The use of saliva to measure oxidative stress (OS) is increasing (1), since researchers have demonstrated that saliva contains oxidation biomarkers similar to those in blood (2-5). The analysis of OS biomarkers in saliva is potentially valuable because its collection is simple and may provide a cost-effective approach for the screening of large populations (6).
Nevertheless, sometimes saliva samples are required to be stored for several days and different results can be obtained because salivary constituents may degrade when processed after multiple freeze–thaw cycles or after different time and temperature of storage (7).
Since there is little consistency in the literature regarding the feasibility of saliva storage before OS markers analysis, the aim of this study was to compare the effects of short-term storage in two different temperatures on stability of salivary total antioxidant capacity (TAC), total oxidant status (TOS) and oxidant stress index (OSI).
Materials and methods
Ethics Committee Approval
The study protocol was approved by the Research Ethics Committee of the Federal University of Paraná, Brazil (Project CEP:872583/2014). All the participants received detailed information concerning the nature and the procedures involved in the study and signed informed consent forms.
This analytic study included saliva samples from 20 healthy volunteers, with an age range of 28–38 years, recruited from February until July 2015.
Saliva Collection and Storage
Saliva production was stimulated by chewing a paraffin gum and total saliva samples were collected in plastic tubes on ice, after 2 hr-fasting, always between 8:00 and 10:00 a.m. Each participant collected saliva for approximately 10 minutes. All the samples were centrifuged at 2,600g for 10 min at 4°C to remove cellular and food debris and none of them were contaminated with blood.
Each saliva sample was divided in 13 identical aliquots (500µL). One aliquot was used immediately to analyze fresh saliva and two series of 6 identical aliquots were used to analyze after 15, 30, 45, 60, 90 and 120 days of storage at -20°C or -80°C.
Measurement of the TAC of the saliva
Saliva TAC was determined using the automated colorimetric measurement method of Erel (8). In this method hydroxyl radicals react with O-dianisidine to produce the bright yellowish brown dianisyl radical and after the addition of saliva, the oxidative reactions are suppressed by the antioxidant components of saliva thus preventing the color change. The results were expressed as millimolar Trolox equivalent per liter (mmol Trolox equiv /L).
Measurement of the TOS of the saliva
Saliva TOS was measured using the fully automated colorimetric method of Erel (9). In this method, the oxidants present in saliva oxidize the ferrous ion –o-dianisidine complex to ferric ion. The results were expressed in terms of micromolar hydrogen peroxide equivalent per liter (µmol H2O2 Equiv/L).
Oxidative stress index
The percent ratio of the TOS to the TAS gives the oxidative stress index (OSI). To perform the calculation, the result unit of TAS, mmol Trolox Equivalent/L, was changed to µmol Trolox Equivalent/L, and the OSI value was calculated as follows: OSI = [(TOS, µmol/L)/TAS, µmol/L)/100]
The results were expressed as mean ± standard error of mean. Statistical analysis was performed using the repeated-measures analysis of variance (ANOVA), the Bonferroni and t-test. A p-value <0.05 was accepted to be statistically significant. Statistical analysis was performed with Statistical Package for the Social Sciences for Windows (SPSS, version 20.0, SPSS Inc., Chicago, IL, USA).
Compared with fresh saliva samples, salivary TAC and TOS levels were significantly lower after 90 days of storage at both temperatures (Figure 1 and Figure 2). Saliva TOS concentration presented a long term decline during the study at both temperatures. There was a statistically significant effect of time on TOS levels at -20°C, F(11.26, 2.21)=5.09, p=0.002, η2p=0.253; with difference between T0 and T90 (p=0.02) and at -80°C, F(12.08, 2.11)=5.716, p=0.001, η2p=0.276; with difference between T0 and T90 (p=0.03).
Saliva TAC concentration also presented a long term decline at both temperatures with statistical significant effect, at -20°C, F(5.71, 2.13)=2.67, p=0.02, η2p=0.151; with difference between T0 and T90 (p=0.012) and at -80°C, F(17.025, 2.50)=6.80, p<0.001, η2p=0.312; with difference between T0 and T90 (p=0.02).
