Electric-field-assisted proton coupling enhanced oxygen evolution reaction.

The discovery of Mn-Ca complex in photosystem II stimulates research of manganese-based catalysts for oxygen evolution reaction (OER). However, conventional chemical strategies face challenges in regulating the four electron-proton processes of OER. Herein, we investigate alpha-manganese dioxide (α-MnO2) with typical MnIV-O-MnIII-HxO motifs as a model for adjusting proton coupling. We reveal that pre-equilibrium proton-coupled redox transition provides an adjustable energy profile for OER, paving the way for in-situ enhancing proton coupling through a new "reagent"- external electric field. Based on the α-MnO2 single-nanowire device, gate voltage induces a 4-fold increase in OER current density at 1.7 V versus reversible hydrogen electrode. Moreover, the proof-of-principle external electric field-assisted flow cell for water splitting demonstrates a 34% increase in current density and a 44.7 mW/cm² increase in net output power. These findings indicate an in-depth understanding of the role of proton-incorporated redox transition and develop practical approach for high-efficiency electrocatalysis.

Diffusiophoresis-Driven Stratification in Pressure-Sensitive Adhesive Films from Bimodal Waterborne Colloids.

The uses of pressure-sensitive adhesives (PSAs) are wide ranging, with applications including labels, tapes, and graphics. To achieve good adhesion, a PSA must exhibit a balance of viscous and elastic properties. Previous research has found that a thin, elastic surface layer on top of a softer, dissipative layer resulted in greater tack adhesion compared with the single layers. Superior properties were achieved through a bilayer obtained via successive depositions, which consume energy and time. To achieve a multilayered structure via a single deposition process, we have stratified mixtures of waterborne colloidal polymer particles with two different sizes: large poly(acrylate) adhesive particles (ca. 660 nm in diameter) and small poly(butyl acrylate) (pBA) particles (ca. 100 nm). We used two types of pBA within the particles: either viscoelastic pBA without an added cross-linker or elastic pBA with a fully cross-linked network. Stratified surface layers of deuterium-labeled pBA particles with thicknesses of at least 1 µm were found via elastic recoil detection and qualitatively verified via the analysis of surface topography. The extent of stratification increased with the evaporation rate; films that were dried slowest exhibited no stratification. This result is consistent with a model of diffusiophoresis. When the elastic, cross-linked pBA particles were stratified at the surface, the tack adhesion properties made a transition from brittle failure to tacky. For pBA without an added cross-linker, all adhesives showed fibrillation during debonding, but the extent of fibrillation increased when the films were stratified. These results demonstrate that the PSA structure can be controlled through the processing conditions to achieve enhanced properties. This research will aid the future development of layered or graded single-deposition PSAs with designed adhesive properties.

Safety, Efficacy, and Ease of Insertion of Gnana Laryngeal Airway (GLA-4): A Prospective Clinical Study Utilizing the Unique Laryngeal Mask Airway With a Suction Tubing.

INTRODUCTION: Utilizing laryngeal mask airways to maintain patients' airways is advantageous because it enables the anesthesiologist to keep the patient spontaneously inhaling and is less traumatic to the airway than intubation. Newer designs such as the Gnana laryngeal mask airway design permit real-time suctioning while the mask is on a patient. METHODS: This is a prospective observational study of the efficacy of Gnana laryngeal airway 4 (GLA-4) in 50 patients undergoing colonoscopy. Induction and maintenance of anesthesia were provided with propofol; GLA-4 was applied to secure the airway; and correct placement was verified. RESULTS: Fifty patients were included in the study (44% female, 56% male, mean age: 56.5 years, mean BMI: 33.3). Twelve patients were assigned American Society of Anesthesiologists (ASA) class 2, and 38 were assigned ASA class 3. The first attempt of GLA-4 insertion was successful in 47 patients, and two attempts were required for the successful placement of the GLA-4 in two patients. The successful placement was not achieved in one patient. The average time to successful insertion was 27.1 ± 3.9s. The average volume of oropharyngeal secretions suctioned through the suction catheter was 9.96 ± 2.31 mL. No intraoperative or postoperative complications occurred in the 50 patients. There were no reports of sore throat, hoarseness, dysphagia, or cough immediately postop. CONCLUSION: GLA-4 can be inserted safely with adequate periglottic occlusion. This laryngeal mask is unique and desirable due to its ability to evacuate oropharyngeal secretions while in place to prevent laryngospasm. To establish the role of GLA-4 in broader clinical situations, additional clinical trials and studies are required.

