from International Chemical and
Nuclear Corp.; and [Me-^C]methyl iodide (10 uCi/ymole) from the New
England Nuclear Corp.

MeB-12, [Me-11+C]MeB-12, and [Me-3H]MeB-12 were synthesized in the dark
from vitamin B-12 and either unlabeled or labeled methyl iodide following
reduction of the vitamin with sodium borohydride  (12).  Ethyl B-12 and
propyl B-12 were prepared in the same manner.  Alkyl cobinamides were
synthesized from the corresponding alkyl iodide and diaquocobinamide
(13).   The concentration of each alkyl corrinoid solution was determined
from the absorbance of its a-band and published molar extinction
coefficients (14).  All light absorption spectra were taken at22Cwith
a Gary Model 15 recording spectrophotometer using 1.0 ml solution
volumes and a 1-cm light path.  Complete phytolysis of any unreacted
MeB-12 in solution was achieved by a 30 rain exposure at 22Cto a 40 W
tungsten lamp at a distance of 10 cm.  Rates of absorbance change at
350 nm were monitored with a Gilford Model 240 spectrophotometer
equipped with a Honeywell recorder.  All pH measurements were made with
a Beckman Model 1019 research pH meter.  Radioactivity determinations
were made at 5C in a Packard Model 3320 liquid scintillation spectro-
meter using a water-miscible counting fluid.  The platinum content of
small segments of paper (chromatography and electrophoresis runs) and
of unknown solutions was determined by X-ray fluorescence.  The method
used was energy dispersive analysis  (Si detector, pulse height analysis)
and the detection limit was about 0.1 nmole with a counting time of
15 min.  Proton nuclear magnetic resonance spectra were taken at 5C
with a spectrometer designed in the General Chemistry Division of
Lawrence Livermore Laboratory.  It operates at 60 MHz and utilizes a
Varian magnet and power supply.  The spectrometer was locked to water
solvent and signal to noise improvement was achieved by ensemble
averaging methods.  Sodium 2,2-dimethyl-2-silapentane-5-sulfonate  (DSS)
was used as an internal Me group reference.

Except where specifically stated otherwise, reaction-mixtures contained
40 pM concentrations of unlabeled or Me-labeled MeB-12 and they were
carried out in the dark at 22C at pH 2.0 in 0.01 M HC1.  Other
essential experimental details are given in legends to the corresponding
figures and tables.

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                              SECTION V

                        RESULTS and DISCUSSION

PLATINUM

We confirmed that in the presence of K2PtClg  C+J^PtCliJ raicromolar
concentrations of MeB-12 are rapidly demethylated.  The characteristics
of this process were then studied in more detail.  As  seen  in  Fig.  1A,
MeB-12 is converted to aquoB-12 with no other discernible corrinoid
intermediates accumulating on the reaction pathway.  Evidence  for this
is the presence of isosbestic points at 490 nm, 367 nm, and 335 nm
(Fig. 1A) which almost exactly match the spectral cross-over wavelengths
when MeB-12 is photolyzed with light directly to aquoB-12 (Fig. 1C).
Figure IB shows that K2PtClit alone is unreactive, while K2PtClg added
alone results in a small amount of demethylation after 5 rain.  Based on
the initial rates of 350 nm absorbance increase, the optimal pH for the
reaction with K2PtCl6 (+K2PtClit) is about 2 (Fig. 2).  Reactivity
diminishes rapidly below and above pH 2.  Demethylation is  negligible
a,t pH 7 even after 2 hrs of incubation.  The initial reaction  rate  is
influenced somewhat by the acid solvent used, but for  any acid chosen
the fastest rate occurs at pH 2.  The inset in Fig. 2  shows that very
small concentrations of K2PtClt,, far less than the MeB-12 and  the
K2PtClg levels, markedly accelerate the initial reaction rate.  Initial
reaction rates were measured on a recorder within seconds after rapid
nixing of the components.