On the other hand, no statistical difference was found between the two temperatures of storage at each different time points for TAC and TOS, as shown on Table 1 and Table 2. Also, no statistical difference was found in the OSI level between the two storage temperatures, remaining practically constant during the period of the study (Table 3).
Saliva has been increasingly used as diagnostic medium because its collection method is easy, non-invasive, can do repeated sampling and suitability for research single analyte or complex measurements (10). Saliva has also been reported to be suitable to detect the body’s oxidative stress levels, helping with diagnosis, prognosis, and therapeutic response of human diseases (10-12). However, some saliva analytes are not stable at room temperature and in many cases, storage of samples for a day prior to analysis is necessary (10). As far as we know, this is the first study to evaluate the stability of the TAC, TOS and OSI of saliva during freeze storage at 2 temperatures (–20º and –80ºC), assessing the variations occurring during 120 days of storage.
According to our results, storage periods led to some changes in both TAC and TOS, regardless of the temperature of storage. It seems that saliva can be frozen and stored at –20ºC or –80ºC for two months, in order to have similar results to the ones found in fresh saliva samples.
In this study, the saliva TAC decreased with a similar pattern, regardless of the storage temperature. This decrease can be explained by the fact that some of the antioxidants comprise proteins and it is a known fact that temperature has a strong influence on the activity of proteins in which lower temperatures result in lower activity. One limitation of the present study is that we did not quantify proteins in the samples; therefore we cannot confirm that this did not interfere with the results. One of the reasons for the decrease in TAC measurements may be the loss of protein activity or protein degradation. Hubel et al. (13), on a review evaluated various fluids, including urine, saliva, blood, among others, shows that different conditions of collection and storage have great effects on the stability of proteins, leading to misinterpretation of the results and invalid conclusions. Emekli-Alturfan et al. (7, 14) evaluated the levels of GSH and lipid peroxidation in saliva samples stored at -20°C, in 30, 60, 90, 120, 150 and 180 days after collection. They suggest that saliva can be stored for 30 days at -20°C. Also, Ng et al. (15) analyzed salivary IgA and lysozyme and they observed that the concentrations remain stable for up to 3 months when stored at -30°C.
The evaluation of oxidative status during storage has been made in other body fluids such as urine, breast milk and blood. Remer et al. (16) used urine which was stored for a period of 15 years and they found that some substances have high stability at -22°C, such as uric acid, a non-enzymatic antioxidant. However, other substances such as oxalate had losses over time and to prevent this, the use of preservatives has been proposed. Nevertheless, adding preservatives can interfere with other saliva compounds.
Akdag et al. (17) assessed the stability of TAC in breast milk stored at -80°C for 3 months and found that it remains stable without significant losses. Silvestre et al. (18) evaluated the activity of glutathione peroxidase and the concentration of MDA in breast milk in 2 temperatures (-20°C and -80°C), at 15, 30 and 60 days of storage. They found that freezing induces losses in the antioxidant properties of breast milk and that such losses increase with duration of storage and differ in intensity according to temperature. They suggests that in order to maximally preserve the antioxidant properties of breast milk, the latter should be stored at -80 °C degrees for a period of less than 30 days, rather than for shorter time periods at usual temperature of -20 °C degrees (18).
In the present study, regardless of the storage temperature, the saliva TAC and TOS decreased with a similar pattern. However, saliva stored at –80 ºC seems to be more stable than saliva stored at -20 ºC. Consequently, the general and widely accepted rule of storing at the lowest temperature possible seems to apply to the saliva TAC and TOS, as might be expected. On the other hand, if saliva cannot be stored at –80ºC, it can be stored at -20°C for 60 days without interfering with the TAC, TOS and OSI levels.
The limitations of our study are a relatively small sample size and the fact that only TAC and TOS were analyzed. Although specific biomarkers should be examined to clearly identify oxidative stress, the assessment of oxidative stress through the evaluation of the TAC and TOS may be sensible to elucidate the oxidant status and the preservation of the samples.