**Fatty Acid Lingual Application Activates Gustatory and Reward Brain Circuits in the Mouse**

#### **Yvan Peterschmitt 1, Souleymane Abdoul-Azize 2, Babar Murtaza 3, Marie Barbier 1, Amira Sayed Khan 3, Jean-Louis Millot 1,† and Naim Akhtar Khan 3,\***


Received: 12 August 2018; Accepted: 4 September 2018; Published: 6 September 2018

**Abstract:** The origin of spontaneous preference for dietary lipids in humans and rodents is debated, though recent compelling evidence has shown the existence of fat taste that might be considered a sixth taste quality. We investigated the implication of gustatory and reward brain circuits, triggered by linoleic acid (LA), a long-chain fatty acid. The LA was applied onto the circumvallate papillae for 30 min in conscious C57BL/6J mice, and neuronal activation was assessed using c-Fos immunohistochemistry. By using real-time reverse transcription polymerase chain reaction (RT-qPCR), we also studied the expression of mRNA encoding brain-derived neurotrophic factor (BDNF), Zif-268, and Glut-1 in some brain areas of these animals. LA induced a significant increase in c-Fos expression in the nucleus of solitary tract (NST), parabrachial nucleus (PBN), and ventroposterior medialis parvocellularis (VPMPC) of the thalamus, which are the regions known to be activated by gustatory signals. LA also triggered c-Fos expression in the central amygdala and ventral tegmental area (VTA), involved in food reward, in conjunction with emotional traits. Interestingly, we noticed a high expression of BDNF, Zif-268, and Glut-1 mRNA in the arcuate nucleus (Arc) and hippocampus (Hipp), where neuronal activation leads to memory formation. Our study demonstrates that oral lipid taste perception might trigger the activation of canonical gustatory and reward pathways.

**Keywords:** linoleic acid; gustation; hedonic; BDNF; fat taste; c-Fos; Zif-268; Glut-1

#### **1. Introduction**

Taste modality serves as an important factor for food choice and for appreciating its hedonic value [1]. There are five basic taste qualities known hitherto in rodents and humans: sweet, sour, bitter, salty, and umami [2]. The specific receptors and cells for each of the five basic taste modalities have been identified and characterized [3]. The series of events that occur before and after the ingestion of food, leading to taste perception and preference, are a topic of wide interest. Recently, convincing evidence has started to accumulate in favor of fat as the sixth fat taste quality in rodents and humans [4]. The two principal receptors of fat taste, CD36 and GPR120, have finally been identified in human taste bud cells, and their sensitivity to fatty acid stimuli has been shown to be altered in obesity [5]. The cellular and molecular mechanisms of fat taste perception have recently been elucidated [4,6].

There are a few studies that have shed light on fat-eating behavior and brain activity. Our laboratory has demonstrated that the addition of a fatty acid on mouse tongues induced the expression of c-Fos immunoreactivity in the nucleus of the solitary tract (NST), the first gustatory relay in the brain [7]. We did not address the question of whether other parts of the brain are also activated during this experimental approach, though several investigators, by employing different methods, have concluded that the primary taste cortex, orbitofrontal cortex, and amygdala are activated by the perception of dietary lipids [8–11]. Tzieropoulos et al. (2013) reported that dietary fat was able to induce sustained reward response in human brains. Eldeghaidy et al. [12] used functional magnetic resonance imaging (fMRI) in human subjects, and suggested that taste, appetite, and reward-related brain areas were responsive to nutritional status and received sensory and interoceptive signals of motivation and hedonic value in response to a fat-rich diet. Other fMRI studies in humans have also demonstrated that administration of dietary lipids activates cerebral taste, texture, and reward areas [13–16].

The abovementioned studies show that different brain areas might be activated by dietary fat; however, there is a dearth of information on the identification of sequential activation of cerebral areas/pathways that are activated in response to taste bud stimulation by dietary fat prior to ingestion of the bolus. Information on this subject would be crucial not only to better understand the fundamental mechanisms of fat intake and its related addiction, but also to modulate fat-eating behavior that is altered in obese subjects [17]. Keeping this argument in view, we designed the present study wherein we added linoleic acid (LA), a long-chain fatty acid, on the circumvallate papillae, which are rich in fat taste receptors, of conscious mice and assessed the neural activity using immunocytochemical localization of c-Fos protein in different brain areas. We also analyzed the mRNA expression of brain-derived neurotrophic factor (BDNF), involved in synaptic plasticity and memory processes, Zif-268, an immediate-early gene, and Glut-1, another marker of neuronal activation during enhanced glucose demand in three brain areas of these animals [18].

#### **2. Materials and Methods**

#### *2.1. Animals and Experimental Set-Up*

Experiments were carried out on 6–10-week-old C57BL/6J male mice (Janvier, Le Genest-St-Isle, Mayenne, France). Animals were group-housed under standard laboratory conditions (12 h:12 h, light/dark cycle; 22 ± 2 ◦C, 50–60% humidity) and fed with standard pelleted food (Scientific Animal Food & Engineering, Augy, France) and water ad libitum. All experiments were designed to minimize animal suffering and the number of animals. The protocols were approved by the regional ethical committee of Burgundy University in compliance with European guidelines for the use and care of laboratory animals.

In the first set of experiments, the neuronal activation along the canonical gustatory pathway was systematically assessed after oral lipid stimulation using c-Fos immunohistochemistry. In order to avoid any stressful situation, the mice were accustomed to gentle handling for 5 days in order to apply the fatty acid. On the 6th day, they were gently handled similarly and linoleic acid (LA, 18:2 n-6) (Sigma-Aldrich, St. Louis, MO, USA) at 50 μM in 70 μl (*w*/*v*) was slowly placed with the help of a spatula for 30 min on the circumvallate papillae. Xanthan gum (0.3% *w*/*v*, Sigma-Aldrich USA), which mimics lipid texture, was similarly applied onto the circumvallate papillae of control animals. At the end of oral stimulation, the animals were injected, intraperitoneally, with sodium pentobarbital (40 mg/kg), the thoracic cavity was opened, and mice were perfused intracardially with 50 mL of ice-cold saline (NaCl, 0.9%), followed by 50 ml of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The entire brain was removed and post-fixed by incubation in 4% paraformaldehyde for 2 h. Samples were cryoprotected by overnight incubation in 30% sucrose in 0.1 M phosphate buffer. Brain samples were embedded in a Tissue-Tek®OCT compound (Sakura FineTek, Torrance, CA, USA), frozen on dry ice, and stored at −20 ◦C before c-Fos immunohistochemical processing.

In the second set of experiments, using real-time reverse transcription polymerase chain reaction (RT-qPCR), we investigated the downstream molecular pathways/candidates in NTS, arcuate nucleus, and hippocampus at mRNA level. The total RNA from different brain areas was isolated by Trizol Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The quality of isolated RNA was determined using denaturing agarose gel electrophoresis. Then, the RNA was quantified by determining its UV absorbance at 260 nm. Five hundred nanograms of total RNA was reverse-transcribed with an iScript cDNA synthesis kit according to the manufacturer's instructions (Bio-Rad, Berkeley, CA, USA). RT-PCR was performed on the iCycler iQ real-time detection system, and amplification was undertaken using SYBR Green detection. Primers against the genes of interest were as follow: BDNF (forward 5 -TTGGATGCCGCAAACATGTC-3 ; reverse, 5 -CTGCCGCTGTGACCCACTC-3 ), Zif-268 (forward, 5 -GCGAACAACCCTATGAGC-3 ; reverse, 5 -GGTCGGAGGATTGGTC-3 ), Glut-1 (forward, 5 -GCTGTGCTTATGGGCTTCTC-3 ; reverse, 5 -CACATACATGGGCACAAAGC-3 ). The cycling conditions used were: 95 ◦C for 10 min; and 40 cycles of 95 ◦C for 15 s, 60 ◦C for 1 min. The amplification was carried out in a total volume of 20 μL, which contained 10.0 μL SYBR Green Supermix buffer (50 mMKCl; 20 mMTris-HCl (pH 8.4); 3 mM MgCl2; 0.2 mM of each dNTP, 0.63 U iTaq DNA polymerase, and SYBR Green 1.0 nM fluorescein), 0.75 μL (0.3 mM) of each primer, and 1.5 μL diluted cDNA. Results were evaluated using iCycleriQ software, and a relative quantification of mRNA in different groups was determined. The relative amounts of RNA were normalized to the amount of the endogenous control 18S using StepOne software version 2.2-2010 (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) and the Δ Ct method.

#### *2.2. Immunohistochemistry*

#### Fos Immunostaining

Fifty μm coronal sections were cut with a cryostat at −20 ◦C through the NST (nucleus of the solitary tract, bregma −0.48 to −7.08 mm), PBN (parabrachial nucleus, bregma −4.96 to −5.32 mm), VPM (ventral posteromedial thalamic nucleus, bregma −1.58 to −2.46 mm), VPMPC (ventroposterior medialis parvocellularis, bregma −1.94 to −2.30 mm), CeA (central amygdaloid nucleus, bregma −0.94 to −1.82 mm), PSTN/CbN (parasubthalamic nucleus/calbindin nucleus, bregma −2.06 to −2.54 mm), AI (agranular insular cortex, bregma −0.94 to +1.54 mm), GI/DI (granular/dysgranular insular cortex, bregma −0.94 to +1.54 mm), Hipp (hippocampus, bregma −1.22 to −2.18 mm), BLA (basolateral amygdaloid nucleus, bregma −1.70 to +1.82 mm), VTA (ventral tegmental area, bregma −2.92 to +3.88 mm), Acb (accumbens nucleus, bregma +1.70 to +0.98 mm), mPFC (medial prefrontal cortex, bregma +1.98 to +1.54 mm), Arc (arcuate nucleus, −1.46 to −2.46 mm caudal to bregma), and Hbn (habenula, bregma −1.22 to −2.18 mm), according to the atlas of Paxinos & Franklin (2001) [19]. Free-floating sections were then collected in 0.1 M phosphate buffer, pH 7.4, and processed for c-Fos immunohistochemistry. Sections were incubated overnight with rabbit anti-c-Fos (1:20,000, Calbiochem®, Paris, France) primary antibodies diluted in 0.1 M phosphate buffer at pH 7.4, containing 0.3% Triton X100 and 3% normal goat serum (*v*/*v*). Subsequently, sections were incubated for 2 h at room temperature with the biotinylated goat antirabbit secondary antibodies (1:4000, Vector laboratories, Burlingame, CA, USA). The formed antigen–antibody complexes were visualized through the avidin–biotin–horseradish peroxidase procedure (Vectastain Elite ABC kit; Vector Laboratories, USA), using 3,3 -diaminobenzidine (0.04%) as the chromogen. Sections were mounted on gelatin-coated slides, dehydrated, and coverslipped with DePeX (VWR International Ltd., Poole, UK) mountant.

#### *2.3. Quantification of c-Fos Immunopositive Neurons*

Sections were analyzed under a 40× objective of a light-optical microscope (Nikon Eclipse E600, Nikon Instruments Inc., Melville, NY, USA) equipped with a digital camera (Nikon Digital Sight DS-Fi1). c-Fos immunoreactive nuclei were quantified on photomicrographs of the regions of interest (ROI) using the imaging software Image J (National Institutes of Health, Bethesda, MD, USA). A cell was considered as labeled (positive) for c-Fos when the brown-black DAB-staining was unambiguously darker than the background, and this included all cells from low to high intensities of staining. Thresholds over the background section and the size of the particles were determined by the experimenter. The entire region for each area was traced, and mean densities of c-Fos-immunopositive neurons (number of c-Fos positive cells/mm2) for each ROI were calculated according to their respective areas.

#### *2.4. Statistical Analysis*

Data are presented as means ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, La Jolla, CA, USA). For the immunohistochemical experiment, the Kolmogorov–Smirnov test revealed parametric (normal) distribution for the c-Fos data for most brain regions. Therefore, differences between groups were assessed using unpaired one-sided *t*-tests. The level of significance was set at *p* < 0.05.

#### **3. Results**

#### *3.1. Lingual LA Stimulation Triggers Neuronal Activation of the Canonical Central Cerebral Gustatory Reward Pathway*

Figure 1A shows the c-Fos expression in the NST, arcuate nucleus (Arc), and hippocampus (Hipp) in LA-stimulated and control mice. Quantitative analysis of the immunohistochemical data revealed that oral LA application produced a robust increase in c-Fos expression in most brain regions explored along the putative pathway for gustatory lipid perception (Figures 1A,B and 2). More precisely, unpaired one-sided *t*-tests show a significant effect of the treatment in the NST (*t*(10) = 2.252; *p* = 0.0240), PBN (*t*(9) = 3.992; *p* = 0.0016), VPMPC (*t*(9) = 3.153; *p* = 0.0058), CeA (*t*(10) = 2.380; *p* = 0.0193), PSTN/CbN (*t*(9) = 3.037; *p* = 0.0070), and VTA (*t*(10) = 2.309; *p* = 0.0218), but the differences did not reach statistical significance either at the cortical level (AI (*t*(10) = 0.3142; *p* = 0.3799), GI/DI (*t*(10) = 0.05505; *p* = 0.4786), mPFC (*t*(10) = 0.8558; *p* = 0.2061) and HIPP (*t*(9) = 0.3831; *p* = 0.3553)) or in the non-gustatory-related thalamus VPM(*t*(9) = 1.377; *p* = 0.1009), BLA (*t*(10) = 0.2419; *p* = 0.4069), Acb (*t*(10) = 0.06695; *p* = 0.4740), Hbn (*t*(10) = 0.4021; *p* = 0.3480) or Arc (*t*(9) = 1.052; *p* = 0.1602) (Figures 1A,B and 2).

