Chromatographic strategies for the determination of aminothiols in human saliva
Introduction
Nowadays, a significant development in every area of life can be observed. This phenomenon is generally considered to be highly beneficial and desirable from the standpoint of well-being of humans. However, the rapid progress of civilization has also led substantially to widespread the occurrence of adverse effects on human health affecting the development of severe pathological conditions in human body. As a result, more and more reports concerning appallingly growing number of people suffering from civilization diseases can be found in the literature. Undoubtedly, this phenomenon have indicated new directions in bioanalysis in recent years. Thus, scientists have started to put considerable effort into (1) searching new compounds, which presence and concentration in biological fluids could be correlated with these pathologies; (2) investigating new sources of such compounds, which can be obtained in a non-invasive and non-intrusive way as well as (3) developing new, simple and low-price analytical tools in order to encourage general public to get through regular checkups and facilitate large scale screening of civilization diseases.
To the best of our knowledge, this review is the first report devoted to methods for salivary aminothiols determination. It summarizes analytical methods published since 1990 till the end of 2019 which could be applied to human saliva. In particular, work places emphasis on pre-analytical treatment strategies and assays based on separation techniques. Moreover, it outlines an association between civilization diseases and biothiols’ metabolism disorders as well as potentialities and limitations of the use of saliva as a media for certain human disease diagnosis and monitoring as these subjects have been already covered in great detail in numerous papers. Hopefully, this review will contribute to widespread interest in the topic to be discussed.
Sulfur-containing amino acids, including homocysteine (Hcy) and other metabolically related thiols such as cysteinyl-glycine (CysGly), γ-glutamyl-cysteine (γ-GluCys), cysteine (Cys) and glutathione (GSH) (Fig. 1), comprise one of the classes of compounds which association with development of several civilization diseases is well-established [[1], [2], [3], [4]]. So far, the least is known about physiological and pathological role of CysGly and γ-GluCys in human body. Their participation in sulfur metabolic pathway in humans have pointed toward a simultaneous quantification of these compounds with other biologically relevant Hcy-related thiols in biofluids for the purpose of certain disease diagnostic and monitoring process. Nevertheless, it need to be emphasized that in spite of all extensive research is being done into the connection between the several diseases and aminothiols’ metabolism disorders, too little is still known about their physiological and pathological role in living systems [[1], [2], [3], [4]]. Therefore, it seems to be essential to elaborate a new, robust and versatile methodologies to increase knowledge about the role of Hcy and other sulfur-containing amino acids in human body.
Currently, blood (plasma/serum) and urine are gold standards in the field of clinical, toxicological and forensic science. Nevertheless, saliva has received continuous attention over the last decades whereas it has multiple distinct advantages over blood and urine as a diagnostic specimen [[5], [6], [7], [8], [9]]. Contrary to them, human saliva is readily accessible biofluid which collection can be performed in non-invasive and non-intrusive way with minimal risk of contracting infections to medical staff. In addition, saliva offers practical advantage of being easy to collect by individuals, when received basic training on correct sampling method as no special equipment is needed to obtain samples. Nonetheless, the attractiveness of this body fluid is particularly attributed to its unique composition.
Saliva is a slightly acidic liquid secreted by three major salivary glands as well as numerous minor salivary glands located throughout the oral cavity. The secretion primarily consists of water, that makes up roughly 99.5% of saliva, while the remainder constitutes compounds produced locally in salivary glands as well as many constituents circulating around body through bloodstream [[5], [6], [7], [8], [9], [10], [11], [12], [13], [14]]. Saliva can be considered as a gland-specific or whole saliva (oral fluid), while the latter is of increasing practical value as an easily accessible diagnostic specimen. Nevertheless, it should be noted that the term saliva is predominantly used in scientific literature, despite the fact that oral fluid more accurately describes the biological characteristics of this matrix. Oral fluid refers to a complex mixture of not only salivary glands’ secretions but also many constituents of non-salivary origin such as gingival crevicular fluid, bronchial/nasal secretions, buccal/mucosal/blood (plasma) transudate, cellular components, microorganisms/viruses and their metabolism products, food debris as well as traces of exogenous chemicals/medicaments [[5], [6], [7], [8], [9], [10], [11], [12],14]. Since they may cause several interferences in analytical assays, this fact could not be neglected during new methods development. In the following paragraphs of the review, terms saliva and oral fluid are used in an interchangeable manner, but they always designate the same biological specimen obtained from the oral cavity.