The stoichiometry of the demethylation by chloroplatinate clearly
involves a 1:1 consumption of MeB-12 and the Ft*** salt K2PtCl6 (Fig. 3).
When the ratio of K2PtCl6 to MeB-12 is reduced to 0.25, the MeB-12  is
demethylated to the extent of 25% and complete conversion to aquoB-12
can only be achieved by photolysis of the Me-cobalt bond with  light
(Fig. 3).  The Pt2* salt K2PtClt acts only catalytically to speed up the
rate of the reaction.  The initial reaction rate depends on the
concentration of all three components (MeB-12, K2PtClg, and K2PtClt),
but MeB-12 can be quantitatively demethylated after 2  hrs by the
addition of K2PtClg alone (Fig. 4).  An initial lag in the  reaction when
K2PtCl1+ is omitted may represent the time required for traces  of Pt2"1" to
be formed in the system.  A lag followed by eventual complete  demethyl-
ation is consistent with the suggestion (9) that the Pt2+ ion  plays an
essential role in the reaction mechanism as depicted below.


           MeB-12 + Pt2+ -* Pt2+'MeB-12                               (1)


           Pt2+-MeB-12 + *Pt**+ -* Pt^+'Me + *Pt2+ + aquoB-12         (2)


The net overall reaction is then

           MeB-12 + Pt"+ * Pt"*+Me + aquoB-12                        (3)

                                   6

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                                1. MeB-12 in 0.01M HC1, pH 2.0_
                                2. After 1.5 min with K-PtCl,.
                                   _i_ I/ n-i-r11                0  
                                3. After 5 min
                                4. After 15 min
                                1. MeB-12 in 0.01M HC1, pH 2.0
                                2. After 15 min with
                                3. After 5 min with
                             2  1. MeB-12 in 0.01M HC1, pH 2.0
                                2. After photolysis
               250
       300    350    400    450    500
                Wavelength, nm
Figure 1.
Demethylation of MeB-12 with  K2PtCl6  in the presence of
^PtClij.  Each platinum salt  was present at a concentration
of 100 pM.

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oo
            0.30
            0.25-
            0.20-
       CO

       <1
            0.10 -
 0.01M HC1, pH 2.0
            0.15 -
            0.05 -
                                                               pH
                     Figure  2.     pH Dependence  of the  demethylation  of MeB-12 with K2PtCl6
                                  (100 yM)  in presence  of  K2PtCl^  (100 yM).   Inset -
                                  catalytic effect of low  concentrations of K2PtClJ|.

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               0.9





               0.8





               0.7
            g  0.6
            o
            CO
               0.5
               0.4
               0.3
                                                   1
                       10   20  30   40   50   60   70  80   90


                                  Minutes, 22
Figure 3.     Stoichiometry of MeB-12 demethylation with the amount of

              K2PtCl6 added.  Closed circles - 40 yM MeB-12 + 40 yM

              K2PtCl6 + 10 yM K2PtClJ|; open triangles - 40 yM MeB-12 +
              10 yM K2PtCl6
+ 40 yM K2PtCltt; open circles - photolysis
              for 10, 20, and 30 min of the partially reacted mixture.

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                                        MeB-12 in 0.01M HC1,  pH 2.0
                                        After
3 min with K-PtClg
                                        After 15 min
                                        After 30 min
     0
                              400     450     500

                              Wavelength, nm
       550
600
650
Figure 4.     Time-dependent quantitative demethylation upon the
              addition of 100 yM K2PtCl6  alone.

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and the methyl transfer is actually a 2-electron redox switch.  In
reaction 3, the stoichiometry does not reflect the pivotal function of
the Pt2+ ion.  Thus, K2PtCllt would be required only in catalytic  (trace)
amounts.  Its concentration would influence the initial reaction rate as
in Fig. 2 (inset), but not the ultimate final extent of demethylation
(Fig. 1A versus Fig. 4).

