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Proteins in Oral Ecosystem Maintenance

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1-4-1 Amylases:

Salivary α-amylase is the first enzyme in the gastrointestinal tract for extracting caloric value from food. However, beyond the primary role of α-amylase to begin digestion of sugars, carbohydrates and complex starches,. salivary α-amylase is known be a important marker of stress. It has also been found that salivary α-amylase may be influenced by psychological and behavioral factors and processes (Kivlighan, 2006).

Human salivary amylase hydrolyses ct-I- 4 glycoside bonds in starch, yelding maltotriose, maltose, glucose and dextrins as final products. In spite of and having similar composition and immunological activity and playing the same role as pancreatic amylase (Liang et al., 1999), these enzymes have different molecular weights, catalytic properties and isoelectric points, (Liang et al., 1999). Salivary amylase exists in two families: family A is glycosylated while family B is nonglycosylated. At least six izoenzymes have been recognized (Liang et al., 1999).

Although playing an important role in the initial digestion of starch (Tseng et at., 1999), the importance of salivary amylase in digestion has been shown to be minor compared to pancreatic, as people who lack it jul to show any digestive perturbations. However, salivary amylase has many important intra-oral functions such as participation in ACDP, modulation of intra-oral microflora and affimity for hydroxyapatite, (Scannapieco et al., 1995; Gong et al., 2000). The catalytic activity of salivary amylase also participate in degradation of sticky starch rich foods which are retained in dental surfaces and theft transformation in slow glucose releasing devices which may play quite a role in dental caries pathogenesis (Tseng et al., 1999).

It has been suggested that amylase represent between 40 to 50% of the total protein produced by salivary gland, most of the enzyme being synthesized in the parotid gland (Noble, 2000). Human submandibular saliva and parotid saliva contain about 45 mg and 30 mg of amylase, respectively, per 100 mg of protein However, it has also been suggested that amylase makes up about 1/3 of the total protein content in parotid saliva, and the content would be lower in whole saliva (Pedersen et al., 2002). The concentration of amylase increases with the increase of salivary flow rate, and it is generally considered to be a reliable marker of serous cell function (Almståhl et al., 2001).

Amylase is also present in human acquired pellicle in vivo (Yao et al., 2001). Fasting has been found to decrease whole saliva amylase levels and activity (Mäkinen, 1989). The amylase concentrations has been found to be reduced in radiation-induced hyposalivation (Almståhl et al., 2001).

During chewing, some starch is hydrolyzed into dextrins and glucose by salivary α-amylase but the degree of hydrolysis ranges considerably (1 to 27%) depending on the type of food (Woolnough et al., 2010). variation in human salivary α-amylase activity has been reported, with values ranging between 50 and 400 U.mL-1 60 (Kivela et al., 1997; Mandel et al., 2010). An indirect measure of α-amylase activity, which is particularly relevant to food application (Gonzalez et al., 2002), can be obtained by measuring the decrease in viscosity of starch pastes with the addition of α-amylase (Collado & Corke, 1999). This assay has been used to study the relationship between sensory analysis of starch thickness perception, α-amylase activity, starch paste and mechanical properties (Evans et al., 1986; de Wijk et al., 2004; Mandel et al., 2010).

Furthermore, the effect of decreased starch viscosity (due to α-amylase activity) affects saltiness perception (Ferry et al., 2006) and aroma release (Ferry et al., 2004; Tietz et al., 2008).

Amylomaltase-treated starches were found to be particularly good fat substitutes in yoghurts and a loss of instrumentally-measured firmness thats because α-amylase was reported in those systems (Alting et al., 2009). It is therefore accepted that α-amylase has a significant 70 impact on a number of critical starch attributes during eating (Engelen & Van Der Bilt, 2008), thickness perception being the main one. In literature reviews, there appeared to be a great variation in sensory analysis of thickness perception for the same starch-thickened food system which could be due to the natural variation of α-amylase activity between donors. Recently, α-amylase concentration variations in saliva has been linked to genetic differences (Mandel et al., 2010) and this was suggested as an explanation for the natural variation observed in thickness perception of starch-thickene systems.

Moreover, sAA levels are influenced by numerous factors which may lead to variability among individual, thus again undermining the accuracy of sAA as a biomarker for fatigue. For instance, studies have shown that cigarette smoking decreases basal α-amylase activity in saliva and that people who chronically drink alcohol have decreased levels of amylase (Rohleder and Nater, 2009).

Activity of amylase was decreased in passive smokers compared to healthy group (Rezaei and Sariri 2011). Similar results have been reported by Granger et al who found lower salivary amylase activity for mothers, not for infants as a result of tobacco smoking exposure (Granger et al., 2007). The results showed also a decrease in salivary amylase smokers as compared to non-smokers were recorded by (Sariri et al., 2008). It was explained that inhibition of salivary amylase by cigarette smoke may be caused by the interaction between SH groups of the enzyme moleculesand smoke aldehydes. Moreover, the percentage of the enzymatic inhibition showed a negative correlation with the basal level of salivary reduced gluthation (GSH). Regular exposure of passive smokers to cigarette smoke may accumulate in their saliva a smoke aldehydes leading to their interaction with –SH group of amylase.

