Salivary Lactoperoxidase Research Articles

The impact of salivary lactoperoxidase and histatin-5 on early childhood caries severity in relation to nutritional status

IntroductionEarly childhood caries is a multifactorial disease. Saliva plays an important role in initiation and protection against caries, and its composition is greatly affected by nutritional status. This study was conducted to determine the impact of salivary lactoperoxidase and histatin-5 on the severity of ECC in relation to nutritional status. Materials and methodsThe sample consisted of 120 children aged 5 years, classified into eight groups: mild ECC in underweight children, mild ECC in normalweight children, moderate ECC in underweight children, moderate in ECC normal weight children, severe ECC in underweight children, severe ECC in normalweight, caries-free (control) underweight children and caries-free normalweight children. Each group consisted of 15 children. Stimulated saliva was collected. Salivary lactoperoxidase was analysed using Human LPO/ Lactoperoxidase ELISA Kit (CLIA)-LS-F29892, and salivary histatin-5 was analysed using Human Histatin-5 ELISA Kit MBS705083_48T. ResultsLactoperoxidase and histatin-5 concentrations were significantly higher in caries-free children than in children with ECC, and they were higher in children with mild ECC than in children with moderate ECC or in children with severe ECC. They were significantly higher among children with normal weight than among those who were underweight (p < 0.01). ECC and nutritional status recorded non-significant interactions with both LPO and HST-5 (p > 0.01), but there was significant interaction between these two variables and LPO and HST-5 together (p < 0.01). The Pearson's correlation coefficient test recorded significant negative correlations between ECC severity and both salivary lactoperoxidase and histatin-5 among the eight study groups, whereas significant positive correlations were recorded between BMI values and both salivary lactoperoxidase and histatin-5 among the eight study groups. ConclusionSalivary lactoperoxidase and histatin-5 may be affected by nutritional status, and these two parameters may play an important role in caries prevention at high concentrations. There is interaction between these two parameters and ECC severity and nutrition.

EFFECT OF HONEY PROPOLIS HARD CANDY ON LACTOPEROXIDASE ACTIVITY IN UNSTIMULATED SALIVA

Objective: We determined the effectiveness of honey propolis hard candy on salivary lactoperoxidase activity in unstimulated saliva.Methods: In this pretest-posttest laboratory experimental design study, saliva collected from 120 subjects was reacted with potassium iodide,phosphate buffer, and hydrogen peroxide. Salivary lactoperoxidase activity was calculated by absorbance value using a microplate reader atwavelength of 340 nm.Results: Honey propolis hard candy increased salivary lactoperoxidase activity, although the increase was not statistically significant (p&gt;0.05).Conclusions: Honey propolis hard candy tends to increase salivary lactoperoxidase activity.

The Significance of Lactoperoxidase System in Oral Health: Application and Efficacy in Oral Hygiene Products.

Lactoperoxidase (LPO) present in saliva are an important element of the nonspecific immune response involved in maintaining oral health. The main role of this enzyme is to oxidize salivary thiocyanate ions (SCN−) in the presence of hydrogen peroxide (H2O2) to products that exhibit antimicrobial activity. LPO derived from bovine milk has found an application in food, cosmetics, and medical industries due to its structural and functional similarity to the human enzyme. Oral hygiene products enriched with the LPO system constitute an alternative to the classic fluoride caries prophylaxis. This review describes the physiological role of human salivary lactoperoxidase and compares the results of clinical trials and in vitro studies of LPO alone and complex dentifrices enriched with bovine LPO. The role of reactivators and inhibitors of LPO is discussed together with the possibility of using nanoparticles to increase the stabilization and activity of this enzyme.

Dynamic Proteome Alteration and Functional Modulation of Human Saliva Induced by Dietary Chemosensory Stimuli.