Relative to other alkylcorrinoids MeB-12 is by far the most reactive
alkyl group donor.  Ethyl B-12 and Mecobinamide are demethylated only
1-2% as rapidly as MeB-12 at pH 2.0 (Table 1).  This specificity pattern
     Table 1.  ALKYLCORRINOID SPECIFICITY OF THE DEMETHYLATION BY
               K2PtCl6a
     Alkylcorrinoid,      .  .      , .       Relative demethylation
          40 UM           * A350nm/min
MeB-12
EthylB-12
PropylB-12
Mecobinamide
Propylcobinamide
0.336
0.004
0.002
0.007
0.0004
1.0
0.012
0.006
0.02
0.001
     Reaction mixtures contained 100 \M K2PtCl6 + 100 yM
is similar to what was observed several years ago in the reactivity of
alkylcorrinoids with HgCl2-  It indicates that just as in the reaction
with Hg2*, the Me group of MeB-12 is being transferred to Pt as a
carbanion (i.e. Cti$ ).  Among other halogen Pt1*"1" compounds 100 \iM levels
of K2PtBr6, K2PtI6, and chloroplatinic acid demethylated MeB-12 100%,
85%, and 65%, respectively, as fast as K2PtCl5-  When reactions between
MeB-12 and K2PtCl6 were carried out at pH 1.0 in 0.1 M HC1 + 1.0 M NaCl,
37% of the Me groups were released as MeCl.  This indicates an overall
transfer from MeB-12 to Pt'*'1" to Cl0 under very high acidic-salt
conditions.

While the reaction with the hologen platinates requires slightly acidic
conditions, the demethylation of MeB-12 by Pt(SOi+)2 does not.  Raising
the pH from 2.0 to 7.0 instead increased the reaction rate by two fold
(Table 2).  Moreover, the reaction rate at pH 2.0 in both HCIO^ (Table 2)
and dilute H250^ (Fig. 5) was not markedly dependent on the simultaneous
addition of the Pt2+ ion' added as K2PtCli+.  Depending on the acid


                                    11

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                           1. MeB-12  in 0.01N
                           2. After 1.5 min with Pt(SOj
                           3. After 15 min
                           4. After 1 hr
                                   pH 2.0
            After 1.5 min with Pt(S04)2
                             MeB-12 in 0.01N. H2S04
                             After 15 min
                             After 1  hr
                             After 16 hr
         300
350    400    450     500    550    600     650
      Wavelength, nm
Figure 5.     Demethylation of MeB-12 by 100 yM Pt(SOi)2.
                           12

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  Table 2.  RELATIVE RATES OF MeB-12 DEMETHYLATION BY K2PtCl6 VERSUS
   Ft1** Compounds,
       100 yM
                              A350nm/rain
Relative demethylation
         rate
K2PtCl6 + K2PtClit
K2PtCl6
Pt(SOiJ2 + K2PtClif
Pt(S002
Pt(SO,)2
Pt(S04)2
Pt(SOJ2
Pt (50^)2
2.0a
2.0a
2.0a
2.0a
2.0b
3.0b
4.5C
7.0d
0.033
0.001
0.029
0.015
0.024
0.037
0.033
0.045
1.0
0.03
0.88
0.46
0.73
1.11
1.0
1.4
Incubation solvent was HCIO^ and K

Incubation solvent was 0.01 N
                                            when present was 1.0 yM.
   'Incubation buffer was 0.1 M Na-acetate.

   Incubation buffer was 0.1 M K-phosphate.
solvent selected, Pt(SOi^ will demethylate MeB-12 at 46-73% of the rate
obtained with 100 yM K2PtCl5 + 1.0 yM K2PtCltt.  In contrast, finely-
divided suspensions of Pt02 did not demethylate MeB-12 at any pH even
after 48 hrs of incubation.

