Application of 15N-Edited 1H-13C Correlation NMR Spectroscopy─Toward Fragment-Based Metabolite Identification and Screening via HCN Constructs.

Many key building blocks of life contain nitrogen moieties. Despite the prevalence of nitrogen-containing metabolites in nature, 15N nuclei are seldom used in NMR-based metabolite assignment due to their low natural abundance and lack of comprehensive chemical shift databases. However, with advancements in isotope labeling strategies, 13C and 15N enriched metabolites are becoming more common in metabolomic studies. Simple multidimensional nuclear magnetic resonance (NMR) experiments that correlate 1H and 15N via single bond 1JNH or multiple bond 2-3JNH couplings using heteronuclear single quantum coherence (HSQC) or heteronuclear multiple bond coherence are well established and routinely applied for structure elucidation. However, a 1H-15N correlation spectrum of a metabolite mixture can be difficult to deconvolute, due to the lack of a 15N specific database. In order to bridge this gap, we present here a broadband 15N-edited 1H-13C HSQC NMR experiment that targets metabolites containing 15N moieties. Through this approach, nitrogen-containing metabolites, such as amino acids, nucleotide bases, and nucleosides, are identified based on their 13C, 1H, and 15N chemical shift information. This approach was tested and validated using a [15N, 13C] enriched Daphnia magna (water flea) metabolite extract, where the number of clearly resolved 15N-containing peaks increased from only 11 in a standard HSQC to 51 in the 15N-edited HSQC, and the number of obscured peaks decreased from 59 to just 7. The approach complements the current repertoire of NMR techniques for mixture deconvolution and holds considerable potential for targeted metabolite NMR in 15N, 13C enriched systems.

Development of Low-Magnetic Susceptibility Microcoils via 5-Axis Machining for Analysis of Biological and Environmental Samples.

In environmental research, it is critical to understand how toxins impact invertebrate eggs and egg banks, which, due to their tiny size, are very challenging to study by conventional nuclear magnetic resonance (NMR) spectroscopy. Microcoil technology has been extensively utilized to enhance the mass-sensitivity of NMR. In a previous study, 5-axis computer numerical control (CNC) micromilling (shown to be a viable alternative to traditional microcoil production methods) was used to create a prototype copper slotted-tube resonator (STR). Despite the excellent limit of detection (LOD) of the resonator, the quality of the line shape was very poor due to the magnetic susceptibility of the copper resonator itself. This is best solved using magnetic susceptibility-matched materials. In this study, approaches are investigated that improve the susceptibility while retaining the versatility of coil milling. One method involves machining STRs from various copper/aluminum alloys, while the other involves machining ones from an aluminum 2011 alloy and electroplating them with copper. In all cases, combining copper and aluminum to produce resonators resulted in improved line shape and SNR compared to pure copper resonators due to their reduced magnetic susceptibility. However, the copper-plated aluminum resonators showed optimal performance from the devices tested. The enhanced LOD of these STRs allowed for the first 1H-13C heteronuclear multiple quantum coherence (HMQC) of a single intact 13C-labeled Daphnia magna egg (∼4 µg total biomass). This is a key step toward future screening programs that aim to elucidate the toxic processes in aquatic eggs.

Exploring the Applications of Carbon-Detected NMR in Living and Dead Organisms Using a 13C-Optimized Comprehensive Multiphase NMR Probe.

