The complexity of oral physiology and its impact on salivary diagnostics.

OBJECTIVES: Saliva contains biomarkers for systemic as well as oral diseases. This study was undertaken to assess the variability in the sources of such biomarkers (plasma, cells) and attempted to identify saliva deterioration markers in order to improve saliva diagnostic outcomes. MATERIALS AND METHODS: Inter- and intrasubject variations in salivary gingival crevicular fluid levels were determined by measuring salivary albumin and transferrin levels. The purity of collected glandular secretions was determined by bacterial culture, and the variability in epithelial cell numbers by cell counting and optical density measurement. Saliva sample deterioration markers were identified by RP-HPLC and LC-ESI-MS/MS. RESULTS: Tenfold variations were observed in plasma-derived albumin and transferrin levels, emphasizing the need for biomarker normalization with respect to plasma contributions to saliva. Epithelial cell levels varied 50-fold in samples collected before and after a meal. Salivary fungal levels varied within subjects and among subjects from 0 to >1,000 colony-forming units per milliliter. In saliva samples incubated for various time intervals at 37°C, five peptides were identified that steadily increased in intensity over time and which could be explored as "deterioration markers." CONCLUSION: Taking saliva characteristics appropriately into account will help realize the promise that this body fluid is suitable to be exploited for reliable healthcare monitoring and surveillance.

Late responses to adenoviral-mediated transfer of the aquaporin-1 gene for radiation-induced salivary hypofunction.

We evaluated late effects of AdhAQP1 administration in five subjects in a clinical trial for radiation-induced salivary hypofunction (http://www.clinicaltrials.gov/ct/show/NCT00372320?order=). All were identified as initially responding to human aquaporin-1 (hAQP1) gene transfer. They were followed for 3-4 years after AdhAQP1 delivery to one parotid gland. At intervals we examined salivary flow, xerostomic symptoms, saliva composition, vector presence and efficacy in the targeted gland, clinical laboratory data and adverse events. All displayed marked increases (71-500% above baseline) in parotid flow 3-4.7 years after treatment, with improved symptoms for ~2-3 years. There were some changes in [Na+] and [Cl-] consistent with elevated salivary flow, but no uniform changes in secretion of key parotid proteins. There were no clinically significant adverse events, nor consistent negative changes in laboratory parameters. One subject underwent a core needle biopsy of the targeted parotid gland 3.1 years post treatment and displayed evidence of hAQP1 protein in acinar, but not duct, cell membranes. All subjects responding to hAQP1 gene transfer initially had benefits for much longer times. First-generation adenoviral vectors typically yield transit effects, but these data show beneficial effects can continue years after parotid gland delivery.

Saliva and Serum Protein Exchange at the Tooth Enamel Surface.

The acquired enamel pellicle is an oral, fluid-derived protein layer that forms on the tooth surface. It is a biologically and clinically important integument that protects teeth against enamel demineralization, and abrasion. Tooth surfaces are exposed to different proteinaceous microenvironments depending on the enamel location. For instance, tooth surfaces close to the gingival sulcus contact serum proteins that emanate via this sulcus, which may impact pellicle composition locally. The aims of this study were to define the major salivary and serum components that adsorb to hydroxyapatite, to study competition among them, and to obtain preliminary evidence in an in vivo saliva/serum pellicle model. Hydroxyapatite powder was incubated with saliva and serum, and the proteins that adsorbed were identified by mass spectrometry. To study competition, saliva and serum proteins were labeled with CyDyes, mixed in various proportions, and incubated with hydroxyapatite. In vivo competition was assessed using a split-mouth design, with half the buccal tooth surfaces coated with serum and the other half with saliva. After exposure to the oral environment for 0 min, 30 min and 2 h, the pellicles were analyzed by SDS-PAGE. In pure saliva- or serum-derived pellicles, 82 and 84 proteins were identified, respectively. When present concomitantly, salivary protein adsorbers effectively competed with serum protein adsorbers for the hydroxyapatite surface. Specifically, acidic proline-rich protein, cystatin, statherin and protein S100-A9 proteins competed off apolipoproteins, complement C4-A, haptoglobin, transthyretin and serotransferrin. In vivo evidence further supported the replacement of serum proteins by salivary proteins. In conclusion, although significant numbers of serum proteins emanate from the gingival sulcus, their ability to participate in dental pellicle formation is likely reduced in the presence of strong salivary protein adsorbers. The functional properties of the acquired enamel pellicle will therefore be mostly dictated by the salivary component.

