Измерение эстрогенов в тканях молочной железы методом жидкостной хроматографии и тандемной масс-спектрометрии

Although estrogen contribution estrogen to breast cancer development is not fully understood, an effective method of their measurement, in the mammary gland might provide additional insight. In this study, we have developed a LC-MS/MS method of simultaneous quantification of estrone and estradiol in breast tissue samples. Analytes were extracted with methyl tert-butyl ether by sonication and derivatized with dansyl chloride. Estrogens were analyzed by liquid chromatography-tandem mass spectrometry with an electrospray ionization source. Accuracy and precision were better than 20% for most concentrations. Although estrone and estradiol levels in normal and malignant breast tissue samples analyzed using our method insignificantly differed. The method developed may be used in further studies aimed at evaluating a role estrogens in breast cancer risk.Воздействие эстрогенов считается одним из основных факторов риска развития рака молочной железы, однако механизмы действия этого фактора до конца не изучены. В связи с этим актуальным является создание методов оценки содержания эстрогенов в тканях молочной железы. В данной работе представлены результаты разработки метода количественного определения эстрона и эстрадиола в образцах ткани молочной железы с помощью высокоэффективной жидкостной хроматографии с масс-спектрометрической детекцией. Аналиты экстрагировали метил-трет-бутиловым эфиром в ультразвуковой бане и дериватизировали дансилхлоридом. Были рассмотрены варианты нормировки количественных данных на исходную массу ткани и на массу экстрагированных липидов. Точность и прецизионность конечного метода были выше 20% для большинства концентраций. С помощью метода были проанализированы уровни эстрона и эстрадиола в образцах нормальной и злокачественной ткани молочной железы. Разработанный метод может быть использован в дальнейших исследованиях влияния эстрогена на риск развития рака молочной железы

Алгоритм обработки масс-спектрометрических данных для получения диагностической панели молекулярных соединений на примере поиска маркеров метастазирования при раке молочной железы

A pathology diagnostic using molecular marker is a perspective direction of clinical medicine. Mass-spectrometry (MS) is a one of methods, which are used for obtaining information about molecular profiles. Selection of species, essential for classification “case/control is an important task for data processing. Pipeline of data processing has been proposed using MS data, obtained during analysis of tumor breast tissue samples and health breast tissue samples, with the aim of metastasis marker selection. As a result, selection of lipid markers that belong to classes, related to metastasis and proliferation processes, makes it possible to create high sensitivity diagnostic model, based on logistic regression. The proposed method is applicable for data processing, obtained by MS analysis of other “omics”: metabolome, proteome, glycome.Диагностика патологии по молекулярным маркерам является перспективным направлением клинической медицины, в котором масс-спектрометрия (МС) яслужит одним из методов, используемых для получения информации о молекулярных профилях. В контексте извлечения соединений, играющих ключевую роль для классификации патология/болезнь, важное значение имеет обработка полученных данных, часто включающих несколько сотен детектированных соединений. В данной работе предложен алгоритм обработки данных на примере данных МС, полученных при анализе опухолевой ткани и здоровой молочной железы, с целью выделения липидных маркеров метастазирования. В результате его реализации получен набор соединений, относящихся к классам липидов, связанных с процессами метастазирования и пролиферации, и позволяющих построить высокочувствительную диагностическую модель на основе логистической регрессии. Предложенный метод потенциально пригоден для обработки данных МС, полученных при анализе молекулярного профиля биоматериала другого базиса (метаболом, протеом, гликом)

METABOLIC "FOOTPRINTS" OF THE CIRCULATING CANCER MUCINS: CA125 IN THE HIGH-GRADE OVARIAN CANCER

Mucins are large glycoproteins characterized by the abundant O-linked oligosaccharides (O-glycans) clustered on a protein backbone. Most of the circulating mucins are rapidly cleared by glycan-recognizing hepatic clearance receptors in the liver. Those mucins that remain in the bloodstream are most commonly used as markers in clinical diagnostics. One of such circulating mucins is MUC16; a peptide epitope of which is known as CA125 antigen - a marker for ovarian cancer. Here, using a targeted 1H-NMR profiling of plasma we are exploring a link between the measured CA125 values and the systemic metabolism of the patients within a group with confirmed high-grade ovarian cancer. The study allowed identifying statistically significant associations between the measured values of CA125 epitope and the plasma concentrations of glucose, glutamine, alanine, betaine and serine. The significance of the identified associations for the listed compounds is below 0.01. This, in turn, enables us to hypothesize about a possibility of including the metabolic measures into a composite score of the ovarian cancer based on the CA125 epitope of MUC16.Proteomic