Comprehensive multiphase-nuclear magnetic resonance (CMP-NMR) is a non-invasive approach designed to observe all phases (solutions, gels, and solids) in intact samples using a single NMR probe. Studies of dead and living organisms are important to understand processes ranging from biological growth to environmental stress. Historically, such studies have utilized 1H-based phase editing for the detection of soluble/swollen components and 1H-detected 2D NMR for metabolite assignments/screening. However, living organisms require slow spinning rates (∼500 Hz) to increase survivability, but at such low speeds, complications from water sidebands and spectral overlap from the modest chemical shift window (∼0-10 ppm) make 1H NMR challenging. Here, a novel 13C-optimized E-Free magic angle spinning CMP probe is applied to study all phases in ex vivo and in vivo samples. This probe consists of a two-coil design, with an inner single-tuned 13C coil providing a 113% increase in 13C sensitivity relative to a traditional multichannel single-CMP coil design. For organisms with a large biomass (∼0.1 g) like the Ganges River sprat (ex vivo), 13C-detected full spectral editing and 13C-detected heteronuclear correlation (HETCOR) can be performed at natural abundance. Unfortunately, for a single living shrimp (∼2 mg), 13C enrichment was still required, but 13C-detected HETCOR shows superior data relative to heteronuclear single-quantum coherence at low spinning speeds (due to complications from water sidebands in the latter). The probe is equipped with automatic-tuning-matching and is compatible with automated gradient shimming─a key step toward conducting multiphase screening of dead and living organisms under automation in the near future.

Evaluation of double-tuned single-sided planar microcoils for the analysis of small 13 C enriched biological samples using 1 H-13 C 2D heteronuclear correlation NMR spectroscopy.

Microcoils provide a cost-effective approach to improve detection limits for mass-limited samples. Single-sided planar microcoils are advantageous in comparison to volume coils, in that the sample can simply be placed on top. However, the considerable drawback is that the RF field that is produced by the coil decreases with distance from the coil surface, which potentially limits more complex multi-pulse NMR pulse sequences. Unfortunately, 1 H NMR alone is not very informative for intact biological samples due to line broadening caused by magnetic susceptibility distortions, and 1 H-13 C 2D NMR correlations are required to provide the additional spectral dispersion for metabolic assignments in vivo or in situ. To our knowledge, double-tuned single-sided microcoils have not been applied for the 2D 1 H-13 C analysis of intact 13 C enriched biological samples. Questions include the following: Can 1 H-13 C 2D NMR be performed on single-sided planar microcoils? If so, do they still hold sensitivity advantages over conventional 5 mm NMR technology for mass limited samples? Here, 2D 1 H-13 C HSQC, HMQC, and HETCOR variants were compared and then applied to 13 C enriched broccoli seeds and Daphnia magna (water fleas). Compared to 5 mm NMR probes, the microcoils showed a sixfold improvement in mass sensitivity (albeit only for a small localized region) and allowed for the identification of metabolites in a single intact D. magna for the first time. Single-sided planar microcoils show practical benefit for 1 H-13 C NMR of intact biological samples, if localized information within ~0.7 mm of the 1 mm I.D. planar microcoil surface is of specific interest.

Association of emergency department boarding times on hospital length of stay for patients with psychiatric illness.

BACKGROUND: Extended periods awaiting an inpatient bed in the emergency department (ED) may exacerbate the state of patients with acute psychiatric illness, increasing the time it takes to stabilise their acute problem in hospital. Therefore, we assessed the association between boarding time and hospital length of stay for psychiatric patients. METHODS: ED clinical records were linked to inpatient administrative records for all patients with a primary psychiatric diagnosis admitted to a Calgary, Alberta hospital between April 2014 and March 2018. The primary exposure was boarding time (admission decision to inpatient bed transfer), and primary outcome was inpatient length of stay. Confounders for this relationship, including indicators of illness severity, were selected a priori then the association was assessed using hierarchical Bayesian Poisson regression, which accounts for repeat observations of the same patient and differences between hospital sites. Changes in length of stay were measured using a rate ratio (ie, expected change in length of stay for each 1 hour increase in boarding time). RESULTS: A total of 19 212 admissions (14 261 unique patients) were included in the analysis. The average boarding time was 14 hours (range: 0-186 hours). Patients who were boarded for greater than 14 hours more frequently required a high-observation bed (14% vs 3.5%), received an antipsychotic (44% vs 14%) or received sedation (55% vs 33%) while in the ED. The probability that boarding time increased hospital length of stay (rate ratio: >1) was 92%, with a median increase for a patient boarded for 24 hours of 0.01 days. CONCLUSION: Boarding in the ED was associated with a high probability of increasing the hospital length of stay for psychiatric patients; however, the absolute increase is minimal. Although slight, this signal for longer length of stay may be a sign of increased morbidity for psychiatric patients held in the ED.

