JDR JDR Most Cited Articles
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow Electronic Appendix
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Proctor, G.B.
Right arrow Articles by Rees, G.D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Proctor, G.B.
Right arrow Articles by Rees, G.D.
J Dent Res 84(1):73-78, 2005
© 2005 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Salivary Proteins Interact with Dietary Constituents to Modulate Tooth Staining

G.B. Proctor*, R. Pramanik, G.H. Carpenter, and G.D. Rees1

Salivary Research Unit, King’s College London, Floor 17, Guy’s Tower, London SE1 9RT, UK; and
1 GlaxoSmithKline R & D, St George’s Avenue, Weybridge, Surrey KT13 ODE, UK;

* corresponding author, Gordon.proctor{at}kcl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dietary components rich in polyphenols—for example, tea and red wine—are thought to cause tooth staining. In the present study, hydroxyapatite was used as a model of enamel for study of the influence of salivary proteins on the binding of different polyphenols to hydroxyapatite in vitro. Neither salivary protein pellicles nor salivary proteins in solution significantly altered the binding of the small polyphenol epigallocatechin to hydroxyapatite. However, hydroxyapatite binding of anthocyanin, a small grape-skin-derived polyphenol, or the larger polyphenols of black tea was increased by the presence of salivary proteins, either as a pellicle or in solution. Proline-rich proteins were enriched from parotid saliva and found to increase binding of anthocyanin and black tea polyphenols to hydroxyapatite, while enriched histatins did not increase binding. It is concluded that some salivary proteins, including proline-rich protein, can mediate increased staining of enamel by red-wine- and black-tea-derived polyphenols.

KEY WORDS: saliva • tooth staining


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Some individuals appear to be more susceptible to extrinsic tooth staining than others, but the reasons for such differences are unclear (Watts and Addy, 2001). It is likely that diet, smoking of tobacco, and oral hygiene play a role in tooth staining. Tea, coffee, red wine, and other foods rich in polyphenols are thought to be responsible for dietary tooth staining. Polyphenols are a diverse group of substances commonly found in plants. Polyphenols range from small flavanoids, such as the green tea catechins and grape-skin anthocyanins (molecular weight, 300), to highly polymerized structures containing more than 50 flavanol molecules, such as the black tea theaflavins (1 kDa) and thearubigins (> 1 kDa) (Bravo, 1999; Bennick, 2002).

Since saliva coats all oral surfaces in a mobile fluid layer and is responsible for the adherent protein pellicle on tooth enamel, it is very likely to influence the process of tooth staining. Saliva contains a high proportion (up to 70%; Kauffman and Keller, 1979) of proline-rich proteins. Acidic proline-rich proteins, along with statherins and histatins, have a high affinity for hydroxyapatite and are prominent components of the tooth enamel protein pellicle (Lamkin et al., 1996). Proline-rich proteins, particularly basic proline-rich proteins, have a particularly high affinity for dietary polyphenols (Hagerman and Butler, 1981), as do histatins (Bennick and Yan, 1995).

In the present study, we examined whether the salivary protein pellicle on teeth promotes tooth-staining and/or whether salivary proteins in solution might promote or reduce tooth staining. We used parotid saliva, since it contains histatins and proline-rich proteins and, since it is ductal saliva, interpretation of results is not complicated by the presence of exogenous components found in whole-mouth saliva.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Saliva
Parotid saliva was collected from volunteers from among the staff in the Department of Oral Pathology, King’s Dental Institute, according to a protocol approved by the local Ethical Review Committee. Salivation was stimulated with the use of a sugar-free lemon drop of known composition (Simpkins Ltd., Sheffield, UK) and collected by means of a sterilized Lashley suction cup placed over the orifice of each volunteer’s Stenson’s duct on the ipsilateral buccal mucosa. The first 5 drops of saliva were discarded and the rest collected in pre-weighed tubes on ice.

