NMR metabolomics study of chronic low-dose exposure to a cocktail of persistent organic pollutants.

Nowadays, exposure to endocrine-disrupting chemicals (EDCs), including persistent organic pollutants (POPs), is one of the most critical threats to public health. EDCs are chemicals that mimic, block, or interfere with hormones in the body's endocrine system and have been associated with a wide range of health issues. This innovative, untargeted metabolomics study investigates chronic low-dose internal exposure to a cocktail of POPs on multiple tissues that are known to accumulate these lipophilic compounds. Interestingly, the metabolic response differs among selected tissues/organs in mice. In the liver, we observed a dynamic effect according to the exposure time and the doses of POPs. In the brain tissue, the situation is the opposite, leading to the conclusion that the presence of POPs immediately gives a saturated effect that is independent of the dose and the duration of exposure studied. By contrast, for the adipose tissues, nearly no effect is observed. This metabolic profiling leads to a holistic and dynamic overview of the main metabolic pathways impacted in lipophilic tissues by a cocktail of POPs.

Present and future of pure shift NMR in metabolomics.

NMR is one of the most powerful techniques for the analysis of biological samples in the field of metabolomics. However, the high complexity of fluids, tissues, or other biological materials taken from living organisms is still a challenge for state-of-the-art pulse sequences, thereby limiting the detection, the identification, and the quantification of metabolites. In this context, the resolution enhancement provided by broadband homonuclear decoupling methods, which allows for simplifying 1 H multiplet patterns into singlets, has placed this so-called pure shift technique as a promising approach to perform metabolic profiling with unparalleled level of detail. In recent years, the many advances achieved in the design of pure shift experiments has paved the way to the analysis of a wide range of biological samples with ultra-high resolution. This review leads the reader from the early days of the main pure shift methods that have been successfully developed over the last decades to address complex samples, to the most recent and promising applications of pure shift NMR to the field of NMR-based metabolomics.

Ultrahigh-Resolution NMR with Water Signal Suppression for a Deeper Understanding of the Action of Antimetabolic Drugs on Diffuse Large B-Cell Lymphoma.

Ultrahigh-resolution NMR has recently attracted considerable attention in the field of complex samples analysis. Indeed, the implementation of broadband homonuclear decoupling techniques has allowed us to greatly simplify crowded 1H spectra, yielding singlets for almost every proton site from the analyzed molecules. Pure shift methods have notably shown to be particularly suitable for deciphering mixtures of metabolites in biological samples. Here, we have successfully implemented a new pure shift pulse sequence based on the PSYCHE method, which incorporates a block for solvent suppression that is suitable for metabolomics analysis. The resulting experiment allows us to record ultrahigh-resolution 1D NOESY 1H spectra of biofluids with suppression of the water signal, which is a crucial step for highlighting metabolite mixtures in an aqueous phase. We have successfully recorded pure shift spectra on extracellular media of diffuse large B-cell lymphoma (DLBCL) cells. Despite a lower sensitivity, the resolution of pure shift data was found to be better than that of the standard approach, which provides a more detailed vision of the exo-metabolome. The statistical analyses carried out on the resulting metabolic profiles allow us to successfully highlight several metabolic pathways affected by these drugs. Notably, we show that Kidrolase plays a major role in the metabolic pathways of this DLBCL cell line.

Metabolic NMR mapping with microgram tissue biopsy.

This study explores the potential of profiling a microgram-scale soft tissue biopsy by NMR spectroscopy. The important elements of high resolution and high sensitivity for the spectral data are achieved through a unique probe, HR-µMAS, which allowed comprehensive profiling to be performed on microgram tissue for the first time under MAS conditions. Thorough spatially resolved metabolic maps were acquired across a coronal brain slice of rat C6 gliomas, which rendered the delineation of the tumor lesion. The results present a unique ex vivo NMR possibility to analyze tissue pathology that cannot be fully explored by the conventional approach, HR-MAS and in vivo MRS. Aside from the capability of analyzing a small localized region to track its specific metabolism, it could also offer the possibility to carry out longitudinal investigations on live animals due to the feasibility of minimally invasive tissue excision.

Characterizing extracellular diffusion properties using diffusion-weighted MRS of sucrose injected in mouse brain.

