Rheological parameters of saliva in comparison with taste examination

Abstract

BACKGROUND:

It is generally unknown if taste sensitivity is dependent upon saliva viscosity. The rheological properties of saliva result from many factors and it has been shown to behave as a non-Newtonian fluid whose viscosity decreases with increasing shear rate. Taste sensitivity may be quantitatively assayed by electro-gustometry.

OBJECTIVES:

The aim of this work was to compare saliva rheological properties, obtained with a rotary-oscillating rheometer, to quantitative measures of taste sensitivity, using electro-gustometry.

METHODS:

Saliva samples were taken from 27 healthy non-smoking donors – 7 men and 20 women aged 18–65 years (mean age – 37 years). After thresholds of taste sensation were measured, the saliva samples were taken and characterized in terms of their rheological properties and pH. Saliva viscosity was measured in the order of decreasing shear rate in the range 100–0.01 s⁻¹. Viscoelastic properties were examined under constant frequency oscillations (with f = 0.5 Hz) and with decreasing shear effective amplitude ${\gamma}_{eff}^{\prime }$ .

RESULTS and CONCLUSIONS:

Saliva viscosity was found to decrease with increasing shear rate and varied with time. Analysis of the dependence of the viscosity values of saliva and components of complex viscosity did not show a significant correlation with taste sensation. A dependency of taste sensation on pH could not be discerned due to the narrow range of naturally occurring pH.

Keywords

Saliva viscosity gustometry viscoelastic properties

1. Introduction

Saliva is produced by salivary glands and has many physiologically important functions in the body [1–3]. The rheological properties of saliva result from many factors such as consistency, proteins profile, pH, presence of oral microorganisms, sex [1–5]. Rheological characteristics of every material must include two major properties: viscosity and elasticity. Viscosity is a parameter determining the resistance of the material to flow, while elasticity expresses the material resistance against deformation. Rheological studies of bio-materials like saliva are typically performed both by rotary measurements (flow curve) and oscillatory experiments (Dynamic Mechanical Analysis – DMA) [2,4,6–9]. Saliva is a non-Newtonian fluid whose viscosity decreases with increasing shear rate [2,4,6–9]. Apart from viscosity measurements, additional information about saliva rheology can be achieved from non-complex-viscometric oscillatory measurements. A measurement performed by means of oscillatory methods provides information about its viscoelastic properties, which can be expressed in terms of a complex viscosity: $\begin{eqnarray}\displaystyle {\eta}^{\ast }={\eta}^{\prime }+i{\eta}^{\prime \prime }, & & \displaystyle\end{eqnarray}$ 1 where: 𝜂^∗ - complex viscosity, 𝜂′ – viscous component, 𝜂′′ – elastic component.

Viscoelastic properties of human saliva is connected with the presence of proteins, glycoproteins and in particular mucins [4,7,9].

There are many methods of studying physiological oral taste sensation in humans [10,11]. As shown in the literature, the sense of taste is not only dependent on the disease processes but it can also be distorted because of using metal dentures, insufficient secretion of saliva, or adapting the receptors to previously taken liquids or food [12–14]. Prior to 1958, taste sensitivity had been tested by tasting calibrated solutions of standard substances (salt, sugar, etc.). Those qualitative methods were not suitable for quantitative analysis. In 1958, Krarup had published the first and basic article on the use of galvanic current for the quantitative examination of taste [10,11,15–18]. The instrument he constructed (and called an electro-gustometer) consisted of a source of electric current (a battery pack giving ∼100 V voltage), two electrodes (one on the tongue), a set of resistors reducing the effect of skin-electrode junction resistance variability, and a microamperometer. It is interesting that it is not completely clear yet what the origin of the taste felt on the tongue stimulated by an electrode really is. It was claimed that when anode had a contact with the tongue, H⁺ ions were produced, giving the characteristic acidic 'metallic’ taste. It was shown by Krarup that the taste thresholds obtained with his instrument as the minimum current required to invoke a taste sensation in patients correlated well with the subjective qualitative taste sensibility based on standard solutions. The taste threshold data showed distributions close to normal in given age groups and shifting with age towards higher values. Within the age groups the distributions were surprisingly broad, however for a given person the measured values were constant in time and almost identical on both sides of the tongue. Such results suggested that the electro-gustometer was a precise instrument capable of measuring the taste threshold. However the measured value was not a good indicator of taste impairment, unless it substantially deviated from the values characterizing the age group. Still, it turned out to be the first precise tool for quantitative characterization of the taste threshold. Despite the natural broad distribution of measured threshold values even in groups of healthy objects, the instrument could be successfully used for detecting changes in distribution parameters in groups of patients suffering from certain disorders or testing the changes of the taste threshold of an individual patient in response to environmental stimuli. Since that time, many instruments for that purpose have been constructed [17,18]. The amperage needed to evoke the sensation of taste is expressed in μA, in electric gust units (according to Krarup) or on a logarithmic scale in dB (Tomita, Rollin) [15]. The time of stimulation should be from 0.5 to 1 second. When the stimulation is longer or shorter, the threshold of taste elevates. In the analysis of taste by means of an electro-gustometer the effect of pH on the surface of the tongue should be taken into account [19].