Microbial Diversity in the Early In Vivo-Formed Dental Biofilm.

Although the mature dental biofilm composition is well studied, there is very little information on the earliest phase of in vivo tooth colonization. Progress in dental biofilm collection methodologies and techniques of large-scale microbial identification have made new studies in this field of oral biology feasible. The aim of this study was to characterize the temporal changes and diversity of the cultivable and noncultivable microbes in the early dental biofilm. Samples of early dental biofilm were collected from 11 healthy subjects at 0, 2, 4, and 6 h after removal of plaque and pellicle from tooth surfaces. With the semiquantitative Human Oral Microbiome Identification Microarray (HOMIM) technique, which is based on 16S rRNA sequence hybridizations, plaque samples were analyzed with the currently available 407 HOMIM microbial probes. This led to the identification of at least 92 species, with streptococci being the most abundant bacteria across all time points in all subjects. High-frequency detection was also made with Haemophilus parainfluenzae, Gemella haemolysans, Slackia exigua, and Rothia species. Abundance changes over time were noted for Streptococcus anginosus and Streptococcus intermedius (P = 0.02), Streptococcus mitis bv. 2 (P = 0.0002), Streptococcus oralis (P = 0.0002), Streptococcus cluster I (P = 0.003), G. haemolysans (P = 0.0005), and Stenotrophomonas maltophilia (P = 0.02). Among the currently uncultivable microbiota, eight phylotypes were detected in the early stages of biofilm formation, one belonging to the candidate bacterial division TM7, which has attracted attention due to its potential association with periodontal disease.

Nanoscale adhesion forces between enamel pellicle proteins and hydroxyapatite.

The acquired enamel pellicle (AEP) is important for minimizing the abrasion caused by parafunctional conditions as they occur, for instance, during bruxism. It is a remarkable feature of the AEP that a protein/peptide film can provide enough protection in normofunction to prevent teeth from abrasion and wear. Despite its obvious critical role in the protection of tooth surfaces, the essential adhesion features of AEP proteins on the enamel surface are poorly characterized. The objective of this study was to measure the adhesion force between histatin 5, a primary AEP component, and hydroxyapatite (HA) surfaces. Both biotinylated histatin 5 and biotinylated human serum albumin were allowed to adsorb to streptavidin-coated silica microspheres attached to atomic force microscope (AFM) cantilevers. A multimode AFM with a Nanoscope IIIa controller was used to measure the adhesion force between protein-functionalized silica microspheres attached to cantilever tips and the HA surface. The imaging was performed in tapping mode with a Si3N4 AFM cantilever, while the adhesion forces were measured in AFM contact mode. A collection of force-distance curves (~3,000/replicate) was obtained to generate histograms from which the adhesion forces between histatin 5 or albumin and the HA surface were measured. We found that histatin 5 exhibited stronger adhesion forces (90% >1.830 nN) to the HA surface than did albumin (90% > 0.282 nN). This study presents an objective approach to adhesion force measurements between histatin 5 and HA, and provides the experimental basis for measuring the same parameters for other AEP constituents. Such knowledge will help in the design of synthetic proteins and peptides with preventive and therapeutic benefits for tooth enamel.

The cultivable human oral gluten-degrading microbiome and its potential implications in coeliac disease and gluten sensitivity.