Brain water and some critically important energy metabolites, such as lactate or glucose, are present in both intracellular and extracellular spaces (ICS/ECS) at significant levels. This ubiquitous nature makes diffusion MRI/MRS data sometimes difficult to interpret and model. While it is possible to glean information on the diffusion properties in ICS by measuring the diffusion of purely intracellular endogenous metabolites (such as NAA), the absence of endogenous markers specific to ECS hampers similar analyses in this compartment. In past experiments, exogenous probes have therefore been injected into the brain to assess their apparent diffusion coefficient (ADC) and thus estimate tortuosity in ECS. Here, we use a similar approach in mice by injecting sucrose, a well-known ECS marker, in either the lateral ventricles or directly in the prefrontal cortex. For the first time, we propose a thorough characterization of ECS diffusion properties encompassing (1) short-range restriction by looking at signal attenuation at high b values, (2) tortuosity and long-range restriction by measuring ADC time-dependence at long diffusion times and (3) microscopic anisotropy by performing double diffusion encoding (DDE) measurements. Overall, sucrose diffusion behavior is strikingly different from that of intracellular metabolites. Acquisitions at high b values not only reveal faster sucrose diffusion but also some sensitivity to restriction, suggesting that the diffusion in ECS is not fully Gaussian at high b. The time evolution of the ADC at long diffusion times shows that the tortuosity regime is not reached yet in the case of sucrose, while DDE experiments suggest that it is not trapped in elongated structures. No major difference in sucrose diffusion properties is reported between the two investigated routes of injection and brain regions. These original experimental insights should be useful to better interpret and model the diffusion signal of molecules that are distributed between ICS and ECS compartments.

Intact NMR spectroscopy: slow high-resolution magic angle spinning chemical shift imaging.

High-Resolution Magic-Angle Spinning Chemical Shift Imaging (HR-MAS CSI) has recently been explored for nuclear magnetic resonance (NMR) metabolomics and shows considerable promise in organism research. This is due to its ability to offer a supplemental dimension - spatial metabolic distribution - for profiling. However, HR-MAS CSI suffers from the large centrifugal stress exerted on the sample, which inevitably hinders the metabolic assessment. Herein, a slow sample spinning strategy was implemented and evaluated. The results demonstrate its potential as a highly informative profiling approach for intact specimens, with high quality data and feasibility.

Simultaneous metabolic mapping of different anatomies by 1H HR-MAS chemical shift imaging.

Localized information on a specimen is considered indispensable for deciphering biological activity. Magnetic resonance spectroscopy is a notable method because of its versatility; however, one limitation is the spectral quality on a static sample. This study explores an amalgamated method with two magnetic resonance experiments: high-resolution magic-angle spinning (HR-MAS) for high-quality spectral acquisition from a spinning sample and chemical shift imaging (CSI) for spatial localization. The advantage of HR-MAS CSI is its amenity for simultaneously profiling the metabolome-with good spectral data-at different spatial regions in a single experiment. Herein, 1H HR-MAS CSI (including a T2-contrast CSI) was described and performed on various food tissues and an intact organism. Different data analyses such as multivariate and quantification were explored to identify the metabolic variants in different anatomical regions and in one case, to assist in a spatial allocation. The limitation and drawback of the experiment are also discussed. Graphical abstract.

Determinants of Informal Care, Burden, and Risk of Burnout in Caregivers of Stroke Survivors: The CONOCES Study.

BACKGROUND AND PURPOSE: The aim of this article was to analyze the likelihood of receiving informal care after a stroke and to study the burden and risk of burnout of primary caregivers in Spain. METHODS: The CONOCES study is an epidemiological, observational, prospective, multicenter study of patients diagnosed with stroke and admitted to a Stroke Unit in the Spanish healthcare system. At 3 and 12 months post-event, we estimated the time spent caring for the patient and the burden borne by primary caregivers. Several multivariate models were applied to estimate the likelihood of receiving informal caregiving, the burden, and the likelihood of caregivers being at a high risk of burnout. RESULTS: Eighty percent of those still alive at 3 and 12 months poststroke were receiving informal care. More than 40% of those receiving care needed a secondary caregiver at 3 months poststroke. The likelihood of receiving informal care was associated with stroke severity and the individual's health-related quality of life. When informal care was provided, both the burden borne by caregivers and the likelihood of caregivers being at a high risk of burnout was associated with (1) caregiving hours; (2) the patient's health-related quality of life; (3) the severity of the stroke measured at discharge; (4) the patient having atrial fibrillation; and (5) the degree of dependence. CONCLUSIONS: This study reveals the heavy burden borne by the caregivers of stroke survivors. Our analysis also identifies explanatory and predictive variables for the likelihood of receiving informal care, caregiver burden, and high risk of burnout.

