HPLC method for measurement of human salivary α-amylase inhibition by aqueous plant extracts.

Control of hyperglycemia is an important treatment in metabolic disorders such as type II diabetes and obesity. α-Amylase, as the first enzyme of glucose release from dietary polysaccharides, is a potential target to identify new sources of novel anti-obesity and anti-diabetic drugs. In this work, different herbal extracts as α-amylase inhibitors were studied by measuring the rate of the cleavage of a maltooligomer substrate 2-chloro-4-nitrophenyl-ß-D-maltoheptoside. Measurement of chromophore containing products after reversed phase HPLC separation was used for α-amylase activity measurement. Rates of hydrolysis catalysed by human salivary α-amylase were determined in the presence and absence of lyophilised water extracts of eleven herbs. Remarkable bioactivities were found for extracts of Cinnamomum zeylanicum Blume (bark), Camellia sinensis L. (leaf), Ribes nigrum L. (leaf), Laurus nobilis L. (leaf), Vaccinium macrocarpon Aiton (fruit) and Syzygium aromaticum L. (bud). Determined IC50 values were in 0.017-41 µg/ml range for these six selected plant extracts. Our results confirm the applicability of this HPLC-based method for the quick and reliable comparison of plants as α-amylase inhibitors.

Gingipains of Porphyromonas gingivalis Affect the Stability and Function of Serine Protease Inhibitor of Kazal-type 6 (SPINK6), a Tissue Inhibitor of Human Kallikreins.

Periodontitis, a chronic inflammation driven by dysbiotic subgingival bacterial flora, is linked on clinical levels to the development of a number of systemic diseases and to the development of oral and gastric tract tumors. A key pathogen, Porphyromonas gingivalis, secretes gingipains, cysteine proteases implicated as the main factors in the development of periodontitis. Here we hypothesize that gingipains may be linked to systemic pathologies through the deregulation of kallikrein-like proteinase (KLK) family members. KLKs are implicated in cancer development and are clinically utilized as tumor progression markers. In tissues, KLK activity is strictly controlled by a limited number of tissue-specific inhibitors, including SPINK6, an inhibitor of these proteases in skin and oral epithelium. Here we identify gingipains as the only P. gingivalis proteases responsible for SPINK6 degradation. We further show that gingipains, even at low nanomolar concentrations, cleaved SPINK6 in concentration- and time-dependent manner. The proteolysis was accompanied by loss of inhibition against KLK13. We also mapped the cleavage by Arg-specific gingipains to the reactive site loop of the SPINK6 inhibitor. Moreover, we identified a significant fraction of SPINK6-sensitive proteases in healthy saliva and confirmed the ability of gingipains to inactivate SPINK6 under ex vivo conditions. Finally, we demonstrate the double-edge action of gingipains, which, in addition, can activate KLKs because of gingipain K-mediated proteolytic processing of the zymogenic proform of KLK13. Altogether, the results indicate the potential of P. gingivalis to disrupt the control system of KLKs, providing a possible mechanistic link between periodontal disease and tumor development.

Inactivation of human salivary glutathione transferase P1-1 by hypothiocyanite: a post-translational control system in search of a role.

Glutathione transferases (GSTs) are a superfamily of detoxifying enzymes over-expressed in tumor tissues and tentatively proposed as biomarkers for localizing and monitoring injury of specific tissues. Only scarce and contradictory reports exist about the presence and the level of these enzymes in human saliva. This study shows that GSTP1-1 is the most abundant salivary GST isoenzyme, mainly coming from salivary glands. Surprisingly, its activity is completely obscured by the presence of a strong oxidizing agent in saliva that causes a fast and complete, but reversible, inactivation. Although salivary α-defensins are also able to inhibit the enzyme causing a peculiar half-site inactivation, a number of approaches (mass spectrometry, site directed mutagenesis, chromatographic and spectrophotometric data) indicated that hypothiocyanite is the main salivary inhibitor of GSTP1-1. Cys47 and Cys101, the most reactive sulfhydryls of GSTP1-1, are mainly involved in a redox interaction which leads to the formation of an intra-chain disulfide bridge. A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein. The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function. In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.

Sodium fluoride inhibits MMP-2 and MMP-9.

