Quantification Of Metabolites Research Articles

Simultaneously quantification of eight marker compounds by HPLC, and HPTLC analysis for the marker-based shelf-life kinetic study for the standardization of polyherbal drug AYUSH SG-5, medicinal properties and computational studies

The herbal or alternative medicines having their own significances in each country. The major problem behind the non-acceptance of the alternative medicines is the poor standardization and quality control. Maximum times, it’s impossible to quantify the complex matrix of polyherbal medicines. The pharmacopeial standards of these medicines are limited to physicochemical and pharmacognostic analysis only, which are insufficient for the proper quality control of ingredients used for the preparation of polyherbal formulations. Multi-markers-based analysis of alternative medicines and their polyherbal formulations by using the sophisticated analytical techniques like HPLC, and LC-MS is the demand of current scenario. In our recent report we have reported pharmaceutical standard operating procedure for a coded polyherbal formulation (CPF i.e. AYUSH-SG-5) which was prepared for the rheumatoid arthritis. The marker-based quantification and identification of major metabolites and shelf-life (marker-based) studies were not reported in that study. In this report we have quantified eight markers (6-Gingerol, 8-Gingerol, Guggulsterone-E, Guggulsterone-Z, Resveratrol, Salicin, Colchicine, and Caffeic acid) simultaneously by HPLC chromatography. To identify the phytochemicals in polyherbal formula (CPF), LC-QTOF-MS/MS analysis was performed, where 32 main phytochemicals were identified out of 80 peaks. In herbal industry, HPTLC is the main tool for drug quantification, so we have reported a HPTLC method for the real time shelf-life study. In HPTLC study, we have used three major markers (6-Gingerol, Guggulsterone-E, Guggulsterone-Z) that are reproducible in hydroalcoholic extract and not in aqueous and methanolic extracts of CPF. The kinetic study-based shelf-life of CPF formulation was performed, the observed shelf-life of CPF was more than 41 months. In addition, evaluation of antioxidant activity like total phenolic (TPC), total flavonoid (TFC) and DPPH assays were performed, and results exhibited that hydroalcoholic extract having more values than aqueous and methanolic extracts of CPF. The phytochemicals observed in LC-QTOF-MS/MS studies were tested for in silico anti-inflammatory activity and anti-arthritic activities, which need to be explored after the detailed pharmacological and biological studies. Overall, analytical studies result of present study could use to standardize the CPF as well as the all relevant polyherbal formulations where these raw drugs have been used.

In vitro batch fermentation of (un)saturated homogalacturonan oligosaccharides

Pectin, predominantly present within plant cell walls, is a dietary fiber that potentially induces distinct health effects depending on its molecular structure. Such structure-dependent health effects of pectin-derived galacturonic acid oligosaccharides (GalA-OS) are yet largely unknown. This study describes the influence of methyl-esterification and ∆4,5-unsaturation of GalA-OS through defined sets of GalA-OS made from pectin using defined pectinases, on the fermentability by individual fecal inocula. The metabolite production, OS utilization, quantity and size, methyl-esterification and saturation of remaining GalA-OS were monitored during the fermentation of GalA-OS. Fermentation of all GalA-OS predominantly induced the production of acetate, butyrate and propionate. Metabolization of unsaturated GalA-OS (uGalA-OS) significantly increased butyrate formation compared to saturated GalA-OS (satGalA-OS), while satGalA-OS significantly increased propionate formation. Absence of methyl-esters within GalA-OS improved substrate metabolization during the first 18 h of fermentation (99 %) compared to their esterified analogues (51 %). Furthermore, HPAEC and HILIC-LC-MS revealed accumulation of specific methyl-esterified GalA-OS, confirming that methyl-esterification delays fermentation. Fermentation of structurally distinct GalA-OS results in donor specific microbiota composition with uGalA-OS specifically stimulating the butyrate-producer Clostridium Butyricum. This study concludes that GalA-OS fermentation induces highly structure-dependent changes in the gut microbiota, further expanding their potential use as prebiotics.

