Proteins in whole saliva during the first year of infancy.

During the first year of an infant's life, the oral environment is subject to drastic changes that coincide with the eruption of teeth. Proteins in saliva are important for protecting oral surfaces and provide receptors for bacterial adhesins. The objective of this longitudinal study was to monitor the general composition and expression of proteins in whole saliva of infants, to prove the hypothesis that expression of certain proteins changes during infant development, and might be associated with tooth eruption. The results showed a remarkable constancy in the overall pattern of salivary proteins and glycoproteins during infancy. Exceptions were the mucins and albumin. The mucins are expressed differentially, with first MUC7 and later MUC5B being predominant. Albumin, a marker of serum leakage, started to rise in whole saliva preceding tooth eruption. Thus, the expression of only few proteins appears to be changed during infant development.

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.

Pro-inflammatory cytokines up-regulate MUC1 gene expression in oral epithelial cells.

The membrane-bound mucin MUC1 is expressed ubiquitously on epithelial surfaces and is thought to provide protection from bacterial and chemical injury. The present study was undertaken to determine whether MUC1 was expressed in cultured oral epithelial cells and whether expression is modulated by pro-inflammatory mediators released as part of the host response to infection by oral pathogens. Northern and Western blotting experiments showed that KB cells express MUC1 mRNA and protein. When cells were treated with interleukins (IL-1beta, IL-6), tumor necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma), or combinations of these, real-time PCR demonstrated that MUC1 mRNA increased 1.4- to 3.2-fold. Interestingly, a significant increase in levels of MUC1 protein was also observed. While no effect was observed when KB cells were incubated with LPS from Porphyromonas gingivalis, infection of KB monolayers with this oral pathogen caused a 2.85-fold increase in MUC1 transcript levels. These results suggest that increased MUC1 synthesis may be a key element in the host response to infection with oral pathogens.

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.

MG2 and lactoferrin form a heterotypic complex in salivary secretions.

Protein-protein interactions are necessary for homeostasis to be maintained and for biological systems to be integrated. Heterotypic complexes occur in saliva, and a complex between MG2 and SIgA has been suggested to promote microbial clearance from the oral cavity. In this study, we used a peptide display library to investigate previously unrecognized heterotypic complexes involving MG2 and other proteins. The library was panned with MG2 12 times, and analyses of clones identified the sequence Ala-Leu-Leu-Cys-, which occurs in salivary lactoferrin. Blotting experiments confirmed that MG2 and lactoferrin form a heterotypic complex in vitro and in vivo. Periodate treatment of MG2 did not affect the interaction. A synthetic lactoferrin peptide containing the motif Ala-Leu-Leu-Cys-blocked the interaction between MG2 and lactoferrin, confirming the specificity of the interaction identified by panning. This complex may enhance the properties of these salivary components in the oral environment.

Physical parameters of hydroxyapatite adsorption and effect on candidacidal activity of histatins.

Histatins 1, 3 and 5 are the major members of a histidine-rich protein family present in human salivary secretions. These proteins are distinct from many salivary proteins in their high positive charge density at neutral pH, and their antibacterial and antifungal properties. In this study, the hydroxyapatite adsorption characteristics of histatin 1, containing a single phosphoserine residue, recombinantly expressed histatin 1, native histatin 3, synthetic histatin 5 and an internal 12-residue sequence of histatin 5 were investigated. A Langmuir-type model was used to analyse the adsorption. A comparison of the affinities and binding sites of phosphorylated and recombinant histatin 1 provided an estimate of the positive influence of the single phosphoseryl group on mineral adsorption. Furthermore, an apparent correlation was shown to exist between peptide chain length and the number of binding sites. The influence of histatin 5 adsorption on its anticandidal activity was also investigated by performing Candida albicans killing assays with histatin 5 and histatin 5/hydroxyapatite suspensions. A decrease in killing activity was observed with the increase of hydroxyapatite present. The results suggest that the anticandidal properties of histatin 5 could be impaired by the conformations resulting from mineral adsorption, or that putative cellular receptors necessary for candidacidal activity are inaccessible when histatin 5 is adsorbed on hydroxyapatite.

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.

The effects of duration and intensity of stimulation on total protein and mucin concentrations in resting and stimulated whole saliva.

