Zinc Distribution in Human Saliva

Abstract

The aim of this study is to check the presence of zinc in salivary proteins and to define a relationship between zinc bonded with proteins and zinc free or bonded with low molecular weight substances.In order to avoid contamination of the examined material, we washed all glassware with HNO3 and then rinsed with bidistilled zinc-free water. Plastic equipment was used whenever possible. Previous atomic absorption spectrophotometric analysis of all the reagents and buffers demonstrated an insignificant content of zinc. We selected a group of 20 healthy male human subjects with good oral hygiene, aged from 20 to 40 years, previously clinically and radiologically checked. The sample collection took place in the morning: All subjects were instructed to refrain from breakfast, smoking, brushing of teeth; the samples were collected in sterile polystyrene test tubes fitted with plastic caps and immediately centrifuged at 20,000 x g for 30' at 00, thus separating pellet and supernatant, which were immediately frozen.When the required volume of saliva was reached, the samples were unfrozen and mixed obtaining two fractions: the 30 ml pellet and the 635 ml supernatant. Five hundred milliliters of supernatant were concentrated by ultrafiltration with Diaflo with PM 10 Arhicon membrane at 3 atm into 7 ml. We preferred to use the concentration by ultrafiltration to the lyophilization technique in order to avoid the increased danger of altering the proteins. From the ultrafiltrated 492 ml, 220 ml were again ultrafiltrated by using um 0.5 Amicon membrane in order to have the most exact evaluation for the zinc free or bonded with very low molecular weight substances. The quantitative analysis was made with an atomic absorption spectrophotometer Perkin Elmer 403 equipped with an airacetylene burner, and the zinc concentration was calculated by comparison with a standard zinc curve. We have also analyzed the specimens with two other techniques: mineralization at 2000 -3000 adding HNO3 plus H2SO4 and H202, and mineralization after lyophilization.We found no significant difference in the results obtained from the three different techniques. We found the following results: Zinc concentration in saliva pellet Concentration of zinc free or bonded with very low MW substances Concentration of zinc bonded with proteins with MW >~10,000 206 ppb (72.6%) 56 ppb (19.7%) 22 ppb ( 7.7%)We wish to point out the importance of the analysis of zinc concentration in the pellet, which has not been considered in other investigations.In order to study the distribution of zinc bonded with proteins in the various proteinic fractions, five milliliters (about 275 mg of proteins) of the material concentrated by ultrafiltration with PM 10 Amicon membrane were applied on a Sephadex G-150 column (3 x 100 cm), which was equilibrated with 0.10 M phosphate buffer (pH 6.8). The proteins were measured by two spectroscopic methods that reflect distinctive protein properties, absorbance at 215 nm and at 280 nm. Absorbance at 280 nm is determined principally by the amou...

Dose-Response Effects of Zinc and Fluoride on Caries Lesion Remineralization

“…[Zn] values represent those found naturally in saliva and after the use of Zn-containing oral care products [Burguera-Pascu et al, 2007]. Citrate addition to the artificial saliva was (a) a necessity to prevent Zn(OH) 2 precipitation, (b) highlights the fact that the majority of Zn-containing oral care products contain Zn-citrate, and (c) mimics an earlier study [Baratieri et al, 1979] showing that only a very low percentage of Zn is present in ionic form in saliva (citrate chelates Zn).…”