The effect of saliva viscosity on the taste threshold has not been analyzed so far. The taste threshold should depend (in the first approximation) on the ion current density i. In such a case a simple relation holds between the current density i and the ion velocity v: i = cqv, where c is the ion concentrations and q is the ion’s electric charge. In principle, saliva viscosity should matter only if the ions created by the electric current have to travel a substantial distance until they are detected by the taste receptors, because only their velocity v depend on the medium viscosity, in contrast to the concentration c of generated ions. Thus, correlating the saliva viscosity with the taste threshold should provide information about the distance that the ions have to travel in the medium to reach the taste receptors. The aim of this work is to analyze the rheological properties of saliva using a Contraves LS40 rheometer and comparison of the results with taste threshold measurements performed by means of an electro-gustometer.

2. Material and methods

Saliva samples (2 ml) were taken from 27 health non-smoking donors – 7 men and 20 women aged 18–65 years (mean age – 37 years). Right before sample collection the thresholds of taste sensation were measured. Saliva was collected into small glass vials within 2–15 minutes, depending on the saliva production efficiency of the patients. The saliva samples were characterized in terms of their rheological properties and pH. All viscosity measurements were performed by means of a rotary-oscillating rheometer Contraves LS40 at the temperature of 37 °C in a Couette DIN 412 system. Saliva shear dependent viscosity (flow curve) was measured in the order of decreasing shear rate in the range 100–0.01 s⁻¹ within 5 minutes after short pre-shearing at 𝛾′ =100 s⁻¹. The viscoelastic properties were examined by applying constant frequency oscillations of frequency f =0.5 Hz and decreasing effective shear amplitude, ${\gamma}_{eff}^{\prime }=\sqrt{2}∼{\Gamma}∼f$ , where 𝛤 is the shear amplitude. For twenty donors the threshold of taste sensation was measured by means of a home-made electro-gustometer [18]. This new electronic device can stimulate small regions of tong by electrodes maintaining a stable electric contact despite the lability of resistance. One of the electrodes was placed on the upper surface of the tongue, about 25 mm from the tip once on the right side and once on the left side of the tongue. The other electrode was held in hand by the patient to close the electric circuit. For all patients no more than 20 μA difference between the left and the right side of the tongue was found which guaranteed the elimination of pathologies in the ability to differentiate the taste threshold. In all cases both polarities were applied (anode first, than cathode). The labels ‘cathode’ and ‘anode’ refer to the electrode placed on the tongue. For the pH measurements, the indicator stripes of pH 1–10 were used, catalog number 715755289 manufactured by Polskie Odczynniki Chemiczne SA. The indicator stripes were placed for 2 seconds on the opposite edges of the tongue. Immediately after the pH was measured, the threshold of taste was determined underneath the positive and the negative electrodes placed in central left and central right area of the tongue.