Saliva flow measurements and SDS-PAGE separation of human whole saliva freshly collected after oral stimulation with citric acid (sour), aspartame (sweet), iso-α-acids (bitter), mono sodium l-glutamate (umami), NaCl (salty), 6-gingerol (pungent), hydroxy-α-sanshool (tingling), and hydroxy-β-sanshool (numbing), followed by tryptic digestion, nano-HPLC-MS/MS, and label-free protein quantitation demonstrated a stimulus- and time-dependent influence of the dietary chemosensates on salivation and the salivary proteome composition. Gene ontology enrichment analysis showed evidence for stimulus-induced alterations of the saliva proteome to boot an efficient molecular defense network of the oral cavity, e.g., 6-gingerol increased salivary lactoperoxidase activity, catalyzing the oxidation of thiocyanate to produce the antimicrobial and antifungal hypothiocyanate, from 0.37 ± 0.02 to 0.91 ± 0.05 mU/mL 45 s after stimulation. In comparison, oral citric acid stimulation induced an increase of myeloperoxidase activity, catalyzing the chloride oxidation to generate antimicrobial hypochloride in saliva, from 0.24 ± 0.04 to 0.70 ± 0.1 mU/mL as well as an increase of salivary levels of lysozyme, exhibiting antimicrobial activity on Gram-positive bacteria, from 6.0-10 to 100-150 μg/mL. Finally, microbial growth experiments clearly demonstrated for the first time that the increase of the salivary lysozyme abundance upon oral citric acid stimulation translates into an enhanced biological function, that is an almost complete growth inhibition of the two lysozyme-sensitive Gram-positive bacteria tested.

Clinical concentrations of peroxidases cause dysbiosis in invitro oral biofilms.

Little is known about the initiation of dysbiosis in oral biofilms, a topic of prime importance for understanding the etiology of, and preventing, periodontitis. The aim of this study was to evaluate the effect of different concentrations of crevicular and salivary peroxidase and catalase on dysbiosis in multispecies biofilms in vitro. The spotting technique was used to identify the effect of different concentrations of myeloperoxidase, lactoperoxidase, erythrocyte catalase, and horseradish peroxidase in salivary and crevicular fluid on the inhibitory effect of commensals on pathobiont growth. Vitality-quantitative real-time PCR was performed to quantify the dysbiotic effect of the peroxidases (adjusted to concentrations found in periodontal health, gingivitis, and periodontitis) on multispecies microbial communities. Agar plate and multispecies ecology experiments showed that production of hydrogen peroxide (H2 O2 ) by commensal bacteria decreases pathobiont growth and colonization. Peroxidases at concentrations found in crevicular fluid and saliva neutralized this inhibitory effect. In multispecies communities, myeloperoxidase, at the crevicular fluid concentrations found in periodontitis, resulted in a 1-3 Log increase in pathobionts when compared with the crevicular fluid concentrations found in periodontal health. The effect of salivary lactoperoxidase and salivary myeloperoxidase concentrations was, in general, similar to the effect of crevicular myeloperoxidase concentrations. Commensal species suppress pathobionts by producing H2 O2 . Catalase and peroxidases, at clinically relevant concentrations, can neutralize this effect and thereby can contribute to dysbiosis by allowing the outgrowth of pathobionts.

Pt-Decorated MWCNTs-Ionic Liquid Composite-Based Hydrogen Peroxide Sensor To Study Microbial Metabolism Using Scanning Electrochemical Microscopy.

Hydrogen peroxide (H2O2) is a highly relevant metabolite in many biological processes, including the oral microbiome. To study this metabolite, we developed a 25 μm diameter, highly sensitive, nonenzymatic H2O2 sensor with a detection limit of 250 nM and a broad linear range of 250 nM to 7 mM. The sensor used the synergistic activity of the catalytically active Pt nanoparticles on a high surface area multiwalled carbon nanotube and conducting ionic liquid matrix to achieve high sensitivity (2.4 ± 0.24 mA cm-2 mM-1) for H2O2 oxidation. The unique composite allowed us to miniaturize the sensor and couple it with a Pt electrode (25 μm diameter each) for use as a dual scanning electrochemical microscopy probe. We could detect 65 ± 10 μM H2O2 produced by Streptococcus gordonii (Sg) in a simulated biofilm at 50 μm above its surface in the presence of 1 mM glucose and artificial saliva solution (pH 7.2 at 37 °C). Because of its high stability and low detection limit, the sensor showed a promising chemical image of H2O2 produced by Sg biofilms. We were also able to detect 30 μM H2O2 at 50 μm above the biofilm in the presence of the H2O2-decomposing salivary lactoperoxidase and thiocyanate, which would not otherwise be possible using an existing H2O2 assay. Thus, this sensor can potentially find applications in the study of other important biological processes in a complex matrix where circumstances demand a low detection limit in a compact space.

