Visual and colorimetric assessments of increased urinary sarcosine levels
Authors:
Z. Tóthová 1; M. Dočekalová 1; M. Staňková 1; D. Uhlířová 1; J. Růžička 1; R. Kizek 1,3
Authors‘ workplace:
Prevention Medicals, s. r. o., Oddělení výzkumu a vývoje, Vedoucí: Ing. Miroslav Dosoudil
1; Masarykova univerzita, Farmaceutická fakulta, Ústav humánní farmakologie a toxikologie, Přednosta: doc. MVDr. Pavel Suchý, Ph. D.
2; Wroclaw Medical University, Wroclaw, Polsko, Ústav biomedicínských a environmentálních analýz, Přednosta: prof. dr. hab. Halina Milnerowicz, MD, Ph. D.
3
Published in:
Prakt. Lék. 2020; 100(5): 251-256
Category:
Of different specialties
Overview
Aim: Prostate carcinoma is the most common malignant tumor in men. Its occurrence varies considerably geographically and increases with age. For the rapid diagnosis, suitable tumor markers are sought. A very promising candidate is the amino acid sarcosine (SAR), which is increased in the urine from patients with tumor. The aim of this work was to develop a simple, rapid, and reliable method for the detection of SAR in human urine.
Material and methods: Artificial urine samples (15 types) were prepared according to available protocols. An electrochemical analysis was performed potentiometrically and voltammetrically. Temperature stability tests of sarcosine oxidase (SOX) were performed at –5, 25, 30, 35, 40, 45, and 60 °C. Lyophilization was carried out for 72 hours (0.1 mbar, –80 °C). At the beginning and at the end of the experiment, the test was evaluated visually. The obtained photographs were subjected to mathematical analysis (evaluation of the color intensity of the signal).
Results: The urine composition is very variable, it can contain a variety of waste metabolites, drugs, and other interferents. Artificial urine is a suitable matrix for studying changes in SOX activity. SOX activity (1 U/mL) was monitored by Trinder reaction as sarcosine hydrolysis (60 min, 125 μM SAR, 0.4 mM AAP). The effect of the addition of interferents (Cd, Pb, Zn, and drugs) was observed in the experiment, with SOX inhibition ranging from 10 to 20 %. The SOX enzyme was heated, freeze-dried, and lyophilized. The results showed that the enzyme was stable at temperatures from 5 to 60 °C (signal drop by 10% in 200 hours). Temperatures above 60 °C led to inactivation of the enzyme (signal drop by 90% at 120 min). Low temperatures (–5 °C to –20 °C) did not lead to a signal decrease after 5 weeks. The visual results were summarized as the average value of the RGB signal density in the studied sample group (n = 10). The results obtained showed that the average variability of the RGB signal among tested samples was 7.4%. From these values, the ROC curve of individual experiments (n = 5) was determined. Using ROC curves, the sensitivity (from 0.59 to 0.83) and the specificity (1) were calculated by the type of artificial urine. The ROC curves were evaluated as follows: excellent – 26.6%, very good – 66.6%, and good curves – 6.6%; no curve was unsatisfactory.
Conclusion: A study of stability in the detection of the amino acid sarcosine using the enzymatic reaction was performed. The visual evaluation of the test exhibited a success rate of 100% in identifying the sarcosine presence in artificial urine. The data obtained show the potential of the method for visual evaluation of the presence of sarcosine in the urine.
Keywords:
sarcosine – malignant prostate tumors – biosensor – visual test
Sources
1. Barbieri CE, Chinnaiyan AM, Lerner SP, et al. The Emergence of Precision Urologic Oncology: A Collaborative Review on Biomarker-driven Therapeutics. Eur Urol 2017; 71: 237–246.
2. Altwaijry N, Somani S, Dufes Ch. Targeted nonviral gene therapy in prostate cancer. Int J Nanomed 2018; 13: 5753–5767.
3. Trinder P. Determination of blood glucose using 4-amino phenazone as oxygen acceptor. J Clin Pathol 1969; 22.
4. Sawyers CL. The cancer biomarker problem. Nature 2008; 452: 548–552.
5. Gil J, Ramirez-Torres A, Encarnacion-Guevara S. Lysine acetylation and cancer: A proteomics perspective. J Proteomics 2017; 150: 297–309.
6. Maurer T, Eiber M. Practice changing for prostate cancer: a vision of the future. Nat Rev Urol 2019; 16: 71–72.
7. Heitzer E, Haque IS, Roberts CES, et al. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat Rev Genetics 2019; 20: 71–88.
8. Zachoval R, Dusek L, Babjuk M. Screening karcinomu prostaty v České republice. Prakt. Lék. 2019; 99: 102–109.
9. Sreekumar A, Poisson LM, Rajendiran TM, et al. Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression. Nature 2009; 457: 910–914.
10. Cernei N, Heger Z, Gumulec J, et al. Sarcosine as a potential prostate cancer biomarker – a review. Int J Mol Sci 2013; 14: 13893–13908.
11. Kanehisa M, Goto S. KEGG PATHWAY: Glycine, serine and threonine metabolism. Encyclopedia of Genes and Genomes 2010.
12. Gkotsos G, Virgiliou C, Lagoudaki I, et al. The role of sarcosine, uracil, and kynurenic acid metabolism in urine for diagnosis and progression monitoring of prostate cancer. Metabolites 2017; 7: 14.