The importance of fluoride (F) in preventing dental caries by favorably interfering in the demineralization-remineralization processes is well-established, but its ability to inhibit matrix metalloproteinases (MMPs), which could also help to prevent dentin caries, has not been investigated. This study assessed the ability of F to inhibit salivary and purified human gelatinases MMPs-2 and -9. Saliva was collected from 10 healthy individuals. Pooled saliva was centrifuged, and supernatants were incubated for 1 hr at 37°C and subjected to zymography. Sodium fluoride (50-275 ppm F) was added to the incubation buffer. The reversibility of the inhibition of MMPs-2 and -9 by NaF was tested by the addition of NaF (250-5,000 ppm F) to the incubation buffer, after which an additional incubation was performed in the absence of F. F decreased the activities of pro- and active forms of salivary and purified human MMPs in a dose-response manner. Purified gelatinases were completely inhibited by 200 ppm F (IC50 = 100 and 75 ppm F for MMPs-2 and -9, respectively), and salivary MMP-9 by 275 ppm F (IC50 = 200 ppm F). Inhibition was partially reversible at 250-1,500 ppm F, but was irreversible at 5,000 ppm F. This is the first study to describe the ability of NaF to inhibit MMPs completely.

Secreted trypanosome cyclophilin inactivates lytic insect defense peptides and induces parasite calcineurin activation and infectivity.

The mechanisms by which Trypanosoma cruzi survives antimicrobial peptides and differentiates during its transit through the gastrointestinal tract of the reduviid vector are unknown. We show that cyclophilin, a peptidyl-prolyl isomerase secreted from T. cruzi epimastigotes, binds to and neutralizes the reduviid antimicrobial peptide trialysin promoting parasite survival. This is dependent on a singular proline residue in trialysin and is inhibited by the cyclophilin inhibitor cyclosporine A. In addition, cyclophilin-trialysin complexes enhance the production of ATP and reductase responses of parasites, which are inhibited by both calcineurin-specific inhibitors cyclosporine A and FK506. Calcineurin phosphatase activity of cyclophilin-trialysin-treated parasites was higher than in controls and was inhibited by preincubation by either inhibitor. Parasites exposed to cyclophilin-trialysin have enhanced binding and invasion of host cells leading to higher infectivity. Leishmanial cyclophilin also mediates trialysin protection and metabolic stimulation by T. cruzi, indicating that extracellular cyclophilin may be critical to adaptation in other insect-borne protozoa. This work demonstrates that cyclophilin serves as molecular sensor leading to the evasion and adaptive metabolic response to insect defense peptides.

An insight into the Phlebotomus perniciosus saliva by a proteomic approach.

Sand fly saliva is known to play an important role in the establishment of Leishmania spp. infection. As a consequence, identifying antigenic salivary proteins of different leishmaniasis vectors has currently become a major task in the field of anti-Leishmania vaccine development. The purpose of this work was to improve the knowledge of Phlebotomus perniciosus salivary proteins by combining two-dimensional gel electrophoresis (2DE) methodology, mass spectrometry and Western blotting (WB). Salivary protein profiles of three P. perniciosus colonies from different geographic origins in Spain were compared through SDS-PAGE, leading to a similar pattern with no qualitatively noticeable differences. A gradual increase of the protein content was significantly detected with the age of sand flies, reaching the complete salivary protein profiles at day four. The 2DE revealed a reproducible protein profile that matched the classic monodimensional SDS-PAGE pattern (1DE). More spots rather than protein bands (19 versus 11) were visualized by 2DE and 1DE, respectively, suggesting the presence of either protein isoforms or posttranslational modifications. Sera of mice and hamsters immunized through exposure to sand fly bites following different immunization schedules showed elevated anti-saliva IgG levels. These sera allowed the detection of 5 bands and 16 immunogenic spots in 1DE and 2DE, respectively, followed by WB. These antigens were identified by MALDITOF/TOF as SP03, SP03B, SP08, SP01, SP01B, SP04, SP04B, SP02, Phlebotomus ariasi SP16, and Phlebotomus argentipes SP13. This work is assumed to be the first attempt to establish 2DE proteomic maps of P. perniciosus saliva. All spots were identified as salivary proteins, confirming this technology as an interesting tool to improve sand fly salivary knowledge.

Role of lipid oxidation, chelating agents, and antioxidants in metallic flavor development in the oral cavity.