Molecular mechanisms of taste recognition: considerations about the role of saliva.

The gustatory system plays a critical role in determining food preferences and food intake, in addition to nutritive, energy and electrolyte balance. Fine tuning of the gustatory system is also crucial in this respect. The exact mechanisms that fine tune taste sensitivity are as of yet poorly defined, but it is clear that various effects of saliva on taste recognition are also involved. Specifically those metabolic polypeptides present in the saliva that were classically considered to be gut and appetite hormones (i.e., leptin, ghrelin, insulin, neuropeptide Y, peptide YY) were considered to play a pivotal role. Besides these, data clearly indicate the major role of several other salivary proteins, such as salivary carbonic anhydrase (gustin), proline-rich proteins, cystatins, alpha-amylases, histatins, salivary albumin and mucins. Other proteins like glucagon-like peptide-1, salivary immunoglobulin-A, zinc-α-2-glycoprotein, salivary lactoperoxidase, salivary prolactin-inducible protein and salivary molecular chaperone HSP70/HSPAs were also expected to play an important role. Furthermore, factors including salivary flow rate, buffer capacity and ionic composition of saliva should also be considered. In this paper, the current state of research related to the above and the overall emerging field of taste-related salivary research alongside basic principles of taste perception is reviewed.

Quantitative Salivary Proteomic Differences in Oral Chronic Graft-versus-Host Disease

PurposeChronic graft-versus-host disease (cGVHD) is a severe immunological complication that occurs after allogeneic hematopoietic stem cell transplantation (HSCT). Although oral cGVHD occurs in >25 % of cGVHD patients and leads to decreased quality of life, its etiology is poorly understood. The present retrospective cross-sectional analysis of oral cGVHD patients sought to (1) test the feasibility of liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify protein biomarkers of oral cGVHD and (2) to gain a clearer understanding of salivary proteins impacted by oral cGVHD.MethodsUsing unstimulated whole saliva, we compared pooled saliva from five patients with a diagnosis of moderate or severe oral cGVHD, with a gender-and age- matched pool of five cGVHD patients with no oral mucosal findings. LC-MS/MS was used to identify salivary proteins, followed by Ingenuity Pathway Analysis (IPA). Selected mass spectrometric findings, including lactotransferrin, lactoperoxidase, and albumin, were confirmed by targeted label-free quantification.ResultsLC-MS/MS led to confident identification of 180 proteins. Of these proteins, 102 changed in abundance at least 2 fold, including 12 proteins identified only in the No oral cGVHD group. Downregulation of ~0.4 fold was confirmed for both lactotransferrin and lactoperoxidase in Oral cGVHD saliva using targeted label-free quantification. IPA analysis implicated pathways involved in cellular metabolism and immunoregulation.ConclusionsReduction of salivary lactoperoxidase, lactotransferrin, and several cysteine proteinase inhibitor family proteins suggests impaired oral antimicrobial host immunity in cGVHD patients. This shotgun proteomic analysis of oral cGVHD saliva using targeted label-free quantification of select proteins supports the use of mass spectrometry for future validation in a large patient population as noninvasive tests for screening, early detection, and monitoring of cGVHD.Electronic supplementary materialThe online version of this article (doi:10.1007/s10875-012-9738-4) contains supplementary material, which is available to authorized users.

