Saliva as a Complex and Important Body Fluid - Case Study Example

ANTIOXIDANT PROJECT By Saliva is a complex, versatile, and important body fluid secreted bymajor salivary glands. Saliva protects oral cavity against dangerous agents such as microorganisms, toxins, and various oxidants. In order to understand what antioxidants are, it is imperative to know the meaning. Oxidants are molecules that initiate a chemical reaction that entails the transmission of electrons from one matter to an oxidizing proxy. Antioxidants therefore prevent the oxidation process from occurring .They achieve this fete by being oxidized in place of other molecules. Antioxidants are traceable in all biological species and body fluids including saliva. Periodontal diseases constitute the largest spread chronic infections and are usually characterized by the damage of the tooth-supporting edifices (Acton, 2012, p. 21). Moreover, periodontal infections are considered the most widespread microbial diseases affecting the human population. Periodontal diseases are initiated by the complex micro biota found as dental plaque, which is a complex microbial biofilm (Touger-Decker, Sirois & Mobley, 2004, p. 66). Tissue destruction is largely mediated by an abnormal host response to specific bacteria and their products. Tobacco use is also known to be a cause of periodontal disease. Pathogens have the capacity to activate host defense mechanisms while at the same time prompt the inactivation of repair systems (National Research Council, 2014, p. 90). Free radicals are atomic or molecular intermediates with one or more unpaired electrons. Recently, it has been demonstrated that the imbalances in free radical levels and reactive oxygen species with antioxidants may play a key role in the onset and development of several inflammatory oral pathologies (Limeback, 2012, p. 17). Oxidative free radicals have been found to be enhanced in individuals with periodontal diseases (National Research Council & Toverud, 1952, p. 53). Human saliva has an antioxidant system, which plays a pivotal role in protecting such inflammatory conditions as it is capable of inhibiting the oxidative reactions of free radicals. In normal individuals, there is a dynamic equilibrium between free radicals and antioxidants. The purpose of this study is to evaluate the FRAP assay of unknown antioxidants. The study will also seek to evaluate the antioxidant strength of certain foods drinks. . Objectives of the Study Week1 To evaluate the FRAP assay of unknown antioxidants (Week 2) To evaluate the antioxidant strength of certain foods drinks (Pineapple and apple juice). . (Week 3) To establish the appropriate levels antioxidants in the human saliva Statistical analysis Data presented will be analysed using a simple correlation analysis given the small sample under study. Two sets of variables will be outlined and a correlation between them will be established. Materials and Methods Study population Total Antioxidant Capacity (TAC) looks at the aggregate action of every antioxidants existent in the plasma, hence presenting a cohesive parameter as opposed to the simple summation of quantifiable antioxidants. The volume of recognized and unknown antioxidants should be evaluated in order to derive meaningful insights into the equilibrium existing in oxidants and antioxidants. FRAP is an abbreviation for Ferric reducing ability of Plasma. It is an antioxidant capacity assay that employs Trolox as a threshold (Benzie, 1996, p. 70). Studies indicate that there numerous unidentified and unknown plants and fruits that contain phytochemicals. Phytochemicals are the bioactive non-nutrient plant compounds in fruit, vegetables, grains, and other plant foods (Liu, 2003, p. 517). These compounds contained in the above plants and fruits have the ability to maintain a balance between oxidants and antioxidants. Certain unknown antioxidants apart from the mainstream ones like vitamins C and E have been found to be beneficial in the treatment of diseases and subsequent protection of the body from attacks. An example of an unidentified oxidant is the alpha-lipoic acid (Lima et al, 2010, p. 90). Methods Study 1: An analysis on a number of plants and fruits was carried out in order to establish the FRAP assay on them. The levels of the antioxidants were compared to a previous study that was conducted using FRAP assay and estimated protein oxidation level in saliva carbonyl assay of Levine. It is imperative to note that the saliva extracts were used as the control experiment in order to ascertain the FRAP assay of the unknown oxidants. In the control experiment, the saliva extracts were collected after rinsing the mouth with water (Packer, 1999, p. 4). FRAP Determination Salivary FRAP levels were tested according to Benzie and Strain. 