Enrichment of Salivary Proteins
Stimulated parotid saliva was boiled in the absence of detergent for 20 min and clarified by centrifugation. Histatins were isolated by zinc chloride precipitation under alkaline conditions (Flora et al., 2001). The supernatant obtained following zinc chloride precipitation contained predominantly proline-rich proteins and statherin. We obtained an enriched proline-rich protein fraction by precipitating statherin from supernatants using reversible trichloroacetic acid (TCA) precipitation (PlusOne 2-D Clean-up kit; Amersham Bioscience, Chalfont St Giles, UK). Briefly, TCA was added to a final concentration of 1%, samples were incubated on ice for 10 min, and the protein precipitate was collected by centrifugation at 14,000 rpm for 5 min at room temperature. The pH of the supernatant was increased to 7.0 with 50 mM sodium hydroxide and vortexed gently.

Following overnight dialysis against water, protein concentrations of the proline-rich protein and histatin fractions were determined by absorbance 215 nm assay with bovine serum albumin as a standard (Arneberg, 1971). The amount of histatin in the enriched fraction was also determined by comparison of staining intensity on electrophoresis gels with staining of known amounts of histatin 5 (Sigma Chem. Co. Ltd., Poole, UK).

Preparation of Protein Pellicles
A commercial hydroxyapatite (suspension in 0.001 M phosphate buffer at pH 6.8 with a solid content of 27%; Sigma Ltd.) was used routinely as follows. A volume of suspension equivalent to 5 mg of wet solid was centrifuged to remove fluid, and a 50-µL quantity of freshly collected parotid saliva or enriched proline-rich protein or histatins (see above) was added and made up to a final volume of 100 µL with distilled water. The mixture was vortexed and incubated for 1 hr at 37°C. Residual unadsorbed protein was removed and the remaining sediment washed once in 100 µL distilled water. The amount of salivary protein binding to hydroxyapatite was assayed by a modified Lowry assay (Petersen, 1977). Pellicles analyzed by SDS-PAGE were removed from hydroxyapatite by being boiled in LDS (lithium dodecyl sulphate) sample buffer (Invitrogen, Paisley, UK) containing 0.08% LDS and 0.006% EDTA (ethylenediaminetetraacetic acid).

Preparation of Black Tea Solution
Two grams of black tea leaves (Extra Strong Tea; Marks & Spencer Ltd., London, UK) were added to 100 mL of double-distilled water and boiled, stirred for 3 min, left to settle, and finally centrifuged (14,000 rpm for 5 min) and filtered to remove sediment, then used at room temperature. Tea was diluted 1:1 in distilled water.

Binding of Salivary Proteins to Black Tea Solution
A 50-µL quantity of parotid saliva was placed in an Eppendorf tube and a 1-, 10-, or 50-µL quantity of black tea solution (prepared as described above) was added; the total volume was made up to 50 µL with double-distilled water. The mixture was incubated for 1 hr at 37°C and centrifuged (14,000 rpm in a bench-top Microfuge) before the supernatent was prepared for electrophoresis (see above).

Assay of Polyphenol Binding to Hydroxyapatite
Epigallocatechin (Sigma Ltd.) binding to hydroxyapatite was assayed with Folin & Ciocalteu’s reagent according to a method adapted from Hagerman et al.(2000). A 50-µL quantity of double-distilled water or salivary protein solution was added to an Eppendorf tube containing 5 mg of hydroxyapatite (Sigma Ltd.) in 50 µL of double-distilled water. The mixture was incubated at 37°C for 1 hr and pelleted by centrifugation. The pellet was washed with 100 µL of double-distilled water and re-pelleted. A 50-µL quantity of a fresh solution of epigallocatechin (500 µg/mL), prepared in distilled water, was added to the re-suspended pellet, incubated, and pelleted as above. The pellet was washed with 100 µL of double-distilled water and re-pelleted. Finally, a 100-µL quantity of a mixture (80:20) of 20% sodium carbonate and Folin phenol reagent was added to the re-suspended pellet. After 10 min, the mixture was pelleted by centrifugation, and absorbance at 655 nm was measured in a spectrophotometer. In other permutations of the assay, hydroxyapatite was incubated with salivary protein and epigallocatechin simultaneously.