Leukocyte Myeloperoxidase and Salivary Lactoperoxidase: Identification and Quantitation in Human Mixed Saliva

Human salivary lactoperoxidase (HS-LP) is synthesized and secreted by the salivary glands, whereas myeloperoxidase (MPO) is found in PMN leukocytes, which migrate into the oral cavity at gingival crevices. HS-LP levels vary with changes in salivary gland function, but increased numbers of MPO-containing leukocytes indicate infection or inflammation of oral tissues. To determine the contribution of each enzyme to the peroxidase activity of mixed-saliva samples, activity was assayed at pH 5.4 with tetramethylbenzidine as the substrate, with and without the inhibitor dapsone (4,4'-diaminodiphenylsulfone). Dapsone blocked the activity of HS-LP but not MPO. The enzymes were also separated and partially purified from the soluble portion of saliva samples and from detergent extracts of the saliva sediment. Chromatographic properties of the proteins were similar to those of LP from bovine milk (BM-LP) and MPO from human leukocytes. The identity and amounts of the enzymes were confirmed by the absorption spectra and by immunoblotting with antibodies to BM-LP and human MPO. Eosinophil peroxidase (EPO), a distinct enzyme found in eosinophilic leukocytes, was not detected by chromatography or with antibodies to human EPO. On average, 75% of the activity in samples from normal donors was due to MPO and 25% to HS-LP. When corrected for the lower specific activity of HS-LP in this assay, the average amount of MPO (3.6 micrograms/mL) was twice the amount of HS-LP (1.9 micrograms/mL). The amount of MPO corresponded to 1 x 10(6) PMN leukocytes/mL of saliva. The enzymes were distributed differently: Eighty-nine percent of the HS-LP was in the soluble portion of saliva, and 78% of the MPO was in the sediment, which contained 51% of the total activity. In contrast to results obtained with PMN leukocytes from blood, detergent was not required for MPO activity to be measured in saliva, indicating that the enzyme was accessible to peroxidase substrates. The results indicate that MPO is responsible for a large portion of peroxidase-catalyzed reactions in mixed saliva. The unique function of HS-LP may be carried out within the salivary glands, prior to secretion into the oral cavity.

The effect of malnutrition on caries development and saliva composition in the rat.

The purpose of this study was to investigate the effect in rats of malnutrition on caries development and on some antibacterial substances in saliva. By gastric intubation, two groups of ten rats each were fed a liquid basic diet (100% diet), and two similar groups were fed the basic diet diluted with an equal volume of water to contain 50% of the nutrients of the basic diet (50% diet). One group on undiluted and one group on the diluted basic diet were offered sucrose as a supplement by mouth 17 times daily, using a programmed feeding machine, and the other two groups were offered starch in a similar manner. The extent and depth of the caries lesions were five times greater in the low-nourished (50% diet) sucrose group in comparison with the group given the undiluted diet supplemented with sucrose. The 50% diet group ate more frequently from the offered supplement. The 50% diet induced lower salivary lactoperoxidase activities, and there was an additive effect of starch as supplement. Higher concentrations of sIgA were found in the saliva of animals fed the 50% diet supplemented with sucrose than in those fed sucrose and the 100% diet. Significantly greater levels of sIgA and IgG were found in the saliva from the 50% diet-sucrose group than in the 50% diet-starch group. Plaque scores differed slightly but not significantly between the groups. The starch group receiving 100% diet showed a slightly lower level of S. mutans infection as compared with the 50% diet-starch group; the caries scores in the starch groups did not differ.

Xylitol, polyol molasses and glucose in the diet of newborn calves: II. Content of antimicrobial factors in blood and saliva, bacteria in faeces and health status

