Advancing Biomarker Research: In Situ Cu Isotope Analysis in Liver Tumors by LA-MC-ICP-MS

Stable metal isotopes have received increasing attention as medical biomarkers due to their potential to detect changes in metal metabolism related to diseases. In particular, copper stable isotopes are a powerful tool to identify isotopic variation between tumors and healthy tissue, suggesting application in cancer diagnosis. However, potential mechanisms causing isotope fractionation, such as redox- or bond-forming reactions and interactions of metals during transmembrane import and export, are less well understood. Here, we established an <i>in situ</i> method using laser ablation-multicollector-inductively coupled plasma-mass spectrometry (LA-MC-ICP-MS) to advance our understanding of the underlying processes responsible for isotope fractionation between normal and diseased tissues. Gelatin-based bracketing standards and quality control reference materials, crucial for laser ablation analysis, were developed to allow correction for instrumentally induced isotope fractionation during LA-MC-ICP-MS analysis. Using such matrix-matched standards, the method achieved intermediate precisions for delta values of better than 0.15 ‰ (2 <i>s</i>) for inorganic reference materials and of better than 0.17 ‰ (2 <i>s</i>) for biological reference materials. The developed routine was tested on rabbit VX2 liver tumor samples, a model system resembling human hepatocellular carcinoma (HCC) used to study liver cancer. <i>In situ</i> Cu isotope compositions between healthy (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msubsup><mml:mrow><mml:mi>δ</mml:mi></mml:mrow><mml:mrow><mml:mi>NIST</mml:mi><mml:mn>976</mml:mn></mml:mrow><mml:mrow><mml:mi>65/63</mml:mi></mml:mrow></mml:msubsup><mml:mrow><mml:mo>(</mml:mo><mml:mi>Cu</mml:mi><mml:mo>)</mml:mo></mml:mrow></mml:math> = -1.5 ‰ to 0.2 ‰) and tumorous (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msubsup><mml:mrow><mml:mi>δ</mml:mi></mml:mrow><mml:mrow><mml:mi>NIST</mml:mi><mml:mn>976</mml:mn></mml:mrow><mml:mrow><mml:mi>65/63</mml:mi></mml:mrow></mml:msubsup><mml:mrow><mml:mo>(</mml:mo><mml:mi>Cu</mml:mi><mml:mo>)</mml:mo></mml:mrow></mml:math> = 0.0 ‰ to 1.3 ‰) liver tissue show distinct differences in their isotope ratios. The observed isotopic dichotomy is consistent with previous solution-based MC-ICP-MS work, showing enrichment of heavy ⁶⁵Cu in cancer biopsies relative to healthy tissue.