**Figure 1.** Linoleic acid deposition on the tongue induces c-Fos expression in the major cerebral structures of the canonical gustatory pathway. (**A**) Typical photomicrographs of the NST, Arc, and Hipp, showing c-Fos immunoreactivity in mice subjected to oral stimulation with linoleic acid or xanthan gum (XG, 0.3%, *w*/*v*) to mimic the texture of lipids. The dotted lines circumscribe the regions

of interest. Arrowheads point to representative c-Fos immunopositive nuclei. The boxes show higher magnification (×4) of representative c-Fos immunopositive nuclei. Scale bar, 100 μm. The square windows indicate the area shown in the photomicrographs. (**B**) Bar graph representation of the density of c-Fos immunopositive cells (number of c-Fos positive cells/mm2) in mice subjected to oral stimulation with linoleic acid (LA) or xanthan gum (XG). Values are means ± standard error of the mean (SEM); *n* = 6; for each structure studied, treatment effects on c-Fos expression were assessed using unpaired one-sided *t*-tests. \*, *p* < 0.05; \*\*, *p* < 0.01. AI, agranular insular cortex; Arc, arcuate nucleus; Hipp, hippocampus; NST, nucleus of the solitary tract; PBN, parabrachial nucleus; VPM, ventral posteromedial thalamic nucleus; VPMPC, ventroposterior medialis parvocellularis.

**Figure 2.** Linoleic acid deposition on the tongue activates c-Fos expression in the major cerebral structures related to emotional and reward traits. Bar graph representation of the density of c-Fos immunopositive cells (number of c-Fos positive cells/mm2) in mice subjected to oral stimulation with linoleic acid (LA) or xanthan gum as control solution. Values are means ± SEM; *n* = 6; for each structure studied, treatment effects on c-Fos expression were assessed using unpaired one-sided *t*-tests. \* *p* < 0.05; \*\* *p* < 0.01. Acb, accumbens nucleus; Arc, arcuate nucleus; BLA, basolateral amygdaloid nucleus; CeA, central amygdaloid nucleus; GI/DI, granular/dysgranular insular cortex; Hbn, habenula; Hipp, hippocampus; mPFC, medial prefrontal cortex; PSTN/CbN, parasubthalamic nucleus/calbindin nucleus; VTA, ventral tegmental area.

#### *3.2. Lingual LA Stimulation Modulates the Expression of mRNA Encoding BDNF, Zif-268 and Glut-1*

The RT-qPCR results show that the relative expression of Zif-268 mRNA was significantly increased (*p* < 0.001) in the NST, arcuate nucleus, and hippocampus by lingual application of LA in mouse brains (Figure 3A). To our surprise, LA induced a nearly fivefold higher increase in Zif-268 mRNA in the NST than controls. LA also resulted in a significantly higher increase (*p* < 0.001) in Glut-1 mRNA expression than control in the nucleus of solitary tract (NST), arcuate nucleus (Arc), and hippocampus (hipp) (Figure 3B). Hence, the increase in Glut-1 mRNA expression was more evident (about a threefold increase) in the NST and hippocampus, as compared to the control solution, after lingual application of LA. The BDNF mRNA expression was increased in the arcuate nucleus and hippocampus, but not in the NTS, after fatty acid lingual application (Figure 3C).

**Figure 3.** Lingual application of LA modulates brain-derived neurotrophic factor (BDNF), Zif-268, and Glut-1 mRNA expression in the mouse brain. Bar graphs represent the relative increase in mRNA expression (Zif-268 in (**A**), Glut-1 in (**B**), BDNF in (**C**)) in mice subjected to oral stimulation with linoleic acid (LA) or xanthan gum as control solution. Values are means ± SEM; *n* = 6; for each structure studied, treatment effects on Zif-268 and Glut-1 mRNA expression were assessed using unpaired one-sided *t*-tests. NST, nucleus of solitary tract; Arc, arcuate nucleus; Hipp, hippocampus.

#### **4. Discussion**

Gene transcription during memory consolidation is a very dynamic and complex process, depending on the type of learning involved. Hence, many types of mRNAs are transcribed, such as for the transcription factors, c-Fos, Zif-268, and the effector genes, like BDNF [20]. The c-Fos, both at mRNA and protein levels, is generally among the first to be expressed and, therefore, referred to as an immediate early gene (IEG) and considered to serve as a marker of the neuronal activity in the neuroendocrine systems [21].

Central gustatory pathways have been well studied in murine models [22]. Branches of the facial (chorda tympani and greater superficial petrosal), glossopharyngeal, and vagus (superior laryngeal) nerves, which establish synaptic contacts with receptor cells in the taste buds, convey taste messages to the first relay nucleus, that is, the rostral part of the nucleus of the solitary tract (NST). The second relay nucleus for ascending taste inputs is the parabrachial nucleus (PBN) of the pons. The third relay station is the ventroposterior medialis parvocellularis (VPMPC) of the thalamus. This thalamic nucleus sends taste information to the insular cortex (IC) [22]. In most of the experiments/observations reported so far, the prominent neurochemical changes in the brain areas take place immediately following training, but in some instances, there are waves from 3 h to 6 h and/or at 24 h following training. In the present study, we were interested in elucidating the early brain responses to short-term application, that is, 30 min, of a long-chain fatty acid, that is, linoleic acid (LA). We applied LA onto the circumvallate papillae as they have been reported to contain more CD36, a lipid receptor, than fungiform papillae [23]. Our results demonstrate that acute lingual application of LA resulted in a significant increase in c-Fos expression in the NST, PBN, and VPMPC in the mouse brain. However, the increase in c-Fos expression in VPM was not significantly higher in fatty-acid-treated mice than control animals, suggesting that VPMPC, but not VPM, is involved in the transfer of lipid taste messages from the PBN to the insular cortical areas (AI, GI/DI), though LA failed to induce c-Fos expression in latter areas of the brain.

The ventral tegmental area (VTA), nucleus accumbens (NAcb), and ventral pallidum (situated between the NAcb and lateral hypothalamus) are the essential components of the brain reward system [24]. Acute application of LA resulted in a significant increase in c-Fos expression in the VTA, which constitutes the mesolimbic dopaminergic system. However, no significant difference in c-Fos expression was observed in the NAcb after the application of the fatty acid. Our observations can be

supported by the study of Dela Cruz et al. [25], who have shown that fat intake is associated with the activation of the VTA in the mouse brain [25]. Reward is closely related to hedonic stimuli and our results, showing c-Fos activation in the CEA and PSTN, support previous reports describing the activation of the PSTN, the major target for projection from the CeA [26] in response to hedonic taste [27].

In order to correlate c-Fos findings, we further quantified the mRNA expression of BDNF, Zif-268, and Glut-1 in three areas of the brain, that is, the NTS, arcuate nucleus, and hippocampus, as peripheral signals go through the NTS and arcuate nucleus to the hippocampus, involved in multimodal learning and memory. BDNF is a small dimeric protein that is widely expressed in the adult mammalian brain and extensively involved in synaptic plasticity and memory processes [28]. We observed that BDNF mRNA was highly induced in the arcuate nucleus and hippocampus, but not in the NTS. In fact, this growth factor is more involved in learning and memory rather than the transfer of peripheral signals, as is the case of the NTS. Alonso et al. [29] have demonstrated that BDNF is involved not only in memory consolidation, but also in long-term memory formation in the CA1 region of the hippocampus. Genoud et al. [30] have clearly shown that BDNF is mandatory to induce formation of activity-dependent synapses in cerebral cortex. However, there are regional and task-dependent differences underlying differential mechanisms of BDNF and its receptor function [28].

Zif-268 belongs to the regulatory transcription factor family, responsible for inducing transcription of late-response genes [31]. Induction of memory in the hippocampus has been shown to increase the expression of Zif-268 mRNA, and *Zif-268* knock-out mice had deficits in long-term memory for socially transmitted food preference and object recognition [32]. As far as Glut-1 is concerned, we would like to state that glucose is an important source of energy for the brain and glucose transporters (Glut) enable passage of glucose across both the endothelial cells of the blood–brain barrier and the plasma membranes of neurons and glia [33]. Glut-1 is present at high levels in brain endothelial cells [34]. We observed that lingual application of LA resulted in a significantly increased expression of Zif-268 and Glut-1 mRNA in the NST and arcuate nucleus, as well as in the hippocampus. Surprisingly, the number of c-Fos immunopositive neurons was not significantly increased in the arcuate nucleus and hippocampus by acute application of LA. We would like to mention that, under some conditions, the concomitant activation of these two markers (c-Fos and Zif-268) is not seen. Barbosa et al. [35] have shown that Zif-268 was increased in the dorsal CA1 region of the hippocampus, while there was no c-Fos activation in the experiments conducted to assess episodic-like memory in rats. Furthermore, we elucidated the Zif-268 expression in the whole hippocampus, and it is possible that, in different subregions, there might be a differential expression of Zif-268 mRNA. It is also worth mentioning that c-Fos is well correlated with neuronal activity, whereas Zif-268 is more related to memorization mechanisms, such as long-term potentiation [36,37]. As far as Glut-1 is concerned, we would like to state that sometimes there is no direct correlation between Glut-1 and c-Fos expression. Glut-1 is activated immediately as per energetic demand of the cells, whereas c-Fos might be activated at a later stage. Indeed, Hauguel-de Mouzon et al. [38] have shown that Glut1 mRNA, but not c-Fos levels, is subjected to the variations in glucose concentrations in human placental cells, and this differential regulation of Glut1 and c-Fos genes could be relevant to, respectively, metabolic and mitogenic pathways. In fact, high glucose concentrations are supposed to upregulate c-Fos expression [39] and downregulate Glut-1 levels [36]. It is possible that, in response to lipid gustatory information coming from tongue to brain, the hippocampus is in high requirement of glucose due to high glucose utilization, and this would result in high GLUT-1 and low c-Fos mRNA expression in this region of the brain.

On the basis of our observations, we provide a schematic representation of the gustatory pathway, depicting the major central synaptic relays and its connections with structures involved in metabolic, reward, learning, and memory processes in response to fatty acid stimulation (Figure 4). Thus, lipid taste perception relies on systematic activation of the major cerebral structures of the canonical gustatory pathway, ranging from the first central synaptic relay NST in the brain stem to the PBN, reaching the gustatory part of the thalamus (VPMPC), up to the gustatory insular cortical areas (AI, GI/DI) with modulatory influences of the central amygdaloid nucleus (CeA) and the posterior part of the lateral hypothalamus, that is, the parasubthalamic nucleus/calbindin nucleus (PSTN/CbN). It is worth noting that, at this early stage of lipid oral stimulation, the reward circuit is already involved, mainly through the VTA. It is not well understood, in the present study, how the arcuate nucleus (Arc), which is sensitive to peripheral postingestive signals, is activated (as evidenced by high BDNF, Zif-268 and Glut-1 mRNA levels). Indeed, the feeding behavior is also regulated by circulating hormonal signals, released by nutrients in the gut, such as cholecystokinin (CCK) and glucagon-like-peptide 1 (GLP-1), released as a result of postingestive/postoral activation of the gastrointestinal tract. Further physiological studies are required to assess the effects of these and other circulating factors that might regulate fat-eating behavior either via the arcuate nucleus or via the vagal nerve X. Nonetheless, ours is the first report to demonstrate that the application of a long-chain fatty acid like linoleic acid would activate a long chain of events in the conscious brain of the mouse.

**Figure 4.** Schematic representation of the gustatory pathway, depicting the major central synaptic relays and their connections with structures involved in metabolic, reward and learning, and memory processes. The lingual application of a long-chain fatty acid will trigger signaling events via CD36, localized in the circumvallate papillae. The gustatory information on dietary lipids will be conveyed to the NST via cranial nerves VII and IX. The NST that serves as the relay structure of the peripheral information will send the gustatory information to different brain areas, as mentioned in the Discussion section. Acb, accumbens nucleus; AI, agranular insular cortex; Arc, arcuate nucleus; BLA, basolateral amygdaloid nucleus; CeA, central amygdaloid nucleus; GI/DI, granular/dysgranular insular cortex; Hbn, habenula; Hipp, hippocampus; mPFC, medial prefrontal cortex; NST, nucleus of the solitary tract; PBN, parabrachial nucleus; PSTN/CbN, parasubthalamic nucleus/calbindin nucleus; VPM, ventral posteromedial thalamic nucleus; VPMPC, ventroposterior medialis parvocellularis; VTA, ventral tegmental area (adapted from Reference [27]).

