The main results of the presented study indicate that although salivary total antioxidant status (TAC) in AD patients was unaltered, we observed increased oxidative modification of cellular elements of the salivary glands and structures present in the oral cavity. Saliva is not only the secretion of the salivary glands, but it also contains exfoliated oral mucosal epithelium and gingival fluid reflecting the redox status of periodontal tissues. The finding that unstimulated salivary flow from the submandibular glands is impaired in AD patients has already been published 27. However, it can be concluded that in the AD patients from our experiment the submandibular glands were completely dysfunctional. Despite using several methods of collecting unstimulated saliva, we were unable to collect the material. Even gentle attempts to collect saliva (using a pipette) from the mucous membranes of the lips, palate and cheeks failed. Moreover, we demonstrated high secretory failure of the parotid glands that secrete stimulated saliva. Despite providing hydration of the patient’s oral cavity (each patient drank a glass of water, and nurses reported that patients regularly drink various types of liquids) and collecting the material in autumn and winter (at room temperature of 20–21°C), secretion of stimulated saliva in AD patients was reduced compared to their peers of the control group, as normal stimulated saliva secretion starts with the value of 0.7 mL/min. Stimulated secretion was at the level of 0.12 mL/min, which indicates severe hyposalivation. The cause of reduced salivary secretion in AD patients is unknown. Saliva secretion is initiated by reflex-induced nerve impulses. Controlling this process depends on neurotransmitters released at nerve ends in the salivary glands. Typical neurotransmitters associated with water secretion are acetylcholine (ACh) and numerous neuropeptide neurotransmitters 28. In the brain of AD patients we observed increased activity of acetylcholinesterase, an enzyme which breaks down ACh, followed by decreased ACh levels 29. This deficit in the cholinergic function was connected with loss of memory as well as cognitive and learning ability in AD individuals 29. Sayer et al. 30 reported decreased salivary concentration of ACh in AD patients who did not respond to AChE-I treatment. The positive correlation between SWS and Mini-Mental State Examination may prove that declined cholinergic conductivity could be a cause of hyposalivation. The negative correlation between AOPP levels and SWS may also be considered as some kind of explanation. It was demonstrated that protein oxidation can accelerate the formation of toxic protein aggregates in the nucleus and cytoplasm of the nerves 31. This may inhibit neurotransmitter release or reduce salivary gland innervation and thus decrease secretory response of the salivary glands. Decreased protein levels in the SWS of patients with AD vs healthy controls may evidence reduced activity of the sympathetic nervous system, whose stimulation determines protein synthesis in the salivary glands. Due to the selection of the control group (age of patients), we excluded age-related salivary gland changes (fatty and fibrous degeneration) as a cause of salivary gland dysfunction. With age, the number of medications taken increases, and many of them may affect saliva secretion and composition. Patients in both study groups had similar general diseases (hypertension, diabetes, coronary heart disease, atherosclerosis, osteoporosis) and were taking similar groups of drugs, so connecting hyposalivation with a particular disease or drug-related condition would be an oversimplification. With such significant deficiency of saliva, it is not surprising to find poor hydration of the vermilion zone, buccal mucosa or tongue condition. The positive correlation between buccal mucosal hydration and AOPP concentration in the control group may result from increased oxidative modification of moisturizing proteins, i.e., mucins and other glycoproteins, accompanying the older age, which entails changes in the coating properties of saliva. Assessment of xerostomia (subjective perception of dryness in the oral cavity) was not possible in every case; therefore, we refrained from presentation of the results.
Analysis of the results obtained in plasma and red cell lysate revealed a significant decrease in SOD and GPx activity (∃17%, ∃39%, respectively), increase in CAT activity (#17%), decrease in the concentration of non-enzymatic antioxidants (∃38% UA, ∃25% GSH, ∃39% TAC) and the existence of general OS (#95% TOS, #227% OSI, #26% MDA, #47% AGE, #42% AOPP) and nitrosative stress (#4% peroxynitrite, #94% nitrotyrosine). TAC is the resultant capacity of a given biological material to counteract specific oxidation reactions 32. It is believed that TAC level corresponds to the antioxidant capacity of non-enzymatic antioxidants in the sample. TOS reflects the level of all free radicals and non-free radical molecules. OSI is the so-called oxidative stress index, that is the ratio of antioxidants to oxidants present in the assayed material 33.
