Detection and scavenging of hydroxyl radical via D-phenylalanine hydroxylation in human fluids

Hydroxyl radical (center dot OH) is highly reactive, and therefore very short-lived. Finding new means to accurately detect center dot OH, and testing the ability of known center dot OH scavengers to neutralize them in human biological fluids would leverage our ability to more effectively counter oxidative (center dot OH) stress-mediated damage in human diseases. To achieve this, we pursued the evaluation of secondary products resulting from center dot OH attack, using a detection system based on Fenton reaction-mediated D-phenylalanine (D-Phe) hydroxylation. This reaction in turn generates o-tyrosine (o-tyr), m-tyrosine (m-tyr) and p-tyrosine (p-tyr). Here, these isomers were separated by HPLC, equipped with fluorescence detectors due to the natural fluorescence of these hydrotyrosines. By extension, we found that, adding radical scavengers competed with D-Phe on center dot OH attack, thus allowing to determine the center dot OH quenching capacity of a given compound expressed as inhibition ratio percent (IR%). Using a kinetic approach, we then tested the center dot OH scavenging capacity (OHSC) of well-known antioxidant molecules. In a test tube, N,N'-dimethylthiourea (DMTU) was the most efficient scavenger as compared to Trolox and N-Acethyl-L-cysteine, with NAC being the less effective. OHSC assay was then applied to biological fluid samples as seminal plasma, human serum from normal subjects and patients undergoing hemodialysis (HD), colostrum and human breast milk from mothers that received daily doses of 30 g of chocolate (70% cocoa) during pregnancy. We found that a daily administration of dark chocolate during pregnancy almost doubled OHSC levels in breast milk (1.88 +/- 0.12 times, p < 0.01). Furthermore, HD treatment determined a significant reduction of serum OHSC concentration (54.63 +/- 2.82%, p < 0.001). Our results provide evidence that Fenton reaction-mediated D-Phe hydroxylation is a suitable method for routine and non-invasive evaluation of center dot OH detection and its scavenging in human biological fluids.