METABOLIC "FOOTPRINTS" OF THE CIRCULATING CANCER MUCINS: CA125 IN THE HIGH-GRADE OVARIAN CANCER

Mucins are large glycoproteins characterized by the abundant O-linked oligosaccharides (O-glycans) clustered on a protein backbone. Most of the circulating mucins are rapidly cleared by glycan-recognizing hepatic clearance receptors in the liver. Those mucins that remain in the bloodstream are most commonly used as markers in clinical diagnostics. One of such circulating mucins is MUC16; a peptide epitope of which is known as CA125 antigen - a marker for ovarian cancer. Here, using a targeted 1H-NMR profiling of plasma we are exploring a link between the measured CA125 values and the systemic metabolism of the patients within a group with confirmed high-grade ovarian cancer. The study allowed identifying statistically significant associations between the measured values of CA125 epitope and the plasma concentrations of glucose, glutamine, alanine, betaine and serine. The significance of the identified associations for the listed compounds is below 0.01. This, in turn, enables us to hypothesize about a possibility of including the metabolic measures into a composite score of the ovarian cancer based on the CA125 epitope of MUC16

METABOLIC “FOOTPRINTS” OF THE CIRCULATING CANCER MUCINS: CA125 IN THE HIGH-GRADE OVARIAN CANCER [МЕТАБОЛОМНАЯ ПОДПИСЬ СВОБОДНЫХ МУЦИНОВ ПРИ ОНКОЛОГИЧЕСКИХ ЗАБОЛЕВАНИЯХ: СА125 И РАК ЯИЧНИКОВ ВЫСОКОЙ СТЕПЕНИ ЗЛОКАЧЕСТВЕННОСТИ]

Mucins are large glycoproteins characterized by the abundant O-linked oligosaccharides (O-glycans) clustered on a protein backbone. Most of the circulating mucins are rapidly cleared by glycan-recognizing hepatic clearance receptors in the liver. Those mucins that remain in the bloodstream are most commonly used as markers in clinical diagnostics. One of such circulating mucins is MUC16; a peptide epitope of which is known as CA125 antigen — a marker for ovarian cancer. Here, using a targeted 1H-NMR profiling of plasma we are exploring a link between the measured CA125 values and the systemic metabolism of the patients within a group with confirmed high-grade ovarian cancer. The study allowed identifying statistically significant associations between the measured values of CA125 epitope and the plasma concentrations of glucose, glutamine, alanine, betaine and serine. The significance of the identified associations for the listed compounds is below 0.01. This, in turn, enables us to hypothesize about a possibility of including the metabolic measures into a composite score of the ovarian cancer based on the CA125 epitope of MUC16. © 2021 Pirogov Russian National Research Medical University. All rights reserved

Genetic diversity of SAD and FAD genes responsible for the fatty acid composition in flax cultivars and lines

Background: Flax (Linum usitatissimum L.) is grown for fiber and seed in many countries. Flax cultivars differ in the oil composition and, depending on the ratio of fatty acids, are used in pharmaceutical, food, or paint industries. It is known that genes of SAD (stearoyl-ACP desaturase) and FAD (fatty acid desaturase) families play a key role in the synthesis of fatty acids, and some alleles of these genes are associated with a certain composition of flax oil. However, data on genetic polymorphism of these genes are still insufficient. Results: On the basis of the collection of the Institute for Flax (Torzhok, Russia), we formed a representative set of 84 cultivars and lines reflecting the diversity of fatty acid composition of flax oil. An approach for the determination of full-length sequences of SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes using the Illumina platform was developed and deep sequencing of the 6 genes in 84 flax samples was performed on MiSeq. The obtained high coverage (about 400x on average) enabled accurate assessment of polymorphisms in SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes and evaluation of cultivar/line heterogeneity. The highest level of genetic diversity was observed for FAD3A and FAD3B genes – 91 and 62 polymorphisms respectively. Correlation analysis revealed associations between particular variants in SAD and FAD genes and predominantly those fatty acids whose conversion they catalyze: SAD – stearic and oleic acids, FAD2 – oleic and linoleic acids, FAD3 – linoleic and linolenic acids. All except one low-linolenic flax cultivars/lines contained both the substitution of tryptophan to stop codon in the FAD3A gene and histidine to tyrosine substitution in the FAD3B gene, while samples with only one of these polymorphisms had medium content of linolenic acid and cultivars/lines without them were high-linolenic. Conclusions: Genetic polymorphism of SAD and FAD genes was evaluated in the collection of flax cultivars and lines with diverse oil composition, and associations between particular polymorphisms and the ratio of fatty acids were revealed. The achieved results are the basis for the development of marker-assisted selection and DNA-based certification of flax cultivars. © 2020, The Author(s)