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 Table of Contents  
DPU: INTERDISCIPLINARY CONFERENCE
Year : 2020  |  Volume : 7  |  Issue : 5  |  Page : 36-40

Detection of urinary metabolites of metabolic pathway disorders using vertical tube gel electrophoresis and liquid chromatography–high-resolution mass spectrometry techniques


1 Department of Oral Pathology and Microbiology, Dr. D. Y. Patil Dental College and Hospital, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India
2 Biotechnology, Cancer and Translational Research Lab, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India

Date of Web Publication26-Feb-2020

Correspondence Address:
Nilesh Kumar Sharma
Department of Biotechnology, Cancer and Translational Research Lab, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune - 411 033, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdrr.jdrr_65_19

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  Abstract 


Background: In recent times, various human health disorders including cancer, diabetes, neurodegenerative, and metabolic diseases are noticed among human populations. Currently, genetic and proteomic approaches are highly reported to detect metabolic disorders that also include inborn error of metabolisms. These existing detection methods are faced with cost issue and time-consuming factors. Therefore, metabolites as biomarkers are one of the potential avenues to detect metabolic disorders. Further, exploitation of urine as potential source of metabolite biomarkers, there are limitations in this area of research due to abundance of nonmetabolite components such as proteins and nucleic acids. Hence, methods and processes are required to precisely fractionate metabolites from urine of inborn error of metabolism patients and are then identified by analytical tools such as liquid chromatography–high-resolution mass spectrometry (LC-HRMS) and gas chromatography-tandem mass spectrometry. Materials and Methods: Sterile filtered urine samples (750 μl) mixed with (250 μl) loading buffer were electrophoresed on VTGE that uses acrylamide gel (acrylamide: bisacrylamide, 30:1) as a matrix of 15%. Further, vertical tube gel electrophoresis (VTGE) technique combined with LC-HRMS to identify metabolites that are known as the biomarkers of metabolic disorders was carried out. Results and Discussion: The authors provide evidence on the use of novel VTGE coupled with LC-HRMS to detect metabolites among metabolic disorders. Data suggest the applicability of VTGE coupled with LC-HRMS technique to detect metabolites such as 2-methyluridine, 2-methylglutaric acid, 2-methylcitric acid, and 2-hydroxyglutaric acid in case of metabolic disorders. Conclusion: This preliminary work is suggested to be extended to large clinical samples to validate application of this method to detect metabolic disorders including the inborn error of metabolisms.

Keywords: Biomarkers, mass spectrometry, metabolic disorders, metabolites, urine


How to cite this article:
Kumar A, Kothari J, Bhatkar D, Mitruka M, Pal R, Sarode SC, Sharma NK. Detection of urinary metabolites of metabolic pathway disorders using vertical tube gel electrophoresis and liquid chromatography–high-resolution mass spectrometry techniques. J Dent Res Rev 2020;7, Suppl S2:36-40

How to cite this URL:
Kumar A, Kothari J, Bhatkar D, Mitruka M, Pal R, Sarode SC, Sharma NK. Detection of urinary metabolites of metabolic pathway disorders using vertical tube gel electrophoresis and liquid chromatography–high-resolution mass spectrometry techniques. J Dent Res Rev [serial online] 2020 [cited 2020 Jul 10];7, Suppl S2:36-40. Available from: http://www.jdrr.org/text.asp?2020/7/5/36/278904

Editor: Dr. Sarika Chaturvedi





  Introduction Top


Among various classes of metabolic disorders, organic acidurias, a type of inherited metabolic disorders, are known to surface as intermediate steps in a metabolic pathways of anabolism and catabolism of lipid, carbohydrate, nucleic acids, and amino acids.[1],[2],[3],[4],[5] In essence, these metabolic disorders are indicated to lead to abundance of organic acids including 2-methyluridine, 2-methylglutaric acid, 2-methylcitric acid, and 2-hydroxyglutaric acid in a relevant tissues and these metabolites are potentially excreted in the urine.[5],[6],[7],[8],[9],[10],[11],[12],[13]