150 μL of pre-warmed 37◦C FRAP reagent was blended with 20 μL of saliva samples. Absorbance was recorded at 593 nm. Note: Ferrous sulphate was employed as a standard measure and the consistency of FRAP was displayed in μmol/L. TAC Determination Salivary TAC was determined according to Erel. Saliva was mixed with 0.4 mol/L acetate buffer (pH 5.8), incubated with ABTS (2, 2’-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) and oxidized with hydrogen peroxide in 0.03 mol/L acetate buffer (pH 3.6). Absorbance was taken at 660 nm. TAC was expressed in μmol/L on the basis of the calibration curve of Trolox. Results and Discussion Two sets of experiments were conducted and compared subsequently. One set was that with unknown antioxidants while the other set was from the antioxidants secreted in the saliva. The two different samples, that is the unknown antioxidant samples and the saliva extracts were compared. There were traces of antioxidants in both the samples. An assumption made in the control experiment is that the antioxidants found therein (in the saliva) were secreted naturally in the body. The levels of the antioxidants were varied and fairly different between the two sets of experiments. The unknown antioxidant samples had fairly lower levels of FRAP as opposed to the saliva extracts. Several studies have addressed the role of oxidative stress in the pathology of oral and dental diseases especially periodontitis (Rajendran, 2010, p. 41). The hypothesis that good oral hygiene may decrease oxidative stress in saliva was proposed. Therefore, the variances in the FRAP assays between the two samples might have been caused due to good dental hygiene practised by the participants who forwarded the saliva extracts. The removal of bacteria potentially producing free radicals or reduced response of host inflammatory system could lead to decreased oxidative stress in saliva (Mandel, 1993, p. 89). On the other hand, more recent studies indicate that mechanical stimulation promotes cell proliferation of basal cells and fibroblasts as well as reduces inflammatory processes rather than removal of periodontal pathogens (Nagel, 2009, p. 78). Study 2 This study was carried out to ascertain the antioxidant strength of certain foods and drinks particularly (Pineapple and apple juice). A variety of foods and drinks contain varied levels of antioxidants. The study was conducted on pineapple extracts and apple extracts compared to other foods and drinks and conclusions made. It is imperative to note that the statistics were obtained from previously conducted research. This segment is to compare the strengths of these fruits and drinks and to make relevant deductions. FRAP assay of Benzie and Strain was employed with slight alterations that permitted quantization of both water- and fat-soluble antioxidants (Steinar et al, 2002, p. 1286). A Technicon RA 1000 structure was employed for the measurements of absorption changes that appear when the TPTZ-Fe3 complex reduces to the TPTZ-Fe2form in the presence of antioxidants (Steinar et al, 2002, 1286). As stated earlier different fruits have different antioxidant levels. This segment will highlight the Total antioxidant capacities (TAC) of certain fruits. The table below shows the different TAC of a select fruits. Table 1.0 Fruit Value (mmol Fe2_/kg FW3) (FRAP) Rank Apple 3.84 24 Pineapple 15.73 10 Blackberry 51.53 1 Watermelon 1.13 30 It is evident from the table 1.0 above that the higher the value of FRAP the higher up the rank falls in terms of the antioxidants it contains. At the top of the rank is the black berry with a high level of mmol Fe2_/kg FW3 hence high levels of antioxidants. At the lowest rank is the apple fruit with low levels mmol Fe2_/kg FW3 hence low levels of antioxidants. A graphical representation of the above data is shown below. It is evident from the findings that different fruits and consequently drinks contain different levels of antioxidants as shown by the comparative study conducted by Steinar et al (2002, 1286). The fruits above are just a select few with others demonstrating various levels of antioxidants. It is important to note that no single method can be used to measure the TAC of foods (Pellegrini et al, 1999, 383). This study looked at FRAP approach. There is a strong positive correlation between TAC and FRAP. Fruits that have low FRAP assays consequently have low TAC while those that have high FRAP assays have high TAC. Graph 1.0 Saliva Sampling Un-stimulated saliva will be collected between 8.30 and 10.30 am over a 5 minute period. This would act as the control. The directive to hold saliva in the bottom of the