Analytical variables affecting exchangeable copper determination in blood plasma.

Area of Science:

• Clinical Chemistry
• Trace Element Analysis
• Biometals Research

Background:

• Discrepancies exist in measuring plasma exchangeable copper (Cu) using various methods.
• Understanding plasma storage effects and optimizing stable isotope dilution (SID) are crucial for accurate Cu determination.
• Exchangeable Cu, also known as direct reacting or loosely bound Cu, plays a role in biological processes.

Purpose of the Study:

• To resolve discrepancies in plasma exchangeable Cu determination.
• To evaluate plasma storage techniques for accurate Cu analysis.
• To optimize and validate a stable isotope dilution procedure for exchangeable Cu.

Main Methods:

• Evaluation of plasma storage conditions: room temperature, 5°C, freeze/thaw cycles, and -65°C.
• Application of a stable isotope dilution (SID) procedure using 65Cu2+ tracer.
• Assessment of analytical factors: shaking method, sample size, heparin presence, sodium diethyldithiocarbamate concentration, and pH.

Main Results:

• Plasma exchangeable Cu increased with storage at room temperature, 5°C, and freeze/thaw cycles; -65°C storage was safe.
• The SID procedure showed rapid exchange of 65Cu2+ with endogenous plasma Cu.
• Exchangeable Cu determination was stable within a specific pH range (7.2-8.5) and sodium diethyldithiocarbamate concentration, indicating specificity for loosely bound Cu.

Conclusions:

• Stable isotope dilution (SID) provides reliable and physiologically meaningful results for plasma exchangeable copper.
• Optimal sample storage at -65°C is essential to prevent artefactual increases in exchangeable Cu.
• The SID method accurately quantifies exchangeable Cu, distinguishing it from tightly bound forms like ceruloplasmin-bound Cu.