Another study by Greabu et al. Concluded that exposure to cigarette smoke caused a significant decrease in salivary uric acid and amylase. (Greabu et al., 2007).

1-4-2 Proteins:

Human whole saliva has a protein content of about 0.5 to 3 mg/mL, and parotid saliva has a protein content of about 0.4 to 4 mg/mL, while sublingual and submandiblar saliva of about 0.6 to 1.5 mg/mL. The protein concentration is independent from the flow rateand is rather stable, Besides maintaining buffer capacity and osmolarity, salivary proteins are also involved in several specific functions. The number of distinct salivary proteins is roughly between 100 and 140 (Wilmarth et al., 2004; Yao et al., 2003), from which 30.40 % are produced by the salivary glands, whereas other proteins are originated from serum, from mucosal and/or immune cells, or from microorganisms (Wilmarth et al., 2004). The most important proteins of glandular origin are alpha-amylase, glycoproteins with blood-group substances, cystatins, epidermal growth factor (EGF), gustin, histatins (HRPs), lactoferrine, lysozyme, mucins, salivary peroxi dase, proline-rich proteins (PRPs) and statherin. The most important serum derived proteins are albumin, alpha1-antitrypsin, blood-clotting factors (VIII; IXa; XI) and members of the fibri- nolytic system (proactivators, traces of plasminogen activator). Most important proteins that originate from immune cells are myeloperoxidase, calprotectin (Ca2+ binding L1 leukocyte pro- tein), cathepsin G, defensins, elastase, immunoglobulins (90% to 98% sIgA, 1% to 10% IgG, a few IgM, IgD, IgE). Finally, the most important protein constituents of microbial (unknown) or mixed origin are fibronectin, alpha2-macroglobulin, , DNases, RNases, kallikrein, streptococcal inhibitor, secretory leukocyte protease inhibitor (SLPI), , molecular chaperone (Hsp70), and cystein peptidases. (Data are summarized in Table 1-2).

The most important proteins involved in oral ecosystem maintenance are, lysozyme, agglutinins and histidine , lactoferrin, peroxidases, proline-rich proteins, as well as secretory immunoglobulin A and immunoglobulins G and M (Liébana et al., 2002), Moreover, saliva contains a many types of proteins and some of them might have protective properties. Additionaly, proteins can protect the tooth structure by the formation of a salivary pellicle when tooth are exposed to saliva (Siqueira et al., 2007). This pellicle may act as a barrier for acids (Dawes, 2008). In hyposalivation, caries process and erosive wear are phenomena that occur simultaneously (Lajer et al., 2009).

With respect to the development of caries it was proposed that the salivary pellicle derived from whole saliva has a preventive role (Featherstone et al., 1993). Concentration of salivary total protein did not show considerable variation in passive smokers compared to control (Rezaei and Sariri2011). A similar result was obtained for salivary protein concentration in school children with smoker parents (Granger, 2007).

1-4-3 Calcium:

Saliva contains many defense factors and it plays an important role in oral metabolism. Due to the affinity of salivary calcium to be taken up readily by dental plaque, it considered as an important factor not only with respect to the onset of periodontitis but also significantly with regard to dental health. The availability of calcium ion, which can be delivered from the saliva, enamel, and dental plaque fluid, is a critical factor for calcium fluride formation (Rosin-Grget et al., 2007). Several agents such as calcium phosphate, calcium glycerophosphate , calcium chloride, α-tri calcium phosphate , and calcium lactate have been introduced in forms of chewing mouthwash , gum, dentifrice and at different concentrations (Pessan, 2006).

Excess concentrations of calcium were involved in specific inter α-amylase molecular interactions but there is no indication of the effect on α-amylase activity was given. Calcium concentration in human saliva varies greatly and published values are: 45 ± 22 ppm (Larsen et al., 1999), 45 – 172 ppm (Salvolini et al., 1999) and 68 ± 16 ppm (Sewon et al., 2004) .

Multiple causative factors are affect Dental caries (Yeung et al., 2005; Roberson et al., 2008). The significant point of dental caries is the demineralization of tooth structure which is initiated by acidogenic plaque microorganisms and low salivary flow leading to decreased supply of calcium ions to repair the altered dental tissues, slow clearance and poor buffering (Dawes, 2003; Andersson et al., 2007). An negative relationship between caries status and rate of secretion of saliva has been reported (Hick et al., 2003; Bots et al., 2004). Thus, Epidemiological studies have supported an evidence that adequate calcium level in saliva might decrease dental carries by reverse the process of demineralization (Pearce et al., 2002; Chalmers, 2008).

