N-acetylglucosamine supplementation fails to bypass the critical acetylation of glucosamine-6-phosphate required for Toxoplasma gondii replication and invasion.

The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). The glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositols (GPIs), leading to impairment of the parasite's ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these vital functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc-supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the importance of glucosamine-6-phosphate acetylation in governing T. gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies.

Hyphenation of microflow chromatography with electrospray ionization mass spectrometry for bioanalytical applications focusing on low molecular weight compounds: A tutorial review.

Benefits of miniaturized chromatography with various detection modes, such as increased sensitivity, chromatographic efficiency, and speed, were recognized nearly 50 years ago. Over the past two decades, this approach has experienced rapid growth, driven by the emergence of mass spectrometry applications serving -omics sciences and the need for analyzing minute volumes of precious samples with ever higher sensitivity. While nanoscale liquid chromatography (flow rates <1 µL/min) has gained widespread recognition in proteomics, the adoption of microscale setups (flow rates ranging from 1 to 100 µL/min) for low molecular weight compound applications, including metabolomics, has been surprisingly slow, despite the inherent advantages of the approach. Highly heterogeneous matrices and chemical structures accompanied by a relative lack of options for both selective sample preparation and user-friendly equipment are usually reported as major hindrances. To facilitate the wider implementation of microscale analyses, we present here a comprehensive tutorial encompassing important theoretical and practical considerations. We provide fundamental principles in micro-chromatography and guide the reader through the main elements of a microflow workflow, from LC pumps to ionization devices. Finally, based on both our literature overview and experience, illustrated by some in-house data, we highlight the critical importance of the ionization source design and its careful optimization to achieve significant sensitivity improvement.

Substantial benefits of an inert biphenyl column for the analysis of steroids and their phase II metabolites in biological samples.

Steroids can be used as biomarkers in clinical metabolomics and other fields related to human toxicology. This chemical group is known for its complexity, considering its number of isobaric compounds and the wide variety of phases I and II metabolic pathways that parent compounds can undergo. For a successful analysis of steroids in biological samples, liquid chromatography separation must be finely tuned. It is especially challenging for glucuronidated and sulfated steroids derivatives that bear polar heads and can be affected by non-specific adsorption. The benefits of a biphenyl stationary phase chemistry for the selectivity of the separation of steroids and their phase II metabolites and the extent to which nonspecific adsorption phenomena could degrade chromatographic performance were investigated. Replacing a conventional hardware by a passivated hardware allowed to considerably reduce peaks width and asymmetry of sulfated species. The addition of weak ion pairing agents in the mobile phase could also help to reduce non-specific adsorption but are detrimental to mass spectrometry detection. As confirmed by the successful detection of 52 steroids in plasma, the use of a biphenyl stationary phase complemented by a passivated column hardware is of great help for a successful biomedical analysis of steroids and their phase II metabolites.

Assessment of the surface contamination of the primary packaging of oral antineoplastic drugs and secondary packaging of chemotherapy preparations at a Swiss hospital.

INTRODUCTION: Due to the high toxicity of antineoplastic drugs, handling their packaging could lead to the chemical contamination of hospital environments and exposure risks to healthcare professionals and patients. This study aimed to assess the contamination of two main surfaces: the outer primary packaging of oral antineoplastic drug formulations (n = 36) available on the Swiss market and the surface of secondary packaging of injectable antineoplastic drug preparations (n = 60) produced by the pharmacy of a Swiss hospital and carriers used for transport (n = 5). METHODS: Samples were collected using a validated wipe sampling method. The simultaneous analysis of 24 antineoplastic drugs: 5-fluorouracil, busulfan, carboplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, gemcitabine, idarubicin, ifosfamide, irinotecan, methotrexate, oxaliplatin, paclitaxel, pemetrexed, raltitrexed, topotecan, treosulfan, vinblastine, vincristine) and 1 antiviral compound (ganciclovir) was performed by UHPLC-MS/MS. RESULTS: A total of 58% and 90% positive results were obtained for the primary packaging of oral chemotherapies and for the secondary packaging of injectable preparations, respectively. The highest quantities found on the primary packaging for oral chemotherapies and on the surface of closed leak-proof bags were 111 ng of methotrexate and 19 ng of gemcitabine, respectively. Gemcitabine (69%) and cyclophosphamide (38%) were the two most common contaminants found on the packaging of injectable preparations and carriers, regardless of the chemotherapy preparations. CONCLUSION: Trace levels (ng) of antineoplastic drugs can be found on most surfaces of all evaluated pharmaceutical products. Thus, suitable personal protective equipment is mandatory for healthcare professional handling antineoplastic drugs.

