Changes Occurring on the Activity of Salivary Alpha-Amylase Proteoforms in Two Naturalistic Situations Using a Spectrophotometric Assay

Abstract

This study aimed to evaluate the changes in the activity of total salivary alpha-amylase (TsAA) and both the non-glycosylated and glycosylated salivary alpha-amylase proteoforms (NGsAA and GsAA, respectively) in physical and psychological stress models, estimated using a simple and easily set-up method. The method used was a spectrophotometric assay with 2-chloro-4-nitrophenyl-α-D-maltotriose (CNPG3) as a substrate, incubated with Concanavalin A (ConA) to remove most of the glycosylated protein from the sample. This method allowed the measurement of TsAA and estimation of NGsAA and GsAA activities with imprecision lower than 10%. When this method was applied to two different stress models, differences in the responses of the proteoforms were observed, with the NGsAA activity showing changes of higher magnitude after stress induction than the GsAA activity, and the highest correlation with the State–Trait Anxiety Inventory Scale in the Trier Social Stress Test (TSST). In conclusion, the activity of the two main sAA proteoforms can be easily estimated in saliva, and their measurement can provide additional information on TsAA activity in physical or psychological stress situations.

Validation of two immunoassays for oxytocin measurements in human saliva

“…The samples used for the CrossFit trial and TSST were samples from previous studies that were stored at -80°C. The CrossFit trial involved 11 male participants who performed a CrossFit workout in a CrossFit training center (Murcia, Spain), according with previous study by Contreras-Aguilar et al [ 27 ]. Saliva samples were collected at three time points: 5 minutes before the exercise (TBe), immediately after completing the exercise (T+0e), and 10 minutes after completion (T+10e).…”

“…Saliva samples for the TSST were collected from 14 female university students, with an average age of 26.2 ± 6.26 years, and the TSST was conducted according to a previous study by Contreras-Aguilar et al [ 27 ]. The participants performed the experimental procedure between 17:30 and 18:30.…”

“…Participants rinsed their mouths carefully with tap water 1 hour before the collection. Saliva samples were collected by passive flow for 1 minute using standard micro-centrifuge polystyrene tubes with a volume of 5 mL, following the method described by Contreras-Aguilar et al [ 27 ]. The samples were stored on ice until they arrived at the laboratory.…”

Enhancing saliva diagnostics: The impact of amylase depletion on MALDI-ToF MS profiles as applied to COVID-19

“…Salivary α-amylase, the most abundant protein in saliva, is an enzyme involved in breaking down starch and other polysaccharides. It exists in two proteoforms: non-glycosylated (∼56 kDa) and glycosylated (∼59 kDa) [31] , [32] , [33] . Various techniques are employed for α-amylase depletion, including the use of a syringe-based potato starch device [34] , gel filtration [35] , affinity chromatography with lectin ConA [31] , co-precipitation and paper-based chips [36] .…”

Amylase Depletion Significantly Improves the Maldi-Tof Profile of Saliva – Application to Covid-19 Diagnostics

“…It exists in two proteoforms: non-glycosylated (~56 kDa) and glycosylated (~59 kDa). [31], [32], [33] Several techniques are currently utilized for α-amylase depletion including a syringe-based potato starch device [34], gel filtration [35], affinity chromatography by lectin ConA [31], co-precipitation and paper-based chips [36]. Deutsch et al designed and patented an amylase depleting device to separate salivary α-amylase from the whole saliva by affinity adsorption to potato starch.…”