Method of salivary histatins determination

Histatins are the most significant antimicrobial peptides (AMP) of saliva and there are 12 types of such AMP. Histatin molecules contain relatively high percent of histidine and tyrosine residues. This property allows to use well known from organic chemistry Pauly reaction for detection of protein bounded histidine and tyrosine residues (BHT), which are in fact characterize the summary content of all histatins in saliva. Aim of the present study was comparison of BHT with antimicrobial activity of salivary AMP fraction in patients with inflammatory diseases of upper airways (IDUA). Group of examined persons include 28 patients with different diagnoses: chronic pharyngitis (n=11), chronic tonsillitis (n=7), nasopharyngitis (n=5), pollinosis (n=5). Degree of intensity of inflammatory symptoms was estimated in balls. The algorithm of BHT analysis include following steps: freezing - thawing of saliva; removal of microparticles by centrifugation; separation of fraction lower than100 kDa; dialysis for free amino acids removal; Pauly reaction carrying out. Antimicrobial activities of saliva and its low molecular fractions were estimated towards Candida albicans cells by the spectrophotometric method with bromocresol purpur. Analysis of saliva sediments for coccoid microbiota was carried out by PCR method. Pauly reaction for histatins estimation in saliva of IDUA patients use here for the first time. The histatins levels (BHT) were significantly correlated with the intensity of inflammatory symptoms (r=0,975) and activity of low molecular salivary fraction (AMP activity) (r=0,824). The AMP activity/ BHT ratio, i.e. antimicrobial activity of histatin unit, decreased together with growth of inflammatory symptoms intensity (r=-0,944). Any considerable differences in coccoid microbiota frequency of finding at different diagnoses were not detected. The S. aureus frequency of occurrence was connected neither with inflammatory symptoms intensity (r=0,118), nor with BHT concentration (r=0,318). However S. pyogenes and S. pneumoniae frequencies of occurrence demonstrated the invert correlation towards these indexes: (r=-0,627/-0,614) and (r=-0,827/-0,864). Probably at the exacerbation forms of IDUA the S. pyogenes and S. pneumoniae growth controlled by high levels of histatins.