Coeliac disease is characterized by intestinal inflammation caused by gluten, proteins which are widely contained in the Western diet. Mammalian digestive enzymes are only partly capable of cleaving gluten, and fragments remain that induce toxic responses in patients with coeliac disease. We found that the oral microbiome is a novel and rich source of gluten-degrading organisms. Here we report on the isolation and characterization of the cultivable resident oral microbes that are capable of cleaving gluten, with special emphasis on the immunogenic domains. Bacteria were obtained by a selective culturing approach and enzyme activities were characterized by: (i) hydrolysis of paranitroanilide-derivatized gliadin-derived tripeptide substrates; (ii) gliadin degradation in-gel (gliadin zymography); (iii) gliadin degradation in solution; (iv) proteolysis of the highly immunogenic α-gliadin-derived 33-mer peptide. For selected strains pH activity profiles were determined. The culturing strategy yielded 87 aerobic and 63 anaerobic strains. Species with activity in at least two of the four assays were typed as: Rothia mucilaginosa HOT-681, Rothia aeria HOT-188, Actinomyces odontolyticus HOT-701, Streptococcus mitis HOT-677, Streptococcus sp. HOT-071, Neisseria mucosa HOT-682 and Capnocytophaga sputigena HOT-775, with Rothia species being active in all four assays. Cleavage specificities and substrate preferences differed among the strains identified. The approximate molecular weights of the enzymes were ~75 kD (Rothia spp.), ~60 kD (A. odontolyticus) and ~150 kD (Streptococcus spp.). In conclusion, this study identified new gluten-degrading microorganisms in the upper gastrointestinal tract. A cocktail of the most active oral bacteria, or their isolated enzymes, may offer promising new treatment modalities for coeliac disease.

Whole-saliva proteolysis and its impact on salivary diagnostics.

There is growing interest in the use of human whole saliva for diagnostics and disease monitoring as an alternative to blood samples. In contrast to blood, whole saliva is a non-sterile body fluid. Proper hand-ling and storage are required to preserve the integrity of potential biomarkers. We investigated salivary autoproteolytic degradation using a variety of approaches. We determined inhibition of protease activities by monitoring the endogenous proteome. In addition, the stability of highly protease-susceptible proteins-histatin 5, statherin, and PRP1-was assessed. Experimental variables included (a) protease inhibitors, (b) salivary pH, (c) incubation temperatures, and (d) sample heating. A cocktail containing AEBSF, aprotinin, pancreatic trypsin inhibitor, leupeptin, antipain, and EDTA could not prevent histatin 5, statherin, or PRP1 degradation in whole saliva. Among the other treatments evaluated, short-term storage of freshly collected samples on ice was effective without interfering with the chemistry of the proteome. In conclusion, whole saliva contains a unique mixture of enzymes as evidenced from their resilience to protease inhibition. Analytical evidence on protein stability is needed to ensure the validity of salivary biomarker study outcomes. Analysis of the data presented will provide help and guidance for the use of saliva samples for diagnostic purposes.

Evidence of intact histatins in the in vivo acquired enamel pellicle.

Understanding the composition and function of the acquired enamel pellicle (AEP) has been a major goal in oral biology. The aim of this study was to test the hypothesis that intact histatins are part of the in vivo AEP and that histatins after adsorption to HA have effects on in vitro enamel demineralization. This is the first study demonstrating the presence of intact histatins in vivo in the AEP. The in vitro experiments show that all naturally occurring histatins in the AEP have the potential to provide some level of protection against acid injury.

Multiple components contribute to ability of saliva to inhibit influenza viruses.

INTRODUCTION: Saliva is a potentially important barrier against respiratory viral infection but its mechanism of action is not well studied. METHODS: We tested the antiviral activities of whole saliva, specific salivary gland secretions, and purified salivary proteins against strains of influenza A virus (IAV) in vitro. RESULTS: Whole saliva or parotid or submandibular/sublingual secretions from healthy donors inhibited IAV based on hemagglutination inhibition and neutralization assays. This differs from human immunodeficiency virus (HIV), for which only submandibular/sublingual secretions are reported to be inhibitory. Among purified salivary proteins, MUC5B, scavenger receptor cysteine-rich glycoprotein 340 (salivary gp-340), histatins, and human neutrophil defensins (HNPs) inhibited IAV at the concentrations present in whole saliva. In contrast, some abundant salivary proteins (acidic proline-rich proteins and amylase) had no activity, nor did several other less abundant salivary proteins with known activity against HIV (e.g. thrombospondin or serum leukocyte protease inhibitor). Whole saliva and MUC5B did not inhibit neuraminidase activity of IAV and viral neutralizing and aggregating activity of MUC5B was potentiated by the neuraminidase inhibitor oseltamivir. Hence, MUC5B inhibits IAV by presenting a sialic acid ligand for the viral hemagglutinin. The mechanism of action of histatins requires further study. CONCLUSIONS: These findings indicate that saliva represents an important initial barrier to IAV infection and underline the complexity of host defense activity of oral secretions. Of interest, antiviral activity of saliva against IAV and HIV differs in terms of specific glandular secretions and proteins that are inhibitory.