A commercial black grape-skin extract consisting of a mixture of anthocyanins (Warner Jenkinson, King’s Lynn, UK) was used at a concentration of 160 µg/mL in distilled water (a concentration similar to that estimated for some red wines; Sanchez-Moreno et al., 2003). Anthocyanin binding to hydroxyapatite was assayed in different permutations, as described for epigallocatechin (see above). The final hydroxyapatite with bound anthocyanin was dissolved in a volume of hydrochloric acid (1 M), and absorbance was measured spectrophotometrically at 525 nm. Binding of black tea (prepared as described above) to hydroxyapatite was assayed as described for anthocyanin, and absorbance was measured at 415 nm.

In all assays, values obtained for hydroxyapatite protein pellicles alone were subtracted from those obtained following both protein and polyphenol. Results are expressed as means ± standard deviation (SD). For statistical comparisons, a one-way ANOVA was used where appropriate, followed by paired Student’s t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Binding of Salivary Proteins to Tea
As the amount of black tea added to parotid saliva was increased, so certain bands resolved by SDS PAGE were reduced in staining intensity, suggesting that an interaction between polyphenols and salivary proteins had occurred (Fig. 1AGo). However, as more tea was added, the stained gels became difficult to interpret, since tea was itself stained with Coomassie Brilliant Blue, appearing as a blue smear.



View larger version (51K):
[in this window]
[in a new window]
  		Figure 1. SDS-PAGE of parotid salivary proteins in 16% tricine electrophoresis gels (Invitrogen, Paisley, UK). Resolved proteins were stained (with 0.2% Coomassie Brilliant Blue R250 in 25% methanol, 10% acetic acid) in gels or transferred onto nitrocellulose for antibody binding (see online Appendix). (A) Interaction of parotid salivary proteins (S) with tea (T). Increasing amounts of tea (1, 10, and 50 µL) were added to saliva (50 µL), and interacting salivary proteins were depleted from the electrophoretic profile. Ten µL of sample (equivalent to approximately 25 µg of salivary protein) were added to sample wells. Arrows indicate some protein bands showing a noticeable depletion. Preliminary identities, assigned on the basis of electrophoretic mobility and staining characteristics, are proline-rich protein (A) and histatin (B). The mobilities of standard proteins (M), with relative molecular weights ranging from 210 down to 3 kDa, are shown. (B) The salivary proteins binding to hydroxyapatite (HA) from a sample of parotid saliva (S) under assay conditions (1 hr at 37°C). The presence of statherin binding to hydroxyapatite is shown by a Western blot (W) of the proteins eluted from hydroxyapatite, probed with an anti-statherin antibody. (C) Histatin (Hp) and proline-rich protein (Pp) enriched fractions from saliva and the profiles of enriched proteins binding to hydroxyapatite under assay conditions. For comparison with the enriched histatin fraction, a sample of synthetic histatin 5 (Hs Sigma Chem. Co. Ltd.) is shown.

 
Formation of a Salivary Protein Pellicle on Hydroxyapatite
Electrophoresis demonstrated that a profile of proteins was bound to hydroxyapatite under the conditions used for assaying polyphenol binding (Fig. 1BGo). Comparison of mobility and staining characteristics of protein bands with synthetic histatin 5 (see Fig. 1CGo) and use of an anti-statherin anti-serum suggested that the main pellicle proteins were proline-rich proteins, histatins, and statherins. When parotid saliva samples from four individuals were compared, it was found that a broadly similar profile of proteins was bound to hydroxyapatite (data not shown).

