CC BY
4Tomsk State University Journal of Chemistry, 2024, 34,155-164
Original article
UDC 612.357.15-047.44:616.995.122 doi: 10.17223/24135542/34/13
Pilot Study on LC-MS Analysis of Bile Acids and Fatty Acids in Duodenal Content of Patients with Chronic Opisthorchiasis
Ksenia I. Kazantseva1, Vladimir S. Sidelnikov2, Daria A. Kokova3, Vladimir E. Frankevich4, Vitaliy V. Chagovets5, Lubov V. Domracheva6, Oleg A. Mayboroda7, Elena E. Ivanyuk8
12, 3, 6 7, 8 Tomsk State University, Tomsk, Russia 44 5 National Medical Research Center for Obstetrics Gynecology and Perinatology named after Academician V.I. Kulakov of the Ministry of Healthcare of Russian Federation,
Moscow, Russia 4 Siberian State Medical University, Tomsk, Russia 1 xenia. caz@yandex. ru 2 [email protected]
3 daria_kokova@mail. ru
4 vfrankevich@gmail. com
5 vvchagovets@gmail. com
6 lvdomracheva42@gmail. com 7 oleg00m2work@gmail. com
8 elenakremer@yandex. ru
Abstract. We present a pilot study utilizing Liquid Chromatography-Mass Spectrometry (LC-MS) to analyze bile acids (BA) and fatty acids (FA) in the duodenal content of patients diagnosed with chronic Opisthorchiasis. By using LC-MS analysis, our study aims to shed light on the alterations in bile acids and fatty acids in the duodenal content, providing insights into the metabolic disruptions caused by chronic opisthorchiasis. Our correlation analysis demonstrated a clear re-wiring of the BA-FA balance. Notably, the key "hubs" in the fatty acid data, such as nervonic, arachidic, stearic, and linoleic acids, are present in both groups. Our findings highlight the importance of exploring the correlation relationships between metabolic compounds to understand the underlying disruptions in metabolic homeostasis in chronic opisthorchiasis.
Keywords: metabolic homeostasis, metabolic compounds, bile acids, fatty acids, duodenal juice, chronic Opisthorchiasis
Acknowledgments: The work was carried out with the financial support of the Russian Foundation for Basic Research grant No. 21-54-14006 ANF_a (Project director -O.A. Mayboroda).
For citation: Kazantseva K.I., Sidelnikov V.S., Kokova D.A., Frankevich V.E., Chagovets V.V., Domracheva L.V., Mayboroda O.A., Ivanyuk E.E. Pilot Study on LC-MS Analysis of Bile Acids and Fatty Acids in Duodenal Content of Patients with Chronic Opisthorchiasis. Vestnik Tomskogo gosudarstvennogo universiteta. Chimia -Tomsk State University Journal of Chemistry, 2024, 34, 155-164. doi: 10.17223/24135542/34/13
© K.I. Kazantseva, V.S. Sidelnikov, D.A. Kokova et al., 2024
Научная статья
doi: 10.17223/24135542/34/13
Анализ желчных и жирных кислот методом жидкостной хромато-масс-спектрометрии: пилотное исследование дуоденального содержимого у больных хроническим
описторхозом
Ксения Игоревна Казанцева1, Владимир Сергеевич Сидельников2, Дарья Алексеевна Кокова3, Владимир Евгеньевич Франкевич4, Виталий Викторович Чаговец5, Любовь Васильевна Домрачева6, Олег Анатольевич Майборода7, Елена Эдуардовна Иванюк8
12, з, б, т, 8 Томский государственный университет, Томск, Россия 4•5 НМИЦ АГП им. В.И. Кулакова Министерства здравоохранения Российской Федерации, Москва, Россия 4 Сибирский государственный медицинский университет, Томск, Россия 1 xenia. caz@yandex. ru 2 [email protected]
3 daria_kokova@mail. ru
4 vfrankevich@gmail. com
5 vvchagovets@gmail. com
6 lvdomracheva42@gmail. com 7 oleg00m2work@gmail. com
8 elenakremer@yandex. ru
Аннотация. Представлено пилотное исследование анализа желчных и жирных кислот в дуоденальном содержимом с использованием жидкостной хромато-масс-спектрометрии у пациентов с хроническим описторхозом. Цель работы -выявление изменений баланса желчных и жирных кислот в содержимом двенадцатиперстной кишки, ассоциированных с метаболическими нарушениями, вызванными хроническим описторхозом. Проведенный корреляционный анализ продемонстрировал четкую корреляционную перестройку баланса жирных и желчных кислот в дуоденальном содержимом при хроническом описторхозе. При этом такие жирные кислоты, как нервоновая, арахиновая, стеариновая и ли-нолевая, присутствовали в обеих группах. Полученные данные являются результатом пилотного исследования и служат основой для последующего изучения метаболических соединений и понимания основных нарушений метаболического гомеостаза при хроническом описторхозе.