This study investigated the production of metallic flavor, which is a combination of taste and retronasal odor. Chemical reactions in the oral cavity and saliva of healthy subjects were investigated after ingesting iron and copper solutions above and near threshold levels. Significant increase in lipid oxidation (p < 0.001) occurred after metal ingestion, detected as TBARS values. Ferrous ion caused the greatest flavor sensation and lipid oxidation, followed by cupric and cuprous ions. Ferric ion did not cause metallic sensation. Occurrence of oxidation was supported by damage to salivary proteins, detected as protein-carbonyls, and by a significant increase of odorous lipid oxidation related aldehydes. Sensory evaluation demonstrated that antioxidants (vitamins E and C) minimally reduced metallic flavor but that chelating agents (EDTA and lactoferrin) removed the metallic flavor. The role of lipid oxidation is essential for the production of a metallic flavor from ingestion of ferrous, cupric, and cuprous ions.

Longistatin is an unconventional serine protease and induces protective immunity against tick infestation.

Classical serine proteases use the conserved Ser/His/Asp catalytic triad to hydrolyze substrates. Here, we show that longistatin, a salivary gland protein with two EF-hand domains from the vector tick Haemaphysalis longicornis, does not have the conserved catalytic triad, but still functions as a serine protease. Longistatin was synthesized in and secreted from the salivary glands of ticks, and is injected into host tissues during the acquisition of blood-meals. Longistatin hydrolyzed fibrinogen, an essential plasma protein in the coagulation cascade, and activated plasminogen, into its active form plasmin, a serine protease that dissolves fibrin clots. Longistatin efficiently hydrolyzed several serine protease-specific substrates showing its specificity to the amide bond of Arg. Longistatin did not hydrolyze synthetic substrates specific for other groups of proteases. The enzyme was active at a wide range of temperatures and pHs, with the optimum at 37°C and pH 7. Its activity was efficiently inhibited by various serine protease inhibitors such as phenylmethanesulfonyl fluoride (PMSF), aprotinin, antipain, and leupeptin with the estimated IC(50) of 278.57 µM, 0.35 µM, 41.56 µM and 198.86 µM, respectively. In addition, longistatin was also potently inhibited by Zinc (Zn(2+)) in a concentration-dependent manner with an IC(50) value of 275 µM, and the inhibitory effect of Zn(2+) was revived by ethylenediaminetetra acetic acid (EDTA). Immunization studies revealed that longistatin sharply induced high levels of protective IgG antibodies against ticks. Immunization with longistatin reduced repletion of ticks by about 54%, post engorgement body weight by >11% and molting of nymphs by approximately 34%; thus, the vaccination trial was approximately 73% effective against tick infestation. Taken together, our results suggest that longistatin is a new potent atypical serine protease, and may be an interesting candidate for the development of anti-tick vaccines.

Longistatin, a novel plasminogen activator from vector ticks, is resistant to plasminogen activator inhibitor-1.

Thrombo-occlusive diseases are major causes of morbidity and mortality, and tissue-type plasminogen activator (t-PA) is recommended for the treatment of the maladies. However, both t-PA and u-PA are rapidly inactivated by plasminogen activator inhibitor-1 (PAI-1). Here, we show that longistatin, a novel plasminogen activator isolated from the ixodid tick, Haemaphysalis longicornis is resistant to PAI-1. Longistatin was relatively less susceptible to the inhibitory effect of SDS-treated platelet lysate than physiologic PAs. Platelet lysate inhibited t-PA and tcu-PA with the IC(50) of 7.7 and 9.1 µg/ml, respectively, whereas for longistatin inhibition IC(50) was 20.1 µg/ml (p<0.01). Similarly, activated PAI-1 (20 nM) inhibited only 21.47% activity of longistatin but almost completely inhibited t-PA (99.17%) and tcu-PA (96.84%). Interestingly, longistatin retained 76.73% initial activity even after 3h of incubation with 20 nM of PAI-1. IC(50) of PAI-1 during longistatin inhibition was 88.3 nM while it was 3.9 and 3.2 nM in t-PA and tcu-PA inhibition, respectively. Longistatin completely hydrolyzed fibrin clot by activating plasminogen efficiently in the presence of 20 nM of PAI-1. Importantly, unlike t-PA, longistatin did not form complex with PAI-1. Collectively, our results suggest that longistatin is resistant to PAI-1 and maybe an interesting tool for the development of a PAI-1 resistant effective thrombolytic agent.

