Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury.

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

Characterization of constitutive ER-phagy of excess membrane proteins.

Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Whereas endogenously expressed ER resident proteins serve as cargos at a basal level, this level can be induced by overexpression of membrane proteins that are not ER residents. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.

Deficiencies in vesicular transport mediated by TRAPPC4 are associated with severe syndromic intellectual disability.

The conserved transport protein particle (TRAPP) complexes regulate key trafficking events and are required for autophagy. TRAPPC4, like its yeast Trs23 orthologue, is a core component of the TRAPP complexes and one of the essential subunits for guanine nucleotide exchange factor activity for Rab1 GTPase. Pathogenic variants in specific TRAPP subunits are associated with neurological disorders. We undertook exome sequencing in three unrelated families of Caucasian, Turkish and French-Canadian ethnicities with seven affected children that showed features of early-onset seizures, developmental delay, microcephaly, sensorineural deafness, spastic quadriparesis and progressive cortical and cerebellar atrophy in an effort to determine the genetic aetiology underlying neurodevelopmental disorders. All seven affected subjects shared the same identical rare, homozygous, potentially pathogenic variant in a non-canonical, well-conserved splice site within TRAPPC4 (hg19:chr11:g.118890966A>G; TRAPPC4: NM_016146.5; c.454+3A>G). Single nucleotide polymorphism array analysis revealed there was no haplotype shared between the tested Turkish and Caucasian families suggestive of a variant hotspot region rather than a founder effect. In silico analysis predicted the variant to cause aberrant splicing. Consistent with this, experimental evidence showed both a reduction in full-length transcript levels and an increase in levels of a shorter transcript missing exon 3, suggestive of an incompletely penetrant splice defect. TRAPPC4 protein levels were significantly reduced whilst levels of other TRAPP complex subunits remained unaffected. Native polyacrylamide gel electrophoresis and size exclusion chromatography demonstrated a defect in TRAPP complex assembly and/or stability. Intracellular trafficking through the Golgi using the marker protein VSVG-GFP-ts045 demonstrated significantly delayed entry into and exit from the Golgi in fibroblasts derived from one of the affected subjects. Lentiviral expression of wild-type TRAPPC4 in these fibroblasts restored trafficking, suggesting that the trafficking defect was due to reduced TRAPPC4 levels. Consistent with the recent association of the TRAPP complex with autophagy, we found that the fibroblasts had a basal autophagy defect and a delay in autophagic flux, possibly due to unsealed autophagosomes. These results were validated using a yeast trs23 temperature sensitive variant that exhibits constitutive and stress-induced autophagic defects at permissive temperature and a secretory defect at restrictive temperature. In summary we provide strong evidence for pathogenicity of this variant in a member of the core TRAPP subunit, TRAPPC4 that associates with vesicular trafficking and autophagy defects. This is the first report of a TRAPPC4 variant, and our findings add to the growing number of TRAPP-associated neurological disorders.

Quantitation of γ-aminobutyric acid in equine plasma by hydrophilic interaction liquid chromatography with tandem mass spectrometry.

γ-Aminobutyric acid is the principal inhibitory neurotransmitter in the central nervous system and regulates the neuronal excitability. There has been anecdotal evidence that γ-aminobutyric acid has been used within a few hours prior to competition in equine sports to calm down nervous horses. However, regulating the use of γ-aminobutyric acid is challenging because it is an endogenous substance in the horse. γ-Aminobutyric acid is usually present at low ng/mL levels in equine plasma; therefore, a sensitive method has to be developed to quantify these low background levels. Measuring low concentrations of endogenous γ-aminobutyric acid is essential to establish a threshold that can be used to differentiate levels attributable to exogenous administrations of γ-aminobutyric acid. A hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the quantitation of γ-aminobutyric acid in equine plasma. Calibrators were prepared in artificial surrogate matrix consisting of 35 mg/mL equine serum albumin in phosphate buffered saline. Samples were prepared by protein precipitation with acetonitrile. Utilizing this methodology, a total of 403 equine plasma samples collected post-competition from horses participating in equestrian events in Canada were analyzed.

Development and validation of a hydrophilic interaction liquid chromatography with tandem mass spectrometry method for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine.