Ключевые слова: метаболический гомеостаз, метаболические соединения, желчные кислоты, жирные кислоты, дуоденальное содержимое, хронический описторхоз
Благодарности: Работа выполнена при финансовой поддержке гранта Российского фонда фундаментальных исследований № 21-54-14006 ANF_a (руководитель проекта О.А. Майборода).
Для цитирования: Казанцева К.И., Сидельников В.С., Кокова Д.А., Франкевич В.Е., Чаговец В.В., Домрачева Л.В., Майборода О.А., Иванюк Е.Э. Анализ желчных и жирных кислот методом жидкостной хромато-масс-спектрометрии: пилотное исследование дуоденального содержимого у больных хроническим
описторхозом // Вестник Томского государственного университета. Химия. 2024.
№ 34. С. 155-164. doi: 10.17223/24135542/34/13
Introduction
The chemical and metabolic homeostasis of duodenal juice is crucial for the health of an organism, a fact recognized since the early days of human physiology [1]. This subtle balance plays an important role in the digestive process, influencing the overall functioning of the gastrointestinal system. Maintaining this homeostasis ensures the efficient breakdown and absorption of nutrients, vital for sustaining metabolic functions and systemic energy balance.
Bile acids and fatty acids are fundamental components of duodenal juice. Bile acids, synthesized in the liver from cholesterol and secreted into the duodenum, are essential for the emulsification of dietary fats [2]. This emulsification process increases the surface area of fat droplets, facilitating the action of pancreatic lipases. In turn, these enzymes are breaking down triglycerides into free fatty acids and monoglycerides. The formation of micelles by bile acids is another crucial step in the digestive process. Micelles are microscopic aggregates that encapsulate fatty acids and monoglycerides, making them soluble in the aqueous environment of the intestinal lumen. This solubilization is vital for the transport of fatty acids to the intestinal mucosa, where they can be absorbed by enterocytes. The formation of micelles ensures that the hydrophobic fatty acids are efficiently delivered to the absorptive surfaces of the intestine, a process essential for the assimilation of dietary fats [3].
Therefore, the interplay between bile acids and fatty acids is fundamental for three key processes: emulsification, micelle formation, and the subsequent absorption of fatty acids. This relationship underscores the importance of maintaining a delicate balance between these components to ensure optimal digestive function [4]. Disruption of this homeostasis can lead to serious health issues. For instance, a shift in the balance between bile acids and fatty acids can result in fat malabsorption, leading to deficiencies in essential fatty acids and fat-soluble vitamins. Furthermore, an imbalance can contribute to the formation of gallstones, which can obstruct bile flow and further complicate fat digestion and absorption. Despite the clear physiological and clinical implications, the interplay between bile acids and fatty acids remains an understudied area in human physiology. Although the basic mechanisms are known, more detailed research is needed to clarify the nuances of this relationship in context of the specific diseases.
