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Rapid LC-MS/MS method for the determination of 4-hydroxycholesterol/cholesterol ratio in serum as endogenous biomarker for CYP3A activity in human and foals.
Abstract: Cytochrome P450 3A (CYP) enzymes are involved in the elimination of many drugs and are known to be regulated by several environmental factors. Thus, it was the aim of this study to develop and validate an analytical method allowing estimation of the hepatic CYP3A enzyme activity using the 4-hydroxycholesterol to cholesterol ratio as an endogenous biomarker in serum. Both compounds were isolated from the biological matrix by liquid-liquid extraction using n-hexane after saponification with ethanolic sodium methoxide solution (2M) to cleave the steroids from their esterified forms without any kind of further derivatization. Chromatographic separation was achieved on a reversed-phase column (SupelcoAcsentis(®), C8) within 7min using an isocratic elution with ammonium acetate 5mM (pH=3.8, 10%) and acetonitrile (90%) at a flow rate of 300μl/min. d6-cholesterol and d7-4β-hydroxycholesterol were used as internal standards. Detection was done on a triple quadrupole mass spectrometer using the following mass transitions: 369.3/161.5, 369.3/147.1 and 369.3/95.2 for cholesterol; 385.2/367.4, 385.2/109.1 for 4-hydroxycholesterol; 374.4/152.7 and 392.2/108.9 for d6-cholesterol and d7-4-hydroxycholesterol, respectively as the internal standards. The method was validated according to current bioanalytical guidelines considering selectivity, linearity, accuracy, precision, recovery, stability. The analytical range was 5-250 and 50-1000ng/ml, for 4-hydroxycholesterol and cholesterol, respectively. The method was shown to be selective for both compounds with good linearity over the selected range (r>0.99) as well as good within- and between day accuracy (error: -1.2-3.7% for 4-hydroxycholesterol and -7.7-9.5% for cholesterol) and within- and between day precision (2.1-14.6% for 4-hydroxycholesterol and 1.1-14.9% for cholesterol). Recovery was found to be over 80% for both analytes while significant stability issues could not be observed. Finally, the validated assay was applied to measure 4-hydroxycholesterol and cholesterol in serum samples of clinical studies in humans and foals that could verify induction of hepatic CYP3A4 (human) and CYP3A89 (foals) after premedication with the known enzyme inducer rifampicin.
Copyright © 2016 Elsevier B.V. All rights reserved.
Publication Date: 2016-08-05 PubMed ID: 27565568DOI: 10.1016/j.jchromb.2016.08.006Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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This research is aimed at developing and testing a method for estimating the activity of the Cytochrome P450 3A (CYP3A) enzyme in the liver, using the ratio of 4-hydroxycholesterol to cholesterol in serum as a natural marker.
Understanding the Research
The study revolves around the role of enzymes known as Cytochrome P450 3A (CYP). These enzymes are crucial in the body as they aid in getting rid of numerous drugs. Their functioning often hinges on a range of environmental factors. In this study specifically, the investigators were bent on coming up with a new approach to approximate the activity level of CYP3A in the liver.
- The method relies on the ratio of 4-hydroxycholesterol to cholesterol present in the serum, viewing it as an endogenous biomarker.
- For isolation of these two compounds, a liquid-liquid extraction process was deployed to initially separate them from the biological matrix. This was followed by saponification aided by an ethanolic sodium methoxide solution, which was purposed to deconstruct the steroids from their esterified forms.
- After this, the process didn’t require any further derivatization. The scientific team exploited a reversed-phase column to achieve chromatographic separation, and they used an isocratic elution strategy.
Mass Spectrometry
Following up on the chromatographic separation, the researchers undertook detection using a three-stage quadrupole mass spectrometer.
- Specific mass transitions were required for cholesterol and 4-hydroxycholesterol, alongside their respective internal benchmarks, d6-cholesterol, and d7-4-hydroxycholesterol.
- The method went through several validation stages, in accordance with current bioanalytical guidelines. The team went ahead to scrutinize various factors in regards to the proposed model, namely its selectivity, linearity, accuracy, precision, recovery, and stability.
- This approach revealed selectivity towards the two compounds at hand and exhibited positive linearity over the selected range. Its within- and between-day precision, as well as accuracy, were good. Both analytes were recovered at more than 80%; furthermore, it was discovered that significant stability issues were not present.