The concentrations of various antimicrobial factors in the saliva and plasma of newborn calves with special reference to possible effects of a diet supplemented with different sugar alcohols were studied. Eighteen calves were assigned alternately at birth to three groups, each comprising six animals. All calves were fed a pooled colostrum diet for the first four days, thereafter whole milk plus milk replacer. Concentrates and hay were given ad libitum. The diets of the different groups were supplemented with the following test substrates: xylitol, polyol molasses or glucose(control). The daily doses of each substrate were progressively increased from 0.5 to 1.0 g/kg of live weight for the last four weeks. Plasma and saliva samples were taken from all calves before colostrum feeding (day 0) and on days 1, 2, 4, 7, 21 and 35 after birth. The following factors were determined quantitatively: lactoferrin (LF), lactoperoxidase (LP), lysozyme (LZM) and immunoglobulins IgG1, lgG2, IgM and IgA (only in plasma). Further determinations included weekly counts of total aerobic bacteria, aerobic haemolytic bacteria, coliforms, lactobacilli and clostridia in faeces. Salivary LF increased from the average 0 day level of 3.6—17.0 /µg/ml in the different groups to a maximum of 38.7—55.6 µg/ml within one week, and declining thereafter slowly in all groups. LF was not found in the saliva of all calves at birth but was consistently present later on. No LF was detected in plasma. Salivary LP increased from the average 0 day level of 56.3—86.6 /µg/ml in the different groups to a maximum of 228—296 /µg/ml within three weeks and declined markedly by day 35. LP was found in all saliva samples, but the plasma concentrations were very low and not always detectable. Salivary LZM was high at birth compared to the plasma level (average 0.8—2.1 /µg/ml vs. 0.3—0.5µg/ml in the different groups).

Inhibition of dental plaque acid production by the salivary lactoperoxidase antimicrobial system.

Resting human whole saliva inhibited acid production by glucose-stimulated, homologous plaque. The degree of inhibition of plaque acid production correlated positively with the concentration of hypothiocyanite (OSCN-) ions in saliva. Supplementation of saliva with an appropriate combination of thiocyanate and hydrogen peroxide resulted in a significant increase in the concentration of OSCN- ions and in more effective inhibition of plaque acid production. In most cases, the inhibition was complete when the supplements were added directly to saliva-plaque mixtures. Acid production resumed when the inhibitory effect of OSCN- was reversed by addition of thiols. Among the oral defense factors, the salivary lactoperoxidase system seems to play an important role by producing highly reactive antibacterial products (including OSCN-) which can regulate bacterial metabolism in the human mouth. The concentration of OSCN- in normal human whole saliva seems to be just below the threshold level required for plaque inhibition. Therefore, enhancement of this system in vivo may be effective in the regulation of plaque acid production.

Different molecular forms of human salivary lactoperoxidase

The two different molecular forms shown to exist probably represent a monomer and an aggrsgate. The pI of lactoperoxidase (8.1) did not change during aggregation. The presence of substrate (SCN −) could cause some disaggregation suggesting that the mechanism of the dissociation is influenced by the substrate. Purified milk lactoperoxidase had a similar Chromatographic pattern to salivary lactoperoxidase. Another peroxidase with pI 4.3 occurs in whole saliva but not in parotid secretions, and is possibly of leukocytic origin. However, its characteristics differ from those of neutrophil myeloperoxidase but resemble those of peroxidases of inflammatory exudate and gingival fluid.

Formation of dityrosine by human salivary lactoperoxidase in vitro.

The study dealt with the formation of dityrosine - a cross-link in some proteins including collagen - by human salivary lactoperoxidase. Dityrosine formation was found at pH range 6.6 to 9.3 with maximum reaction velocity at pH 8.5. However, thiocyanate ions at physiological salivary concentrations inhibited dityrosine formation by 70 to 80 per cent compared with the optimum rate. The inhibition seemed to result from the competition of SCN ions and L-tyrosine for the same binding site on enzyme surface. The possibility of dityrosine cross-linking in vivo in human oral fluid seems to be limited compared with e.g. human milk or macaque saliva where the concentration of SCN ions is low but the activity of lactoperoxidase is considerably high.

Effect of Peroral Administration of Xylitol on Exocrine Secretions of Monkeys

The effects of the administration of xylitol on the biochemical properties of monkey parotid and submandibular saliva and lacrimal fluid were investigated. Monkeys (Macaca mulatta) were fed either a sucrose or xylitol diet for 3 days. Ingestion of xylitol was associated with a significant increase of the activity of the salivary lactoperoxidase. The concentration of protein was also increased. Higher lactoperoxidase activity was found in parotid than in submandibular saliva. The concentrations of inorganic phosphate, calcium and SCN ions were practically unaffected. The concentration of SCN ions in pilocarpine stimulated monkey saliva was low (less than 5.5 mg/liter). Peroral administration of 2.5 g of xylitol or sorbitol per day to M. fascicularis resulted in almost similar levels of salivary lactoperoxidase activity. The administration of xylitol orally or by gastric intubation was not found to affect the concentration of lactoperoxidase, protein, phosphate, and SCN and iodine ions in lacrimal fluid. The results suggest that specific dietary sugars have a selective effect on the biochemical properties of saliva.

