Phenolic enrichment of foods curated in olive oil: Kinetics and chemical evaluation.

Since ancient times food has been preserved in vegetable oils for curation. Nevertheless, the transfer of bioactive compounds from these oils to curated foods has not been studied. This research has evaluated the phenolic enrichment of foods curated in olive oil. For this purpose, six foods (fish, vegetables, and cheese) were immersed in olive oil for 30 days and analyzed to determine these antioxidant phenols by LC-MS/MS. Oleuropein aglycone, hydroxytyrosol and tyrosol were the main phenols quantitatively enriched in the foods (up to 42.1, 26.2 and 53.0 mg/kg, respectively). The total phenolic content ranged from 5.8 to 12.1 mg in the evaluated foods taking as reference the recommended daily intake (150 g for fish, 200 g for vegetables, and 50 g for cheese). This research proves the phenolic enrichment of foods curated in olive oil, which can hypothetically increase their antioxidant and bioactive properties.

The dynamic changes in olive fruit phenolic metabolism and its contribution to the activation of quiescent Colletotrichum infection.

Anthracnose, the most critical disease affecting olive fruits, is caused by Colletotrichum species. While developing olive fruits are immune to the pathogen regardless of the cultivar, the resistance level varies once the fruit ripens. The defense mechanisms responsible for this difference in resistance are not well understood. To explore this, we analyzed the phenolic metabolic pathways occurring in olive fruits and their susceptibility to the pathogen during ripening in two resistant cultivars ('Empeltre' and 'Frantoio') and two susceptible cultivars ('Hojiblanca' and 'Picudo'). Overall, resistant cultivars induced the synthesis of aldehydic and demethylated forms of phenols, which highly inhibited fungal spore germination. In contrast, susceptible cultivars promoted the synthesis of hydroxytyrosol 4-O-glucoside during ripening, a compound with no antifungal effect. This study showed that the distinct phenolic profiles between resistant and susceptible cultivars play a key role in determining olive fruit resistance to Colletotrichum species.

Lipidomics signature in post-COVID patient sera and its influence on the prolonged inflammatory response.

BACKGROUND: The ongoing issues with post-COVID conditions (PCC), where symptoms persist long after the initial infection, highlight the need for research into blood lipid changes in these patients. While most studies focus on the acute phase of COVID-19, there's a significant lack of information on the lipidomic changes that occur in the later stages of the disease. Addressing this knowledge gap is critical for understanding the long-term effects of COVID-19 and could be key to developing personalized treatments for those suffering from PCC. METHODS: We employed untargeted lipidomics to analyze plasma samples from 147 PCC patients, assessing nearly 400 polar lipids. Data mining (DM) and machine learning (ML) tools were utilized to decode the results and ascertain significant lipidomic patterns. RESULTS: The study uncovered substantial changes in various lipid subclasses, presenting a detailed profile of the polar lipid fraction in PCC patients. These alterations correlated with ongoing inflammation and immune response. Notably, there were elevated levels of lysophosphatidylglycerols (LPGs) and phosphatidylethanolamines (PEs), and reduced levels of lysophosphatidylcholines (LPCs), suggesting these as potential lipid biomarkers for PCC. The lipidomic signatures indicated specific anionic lipid changes, implicating antimicrobial peptides (AMPs) in inflammation. Associations between particular medications and symptoms were also suggested. Classification models, such as multinomial regression (MR) and random forest (RF), successfully differentiated between symptomatic and asymptomatic PCC groups using lipidomic profiles. CONCLUSIONS: The study's groundbreaking discovery of specific lipidomic disruptions in PCC patients marks a significant stride in the quest to comprehend and combat this condition. The identified lipid biomarkers not only pave the way for novel diagnostic tools but also hold the promise to tailor individualized therapeutic strategies, potentially revolutionizing the clinical approach to managing PCC and improving patient care.

Comprehensive profiling of ceramides in human serum by liquid chromatography coupled to tandem mass spectrometry combining data independent/dependent acquisition modes.