HR-µMAS NMR-Based Metabolomics: Localized Metabolic Profiling of a Garlic Clove with µg Tissues.

The localization of metabolic profiles within a tissue sample is of particular interest when the sampling size is considerably small, i.e., in the order of a microgram (µg) scale. Small sampling size is inevitable when the sample availability is limited, or when different metabolic profiles are suspected in small disparate sample regions. Capitalizing a recently introduced high-resolution micro-MAS probe (HR-µMAS) for its capability of high-quality NMR data acquisition of µg samples, this study explores the localized metabolic NMR profiling of a single garlic clove and compares the methodology and results with the standard HR-MAS. One advantage of HR-µMAS is the feasibility of analyzing homogeneous µg samples within a large heterogeneous tissue. As a result, the sampling mass (<0.5 mg) allows to selectively profile four homogeneous anatomical garlic regions by HR-µMAS (skin, flesh, inner epidermis, and sprout), in contrast to three regions (skin, flesh, and core ≡ inner epidermis and sprout) by HR-MAS, with a sampling mass of ca. 8 mg. Discriminant analysis was carried out to identify the significant variables in the different regions. It found a significant presence of fructose in both skin and flesh, while sucrose and glucose are predominant in the core. Among the garlic characteristic sulfur compounds, allicin is dominant in the core and allyl mercaptan in both skin and flesh. Quantification analysis was conducted and demonstrated its potential by quantifying metabolites at the µg-level. This study offers an important basis for designing HR-µMAS NMR-based metabolomics studies that can benefit from profiling µg-scale samples.

Cost-effectiveness of a hepatitis B virus screening strategy to prevent reactivation in patients with hematologic neoplasms.

INTRODUCTION: The effectiveness of a screening strategy for the detection of a hepatitis B virus (HBV) infection followed by prophylaxis in order to prevent HBV reactivation was assessed in patients with hematologic neoplasms. MATERIAL AND METHODS: A decision tree was developed to compare the cost and effectiveness (prevented reactivations) over an 18 month period of a screening strategy prior to chemotherapy with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) with a non-screening approach. HBsAg+ (hepatitis B surface antigen) and/or anti-HBc+ (antibodies to hepatitis B core antigen) and HBV-DNA+ patients received oral antiviral prophylaxis with tenofovir disoproxil (245 mg once daily) from chemotherapy baseline until one year after chemotherapy completion. Non-screened patients received tenofovir in case of a reactivation. Model probabilities were obtained from the literature. The total cost (€, 2015) included: antiviral prophylaxis, R-CHOP, screening tests (HBsAg, anti-HBc, HBV-DNA) and liver function tests. Drug therapy costs were estimated using ex-factory prices with mandatory deductions. The incremental cost-effectiveness ratio (ICER) was calculated in order to assess the cost-effectiveness of this intervention in terms of cost per reactivation averted versus no screening. RESULTS: In a hypothetical cohort of 1,000 patients, screening prevented 7.36 reactivations when compared to the non-screening approach (14.9 versus 22.3). Total cost/patient (including €8,282 for R-CHOP) was €8,584 for the screening strategy and €8,449 for the non-screening approach. The ICER for screening versus non-screening was €18,376/prevented reactivation. CONCLUSION: HBV screening followed by oral antiviral prophylaxis yielded more health benefits than non-screening, reducing HBV reactivation in patients with hematologic neoplasms on chemotherapy.

Outcomes measured by mortality rates, quality of life and degree of autonomy in the first year in stroke units in Spain.