Lactoperoxidase-catalysed iodine metabolism in human saliva

The oxidation of iodide to iodine and the iodination of tyrosine and some other substances catalysed by partially-purified human salivary peroxidases was studied in conditions as physiological as possible. The oxidation of iodide by the enzymes had a pH optimum at 6.2, and the iodination rate was highest at pH 3.4. Salivary lactoperoxidase in the presence of iodine was capable of iodinating tyrosine, monoiodotyrosine and bovine serum albumin. However, thiocyanate ions at concentrations present in human saliva effectively inhibited both the oxidation and the iodination reactions. On the other hand, thiocyanate had a preserving influence on the enzyme activity. This may be due to a formation of an enzyme-substrate complex before the catalysis can occur, after the addition of hydrogen peroxide. The observations indicate that, owing to the presence of thiocyanate ions and unfavourable pH values in human oral fluid, the metabolism of iodine by salivary peroxidases in vivo cannot be significant, compared to the metabolism occurring in the thyroid gland, although all the necessary components are present in the human mouth.

Antibacterial Effect of Salivary Peroxidases on a Cariogenic Strain of Streptococcus mutans

The antibacterial effect of purified human salivary lactoperoxidase on a cariogenic strain of Streptococcus mutans was demonstrated while another oral peroxidase, probably of leukocytic origin, did not affect the growth. Lactoperoxidase was rapidly adsorbed by bacterial cells indicating the necessity of the contact between the enzyme and the cells before inhibition.

Enzyme activity of salivary lactoperoxidase adsorbed to human enamel

Human saliva was incubated with human enamel powder, and the lactoperoxidase activity of the saliva was measured before and after incubation. Liquidphase lactoperoxidase activity was reduced in direct proportion to the weight of enamel powder added. Lactoperoxidase molecules were adsorbed to the enamel surface in an enzymatically active conformation, and this enamel-bound lactoperoxidase activity was also measured. The adsorption of lactoperoxidase was irreversible and produced at least a 40% increase in the concentration of lactoperoxidase in the enamel surface phase as compared with its concentration in the liquid phase. Enamel-bound lactoperoxidase, as well as the free enzyme, was capable of inactivating the key glycolytic enzyme hexokinase. The implications of the adsorption phenomenon for bacterial colonization are discussed.

Xylitol-induced increase of lactoperoxidase activity.

Whole saliva samples of volunteers who ate a strict diet for two years with regard to the type of sweeteners used (sucrose, fructose, and xylitol) showed considerable differences in the lactoperoxidase activity. The consumption of a xylitol diet increased the activity of this enzyme fourfold to tenfold when compared to the other two test groups. Lactoperoxidase belongs to the natural defense mechanisms of the oral cavity. However, the consumption of a xylitol diet also leads to a strong reduction in the incidence of dental caries. It is suggested that the xylitol-induced elevation of the salivary lactoperoxidase activity and the cariostatic properties of xylitol are partly interrelated phenomena.

The variation of salivary peroxidase activities in persons of different oral health.

18 subjects (10 males, 8 females) with considerable differences in oral health conditions were examined regarding the peroxidase activity of oral fluid samples. Subjects with high caries prevalence and/or severe gingivitis displayed a significantly higher peroxidase activity than control subjects. Chromatographic experiments revealed that the increased activity was caused by salivary lactoperoxidase. This elevated activity does not, however, necessarily lead to better oral health because the antibacterial activity of lactoperoxidase also depends on the concentrations of thiocyanate ions and hydrogen peroxide. Hence the specific activity of lactoperoxidase, determined in vitro, is not as such as adequate measure of its antibacterial efficacy.