Microbiome metabolite quantification methods enabling insights into human health and disease

Many of the health-associated impacts of the microbiome are mediated by its chemical activity, producing and modifying small molecules (metabolites). Thus, microbiome metabolite quantification has a central role in efforts to elucidate and measure microbiome function. In this review, we cover general considerations when designing experiments to quantify microbiome metabolites, including sample preparation, data acquisition and data processing, since these are critical to downstream data quality. We then discuss data analysis and experimental steps to demonstrate that a given metabolite feature is of microbial origin. We further discuss techniques used to quantify common microbial metabolites, including short-chain fatty acids (SCFA), secondary bile acids (BAs), tryptophan derivatives, N-acyl amides and trimethylamine N-oxide (TMAO). Lastly, we conclude with challenges and future directions for the field.

Fabrication of silastic testosterone enanthate implants to achieve virilizing levels of serum testosterone in swine

The aim of this study was to create a continuous drug delivery system that would yield serum testosterone levels in sexually mature, female pigs consistent with serum testosterone levels of human transgender men. Testosterone enanthate was mixed with medical grade silicone at a ratio of 20 % by weight, placed in silastic tubing to cure at room temperature for 24 h, then removed from the tubing mold and stored at 4 °C until surgical implantation. Testosterone enanthate oxidizes at high temperatures which is why the implants had to be stored cold. Each implant was 9 cm long and contained 0.56 g of testosterone enanthate. A minimum of one implant (0.56 g testosterone enanthate) and a maximum of four implants (2.24 g testosterone enanthate) were placed in the cervical subcutaneous fat of each pig. After implantation, serum testosterone was assessed over 40 days. Silastic testosterone enanthate implants increase serum testosterone and maintain it in a relatively constant state in a dose-dependent manner for ∼ 21–25 days post-implantation.•Effective method for subcutaneous delivery of large quantities of testosterone or testosterone metabolites in compact implants to large animal biomedical model species.•Maintains increased circulating testosterone levels for up to 3, 4 weeks post-implantation in pigs.

Metabolomic approaches to explore chemodiversity in seeds of guaraná (Paullinia cupana) using UPLC-QTOF-MSE and NMR analysis.

The growing interest in health and well-being has spurred the evolution of functional foods, which provide enhanced health benefits beyond basic nutrition. Guaraná seeds (Paullinia cupana) have been widely studied and used as a functional food due to their richness in caffeine, phenolic compounds, amino acids, and other nutrients. This has established guaraná as a significant food supplement, with Brazil being the largest producer of the world. This study aims to propose a set of analytical methods to chemically evaluate fifty-six different guaraná clones, from the Guaraná Germplasm Active Bank, to accommodate the diverse requirements of the food industry. Metabolomic approaches were employed, in which a non-target metabolomic analysis via UPLC-QTOF-MSE led to the annotation of nineteen specialized metabolites. Furthermore, targeted metabolomics was also used, leading to the identification and quantification of metabolites by NMR. The extensive data generated were subjected to multivariate analysis, elucidating the similarities and differences between the evaluated guaraná seeds, particularly concerning the varying concentration levels of the metabolites. The metabolomics approach based on the combination of UPLC-QTOF-MSE, NMR and chemometric tools provided sensitivity, precision and accuracy to establish the chemical profiles of guaraná seeds. In conclusion, evaluating and determining the metabolic specificities of different guarana clones allow for their application in the development of products with different levels of specific metabolites, such as caffeine. This caters to various purposes within the food industry. Moreover, the recognized pharmacological properties of the annotated specialized metabolites affirm the use of guarana clones as an excellent nutritional source.

Microfluidic Cartridge for Bead-Based Affinity Assays.

Within the vast field of medical biotechnology, the biopharmaceutical industry is particularly fast-growing and highly competitive, so reducing time and costs associated to process optimization becomes instrumental to ensure speed to market and, consequently, profitability. The manufacturing of biopharmaceutical products, namely, monoclonal antibodies (mAbs), relies mostly on mammalian cell culture processes, which are highly dynamic and, consequently, difficult to optimize. In this context, there is currently an unmet need of analytical methods that can be integrated at-line in a bioreactor, for systematic monitoring and quantification of key metabolites and proteins. Microfluidic-based assays have been extensively and successfully applied in the field of molecular diagnostics; however, this technology remains largely unexplored for Process Analytical Technology (PAT), despite holding great potential for the at-line measurement of different analytes in bioreactor processes, combining low reagent/molecule consumption with assay sensitivity and rapid turnaround times.Here, the fabrication and handling of a microfluidic cartridge for protein quantification using bead-based affinity assays is described. The device allows geometrical multiplexed immunodetection of specific protein analytes directly from bioreactor samples within 2.5h and minimal hands-on time. As a proof-of-concept, quantification of Chinese hamster ovary (CHO) host cell proteins (HCP) as key impurities, IgG as product of interest, and lactate dehydrogenase (LDH) as cell viability marker was demonstrated with limits of detection (LoD) in the low ng/mL range. Negligible matrix interference and no cross-reactivity between the different immunoassays on chip were found. The results highlight the potential of the miniaturized analytical method for PAT at reduced cost and complexity in comparison with sophisticated instruments that are currently the state-of-the-art in this context.