We used the Folin phenol assay to estimate the salivary protein that was bound to 5 mg of hydroxyapatite. A linear standard graph (R2 = 0.98) for the Folin phenol assay of salivary protein in solution was compared with a curve for hydroxyapatite-bound protein. It was estimated that a maximum of approximately 40 µg of salivary protein was bound to 5 mg of hydroxyapatite, and that maximal binding was obtained when more than 100 µg salivary protein was added to hydroxyapatite.

Enrichment of Histatins and Proline-rich Proteins
SDS PAGE revealed that greatly enriched samples of histatins and proline-rich proteins were obtained (Fig. 1CGo). From a total of 27 mL of parotid saliva, approximately 500 µg of histatin and 59 mg of proline-rich protein were isolated, as determined by absorbance at 215 nm with a bovine serum albumin standard. When these samples of enriched proteins were used to form pellicles, it was found that bound histatins had an electrophoretic profile similar to that of the purified proteins when compared by SDS-PAGE. A subgroup of proline-rich proteins bound from the purified proline-rich protein fraction (see Fig. 1CGo).

Folin Phenol Assay of Epigallocatechin
There were linear relationships between absorbance at 655 nm and the amount of epigallocatechin in the presence or absence of hydroxyapatite (Fig. 2Go). The presence of mixed salivary protein pellicles on hydroxyapatite did not significantly alter the binding of epigallocatechin, since 1.1 ± 0.67 µg (mean ± standard deviation, n = 5) bound in the presence and 1.3 ± 0.33 µg (n = 5) bound in the absence of a salivary protein pellicle. When salivary protein and epigallocatechin were simultaneously added to the hydroxyapatite, similar results were obtained.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 2. Folin phenol assay of epigallocatechin (egc). (A) A standard curve of increasing amounts of epigallocatechin up to 25 µg in solution. (B) Increasing amounts of epigallocatechin (up to 25 µg) were incubated with 5 mg of hydroxyapatite, and the bound epigallocatechin was assayed. Approximately 0.25 µg of epigallocatechin bound to hydroxyapatite from an aqueous solution containing 50 µg of epigallocatechin. Values are means ± standard deviation of 6 assays.

 
Anthocyanin Binding to Hydroxyapatite
A linear relationship was obtained for increasing amounts of anthocyanin and absorbance at 525 nm under acidic conditions (Fig. 3AGo). When hydroxyapatite was coated in a salivary protein pellicle, anthocyanin binding was increased compared with anthocyanin binding to hydroxyapatite alone. A similar increase occurred if both protein and anthocyanin were added simultaneously (Fig. 3BGo). Pellicles formed on hydroxyapatite from 0.42 µg of histatins had no effect on the binding of anthocyanin to hydroxyapatite, although higher amounts (1.7 µg) reduced anthocyanin binding (compare H/AN with AN only in Fig. 3CGo). Pellicles formed from 25 or 100 µg proline-rich protein increased anthocyanin binding. Similar effects were seen when histatins or proline-rich proteins were incubated with anthocyanin and hydroxyapatite simultaneously (Figs. 3CGo, 3DGo). The amounts of purified histatins and proline-rich proteins used reflected approximately those present in saliva, 1.9 µg and 220 µg per 100 µL of saliva in the present study.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 3. Anthocyanin assay. (A) Standard curve of increasing amounts of anthocyanin in solution measured in the presence of 1 M HCl at 525 nm. Mean ± SD of 5 assays. Eight µg of anthocyanin gave an absorbance reading of approximately 1.00, and therefore this amount was used in the hydroxyapatite binding assay (Fig. 3BGo). (B) Anthocyanin (AN) and parotid saliva (S) sequentially or simultaneously bound to hydroxyapatite. Mean ± SD of 6 assays. Saliva first, then anthocyanin sequentially (S/AN); anthocyanin first, then saliva sequentially (AN/S); saliva and anthocyanin simultaneously (S+AN). Background absorbance due to saliva has been subtracted from the results. aS/AN and S+AN were statistically significantly greater than anthocyanin alone, p < 0.05 by paired t test. (C,D) Anthocyanin and salivary fractions sequentially or simultaneously bound to hydroxyapatite. Total amount of histatin (H) fraction in solution added to hydroxyapatite was 0.42 µg or 1.7 µg. Proline-rich protein (P) fraction in solution added to hydroxyapatite was 25 µg or 100 µg. Total amount of anthocyanin added to hydroxyapatite was 8 µg. Histatin first, then anthocyanin sequentially (H/AN); histatin and anthocyanin simultaneously (H+AN); anthocyanin only (AN). Proline-rich proteins first, then anthocyanin sequentially (P/AN); proline-rich proteins and anthocyanin simultaneously (P+AN); anthocyanin only (AN). Mean ± SD of 6 assays. Background absorbance due to histatins and proline-rich proteins has been subtracted from the results. Binding of anthocyanin was statistically different by ANOVA (p < 0.05) in the presence of histatins or proline-rich proteins. bAt 1.7 µg protein, H/AN and H+AN were statistically significantly lower than anthocyanin alone (p < 0.05, paired t test). aP/AN and P+AN were statistically significantly greater than anthocyanin alone (p < 0.05, paired t test).