A sensitive hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine. The method is highly sensitive and specific with good precision and accuracy. In plasma the limit of detection and limit of quantification are 0.03 and 0.1 ng/mL, respectively, for both analytes. In urine the limit of detection and limit of quantification are 0.3 and 1 ng/mL, respectively, for both analytes. The suitability of the method for doping control analysis in equine species is demonstrated by analyzing postadministration samples collected after a single intravenous administration of 50 mg etilefrine to a standardbred mare. Etilefrine was detected up to 120 h in urine and up to 48 h in plasma. Etilefrine is highly conjugated in equine urine whereas it exists in the free form in equine plasma. Therefore, enzyme hydrolysis prior to sample preparation is recommended for the detection and quantification of etilefrine and oxilofrine in equine urine.

Parents Experiences of Racism in the Neonatal Intensive Care Unit.

Few studies have investigated parent's experiences with racism in the neonatal intensive care unit (NICU). Our objective was to explore how parents perceive their interactions with NICU staff and if/how racism in the NICU was experienced. Parents of infants receiving care in an urban NICU completed fixed choice surveys regarding their experiences and demographics, with 6 open-ended questions to elaborate on their fixed-choice responses. Using a constant comparative method informed by Constructivist Grounded Theory, we identified 3 main themes from the comments provided by 97 respondents: Care and harm coexisting, racism often manifesting as neglectful care, and the power differential is most impactful during times of parent advocacy. Parents spoke positively regarding their experiences and also reported disparate treatment attributed to their racial/ethnic identity. Racism was experienced by inappropriate comments and apathy toward parent requests, occurring during intimate interactions between staff and parents. Descriptions of parental advocacy efforts highlighted the lack of power they held in relation to the NICU staff. We recommend strengthening the focus on equity and mitigating power imbalances in the NICU.

Detection and elimination profile of cathinone in equine after norephedrine (Propalin®) administration using a validated liquid chromatography-tandem mass spectrometry method.

Cathinone is the principal psychostimulant present in the leaves of khat shrub, which are widely used in East Africa and the Arab peninsula as an amphetamine-like stimulant. Cathinone readily undergoes metabolism in vivo to form less potent cathine and norephedrine as the metabolites. However, the presence of cathine and norephedrine in biological fluids cannot be used as an indicator of cathinone administration. The metabolism of pseudoephedrine and ephedrine, commonly used in cold and allergy medications, also produces cathine and norephedrine, respectively, as the metabolites. Besides, cathine and norephedrine may also originate from the ingestion of nutritional supplemental products containing extracts of Ephedra species. In Canada, ephedrine and norephedrine are available for veterinary use, whereas cathinone is not approved for human or veterinary use. In this article, the detection of cathinone in equine after administration of norephedrine is reported. To the best of our knowledge, this is the first such report in any species where administration of norephedrine or ephedrine generates cathinone as the metabolite. This observation is quite significant, because in equine detection of cathinone in biological fluids could be due to administration of the potent stimulant cathinone or the nonpotent stimulant norephedrine. A single oral dose of 450 mg norephedrine was administered to four Standardbred mares. Plasma and urine samples were collected up to 120 h after administration. The amount of cathinone and norephedrine detected in post administration samples was quantified using a highly sensitive, specific, and validated liquid chromatography-tandem mass spectrometry method. Using these results, we constructed elimination profiles for cathinone and norephedrine in equine plasma and urine. A mechanism that generates a geminal diol as an intermediate is postulated for this in vivo conversion of norephedrine to cathinone. Cathinone was also detected in samples collected after a single intramuscular administration of 200 mg ephedrine and oral administration of 300 mg ephedrine in equine.

Developing Bespoke High Volume Low Complexity (HVLC) Theatre Lists With a Focus on Training to Address the Impact of COVID-19: A Pilot Study.

