Abstract
Background: Occlusal raising method (so-called ‘Template therapy’) has been reported to alleviate various diseases and symptoms, but the underlying mechanism is not clear. We searched the low-molecular weight metabolite(s) in the saliva, the concentration of which is significantly changed by the template therapy. Materials and Methods: One female patient with headache underwent the template therapy for 12 days, and her total saliva was subjected to non-targeted analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). Results: One hundred and thirteen substances were identified in the saliva. Glycine was the most abundant amino acid in the saliva, followed by alanine, serine and proline. After the start of the template therapy, her headache was alleviated, accompanied by a significant (p=0.042) increase of salivary concentration of glycine, as compared with total amino acids whereas that of other amino acids was not significantly changed. In the metabolomics profile, salivary concentration of large number of metabolites as compared with total metabolite concentration decreased, including N-acetylneuraminate (p=0.025) and p-hydroxyphenylacetate (p=0.039). Conclusion: This pilot study demonstrated, to our knowledge for the first time, that only glycine exhibited unique changes among total metabolites, suggesting its significant role in template therapy.
There have been many clinical reports of various systemic symptoms such as headache, shoulder stiffness and mal-posture due to changes in the occlusal position (1-3). Several reports have attributed the artificial occlusal abnormalities to tooth extraction, bite raising and tooth grinding in experimental animals (4-9). These studies suggest that the trigeminal responses to occlusal changes induce various systemic symptoms by as yet -unidentified mechanisms. Out of these symptoms, the head drop and the drooping of the submandibular mental area to the ground were observed about one week after grinding the maxillary posterior teeth of guinea pigs to the cervical area. Abnormal waveforms including T-wave inversion were also observed on electrocardiogram (ECG) (9). We have recently reported that the decrease of the occlusal vertical dimension (OVD) in guinea pig models resulted in two-phase wave of heart rate fluctuations, with the first peak occurring 0-2 days after tooth grinding and the second peak starting from 4 days after teeth grinding until sudden death (usually 12th day), accompanied by the head drop, and that when the OVD was increased, such heart rate fluctuations disappeared (10). Although the occlusal raising method (the so-called ‘Template therapy’) has been reported to improve such systemic symptoms, the fundamental mechanism is not clear. In order to identify the biomarkers of compounds responsible for the efficacy of template therapy, we searched for low-molecular weight metabolites in the saliva, the concentration of which is significantly changed by template therapy, using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS).
Materials and Methods
Sample collection. A female patient (30 years old) had headache or tension-type headache, low back pain and shoulder stiffness, and had a medical examination in Maehara Dental Clinic, Shinjuku-branch, Tokyo. She wore the template there on August 6, 2011 during her summer-time holiday, as previously reported (1). She used the template during sleeping and 2 h exercise for over 3 months. Her symptoms such as non-specific complaints, had disappeared after a week. Whole saliva (2-5 ml) was collected in a 50 ml centrifugation tube and immediately frozen at −20°C six times at 15, 14, 12, 10, 8, 4 days before the therapy and six times at 1, 3, 8, 14, 18, 22 days after the therapy. The CE-TOF-MS analysis of the collected saliva has been performed, according to the Guideline of the Intramural Ethics Committee (approved as no. A1113), as described below.
Sample preparation. Saliva was thawed and centrifugally-filtered through a 5-kDa cut-off filter (Millipore, Bedford, MA, USA) at 9,100 ×g for at least 2.5 h at 4°C to remove macromolecules. Five microliters of Milli-Q water containing internal standards (2 mmol/l each of methionine sulfone, 2-[N-morpholino]-ethanesulfonic acid, D-camphor-10-sulfonic acid, 3-aminopyrrolidine, and trimesate) was added to 45 μl of the filtrate and mixed immediately before CE-TOF-MS analysis.
CE-TOF-MS analysis. The instrumentation and measurement conditions used for CE-TOF-MS are described elsewhere (12, 13) with slight modifications. Briefly, cation analysis was performed using an Agilent CE capillary electrophoresis system, an Agilent G6220A LC/MSD TOF system, an Agilent 1100 series isocratic HPLC pump, a G1603A Agilent CE-MS adapter kit, and a G1607A Agilent CE-ESI-MS sprayer kit (Agilent Technologies, Waldbronn, Germany). Anion analysis was performed using an Agilent CE capillary electrophoresis system, an Agilent G6210A LC/MSD TOF system, an Agilent 1200 series isocratic HPLC pump, a G1603A Agilent CE-MS adapter kit, and a G1607A Agilent CE-electrospray ionization (ESI) source-MS sprayer kit (Agilent Technologies). For the cation and anion analyses, the CE-MS adapter kit includes a capillary cassette that facilitates thermostatic control of the capillary. The CE-ESI-MS sprayer kit simplifies coupling of the CE system with the MS system, and is equipped with an electrospray source. For system control and data acquisition, we used G2201AA Agilent ChemStation software for CE and the Agilent MassHunter software for TOF-MS. The original Agilent SST316Ti stainless steel ESI needle was replaced with a passivated SST316Ti stainless steel and platinum needle (passivated with 1% formic acid and 20% aqueous solution of isopropanol at 80°C for 30 min) for anion analysis.