Multitargeted Internal Calibration for the Quantification of Chronic Kidney Disease-Related Endogenous Metabolites Using Liquid Chromatography-Mass Spectrometry.

Accurate quantitative analysis in liquid chromatography-mass spectrometry (LC-MS) benefits from calibration curves generated in the same matrix as the study sample. In the case of endogenous compound quantification, as no blank matrix exists, the multitargeted internal calibration (MTIC) is an attractive and straightforward approach to avoid the need for extensive matrix similarity evaluation. Its principle is to take advantage of stable isotope labeled (SIL) standards as internal calibrants to simultaneously quantify authentic analytes using a within sample calibration. An MTIC workflow was developed for the simultaneous quantification of metabolites related to chronic kidney disease (CKD) using a volumetric microsampling device to collect 20 µL of serum or plasma, followed by a single-step extraction with acetonitrile/water and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Since a single concentration of internal calibrant is necessary to calculate the study sample concentration, the instrument response function was investigated to determine the best SIL concentration. After validation, the trueness of 16 endogenous analytes in authentic human serum ranged from 72.2 to 116.0%, the repeatability from 1.9 to 11.3%, and the intermediate precision ranged overall from 2.1 to 15.4%. The proposed approach was applied to plasma samples collected from healthy control participants and two patient groups diagnosed with CKD. Results confirmed substantial concentration differences between groups for several analytes, including indoxyl sulfate and cortisone, as well as metabolite enrichment in the kynurenine and indole pathways. Multitargeted methodologies represent a major step toward rapid and straightforward LC-MS/MS absolute quantification of endogenous biomarkers, which could change the paradigm of MS use in clinical laboratories.

Low-polarity untargeted metabolomic profiling as a tool to gain insight into seminal fluid.

INTRODUCTION: A decrease in sperm cell count has been observed along the last several decades, especially in the most developed regions of the world. The use of metabolomics to study the composition of the seminal fluid is a promising approach to gain access to the molecular mechanisms underlying this fact. OBJECTIVES: In the present work, we aimed at relating metabolomic profiles of young healthy men to their semen quality parameters obtained from conventional microscopic analysis. METHODS: An untargeted metabolomics approach focusing on low- to mid-polarity compounds was used to analyze a subset of seminal fluid samples from a cohort of over 2700 young healthy men. RESULTS: Our results show that a broad metabolic profiling comprising several families of compounds (including acyl-carnitines, steroids, and other lipids) can contribute to effectively distinguish samples provided by individuals exhibiting low or high absolute sperm counts. CONCLUSION: A number of metabolites involved in sexual development and function, signaling, and energy metabolism were highlighted as being distinctive of samples coming from either group, proving untargeted metabolomics as a promising tool to better understand the pathophysiological processes responsible for male fertility impairment.

Network principal component analysis: a versatile tool for the investigation of multigroup and multiblock datasets.