Ceramides are sphingolipids with a structural function in the cell membrane and are involved in cell differentiation, proliferation and apoptosis. Recently, these chemical species have been pointed out as potential biomarkers in different diseases, due to their abnormal levels in blood. In this research, we present an overall strategy combining data-independent and dependent acquisitions (DIA and DDA, respectively) for identification, confirmation, and quantitative determination of ceramides in human serum. By application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in DIA mode we identified 49 ceramides including d18:1, d18:0, d18:2, d16:1, d17:1 and t18:0 species. Complementary, quantitative determination of ceramides was based on a high-throughput and fully automated method consisting of solid-phase extraction on-line coupled to LC-MS/MS in DDA to improve analytical features avoiding the errors associated to sample processing. Quantitation limits were at pg mL-1 level, the intra-day and between-days variability were below 20 and 25 %, respectively; and the accuracy, expressed as bias, was always within ±25 %. The proposed method was tested with the CORDIOPREV cohort in order to obtain a qualitative and quantitative profiling of ceramides in human serum. This characterization allowed identifying d18:1 ceramides as the most concentrated with 70.8% of total concentration followed by d18:2 and d18:0 with 13.0 % and 8.8 %, respectively. Less concentrated ceramides, d16:1, d17:1 and t18:0, reported a 7.1 % of the total content. Combination of DIA and DDA LC-MS/MS analysis enabled to profile qualitative and quantitatively ceramides in human serum.

Influence of genetic and interannual factors on the fatty acids profile of virgin olive oil.

Among olive oil nutritional benefits, it is worth mentioning its fatty acids composition with predominance of monounsaturated fatty acids (MUFAs). We have evaluated the influence of the cultivar and interannual factors on the fatty acids profile of virgin olive oil samples obtained from 45 and 71 cultivars along three and two consecutive crop seasons, respectively. The cultivars were classified in two groups according to the fatty acids composition: (1) high content in MUFAs and moderate content in saturated and polyunsaturated fatty acids (SFAs and PUFAs, respectively) and (2) moderate content in MUFAs and high content in SFAs/PUFAs. We also observed variations in the fatty acids content with the climate conditions, which can significantly alter the saturated and unsaturated profiles. Thus, a significant decrease in MUFAs and an increase in SFAs/PUFAs concentrations was found when the precipitation accumulated within the June-October period was reduced.

Combining data acquisition modes in liquid-chromatography-tandem mass spectrometry for comprehensive determination of acylcarnitines in human serum.

Acylcarnitines (ACs) are metabolites involved in fatty acid ß-oxidation and organic acid metabolism. Metabolic disorders associated to these two processes can be evaluated by determining the complete profile of ACs. In this research, we present an overall strategy for identification, confirmation, and quantitative determination of acylcarnitines in human serum. By this strategy we identified the presence of 47 ACs from C2 to C24 with detection of the unsaturation degree by application of a data-independent acquisition (DIA) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Complementary, quantitative determination of ACs is based on a high-throughput and fully automated method consisting of solid-phase extraction on-line coupled to LC-MS/MS in data-dependent acquisition (DDA) to improve analytical features avoiding the errors associated to sample processing. Quantitation limits were at pg mL-1 level, the intra-day and between-day variability were below 15-20%, respectively; and the accuracy, expressed as bias, was always within ± 25%. The proposed method was tested with 40 human volunteers to determine the relative concentration of ACs in serum and identify predominant forms. Significant differences were detected by comparing the ACs profile of obese versus non-obese individuals.

The secoiridoid profile of virgin olive oil conditions phenolic metabolism.

The European Food Safety Authority highlights the beneficial effects of olive oil phenols, mainly, secoiridoids. Nevertheless, the metabolism of secoiridoids in humans has not been fully elucidated. This research evaluated the metabolism of secoiridoids in humans after intake of olive oils with diverse phenolic profiles. For this purpose, three extra virgin olive oils (EVOOs) were ingested by six volunteers at scheduled meals, and urine samples were collected the following morning for subsequent LC-MS/MS analysis. Using untargeted analysis, urinary metabolites revealed representative patterns associated with the various olive oil phenolic contents in absolute and relative terms. We were able to identify metabolites obtained through phase I, phase II, and microbial metabolism with discrimination between tyrosol and hydroxytyrosol derivatives. Metabolism of phenols is differentially activated as a function of the olive oil secoiridoids content, and this proof-of-concept study shows how urinary metabolites represent olive oil phenolic content.

