Changes in saliva of epileptic patients A. V. Nieuw Amerongen\ H. Strooker', C. H. Oderkerk', R. A. Bank', Y. M. C. Henskens', L. C. P. M. Schenkels\ A. J. IVI. Ligtenberg^ and E. C. I. Veerman^ Departments of ^Oral Biochemistry, 'Human Genetics, Academic Center for Dentistry, ACTA, Vrije Universiteit, Amsterdam, The Netherlands Nieuw Amerongen AV, Strooker H, Oderkerk CH, Bank RA, Henskens YMC, Sehenkels LCPM, Ligtenberg AJM, Veerman ECI; Changes in saliva of epileptic patients. J Oral Pathol Med 1992; 21; 203-8. Unstimulated whole saliva samples of 27 indoor epileptic patients were studied on their protein composition using biochemical and immunochemical methods. A number of salivary proteins appeared at least partially to be hydrolyzed. In a number of saliva samples the concentration of carbohydrate-containing isoen- zymes of amylase was reduced. In addition, the concentration of the 20 kD glycoprotein EP-GP was reduced by 60%. Sialic acid, the terminal sugar of the glycoproteins and mucins, was released for about 50% and in three salivas even nearly completely. Moreover, sialic acid- and fucose-containing epitopes could hardly be detected by monoclonal antibodies to human salivary mucins. As a consequence of this hydrolytic breakdown the saliva mediated aggregation of two S. sanguis strains had been reduced. In contrast, the aggregation of S. oralis had been maintained. Key words: amylase; bacterial aggregation; epileptic patients; saliva; sialic acid. A. V. Nieuw Amerongen, Academic Center for Dentistry, ACTA, Vrije Universiteit, Dept. of Oral Biochemistry, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands Accepted for publication January 12, 1992. Saliva plays a crucial role in maintain- ing the oral tissues healthy. Particularly the salivary protein components in- volved in defense mechanisms to micro- organisms are essential. Among the major components of saliva protecting the teeth surfaces and oral epithelial tissues against bacterial acidic attacks and enzymatic hydrolysis are the salivary mucins (1, 2). Salivary mucins in the soluble state induce the aggregation of a great number of oral micro-organisms (3) and are therefore involved in their oral clearance. Recently, we found that in saliva of patients with moderate periodontal dis- ease a specific group of salivary proteitis had been affected, namely the proline- rich proteins and glycoproteins (4). To study the infiuence of severe gingivitis and of periodontitis on salivary (glyco)- proteins, salivas of institutionalized epi- leptic patients, having a poor oral hy- giene, were tested. Particular attention was paid to the hydrolysis of covalently bound sialic acid and to the bacterial interaction with the salivary mucins. Material and methods Group of epileptic patients - The group of patients studied consisted of 27 indi- viduals with severe epileptic symptoms. hospitalized in an epilepsy centre in Heemstede, NL. The group was com- posed of 12 tnen and 15 women of an average age of 36+10 years, ranging from 17 to 57. Nearly all patients used a combined set of drugs, with predomi- nating Tegretol, Diphantoin, Propymal and Frisium. More than 70% of the pa- tients used one or more drugs with as side effect inhibition of salivary secre- tion. Collection of salivary samples - From twenty-seven institutionalized patients of an epilepsy centre unstimulated whole saliva samples were collected by expectoration into ice-chilled vessels. Immediately after coUectioti (usually within 30 min) salivas were frozen at -20°C, and stored until use. After thawing, salivas were clarified by cen- trifugation at 3,000 r.p.m. in a Sigma 2K table centrifuge at 4°C for 30 min. Each salivary sample was dispensed in aliquouts, which were stored at — 20°C until use in one of the various assays applied. As controls served salivary samples of healthy individuals and of edentate individuals, collected and treated essentially the same way. Electrophoretic protein pattern - To visualize the protein composition, par- ticularly the presence of proline-rich proteins, 10 typical salivary samples of epileptic patients were analyzed using an SDS-PAAGE system according to (5). The proline-rich proteins stained as pink-violet bands by Coomassie Bril- liant Blue R250 (4). Detection of a-amylase isoenzymes - To detect the pattern of a-amylase iso- enzymes, the saliva samples were elec- trophoresed on a polyacrylamide gel using the Biorad Protean apparatus, as described previously (6). Zymograms were developed using an atnyloclastic method, staining residual starch after complexing it with iodine. The amylase isoenzymes were visualized as white batids on a dark blue background, sta- bilized in 0.7% (v/v) solution of per- chloric acid (7). Quantification of the salivary glyco- protein EP-GP - To check whether sali- vary proteins had been submitted to partial proteolytic breakdown in saliva of epileptic patients, EP-GP, a 20 kD glycoprotein from the submandibular and sublingual gland, was used as an example (8). Quantification of this 20 kD salivary glycoprotein was performed by an ELISA using a specific monoclo- nal antibody (7E5) to EP-GP (9, 10). Quantification of salivary mucins - To obtain information on immunochemical properties of the salivary mucin fraction in saliva of epileptic patients, ELISA's