Saliva can minimize incidence of dental caries in many approaches, primarily, it act as cleansing agent to reduce the accumulation of dental plaque, secondly, by minimizing the solubility of enamel by continuous supply of minerals, especially calcium, and finally, by antibacterial and buffering activity (Yazeed et al., 2009).

The constant increased level of salivary calcium ions in low concentration is important in decreasing the dental caries formation (Hicks and Flaitz, 2000; Picu, 2010). The salivary pH is considered asthe mechanism that regulates the deposition of salivary calcium. Significant decrease in local pH changes the chemical balance of the tooth surface and increases the solubility of hydroxyapatite (Dawes, 2004). Calcium in saliva acts as major mineral to prevent dissolution of teeth through its continuous supply to affected areas of teeth and its solubility constant (Ten Cate, 2008; Jawed et al., 2011).

All inorganic minerals which present in serum are continuously in exchange phase with saliva around dental plaque and acting as reservoir of calcium in order to keep adequate saturation level (Chalmers, 2008; Chu et al., 2008). The demineralization process has been checked by the suitable concentration of calcium, phosphate, and other inorganic ions around the affected tooth surface (Pearce eta l., 2002; Ten Cate, 2008).

Optimum concentration of calcium in saliva promotes remineralization and prevents dental caries, by giving perfectness and strength to the structure of teeth (Jawed et al., 2012).

Calcium is found in greater quantities in unstimulated than in stimulated saliva as its main source is the saliva secreted by the submaxillary and sublingual glands, whereas when stimulation occurs, it is the parotid gland which produces the greatest volume of secretion. The phosphate concentration in saliva from the submaxillary glands is approximately 1/3 of that in parotid saliva but is six times higher than that of the saliva produced by the minor salivary glands (Nauntofte et al., 2003).

The high concentrations of calcium in saliva ensure ionic exchanges to be directed towards the tooth surfaces that start with tooth eruption leading to post-eruptive maturation.It is possible for remineralization of a carious tooth before cavitation occurs to be happened, mainly because of the availability of calcium ions in saliva. (Stack and Stack, 2001).

The concentration calcium ions in saliva varies with the salivary flow rate and is not affected by diet. However, some medications such as pilocarpine and diseases such as cystic fibrosis cause an increase in calcium levels. Depending on the pH, salivary calcium can be linked or ionized. Ionized calcium has an essential role in establishing the equilibrium between the calcium phosphates of tooth enamel and its adjacent liquid. Inorganic ions can be linked to non-ionized calcium (fluoride , inorganic phosphate, bicarbonate), to small organic ions (citrate), and to macromolecules (proline-rich proteins, statherin and histidine-rich peptides).

An example of the combination of calcium is its potent link with α-amilase, where it acts as a co-factor essential for the enzyme function. (Axelsson, 2000).

Epidemiological studies have confirmed the evidence that adequate calcium level in saliva might inhibit dental carries by enhancing the process of remineralization (Pearce et al., 2002; Chalmers, 2008).

The process of demineralization is the main mechanism leading to dental caries. Saliva has many defensive roles against demineralization like optimum level of salivary pH, flow rate, and optimum concentration of calcium. The problem of dental caries can be aggravated by calcium deficiency especially in diabetes mellitus patients type 2.

Suitable concentration of calcium in saliva inhibit dental caries and promotes remineralization, by provididng perfectness and strength to the structure of teeth, icreased severity of dental caries is associated with decreased salivary flow rate level (Jawed, 2012).

Saliva plays a major role in maintaining the integrity of enamel. While many inorganic ions are present in saliva (e.g., calcium, , fluoride, sodium magnesium, phosphates, chloride, and potassium), many salivary components enhance supersaturation of calcium ions and phosphate ions in saliva. The elevated concentrations of phosphate and calcium ions in saliva enhance both remineralization of carious teeth and post-eruptive maturation of enamel before carious leision occurs. (Kaufman and Lamster, 2002; Almeida et al., 2008).

Ghulam et al (2005) concluded that higher levels of calcium are present in the saliva of long-term tobacco users than non-users and the calcium concentration decreases as the flow of saliva increases.

Electrolyte variations are one set of changes occurring in response to smoking. Change in electrolytes occurs at systemic as well as molecular and cellular level. Electrolyte variations in smoking have been studied by several investigators (Laine et al., 2002; Erdemir and Erdemir, 2006; AvÅŸar et al., 2009; Padmavathi et al., 2010).

Erdemir and Erdemir (2006) showed non-significant difference for salivary calcium concentrations in smokers and nonsmokers. Similar results for salivary electrolytes in smokers were obtained in an earlier (Laine et al., 2002) and later report (AvÅŸar et al., 2009).

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