Comparison of methods for rapid determination of cholesterol concentration in human sperm membrane in clinical laboratory practice

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Abstract

This study proposes a rapid method for the determination of cholesterol in human sperm membranes suitable for use in the clinical laboratory. Four physicochemical methods for the quantitative measurement of cholesterol were selected for comparison: the enzymatic cholesterol assay, the Liberman–Burkhardt method, the infrared spectroscopy and the high-performance liquid chromatography. The following cholesterol concentrations were obtained: 1.0 ± 0.3, 1.32 ± 0.15, 5.1 ± 1.8, and 1.53 ± 0.18 nmol/106 cells, respectively. The following criteria of the applicability of the method were chosen: the amount of material to be analyzed, determined by the number of spermatozoa in the seminal fluid of a single ejaculate of a patient, the number of sample preparation steps that account for the systematic error of the analysis, and the total time of the analysis. The infrared spectroscopy method requires at least 20 mg of cellular sample, which is unrealizable for estimating cholesterol in sperm membranes of a single patient. The Liberman–Burkhardt and high-performance liquid chromatography methods require multi-step sample preparation and the use of aggressive volatile reagents. In turn, the enzymatic assay is optimal for the considered criteria, it allows rapid analysis of cholesterol in the sperm membrane of a single patient, and is suitable for use within the in vitro fertilization laboratory.

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About the authors

A. G. Mironova

Human Reproduction Clinic “Altravita” (LLC “ECO CENTER”); N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences

Author for correspondence.
Email: agm90@mail.ru
Russian Federation, Moscow; Moscow

S. I. Afanasyeva

Lomonosov Moscow State University, Faculty of Physics, Department of Biophysics

Email: agm90@mail.ru
Russian Federation, Moscow

A. V. Sybachin

Lomonosov Moscow State University, Faculty of Chemistry, Department of High Molecular Compounds

Email: agm90@mail.ru
Russian Federation, Moscow

V. V. Spiridonov

Lomonosov Moscow State University, Faculty of Chemistry, Department of High Molecular Compounds

Email: agm90@mail.ru
Russian Federation, Moscow

M. A. Bolshakov

FSBI “Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”

Email: agm90@mail.ru
Russian Federation, Pushchino

E. Yu. Simonenko

Lomonosov Moscow State University, Faculty of Physics, Department of Biophysics

Email: agm90@mail.ru
Russian Federation, Moscow

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2. Fig. 1. Structure of the cholesterol molecule. All four rings of the sterol group are in the trans conformation, which makes the cholesterol molecule planar. The double bond between the fifth and sixth carbon atoms of the chain provides rigidity to the cholesterol molecule [2]. For the formation of an idea of ​​the three-dimensional structure of cholesterol, it is important to note that the OH group, two methyl groups and the side chain are located on one side of the ring skeleton (β-configuration) [1]. The hydroxyl group in the cholesterol molecule gives the compound an amphiphilic character and contributes to the orientation of the cholesterol molecule in the bilayer.

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