Comprehensive Multiphase NMR Probehead with Reduced Radiofrequency Heating Improves the Analysis of Living Organisms and Heat-Sensitive Samples.

Comprehensive multiphase (CMP) NMR, first described in 2012, combines all of the hardware components necessary to analyze all phases (solid, gel, and solution) in samples in their natural state. In combination with spectral editing experiments, it can fully differentiate phases and study the transfer of chemical species across and between phases, providing unprecedented molecular-level information in unaltered natural systems. However, many natural samples, such as swollen soils, plants, and small organisms, contain water, salts, and ionic compounds, making them electrically lossy and susceptible to RF heating, especially when using high-strength RF fields required to select the solid domains. While dedicated reduced-heating probes have been developed for solid-state NMR, to date, all CMP-NMR probes have been based on solenoid designs, which can lead to problematic sample heating. Here, a new prototype CMP probe was developed, incorporating a loop gap resonator (LGR) for decoupling. Temperature increases are monitored in salt solutions analogous to those in small aquatic organisms and then tested in vivo on Hyalella azteca (freshwater shrimp). In the standard CMP probe (solenoid), 80% of organisms died within 4 h under high-power decoupling, while in the LGR design, all organisms survived the entire test period of 12 h. The LGR design reduced heating by a factor of ∼3, which allowed 100 kHz decoupling to be applied to salty samples with generally ≤10 °C sample heating. In addition to expanding the potential for in vivo research, the ability to apply uncompromised high-power decoupling could be beneficial for multiphase samples containing true crystalline solids that require the strongest possible decoupling fields for optimal detection.

Expanding current applications and permitting the analysis of larger intact samples by means of a 7 mm CMP-NMR probe.

Comprehensive multiphase NMR combines the ability to study and differentiate all phases (solids, gels, and liquids) using a single NMR probe. The general goal of CMP-NMR is to study intact environmental and biological samples to better understand conformation, organization, association, and transfer between and across phases/interfaces that may be lost with conventional sample preparation such as drying or solubilization. To date, all CMP-NMR studies have used 4 mm probes and rotors. Here, a larger 7 mm probehead is introduced which provides ∼3 times the volume and ∼2.4 times the signal over a 4 mm version. This offers two main advantages: (1) the additional biomass reduces experiment time, making 13C detection at natural abundance more feasible; (2) it allows the analysis of larger samples that cannot fit within a 4 mm rotor. Chicken heart tissue and Hyalella azteca (freshwater shrimp) are used to demonstrate that phase-based spectral editing works with 7 mm rotors and that the additional biomass from the larger volumes allows detection with 13C at natural abundance. Additionally, a whole pomegranate seed berry (aril) and an intact softgel capsule of hydroxyzine hydrochloride are used to demonstrate the analysis of samples too large to fit inside a conventional 4 mm CMP probe. The 7 mm version introduced here extends the range of applications and sample types that can be studied and is recommended when 4 mm CMP probes cannot provide adequate signal-to-noise (S/N), or intact samples are simply too big for 4 mm rotors.

Targeting the Lowest Concentration of a Toxin That Induces a Detectable Metabolic Response in Living Organisms: Time-Resolved In Vivo 2D NMR during a Concentration Ramp.

