Salivary oxidative stress markers represent a promising tool for monitoring of oral diseases. Saliva can often be contaminated by blood, especially in patients with periodontitis. The aim of our study was to examine the impact of blood contamination on the measurement of salivary oxidative stress markers. Saliva samples were collected from 10 healthy volunteers and were artificially contaminated with blood (final concentration 0.001–10%). Next, saliva was collected from 12 gingivitis and 10 control patients before and after dental hygiene treatment. Markers of oxidative stress were measured in all collected saliva samples. Advanced oxidation protein products (AOPP), advanced glycation end products (AGEs), and antioxidant status were changed in 1% blood-contaminated saliva. Salivary AOPP were increased in control and patients after dental treatment (by 45.7% and 34.1%,
Markers of oxidative stress in saliva have become an attractive tool for analyzing the pathogenesis and monitoring of oral and dental diseases. Cross-reacting substances in the mouth and saliva collection methods could influence assay validity of oxidative stress markers [
The aim of our study was to analyze the effect of artificial whole blood contamination and the effect of contamination with individual blood components (plasma, red blood cells, and hemoglobin) on salivary concentrations of markers of oxidative stress in healthy probands. In addition, the impact of blood contamination should be studied in a case-control study comparing the salivary markers of oxidative stress in patients with gingivitis and healthy controls after dental hygiene treatment.
In study I 10 young periodontally healthy volunteers (5 females and 5 males) with an average age of 23.5 ± 1.9 years were enrolled. In study II a total of 22 subjects were enrolled in the dental ambulance in Bratislava, Slovakia. Twelve subjects were male patients with gingivitis with an average age of 35.3 ± 8.0 years and 10 male subjects were age-matched healthy controls with an average age of 38.2 ± 4.9 years. In study II subjects underwent an examination of their periodontal status using plaque index (PI) [
Whole unstimulated saliva samples were collected in the morning before eating. Collected saliva samples were stored at −20°C until analyses. On the day of testing, samples were brought to room temperature and centrifuged at 1000 g for 10 min and the supernatant was used for testing.
In study I saliva samples were artificially contaminated with blood. Samples of saliva were divided into aliquots. One aliquot from each individual was used as a control (no blood added). The remaining salivary aliquots were contaminated by venous blood and serially diluted to obtain saliva samples with the following concentrations of blood: 10%, 5%, 2.5%, 1%, 0.1%, 0.01%, and 0.001%. Similar to contamination of saliva with whole blood other aliquots were contaminated with plasma, red blood cells, or hemoglobin (Sigma Aldrich, Steinheim, Germany) ranging from 10% to 0.001%.
In study II a baseline saliva sample was collected from the participants. Dental hygiene treatment was performed by a dentist (LB). Saliva samples were collected again after treatment. Dental hygiene treatment was used as a model of blood leakage due to microinjury.
All reagents or chemicals used in our experiments were purchased from Sigma-Aldrich (Steinheim, Germany). Salivary advanced oxidation protein products (AOPP) as markers of protein oxidation were determined using a spectrophotometric method. Two hundred
Salivary advanced glycation end products (AGEs) as markers of carbonyl stress were measured using spectrofluorometric method. Saliva samples were diluted 10-fold with phosphate buffered saline (PBS, pH = 7.2) and measured at
Ferric reducing antioxidant power (FRAP), marker of antioxidant status, was determined according to Benzie and Strain [
Total antioxidant capacity (TAC), marker of antioxidant status, was measured using spectrophotometric method. Saliva was mixed with acetate buffer (pH = 5.8), incubated with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and oxidized with hydrogen peroxide in acetate buffer (pH = 3.6). Absorbance was measured at 660 nm. Trolox was used as standard in calibration curve [
Total proteins were quantified using BCA protein assay kit (Sigma Aldrich, Steinheim, Germany). Briefly 10
Analysis was performed with XLStatistics 10.05.30 (Carr, R., XLent Works, Australia) and GraphPad Prism 5.03 (GraphPad Software, San Diego, California). In study I two-way repeated measures (RM) ANOVA was used to analyze oxidative stress markers in saliva artificially contaminated with blood. Based on the results from two-way RM ANOVA data from both genders were combined and analyzed using one-way RM ANOVA and Tukey’s multiple comparison test. In study II the effect of microinjury on salivary markers of oxidative stress before and after dental hygiene was determined using Wilcoxon matched-pairs signed rank test for control and gingivitis group separately. Data are presented as mean + SD. Level
Saliva samples contaminated by whole blood with a final concentration of 0.1% blood and higher are visibly colored (Figure
The effect of gender and blood contamination on oxidative stress markers was analyzed using two-way RM ANOVA. Separate ANOVAs were computed for whole blood, plasma, red blood cells (RBC), and hemoglobin contamination.