Introduction The COVID-19 pandemic has had an unprecedented impact on both healthcare delivery and surgical training. There have been significant efforts to manage the growing elective waiting list backlog whilst addressing the training deficit. We outline a successful pilot high volume low complexity (HVLC) program held at the Croydon Elective Centre between 2021-2022 which aimed to amalgamate training and elective recovery. Methods Two pilot HVLC training lists were carried out in June 2021 and March 2022. Three parallel theatre lists on each date were supervised by a single consultant floor trainer. All lists followed a standard pre-defined HVLC protocol. Trainees and trainers were invited to participate and encouraged to utilize these lists to sign off relevant work-based assessments. HVLC cases included hernia repairs and simple lesion excisions. Patient, theatre staff, and trainee experiences were collated via questionnaires. Results A total of one consultant supervisor, six trainers, and eight trainees participated in the pilot with a total of 34 elective procedures performed on 29 patients. The mean patient age was 52.4 years with 8 out of 29 patients being female. Of these patients 41.4% were American Society of Anaesthesiologists (ASA) Classification one, 51.72% were ASA two and 6.9% were ASA three. No patients to date were readmitted to the hospital post-operatively or presented with post-operative complications. One hundred percent of trainees felt satisfied with the training and would recommend it to a colleague. Conclusion The training deficit that developed during the first COVID-19 pandemic wave has been compounded by the second and third waves, and trainees are concerned that further waves are anticipated. Returning to operating is vital and our approach has been shown to improve training, whilst maintaining patient safety and accelerating elective waiting list recovery.

Dual function of Rab1A in secretion and autophagy: hypervariable domain dependence.

We currently understand how the different intracellular pathways, secretion, endocytosis, and autophagy are regulated by small GTPases. In contrast, it is unclear how these pathways are coordinated to ensure efficient cellular response to stress. Rab GTPases localize to specific organelles through their hypervariable domain (HVD) to regulate discrete steps of individual pathways. Here, we explored the dual role of Rab1A/B (92% identity) in secretion and autophagy. We show that although either Rab1A or Rab1B is required for secretion, Rab1A, but not Rab1B, localizes to autophagosomes and is required early in stress-induced autophagy. Moreover, replacing the HVD of Rab1B with that of Rab1A enables Rab1B to localize to autophagosomes and regulate autophagy. Therefore, Rab1A-HVD is required for the dual functionality of a single Rab in two different pathways: secretion and autophagy. In addition to this mechanistic insight, these findings are relevant to human health because both the pathways and Rab1A/B were implicated in diseases ranging from cancer to neurodegeneration.

Bouveret syndrome: A challenging case of impacted gallstone within the fourth part of the duodenum.

INTRODUCTION AND IMPORTANCE: Bouveret syndrome is a rare condition characterised by gastric outlet obstruction secondary to a gallstone fistulating into the proximal duodenum or pylorus. Although rare, this condition carries a high mortality rate and no current standardised guidelines for management. CASE PRESENTATION: We present a case of a patient in their 60s with recurrent small bowel obstruction secondary to a cholecysto-duodenal fistula and large gallstone which became impacted in the fourth part of the duodenum. The patient had a P-POSSUM Score of 14% mortality and 60% morbidity risk, had multiple co-morbidities, was bedbound, BMI 59 and had been deemed high risk for general anaesthetic at oncology centre for a 10 × 10 cm likely gynaecological malignancy a month prior to this admission. CLINICAL DISCUSSION: In contrast to existing literature, endoscopic lithotripsy was considered but not attempted due to unavailability of this service locally. Surgical intervention was decided based on radiological features of impending duodenal perforation on CT imaging and multiple disciplinary team discussion. The patient was managed with open enterolithotomy at the duodeno-jejunal (DJ) flexure and discharged 3 weeks post-operatively at her pre-operative baseline. CONCLUSION: This is the first report to our knowledge to describe successful surgical management of a gallstone impacted in the fourth part of the duodenum. In cases where anatomical location of impaction precludes retrieval via simple gastrostomy, we suggest using high pressure flush to mobilise the stone to more favourable location distally. We emphasise that stone size should be considered when planning surgical management.

Post-operative antibiotics for cutaneous abscess after incision and drainage: Variations in clinical practice.