In vivo nuclear magnetic resonance (NMR) is a powerful analytical tool for probing complex biological processes inside living organisms. However, due to magnetic susceptibility broadening, which produces broad lines in one-dimensional NMR, 1H-13C two-dimensional (2D) NMR is required for metabolite monitoring in vivo. As each 2D experiment is time-consuming, often hours, this limits the temporal resolution over which in vivo processes can be monitored. Furthermore, to understand concentration-dependent responses, studies are traditionally repeated using different contaminant and toxin concentrations, which can make studies prohibitively long (potentially months). In this study, time-resolved non-uniform sampling NMR is performed in the presence of a contaminant concentration sweep. The result is that the lowest concentration that elicits a metabolic response can be rapidly detected, while the metabolic pathways impacted provide information about the toxic mode of action of the toxin. The lowest concentration of bisphenol A (BPA) that induces a response was ∼0.1 mg/L (detected in just 16 min), while changes in different metabolites suggest a complex multipathway response that leads to protein degradation at higher BPA concentrations. This proof of concept shows it is possible, on the basis of "real-time" organism responses, to identify the sublethal concentration at which a toxin impacts an organism and thus represents an essential analytical tool for the next generation of toxicity-based research and monitoring.

5-Axis CNC Micromilling for Rapid, Cheap, and Background-Free NMR Microcoils.

The superior mass sensitivity of microcoil technology in nuclear magnetic resonance (NMR) spectroscopy provides potential for the analysis of extremely small-mass-limited samples such as eggs, cells, and tiny organisms. For optimal performance and efficiency, the size of the microcoil should be tailored to the size of the mass-limited sample of interest, which can be costly as mass-limited samples come in many shapes and sizes. Therefore, rapid and economic microcoil production methods are needed. One method with great potential is 5-axis computer numerical control (CNC) micromilling, commonly used in the jewelry industry. Most CNC milling machines are designed to process larger objects and commonly have a precision of >25 µm (making the machining of common spiral microcoils, for example, impossible). Here, a 5-axis MiRA6 CNC milling machine, specifically designed for the jewelry industry, with a 0.3 µm precision was used to produce working planar microcoils, microstrips, and novel microsensor designs, with some tested on the NMR in less than 24 h after the start of the design process. Sample wells could be built into the microsensor and could be machined at the same time as the sensors themselves, in some cases leaving a sheet of Teflon as thin as 10 µm between the sample and the sensor. This provides the freedom to produce a wide array of designs and demonstrates 5-axis CNC micromilling as a versatile tool for the rapid prototyping of NMR microsensors. This approach allowed the experimental optimization of a prototype microstrip for the analysis of two intact adult Daphnia magna organisms. In addition, a 3D volume slotted-tube resonator was produced that allowed for 2D 1H-13C NMR of D. magna neonates and exhibited 1H sensitivity (nLODω600 = 1.49 nmol s1/2) close to that of double strip lines, which themselves offer the best compromise between concentration and mass sensitivity published to date.

Screening strategies to identify sepsis in the prehospital setting: a validation study.

BACKGROUND: In the prehospital setting, differentiating patients who have sepsis from those who have infection but no organ dysfunction is important to initiate sepsis treatments appropriately. We aimed to identify which published screening strategies for paramedics to use in identifying patients with sepsis provide the most certainty for prehospital diagnosis. METHODS: We identified published strategies for screening by paramedics through a literature search. We then conducted a validation study in Alberta, Canada, from April 2015 to March 2016. For adult patients (≥ 18 yr) who were transferred by ambulance, we linked records to an administrative database and then restricted the search to patients with infection diagnosed in the emergency department. For each patient, the classification from each strategy was determined and compared with the diagnosis recorded in the emergency department. For all strategies that generated numeric scores, we constructed diagnostic prediction models to estimate the probability of sepsis being diagnosed in the emergency department. RESULTS: We identified 21 unique prehospital screening strategies, 14 of which had numeric scores. We linked a total of 131 745 eligible patients to hospital databases. No single strategy had both high sensitivity (overall range 0.02-0.85) and high specificity (overall range 0.38-0.99) for classifying sepsis. However, the Critical Illness Prediction (CIP) score, the National Early Warning Score (NEWS) and the Quick Sepsis-Related Organ Failure Assessment (qSOFA) score predicted a low to high probability of a sepsis diagnosis at different scores. The qSOFA identified patients with a 7% (lowest score) to 87% (highest score) probability of sepsis diagnosis. INTERPRETATION: The CIP, NEWS and qSOFA scores are tools with good predictive ability for sepsis diagnosis in the prehospital setting. The qSOFA score is simple to calculate and may be useful to paramedics in screening patients with possible sepsis.

