8 ohdg

For this study, 8-OHdG was purchased from Sigma-Aldrich (St. Louis, MO, USA); 15N5-8-OHdG was obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA), and 8-iso-PGF2α and 8-iso-PGF2α-d4 were from Cayman Chemicals Co. (Ann Arbor, MI, USA). Amicon^® ultracentrifugal filters (Ultracel^®-30 K) were purchased from Merck Millipore, Ltd. (Cork, Ireland), and Sep-Pak^® C18 solid-phase extraction (SPE) cartridges (3 cc, 500 mg) were purchased from Waters (Milford, MA, USA). Purified water was obtained from the Milli-Q^® Integral system (Merck Millipore Ltd., Cork, Ireland). All other chromatography-grade chemicals were obtained from Sigma-Aldrich.

HNE-MA (1mg in 100 μl ethanol), HNE-MA-d₃ (100 μg in 100 μl ethanol), 8-isoPGF_2α, and 8-isoPGF_2α-d₄ were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Maleic anhydride, 8-OHdG, and 7.5M of ammonium acetate solution (NH₄Ac_(aq)), 0.1 N sodium hydroxide (NaOH) standard solutions, and were purchased from Sigma–Aldrich (St. Louis, MO, USA). The internal standard, ¹⁵N₅-8-OHdG (99% purity), was acquired from Cambridge Isotope Laboratories (Andover, MA, USA). Unlabeled 8-NO₂Gua and its internal standard, 8-NO₂Gua-4, 8-¹³C₂-7-¹⁵N, were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). HPLC-grade methanol (MeOH) was procured from MACRON Chemicals (Center Valley, PA). For all subsequent steps, Milli-Q water was produced by a Millipore Elix 10 RO system and a Millipore Synergy UV system (Millipore SAS, Molsheim, France).

We obtained 8-OHdG from Sigma (St. Louis, MO). In addition, we obtained dl-isoproterenol hydrochloride, which was used as an internal standard (IS); sodium dihydrogen phosphate dehydrate; and disodium hydrogen phosphate 12-water from Wako Pure Chemical Industries (Osaka, Japan). We purchased ethylene diamine tetra-acetic acid disodium salt (EDTA-2Na) from Dojindo Laboratories (Kumamoto, Japan). Methanol of chromatographic grade was produced by Kanto Chemical Co., Inc., (Tokyo, Japan). Ultra-pure water was produced using the Milli-Q water purification system (Millipore, Bedford, MA). Other chemicals and solvents were of high-performance liquid chromatography (HPLC) grade or reagent grade quality.

A total of four biomarkers were selected for method development due to their acknowledged indication of oxidative stress within urine [28 (link)]. The standard 8-OHdG was bought from Sigma–Aldrich (UK), its respective internal standard ¹⁵N₅-8-OHdG along with 8-NO₂Gua were purchased from Santa Cruz Biotechnologies (UK). The standards 8-iso-PGF_2β, HNE-MA and the internal standard HNE-MA-d3 were bought from Cayman Chemicals (US). Stock solutions of selected biomarkers were made up by dissolving solid samples in MeOH and all stock solutions were kept in the dark at − 80 °C. Working solutions were diluted from the stock solutions to make up the desired concentrations in 80:20 H₂O:MeOH. Solvents such as MeOH and toluene were HPLC grade and purchased from Sigma–Aldrich. To remove the risk of basic functional groups reacting with silanols on glass surfaces, all glassware was deactivated using 5% dimethylchlorosilane (DMDCS) in toluene. The silanisation of glass occurred by rinsing with DMDCS before washing twice with toluene and three times with MeOH.

Methanol (MeOH) of HPLC grade was obtained from Merck KGaA (Darmstadt, Germany). Formic acid (HCOOH), acetic acid (CH₃COOH), and 8-OHdG were bought from Sigma-Aldrich (St Louis, MO, USA). [¹⁵N₅]8-Hydroxy-2′-deoxyguanosine ([¹⁵N₅]8-OHdG) was purchased from Cambridge Isotope Laboratories Inc. (Andover, MA, USA). 8-OHG and [¹³C¹⁵N₂]8-hydroxyguanosine ([¹³C¹⁵N₂]8-OHG) were bought from Toronto Research Chemical (Toronto, Canada). Water was gained from a Milli-Q water purification apparatus (Millipore, Milford, MA, USA).

Specifically, 8-OHdG, bovine serum albumin (BSA), citric acid, diethylenetriamine (EDTA), (3-glycidyloxypropyl)trimethoxysilane (GPMS), glutaraldehyde, methylbenzene, chloroauric acid, 3-mercaptopropionic acid (MPA), zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O), methanol (99.8%), sodium chloride, potassium chloride, calcium chloride, acetone, N,N-dimethylformamide (DMF), sodium borohydride (NaBH₄, 99.99%), and dehydrated alcohol were obtained from Sigma Aldrich (St. Louis, Missouri (Mo), USA). NaCl, KCl, CaCl₂, MgCl₂, thymine, cytosine, adenine, guanine, and hydrogen peroxide were ordered from ALADDIN Reagent (Shanghai, China). Nanoporous alumina membranes were purchased from Whatman (Boston, Massachusetts (Ma), USA). Alongside, 8-OHdG antibody was bought from Abcam (Cambridge, UK). In addition, 2-methylimidazole (2-MeIM, 99%), 1-dodecanethiol (DDT, ≥98%), hexadecyltrimethyl ammonium bromide (CTAB, 98%), silver nitrate (AgNO₃, ≥99.0%), 11-mercaptoundecanoic acid (MUA, 95%), and 4-nitrophenol (4-NP, ≥99.5%) were purchased from Sigma Aldrich (St. Louis, Missouri (Mo), USA). The chemicals were used as received without further purification.

The method described previously [23 (link)] was followed with some modifications. In brief, cells were treated with 1 mM H₂O₂ for 1 h and harvested at the indicated time points for genomic DNA extraction. Each DNA sample was denatured at 95°C for 5 min and was then chilled on ice followed by incubation with 2 units of alkaline phosphatase (BioLabs, Ipswich, MA, USA) and 5 units of DNase I (Sigma-Aldrich) in 50 mM Tris, pH 7.3, and 1 mM MgCl₂ (Merck, Germany) buffer at 37°C for 2 h. 96-well plates were first coated with 0.003% protamine sulfate (Sigma-Aldrich) and then with 100 ng 8-OHdG (Sigma-Aldrich). Coated wells were added in a series of concentrations of pure 8-OHdG or DNA samples. The antibody to 8-OHdG (1 : 500; Trevigen), biotin goat anti-mouse IgG (1 : 1000; Zymed Laboratories, Inc., San Francisco, CA, USA), and peroxidase-streptavidin (1 : 10000; Sigma-Aldrich) were used sequentially for the detection of 8-OHdG. O-Phenylenediamine (Pierce, Thermo Scientific, Rockford, IL, USA) dissolved in citrate phosphate buffer (5.103 g citrate acid monohydrate, 7.297 g Na₂PO₄ in 1 L ddH₂O adjusted to pH 5.0 with citric acid) was used as a substrate for peroxidase. The absorbance was read at 492 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).