Influence of genetic and interannual factors on bioactive compounds of olive pomace determined through a germplasm survey.

Olive mill wastes, generated in the extraction of virgin olive oil (VOO), are of important concern for the industry owing to the produced volume and polluting load, mainly associated with the presence of organic compounds. Among them, it is worth mentioning bioactive compounds, mainly phenols and triterpenes, which could be potentially isolated for further use in the cosmetic, pharmaceutical, or food industries. This research analyzed the olive pomace after extraction of VOO from fruits harvested of 43 international olive cultivars during three consecutive seasons. The cultivar was identified as the most determinant factor to explain the variability in the relative concentration of phenols and terpenic acids in the extracts. In addition, the characterization of olive pomace extracts allowed clustering cultivars according to the profile of bioactive compounds. Finally, we identified the components responsible for the observed discrimination that was explained according to biosynthetic metabolic pathways.

Vitamin D3 levels in women and factors contributing to explain metabolic variations.

The elucidated metabolism of vitamin D3 in humans has been the support to explain the high involvement of this liposoluble vitamin in physiological functions. Clinical studies have associated levels of vitamin D3 metabolites with several disorders. Despite this knowledge, there is a controversy regarding the estimation of deficiency and the physiological and supraphysiological levels of vitamin D3 metabolites. The association between serum concentrations of vitamin D3 metabolites and several potentially influential factors (namely, age and anthropometric, seasonal, spatial and metabolic factors) is analyzed in this study. For this purpose, 558 women were recruited and interviewed in several Spanish provinces before blood sampling. Serum vitamin D3 and its metabolites were determined using an SPE-LC-MS/MS platform. The concentration range for vitamin D3 was 1.7-21.1 nmol/L and was influenced by body mass index (BMI), waist-to-hip ratio (WHR) and seasonal period. 25-hydroxyvitamin D3 levels were within 4.8-147.2 nmol/L and were related to WHR, season, latitude and calcium intake. The range of 24,25-dihydroxyvitamin D3, 0.3-15.0 nmol/L, was associated to BMI, WHR, season, latitude and calcium intake. Finally, energy intake influenced the vitamin D 25-hydroxylase through the 25-hydroxyvitamin D3/vitamin D3 ratio, which regulates the synthesis of the circulating form. According to these results, it is worth emphasizing the relevance of all these factors to explain the variability in serum levels of vitamin D3 and its metabolites. All these factors should be considered in future studies assessing the alteration of vitamin D3 metabolism.

Influence of genetic and interannual factors on the phenolic profiles of virgin olive oils.

Phenolic compounds in virgin olive oil (VOO) contribute to its health properties, organoleptic features and oxidative stability. In this study, a total of 44 olive tree cultivars categorized by the International Olive Council to be among the most internationally widespread varieties were exhaustively and homogenously evaluated by analysis of the VOO phenolic profile during three consecutive crop seasons. Differences among cultivars resulted in up to 15-fold variations in the total phenol concentration. The 'cultivar' factor contributed the most to the variance (66.8% for total phenolic concentration) for almost all the phenols. However, the 'interannual variability' factor and the interaction 'cultivar x interannual variability' exhibited significant influences on specific phenols. According to the phenolic profile of the VOOs, we determined the presence of three groups of cultivars marked by the predominance of secoiridoid derivatives, which supports the phenolic profile as a criterion to be considered in olive breeding programs.

Fully automated method for quantitative determination of steroids in serum: An approach to evaluate steroidogenesis.