To this end, we present a pilot study utilizing Liquid Chromatography-Mass Spectrometry (LC-MS) to analyze bile acids (BA) and fatty acids (FA) in the duodenal content of patients diagnosed with chronic Opisthorchiasis. Opisthorchiasis is a parasitic disease caused by the trematode species Opisthorchis felineus [5]. Chronic opisthorchiasis is known to trigger the formation of periportal fibrosis and ulcerative erosions in the gastroduodenal area. Additionally, this condition frequently results in recurrent pancreatitis, obstructive cholangitis, cholecystitis, cholelithiasis, and cholangiocarcinoma. By using LC-
MS analysis, our study aims to shed light on the alterations in bile acids and fatty acids in the duodenal content, providing insights into the metabolic disruptions caused by chronic opisthorchiasis.
Material and Methods
Study design and sample collection
The study was approved by the Ethical Committee of the Siberian State Medical University (SSMU, № 4815, 27.06.2016). All patients gave written informed consent, which included sample storage and use for future studies. Twenty patients with a clinically confirmed opisthorchiasis infection and twenty uninfected patients were included in the study. Duodenal content and stool samples were obtained from each patient; all samples were weighed and encrypted before being stored at -80 °C.
Reagents, instruments and software for HPLC analysis
The solvents of the HPLC grade and chemicals of the ultra-pure grade were used for LC-MS analysis. Two internal standards were added to samples for bile acids analysis: P-muricholic acid (>98 %) and oleoyl(d7)-sn-glycero-3-phospho-choline (18:1-d7, >99 %); both were purchased from Sigma-Aldrich (US). A bile acids model solution consisted of the following compounds: glycochenodeoxy-cholic acid - GCDCA; glycocholic acid - GCA; taurolithocholic acid - TLCA; taurocholic acid - TCA; deoxycholic acid - DCA; glycodeoxycholic acid - GDCA; taurodeoxycholic acid - TDCA; chenodeoxycholic acid - CDCA; ursodeoxycholic acid - UDCA; cholic acid - CA) all purchased from Sigma-Aldrich (US). A model solution for fatty acids analysis consists of the following structures: myristic acid - FA 14:0; palmitic acid - FA 16:0; margaric acid - FA 17:0; stearic acid - FA 18:0; oleic acid - FA 18:1; linoleic acid - FA 18:2; linolenic acid -FA 18:3; arachidic acid - FA 20:0, all from Sigma-Aldrich.
Sample preparation of Duodenal Content Samples
Bile acids. 50 ^l of internal standard, and 100 ^L of the homogenized duodenal content, 200 ^l of formate-acetate buffer (pH 5), and 25 ^L of methanol were added to a 1.5 mL eppendorf tube. Next, the mixture was vortexed and centrifuged for 5 min at 15000 rpm. Afterwards, we performed solid phase extraction of the samples in order to remove the more hydrophobic components from the sample. SPE C18 cartridge was activated with 3 mL of methanol and conditioned with 3 mL of water. The sample was transferred to the cartridge and eluted with 100% methanol. To concentrate and bring the extract to a constant volume, the sample was dried in a nitrogen stream and redissolved in 500 ^l of 100% methanol.
Fatty Acids. Fatty acids were extracted with tert-butyl methyl ether. 1000 ^L of sample and 20 ^L of the internal standard were placed in a 2 mL eppendorf tube №1, then 1000 ^L of tert-butyl methyl ether was added, and the mix was vortexed. 900 ^l of the extract was transferred into a 5 ml Eppendorf tube №2. Extraction was repeated three times. The combined extract was dried in a flow of nitrogen at 50 °C and redissolved in 100 ^L of methanol. The sample was transferred to a glass vial insert.
