Analysis of Salivary Flow , pH , Buffer Capacity , and Creatinine in Individuals Undergoing Hemodialysis

Objective: To quantify salivary creatinine levels patients with end-stage renal disease (ESRD) before, during, and after hemodialysis sessions. Material and Methods: Sixtyeight individuals, aged between 4 and 25 years, were selected, and among these, 34 were diagnosed with ESRD and were undergoing hemodialysis (Group 1) and 34 were clinically healthy patients (Group 2). Saliva samples were collected before, during, and after hemodialysis sessions for Group 1 and compared with those of Group 2. Stimulated saliva flow rate (SSFR), buffer capacity (BC), pH, and salivary creatinine levels were determined. Shapiro-Wilk test, followed by the Friedman, Mann-Whitney and ANOVA tests were used to analyze the variables. Results: Mean SSFR values of Group 1 at the three stages of hemodialysis sessions did not differ from those of Group 2. Furthermore, BC and pH values were within their normal limits, and no difference was detected between the two groups. Mean salivary creatinine levels at baseline and during hemodialysis were significantly higher in Group 1 that in Group 2, but these values were not different between Groups at the end of hemodialysis. Conclusion: Salivary creatinine levels reduce significantly after hemodialysis sessions suggesting that saliva may be used to monitor the efficiency of hemodialysis or even indicate the moment at which it should start.


Introduction
Saliva provides several innate and acquired defense factors that can inhibit bacterial invasion, growth, and metabolism by different mechanisms [1,2] such as bacterial adherence and streptococci acid production [3].Several studies have so far investigated many biological determinants that may influence biofilm cariogenicity [4,5], such as saliva flow and composition [6].A constant salivary flow efficiently eliminates microorganisms from the oral cavity; conversely, a reduced flow may easily favor microbial growth, followed by teeth deterioration [7].Some salivary proteins such as lysozymes, lactoperoxidase, immunoglobulins, agglutinines, mucins, and lactotransferrin have antibacterial effects [8] and hence help in preventing oral diseases.
Saliva is also an extremely useful biological fluid for prognosis, laboratory, or clinical diagnosis and monitoring systemic diseases [9].Salivary biomarkers are employed for screening purposes in epidemiological studies [10] and are being used to monitor and detect various oral and systemic diseases such as breast cancer [11], lung cancer [12], celiac disease [13], and chronic renal failure [14].Recent studies on end-stage renal disease (ESRD) reported a series of salivary markers associated with ESRD.The list of markers included cortisol, nitrite, uric acid, sodium, chloride, pH, amylase, and lactoferrin [15].
In the case of ESRD, measurement of serum creatinine in individual patients provides an estimate of disease progression and may determine the effects of therapy.It may be used to predict when dialysis becomes necessary and also indicate progress of ESRD [16].Since serum constituents may reach the saliva [17], the goal of this study was to quantify the salivary creatinine in patients with ESRD before, during, and after hemodialysis sessions.Saliva samples from both groups were obtained by spitting [18].For the G1, saliva was collected in the hemodialysis room before connecting the hemodialysis machine, during the hemodialysis, and after switching off the hemodialysis machine.Saliva samples for G2 were collected before the dental clinical care at the Health Unit Nossa Senhora da Conceição, as they sat in the dental chair.Salivary flow was evaluated by means of stimulated saliva collection, or rather, by mechanical masticatory stimulation using a standard (1.5 cm) piece of sterile rubber tourniquet during continuous mastication by the patient for 6 minutes.Saliva produced during the first minute of stimulation was discarded.During the following 5 minutes, individuals expelled saliva into a sterile universal collector vial that had been previously weighed with Marte® analytical scales, model AL 500 (São Paulo SP, Brazil).Stimulated saliva flow rate (SSFR) was evaluated using the gravimetric method and expressed in mL/min [19].Furthermore, pH was measured by a portable pH meter (Digimed Analytical Instrumentation Inc., São Paulo, SP, Brazil).Salivary buffering capacity (BC) was determined with Caritest ® -SL kit (Technew Com. e Ind. Ltda., Rio de Janeiro, RJ, Brazil).Moreover, 1 mL of the collected saliva was added to a flask containing 3 mL of 0.005N HCl solution.Sample readings were performed following the manufacturer's instructions.

All individuals included in
The remaining saliva samples were centrifuged (3,000 g for 10 min).Salivary creatinine was measured using the alkaline picrate colorimetric method, Creatinina K -Labtest Diagnóstica, on a Bioplus spectrophotometer (Hoffmann-La Roche, Basel, Switzerland).Creatinine reacts with picrate in an alkaline medium to form a red complex measured photometrically.The addition of an acidulant lowers pH to 5.0 and causes the decomposition of creatinine picrate.The reading of the samples takes place at 30 and 90 seconds and the difference between the two readings provides the true creatinine rate.All tests were performed three times.
The Shapiro-Wilk Test was employed for the normality analysis.Friedman test, followed by nonparametric multiple comparisons, at 0.05 significance level, was used.The Mann-Whitney and ANOVA tests were employed when statistically significant rates (p ≤ 0.05 and confidence interval [CI] 95%) occurred.