Lorazepam Versus Diazepam in the Management of Emergency Department Patients With Alcohol Withdrawal.

STUDY OBJECTIVE: Alcohol withdrawal is a common emergency department (ED) presentation. Although benzodiazepines reduce symptoms of withdrawal, there is little ED-based evidence to assist clinicians in selecting appropriate pharmacotherapy. We compare lorazepam with diazepam for the management of alcohol withdrawal to assess 1-week ED and hospital-related outcomes. METHODS: From January 1, 2015, to December 31, 2018, at 3 urban EDs in Vancouver, Canada, we studied patients with a discharge diagnosis of alcohol withdrawal. We excluded individuals presenting with a seizure or an acute concurrent illness. We performed a structured chart review to ascertain demographics, ED treatments, and outcomes. Patients were stratified according to initial management with lorazepam versus diazepam. The primary outcome was hospital admission, and secondary outcomes included in-ED seizures and 1-week return visits for discharged patients. RESULTS: Of 1,055 patients who presented with acute alcohol withdrawal, 898 were treated with benzodiazepines. Median age was 47 years (interquartile range 37 to 56 years) and 73% were men. Baseline characteristics were similar in the 2 groups. Overall, 69 of 394 patients (17.5%) receiving lorazepam were admitted to the hospital compared with 94 of 504 patients receiving diazepam (18.7%), a difference of 1.2% (95% confidence interval -4.2% to 6.3%). Seven patients (0.7%; 95% confidence interval 0.3% to 1.4%) had an in-ED seizure, but all seizures occurred before receipt of benzodiazepines. Among patients discharged home, 1-week return visits occurred for 78 of 325 (24.0%) who received lorazepam and 94 of 410 (23.2%) who received diazepam, a difference of 0.8% (95% confidence interval -5.3% to 7.1%). CONCLUSION: In our sample of ED patients with acute alcohol withdrawal, patients receiving lorazepam had an admission rate similar to that of those receiving diazepam. The few in-ED seizures occurred before medication administration. For discharged patients, the 1-week ED return visit rate of nearly 25% could warrant enhanced follow-up and community support.

Classification versus Prediction of Mortality Risk using the SIRS and qSOFA Scores in Patients with Infection Transported by Paramedics.

Objective: Identifying patients with sepsis in the prehospital setting is an important opportunity to increase timely care. When assessing clinical tools designed for paramedic sepsis identification, predicted risk may provide more useful information to support decision-making, compared to traditional estimates of classification accuracy (i.e., sensitivity and specificity). We sought to contrast classification accuracy versus predicted risk of a modified version of the Systemic Inflammatory Response Syndrome score (i.e., excluding white blood cell measure which is often unavailable to paramedics; mSIRS) and quick Sepsis Related Organ Failure Assessment (qSOFA) for determining mortality risk among patients with infection transported by paramedics. Methods: A one-year cohort of patients with infections transported to the Emergency Department (ED) by paramedics was linked to in-hospital administrative databases. Scores were calculated using the first reported vital sign measure for each patient. We calculated sensitivity and specificity of mSIRS and qSOFA for classifying hospital mortality at different score thresholds, and estimated discrimination (using the C-statistic) and calibration (using calibration curves). Regression models for predicting hospital mortality were constructed using the mSIRS or qSOFA scores for each patient as the predictor. Results: A total of 10,409 patients with infection who were transported by paramedics were successfully linked, with an overall mortality rate of 9.2%. The mSIRS score had higher sensitivity estimates than qSOFA for classifying hospital mortality at all thresholds (mSIRS ≥ 1: 0.83 vs. qSOFA≥ 1: 0.80, mSIRS ≥ 3: 0.11 vs. qSOFA ≥ 3: 0.08), but the qSOFA score had better discrimination (C-statistic qSOFA: 0.72 vs. mSIRS: 0.63) and calibration. The risk of hospital mortality predicted by the mSIRS score ranged from 8.0 to 19% across score values, whereas the risk predicted by the qSOFA score ranged from 10 to 51%. Conclusion: Assessing the predicted risk for the mSIRS and qSOFA scores instead of classification accuracy reveals that the qSOFA score provides more information to clinicians about a patient's mortality risk, supporting its use in clinical decision-making.

