Special issue: Original articles Responsible writing in science Changes of salivary biomarkers under different storage conditions: effects of temperature and length of storage Tomás Barranco1, Camila P Rubio1, Asta Tvarijonaviciute1, Mónica Rubio2, Elena Damia2, Elsa Lamy3, Ramón Cugat4, José J Cerón1, Fernando Tecles*1, Damián Escribano5 1Interdisciplinary Laboratory of Clinical Analysis (Interlab-UMU), Campus of Excellence Mare Nostrum, University of Murcia, Murcia, Spain 2Department of Animal Medicine and Surgery, University CEU Cardenal Herrera, Valencia, Spain 3Institute of Mediterranean Agricultural and Environmental Sciences (ICAAM), University of Évora, Évora, Portugal 4Arthroscopy and Traumatology Unit of the Quirón Hospital, Barcelona, Spain 5Department of Animal and Food Science, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Barcelona, Spain The first two authors contributed equally to this work. *Corresponding author: ftecles@um.es Abstract Introduction: In this report, we aimed to examine the stability of various analytes in saliva under different storage conditions. Materials and methods: Alpha-amylase (AMY), cholinesterase (CHE), lipase (Lip), total esterase (TEA), creatine kinase (CK), aspartate aminotran- sferase (AST), lactate dehydrogenase (LD), lactate (Lact), adenosine deaminase (ADA), Trolox equivalent antioxidant capacity (TEAC), ferric reducing ability (FRAS), cupric reducing antioxidant capacity (CUPRAC), uric acid (UA), catalase (CAT), advanced oxidation protein products (AOPP) and hydro- gen peroxide (H2O2) were colorimetrically measured in saliva obtained by passive drool from 12 healthy voluntary donors at baseline and after 3, 6, 24, 72 hours, 7 and 14 days at room temperature (RT) and 4 ºC, and after 14 days, 1, 3 and 6 months at – 20 ºC and – 80 ºC. Results: At RT, changes appeared at 6 hours for TEA and H2O2; 24 hours for Lip, CK, ADA and CUPRAC; and 72 hours for LD, Lact, FRAS, UA and AOPP. At 4 ºC changes were observed after 6 hours for TEA and H2O2; 24 hours for Lip and CUPRAC; 72 hours for CK; and 7 days for LD, FRAS and UA. At – 20 ºC changes appeared after 14 days for AST, Lip, CK and LD; and 3 months for TEA and H2O2. At – 80 ºC observed changes were after 3 months for TEA and H2O2. Conclusions: In short-term storage, the analytes were more stable at 4 ºC than at room temperature, whereas in long-term storage they were more stable at - 80 ºC than at – 20 ºC. Keywords: biomarker; enzyme stability; oxidative stress; saliva; temperature Received: September 07, 2018 Accepted: December 21, 2018 Introduction Interest in saliva assays for clinical purposes has in- The use of salivary biomarkers for diagnostic pur- creased during recent years because this fluid has poses would be of benefit if standardised proce- important advantages: its collection is easy, does dures for saliva collection were used, as well as the not produce evident stress or pain and does not validation/verification of the methods performed in require expensive material or medical personnel. saliva. In addition, knowledge of how the storage The main areas in which saliva can be used for conditions can affect the stability of measured ana- testing are psychology and stress research, endo- lytes is of major importance, especially as saliva is crinology, occupational and sports medicine, drug usually less stable than plasma (due to bacterial monitoring, metabolism and oxidative status eval- multiplication, viscosity or extensive proteolytic uation, immunology and inflammation (1). cleavage by endogenous and exogenous proteas- https://doi.org/10.11613/BM.2019.010706 Biochem Med (Zagreb) 2019;29(1):010706 1 Barranco T. et al. Storage conditions for salivary biomarkers es) (2). Stability would be of particular importance erated in the assay (14). The Trolox equivalent anti- when retrospective studies or studies involving oxidant capacity measures the capacity of the multiple experimental sampling time-points are de- sample to reduce the 2,2’-azino-bis(3-ethylbenz- signed, since they usually involve the storage of thiazoline-6-sulfonic acid) (ABTS) radical, whereas samples, and an inappropriate temperature can af- FRAS and CUPRAC measure the ability of a sample fect enzymatic activities in saliva samples during to reduce Fe3+ and Cu2+ to Fe2+ and Cu1+, respec- sampling and storage (3). tively. They have been evaluated in saliva in pa- Salivary α-amylase (AMY) increases in situations of tients with diseases such as oral lichen planus (13). acute stress and activation of the sympathetic In addition, individual components of the antioxi- nervous system (SNS), due to different psychologi- dant system such as uric acid (UA) and catalase cal causes or physical efforts (4). Changes in sali- (CAT) can be measured. Uric acid is the final me- vary cholinesterase (CHE) activity have been de- tabolite of purines and represents approximately scribed in Alzheimer’s disease and they have been 70% of salivary TAC (15). It can increase in hypoxia related