For cationic metabolite analysis using CE-TOF-MS (12), sample separation was performed in fused silica capillaries (50 μm i.d. × 100 cm total length) filled with 1 mol/l formic acid as the reference electrolyte. Sample solutions (3 nl) were injected at 50 mbar for 5 s and a voltage of 30 kV was applied. The capillary temperature was maintained at 20°C and the temperature of the sample tray was kept below 5°C. The sheath liquid, composed of methanol/water (50% v/v) and 0.1 μmol/l hexakis(2,2-difluoroethoxy) phosphazene (Hexakis), was delivered at 10 μl/min. ESI-TOF-MS was conducted in the positive ion mode. The capillary voltage was set at 4 kV and the flow rate of nitrogen gas (heater temperature=300°C) was set at 7 psig. In TOF-MS, the fragmentor, skimmer and OCT RF voltages were 75, 50 and 125 V, respectively. Automatic recalibration of each acquired spectrum was performed using reference standards {[13C isotopic ion of protonated methanol dimer (2MeOH+H)]+, m/z 66.0632)} and {[protonated Hexakis (M+H)]+, m/z 622.0290]}. Mass spectra were acquired at the rate of 1.5 cycles/s over a m/z range of 50-1,000.
For anionic metabolite analysis using CE-TOF-MS (14), a commercially available COSMO(+) capillary (50 μm i.d. × 105 cm, Nacalai Tesque, Kyoto, Japan), chemically-coated with a cationic polymer, was used for separation. Ammonium acetate solution (50 mmol/l; pH 8.5) was used as the electrolyte for separation. Before the first use, the new capillary was flushed successively with the running electrolyte (pH 8.5), 50 mmol/l acetic acid (pH 3.4), and then the electrolyte again for 10 min each. Before each injection, the capillary was equilibrated for 2 min by flushing with 50 mM acetic acid (pH 3.4) and then flushed for 5 min with the running electrolyte. A sample solution (30 nl) was injected at 50 mbar for 30 s, and a voltage of −30 kV was applied. The capillary temperature was maintained at 20°C and the sample tray was cooled below 5°C. An Agilent 1100 series pump, equipped with a 1:100 splitter was used to deliver 10 μl/min of 5 mM ammonium acetate in 50% (v/v) methanol/water, containing 0.1 μM Hexakis, to the CE interface. Here, it was used as a sheath liquid surrounding the CE capillary to provide a stable electrical connection between the tip of the capillary and the grounded electrospray needle. ESI-TOF-MS was conducted in the negative ionization mode at a capillary voltage of 3.5 kV. For TOF-MS, the fragmentor, skimmer and OCT RF voltages were set at 100, 50 and 200 V, respectively. The flow rate of the drying nitrogen gas (heater temperature=300°C) was maintained at 7 psig. Automatic recalibration of each acquired spectrum was performed using reference standards {[13C isotopic ion of de-protonated acetic acid dimer (2 CH3COOH-H)]−, m/z 120.03841}, and {[Hexakis+ deprotonated acetic acid (M+CH3COOH-H)]−, m/z 680.03554}. Exact mass data were acquired at a rate of 1.5 spectra/s over a m/z range of 50-1,000.
Data analysis and statistical analysis. Raw data were analyzed by our proprietary software MasterHands (13), which follows typical data processing flows including detecting all possible peaks, eliminating noise and redundant features, and generating the aligned data matrix with annotated metabolite identities and relative area (peak areas normalized by those of internal standards) (15). Concentrations were calculated using external standards based on relative area. To eliminate the variation of overall concentration, the concentration of each metabolite was divided by the concentration of total metabolites (relative concentration), or that of each amino acid was divided by the concentration of total amino acids. Only the metabolites frequently observed in four, or more, out of six samples before or after template therapy were used for the data analysis. The student's t-test (two-tailed) was used for statistical comparisons.
Results
In total, 113 substances were identified in the saliva and, out of these, 56 metabolites frequently observed were used for the data analysis. Glycine was the most abundant amino acid in the saliva, followed by proline, alanine and serine (Figure 1A). After the start of the template therapy, the patient's headache was alleviated, accompanied by significant (p=0.042) increase of the salivary concentration of glycine, as compared with total amino acids whereas that of other amino acids was not significantly changed (Figure 1B).