3. Results

For a few saliva samples a more detailed time dependence of their rheological behavior was studied. A flow curve was measured every ∼30 min for a period of ∼3 hours. The eight flow curves obtained in this way are shown in Fig. 1 with the time after saliva collection indicated in the legend. It is clear that that the viscosity at low shear rates drops quickly with time. Only for two first measurements the viscosity at this range could be reliably measured. All subsequent curves for 𝛾′ < 1 s⁻¹ contained data below the detection level of the instrument. At high shear rate values saliva behaves almost like a Newtonian liquid. In order to quantify this viscous behavior, viscosity values for highest reliable shear rate 𝛾′ = 100 s⁻¹ were plotted in Fig. 2 as a function of time after saliva collection. The visco-elastic behavior time evolution in the form of viscous (𝜂′) and elastic (𝜂′′) components of complex viscosity (𝜂^∗) as a function of effective shear amplitude (𝛾′ _eff) at constant frequency 𝜈 = 0.5 Hz is shown in Fig. 3. The influence of storing temperature on the rheological properties of saliva was investigated for three saliva samples. Immediately after collection, each of them was divided into two parts, one of which was stored in refrigerator at 4 °C, while the other part was immediately transferred to the rheometer, measured at 37 °C and left at this temperature. After two hours both parts were measured again at 37 °C. The part transferred from refrigerator was allowed to equilibrate in the rheometer for 2 minutes at 37 °C before the measurement. The mean values of the most reliable viscosity values for 𝛾′ = 100 s⁻¹, calculated for the three pairs of samples were both equal to 𝜂 = 2.2 ± 0.2 mPa s (mean values for cooled and non-cooled parts).

Fig. 1.

Flow curves for the same saliva sample measured after indicated periods of time after sample collection. Except for two first measurements, the data for 𝛾′ < 1 s⁻¹ are beyond or at the edge of instrument sensitivity.

Fig. 2.

Time dependence of saliva viscosity measured at the shear rate 𝛾′ = 100 s⁻¹.

Fig. 3.

Time evolution of the viscous (𝜂′) and elastic (𝜂′′) components of complex viscosity (𝜂*) as a function of effective shear amplitude (𝛾′ _eff). The measurements were taken every 30 minutes.

Collected saliva flow curves measured right after saliva collection are shown in Fig. 4. The substantial scatter of the flow curves results both from individual features of the persons involved in the study and from the time needed by a person to deliver sufficient amount of sample (2 ml). In some cases it took more than 15 minutes.

Fig. 4.

A mean flow curve of saliva samples measured right after their collection.

The values of viscous (𝜂′) and elastic (𝜂′′) components of complex viscosity (𝜂^∗) measured immediately after collecting the saliva samples are presented in Fig. 5.

Fig. 5.

The values of viscous (𝜂′) and elastic (𝜂′′) components of complex viscosity (𝜂^∗) measured immediately after collecting the saliva samples. Only mean values and standard deviations are shown.

Table 1

Values of saliva rheological parameters

𝛾′ [s⁻¹]	𝜂 [mPa s]
100	5.4 ± 0.8
10	8.2 ± 1.5
1	21 ± 3
0.1	121 ± 11
𝛾′ _eff [s⁻¹]	𝜂′ [mPa s]
34	4.0 ± 0.5
10	4.4 ± 0.5
1	7 ± 1
𝛾′ _eff [s⁻¹]	𝜂′′ [mPa s]
34	1.4 ± 0. 2
10	1.7 ± 0.2
1	5 ± 1

The values of rheological parameters: saliva viscosity for four shear rate values and viscous (𝜂′) and elastic (𝜂′′) components of complex viscosity (𝜂^∗) are shown in Table 1.

The results of the taste threshold value for 20 persons are presented in Fig. 6. The mean values for anode measurement is equal to 8.9 ± 1.5 μA and for cathode measurement is equal to 17 ± 3 μA. The pH value ranged from 6.25 to 7.5 with the mean value of 6.86 ± 0.03.

Fig. 6.

The values of threshold of taste for donors.

Figure 7 shows the dependence of the saliva viscosity for 𝛾′ = 100 s⁻¹ on the threshold of taste sensation immediately after sampling. Figures 8 (a) and (b) present the dependence of the viscous (𝜂′) and elastic (𝜂′′) components of complex viscosity (𝜂^∗) of saliva for 𝛾′ _eff = 34 s⁻¹ on the threshold of taste sensation measured immediately after sampling.