Results
Sixty-eight participants, 34 patients with ESRD (Group 1) and 34 healthy subjects (Group 2), participated in the current analysis.Twenty-three subjects were male (67.6%) and 11 were female (32.4%) in each group.The average age was 13.4 years in both groups.Five of the 34 patients in the study did not participate in the analysis due to the following causes: two of the patients were unable to produce a salivary flow with stimulation, two displayed behavioral resistance, and one died before the collection date.
Mean SSFR rates of ESRD patients at the three stages of hemodialysis sessions did not differ significantly from those of healthy patients (Group 2).In addition, BC was normal in the three stages of hemodialysis sessions and did not differ between both groups.Initial salivary pH of the ESRD group was higher at the start of the hemodialysis sessions and decreased during and after the sessions.Nonetheless, it remained higher than the salivary pH of the Group 2, albeit without statistically significant differences.
Mean salivary creatinine levels before and during hemodialysis were significantly higher in ESRD patients compared to healthy subjects (p = 0.0063 and p = 0.0409, respectively), but these values were not different between groups at the end of hemodialysis (p = 0.4569).ANOVA test showed a difference between the three sessions of hemodialysis for creatinine (p = 0.0531).
Descriptive statistics of the variables analyzed according to the groups are given in Tables 1 and 2.

Discussion
The complex oral fluid called saliva has long been acknowledged as a clinically informative biological fluid that is highly useful for novel approaches in the diagnosis of diseases.The many advantages of saliva comprise good cooperation with patients, non-invasive collection, low cost, easy transportation and storage, early detection of disease [20], and correlation with levels of the same metabolites in blood [21].
Biochemical analysis of saliva and blood have two main objectives: to identify individuals with disease and evaluate the prognosis of the same during treatment [22].Salivary fluid composition may be affected by different forms of stimulation, time of day, diet, age, gender, a variety of disease conditions, and several pharmacological agents [17,23].Whole saliva is a mixed fluid that is derived predominantly from major and minor salivary glands.It contains gingival crevicular fluid, mucosal transudations, expectorated bronchial, and nasal secretions, serum and blood derivatives from oral wounds, bacteria and bacterial products, viruses and fungi, desquamated epithelial cells, cellular components, and food debris [8,21] besides urea, ammonia, uric acid, glucose, cholesterol, fatty acids, triglycerides, phosphorus, neutral lipids, glycolipids, amino-acids, and steroid hormones [24].
Brazilian Research in Pediatric Dentistry and Integrated Clinic 2016, 16(1):141-147 Earlier researchers had reported a series of salivary markers associated with ESRD.The list included cortisol, nitrite and uric acid, sodium, chloride, pH, amylase, and lactoferrin [15].Salivary phosphate has also been widely used as a clinical biomarker for hyperphosphatemia and these levels correlated well with serum creatinine and glomerular filtration rate [25].Some authors reported that salivary creatinine concentration in individuals with chronic renal failure was associated with increased serum creatinine levels, which is not observed in healthy subjects [26].Therefore, salivary creatinine may be a marker for kidney diseases.
The current assay showed that initial salivary creatinine of the ESRD patients was significantly higher before hemodialysis.In the course of the session and after that there was a significant decrease in creatinine.At the end of the hemodialysis session, salivary creatinine concentration remained higher but near the values observed in healthy individuals.The hemodialysis session clearly caused a significant decrease in the amount of salivary creatinine in ESRD patients.
Previous studies tested colorimetric test strips to monitor salivary nitrate and uric acid before and after hemodialysis [27].At this point, the development of test strips to monitor the salivary creatinine may be interesting to indicate when hemodialysis session must begin or end, saving the patient time and providing great benefits to health services, particularly in primary health care.
Since the current research is related to SSFR, BC and pH, an inert material was employed to avoid interfering with the patient's salivary flow.This is why a piece of latex hose was used.During the acquisition of saliva samples, the patients from the two groups were seated.ESRD patients were sitting on the same chairs used for the hemodialysis sessions.Results on SSFR, BC, and pH revealed that the variables at the three sessions of hemodialysis presented no significant difference from the results of healthy subjects.These findings were unexpected as they are in direct contrast with the findings from most studies in the literature [28,29], which have reported a significantly reduced mean salivary flow in uremic patients.According to these authors, the quantitative change in salivary flow is directly associated with uremia involving salivary glands and a lower degree of hydration due to the restriction of fluid ingestion.
Finally, salivary research is a dynamic field.General and oral health will largely benefit from the development and improvement of new research techniques in this field.

Conclusion
Mean rates for SSFR, BC, and pH were not different between ESRD patients and healthy subjects.Salivary creatinine decreased significantly after hemodialysis sessions suggesting that saliva may be used to monitor the efficiency of hemodialysis or even indicate the moment at which it should start.

References
the study signed informed consent form, according to norms of the Ethical Committee on Research of the Center for Health and Biological Sciences of the São

Table 1 . Descriptive statistics of the variables in the two groups. ESRD Patients Mean Value Median Standard Deviation Standard Error SSFR (mL/min) Initial
Ssfr: Stimulated Salivary Flow Rate; Cre: Creatinine.

Table 2 . Distribution of average creatinine values for each group according to the time of saliva collection.
CI: Confidence Interval; LL: lower limit; UL: Upper limit; The same small letters in the lines and the same capital letters in the columns show no statistical difference; Mann-Whitney U and ANOVA tests.