to situations of stress, although it is tradi- due to the appearance of oxidant metabolites and tionally measured as a biomarker of anti-CHE in- there is evidence that salivary UA is altered in dis- secticides exposure (5,6). Salivary lipase (Lip) secre- eases such as oral lichen planus or diabetes (13,16). tion also seems to be promoted by the activation Catalase is an enzyme capable of removing ROS of the SNS (7). The total esterase activity (TEA) of from saliva and its activity is altered in patients saliva comprises several enzymatic activities, and with different diseases such as human immunode- it is increased in situations of physical stress (8). ficiency virus (17). Components of the oxidant sys- Creatine kinase (CK), aspartate transaminase (AST) tem can also be measured in saliva, namely the ad- and lactate dehydrogenase (LD) can increase in vanced oxidation protein products (AOPP) and hy- human saliva in conditions such as intensive exer- drogen peroxide (H2O2). Advanced oxidation pro- cise (9). Lactate (Lact) is considered a marker of tein products represent a sensitive biomarker of muscle function and its quantification in saliva is oxidative-modified proteins and it has been meas- important in internal and sport medicine to moni- ured in human saliva before and after acute resist- tor the maximum performance level of athletes ance exercise (18). Hydrogen peroxide is a reactive (10). Adenosine deaminase (ADA) is involved in species produced during normal metabolism, with various processes related with the immune sys- increased concentrations found in situations of in- tem, it is considered a biomarker of inflammation flammation and tissue damage (19). and it has been found to change in saliva in oral Studies on the stability for some of these analytes diseases (11). already have been published; such as stability of In situations of oxidative stress, reactive oxygen AMY at room temperature (RT), 4 ºC and after species (ROS) are produced in high amounts that freeze-thaw cycle; stability of LD and AST at RT, 4 cannot be removed by antioxidants. Total antioxi- ºC and – 20 °C for 3 months and at – 80 ºC for 28 dant capacity (TAC) represents the antioxidant sta- days, or the stability of AOPP for 4 weeks at – 20 ºC tus of a sample and comprises the sum of the con- and – 80 ºC (2,3,20-25). However, long-term stud- centrations of almost all non-enzymatic antioxi- ies longer than 3 months in which different stor- dants (12). It can be evaluated in saliva by different age conditions were compared have not been assays, such as ferric reducing ability of saliva published. In addition, to the authors’ knowledge, (FRAS), cupric reducing antioxidant capacity (CU- there have not been studies about the stability of PRAC), and the 6-hydroxy-2,5,7,8-tetramethylchro- CHE, Lip, TEA, CK, Lact, ADA, TEAC, UA, CAT and man-2-carboxylic acid (Trolox) equivalent antioxi- H2O2 in human saliva under different conditions of dant capacity (TEAC) (13). The Trolox equivalent storage. antioxidant capacity, FRAS and CUPRAC are assays Our hypothesis was that the analytes in saliva can based on the ability of antioxidants present in a have different changes depending of the sample sample to reduce or inhibit oxidized products gen- storage conditions and used times. In this report, Biochem Med (Zagreb) 2019;29(1):010706 https://doi.org/10.11613/BM.2019.010706 2 Barranco T. et al. Storage conditions for salivary biomarkers we aimed to examine the stability of various ana- avoid coughing or clearing the throat into the col- lytes (AMY, CHE, Lip, TEA, CK, AST, LD, Lact, ADA, lection tube and were to abstain from brushing TEAC, FRAS, CUPRAC, UA, CAT, AOPP and H2O2) in teeth or using mouthwash, ingesting any food or saliva under different storage conditions. chewing gum for 1 hour before saliva collection. The participants rinsed their mouth with water Material and methods five minutes before saliva collection. Then, unstim- ulated saliva was collected by passive drool in the Subjects absence of chewing movements into 10 mL plain tubes (Proquilab, Murcia, Spain). Collection started This experimental study was conducted at the In- in all subjects at 9:30 am and lasted between 2 terdisciplinary Laboratory of Clinical Analyses of and 5 minutes. The volunteers sat in a relaxed po- the University of Murcia, Spain, from June to De- sition throughout the sampling procedure to cember 2017. The experimental protocol was ap- avoid any stress. Between 3 to 5 mL of saliva was proved by the Investigation Ethics Committee of obtained from each participant, all samples were the University of Murcia (Spain) and followed the checked for blood contamination by visual inspec- Declaration of Helsinki of the World Medical Asso- tion and no reddish samples indicating blood con- ciation for research with humans. tamination were included in the study. Immediate- Twelve subjects, six men (29-58 years) and six ly after collection, the saliva samples were centri- women (28-56 years) participated