**Author Contributions:** Y.P., J.-L.M. and N.A.K. designed the study. S.A.-A. and Y.P. conducted the experiments. Y.P., N.A.K., B.M. and A.S.K. were involved in writing and statistical analysis.

**Funding:** This work was financed by a contingent grant from Bourgogne-Franche-Comté Région to encourage the bilateral collaboration between two universities (AAP/BQR/PRES).

**Acknowledgments:** Authors are thankful to the PRES–Bourgogne-Franche-Comté for the contingent grant.

**Conflicts of Interest:** All the authors declare that they have nothing to disclose.

#### **References**


© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Umami as an 'Alimentary' Taste. A New Perspective on Taste Classification**

#### **Isabella E Hartley, Djin Gie Liem and Russell Keast \***

Centre for Advanced Sensory Science, School of Exercise and Nutrition Sciences, Deakin University,

1 Gheringhap Street, Geelong 3220, Australia; iehartle@deakin.edu.au (I.E.H.); gie.liem@deakin.edu.au (D.G.L.)

**\*** Correspondence: russell.keast@deakin.edu.au; Tel.: +61-3-9244-6944

Received: 15 December 2018; Accepted: 10 January 2019; Published: 16 January 2019

**Abstract:** Applied taste research is increasingly focusing on the relationship with diet and health, and understanding the role the sense of taste plays in encouraging or discouraging consumption. The concept of basic tastes dates as far back 3000 years, where perception dominated classification with sweet, sour, salty, and bitter consistently featuring on basic taste lists throughout history. Advances in molecular biology and the recent discovery of taste receptors and ligands has increased the basic taste list to include umami and fat taste. There is potential for a plethora of other new basic tastes pending the discovery of taste receptors and ligands. Due to the possibility for an ever-growing list of basic tastes it is pertinent to critically evaluate whether new tastes, including umami, are suitably positioned with the four classic basic tastes (sweet, sour, salty, and bitter). The review critically examines the evidence that umami, and by inference other new tastes, fulfils the criteria for a basic taste, and proposes a subclass named 'alimentary' for tastes not meeting basic criteria.

**Keywords:** basic tastes; taste; taste reception; umami

#### **1. Introduction**

The relationship between individual variation in taste perception, food choice and intake, and ultimately diet related disease, provides a framework for applied taste research. A taste perception arises from the interaction of non-volatile, saliva soluble chemicals with taste receptors on the tongue within the oral cavity. This interaction initiates a signal transduction to processing regions of the brain, resulting in the formation of a taste perception. The taste perception formed could include the perception of one of the basic tastes: sweet, sour, salty, bitter, umami [1], or fat [2]; or a perception of other putative taste qualities including, but not limited to, kokumi (rich, mouthful, thick, delicious taste) [3,4], carbohydrate [5], calcium [6], or metallic tastes [7]. Detection and perception of basic tastes is hypothesised to exist for species' survival throughout evolution to prevent the consumption of potential noxious food, and promote consumption of nutritious food, a nutrient–toxin detection system [8–11].

Basic tastes have perceptual independence, that is, they do not elicit a taste perception similar to that of any other basic tastes and cannot be produced from a combination of other tastes [12–14]. The concept of basic tastes dates back as far 384–322 B.C. when Aristotle originally listed the seven tastes he proposed as basics, these included sweet, sour, salty, and bitter as well as astringent, pungent, and harsh [15]. Throughout history the lists of tastes have been extended, or reduced, depending on the prevailing thought of the time, with the only consistency being the inclusion of sweet, sour, salty, and bitter in basic taste lists [15]. Research during the 1800's separated olfaction and tactile perceptions from tastes. The development and advancement of science and technology, including psychophysical testing led to sweet, sour, salty, and bitter tastes being confirmed as basics as evidenced throughout published literature [16].

Advances in molecular biology, and the recent discovery of taste receptors and ligands has increased the basic taste list to include umami and fat taste [17–20]. The existence of specific taste receptors responsive to a single compound that elicits a taste is often suggested as a key piece of evidence for basic tastes classification, with literature citing this as the key evidence in the case of umami [14]. Due to these recent advances, there is the potential for a plethora of new tastes to be discovered, and potentially classified as a basic taste if receptors on taste cells are found to respond to ligands. The current possibilities include, but are certainly not limited to kokumi, carbohydrate, calcium, and metallic tastes.

Thus, due to advances in knowledge, and the possibility for an ever-growing list of basic tastes, it is pertinent to critically evaluate whether new tastes, including umami, are suitably positioned with the four classic basic tastes (sweet, sour, salty, and bitter). This is of importance for three predominant reasons, first, there is historical and academic relevance to determine whether umami, and by inference other new tastes, should in-fact be considered in the same category as sweet, sour, salty, and bitter. Second, understanding individual variation in taste perception, perceptual associations with other basic tastes, and physiological responses resulting from detection of specific tastants, may enhance our understanding of the complex relationship between taste, dietary choice and intake, and diet related health related outcomes. Third, the classic basic tastes have significant immediate influence on whether to swallow or not swallow a potential food, while the more recent tastes such as umami and fat may have more post-ingestive relevance and determine extent of consumption and ultimately health. The review that follows will critically evaluate the evidence that umami fulfils criteria for classification as a basic taste.

#### **2. Basic Taste Criteria**

Criteria that a stimulus must fulfil for it to be classified as a basic taste has been proposed, although these criteria have not been consistent [12,13]. Kurihara and Kashiwayanagi (2000) suggested that for a compound to be considered a basic taste it should fulfil the following criteria. The proposed basic taste is (1) different to any other basic taste; (2) not replicated by combining other basic tastes and; (3) a taste which is commonly consumed and induced by common components of food [13]. The requirement for a basic taste to have an identified receptor was recently added to this set of criteria [14]. The argument could then be put forward that the discovery of a receptor-ligand complex alone is not reason to justify a taste as a basic taste. Whether the detection of the stimulus from that receptor is transduced and forms a unique perceptible experience may be of higher importance. That is, if a taste receptor is identified, but no unique perceptible experience occurs from the activation of that receptor, then is it appropriate to classify the stimulus as eliciting a basic taste?

A more comprehensive set of criteria have been outlined, covering both unique effective stimuli, transduction (receptors), neurotransmission, and finally, perception [12]. These criteria have been used previously to investigate the appropriateness of other new tastes, specifically fat taste; in this review the following criteria will be used to specifically investigate umami as a basic taste [10,12]. We extend this criterion to involve hedonic responses occurring from tasting the effective stimuli. The criteria are as follows:


#### **3. Umami Taste and Unique Class of Umami Effective Stimuli**

Umami was initially discovered by Ikeda who isolated glutamic acid from kombu (seaweed), finding that the salts of glutamic acid, particularly the sodium salt, monosodium glutamate (MSG), gave the seaweed its specific flavour (translated in [21]). Thus, free L-glutamate (glutamic acid) is the predominant umami effective stimuli, and MSG is the predominant prototypical umami stimuli used in psychophysical testing. It was later discovered that the taste of L-glutamate could be synergistically increased through the addition of disodium 5'ribonucleic acids, specifically disodium 5'inosinate monophosphate (IMP) and disodium 5'guanylate monophosphate (GMP) [22]. When tasted in isolation IMP elicits a minimal to weak umami taste hypothesised to occur due to the interaction of IMP with subthreshold concentrations of L-glutamate in humans' saliva, demonstrating that IMP and GMP require L-glutamate for an umami taste perception to occur [23]. In human psychophysical studies, the addition of 0.5 mM IMP significantly reduces the concentration of MSG required for participants to reach RT (7.66 mM and 0.20 mM MSG respectively) due to the taste potentiation produced when IMP is applied with MSG [24].

Free L-glutamate is naturally present in high concentrations in a wide variety of foods including certain vegetables (and fruits i.e., tomato), seaweeds, aged cheese, seafood, fish and soy sauce, egg yolks, and human breast milk [14,25–27]. Whereas, IMP is found predominately in animal products such as chicken, pork, beef, and tuna, and GMP in dried mushrooms such as shitake [14,25,26]. The curing, ageing, heat treatment, and fermenting of certain foods results in an increase in free amino acids, including L-glutamate, and often an increase in umami potentiating ribonucleotides (IMP and/or GMP) [14,26]. Specifically, in animal products such as beef, pork, chicken, and fish that contain high concentrations of protein, which is essentially tasteless, proteolysis occurring from fermentation, curing, or heat treatment releases a complex mixture of amino acids, including L-glutamate. As these animal products naturally contain high concentrations of IMP the umami taste potentiation between IMP and L-glutamate can occur [14,26]. For example, during the process of ageing beef the concentration of free L-glutamate has been shown to increase by approximately 33% over eight days, and when this is combined with naturally present IMP, umami taste potentiation can occur [26].

The increase of tastants occurring from fermenting, curing, heat treatment or ageing is not unique to specifically umami taste quality, with kokumi taste (rich, mouthful, thick, delicious taste) peptides similarly increasing through these same processes. Kokumi tasting compounds include certain γ-glutamyl peptides and during the ageing of dairy products [28] and in fermented products including soy or fish sauce [4,29] the concentrations of γ-glutamyl peptides increases. This ageing/fermenting process results in an accumulation of peptides including γ-glutamic acid that forms a peptide bond with an amino group of a non-polar amino acid [30]. Additionally, glutathione (GSH) a tripeptide made up of glutamic acid, cysteine, and glycine, which elicits a kokumi taste, similarly increases in concentration through fermentation of certain foods. Kokumi stimuli including GSH, similar to IMP, elicit little taste in isolation, but when combined with an umami solution enhances the mouthful, continuity, and thickness, thus enhancing the kokumi aspects of the umami solution [4]. Similar to umami effective stimuli, GSH and other γ-glutamyl peptides are common in a number of high protein foods, such as beef, chicken, and ham, and in low protein foods such as tomato juice, and red wine, at concentrations above GSH DTs [4]. This suggests that there is an association between the effective stimuli eliciting both umami and kokumi tastes in food, namely the involvement of glutamic acid derivatives in both umami and kokumi effective stimuli [30].

#### **4. Umami Taste from an Evolutionary Perspective**

In humans, the ability to detect chemicals in the oral cavity prior to ingestion, and interpret salient perceptions of sweet, sour, salty, and bitter allows for rapid evaluation of a food, identifying whether it is acceptable (swallow), or unacceptable and potentially harmful (expectorate), which was essential for species survival [11,31–33]. Sweet taste is stimulated by simple carbohydrates, believed to indicate the presence of readily usable energy, eliciting appetitive hedonic responses

and thereby encouraging consumption [11,31]. Similarly, the detection of complex carbohydrates may also encourage consumption by signaling information regarding the carbohydrate and energy content of food [20]. Excess bitterness indicates the presence of potential toxins within food, ultimately encouraging rejection of the food [8,11,31]. Excess sourness can indicate off or spoilt foods, and is avoided to ensure the body's acid–base balance is maintained [8,31]. Salt taste perception is posited to be for maintenance of the body's electrolyte balance [9], for example, at high concentrations salt taste may play a role in the immediate analysis of whether to swallow or expectorate food, perhaps to avoid acute disturbance in the body's osmotic balance [11]. The ability to detect fat taste may be less important for the rapid evaluation of food and more closely related to activating physiological responses related to digestion and food intake regulation [18,34].

Umami taste has previously been hypothesised to signal the presence of amino acids and protein, promoting consumption of certain protein containing foods. Conversely, many foods naturally high in free L-glutamate are not typically high in protein, for example, peas, corn, red grapes, and tomatoes [14,35]. Along the same line, high protein foods, including beef, pork, and chicken do not contain high concentrations of free L-glutamate [26]. As previously mentioned, protein is essentially tasteless, it is the proteolysis of protein within these foods occurring from fermentation, curing, or heat treatment, that releases amino acids and peptides that can stimulate taste responses. Thus, umami taste perception may indicate the presence of accessible, rather than protein bound amino acids, in foods that have been released during proteolysis occurring through various cooking processes [11]. During proteolysis it is important to consider that the amino acids released are not solely umami tasting (L-glutamate), the release of bitter tasting (i.e., L-Leucine, -Phenylalanine, -Tryptophan) and sweet tasting (i.e., L-Glycine, -Alanine, -Proline) amino acids also occurs in different concentrations depending on the specific food [36]. Thus raising the question of whether it is appropriate to designate the evolutionary purpose of umami taste perception to signal protein content of food, when proteolysis in certain foods results in a complex mixture of taste active amino acids, including sweet and bitter tasting amino acids.