How Particle Deformability Influences the Surfactant Distribution in Colloidal Polymer Films.

The distribution of surfactants in waterborne colloidal polymer films is of significant interest for scientific understanding and defining surface properties in applications including pressure-sensitive adhesives and coatings. Because of negative effects on appearance, wetting, and adhesion, it is desirable to prevent surfactant accumulation at film surfaces. The effect of particle deformation on surfactant migration during film formation was previously investigated by Gromer et al. through simulations, but experimental investigations are lacking. Here, we study deuterium-labeled sodium dodecyl sulfate surfactant in a poly(butyl acrylate) latex model system. The particle deformability was varied via cross-linking of the intraparticle polymer chains by differing extents. The cross-linker concentration varied from 0 to 35 mol % in the copolymer, leading to a transition from viscoelastic to elastic. Ion beam analysis was used to probe the dry films and provide information on the near-surface depth distribution of surfactant. Films of nondeformable particles, containing the highest concentration of cross-linker, show no surfactant accumulation at the top surface. Films from particles partially deformed by capillary action show a distinct surfactant surface layer (ca. 150 nm thick). Films of coalesced particles, containing little or no cross-linker, show a very small amount of surfactant on the surface (ca. 20 nm thick). The observed results are explained by considering the effect of cross-linking on rubber elasticity and applying the viscous particle deformation model by Gromer et al. to elastically deformed particles. We find that partially deformed particles allow surfactant transport to the surface during film formation, whereas there is far less transport when skin formation acts as a barrier. With elastic particles, the surfactant is carried in the water phase as it falls beneath the surface of packed particles. The ability to exert control over surfactant distribution in waterborne colloidal films will aid in the design of new high-performance adhesives and coatings.

Generation of pro-and anti-inflammatory mediators after acetaminophen overdose in surviving and non-surviving patients.

Acetaminophen (APAP) overdose causes liver injury in animals and humans. Although well-studied in animals, limited longitudinal data exist on cytokine release after APAP overdose in patients. The purpose of this study was to quantify concentrations of cytokines in APAP overdose patients to determine if early cytokine or complement measurements can distinguish between surviving and non-surviving patients. Plasma was obtained from healthy controls, APAP overdose patients with no increase in liver transaminases, and surviving and non-surviving APAP overdose patients with severe liver injury. Interleukin-10 (IL-10), and CC chemokine ligand-2 (CCL2, MCP-1) were substantially elevated in surviving and non-surviving patients, whereas IL-6 and CXC chemokine ligand-8 (CXCL8, IL-8) had early elevations in a subset of patients only with liver injury. Day 1 IL-10 and IL-6 levels, and Day 2 CCL2, levels correlated positively with survival. There was no significant increase in IL-1α, IL-1ß or TNF-α in any patient during the first week after APAP. Monitoring cytokines such as CCL2 may be a good indicator of patient prognosis; furthermore, these data indicate the inflammatory response after APAP overdose in patients is not mediated by a second phase of inflammation driven by the inflammasome.

Effect of Long-term Nicotine Replacement Therapy vs Standard Smoking Cessation for Smokers With Chronic Lung Disease: A Randomized Clinical Trial.