Most of our effort with Pt has been directed at the fate of the Me group
when equimolar levels of MeB-12 and K2PtCl6 react at pH 2.0 in the
presence of low Cl0 concentrations.  To facilitate this study, extensive
use was made of the [Me-ll+C]MeB-12 and the [Me-3H]MeB-12 that we
synthesized.  When either 40 yM [Me-^C]MeB-12 or [Me-3H]MeB-12 are
reacted with 40 yM K2PtCl6 about 75% of the label is retained in the
incubation residue after lyophilization to near dryness.  Upon subsequent
paper chromatography (Fig. 6) and paper electrophoresis (Fig. 7) much
of the radioactivity coincides with a major zone of Pt.  There was no
selective loss of 3H relative to lttC in mixed labeled reaction mixtures
(Fig. 7).  Large amounts of ^C-Pt product were then prepared by reacting
50 ymoles each of [Me-14C]MeB-12 and K2PtCl6.  Purification of the
l^C-Pt product was achieved by three successive column chromatography
steps (Table 3), the material being monitored by its radiolabel and its
UV absorbance.  The isolated product has a lkC/Pt ratio of 1.21 and is

                                  13

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                   (	j  S9LOU1U uinu|4B[cj
                in    o    in
                            
                CM    CM    i
Figure 6.     Ascending paper chromatography of the reaction products
              from a 2 hr incubation of 40 yM  [Me-llfC]MeB-12 +  40  yM
              KaPtClg.  After lyophilization the residue was dissolved
              in 0.1 ml of water and aliquots were subjected to paper
              chromatography in the dark in two solvent systems.
              A.  H20:n-butanol:isopropanol:acetic acid (100:100:70:1)
              and B.  n-butanol:ethanol:H20 (50:15:35).  The chromato-
              grams were then cut into 1 cm sections for radioactive
              counting and Pt analysis.

                                    14

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-------
       Table 3.  PURIFICATION SUMMARY OF THE [Me-^CjPt PRODUCT
                                      la|C           l^C        Yield
              Step                   (cpm)        (pmoles)      (%)

    Original reaction mixture     13.2  x io6       50          100

    LyophiHzed incubation         9.14 x io6       34.5         69
      mixture

    1st Sephadex G-15              7.49 x IO6       28.5         57
    '  column

    2nd Sephadex G-15              5.39 x io6       20.5         41
      column

    CM-cellulose column            4.80 * io6       18           36
characterized by absorption maxima at 260 nm and 208 ran with a minimum
at 240 nm (Fig. 8).  Spectrally, the 11+C-Pt product resembles a Pt1**
salt (K2PtCl6) rather than a Pt*+ salt (K2PtCllt) (Fig. 8B), but the
actual Pt valence state remains to be determined definitively.
Generally, the overall recovery of 14C relative to the original
incubation mixture was 36-42% (Table 3).  Similarly, a purified 3H-Pt
compound was obtained in the same overall yield starting from 50 ymoles
of [Me-3H]MeB-12.  It exhibited an identical absorption spectrum and
the 3H/Pt ratio was 1.3.  This information, in combination with our
paper chromatographic-electrophoretic results (Figs. 6 and 7) and the
release of MeCl under extreme acid-salt conditions, proves that the
isolated ll*C-Pt product contains intact Me groups.

To determine the nature of the bonding between carbon and Pt, the
isolated compound was studied by proton-NMR spectroscopy (Fig. 9).  It
yields a three banded spectrum with a J (coupling constant for 1H, 195Pt)
of 78.2 Hz and a T for l^Pt-Me + 196Pt-Me of 6.956.  The NMR spectrum
confirms the presence of an H-C-Pt covalent bonding pattern in the
product.  When considered with other data that the carbon  is present in
intact Me groups, the NMR spectrum (Fig. 9) provides definitive evidence
that the product is an Me-Pt compound.