Two-way RM ANOVA |
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Blood contamination | |||
Effect of blood | 49.86 | <0.0001 |
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Effect of gender | 0.51 | 0.49 | ns |
Plasma contamination | |||
Effect of plasma | 33.59 | <0.0001 |
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Effect of gender | 3.68 | 0.09 | ns |
RBC contamination | |||
Effect of RBC | 296.3 | <0.0001 |
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Effect of gender | 0.20 | 0.66 | ns |
Hemoglobin contamination | |||
Effect of hemoglobin | 28.31 | <0.0001 |
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Effect of gender | 1.36 | 0.28 | ns |
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Blood contamination | |||
Effect of blood | 21.39 | <0.0001 |
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Effect of gender | 5.42 | 0.0483 |
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Plasma contamination | |||
Effect of plasma | 20.75 | <0.0001 |
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Effect of gender | 4.48 | 0.07 | ns |
RBC contamination | |||
Effect of RBC | 68.02 | <0.0001 |
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Effect of gender | 5.25 | 0.05 | ns |
Hemoglobin contamination | |||
Effect of hemoglobin | 19.17 | <0.0001 |
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Effect of gender | 6.52 | 0.0340 |
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Blood contamination | |||
Effect of blood | 12.00 | <0.0001 |
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Effect of gender |
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0.99 | ns |
Plasma contamination | |||
Effect of plasma | 23.59 | <0.0001 |
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Effect of gender | 0.27 | 0.62 | ns |
RBC contamination | |||
Effect of RBC | 9.772 | <0.0001 |
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Effect of gender | 0.18 | 0.68 | ns |
Hemoglobin contamination | |||
Effect of hemoglobin | 18.32 | <0.0001 |
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Effect of gender | 0.8376 | 0.39 | ns |
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Blood contamination | |||
Effect of blood | 24.31 | <0.0001 |
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Effect of gender | 0.20 | 0.67 | ns |
Plasma contamination | |||
Effect of plasma | 33.02 | <0.0001 |
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Effect of gender | 0.51 | 0.49 | ns |
RBC contamination | |||
Effect of RBC | 24.37 | <0.0001 |
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Effect of gender | 0.25 | 0.63 | ns |
Hemoglobin contamination | |||
Effect of hemoglobin | 43.80 | <0.0001 |
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Effect of gender | 0.15 | 0.71 | ns |
AOPP, advanced oxidation protein products; AGEs, advanced glycation end products; FRAP, ferric reducing antioxidant power; TAC, total antioxidant capacity; ns, nonsignificant.
Saliva samples of two probands (columns P1 and P2) contaminated by venous blood with the following final concentrations of blood: 10%, 5%, 2.5%, 1%, 0.1%, 0.01%, 0.001%, and 0%. Note the visible discoloration of saliva with blood contamination from 0.1%.
Oxidative stress and antioxidant status markers in saliva contaminated with blood. (a) Salivary AOPP concentrations, biomarker of oxidative damage to proteins. (b) Salivary AGEs concentrations, biomarker of carbonyl stress. (c) Salivary FRAP concentrations, biomarker of antioxidant status. (d) Salivary TAC concentrations, biomarker of antioxidant status. Data are presented as mean + SD;
To determine which blood component is responsible for changes in measured salivary markers, the impact of plasma, red blood cells, and hemoglobin was studied. Salivary AOPP concentrations were decreased proportionally in the presence of 0.1–10% plasma contamination in saliva by 27.3–85.0% (Figure
Oxidative stress and antioxidant status markers in saliva contaminated with plasma. (a) Salivary AOPP concentrations, biomarker of oxidative damage to proteins. (b) Salivary AGEs concentrations, biomarker of carbonyl stress. (c) Salivary FRAP concentrations, biomarker of antioxidant status. (d) Salivary TAC concentrations, biomarker of antioxidant status. Data are presented as mean + SD;
Oxidative stress and antioxidant status markers in saliva contaminated with red blood cells (RBC). (a) Salivary AOPP concentrations, biomarker of oxidative damage to proteins. (b) Salivary AGEs concentrations, biomarker of carbonyl stress. (c) Salivary FRAP concentrations, biomarker of antioxidant status. (d) Salivary TAC concentrations, biomarker of antioxidant status. Data are presented as mean + SD;
Oxidative stress and antioxidant status markers in saliva contaminated with hemoglobin. (a) Salivary AOPP concentrations, biomarker of oxidative damage to proteins. (b) Salivary AGEs concentrations, biomarker of carbonyl stress. (c) Salivary FRAP concentrations, biomarker of antioxidant status. (d) Salivary TAC concentrations, biomarker of antioxidant status. Data are presented as mean + SD;
To study the effect of blood contamination in a real clinical situation the impact of microinjury in gingivitis and age-matched healthy control patients was modeled. Clinical parameters of both study groups are summarized in Table
Comparison of clinical parameters between control and gingivitis patients.
