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Rizayev Jasur Alimjanovich, Ph D., professor of faculty therapeutic dentistry department Tashkent State dental Institute E-mail: Dr. Jasur@gmail.com Asadullaev Nurulloxon Sagdullayevich, assistant teacher of department hospital prosthetic dentistry
Tashkent State dental institute
OXIDIZING STRESS FACTORS AND THE ANTIOXIDANT PROTECTION SYSTEM OF ORAL FLUID IN ELDERLY AND SENILE PEOPLE
Abstract: Studying the processes ofthe LPO-OAS in oral fluid ofpeople ofmiddle age (40 people aged 45-60years); 43 people of advanced age (61-74 years) (28 women and 15 men); 35senile ones aged 74-89 years (19 women and 16 men) and 15 long-livers (10 women and 5 men over 90 years) and the index age-group (35-44 years) - 42 people (24 women and 14 men) showed that, with an increase in age, accumulation of the LPO products was accompanied by a decrease in activity of the AOS enzymes. It makes relevant application of the measures capable to influence oxidizing homeostasis of oral fluid and eliminate changes in pro-oxidant-antioxidant system.
Keywords:
Dental pathology in people of the senior age-groups is various; patients of this category demonstrate an increase in severity and intensity of inflammatory and destructive lesions of the periodontium, caries and non-caries damages of the teeth as well as pathology of the salivary glands and oral cavity mucous membrane, orthopedic stomatitis etc. [1-4]. Treatment of these diseases require application of a complex of diagnostic, therapeutic, preventive and rehabilitation interventions, long treatment, and considerable financial expenses as well [5-8].
Currently the peroxide pathogenesis concept of many diseases including dental ones is acknowledged; according to it some imbalance develops between the products of lipid peroxidation (LPO) and the antioxidant protection system components [9; 10; 11]. The LPO is a chain-reaction during which a breakage of the oxidation chain is initiated, continued, branched and followed by formation of free radicals of toxic products: ketones, aldehydes, hydroperoxides and others. The LPO intensity is regulated by the balanced correlation ofpro-oxidizers and antioxidants (oxidative homeostasis). With ageing, a sharp strengthening ofthe LPO activity occurs that is beyond the physiological process frame and gets a destructive impact.
Thus, studying oxygen-dependent metabolism and antioxidant systems (AOS) is relevant as it enabled not only to estimate the inflammatory response intensity and its role in the pathogenesis of oral cavity diseases, but also to predict their course and outcome. All set forth above manifests necessity of studying the dynamics of the LPO-AOS processes in oral fluid of people of senior age-groups. Being the links of one chain these processes are interdependent and associated with each other. The prospect of therapeutic impact on these processes playing a key role in the system inflammation, lesions
of the mucous membrane of the oral cavity and paradontium is doubtless.
Research objective is to reveal clinical and pathogenetic trends of expression of factors of oxidizing stress and activity of the system of oral fluid antioxidant protection in elderly and senile people.
Materials and methods. According to the WHO classification 42 people (24 women and 14 men) composed the index age-group (35-44 years); 40 middle-aged people of (45-60 years) formed group 1; 43 people of advanced age (61-74 years) composed group 2 that included 28 women and 15 men; 35 senile people aged 74-89 years (19 women and 16 men) formed group 3 and 15 long-livers (over 90 years) composed group 4(10 women and 5 men).
The LPO-AOS system indicators were determined in non-stimulated oral fluid (NSOF). The fluid was collected in the graduated plastic test-tube by spitting it within 6 minutes (Redinova T. L., Pozdeev A. R., 1994); the sample was cen-trifuged at 3000 rpm; the lypoperoxidation products were determined in the supernatant [12].
The NSOF oxidant activity parameters were determined by V. S. Kamyshnikova's technique. The method reveals the relative level of the isolated double bonds, and also primary and end-products of the LPO; it is based on absorption of the DC, TC, SB extracted from the oral fluid by heptan-izopro-panol mixture in the UV-spectrum: diene conjugates (DC), triene conjugates (TC) and Schiff's bases (SB); the concentration of malonic dialdehyde (MDA) determined by reaction with thiobarbituric acid (Kamyshnikov V. S, 2004) [12].