Endometrial cysteine-rich secretory protein 3 is inhibited by human chorionic gonadotrophin, and is increased in the decidua of tubal ectopic pregnancy.

Ectopic pregnancy (EP) remains a considerable cause of morbidity and occasional mortality. Currently, there is no reliable test to differentiate ectopic from intrauterine gestation. We have previously used array technology to demonstrate that differences in gene expression in decidualized endometrium from women with ectopic and intrauterine gestations could be used to identify candidate diagnostic biomarkers for EP. The aim of this study was to further investigate the decidual gene with the highest fold increase in EP, cysteine-rich secretory protein-3 (CRISP-3). Decidualized endometrium from gestation-matched women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6) and surgery for EP (n = 11) was subjected to quantitative RT-PCR, morphological assessment, immunohistochemistry and western blot analysis. Sera were analysed for progesterone and human chorionic gonadotrophin (hCG) levels. Immortalized endometrial epithelial cells were cultured with physiological concentrations of hCG. CRISP-3 mRNA and protein expression were greater in endometrium from ectopic when compared with intrauterine pregnancies (P < 0.05). CRISP-3 protein was localized to epithelium and granulocytes of endometrium. CRISP-3 serum concentrations were not different in women with ectopic compared with intrauterine pregnancies. CRISP-3 expression in endometrium was not related to the degree of decidualization or to serum progesterone levels. Endometrial CRISP-3 expression was inversely proportional to serum hCG concentrations (P < 0.001). Stimulation of endometrial epithelial cells with hCG in vitro caused a reduction in CRISP-3 expression (P < 0.01). The measurement of CRISP-3 in endometrium could provide an additional tool in the diagnosis of failing early pregnancy of unknown location. The absence of a local reduction in expression of CRISP-3 in decidualized endometrium of women with EP may be due to reduced exposure to hCG due to the ectopic location of the trophoblast.

A peptide domain of bovine milk lactoferrin inhibits the interaction between streptococcal surface protein antigen and a salivary agglutinin peptide domain.

The peptide domain of salivary agglutinin responsible for its interaction with cell surface protein antigen (PAc) of Streptococcus mutans or bovine lactoferrin was found in the same peptide, scavenger receptor cysteine-rich domain peptide 2 (SRCRP2). Inhibition studies suggest that PAc and lactoferrin, of which residues 480 to 492 seem important, competitively bind to the SRCRP2 domain of salivary agglutinin.

Calcium blocks fungicidal activity of human salivary histatin 5 through disruption of binding with Candida albicans.

Salivary histatin 5 (Hst 5) kills the fungal pathogen C. albicans via a mechanism that involves binding and subsequent efflux of cellular ATP. Our aims were to identify inorganic ions found in saliva that influence Hst 5 fungicidal activity. Increasing ionic strength with relevant salivary anions (Cl(-) and CO(3)(-)) did not reduce Hst 5 binding or uptake by yeast cells, but reduced the Hst-induced efflux of ATP. Extracellular MgCl(2) (25 mM) maximally inhibited 30-40% of Hst 5 killing with 40% reduction in ATP efflux, while pre-treatment of cells with only 2 mM CaCl(2) inhibited 80-90% of killing, and prevented ATP efflux. Loss of fungicidal activity by the addition of CaCl(2) or MgCl(2) was a result of inhibition of binding of Hst 5 to C. albicans cells. Calcium is a potent inhibitor of Hst 5 candidacidal activity at physiological concentrations and may be the primary salivary ion responsible for the masking effect of saliva.

A sulfated proteoglycan is necessary for storage of exocrine secretory proteins in the rat parotid gland.

Sulfated proteoglycans have been proposed to play a role in the sorting and storage of secretory proteins in exocrine secretory granules. Rat parotid acinar cells expressed a 40- to 60-kDa proteoglycan that was stored in secretory granules. Treatment of the tissue with the proteoglycan synthesis inhibitor paranitrophenyl xyloside resulted in the complete abrogation of the sulfated proteoglycan. Pulse-chase experiments in the presence of the xyloside analog showed a significant reduction in the stimulated secretion and granule storage of the newly synthesized regulated secretory proteins amylase and parotid secretory protein. Inhibition of proteoglycan sulfation by chlorate did not affect the sorting of these proteins. The effect of proteoglycan synthesis inhibition on protein sorting was completely reversed upon treatment with a weak acid. These results suggest that the sulfated proteoglycan is necessary for sorting and storage of regulated secretory proteins in the exocrine parotid gland. Preliminary evidence suggests that the mechanism involves the modulation of granule pH by the proteoglycan rather than a direct interaction with other granule components.

