Hydroxyl radical (.OH) is highly reactive, and therefore very short-lived. Finding new means to accurately detect .OH, and testing the ability of known .OH scavengers to neutralize them in human biological fluids would leverage our ability to more effectively counter oxidative (.OH) stress-mediated damage in human diseases. To achieve this, we pursued the evaluation of secondary products resulting from .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 .OH attack, thus allowing to determine the .OH quenching capacity of a given compound expressed as inhibition ratio percent (IR%). Using a kinetic approach, we then tested the .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 .OH detection and its scavenging in human biological fluids.

羟基自由基（.OH）具有很高的反应活性，因此寿命很短。寻找准确检测.OH的新方法，并测试已知.OH清除剂在人类生物流体中中和它们的能力，将利用我们的能力更有效地抵抗人类疾病中氧化（.OH）应激介导的损害。为此，我们使用基于Fenton反应介导的D-苯丙氨酸（D-Phe）羟基化。该反应继而产生邻酪氨酸（o-tyr），间酪氨酸（m-tyr）和对酪氨酸（p-tyr）。在这里，由于这些水酪氨酸的天然荧光，这些异构体通过配备有荧光检测器的HPLC进行了分离。通过扩展，我们发现，添加自由基清除剂可与D- Phe竞争.OH攻击，从而可以确定以抑制率百分比（IR％）表示的给定化合物的.OH猝灭能力。然后，我们使用动力学方法测试了著名的抗氧化剂分子的.OH清除能力（OHSC）。在试管中，与Trolox和N-Acethyl- L相比，N，N'-二甲基硫脲（DMTU）是最有效的清除剂-半胱氨酸，NAC效果较差。然后将OHSC测定法应用于精液，精浆，正常受试者的人血清以及接受血液透析（HD）的母亲的初乳和人母乳患者的生物液体样品，这些母亲在怀孕期间每天接受30 g巧克力（70％可可）的剂量。我们发现，怀孕期间每天服用黑巧克力几乎会使母乳中的OHSC水平增加一倍（1.88±0.12倍，p <0.01）。此外，HD治疗确定血清OHSC浓度显着降低（54.63±2.82％，p <0.001）。我们的结果提供了证据，表明Fenton反应介导的D - Phe羟基化是常规和非侵入性评估.OH检测及其在人体生物流体中清除的合适方法。