Following on from this, nutritional status, specifically protein-calorie deficiency does not appear to feedback onto preferences for umami tasting stimuli, as both malnourished (protein-calorie malnourished) and healthy infants showed preference for soup containing MSG to the same soup without MSG [37,38]. When the soup was provided in combination with casein hydrolysate, which contains a mixture of amino acids where a bitter taste dominates, the malnourished infants preferred the casein hydrolysate soup whereas the healthy infants did not [38]. Protein deficiency in infants increases consumption of protein containing food independent of the taste profile; the hypothesis that umami taste exists to signal the presence of protein is not supported.

Glutamate receptors (T1R1/T1R3 and mGluR1) exist throughout the gastrointestinal tract [39,40], and stimulation of these receptors has been suggested to affect nutrient absorption through regulating satiety hormones including cholecystokinin (CCK) [40–42]. Moreover, consumption of umami stimuli (MSG) appears to be involved in appetite stimulation and satiety regulation regardless of the macronutrient (i.e., protein and carbohydrate) consumed in human behavioural studies [43] (for further discussion please see section *Behavioural and physiological responses to umami effective stimuli*). Perhaps umami is less involved in the rapid analysis of food in the oral cavity, and more involved in increasing appetite to promote consumption, whilst simultaneously assisting in regulation of protein digestion through signaling mechanisms that promote gastric secretion.

#### **5. Unique Receptor and Neural Transmission of Umami Effective Stimuli**

#### *5.1. Unique Receptors for* L*-glutamate*

As previously discussed, taste receptors on the tongue detect saliva soluble, non-volatile chemicals from foods in the oral cavity. Of the basic tastes, sweet, bitter, and umami tastes are mediated via G-protein-coupled receptors, T1Rs and T2Rs, found in type II taste receptor cells [44]. Bitter ligands are detected by T2R of which there are currently over 25 genes encoding the T2Rs [44,45]. Salty and sour taste have been suggested to be modulated by specialised ion channels. Salty taste has been proposed to involve the selective epithelial type sodium channel (ENaC), and putative sour taste receptors include H+ ions permeating type III sour sensing cells resulting in type III sour cells depolarising and reaching action potential [8,44,45], see Roper et al (2017) for a recent comprehensive review on taste receptor mechanisms.

Umami was widely accepted as a basic taste based on the discovery that the heterodimeric G-protein-coupled receptors, T1R1/T1R3 mediate umami taste detection [17,46,47]. The umami taste heterodimer complex, T1R1/T1R3, shares a common receptor subunit (T1R3) with sweet taste detected by the heterodimeric G-protein-coupled receptors, T1R2/T1R3 [9,44]. T1R1/T1R3 heterodimeric receptor is specific to detecting umami-tasting stimuli (L-amino acids), as it is non-responsive to sweet stimuli but responsive to umami stimuli (MSG and L-glutamate) *in vitro* [17,46]. T1R1/T1R3 was confirmed to respond to umami-tasting stimuli upon the discovery that T1R1, T1R3, and T1R1/T1R3 knockout mice lack, or have attenuated taste responses to umami stimuli (MSG) [47], and human T1R1/T1R3 receptors responded when L-glutamate was applied *in vitro* [46]. Although, studies have found in T1R1 and T1R3 knockout mice that a reduced, but not abolished, taste response to umami stimuli (MSG and MPG) occurs, indicating that other receptors responding to umami stimuli exist [48–51].

When investigating the umami taste synergism occurring from the mixing of IMP/GMP with MSG, T1R3 knockout mice had only moderately reduced taste responses, both neural and behavioural, although the contribution of Na+ was not eliminated in this study, so remaining taste responses in these knockout mice is likely due to the Na+ [48]. Zhao and colleagues showed, in independently generated T1R3 knockout mice, that when the contribution of Na+ was reduced with amiloride, the T1R3 knockout mice lacked responses to IMP with MSG, where responses in control mice remained, highlighting the importance of the T1R3 subunit in umami taste synergism [47]. Similarly, in T1R1 knockout mice the umami synergy when IMP was applied with MSG was abolished [52]. All of this shows that the T1R1/T1R3 umami receptors are important, if not essential, for the synergistic effect of IMP/GMP when applied with MSG, but for MSG in isolation an umami taste response, albeit reduced, remains in the absence of the T1R1/T1R3 umami receptors [53]. This suggests that additional receptors respond to umami taste stimuli, which was supported by studies finding putative umami receptors, metabotropic glutamate receptor 1 and 4 (mGluR1, mGluR4) were activated by concentrations of umami stimuli (MPG) commonly found in food in an *in vitro* assay [51], and mGluR4 knockout mice had reduced neural responses *in vivo* to umami stimuli (MPG) [54].

Finally, the discovery of single nucleotide polymorphisms on human TAS1R1, and TAS1R3 receptor genes, and their association with individual variation in umami (MSG, MPG, and MSG+IMP) taste perception phenotypes, provided further evidence for T1R1/T1R3 contributing to umami taste detection in humans [24,55,56].

#### *5.2. Neural Responses to Umami Stimuli*

When taste receptor cells detect chemicals in the oral cavity a neurotransmitter (ATP) is released onto afferent gustatory fibres, three predominant gustatory afferent nerves transmit information from taste buds to the brain [8]. The 7th cranial nerve, chorda tympani (CT), innervates the anterior two thirds of the tongue, and the 9th cranial nerve, glossopharyngeal (GL), innervates the posterior third, and the 10th cranial nerve, vagus nerve, similarly innervates the posterior of the tongue. The information transmitted for umami taste is then processed in the primary and secondary gustatory cortex [57].

Studies investigating responses of the CT in both wild-type mice (not genetically modified), and T1R3 knockout mice, have shown that there are two predominant fibre groups in the CT [58]. These fibres noted are sucrose best (S) and MPG best, or L-glutamate best (M) fibres, each of these fibres have sub-groups (S1, S2, and M1, M2) [58]. S1 and M1 show synergism between L-glutamate and IMP,

whereas S2 and M2 do not display this synergism [58]. In T1R3 knockout mice S1 fibres were lacking, and no synergistic effect between MPG and IMP was observed [58]. Similarly, whole CT responses in T1R3 knockout mice showed the synergism between IMP mixed with MSG is attenuated [48], or eliminated [47], demonstrating the importance of the T1R3 subunit for the synergistic effect between L-glutamate and IMP in the CT nerve. In response to MSG in isolation, T1R3 knockout mice showed reduced CT responses only at the highest MSG concentration [48]. This reduced response did not occur in the GL nerve, indicating that perhaps other receptors mediate umami responses from the GL nerve, for example, the mGluR4 receptor [48]. Supporting this, mGluR4 knockout mice displayed reduced responses to umami stimuli (MPG) in both the CT and GL nerves, these receptors may not be innervated by S1, or S2 fibres [54]. Whether the transduction of umami taste results in a uniquely perceptible experience will be discussed below.

#### **6. Perceptual Independence of Umami Taste**

Perception is input from the senses giving rise to a conscious experience of the particular stimulus [11]. Basic tastes should elicit perceptions independent to other basic tastes, and should not be produced by combination of existing basic tastes, or other sensory systems, such as the somatosensory system (i.e., mouthfeel or mouthfullness) [10,12]. The detection of a compound by taste receptors, and transduction to gustatory processing areas may produce a taste perception, but this taste perception may not always be a perceptually salient experience.

An important point to note regarding perceptual independence of umami is that the compound responsible, L-glutamate, is not used in the glutamic acid form as it is sour, so the sodium salt form is primarily used in psychophysical studies, meaning some potential overlap with salt taste (please see *Umami and salty* section).

Describing the perception arising from tasting umami effective stimuli becomes difficult due to the absence of a clear set of lexicon for describing umami, thus, whether umami is perceptually salient is not clear. Throughout the literature, a multitudinous lexicon has been used to describe umami taste, ranging from meaty, savoury, brothy, mouthfullness, and delicious [35,55,59]. Familiarisation or a learning effect for umami taste perception is not consistent within the literature, for example, a learning effect for umami hypotasters occurred after repeated exposure [60], contrary to this, umami taste sensitivity either increased or decreased depending on participants' age over repeated measures [61]. Using familiarisation or repeated exposure for improving perceptual salience of umami taste requires further research, as the current literature is inconclusive. The question that remains is whether a basic taste should require familiarisation for a perceptually salient experience to occur? Additionally, the common description of umami flavour as 'mouthfeel' [62] implies a tactile component to umami taste, similar to the description used for kokumi taste. Descriptions for umami taste cited within the literature are similar to those used to describe kokumi taste. Kokumi descriptions include deliciousness, rich, continuity, and mouthfullness [63]. Although kokumi is not a basic taste, there are similarities in both effective stimuli (glutamic acid derivatives) and descriptions of perceptual experiences arising from tasting both kokumi and umami stimuli suggests these taste qualities have perceptual similarities.

Considering L-glutamate is the predominant umami stimuli that is detected by glutamate receptors [17], it may be suitable to predict that foods containing high concentrations of free L-glutamate would ultimately lead to an experience that is perceived, and described, as umami or savoury. Although, it is important to note that taste perception of whole foods is indeed complex, with contribution of many tastants within the one food resulting in the overall taste perception produced. Nevertheless, in foods including seaweed and specific mushrooms, for example shitake, that contain high concentrations of L-glutamate, these are typically described as umami tasting. Contrary to this, there is a number of natural foods containing high concentrations of free L-glutamate that are not described as having an umami taste, for example peas (200 mg/10 g), corn (140 mg/100 g), red grapes (184 mg/100 g), or tomatoes (140 mg/100 g) [14,64,65]. Similar to sweet and sour tastants in the

previously mentioned foods, the presence of L-glutamate in these foods is an important compound to produce the overall flavour of these foods, rather than eliciting a clear perceptible umami taste [36,66].

Umami (MSG) has been shown to exhibit partially independent taste perception, as previous studies using multidimensional scaling have found that umami lies perceptually outside of the four basic tastes (sweet, sour, salty, and bitter) (cited in [25]), and that the taste perception of umami is predominately due to the anion (L-glutamate), albeit a small effect of the cation needs to be considered [23]. Perceptual associations between umami and salty taste exist as thresholds for the two tastes were found to correlate in participants classified as umami hypotasters [60]. Perceptual associations may similarly exist for umami and sweet tastes possibly due to the shared taste receptor subunit (T1R3). For example, rodents have reduced discrimination ability between sucrose and MSG when the sodium in MSG is neutralised using a salt taste blocker (amiloride) [49]. Furthermore, in humans, perceptual associations have been found between umami and sweet tastes in umami hyposensitive participants [67]. Therefore, it is pertinent to consider the perceptual relationship between umami taste and basic tastes, specifically salty and sweet tastes.

#### *6.1. Umami and Salty*

Glutamate in isolation from the sodium ion is glutamic acid, and has been described as having a sour taste [14]. The sodium salt of L-glutamate, MSG, produces an umami taste, and is the predominant prototypical umami stimulus used in psychophysical testing [60,67–69]. MSG potentiating 5'ribonucleotides are similarly tasted in their disodium salt form [13,22] complicating the perceptual independence of umami from salt taste in psychophysical testing [60,67–69]. Participants confuse umami with salty taste [68], and food (soup) containing MSG+IMP has been perceived as saltier, but not more savoury, than soups without MSG+IMP [43]. To overcome the sodium component of MSG, MPG is used in some psychophysical studies as a sodium free umami stimuli [55], although potassium also imparts salty, bitter, and metallic tastes, thus for all psychophysical testing L-glutamate requires a cation to produce a perceptible umami taste [14,23].

Through measuring DT and suprathreshold intensity for umami (MSG) and salty (NaCl), Lugaz, Pillias and Faurion (2002) found that 27% of their study population were classified as putative umami hypotasters. This proportion of umami hypotasters is consistent throughout the literature, with 28% of female participants [67], and 21% of participants [55], having a reduced ability to discriminate between 29 mM MSG and 29 mM NaCl. Using a filter paper disk method, 24% of female Japanese subjects had umami taste thresholds above 50 mM MSG, and were considered hypotasters [69]. The remainder of participants were classified as either semi-discriminators [67], or were able to discriminate between NaCl and MSG at the level of significance and were considered umami tasters [55,60]. Although a very low percentage of the population, 3.5% of a French [60], 3.2% of a German, and 4.6% of a Norwegian population [70] had no ability to discriminate between 29 mM NaCl and 29 mM MSG. These participants were unable to taste the L-glutamate in MSG and were considered non-tasters. Whether a similar proportion of subjects would be found to be umami non-tasters and tasters in non-European populations, or populations with high MSG intake, requires further research.

