Short communication Toothbrushing affects the protein composition of whole saliva G. Henk Hoek, Henk S. Brand, Enno C. I. Veerman, Arie V. Nieuw Amerongen Department of Basic Dental Sciences (section Oral Biochemistry), Academic Center for Dentistry (ACTA), Amsterdam, the Netherlands In the past two decades, saliva has been increasingly in- vestigated as an alternative to serum for a number of di- agnosticpurposes(1).Salivacanbecollectedinapainless and non-invasive manner, with low risk of infection (2), and many sensitive assays have been published for meas- uringsubstancesinsaliva(1).Therefore,itisexpectedthat a considerable number of blood tests will be replaced by salivatestsinthenearfuture.However,theuseofsalivafor diagnostic purposes may have some limitations. The composition of saliva shows considerable diurnal varia- tions and depends on mechanical and chemical stimuli of the secretion rate. In addition, a very small amount of blood, e.g. induced by toothbrushing, can influence the composition of saliva. Indeed, previous studies have re- portedthatbrushingoftheteethincreasedsalivaryplatelet activating factor (PAF) and testosterone concentrations (3,4).Toothbrushingalsoaffectedthetiterofhepatitis-A immunoglobulinsinsaliva(5).Theaimofthisstudywasto investigate the effect of toothbrushing according to a standardized protocol on the protein composition of sa- liva. Material and methods Fourteen volunteers (seven men and seven women, mean age 32 ± 11 yr) participated in this study. All volunteers were over the age of 18 yr and reported to have no systemic disease. None of the volunteers used prescribed or non- prescribed medication, except for the use of oral contra- ceptives in most women. All volunteers received written and oral instructions on the Bass method for toothbrushing. They were instructed to abstain from smoking, eating, drinking caffeine-containing beverages and toothbrushing at least 1 h prior to the experiment. The experiment took place between 1000 h and 1100 h. First, unstimulated whole saliva was collected for 5 min The collection period started with the instruction to swallow to minimize the volume of saliva in the oral cavity. Then saliva was allowed to accumulate in the floor of the mouth, and the subjects spat into preweighed ice-chilled test tubes every 60 s. During the collection period the volunteers were comfortably seated with eyes open, head tilted slightly for- ward and making minimal orofacial movements. Next, the volunteers brushed their teeth according to the Bass method for 2 min, using a new Lactona IQ softtip without toothpaste (Lactona, Warminster, USA). Immedi- ately after toothbrushing and 15, 30 and 45 min later, unstimulated saliva was collected for 5 min The secretion rates were determined gravimetrically. The salivary samples were transferred to Eppendorf vials, centrifuged at 10 000 g for 5 min and stored at )20°C. Amylase activity was determined using the quantitative kinetic determination kit (no 577 from Sigma Diagnostics, St Louis, MO, USA) as described by Henskens et al. (6). Proteinconcentrationwasquantifiedusingthebicinchoninic acidmethodwithbovineserumalbuminasastandard(7).A modifiedversionoftheenzyme-linkedimmunosorbentassay (ELISA) described by Armstrong et al. (8) was used to quantifyimmunoglobulinA(IgA)(6).UsingtheSpearman– Brownformula,thesplit-halfreliabilityoftheamylaseassay, protein assay and IgA ELISA were 0.98, 0.99 and 0.99, respectively (7). Albumin was quantified with a sandwich ELISA, as fol- lows: high-affinity microtiter plates (Greiner, Hannover, Germany) were coated with 0.15 lg ml )1 of purified immunoglobulins against human albumin (Dakopatts, Glostrup, Denmark). After this and each following Hoek GH, Brand HS, Veerman ECI, Nieuw Amerongen AV. Toothbrushing affects the protein composition of whole saliva. Eur J Oral Sci 2002; 110: 480–481. Ó Eur J Oral Sci, 2002 Saliva is increasingly used for diagnostic purposes, but little is known about the potential effect of toothbrushing on the composition of saliva. Therefore, saliva col- lected before and 0, 15, 30 and 45 min after toothbrushing according to the Bass method was analysed for relevant protein composition. The flow rate initially increased (+15%) after toothbrushing, followed by a decrease after 15 min ()15%). The concentration of salivary immunoglobulin A (S-IgA) decreased ()40%) whereas the albumin concentration increased (+400%) within 20 min. Flow rate and S-IgA concentration returned to baseline values within 30 min. The albumin concentration, however, was still increased after 45 min. No significant increases were observed in the concentrations of total protein and amylase. Our results show that toothbrushing has a significant effect on the protein composition of saliva, by contamination with serum constituents. This should be taken into account when saliva is used for diagnostic purposes. Henk S. Brand, Department of Basic Dental Sciences (section Oral Biochemistry), Academic Center for Dentistry (ACTA), v.d. Boechorststraat 7, NL – 1081 BT Amsterdam, The Netherlands Telefax: +31–20–4448685 E-mail: hs.brand.obc.acta@med.vu.nl Key words: albumin; amylase; immunoglobulin; protein; saliva Accepted for publication July 2002 Eur J Oral Sci 2002; 110: 480–481 Printed in UK. All rights reserved Copyright Ó Eur J Oral Sci 2002 European Journal of Oral Sciences ISSN 0909-8836