 
Binding of Black Tea Polyphenols to Hydroxyapatite
There was a linear relationship between increasing amounts of black tea in solution and absorbance at 415 nm (Fig. 4AGo). Similarly, binding of black tea stain to hydroxyapatite, as assessed by the dissolution of hydroxyapatite with its bound black tea, showed a linear relationship between absorbance at 415 nm and the amount of black tea added (R2 = 0.94; results not shown). A salivary protein pellicle on hydroxyapatite increased the amount of black tea bound subsequently (Fig. 4BGo), as did the simultaneous addition of both black tea and salivary proteins (data not shown). Furthermore, histatins caused no change in binding of tea stain, whereas proline-rich protein increased tea stain binding.



View larger version (11K):
[in this window]
[in a new window]
  		Figure 4. Assay of black tea. (A) Standard curve with increasing amounts of black tea in solution measured in the presence of 1 M HCl at 415 nm. Mean ± SD of 5 assays. The amount of black tea polyphenols is unknown, but the figures shown are based on the volume of tea stock solution (2 g in 100 mL water) and published figures suggesting {approx} 1 mg polyphenol per mL tea (Bravo, 1999). (B) Assay of black tea and salivary protein added sequentially to hydroxyapatite. Total amounts of protein added were: parotid saliva only (S), 400 µg; histatin (H) fraction, 1.7 µg; and proline-rich protein (P) fraction, 100 µg. Mean ± SD of 3 assays. Saliva first, then tea sequentially (S/T); histatin first, then tea sequentially (H/T); proline-rich proteins first, then tea sequentially (P/T). aS/T and P/T were statistically significantly greater than black tea alone (p < 0.05, paired t test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A high proportion of human parotid salivary proteins is proline-rich protein (Bennick, 2002). Basic proline-rich proteins, in particular, have been found to interact strongly with dietary polyphenols (Lu and Bennick, 1998), and binding is thought to occur via the proline rings (Williamson, 1994). Low-molecular-weight histatins, another prominent group of salivary proteins, also bind dietary polyphenols such as those found in tea and red wine (Wroblewski et al., 2001). In our studies, such interactions were shown when we added tea to parotid saliva and analyzed proteins by SDS-PAGE. The disappearance of certain proline-rich proteins and histatins from electrophoretic profiles under non-reducing conditions can be explained by the formation of large soluble protein-polyphenol complexes that precede eventual precipitation (Williamson, 1994). However, the interactions that we observed did not result in significant precipitation, suggesting that complexes remained soluble under these conditions but were large enough to be excluded from the SDS gel. Electrophoresis of the same samples under reducing conditions (results not shown) produced a normal profile of salivary proteins, indicating that the disappearance of salivary proteins was not due to proteolytic degradation.