MOTIVATION: Complex data structures composed of different groups of observations and blocks of variables are increasingly collected in many domains, including metabolomics. Analysing these high-dimensional data constitutes a challenge, and the objective of this article is to present an original multivariate method capable of explicitly taking into account links between data tables when they involve the same observations and/or variables. For that purpose, an extension of standard principal component analysis called NetPCA was developed. RESULTS: The proposed algorithm was illustrated as an efficient solution for addressing complex multigroup and multiblock datasets. A case study involving the analysis of metabolomic data with different annotation levels and originating from a chronic kidney disease (CKD) study was used to highlight the different aspects and the additional outputs of the method compared to standard PCA. On the one hand, the model parameters allowed an efficient evaluation of each group's influence to be performed. On the other hand, the relative relevance of each block of variables to the model provided decisive information for an objective interpretation of the different metabolic annotation levels. AVAILABILITY AND IMPLEMENTATION: NetPCA is available as a Python package with NumPy dependencies. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Challenges in ESI-MS-based Untargeted Metabolomics.

Untargeted metabolomics is now widely recognized as a useful tool for exploring metabolic changes taking place in biological systems under different conditions. In this article, we aim to provide a short overview of the liquid-phase separation methods hyphenated to MS to perform untargeted metabolomics of biological samples. Each approach is complemented by up-to-date literature to guide readers, as well as practical information for avoiding or fixing some of the most frequently encountered pitfalls. This article covers mainly data acquisition, but a short discussion is provided regarding signal processing and data treatment, as well as data analysis and its biological interpretation in the context of metabolomic studies.

Capillary Electrophoresis Instruments for Medical Applications and Falsified Drug Analysis/Quality Control in Developing Countries.

The implementation of integrated analytical techniques to meet stringent requirements in the life sciences requires a well-developed analytical capacity. New technology in analytical equipment for the analysis of large and small molecules is continuously being developed. However, developing countries frequently struggle to keep pace with technological advancements. Hence, it is of utmost importance to better invest in optimizing existing and proven methodologies to tackle life-saving challenges in developing countries. In this regard, capillary electrophoresis is a promising candidate for solving multiple analytical problems compared to its chromatographic and spectroscopic counterparts due to its fast analytical response time and notable cost efficiency. In the following, we summarize various issues and opportunities for capillary electrophoresis to be the technique of choice for the unresolved bottlenecks in analytical equipment in developing countries for drug quality control. This perspective demonstrates that the ongoing quest for the design of new, impactful analytical techniques is a dynamic and rapidly developing research area and mentions some directions and opportunities that have arisen during the recent pandemic.

Evaluation of ion mobility in capillary electrophoresis coupled to mass spectrometry for the identification in metabolomics.

Currently, feature annotation remains one of the main challenges in untargeted metabolomics. In this context, the information provided by high-resolution mass spectrometry (HRMS) in addition to accurate mass can improve the quality of metabolite annotation, and MS/MS fragmentation patterns are widely used. Accurate mass and a separation index, such as retention time or effective mobility (µeff ), in chromatographic and electrophoretic approaches, respectively, must be used for unequivocal metabolite identification. The possibility of measuring collision cross-section (CCS) values by using ion mobility (IM) is becoming increasingly popular in metabolomic studies thanks to the new generation of IM mass spectrometers. Based on their similar separation mechanisms involving electric field and the size of the compounds, the complementarity of DT CCSN2 and µeff needs to be evaluated. In this study, a comparison of DT CCSN2 and µeff was achieved in the context of feature identification ability in untargeted metabolomics by capillary zone electrophoresis (CZE) coupled with HRMS. This study confirms the high correlation of DT CCSN2 with the mass of the studied metabolites as well as the orthogonality between accurate mass and µeff , making this combination particularly interesting for the identification of several endogenous metabolites. The use of IM-MS remains of great interest for facilitating the annotation of neutral metabolites present in the electroosmotic flow (EOF) that are poorly or not separated by CZE.

New insights into the conversion of electropherograms to the effective electrophoretic mobility scale.