The present investigation has characterized the influence of the duration and intensity of stimulation on the secretion pattern of total protein and salivary mucins MG1 and MG2 in whole saliva. Resting and stimulated whole saliva was collected from six healthy subjects on 2 consecutive days. Whole saliva was collected for 2 five-minute intervals under resting conditions followed by collection under masticatory stimulation induced by the chewing of parafilm (1 g) at 10 or 60 strokes/min for 15 min. Flow rates were different under the 2 levels of stimulation. The concentration of total protein was different in resting and stimulated whole saliva but was not affected by the duration or intensity of stimulation. Analysis of mucin concentrations determined by capture ELISAs revealed that the pattern of MG1 secretion was similar to that of total protein. The pattern of MG2 secretion was unique in that no differences were observed in the concentration of this mucin under resting and stimulated conditions. This study shows that the pattern of protein secretion in whole saliva does not reflect the combined pattern observed for protein secretion in parotid and submandibular/sublingual glands, and that the secretion patterns of MG1 and MG2 in whole saliva are quite different from one another.

Is salivary histatin 5 a metallopeptide?

Histatins are small histidine-rich salivary polypeptides which exhibit antimicrobial activity against Candida albicans. This antimicrobial activity has been ascribed in part to a high content of basic amino acids. However, unlike most other antimicrobial proteins histatins have a high content of histidine, tyrosine and acidic amino acids known to participate in metal ion coordination. This study was conducted to test whether histatin 5 could bind zinc and copper which are metals present in salivary secretions and whole saliva. Physical binding parameters and spectral properties of zinc- and copper-histatin complexes were investigated in order to obtain direct evidence of these interactions. A spectrophotometric competition assay using the metallochromic indicator murexide showed that histatin 5 dissociates metal indicator complexes containing zinc or copper ions. Absorption spectra of histatin 5 at increasing copper chloride concentrations resulted in higher absorbance in the 230-280 nm wavelength range and this spectral change was saturated at a peptide:metal molar ratio of approx. 1:1. A corresponding band was observed in the visible range of the spectrum with a maximum and molar extinction coefficient corresponding to that of copper binding to an ATCUN motif. Quantitative assessment of zinc and copper binding to histatin 5 using isothermal titration calorimetry revealed at least one high affinity site for each metal, with binding constants of 1.2x10(5) and 2.6x10(7) M(-1), respectively. These results indicate that histatin 5 exhibits metallopeptide-like properties. The precise biological significance of this has not yet been established but histatins may contribute significantly to salivary metal binding capacity.

Salivary histatin 5 is a potent competitive inhibitor of the cysteine proteinase clostripain.

Histatin 5 is a low molecular weight salivary protein which is known to exhibit inhibitory activity against several proteinases, including the cysteine proteinases gingipains. The purpose of this study was to characterize the effect of salivary histatin on the proteolytic activity of the cysteine proteinase clostripain derived from the pathogen Clostridium histolyticum. Using a synthetic nitroanilide substrate, we studied in detail the inhibition of clostripain by histatin 5 and compared the effect of this peptide to that of leupeptin, a known competitive inhibitor of clostripain. It was found that the concentration of histatin 5 required to inhibit 50% of clostripain activity was 23.6+/-1.6 nM. Kinetic analysis revealed that histatin 5 is a competitive inhibitor of clostripain with an inhibition constant (K(i)) of 10 nM. The K(i) for the inhibition of clostripain activity against nitroanilide substrate by leupeptin was found to be 60 nM, significantly higher than that of histatin 5. Thus, histatin 5 inhibits clostripain more effectively than leupeptin and other cysteine protease inhibitors studied here. No significant proteolysis of histatin 5 was observed when histatin 5 was incubated at physiologic concentrations with clostripain. The potent inhibition of clostripain by histatin 5 points towards the possibility that this protein may prevent establishment of clostridial infections and therefore may have significant potential for the treatment of diseases associated with this enzyme.

New in vitro model for the acquired enamel pellicle: pellicles formed from whole saliva show inter-subject consistency in protein composition and proteolytic fragmentation patterns.

The present investigation was undertaken to investigate the variability of proteins in whole saliva which adsorb to hydroxyapatite and are thus likely to be precursors of the acquired enamel pellicle. Whole-saliva proteins from 18 subjects were absorbed to hydroxyapatite, and the gel filtration patterns of released proteins revealed four major peaks and three minor peaks eluting between the major peaks. Amino acid analysis indicated that minor peaks contained fragments of proteins in major peaks, and this was confirmed by sequence analysis. Major peaks comprised 95% and minor peaks comprised 5% of protein absorbed to hydroxyapatite, suggesting a limited proteolytic capacity of whole saliva. HPLC elution patterns of components in minor peaks suggested that proteolysis is not totally random but is an orderly and consistent process. These studies suggest that whole saliva may be suitable as a model system for the investigation of post-secretory modifications of salivary proteins important for the formation of the acquired enamel pellicle.