In individuals with an ability to taste L-glutamate, umami and salty taste perception are independent, conversely, in participants considered umami hypotasters, umami and salty taste perception are associated. Lugaz and colleagues (2002) found a positive correlation (*r* = 0.75) between individual salty (NaCl), and umami taste (MSG) thresholds in hypotasters, indicating that the hypotasters were likely to be perceiving only the sodium cation of the MSG. Along the same line, Pepino and colleagues (2010) found that participants classified as umami hypotasters (referred to as non-discriminators), perceived significantly more saltiness, and significantly less savouriness in MSG at suprathreshold concentrations, than umami tasters. There was no significant difference between umami tasters and hypotasters umami (MSG) DT [67], suggesting different mechanisms may mediate umami DT and suprathreshold taste dimensions supporting Lugaz and colleagues' (2002) findings that thresholds and intensity perception for umami do not necessarily co-vary. Associations between

umami DT and salty DT were not investigated, therefore, it is unknown if an association would have occurred between salty and umami DT in this female population group [67].

The difficulty with confirming umami taste perception independent from salt taste perception lies in the sodium cation in MSG. This can be overcome in part by the use of MPG, potassium, however has salty, bitter, and metallic taste characteristics [71], and therefore MPG taste perceptions cannot be solely attributed to L-glutamate. The contribution of the sodium ion in MSG can be reduced with the addition of IMP, nevertheless, IMP is tasted in disodium form and although IMP is added to MSG/MPG at subthreshold NaCl concentrations, the presence of sodium cannot be completely negated. Perceptual associations between umami and salty tastes appear to occur specifically in participants classified as umami hypotasters, but not in umami tasters. That is, for umami hyposensitive or non-tasting participants they are predominately sensing the sodium cation within MSG, resulting in associations between salty and umami taste perception for these participants.

#### *6.2. Umami and Sweet*

Umami and sweet taste share a common taste receptor subunit, T1R3, therefore there is potential for perceptual associations to exist [67,69]. Mice are capable of discriminating between MSG and sucrose [49], but when amiloride (a sodium blocker) is applied to neutralise the Na+ in MSG, a significant reduction in discrimination ability occurs [49]. Although significantly reduced, discrimination ability of the mice was still above chance, nevertheless, MSG and sucrose have some perceptual associations in rodents when the perceptual influence of Na+ is eliminated [49].

Interestingly, in human studies, umami hypotasters (non-discriminators) (27%, *n* = 16), have both significantly lower umami, and significantly lower sweet taste perception at suprathreshold concentrations, compared to umami tasters (discriminators) [67]. At DT, no association between umami (MSG) and sweet (sucrose) DT was found, again indicating different mechanisms may mediate suprathreshold and DT taste perception [67]. Similarly, umami (MSG) hypotasters (23.8%, *n* = 10) have a significantly lower sweet taste sensitivity at RT than umami tasters (*n* = 32) [69]. This suggests a relationship between umami and sweet taste perception. Contrary to these studies, Chen and colleagues (2009) found no significant difference in sweet taste intensity ratings in umami (MPG) insensitive (*n* = 5), and umami sensitive (*n* = 5) subjects. Mixed results could be attributed to different prototypical umami stimulus used (MPG or MSG), or due to the relatively low number of participants (*n* = 10), compared to previous psychophysical studies [67,69]. Similar to the associations between salty and umami taste, sweet and umami taste perception has been found to be perceptually associated in participants considered umami hypotasters, but not in umami tasters. Although the literature is not consistent, the associations found in some studies could conceivably be due to the shared receptor subunit between sweet and umami tastes, T1R3.

There is enough evidence to question whether umami is perceptually salient, particularly owing to the lexicon used to describe umami taste perception, and the similarities of these descriptions to kokumi taste. Perceptual independence of umami from salty, and sweet, is also unclear as associations exist between umami and salty, and umami and sweet tastes specifically in umami hypotasters. These associations are found across multiple taste dimensions, including DT, RT, and suprathreshold intensity.

#### **7. Umami and Hedonics**

MSG in an aqueous solution does not taste pleasant, however, when added to a complex food such as broth, it enhances palatability. For example, in infants the presence of high concentrations of L-glutamate in a breast milk matrix may increase the milks palatability and acceptability [25], and MSG added to a food matrix (soup) is preferred, but in an aqueous solution MSG is aversive [37]. As previously mentioned, free L-glutamate and IMP/GMP are naturally present in a range of foods. Across many cuisines mixtures of foods containing high concentrations of free L-glutamate are combined with foods containing high concentrations IMP/GMP thereby promoting the umami taste synergism and palatability [1]. For example, in Italian cuisine the combination of parmesan

(1200 mg/100 g L-glutamate) and beef/pork mince sauce (70 mg and 200 mg/100 g IMP respectively) or parmesan and tomato (120 mg/100 g L-glutamate). Or in Asian cuisines the combination of fish sauce (ranging from 620–1380 mg/100 g L-glutamate) and meat or fish products (ranging from 70–285 mg/100 g IMP) is frequently seen (see [25] for further examples). This combining of foods for enhanced palatability does not often come independent of sodium and kokumi peptides. Taking the previous example, parmesan contains high concentrations of sodium, and kokumi peptides [72], as does soy sauce [73]. Likewise, in studies where ingredients were omitted to determine key taste active compounds within a food, sodium, and free L-glutamate (along with other taste active amino acids) were common key tastants (reviewed in [74]).

The combination of added MSG and salt (NaCl) increases the acceptance of some foods, including various soup/stocks [75] and rice dishes [76] at certain ratios (usually between 0.1% and 0.8% by weight [66]) depending on the foodstuff and culture. For example, in European populations this may be higher (between 0.6 to 1.2%), possibly owing to the reduced familiarity of umami taste in Western populations [77]. The addition of MSG to improve palatability has been successfully used to reduce the sodium concentration in food without implicating the sensory properties of the foods [75,76], thus, displaying that in certain foods L-glutamate, IMP, sodium and kokumi effective peptides all contribute to the development of flavour and palatability in commonly consumed foods globally.

#### **8. Relationship between Receptor, Perception, and Behavioural Responses of Umami and Sweet Taste**

Perceptual associations between sweet and umami taste exist and may be due to behavioural factors including MSG and sucrose consumption [69,78], potentially owing to expression or sensitivity of the shared common receptor subunit T1R3 [17]. *In vitro* when MSG and sucrose are co-applied to sweet taste receptor cells, the response of the sweet taste receptor cells to sucrose is weakened [79]. Response from sweet taste receptor cells is also significantly weakened when glutamyl dipeptides are co-applied with sucrose [79]. When the umami tasting compounds are applied with lactisole, which inhibits activation of T1R3, a more severe reduction in the response from sweet taste receptors occurs. If umami tasting compounds and lactisole interacted with the same transmembrane domain of the T1R3, a synergistic reduction would not be expected, as the two stimuli would be competing for the same transmembrane domain. This suggests an interaction between umami peptides and MSG with sweet taste receptors, preventing sweet substances binding to an alternative domain, potentially T1R2 extracellular domain rather than the T1R3 domain [79].

Interestingly, in a human intervention study, prolonged consumption of MSG significantly reduced female participants' umami suprathreshold intensity perception, and similarly reduced (trending towards significant, *p* = 0.06) sweet taste suprathreshold intensity perception [78]. Similarly, Kubota and colleagues (2018) found that umami hypotasters also had a decreased sweet taste sensitivity, and consumed more sugar than umami tasters, although causation cannot be inferred between umami perception, sweet taste perception and sugar intake. It was not investigated whether umami hypotasters simply had a lower taste sensitivity overall compared to umami tasters, although this is unlikely as no significant differences in bitter taste sensitivity between umami tasters, and non-tasters was found [69].

Increased consumption has been linked with decreased receptor expression for other basic tastes, for example, increased consumption of fat was associated with decreased fat taste perception and decreased expression of fat taste receptor CD36 [18,80]. It would be interesting to know if increased intake of umami and sweet tastes decreases both taste perception of sweet and umami tastes and expression of the shared receptor subunit, T1R3. Alternatively, it is possible that increased intake of L-glutamate may decrease expression of T1R1, mGluR1, or mGluR4 taste receptors, although this does not account for the reduction in sweet taste perception found in previous psychophysical studies [69,78]. Considering umami stimuli may interact with the T1R2 extracellular domain [79], it would be interesting to investigate the influence of oral exposure to umami on T1R2 receptor

expression. Although further research into receptor expression is required, dietary intake of both sweet and umami stimuli appear to influence both umami and sweet taste perception in a similar direction, showing an interesting association between receptor, perception, and intake for umami and sweet tastes.

#### **9. Behavioural and Physiological Responses to Umami Effective Stimuli**

A key aspect of taste and taste receptor activation is the physiological responses initiated from oral taste receptor activation, and the influence on behavioural responses in human studies, for example increasing satiation and satiety [41,81]. The commencement of digestion is initiated through the secretion of saliva, the presence of MSG in the oral cavity stimulates a strong response of salivary release through a vagal efferent activation, assisting in initiating this digestion [40,82]. L-glutamate is not only detected in the oral cavity, but also in the gastrointestinal tract where T1R1/T1R3 are found [40,42,83]. T1R1/T1R3 heterodimer has been suggested to affect nutrient absorption through regulation of a peptide transporter through the activation by L-glutamate (reviewed in [42]). Daly and colleagues (2013) found in rodents' gastrointestinal tract T1R1/T1R3 are expressed and activation by L-glutamate results in CCK secretion *in vitro*, which is enhanced by IMP. CCK is involved in digestive processes, including slowing gastric emptying, and has also been suggested to inhibit food intake, thus has a satiety-like action through activating vagal afferent fibres that innervate the stomach and upper intestine (reviewed in [84]).

This enhanced satiety-like action from glutamate consumption has been demonstrated in human behavioural studies where the return of hunger after eating is slowed down after participants consume soup containing MSG, compared to soup without MSG [85], and soup containing protein and MSG compared to other treatments [86]. Similarly, in infants, consumption is decreased and satiation and satiety is increased when infants are fed formula supplemented with MSG, compared with standard cow's milk formula with the same concentration of protein [87]. Masic et al (2014) postulated that umami flavour may play a role in the satiating effects of protein, through sensory-nutrient interactions. Conversely, pre-load soups all containing MSG + IMP in conjunction with either low-energy, high-energy carbohydrate, or high-energy protein, all reduced consumption at a subsequent test meal compared to the same pre-load soups without added MSG + IMP [43]. The presence of MSG + IMP alone reduced consumption, irrespective of the protein content of the preload soup [43]. It is plausible that the presence of MSG + IMP enhanced the post-ingestive release of CCK in the gastrointestinal tract, influencing gastric emptying for all soup pre-load conditions, enhancing satiety, and reducing subsequent intake, although this requires further research. The effect of MSG on satiety is not consistent in the literature, as pre-load soups containing MSG improved energy compensation at a subsequent test meal but did not reduce hunger ratings or total energy intake compared to pre-load soups without MSG [88]. This indicates the importance of IMP in conjunction with MSG, for satiety-like responses and potentially CCK secretion, and considering MSG and IMP are often consumed together in animal protein and other common food combinations, this provides evidence for the importance of umami taste detection and perception in physiological processes and behavioural outcomes.

Interestingly, when a combination of tastants (sweet, bitter, and umami) were infused directly into the duodenum, increased satiety and decreased hunger responses were observed, as was a reduction in consumption of an *ad libitum* meal [89]. When umami was infused in isolation this reduction in hunger and increase in satiety was still observed, but not when sweet or bitter were infused alone, demonstrating the results from the combination of tastants were predominately driven by umami, with the exception of reducing energy intake at an *ad libitum* meal. Interestingly, the infusion of all individual tastants and combination of tastants did not influence the secretion of gastrointestinal peptides in comparison to the placebo infusion. It would be interesting to investigate the interaction of umami stimuli with taste receptors in the oral cavity without subsequent consumption, and whether individual variation in umami taste sensitivity is associated with the previously discussed physiological (satiety hormone release) and behavioural responses (satiety, satiation and intake). Although further

research is required, the consumption of MSG and IMP and the discovery of glutamate receptors in the gastrointestinal tract provides evidence for the role of umami effective stimuli detection stimulating physiological responses which may translate into behavioural responses in human studies.

#### **10. Summary—Is Umami A Basic Taste?**

For the past 3000 years four tastes (sweet, sour, salty, and bitter) have been included in all lists of basic tastes, predominantly based on perception. While these lists changed significantly in other attributes listed, often dependent on current thinking at that time, sweet, sour, salty, and bitter have been consistent. The recent advancements of technology and knowledge has led to the discovery of taste receptors and ligands, extending this basic taste list to include umami and fat as basic tastes. Thus, the basic taste list has grown and has the potential to include a plethora of other tastes including kokumi, carbohydrate, calcium and metallic tastes. With the potential of an ever-growing list of basic tastes in the current day it is pertinent to evaluate the current evidence and the 'moment in time' approach to naming basic tastes. It seems reasonable for new basic tastes, including umami, to consider if they belong in the same category as sweet, sour, salty, and bitter. Below is a summary of the evidence of umami as a basic taste, including an overview of basic and new tastes, against the proposed taste criteria, see Figure 1.


saltiness perception of MSG at suprathreshold concentrations [67]. For umami and sweet taste, associations have similarly been found at DT [67] and RT [69], possibly owing to the shared receptor subunit T1R3. Considering current research finds perceptual associations between umami, other basic tastes (salty and sweet) and putative tastes (kokumi), it is relevant to question umami's classification as a basic taste. Perhaps umami taste fits into a taste classification with other basics (fat) or putative tastes including carbohydrate, kokumi, metallic, and calcium tastes that do elicit a taste perception when presented at high enough concentrations in the oral cavity but this is not necessarily a unique or perceptually salient taste experience.


