Visual and colorimetric assessments of increased urinary sarcosine levels

Česká verzia

Authors: Z. Tóthová ¹; M. Dočekalová ¹; M. Staňková ¹; D. Uhlířová ¹; J. Růžička ¹; R. Kizek ^1,3
Authors place of work: 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 the journal: Prakt. Lék. 2020; 100(5): 251-256
Category: Of different specialties

Summary

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

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