Background: Acute cutaneous abscess is a common surgical condition that mostly requires incision and drainage. Despite this, there is no standardized national or international guidance on post-operative antibiotics prescription. Traditionally, antibiotics are not indicated unless complications and/or risk factors such as immunocompromisation, diabetes or cellulitis exist. We aimed to study the local practice for post-operative antibiotics prescription for cutaneous abscesses in a UK university teaching hospital. Methods: Retrospective data collection for emergency general surgical admissions for a period of 6 months was carried out. All patients with cutaneous abscesses were included in this analysis. Scrotal, breast and limb abscesses were excluded. Patients' demographics, co-morbidities and complications, including local (cellulitis, necrosis) and systemic (e.g sepsis), were studied. Approval for access to patient data was granted by the local clinical governance department prior to the commencement of this study. Computations were performed using IBM SPSS version 26. Chi square (X 2), Pearson correlation (r), one or two samples t-test (one or two tailed) were applied. Results: A total of 148 patients were included. The mean age was 40 years (55 % males). The most common site of abscess was perianal (27.7 %), followed by pilonidal (20.3 %) and axilla (16.9 %). A total of 107 (73 %) were managed surgically with incision and drainage, and of these 92 (86 %) were managed within 24 h. Altogether, 83 (76 %) were prescribed post-operative antibiotics, while only 25 (23 %) had indications. The most used post-operative empirical antibiotics was co-amoxiclav (59 %). There was a significant relationship between 'abscess site' × 'antibiotics' [X 2 (36)=54.8, P=0.023]. A total of 103 patients' average duration of post-operative antibiotics was 7.2 (sd 2.9) days. Ten patients subject to readmission spent an average of 8.4 (sd 3.8) days on antibiotics. Conclusions: There were variations in clinical practice regarding post-operative antibiotic prescription for cutaneous abscesses. Research is required in the future in cooperation with microbiologists to develop a standardized evidence-based treatment protocol for the management of such a common surgical condition.

Internalization and trafficking of CSPG-bound recombinant VAR2CSA lectins in cancer cells.

Proteoglycans are proteins that are modified with glycosaminoglycan chains. Chondroitin sulfate proteoglycans (CSPGs) are currently being exploited as targets for drug-delivery in various cancer indications, however basic knowledge on how CSPGs are internalized in tumor cells is lacking. In this study we took advantage of a recombinant CSPG-binding lectin VAR2CSA (rVAR2) to track internalization and cell fate of CSPGs in tumor cells. We found that rVAR2 is internalized into cancer cells via multiple internalization mechanisms after initial docking on cell surface CSPGs. Regardless of the internalization pathway used, CSPG-bound rVAR2 was trafficked to the early endosomes in an energy-dependent manner but not further transported to the lysosomal compartment. Instead, internalized CSPG-bound rVAR2 proteins were secreted with exosomes to the extracellular environment in a strictly chondroitin sulfate-dependent manner. In summary, our work describes the cell fate of rVAR2 proteins in tumor cells after initial binding to CSPGs, which can be further used to inform development of rVAR2-drug conjugates and other therapeutics targeting CSPGs.

iCn3D: From Web-Based 3D Viewer to Structural Analysis Tool in Batch Mode.

iCn3D was initially developed as a web-based 3D molecular viewer. It then evolved from visualization into a full-featured interactive structural analysis software. It became a collaborative research instrument through the sharing of permanent, shortened URLs that encapsulate not only annotated visual molecular scenes, but also all underlying data and analysis scripts in a FAIR manner. More recently, with the growth of structural databases, the need to analyze large structural datasets systematically led us to use Python scripts and convert the code to be used in Node. js scripts. We showed a few examples of Python scripts at https://github.com/ncbi/icn3d/tree/master/icn3dpython to export secondary structures or PNG images from iCn3D. Users just need to replace the URL in the Python scripts to export other annotations from iCn3D. Furthermore, any interactive iCn3D feature can be converted into a Node. js script to be run in batch mode, enabling an interactive analysis performed on one or a handful of protein complexes to be scaled up to analysis features of large ensembles of structures. Currently available Node. js analysis scripts examples are available at https://github.com/ncbi/icn3d/tree/master/icn3dnode. This development will enable ensemble analyses on growing structural databases such as AlphaFold or RoseTTAFold on one hand and Electron Microscopy on the other. In this paper, we also review new features such as DelPhi electrostatic potential, 3D view of mutations, alignment of multiple chains, assembly of multiple structures by realignment, dynamic symmetry calculation, 2D cartoons at different levels, interactive contact maps, and use of iCn3D in Jupyter Notebook as described at https://pypi.org/project/icn3dpy.