Changes in the redox balance in SWS are shifted towards oxidative processes similarly to plasma/blood cells, although not exactly to the same extent. In stimulated saliva of AD patients, we observed significantly decreased activity of all antioxidant enzymes: SOD, CAT and GPx (∃42%, ∃80%, ∃15%, respectively) compared to the control group. Decreased parameters of the these components of the antioxidant barrier may result mainly from highly increased production of ROS (98% #TOS) which consistently utilize antioxidants in their combating. It may also be due to oxidative modifications of protein chains of the mentioned enzymes (12% #AOPP, 20% #AGE), leading to their inactivation, as observed in the brain of AD patients 34. Decreased GPx activity demonstrates antioxidant failure as well as impairment of other functions (not related to redox balance) of the parotid glands. As the only type of proteins studied, GPx is synthesized exclusively in the acinar cells of the salivary glands and is recognized as a marker of proper function of the parotid glands 35,36. Interestingly, reduction of SOD and GPx activity in the SWS of AD patients correlated positively with the time elapsed from AD diagnosis; in addition, decreased SOD activity correlated positively with Aβ levels in the SWS. The latter correlation may be caused by high affinity of Aβ to bind Cu2+, which deprives saliva of this trace element 37. Cu2+ is a cofactor of the dismutation reaction catalyzed by SOD 38.
GSH deficiency (∃11%) accompanying negative correlation between GSH and AOPP may be due to increased oxidation of proteins that form oral cavity structures. Indeed, the main function of GSH is to maintain a reduced state of the thiol groups of proteins. The reduction of GSH may also result from deficiency of substrates involved in the regeneration of GSH. Another cause may be oxidative damage to GSH reductase, an enzyme that catalyzes the conversion of GSSG (oxidized form of glutathione) to GSH, with NADPH as the reducing cofactor 39. A decrease in NADPH generation observed in the brain of AD patients 40 could hinder the re-synthesis of salivary GSH. Reduction in GSH concentration in the saliva may enhance OH. formation, thus increasing the ROS load and be the cause of the increase in the reported concentrations of peroxynitrite (#15%) and nitrotyrosine (#16%) in the SWS of AD patients. GSH is known as the more potent detoxification agent of peroxynitrite 39. It was evidenced that peroxynitrite is involved in the pathogenesis of AD. It was observed that peroxynitrite simultaneously induces hyperphosphorylation, nitration and accumulation of tau protein 41. The thus modified tau protein (p-tau) aggregates to form intracellular neurofibrillary tangles, exerting a toxic effect 41. It is noteworthy that there was greater reduction in plasma GSH level (∃25%) than in the SWS level (∃11%) in AD patients, with simultaneous greater increase in plasma nitrosative stress expressed by nitrotyrosine levels (#94% vs #16%).
Only UA and TAC in stimulated saliva of AD patients did not differ compared to the controls. Despite unchanged TAC levels, salivary antioxidant systems were unable to counterbalance the increased ROS/RNS formation (#TOS) and prevent the development of OS (#91% OSI, #AOPP, #AGE, #180% MDA). The reason for the accumulation of products of cellular elements oxidation/peroxidation could undoubtedly be malfunctioning of the repair systems responsible for the removal of defective macromolecules, which is typical in the course of AD 42. Increased MDA concentration in SWS suggests the existence of advanced stages of free radical processes in the cells of salivary glands as well as oral cavity structures of AD patients compared to the control group 43, as membrane lipid molecules are thought to undergo oxidative modification with generation of reactive aldehydes at higher concentrations of ROS than proteins 44. Interestingly, we demonstrated a positive correlation between serum MDA concentration and MMSE. It can be assumed that the level of MDA reflects the degree of peroxidation of unsaturated fatty acids of the biological membranes and thus changes of membrane fluidity. Membrane fluidity determines the normal functioning of cells, including neurons, so an increase in serum MDA content could be linked to neuronal loss in AD.
A weakness of the presented study was its small panel of parameters examined, which was determined by the very small amount of saliva available for assays.