HPLC-MS Analysis
Bile acids. Bile acids were analyzed by HPLC-HRMS (high-resolution mass spectrometry) using Luna® Phenyl-Hexyl column (100 A 4.6 X 150 mm (3 ^m), Phenomenex). We used pH 4.5 buffer (10 mM ammonium acetate, formic acid) as mobile phase A, and methanol with 0.024% formic acid as mobile phase B. We used gradient chromatography mode. The gradient started with 70% mobile phase B, the phase ratio was maintained for 4 minutes, after which the B content increased to 100% in 5 minutes, the column was washed for 3 minutes, after which the B content decreased to 70% for reequilibration.
The analysis was performed on Agilent 6550 Q-TOF mass spectrometer in the MS mode of negative electrospray ionization with a scanning range of300-900 m/z. Capillary and nozzle voltages were 3000 and 1000 V, respectively; drying gas and sheath gas temperature was 250 °C; drying gas flow was 15 L/min and sheath gas flow was 12 L/min. The conditions were optimized using a model solution of bile acid standards. The injection volume was 100 ^L for fecal samples and 20 ^L for duodenal samples.
Fatty acids. Fatty acids were analyzed by HPLC-MS mode with Zorbax using SB-C18 column (2.1 X50 mm (3.5 ^m), Agilent). We used water: acetonitrile (55/45, v/v), 0.02% acetic acid as mobile phase A, and methanol/isopropyl alcohol (50/50, v/v) as mobile phase B. The column temperature was set to 30 °C with a flow rate of 0.8 mL/min. Gradient chromatography mode was also used.
The gradient was started with 10% mobile phase B, after which the B content increased to 98% in 3 min, the column was washed with 100% mobile phase B for 2 min, after which the B content decreased to 10% and remained unchanged for 2.5 min. Analysis was performed on an Agilent Triple Quad 6460 mass spectrometer in MS2 negative electrospray ionization mode with a scan range of 100-1000 m/z at a fragmenter voltage of 135 V.
Data analysis and visualization
All data analysis was conducted in the R (4.4.1) environment. For comparison between groups, the Mann-Whitney (MW) test was utilized due to its non-parametric nature. To quantify the effect sizes, Cliff's delta was employed, providing a measure of the difference between groups that is independent of sample size. Additionally, the R package "circlize" was used for visualizing the correlations between the datasets, allowing for an intuitive representation of the correlational relationships [6].
Results
Descriptive Analysis of Bile Acids and Fatty Acids in Duodenal Content
After data pre-processing, filtering and annotation, we obtained two datasets for the same patient selection: one dataset with the 17 most abundant fatty acids and another with 27 bile acid structures, each with varying degrees of confidence in structural assignment. For some bile acid structures, e.g. taurocholic acid (TCA) or deoxycholic acid (DCA), the annotations were confirmed using analytical standards. Other structures were matched to database entries based
on m/z values and retention times. However, a few structures exhibited clear, compound-like chromatographic behavior but could not be unequivocally matched to known database entries with the current data. Considering the exploratory nature of this report, we decided to report these structures as m/z values. To explore the differences between patients with chronic opisthorchiasis and controls within each dataset, we used a nonparametric test. Figure 1 shows the forest plots for bile acids (A) and fatty acids (B); the x-axis represents the effect sizes and confidence intervals, while the color bar indicates the -log10 of the p-values. The data do not show striking or strong differences. For fatty acids, only adrenic acid, a long-chain fatty acid, is significantly higher in the controls. In the case of bile acids, two structures are more abundant in the opisthorchiasis (OPI) group, and a single structure is more abundant in the control group.
b
Fig. 1. The forest plots for bile acids (a) and fatty acids (b). The x-axis represents the effect sizes and confidence intervals, while the color bar indicates the -log10 of the p-values. We used Cliffs delta, a non-parametric effect size measure, to quantify the amount of overlap between two distributions. It ranges from -1 to 1, where values close to -1 or 1 indicate less overlap and a stronger effect, while values near 0 suggest greater overlap and a weaker effect. The abbreviations used in the figure are: UCHA - ursocholic acid, TUDCA - tauroursodeoxycholic acid, GUDCA - glycoursodeoxycholic acid, GCA - glycocholic acid, CDGCA -chenodeoxyglycocholic acid, and aLinoleic_acid - alpha linoleic acid
Exploring Correlations Between the Datasets Within Patient Subgroups Thus, with the exception of adrenic acid, a standard statistical inference approach reveals no strong changes between patients with chronic opisthorchiasis and controls.