INTRODUCTION: The primary objective of this sub analysis of the CONOCES study was to analyse outcomes in terms of mortality rates, quality of life and degree of autonomy over the first year in patients admitted to stroke units in Spain. The secondary objective was to identify the factors determining good prognosis. METHODS: We studied a sample of patients who had suffered a confirmed stroke and been admitted to a Stroke Unit in the Spanish healthcare system. Socio-demographic and clinical variables and variables related to the level of severity (NIHSS), the level of autonomy (Barthel, modified Rankin) and quality of life (EQ-5D) were recorded at the time of admission and then three months and one year after the event. Factors determining prognosis were analysed using logistic regression and ROC curves. RESULTS: A total of 321 patients were recruited, 33% of whom received thrombolytic treatment, which was associated with better results on the Barthel and the modified Rankin scales and in terms of the risk of death. Mean quality of life measured through EQ-5D improved from 0.57 at discharge to 0.65 one year later. Full autonomy level measured by Barthel index increased from 30.1% at discharge to 52.8% at one year and by the modified Rankin scale from 51% to 71%. The rates for in-hospital and 1-year mortality were 5.9% and 17.4% respectively. Low NIHSS scores were associated with a good prognosis with all the outcome variables. The three instruments applied (NIHSS, Barthel and modified Rankin scales) on admission showed good discriminative ability for patient prognosis in the ROC curves. CONCLUSIONS: There has been a change in the prognosis for stroke in Spain in recent years as the quality of life at 1 year observed in our study is clearly higher than that obtained in other Spanish studies conducted previously. Moreover, survival and functional outcome have also improved following the introduction of a new model of care. These results clearly promote extension of the model based on stroke units and reinforced rehabilitation to the majority of the more than 100,000 strokes that occur annually in Spain.

Healthcare Resource Utilization and Associated Costs among Patients with Advanced Non-Small-Cell Lung Cancer Receiving Chemotherapy or Immunotherapy in Spain: A Single-Center, Real-World, Exploratory Study.

The objective of this observational, single-center, retrospective study conducted in a Spanish tertiary hospital was to describe the real-world (RW) healthcare resource utilization (HCRU) among patients with advanced non-small-cell lung cancer (aNSCLC) who received chemotherapy (CT) or immunotherapy (IT) as first and second lines of treatment. A total of 173 patients diagnosed with aNSCLC and treated between January 2016 and August 2020 were included. The standardized average costs per patient/year were EUR 40,973.2 and EUR 22,502.4 for first-line CT and IT and EUR 140,601.3 and EUR 20,175.9 for second-line CT and IT, respectively. The average annual costs per patient associated with adverse-event (AE) onset were EUR 29,939.7 and EUR 460.7 for first-line CT and IT and EUR 35,906.4 and EUR 3206.1 for second-line CT and IT, respectively. The costs associated with disease management were EUR 33,178.0 and EUR 22,448.4 for first-line CT and IT and EUR 127,134.2 and EUR 19,663.9 for second-line CT and IT, respectively. In conclusion, IT use showed a lower average annual cost per patient, which was associated with lower HCRU for both disease and AE management, compared to the use of CT. However, these results should be further confirmed in the context of the currently implemented treatment schemes, including the combination of CT with single or dual IT.

Cost-Effectiveness Analysis of Triple Therapy with Budesonide/ Glycopyrronium/ Formoterol Fumarate versus Dual Therapy in Patients with Chronic Obstructive Pulmonary Disease in Spain.