Among various classes of metabolic disorders, inborn errors of metabolism are seen as one of a kind of genetic disorders. In this class of metabolic disorders, lack or altered enzyme activity can lead to the disrupted biochemical processes that lead to the pathophysiological conditions.[5],[6],[7],[8],[9],[10],[11],[12],[13] In essence, such metabolic disorders display a condition of either as the aberrant abnormal accumulation of a substrate, and on the other hand, a deficit of the product is noticed in a target clinical patient. Further, it is also understood that all such metabolic disorders are inherited in an autosomal recessive manner.[5],[6],[7],[8],[9],[10],[11],[12],[13] Based on the literature, more than 500 human diseases depicted as the metabolic disorders are reported in clinical settings. Predominantly, these inborn errors of metabolism are shown to affect more than one child among 1000 population. Interestingly, in the Indian settings, lack of awareness and established method to adopt genetic and metabolic approaches, these metabolic disorders have remained unnoticed and undetected. Such unnoticed and undetected metabolic disorders from the early stage of life to the adult stage are documented to be implicated in many human diseases including diabetes and cancer.[10],[11],[12],[13] Hence, metabolic screening is recommended during the early stage of life and may also be extended to the later part of life. Among various markers, metabolic acidosis is suggested as one of the important displays during such metabolic disorders.[10],[11],[12],[13] Currently, various modalities including genetic and metabolomics approaches are reported in the literature. However, these methods and processes are faced with limitations in terms of validation accuracy and feasibility of translation at bedside approaches.

In this article, the authors present an approach that combines the vertical tube gel electrophoresis (VTGE)-based fractionation of urine samples and liquid chromatography–high-resolution mass spectrometry (LC-HRMS) identification of metabolites in the urine samples of potential human subjects on a pilot experimental basis.


  Materials and Methods Top


Collection and preparation of urine samples

Fresh urine samples from healthy clinical subjects were collected in the sterile collection tube. Formal approval was obtained through the institutional ethics committee and institutional scientific committee in accordance with the standard protocol to conduct research on healthy clinical subjects. Informed consent was obtained from the participating healthy clinical subjects. Further, these urine samples were centrifuged twice at 12,000 ×g for 30 min to get rid of debris, dead cells, insoluble particulates, and other interfering precipitates. Next, centrifuged urine samples were filtered using 0.45 μ syringe filter to get clear and sterile urine filtrate for subjected to VTGE-based fractionation of metabolites.

Vertical tube gel electrophoresis-based fractionation of metabolites from urine

Sterile filtered urine samples (750 μl) mixed with (250 μl) loading buffer were electrophoresed on VTGE that uses acrylamide gel (acrylamide: bisacrylamide, 30:1) as a matrix of 15%. The fractionated elute was collected in electrophoresis running buffer that contains water and glycine and excludes traditional sodium dodecyl sulfate (SDS), and other reducing agents. A flow diagram of VTGE method is presented in [Figure 1]a and [Figure 1]b that shows the assembly and design of VTGE system.[14],[15]
Figure 1: (a) A flow diagram of VTGE. (b) A working model of VTGE

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Urine metabolites from healthy clinical subjects were eluted in the same running buffer, which is referred as elution buffer. After loading of urine samples along with loading buffer, power supply was connected and voltage and current ratio was maintained to generate 1500–2500 MW of power to achieve the electrophoresis of urine samples. The total run time has been allowed for 2 h, and at the end of 2 h, lower collecting buffer was collected in the tube for LC-HRMS characterization. At the end of run, inner tube containing polyacrylamide gel was removed and put for Coomassie brilliant blue dye staining to ensure that protein components of urine samples were trapped in the polyacrylamide gel electrophoresis. The eluted urine metabolites in the elution buffer were measured for pH value and were found to be in the range of 2.5–3.0. Further, these eluted fractions from different healthy clinical subjects were stored at −20°C for the LC-HRMS analysis and identification of urine metabolites.