The stability of our purified [Me-^CJPt product with respect to its
260 nm absorbance was studied under a variety of conditions.  Under each
condition tested the loss of absorbance at 260 nm plotted  as a first
order decomposition process with respect to time (e.g. Fig. 10).  In
Table 4 we have summarized the times required for 50% decay of 180 yM
solutions of the [Me-ll+C]Pt compound.  These half-lives indicate that
although our Me-Pt derivative is moderately light-sensitive, it is
sufficiently stable with respect to temperature, NaCl, and pH to exist
                                   16

-------
   1.0

   0.9

   0.8

   0.7

a,  0-6
u
.2  0.5
i.
o
VI
3  0.4

   0.3

   0.2

   0.1

     0
         I        I        I         I       I

   1. Eluate off CM cellulose column
   2. Eluate after rotary evaporation at 30
          250
300
350
400     450     500     550      600
               Wavelength, nm
650 200
                                                                    1.  30 yM [Me-14C]Pt
                                                                    2.  50 yM K2PtCl4
                                                                    3.  15 yM K2PtClg
250
                  Figure 8.
              Absorption spectrum of the [Me-llfC]Pt reaction product
              formed from [Me-1'*C]MeB-12 and K2PtCl6.  A.  Spectrum
              after CM-cellulose column purification step and a
              subsequent flash evaporation step to concentrate the
              compound in solution.  B.  Comparison of the light
              absorption spectrum to two chloroplatinates which contain
              Pt2* and Pt1* , respectively.  All spectra were taken at
              pH 2.0 in 0.01 M HC1.
300
350

-------
00
                                                                                                             10
                    Figure 9.      Proton NMR spectrum of the  [Me-ll*C]Pt product dissolved in
                                  water at  a **C concentration of 33 mM and containing a
                                  5 mM level of DSS internal  standard.

-------
           o

           OJ
           N
           0>
           U

           O)
           a.
           o
           vo
           CVJ
Figure 10.    Effect of  light  and temperature on the UV absorption of

              the  [Me-ll+C]Pt product.   Temperatures shown are in C.

              Light is a 40 W  tungsten lamp at a distance of 10 cm.
                                   19

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             Table 4.  STABILITY OF THE [Me-1'*C]Pt PRODUCT
                       UNDER VARIOUS CONDITIONS
               Storage condition in                  Half
                 aqueous solution                    nair
        0C, Dark, H20                              21   days
       22C, Dark, H20                                10.2 days
       37C, Dark, H20                                 1.3 days
       45C, Dark, H20                                 9.7 hrs 
       62C, Dark, H20                                 1.3 hrs
        0WC, Light, H20d                               9.0 hrs
       37C, Light, H20a                               6.0 hrs
       22C, Dark, 4 mM NaCl                          10.2 days
       22C, Dark, 0.12 M NaCl                         2.8 days
       22C, Dark, 0.1 M HC1                           2.8 days
       22C, Dark, 0.01 M HC1                          9.0 days
       22C, Dark, 0.1 M acetic acid                   6.2 days
       22C, Dark, 0.1 M Na-acetate (pH 4.5)           7.0 days
       22C, Dark, 0.1 M K-phosphate (pH 7.0)          3.3 days
       22C, Dark, 0.1 M Na-pyrophosphate (pH 8.4)     2.3 days
       aLight = 40 W tungsten lamp at a distance of 10 cm.
        Half-life is the time required for the 260 nm absorbance
        peak of a 180 yM solution of [Me-1<4C]Pt  to decrease by
        one-half of the maximal possible amount.
in freshwater ecosystems.  It would also appear to be stable enough to
have biological activity.
PALLADIUM
When 40 yM MeB-12 is mixed with 40-100 yM I^PdClg at pH 2.0, a complex
forms immediately.  It has absorption maxima at 350 nm, 495 nm, and
525 nm and is stable for about 30 min in the dark.  No evidence for a
spectrally distinct Complex between 40 yM MeB-12 and 40-100 yM K2PdCl6
was observed at pH 7.0 and no complex with I^PdCl^ was detectable at any
pH.  Spectrophotometric titration at pH 2.0 revealed that two equivalents
of K2PdC]gwere necessary to convert MeB-12 completely into a complex.
                                   20