Characterization and cDNA cloning of halyxin, a heterogeneous three-chain anticoagulant protein from the venom of Agkistrodon halys brevicaudus.

We report upon the isolation, characterization, and cDNA cloning of an anticoagulant protein, halyxin from Agkistrodon halys brevicaudus venom. The protein exists as a 29kDa protein, and is separated into three chains on SDS-PAGE under reducing conditions. However, we cloned only two cDNAs encoding halyxin from the cDNA library of the snake venom gland, on the basis of the determined amino acid sequences. The complete amino acid sequences were deduced from their nucleotide sequences and named halyxin A (129 amino acid residues) and B chain (123 amino acid residues). The deduced amino acid sequence of halyxin A chain corresponds to the two smaller chains. Thus, it is considered that halyxin A chain could be synthesized as a single-chain protein that is subsequently cleaved to yield the mature two-chain protein. The amino acid sequence of halyxin is similar to that of other snake venom proteins of the C-type lectin superfamily, and prolongs plasma-clotting time. In the presence of Ca(2+) ions, halyxin binds to coagulation factors IX, X, IXa, and Xa, but not to other vitamin K-dependent coagulation factors. It also inhibits factor Xa in a non-competitive manner but does not affect other activated coagulation factors.

Saliva flow rate, amylase activity, and protein and electrolyte concentrations in saliva after acute alcohol consumption.

OBJECTIVE: The aim of our study was to evaluate the effects of acute alcohol consumption on saliva secretion rate and selected salivary parameters in healthy nonalcoholic volunteers. STUDY DESIGN: Twenty-four volunteers (37.7 +/- 9.6 years, mean +/- SD) consumed 0.6 g or 0.7 g alcohol/kg of body weight (for women and men, respectively) in a soft drink. Saliva samples were collected, first (S0) before any alcohol was consumed, 45 minutes after consumption (S1) and, finally, 60 minutes after S1 (S2). Flow rates of both resting whole saliva and paraffin-stimulated (SWS) whole saliva were assessed. SWS was assessed for amylase, total protein, inorganic phosphate (PO4(3-)), sodium (Na+), potassium (K+), and calcium (Ca2+) content. RESULTS: SWS, but not resting whole saliva (in milliliters/minute), decreased significantly after consumption of alcohol. Amylase activity (P =.010) and the concentrations of Na+ (P =.000) and Ca2+ (P =.002) decreased significantly between S0 and S1. When SWS was analyzed for output, the total protein concentration (S0 to S1, P =.000; S0 to S2, P =.033) and amylase activity (S0 to S1, P =.000) decreased significantly. Further, the output of all the studied electrolytes decreased significantly as blood alcohol concentration increased. CONCLUSIONS: We conclude that acute alcohol consumption causes a decrease in SWS flow rate. The decrease in flow rate also results in impaired output of total protein and amylase, as well as in a decrease in the output of electrolytes.

The effects of MMP inhibitors on human salivary MMP activity and caries progression in rats.

Previous studies suggest that salivary and pulp-derived host enzymes, matrix metalloproteinases (MMPs), may be involved in dentin caries pathogenesis. To study the inhibition of acid-activated human salivary MMPs by non-antimicrobial chemically modified tetracyclines (CMTs), we used a functional activity assay with 125I-labeled gelatin as a substrate. To address the role of MMPs in the progression of fissure caries in vivo, we administered the MMP inhibitors CMT-3 and zoledronate to young rats per os for 7 weeks, 5 days a week. Caries lesions were visualized by Schiff reagent in sagittally sectioned mandibular molars. Marked reduction in gelatinolytic activity of human salivary MMPs was observed with CMT-3. CMT-3 and zoledronate, both alone and in combination, also reduced dentin caries progression in the rats. These results suggest that MMPs have an important role in dentin caries pathogenesis, and that MMP inhibitors may prove to be useful in the prevention of caries progression.