The activity of the enzymes of anti-oxidizing system (AOS) was evaluated by superoxide dismutase activity (SOD)
by Nishkimi method modified by P. G. Storozhuk, A. P. Sto-rozhuk (1998) based on SOD ability to compete with nitro-blue tetrazolium (NBT) for superoxide anions formed as a result of aerobic interaction of restored nicotinamide adenine dinucleotide (NAD) (a coenzyme of nucleotide origin) and phenozine methosulfate (PMS); SOD inhibits NBT restoration [12]. Catalase (CT) was determined by Korolyuk method (M. A. Korolyuk et al.) modified by P. G. Storozhuk and S. P. Korochansky (P. G. Storozhuk, S. P. Korochansky, 2001). The method is based on ability of hydrogen peroxide to form the stable colored complex with molybdenum salts. The antioxidant activity was evaluated by registration of rate of oxidation of the restored form of 2,6-dichlorfenolindofenol (2,6-DHFIF) by oxygen dissolved in the reactive serum (Kon-drakhin I. P., 2004) while the concentration of ceruloplasmin (CP) was determined by Ravin's (Ravin,) method based on p-fenilendiamin oxidation by ceruloplasmin [12].
Statistical analysis was performed by the methods ofvari-ation statistics (mean arithmetic values - M, standard error for the average - m, Student's t-test, degrees of difference reliability - p). Statistical processing of the findings was carried out
using the standard software package of the applied statistical analysis (Statistica for Windows v. 7.0).
Results and their discussion. The results of research on oral fluid biochemical indicators in the people under study in the dynamics of their values increasing with age and in the index age-group are presented in the Table and Figure. They demonstrate that an increase in the level of inflammation markers is observed in 75-100% of the people of the senior age- groups.
The MDA role as a marker of oxidizing stress is universally recognized; a change in MDA concentration is a typical process of a number of physiological conditions including ageing, adaptation, and stress. The MDA concentration, being an end-product of LPO degradation, was found to be 0.53 ± ± 0.02 nmol/ml in the index age-group (35-44 years). In elder age it was associated with the MDA concentration growth in oral fluid compared to the index age-group (35-44 years): by 22.64% (p < 0.5) in people of middle age (45-60 years); by 45.28% (p <0.05) in elderly ones (61-74 years); senile people (75-89 years) had 56.60% (p < 0.05) growth while in the long-livers (90 years and elder) it increased by 83.02% (p < 0.05) (Table, Figure).
Table 1. - Products of peroxidation recurrence and antioxidant enzymes in oral fluid of people of elderly and senile age (M±m)
Lipids, enzymes Age, years
35-44 45-60 61-74 75-89 90 & more
DC, ^mol/L 0.88 ± 0.03 0.97 ± 0.04" 0.02 ± 0.05"° 12.2 ± 0.05"°x 1.43 ± 0.06"°x^
TC ^mol/L 0.37 ± 0.01 0.44 ± 0.02" 0.50 ± 0.02"° 0.55 ± 0.01"°x 0.60 ± 0.02"°XA
SB ^mol/L 20.71 ± 0.93 29.62 ± 1.03" 27.31 ± 1.27"° 29.42 ± 1.21"°x 31.44 ± 1.42"° XA
MDA, ^mol/L 0.53 ± 0.02 0.65 ± 0.03" 0.77 ± 0.03"° 0.83 ± 0.04"°x 0.97 ± 0.04"°xA
Catalase^cat/L 3.25 ± 0.13 3.00 ± 0.01" 0.65 ± 0.02"° 2.45 ± 0.05"°x 2.03 ± 0.08"°XA
SOD,c.u. 93.25 ± 3.65 85.31 ± 3.75" 80.27 ± 3.77"° 70.62 ± 3.21"°x 63.08 ± 2.65"° XA
AOA,% 2.06 ± 0.09 1.90 ± 0.03" 1.81 ± 0.04"° 1.42 ± 0.04"°x 1.22 ± 0.04"°xA
Note: • - p<0.05 compared to the index group (35-44 years); ° - p <0.05 compared to the group of45-69-year old; x- p <0.05 compared to the 60-74-year old people; A - p <0.05 compared to the group of 78-89-year old people
The levels of DC and TC, the LPO in NSOF primary products, were the following in the index age-group: DC -from 0.88 ± 0.03 ^mol/L; TC - from 0.37 ± 0.01^mol/L. The concentration of SB, an end-product of peroxidation of free and membrane-linked lipids in the mixed saliva, makes from 20.71 ± 0.93 to 31.44 ± 1.42 ^mol/l. An increase in the primary and end-products of the LPO in age- groups under study was found, for instance, the DC level in people of middle age (45-60 years) increased in comparison to the index age-group (35-44 years) by 10.02%(p > 0.05); in elderly people (61-74 years) by 15.91% (p < 0.05); in senile ones (75-89 years) by
38.64%(p < 0.05) and in long-livers (90 years and more) by 61.36%(p < 0.05). The corresponding TC dynamics made 18.92% (p < 0.05); 35.14%(p < 0.05); 48.65% (p < 0.05) and 62.62 (p < 0.05), respectively, while the peroxidation end-products of Schiff's bases were 18.88%(p < 0.05); 31.87% (p < 0.05); 42.06% (p < 0.05) and 51.818%(p < 0.05), respectively (Table, Figure).
