Reference intervals for saliva analytes collected by a standardized method in a physically active population Lázaro Alessandro Soares Nunes a, ⁎, René Brenzikofer b , Denise Vaz Macedo a a Laboratory of Exercise Biochemistry — LABEX, Biochemistry Department, Biology Institute, State University of Campinas, Cidade Universitária Zeferino Vaz, s/n, Barão Geraldo, CEP: 13083-970, CP6109, Campinas, SP, Brazil b Laboratory of Instrumentation for Biomechanics, Faculty of Physical Education, State University of Campinas, Campinas, SP, Brazil abstract article info Article history: Received 18 June 2011 Received in revised form 6 September 2011 Accepted 22 September 2011 Available online 2 October 2011 Keywords: Oral fluid Reference value Antioxidant capacity Athletic monitoring Objectives: Our aims were to test a liquid-based saliva collection system for total antioxidant status (TAS), uric acid (UA), total protein concentration (TP) and salivary alpha-amylase (SAA) activity; to determine if these analytes in serum and saliva are correlated in a physically active population and to establish reference intervals for these parameters. Design and methods: Participants in this study were 115 physically active males (18–20 years old). Saliva samples were collected using the Saliva Collection System (Greiner Bio-One®) immediately before obtaining blood. Biochemical analyses were conducted using an Autolab Boehringer analyzer. Results: We found a correlation between UA and TP concentrations in serum and saliva samples. The refer- ence intervals for TP and SAA activity in the morning were lower than in the afternoon (p b 0.0001). The ref- erence intervals for UA and TAS did not vary with the time of collection. Conclusion: The establishment of reference intervals for these saliva constituents increases their diagnostic utility and allows for detection of physiological or pathological states. © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. 1. Introduction Monitoring biochemical blood parameters can be a powerful methodology for evaluating the physical stress thresholds of athletes during a competitive season. Such evaluations, however, require fre- quent and successive blood samplings. These samplings are normally done by venous puncture, which is stressful for some individuals [1]. Saliva presents a potential application as a substitute for blood in the diagnosis and monitoring of different metabolic states because it can be collected by an easier, noninvasive method that does not require a trained specialist [2]. The term “saliva” is generally used to designate the fluid present in the oral cavity, which mainly originates from three salivary glands: parotid, submandibular, and sublingual [3]. Saliva is consists of 98% water and 2% proteins and peptides (e.g., α-amylase, immunoglobu- lins, mucins, cystatins, and proline-rich peptides), electrolytes (e.g., sodium, potassium, chlorides, and bicarbonate), microorganisms, lipids [4] and metabolites (e.g., uric acid, urea, and lactate) [5,6]. Saliva composition can be altered in the course of circadian rhythm and by sympathetic nervous system activity [3,7], medications [8], and physical exercise [5]. Previous studies have used saliva to diagnose and monitor renal diseases by measuring the concentration urea and creatinine [9]. Sali- va has also been used in therapeutic drug management [10] to mon- itor lactate concentrations [5] and antioxidant status after exercise [11], and to measure cortisol concentrations and alpha-amylase activ- ity under stress [12]. Exercise is associated with stress and increased production of re- active oxygen species (ROS) [13]. Enzymatic and low-molecular- weight antioxidants minimize the deleterious actions of ROS through redox reactions. Athletes and physically active individuals displayed enhanced antioxidant capacity [14] and increased plasma concentra- tions of uric acid (UA), which is a major antioxidant in plasma and sa- liva [15]. Thus, assays of the antioxidant capacity of either plasma or saliva can be useful tools for monitoring oxidative stress during train- ing [11]. Saliva can be collected using a non-stimulated method, such as spitting directly from the mouth into a receptacle; however, only a small volume is obtained using this method [2]. Stimulated saliva can be collected through masticatory action (e.g., from a subject chewing on paraffin) or gustatory stimulation with citric acid applica- tion [2]. Collection can also be done using cotton rolls; however, cot- ton may interfere with some analyses [16]. The main problem with all Clinical Biochemistry 44 (2011) 1440–1444 Abbreviations: TAS, total antioxidant status; UA, uric acid; TP, total protein concen- tration; SAA, salivary alpha-amylase. ⁎ Corresponding author at: Biology Institute, State University of Campinas, Cidade Universitária Zeferino Vaz, s/n, Barão Geraldo, CEP: 13083-970, CP6109, Campinas, SP, Brazil. Fax: +55 19 3521 6129. E-mail address: lazaroalessandro@yahoo.com.br (L.A.S. Nunes). 0009-9120/$ – see front matter © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2011.09.012 Contents lists available at SciVerse ScienceDirect Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem