Antioxidants in Humans Saliva - Case Study Example

ANTIOXIDANT PROJECT by The of the The of the School The and where it is located The Date Antioxidant Project Saliva is a complex, versatile, and important body fluid secreted by major salivary glands. Saliva protects oral cavity against dangerous agents such as microorganisms, toxins, and various oxidants. Antioxidants are found in all biological species and body fluids including saliva. Currently, periodontal diseases are among the widest spread chronic infectious inflammatory conditions characterized by the destruction of the tooth-supporting structures (Acton, 2012, p. 21). In addition, periodontal diseases are the most prevalent 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. Oxidative free radicals have been found to be enhanced in individuals with periodontal diseases (National Research Council (U.S.), & 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 association between periodontal diseases and total salivary antioxidant status. Objectives of the Study Week1 To estimate the total salivary antioxidant capacity in a healthy individual over a 3 week period of tooth-brushing instruction (TBI) (Week 2) To evaluate the association between periodontal disease and total salivary antioxidant status (Week 3) Comparison of salivary antioxidants in healthy smoking and non-smoking individuals Statistical analysis Data presented as the mean (±SD) in the tables and figures and the salivary antioxidant capacity were analyzed by one-way ANOVA. Materials and Methods Study population Eighty individuals comprising of 40 non-smokers who range between 30-48 years old and a mean age of 38 Ų 5.3 years were enrolled in this study. The other population include 40 smokers who had an age range of 31-50 years old and a mean age 40 Ų 4.6 years with a daily consumption of 20 cigarettes for at least 10 years were also used in the research. All subjects were clinically healthy and had moderate periodontitis clinical attachment loss of 3 to 4 mm. The test group included 25 patients diagnosed with severe chronic periodontitis who had more than two areas with a 5 mm or more probing depth (PD) in the quadrant and bone loss of more than 30% of the root length on the radiographs. In a control group, 25 patients who did not have attachment loss or sites showing a PD of 3 mm or more with a sulcus bleeding index (SBI) less than10% were enrolled. The exclusion criteria were as follows: People with other medical conditions that could affect the results, for instance, people who had taken anti-inflammatory drugs or antibiotics within the previous 3 months Pregnant women People who use mouth-washes regularly People who had taken vitamins in the previous 3 months There were some drop-outs during the study resulting in a final control and test group, which consisted of 30 smoking (22 men, 8 women) and 25 non-smoking individuals (15 men, 10 women). The test group of patients diagnosed with severe chronic periodontitis had 18 patients while the control group had 12 patients (Lima et al., 2010, p. 90). Note: Both the control and test groups had more than 20 functional teeth. Methods Study 1: A study was conducted on 30 patients to assess the association of periodontal diseases and total antioxidant capacity on saliva in a healthy individual over a 3-week period of tooth-brushing instruction (TBI). Week1 was assessed quantitatively using FRAP assay and estimated protein oxidation level in saliva by carbonyl assay of Levine. On the three days, the sampling was performed at 08:00, 14:00 and 21:00 before tooth brushing. An additional sampling was carried in the morning 10 minutes after tooth brushing. It should be taken to consideration that this would create a mean value for the patients. On the last three days, the saliva samples were collected before and 30 minutes after an oral administration of 250 mg of vitamin C. Specifically, powder vitamin C was dissolved in water to final concentration 125 g/L. The patients were asked to drink 2 ml of this vitamin C solution provided in micro tube. . Collected saliva samples were stored at −20◦C until analyses were performed. All saliva samples were collected after rinsing the mouth with water and the volunteers were instructed not to eat or drink 30 minutes before any sampling (Packer, 1999, p. 4). Note: Smokers and people with systemic disorders were excluded from the study 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 No significant differences in salivary levels of oxidative stress markers were observed between men and women. Based on these results, data from both genders was combined for further analyses. Significant increase was detected in salivary FRAP after tooth-brushing in 14 out of 19 participants (on average by 89%). However, the salivary TAC levels were not significantly influenced analogously to the influence of daily variations. Salivary TAC was decreased in 13 out of 19 participants after tooth brushing. 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. In our study, saliva samples collected before and 10 minutes after tooth brushing were monitored. Traditionally, tooth brushing is viewed as a procedure for mechanical removing dental plaque. 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 As explained before, dental caries is the most well documented infectious illnesses globally. Saliva has numerous purposes in the oral fissure and is the headline against dental caries. Oxidative strain can trigger the start and development of various inflammatory and infectious illnesses such as dental caries. Thus, the purpose of this part of the study was to assess the relationship concerning saliva’s total antioxidant capacity (TAC) as well as dental method caries. The population of the study consisted of 50 healthy high school students, 25 female and 25male, with an age range of 14 -18 years who were arbitrarily chosen and contributed in this part of the study (Limeback, 2012, p. 17). Saliva Sampling Unstimulated saliva will be collected between 8.30 and 10.30 am over a 5 minute period. 