Metabolomics: Paving the path for personalized periodontics - A literature review.

The pathogenesis of periodontal disease is governed by a multitude of factors ranging from the macroscopic to the microscopic scale. Among the factors that constitute the etiological agents of the disease, a major element is the role played by the body's metabolome-i.e., the complete collection of microscopic molecules and metabolic products of cells and tissues in the body. Being of a regulatory nature, the interplay of these molecules exerts a considerable effect on the development as well as the progression of disease, which differs in each individual based on their phenotype. Exploring this connection and application into the field of diagnostic as well as prediction of risk for periodontitis will ultimately result in a personalized standard of care for patients in the future. Copyright:

INTRODUCTION

Periodontitis is a disease influenced by the actions and interactions of intrinsic as well as extrinsic elements. Periodontitis is an acute inflammatory state and affects both the hard as well as soft tissues of the periodontium. The disease proceeds with the formation and exponential growth of colonies of putative micro-organisms in the periodontal pocket. The presence of these microbes, coupled with deficits in the body's immune response, act together with other established risk factors and play a key role in the pathobiology of periodontal disease.[1] Due to its pro-inflammatory characteristic, periodontal disease can be quite cumbersome to treat definitively with sustainable results.

BEHIND THE METABOLOMIC APPROACH—THE WHAT

Background—is it required to look beyond traditional diagnostics?

The most significant drawback of diagnostic methods available conventionally is that they are time-consuming and might lead to a late detection of the disease, as a result of which irreversible damage to the periodontal tissues and tooth loss may occur. Signs and symptoms for examination also vary in their order and degree of appearance from person to person, causing further delay. Apart from demographic and sociographic risk factors, these clinical signs are also governed by the presence or absence of comorbidity or adverse habits such as smoking. Being at the behest of such a variety of elements can ultimately be unfavorable to accurate diagnosis.[2] The search for a method that is as inclusive and productive as genome-sequencing led to the advent of “omic”-based methods that exist currently.[3] However, unlike other similar approaches using transcriptomes, proteomes as well as genomes, analysis of the metabolites in the body, or the “metabolome” is capable of drawing direct links to bodily function in both health and disease. This method provides support to the hypothesis that analysis and drawing inference via metabolomics may be a valid diagnostic approach for periodontal diseases,[4] and hence should be able to help cater diagnosis and treatment outcomes in the field of personalized periodontics.[5]

Qualitative and quantitative measurement and characterization of small molecules (1 kDa) in biological samples at a defined state are referred to as metabolomics. Small molecules or metabolites may be present inside the body's tissues and cells or flowing freely through bio-organic fluids. Essentially chemical compounds, these can be either produced within the body itself or sourced externally. Scrutiny of a “profile” of these metabolites of a person's body can help reveal semi-quantitative data of their entire metabolome.[6] The method evaluates sugars, amino acids, lipids, and their associated by-products via intracellular reactions and measures them. These values, or levels, signify whether the body's metabolism is functioning effectively. Deviation from the normal for any of these values indicates underlying disease, including but not limited to the periodontium.

Given the regulatory functions of these metabolites inside cells, the metabolome is an established connection between both the genotype as well as the phenotype of the individual.[7] This allows a unique insight into the inter-relationships between genes, their expression, their influence on and from the body's microenvironment, microbiomes, and any underlying dysbiosis if occurring. Due to its close relationship to the phenotype of the individual, it has the potential to be a valid resource to draw inferences from and conclude about the progression of disease and the response to external factors such as chemotherapeutics, nutrition, exercise, etc.

ANALYZING THE NEED FOR METABOLOMICS—WHY IS IT REQUIRED IN CONTEMPORARY PERIODONTICS?

A multifaceted rather than streamlined approach has always been at the forefront of periodontal diagnostics. With the evolution of how diagnostic medicine is applied and executed, the ability to accurately anticipate the probability of dysbiosis in a patient has become the need of the hour. For a disease of infectious origin such as periodontitis, the treatment plan should be able to prevent as well as overcome a recurrence to sustain the results of the treatment over the long term.[8]

Although periodontitis is one of the most common diseases of infectious nature, affecting approximately 10% of the population all over the world,[9] conclusive diagnosis is reached usually with a delay once signs of tissue breakdown are clinically apparent. Hence, there is a need for the development of a diagnostic technology which enables clinicians to diagnose periodontitis much earlier than is possible via traditional diagnostic methods. These include the clinical and microbiological examination and radiological interpretation. Several pitfalls exist in relation to either of them, as both methods rely heavily on the examiner's assessment and elucidation. They can therefore be deceptive as well as inconclusive.