Effects of media composition and light exposure on the electrochemical current response during scanning electrochemical microscopy live cell imaging.

Scanning Electrochemical Microscopy (SECM) has been used as a non-invasive electrochemical technique for studying cellular processes. SECM enables the quantification of cellular metabolites in real-time providing a deeper understanding of cellular responses to external stimuli. SECM imaging of living cells requires maintaining an ideal physiological environment to ensure reliable data collection on cellular reactivity. The cellular response can be directly influenced by physicochemical parameters including cell media composition, temperature and light exposure. This research demonstrates the effect of media composition on the electrochemical current signal of adenocarcinoma cervical cancer (HeLa) cells during SECM measurements using ferrocenemethanol as a redox mediator. Investigated media that are commonly used as electrolyte, are phosphate buffered saline (PBS), and Dulbecco's modified Eagle's medium (DMEM) in the absence and presence of fetal bovine serum (FBS). In addition, this research demonstrates that fluctuating light illumination impacts the stability of the cellular electrochemical current response. Our findings reveal that media composition and illumination are important parameters that must be carefully considered and monitored during SECM live cell imaging.

The impact of nitrogen fertilization on the alkaloid content and growth traits of Datura (Datura stramonium L.)

D. stramonium is a wild plant species belonging to the nightshade family Solanaceae and grows on the edges of cotton fields, empty lands and roadsides. The plant is one of the most important economic weed plants that contain both toxic and medicinal properties. Studies have shown that the plant contains toxic tropane alkaloids such as atropine, hyoscyamine, and scopolamine. It is known that cultivation techniques have a great influence on the quality and quantity of secondary metabolites. For this reason, it is important to determine the optimal values of the agronomic factors that influence plant growth and production. This study was conducted in the experimental area of the Agricultural Faculty of Dicle University in the 2010 and 2011 growing seasons so as to evaluate the impact of nitrogen fertilizer rates (0, 50, 100 and 200 kg ha-1) on some agronomical characteristics such as fresh and dry herb yield and total alkaloid content of Datura (Datura stramonium L.). The results revealed that significant (P?0.05) differences were observed between nitrogen doses in the 2010 and 2011 growing seasons in terms of seed yield, fresh and dry herb yield and total alkaloid yield of seeds. However, seed yield changed between 545 kg ha-1 and 625 kg ha-1, fresh herb yield between 8000 and 24483 kg ha-1, dry herb yield between 2190 kg ha-1 and 5083 kg ha-1 and alkaloid content between 0.259% and 0.366%, respectively. The results showed that fresh and dry herb yields increased with increasing nitrogen doses.

Unveiling Amaryllidaceae alkaloids: from biosynthesis to antiviral potential - a review.

Covering: 2017 to 2023 (now)Amaryllidaceae alkaloids (AAs) are a unique class of specialized metabolites containing heterocyclic nitrogen bridging that play a distinct role in higher plants. Irrespective of their diverse structures, most AAs are biosynthesized via intramolecular oxidative coupling. The complex organization of biosynthetic pathways is constantly enlightened by new insights owing to the advancement of natural product chemistry, synthetic organic chemistry, biochemistry, systems and synthetic biology tools and applications. These promote novel compound identification, trace-level metabolite quantification, synthesis, and characterization of enzymes engaged in AA catalysis, enabling the recognition of biosynthetic pathways. A complete understanding of the pathway benefits biotechnological applications in the long run. This review emphasizes the structural diversity of the AA specialized metabolites involved in biogenesis although the process is not entirely defined yet. Moreover, this work underscores the pivotal role of synthetic and enantioselective studies in justifying biosynthetic conclusions. Their prospective candidacy as lead constituents for antiviral drug discovery has also been established. However, a complete understanding of the pathway requires further interdisciplinary efforts in which antiviral studies address the structure-activity relationship. This review presents current knowledge on the topic.

