Biomarkers known to be influenced by n-3 PUFA were analysed. Serum lipids were analysed using the I-LAB 650 Chemical Analyser (Instrumentation Laboratories, Warrington, Cheshire, UK). LDL cholesterol was calculated using the Friedewald formula (Total cholesterol – HDL – (triglycerides/2.2)). The I-LAB 650 Chemical Analyser was also used to measure high-sensitivity C-reactive protein (hsCRP) in serum samples. The inflammatory markers interleukin (IL)-5, IL-10, IL-1β, IL-6 and tumour necrosis factor-alpha (TNF-α) were measured using an immunoassay from Meso Scale Discovery (MSD, Meso Scale Diagnostics, LLC, Maryland, USA). Samples with concentrations of inflammatory markers below the lower limit of detection (LLOD) had the LLOD/√2 inputted. Samples of whole blood were analysed for glutathione peroxidase (GPx), a marker of oxidative stress, using the Ransal Assay (Randox, UK) on the I-LAB 650 Chemical Analyser. At the time of processing, an aliquot of plasma had 0.005% BHT to allow for analysis of 8-isoprostanes. The plasma samples were then purified using 8-isoprostane affinity sorbent (Cayman Chemical, Ann Arbor, MI, USA), and analysed using an 8-isoprostane ELISA kit supplied by Cayman Chemical.
8 isoprostane affinity sorbent
Manufactured by Cayman Chemical
Sourced in United States
8-isoprostane affinity sorbent is a laboratory product that can be used to isolate and purify 8-isoprostane, a biomarker of oxidative stress, from biological samples. It provides a consistent and reproducible method for the extraction and concentration of 8-isoprostane prior to analysis.
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Lab products found in correlation
7 protocols using 8 isoprostane affinity sorbent
1
Biomarkers of n-3 PUFA Supplementation
2
Quantifying Alzheimer's Biomarkers
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Immunoassays for Aβ1-40 (ThermoFisher, KHB3481), Aβ1-42 (ThermoFisher KHB3441), aggregated Aβ (ThermoFisher, KHB3491), and sAβPP-α (MyBioSource, MBS9358454) in conditioned media was measured via ELISA according to manufacturer’s instruction. Total 8-isoprostanes were measured via ELISA (516351, Cayman Chemical) in cell lysates following purification using the 8-isoprostane affinity sorbent (401113, Cayman Chemical) suspended in Eicosanoid Column Affinity buffer (400220, Cayman Chemical). Protein carbonylation was assessed by the method of Carty et al. (2000).
3
Plasma 15-F2t-IsoP Quantification Protocol
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Blood plasma sample purification and plasma 15-F2t-IsoP analysis were performed according to the protocol for 8-isoprostane ELISA kit from Cayman Chemical Company (Ann Arbor, MI, USA). Each sample (125 μL) was first hydrolyzed and further purified using 8-Isoprostane Affinity Sorbent (Cayman, Ann Arbor, MI, USA). Following evaporation of the elution solution, purified samples were dissolved in ELISA buffer (330 μL) and analyzed in technical duplicates using 50 μL sample/well. Absorbance was measured by SpectraMax®iD3 (Molecular Devices, San Jose, CA, USA) at 405 nm, and plasma 15-F2t-IsoP concentration were expressed as pg 15-F2t-IsoP/mL plasma.
4
Urinary 8-isoprostane quantification
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24-h urinary 8-isoprostane concentration was measured by a specific affinity sorbent. Urine samples were centrifuged to remove sediment (10 min at 800 g); aliquots of supernatants were incubated with 8-isoprostane affinity sorbent for 60 min according to the manufacturer’s instructions (Cayman Chemical, Ann Harbor, MI, USA).
5
Exhaled Breath Condensate Analysis
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We collected 1–2 ml of EBC using an EcoScreen device (Jaeger, Wurzburg, Germany) with 20 minutes of tidal breathing. We triple rinsed all surfaces with nitrite-free water prior to contacting the EBC, and froze samples at −80 C for later analysis. We measured concentrations of nitrite and nitrite + nitrate using selective catalytic reduction and chemiluminescence detection (NOA 280i, GE Analytics, Boulder Co.). The detection limits and precision for nitrite and nitrite + nitrate were 1 μM and 8.2% (measured as %RSD from 10 replicates) and 2.5 μM and 12.0% (6 replicates), respectively.
For MDA analysis, a mixture of EBC, phosphoric acid, and thiobarbituric acid, was heated and injected into an HPLC-fluorescence system [50 (link)]. The detection limit, recovery and precision of this method were 1.8 nM, 75.9%, and 2.2% (8 replicates), respectively.
Concentrations of 8-isoprostane in EBC samples were analyzed using a modified immunoaffinity purification combined with a LC-MS/MS method [51 (link)]. After addition of 0.25 ng of 8-iso-PGF2α-D4, the extract from 8-isoprostane affinity sorbent (Cayman, Ann Arbor, MI, USA) was analyzed on a quadrupole mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA). The method recovery was 98.4% with analytic precision (8 replicates) of 11.2% and a detection limit of 2.5 pg/mL.