Correlating with current paradigms

The pathogenesis of chronic periodontitis is linked to the concept of a microbial shift in the body in which a part of or the entire population of bacterial flora loses its homeostasis. The microbial population undergoes a reversal, with the species which usually dominate decreasing in number whereas the contained or noxious flora rapidly multiplying to adjust the imbalance created.[10] Although first discovered and mostly associated with disorders of the gastrointestinal system such as gastroesophageal reflux disease, inflammatory bowel diseases, and inter alia in recent years, it has been recognized that dysbiosis in the oral microenvironment or “-biome” is implicated in the pathogenesis of periodontitis. This occurs gradually over a considerable amount of time, following which the relationship between the host and the microbial population degrades from symbiosis to a pathologic one.[11] This “sub-version” of homeostasis in the host body leads to periodontal destruction.

To gain a comprehensive understanding of periodontal pathobiology, it is imperative to attempt and establish a chain of causality between the inflammation that is clinically visible, and the composition of the periodontal microflora. There exists a bi-directional relationship between the effect (inflammation) and the cause (microbial population), and these play a part in the etiopathogenesis of periodontitis. Recently, a new model called inflammation-mediated polymicrobial emergence and dysbiotic exacerbation was proposed by Van Dyke et al.[12] This characteristic dysbiosis does not involve changes in microbial population solely but also receives input from changes in the metabolomics of the individual[13] [Figure 1].

Figure 1

Clinical application of a personalized approach towards the treatment of periodontitis. GC – Gas chromatography; MS – Mass spectrometry

Proof of hypothesized correlation via research

In 2017, Sakanaka et al.[14] were successful at establishing a clear association between the degree of inflammation in periodontitis and the presence of certain metabolites. After conducting enrichment analysis of the metabolic compounds, it helped put together a characteristic metabolomic “blueprint” or a signature of periodontitis [Table 1]. Other than investigating the role of certain metabolites and their levels influencing the pathogenesis of the periodontal disease, this study also aimed to draw inferences from decrease or increase in these levels and whether that had any impact on the severity of disease as well as the degree of inflammation present. This degree, or status of inflammatory burden, was measured using an index called the Periodontal Inflamed Surface Area (PISA), coined by Nesse et al. in 2008.[15] According to the study, following the creation of a metabolomic “profile” of samples collected both preceding and following removal of plaque and calculus and subjecting them to metabolomic analysis following protocol, several incriminatory metabolic molecules were recognized [Table 2]. The results of the analyses divulged that out of all the metabolites that were detected, a few metabolites were found to be higher and/or lower in amount specific to the inflammation even after removal of plaque had been done. The authors implied that the metabolic pathways involving each of these chemicals are upregulated in a more severe form of periodontitis, i.e., individuals having a greater PISA score.

Table 1

Review of most recent literature supporting the use of metabolomics to advance the field of personalized medicine including periodontics

Author (s)	Chief inferences
Sakanaka et al., 2017	The study elicited a characteristic metabolomic “blueprint” or a signature, which could be definitively used to predict or detect periodontal disease activity. These chemicals included arginine, proline, butanoic acid, along with degraded lysine. Apart from these molecules, cadaverine and hydrocinnamate were two metabolites that were majorly implicated in altering not only the degree of inflammation but also the severity of periodontal disease activity in the patients
Bartold et al., 2018	Based on an idea of a model for periodontal treatment, their proposal suggested a carefully systematized angle wherein the patient population is divided and sub-divided into several groups/strata, and a profile consisting of custom-made clinical decision-making, practice, and treatment options is formulated. Such a model will also require input from the patient’s sociologic, physiologic, molecular, and cellular analyses, apart from genetic and epidemiologic data. The cumulative effect of all these approaches ultimately will help to create a comprehensive data-set and a corresponding treatment plan for the concerned individual and the level of severity of disease in them
Van Dyke et al., 2020	The massive amount of data that is obtained via metabolomic analysis may be subjected to stratification of various case phenotypes using machine learning or artificial intelligence, to eliminate personnel error. Based on the results thereof, blueprints can be created for diagnosis, treatment planning as well as determining the prognostic risk of periodontal disease in an individual

Table 2

Metabolites reduced via removal of plaque and calculus in gingival blood in relation to periodontal disease severity

