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

Abstract

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 30g 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.

Hemodialysis raises oxidative stress through carbon-centered radicals despite improved biocompatibility

“…(31) Although contradictory results have been reported on the effect of HD on • OH scavenging activity, previous studies using ESR have shown an increase in the scavenging activity, regardless of the generation method of the hydroxyl radicals. (32)(33)(34)(35)(36) Thus, we strongly suggest that the • OH scavenging activity may be restored by HD.…”

A New Source of Diterpene Lactones From Andrographis paniculata (Burm. f.) Nees—Two Endophytic Fungi of Colletotrichum sp. With Antibacterial and Antioxidant Activities

“…The antioxidant activity of EXEs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), hydroxyl radical (OH), 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO), and ferric reducing antioxidant power (FRAP) methods, which were improved according to Li (2017) , Biondi et al (2018) , and Gulcin (2020) . The EXE samples with high antioxidant data were selected, gradient diluted, and analyzed for IC 50 values (the concentration at which 50% scavenging of DPPH, ABTS, hydroxyl, PTIO, and ferric was achieved) to evaluate the antioxidant activity of EXEs.…”

Impact of Increased Oxidative Stress on Cardiovascular Diseases in Women With Polycystic Ovary Syndrome

“…A change in the balance between oxidizing and antioxidant substances in favor of excess oxidants leads to oxidative stress. These systems include enzymes (superoxide dismutase, catalase, and glutathione peroxidase), antioxidant macromolecules (albumin, ceruloplasmin, and ferritin), antioxidant micro-molecules (ascorbic acid, α-tocopherol, β-carotene, ubiquinone, flavonoids and glutathione, methionine, uric acid) bilirubin ( 1 , 158 – 161 ).…”

“…ROS are highly reactive molecules with a very short lifespan, and are classified into two categories, namely non-radical species, including hydrogen peroxide (H2O2), hypochlorous acid (HOCl-), ozone (O3-), lipid peroxides (LOOH), along with hydroperoxides (ROOH), and radical species, consisting of superoxide anion (O2•−), singlet oxygen (1O2), hydroperoxyl radical (HOO•), hydroxyl radical (•OH), with •OH being considered the most important ROS ( 158 ). RNS on the other hand comprise a range of various chemical compounds derived from nitric oxide (NO) in the reaction of biologically generated free radicals that tend to form more stable species, a process that generates multiple biological effects ( 162 ).…”