Comparison of Methods for Rapid Determination of Cholesterol Concentration in Human Sperm Membrane in Clinical Laboratory Practice

IF 1.1 4区 化学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY
A. G. Mironova, S. I. Afanasyeva, A. V. Sybachin, V. V. Spiridonov, M. A. Bolshakov, E. Yu. Simonenko
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Abstract

Objective: Cholesterol is an important structural component of the plasma membrane of mammalian cells. Cholesterol, among other important roles, plays a special role in sperm membranes. Change in the lipid composition of the sperm membrane, particularly the outflow of cholesterol, is an integral part of the process of capacitation and subsequent acrosomal reaction necessary for the sperm to fertilize an egg. Deviations in cholesterol concentration in sperm membrane may indicate a decrease in the fertilizing potential of sperm. To determine the optimal method for rapid analysis of the cholesterol content in human sperm membranes in the IVF laboratory, four methods of quantitative determination of cholesterol were compared in terms of practicality and effectiveness of their use to assess the concentration of cholesterol in human sperm membranes: the method of enzymatic colorimetric detection, the Lieberman–Burchard method, infrared spectroscopy and high-performance liquid chromatography. Methods: 101 ejaculates of patients with established normozoospermia (according to WHO criteria) were used in the work. Spermatozoa were separated from the semen by double centrifugation with the addition of DPBS medium. The resulting cellular pellet was used to determine the concentration of cholesterol in a sample by one of four methods: enzymatic colorimetric detection (FCD), HPLC, Lieberman-Burchard method, infrared spectroscopy. Results and Discussion: The following cholesterol concentrations were obtained by enzymatic colorimetric detection, Lieberman–Burchard, infrared spectroscopy and high-performance liquid chromatography: 1.0 ± 0.3, 1.32 ± 0.15, 5.1 ± 1.8 and 1.53 ± 0.18 nmol/106 cells, respectively. The Lieberman–Burchard method, enzymatic colorimetric detection and HPLC showed similar results, the obtained average cholesterol concentrations coincide within the error. The mean cholesterol concentration in sperm membranes obtained using infrared spectroscopy method significantly exceeds the values presented in the literature and the values obtained using other methods. In addition, this method requires an amount of analyzed material that significantly exceeds the volume of one ejaculate. Conclusions: As a result of comparing four methods of quantitative cholesterol analysis, the method of enzymatic colorimetric detection is proposed as a method of rapid analysis of cholesterol in human sperm membranes suitable for routine use in a clinical laboratory. The advantages of this method include the low toxicity of the method, it’s cost-effectiveness and a significant reduction in the time of complete analysis: from sample preparation to obtaining the result.

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来源期刊
Russian Journal of Bioorganic Chemistry
Russian Journal of Bioorganic Chemistry 生物-生化与分子生物学
CiteScore
1.80
自引率
10.00%
发文量
118
审稿时长
3 months
期刊介绍: Russian Journal of Bioorganic Chemistry publishes reviews and original experimental and theoretical studies on the structure, function, structure–activity relationships, and synthesis of biopolymers, such as proteins, nucleic acids, polysaccharides, mixed biopolymers, and their complexes, and low-molecular-weight biologically active compounds (peptides, sugars, lipids, antibiotics, etc.). The journal also covers selected aspects of neuro- and immunochemistry, biotechnology, and ecology.
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