Nowadays, it is well-known that saliva is a complex biofluid harboring a wide spectrum of compounds which relation to health status, well-being as well as certain diseases and circumstances related to health problems have been indicated [[6], [7], [8],[11], [12], [13],15]. Importantly, these studies have also shown significant positive correlation between salivary concentration of analytes and their blood (plasma/serum) level [[7], [8], [9],13]. Hence, saliva providing information from several organs and systems is more and more frequently termed “the mirror of the body” in the literature.
Importantly, the complexity of saliva is lower in comparison to blood/urine, what makes easier to provide sufficient resolution between all sample components, while lower concentration of proteins reduces the risk of analytes binding by large biomolecules. On the other hand, both lower complexity and media dilution attribute to considerably lower concentration of all components including analytes in saliva by comparison with their blood levels [9]. Moreover, salivary secretion and composition vary notably from individual to individual and the same person under different conditions. Generally, it is assumed that healthy adult produces up to 1.5 L of resting saliva per day at an average flow rate of 0.1–0.3 mL min−1 [11,14] while several physiological and pathological conditions in human body may contribute to quantitative and qualitative salivary changes [5,7,10,12,15]. However, from the practical point of view, the most challenging problem seems to refer to representative sample collection. Based upon scientific literature studies, it can be concluded that a system in particular capable of automatically monitoring saliva output (the amount of saliva and time it took to produce) and measuring some physicochemical properties, namely pH, density and turbidity, is desirable in order to get meaningful results and facilitate comparisons between experimental data collected from particular cases. To the best of our knowledge, such kind of multifunctional device is not available to widespread usage as yet.
Section snippets
Pre-separation consideration
Biological fluids, including saliva are known to contain a large number of components representing wide range of structures and chemical properties. Such complexity of matrices make their analysis quite challenging. Moreover, the presence of some matrix components, like proteins, may cause analytes’ conjugation resulting in poor method recovery. It could also contribute to analytical system destruction since majority of instruments are designed to handle analytes but are usually incapable of
Analytical methods for the determination of biothiols in human saliva
A growing interest in saliva as a diagnostic specimen, regarding to biological aminothiols determination, dates from the end of 20th century. So far, several assays have been developed or adopted for the determination of salivary Hcy and related compounds. Among them HPLC [[19], [20], [21], [22], [23], [24], [25], [26], [27], [28],[30], [31], [32], [33], [34]] and CE [29] based methods, utilizing a derivatization with a suitable labeling reagent followed by separation and UV–Vis [[19], [20],
Final conclusions and perspectives
Over the recent decades, biologically active low molecular mass thiols, including Hcy, Cys, γ-GluCys, GSH and CysGly have received continuing attention due to their physiological importance and well-documented association with several human diseases. Nowadays, blood (plasma/serum) and urine are the most commonly analyzed biological fluids for aminothiols determination. However, non-invasive and non-intrusive nature of saliva sampling process as well as the fact that various drugs and
Author contributions
JP has created the main concept of the work, wrote the manuscript and had primary responsibility for the final content. MW contributed the input into manuscript by preparing figures and tables. RG has co-created work concept and critically revised the manuscript. All authors have read and given their final approval of the version of the manuscript to be published.
Acknowledgements
This work was supported by the National Science Centre, Poland (grant numbers 2018/02/X/ST4/00779, 2017/27/B/ST4/01476).
References (45)
- et al.
Saliva as an alternative specimen to plasma for drug bioanalysis. A review
Trends Anal. Chem.
(2016) - et al.
Saliva: an all-rounder of our body
Eur. J. Pharm. Biopharm.
(2019) - et al.
Clinical validity of saliva and novel technology for cancer detection
Biochim. Biophys. Acta Rev. Canc
(2019) - et al.
Recent trends in the development of diagnostic tools for diabetes mellitus using patient saliva
Trends Anal. Chem.
(2017) - et al.
A review of saliva: normal composition, flow and function
J. Prosthet. Dent
(2001) - et al.
Ultraviolet derivatization of low-molecular-mass thiols for high performance liquid chromatography and capillary electrophoresis analysis
J. Chromatogr. B
(2011) Recent advances in separation and detection methods for thiol compounds in biological samples
J. Chromatogr. B
(2009)- et al.
A versatile method for analysis of saliva, plasma and urine for total thiols using HPLC with UV detection
Talanta
(2016) - et al.