Importance: Smokers with chronic obstructive pulmonary disease (COPD) have particular difficulty quitting. Long-term nicotine replacement therapy (LT-NRT) might offer a strategy for reducing harm from cigarettes and provide a pathway for later cessation. Objective: To compare the effect of LT-NRT vs standard smoking cessation (SSC) on exposure to cigarette smoke, harm related to smoking, and cessation among smokers with COPD. Design, Setting, and Participants: This unblinded, randomized clinical trial recruited smokers who self-reported a diagnosis of COPD at any level of readiness to quit from May 23, 2014, through November 30, 2015. The 12-month follow-up was completed December 6, 2016. Patients were recruited at a clinical research unit of an academic medical center. Analysis was based on intention to treat and performed from March 8 through November 30, 2017. Interventions: Standard smoking cessation treatment included 10 weeks of NRT and 4 follow-up counseling sessions for those willing to make a quit attempt. Long-term NRT included 12 months of NRT and 6 follow-up counseling sessions regardless of initial willingness to quit. Overall, 198 patients were randomized to SSC, and 197 were included in the primary analysis; 200 patients were randomized to LT-NRT, and 197 were included in the primary analysis. Main Outcomes and Measures: The primary outcome was 7-day abstinence verified by carbon monoxide (CO) levels at 12 months. Secondary outcomes included cigarettes smoked per day (CPD), exposure to CO, urinary excretion of 4-methylnitrosamino-1-3-pyridyl-1-butanol (NNAL) (a smoking-related carcinogen), and adverse events. Results: Among 398 patients who were randomized (59.8% female; mean [SD] age, 56.0 [9.3] years), the mean (SD) CPD was 23.1 (12.3). Twelve-month follow-up was completed by 373 participants (93.7%), and 394 (99.0%) were included in the primary analysis. At 12 months, CO-verified abstinence occurred in 23 of 197 participants (11.7%) in the SSC arm and 24 of 197 (12.2%) in the LT-NRT arm (risk difference, 0.5%; 95% CI, -5.9% to 6.9%). Continuing smokers in the SSC and LT-NRT arms had similar, significantly reduced harms caused by smoking, including cigarette consumption by 12.4 and 14.5 CPD, respectively, exhaled CO level by 5.5 and 7.8 ppm, respectively, and mean urinary NNAL excretion by 21.7% and 23.0%, respectively. In multivariate analyses, continuing smokers with greater adherence to NRT experienced less reduction in NNAL exposure. The frequency of major adverse cardiac events was similar in both groups. Conclusions and Relevance: Similar rates of cessation and similar reductions in exposure to tobacco smoke resulted with LT-NRT and SSC. Among continuing smokers, ongoing use of NRT was not associated with reductions in smoke exposure. Trial Registration: ClinicalTrials.gov Identifier: NCT02148445.

A Case of Sevoflurane Use during Pregnancy in the Management of Persistent Status Asthmaticus.

BACKGROUND: Sevoflurane is rarely used for the treatment of status asthmaticus. We report a case of sevoflurane hepatotoxicity in pregnancy with presentation similar to HELLP syndrome. CASE: A G2P1001 at 23 weeks in status asthmaticus presented with pCO2 > 130 and pH < 7. She was nonresponsive to traditional therapy. Sevoflurane was added for a 24 hr period. Respiratory status improved. Extubation occurred on day 12. Workup for preeclampsia spectrum disorders occurred due to maternal hypertension. Given the atypical presentation and hepatotoxicity, a liver biopsy was performed. Histologic features suggested drug induced hepatic injury. Liver function subsequently normalized. She delivered a term neonate without short-term complications. CONCLUSION: The use of sevoflurane is a treatment option of status asthmaticus during pregnancy. Providers should be aware of the potential for hepatotoxicity.

Plasma biomarkers to study mechanisms of liver injury in patients with hypoxic hepatitis.