Proteome of human minor salivary gland secretion.

Recent research efforts in oral biology have resulted in elucidation of the proteomes of major human salivary secretions and whole saliva. One might hypothesize that the proteome of minor gland secretions may show significantly different characteristics when compared with the proteomes of parotid or submandibular/sublingual secretions. To test this hypothesis, we conducted the first exploration into the proteome of minor salivary gland secretion. Minor gland secretion was obtained from healthy volunteers, and its components were subjected to liquid-chromatography-electrospray-ionization-tandem-mass-spectrometry. This led to the identification of 56 proteins, 12 of which had never been identified in any salivary secretion. The unique characteristics of the minor salivary gland secretion proteome are related to the types as well as the numbers of components present. The differences between salivary proteomes may be important with respect to specific oral functions.

Saliva: a dynamic proteome.

The proteome of whole saliva, in contrast to that of serum, is highly susceptible to a variety of physiological and biochemical processes. First, salivary protein secretion is under neurologic control, with protein output being dependent on the stimulus. Second, extensive salivary protein modifications occur in the oral environment, where a plethora of host- and bacteria-derived enzymes act on proteins emanating from the glandular ducts. Salivary protein biosynthesis starts with the transcription and translation of salivary protein genes in the glands, followed by post-translational processing involving protein glycosylation, phosphorylation, and proteolysis. This gives rise to salivary proteins occurring in families, consisting of structurally closely related family members. Once glandular secretions enter the non-sterile oral environment, proteins are subjected to additional and continuous protein modifications, leading to extensive proteolytic cleavage, partial deglycosylation, and protein-protein complex formation. All these protein modifications occur in a dynamic environment dictated by the continuous supply of newly synthesized proteins and removal by swallowing. Understanding the proteome of whole saliva in an environment of continuous turnover will be a prerequisite to gain insight into the physiological and pathological processes relevant to oral health, and be crucial for the identification of meaningful biomarkers for oral disease.

Acquired enamel pellicle and its potential role in oral diagnostics.

The acquired enamel pellicle (AEP) is a protein film with unique composition and properties, which is formed by the selective adsorption of a variety of oral fluid-derived proteins onto tooth enamel surfaces. Since events leading to caries and periodontal disease occur in close proximity to the tooth surface, pellicle constituents are likely to contain biomarkers valuable for diagnostic applications. Despite the importance of this oral structure, progress in understanding its formation and composition has been slow because of difficulties in efficient pellicle collection methods and limitations of biochemical techniques for the characterization of microgram amounts of proteins/peptides. Recent developments in both pellicle collection methods and nanoscale sensing technologies have brought the exploitation of pellicle analysis into the realm of point-of-care oral diagnostics.

Oral fluid proteolytic effects on histatin 5 structure and function.

Histatins are human salivary antifungal proteins that are prone to extensive enzymatic degradation upon their release into the oral cavity. Histatin proteolysis, leading to the disappearance of the intact protein can be expected to have functional consequences. Histatin 5, comprising 24 residues, is the smallest of the major salivary histatins and the most active in terms of its antifungal properties. The rate and mode of histatin 5 degradation were determined by incubating the protein in whole saliva supernatant for various time intervals. Fragmentation products were collected by reversed-phase high performance liquid chromatography (RP-HPLC), characterised structurally by matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry and functionally in a fungal growth inhibition assay. Of the 19 fragments identified, 16 were derived from single proteolytic cleavage events in histatin 5. A remarkable finding was the inter-subject consistency in the histatin 5 degradation pattern. Added histatin 5 disappeared from whole saliva supernatant at an average rate of 105+/-22 microg/ml/h, which in part could explain the virtual absence of histatin 5 in whole saliva. Despite the rapid proteolysis of histatin 5, the early degradation mixture was as active in antifungal assays as intact histatin 5. These data demonstrate that the oral-fluid mediated proteolysis of histatin 5 represents an intrinsic biological property of whole saliva. The data also reveal that the early proteolysis phase of histatin 5 does not abolish the antifungal properties associated with this protein.

Identification of early microbial colonizers in human dental biofilm.