-------
Upon extensive incubation of the complex the absorbance at 350 nm
slowly increased and its spectrum became identical to that of aquoB-12.
However, since both the complex and aquoB-12 have maxima at 350 nm, it
is difficult to determine spectrophotometrically the rate of complex
demethylation.  Using 40 yM [Me-1^C]MeB-12 we estimated that after 24
hrs at 22C at least 80% of the MeB-12 was demethylated by 100 yM
K2PdCl6 and 23% was demethylated in the presence of K2PdClit.  Thus,
Pd1** and Pd2+ can react with MeB-12 at pH 2.0, but the rates are much
slower than for 100 yM Ft1*"1".  Also, Pd2+ does not significantly increase
the rate of complex formation between K2PdCl6 and MeB-12 or its rate of
breakdown to aquoB-12.

In view of the reactivity of Pt (80^)2 we tested the effect of 100 yM
PdSOjj on 40 yM MeB-12.  Incubations were made at pH 2.0, 4.5, and 7.0.
After 48 hrs the extents of demethylation were 72%, 32%, and 0%,
respectively.  Since K2PdCl6 reacts faster than K2PdClit, it is possible
that the demethylation observed with Pd2+ salts is due to their slow
oxidation to Pd^+.

LEAD

A study was made of the reactivity between 40 yM MeB-12 and various Pb
compounds at pH 2.0 (0.01 M HC1) and at pH 4.5 (0.1 M Na-acetate) .  The
increase in absorbance at 350 nm served as a measure of the extent of
MeB-12 demethylation to aquoB-12.  No significant reaction occurred at
either pH between MeB-12 and 100 yM PbCl2, PbBr2, and Pb(Ac)2 after 24
hrs at 22C.  However, PbfAc)^ altered the spectrum of MeB-12 and
formed a complex over a period of 0-30 min.  Upon prolonged incubation
for 24 hrs, the conversion of MeB-12 to aquoB-12 was 57-64% at pH 2.0
(Fig. 11).  The extent of demethylation at pH 4.5 was 31% for Pb(Ac)u;
at pH 7.0 it was 0%.  Since Pt2* stimulates the reaction between Ptk+
and MeB-12, we examined the possibility that Pb2+ might behave similarly
with respect to Pb**+.  In contrast, no evidence was found that Pb(Ac)2
promotes a faster reaction between MeB-12 and Pb(Ac)it.
Both Pb(Ac)i and PbPi^ are rapidly hydrolyzed by water forming a brown
precipitate of Pb^O^ and Pb02.  At 100-200 nmole/ml levels these fine
suspensions of Pb$Q^ and Pb02 are barely visible so that the reaction
solutions still appear quite clear.  For comparison, 40 yM solutions of
MeB-12 were incubated with 100 nmole/ml fine suspensions of PbaOi^ and
Pb02 at pH 2.0 (Table 5).  After 24 hrs the extents of demethylation
were 57% and 37%, respectively.  At pH 4.5 they were both 25% and at
pH 7.0 they were 0%.  Thus, from the similar extents of demethylation,
it is likely that when one adds Pb(Ac)it or Pbfi^ to aqueous solutions
the actual Pb compounds being tested for reactivity are Pb^Oi^ and Pb02.
Apparently, suspensions of Pb oxide are in equilibrium with traces of
Pb*+ that are reactive with MeB-12.  The rate of MeB-12 demethylation
is quite slow due to the extremely low solubility of Pb^O^ and Pb02 in
water; nonetheless, it is significant over an extended period of time.
It should be noted in Table 5 that a close correspondence was observed
                                   21

-------
                               1. MeB-12 In 0.01M HC1, pH 2.0
                               2. After 1.5 min with PbAc
                               3. After 30 min
                               4. After 24 hrs
                              400     450     500
                                 Wavelength,  nm
Figure 11.    Partial demethylation of MeB-12 with Pb(Ac)u at a
              concentration of 100 nmoles/ml.
                                 22