Specific salivary proteins bind to teeth and hydroxyapatite, the main mineral component of teeth. The salivary protein pellicle on teeth is often seen as a protective layer that maintains structural integrity by reducing mineral dissolution and mechanical abrasion. However, if polyphenol-binding proteins also bind to teeth, there is a potential for tooth staining (Nathoo, 1997). Under the present assay conditions, hydroxyapatite specifically bound statherin, proline-rich proteins, and some histatins, which agrees with the profile of proteins found by others (Lamkin et al., 1996; Schüpbach et al., 2001).

Our studies indicated that a protein pellicle had no quantitative effect on the binding of epigallocatechin, at a concentration of 500 µg/mL, to hydroxyapatite. This result is in contrast to that of Bacon and Rhodes (1998), who found that parotid salivary protein greatly increased binding of horseradish-peroxidase-labeled epigallocatechin to plastic microplates. The difference between the 2 studies may be due to the method of detecting epigallocatechin, since horseradish peroxidase is a large molecule (60 kDa) that might interfere with epigallocatechin (306 Da) binding to salivary proteins. In contrast, the binding of anthocyanin or black tea polyphenols to hydroxyapatite increased two-fold and four-fold, respectively, in the presence of parotid protein, either as a pellicle or in solution. The differences observed may be due to structural characteristics of the polyphenols, since epigallocatechin is small and neutral, anthocyanin is small and charged, and tea polyphenols are larger and more polar.

To investigate further whether specific salivary polyphenol-binding proteins can mediate increased polyphenol binding to hydroxyapatite, we studied histatins and proline-rich proteins. The enrichment of these proteins utilized their resistance to precipitation during boiling (personal communication). A pellicle of histatins on hydroxyapatite, formed at amounts that reflect those in saliva, did not increase black tea binding and even reduced anthocyanin binding. In contrast, a pellicle of proline-rich proteins increased binding of both anthocyanin and black tea. This is the first indication that, although both groups of proteins readily interact with polyphenols, their action, at the concentrations used, appears to differ in the presence of hydroxyapatite. These results suggest that proline-rich proteins, but not histatins, promote stain formation. Furthermore, when these proteins were incubated in solution with polyphenols in the presence of hydroxyapatite, proline-rich proteins promoted tooth staining but histatins did not. How might these results be explained? It may be that interaction with hydroxyapatite causes a conformational change in histatins, resulting in a reduced affinity for polyphenols. Acidic proline-rich proteins interact with hydroxyapatite and have lesser affinity for polyphenols than do basic proline-rich proteins (Bennick, 2002). However, it may be that interaction with hydroxyapatite increases the affinity of acidic proline-rich proteins for polyphenols.

The precipitation of higher-molecular-weight polyphenols with salivary proteins is thought to have a protective role in preventing the potential toxic effects of these molecules on the digestive system (Bennick, 2002). However, it may be that such interactions increase tooth staining, which is generally regarded as an unwanted occurrence (Nathoo, 1997). Lower-molecular-weight polyphenols, such as the flavanoids, are thought to have health-enhancing properties (Sakagami et al., 1999), including anti-cariogenic properties (Hamilton-Miller, 2001), which are almost certainly dependent on tooth binding, a mechanism influenced by salivary proteins.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge funding from GlaxoSmithKline Ltd., and helpful discussions with Dr. Graham Jackson and communication with Dr. Anders Bennick.


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received September 12, 2003; Last revision September 10, 2004; Accepted September 16, 2004


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Arneberg P (1971). Quantitative determination of protein in saliva. Scand J Dent Res 79:60–64.[Medline]

Bacon JR, Rhodes MJC (1998). Development of a competition assay for the evaluation of the binding of human parotid salivary proteins to dietary complex phenols and tannins using a peroxidase labelled tannin. J Agric Food Chem 46:5083–5088.