Endometriosis of the Small Bowel: A Diagnostic Enigma.

Endometriosis refers to the implantation and proliferation of endometrial tissue outside the uterus. Small bowel endometriosis is an uncommon location for endometrial deposits and when present, it can pose diagnostic difficulty. Here, we present a case of a 50-year-old female with small bowel endometriosis who suffered from recurrent attacks of colicky abdominal pain for few months. Her cross-sectional investigations remained largely inconclusive. Ultimately, she underwent diagnostic laparoscopy which was diagnostic and therapeutic.

Power-Tool Use in Orthopaedic Surgery: Iatrogenic Injury, Its Detection, and Technological Advances: A Systematic Review.

BACKGROUND: Power tools are an integral part of orthopaedic surgery but have the capacity to cause iatrogenic injury. With this systematic review, we aimed to investigate the prevalence of iatrogenic injury due to the use of power tools in orthopaedic surgery and to discuss the current methods that can be used to reduce injury. METHODS: We performed a systematic review of English-language studies related to power tools and iatrogenic injuries using a keyword search in MEDLINE, Embase, PubMed, and Scopus databases. Exclusion criteria included injuries related to cast-saw use, temperature-induced damage, and complications not clearly related to power-tool use. RESULTS: A total of 3,694 abstracts were retrieved, and 88 studies were included in the final analysis. Few studies and individual case reports looked directly at the prevalence of injury due to power tools. These included 2 studies looking at the frequency of vascular injury during femoral fracture fixation (0.49% and 0.2%), 2 studies investigating the frequency of vertebral artery injury during spinal surgery (0.5% and 0.08%), and 4 studies investigating vascular injury during total joint arthroplasty (1 study involving 138 vascular injuries in 124 patients, 2 studies noting 0.13% and 0.1% incidence, and 1 questionnaire sent electronically to surgeons). There are multiple methods for preventing damage during power-tool use. These include the use of robotics and simulation, specific drill settings, and real-time feedback techniques such as spectroscopy and electromyography. CONCLUSIONS: Power tools have the potential to cause iatrogenic injury to surrounding structures during orthopaedic surgery. Fortunately, the published literature suggests that the frequency of iatrogenic injury using orthopaedic power tools is low. There are multiple technologies available to reduce damage using power tools. In high-risk operations, the use of advanced technologies to reduce the chance of iatrogenic injury should be considered. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Controlling the spread of COVID-19 on college campuses.

This research was done during the DOMath program at Duke University from May 18 to July 10, 2020. At the time, Duke and other universities across the country were wrestling with the question of how to safely welcome students back to campus in the Fall. Because of this, our project focused on using mathematical models to evaluate strategies to suppress the spread of the virus on campus, specifically in dorms and in classrooms. For dorms, we show that giving students single rooms rather than double rooms can substantially reduce virus spread. For classrooms, we show that moving classes with size above some cutoff online can make the basic reproduction number $ R_0 < 1 $, preventing a wide spread epidemic. The cutoff will depend on the contagiousness of the disease in classrooms.

Measuring bone stiffness using spherical indentation.