a
Fig. 3
Fig. 2, 3. The circular correlation plots for the control group (Fig. 2) and the group of patients with chronic opisthorchiasis (Fig. 3). The thickness of the lines indicates the strength of the correlations, the color shows the direction of the correlations (blue for negative and red for positive), and the opacity emphasizes the statistical significance of the correlations (more transparent connections correspond to less significant p-values)
In the fatty acid's dataset, adrenic acid was the only structure above the significance threshold, while in the bile acid dataset, three structures in total were just barely above the significance cut-off. Given these findings, we decided to apply a different strategy. Instead of using statistical inference based on the comparison of central tendencies, we explored the correlation relationships between the individual elements of the datasets within patients and controls. While this approach lacks an easily interpreted statistical measure like a p-value, it is compensated by the clear visual presentation of the data, which complements the exploratory nature of this report. Figures 2 and 3 display the circular correlation plots for the control group (Figure 2) and the group of patients with chronic opisthorchiasis (Figure 3). In these plots, the thickness of the lines indicates the strength of the correlations, color shows a direction of the correlations (blue - negative, red -positive), while the opacity emphasizes the statistical significance of the correlations (with more transparent connections corresponding to less significant p-values). Even a superficial comparison of the figures shows that the main difference between the conditions is the degree of significance of the visualized correlations. In the control group, the majority of the correlations are statistically significant, whereas this is less so in the group of patients with chronic opisthorchiasis. Thus, this method of visualization highlights the complex interplay between bile acids and fatty acids in the duodenal content and underscores the potential differences in metabolic homeostasis between the two groups.
Conclusions
In this pilot study, we explored the metabolic homeostasis of bile acids and fatty acids in the duodenal content of patients with chronic opisthorchiasis. While standard methods of analysis based on statistical inference did not reveal striking or strong differences, our correlation analysis demonstrated a clear re-wiring of the BA-FA balance. Specifically, we observed a shift from a situation with strong, statistically significant correlational interdependencies in the normal samples to much weaker and less significant ones in the samples from patients with chronic opisthorchiasis. Notably, the key "hubs" in the fatty acid data, such as nervonic, arachidic, stearic, and linoleic acids, are present in both groups. However, their bile acid counterparts exhibit notably different strengths and directions of correlations. We fully acknowledge the potential weaknesses of this study, including the limited sample size and technical constraints of the analytical workflow, particularly in the structural annotation of bile acids. Consequently, we refrain from making mechanistic interpretations or far-reaching biological statements. Yet, despite these limitations, we believe that our study clearly demonstrates an effect of the homeostatic shift. The current data, with all its limitations, offer a fresh perspective on the problem and provide a solid basis for follow-up experiments.
To summarize, our findings highlight the importance of exploring the correlation relationships between metabolic compounds to understand the underlying disruptions in metabolic homeostasis in chronic opisthorchiasis. Further research with larger sample sizes and improved analytical techniques is warranted to build
on these initial observations and deepen our understanding of this complex interplay.