CE-MS is increasingly gaining momentum as an analytical tool in metabolomics, due to its ability to obtain information about the most polar elements in biological samples. This has been helped by improvements of robustness in peak identification by means of mobility-scale representations of the electropherograms (mobilograms). As a necessary step toward facilitating the use of CE-MS for untargeted metabolomics data, the authors previously developed and introduced ROMANCE, a software automating mobilogram generation for large untargeted datasets through a simple and self-contained user interface. Herein, we introduce a new version of ROMANCE including new features such as compatibility with other types of data (targeted MS data and 2D UV-Vis absorption-like electropherograms), and the much needed additional flexibility in the transformation parameters (including field ramping and the use of secondary markers), more measurement conditions (depending on detection and integration modes), and most importantly tackling the issue of quantitative peak conversion. First, we present a review of the current theoretical framework with regard to peak characterization, and we develop new formulas for multiple marker peak area corrections, for anticipating peak position precision, and for assessing peak shape distortion. Then, the new version of the software is presented and validated experimentally. We contrast the multiple marker mobility transformations with previous results, finding increased peak position precision, and finally we showcase an application to actual untargeted metabolomics data.

Determination of antiretroviral drugs for buyers' club in Switzerland using capillary electrophoresis methods.

Human immunodeficiency virus-acquired immunodeficiency syndrome continues to be a major global public health issue, having claimed almost 33 million lives to date. Due to the high cost of antiretroviral treatment, access to these drugs remains difficult for vulnerable populations, such as migrants and people living in prisons, who often do not have health insurance. These factors lead to poorer health outcomes and higher transmission rates. The personal importation scheme for unapproved generics from foreign countries is one option to access affordable human immunodeficiency virus treatment. However, the risk of importing falsified medicine remains high, and the quality control of unapproved drugs is lacking. In this context, three CE methods for the analysis of nine antiviral drugs found in commercial pharmaceutical formulations were evaluated. The selected compounds were emtricitabine, tenofovir disoproxil, tenofovir alafenamide, rilpivirine, efavirenz, raltegravir, dolutegravir, abacavir, and lamivudine. The developed methods were successfully applied to determine the active pharmaceutical ingredients of commercial formulations and unapproved generics. The quality control of unapproved generics by CE is an attractive approach due to its good standard of quality, low cost, ecofriendliness, and ease of implementation.

Approaches in metabolomics for regulatory toxicology applications.

Innovative methodological approaches are needed to conduct human health and environmental risk assessments on a growing number of marketed chemicals. Metabolomics is progressively proving its value as an efficient strategy to perform toxicological evaluations of new and existing substances, and it will likely become a key tool to accelerate chemical risk assessments. However, additional guidance with widely accepted and harmonized procedures is needed before metabolomics can be routinely incorporated in decision-making for regulatory purposes. The aim of this review is to provide an overview of metabolomic strategies that have been successfully employed in toxicity assessment as well as the most promising workflows in a regulatory context. First, we provide a general view of the different steps of regulatory toxicology-oriented metabolomics. Emphasis is put on three key elements: robustness of experimental design, choice of analytical platform, and use of adapted data treatment tools. Then, examples in which metabolomics supported regulatory toxicology outputs in different scenarios are reviewed, including chemical grouping, elucidation of mechanisms of toxicity, and determination of points of departure. The overall intention is to provide insights into why and how to plan and conduct metabolomic studies for regulatory toxicology purposes.

Progress towards an OECD reporting framework for transcriptomics and metabolomics in regulatory toxicology.

Omics methodologies are widely used in toxicological research to understand modes and mechanisms of toxicity. Increasingly, these methodologies are being applied to questions of regulatory interest such as molecular point-of-departure derivation and chemical grouping/read-across. Despite its value, widespread regulatory acceptance of omics data has not yet occurred. Barriers to the routine application of omics data in regulatory decision making have been: 1) lack of transparency for data processing methods used to convert raw data into an interpretable list of observations; and 2) lack of standardization in reporting to ensure that omics data, associated metadata and the methodologies used to generate results are available for review by stakeholders, including regulators. Thus, in 2017, the Organisation for Economic Co-operation and Development (OECD) Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) launched a project to develop guidance for the reporting of omics data aimed at fostering further regulatory use. Here, we report on the ongoing development of the first formal reporting framework describing the processing and analysis of both transcriptomic and metabolomic data for regulatory toxicology. We introduce the modular structure, content, harmonization and strategy for trialling this reporting framework prior to its publication by the OECD.

