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Depending on the oxygenation state, NO may be released from hemoglobin [173]

Concurrently, mitochondrial ROS (mtROS) amplifies vascular dysfunction by oxidizing BH4, uncoupling endothelial eNOS, and reducing NO bioavailability while also activating pro-inflammatory pathways (e.g., NLRP3 inflammasome) and stimulating NOX activity, creating continuous oxidative damage (Penna and Pagliaro, 2025)

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The whole mixture was incubated at 37 C temperature for 1 h and read the absorbance at 525 nm