Effect of smoking one cigarette on antioxidant metabolites in the saliva of healthy smokers
Arch. Oral Biol.
(1999) - et al.
HPLC analysis of some sulphur compounds in saliva: comparison between healthy subjects and periodontopathic patients
Clin. Chim. Acta
(2003)
Salivary thiols and enzyme markers of cell damage in periodontal disease
Clin. Biochem.
Sensitive determination of glutathione in biological samples by capillary electrophoresis with green (515nm) laser-induced fluorescence detection
J. Chromatogr., A
In syringe hybrid monoliths modified with gold nanoparticles for selective extraction of glutathione in biological fluids prior to its determination by HPLC
Talanta
Analysis of glutathione and glutathione disulfide in human saliva using hydrophilic interaction chromatography with mass spectrometry
J. Chromatogr. B
A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome
Redox Biol.
Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection
J. Chromatogr., A
Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with fluorescence detection
J. Chromatogr. B Biomed. Sci. Appl.
The preferences of Aault outpatients in medical or dental care settings for giving saliva, urine or blood for clinical testing
J. Am. Dent. Assoc.
Implications of plasma thiol redox in disease
Clin. Sci.
The controversial role of homocysteine in neurology: from labs to clinical practice
Int. J. Mol. Sci.
Disturbed homocysteine metabolism is associated with cancer
Exp. Mol. Med.
The current status of homocysteine as a risk factor for cardiovascular disease: a mini review
Expert Rev. Cardiovasc Ther.
Cited by (13)
One-pot sample preparation procedure for the determination of protein N-linked homocysteine by HPLC-FLD based method
2023, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesUp-to-date knowledge about analytical methods for homocysteine thiolactone determination in biological samples
2023, TrAC - Trends in Analytical ChemistryCitation Excerpt :Since majority of instruments are incapable of accommodating such kinds of biomolecules, it could primarily cause practical problems. Therefore, classical methods designed for the determination of HTL, using liquid and gas phase separation techniques, include deproteinization step [16,25–29] despite the fact that plasma, saliva and urine are usually considered a low protein abundance matrices [37–39]. In relation to HTL assays, the protein removal, excepting that methods based on extraction, include the addition of water-miscible organic solvent [26,28,29] and ultrafiltration [16,25,27].
A highly chromogenic selective Rhodamine-chloride-based fluorescence probe activated by cysteine and application in living cells and zebrafish
2022, Spectrochimica Acta - Part A: Molecular and Biomolecular SpectroscopyCitation Excerpt :Briefly speaking, the achievement for measuring the level of Cys could have a tremendous assistance to reveal the complex interactions of the sulfur metabolism and provide the key parameters of pre-clinical diagnosis such as in tumor progression. For detecting Cys, there were many the traditional approaches such as high performance liquid chromatography (HPLC) [21], mass spectrometry (MS) [22], and capillary electrophoresis [23]. However, the major disadvantages of these approaches were high cost, complex sample preparation and sophisticated operations [24].
Optical nanoprobes for aminothiols sensing in real-world samples
2022, Sensors and Actuators ReportsCitation Excerpt :The main drawbacks of the separation technique include long analysis time, sophisticated operation, and high-cost equipment. Up to now, several comprehensive and well-written reviews have included synthetic routes of aminothiol-sensitive organic dyes [17–22], the design of separation and extraction processes and strategies [23–26], and the fabrication of metal nanoparticle-based aminothiol sensors [27,28]. In this review, we shed light on detailed concepts and sensing procedure related to the design of colorimetric, fluorometric and Raman nanoprobes to identify aminothiols in biological fluids and use them for in vivo and in vitro imaging studies.
An ultrasensitive aptamer-antibody sandwich cortisol sensor for the noninvasive monitoring of stress state
2021, Biosensors and BioelectronicsCitation Excerpt :Therefore, the cortisol sensor based on the aptamer-antibody sandwich pattern is an effective and reliable detection platform. In this study, the selectivity of the aptamer-antibody sandwich sensor to cortisol among several common salivary interfering compounds was investigated (Coyle et al., 2019; Piechocka et al., 2020). Specifically, these compounds were ascorbic acid, uric acid, urea, and glucose, and their concentrations were controlled to be similar or higher than those in the saliva of healthy people (Ngamchuea et al., 2018; Xia et al., 2012; Jurysta et al., 2009), which were 10 mg/L, 50 mg/L, 100 mg/L, and 800 mg/L, respectively.