BACKGROUND & AIMS: Hypoxic hepatitis is a clinical condition precipitated by prolonged periods of oxygen deprivation to the liver. It can have several underlying causes. Despite its prevalence in critically ill patients, which can reach upwards of 10%, very little is known about the mechanisms of injury. Thus, we set out to measure previously identified circulating biomarkers in an attempt to describe mechanisms of injury following hypoxic hepatitis. METHODS: Plasma from patients diagnosed with hypoxic hepatitis was collected for this study. Biomarkers of hepatocellular injury, mitochondrial damage and cell death were measured. These results were compared against results obtained from well-characterized acetaminophen overdose patients. RESULTS: At peak injury, ALT measured 4082±606 U/L and gradually decreased over 5 days, corresponding to the clinically observed pattern of hypoxic hepatitis. Levels of GDH showed a similar pattern, but neither ALT nor GDH were significantly higher in these patients than in acetaminophen patients. Plasma levels of DNA fragments mimicked hepatocellular injury as measured by ALT and miRNA-122. Interestingly, we found a significant increase in caspase-cleaved cytokeratin-18; however, the full-length form greatly exceeded the cleaved form at the time of maximum injury (45837±12085 vs 2528±1074 U/L). We also found an increase in acHMGB1 at later time points indicating a possible role of inflammation, but cytokine levels at these times were actually decreased relative to early time points. CONCLUSIONS: The mechanism of injury following hypoxic hepatitis involves mitochondrial damage and DNA fragmentation. Importantly, necrosis, rather than apoptosis, is the main mode of cell death.

Perception of Exercise-Induced Bronchoconstriction in College Athletes.

BACKGROUND: Exercise-induced bronchoconstriction (EIB) can lead to long-term respiratory illness and even death. EIB prevalence rates are both high and variable in college athletes. Also, prevalence rates may be underestimated due to ineffective screening. The purpose of this study is to investigate the prevalence of EIB and the perceived impact of EIB in college athletes via a self-report questionnaire. METHODS: A self-report EIB questionnaire was administered to college athletes on 8 different sports teams. Information collected was used to identify athletes who self-reported: (1) a history of EIB and/or asthma, (2) respiratory symptoms during exercise, (3) medication use, and (4) concern about EIB. RESULTS: Results showed that 56 of 196 athletes (28.6%) self-reported a history of EIB or asthma. Over half (52%) reported a history of EIB/asthma or current EIB symptoms. Forty-six of the 140 athletes (32.9%) who did not report a history of EIB or asthma indicated symptoms of EIB during sports, training, or exercise. Fourteen of 56 athletes (25%) self-reporting a history of EIB or asthma did not report the use of a respiratory medication. Nineteen of 196 athletes (9.7%) reported being concerned that EIB was adversely affecting their sports performance. CONCLUSIONS: College athletes self-report a high prevalence of EIB or asthma. Although college athletes may not report a history of EIB or asthma, they indicate symptoms of EIB. A majority of athletes reported a history or current symptoms related to EIB or asthma. Many athletes with a history of EIB or asthma are not taking any asthma medication. Last, athletes report concern about EIB adversely affecting their sports performance. More work is needed using a combination of a screening questionnaire and standardized EIB testing to develop a validated tool for accurately screening and diagnosing EIB in college athletes.

Prevalence of Exercise-Induced Bronchoconstriction Measured by Standardized Testing in Healthy College Athletes.

BACKGROUND: Exercise-induced bronchoconstriction (EIB) can lead to long-term respiratory illness and even death. EIB prevalence rates are both high and variable in college athletes. Prevalence rates may be underestimated due to ineffective testing and screening. The purpose of this study was to investigate the prevalence of EIB in college athletes by a standardized EIB test that can be used on many college campuses. In addition, we assessed the usefulness of self-reporting EIB/asthma (1) history, (2) symptoms, and (3) respiratory medication obtained from a simple screening questionnaire for predicting an EIB-positive athlete. METHODS: A standardized EIB test and self-report questionnaire were administered to college athletes on 10 different sports teams. Information collected included pulmonary function (spirometry), expired gas analysis (maximal oxygen uptake), CO2 production, minute ventilation, EIB/asthma history, current symptoms, and medication use. RESULTS: Results showed that 34 of 80 athletes (42.5%) were EIB-positive by standardized exercise testing. The majority (76.5 and 58.8%) of the 34 athletes who tested positive self-reported a negative history or no symptoms, respectively. Also, 79.4% of the athletes who tested positive for EIB reported not using a respiratory medication. There were no significant differences in a positive EIB test when assessing interactions for history (P = .93), current symptoms (P = .12), or respiratory medication use (P = .66). CONCLUSIONS: A high proportion of college athletes tested positive for EIB when using a standardized test. Positive history, current symptoms of EIB/asthma, and respiratory medication use were not predictive of a positive test. Many EIB-positive athletes are not using a respiratory medication. More work is needed to develop an effective screening tool and improve education for EIB in college athletes.