AIMS: To elucidate the first colonizers within in vivo dental biofilm and to establish potential population shifts that occur during the early phases of biofilm formation. METHODS AND RESULTS: A 'checkerboard' DNA-DNA hybridization assay was employed to identify 40 different bacterial strains. Dental biofilm samples were collected from 15 healthy subjects, 0, 2, 4 and 6 h after tooth cleaning and the composition of these samples was compared with that of whole saliva collected from the same individuals. The bacterial distribution in biofilm samples was distinct from that in saliva, confirming the selectivity of the adhesion process. In the very early stages, the predominant tooth colonizers were found to be Actinomyces species. The relative proportion of streptococci, in particular Streptococcus mitis and S. oralis, increased at the expense of Actinomyces species between 2 and 6 h while the absolute level of Actinomyces remained unaltered. Periodontal pathogens such as Tannerella forsythensis(Bacteroides forsythus), Porphyromonas gingivalis and Treponema denticola as well as Actinobacillus actinomycetemcomitans were present in extremely low levels at all the examined time intervals in this healthy group of subjects. CONCLUSION: The data provide a detailed insight into the bacterial population shifts occurring within the first few hours of biofilm formation and show that the early colonizers of the tooth surface predominantly consist of beneficial micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The early colonizers of dental plaque are of great importance in the succession stages of biofilm formation and its overall effect on the oral health of the host.

Statherin is an in vivo pellicle constituent: identification and immuno-quantification.

Recently, we demonstrated that anti-statherin monoclonal antibodies could be generated upon immunisation of mice with in vivo formed human acquired enamel pellicle, indicating that statherin is a constituent of pellicle. To gain insight in the in vivo adsorption behaviour of statherin we tested the abundance of statherin in pellicle and investigated the relationship between statherin and protein levels in salivary secretions and pellicle using a capture ELISA. Statherin levels were approximately 20-fold higher in parotid and submandibular-sublingual secretions than in cleared whole saliva supernatant or pellicle, suggesting the rapid degradation of statherin in the oral cavity. A strong positive correlation was observed between statherin and protein levels in pellicle but not in saliva indicating that statherin and protein adsorption to pellicle are related processes. This indicates that statherin represents the integral part of proteins that constitute the pellicle structure and may play a key role in its formation.

Identification of in vivo pellicle constituents by analysis of serum immune responses.

Human acquired enamel pellicle is composed of molecules that selectively adsorb from saliva onto tooth surfaces and provides a protective interface between the tooth enamel and the oral environment. To identify the micro-amounts of components present in pellicle, we immunized mice with in vivo-formed human acquired enamel pellicle and analyzed the serum immune responses. Selective reactivities of the serum (OD > 1.0 above background) against albumin, amylase, carbonic anhydrase II, sIgA, IgG, IgM, lactoferrin, lysozyme, proline-rich proteins, statherin, histatin 1, and mucous glycoprotein 1 were observed. We further confirmed the presence of proline-rich proteins, lactoferrin, lysozyme, and carbonic anhydrase II by probing in vivo pellicle with specific polyclonal anti-sera. The polyclonal antibody approach provided a powerful method for the identification of various pellicle proteins, including some which show mineral homeostasis or antimicrobial activity.

Characterization of the immunologic responses to human in vivo acquired enamel pellicle as a novel means to investigate its composition.

Human acquired enamel pellicle is formed by molecules selectively adsorbed onto tooth surfaces. The present work describes the use of monoclonal antibody (mAb) technology as a novel approach to identify micro amounts of components present in pellicle. MAbs were obtained with reactivities against statherin, histatin, mucous glycoprotein 1(MGI), albumin, amylase and human immunoglobulins (Igs), indicating that these are pellicle components, which was further confirmed by immunoblotting. No mAbs against proline-rich proteins (PRPs), lysozyme, mucous glycoprotein 2 (MG2), carbonic anhydrase, lactoferrin or peroxidase were obtained, suggesting that these components are absent, present in low amounts, or exhibit low antigenicity. Further characterization of the binding epitopes of some of th e obtained anti-MGO, anti-statherin and anti-histatin mAbs were carried out and the biological relevance is discussed. The results open up the possibility that immunization with human pellicle and mAbs production can be employed to identify hitherto unknown constituents of pellicle.