Neuroinflammatory Response to TNFα and IL1ß Cytokines Is Accompanied by an Increase in Glycolysis in Human Astrocytes In Vitro.

Astrogliosis has been abundantly studied in rodents but relatively poorly in human cells due to limited access to the brain. Astrocytes play important roles in cerebral energy metabolism, and are also key players in neuroinflammation. Astroglial metabolic and inflammatory changes as a function of age have been reported, leading to the hypothesis that mitochondrial metabolism and inflammatory responses are interconnected in supporting a functional switch of astrocytes from neurotrophic to neurotoxic. This study aimed to explore the metabolic changes occurring in astrocytes during their activation. Astrocytes were derived from human ReN cell neural progenitors and characterized. They were activated by exposure to tumor necrosis factor alpha (TNFα) or interleukin 1ß (IL1ß) for 24 h. Astrocyte reaction and associated energy metabolic changes were assessed by immunostaining, gene expression, proteomics, metabolomics and extracellular flux analyses. ReN-derived astrocytes reactivity was observed by the modifications of genes and proteins linked to inflammation (cytokines, nuclear factor-kappa B (NFκB), signal transducers and activators of transcription (STATs)) and immune pathways (major histocompatibility complex (MHC) class I). Increased NFκB1, NFκB2 and STAT1 expression, together with decreased STAT3 expression, suggest an activation towards the detrimental pathway. Strong modifications of astrocyte cytoskeleton were observed, including a glial fibrillary acidic protein (GFAP) decrease. Astrogliosis was accompanied by changes in energy metabolism characterized by increased glycolysis and lactate release. Increased glycolysis is reported for the first time during human astrocyte activation. Astrocyte activation is strongly tied to energy metabolism, and a possible association between NFκB signaling and/or MHC class I pathway and glycolysis is suggested.

Comprehensive Examination of the Mouse Lung Metabolome Following Mycobacterium tuberculosis Infection Using a Multiplatform Mass Spectrometry Approach.

The mechanisms whereby Mycobacterium tuberculosis (Mtb) rewires the host metabolism in vivo are surprisingly unexplored. Here, we used three high-resolution mass spectrometry platforms to track altered lung metabolic changes associated with Mtb infection of mice. The multiplatform data sets were merged using consensus orthogonal partial least squares-discriminant analysis (cOPLS-DA), an algorithm that allows for the joint interpretation of the results from a single multivariate analysis. We show that Mtb infection triggers a temporal and progressive catabolic state to satisfy the continuously changing energy demand to control infection. This causes dysregulation of metabolic and oxido-reductive pathways culminating in Mtb-associated wasting. Notably, high abundances of trimethylamine-N-oxide (TMAO), produced by the host from the bacterial metabolite trimethylamine upon infection, suggest that Mtb could exploit TMAO as an electron acceptor under anaerobic conditions. Overall, these new pathway alterations advance our understanding of the link between Mtb pathogenesis and metabolic dysregulation and could serve as a foundation for new therapeutic intervention strategies. Mass spectrometry data has been deposited in the Metabolomics Workbench repository (data-set identifier: ST001328).

Interlaboratory and Interplatform Study of Steroids Collision Cross Section by Traveling Wave Ion Mobility Spectrometry.