Metabolites reduced posthebridement in periodontitis	Metabolites reduced posthebridement in high PISA	Metabolites reduced posthebridement in low PISA
5-oxoproline	4-aminobutyricacid	Tryptophan
Aspartic acid	Cadaverine	Glutamine
Fucose 2	Phenylalanine	Isoleucine_1TMS
Glutamic acid	5-aminovaleric acid	Fucose_1
Indole 3 acetic acid	Succinic acid	Ethanolamine
N-acetyl ornithine	Putrescine	Alanine_2TMS
Leucine	Hydrocinnamate
Alanine_3TMS	Fructose 6-phosphate
Hypotaurine

PISA – Periodontal inflamed surface area

With substantial evidence about the role, that metabolomic analysis has to play in the dysbiosis and subsequent destruction related to periodontitis, the paradigm shifts from a generalized to a personalized approach in the treatment of periodontal conditions. This approach, as elucidated by Bartold in 2018,[5] is based on the key idea of a treatment model that enables the periodontist to plan and provide a catered or tailor-made approach towards treatment that varies between each patient. To bring this concept to fruition, sophisticated diagnostic methodologies are required that take into consideration all or most of these elements, and metabolomics analysis shows considerable promise for the same [Table 1].

THE HOW—USING SALIVA FOR OMICS-BASED PERIODONTAL DIAGNOSTICS

Several methods are available to collect saliva for the purposes of processing and research. These include the use of specialized kits which employ a swabbing technique, collection via active expectoration, inadvertent drooling, both spontaneous as well as stimulated, etc. However, it is important to have a clear understanding at this juncture of the fact that “saliva” may actually be an umbrella term under which all and any different fluids or elements that exist or are produced in the oral cavity may be considered.

Once the adequate amount of saliva is collected, it usually undergoes processing via a cascade of steps[16] that are executed meticulously. The sequence proceeds from the initial design of the sample subset and the research experiment to processing and analysis wherein the discriminating metabolites are elucidated using biostatistics, and the untreated data derived so far is examined to identify the metabolites present in the panel. This is done to avoid any contamination of the sample, loss of dilution factor, and to anticipate and sidestep any errors, be it with respect to processing or analysis, that may influence the result of the investigative analysis.

Once the collection of samples is performed and ready to undergo processing, it can be subjected to by either of several approaches that are currently available. These techniques include either a targeted or nontargeted approach.[16] The former includes techniques that are driven by the biology of the sample, meaning it would be apt to use such a method that would give more accurate and most importantly, sensitive results. They are based on the rationale of supervising levels of known metabolites. Examples include liquid or gas chromatography/mass spectrometry (MS), etc. These techniques provide complete and thorough appraisal of the values of the metabolites and are considered to be high-throughput mechanisms with a low limit of detection. However, the analysis of untargeted compounds or a more comprehensive “global” metabolomic profiling is not possible with these techniques. This drawback is addressed through the untargeted metabolomics approach which includes proton nuclear magnetic resonance spectroscopy (1H-NMR). This is at present one of the most well-tested method of use for metabolomic profiling of saliva. The benefit of using NMR spectroscopy for analyzing whole saliva is that it does not require any form of pre-treatment. On the other hand, variants of techniques using MS do not need postextraction separation of the metabolites found, and hence have gained wide acceptance and use in salivary metabolomic studies.[17]

It is now quite an established fact that all underlying, as well as apparent diseases, have a significant effect on the body's metabolomic profile at varying extents and rates, which may result in an overhaul of this profile in the organism. Hence, an exploration into these changes can lead to a faster and more superior identification of patterns of metabolomics and/or biological markers that may be associated with the path-biogenesis of periodontal diseases. In order to do so, there are several devices and armamentaria either present or under development currently such as smartphone-based applications, handheld meter device, and miniature laboratory devices.[18]

Finally, once the armamentarium, analytic workflow, and method of diagnostic testing are selected, each analysis pattern must be governed by a set protocol of functioning [Figure 2] that should be followed diligently. Doing so avoids any experimental errors that may occur inadvertently, apart from ensuring a smooth completion of each cycle. These steps can be elucidated as a systematized sequence, beginning when a patient reports with clinical findings and culminates into diagnosis and management of the disease.

