Cory Holder (Presenter)
Oak Ridge Institute of Science and Education/ Centers for Disease Control and Prevention
Bio: Cory Holder is an Oak Ridge Institute of Science and Education Fellow at the Centers for Disease Control and Prevention in the Tobacco and Volatiles Branch of the Division of Laboratory Sciences in the National Center for Environmental Health.
Authorship: Cory Holder MS (1,2), James E. McGuffey BS (1), Aaron Adams BS (1,2), Ernest McGahee MPH MHSA (1), and Lanqing Wang PhD (1)
(1) Tobacco and Volatiles Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA, (2) Oak Ridge Institute for Science and Education, Oak Ridge, TN 37831, USA
| Short Abstract A non-enzymatic peroxidation product of arachidonic acid, 8-isoprostane, is a known biomarker for estimating oxidative stress. We have developed a robust automated method for measuring urinary 8-isoprostane using polymeric weak anion exchange solid phase extraction isotope dilution ultra-high performance liquid chromatography atmospheric pressure ionization tandem mass spectrometry assay. Since 8-isoprostane exists in urine as glucuronide conjugates and free acids, we enzymatically hydrolyzed the samples with β-glucuronidase prior to UPLC-MS/MS quantification. Using 400 µL sample volume, this method returned a limit-of-detection below 8 pg/mL and a coefficient of variation (CV) below 10% with cycle times of less than 10 min. |
Long Abstract
Introduction
Oxidative stress (OS) has been linked to several human pathologies; such as cancer, [1, 2, 3] cardiovascular diseases, [4, 5] respiratory diseases, [6] neurodegenerative disorders, [7] as well as the aging process. [8, 9] OS is characterized as an imbalance between pro-oxidant and anti-oxidant defenses caused by overproduction of reactive oxygen species (ROS) or deficient levels of anti-oxidants. ROS are short-lived chemical species generated endogenously during mitochondrial metabolism and immune response, and exogenously produced by harmful radiation or exposure to environmental toxicants, such as tobacco smoke. [10] Being highly reactive and short lived makes ROS difficult to monitor directly; therefore it is more reasonable to monitor biomarkers produced by ROS. [11] One of the most abundant and stable biomarkers for OS, 8-isoprostane, has been previously quantified in urine and plasma using immunoassays, gas chromatography-mass spectrometry (GC-MS), GC-tandem mass spectrometry (GC-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS).[8, 11, 12, 13]
Urinary 8-isoprostane exists in the free acid form and as glucuronide conjugates. However, there is significant variability in the amount of glucuronide conjugation that occurs between individuals, 30-80%. [14, 15, 16, 17] Some commercially available EIA kits contain glucuronidase to measure ‘total’ levels of 8-isoprostane, however, most GC- or LC-MS/MS methods have been developed for measuring only ‘free’ 8-isoprostane. We have developed an analytical method that measures the ‘total’ concentration of urinary 8-isoprostane by hydrolyzing the urine samples with β-glucuronidase followed by SPE-UPLC-MS/MS analysis. This method has also been automated using a Hamilton STAR liquid robot handling system to reduce sample preparation errors and streamline the analytical process for large population studies.
Methods
We report urinary 8-isoprostane levels as ‘total’. Water blanks, QCs, and urine samples were all prepared following the same procedure. Samples stored at – 20° C or below were gradually thawed and manually shaken at room temperature. To quantify ‘total’ 8-isoprostane we added 40µL of an isotopically labeled internal standard, 800 µL of HPLC water, 160 µL of enzyme solution (2000 units Escherichia coli, type IX-A glucuronidase—dissolved in 0.5 M phosphate buffer pH 6.4), and 400 µL of urine specimen was dispensed into glass 12 x 75 mm test tubes, capped and incubated overnight in a water bath for about 21 h at 37° C. After incubation, 400 µL of methanol was added to each sample tube and the contents were transferred to a 96-well anion exchange SPE plate. Nitrogen gas was used to push the samples through the SPE plate while the analyte, 8-isoprostane, was retained on the resin. The SPE plate was first washed with 1.8 mL of HPLC water, then 3.6 mL (2 washes of 1.8 mL) of methanol in water (v/v 1:3), followed by 1.8 mL of acetonitrile. 8-Isoprostane was eluted from the resin with 1.8 mL methanol into a 96-well collection plate. Subsequently, the methanol was evaporated under nitrogen flow at 37° C. The sample was then reconstituted using 50 µL of methanol in water (v/v 1:3) and vortexed before placing the plate in the LC autosampler.
Sample aliquots were performed by a Hamilton Microlab Star liquid handling robot using Hamilton CO-RE tips (50 µL, 300 µL, and 1000 µL) (Reno, NV, USA). For transferring urine from the source tubes we used clear pressure-based Liquid Level Detection (pLLD) 1000 µL tips and black capacitive Liquid Level Detection (cLLD) for all other liquid transfers on the Microlab Star. We conducted a preliminary experiment for assessing the recovery of the analyte using different tips and observed the cLLD tips retained more sample than pLLD. After incubation, samples were placed into the Microlab Star; 400 µL of methanol was added to each sample and the resulting mixture was transferred to a Strata-X-AW 33 µm Polymeric Weak Anion, 60 mg/ 96-well plate from Phenomenex (Torrance, CA, USA). A Biotage Pressure +96 positive pressure manifold using nitrogen gas generated in-house using a NM20ZA Peak Generator for SPE. Sample collection and injection was done using an Analytical Sales and Services Inc. (Flanders, NJ, USA) 2 mL 96 deep square well, with a tapered V-bottom collection plate.