Assessing Severity of Illness in Patients Transported to Hospital by Paramedics: External Validation of 3 Prognostic Scores.

Introduction: Emergency Medical Services (EMS) are the first healthcare contact for the majority of severely ill patients. Physiologic measures collected by EMS, when incorporated into a prognostic score, may provide important information on patient illness severity. This study compares the predictive ability of 3 common prognostic scores for predicting clinical outcomes in EMS patients. Methods: Discrimination and calibration for predicting the primary outcome of hospital mortality, and secondary outcomes of 2-day mortality and ED disposition, were assessed for each of the scores using a one-year cohort of patients transported to hospital by EMS in Alberta, Canada. For each score, binary logistic regression was used to predict hospital mortality and 2-day mortality and ordinal logistic regression was used to predict ED disposition. Discrimination for each outcome was assessed using C-statistics, and calibration was assessed using calibration curves comparing predicted versus observed outcomes. Results: The Critical Illness Prediction [CIP], Modified Early Warning Score [MEWS], and National Early Warning Score [NEWS] were compared using 121,837 adult patients who were transported by paramedics. All scores had good discrimination for hospital mortality (C-statistic CIP: 0.79, MEWS: 0.71, NEWS: 0.78) and 2-day mortality (CIP:0.85, MEWS: 0.80, NEWS:0.85) but only moderate discrimination for ED disposition (CIP: 0.68, MEWS: 0.61, NEWS: 0.66). Calibration was reliable for hospital mortality in all scores but over-predicted risk for 2-day mortality at higher scores. Overall, the CIP score had the best discrimination, good calibration, and the greatest range of predicted probabilities (0.01 at a CIP score of 0 to 0.92 at a CIP score of 8) for hospital mortality. Conclusions: Prognostic scores using physiologic measures assessed by paramedics have good predictive ability for hospital mortality. These scores, particularly the CIP score, may be considered as a tool for mortality risk stratification or as a general measure of illness severity for patients included in EMS studies.

Strategy to Identify Paramedic Transported Sepsis Cases in an Emergency Department Administrative Database.

Background: To evaluate a new strategy for identifying sepsis in Emergency Department (ED) patients that combines administrative diagnosis codes with clinical information from the point of first contact. Methods: This study linked clinical data from adult patients transported by a provincial Emergency Medical Services (EMS) system to ED and inpatient administrative databases. Sepsis cases were identified by searching ED databases for diagnosis codes consistent with infection and organ dysfunction. Organ dysfunction was further assessed using a partial Sequential Organ Failure Assessment (SOFA) score derived from EMS clinical information. Reliability was evaluated by comparing patients' ED diagnosis codes (ICD-10CA) to their inpatient diagnosis codes; criterion validity by comparing cases classified by the new strategy to an existing inpatient administrative algorithm; and construct validity by assessing for clinical characteristics typically associated with sepsis (e.g., mortality). Results: A total of 43,297 patients were included. ED infection codes were more reliable for classifying patients with infection than using ED sepsis codes alone (proportion of agreement with inpatient codes 79% vs. 74%; p-value < 0.001). The novel strategy requiring the presence of an infection code and either an organ dysfunction code or 2 or more SOFA points from EMS clinical information identified 1,379 more ED patients as having sepsis than the inpatient algorithm. These patients had high mortality supporting construct validity. Conclusions: Incorporation of a broader range of diagnostic codes and linking to an electronic database to obtain initial clinical information for the assessment of organ dysfunction improves reliability, criterion, and construct validity for identifying sepsis in ED patients.

In vivo comprehensive multiphase NMR.