-------
    Table 5.  EXTENTS OF [Me-1!C]MeB-12 DEMETHYLATION AND THE
              RECOVERY OF 14C AFTER PROLONGED INCUBATION WITH VARIOUS
              LEAD SALTS AND OXIDES3
    Compound    Concentration    % Demethylation
% of the initial
     recovered
PbCl2
PbBr2
Pb(Ac)2
Pb(Ac)^
Pb(Ac)4
Pb30t
Pb304
Pb02
Pb02
100 yM
100 yM
100 yM
100 nmoles/ml
200 nmoles/ml
100 nmoles/ml
200 nmoles/ml .
100 nmoles/ml
200 nmoles/ml
0-2
0-2
0-2
59
100
57
90
37
61
97
97
98
40
2
33
2
61
31
    alncubations were for 24 hrs at 22C in the dark at pH 2.0.

     After the incubation the reaction mixtures were lyophilized to
     dryness in the dark and then reconstituted in water to
     determine the recovery of non-volatile 1I+C.
between the extents of demthylation by several lead compounds and the
disappearance (volatization) of 11+C.  Several paper electrophoresis runs
of the lyophilized Pb reaction mixtures (analogous to Fig. 7) revealed
the presence of ^C associated only with the remaining unreacted
[Me-^C]MeB-12.

MANGANESE

We also examined the reactivity of several Mn2+ salts and Mn02 with
MeB-12.  After incubation of 40 yM MeB-12 with 100 yM MnCl2, MnBr2,
MnSoij, and a fine suspension (100 nmoles/ml) of Mn02 for 24-48 hrs, we
could detect no demethylation.  No reactivity was observed at pH 2.0,
4.5, or 7.0.
                                   23

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                              SECTION IV

                              REFERENCES

 1.  Barnes, J.M., and Magos, L., "The Toxicology of Organometallic
     Compounds," Organometallic Chem. Rev. 3, 137-150 (1968).

 2.  Wood, J.M., Kennedy, F.S., and Rosen, C.G., "Synthesis of Methyl-
     mercury Compounds by Extracts of a Methanogenic Bacterium," Nature
     220, No. 5163, 173-174 (1968).

 3.  Imura, N., Sukegawa, E., Pan, S.-K., Nagao, K., Kim, J.-Y.,
     Kwan, T., and Ukita, T., "Chemical Methylation of Inorganic Mercury
     with Methylcobalamin, a Vitamin B-12 Analog," Science 172, No. 5989,
     1248-1249 (1971).

 4.  Bertilsson, L.,  and Neujahr, H.Y., "Methylation of Mercury Compounds
     by Methylcobalamin," Biochemistry 10, No.  14, 2805-2808 (1971).

 5.  DeSimone, R.E,  Penley, M.W., Charbonneau, L., Smith, S.G.,
     Wood, J.M., Hill, H.A.O., Pratt, J.M., Ridsdale', S., and
     Williams, R.J.P., "The Kinetics and Mechanism of Cobalamin-
     Dependent Methyl and Ethyl Transfer to Mercuric Ion," Biochim.
     Biophys. Acta 304, No. 3, 851-863 (1973).

 6.  Pan, S.-K., Imura, N., and Ukita, T., "Fractionation and
     Characterization of Mercury-Methylation Factor in Tuna Liver,"
     Chemosphere 2, No. 6, 247-252 (1973).

 7.  Holbrook, Jr., D.J., Washington, M.E., Leake, H.B., and Brubaker, P.E.,
     "Studies on the Evaluation of the Toxicity of Various Salts of Lead,
     Manganese, Platinum, and Palladium," Environmental Health
     Perspectives.  10, 95-101 (1975).

 8.  Brubaker, P.E.,  Moran, J.P., Bridbord, K., and Hueter, F.G.,
     "Noble Metals:  A Toxicological Appraisal  of Potential New
     Environmental Contaminants," Environmental Health Perspectives 10,
     39-56 (1975).

 9.  Agnes, G., Bendle, S., Hill, H.A.O., Williams, F.R., and
     Williams, R.J.P., "Methylation by Methyl Vitamin B-12," Chemical
     Communications (London) pp 850-851 (1971).

