# Direct Observation of Ascorbyl Free Radicals in Whole Blood of Goats During Cardiac Surgery With Cardiopulmonary Bypass

## TL;DR

This study observed ascorbyl free radicals in goat blood during heart surgery, showing increased levels during cardiopulmonary bypass and reperfusion.

## Contribution

The study directly measured ascorbyl free radicals in whole blood without spin traps, enabling real-time oxidative stress assessment during surgery.

## Key findings

- AFR levels in goat blood significantly increased during cardiopulmonary bypass and remained elevated during aortic clamping.
- Plasma scavenging activity against hydroxyl radicals increased during surgery but not against DPPH.
- AFR levels returned to preoperative levels after aortic declamping, suggesting a link to oxidative stress phases.

## Abstract

Background: Ascorbate is located at the most downstream end of radical scavenging reactions and reacts with various free radicals to form ascorbyl free radical (AFR). In this study, we assessed AFR levels in whole blood samples obtained from perioperative goats under cardiac surgery with cardiopulmonary bypass (CPB) using electron spin resonance (ESR) spectrometry without adding any spin trap, which the stability of AFR made possible. The direct radical scavenging activity of the plasma was evaluated for hydroxyl radicals by the spin trapping method.

Materials and methods: This is an experimental animal study on goats (n = 19). Blood samples were collected at eight time points during the heart surgery using CPB. The whole blood sample was aspirated in a disposable ESR flat cell, and 95% confidence intervals (95% CIs) of the amount of AFR relative to those of the induction of anesthesia were quantified using ESR spectroscopy at each time point of the heart surgery. The scavenging activity of the plasma against hydroxyl radical and DPPH (2,2-diphenyl-1-picrylhydrazyl) was also evaluated using ESR spectroscopy with the spin trapping method; a dose-response curve for each free radical was drawn to estimate the half maximal inhibitory concentration (IC50) of plasma during heart surgery. The reciprocals of IC50 were used as the indicators of scavenging activities.

Results: The AFR level detected in goat blood significantly increased from the start of CPB (95% CI: 1.09-1.39 ) and remained significantly higher than the preoperative level during aortic clamping (1.15-1.73 at aortic cross-clamping, 1.17-1.74 one hour after clamping, and 1.16-1.52 two hours after clamping). The AFR returned to the preoperative levels after aortic declamping. The free radical scavenging activities of plasma sampled at aortic cross-clamping and from two hours after clamping to the end of surgery significantly increased against hydroxyl radicals (p < 0.05). Scavenging activity was not observed against DPPH.

Conclusion: Real-time assessment of oxidative stress was successfully conducted by ESR spectroscopy of whole blood without a spin trap. The biphasic increase in AFR during the open-heart surgery might reflect the production of free radicals under oxidative stress due to the cardiac surgery. The first phase might be due to inflammatory responses after CPB induction, and the second might reflect production of free radicals after reperfusion. We believe that measuring AFR levels in fresh whole blood would be a simple but informative indicator of oxidative stress during surgery.