OBJECTIVES: Bone material properties are a major determinant of bone health in older age, both in terms of fracture risk and implant fixation, in orthopaedics and dentistry. Bone is an anisotropic and hierarchical material so its measured material properties depend upon the scale of metric used. The scale used should reflect the clinical problem, whether it is fracture risk, a whole bone problem, or implant stability, at the millimetre-scale. Indentation, an engineering technique involving pressing a hard-tipped material into another material with a known force, may be able to assess bone stiffness at the millimetre-scale (the apparent elastic modulus). We aimed to investigate whether spherical-tip indentation could reliably measure the apparent elastic modulus of human cortical bone. MATERIALS AND METHODS: Cortical bone samples were retrieved from the femoral necks of nineteen patients undergoing total hip replacement surgery (10 females, 9 males, mean age: 69 years). The samples underwent indentation using a 1.5 mm diameter, ruby, spherical indenter tip, with sixty indentations per patient sample, across six locations on the bone surfaces, with ten repeated indentations at each of the six locations. The samples then underwent mechanical compression testing. The repeatability of indentation measurements of elastic modulus was assessed using the co-efficient of repeatability and the correlation between the bone elastic modulus measured by indentation and compression testing was analysed by least-squares regression. RESULTS: In total, 1140 indentations in total were performed. Indentation was found to be repeatable for indentations performed at the same locations on the bone samples with a mean co-efficient of repeatability of 0.4 GigaPascals (GPa), confidence interval (C.I): 0.33-0.42 GPa. There was variation in the indentation modulus results between different locations on the bone samples (mean co-efficient of repeatability: 3.1 GPa, C.I: 2.2-3.90 GPa). No clear correlation was observed between indentation and compression values of bone elastic modulus (r = 0.33, p = 0.17). The mean apparent elastic modulus obtained by spherical indentation was 9.9 GPa, the standard deviation for each indent cycle was 0.11 GPa, and the standard deviation between locations on the same sample was 1.01 GPa. The mean compression apparent elastic modulus was 4.42 GPa, standard deviation 1.02 GPa. DISCUSSION: Spherical-tip indentation was found to be a repeatable test for measuring the elastic modulus of human cortical bone, demonstrated by a low co-efficient of repeatability in this study. It could not, however, reliably predict cortical bone elastic modulus determined by platens compression testing in this study. This may be due to indentation only probing mechanical properties at the micro-scale while platens compression testing assesses millimetre length-scale properties. Improvements to the testing technique, including the use of a larger diameter spherical indenter tip, may improve the measurement of bone stiffness at the millimetre scale and should be investigated further.

Detection of myo-inositol tris pyrophosphate (ITPP) in equine following an administration of ITPP.

Myo-Inositol tris pyrophosphate (ITPP) is a powerful allosteric modulator of haemoglobin that increases oxygen-releasing capacity of red blood cells. It is capable of crossing the red blood cell membrane unlike its open polyphosphate analog myo-inositol hexakisphosphate (IHP). Systemic administration of ITPP enhanced the exercise capacity in mice. There have been rumours of its abuse in the horse racing industry to enhance the performance of racing horses. In this paper, the detection of ITPP in equine plasma and urine after an administration of ITPP is reported. A Standardbred mare was administered 200 mg of ITPP intravenously. Urine and plasma samples were collected up to 120 h post administration and analyzed for ITPP by liquid chromatography-tandem mass spectrometry. ITPP was detected in post administration plasma samples up to 6 hours. The peak concentration was detected at 5 min post administration. In urine, ITPP was detected up to 24 h post administration. The peak concentration was detected at 1.5 h post administration.

Detection of efaproxiral (RSR13) and its metabolites in equine by liquid chromatography tandem mass spectrometry.

Efaproxiral (RSR 13) is an experimental synthetic allosteric modifier of haemoglobin (Hb) that acts by increasing the release of oxygen from Hb to the surrounding tissues. It has been shown to increase maximum oxygen uptake (VO(2max)) in a canine skeletal muscle model. The ability to increase maximal muscle oxygen uptake makes efaproxiral a potential performance-enhancing agent and is therefore prohibited by the World Anti-Doping Agency. In this study, a method for the detection and elimination of efaproxiral in equine plasma and urine after a 2.5 g intravenous administration of efaproxiral is described. Post administration plasma and urine samples were collected up to 120 h. Efaproxiral was detected up to 120 h in urine and up to 78 h in plasma. In plasma, the peak concentration was 42 µg/ml and detected at 5 min post administration. In urine, the peak concentration was 2.8 mg/ml and detected at 0-1 h post administration. A validated liquid chromatography tandem mass spectrometry method was used for the quantitation of efaproxiral in equine plasma and urine. The limit of detection of the method is 0.05 ng/ml in plasma and 0.1 ng/ml in urine. The method is highly sensitive and specific with good precision, accuracy and recovery. The manuscript also describes the systematic identification of efaproxiral metabolites detected in post administration equine urine samples. The metabolites were identified by use of enhanced mass spectra and enhanced product ion scans. Both positive and negative mode ionizations were utilized for metabolite identification and plausible fragmentation pathways were proposed for the phase 1 metabolite identified. In addition to free efaproxiral, one phase 1 metabolite and two phase 2 metabolites were identified in post administration urine.