Inhibitory effect of synthetic histatin 5 on leukotoxin from Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is a gram-negative bacterium strongly implicated in the pathogenesis of juvenile periodontitis. This periodontal pathogen synthesizes a leukotoxin that destroys human polymorphonuclear leukocytes (PMNs), and this toxin is thought to be responsible for the virulence of A. actinomycetemcomitans. It was therefore of interest to assess whether major virulence factors of periodontal pathogens were neutralized by salivary components. This study focuses on the effect of histatins, components of the nonimmune oral defense system, on leukotoxin activity. Leukotoxin was extracted with polymyxin B from freshly grown anaerobic cultures of A. actinomycetemcomitans strain Y4. PMNs isolated from blood of healthy human volunteers were incubated in a cytotoxicity assay containing PMNs (10(7) cells/ml) and leukotoxin preparation (0-500 microg/ml) in Hanks' balanced salt solution at 37 degrees C for 0-120 min with or without synthetic histatin 5 (0-500 microM). Cytotoxicity was measured by release of lactate dehydrogenase (LDH) at different time intervals. Histatin 5 neutralized the toxic effect of the leukotoxin preparation in a concentration-dependent manner, with an IC(50) value of 150 microM. When PMNs were preincubated with histatin 5 (300 microM), washed and subsequently exposed to leukotoxin, no protective effect was observed. This observation suggests a mechanism of inhibition whereby histatin 5 either directly neutralizes the leukotoxin or interferes with the leukotoxin-PMN interaction. The inhibitory effect of histatin 5 on leukotoxic activity may suggest a new biological function of histatins in the oral cavity as a naturally occurring secondary antibiotic.

The human salivary peptide histatin 5 exerts its antifungal activity through the formation of reactive oxygen species.

Previous studies have shown that the human salivary antifungal peptide histatin 5 is taken up by Candida albicans cells and associates intracellularly with mitochondria. The purpose of the present study was to investigate the biological consequence of this specific subcellular targeting. Histatin 5 inhibited respiration of isolated C. albicans mitochondria as well as the respiration of intact blastoconidia in a dose and time-dependent manner. A nearly perfect correlation was observed between histatin-induced inhibition of respiration and cell killing with either logarithmic- or stationary-phase cells, but stationary-phase cells were less sensitive. Because nonrespiring yeast cells are insensitive to histatin 5, the potential mechanistic relationship between histatin 5 interference with the respiratory apparatus and cell killing was explored by using an oxygen radical sensitive probe (dihydroethidium). Fluorimetric measurements showed that histatin 5 induced the formation of reactive oxygen species (ROS) in C. albicans cells as well as in isolated mitochondria and that ROS levels were highly correlated with cell death. In the presence of an oxygen scavenger (l-cysteine), cell killing and ROS formation were prevented. In addition, the membrane-permeant superoxide dismutase mimetic 2,2,6,6-tetramethylpiperidine-N-oxyl, abolished histatin-induced ROS formation in isolated mitochondria. In contrast to histatin 5, the conventional inhibitors of the respiratory chain, sodium cyanide or sodium azide, neither induced ROS nor killed yeast cells. These data provide strong evidence for a comprehensive mechanistic model of histatin-5-provoked yeast cell death in which oxygen radical formation is the ultimate and essential step.

A new method for the isolation of histatins 1, 3, and 5 from parotid secretion using zinc precipitation.

Histatins, a family of small-molecular-weight, histidine-rich cationic salivary proteins, have been difficult to isolate in an efficient way by conventional procedures due to their anomalous interactions with chromatographic resins. In the present study we explored the possibility of developing a new isolation procedure based on recent observations that histatins associate with various metal ions, including zinc. Since solubility studies showed that histatin 5 forms precipitates with zinc under alkaline conditions, we investigated whether this characteristic could be exploited for the preparative isolation of histatins from salivary secretions. A fast and efficient two-step procedure was developed using zinc precipitation of histatins from human parotid secretion followed by final purification using reversed-phase high-performance liquid chromatography (HPLC). Analysis of zinc precipitates by Tricine-SDS-PAGE, cationic PAGE, HPLC, and mass spectrometry revealed the presence of the three major histatins, 1, 3, and 5, as well as statherin. The histatin yield obtained by the precipitation step was approximately 90%. Therefore, zinc precipitation of histatins from glandular salivary secretions is a novel, rapid, and effective means for the isolation of these proteins.