mouth and drained to collection tube is equated to the time during which the additives are able to significantly reduce the rate of an ongoing free radical process. Saliva will be stored at 4°C before salivary antioxidant estimation (Dasgupta & Klein, 2014, p. 20). The saliva samples will be prepared on days zero, week 1 (day seven), week 2 (day 14) and week 3 (day 21). The samples were later kept at −20°C in the freezer, and transported to the laboratory (Dodds, Johnson & Yeh, 2005, p. 67). The saliva samples underwent centrifugation at 3000 rpm for 15 min and supernatant transferred to the test tubes after deposition of saliva impurities. The total antioxidant activity was calculated using the FRAP (Ferric reducing antioxidant power) technique (Fredricks, 2008, p. 56). . Results The samples from the saliva extracts were analysed to ascertain the optimum levels of antioxidants in the human saliva. From the foregoing, it has been established that saliva has antioxidant traces that are essential in combating reactive oxygen species. There should be a balance that should prevent oxidation from taking place. The optimum PH level of saliva should fall between 6.5 and 7.0. It is important to note that a number of dynamics affect the antioxidant levels of the saliva in human beings ranging from age, foods and beverage intake and sometimes even gender. It is therefore difficult to ascertain the optimum level of antioxidants in the saliva. In order to effectively get a perspective on the optimum levels of antioxidants in the saliva, a number of variables were put into place. These were gender and other external influences such as smokers. The experiment had to be done on different control groups in order to draw reasonable comparisons of the antioxidant levels. The different age groups were varied in terms of age groups and were all male. The age groups ranged from, 18 (p1), 19 (p2), 20(P3) and 43(P4). The different groups were given different substances at breakfast. P1 was given tea and a spoon of honey, P2 was given water and sweets, P3 was given pineapple juice and tea while P4 was given Pepsi and noodles. The absorbance readings are shown in the table below. Table 1.1 Student Substance R1 R2 R3 R(R1+ R2 +R3)/3 P1 Tea and honey 0.564 0.591 0.547 0.567 P2 Water and sweets 0.649 0.611 0.631 0.630 P3 Pineapple juice and tea 0.553 0.512 0.497 0.521 P4 Pepsi and noodles 0.970 0.950 0.960 0.960 The results in the table above show a number of findings. Rx refer to the reading, for instance R1 refers to the first reading. R is the mean of the readings, that is, sum of R1, R2 and R3 divided by 3.P4 had the highest absorbance reading 0.960 after taking Pepsi and noodles. The lowest reading was recorded by p3, 0.521 after taking pineapple juice and tea. These results are a manifestation of the antioxidant levels in the substances that the students took. The different substances taken had different levels of antioxidants as exhibited in the table above. A different study conducted on different males with caries also exhibited different results (Kumpulainen & Salonen, 1996, p. 56). The study provides a good basis for comparison between the study conducted on the males on this paper and the one earlier conducted on both males and females. The results exhibited that CA set had greater TAC than CF the group. CA males had statistically noteworthy greater TAC. Statistical examination for male and females clusters presented a statistically noteworthy lesser value of TAC in feminine group. CA females had lesser TAC than their CA males counterparts (p= 0.000) also related result was detected in CF group signifying lesser TAC in CF female linked to CF males. Nonetheless, the dissimilarity was not statistically noteworthy (Kumpulainen & Salonen, 1996, p. 56). The average and standard nonconformities of each group is displayed below. Group male Total P value Caries active (n = 25) Caries free (n = 25) 58.75±11.95 41.32±9.92 49.52±16.69 40.74±10.85 0.000 0.710 Total (n = 50) 53.35±14.38 42.90±15.71 0.001 P value 0.000 0.001 Comparison between CA and CF Discussion The results showed that there is a association between TAC of saliva in addition to dental. Non-enzymic and enzymic salivary antioxidants for instance, glutathione peroxidase, Uric acid, peroxidase, vitamin E as well as vitamin C may be related to dental caries. Other revisions must be done to find the connection between these antioxidants in relation to the outcome of employing antioxidant in nourishment regimen as well as dental caries (Amerongen & Veerman, 2002, p. 35). Study3 This was to establish the appropriate levels of antioxidants in saliva. Antioxidant capacity of saliva was determined by using different methods in the