**Figure 1.** Criteria for tastes to fulfil to be classified as either basic tastes, or within a new taste subgroup. At the first criteria that a taste does not fulfil it is placed on the left-hand side of the model in the 'NO' section, those that fulfil the criteria remain on the right-hand side in the 'YES' criteria. \* ENaC knockout mice have eliminated taste and neural responses to NaCl providing evidence for ENaC as the salt taste receptor [90], human studies have not yet confirmed the ENaC channel for salt taste detection. For the receptor criterion the ENaC receptor for salt taste, albeit in mice, has supporting evidence. \* Type III sour sensing cells have been shown depolarise and reach action potential due to influx of H+ ions, providing evidence for sour taste detection, the specific proton channel responsible for this remains to be confirmed [45].

#### **11. A New Class for New Tastes: Alimentary Taste**

Current advances in knowledge and technology has led to the discovery of taste receptors, which has broadened the stimuli that could potentially be considered basic tastes, including kokumi, calcium, and likely many more to be discovered, for example, receptors responding to carbohydrate and metallic taste ligands. So, this 'moment in time' list of basic tastes has begun to expand with the addition of umami and fat, and others on the horizon such as carbohydrate and kokumi. Should kokumi, fat, or even umami be classified in the same category as sweet, sour, salty, and bitter, all of which have lingered throughout history? Is it enough to have identified receptors on taste cells for new tastes to be considered a basic taste, if the activation of these receptors does not result in a perceptually independent (umami) or perceptually salient (umami and fat) experience? An example of the identification of receptors with an absence of perceptual salience is fat taste, conceivably umami may fall into a similar category. Due to their unquestionable perceptual salience, sweet, sour, salty, and bitter have importance for immediate decision making; do we ingest or reject, that is, these tastes are critical during pre-ingestive taste detection. Many of the new and putative tastes may have far greater importance on post-ingestive consequences of nutrients that are detected not only in the oral cavity, but throughout the alimentary canal. Perhaps it is important, particularly in the context of applied taste research, that we consider umami and fat in a new subgroup of tastes. We propose a new structure of taste classification, with the four traditional tastes remaining as basic tastes due to their critical function during pre-ingestive taste detection, and new tastes becoming 'alimentary' tastes, including umami and fat, which have greater importance for post-ingestive functioning.

**Author Contributions:** R.K. Conceptualisation of the manuscript; I.E.H. Writing—original draft preparation; R.K., D.G.L. Writing—reviewing and editing; all authors reviewed the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Distorted Taste and Impaired Oral Health in Patients with Sicca Complaints**

**Preet Bano Singh 1,\*, Alix Young 2, Amin Homayouni 2, Lene Hystad Hove 2, Beáta Éva Petrovski 3, Bente Brokstad Herlofson 1, Øyvind Palm 4, Morten Rykke <sup>2</sup> and Janicke Liaaen Jensen <sup>1</sup>**


Received: 15 December 2018; Accepted: 21 January 2019; Published: 24 January 2019

**Abstract:** Senses of smell and taste, saliva flow, and dental status are considered as important factors for the maintenance of a good nutritional status. Salivary secretory rates, chemosensory function, burning mouth sensation, halitosis and dental status were investigated in 58 patients with primary Sjögren's syndrome (pSS), 22 non-Sjögren's syndrome sicca (non-SS) patients, and 57 age-matched healthy controls. A significantly greater proportion of patients with pSS and non-SS had ageusia, dysgeusia, burning mouth sensation, and halitosis compared to controls. Patients with pSS had significantly lower olfactory and gustatory scores, and significantly higher caries experience compared to controls. Patients with pSS and non-SS patients had significantly lower unstimulated and stimulated whole saliva secretory rates compared to controls. The findings indicated that several different aspects of oral health were compromised in both, patients with pSS and non-SS, and this may affect their food intake and, hence, their nutritional status. Although non-SS patients do not fulfill Sjögren's syndrome classification criteria, they have similar or, in some cases, even worse oral complaints than the patients with pSS. Further studies are needed to investigate food preferences, dietary intake, and nutritional status in these two patient groups in relation to their health condition.

**Keywords:** taste; smell; dysgeusia; burning sensation; halitosis; saliva; caries; primary Sjögren's syndrome; non-SS sicca syndrome

#### **1. Introduction**

Nutritional status is closely associated with health status, and decline in dietary intake can lead to weight loss and increased risk for disease [1]. The senses of smell and taste are important for nutrition—smell is vital in identifying potential dietary substances in the environment, while taste is instrumental in voluntary ingestion and early digestion of these dietary substances [2]. Saliva and nasal mucus are important for maintaining normal function of the taste buds imbedded in the oral epithelium and olfactory cells found in the nasal cavity [3]. Patients with reduced salivary secretion are known to have taste and smell abnormalities [3,4]. Furthermore, nutritional status is impaired in patients with taste and smell disorders [5]. Most studies showing taste and smell abnormalities in patients with dry mouth are reported from patients with Sjögren's syndrome. Little is known about patients having similar symptoms of severe dry mouth and dry eyes, but not fulfilling the classification criteria for Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune connective tissue disorder of the exocrine glands, primarily the salivary and lacrimal glands [6]. A long-lasting inflammatory process in glandular tissue can lead to the loss of glandular cells, resulting in reduction or, in the worst cases, even complete loss of saliva and tear secretions [7]. The disorder has an unknown etiology, and mainly affects women [8]. The female to male ratio has been reported to be nine to one [8].

To be classified for SS diagnosis, patients have to fulfill at least four out of six classification criteria [9]. These criteria include symptoms of dry mouth and dry eyes; reduced tear secretion; reduced saliva secretion; histopathology of minor salivary glands showing infiltrates of lymphocytes; and the presence of autoantibodies directed against Ro/SSA (anti-Sjögren's-syndrome-related antigen A, also called anti-Ro) and/or La/SSB (anti-Sjögren's-syndrome-related antigen B, also called anti-La) [9]. As long as either serological or histopathological tests are positive, the presence of any four out of six symptoms indicates SS. If three out of four objective symptoms are present, it also justifies classifying the patient with SS. Patients complaining of dry eyes and dry mouth, but not fulfilling all the required criteria, are referred to as non-Sjögren's syndrome sicca (non-SS) patients.

Sjögren's syndrome can be subdivided into primary and secondary Sjögren's syndrome. Primary Sjögren's syndrome (pSS) is a diagnosis given to patients with manifest symptoms of dryness in the absence of other connective tissue diseases. Secondary Sjögren's syndrome (sSS) describes patients with symptoms of dryness, in the setting of another connective tissue disease or chronic inflammatory process, such as rheumatoid arthritis, systemic lupus erythematosus, diagnosed prior to developing SS symptoms [10]. The prevalence of pSS has been reported to range from 0.03% to 2.7% worldwide when different classification criteria were applied [11]. When applying the criteria of the American–European Consensus Group, the prevalence of pSS in the Norwegian population is estimated at 0.05% [12].

Patients with pSS and non-SS display a wide range of similar symptoms; among these are xerostomia—the subjective sensation of oral dryness. Symptoms of dry mouth often include frequent feeling of thirst, feeling of dryness in the mouth and throat, and ulcers may occur in the oral cavity [13]. Patients with dry mouth often have problems with decreased taste sensitivity and chewing in addition to difficulties with articulation [14]. Although, patients categorized as non-SS have similar complaints as patients with pSS, there is a risk that they do not receive appropriate medical care by the health authorities because of lacking diagnosis of SS.

Olfactory and gustatory disorders, also known as chemosensory disorders, are the disorders affecting the senses of smell and taste. Chemosensory disorders are categorized into quantitative and qualitative disorders, depending on whether the senses are reduced or distorted, respectively. Following this categorization, olfactory disorders are classified into anosmia (complete loss of smell), hyposmia (reduced ability to smell), and dysosmia (distorted sense of smell) [15]. Similarly, gustatory disorders are classified as ageusia (complete loss of taste), hypogeusia (reduced ability to taste), and dysgeusia (distorted taste, for example, metallic taste perception) [16]. Patients with a normal sense of smell and taste are categorized as normosmic and normogeusic, respectively. Other oral disorders, like halitosis/oral malodor and burning sensation/numbness in the oral cavity, are often observed in patients with chemosensory disorders [4]. About 50% of patients with chemosensory disorders have reported a negative impact on (i) appetite and body weight, (ii) quality of life, and (iii) psychological well-being [17].

There is evidence that patients with SS have a poor dental status [18]. In a cross-sectional study of Chilean SS-patients, as many as 60% had dental caries, a higher prevalence than the general population [18]. However, in another study, no significant differences could be detected in the dental caries experience of Swedish SS patients compared to dry mouth controls [18,19]. Patients with pSS are also reported to have a significantly higher dental caries experience, also called DMFT (DMFT: decayed, missing, and filled teeth) than healthy controls, mainly due to a higher number of filled and missing teeth [20]. A change in a patient's dental caries status has been suggested as one of several potential markers of the extent of autoimmune-mediated salivary gland dysfunction in pSS [20].

The aim of this study was to compare salivary flow, olfactory and gustatory function, burning mouth sensation, halitosis, and dental status in patients with pSS, non-SS sicca patients, and healthy age-matched controls, to gain more insight into the oral status of non-SS sicca patients.

#### **2. Materials and Methods**

#### *2.1. Participants*

The study was conducted at the Dry Mouth Clinic at the University of Oslo (UiO), Norway, and was approved by the Norwegian Regional Committee for Research Ethics (REK 2015/363). Another study has previously been published with the same REK number which includes 31 female patients with pSS and 33 gender-matched controls [4]. The present study presents an additional patient group (22 non-SS patients), and a higher number of patients, in both pSS group (58 patients with pSS) and healthy control group (57 healthy controls). Moreover, different parameters have been investigated in the two studies. The data presented in this study has not been published before. Participant characteristics are presented in Table 1. Written informed consent was obtained from all participants prior to examination. Most patients with pSS were referred from the Department of Rheumatology at Oslo University Hospital (OUS), where they were classified according to the American–European Consensus Group criteria (13). Non-SS patients were referred to the last author J.L.J for salivary gland biopsies [13]. They all had sicca complaints, but anti-Ro/SSA were absent, and the histopathology of their salivary gland biopsies were not consistent with pSS. The exclusion criteria for controls were mouth and eye dryness, chronic diseases, and use of medications that could affect the salivary glands. The participants were instructed to refrain from eating, drinking, and smoking one hour prior to examination. The assessments of salivary secretory rates, olfaction, gustation, oral malodor, and dental status were carried out by a team of calibrated dental practitioners and specialists.


**Table 1.** Participant characteristics.

non-SS: non-Sjögren's sicca patients, pSS: primary Sjögren's syndrome patients; Fischer's-exact test, One-Way ANOVA, NS = Not Significant.

#### *2.2. Saliva Assessment*

Summated Xerostomia Inventory-Dutch (SXI-D) version was used to assess participants' self-reported perception of dry mouth [21]. SXI-D is a shortened version of the Xerostomia Inventory

(XI) [22] and consists of five statements that are used to determine the severity of xerostomia. The SXI-D sum score ranges from 5 to 15, where 15 = very severe problems related to xerostomia. Thereafter, unstimulated (UWS) and chewing-stimulated (SWS) whole saliva were collected from all participants to determine salivary secretory rates. Unstimulated whole saliva was collected first for 15 min, and then SWS for 5 min. Saliva samples were weighed and secretory rates were calculated for UWS and SWS (g/min = mL/min). UWS secretory rate was considered normal if ≥ 0.1mL/min, and SWS secretion rate was considered normal if ≥ 0.7 mL/min [23].

#### *2.3. Olfactory Assessment*

Self-reported perception of sense of smell was obtained prior to olfactory testing. Participants were asked to score their own subjective smell perception on a visual analogue scale (VAS) from 0 to 10, where 0 = no smell perception, and 10 = very good smell perception. Cognitive olfactory function was measured using twelve-stick identification test (Burghart Messtechnik, Wedel, Germany). The participants were instructed to choose from four possible answers on a multiple choice-scoring card. The answers were recorded, and the data were summarized for each participant. A normative classification [24] was used to define anosmic (score 0–5), hyposmic (score 6–9), and normosmic (score 10–12) participants.