Bennick A (2002). Interaction of plant polyphenols with salivary proteins. Crit Rev Oral Biol Med 13:184–196.[Abstract/Free Full Text]

Bennick A, Yan Q (1995). Identification of histatins as tannin-binding proteins in human saliva. J Biochem 311:341–347.

Bravo L (1999). Polyphenols: chemistry, dietary sources, metabolism and nutritional significance. Nutrit Rev 56:317–333.

Flora B, Gusman H, Helmerhorst EJ, Troxler RF, Oppenheim FG (2001). A new method for the isolation of histatins 1, 3 and 5 from parotid secretion using zinc precipitation. Prot Exp Purif 23:198–206.[ISI][Medline]

Hagerman AE, Butler LG (1981). The specificity of proanthocyanidin-protein interactions. J Biol Chem 256:4494–4497.[Abstract/Free Full Text]

Hagerman AE, Mueller-Harvey I, Makkar HP (2000). Quantification of tannins in tree foliage: a laboratory manual for the FAO/IAEA co-ordinated research project on "The use of nuclear and related techniques to develop simple tannin assays for predicting and improving the safety and efficiency of feeding ruminants on tanninferous tree foliage". Jt FAO/IAEA Div Nuclear Rel Techn Food Agric, http://www.iaea.org/programmes/nafa/d3/crp/pubd31022manual-tannin.pdf

Hamilton-Miller JMT (2001). Anti-cariogenic properties of tea (Camellia sinesis). J Med Microbiol 50:299–302.[Abstract/Free Full Text]

Kauffman DL, Keller PJ (1979). The basic proline-rich proteins in human parotid saliva from a single subject. Arch Oral Biol 24:249–256.[ISI][Medline]

Lamkin MS, Arancillo AA, Oppenheim FG (1996). Temporal and compositional characteristics of salivary protein adsorption to hydroxyapatite. J Dent Res 75:803–808.[Abstract/Free Full Text]

Lu Y, Bennick A (1998). Interaction of tannin with human salivary proline-rich proteins. Arch Oral Biol 43:717–728.[ISI][Medline]

Nathoo SA (1997). The chemistry and mechanisms of extrinsic and intrinsic discoloration. J Am Dent Assoc 128:6S–10S.[Abstract]

Petersen GL (1977). A simplification of the protein assay method of Lowry et al., which is more generally applicable. Anal Biochem 83:346–356.[ISI][Medline]

Sakagami H, Takeshi O, Satoh K (1999). Prevention of oral diseases by polyphenols (review). In Vivo 13:155–172.[ISI][Medline]

Sanchez-Moreno C, Chao G, Ou B, Prior RL (2003). Anthocyanin and proanthocyanin content in selected white and red wines. Oxygen radical absorbance capacity comparison with nontraditional wines obtained from highbush blueberry. J Agric Food Chem 51:4889–4896.[ISI][Medline]

Schüpbach P, Oppenheim FG, Lendenmann U, Lamkin MS, Yao Y, Guggenheim B (2001). Electron microscopic demonstration of proline-rich proteins, statherin and histatins in acquired enamel pellicles in vitro. Eur J Oral Sci 109:60–68.[ISI][Medline]

Watts A, Addy M (2001). Tooth discolouration and staining: a review of the literature. Br Dent J 190:309–316.[ISI][Medline]

Williamson MP (1994). The structure and function of proline-rich regions in proteins. Biochem J 297:249–560.

Wroblewski K, Muhandiram R, Chakrabartty A, Bennick A (2001). The molecular interaction of human salivary histatins with polyphenolic compounds. Eur J Biochem 268:4384–4397.[ISI][Medline]





This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow Electronic Appendix
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Proctor, G.B.
Right arrow Articles by Rees, G.D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Proctor, G.B.
Right arrow Articles by Rees, G.D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
IADR Journals Advances in Dental Research ®
Journal of Dental Research ® Critical Reviews (1990-2004)