Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Effect of saliva collection methods and oral hygiene on salivary biomarkers.

Author(s): Justino AB, Teixeira RR, Peixoto LG, Jaramillo OLB, Espindola FS

Publication: Scand J Clin Lab Invest, 2017, Vol. , Page 1-8

PubMed ID: 28613965 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of six different saliva collection methods and tooth-brushing on saliva volume, total protein, nitrite and alpha-amylase secretion, and salivary antioxidant capacity. Saliva was collected between 8 and 9 AM from healthy non-smokers with no history of cavities or periodontal disease. Volunteers refrained from eating for at least 2 h and from drinking (including water) for 30 min before collection and volunteers rinsed their mouths with distilled water five minutes before saliva collection. From each volunteer, saliva was collected by three unstimulated (US) methods and three stimulated (SS) methods with a 5 min break between collections. US saliva was collected by accumulation of saliva in the mouth for 30 sec followed by continuous spitting for 90 sec (US1), accumulation of saliva in the mouth for 60 sec followed by spitting out (US2), or by spitting in the tube for 90 sec with no accumulation (US3). Stimulated saliva (SS) was collected by the Salivette method in which cotton was circulated in the mouth for 90 sec and then placed in a tube (SSI), chewing on Parafilm for 30 sec and then continuous spitting for 90 sec (SS2), or by  chewing mint-flavored gum for 30 sec then continuous spitting for 90 sec (SS3). To assess the effect of tooth-brushing, saliva was collected by the US1 method before tooth-brushing at 8 AM, teeth were brushed for 2 min with Colgate Total, and then a second specimen was collected by US1 at 8 AM. Saliva was stored at 4˚C until all specimens from an individual could be centrifuged at 1976 x g at 4˚C for 15 min. Supernatants were stored at -80˚C until analysis. Protein concentrations were determined by Bradford assay, alpha-amylase concentrations were determined by Western Blot, and nitrite concentrations were determined colorimetrically. Antioxidant capacity was determined by the spectrophotometric measurement of iron reduction.

   Summary of Findings:

   The flow rate of saliva (mL collected/min) was not significantly different among unstimulated (US) collection methods but was significantly higher when saliva was collected following chewing gum (SS3) than when collected by Salivette cotton (SS1), after chewing Parafilm (SS2), or by any of the unstimulated methods (P<0.001, all). Although the salivary flow rate was higher for collection by SS1 or SS2 thaen by accumulation of saliva in the mouth for 30 sec followed by continuous spitting for 90 sec (US1)(P<0.05, both) or by accumulation of saliva in the mouth for 60 sec followed by spitting out (US2) (P<0.05, both), it was not significantly higher than when collected by spitting in the tube for 90 sec with no accumulation (US3).

   Salivary secretion of total protein and nitrite and antioxidant capacity were significantly higher when saliva was collected by SS3 than by all other methods (P<0.001, P<0.001, and P<0.05, respectively). Salivary secretion of total protein was also higher when collected by SS2 than by SS1, US1, or US2.  Interestingly, salivary alpha-amylase was comparable among the stimulated saliva collection methods but was lower when collected by US3 than by US1 or by stimulated methods (P<0.05, all) and when collected by US2 than by SS3 (P<0.05).

   Salivary flow was lower 30 min after tooth-brushing than before (1.06 mL/min versus 1.31 mL/min, P<0.05) and specimens collected 30 min after rather than before tooth-brushing had a lower secretion rate of salivary protein (P<0.05) and higher alpha-amylase concentrations (P<0.05) but comparable amounts of nitrite and antioxidant capacity. 

   Biospecimens

   • Bodily Fluid - Saliva

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Colorimetric assay
   Protein	Western blot
   Small molecule	Spectrophotometry
   Small molecule	Enzyme assay
   Small molecule	Colorimetric assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Western blot Specific	Targeted peptide/protein	Alpha-amylase
   Biospecimen Acquisition	Method of fluid acquisition	Non-stimulated saliva Stimulated saliva (chewing prior to collection) Sublingual cotton roll Toothbrushing prior to collection Expectoration

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