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Information about the authors:
Kazantseva Ksenia I. - laboratory assistant, Laboratory for the Study and Application of Supercritical Fluid Technologies in Agro-Food Biotechnology, Tomsk State University (Tomsk, Russia). E-mail: [email protected]
Sidelnikov Vladimir S. - Laboratory for the Study and Application of Supercritical Fluid Technologies in Agro-Food Biotechnology, junior researcher, Tomsk State University (Tomsk, Russia). E-mail: [email protected]
Kokova Daria A. - Candidate of Chemical Sciences, senior researcher, Center for Research in Materials and Technology, Faculty of Chemistry, Tomsk State University (Tomsk, Russia). E-mail: [email protected]
Frankevich Vladimir E. - Dr. Habil. of Physical and Mathematical Sciences, Head of the Department of Systems Biology and Reproduction at the National Medical Research Center for Obstetrics Gynecology and Perinatology Named after Academician V.I. Kulakov of the Ministry of Healthcare of Russian Federation (Moscow, Russia); Siberian State Medical University (Laboratory of Translational Medicine) (Tomsk, Russia). E-mail: [email protected] Chagovets Vitaliy V. - Candidate of Physical and Mathematical Sciences, Senior Researcher at the Laboratory of Metabolomics and Bioinformatics at the National Medical Research Center for Obstetrics Gynecology and Perinatology Named after Academician V.I. Kulakov of the Ministry of Healthcare of Russian Federation (Moscow, Russia). E-mail: [email protected] Domracheva Lubov V. - Laboratory of Translational Cellular and Molecular Biomedicine, Junior Researcher, Tomsk State University (Tomsk, Russia). E-mail: [email protected] Mayboroda Oleg A. - Associate Professor of the Department of Natural Compounds, Pharmaceutical and Medical Chemistry, Faculty of Chemistry, Candidate of Biological Sciences, Tomsk State University (Tomsk, Russia). E-mail: [email protected] Ivanyuk Elena E. - Associate Professor of the Department of Natural Compounds, Pharmaceutical and Medical Chemistry, Faculty of Chemistry, Candidate of Medical Sciences, Tomsk State University (Tomsk, Russia). E-mail: [email protected]
Contribution of the authors: the authors contributed equally to this article. The authors declare no conflicts of interests.
Информация об авторах:
Казанцева Ксения Игоревна - лаборант лаборатории исследования и применения сверхкритических флюидных технологий в агропищевых биотехнологиях Томского государственного университета (Томск, Россия). E-mail: [email protected] Сидельников Владимир Сергеевич - младший научный сотрудник лаборатории исследования и применения сверхкритических флюидных технологий в агропищевых биотехнологиях Томского государственного университета (Томск, Россия). E-mail: vladimir. svibla. sidelnikov@gmail. com
Кокова Дарья Алексеевна - кандидат химических наук, старший научный сотрудник центра исследований в области материалов и технологий химического факультета Томского государственного университета (Томск, Россия). E-mail: [email protected] Франкевич Владимир Евгеньевич - доктор физико-математических наук, заведующий отделом системной биологии и репродукции Института трансляционной медицины НМИЦ АГП им. В.И. Кулакова Министерства здравоохранения Российской Федерации (Москва, Россия); ведущий научный сотрудник лаборатории трансляционной медицины Сибирского государственного медицинского университета (Томск, Россия). E-mail: [email protected]
Чаговец Виталий Викторович - кандидат физико-математических наук, старший научный сотрудник лаборатории метаболомики и биоинформатики НМИЦ АГП им. В.И. Кулакова Министерства здравоохранения Российской Федерации (Москва, Россия). E-mail: [email protected]
Домрачева Любовь Васильевна - младший научный сотрудник лаборатории трансляционной клеточной и молекулярной биомедицины Томского государственного университета (Томск, Россия). E-mail: [email protected]
Майборода Олег Анатольевич - кандидат биологических наук, доцент кафедры природных соединений, фармацевтической и медицинской химии химического факультета Томского государственного университета (Томск, Россия). E-mail: [email protected] Иванюк Елена Эдуардовна - кандидат биологических наук, доцент кафедры природных соединений, фармацевтической и медицинской химии химического факультета Томского государственного университета (Томск, Россия). E-mail: [email protected]
Вклад авторов: все авторы сделали эквивалентный вклад в подготовку публикации. Авторы заявляют об отсутствии конфликта интересов.
The article was submitted 01.06.2024; accepted for publication 16.08.2024 Статья поступила в редакцию 01.06.2024; принята к публикации 16.08.2024