A circular network of adenosine-mediated mitochondrial dysfunction as coregulators of acute myocardial infarction.

This study aims to explore the molecular mechanisms and associated pathways of myocardial infarction (MI). We employed a variety of analytical methods, including Mendelian Randomization (MR) analysis, transcriptome microarray data analysis, gene function and pathway enrichment analysis, untargeted metabolomic mass spectrometry analysis, and gene-metabolite interaction network analysis. The MR analysis results revealed a significant impact of mitochondrial DNA copy number on MI and coronary artery bypass grafting. Transcriptome analysis unveiled numerous differentially expressed genes associated with myocardial ischemia, with enrichment observed in cardiac function and energy metabolism pathways. Metabolomic analysis indicated a significant downregulation of mitochondrial regulation pathways in ischemic myocardium. T500 metabolite quantification analysis identified 90 differential metabolites between MI and Sham groups, emphasizing changes in metabolites associated with energy metabolism. Gene-metabolite interaction network analysis revealed the significant roles of key regulatory molecules such as HIF1A, adenosine, TBK1, ATP, NRAS, and EIF2AK3, in the pathogenesis of myocardial ischemia. In summary, this study provides important insights into the molecular mechanisms of MI and highlights interactions at multiple molecular levels, contributing to the establishment of new theoretical foundations for the diagnosis and treatment of MI.

A new adsorptive 3D–printed sampling device for simultaneous determination of 63 urinary organic acids by LC–MS/MS

BackgroundThe detection and quantification of urinary metabolites play an important role in disease diagnosis. In most cases, urinary analyses are done with liquid urine samples, which must be quickly transported to the laboratory to avoid metabolites degradation that is associated with temperature fluctuations. Consequently, dried sampling devices have emerged to minimize analyte degradation. However, most commercial dried sampling devices are expensive, aggregate low volumes, and need better analytical sensitivity. Therefore, a new dry urine sampling device that is inexpensive, suitable for domestic sampling operation, and efficient for quantifying metabolites without requiring high–resolution instruments is proposed in the present study. ResultsThe newly designed dry urine sampling device was produced by 3D printing that efficiently determines 63 urinary organic acids using liquid chromatography coupled with mass spectrometry (LC–MS/MS). The system's efficiency was demonstrated with analytical figures of merit, such as precision, accuracy, and stability of analytes after the sampling and storing of ordinary urine samples. The limits of quantification ranged from 0.01 to 0.42 ng mL–1. Precision and accuracy tests showed relative standard deviations of less than 15 %. The urine stability in the sampling device was high within seven days without any significant degradation of the metabolites. The method was applied to the analysis of 10 human urine samples and compared to a conventional method without the use of the sampling device. The results showed no statistically significant differences, demonstrating the method's efficiency. SignificanceThe proposed 3–D printing device was developed with fast, low–cost manufacturing features and can be manufactured with different volumetric capacities, adaptable to the needs of each user. Furthermore, it is innovative because this is the first sampling device that is effective for the simultaneous storage and preservation of several important urinary metabolites. Thus, it is anticipated that its application would contribute significantly to the identification of metabolic disorders.

Telomere-to-Telomere Genome Assembly of Tibetan Medicinal Mushroom Ganoderma leucocontextum and the First Copia Centromeric Retrotransposon in Macro-Fungi Genome.

A complete telomere-to-telomere (T2T) genome has been a longstanding goal in the field of genomic research. By integrating high-coverage and precise long-read sequencing data using multiple assembly strategies, we present here the first T2T gap-free genome assembly of Ganoderma leucocontextum strain GL72, a Tibetan medicinal mushroom. The T2T genome, with a size of 46.69 Mb, consists 13 complete nuclear chromosomes and typical telomeric repeats (CCCTAA)n were detected at both ends of 13 chromosomes. The high mapping rate, uniform genome coverage, a complete BUSCOs of 99.7%, and base accuracy exceeding 99.999% indicate that this assembly represents the highest level of completeness and quality. Regions characterized by distinct structural attributes, including highest Hi-C interaction intensity, high repeat content, decreased gene density, low GC content, and minimal or no transcription levels across all chromosomes may represent potential centromeres. Sequence analysis revealed the first Copia centromeric retrotransposon in macro-fungi genome. Phylogenomic analysis identified that G. leucocontextum and G. tsugae diverged from the other Ganoderma species approximately 9.8-17.9 MYA. The prediction of secondary metabolic clusters confirmed the capability of this fungus to produce a substantial quantity of metabolites. This T2T gap-free genome will contribute to the genomic 'dark matter' elucidation and server as a great reference for genetics, genomics, and evolutionary studies of G. leucocontextum.