Liquid chromatography–high-resolution mass spectrometry analysis of vertical tube gel electrophoresis fractionated urine metabolite elute

LC-HRMS analysis of VTGE metabolite elutes was performed in negative electrospray ionization (ESI) M-H adduct mode. For the analysis of urine metabolites in LC-HRMS, a flow rate of 0.2 mL/min and a gradient were formed by mixing mobile Phase A (water containing 5 mM ammonium acetate) and B (0.2% formic acid). The high-performance liquid chromatography column effluent was allowed to move onto an ESI triple quadrupole mass spectrometer (Agilent Technologies). Here, urine metabolites were analyzed using negative ESI in the multiple reaction monitoring modes.[1],[2],[3],[15] During LC-HR-MS analysis of urine metabolites, mass spectrometer component was used as quadrupole time-of-flight mass spectrometry (Q-TOF-MS) (Agilent Technologies, 6500 Series Q-TOF LC/MS system) with dual Agilent Jet Stream (AJS) ESI mode. For liquid chromatography component, RP-C18 column Zorbax, 2.1 mm × 50 mm, 1.8 μm was used to separate the urine metabolite components. The acquisition mode of mass analysis1 (MS1) is maintained at a minimum range of m/z at 60 and maximum range of m/z at 1700. For the run of sample, an injection volume was maintained at 25 μl and a flow rate of solvent was at 0.3 ml/min.


  Results Top


In view of the need for the detection of potential metabolites of metabolic disorders, a VTGE method combined with LC-HRMS is used to detect these metabolites as biomarkers in urine samples of healthy clinical subjects. In this article, VTGE method that employs 15% polyacrylamide gel matrix purifies metabolites in the range of <500 Da from urine samples. These fractionated urine metabolites were subjected to LC-HRMS to detect the nature of metabolites. Here, nature of identified metabolites is shown as ESI chromatogram of 3-methyluridine [Figure 2], 2-methylcitric acid [Figure 3] and 2-methyglutaric acid [Figure 4] and 2-hydroxyglutaric acid [Figure 5].
Figure 2: A liquid chromatography–high-resolution mass spectrometry-based electrospray ionization chromatogram of vertical tube gel electrophoresis fractionated urine metabolites from healthy clinical subjects. In this electrospray ionization chromatogram, 3-methyluridine is detected as potential urine metabolite

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Figure 3: A liquid chromatography–high-resolution mass spectrometry-based electrospray ionization chromatogram of vertical tube gel electrophoresis fractionated urine metabolites from healthy clinical subjects. In this electrospray ionization chromatogram, 2-methylcitric acid is shown as potential urine metabolite

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Figure 4: A liquid chromatography–high-resolution mass spectrometry-based electrospray ionization chromatogram of vertical tube gel electrophoresis fractionated urine metabolites from healthy clinical subjects. In this electrospray ionization chromatogram, 2-methyglutaric acid is shown as potential urine metabolite

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Figure 5: A liquid chromatography–high-resolution mass spectrometry-based electrospray ionization chromatogram of vertical tube gel electrophoresis fractionated urine metabolites from healthy clinical subjects. In this electrospray ionization chromatogram, 2-hydroxyglutaric acid is shown as potential urine metabolite

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A detailed analysis of molecular structure, mass, and polarity of these potential metabolites is given in [Table 1]. This table clearly signifies the detection of 3-methyluridine, 2-methylcitric acid, 2-methyglutaric acid, and 2-hydroxyglutaric acid. In this article, all detected metabolites are in the range of 130–258 Da. M.W. In other way, these data substantiate the working of the VTGE method that is configured to fractionate metabolites with <500 Da MW.
Table 1: List of detected urine metabolites as potential biomarkers of metabolic disorders