Fig. 7.

The dependence of saliva viscosity for 𝛾′ =100 s⁻¹ on the threshold of taste sensation immediately after sampling. The lines represents linear fits to the respective data, however statistically both slopes are not significantly different from zero (p = 0.34 for anode and p = 0.27 for cathode).

Fig. 8.

The dependence of the viscous (𝜂′) and elastic (𝜂′′) components of complex viscosity (𝜂^∗) of saliva for 𝛾′ _eff = 34 on the threshold of taste sensation immediately after sampling. No statistically significant correlations were found.

4. Discussion

This work examines whether there is a correlation between the viscosity of saliva and the threshold of taste sensation. There are many factors which can affect the rheological properties of saliva such as sex, biochemical and physicochemical properties of saliva, health, drugs, food and exercise [1,2,4–9,20]. Studies of saliva viscosity compared to other factors may help understand the physiological basis of taste sensation [1,2,5,16,21].

The first step was to explore the possibility of using an oscillating-rotational rheometer to measure the viscosity of saliva and finding the best protocol of saliva viscosity measurements. The results presented in figures from 1 to 4 show that saliva is not only a non-Newtonian fluid but also its rheological properties change with time. This effect is well known and attributed to decomposition of mucins in saliva, which leads to a loss of the structures being the source of non-Newtonian character of saliva [3]. It seems that to keep comparison to taste sensitivity realistic, saliva viscosity should be measured immediately after collection. The observed decrease in viscosity over time is consistent with the reports of other authors [2,4,7–9,21]. Placing a sample of saliva in the refrigerator did not slow down these processes (Fig. 2).

Saliva samples were also characterized by oscillatory measurement shear responses. The two components of the complex viscosity, viscous (𝜂′) and elastic (𝜂′′) shown in Fig. 5 reveal shapes typical for shear thinning fluids. Clearly, for small values of 𝛾^′ the elastic component becomes dominant, while with increasing shear rate saliva becomes a regular Newtonian fluid.

The complementary part of study included measurements of taste threshold values for twenty donors. The results presented in Fig. 6 show clear differences between the persons. The range of measured values is consistent with literature reports [16–18].

In an attempt to correlate the taste sensitivity with the viscosity values, plots of high-shear-rate viscosity (Fig. 7) and high-shear-rate components of the complex viscosity (Figs. 8 a, b) were made as a function of the taste sensation threshold (anode and cathode). As one can see in all these plots, no correlations have been found for these parameters. The suggested mechanism of gustometer principle of operation is the production of ions that can be sensed by the taste receptors. In the first approximation the flux of those ions depends on medium viscosity, but only if the place of their creation is distant from the receptors, so that the viscous resistance can become effective. Within this approximation the negative correlation of the taste threshold values with saliva viscosity suggests that the ions are created in the direct vicinity of the receptors. Another explanation of the negative correlation could be the concept of nano-viscosity [22] which attributes different viscosities to probes of different size immersed in a complex medium. According to this concept, a small ion may sense viscosity much smaller than the macroscopic viscosity measured by a rheometer and that nano-viscosity could be correlated with the taste threshold. Unfortunately, diffusion of small ions cannot be easily measured in such complex environment as saliva, however an attempt to measure small dyes diffusion in saliva is planned in near future to check the nano-viscosity of a probe intermediate in size between an ion and a macroscopic object.

The pH distribution turned out to be so narrow, that no effects due to pH variations could be detected. In former studies [18] special procedures were applied to induce acidic or basic environment in mouth. As this was not the main aim of the study, such procedures were not applied in the current study.

5. Conclusion

In summary, based upon the measurements conducted herein, it can be stated that:

–

Saliva is a shear thinning fluid.

–

The viscosity of saliva and its non-Newtonian character decreases over time.

–

The taste sensation threshold measured by means of an electro-gustometer does not correlate with the saliva viscosity.

–

Two interpretations of this finding were proposed: i) The ions produced by the electric field of the instrument appear so close to the receptors that viscous resistance does not influence the ion flux; and ii) the ion movement is more dependent upon nano-viscosity than macroscale bulk viscosity and hence correlations could occur with the former one and not the latter.

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