Sterol extraction and analysis are essential techniques for studying pathogenic fungi, as sterols, particularly ergosterol, play a pivotal role in fungal pathogenesis. Ergosterol is a key component of the fungal cell membrane, crucial for maintaining its integrity and fluidity, which are essential for cell growth and survival (Choy et al, mBio 14:e01353, 2023). This sterol enables important functions such as nutrient uptake, signal transduction, and protection against environmental stressors. Disruption of ergosterol biosynthesis has become a primary target for antifungal therapies, as it compromises membrane function, ultimately impairing fungal virulence and reducing the ability of the pathogen to infect its host. The method described in this chapter focuses on extracting sterols from fungal cells and quantifying their content to assess fungal membrane composition. In this process, fungal cells are first cultured overnight and subjected to alkaline hydrolysis using alcoholic potassium hydroxide (KOH), which breaks down cellular structures and releases the sterols from the cell membranes (Arthington-Skaggs et al, J Clin Microbiol 37(10):3332–3337, 1999). The sterols are subsequently extracted using petroleum ether, a non-polar solvent that effectively dissolves lipophilic compounds, including sterols. The extraction process ensures the separation of sterols from other cell debris and impurities. Once extracted, the sterol content is quantified using UV-VIS spectrophotometry (Pasrija et al, J Antimicrob Chemother 55:905–913, 2005). The absorbance of the sample is measured at specific wavelengths that correspond to the characteristic absorbance peaks of these sterols. This technique allows for the quantification of ergosterol and related sterols, which are integral components of fungal cell membranes and play a critical role in membrane fluidity and function (Pasrija et al, Int Microbiol 27:1–12, 2023). The method provides a reliable, reproducible approach for quantifying ergosterol, offering valuable insights into fungal membrane composition and the effects of antifungal treatments. By assessing the sterol profile, researchers can gain a better understanding of how antifungal drugs interact with the fungal membrane and potentially lead to resistance. The chapter outlines a straightforward yet comprehensive protocol that can be used to study ergosterol biosynthesis in pathogenic fungi, providing a basis for the development of new antifungal strategies.

Sterols Extraction, Analysis and Quantification in Pathogenic Fungi

章节
发表日期：

2025年

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Ritu Pasrija,
Deepika Kumari

摘要

Sterol extraction and analysis are essential techniques for studying pathogenic fungi, as sterols, particularly ergosterol, play a pivotal role in fungal pathogenesis. Ergosterol is a key component of the fungal cell membrane, crucial for maintaining its integrity and fluidity, which are essential for cell growth and survival (Choy et al, mBio 14:e01353, 2023). This sterol enables important functions such as nutrient uptake, signal transduction, and protection against environmental stressors. Disruption of ergosterol biosynthesis has become a primary target for antifungal therapies, as it compromises membrane function, ultimately impairing fungal virulence and reducing the ability of the pathogen to infect its host. The method described in this chapter focuses on extracting sterols from fungal cells and quantifying their content to assess fungal membrane composition. In this process, fungal cells are first cultured overnight and subjected to alkaline hydrolysis using alcoholic potassium hydroxide (KOH), which breaks down cellular structures and releases the sterols from the cell membranes (Arthington-Skaggs et al, J Clin Microbiol 37(10):3332–3337, 1999). The sterols are subsequently extracted using petroleum ether, a non-polar solvent that effectively dissolves lipophilic compounds, including sterols. The extraction process ensures the separation of sterols from other cell debris and impurities. Once extracted, the sterol content is quantified using UV-VIS spectrophotometry (Pasrija et al, J Antimicrob Chemother 55:905–913, 2005). The absorbance of the sample is measured at specific wavelengths that correspond to the characteristic absorbance peaks of these sterols. This technique allows for the quantification of ergosterol and related sterols, which are integral components of fungal cell membranes and play a critical role in membrane fluidity and function (Pasrija et al, Int Microbiol 27:1–12, 2023). The method provides a reliable, reproducible approach for quantifying ergosterol, offering valuable insights into fungal membrane composition and the effects of antifungal treatments. By assessing the sterol profile, researchers can gain a better understanding of how antifungal drugs interact with the fungal membrane and potentially lead to resistance. The chapter outlines a straightforward yet comprehensive protocol that can be used to study ergosterol biosynthesis in pathogenic fungi, providing a basis for the development of new antifungal strategies.