Collision cross section (CCS) databases based on single-laboratory measurements must be cross-validated to extend their use in peak annotation. This work addresses the validation of the first comprehensive TWCCSN2 database for steroids. First, its long-term robustness was evaluated (i.e., a year and a half after database generation; Synapt G2-S instrument; bias within ±1.0% for 157 ions, 95.7% of the total ions). It was further cross-validated by three external laboratories, including two different TWIMS platforms (i.e., Synapt G2-Si and two Vion IMS QToF; bias within the threshold of ±2.0% for 98.8, 79.9, and 94.0% of the total ions detected by each instrument, respectively). Finally, a cross-laboratory TWCCSN2 database was built for 87 steroids (142 ions). The cross-laboratory database consists of average TWCCSN2 values obtained by the four TWIMS instruments in triplicate measurements. In general, lower deviations were observed between TWCCSN2 measurements and reference values when the cross-laboratory database was applied as a reference instead of the single-laboratory database. Relative standard deviations below 1.5% were observed for interlaboratory measurements (<1.0% for 85.2% of ions) and bias between average values and TWCCSN2 measurements was within the range of ±1.5% for 96.8% of all cases. In the context of this interlaboratory study, this threshold was also suitable for TWCCSN2 measurements of steroid metabolites in calf urine. Greater deviations were observed for steroid sulfates in complex urine samples of adult bovines, showing a slight matrix effect. The implementation of a scoring system for the application of the CCS descriptor in peak annotation is also discussed.

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

[Buyers' club†: an alternative for access to treatment†?] / Buyers' club†: une alternative pour l'accès au traitement†?

Rapid medication management for patients infected with HIV, HCV or HBV is key in optimizing a more favourable clinical response, in terms of morbidity, mortality, quality-of-life and reduced risk of transmission. If a drug is expensive, access to treatment for an uninsured patient with limited resources can be a hurdle that leads to forgoing healthcare for economic reasons. The buyers' club's objective is to provide logistics and/or financial assistance to a patient aiming to import qualitative generics for his personal use at an affordable price oversea. The drug is purchased on the internet.

La prise en charge médicamenteuse rapide pour les patients infectés par le VIH, le VHC ou le VHB est un élément clé pour obtenir une réponse clinique favorable en termes de morbidité, mortalité, et qualité de vie, et elle permet de diminuer les risques de transmission. Lorsqu'un médicament est cher, l'accès aux traitements pour un·e patient·e sans assurance avec des ressources limitées est une barrière qui peut conduire à renoncer aux soins pour des raisons économiques. Un buyers' club est une structure dont l'objectif est d'apporter une aide logistique et/ou financière à un·e patient·e qui souhaite importer à titre personnel un médicament de qualité et efficace à des conditions économiquement plus favorables. L'achat du médicament se fait par internet.

Toxic doses of caffeine are needed to increase skeletal muscle contractility.

Discrepant results have been reported regarding an intramuscular mechanism underlying the ergogenic effect of caffeine on neuromuscular function in humans. Here, we reevaluated the effect of caffeine on muscular force production in humans and combined this with measurements of the caffeine dose-response relationship on force and cytosolic free [Ca2+] ([Ca2+]i) in isolated mouse muscle fibers. Twenty-one healthy and physically active men (29 ± 9 yr, 178 ± 6 cm, 73 ± 10 kg, mean ± SD) took part in the present study. Nine participants were involved in two experimental sessions during which supramaximal single and paired electrical stimulations (at 10 and 100 Hz) were applied to the femoral nerve to record evoked forces. Evoked forces were recorded before and 1 h after ingestion of 1) 6 mg caffeine/kg body mass or 2) placebo. Caffeine plasma concentration was measured in 12 participants. In addition, submaximal tetanic force and [Ca2+]i were measured in single mouse flexor digitorum brevis (FDB) muscle fibers exposed to 100 nM up to 5 mM caffeine. Six milligrams of caffeine per kilogram body mass (plasma concentration ~40 µM) did not increase electrically evoked forces in humans. In superfused FDB single fibers, millimolar caffeine concentrations (i.e., 15- to 35-fold above usual concentrations observed in humans) were required to increase tetanic force and [Ca2+]i. Our results suggest that toxic doses of caffeine are required to increase muscle contractility, questioning the purported intramuscular ergogenic effect of caffeine in humans.