in this study. fuged (Universal 320R, Hettich, Tuttlingen, Germa- They were healthy volunteers from the personnel ny) at 5000xg and 4 ºC for 5 minutes, then the su- of Murcia University. All participants filled a ques- pernatant was collected and divided into aliquots, tionnaire in which they were asked about habits, discarding the sediment. To evaluate the effects of the presence and description of any acute or different storage conditions, 19 aliquots of each chronic disease, any type of symptom in the days sample were prepared: (a) 1 aliquot was freshly an- before the experiment took place, or whether they alysed (baseline) and used as the reference value were receiving or they had recently received any for all analytes; (b) 4 aliquots of each sample were medical treatment. The inclusion criteria for the stored at room temperature (RT); (c) 6 aliquots participants were that they should be adults with- were refrigerated at 4 ºC, (d) 4 were frozen at – 20 out any acute or chronic disease, not under physi- ºC and (e) 4 were frozen at – 80 ºC. The aliquots cian’s care for any disease for the last 6 months, stored at RT were analysed at 3 (T1), 6 (T2), 24 (T3) not receiving any medication (including hor- and 72 (T4) hours after sampling; the refrigerated mones, steroids or non-steroidal anti-inflammato- aliquots were analysed at T1, T2, T3 and T4, and in ries), food supplements (vitamins or sport supple- addition 7 (T5) and 14 days (T6) from collection. Fi- ments) and not being smokers or alcohol consum- nally, the aliquots stored at – 20 ºC and at – 80 ºC ers. In addition, subjects should not have oral dis- were analysed 14 days (T6), 1 (T7), 3 (T8) and 6 eases, such as gingivitis or periodontitis, after months (T9) from collection. complete examination of the oral cavity per- The analytical methods used, which were adapted formed by an oral medicine professional. in the authors’ laboratory for saliva samples, as well as their fundamentals, details of the reagents Methods and analytical performance, appear in Table 1. All The participants themselves performed sample assays were performed on an automated bio- collection. All participants received detailed infor- chemistry analyser (Olympus AU400, Olympus Di- mation by oral communication and written guide- agnostica GmbH, Ennis, Ireland) at 37 ºC. Manufac- lines about the aims and experimental protocol, turers’ control solutions of two different values the saliva collection procedure, and signed written were used for the quality control analysis of AMY, consent. They were informed about the need to Lip, CK, AST, LD, Lact and UA (Beckman Coulter, lot 0037 and 0038) and one control solution was used https://doi.org/10.11613/BM.2019.010706 Biochem Med (Zagreb) 2019;29(1):010706 3 4 Reference in which the method was validated for saliva or basis of the method (in those Manufacturer or reagents (in Intra-assay Inter-assay Linearity Method LLOD assays in which validation was performed home-made assays) CV (%) CV (%) (R2) specifically for this research) Barranco T. et al. R1 R2 α-amylase (24) Beckman Coultera < 3.0 < 3.0 > 0.99 11.7 U/L Hydrolysis of ATCI to thiocholine in presence 1mM DTNB, in 0.1M 1.4d µM/ Cholinesterase DTNB; non-enzymatic hydrolysis of ATCI must be phosphate buffer 55mM ATCI 4.5b 6.2b 0.90c mL/min subtracted pH 7.5 1,2-Diglyceride is hydrolyzed to 2-monoglyceride and fatty acid. The 2- monoglyceride is then measured by coupled Lipase Beckman Coultera 4.3b 5.7b > 0.99c 1.0d U/L enzyme reactions catalyzed by monoglyceride lipase, glycerol kinase, glycerol phosphate oxidase and peroxidase 4.55mM Total esterase 0.1M Tris-HCl buffer (8) 4-nitrophenyl 2.46 5.18 > 0.99 0.8 U/L activity pH 8.0 acetate Creatin kinase (25) Beckman Coultera < 8.0 < 9.0 > 0.99 2.0 U/L Aspartate (25) Beckman Coultera < 6.0 < 11.0 > 0.99 3.0 U/L transaminase Lactate (25) BioSystemse < 1.0 < 3.0 > 0.99 10.0 U/L dehydrogenase Table 1. Analytical methods used and results of their analytical performance in human saliva Deamination of adenosine to inosine, which is converted to hypoxanthine by purine nucleoside phosphorylase (PNP). Hypoxanthine is then converted to uric acid and hydrogen peroxide Adenosine (H2O2) by xanthine oxidase (XOD). H2O2 is Diazymef 7.3b 6.1b > 0.99c 0.07d U/L deaminase further reacted with N-Ethyl-N-(2-hydroxy- 3-sulfopropyl)-3-methylaniline (EHSPT) and 4-aminoantipyrine (4-AA) in the presence of peroxidase (POD) to generate quinone dye Lactate (10) Beckman Coultera 2.86 5.23 0.99 0.01 mM Biochem Med (Zagreb) 2019;29(1):010706 https://doi.org/10.11613/BM.2019.010706 Storage conditions for salivary biomarkers Reference in which the method was validated for saliva or basis of the method (in those Manufacturer or reagents (in Intra-assay Inter-assay Linearity Method LLOD assays in which validation was performed home-made assays) CV (%) CV (%) (R2) specifically for this research) Barranco T. et al. R1 R2 0.7mM of TPTZ, Reduction of Fe3+ to Fe2+ by the antioxidants and 1.5mM of FRAS None 0.89b 