Chromatography was achieved with a Waters Acquity reversed-phase column (150 mm x 2.1 mm, particle size 1.8 µm, C18) and a Waters Acquity reversed-phase pre-column (5 mm x 1 mm, particle size 1.7 µm, C18) (Milford, MA, USA) using a Shimadzu UPLC system (Columbia, MD, USA) at a flow rate of 0.65 mL/min. Column temperature was maintained at 60 °C during the entire analysis. The gradient program contained acidified water (0.15% formic acid) (mobile phase A) and acetonitrile in acidified water (0.15% formic acid) (v/v 1:1) (mobile phase B).
MS/MS analysis was performed on a Sciex triple quadrupole 6500 with a Turbo IonSpray source (Foster City, CA). ESI- mode was used to obtain MRM transition data. The MS source parameters were: curtain gas, 30 psi; collision gas, 8 psi; IonSpray voltage, -4000 V; source temperature, 600° C; ion source gas 1, 60 psi; ion source gas 2, 70 psi.
Results
This biomonitoring method can analyze up to 360 samples per week being and is currently being used for a large population study to determine the relationship of 8-isoprostane and tobacco use. Using 400 µL sample volume, this method returned a limit-of-detection below 8 pg/mL and a coefficient of variation (CV) below 10% with cycle times of less than 10 min.
Conclusions & Discussion
We have developed a robust automated method for measuring urinary 8-isoprostane using polymeric weak anion exchange solid phase extraction with isotope dilution ultra-high performance liquid chromatography atmospheric pressure ionization tandem mass spectrometry assay.
References & Acknowledgements:
Notes:
The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service/U.S. Department of Health and Human Service.
Acknowledgements:
This study was funded through an interagency agreement by U.S. Food and Drug Administration Center for Tobacco Products. This project was supported in part by an appointment to the Research Participation Program at the Centers for Disease Control and Prevention administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the Centers for Disease Control and Prevention.
References:
1. Reuter, S.; Gupta, S.C.; Chaturvedi, M.M.; Aggarwal, B.B. Free Radical Biol. Med. 2010, 49 (11), 1603-1616
2. Valko, M.; Rhodes, C.J.; Mocoi, J.; Izakovic, M.; Mazur, M. Chem. Biol. Interact., 2006, 160 (1), 1-40
3. Halliwell, B. Biochem. J., 2007, 401 (1), 1-11
4. Wang, B.; Pan, J.; Wang, L.; Zhu, H.; Yu, R.; Zou, Y. Atherosclerosis, 2006, 184 (2), 425-430
5. Griendling, K.K.; FitzGerald, G.A. Circulation, 2003, 108 (17), 2034-2040
6. Montuschi, P.; Collins, J.V.; Ciabattoni, G.; Lazzeri, N.; Corradi, M.; Kharitonov, S.A.; Barnes, P.J. Am. J. Respir. and Crit. Care Med., 2000, 162(3), 1175-1177
7. Lin, M.T.; Beal, M.F. Nature, 2006, 443, 787-795
8. Milne, G.L.; Yin, H.; Hardy, K.D.; Davies, S.S.; Roberts II, L.J. Chem. Rev. 2011, 111 (10), 5973–5996
9. Sohal, R.S.; Weindruch, R. Science, 1996, 273 (5271), 59-63
10. Ray, P.D.; Huang, B-W.; Tsuji, Y. Cell Signaling, 2012, 24 (5), 981-990
11. Tsikas, D.; Theodoridis, G., J. Chromatogr. B. 2016, 1019, 1-3
12. Morrow, J.D.; Hill, K.E.; Burk, R.F.; Nammour, T.M.; Badr, K.F.; Roberts II, L.J. Proc. Natl. Acad. Sci. 1990, 87 (23), 9383-9387
13. Xiao, Y.; Fu, X.; Pattengale, P; Bard, J.D.; Xu, Y-K; O’Gorman, M.R. Clinica Chimica Acta, 2016, 460, 128-134
14. Morrow, J.D.; Zackert, W.E.; Yang, J.P.; Kurhts. E.H.; Callewaert, D.; Dworski, R.; Kanai, K.; Taber, D.; Moore, K.; Oates, J. A.; Roberts II, L. J. Anal. Biochem. 1999, 269 (2), 326
15. Yan, Z.; Mas, E.; Mori, T.A.; Croft, K.D.; Barden, A.E. Anal. Biochem. 2010, 403, 126-128
16. Tsikas, D.; Suchy, M-S. J. Chromatogr. B. 2016, 1019, 191-201
17. Langhorst, M.L.; Hastings, M.J.; Yokoyama, W.H.; Hung, S-C.; Cellar, N.; Kuppannan, K.; Young, S.A. J Agri. Food Chem. 2010, 58, 6614-6620
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