Stability and Reproducibility of the Measurement of Plasma Nitrate in Large Epidemiologic Studies
Keywords:
nitrate, stability, reproducibility, epidemiologic blood collectionAbstract
Inorganic nitrate has emerged as a therapeutic agent for cardiovascular disease; however, nitrate can also metabolize to carcinogenic nitrosamines under pathologic conditions. Few large epidemiologic studies have examined circulating levels of nitrate in relation to cardiovascular disease and cancer. Data on the validity of nitrate measurement in blood samples collected in typical epidemiologic settings are needed before nitrate can be evaluated as an exposure in large epidemiologic studies.
We measured plasma levels of nitrate in three pilot studies to evaluate its laboratory variability, stability with delayed processing, and reproducibility over time among women from the Nurses’ Health Study and healthy female volunteers.
Laboratory variability of nitrate levels was fairly low, with a coefficient variation (CV) of 7%. Plasma nitrate levels in samples stored as whole blood on ice for up to 48 hrs before processing were very stable; the overall intra-class correlation (ICC) from 0 to 48 hours was 0.89 (95%CI, 0.70-0.97). The within-person reproducibility over a one-year period was modest, with an ICC of 0.49 (95% CI, 0.33- 0.94).
Our results indicate that measurement of nitrate in plasma is reliable and stable in blood samples with delayed processing up to 48 hours. Within-person reproducibility was modest but data from this study can be used for measurement error correction in subsequent analyses. The measurement of nitrate cannot be widely used in epidemiologic research without the documentation of its stability and reproducibility.
[N A J Med Sci. 2013;6(2):82-86. DOI: 10.7156/najms.2013.0602082]
References
Gladwin MT, Schechter AN, Kim-Shapiro DB, et al. The emerging biology of the nitrite anion. Nat Chem Biol. 2005;1(6):308-314.
Bahra M, Kapil V, Pearl V, Ghosh S, Ahluwalia A. Inorganic nitrate ingestion improves vascular compliance but does not alter flow-mediated dilatation in healthy volunteers. Nitric Oxide. 2012;26(4):197-202.
Larsen FJ, Ekblom B, Sahlin K, Lundberg JO, Weitzberg E. Effects of dietary nitrate on blood pressure in healthy volunteers. N Engl J Med. 2006;355(26):2792-2793.
Webb AJ, Patel N, Loukogeorgakis S, et al. Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Hypertension. 2008;51(3):784-790.
Bryan NS, Calvert JW, Elrod JW, Gundewar S, Ji SY, Lefer DJ. Dietary nitrite supplementation protects against myocardial ischemia-reperfusion injury. Proc Natl Acad Sci U S A. 2007;104(48):19144-19149.
Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov. 2008;7(2):156-167.
Bryan NS, Alexander DD, Coughlin JR, Milkowski AL, Boffetta P. Ingested nitrate and nitrite and stomach cancer risk: an updated review. Food Chem Toxicol. 2012;50(10):3646-3465.
Aschebrook-Kilfoy B, Cross AJ, Stolzenberg-Solomon RZ, et al. Pancreatic cancer and exposure to dietary nitrate and nitrite in the NIH-AARP Diet and Health Study. Am J Epidemiol. 174(3):305-315.
Ward MH, Kilfoy BA, Weyer PJ, Anderson KE, Folsom AR, Cerhan JR. Nitrate intake and the risk of thyroid cancer and thyroid disease. Epidemiology (Cambridge, Mass). 2010;21(3):389-395.
Machha A, Schechter AN. Inorganic nitrate: a major player in the cardiovascular health benefits of vegetables? Nutr Rev. 2012; 70(6):367-372.
Lundberg JO, Weitzberg E. NO generation from nitrite and its role in vascular control. Arterioscler Thromb Vasc Biol. 2005; 25(5): 915-922.
Hankinson SE, Manson JE, Spiegelman D, Willett WC, Longcope C, Speizer FE. Reproducibility of plasma hormone levels in postmenopausal women over a 2-3-year period. Cancer Epidemiol Biomarkers Prev. 1995;4(6):649-654.
Wishnok JS, Glogowski JA, Tannenbaum SR. Quantitation of nitrate, nitrite, and nitrosating agents. Methods Enzymol. 1996;268:130-141.
Nagababu E, Rifkind JM. Measurement of plasma nitrite by chemiluminescence without interference of S-, N-nitroso and nitrated species. Free Radic Biol Med. 2007;42(8):1146-1154.
Gordon T, Shurtleff D. The Framingham Study: an epidemiologic investigation of cardiovascular disease. Section 29: Means at eacexamination and inter-examination variation of specified characteristics: Framingham Study Exam 1 to Exam 10. 1973: DHEW Pub No. (NIH) 74-478. 1973.
Tworoger SS, Eliassen AH, Sluss P, Hankinson SE. A prospective study of plasma prolactin concentrations and risk of premenopausal and postmenopausal breast cancer. J Clin Oncol. 2007;25(12):1482-1488.