2.25 b 0.99c 0.031 mM present in the sample FeCl3·6H2O in acetate buffer 2mM of ABTS, 0.25μM of HRP, Reduction of ABTS radical to ABTS by the TEAC and 40μM of None 1.91b 4.51b 0.99c 0.09 mM https://doi.org/10.11613/BM.2019.010706 antioxidants present in the sample H2O2 in 50mM of phosphate buffer 0.25mM of BCS 0.5mM of CuSO4 Reduction of Cu2+ to Cu1+ by the antioxidants CUPRAC in 10mM of in ultrapure 0.60b 1.25b 0.99c 0.003 mM present in the sample phosphate buffer water Uric acid (10) Beckman Coultera 0.57 6.41 >0.99 0.22 µmol/L 2mM of DHBS, 0.5mM of AP, 50mM of Inhibition of colour development in Fe(CN)6·3H2O, 28 500mM of uric 0.03 units/ Catalase 2.09b 13.03b 0.91c a H2O2-producing urate assay U/L of uricase, acid mL and 200 U/L of HRP in 50mM of phosphate buffer Producing solutions which in the presence of 0.059 M of KI in Acetic acid, 50% AOPP 1.48b 6.25b 0.98c 3.67 μM potassium iodide absorb at 340nm ultrapure water (v/v) 0.7mM of TMB Production of diamine (yellow-coloured and 25mU of 500mM of H2O2 3.43b 16.58b 0.95c 0.01 μM oxidation product) which absorbs at 450 nm HRP in 100mM of sulphuric acid phosphate buffer CV - coefficient of variation. R2 - coefficient of determination. LLOD - lower limit of detection. R1 - reagent 1. R2 - reagent 2. ATCI - acetylthiocholine iodide. DTNB - 5,5’- dithiobis-2-nitrobenzoic acid. TPTZ – tripyridyltriazine. FeCl3·6H2O - ferric chloride hexahydrate. FeSO4·7H2O - ferrous sulfate heptahydrate. ABTS - 2,2’-azino-bis-3-eth- ylbenzthiazoline-6-sulfonic acid. HRP - horseradish peroxidase. BCS - bathocuproinedisulfonic acid disodium salt. CuSO4 – copper (II) sulphate. DHBS - 3,5-dichloro- 2-hydroxybenzenesulphonate. AP - 4-aminophenazone. K4Fe(CN)6·3H2O - potassium hexacyanoferrate II. KI - potassium iodide. TMB, - 3,5,3050-tetramethylbenzidine. aBeckman Coulter Inc, Fullerton, CA, USA. bMean of CVs for 6 repeated measurements of three pooled saliva samples of different concentrations as follows: standard deviation from the 6 measurements divided by mean and multiplied by 100. cMean of R2 measured after linearity under dilution of two saliva samples. dCalculated as 5 Biochem Med (Zagreb) 2019;29(1):010706 Storage conditions for salivary biomarkers mean + 2 standard deviations of 20 replicates of the zero standard. eBioSystems, Barcelona, Spain. fDiazyme Laboratories, Poway, CA, USA. Barranco T. et al. Storage conditions for salivary biomarkers for ADA (Diazyme, DZ177A-Con). Daily in-house When samples were stored at - 20 ºC, AMY, CHE, controls of two different values were used for anal- Lact, ADA, TEAC, FRAS, CUPRAC, UA, CAT and ysis of FRAS, TEAC, CUPRAC, CAT, AOPP and H2O2. AOPP were stable for 6 months. Significant de- The control for FRAS was a ferric chloride hexahy- creases were recorded after 14 days of storage for drate solution, for TEAC and CUPRAC a Trolox solu- AST, Lip, CK and LD; and after 3 months for TEA. A tion, an enzyme solution for CAT, Chloramine-T so- significant increase was observed for H2O2 after 3 lution for AOPP, and a H2O2 solution for H2O2 assay. months of storage. A single measurement was made in all cases since At - 80 ºC, AMY, CHE, AST, Lip, CK, LD, Lact, ADA, all analytical methods showed an intra-assay im- TEAC, FRAS, CUPRAC, UA, CAT and AOPP were sta- precision lower than 15%, which indicated ade- ble for 6 months. Significant decreases were ob- quate assay repeatability. served after 3 months for TEA. A significant in- crease was detected for H2O2 at 3 months. Statistical analysis Descriptive statistical procedures and spread- Discussion sheets (Excel 2000, Microsoft Corporation, Red- mond, USA) and software (Graph Pad Prism, Ver- This study found that although there was a varia- sion 5 for Windows, Graph Pad Software Inc, San bility in the results depending on the studied ana- Diego, USA, and IBM SPSS statistic for Windows, lyte, in general, in the short-term storage condi- version 24.0, IBM Corp., Armonk, USA) were used. tions tested, the storage at 4º C provided longer Since only 12 data were included, normality was stability for analytes in saliva than at RT. On the not assumed. Therefore, the values of the analytes other hand, in the long-term storage conditions at different times and conditions after storage tested, - 80º C provided longer stability than - 20º were compared with aliquots analysed immedi- C. In the short-term storage conditions, we also in- ately using the Friedman test, followed by Dunn’s cluded storage for 72 hours at RT and 7 days at 4º multiple comparison test. The results were consid- C. We are aware that samples are not usually ered significant when P < 0.05. stored in these conditions; however, other re- searchers in their stability studies have used simi- lar time points and they were included in our Results study in order to obtain information regarding sta- The results for the stability of the studied analytes, bility in those conditions (26). under different storage conditions, are shown in Regarding individual analytes, AMY, CHE and