Steroidogenesis is a set of metabolic reactions where the enzymes play a key role to control the physiological levels of steroids. A deficiency in steroidogenesis induces an accumulation and/or insufficiency of steroids in human blood and can lead to different pathologies. This issue added to the low levels of steroids (pg mL-1 to ng mL-1) in this biofluid make of their determination an analytical challenge. In this research, we present a high-throughtput and fully automated method based on solid-phase extraction on-line coupled to liquid chromatography with tandem mass spectrometry detection (SPE-LC-MS/MS) to quantify estrogens (estrone and estradiol), androgens (testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone), progestogens (progesterone, pregnenolone, 17-hydroxyprogesterone and 17-hydroxypregnenolone), glucocorticoids (21-hydroxyprogesterone, 11-deoxycortisol, cortisone, corticosterone and cortisol) and one mineralocorticoid (aldosterone) in human serum. The performance of the SPE step and the multiple reaction monitoring (MRM) mode allowed reaching a high sensitivity and selectivity levels without any derivatization reaction. The fragmentation mechanisms of the steroids were complementary studied by LC-MS/MS in high-resolution mode to confirm the MRM transitions. The method was characterized with two SPE sorbents with similar physico-chemical properties. Thus, limits of quantification were at pg mL-1 levels, the variability was below 25% (except for pregnenolone and cortisone), and the accuracy, expressed as bias, was always within ±25%. The proposed method was tested in human serum from ten volunteers, who reported levels for the sixteen target steroids that were satisfactorily in agreement with the physiological ranges reported in the literature.

Lyophilization as pre-processing for sample storage in the determination of vitamin D3 and metabolites in serum and plasma.

Determination of vitamin D levels in human biological specimens has gained a high relevance over the last decades, essentially because low levels have been associated with several biological disorders. In fact, vitamin D deficiency has become a worldwide health concern covering all ages and genders. The storage of biofluids has to be considered for determination of vitamin D and metabolites in order to fully preserve matrices status. This study attempts to evaluate lyophilization of serum and plasma as a pre-processing step for sample storage prior to quantitative analysis of vitamin D3 and its main hydroxylated metabolites -25(OH)D3, 24,25(OH)2D3 and 1,25(OH)2D3. The protocol including sample lyophilization was characterized in terms of analytical features and compared to the same method, based on SPE-LC-MS/MS, without lyophilization. Sensitivity, precision and accuracy were not affected when we operated with lyophilized serum and plasma and results provided by a set of twenty-four serum samples from DEQAS (Vitamin D External Quality Assessment Scheme) were in agreement with reported concentrations for 25(OH)D3 and 1,25(OH)2D3. A stability study programmed for 9 months allowed ensuring that the concentration of vitamin D3 and metabolites in lyophilized serum and plasma stored at room temperature was not affected during this period. This research has demonstrated that the quantitation of target metabolites is not under the influence of lyophilization. Therefore, including lyophilization prior to analysis could reduce shipment and storage costs, avoid delays of sample processing, and increase the stability of the target analytes due to an effective quenching process.

The decrease in the health benefits of extra virgin olive oil during storage is conditioned by the initial phenolic profile.

Phenols are responsible for the only health claim of virgin olive oil (VOO) recognized by the European Commission EU 432/2012 and the European Food Safety Authority. In this research, we studied the decrease in the phenolic content of 160 extra VOOs (EVOOs) after 12 months storage in darkness at 20 °C. Phenolic concentration was decreased 42.0 ± 24.3% after this period and this reduction strongly depended on the initial phenolic profile. Hence, EVOOs with predominance in oleacein and oleocanthal experienced a larger decrease in phenolic content than oils enriched in other phenols. Complementarily, hydroxytyrosol and oleocanthalic acid increased significantly in aged EVOOs, which allowed their discrimination from recently produced EVOOs. These changes are explained by degradation of main secoiridoids during storage due to their antioxidant properties. Hydroxytyrosol and oleocanthalic acid can be considered markers of olive oil ageing, although they can also provide information about quality or stability.

Development of a quantitative method for determination of steroids in human plasma by gas chromatography-negative chemical ionization-tandem mass spectrometry.