The Effect of Model Fidelity on Learning Outcomes of a Simulation-Based Education Program for Central Venous Catheter Insertion.

INTRODUCTION: Simulation-based education for central venous catheter (CVC) insertion has been repeatedly documented to improve performance, but the impact of simulation model fidelity has not been described. The aim of this study was to examine the impact of the physical fidelity of the simulation model on learning outcomes for a simulation-based education program for CVC insertion. METHODS: Forty consecutive residents rotating through the medical intensive care unit of an academic medical center completed a simulation-based education program for CVC insertion. The curriculum was designed in accordance with the principles of deliberate practice and mastery learning. Each resident underwent baseline skills testing and was then randomized to training on a commercially available CVC model with high physical fidelity (High-Fi group) or a simply constructed model with low physical fidelity (Low-Fi group) in a noninferiority trial. Upon completion of their medical intensive care unit rotation 4 weeks later, residents returned for repeat skills testing on the high-fidelity model using a 26-item checklist. RESULTS: The mean (SD) posttraining score on the 26-item checklist for the Low-Fi group was 23.8 (2.2) (91.5%) and was not inferior to the mean (SD) score for the High-Fi group of 22.5 (2.6) (86.5%) (P < 0.0001). Residents in both groups judged the training program to be highly useful despite perceiving a lesser degree of physical realism in the low-fidelity model compared with the high-fidelity model (P = 0.05). CONCLUSIONS: Simulation-based education using equipment with low physical fidelity can achieve learning outcomes comparable with those with high-fidelity equipment, as long as other aspects of fidelity are maintained and robust educational principles are applied during the design of the curriculum.

Time course of acetaminophen-protein adducts and acetaminophen metabolites in circulation of overdose patients and in HepaRG cells.

1. It has been suggested that acetaminophen (APAP)-protein adducts can be measured in circulation to diagnose APAP-induced liver injury. However, the full-time course of plasma adducts has not been studied specifically in early-presenting overdose patients. In fact, surprisingly little work has been done on the metabolism of APAP after overdose in general. 2. We measured APAP, five APAP metabolites and APAP-protein adducts in plasma samples from early- and late-presenting overdose patients, and APAP-protein adducts in culture medium from HepaRG cells. 3. In contrast to earlier rodents studies, we found that APAP-protein adducts were lower at early time points and peaked around the time of peak liver injury, suggesting that these adduct levels may take longer to become elevated or remain elevated than previously thought. 4. APAP and its major metabolites were elevated in plasma at early time points and rapidly decreased. 5. Although clinical measurement of APAP-protein adducts holds promise as a diagnostic tool, we suggest caution in its interpretation in very early-presenting patients. Our data also support the idea that sulfation is saturated even at low doses but glucuronidation has a much higher capacity, highlighting the importance of glucuronidation in APAP metabolism.

Glycodeoxycholic acid levels as prognostic biomarker in acetaminophen-induced acute liver failure patients.

Acetaminophen (APAP)-induced acute liver failure (ALF) remains a major clinical problem. Although a majority of patients recovers after severe liver injury, a subpopulation of patients proceeds to ALF. Bile acids are generated in the liver and accumulate in blood during liver injury, and as such, have been proposed as biomarkers for liver injury and dysfunction. The goal of this study was to determine whether individual bile acid levels could determine outcome in patients with APAP-induced ALF (AALF). Serum bile acid levels were measured in AALF patients using mass spectrometry. Bile acid levels were elevated 5-80-fold above control values in injured patients on day 1 after the overdose and decreased over the course of hospital stay. Interestingly, glycodeoxycholic acid (GDCA) was significantly increased in non-surviving AALF patients compared with survivors. GDCA values obtained at peak alanine aminotransferase (ALT) and from day 1 of admission indicated GDCA could predict survival in these patients by receiver-operating characteristic analysis (AUC = 0.70 for day 1, AUC = 0.68 for peak ALT). Of note, AALF patients also had significantly higher levels of serum bile acids than patients with active cholestatic liver injury. These data suggest measurements of GDCA in this patient cohort modestly predicted outcome and may serve as a prognostic biomarker. Furthermore, accumulation of bile acids in serum or plasma may be a result of liver cell dysfunction and not cholestasis, suggesting elevation of circulating bile acid levels may be a consequence and not a cause of liver injury.