Blockade of isoprenaline-induced fluid and protein secretion by the mandibular glands of the red kangaroo, Macropus rufus, with selective antagonists.

Selective and non-selective beta-adrenoceptor antagonists were used to block the increases in fluid and protein secretion caused by sympathomimetic stimulation of the mandibular gland of red kangaroos during intracarotid infusion of isoprenaline. Atenolol or ICI118551 at antagonist:agonist ratios up to 300:1 caused increasing but incomplete blockade of fluid secretion and protein release. Both selective antagonists had equal potency and both antagonists were more effective at blocking protein release than at blocking fluid secretion. Consequently, the mechanisms underpinning fluid secretion are more sensitive to beta-sympathomimetic stimulation than those causing protein release. Propranolol at antagonist:agonist ratios of 300:1 was more potent than the selective antagonists, almost totally blocking the increases in fluid secretion and protein release. The data are consistent with the acini of the kangaroo mandibular gland having both beta(1)- and beta(2)-adrenoceptors and with the increased fluid secretion and protein release by isoprenaline being mediated by both receptor subtypes.

Inhibition of salivary amylase by black and green teas and their effects on the intraoral hydrolysis of starch.

Tea decoctions prepared from a number of black and green teas inhibited amylase in human saliva. Black teas gave higher levels of inhibition than green teas, and removal of tea tannins with gelatin led to the loss of inhibitory activity from all decoctions. Streptococcal amylase was similarly inhibited by tea decoctions. Fluoride was without effect on amylase. Since salivary amylase hydrolyzes food starch to low molecular weight fermentable carbohydrates, experiments were carried out to determine whether tea decoctions would interfere with the release of maltose in food particles that became entrapped on the dentition. Subjects consumed salted crackers and rinsed subsequently for 30 s with black or green tea decoctions, or water. Maltose release was reduced by up to about 70% after rinsing with the teas. Black tea decoction was significantly more effective than green tea, in agreement with the in vitro data. The observations supported the hypothesis that tea consumption can be effective in reducing the cariogenic potential of starch-containing foods such as crackers and cakes. Tea may reduce the tendency for these foods to serve as slow-release sources of fermentable carbohydrate.

Effects of delmopinol on antimicrobial peroxidase systems and lysozyme in vitro and in human whole saliva.

Delmopinol is a new surface-active agent which can reduce plaque formation and gingivitis. This study was aimed to analyze whether delmopinol (0.0032-0.65 mM) interferes with the activity of two surface-active oral antimicrobial enzymes, salivary peroxidase and lysozyme. In addition to human whole saliva (pH 5.0 and 6.0), the experiments were done in 0.1 M phosphate buffer (pH 6.0) with purified lactoperoxidase (LPO) and myeloperoxidase (MPO). LPO and MPO were significantly inhibited in buffer by delmopinol concentrations > 6.5 mM and > or = 3.2 mM, respectively. No such inhibition was found for total peroxidase activity in mixed saliva. In vitro, delmopinol was found to desorb surface-bound peroxidases in an active form to the liquid phase. In further analyses, the possible effect of delmopinol on peroxidase-generated hypothiocyanite (HOSCN/OSCN-) was studied in saliva and buffer. No effect was found in buffer, but salivary HOSCN/OSCN- declined significantly with 6.5 mM delmopinol. This was obviously due to an enhanced decay of hypothiocyanite, rather than its reduced rate of formation. No delmopinol-related inhibition of lysozyme occurred in saliva or buffer. The results suggest that high concentration (6.4 mM -0.2%) of delmopinol may lower the concentrations of antimicrobial HOSCN/OSCN- in saliva but has no effect on human lysozyme.

The influence of different fluoride salts on fluoride-mediated inhibition of peroxidase activity in human saliva.

Fluoride-mediated inhibition of peroxidase potential activity in human saliva was investigated using NaF, NH4F, CaF2, Na2PO3F (MFP), SnF2 and TiF4. At pH 5.5 and for a 20 mM F concentration, the inhibition percentages increased from 2% for MFP and 5% for CaF2 to 61% for NaF and 65% for NH4F, while a 100% inhibition was observed at 10 mM for TiF4 and at 5 mM for SnF2. The inhibition was enhanced at acid pH and removed at pH 7. The inhibition could be attributed to ionized F-, except for SnF2 and TiF4, in which part of the inhibition could be imputed to the cations.