Indicators of diene conjugate (DC) and triene conjugates (TC), being primary products of lipoperoxidation, and Schiff's bases (SB), i.e. the LPO end-products, indicate the intensity of peroxidation of free and membrane-linked
lipids. One may assume that an increase in DC, TC and SB and MDA values in NSOF is caused by high intensity of some dental pathology, mucous membrane of the oral cavity age degeneration that inevitably leads to higher intensity of pro-oxidant processes. It may be caused by the system
biochemical processes accompanied by metabolism disorder in these patients, and constant medication rendering antioxidant effect as well. The concentration of LPO products in mixed saliva enables to assess reliably and objectively the degree of the AOS activity.
Figure 1. Dynamics of antioxidant system activity and lipoperoxidation in people of elderly and senile age,% (compared to the index age-group of 35-44-year old people)
The antioxidant status of oral fluid was demonstrated by the activity of the first enzyme which is on guard to protect the body from superoxide radicals, superoxide dismutase (SOD), catalyzing the dismutation reaction that results in transformation of a highly reactive anion of oxygen radical (anion superoxide, O-2) into relatively less active hydrogen peroxide and molecular oxygen (O-2 + O-2 + 2H + = H2O2 + O2) as well as in the activity of catalase inactivating the product of SOD reaction, i.e. hydrogen peroxide.
As the presented Table and Figure show, LPO products accumulation was accompanied by a decrease in activity of the AOS enzymes. Reduction of SOD activity in people of middle age compared to the index age-group made 8.51%(p > 0.05); in elder people, a decrease in SOD activity was statistically significant (p < 0.05) and made 13.92%; 24.27% and 32.35%, respectively. The similar dynamics of the second line enzyme of the antioxidant protection of catalase made 7.69%(p > 0.05); 18.45%(p < 0.05); 24.62%(p < 0.05) and 37.54%(p < 0.05), respectively. The corresponding dynamics of the general antioxidant activity of oral fluid was as follows: 7.73%(p >0.05); 12.14%(p > 0.05); 31.07% (p < 0.05) and 40.78%(p < 0.05) (Table, Figure).
It is well-known that the LPO products are found in all tissues and fluids of human body, and an increase in their concentration occurs in various pathological conditions ac-
companied by more intensive peroxidation. The antioxidant system (AOS), developed in the course of evolution, prevents free radical degradation of the phases of membranes and lipoproteins. It maintains free radical oxidation (FRO) at the level at which the processes damaging cells do not develop, keeping homeostasis in dynamic equilibrium. A failure of mechanisms of peroxidation regulation contributes to excessive accumulation of free radicals. It impairs permeability, structure and function of biological membranes and damages lipids, tissues, nucleic acids. In addition it changes a bio-energy potential of cells when regulatory and protective factors of local immunity decrease; it also leads to deterioration of an oral cavity pathology [1; 10; 13].
Age changes in the mucous membrane of the oral cavity impair microcirculation in the same way as in arteriosclerosis; hemodynamic disorders contribute to growth of the tissue hypoxia signs manifested by reduction of antioxidant potential [5; 13]. Weaker antioxidant protection, accumulation of the LPO products in the oral cavity can be also caused by adentia and insufficient supply ofantioxidants associated with it [5; 14]. The general biochemical status plays an important role determining the course of oral diseases and the condition of the major homeostatic mechanisms of the oral cavity, and, hence, the choice of the optimum approach to therapy under all equal conditions [5; 9; 13; 15].
Excessive formation of FRO initiators can exhaust a pool of non-enzymatic antioxidants which, having executed the role of traps of free radicals, turn to inactive products. Deterioration of microcirculation in the parodontium tissue observed in adentia [6], reduces the inflow of antioxidants that strengthens the imbalance in the AOS functioning.
Thus, in oral fluid of people of elderly and senile age, activation of FRO biomolecules, displacement of pro-oxidant-antioxidant balance towards peroxidation, exhaustion of non-enzymatic (thiols) and enzymatic (SOD) links of the AOS
and a compensatory increase in catalase activity are observed. However, currently the problems of biochemical diagnostics and prediction of the course of oral pathology in patients of elderly and senile age as well as determination of the optimum methods of diagnostics, prevention and treatment of patients of these age-groups are still in the shadow. In this view, application of methods capable to influence the condition of oxidizing homeostasis and eliminate changes in the pro-ox-idant-antioxidant system induced by pathological processes is of special interest.
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