1) Exposure Regime
Approximately 100 animals were exposed for 8 weeks to seawater containing (10-11 M) stable isotopic 65Cu2+ and then transferred to
media having a comparable concentration of 63Cu. The free ion activities of Cu2+ were controlled using an NTA metal - chelate buffer
system. Animals were maintained at 15o C and were not fed during the exposure time. The seawater media was replaced weekly.
2) Chemicals and Media
a) Seawater Media:
The concentrations of various compounds in the seawater media were as follows:
(Prepared from a 10x stock solution)
5 x 10-4 M NTA
5 x 10-6 M 65Cu or 63Cu
b) Homogenization buffer:
200mM Tris buffer containing 2mM DDT and 0.51% sodium azide (pH 8.5). Phenylmethlsulfonyl fluoride was added to the sample at a
final concentration of 0.1 mM immediately prior to homogenization.
3) Sample Preparation
Animals were sacrificed after 8 weeks of exposure to 65Cu and after 8 weeks of transfer to 63Cu. Animals maintained in filtered seawater
were used as controls. Changes in the isotopic content of 63Cu and 65Cu were used to determine the rate of excretion of Cu from the
various tissues, and cytoplasmic ligand pools. Fifteen animals were sacrificed at each time period, de-shelled, rinsed in fresh seawater and
blotted dry using a Kimwipe tissue. Ten of the animals were processed for tissue analysis and the remaining 5 for cytosol analysis.
a) Tissue analysis:
Animals were dissected and the visceral complex removed. The tissue was dried to constant weight at 60oC on a pre-cleaned glass
microscope slide. The dried tissue was removed with the aid of a razor blade, weighed, placed in an acid cleaned, metal free glass tube and
then digested by boiling to near dryness in 500µl of ultra-high purity HNO3 for metal analysis by
ICP-MS.
b) Analysis of Cytosols:
The visceral complex of 5 animals were dissected upon ice and pooled. The tissue was weighed and then homogenized individually at 4oC
in 5 volumes of ice cold homogenization buffer. The homogenates were carefully transferred using a glass pipette to microfuge tubes and
were spun at 4oC for 800 x g for 5 minutes, 10,000 x g for 20 minutes and 100,000 x g, for 60 min to produce nuclear, mitochondrial and
microsomal enriched pellets and a cytosolic fraction. The various fractions were then stored frozen (-80oC) for analysis and further
fractionation and metal analysis by SE/IE-HPLC-ICPMS.
4) Instrumental design of SE-IE HPLC/ICP-MS
A schematic of the SE-IE HPLC/ICP-MS system is shown in Figure 1a and the configuration of the interface is shown in Figures 1b and
1c. Aliquots (100µL) of visceral complex cytosol were fractionated at a flow rate of 1ml. min-1 isocratically by size-exclusion (SW 2000
TSK Column; #5) using a mobile phase of 20mM Tris (pH 7.2) degassed with nitrogen (#2).
Total concentrations of protein injected onto the column were 0.138, 0.19 and 0.16 mg for the control and the 65Cu and 65 +63Cu samples respectively. A Rheodyne 7000 switching
valve (#10) is used to re-direct eluting species of interest from the size exclusion to an ion-exchange column (#11; Showdex DEAE-825). A
linear gradient of 20mM Tris (#7; pH 8.2) increasing to 250mM Tris in 500mM NH4Cl (#8) over a stepped 40 minute period was used to
fractionate the proteins from the ion-exchange column (#11).
The UV absorbance of the eluent is monitored at either 254nm or 280nm by two detectors positioned down stream of the two columns (#6 and #12). Spectra on up to 11 masses for up to 8 elements was acquired
simultaneously by scanning the quadrapole of the ICP-MS in the peak hopping mode using time resolved acquisition software (#15). A
250µL flow-injection loop inserted downstream of the columns (#14) was used to quantify the resulting ion chromatograms from the
integrated peak areas of the ion intensity profiles from the MS.
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