Short Communication Long-term Cryoconservation and Stability of Vitamin C in Serum Samples of the European Prospective Investigation into Cancer and Nutrition Mazda Jenab, 1 Sheila Bingham, 2 Pietro Ferrari, 1 Marlin D. Friesen, 1 Wael K. Al-Delaimy, 1 Robert Luben, 3 Nick Wareham, 4 Kay-Tee Khaw, 5 and Elio Riboli 1 1 Nutrition and Hormones Group, IARC-WHO, Lyon, France; 2 Medical Research Council Dunn Human Nutrition Unit; 3 Department of Community Medicine, 4 Institute of Public Health, and 5 Clinical Gerontology Unit, University of Cambridge, Cambridge, United Kingdom Abstract Plasma vitamin C level may be associated with risk of some chronic diseases. The rapid degradability of vitamin C in biological samples necessitates its stabilization with meta- phosphoric acid or similar agents. However, in most cohort studies, prospectively collected biological samples are not treated with stabilizing agents before long-term frozen storage and it is not known whether vitamin C can be properly measured in such samples. The objective of this study was to determine the degree of vitamin C degradation in plasma samples stored without stabilization for 7 to 11 years at À196°C. Spearman’s correlation coefficients indi- cate a moderate correlation between baseline and final plasma vitamin C levels in both men (r = 0.57, P < 0.0001) and women (r = 0.52, P < 0.0001). Samples were also categorized based on low or high baseline levels of plasma vitamin C, with the latter category showing the highest rate of loss per year of frozen storage in men (1.96 Mmol/L, P value for difference <0.0001; percent loss 24.6%) and women (2.35 Mmol/L, P value for difference <0.0001; percent loss 24.2%), as determined by multiple regression analysis adjusted for smoking status, age, and body mass index. In men, both baseline and final plasma vitamin C values were lower in smokers than never smokers, but for both men and women the rate of vitamin C loss during storage was not significantly different between smokers and never smokers. The results of this study show that vitamin C can be measured with reasonable reliability in plasma samples frozen for long periods of time without addition of any stabilizing agents. (Cancer Epidemiol Biomarkers Prev 2005;14(7):1837 – 40) Introduction Vitamin C from the diet has been suggested to be inversely associated with risk of some cancers (1, 2) and cardiovascular disease (2), likely due to its antioxidant properties (3). With the advent of appropriate analysis techniques, measures of vitamin C in biological samples have been used as a biomarker of health status [e.g., in the study of gastric cancer (4, 5) and total mortality (6)]. Because vitamin C in biological samples can easily degrade or be readily oxidized, it is often stabilized before sample storage by the addition of stabilizing agents such as EDTA (a metal chelator), perchloric acid, DTT, or metaphosphoric acid, all of which act as protein-precipitating agents (7). However, in the case of large, multipurpose prospective studies, biological samples are collected and often stored for many years before analysis for a multitude of biomarkers. Thus, blood samples are not always treated specifically for the preservation of vitamin C before storage. Present literature on vitamin C stability deals with losses during the handling, processing, and freezing procedures of blood samples (7-12), after short periods of frozen storage without stabilization (11, 12) or for longer time periods of up to 6 years with stabilization (13, 14). However, there is currently very little information on the feasibility, stability, and validity of vitamin C analysis in blood samples stored by freezing at very low temperatures (À196jC) for very long periods of time without the addition of stabilizing compounds such as metaphosphoric acid. Thus, the objective of this study was to determine the stability of vitamin C in plasma samples stored for 7 to 11 years in liquid nitrogen and not treated with any stabilizing agents during storage compared with samples analyzed at baseline with stabilization. The samples were collected from a subset of subjects enrolled in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Materials and Methods The EPIC Study and the EPIC-Norfolk Subcohort. The EPIC study (15-17) is an ongoing prospective multicenter cohort study with 521,468 subjects from 23 centers in 10 European countries. The EPIC biorepositories are the world’s largest, hosting over 9 million aliquots maintained at À196jC. For the present study, subjects were selected from the Norfolk (Cambridge, United Kingdom) center of the EPIC study (EPIC- Norfolk). The enrollment, recruitment, and sample collection Cancer Epidemiology, Biomarkers & Prevention 1837 Cancer Epidemiol Biomarkers Prev 2005;14(7). July 2005 Received 1/24/ 05; revised 3/29/ 05; accepted 4/14/ 05. Grant support: ‘‘Europe Against Cancer’’ Programme of the European Commission; Ligue contre le Cancer (France); Socie ´te ´ 3M (France); Mutuelle Ge ´ne ´rale de l’Education Nationale; Institut National de la Sante ´ et de la Recherche Me ´dicale; German Cancer Aid; German Cancer Research Center; German Federal Ministry of Education and Research; Danish Cancer Society; Health Research Fund (FIS) of the Spanish Ministry of Health; the participating regional governments and institutions of Spain; Cancer Research UK; Medical Research Council, UK; the Stroke Association, UK; British Heart Foundation; Department of Health, UK; Food Standards Agency, UK; the Wellcome Trust, UK; Greek Ministry of Health; Greek Ministry of Education; Italian Association for Research on Cancer; Italian National Research Council; Dutch Ministry of Public Health, Welfare and Sports; Dutch Ministry of Health; Dutch Prevention Funds; LK Research Funds; Dutch Zorg Onderzoek Nederland; World Cancer Research Fund; Swedish Cancer Society; Swedish Scientific Council; Regional Government of Skane, Sweden; and Norwegian Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Mazda Jenab, Nutrition and Hormones Group, IARC-WHO, Lyon, France. Phone: 33-4-72-738082; Fax: 11-33-4-72-738361. E-mail: jenab@iarc.fr Copyright D 2005 American Association for Cancer Research. on June 17, 2020. © 2005 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from