For MDA analysis, a mixture of EBC, phosphoric acid, and thiobarbituric acid, was heated and injected into an HPLC-fluorescence system [50 (link)]. The detection limit, recovery and precision of this method were 1.8 nM, 75.9%, and 2.2% (8 replicates), respectively.
Concentrations of 8-isoprostane in EBC samples were analyzed using a modified immunoaffinity purification combined with a LC-MS/MS method [51 (link)]. After addition of 0.25 ng of 8-iso-PGF2α-D4, the extract from 8-isoprostane affinity sorbent (Cayman, Ann Arbor, MI, USA) was analyzed on a quadrupole mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA). The method recovery was 98.4% with analytic precision (8 replicates) of 11.2% and a detection limit of 2.5 pg/mL.
6
Plasma 8-isoprostane Quantification by ELISA
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Total 8-isoprostanes were measured using an ELISA kit to assess lipid peroxidation in plasma (8-isoprostane ELISA kit, Cayman Chemical). Prior to this assay the plasma samples were purified following the manufacturer's instructions.
Briefly, plasma (100 µL) was added to the 8-isoprostane affinity sorbent (401113, Cayman Chemical) and incubated for 60 minutes with gentle mixing and then centrifuged at 1500xg for 30 seconds to sediment the sorbent. The supernatant was removed and discarded. Eicosanoid Column Affinity buffer (100μL, 400220, Cayman Chemical) was added and placed in the centrifuge at 1500xg for 30 seconds, the supernatant was then removed. This step was repeated by adding ultrapure water (100µL) before centrifugation and discarding the supernatant. Elution solution (100µL, 95% ethanol) was then added to the sediment and evaporated to dryness under nitrogen. Samples were suspended in the ELISA buffer (100µL). Next, standards were prepared from the assay stock solution to create an 8-point standard curve ranging from 0.8 to 500pg/ml, a blank well was used as the 0pg.ml standard.
Standard or sample (50µL) was added to the appropriate well with of 8-isoprostane tracer (50µL) and antiserum (50µL). This was incubated for 18hours at 4 O C. Ellman's reagent was added to each well (200μL) and left to incubate for 2 hours with gentle agitation. The plate was then read at 420nm.
Briefly, plasma (100 µL) was added to the 8-isoprostane affinity sorbent (401113, Cayman Chemical) and incubated for 60 minutes with gentle mixing and then centrifuged at 1500xg for 30 seconds to sediment the sorbent. The supernatant was removed and discarded. Eicosanoid Column Affinity buffer (100μL, 400220, Cayman Chemical) was added and placed in the centrifuge at 1500xg for 30 seconds, the supernatant was then removed. This step was repeated by adding ultrapure water (100µL) before centrifugation and discarding the supernatant. Elution solution (100µL, 95% ethanol) was then added to the sediment and evaporated to dryness under nitrogen. Samples were suspended in the ELISA buffer (100µL). Next, standards were prepared from the assay stock solution to create an 8-point standard curve ranging from 0.8 to 500pg/ml, a blank well was used as the 0pg.ml standard.
Standard or sample (50µL) was added to the appropriate well with of 8-isoprostane tracer (50µL) and antiserum (50µL). This was incubated for 18hours at 4 O C. Ellman's reagent was added to each well (200μL) and left to incubate for 2 hours with gentle agitation. The plate was then read at 420nm.
7
Quantification of 8-Isoprostane: Lipid Peroxidation Marker
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Total 8-isoprostanes were measured using a commercially available ELISA kit to assess lipid peroxidation (8-isoprostane ELISA kit, Cayman Chemical). Prior to this assay the cell lysate samples were purified following the manufacturer's instructions. Briefly, cell lysate was added to the 8-isoprostane affinity sorbent (401113, Cayman Chemical) and incubated for 60 minutes with gentle mixing. Samples were then centrifuged at 1500xg for 30 seconds to sediment the sorbent, the resulting supernatant was removed and discarded, this was repeated 2 times following the addition Eicosanoid Column Affinity buffer (100μL, 400220, Cayman Chemical) and ultrapure water (100μL). Elution solution (95% ethanol) was then added to the sediment and evaporated to dryness under nitrogen. Samples were suspended in the ELISA buffer. Standards were then prepared from the assay stock solution to create an 8-point standard curve ranging from 0.8 to 500pg/mL, ELISA buffer was used as the 0pg.mL standard. Standard or lysates (50µL) were added to the appropriate well with 8-isoprostane tracer (50μL) and antiserum (50μL) in triplicate, this was incubated for 18hours at 4°C.
Ellman's reagent was added to each well (200μL) and left to incubate for 2 hours with gentle agitation. The plate was then read at 420nm.
Ellman's reagent was added to each well (200μL) and left to incubate for 2 hours with gentle agitation. The plate was then read at 420nm.
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