Traditionally, due to different hardware requirements, nuclear magnetic resonance (NMR) has developed as two separate fields: one dealing with solids, and one with solutions. Comprehensive multiphase (CMP) NMR combines all electronics and hardware (magic angle spinning [MAS], gradients, high power Radio Frequency (RF) handling, lock, susceptibility matching) into a universal probe that permits a comprehensive study of all phases (i.e., liquid, gel-like, semisolid, and solid), in intact samples. When applied in vivo, it provides unique insight into the wide array of bonds in a living system from the most mobile liquids (blood, fluids) through gels (muscle, tissues) to the most rigid (exoskeleton, shell). In this tutorial, the practical aspects of in vivo CMP NMR are discussed including: handling the organisms, rotor preparation, sample spinning, water suppression, editing experiments, and finishes with a brief look at the potential of other heteronuclei (2 H, 15 N, 19 F, 31 P) for in vivo research. The tutorial is aimed as a general resource for researchers interested in developing and applying MAS-based approaches to living organisms. Although the focus here is CMP NMR, many of the approaches can be adapted (or directly applied) using conventional high-resolution magic angle spinning, and in some cases, even standard solid-state NMR probes.

Assessing the potential of quantitative 2D HSQC NMR in 13C enriched living organisms.

In vivo Nuclear Magnetic Resonance (NMR) spectroscopy has great potential to interpret the biochemical response of organisms to their environment, thus making it an essential tool in understanding toxic mechanisms. However, magnetic susceptibility distortions lead to 1D NMR spectra of living organisms with lines that are too broad to identify and quantify metabolites, necessitating the use of 2D 1H-13C Heteronuclear Single Quantum Coherence (HSQC) as a primary tool. While quantitative 2D HSQC is well established, to our knowledge it has yet to be applied in vivo. This study represents a simple pilot study that compares two of the most popular quantitative 2D HSQC approaches to determine if quantitative results can be directly obtained in vivo in isotopically enriched Daphnia magna (water flea). The results show the perfect-HSQC experiment performs very well in vivo, but the decoupling scheme used is critical for accurate quantitation. An improved decoupling approach derived using optimal control theory is presented here that improves the accuracy of metabolite concentrations that can be extracted in vivo down to micromolar concentrations. When combined with 2D Electronic Reference To access In vivo Concentrations (ERETIC) protocols, the protocol allows for the direct extraction of in vivo metabolite concentrations without the use of internal standards that can be detrimental to living organisms. Extracting absolute metabolic concentrations in vivo is an important first step and should, for example, be important for the parameterization as well as the validation of metabolic flux models in the future.

Understanding the Fate of Environmental Chemicals Inside Living Organisms: NMR-Based 13C Isotopic Suppression Selects Only the Molecule of Interest within 13C-Enriched Organisms.

In vivo nuclear magnetic resonance (NMR) is rapidly evolving as a critical tool as it offers real-time metabolic information, which is crucial for delineating complex toxic response pathways in living systems. Organisms such as Daphnia magna (water fleas) and Hyalella azteca (freshwater shrimps) are commonly 13C-enriched to increase the signal in NMR experiments. A key goal of in vivo NMR is to monitor how molecules (nutrients, contaminants, or drugs) are metabolized. Conventionally, these studies would normally involve using a 13C-enriched probe molecule and feeding this to an organism at natural abundance, in turn allowing the fate of the probe molecule to be selectively analyzed. The drawback of such an approach is that there is a limited range of 13C-enriched probe molecules, and if available, they are extremely cost prohibitive. Uniquely, when utilizing 13C organisms, a reverse strategy of isotopic filtering becomes possible. The concept described here uses 1H detection in combination with a 13C filter on living organisms. The purpose is to suppress all 1H signals from the organism (i.e., 1H attached to 13C), leaving only the probe molecule (1H attached to 12C). Because the probe molecule can be selectively observed using this approach, it then makes it possible to follow and discern processes such as bioconversion, bioaccumulation, and excretion in vivo. As the approach uses 1H detection, it provides excellent detection limits in the nanogram range. In this article, the approach is introduced, optimized on standards, and then applied to follow nicotine biotransformation and lipid assimilation in vivo to demonstrate the concept.