Salivary histatin 5 is an inhibitor of both host and bacterial enzymes implicated in periodontal disease.

One of the salient features of periodontitis and gingivitis is the increase in the levels of bacterial and host-derived proteolytic enzymes in oral inflammatory exudates. This study evaluated the potential of histatin 5, a 24-residue histidine-rich salivary antimicrobial protein, to inhibit these enzymes. Using biotinylated gelatin as a substrate, histatin 5 was found to inhibit the activity of the host matrix metalloproteinases MMP-2 and MMP-9 with 50% inhibitory concentrations (IC50s) of 0.57 and 0.25 microM, respectively. To localize the domain responsible for this inhibition, three peptides containing different regions of histatin 5 were synthesized and tested as inhibitors of MMP-9. Peptides comprising residues 1 to 14 and residues 4 to 15 of histatin 5 showed much lower inhibitory activities (IC50, 21.4 and 20.5 microM, respectively), while a peptide comprising residues 9 to 22 showed identical activity to histatin 5 against MMP-9. These results point to a functional domain localized in the C-terminal part of histatin 5. To evaluate the effect of histatin 5 on bacterial proteases, a detailed characterization of histatin 5 inhibition of gingipains from Porphyromonas gingivalis was carried out using purified Arg- and Lys-specific enzymes. Kinetic analysis of the inhibition of the Arg-gingipain revealed that histatin 5 is a competitive inhibitor, affecting only the Km with a K(i) of 15 microM. In contrast, inhibition of Lys-gingipain affected both the Km and Vmax, suggesting that both competitive and noncompetitive competitive processes underlie this inhibition. The inhibitory activity of histatin 5 against host and bacterial proteases at physiological concentrations points to a new potential biological function of histatin in the oral cavity.

Zinc and copper bind to unique sites of histatin 5.

Metal binding has been suggested to be relevant in the antifungal and antibacterial mechanism of histatin 5, a human salivary protein. Proton nuclear magnetic resonance (NMR) spectra were obtained to investigate the specificity of metal binding to the seven histidyl, one aspartyl and one glutamyl amino acid side-chains of histatin 5 in aqueous solutions. Three C(epsilon1)-H histidyl and the C(gamma)-H glutamyl resonances of histatin 5 were selectively altered in spectra of solutions containing three equivalents of zinc. Copper binding to histatin 5 resulted in a reduced intensity of C(beta)-H aspartyl resonances, while no evidence for calcium binding was found. These results indicate that zinc binding to histatin 5 involves His-15 present within the -H-E-X-X-H- zinc binding motif, and copper binding occurs within the N-terminal D-S-H-, ATCUN motif.

Characterization of histatin 5 with respect to amphipathicity, hydrophobicity, and effects on cell and mitochondrial membrane integrity excludes a candidacidal mechanism of pore formation.

Histatin 5 is a 24-residue peptide from human saliva with antifungal properties. We recently demonstrated that histatin 5 translocates across the yeast membrane and targets to the mitochondria, suggesting an unusual antifungal mechanism (Helmerhorst, E. J., Breeuwer, P., van't Hof, W., Walgreen-Weterings, E., Oomen, L. C. J. M., Veerman, E. C. I., Nieuw Amerongen, A. V., and Abee, T. (1999) J. Biol. Chem. 274, 7286-7291). The present study used specifically designed synthetic analogs of histatin 5 to elucidate the role of peptide amphipathicity, hydrophobicity, and the propensity to adopt alpha-helical structures in relation to membrane permeabilization and fungicidal activity. Studies included circular dichroism measurements, evaluation of the effects on the cytoplasmic transmembrane potential and on the respiration of isolated mitochondria, and analysis of the peptide hydrophobicity/amphipathicity relationship (Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595-623). The 14-residue synthetic peptides used were dh-5, comprising the functional domain of histatin 5, and dhvar1 and dhvar4, both designed to maximize amphipathic characteristics. The results obtained show that the amphipathic analogs exhibited a high fungicidal activity, a high propensity to form an alpha-helix, dissipated the cytoplasmic transmembrane potential, and uncoupled the respiration of isolated mitochondria, similar to the pore-forming peptide PGLa (Peptide with N-terminal Glycine and C-terminal Leucine-amide). In contrast, histatin 5 and dh-5 showed fewer or none of these features. The difference in these functional characteristics between histatin 5 and dh-5 on the one hand and dhvar1, dhvar4, and PGLa on the other hand correlated well with their predicted affinity for membranes based on hydrophobicity/amphipathicity analysis. These data indicate that the salivary protein histatin 5 exerts its antifungal function through a mechanism other than pore formation.