Serum mitochondrial biomarkers and damage-associated molecular patterns are higher in acetaminophen overdose patients with poor outcome.

UNLABELLED: Acetaminophen (APAP) overdose is a major cause of acute liver failure (ALF). Numerous studies have shown that APAP hepatotoxicity in mice involves mitochondrial dysfunction, and recent data suggest that this is also the case in humans. We have previously shown that glutamate dehydrogenase (GDH), mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) fragments can be measured in circulation of overdose patients as mechanistic biomarkers of mitochondrial damage and damage-associated molecular patterns. In the present study, our aim was to determine whether these biomarkers are higher in serum from nonsurvivors of APAP-induced ALF (AALF), compared to survivors. GDH, mtDNA, and nDNA fragments were measured in serum from AALF patients who did (n = 34) or did not (n = 35) recover. Importantly, all three were significantly increased in patients who died, compared to those who survived (GDH: 450 ± 73 vs. 930 ± 145 U/L; mtDNA: 21 ± 6 vs. 48 ± 13 and 33 ± 10 vs. 43 ± 7 ng/mL for two different genes; nDNA fragments: 148 ± 13 vs. 210 ± 13% of control). Receiver operating characteristic (ROC) curve analyses revealed that nDNA fragments, GDH, and mtDNA were predictive of outcome (area under the curve [AUC], study admission: 0.73, 0.70, and 0.71 or 0.76, respectively, P < 0.05; AUC, time of peak ALT: 0.78, 0.71, and 0.71 or 0.76, respectively, P < 0.05), and the results were similar to those from the Model for End-Stage Liver Disease (MELD; AUC, peak MELD: 0.77; P < 0.05). CONCLUSIONS: Our data suggest that patients with more mitochondrial damage are less likely to survive, demonstrating that mitochondria are central in the mechanisms of APAP hepatotoxicity in humans. Clinically, serum nDNA fragments, GDH, and mtDNA could be useful as part of a panel of biomarkers to predict patient outcome. (Hepatology 2014;60:1336-1345).

Argininosuccinate synthetase as a plasma biomarker of liver injury after acetaminophen overdose in rodents and humans.

CONTEXT: New biomarkers are needed in acetaminophen (APAP) hepatotoxicity. Plasma argininosuccinate synthetase (ASS) is a promising candidate. OBJECTIVE: Characterize ASS in APAP hepatotoxicity. METHODS: ASS was measured in plasma from rodents and humans with APAP hepatotoxicity. RESULTS: In mice, ASS increased before injury, peaked before alanine aminotransferase (ALT) and decreased rapidly. Fischer rats had a greater increase in ASS relative to ALT. Patients with abnormal liver test results had very high ASS compared to controls. ASS appeared to increase early in some patients, and declined rapidly in all. CONCLUSIONS: ASS may be a useful biomarker of acute cell death in APAP hepatotoxicity.

Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans.

