The changes in the total concentrations of 63Cu and 65Cu in the
visceral complex during the 8-week exposure to 63Cu are shown in
figure 2. Linear regression analysis shows that the rates of
65Cu loss (y = -0.010t +1.121 : R2 = 0.84) is slightly less than
63Cu uptake (y = 0.016t +0.575 : R2 = 0.89). Expressing this
data as isotopic ratios (Figure 3) provides information, independent of
variations in tissue mass, about the integrated rates of 63Cu accumulation,
65Cu loss, and turnover of the metal. Equations based upon linear
rates of change of 63Cu and 65Cu and fitted to the data iteratively by
the method of least squares give an equation of y = (-0.013t + 1.11)/(0.008t
+ 0.06) with a R2 value of 0.96 implying that the movement of metal within
the visceral complex can be described by exchange in a one-compartment
model with an initial instantaneous rate of change in the isotopic ratios
of 65Cu to 63Cu of –0.05 days–1.
Figures 4, 5 and 6 show absorbency data from HPLC fractionation and
elemental analysis of the visceral complex cytosol from control animals
(Figure 4) and experimental animals exposed to 8 weeks 65Cu (Figure 5)
and others exposed to first 65Cu and then for 8 weeks to 63Cu (Figure 6).
Quantification of the various integrated peaks identified in the time resolved
mass profiles was accomplished using the integrated areas obtained from
flow injection analysis responses from 25ng injections of metal standards
taken before and after each chromatographic run. The mass quantities
of each element for the major chromatographic peaks obtained by size exclusion
are shown in Table 1. Similarly, the mass quantities of elements
associated with the various peaks obtained by subsequent ion exchange of
the moieties obtained within a molecular mass range of 30 to 10 kDa by
size exclusion HPLC are shown in Table 2.
Fig. 1 SE-IE/HPLC / ICP-MS:
(a) Chronological order of compartments within the SE-IE/HPLC / ICP-MS
system
(b) Size Exclusion – Ion Exchange High Pressure Liquid Chromatography
Apparatus
(c) Inductively Coupled Plasma Mass Spectrometer
Fig. 2 Time dependent changes in the concentrations of 63Cu (circles)
and 65Cu (squares) in the visceral complex of L. littorea during the 8
weeks of exposure to 63Cu (courtesy of Simone Francis)
Fig. 3 Time dependent changes in the isotopic ratio of 65Cu:63Cu
in the visceral complex of L. littorea during the 8 weeks of exposure to
63Cu (courtesy of Simone Francis)
Fig. 4 CONTROL: (a) UV Profile from Size Exclusion HPLC
(b1-b2) SE–HPLC coupled ICP–MS Elemental Profile
(c) UV Profile from Ion-Exchange of
17 kDa Protein Pool
(d1-d2) IE-HPLC coupled ICP-MS Elemental Profile
Fig. 5 65Cu 8 weeks:
(a) UV Profile from Size Exclusion HPLC
(b1-b2) SE–HPLC
coupled ICP–MS Elemental Profile
(c) UV Profile from Ion-Exchange of 17 kDa Protein Pool
(d1-d2) IE-HPLC coupled ICP-MS Elemental Profile
Fig. 6 65Cu 8 weeks, then 63Cu 8 weeks:
(a) UV Profile from Size Exclusion HPLC
(b1-b2) SE–HPLC coupled ICP–MS Elemental Profile
(c) UV Profile from Ion-Exchange of 17 kDa Protein Pool
(d1-d2) IE-HPLC coupled ICP-MS Elemental Profile
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