Stiftung Tierärztliche Hochschule Hannover (TiHo)

Spectroscopic assessment of oxidative damage in biomolecules and tissues

Oxidative damage is one of the main causes of cryopreservation injury compromising the use of cryopreserved biospecimens. The aim of this study was to evaluate the use of Fourier transform infrared spectroscopy (FTIR) as a non-invasive method to assess changes in biomolecular composition and structure, associated with oxidative stress in isolated biomolecules, acellular heart valve tissues, and ovarian cortex tissues. FTIR spectra of these specimens subjected to various treatments (H<sub>2</sub>O<sub>2</sub>- and Fenton-treatment or elevated temperatures) were vector normalized and selected spectral regions were analyzed by principal component analysis (PCA). Control and damaged biomolecules can easily be separated using PCA score plots. Acellular heart valve tissues that were subjected to different levels of oxidative damage formed separate cluster in PCA score plots. In hydrated ovarian tissue, large variation of the principal components was observed. Drying the ovarian tissues samples resulted in improved cluster separation of treatment groups. However, early signs of oxidative damage under mild stress conditions could not be detected by PCA of FTIR spectra. For the ovarian tissue samples, the standardly used nitro blue tetrazolium chloride (NBT) assay was used to monitor the amount of formazan production, reflecting reactive oxygen species (ROS) production at various temperatures. At 37 °C, formazan staining rapidly increased during the first 30 min, and then slowly reached a saturation level, but also at lower temperatures (i.e. 4 °C) formazan production was observed. In summary, we conclude that ATR-FTIR combined with PCA can be used to study oxidative damage in biomolecules as well as in tissues. In tissues, however, sample heterogeneity makes it difficult to detect early signs of oxidative damage.

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