Extended automated quantification algorithm (AQuA) for targeted 1H NMR metabolomics of highly complex samples: application to plant root exudates

IntroductionThe Automated Quantification Algorithm (AQuA) is a rapid and efficient method for targeted NMR-based metabolomics, currently optimised for blood plasma. AQuA quantifies metabolites from 1D-1H NMR spectra based on the height of only one signal per metabolite, which minimises the computational time and workload of the method without compromising the quantification accuracy.ObjectivesTo develop a fast and computationally efficient extension of AQuA for quantification of selected metabolites in highly complex samples, with minimal prior sample preparation. In particular, the method should be capable of handling interferences caused by broad background signals.MethodsAn automatic baseline correction function was combined with AQuA into an automated workflow, the extended AQuA, for quantification of metabolites in plant root exudate NMR spectra that contained broad background signals and baseline distortions. The approach was evaluated using simulations as well as a spike-in experiment in which known metabolite amounts were added to a complex sample matrix.ResultsThe extended AQuA enables accurate quantification of metabolites in 1D-1H NMR spectra with varying complexity. The method is very fast (< 1 s per spectrum) and can be fully automated.ConclusionsThe extended AQuA is an automated quantification method intended for 1D-1H NMR spectra containing broad background signals and baseline distortions. Although the method was developed for plant root exudates, it should be readily applicable to any NMR spectra displaying similar issues as it is purely computational and applied to NMR spectra post-acquisition.

Improving quality of analysis by suppression of unwanted signals through band-selective excitation in NMR spectroscopy for metabolomics studies.

Nuclear Magnetic Resonance (NMR) spectroscopy stands as a preeminent analytical tool in the field of metabolomics. Nevertheless, when it comes to identifying metabolites present in scant amounts within various types of complex mixtures such as plants, honey, milk, and biological fluids and tissues, NMR-based metabolomics presents a formidable challenge. This predicament arises primarily from the fact that the signals emanating from metabolites existing in low concentrations tend to be overshadowed by the signals of highly concentrated metabolites within NMR spectra. The aim of this study is to tackle the issue of intense sugar signals overshadowing the desired metabolite signals, an optimal pulse sequence with band-selective excitation has been proposed for the suppression of sugar's moiety signals (SSMS). This sequence serves the crucial purpose of suppressing unwanted signals, with a particular emphasis on mitigating the interference caused by sugar moieties' signals. We have implemented this comprehensive approach to various NMR techniques, including 1D 1H presaturation (presat), 2D J-resolved (RES), 2D 1H-1H Total Correlation Spectroscopy (TOCSY), and 2D 1H-13C Heteronuclear Single Quantum Coherence (HSQC) for the samples of dates-flesh, honey, a standard stock solution of glucose, and nine amino acids, and commercial fetal bovine serum (FBS). The outcomes of this approach were significant. The suppression of the high-intensity sugar signals has considerably enhanced the visibility and sensitivity of the signals emanating from the desired metabolites. This, in turn, enables the identification of a greater number of metabolites. Additionally, it streamlines the experimental process, reducing the time required for the comparative quantification of metabolites in statistical studies in the field of metabolomics.

Various cold storage-backslopping cycles show the robustness of Limosilactobacillus fermentum IMDO 130101 as starter culture for Type 3 sourdough production