Sex steroids are involved in biological functions that encompass from the complete sexual development of individuals up to the deregulation of metabolic pathways leading to some pathologies. Steroids are present in blood at low concentration levels from pg mL-1 to ng mL-1. For this reason, a high sensitive and selective method based on gas chromatography-negative chemical ionization-tandem mass spectrometry (GC-NCI-MS/MS) is here proposed to quantify either androgens (androstenedione, dehydroepiandrosterone, dihydrotestosterone and testosterone), estrogens (estrone and estradiol) and a progestogen (progesterone) in human plasma. The sample preparation steps, protein precipitation and solid phase extraction, were optimized to ensure the sample matrix removal and to extract steroids with high efficiency. The NCI-MS/MS detection approach was compared with that based on electron impact to evaluate the incidence of the ionization source in the determination of steroids. The quantification limits for determination of these analytes were in a range from 10 pg mL-1 to 5 ng mL-1, with a high sensitivity for estrogens, typically found at low concentrations. The proposed method was tested for the determination of steroids in male blood samples, in which 6 out of 7 steroids were detected and quantified to report concentration values in agreement with those described in the literature.

Optimization of a MALDI-Imaging protocol for studying adipose tissue-associated disorders.

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is increasingly recognized for its potential in the discovery of novel biomarkers directly from tissue sections. However, there are no MALDI IMS studies as yet on the adipose tissue, a lipid-enriched tissue that plays a pivotal role in the development of obesity-associated disorders. Herein, we aimed at developing an optimized method for analyzing adipose tissue lipid composition under both physiological and pathological conditions by MALDI IMS. Our studies showed an exacerbated lipid delocalization from adipose tissue sections when conventional strategies were applied. However, our optimized method using conductive-tape sampling and 2,5-dihydroxybenzoic acid (DHB) as a matrix, preserved the anatomical organization and minimized lipid diffusion from sample sections. This method enabled the identification of a total of 625 down-regulated and 328 up-regulated m/z values in the adipose tissue from a rat model of extreme obesity as compared to lean animals. Combination of MALDI IMS and liquid chromatography (LC)-MS/MS data identified 44 differentially expressed lipid species between lean and obese animals, including phospholipids and sphingomyelins. Among the lipids identified, SM(d18:0_18:2), PE(P-16:0_20:0), and PC(O-16:0_16:1) showed a differential spatial distribution in the adipose tissue of lean vs. obese animals. In sum, our method provides a valuable new tool for research on adipose tissue that may pave the way for the identification of novel biomarkers of obesity and metabolic disease.

The phenolic profile of virgin olive oil is influenced by malaxation conditions and determines the oxidative stability.

Phenolic compounds largely contribute to the nutraceutical properties of virgin olive oil (VOO), the organoleptic attributes and the shelf life due to their antioxidant capabilities. Due to the relevance of malaxation in the oil extraction process, we tested the effects of malaxation time on the concentrations of relevant phenolic compounds in VOO, and we evaluated the influence of performing malaxation under vacuum. An increase in malaxation time significantly decreased the concentrations of aglycone isomers of oleuropein and ligstroside but, conversely, increased the oleocanthal and oleacein contents. Additionally, malaxation under vacuum led to an increase in phenolic contents compared to standard conditions carried out at atmospheric pressure. Finally, we explored the possibility of predicting the VOO oxidative stability on the basis of the phenolic profile, and a model (R2 = 0.923; p < 0.0001) was obtained by combining the concentration of the VOO phenolic compounds and the main fatty acids.

Dry sweat as sample for metabolomics analysis.

Sweat is gaining popularity in clinical metabolomics as this biofluid is non-invasively sampled and its composition is modified by several pathologies. There is a lack of standardized strategies for collection of human sweat. Most studies have been carried out with fresh sweat collected after stimulation. A promising and simple alternative is sampling dry sweat by a solid support impregnated with a suited solvent. This research was aimed at comparing the metabolomics coverage provided by dry sweat collected by two solid supports (gauzes and filter papers) impregnated with different solvents. The dissolved dry sweat was analyzed by a dual approach: gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among the tested sampling strategies, filter paper impregnated with 1:1 (v/v) ethanol‒phosphate buffer resulted the combination providing the highest metabolomics coverage (tentative identification of one hundred seventy-five compounds). Dry and fresh sweat were compared by using pools from the same individuals to evaluate compositional differences. Families of metabolites such as carnitines, sphingolipids and N-acyl-amino acids, among others, were exclusively identified in dry sweat. Comparison of both samples allowed concluding that dry sweat is better for analysis of low polar metabolites and fresh sweat is more suited for polar compounds. As most of the identified metabolites are involved in key biochemical pathways, this study opens interesting possibilities to the use of dry sweat as a source of metabolite markers for specific disorders. Sampling of dry sweat could provide a standardized approach for collection of this biofluid, thus overcoming the variability limitations of fresh sweat.