Objective: To evaluate the cost-effectiveness of Budesonide/Glycopyrronium/Formoterol (BUD/GLY/FOR) versus LAMA/LABA and ICS/LABA, respectively, in patients with moderate to severe COPD, from the Spanish National Healthcare System (NHS) perspective. Methods: A lifetime Markov model with monthly cycle length was developed with baseline and treatment effect data from ETHOS clinical trial, together with utility values from literature and Spanish healthcare resource costs (€, 2021). A 3% annual discount rate was used for costs and benefits. The model comprised ten health states: nine forced expiratory volume in 1 second (FEV1)-related, which were divided by three levels of severity: moderate (FEV1 ≥50% and <80%); severe (FEV1 ≥30% and <50%) and very severe (FEV1 <30%) and a death state. Each FEV1-health state was divided into no exacerbation, moderate exacerbation, and severe exacerbations. An expert panel validated data and assumptions. Outcomes were measured as incremental cost per exacerbation avoided, per life year (LY) gained, and per quality-adjusted life-year (QALY) gained (ICUR). One-way (OWSA), scenario, and probabilistic sensitivity analyses (PSA) were performed. Results: According to this cost-effectiveness analysis based on a Markov model, BUD/GLY/FOR was associated with a lower totals exacerbation per patient (12.80) compared to LAMA/LABA (13.36) and ICS/LABA (13.23) and higher LYs (10.32 vs 10.14 and 10.06, respectively) and QALYs (7.55 vs 7.41 and 7.32, respectively). The incremental costs were €850.95, and €2422.26, respectively, per exacerbation avoided, €2733.38 and €4111.15, respectively, per LY gained and €3461.19 and €4545.24 per QALY gained. OWSA showed that the model was most sensitive to the costs of treatments following discontinuation, but the ICUR remained below the cost-effectiveness threshold of €25,000 per QALY gained. In the PSA, the probability of BUD/GLY/FOR being cost-effective was 91.32% vs LAMA/LABA and 99.29% vs ICS/LABA. Conclusion: BUD/GLY/FOR is a cost-effective treatment strategy for Spanish NHS patients with COPD compared to dual therapies.

General Guidelines for Sample Preparation Strategies in HR-µMAS NMR-based Metabolomics of Microscopic Specimens.

The study of the metabolome within tissues, organisms, cells or biofluids can be carried out by several bioanalytical techniques. Among them, nuclear magnetic resonance (NMR) is one of the principal spectroscopic methods. This is due to a sample rotation technique, high-resolution magic angle spinning (HR-MAS), which targets the analysis of heterogeneous specimens with a bulk sample mass from 5 to 10 mg. Recently, a new approach, high-resolution micro-magic angle spinning (HR-µMAS), has been introduced. It opens, for the first time, the possibility of investigating microscopic specimens (<500 µg) with NMR spectroscopy, strengthening the concept of homogeneous sampling in a heterogeneous specimen. As in all bioanalytical approaches, a clean and reliable sample preparation strategy is a significant component in designing metabolomics (or -omics, in general) studies. The sample preparation for HR-µMAS is consequentially complicated by the µg-scale specimen and has yet to be addressed. This report details the strategies for three specimen types: biofluids, fluid matrices and tissues. It also provides the basis for designing future µMAS NMR studies of microscopic specimens.

Current Developments in µMAS NMR Analysis for Metabolomics.

Analysis of microscopic specimens has emerged as a useful analytical application in metabolomics because of its capacity for characterizing a highly homogenous sample with a specific interest. The undeviating analysis helps to unfold the hidden activities in a bulk specimen and contributes to the understanding of the fundamental metabolisms in life. In NMR spectroscopy, micro(µ)-probe technology is well-established and -adopted to the microscopic level of biofluids. However, this is quite the contrary with specimens such as tissue, cell and organism. This is due to the substantial difficulty of developing a sufficient µ-size magic-angle spinning (MAS) probe for sub-milligram specimens with the capability of high-quality data acquisition. It was not until 2012; a µMAS probe had emerged and shown promises to µg analysis; since, a continuous advancement has been made striving for the possibility of µMAS to be an effective NMR spectroscopic analysis. Herein, the mini-review highlights the progress of µMAS development-from an impossible scenario to an attainable solution-and describes a few demonstrative metabolic profiling studies. The review will also discuss the current challenges in µMAS NMR analysis and its potential to metabolomics.

Economic impact of patients admitted to stroke units in Spain.