Development and Application of a Low-Volume Flow System for Solution-State in Vivo NMR.

In vivo nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful technique, since it allows samples to be analyzed in their natural, unaltered state, criteria paramount for living organisms. In this study, a novel continuous low-volume flow system, suitable for in vivo NMR metabolomics studies, is demonstrated. The system allows improved locking, shimming, and water suppression, as well as allowing the use of trace amounts of expensive toxic contaminants or low volumes of precious natural environmental samples as stressors. The use of a double pump design with a sump slurry pump return allows algal food suspensions to be continually supplied without the need for filters, eliminating the possibility of clogging and leaks. Using the flow system, the living organism can be kept alive without stress indefinitely. To evaluate the feasibility and applicability of the flow system, changes in the metabolite profile of 13C enriched Daphnia magna over a 24-h period are compared when feeding laboratory food vs exposing them to a natural algal bloom sample. Clear metabolic changes are observed over a range of metabolites including carbohydrates, lipids, amino acids, and a nucleotide demonstrating in vivo NMR as a powerful tool to monitor environmental stress. The particular bloom used here was low in microcystins, and the metabolic stress impacts are consistent with the bloom being a poor food source forcing the Daphnia to utilize their own energy reserves.

13C quantification in heterogeneous multiphase natural samples by CMP-NMR using stepped decoupling.

Many natural and environmental samples contain combinations of liquids, gels, and solids, yet quantification in the intact state and across multiple phases is highly challenging. Comprehensive multiphase nuclear magnetic resonance (CMP-NMR) combines all the capabilities of high-resolution magic angle spinning (HR-MAS), with the addition of full solids power handling, permitting all phases (i.e., mixtures of liquids, gels, and solids) to be studied and differentiated in intact samples without pre-treatment or extraction. Here, quantification in CMP-NMR is considered. As 1H NMR is considerably broadened in the solid-state, quantification is easier to achieve through 13C which can be observed easily in all the phases. Accurate 13C quantification requires effective 1H decoupling for all the phases, but each phase requires different decoupling conditions. To satisfy these conditions, a pulse sequence termed stepped decoupling is introduced. This sequence can be used to study all components under ideal decoupling conditions resulting in high-resolution spectra without truncation artifacts and provides accurate integrals of components in all phases. The approach is demonstrated on standards and then applied to natural samples including broccoli, soil, and Arabidopsis. The approach permits accurate quantification of chemical categories (for example total carbohydrates) as well as individual species (for example glucose). Further, as the samples are studied intact, volatile species such as methanol and ethylene which are normally hard to detect in plants can be easily quantified in Arabidopsis. Graphical abstract ᅟ.

Improvements in lipid suppression for 1 H NMR-based metabolomics: Applications to solution-state and HR-MAS NMR in natural and in vivo samples.

Proton nuclear magnetic resonance (NMR) spectra of intact biological samples often show strong contributions from lipids, which overlap with signals of interest from small metabolites. Pioneering work by Diserens et al. demonstrated that the relative differences in diffusivity and relaxation of lipids versus small metabolites could be exploited to suppress lipid signals, in high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. In solution-state NMR, suspended samples can exhibit very broad water signals, which are challenging to suppress. Here, improved water suppression is incorporated into the sequence, and the Carr-Purcell-Meiboom-Gill sequence (CPMG) train is replaced with a low-power adiabatic spinlock that reduces heating and spectral artefacts seen with longer CPMG filters. The result is a robust sequence that works well in both HR-MAS as well as static solution-state samples. Applications are also extended to include in vivo organisms. For solution-state NMR, samples containing significant amount of fats such as milk and hemp hearts seeds are used to demonstrate the technique. For HR-MAS, living earthworms (Eisenia fetida) and freshwater shrimp (Hyalella azteca) are used for in vivo applications. Lipid suppression techniques are essential for non-invasive NMR-based analysis of biological samples with a high-lipid content and adds to the suite of experiments advantageous for in vivo environmental metabolomics.