Researchers from Prague, Czech Republic have investigated the stability of selenium species during extraction from biological material. By using the most common agent Protease XIV for extraction from biological samples they observed the interconversion of selenocystine to Se-methyl-selenocysteine. The interconversion of these species was suppressed by using a protease XXIII as an alternative.
Selenium (Se) is an essential element as the active site of selenoproteins and selenoenzymes protecting against oxidative stress and is involved in the regulation of thyroid gland function. On the other side, selenium is also toxic at concentrations less than 10 times away from the optimum. For a detailed understanding of Se function in human health knowledge of its speciation is mandatory, because selenium species define its role in the living system. The most often used technique for Se speciation analysis is the hyphenated system of high-performance liquid chromatography (HPLC) for separation of species and inductively coupled plasma-mass spectrometry (ICP-MS) for their detection. Since this technique calls for liquid samples, a suitable extraction procedure must be used for solid samples. It is this extraction procedure, which is the most critical step for speciation analysis, since it has to be efficient with respect to extract the different Se species quantitatively without using too harsh chemistry that could degrade the species being present. Various procedures have been developed for the extraction of Se species from biological materials, out of which the most common is usage of enzymatic extraction with protease XIV. Unfortunately, the stability of Se species during extraction with this agent is not well understood.
The new study:
A group of researchers from the Czech Republic now investigated the stability of Se species during extraction with protease XIV and also with protease XXIII as an alternative. Speciation analysis was based on HPLC-ICP-MS and biological sample materials investigated included animal tissues, fungi, algae, and bacteria.
Portions of the homogenized samples (50 - 250 mg) were sonicated with 2 mL of Tris-HCL buffer containing 0.1 % Triton X-100 or sodium dodecylsulphate. with an ultrasonic homogenizer for 1 min. Then 25 mg of protease was added and the sample was extracted for 24 h at 37°C. After filtration with 0.45 µm syringe filter, the extract was analyzed directly with HPLC-ICP-MS.
The Se compounds were separated by ion-pair chromatography using a reversed phase RP-C8 column and a mobile phase containing 5.4 mmol/L TMAAH, 5.4 mmol/L malonic acid, 7 mmol/l sodium butane-1-sulfonate. Detection by quadrupole ICP-MS with tellurium as internal standard pumped with peristatic pump integrated to ICP-MS and mixed with the column eluent using a Y-piece located just in front of the nebulizer resulted in quantification limits (LOQ) between 0.18 and 0.70 µg/L Se for the different species.
The stability of Se species during extraction process was tested by simulated extraction of aqueous standard solutions of individual Se species. These basic tests revealed that the use of surfactants meant to improve extraction efficiency is problematic since oxidation of many species occurred. The change in speciation was observed also in the case of selenocystine when protease XIV was used: the 29 % (21 %) was changed also to species with retention time corresponding to Se-methyl-selenocysteine. The interconversion of these species was suppressed by using a protease XXIII as an alternative to protease XIV, which resulted in an extraction efficiency only slightly lower (~53%) in comparison to protease XIV (~ 65%). Based on the less disturbing extraction with protease XXIII, a method for the speciation of nine Se species (selenate, selenite, selenomethionine, selenocystine, Se-methylselenocysteine, selenoethionine, trimethylselenonium, methylselenocysteine oxide, and selenomethionine Se-oxide) was developed and validated.
The authors concluded that the new extraction procedure does not alter the main Se species in biological samples, selenomethionine, selenocystine, and Se-methyl-selenocysteine, which were stable for three days at room temperature.
The original publication
Vera Kantorová, Antonín Kana, Gabriela Krausová, Ivana Hyrslová, Oto Mestek, Effect of protease XXIII on selenium species interconversion during their extraction from biological samples,
J. Food Compos. Anal., 105 (2022) 104260. DOI: 10.1016/j.jfca.2021.104260
Instrumentation used: PerkinElmer - ELAN DRC-e ICP-MS PerkinElmer - Series 200 HPLC system Related studies
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last time modified: December 14, 2021