10.  Wong, P.T.S., Chan, Y.K., and Luxon, P.L., "Methylation of Lead in
     the Environment," Nature 253, No. 5489,  263-264 (1975).

11.  Jarvie, A.W.P.,  Markall, R.N., and Potter, H.R., "Chemical
     Alkylation of Lead," Nature 255, No. 5505, 217-218 (1975).

12.  Dolphin, D./'Preparation of the Reduced Forms of Vitamin B-12 and
     of Some Analogs  of the Vitamin B-12 Coenzyme Containing a Cobalt-
     Carbon Bond,"  In:  Methods in Enzymology, Vol. XVIII, part C,
     McCormick, D.B., and Wright, L.D. (eds.),  New York, Academic Press,
     1971, p. 34-54.

                                  24

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13.  Pailes, W.H., and Hogenkamp, H.P.C., "The Photolability of
     Co-alkyicobinamides," Biochemistry 7, No. 12, 4160-4166, (1968).

14.  Taylor, R.T., Smucker, L., Hanna, M.L., and Gill, J., "Aerobic
     Photolysis of Alkylcobalamins:  Quantum Yields and Light-Action
     Spectra," Arch.  Biochem. Biophys. 156, No. 2, 521-533 (1973).
                                  25

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TrCHMOAI. REI'OHT DATA
(I'lcasr read lnunieli"ii': i>n the tcvcnc lic/iirc comi>trlinxl
1. REPORT NO. 2- ;_
EPA-600/1-76-016
.1. TITIE AND SUBTITLE
Comparative Methylation Chemistry of Platinum, Palla-
dium, Lead, and Manganese ;
7. AUTHORISt *
Robert T. Taylor
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Biomedical Division
Lawrence Livermore Laboratory
University nf California
Livermore, CA 94550
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, fl.C. 27711
IS. SUPPLEMENTARY NOTES
1. RECIPIHMTT. ACCESSION-NO.
5. REPORT DATE
March 1976
6. PERFORMING ORGANIZATION CODE '
0. PEAfOHMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
1 1. CONTRACT/GRANT NO.
EPA-IA.G-D4-0439
13. t-Y^'E O.F REPORT AND PERIOD COVERED
 "illy3 1<74 - -I'm" 1Q7S
14. SPONSORING AGENCY CODE 


16. ABSTRACT
A study was carried out to evaluate the potential for platinum, palladium, lead, and
manganese salts and oxides to be biochemically methylated. Methylation is an impor-
tant, well recognized, determinant of metal toxicity.; the striking example being
the extreme health hazard of methylated mercury. The possible biological methyla-
tion of the metals which are associated with emissions arising from the use of auto-
motive fuels, fuel additives, and catalytic control devices is of special concern i
to the Environmental Protection Agency's Catalyst Research Program.
  Salts of platinum, palladium, and  lead,  and oxides of lead all containing the metal  in  j
  a 4+ valence were observed to demethylate  methylcobalamin, a biologically active  form  i
  of vitamin B-12.  Inorganic salts  and  oxides of manganese were unreactive.  No evidence]
  for a stable monomethyl-metal derivative was found using palladium and lead compounds
  as reactants.  However, salts of platinum  4+ do result in the formation of stable      [
  methylation products.  The reaction  product formed from methylcobalamin and hexachloro-i
  platinate was shown definitively to  be a monomethyl-platinum compound  It is suffi-
  ciently stable in aqueous solutions  under  a variety of conditions to exist in fresh-
  water ecosystems and to. exhibit toxic  effects on mammalian cells.
                               KEY WORDS AND DOCUMENT ANALYSIS
                 DESCRIPTORS
  platinum
  palladium -
  manganese
  lead (metal)
  methylation
  fuel additives
  fuels
                                              h.IDENTIFIERS/OPEN ENDED TERMS
C. COSATI I IcIil/CllMip


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