#### *2.4. Gustatory Assessment*

Self-reported perception of sense of taste was obtained prior to gustatory testing. Participants were asked to score their own subjective taste perception on a visual analogue scale (VAS) from 0 to 10, where 0 = no taste perception, and 10 = very good sense of taste. Gustatory function was evaluated using taste strips (Burghart Messtechnik, Wedel, Germany) with four basic taste qualities; sweet, sour, salty, and bitter, each tested at 4 different concentrations. The taste qualities were presented in a random manner, starting with the weakest concentrations. This protocol resulted in a total of 32 values for each participant, as both sides of the tongue were tested. A normative classification [25] was followed to distinguish between ageusic (score 0–12), hypogeusic (score 13–18), and normogeusic (score 19–32) participants.

#### *2.5. Assessment of Dysgeusia, Burning Mouth Sensation, and Halitosis*

A questionnaire was designed for use in this study to assess participants' experience of dysgeusia, burning mouth sensation (BMS), and halitosis (Table 2). The present questionnaire is a modified version of a questionnaire that we have published in a previous study [4]. Both patients with pSS and non-SS reported that they had periods when their disease symptoms were more pronounced ("bad periods") and periods when the symptoms were less pronounced ("good periods").

#### *2.6. Oral Malodor Assessment*

Self-reported perception of halitosis was obtained prior to oral gas sampling. Participants were asked to score their own subjective perception of oral malodor on a scale from 0 to 5, where 0 = no appreciable odor, and 5 = extremely foul odor. Halitosis was measured using both organoleptic and objective methods. The organoleptic measurements were performed by instructing the participants to exhale briefly through the mouth at three different distances (100, 30, and 10 cm) from the nose of the organoleptic judge. The level of malodor was recorded using the same scale as for the self-reported perception of halitosis [26]. Levels of volatile sulfur compounds (VSC) in the mouth air of the participants were measured by gas chromatography (GC: OralChroma™, Nissha FIS, Inc., Osaka, Japan). Mouth air samples from the participants were obtained using a standardized procedure according to the user manual. A 1.0 mL syringe was inserted into the oral cavity until the stopper was in contact with the lips and the syringe could be held gently between the teeth without the tongue touching the tip of the syringe. After the syringe was held in this position for 30 s, a mouth air sample was withdrawn using the syringe, and was immediately injected into the OralChroma™. Analysis of VSC started automatically, and the levels of hydrogen sulfide (H2S) and methyl mercaptan (CH3SH) determined. The olfactory threshold levels (in parts per billion, ppb) indicating oral malodor were considered either high (H2S > 112 ppb and CH3SH > 26 ppb) or low (H2S < 112 ppb and CH3SH < 26 ppb), as recommended by the manufacturer and used in other studies [27].


**Table 2.** Questionnaire used to assess participants' complaints of dysgeusia, burning mouth sensation, and halitosis, and their impact on quality of life.

\* Bad periods: periods when disease symptoms are more pronounced.

#### *2.7. Dental Assessment*

Self-reported perception of dental health and general health was obtained from the participants prior to clinical and radiological examination of the teeth. Participants were asked to score their own subjective assessment of their dental and general health status on a scale from 0 to 5, where 0 = very poor, and 5 = excellent. A thorough dental examination, consisting of clinical and radiological examination of the oral cavity, was conducted by general dental practitioners. The number of decayed, missing, or filled teeth (DMFT) and only filled teeth (FT) were recorded [23].

#### *2.8. Statistical Analyses*

Descriptive statistical analysis was performed, and the results are presented in percentages, median/interquartile range (IQR)/ranges. Normality of continuous variables was tested on histogram, Q–Q plot, and by Shapiro–Wilk test. Due to the low sample size and non-normal distribution of the continuous variables, Kruskal–Wallis ANOVA and Mann–Whitney *U* test was used to detect median differences of continuous, numerical variables between the two or three groups (control, non-SS, pSS). Chi-square (χ2) test and Fischer's-exact test was used to test the differences of the distribution of categorical variables. Point-biserial and Spearman correlations were used to measure the strength and direction of the association between the one continuous and one dichotomous variable, and between two continuous variables respectively. All differences were considered significant at *p* < 0.05. Statistical Package for STATA (Stata version 14.0; College Station, TX, USA) and SPSS (SPSS version 24, IBM, Armonk, NY, USA) were used for the statistical analyses.

#### **3. Results**

#### *3.1. Dysgeusia, Burning Mouth Sensation, and Halitosis*

Self-reported complaints of dysgeusia, burning mouth sensation, and halitosis in the three groups are shown in Table 3. The completion rate for Yes/No questions in the questionnaire was 100% in the three groups. The frequency of dysgeusia, burning sensation, and halitosis was significantly higher in the non-SS and pSS groups versus controls, and these self-reported complaints showed significant association with the disease (*p* < 0.001).

**Table 3.** Overview of self-reported complaints of dysgeusia, burning mouth sensation, and halitosis in the three groups.


non-SS: non-Sjögren's sicca patients, pSS: primary Sjögren's patients; Chi-square test.

Fifteen patients with non-SS, thirty-one patients with pSS and one participant in the control group, who experienced dysgeusia, answered further questions. Metallic taste dysgeusia was the most common complaint both in the non-SS and pSS groups. Other taste distortions were described as "rotten" and "bitter", in addition to "other" taste distortions which the participants were not able to describe in words. Distorted taste was significantly more common in the non-SS and pSS groups, compared to controls (*p* < 0.001) (Table 4).


**Table 4.** Participants experiencing distorted taste.

non-SS: non-Sjögren's sicca patients, pSS: primary Sjögren's patients; Fischer's-exact test.

Some patients with pSS and non-SS described that they had good and bad periods, where the disease symptoms were less pronounced in good periods and more pronounced in bad periods. The duration of good and bad periods varied between individuals. Dysgeusia was experienced either "constantly", "daily", "sometimes, or "in bad periods". The perceived distorted taste was significantly more frequent in the non-SS and pSS groups, compared to controls (*p* < 0.001) (Table 5).

**Table 5.** Overview showing how often participants experienced distorted taste.


non-SS: non-Sjögren's sicca patients, pSS: primary Sjögren's patients; Fischer's-exact test.

When answering the question "is bad taste related to meals?", some participants reported dysgeusia "during meals", others experienced it "in between meals", while some reported "constant" lingering of bad taste in the mouth. Only patients with pSS reported that bad taste was more pronounced "during meals", resulting in foul-tasting meals. One of the patients with pSS reported that even water had a metallic taste. Moreover, "constant" perception of dysgeusia was also found only in the pSS group (Table 6).


**Table 6.** Participants reporting whether dysgeusia was related to meals.

non-SS: non-Sjögren's sicca patients, pSS: primary Sjögren's patients; Fischer's-exact test.

The response rate for the multiple choice questions for burning mouth sensation among the participants experiencing burning mouth, was almost 30% in the non-SS group, 65% in the pSS group and 50.0% controls. Majority of these participants experienced a burning sensation on the "whole tongue", while some patients experienced this only on the "anterior tongue". Only one patient with pSS experienced a burning sensation on the "lips and palate" in addition to the tongue. A significantly higher proportion of participants experienced burning mouth sensation on the tongue, compared to controls (*p* < 0.001) (Table 7). An overview of how often participants in the three groups experienced burning mouth sensation is shown in Table 8.

**Table 7.** Location of burning mouth sensation experienced in the oral cavity.


non-SS: non-Sjögren's sicca patients, pSS: primary Sjögren's patients; Fischer's-exact test.


**Table 8.** Overview showing how often participants experienced burning mouth sensation.

non-SS: non-Sjögren's sicca patients, pSS: primary Sjögren's patients; Fischer's-exact test.

For some of the patients complaining of burning mouth sensation, it was reported to be worst "during meals" in the non-SS and pSS groups (Table 9). Twenty-seven percent of non-SS patients and 24% of patients with pSS reported that they had to refrain from food items like spicy food, sour food items, sour fruits, and beverages like soft drinks, juices, and wine, because of burning mouth sensation.


**Table 9.** Participants reporting whether burning sensation was related to meals.

non-SS: non-Sjögren's sicca patients, pSS: primary Sjögren's patients; Fischer's-exact test.

Among participants complaining of halitosis, non-SS patients were more affected than patients with pSS, while none of controls complained of oral malodor. Table 10 shows how often participants experienced oral malodor. Some patients reported that they avoided drinking tea or coffee because of perceived risk of getting halitosis. When answering "which of the disturbances have a negative effect on your quality of life?", both non-SS patients and patients with pSS reported burning mouth sensation and distorted taste as major factors affecting their quality of life.


**Table 10.** Overview showing how often participants experienced halitosis.

non-SS: non-Sjögren's sicca patients, pSS: primary Sjögren's patients; Fischer's-exact test.

#### *3.2. Gustatory Function*

The results of the Kruskal–Wallis ANOVA and the Mann–Whitney *U* test showed that the measured median gustatory scores (median (IQR), range) were significantly lower in the pSS group (20.0 (16.0–26.0), 2.0–32.0) than in the control group (26.0 (22.0–28.0), 12.0–32.0) (*p* = 0.001). No significant differences were observed between the non-SS (24.0 (20.0–26.0), 2.0–32.0) and the control group (Figure 1a). Participants' self-reported taste scores also revealed a significantly lower mean perception of taste in the pSS group (7.0 (5.0–9.0), 0.0–10.0) compared to the control group (8.0 (8.0–10.0), 3.0–10.0), (*p* = 0.009). No significant difference was found comparing the non-SS group (8.0 (5.0–9.0), 3.0–10.0) with controls (Figure 1b). Chi-square tests showed that a significantly higher percentage of pSS and non-SS patients had ageusia compared to controls (Table 11).

**Figure 1.** Measured and self-reported taste score in the three groups. Boxplots illustrating (**a**) measured taste scores and (**b**) participants' self-reported taste score in controls, primary Sjögren's patients (pSS), and non-Sjögren's sicca patients (non-SS). (Kruskal–Wallis ANOVA and Mann–Whitney *U* test; \*\* *p* < 0.01.). Dots in the figures represent the outliers.

**Table 11.** Percentage of participants with ageusia (no taste perception) and hypogeusia (reduced taste perception) among participants.


non-SS: non-Sjögren's sicca patients, pSS: primary Sjögren's patients; Chi-square test. \* *p* < 0.5.

#### *3.3. Olfactory Function*

The results of the Kruskal–Wallis ANOVA and the Mann–Whitney *U* test showed that the measured median olfactory scores (median (IQR), range) were significantly lower in the pSS group (10.0 (9.0–11.0), 0.0–12.0) than in the control group (11.0 (9.0–11.0), 3.0–12.0), (*p* = 0.007). No significant differences were observed between the non-SS (10.0 (9.0–11.0), 6.0–16.0) and the control group (Figure 2a). Participants' self-reported smell scores did not reveal any significant differences between the three groups (Figure 2b).

**Figure 2.** Measured and self-reported smell score in the three groups. Boxplots illustrating median, interquartile ranges (IQRs), and ranges of (**a**) measured smell scores (0–12) and (**b**) participants' self-reported smell scores (0–10) in controls, primary Sjögren's patients (pSS), and non-Sjögren's sicca patients (non-SS). (Kruskal–Wallis ANOVA and Mann–Whitney *U* test; \*\* *p* < 0.01.). Dots in the figures represent the outliers.

#### *3.4. Oral Malodor Results*

Gas chromatographic analysis (median (IQR), range) revealed the following H2S-values (ppb): control group (33.5 (8.7–141.0), 0.0–2885.0), pSS group (27.5 (15.7–96.2), 0.0–458.0), and non-SS group (41.0 (13.5–84.0), 0–803.0). The results for CH3SH (ppb) were as follows: control group (8.0 (3.0–26.5), 0–193.0), and for the pSS and non-SS groups were (6.0 (2.0–13.2), 0–75.0) and (5.0 (0–13.2), 0–83.0), respectively. There were no significant differences in H2S and CH3SH levels between the groups.

There was no significant correlation between the self-reported perception of halitosis and the organoleptic measurements. The self-reported perceived halitosis scores (median (IQR), range) for the control group were (0.0 (0.0–1.0), 0–3), while the scores for the pSS group and non-SS group were (1.0 (0.0–2.0), 0–4) and (2.0 (1.0–3.0), 0–5), respectively. Organoleptic judge scores were (0.0 (0.0–1.0), 0–2) for the control group, (0.0 (0.0–1.0), 0–3) for the pSS group, and (0.0 (0.0–1.0), 0–2) for the non-SS group. No significant differences were found between groups in self-reported perception of halitosis and organoleptic measurements.