Following acetaminophen (APAP) overdose there is an inflammatory response triggered by the release of cellular contents from necrotic hepatocytes into the systemic circulation which initiates the recruitment of neutrophils into the liver. It has been demonstrated that neutrophils do not contribute to APAP-induced liver injury, but their role and the role of NADPH oxidase in injury resolution are controversial. C57BL/6 mice were subjected to APAP overdose and neutrophil activation status was determined during liver injury and liver regeneration. Additionally, human APAP overdose patients (ALT: >800 U/L) had serial blood draws during the injury and recovery phases for the determination of neutrophil activation. Neutrophils in the peripheral blood of mice showed an increasing activation status (CD11b expression and ROS priming) during and after the peak of injury but returned to baseline levels prior to complete injury resolution. Hepatic sequestered neutrophils showed an increased and sustained CD11b expression, but no ROS priming was observed. Confirming that NADPH oxidase is not critical to injury resolution, gp91(phox)⁻/⁻ mice following APAP overdose displayed no alteration in injury resolution. Peripheral blood from APAP overdose patients also showed increased neutrophil activation status after the peak of liver injury and remained elevated until discharge from the hospital. In mice and humans, markers of activation, like ROS priming, were increased and sustained well after active liver injury had subsided. The similar findings between surviving patients and mice indicate that neutrophil activation may be a critical event for host defense or injury resolution following APAP overdose, but not a contributing factor to APAP-induced injury.

Circulating acylcarnitines as biomarkers of mitochondrial dysfunction after acetaminophen overdose in mice and humans.

Acetaminophen (APAP) is a widely used analgesic. However, APAP overdose is hepatotoxic and is the primary cause of acute liver failure in the developed world. The mechanism of APAP-induced liver injury begins with protein binding and involves mitochondrial dysfunction and oxidative stress. Recent efforts to discover blood biomarkers of mitochondrial damage have identified increased plasma glutamate dehydrogenase activity and mitochondrial DNA concentration in APAP overdose patients. However, a problem with these markers is that they are too large to be released from cells without cell death or loss of membrane integrity. Metabolomic studies are more likely to reveal biomarkers that are useful at early time points, before injury begins. Similar to earlier work, our metabolomic studies revealed that acylcarnitines are elevated in serum from mice after treatment with toxic doses of APAP. Importantly, a comparison with furosemide demonstrated that increased serum acylcarnitines are specific for mitochondrial dysfunction. However, when we measured these compounds in plasma from humans with liver injury after APAP overdose, we could not detect any significant differences from control groups. Further experiments with mice showed that N-acetylcysteine, the antidote for APAP overdose in humans, can reduce acylcarnitine levels in serum. Altogether, our data do not support the clinical measurement of acylcarnitines in blood after APAP overdose due to the standard N-acetylcysteine treatment in patients, but strongly suggest that acylcarnitines would be useful mechanistic biomarkers in other forms of liver injury involving mitochondrial dysfunction.

Lysosomal instability and cathepsin B release during acetaminophen hepatotoxicity.

Acetaminophen (APAP) overdose is currently the most frequent cause of drug-induced liver failure in the United States. Recently, it was shown that lysosomal iron translocates to mitochondria where it contributes to the collapse of the mitochondrial membrane potential. Therefore, the purpose of this study was to investigate whether cathepsin B, a lysosomal protease, is involved in APAP-induced hepatotoxicity. Cathepsin B activity was measured in subcellular liver fractions of C57Bl/6 mice 3 hr after 300 mg/kg APAP treatment. There was a significant increase in cytoplasmic cathepsin activity, concurrent with a decrease in microsomal activity, indicative of lysosomal cathepsin B release. To investigate the effect of cathepsin B on hepatotoxicity, the cathepsin inhibitor AC-LVK-CHO was given 1 hr prior to 300 mg/kg APAP treatment along with vehicle control. There was no difference between groups in serum alanine aminotransferase (ALT) values, or by histological evaluation of necrosis, although cathepsin B activity was inhibited by 70-80% compared with controls. These findings were confirmed with a different inhibitor (z-FA-fmk) in vivo and in vitro. Hepatocytes were exposed to 5 mM acetaminophen. Lysotracker staining confirmed lysosomal instability and cathepsin B release, but there was no reduction in cell death after treatment with cathepsin B inhibitors. Finally, cathepsin B release was measured in clinical samples from patients with APAP-induced liver injury. Low levels of cathepsin B were released into plasma from overdose patients. APAP overdose causes lysosomal instability and release of cathepsin B into the cytosol but does not contribute to liver injury under these conditions.