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  Discussion Top


In the human metabolome database, 2-methylglutaric acid is known as a derivative of leucine metabolite.[1],[2],[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13] It is suggested that the secretion of methylglutaric acid can be found in the urine of patients showing the lack of 3-methylglutaconyl coenzyme a hydratase and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency during a type of inborn error of metabolisms. Another example of metabolite as 2-hydroxyglutaric acid is known to be accumulated in an organic aciduria condition.[1],[2],[3],[4],[5] There is an indication that 2-hydroxyglutaric acid can be generated due to the enzymatic action of hydroxyacid-oxoacid transhydrogenase and further 2-hydroxyglutaric acid is shown to be converted into alpha-ketoglutaric acid by 2-hydroxyglutarate dehydrogenase. There is a known fact that 2-hydroxyglutaric acid is produced due to the gain-of-function mutations of isocitrate dehydrogenase enzyme and this enzyme is a part of tricarboxylic acid cycle.[1],[2],[3],[4],[5],[6],[7]

It is interesting to note that 2-hydroxyglutaric acid may inhibit a range of enzymes such as histone lysine demethylases and members of the ten-eleven translocation family of 5-methylcytosine hydroxylases.[1],[2],[3],[4],[5] In summary, the uses of 2-hydroxyglutaric acid as metabolite biomarkers are having relevance in metabolic disorders and also as a key oncometabolites. In fact, 2-hydroxyglutaric acid is known as an alpha hydroxy acid that belongs to the class of organic compounds like organic acids. There are findings that support that the levels of 2-hydroxyglutaric acid are an indicator of inborn error of metabolism as an organic aciduria. Organic aciduria can be found in both children and adults that shows various symptoms as tremors, sleepiness, headaches, feeling tired, and seizures.[1],[2],[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13] In line with the existing evidence on the potential use of 2-hydroxyglutaric acid and 2-methylglutaric acid, the authors report on a novel methods and processes to detect these urine metabolites as a source of biomarkers for metabolic disorders.

A quantitative approach to estimate hydroxyglutaric acid in plasma and urine is reported by using the LC-MS/MS system to confirm the glutaric aciduria among patients.[1] Other than urine sample, a MS approach is reported on the detection of methylmalonic acid, 2-methylcitric acid, and homocysteine in blood spot samples.[5] In the same direction, another LC-MS/MS-based detection of methylmalonic acid, 2-methylcitric acid, and homocysteine is reported in case of organic aciduria among infant patients.[7] A gas chromatography-tandem MS (GC-MS/MS) technology based analysis of metabolite biomarkers of inborn error of metabolic disorders detects methylmalonic academia.[8] In convergence of earlier reported GC-MS- and LC-MS-based approaches to identify, our data are promising by adopting a new methods by combining VTGE coupled with LC-HRMS for the detection of urine metabolites as potential biomarkers for metabolic disorders.


  Conclusion Top


This article reports on the use of VTGE combined with LC-HRMS technique for the identification of 2-methyluridine, 2-methylglutaric acid, 2-methylcitric acid, and 2-hydroxyglutaric acid as potential metabolite biomarkers in case of metabolic disorders clinical subjects. This is report is of a novel in the sense that uses new method VTGE to fractionate metabolites from urine samples and further identifications of potential metabolites relevant to metabolic disorders. This study is limited to less number of clinical subjects, however, may be extended to large sample size for application in clinics for the detection of metabolic disorders in children and adult subjects.

Acknowledgments

The authors acknowledge inputs and suggestions from Prof. J. K. Pal, Dr. Ramesh Bhonde, and the assessment team of DPU innovation award (2019) to improve the vertical tube gel electrophoresis system for better applicability in health research.