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). 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 results exhibited that CA set had greater TAC than CF the group. TAC in CA female students was greater than those recorded from CF female scholars but the dissimilarity was not statistically weighty. CA males had statistically noteworthy greater TAC than their CF male counterparts. 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 female male total P value Caries active (n = 25) Caries free (n = 25) 41.85±15.48 42.16±11.88 58.75±11.95 41.32±9.92 49.52±16.69 40.74±10.85 0.000 0.710 Total (n = 50) 40.59±15.73 53.35±14.38 42.90±15.71 0.001 P value 0.85 0.000 0.001 Comparison between sex groups 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). Study3 Cigarette smoke is injurious to both oral cavity and internal body environment. Saliva is the first body fluid to encounter the dangerous smoke. The aim of the study was, therefore, to evaluate the influence of smoking in antioxidant activity of saliva healthy smokers. Antioxidant capacity of saliva was determined by using different methods in the centrifuged saliva from 25 male smokers and 25 non-smokers in similar 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 smokers (Nix & Nix, 2013). Based on the results obtained in the present study, we concluded that smoking could significantly affect the antioxidant behaviour of normal human saliva. Thus, smoking may induce several oral diseases as well as respiratory as well as cardiovascular disorders and could cause different cancers (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. In addition, the smoking volunteers were asked not to smoke for one hour prior to the experiment. We employed a collection protocol using the modified Navazesh method. While in a sitting position, the participants were asked to swallow saliva, then stay motionless and allow the saliva to drain passively for 10 minutes over the lower lip into a sterile plastic vial. 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 smoking group and 133.4 Ų 26.59 U/L in the non-smoking group. The level of GPx activity was significantly (p = 0.000) lower in the smoking group than the non-smoking group. The mean concentrations of salivary UA were 2.8 Ų 2.1 mg/dl and 3.7 Ų 2.6 mg/dl in the smoking and non-smoking groups respectively with no significant (p = 0.094) difference between groups. The mean SOD activity in the smoking and non-smoking groups was 6.24 Ų 2.62 and 8.07 Ų 1.30 U/ml respectively (Roda, 2011, p. 89). The level of SOD was significantly (p = 0.000) lower in the smoking than non-smoking group. The mean level POx was 3.67 Ų 2.35 U/mg proteins in the smoking group and 7.53 Ų 6.24 U/mg protein in the non-smoking group. This was (p = 0.036) lower level in the smoking group. Discussion Cigarette smoke is a mixture of chemicals containing over 4000 constituents. Some of the compounds identified include nicotine, ammonia, acrolein, phenols, acetaldehyde, benzopyrine, nitrogen oxides, carbon monoxide, polonium, radium and thorium. In addition, cigarette smoke contains free radicals that can cause cellular damage. It was demonstrated that one cigarette puff contains 1014 free radicals in the tar phase and 1015 radicals in the gas phase. The tar and gas phases are two major phases in cigarette smoke. Pryor and Stone showed the relationship between DNA damage and tar phase free radicals after smoking. These radicals are mostly quinine-hydroquinone and are not highly reactive. On the other hand, gas phase free radicals are generally more reactive. Tobacco smoke can alter the antioxidative capability of saliva, but the causes of these changes are not exactly known. Similarly, it has been noted that the salivary antioxidant defense system appears to be less efficient with age. The results of this study suggest that cigarette smoke is associated with a significant decrease in salivary antioxidant concentrations. Bibliography Acton, Q. A. (2012). Protective agents: advances in research and application. Amerongen, A, V., & Veerman, E, C. (2002). Saliva--the defender of the oral cavity. Oral Dis. Dasgupta, A., & Klein, K. (2014). Antioxidants in food, vitamins and supplements prevention and treatment of disease. Burlington, Elsevier Science. Dodds, M, W., Johnson, D, A., & Yeh C, K. (2005). Health benefits of saliva:a review. J Dent. Forrest, J, L., Hujoel, P, P., & Lieberman, M, 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, J. T., & Salonen, J. T. (1996). Natural antioxidants and food quality in atherosclerosis and cancer prevention. Cambridge, Woodhead Publishing Ltd. Lawrence, H, P. (2002). Noninvasive diagnosis of disease and monitoring of general health. J Can Dent Assoc. Lima, D, P., Diniz, D, G., Moimaz, S, A., Sumida, D, H., Okamoto, A, C. (2010). Saliva: reflection of the body. Int J Infect Dis. Limeback, H. (2012). Comprehensive Preventive Dentistry. Wiley & Sons, Incorporated, John. Mandel, I, 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. 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. Packer, L. (1999). Oxidants and antioxidants. Pt.B Pt.B. San Diego, Calif, Academic. 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