Clinical parameter | Group |
Unpaired |
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Control | Gingivitis | ||||
( |
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BOP (%) | 18.52 ± 4.69 | 74.32 ± 13.00 | 12.9 | <0.0001 |
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SBI (score) | 0.40 ± 0.12 | 1.72 ± 0.33 | 11.9 | <0.0001 |
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PI (score) | 0.58 ± 0.15 | 1.14 ± 0.44 | 3.9 | 0.0010 |
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BOP, bleeding on probing; SBI, sulcus bleeding index; PI, plaque index. Data are presented as mean ± SD.
The effect of blood leakage after dental hygiene treatment on markers of oxidative stress and antioxidant status in saliva of control (a–d) and gingivitis (e–h) patients. (a, e) Salivary AOPP concentrations, biomarker of oxidative damage to proteins. (b, f) Salivary AGEs concentrations, biomarker of carbonyl stress. (c, g) Salivary FRAP concentrations, biomarker of antioxidant status. (d, h) Salivary TAC concentrations, biomarker of antioxidant status.
Schwartz and Granger reported that blood components in saliva invisible to the eye have the potential to bias salivary analytes and the control of blood contamination in saliva was suggested. Transferrin enzymatic immunoassay was designed for quantitative monitoring of blood contamination [
Four markers of oxidative stress and antioxidant status were analyzed in our study. Advanced oxidation protein products (AOPP), a novel oxidative stress biomarker was discovered in the plasma of uremic patients in 1996 [
To determine which blood component is responsible for changes in measured salivary markers, the impact of plasma, red blood cells, and hemoglobin was studied. Based on our results increased AOPP concentrations in blood-contaminated saliva can be explained by the presence of red blood cells and hemoglobin in saliva. Hemoglobin in saliva probably interferes with the colorimetric AOPP assay and artificially increases the AOPP concentrations. AOPP concentrations in plasma of healthy probands were reported as 3 times as high as AOPP concentrations in saliva [
The effect of blood contamination in saliva on concentrations of salivary markers of oxidative stress was not studied in real clinical situation in the past. Concerning that blood contamination in saliva is common in patients with gingivitis the impact of microinjury was modeled in this study group and age-matched healthy controls. The results were similar to saliva artificially contaminated with blood. AOPP concentrations were increased after microinjury in the visual presence of blood in saliva. AOPP concentrations were increased in both control and gingivitis group after dental hygiene treatment. AOPP concentrations were not different between control and gingivitis patients before dental hygiene treatment. Also other measured salivary markers followed the trend observed in saliva artificially contaminated with blood. Salivary AGEs concentrations and also antioxidant markers TAC and FRAP were decreased after dental hygiene treatment due to presence of blood in saliva. Our results have confirmed the concern that dental hygiene treatment could bias the concentrations of oxidative stress markers in saliva. Based on our results we recommend the saliva collection before dental hygiene treatment or clinical examination of oral cavity.
Salivary oxidative stress concentrations are significantly influenced by 1% blood contamination in saliva. Saliva samples with 1% blood contamination are visibly colored and it is possible to easily exclude such contaminated samples from further salivary oxidative stress analyses. Microinjury to the periodontium caused blood leakage into saliva in both gingivitis and control group and biased concentrations of oxidative stress markers in saliva. For the purpose of salivary oxidative stress analyses saliva samples should be collected before dental hygiene treatment or clinical examination of the oral cavity.
The paper is original work and is not under consideration by another journal. There is no conflict of interests to declare. This publication is the result of the implementation of the project of University Science Park of Comenius University in Bratislava (ITMS 26240220086) supported by the Research and Development Operational Programme funded by the European Regional Development Fund.