centrifuged saliva from the four students who had ingested the substances provided at breakfast. The students were from different age groups (Lawrence, 2002, p. 43). Chemical methods and high performance liquid chromatography (HPLC) revealed a significant decrease in total antioxidant capacity and values of non-enzymatic antioxidants in the saliva of people with substance additives in their systems (Nix & Nix, 2013, p. 20). Based on the results obtained in the present study, we concluded that substance intake could significantly affect the antioxidant behaviour of normal human saliva. Increased substance intake and abuse could subsequently lead to cardiovascular diseases and other opportunistic infections (Forrest, Hujoel & Lieberman, 2006, p. 78). In this study, un-stimulated whole saliva was collected from each subject between 8-10 a.m. to avoid circadian variations. No oral stimulus was done for 90 minutes prior to salivary collection.. Saliva samples were immediately centrifuged (1000 g, 10 minutes) at 4°C to remove cell debris. The resulting supernatants were immediately deep-frozen at –80°C and stored for later analysis (Pink et al., 2009, p. 71). Results The mean levels of salivary antioxidants showed that the mean GPx activity was 94.54 Ų 37.47 U/L in the group that had taken additives and 133.4 Ų 26.59 U/L in the group that had not taken any additives. The level of GPx activity was significantly (p = 0.000) lower in the group that had ingested additives than the group which had not. The mean concentrations of salivary UA were 2.8 Ų 2.1 mg/dl and 3.7 Ų 2.6 mg/dl in the group that had ingested additives and the group that had not ingested additives respectively with no significant (p = 0.094) difference between groups. The mean SOD activity in the additive group and the non-additive groups was 6.24 Ų 2.62 and 8.07 Ų 1.30 U/ml respectively (Roda, 2011, p. 89). Intake of additives affects the levels of antioxidants in the saliva as shown by the results above. The group that had ingested preselected additives for instance, pineapple, honey, tea had totally different readings compared to the outcomes deduced from the saliva extracts that had no additives in them. . References Acton, A., 2012. Protective agents: advances in research and application. Amerongen, V., & Veerman, E., 2002. Saliva--the defender of the oral cavity. Oral Dis. Benzie, F., & Strain, J.,1996. The Ferric Reducing Ability of Plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay”. Analytical Biochemistry.239(1):70-6 Dasgupta, A., & Klein, K., 2014. Antioxidants in food, vitamins and supplements prevention and treatment of disease. Burlington, Elsevier Science. Dodds, W., Johnson, A., & Yeh, K., 2005. Health benefits of saliva:a review. J Dent. Forrest, L., Hujoel, P., & Lieberman, B., 2006. Host modulation. In: Newman MG, Takei HH, Carranza FA, editors. Carranzas clinical periodontology. 10th ed. St. Louis: Saunders/Elsevier Fredricks, R., 2008. Healing and wholeness: complementary and alternative therapies for mental health. Bloomington, IN, All Things Well Publications/AuthorHouse. Kumpulainen, T., & Salonen, T.,1996. Natural antioxidants and food quality in atherosclerosis and cancer prevention. Cambridge, Woodhead Publishing Ltd. Lawrence, P.,2002. Noninvasive diagnosis of disease and monitoring of general health. J Can Dent Assoc. Lima, P., Diniz, G., Moimaz, A., Sumida,H., & Okamoto, C.,2010. Saliva: reflection of the body. Int J Infect Dis. Limeback, H., 2012. Comprehensive Preventive Dentistry. Wiley & Sons, Incorporated, John. Mandel, D., 1993. A contemporary view of salivary research. Crit Rev Oral Biol Med. p.604. Nagel, R., 2009. Cure tooth decay: heal & prevent cavities with nutrition. Los Gatos, CA, Golden Child Pub. National Research Council.,2014. Nutrition in Pediatric Pulmonary Disease. Totowa, NJ, Humana Press. National Research Council (U.S.)., & Toverud, G.,1952. A survey of the literature of dental caries. Nix, S., & Nix, S. 2013. Williams basic nutrition and diet therapy. St. Louis, Mo, Elsevier.. Packer,L., 1999. Oxidants and antioxidants. Pt.B Pt.B. San Diego, Calif, Academic. Pellegrini,N., Re, R., Yang, M., & Rice-Evans, A., 1999. Screening of dietary carotenoids and carotenoid 299: 379–389. Pink, R., Simek, J., Vondrakova, J., Faber, E., Michl, P., & Pazdera, J.,2009. Saliva as a diagnostic medium. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub Rajendran, R.,2010. Shafers textbook of oral pathology. [S.l.], Reed Elsevier. Roda, A.,2011. Chemiluminescence and bioluminescence: past, present and future. Cambridge, RSC Publishing. Touger-Decker, R., Sirois, D., & Mobley, C. C. (2004). Nutrition and oral medicine. Totowa, N.J., Humana Press. Read More