Quantitation of Histatins in Human Salivary Secretions by Zinc Precipitation

Histatins are human salivary peptides enriched in histidines that associate with metal ions including zinc. In a previous study we demonstrated that under alkaline conditions, histatin-zinc complexes precipitate, and that this feature can be employed to isolate histatins from parotid salivary secretions. Objective: To investigate the suitability of the zinc precipitation technique to quantify histatins in parotid secretion (PS), submandibular/sublingual secretions (SM/SL) and whole saliva supernatant (WS). Methods: PS, SM/SL and WS were obtained from three individuals, 500 µM zinc chloride was added to 1 ml of each secretion in triplicate, histatins were precipitated by raising the pH to 9, and the pellet was analyzed by reversed-phase HPLC. To some samples, 50 µ g synthetic histatin 5 was added to determine the percent recovery after zinc precipitation. Results: Synthetic histatin 5, added to PS, SM/SL or WS secretion could be recovered by 100%, 80% and 80%, respectively, indicating that zinc precipitates histatins in all major salivary secretions but with different efficiency. Natural histatin levels (histatins 1+3+5) ranged between 4.5-5.4 mg% in PS, 14.0-18.4 mg% in SM/SL and 0.22-0.54 mg% in WS. The experimental standard error of approximately 10% could be drastically reduced to 1% when filters were washed after sample filtration. Conclusion: The zinc precipitation procedure is a fast and reliable method with low standard errors for the quantitation of histatins in human major salivary secretions and this will allow studies to explore the clinical significance of histatins in a variety of physiological and pathological conditions. This study was supported by NIH Grants DE05672 and DE07652.