American Journal of Analytical Chemistry, 2011, 2, 683-688 doi:10.4236/ajac.2011.26078 Published Online October 2011 (http://www.SciRP.org/journal/ajac) Copyright © 2011 SciRes. AJAC Effect of Pre-Analytical Conditions on Salivary Nitrite Levels Rosana Andrea Morelatto 1* , Tomás Enrique Benavidez 2 , Ana María Baruzzi 2 , Velia Matilde Solís 2 , Silvia Adriana López de Blanc 1 1 Department of Oral Pathology, Clinical Stomatology I and II “B”, Faculty of Dentistry, National University of Córdoba, Córdoba, Argentina 2 INFIQC-CONICET, Department of Physical Chemistry, Faculty of Chemical Sciences, National University of Córdoba, Córdoba, Argentina E-mail: * rmorelatto@gmail.com Received June 29, 2011; revised July 29, 2011; accepted August 5, 2011 Abstract The aim of the present paper was to analyze the influence of sampling and storage procedures on nitrite con- centration values in the saliva of healthy persons. The samples were obtained and stored under varied condi- tions, and processed using the Griess method. Results: when the salivary nitrite concentration was measured immediately after collection a significant dependence on the collection time was observed. A mean value of 94 µmol/L (range 3 - 625) was obtained at 8:30 am. This value decreased significantly with time (p < 0.05) reaching a value of 68 at 12:30 noon. Concerning the sample storage, a significant increase in the nitrite concentration was observed after 2 hrs, either at 4˚C or at room temperature (p < 0.05). In spite of the high variability between individuals the values for each individual showed a marked constancy independent of the sampling day. According to our results, by controlling pre-analytical parameters, principally sampling and storage procedures, reproducibility is improved. Keywords: Salivary Nitrite, Saliva, Oral Cancer, Storage Conditions 1. Introduction N-nitroso compounds have been shown to be potent car- cinogens in animals [1]; in the human body they can be formed by the interaction of nitrite and a variety of amine precursors, developing infantile methahemoglobinemia and gastric cancer. Nitrite has been extensively studied in relation to carcinogenesis [2]; it is an important factor for gastric nitrozation, and could contribute to the etiology of lung, stomach, esophagus, nasal cavity, bladder and oral cavity cancer, leukemia and Non Hodgkin lym- phoma [3]. It has mutagenic effects at cellular levels and acts on p53 gene, closely related to the head and neck squamous cell carcinoma [4]. Several studies have confirmed that the level of salivary nitrite is strictly dependent on salivary nitrate, and thus dependent on the dietary nitrate intake [5-8]. Dietary nitrate derives mainly from vegetables and drinking wa- ter [6,7] and in the latter case it could be more hazardous since nitrate in vegetables is counterbalanced by vitamin C and polyphenols that inhibit nitrozation [8]. Ingested nitrate is absorbed from the stomach or intestines and about 25% is secreted in saliva by an anion transport system [5]. As a result, nitrate concentrations in saliva are approximately 10 to 20 times higher than those found in plasma [5]. In addition, it has been estimated that 70% of the orally ingested nitrate is reduced to nitrite by mouth microorganisms, mainly on the posterior surface of the tongue [9,10]. The assay of saliva is an increasing area of research with implications in basic and clinical purposes. Re- cently, saliva has provided a substantial tool investiga- tion of disease processes and disorders. Although this biological fluid is easy to collect and manipulate, special attention must be paid to minimize variation in specimen integrity [11]. Many authors have investigated the concentration of nitrite in saliva considering the advantages of obtaining this biological fluid and its possible role in the etiology and diagnostic relevance of nitro compounds. A common feature of in vivo studies is the large spread in results, both between individuals and for each individual in rela-