After template therapy, the concentration of only six metabolites was increased [fold-change (FC) >1.2), out of which priopinate and urea were the most dominant, and the relative concentrations of other metabolites were less than 1% (Figure 2A). No metabolite significantly increased in concentration. The concentration of many metabolites decreased after the therapy (FC<0.7), where N-acetylneuraminate (p=0.025) and p-hydroxyphenylacetate (p=0.039) was presented significantly decreased (Figure 2B). Out of these, 11 amino acids, namely proline, alanine, glutamate, glutamine, serine, asparagine, valine, phenylalanine, leucine, threonine and isoleucine, tended to decrease although not significantly. The other 23 metabolites, such as lactate and pyruvate, were hardly changed (data not shown).
Salivary amino acid concentrations before and after template therapy. A: Absolute concentration (left) and ratio of each amino acid to total amino acids (relative concentration) (right). B: Bar graph showing mean and standard deviation of the relative concentration of four major salivary amino acids, glycine, proline, alanine and serine. *p<0.05.
Discussion
There are numerous reports that investigated the salivary concentration of amino acids in relation to caries (16, 17), periodontal diseases (18-20), phenylketonuria (21), migraine (22), a lacto-ovo vegetarian diet (23), smoking and gender difference (24), diurinal changes and aging (25). However, to our knowledge, none of these reports, except for ours (26), has attributed attention to the salivary glycine level. We reported that (i) glycine was the most abundant amino acid in the saliva; (ii) glycine and lysine concentrations increased significantly (p<0.05) with aging, regardless of gender difference; and (iii) glycine and lysine were positively correlated (p<0.001), (iv) however, there was no significant correlation between the salivary concentration of glutamic acid or histidine and age, suggesting that salivary amino acid levels may be regarded as markers of aging (26). The present study demonstrated that glycine was also the most abundant amino acid in the saliva of the female patient presented here, and glycine is the only amino acid that increased significantly with template therapy (Figure 1). This does not necessarily mean that the elevation of glycine is the result of accelerating the aging process, since the observed glycine concentration (at most 100 μM) is much smaller than the value reported in aged people (570 μM) (26). It is interesting to note that the plasma concentration of glycine is significantly reduced by occlusal destruction, producing the opposite effect to template therapy (27).
Relative concentrations of metabolites before and after the template therapy. Metabolites increased (fold-change (FC) >1.2) (A) and reduced (FC<0.7) (B) after template therapy. Bar graph indicates mean and standard deviation. *p<0.05.
The biological significance of the increase of the relative concentration of glycine by template therapy is unclear. Recent reports have suggested the possible role of glycine in inflammation. Glycine stimulated production of pro-inflammatory substances such as nitric oxide, prostaglandin E2, tumor necrosis factor-α and cyclooxygenase-2 in macrophages, gingival fibroblasts and microglia (28-30). On the other hand, glycine exerts anti-inflammatory activity via glycine-activated chloride channels that suppress the production of oxidants and pro-inflammatory cytokines (31) and protected the cells from cadmium (32), cyclosporine-induced kidney damage (33) and liver injury (34, 35), suggesting a possible role of glycine in cell survival and activation. These data suggest dual actions of glycine.
In the metabolomics profiles, a large number of metabolites decreased in concentration (n=27) while that of a few metabolites increased (n=6) on template therapy. The decreased metabolites included many compounds related to nervous systems status; taurine was the most abundant and this metabolite acts as an inhibitory neurotransmitter and is used to aid the treatment of epilepsy and excitable brain status (36). Glutamine and asparagine are excitatory neurotransmitters within the central nervous system responsible for normal synaptic neurotransmission, serine acts as both neurotransmitter and neuromodulator, and alanine is also a neurotransmitter in the visual system (36). Only N-acetylneuraminate and p-phydroxyphenylacetate showed significant reduction (p=0.025 and p=0.039, respectively) after template therapy. N-Acetylneuraminate is the most prominent sialic acid that is a terminal sugar and acts as a marker for chronic inflammation in various systemic diseases, such as heart diseases (37, 38) and breast cancer (39). p-Hydroxyphenylacetate is expected to be produced by oral bacteria (40). Thus, the significant decrease of these salivary components might reflect the metabolic change of both systemic and oral physiological conditions. Further extensive studies with more patients are required to elucidate the biological significance of the present findings.
Acknowledgements
This work was supported by research funds from the Yamagata Prefectural Government and the City of Tsuruoka, and in part by a Grant-in-Aid for Scientific Research (C) of the Ministry of Education, Culture, Sports, Science and Technology of Japan (S. Tanaka, no. 24593164).
Footnotes
-
↵* Undergraduate student, Meikai University School of Dentistry
- Received September 10, 2012.
- Revision received October 16, 2012.
- Accepted October 17, 2012.
- Copyright © 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