Untargeted characterization of extracts from Cannabis sativa L. cultivars by gas and liquid chromatography coupled to mass spectrometry in high resolution mode.

Elucidation of Cannabis composition is required to evaluate the potential of this plant for pharmacological uses, but also for implementation in breeding programs with agronomical purposes. The aim of the present study was to develop a method for untargeted analysis of polar and non-polar Cannabis extracts. For this purpose, extracts from 17 cultivars of Cannabis sativa L. were analyzed by gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) and liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOF MS/MS) in high resolution mode. One hundred sixty-nine compounds were identified in the extracts by searching MS and MS/MS information. Among identified families, there were mainly cannabinoids, terpenoids, lipids and flavonoids, but also some interesting compounds such as amino and organic acids, among others. Relative contents of terpenoids and cannabinoids in the same cultivars grown in greenhouse and field were compared. Compositional differences in the profile of terpenoids and cannabinoids between both types of grown conditions were found.

Development of a qualitative/quantitative strategy for comprehensive determination of polar lipids by LC-MS/MS in human plasma.

Polar lipids, especially glycerophospholipids, constitute the main components of cell membranes and are precursors of signaling molecules in many cellular and physiological processes. For this reason, the development of methods with high capability for detection of polar lipids in biological samples is required. In this research, the objective was to develop a method for comprehensive qualitative/quantitative determination of polar lipids in plasma by a combination of acquisition methods with a triple quadrupole mass analyzer. The strategy was optimized in two steps: (a) a first step for detection of lipids by monitoring selective fragmentation patterns representative of each lipid family and (b) a second step for confirmation of lipid species by detection and identification of product ions associated with the conjugated fatty acids. The acquisition list was divided into two multiple reaction monitoring (MRM) methods to ensure the detection of all transitions with suited instrumental sensitivity according to chromatographic retention time and relative abundance in plasma. The combination of the two MRM methods allowed the detection of 398 polar lipids in plasma in 64 min. Precision, estimated as within-day variability, was below 6.8% for all determined lipid families, while between-day variability was below 24.0%. This strategy has been applied to a cohort formed by 384 individuals in order to obtain a qualitative and quantitative distribution of polar lipids in human plasma. The most concentrated lipid families in relative terms were lysophospholipids, plasmalogens, and phosphatydilcholines, with mean relative concentration of 58.0, 17.1, and 8.3%, respectively. Then, sphingomyelins and phosphatidylethanolamines reported a relative concentration of 2.0%, followed by phosphatidylserines, with 1.1%. Graphical abstract.

Determination of glycerophospholipids in vegetable edible oils: Proof of concept to discriminate olive oil categories.

Glycerophospholipids (GPLs) constitute a chemical family within the saponifiable fraction of vegetable oils. GPLs have been scarcely studied in edible oils owing to the lack of sensitive and selective analytical methods. We have developed a method for identification, confirmation and relative quantitation of GPLs in vegetable oils. The method is based on solid-phase extraction (SPE) for isolation of GPLs and determination by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). As proof of concept, the approach has been applied to characterize GPLs in different olive oil categories, thus revealing compositional changes, which could be explained by factors such as the quality of fruits and the extraction process. Families such as glycerophosphatidic acids and phosphatidylglycerides are remarkable because of their capability to discriminate virgin olive oils from the rest of categories. These results open a door to additional studies targeted at the identification of olive oil quality by monitoring these lipids.