Research Application
The tools that the team built didn’t just remain in the lab for theoretical analysis. The researchers applied the validated assay practically to measure 4-hydroxycholesterol and cholesterol in serum samples, collected from human beings and foals involved in clinical studies.
- These tests provided empirical evidence for the induction of hepatic CYP3A4 (in humans) and CYP3A89 (in foals) following premedication with rifampicin, a widely acknowledged enzyme inducer.
Cite This Article
APA
Hasan M, Siegmund W, Oswald S.
(2016).
Rapid LC-MS/MS method for the determination of 4-hydroxycholesterol/cholesterol ratio in serum as endogenous biomarker for CYP3A activity in human and foals.
J Chromatogr B Analyt Technol Biomed Life Sci, 1033-1034, 193-199.
https://doi.org/10.1016/j.jchromb.2016.08.006 Publication
Researcher Affiliations
- Department of Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald, Felix-Hausdorff-Str. 3, 17487 Greifswald, Germany.
- Department of Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald, Felix-Hausdorff-Str. 3, 17487 Greifswald, Germany.
- Department of Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald, Felix-Hausdorff-Str. 3, 17487 Greifswald, Germany. Electronic address: stefan.oswald@uni-greifswald.de.
MeSH Terms
- Adult
- Animals
- Biomarkers / blood
- Cholesterol / blood
- Chromatography, Liquid / methods
- Cytochrome P-450 CYP3A / metabolism
- Horses
- Humans
- Hydroxycholesterols / blood
- Male
- Reference Standards
- Reproducibility of Results
- Rifampin / pharmacology
- Tandem Mass Spectrometry / methods
- Young Adult
Citations
This article has been cited 8 times.- Taya Y, Mizunaga M, Nakao S, Jutanom M, Shimizu N, Nomura Y, Nakagawa K. Clinical Evaluation Based on a New Approach to Improve the Accuracy of 4β-Hydroxycholesterol Measurement as a Biomarker of CYP3A4 Activity. Molecules 2023 Feb 7;28(4).
- Na HW, Kim HS, Choi H, Cha N, Seo YR, Hong YD, Kim HJ. Transcriptome Analysis of Particulate Matter 2.5-Induced Abnormal Effects on Human Sebocytes. Int J Mol Sci 2022 Sep 29;23(19).
- Lee SH, Seo HS, Seo SJ, Kim CD, Hong SP. Screening of Plant-Derived Natural Extracts to Identify a Candidate Extract Capable of Enhancing Lipid Synthesis in Keratinocytes. Ann Dermatol 2022 Oct;34(5):331-339.
- Jung SW, Park GH, Kim E, Yoo KM, Kim HW, Lee JS, Chang MY, Shin KO, Park K, Choi EH. Rosmarinic Acid, as an NHE1 Activator, Decreases Skin Surface pH and Improves the Skin Barrier Function. Int J Mol Sci 2022 Mar 31;23(7).
- Che Y, Yang J, Tang F, Wei Z, Chao Y, Li N, Li H, Wu S, Dong X. New Function of Cholesterol Oxidation Products Involved in Osteoporosis Pathogenesis. Int J Mol Sci 2022 Feb 11;23(4).
- Suzuki Y, Oda A, Negami J, Toyama D, Tanaka R, Ono H, Ando T, Shin T, Mimata H, Itoh H, Ohno K. Sensitive UHPLC-MS/MS quantification method for 4β- and 4α-hydroxycholesterol in plasma for accurate CYP3A phenotyping. J Lipid Res 2022 Mar;63(3):100184.
- Kim BJ, Lee NR, Lee CH, Lee YB, Choe SJ, Lee S, Hwang HJ, Kim E, Lavery GG, Shin KO, Park K, Choi EH. Increased Expression of 11β-Hydroxysteroid Dehydrogenase Type 1 Contributes to Epidermal Permeability Barrier Dysfunction in Aged Skin. Int J Mol Sci 2021 May 27;22(11).
- Vanhove T, Hasan M, Annaert P, Oswald S, Kuypers DRJ. Pretransplant 4β-hydroxycholesterol does not predict tacrolimus exposure or dose requirements during the first days after kidney transplantation. Br J Clin Pharmacol 2017 Nov;83(11):2406-2415.
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