Type 3 sourdoughs, which are starter culture-initiated and subsequently backslopped, are less studied than other sourdough types. Yet, they can serve as a model to assess how competitive starter culture strains for sourdough production are and how the microbial composition of such sourdoughs may evolve over time. In the present study, Limosilactobacillus fermentum IMDO 130101 was used to produce Type 3 sourdoughs, prepared from wheat and wholemeal wheat flours. Therefore, an initial fermentation of the flour-water mixture was performed at 30 °C for 48 h. This was followed by cold storage-backslopping cycles, consisting of refreshments (50 %, v/v), fermentation steps of 16 h, and storage at 4 °C each week, every three weeks, and every six weeks. The microbial dynamics (culture-dependent and -independent approaches) and metabolite dynamics were measured. In all sourdoughs produced, starter culture strain monitoring, following an amplicon sequence variant approach, showed that Liml. fermentum IMDO 130101 prevailed during one month when the sourdoughs were refreshed each week, during 24 weeks when the sourdoughs were refreshed every three weeks, and during 12 weeks when the sourdoughs were refreshed every six weeks. This suggested the competitiveness and robustness of Liml. fermentum IMDO 130101 for a considerable duration but also showed that the strain is prone to microbial interference. For instance, Levilactobacillus brevis and Pediococcus spp. prevailed upon further cold storage and backslopping. Also, although no yeasts were inoculated into the flour-water mixtures, Kazachstania unispora, Torulaspora delbrueckii, and Wickerhamomyces anomalus were the main yeast species found. They appeared after several weeks of storage and backslopping, which however indicated the importance of an interplay between LAB and yeast species in sourdoughs. The main differences among the mature sourdoughs obtained could be explained by the different flours used, the refreshment conditions applied, and the sampling time (before and after backslopping). Finally, the metabolite quantifications revealed continued metabolite production during the cold storage periods, which may impact the sourdough properties and those of the breads made thereof.

Quantification of main secondary metabolites of Satureja icarica P.H. Davis (Lamiaceae) by LC-HRMS and evaluation of antioxidant capacities

Quantification of main secondary metabolites of Satureja icarica P.H. Davis (Lamiaceae) by LC-HRMS and evaluation of antioxidant capacities

Quantification of glucocorticoid and progestogen metabolites in bovine plasma, skimmed milk and saliva by UHPLC-HR-MS with polarity switching

Steroid metabolites are increasingly in focus when searching for novel biomarkers in physiological mechanisms and their disorders. While major steroids such as progesterone and cortisol are well-researched and routinely determined to assess the health, particularly the reproductive status of mammals, the function of potentially biologically active progestogen and glucocorticoid metabolites is widely unexplored. One of the main reasons for this is the lack of comprehensive, sensitive, and specific analytical methods. This is particularly the case when analyzing matrices like milk or saliva obtained by non-invasive sampling with steroid concentrations often below those present in plasma. Therefore, a new UHPLC-HR-MS method based on an Ultimate UHPLC system equipped with an Acquity HSS T3 reversed-phase column and a Q Exactive™ mass spectrometer was developed, enabling the simultaneous chromatographic separation, detection and quantification of eleven isobaric glucocorticoids (11-dehydrocorticosterone (A), corticosterone (B), cortisol (F), cortisone (E), the tetrahydrocortisols (THF): 3α,5α-THF, 3α,5β-THF, 3β,5α-THF, 3β,5β-THF, and the tetrahydrocortisones (THE): 3α,5α-THE, 3α,5β-THE, 3β,5α-THE) and twelve progestogens (progesterone (P4), pregnenolone (P5), the dihydroprogesterones (DHP): 20α-DHP, 20β-DHP, 3α-DHP, 3β-DHP, 5α-DHP, 5β-DHP, and the tetrahydroprogesterones (THP): 3α,5α-THP, 3α,5β-THP, 3β,5α-THP, 3β,5β-THP) in bovine plasma, skimmed milk, and saliva. A simple liquid-liquid extraction (LLE) with MTBE (methyl tert-butyl ether) was used for sample preparation of 500 μL plasma, skimmed milk, and saliva. Heated electrospray ionization (HESI) with polarity switching was applied to analyze steroids in high-resolution full scan mode (HR-FS). The method validation covered the investigation of sensitivity, selectivity, curve fitting, carry-over, accuracy, precision, recovery, matrix effects and applicability. A high sensitivity in the range of pg mL−1 was achieved for all steroids suitable for the analysis of authentic samples.

Multimodal assessment of mitochondrial function in Parkinson's disease.