OBJECTIVES: Stroke is a major social and health problem. However, since the recent incorporation of new advances in its management, little is known about the cost of stroke. The aim of this study is to find out the real cost of stroke in Spain. METHODS: This is an epidemiological, observational, prospective, multicenter study of patients diagnosed with stroke and admitted to a stroke unit. Patients were recruited from 16 hospitals throughout Spain and followed up for 1 year. Sociodemographic, clinical, and economic data were collected. Costs (€ 2012) were estimated from the social perspective and were divided into direct healthcare (inpatient, outpatient, and medication), direct non-healthcare (mainly formal and informal care) and labor productivity losses. RESULTS: A total of 321 patients were included. Mean age was 72.1 years and 176 patients (54.8 %) were male. Total average cost per patient/year was €27,711. Direct healthcare costs amounted to €8491 per patient/year (68.8 % due to inpatient costs) and non-healthcare costs to an average of €18,643 per patient/year (89.5 % due to informal care). Productivity loss costs per patient/year were €276. Total costs of hemorrhagic strokes were slightly higher than ischemic (€28,895 vs. €27,569 per patient/year, p = 0.550) without significant differences. The main variables associated with higher costs were the presence of hypertension (€30,332 vs. €23,234 per patient/year, p < 0.05) and the severity of stroke (p < 0.05), both independently associated after a multivariate analysis. CONCLUSIONS: The cost of patients admitted to stroke units in Spain is €27,711 per patient/year. More than two-thirds are social costs, mainly informal care. Stroke remains a major burden on health systems and society, so additional efforts are needed for its prevention.

Study by ³¹P NMR spectroscopy of the triacylglycerol degradation processes in olive oil with different heat-transfer mechanisms.

The thermal degradation of olive oil using conventional and microwave heating under the same experimental conditions were compared. A powerful identification and quantification technique based on (31)P NMR has been developed to characterise the differences between the minor components including diacylglycerol and free fatty acids in the heated samples. The (31)P NMR spectra of the degraded olive oils, which contain OH groups derivatised with a phosphorus reagent, showed that conventional heating is more detrimental to the oil than microwave technique. Conventional heating leads to a significant increase in the diacylglycerol and free fatty acid contents as well as in the number of degradation compounds, which damage the olive oil quality. However, the main process that takes place on using microwave heating is isomerisation between diacylglycerols, a change that could give a potential longer shelf life to the olive oil.

ACTG-HIV symptoms changes in patients switched to RPV/FTC/TDF due to previous intolerance to CART. Interim analysis of the PRO-STR study.

INTRODUCTION: Tolerability and convenience are crucial aspects for the long-term success of combined antiretroviral therapy (cART). The aim of this study was to investigate the impact in routine clinical practice of switching to the single tablet regimen (STR) RPV/FTC/TDF in patients with intolerance to previous cART, in terms of patients' well-being, assessed by several validated measures. METHODS: Prospective, multicenter study. Adult HIV-infected patients with viral load under 1.000 copies/mL while receiving a stable ART for at least the last three months and switched to RPV/FTC/TDF due to intolerance of previous regimen, were included. Analyses were performed by ITT. Presence/magnitude of symptoms (ACTG-HIV Symptom Index), quality of life (EQ-5D, EUROQoL & MOS-HIV), adherence (SMAQ), preference of treatment and perceived ease of medication (ESTAR) through 48 weeks were performed. RESULTS: Interim analysis of 125 patients with 16 weeks of follow up was performed. 100 (80%) were male, mean age 46 years. Mean CD4 at baseline was 629.5±307.29 and 123 (98.4%) had viral load <50 copies/mL; 15% were HCV co-infected. Ninety two (73.6%) patients switched from a NNRTI (84.8% from EFV/FTC/TDF) and 33 (26.4%) from a PI/r. The most frequent reasons for switching were psychiatric disorders (51.2%), CNS adverse events (40.8%), gastrointestinal (19.2%) and metabolic disorders (19.2%). At the time of this analysis (week 16), four patients (3.2%) discontinued treatment: one due to adverse events, two virologic failures and one with no data. A total of 104 patients (83.2%) were virologically suppressed (<50 copies/mL). The average degree of discomfort in the ACTG-HIV Symptom Index significantly decreased from baseline (21±15.55) to week 4 (10.89±12.36) & week 16 (10.81±12.62), p<0.001. In all the patients, quality of life tools showed a significant benefit in well-being of the patients (Table 1). Adherence to therapy significantly and progressively increased (SMAQ) from baseline (54.4%) to week 4 (68%), p<0.001 and to week 16 (72.0%), p<0.001. CONCLUSIONS: Switching to RPV/FTC/TDF from another ARV regimen due to toxicity, significantly improved the quality of life of HIV-infected patients, both in mental and physical components, and improved adherence to therapy while maintaining a good immune and virological response.