ADA Table 2. were the enzymes that showed fewer changes in At RT, AMY, CHE, AST, TEAC and CAT were stable the different storage conditions. The high stability during the whole experimental period (72 hours). of AMY is in accordance with the results of other The analytes that showed significant decreases studies where, for example, AMY was stable for 5 were: TEA at 6 hours; Lip, CK and CUPRAC at 24 days at RT or for 10 days at RT or 4 ºC (20,21). Cho- hours; and LD, Lact, FRAS, UA and AOPP at 72 linesterase was also stable in all conditions, so it hours. Significant increases were detected for seems that its activity is not affected after storage, H2O2 at 6 hours and for ADA at 24 hours. although there is a lack of previous reports to compare with. Adenosine deaminase was also sta- At 4 ºC, AMY, CHE, AST, Lact, ADA, TEAC, CAT and ble in most of the conditions with the exception of AOPP were stable after 14 days of storage. Signifi- RT, where it showed a significant increase. Stability cant decreases were observed after 6 hours for of ADA has been studied in porcine saliva and was TEA; after 24 hours for Lip and CUPRAC; after 72 considered as stable for up to 1 year at - 80 ºC (27). hours for CK; and after 7 days for LD, FRAS and UA. The reason why the activity of this enzyme in sali- Significant increases were recorded for H2O2 after va increases at RT needs to be further elucidated. 6 hours. Biochem Med (Zagreb) 2019;29(1):010706 https://doi.org/10.11613/BM.2019.010706 6 Analyte T0 Temperature T1 T2 T3 T4 T5 T6 T7 T8 T9 (unit) 58,570 71,340 74,920 79,850 (47,055 – (48,460 – (44,770 – (48,175 - - - - - 101,200) 94,915) 92,445) -100,690) Barranco T. et al. Variation RT 1.9 24.1 30.4 38.9 - - - - - (%) ferent storage conditions P > 0.999 > 0.999 > 0.999 > 0.999 - - - - - 78,810 68,670 76,480 79,970 76,540 81,000 (48,060 – (41,470 – (39,935 – (47,300 – (47,395 – (48,325 – - - - 102,305) 90,655) 95,885) 101,295) 99,160) 100,705) Variation 4 ºC 37.1 19.5 33.1 39.2 33.2 40.9 - - - https://doi.org/10.11613/BM.2019.010706 (%) P 0.494 > 0.999 > 0.999 0.769 > 0.999 > 0.999 - - - 57,470 79,870 68,840 74,160 77,610 AMY (U/L) (39,615 – - - - - - (47,165 – (40,750 – (42,280 – (47,965 – 101,310) 100,730) 89,215) 97,095) 99,245) Variation - 20 ºC - - - - - 39.0 19.8 29.0 35.0 (%) P - - - - - > 0.999 0.054 > 0.999 > 0.999 79,300 68,140 71,250 77,460 - - - - - (47,730 – (39,715 – (42,175 – (49,580 – 100,625) 87,200) 89,315) 104,120) Variation - 80 ºC - - - - - 38.0 18.6 24.0 34.8 (%) P - - - - - > 0.999 0.156 0.248 > 0.999 7.4 (3.7 5.5 (0.1 – 5.3 (2.6 - 5.3 (3.6 – - - - - - -10.3) 7.7 11.1) 11.9) Variation RT 2.8 23.6 26.4 26.4 - - - - - (%) P > 0.999 > 0.999 > 0.999 > 0.999 - - - - - 7.2 (3.6 6.6 (3.2 – 11.2 (7.8 – 7.1 (4.0 – 11.4 (9.3 – 7.4 (6-6 – - - - -10.3) 8.8) 14.5) 10.7) 14.7) 10.3) Variation 4 ºC 0.0 8.3 55.6 1.4 58.3 2.8 - - - (%) P > 0.999 > 0.999 0.223 > 0.999 0.097 > 0.999 - - - CHE (µM/mL/ 7.2 4.9 (3.1 – 6.7 (4.2 – 5.9 (4.2 – 4.0 (0.6 – - - - - - min) (2.6 - 9.5) 8.3) 8.0) 7.2) 4.8) Variation - 20 ºC - - - - - 31.9 6.9 18.1 44.4 (%) 7 Biochem Med (Zagreb) 2019;29(1):010706 Table 2. Stability results for 12 different saliva analytes obtained after measuring samples at different processing time and under dif- Storage conditions for salivary biomarkers 8 Analyte T0 Temperature T1 T2 T3 T4 T5 T6 T7 T8 T9 (unit) P - - - - - > 0.999 > 0.999 > 0.999 > 0.999 7.2 (6.1 – 8.1 (5.8 – 6.9 (4.9 – 4.5 (3.8 – - - - - - Barranco T. et al. 11.3) 11.0) 10.1) 7.3) Variation - 80 ºC - - - - - 0.0 12.5 4.2 37.5 (%) P - - - - - > 0.999 > 0.999 > 0.999 > 0.999 10.6 (4.5 – 11.6 (4.3 – 4.4 (3.6 – 4.1 (3.6 - - - - - 25.5) 15.9) 8.2) -4.7) Variation RT 20.9 13.4 67.2 69.4 - - - - - (%) P > 0.999 > 0.999 < 0.001 < 0.001 - - - - - 14.3 (5.1 15.7 (3.7 - 6.3 (2.4 – 2.1 (0.4 - 1.7 (1.4 - 0.6 (0.5 - - - - -23.3) 22.1) 16) 3.3) 4.1) 3.9) Variation 4 ºC 6.7 17.2 53.0 84.3 87.3 95.5 - - - (%) P > 0.999 > 0.999 < 0.001 < 0.001 <0.001 < 0.001 - - - 13.4 3.8 (0.9 – 2.0 (1.4 – 2.0 (1.2 - 3.5 (2.4 - Lip (U/L) - - - - - (7.3 - 33.2) 8.0) 14.0) 3.5) 10.6) Variation - 20 ºC - - - - - 71.6 85.1 85.1 73.9 (%) P - - - - - 0.018 0.017 < 0.001 0.031 12.5 (3.5 - 9.9 (2.7 - 11.6 (4.1 - 12.9 (4.5 - - - - - - 32.4) 36.4) 34.7) 54.4) Variation - 80 ºC - - - - - 6.7 26.1 13.4 3.7 (%) P - - - - - > 0.999 > 0.999 > 0.999 > 0.999 20.1 (14.4 15.7 (12.4 - 27.7 (23.8 - 18.1 (16.4 - - - - - - -48.8) 39.1) 32.4) 25.4) Variation RT 9.9 29.3 24.5 18.6 - - - - - (%) P > 0.999 0.002 > 0.999 0.035 - - - - - 19.7 (15.7 - 16.4 (13.3 - 28.8 (25.4 - 14.8 (12.2 - 26.5 (20.4 23.2 (18.4 - - - - 51.3) 42.4) 57.4) 27.2) -42.6) 34.2) Variation 4 ºC 11.5 26.5 29.2 33.7 18.9 4.3 - - - (%) P > 0.999 0.041 > 0.999 < 0.001 > 0.999 > 0.999 - - - Biochem Med (Zagreb) 2019;29(1):010706 https://doi.org/10.11613/BM.2019.010706 Storage conditions for salivary biomarkers Analyte T0 Temperature T1 T2 T3 T4 T5 T6 T7 T8 T9 (unit) 22.3 19.9 (15.6 - 24.4 (17 - 16.8 (9.9 21.5 (13.9 TEA (U/L) - - - - - (15.6 - 59.4) 49.7) 56.9) -35.4) -43.4) Barranco