Figure 2

Systematized approach to metabolomics analysis–from processing to results

However, in developing countries, the feasibility of such a large-scale investigation still requires some time to come to fruition. Multiple contributing elements such as clinical parameters, age, sex, presence or absence of comorbidities and variations in lifestyles present a possible road for research to continue. Creating a panel of endogenous metabolites present in or produced by either the periodontal pathogens or the hosts themselves; and measurement of these metabolites using microfluidics are a possible route that metabolomics research can be taken toward. According to the findings described by Derewacz et al., Goodwin and their team,[19] mutations on the metabolic scale in an organism can also predispose them to exhibit resistance towards antibiotic drugs, apart from initiating fundamental changes in both the primary and secondary metabolic pathways of the concerned organism. On the other hand, for systemic diseases that share the inflammatory background of periodontitis, it can impart a degree of co-morbidity, which makes it difficult to provide definitive treatment of either.[20] With an expansive amount of evidence existing in the scientific literature proving the bi-directional relationship between systemic inflammatory disorders such as diabetes, cardiovascular disease, and disease of autoimmune origin to periodontal inflammation, it can be hypothecated that the variability of the presentation of disease differs based on the metabolic phenotype of each patient. As a result, it is difficult to streamline treatment approaches to a certain profile of concomitant diseases occurring simultaneously. In such a case, the only viable option for treatment remains an empirical approach, which, again, cannot resolve the progression of disease substantially. To remedy this situation, the treatment techniques must, therefore, be designed in a way that is catered to every single aspect of the profile of the disease (s), justifying the need for a personalized approach to be inculcated into periodontics.

UPCOMING CHALLENGES AND OPPORTUNITIES

As is likely with the advent of any new technique or concept, metabolomics in periodontics is not without its share of issues that still need to be addressed.

Poor perceptions

The amount of publicity that metabolomics receives is by far a much lesser amount than other-omics-based technologies. Metabolomics is also a relatively new approach and has had much lesser time to rise to prominence.[21] Even though the amount of data that is generated at a single instance of analysis via metabolomics is humongous, due to which, a thorough analysis of every single piece of information gathered may remain compromised.[22]

Experimental costs

The economic efficiency of metabolomics techniques is quite high, which makes it a difficult challenge. For a conclusive investigation, input is required from several different platforms to investigate the compound biochemical profile of the individual.[23] Hence, should an investigator choose to invest in a set-up for a laboratory capable of conducting such an experiment, they need to factor in the monetary feasibility as well.

Acquisition of experimental data

This is the cornerstone of any metabolomics investigation. The most important issue in this regard is the accurate measurement of the entire metabolic biome of the subject by using only one method.[23] Valuable time may be lost when metabolites may undergo transformation/degradation at the time of the transport of collected samples for processing. To prevent this, it may require the presence of resources and armamentarium which may not be available at facilities providing basic health care services.

Identification of metabolites and pathway mapping

Not all the metabolites naturally occurring in organisms can be quantified or assessed commercially for the time being, and the kits for such an assessment still require to be developed and tested.[24]

Quantification of results

This is possibly the most important aspect of the development of any assessment technology, including metabolomics.[25] The increasing requirement to successfully include data from metabolomic analyses with data from other-omics methods, or a “multi-omics interaction,” is hindered with data that is at best semi-quantitative.[23] Considering these issues, a conclusive quantification of the levels of metabolites is now the prime focus of metabolomics-related research going forward, along with being a necessary element at determining applications in clinical scenarios.

Despite all the aforementioned issues, quantified data from metabolite analyses has been tested in recent history as a possible avenue into the discovery of biological markers of disease.[26] Since metabolites can be measured more easily and on a regular basis than other biological units, it can find ideal use in this direction.[27] All of these factors together make a strong case in favor of implementing metabolomics as a solid opportunity in the future of periodontal diagnostics.

Opportunities for implementation of metabolomics in regular clinical settings

As seen in literature published from studies that have so far been conducted to analyze and segregate the metabolites present in periodontal health and disease among various subsets of the population, there is a significant elevation of reactive oxygen species as well as anti-oxidants that are usually present in bodily fluids viz., crevicular fluid, saliva, etc. The presence of all of these metabolic products such as methanol, lactate, acetone, propane-1,23-triol, and others confirms their presence and supports the assumption that they can be contributing elements to chronic inflammatory disease of the periodontium, and hence, to be able to ascertain and predict their changing patterns can become extremely essential at highlighting the exact point at which dysbiosis in the oral/systemic biome occurs, leading to disease.

CONCLUSION

Translational metabolomics has already exhibited an astounding potential in the field of diagnostic medicine, both as an indicator as well as a predictor of disease activity. However, it is currently still an evolving technique and is not without its fair share of pitfalls. Addressing these lacunae and active research toward making it possible for implementation into clinical periodontal practice can be an invaluable resource and should be explored, more so now than ever with the constant evolution of disease pathogenesis and progression as well.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.