13. Lee SY, Chan KY, Chan AYW, et al. A report of two families with sarcosinaemia in Hong Kong and revisiting the pathogenetic potential of hypersarcosinaemia. Annal Acad Med Singapore 2006; 35: 582–584.
14. Cernei N, Zitka O, Ryvolova M, et al. Spectrometric and Electrochemical Analysis of Sarcosine as a Potential Prostate Carcinoma Marker. Int J Electrochem Sci 2012; 7: 4286–4301.
15. Huang Y, Huang XB, Huang LP, et al. Three-phase solvent bar liquid-phase microextraction combined with high-performance liquid chromatography to determine sarcosine in human urine. J Sep Sci 2018; 41: 3121–3128.
16. Narwal V, Kumar P, Joon P, et al. Fabrication of an amperometric sarcosine biosensor based on sarcosine oxidase/chitosan/CuNPs/c-MWCNT/Au electrode for detection of prostate cancer. Enzym Microb Technol 2018; 113: 44–51.
17. Kumar P, Jaiwal R, Pundir CS. An improved amperometric creatinine biosensor based on nanoparticles of creatininase, creatinase and sarcosine oxidase. Anal Biochem 2017; 537: 41–49.
18. Josypcuk O, Barek J, Josypcuk B. Construction and application of flow enzymatic biosensor based of silver solid amalgam electrode for determination of sarcosine. Electroanalysis 2015; 27: 2559–2566.
19. Gonzalez-Solino C, di LorenzoM. Enzymatic fuel cells: towards self-powered implantable and wearable diagnostics. Biosensors-Basel 2018; 8: 18.
20. Samanta S, Rahaman SZ, Roy A, et al. Understanding of multi-level resistive switching mechanism in GeOx through redox reaction in H2O2/sarcosine prostate cancer biomarker detection. Sci Rep 2017; 7: 12.
21. Berthias F, Maatoug B, Glish GL, et al. Resolution and Assignment of differential ion mobility spectra of sarcosine and isomers. J Am Soc Mass Spectrom 2018; 29: 752–760.
22. Uhlirova D, Stankova M, Docekalova M, et al. A rapid method for the detection of sarcosine using SPIONs/Au/CS/SOX/NPs for prostate cancer sensing. Int J Mol Sci 2018; 19: 29.
23. Uhlirova D, Docekalova M, Stankova M, et al. A rapid ELISA method for the detection of sarcosine using pseudoperoxidase activity of gold nanoparticles. 9th International Conference on Nanomaterials – Research & Application 2018: 524–530.
24. Jia J, Liu G, Li S, et al. Urine sample hepatitis B virus covalently closed circular DNA sarcosine quantitative detection method, involves detecting quantitative rate of sarcosine solution, and calculating content of sarcosine in urine sample. Patent CN102662013-A CN10154590 2012: 7.
25. Stankova M, Ruttkay-Nedecky B, Docekalova M, et al. Fotometrická detekce aminokyseliny sarkosinu za využití jeho hydrolýzy sarkosin oxidasou. Chem Listy 2019; 113: 603–609.
26. Wiewiorka O, Dastych M, Cermakova Z. Trinderova reakce v klinické biochemii – přínosy a limity. Chem Listy 2017; 111: 186–191.
27. Lan JM, Xu WM, Wan QP, et al. Colorimetric determination of sarcosine in urine samples of prostatic carcinoma by mimic enzyme palladium nanoparticles. Anal Chim Acta 2014; 825: 63–68.
28. Rebelo TSCR, Pereira CM, Sales MGF, et al. Sarcosine oxidase composite screen-printed electrode for sarcosine determination in biological samples. Anal Chim Acta 2014; 850: 26–32.
29. Pietrzynska M, Voelkel A. Stability of simulated body fluids such as blood plasma, artificial urine and artificial saliva. Microchem J 2017; 134: 197–201.
30. Chutipongtanate S, Thongboonkerd V. Systematic comparisons of artificial urine formulas for in vitro cellular study. Anal Biochem 2010; 402: 110–112.
31. Shmaefsky BR. Artificial urine for laboratory testing. Amer Biol Teacher 1990; 52: 170–172.
32. Yamkamon V, Phakdee B, Yainoy S, et al. Development of sarcosine quantification in urine based on enzyme-coupled colorimetric method for prostate cancer diagnosis. EXCLI J 2018; 17: 467–478.
33. Jones PF, Johnson KE. Estimation of phenols by the 4-aminoantipyrine method: Identification of the colored reaction products by proton magnetic resonance spectroscopy. Canad J Chem 1973; 51: 2860–2868.
34. Vecera M. Detection and identification of organic compounds. Boston, USA: Springer 1971; 1: 1–150.
35. Uhlirova D, Docekalova M, Stankova M, et al. Quantitatively determining sarcosine in biological sample, involves using anti-sarcosine antibodies and peroxidase-like activity gold nanoparticles, or anti-sarcosine antibodies and quantum dots. Patent EP176436.
Labels
General practitioner for children and adolescents General practitioner for adultsArticle was published in
General Practitioner
2020 Issue 5
Most read in this issue
- Electronic sick notes
- Assessment of preoperative fear in patients before elective surgery
- Possibilities of stability evaluation in clinical practice in patients at risk of falls Falls are a serious global public
- Evaluation of motor control disorders in patients with non-specific low back pain in general practitioner surgery Nearly every individual at least