The heterogenous aetiology of Parkinson's disease is increasingly recognized; both mitochondrial and lysosomal dysfunction have been implicated. Powerful, clinically applicable tools are required to enable mechanistic stratification for future precision medicine approaches. The aim of this study was to characterize bioenergetic dysfunction in Parkinson's disease by applying a multimodal approach, combining standardized clinical assessment with midbrain and putaminal 31-phosphorus magnetic resonance spectroscopy (31P-MRS) and deep phenotyping of mitochondrial and lysosomal function in peripheral tissue in patients with recent-onset Parkinson's disease and control subjects. Sixty participants (35 patients with Parkinson's disease and 25 healthy controls) underwent 31P-MRS for quantification of energy-rich metabolites [ATP, inorganic phosphate (Pi) and phosphocreatine] in putamen and midbrain. In parallel, skin biopsies were obtained from all research participants to establish fibroblast cell lines for subsequent quantification of total intracellular ATP and mitochondrial membrane potential (MMP) as well as mitochondrial and lysosomal morphology, using high content live cell imaging. Lower MMP correlated with higher intracellular ATP (r = -0.55, P = 0.0016), higher mitochondrial counts (r = -0.72, P < 0.0001) and higher lysosomal counts (r = -0.62, P = 0.0002) in Parkinson's disease patient-derived fibroblasts only, consistent with impaired mitophagy and mitochondrial uncoupling. 31P-MRS-derived posterior putaminal Pi/ATP ratio variance was considerably greater in Parkinson's disease than in healthy controls (F-tests, P = 0.0036). Furthermore, elevated 31P-MRS-derived putaminal, but not midbrain Pi/ATP ratios (indicative of impaired oxidative phosphorylation) correlated with both greater mitochondrial (r = 0.37, P = 0.0319) and lysosomal counts (r = 0.48, P = 0.0044) as well as lower MMP in both short (r = -0.52, P = 0.0016) and long (r = -0.47, P = 0.0052) mitochondria in Parkinson's disease. Higher 31P-MRS midbrain phosphocreatine correlated with greater risk of rapid disease progression (r = 0.47, P = 0.0384). Our data suggest that impaired oxidative phosphorylation in the striatal dopaminergic nerve terminals exceeds mitochondrial dysfunction in the midbrain of patients with early Parkinson's disease. Our data further support the hypothesis of a prominent link between impaired mitophagy and impaired striatal energy homeostasis as a key event in early Parkinson's disease.

A simple and convenient synthesis of labeled 9-hydroxy[13C6]phenanthrene as internal standard for mass spectrometry quantification of a key phenanthrene metabolite in human urines

A simple and convenient synthesis of 9-hydroxy[13C6]phenanthrene has been developed from one-ring labeled [13C6]phenanthrene in three steps, involving a copper-ethyl acetate catalyzed “Ullmann-type” reaction. The mild conditions of this nucleophilic aromatic substitution are particularly advantageous in the case of expensive labeled material. Stable-labeled 9-hydroxy[13C6]phenanthrene has proven to be an ideal [M + 6]isotopomer for use as an internal standard for quantification of OH-polycyclic aromatic hydrocarbons by gas chromatography with tandem mass spectrometry (GC-MS/MS) in human urines.

Essential oil profiles, improvement of enzymatic and non-enzymatic antioxidant systems in different populations of Salvia subg. Perovskia by irrigation management

Salvia subg. Perovskia is an aromatic medicinal plant from the Lamiaceae family. Essential oil (EO) content and composition, along with enzymatic and non-enzymatic antioxidants, were evaluated in 18 plant populations under three levels of irrigation for two consecutive years. Based on the GC-MS analysis, the main components of EO were borneol (1.18–36.53%), followed by camphor (0.54–32.17%), 1,8-cineole (12.44–29.26%), δ-3-carene (0.39–21.20%), myrcene (0.59–16.28%), and α- pinene (0.79–12.87%) in the studied treatment. Except for malondialdehyde and hydrogen peroxide in leaves and roots, all the examined parameters showed lower values in the first harvest year compared to those of the second year. The activities of antioxidant enzymes, total phenolics, and flavonoids of the extracts were substantially enhanced as stress intensified to reach maximum values in leaves under severe stress and in roots under moderate stress conditions. The maximum root tanshinones content was observed under moderate water deficit conditions, while the highest EO content was obtained in plants exposed to severe stress conditions. Taking into account the evidence provided by this study, it can be inferred that the use of water deficit stress can serve as an effective method to stimulate and improve antioxidant properties, as well as the quantity and quality of secondary metabolites in Salvia subg. Perovskia.