T. et al. Variation - 20 ºC - - - - - 10.3 9.7 24.5 3.4 (%) P - - - - - > 0.999 > 0.999 < 0.001 0.315 20.5 (15.4 24.3 (17.8 - 13.6 (9.0 21.0 (15.6 - - - - - - 50.9) 56.7) -34.2) -53) Variation - 80 ºC - - - - - 8.1 9.0 38.9 5.6 (%) https://doi.org/10.11613/BM.2019.010706 P - - - - - > 0.999 > 0.999 < 0.001 > 0.999 9.1 (3.6 - 10.0 (3.8 - 5.5 (1.5 - 6.0 (2.4 - - - - - - 21.6) 20.4) 9.8) 11.2) Variation RT 45.5 40.1 67.2 63.9 - - - - - (%) P > 0.999 0.248 0.002 0.003 - - - - - 11.7 (5.8 - 11.5 (6.6 - 10.9 (4.7 - 5.7 (2.7 - 4.4 (0.9 - 2.0 ((- 1.0) - - - 21.4) 16.5) 18.0) 10.1) 7.8) -7.9) Variation 4 ºC 29.8 30.7 34.6 66.0 70.8 88.0 - - - (%) P > 0.999 > 0.999 > 0.999 0.006 <0.001 < 0.001 - - - 16.6 7.0 (3.4 - 5.9 (4.7 7.2 (5.7 - 6.9 (4.0 - CK (U/L) (7.00 - - - - - - 10.8) -10.2) 11.5) 8.5) 33.2) Variation - 20 ºC - - - - - 57.8 64.8 56.6 58.4 (%) P - - - - - 0.003 0.043 0.045 0.020 12.9 (6.7 - 13.6 (8.7 - 15.9 (8.1 - 13.6 (10.1 - - - - - - 22.4) 20.7) 20.5) 20.1) Variation - 80 ºC - - - - - 22.6 18.1 4.2 18.4 (%) P - - - - - > 0.999 > 0.999 > 0.999 > 0.999 11.2 (9.0 - 11.5 (10.2 - 15.3 (12.4 - 22.2 (15.3 - - - - - 25.5) 24.0) 24.7) - 32.5) Variation RT 7.1 4.6 27.0 83.8 - - - - - (%) 9 Biochem Med (Zagreb) 2019;29(1):010706 Storage conditions for salivary biomarkers 10 Analyte T0 Temperature T1 T2 T3 T4 T5 T6 T7 T8 T9 (unit) P > 0.999 > 0.999 > 0.999 > 0.999 - - - - - 11.7 (9.5 - 12.7 (10.5 - 12.8 (10.3 - 10.3 (8.3 - 10.7 (8.4 - 10.3 (8.2 - Barranco T. et al. - - - 25.2) 24.4) 23.6) 24.1) 29.3) 24.7) Variation 4 ºC 3.3 5.0 6.2 14.5 11.2 14.9 - - - (%) P > 0.999 > 0.999 > 0.999 0.760 0.508 0.222 - - - 12.1 9.7 (7.7 - 9.1 (5.9 - 7.9 (6.1 - 5.2 (2.8 - AST (U/L) (10.1 - - - - - - 17.2) 11.3) 11.5) 6.0) 26.7) Variation - 20 ºC - - - - - 19.5 24.9 34.4 57.3 (%) P - - - - - 0.028 < 0.001 < 0.001 < 0.001 12.8 (11.0 - 13.1 (11.1 - 13.5 (11.4 - 13.9 (9.7 - - - - - - 25.1) 26.1) 26.1) 25.0) Variation - 80 ºC - - - - - 6.2 8.3 12.0 15.4 (%) P - - - - - > 0.999 > 0.999 > 0.999 0.666 338 (214 339 (212 – 276 (189 – 204 (139 – - - - - - -409) 399) 344) 295) Variation RT 0.3 0.6 18.1 39.3 - - - - - (%) P > 0.999 > 0.999 0.365 0.006 - - - - - 333 (224- 336 (193 – 320 (211 – 220 (172 – 196 (156 – 131 (77 – - - - 402) 404) 383) 338) 302) 261) Variation 4 ºC 1.0 0.1 5.0 34.8 41.7 61.1 - - - (%) P > 0.999 > 0.999 > 0.999 0.222 0.006 < 0.001 - - - 337 69 (27 – 54 (18 – 31 (15 – LD (U/L) - - - - - 27 (5 – 59) (221-403) 168) 97) 59) Variation - 20 ºC - - - - - 79.5 84.0 92.1 90.9 (%) P - - - - - < 0.001 < 0.001 < 0.001 < 0.001 323 (214 – 321 (223 – 335 (234 – 370 (279 – - - - - - 391) 399) 414) 445) Biochem Med (Zagreb) 2019;29(1):010706 https://doi.org/10.11613/BM.2019.010706 Storage conditions for salivary biomarkers Analyte T0 Temperature T1 T2 T3 T4 T5 T6 T7 T8 T9 (unit) Variation - 80 ºC - - - - - 4.0 1.8 0.6 10.0 (%) Barranco T. et al. P - - - - - > 0.999 > 0.999 > 0.999 > 0.999 0.01((- 0.5 (0.4 - 0.5 (0.4 - 0.3 (0.1 - 0.003) - - - - - 1.3) 1.3) 1.2) -0.04) Variation RT 1.0 1.0 58.2 98.1 - - - - - (%) P > 0.999 > 0.999 0.634 0.003 - - - - - https://doi.org/10.11613/BM.2019.010706 0.5 (0.4 - 0.5 (0.4 - 0.4 (0.3 0.4 (0.3 - 0.5 (0.4 0.5 (0.1 - - - - 1.2) 1.2) -1.3) 1.4) -1.5) 0.7) Variation 4 ºC 1.9 1.0 15.5 17.5 8.7 9.7 - - - (%) P > 0.999 > 0.999 > 0.999 > 0.999 >0.999 > 0.999 - - - Lact 0.5 0.5 (0.4 - 0.5 (0.4 - 0.5 (0.4 - 0.5 (0.4 - - - - - - (mmol/L) (0.4 - 1.2) 1.2) 1.1) 1.2) 1.2) Variation - 20 ºC - - - - - 1.0 10.7 5.8 3.9 (%) P - - - - - > 0.999 > 0.999 > 0.999 > 0.999 0.5 (0.4 - 0.5 (0.4 - 0.5 (0.4 - 0.5 (0.4 - - - - - 1.2) 1.2) 1.2) -1.2) Variation - 80 ºC - - - - - 1.0 6.8 1.9 3.9 (%) P - - - - - > 0.999 > 0.999 > 0.999 > 0.999 1.2 (0.8 - 1.3 (1.0 - 2.0 (1.2 1.1 (0.9 - - - - - - 1.7) 1.9) -2.2) 1.8) Variation RT 18.2 35.4 101 6.1 - - - - - (%) P > 0.999 0.059 0.007 > 0.999 - - - - - 1.1 (0.9 - 1.2 (0.8 - 1.0 (0.8 - 0.9 (0.6 - 0.8 (0.6 0.6 (0.5 - - - - 1.6) 1.8) 1.9) 1.7) -1.2) 1.2) Variation 4 ºC 12.1 21.2 4.0 6.1 20.2 37.4 - - - (%) P > 0.999 0.734 > 0.999 > 0.999 >0.999 > 0.999 - - - 11 Biochem Med (Zagreb) 2019;29(1):010706 Storage conditions for salivary biomarkers 12 Analyte T0 Temperature T1 T2 T3 T4 T5 T6 T7 T8 T9 (unit) ADA 1.0 1.0 (0.5 - 0.8 (0.3 - 0.5 (0.2 - 0.6 (0.2 - - - - - - (U/L) (0.7 - 1.6) 1.7) 1.3) 1.1) 0.8) Barranco T. et al. Variation - 20 ºC - - - - - 2.0 15.2 45.5 41.4 (%) P - - - - - > 0.999 > 0.999 0.189 0.064 1.2 (0.8 - 1.2 (0.8 - 1.0 (0.7 - 1.2 (1.0 - - - - - - 1.8) 1.8) 1.5) 2.0) Variation - 80 ºC - - - - - 17.2 16.2 2.0 18.2 (%) P - - - - - > 0.999 > 0.999 > 0.999 0.291 0.2 (0.2 - 0.2 (0.2 - 0.1 (0.1 - 0.2 (0.1 - - - - - - 0.3) 0.3) 0.2) 0.2) Variation RT 31.6 26.2 13.9 9.1 - - - - - (%) P 0.701 0.529 0.582 0.582 - - - - - 0.2 (0.2 - 0.2 (0.2 - 0.2 (0.1 - 0.2 (0.1 - 0.2 (0.2 0.2 (0.1 - - - - 0.3) 0.3) 0.2) 0.2) -0.3) 0.2) Variation 4 ºC 29.5 24.1 4.6 7.4 17.1 10.5 - - - (%) P > 0.999 > 0.999 > 0.999 > 0.999 0.064 > 0.999 - - - TEAC 0.2 0.2 (0.1 - 0.2 (0.1 - 0.2 (0.1 - - - - - - - (mM) (0.1 – 0.2) 0.2) 0.2) 0.3) Variation - 20 ºC - - - - - - 6.0 6.8 33.8 (%) P - - - - - - > 0.999 > 0.999 0.457 0.2 (0.1 - 0.2 (0.1 - 0.2 (0.2 - - - - - - - 0.2) 0.3) 0.3) Variation - 80 ºC - - - - - - 8.3 11.0 32.0 (%) P - - - - - - > 0.999 0.291 0.841 0.4 (0.3 - 0.4 (0.3 - 0.4 (0.3 - 0.3 (0.2 - - - - - - 0.5) 0.5) 0.5) 0.3) Variation RT 3.3 9.1 9.5 13.3 - - - - - (%) Biochem Med (Zagreb) 2019;29(1):010706 https://doi.org/10.11613/BM.2019.010706 Storage conditions for salivary biomarkers Analyte T0 Temperature T1 T2 T3 T4 T5 T6 T7 T8 T9 (unit) P > 0.999 0.669 > 0.999 < 0.001 - - - - - 0.4 (0.2 - 0.3 (0.2 - 0.4 (0.3 - 0.3 (0.3 - 0.3 (0.2 0.4 (0.3 - Barranco T. et al. - - - 0.5) 0.5) 0.5) 0.4) -0.4) 0.4) Variation 4 ºC 3.0 1.3 2.0 4.5 9.4 4.3 - - - (%) P > 0.999 > 0.999 > 0.999 0.555 0.018 0.047 - - - FRAS 0.3 0.3 (0.3 - 0.4 (0.3 - 0.3 (0.3 - 0.3 (0.3 - - - - - - (mM) (0.3 - 0.5) 0.5) 0.5) 0.5) 0.5) Variation https://doi.org/10.11613/BM.2019.010706 - 20 ºC - - - - - 0.1 10.4 4.8 0.3 (%) P - - - - - > 0.999 > 0.999 > 0.999 > 0.999 0.3 (0.2 - 0.3 (0.2 - 0.3 (0.2 - 0.3 (0.2 - - - - - - 0.5) 0.5) 0.5) 0.5) Variation - 80 ºC - - - - - 0.1 0.6 1.3 2.1 (%) P - - - - - > 0.999 > 0.999 > 0.999 0.394 0.2 (0.2 - 0.2 (0.1 - 0.2 (0.1 - 0.2 (0.1 - - - - - - 0.2) 0.2) 0.2) 0.2) Variation RT 1.1 1.5 0.1 27.2 - - - - - (%) P > 0.999 > 0.999 0.010 < 0.001 - - - - - 0.2 (0.2 - 0.2 (0.1 - 0.2 (0.1 - 0.2 (0.1 - 0.2 (0.1 0.2 (0.1 - - - - 0.2) 0.2) 0.2) 0.2) -0.2) 0.2) Variation 4 ºC 0.8 0.1 5.9 12.7 14.3 10.1 - - - (%) P > 0.999 0.794 0.002 < 0.001 <0.001 < 0.001 - - - CUPRAC 0.2 0.2 (0.2 - 0.2 (0.2 - 0.2 (0.2 - 0.2 (0.2 - - - - - - (mM) (0.2 - 0.3) 0.2) 0.2) 0.2) 0.3) Variation - 20 ºC - - - - - 1.2 0.3 3.3 2.1 (%) P - - - - - > 0.999 > 0.999 0.162 > 0.999 0.2 (0.2 - 0.2 (0.2 - 0.2 (0.2 - 0.2 (0.2 - - - - - - 0.2) 0.3) 0.2) 0.3) 13 Biochem Med (Zagreb) 2019;29(1):010706 Storage conditions for salivary biomarkers 14 Analyte T0 Temperature T1 T2 T3 T4 T5 T6 T7 T8 T9 (unit) Variation - 80 ºC - - - - - 0.1 3.7 0.2 2.4 (%) Barranco T. et al. P - - - - - > 0.999 > 0.999 0.131 > 0.999 13.1 (9.5 - 13.7 (9.5 - 11.9 (7.7 - 9.5 (5.9 - - - - - - 16.7) 16.7) 16.1) 12.5) Variation RT 2.6 1.3 12.2 28.6 - - - - - (%) P > 0.999 > 0.999 0.084 < 0.001 - - - - - 13.7 (9.5 - 13.1 (9.5 - 13.1 (8.9 - 12.5 (8.3 - 11.9 7.7 - 10.7 (7.1 - - - - 17.3) 17.3) 16.7) 16.1) 14.3) 13.7) Variation 4 ºC 1.7 1.5 4.6 10.2 12.1 20.6 - - - (%) P > 0.999 > 0.999 > 0.999 0.383 0.014 0.002 - - - Uric acid 13.7 13.7 (9.5 - 13.7 (9.5 - 14.3 (9.5 - 14.3 (10.1 - - - - - - (µmol/L) (9.5 - 16.1) 18.4) 16.7) 17.3) 17.8) Variation - 20 ºC - - - - - 1.5 2.4 2.8 5.6 (%) P - - - - - > 0.999 > 0.999 > 0.999 0.365 13.7 (9.5 - 13.1 (9.5 - 13.7 (9.5 - 14.3 (9.5 - - - - - - 16.7) 16.7) 17.3) 16.7) Variation - 80 ºC - - - - - 0.2 2.8 0.0 2.8 (%) P - - - - - > 0.999 > 0.999 > 0.999 0.666 0.2 (0.2 - 0.2 (0.2 - 0.2 (0.2 - 0.4 (0.2 - - - - - - 0.3) 0.3) 0.4) 0.5) Variation RT 2.2 13.3 12.5 50.0 - - - - - (%) P > 0.999 0.960 > 0.999 > 0.999 - - - - - 0.2 (0.2 - 0.2 (0.2 - 0.3 (0.2 - 0.2 (0.2 - 0.3 (0.2 - 0.2 (0.2 - - - - 0.3) 0.3) 0.3) 0.3) 0.4) 0.4) Variation 4 ºC 8.9 1.2 2.0 6.5 33.5 0.4 - - - (%) P > 0.999 > 0.999 > 0.999 > 0.999 >0.999 > 0.999 - - - Biochem Med (Zagreb) 2019;29(1):010706 https://doi.org/10.11613/BM.2019.010706 Storage conditions for salivary biomarkers Analyte T0 Temperature T1 T2 T3 T4 T5 T6 T7 T8 T9 (unit) CAT (units/ 0.2 0.2 (0.2 - 0.3 (0.2 - 0.3 (0.2 - 0.3 (0.2 - - - - - - mL) (0.2 – 0.3) 0.3) 0.4) 0.4) 0.4) Barranco T. et al. Variation - 20 ºC - - - - - 8.1 14.1 19.4 26.2 (%) P - - - - - > 0.999 > 0.999 > 0.999 > 0.999 0.3 (0.2 - 0.3 (0.2 - 0.2 (0.2 - 0.2 (0.2 - - - - - - 0.3) 0.4) 0.4) 0.4) Variation - 80 ºC - - - - - 4.0 11.3 2.4 4.8 (%) https://doi.org/10.11613/BM.2019.010706 P - - - - - > 0.999 > 0.999 > 0.999 > 0.999 103 (80 – 106 (83 – 121 (79 – 59 (41 – - - - - - 169) 168) 147) 69) Variation RT 6.7 3.7 9.8 46.9 - - - - - (%) P > 0.999 > 0.999 > 0.999 0.001 - - - - - 116 (81 – 119 (89 – 115 (67 – 102 (60 – 109 (56 – 95 (69 – - - - 169) 165) 142) 126) 173) 123) Variation 4 ºC 4.6 8.1 4. 8.1 1.2 14.2 - - - (%) P > 0.999 > 0.999 0.914 0.331 >0.999 > 0.999 - - - AOPP 111 109 (97 – 115 (100 – 102 (81 – 96 (82 – - - - - - (μM) (76 -180) 184) 186) 155) 146) Variation - 20 ºC - - - - - 1.9 4.1 7.3 13.3 (%) P - - - - - > 0.999 > 0.999 > 0.999 > 0.999 114 (90 – 116 (98 – 110 (84 – 124 (83 – - - - - - 185) 182) 171) 207) Variation - 80 ºC - - - - - 2.9 4.7 0.5 11.8 (%) P - - - - - > 0.999 > 0.999 > 0.999 > 0.999 5.4 (2.1 - 6.8 (3.2 5.6 (4.3 4.4 (2.2 - - - - - - 6.6) -10.6) -10.8) 7.2) Variation RT 120.4 178.8 127.3 80.8 - - - - - (%) 15 Biochem Med (Zagreb) 2019;29(1):010706 Storage conditions for salivary biomarkers 16 Analyte T0 Temperature T1 T2 T3 T4 T5 T6 T7 T8 T9 (unit) P > 0.999 0.011 0.001 > 0.999 - - - - - 5.3 (2.4 - 7.3 (3.1 8.6 (5.7 6.1 (3.2 - 4.2 (3.5 4.5 (2.5 - Barranco T. et al. - - - 6.4) -10.2) -12.2) 7.6) -7.2) 8.0) Variation 4 ºC 117.1 198 252.2 146.9 72.2 84.5 - - - (%) P > 0.999 0.012 < 0.001 0.960 >0.999 > 0.999 - - - H2O2 2.5 6.8 (2.1 - 7.5 (3.1 - 9.2 (4.1 7.7 (3.8 - - - - - (μM) (1.7 - 8.9) 9.4) 9.0) -11.8) -10.8) Variation - 20 ºC - - - - - 176.3 206.9 274.7 212.2 (%) P - - - - - > 0.999 0.248 < 0.001 0.002 6.2 (2.5 6.6 (2.8 - 7.2 (3.3 9.6 (3.0 - - - - - -10.8) 9.0) -12.4) -12.2) Variation - 80 ºC - - - - - 153.5 169.8 193.9 291.0 (%) P - - - - - 0.804 > 0.999 < 0.001 0.002 Quantitative data is presented as median and interquartile range. T0 - baseline. T1 - 3 hours. T2 - 6 hours. T3 - 24 hours.T4 - 72 hours T5 - 7 days. T6 - 14 days. T7 – 1 month . T8 – 3 months . T9 – 6months. RT - room temperature. AMY - α-amylase. CHE - cholinesterase. Lip - lipase. TEA - total esterase. CK - creatine kinase. AST - aspartate aminotransferase. LD - lactate dehydrogenase. Lact - lactate. ADA - adenosine deaminase. TEAC - Trolox equivalent antioxidant capacity. FRAS - ferric reducing ability. CUPRAC - cupric reducing antioxidant capacity. CAT - catalase. AOPP - advanced oxidation protein products. H2O2 - hydrogen peroxide. P < 0.05 represents statistical significant difference. Biochem Med (Zagreb) 2019;29(1):010706 https://doi.org/10.11613/BM.2019.010706 Storage conditions for salivary biomarkers Barranco T. et al. Storage conditions for salivary biomarkers Lipase and CK showed a high stability at - 80 ºC; could only be measured after 3 hours at 4 ºC or 1 however, in the rest of the storage conditions they month under freezing conditions. Further studies showed significant changes. To our knowledge, should be made to elucidate the reason for the the stability of Lip and CK in saliva under different production of H2O2 in the saliva samples when storage conditions has not been accessed before, they are stored. and the reason for lipase and CK enzymatic activi- This study has some limitations that should be tak- ty changes in saliva is unknown and should be fur- en into account. Firstly, the study has been made ther studied. Aspartate aminotransferase showed in healthy subjects. It would be of interest in the also the highest stability at - 80 ºC, being stable for future to perform further studies also involving 6 months. The lack of stability of AST in other stor- subjects with different diseases in order to evalu- age conditions, such as - 20 ºC, agrees with previ- ate the possible differences of stability between ous reports (2,3). samples of healthy and sick subjects. Although in Total esterase and LD were the most labile en- line with previous reports, the number of subjects zymes in our stability study. It is difficult to know included in this study can be considered low and the reason for the changes of TEA, it is likely to ideally a higher number of cases should be includ- have been influenced by the instability of any of ed (21,25,30). Therefore, this report should be tak- the various enzymes that integrate in this total ac- en as a pilot study and additional studies would be tivity (8). Lactate dehydrogenase results in our needed prior to making recommendations about study were similar to other reports that showed a the storage conditions. In addition, in the case of significant decrease of this enzyme in saliva after the enzymes, it would have been interesting to only 30 min, 3 days and 2 weeks of storage at - 20 study the stability of the different isoenzymes. ºC (2,3,22). These results could be due to the labili- It can be concluded that in short-term storage the ty of the LD-4 and LD-5 isoenzymes at - 20 ºC (28). analytes were more stable at 4 ºC than at room With the exception of H2O2, the remaining antioxi- temperature, whereas in long-term storage they dants biomarkers showed a high stability under were more stable at - 80 ºC than at - 20 ºC. freezing conditions. Uric acid has been observed to remain relatively stable during storage, in ac- Acknowledgments cordance with previous studies describing stability This research was supported by the Garcia Cugat at - 20 ºC, - 70 ºC and at - 196 ºC in human serum Foundation and the Seneca Foundation of Murcia when stored for 1 year (29). This can also help to Region. The authors also had financial support explain the stability observed for the TEAC and from the Portuguese Foundation for Science and FRAS in saliva, both at - 20 ºC and at - 80 ºC, as UA Technology (FCT) in the form of Elsa Lamy's FCT in- is one of the main contributors to TEAC and FRAS. vestigator contract IF/01778/2013. When oxidant biomarkers were studied, AOPP showed a high level of stability in all conditions, Potential conflict of interest except at RT. Only H2O2 was very unstable and None declared. References 1. Gröschl M. Current status of salivary hormone analysis. 3. Kasim NA, Ariffin SHZ, Shahidan MA, Abidin IZZ, Senafi S, Je- Clin Chem. 2008;54:1759-69. https://doi.org/10.1373/ main AA, et al. Stability of lactate dehydrogenase, aspartate clinchem.2008.108910 aminotransferase, alkaline phosphatase and tartrate resi- stant acid phosphatase in human saliva and gingival crevi- 2. 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