Financial support and sponsorship

DPU funded project to Dr. Nilesh K. Sharma and DPU innovation award (2019) to a team lead by Dr. Nilesh K. Sharma.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Simon GA, Wierenga A. Quantitation of plasma and urine 3-hydroxyglutaric acid, after separation from 2-hydroxyglutaric acid and other compounds of similar ion transition, by liquid chromatography-tandem mass spectrometry for the confirmation of glutaric aciduria type 1. J Chromatogr B Analyt Technol Biomed Life Sci 2018;1097-1098:101-10.  Back to cited text no. 1
    
2.
Stenton SL, Kremer LS, Kopajtich R, Ludwig C, Prokisch H. The diagnosis of inborn errors of metabolism by an integrative “multi-omics” approach: A perspective encompassing genomics, transcriptomics, and proteomics. J Inherit Metab Dis 2019. doi: 10.1002/jimd.12130.  Back to cited text no. 2
    
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Shanmuganathan M, Britz-McKibbin P. New Advances for newborn screening of inborn errors of metabolism by capillary electrophoresis-mass spectrometry (CE-MS). Methods Mol Biol 2019;1972:139-63.  Back to cited text no. 3
    
4.
Villani GR, Gallo G, Scolamiero E, Salvatore F, Ruoppolo M. “Classical organic acidurias”: Diagnosis and pathogenesis. Clin Exp Med 2017;17:305-23.  Back to cited text no. 4
    
5.
Turgeon CT, Magera MJ, Cuthbert CD, Loken PR, Gavrilov DK, Tortorelli S, et al. Determination of total homocysteine, methylmalonic acid, and 2-methylcitric acid in dried blood spots by tandem mass spectrometry. Clin Chem 2010;56:1686-95.  Back to cited text no. 5
    
6.
Wang Y, Sun Y, Jiang T. Clinical application of LC-MS/MS in the follow-up for treatment of children with methylmalonic aciduria. Adv Ther 2019;36:1304-13.  Back to cited text no. 6
    
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Valik D, Jones JD. Hereditary disorders of purine and pyrimidine metabolism: Identification of their biochemical phenotypes in the clinical laboratory. Mayo Clin Proc 1997;72:719-25.  Back to cited text no. 7
    
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Wang H, Wang X, Li Y, Dai W, Jiang D, Zhang X, et al. Screening for inherited metabolic diseases using gas chromatography-tandem mass spectrometry (GC-MS/MS) in Sichuan, China. Biomed Chromatogr 2017;31(4).  Back to cited text no. 8
    
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Schillaci LP, DeBrosse SD, McCandless SE. Inborn errors of metabolism with acidosis: Organic acidemias and defects of pyruvate and ketone body metabolism. Pediatr Clin North Am 2018;65:209-30.  Back to cited text no. 9
    
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Garrod AG. Inborn Error of Metabolism. Oxford: Oxford University Press; 1909.  Back to cited text no. 10
    
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Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler KW, editors. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. New York: McGraw-Hill; 2001.  Back to cited text no. 11
    
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Superti-Furga A, Hoffmann GF. Glutaric aciduria type 1 (glutaryl-CoA-dehydrogenase deficiency): Advances and unanswered questions. Report from an international meeting. Eur J Pediatr 1997;156:821-8.  Back to cited text no. 12
    
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Lee HJ, Kremer DM, Sajjakulnukit P, Zhang L, Lyssiotis CA. A large-scale analysis of targeted metabolomics data from heterogeneous biological samples provides insights into metabolite dynamics. Metabolomics 2019;15:103.  Back to cited text no. 13
    
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Sharma NK, Kumar A, Waghmode A. Design of vertical tube electrophoretic system and method to fractionate small molecular weight compounds using polyacrylamide gel matrix Indian Patent Application Number no: 201921000760.  Back to cited text no. 14
    
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Sharma NK, Sarode SC, Pal R. A Method of Urine Metabolite Profiling by Combining Vertical Tube Gel Electrophoresis and LC-HR-MS for the Detection of Oral Cancer. Indian Patent Application Number: 201921021395.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 3], [Figure 2], [Figure 5], [Figure 4]
 
 
    Tables

  [Table 1]



 

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