Killing of Candida albicans by histatin 5: cellular uptake and energy requirement.

Histatins, a group of histidine-rich proteins in human saliva, exhibit antimicrobial activity and are therefore considered to be important in the prevention of infections in the oral cavity. Although killing of C. albicans by histatins has been extensively studied, little is known about the processes responsible for this antifungal activity. Recent studies show the requirement of metabolic activity and ATP production for histatin 5 killing activity. Therefore, the goal of this study was to investigate the kinetics of histatin 5 interaction at different temperatures with C. albicans wild type cells and with respiratory deficient mutants of C. albicans. Synthetic histatin 5 was labeled with fluorescein-5-isothiocyanate (FITC) and its association with C. albicans cells was followed by epi-fluorescence microscopy and fluorescence confocal microscopy. At 37 degrees C, histatin 5 accumulates intracellularly, and both killing activity and uptake of unlabeled and FITC-labeled histatin 5 are time- and concentration-dependent. At 4 degrees C, no killing is observed and FITC-histatin 5 is only associated with the cytoplasmic membrane. Internalization and killing activity only occurs after cells are transferred to 37 degrees C. In addition, cellular accumulation of histatin 5 is concomitant with a moderate alteration of membrane integrity leading to the release of UV-absorbing cell components into the medium. The uptake of histatin 5, the release of UV-absorbing materials and killing of C. albicans are markedly decreased by the respiratory inhibitor sodium azide. Concomitantly, respiratory deficient mutants of C. albicans are also less susceptible to histatin 5. These results indicated that histatin 5 killing activity could be directly correlated to histatin 5 internalization. Both of these processes are prevented by modulators of cellular metabolic activity.

Salivary mucin: a factor in the lower prevalence of gastroesophageal reflux disease in African-Americans?

OBJECTIVES: Organic and inorganic constituents of saliva have been implicated as protective components in the esophagus, and deficiencies in one or more of these factors in different races may be an important element in the prevalence of gastroesophageal reflux disease (GERD). To determine whether there are differences in the concentration of salivary mucins between different racial groups, we measured the concentration of mucous glycoprotein MG1 and mucous glycoprotein MG2 in whole saliva of African-Americans and Caucasians. METHODS: Whole saliva was collected from 19 African-American (four male, 15 female; mean age 34 yr, range 19-53 yr) and 25 Caucasian (11 male, 14 female; mean age 31 yr, range 20-51 yr) volunteers under masticatory stimulation (1 g Parafilm, 60 strokes/min) between 11:00 AM and 12:00 noon. Total salivary carbohydrate was measured with a periodic acid-Schiff assay and total protein by absorbance at 215 nm. Immunological reagents were employed to quantify MG2 in a combined enzyme-linked immunosorbent assay/enzyme linked lectin assay (ELISA/ELLA) and to quantify MG1 in a capture ELISA. RESULTS: The total carbohydrate, protein, MG1 and MG2 values were 24.4 +/- 11.9, 243.5 +/- 62.7, 21.8 +/- 13.4, and 11.6 +/- 9.5 mg% for African-Americans, and the corresponding values were 23.3 +/- 9.3, 221.7 +/- 39.7, 25.7 +/- 16.2, and 10.9 +/- 8.7 mg% for Caucasians. There was no statistical difference for any of the parameters measured between the two groups. Furthermore, it was shown that no correlation existed between salivary flow rate and the concentration of carbohydrate, protein, or salivary mucins in African-Americans and in Caucasians. These results show that flow rate did not influence the measured values for salivary parameters in the two groups. CONCLUSIONS: No differences were found in the concentration of salivary mucins MG1 and MG2 in whole saliva of African-Americans and Caucasians, and it seems unlikely that variations in mucin levels influence the prevalence of GERD in these groups.