#### *3.5. Saliva and SXI-D*

The results of the Kruskal–Wallis ANOVA and the Mann–Whitney *U* test showed that the UWS secretory rates (mL/min) were significantly lower in the pSS group (0.1 (0.0–0.1), 0.0–0.4) and non-SS group (0.1 (0.0–0.2), 0.0–0.6) compared to the control group (0.3 (0.2–0.4), 0.0–0.8), (*p* < 0.001) (Figure 3a). Also, SWS secretory rates were significantly lower in the pSS group (0.7 (0.4–1.0), 0.0–1.5) and non-SS group (0.9 (0.6–1.3), 0.3–1.8) compared to controls (1.6 (1.1–2.4), 0.5–3.5), (*p* < 0.001) (Figure 3a). The results of participants' self-reported perception of xerostomia showed significantly higher SXI-D scores in both the pSS group (12.0 (10.0–14.0), 6.0–15.0) and the non-SS group (12.0 (11.0–14.0), 9.0–15.0) compared to controls (6.0 (5.0–7.0), 5.0–9.0), (*p* < 0.001) (Figure 3b). No significant differences were observed between pSS and non-SS groups, for either salivary secretory rates or SXI-D score.

**Figure 3.** Measured saliva secretory rate and self-reported perception of xerostomia in the three groups. Boxplots illustrate median, IQRs, and ranges of (**a**) saliva secretion rates (mL/min) and (**b**) SXI-D: Summated Xerostomia Inventory-Dutch scores in controls, primary Sjögren's patients (pSS), and non-Sjögren's sicca patients (non-SS). (Kruskal–Wallis ANOVA and Mann–Whitney *U* test; \*\*\* *p* < 0.001.)

Pathologically low saliva secretory rates for UWS (≤ 0.1 mL/min) and SWS (≤ 0.7 mL/min were analyzed among the participants. A significantly higher proportion of patients with pSS had saliva secretory level below the threshold level for both UWS and SWS (Table 12). Furthermore, moderate, significant correlations were found between salivary secretory values (USW and SWS) and dysgeusia, burning mouth sensation, halitosis, taste score, DMFT, and FT, when all the participants were considered together (Table 13).


**Table 12.** Correlations between pathologically low UWS/SWS and the three groups.

non-SS: non-Sjögren's sicca patients, pSS: primary Sjögren's patients. UWS: unstimulated whole saliva, below threshold (≤0.1 mL/min); SWS: stimulated whole saliva (≤0.7 mL/min); Chi-square test. \*\*\* *p* < 0.001.

**Table 13.** Correlations between UWS and SWS and dysgeusia, burning mouth sensation, halitosis, taste score, DMFT and FT.


non-SS: non-Sjögren's sicca patients, pSS: primary Sjögren's patients. UWS: unstimulated whole saliva, SWS: stimulated whole saliva, Pearson's point-biserial correlation coefficient. \*\*\* *p* <0.001, \*\* *p* < 0.01; \* *p* < 0.05.

#### *3.6. DMFT/FT*

Caries experience as measured by DMFT (median (IQR), range) was significantly higher in the pSS group (18.0 (11.0–23.0), 0.0–28.0) compared to the control group (12.0 (6.5–18.0), 1.0–27.0), (*p* = 0.005). The DMFT in the non-SS group (16.0 (12.8–19.3), 0.0–28.0) did not differ from that of the control group (*p* = 0.3) or the pSS group (*p* = 1.0) (Figure 4a).

**Figure 4.** DMFT and FT results from the three groups. Boxplots illustrate median, IQRs, and ranges of (**a**) DMFT: decayed, missing, and filled tooth surfaces and (**b**) FT: filled teeth in controls, primary Sjögren's patients (pSS), and non-Sjögren's sicca patients (non-SS). (Kruskal–Wallis ANOVA and Mann–Whitney *U* test; \*\* *p* < 0.01.)

Similarly, the FT component of the DMFT index (median (IQR), range) was significantly higher in the pSS group (14.0 (10.0–20.0), 1.0–27.0) than the control group (11.0 (5.0–17.0), 0.0–24.0), (*p* = 0.030). The FT score in the non-SS group (15.5 (11.8–18.2), 0.0–24.0) did not differ from that of the control group (*p* = 0.241) or the pSS group (*p* = 1.0) (Figure 4b).

#### *3.7. General Health Status and Dental Status*

Statistically significant differences were found in self-reported general health status (median (IQR), range) between patients with pSS (2.0 (1.0–3.0), 0–4.0), non-SS patients (1.5 (1.0–2.0), 0–3.0), and controls (4.0 (3.0–4.0), 2.0–4.0), *p* < 0.0001 (Figure 5b). Similar statistically significant differences were found between patients with pSS (2.0 (1.0–3.0), 0–4.0), non-SS patients (1.0 (1.0–2.0), 0–3.0), and controls (3.0 (3.0–4.0), 2.0–4.0), *p* < 0.0001, when participants scored their own dental health status (Figure 5a). Spearman's test showed that when all participants were considered together, participants self-reported dental health status was found to be significantly, negatively correlated to dental status DMFT (*r* = −0.27, *p* = 0.001) and FT (*r* = −0.18, *p* = 0.04). Furthermore, significant positive correlations were found between participants' dental and general health status (*r* = 0.58, *p* < 0.001).

**Figure 5.** Self-reported dental and general health status in the three groups. Boxplots illustrate median, IQRs, and ranges of (**a**) self-reported dental status (0–5) and (**b**) self-reported general health status (0–5) in controls, primary Sjögren's patients (pSS), and non-Sjögren's sicca patients (non-SS). (Kruskal–Wallis ANOVA and Mann–Whitney *U* test; \*\*\* *p* < 0.001.)

#### **4. Discussion**

The present study revealed that the non-SS patients have similar or even worse oral health than patients with Sjögren's syndrome. In general, patients with sicca symptoms, suspected to have SS but not fulfilling the classification criteria for SS, far outnumber the patients who fulfill the criteria. Still, only patients who fulfill the criteria are usually included in studies [28]. Thus, non-SS patients are left both without a diagnosis and are often not considered to be of interest for researchers. Therefore, the main focus in this study was the oral health status of the sicca patients without a Sjögren's diagnosis.

In the present study, we found that complaints of dysgeusia, burning mouth sensation, and halitosis were common in the non-SS group. It has previously been shown that patients with pSS have a high percentage of complaints of dysgeusia, burning sensation on the tongue, and halitosis, and that about 50% of patients with pSS report these disorders [4]. In the present study, when comparing non-SS patients with patients with pSS, it was found that non-SS patients had a much higher occurrence of dysgeusia, burning mouth sensation, and halitosis. In the literature, there are no studies available to compare our current findings with results from other studies on non-SS patients. To our knowledge, this is the first study comprehensively evaluating oral health in patients with sicca symptoms without an SS diagnosis. Since many of the patients avoided certain food items due to problems with dysgeusia and burning sensations, it may affect their dietary intake. This is consistent with the literature, where decreased appetite has been reported in 30% of patients and decreased enjoyment of food in 70% of patients complaining of dysgeusia [29]. About 60% of dysgeusia patients have been reported to change their eating patterns and 40% to modify their use of seasonings [29].

In addition to distorted taste, reduced taste function was observed in both non-SS and patients with pSS. Ageusia, a condition characterizing complete absence of taste perception, is a very seldom condition and accounts for less than 1% of patients referred to taste and smell research centers [5,17]. When taste function was measured, almost 15% of non-SS and patients with pSS were found to have ageusia in this study. Loss of appetite has been reported in patients suffering from ageusia [5]. Furthermore, about 10% of non-SS patients and 26% of patients with pSS were found to be hypogeusic. The incidence of this taste disorder is also low in the general population, and some but not all patients suffering from hypogeusia report decreased enjoyment of food and decreased appetite [5]. However, hypogeusia in combination with other disorders, like dysgeusia and burning sensations, might exacerbate the changes in dietary intake [30]. Therefore, dietary intake monitoring and counselling is very important in those patients with pSS and non-SS patients that suffer from both qualitative and quantitative taste disorders.

Smell and taste disorders are common in the general population, however, patients are frustrated due to the lack of appropriate medical attention and care [17,31]. This may partly be a result of a lack of knowledge and focus on appropriate tools required to assess disorders involving chemical senses among medical practitioners. In this paper, we present a novel questionnaire that can be used to assess (i) patient's chemosensory and trigeminal disorders, (ii) their duration, (iii) their effect on food preferences, and (iv) the effect on patient's quality of life. This questionnaire may be helpful for nutritionists and other health professionals in getting an overview of patients' oral disturbances. This will further be beneficial in managing patients' dietary intake. The questionnaire consists of questions with yes and no answers, supplemented with multiple choice questions, and with the option "other", for open-ended answers. It is easy to fill in and not time-consuming. Therefore, it is practical for use both in clinical and research settings. One of the limitations in the present study is that we did not attain a full rate of completion of the questionnaire, as it was first introduced when we realized that patients were having major issues with dietary intake due to their oral health complications. Further studies are needed to validate this questionnaire.

A large proportion of patients reported dietary limitations because of either dysgeusia, burning mouth sensation, halitosis, dry mouth, or a combination of these different oral problems. A synergetic relationship between oral health and nutrition has been suggested [32], in other words, the relationship may be considered as a positive feedback or a vicious circle. Oral conditions, caused by either local or systemic diseases, impact the functional ability to eat and vice versa, and decline in dietary intake can lead to progression of oral diseases [32]. However, little is known about dietary implications and oral disorders in patients with dry mouth symptoms without SS diagnosis. Further studies are needed to gain better insight into mechanisms leading to oral disorders in this group of patients.

In the present study, there were no significant differences in salivary secretory rates between the two patient groups, indicating that both patient groups have similar problems with dryness of the mouth. Results from self-reported mouth dryness scores and measured salivary secretory rates were also well correlated in this study, indicating severe mouth dryness. Furthermore, significant associations were found in this study, among participants with pathologically low salivary flow rates and oral disorders (chemosensory disturbances, trigeminal disorders, halitosis, and DMFT), consistent with other studies [3,33,34]. These oral disturbances can affect the integrity of the oral cavity and, hence, lead to malnutrition [32].

Patients with pSS had a significantly higher number of decayed, filled, or missing teeth compared to non-SS patients and controls. The dental treatments performed on patients included dental fillings, crowns, and bridges. The reason behind extensive dental treatment may be related to low salivary secretory rates, presence of oral disorders, and/or dietary preferences. Interestingly, non-SS patients share similar symptoms with patients with pSS regarding salivary flow rates and oral disorders, but the same degree of dental treatment was not observed in this group. Other systemic, inflammatory causes in SS may therefore be considered as a potential cause of high caries experience in patients with pSS. These findings are consistent with other studies where dental caries status has been suggested as one of several potential markers of the extent of autoimmune-mediated salivary gland dysfunction in pSS [20].

About 60% of non-SS patients and 40% of patients with pSS reported halitosis when answering the questionnaire. However, no significant differences were observed between these two groups regarding subjective and objective measurements of oral malodor. Halitosis experienced in these patients could therefore neither be confirmed by organoleptic assessment, nor by analysis of VSC levels measured by gas chromatography. The difficulty in the self-assessment of breath odor has been discussed by Rosenberg [35]. Furthermore, organoleptic assessments may assess foul-smelling gases other than those containing sulfur (VSC), and this may explain the difference between organoleptic scores and VSC levels measured by GC. These findings are consistent with other studies where clinicians have reported that one-third of the patients seeking treatment for halitosis do not actually have genuine halitosis [36]. The presence of taste and smell dysfunction has been suggested as an alternative explanation for halitosis [36], which might also be the case in the patient groups in this study.

The main limitation of this study is the small sample size, especially for non-SS patients. The prevalence of SS has been reported to be 0.05% in the Norwegian population [12]. The low prevalence of SS is also reflected in our study with low sample sizes of both patients with pSS and non-SS. For reasons not clear to us, non-SS patients were more difficult to recruit to the study than patients with pSS. Another limitation of this study is the lack of assessments of dietary intake and body composition of the participants. We continue the inclusion of patients in these categories in our studies at the Dry Mouth Clinic and plan to introduce more dietary assessments in the future.

#### **5. Conclusions**

In conclusion, this study demonstrated significantly high occurrence of dysgeusia, burning mouth sensation, halitosis, reduced taste, and mouth dryness in non-SS patients and patients with pSS. Impaired smell function and caries experience were more severe in patients with pSS than non-SS patients. Associations were found between participants' self-reported dental health status and general health status indicating a clear synergy between oral and general health.

**Author Contributions:** Conceptualization J.L.J., P.B.S.; methodology, P.B.S., A.Y., A.H., L.H.H., B.É.P., M.R., B.B.H., Ø.P., J.L.J.; formal analysis, P.B.S., B.É.P.; investigation, P.B.S., A.Y., A.H., L.H.H., M.R., B.B.H., J.L.J.; resources, J.L.J. Ø.P.; writing—original draft preparation, P.B.S.; writing—review and editing, A.Y., A.H., L.H.H., M.R., Ø.P., B.É.P., B.B.H., J.L.J.; visualization, P.B.S. J.L.J.; supervision, J.L.J.; project administration, J.L.J.; funding acquisition, J.L.J.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors express their sincere gratitude to all the staff members involved at the research and clinical institutions for their efforts. Thanks are also due to patients and controls who participated in this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*
