Carolyn V. Coulter

Selective targeting of a redox-active ubiquinone to mitochondria within cells : antioxidant and antiapoptotic properties

With the recognition of the central role of mitochondria in apoptosis, there is a need to develop specific tools to manipulate mitochondrial function within cells. Here we report on the development of a novel antioxidant that selectively blocks mitochondrial oxidative damage, enabling the roles of mitochondrial oxidative stress in different types of cell death to be inferred. This antioxidant, named mitoQ, is a ubiquinone derivative targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation through an aliphatic carbon chain. Due to the large mitochondrial membrane potential, the cation was accumulated within mitochondria inside cells, where the ubiquinone moiety inserted into the lipid bilayer and was reduced by the respiratory chain. The ubiquinol derivative thus formed was an effective antioxidant that prevented lipid peroxidation and protected mitochondria from oxidative damage. After detoxifying a reactive oxygen species, the ubiquinol moiety was regenerated by the respiratory chain enabling its antioxidant activity to be recycled. In cell culture studies, the mitochondrially localized antioxidant protected mammalian cells from hydrogen peroxide-induced apoptosis but not from apoptosis induced by staurosporine or tumor necrosis factor-α. This was compared with untargeted ubiquinone analogs, which were ineffective in preventing apoptosis. These results suggest that mitochondrial oxidative stress may be a critical step in apoptosis induced by hydrogen peroxide but not for apoptosis induced by staurosporine or tumor necrosis factor-α. We have shown that selectively manipulating mitochondrial antioxidant status with targeted and recyclable antioxidants is a feasible approach to investigate the role of mitochondrial oxidative damage in apoptotic cell death. This approach will have further applications in investigating mitochondrial dysfunction in a range of experimental models.

Mitochondrially targeted antioxidants and thiol reagents.

Mitochondrial oxidative damage and dysfunction contributes to a number of cell pathologies. To investigate how this damage affects cell function we have developed mitochondrially targeted antioxidants and thiol reagents by covalently linking them to lipophilic cations. The cation drives the selective accumulation of these reagents into mitochondria within cells where the antioxidants decrease oxidative damage and the thiol reagents enable measurement of the redox status of thiol proteins. In conjunction with cell and animal models of apoptosis, oxidative damage, and nitric oxide signaling, these molecules may provide new insights into the roles of mitochondria in human pathologies.

Red Blood Cell Survival in Long-term Dialysis Patients

BACKGROUND Shortening of red blood cell (RBC) survival contributes to the anemia of chronic kidney disease. The toxic uremic environment accounts for the decreased RBC life span. The contribution of mechanical damage caused by hemodialysis to the shortened life span is unclear. Reductions up to 70% in RBC survival have been reported in uremic patients. To date, no accurate well-controlled RBC survival data exist in dialysis patients treated using different dialysis modalities and receiving erythropoiesis-stimulating agent (ESA) therapy. The aim of this study was to determine RBC survival in hemodialysis (HD) and peritoneal dialysis (PD) patients compared with healthy persons. STUDY DESIGN Observational study. SETTING & PARTICIPANTS 14 HD patients and 5 PD patients were recruited from the dialysis unit. Healthy volunteers (n = 14) age- and sex-matched to HD participants were included. All dialysis patients received either ESA therapy or regular iron supplementation. PREDICTOR Dialysis patients versus age- and sex-matched healthy controls. OUTCOMES RBC survival. MEASUREMENTS RBC survival was determined using radioactive chromium labeling. RESULTS More than 85% of dialysis patients were anemic (hemoglobin, 12.0 ± 1.1 g/dL); hemoglobin concentrations were not significantly different between HD and PD patients. Median RBC survival was significantly decreased by 20% in HD patients compared with healthy controls: 58.1 (25th-75th percentile, 54.6-71.2) versus 72.9 (25th-75th percentile, 63.4-87.8) days (P = 0.02). No difference was shown between the PD and HD groups: 55.3 (25th-75th percentile, 49.0-60.2) versus 58.1 (25th-75th percentile, 54.6-71.2) days (P = 0.2). LIMITATIONS Label loss from RBCs associated with the chromium 51 labeling technique needs to be accounted for in the interpretation of RBC survival data. CONCLUSIONS Despite current ESA therapy, decreased RBC survival contributes to chronic kidney disease-related anemia, although the reduction is less than previously reported. There does not appear to be net mechanical damage associated with HD therapy resulting in decreased RBC life span.

Assessment of markers of glycaemic control in diabetic patients with chronic kidney disease using continuous glucose monitoring.

Aim:  Due to altered red blood cell survival and erythropoietin therapy glycated haemoglobin (HbA1c) may not accurately reflect long‐term glycaemic control in patients with diabetes and chronic kidney disease (CKD). Glycated albumin (GA) and fructosamine are alternative markers of glycaemia. The aim of this study was to investigate the accuracy of HbA1c, GA and fructosamine as indicators of glycaemic control using continuous glucose monitoring.

Methanol and ethylene glycol acute poisonings - predictors of mortality.

Context. Methanol and ethylene glycol cause significant mortality post-ingestion. Predicting prognosis based on the biomarkers osmolal gap, anion gap and pH is beneficial. Objective. To evaluate the relationship between biomarkers, measured post-methanol and ethylene glycol exposure, and clinical outcomes. Methods. A review of the literature identified cases where methanol or ethylene glycol had been ingested and clinical outcomes were recorded. Biomarkers were extracted including osmolal gap, anion gap and pH, with clinical outcomes categorised as recovered, recovered with adverse sequelae and death. Biomarkers were analysed using the Mann–Whitney test for two samples; sensitivity and specificity were evaluated using receiver operating characteristic (ROC) curves. Results. In total, 119 cases of methanol and 88 of ethylene glycol poisoning were identified; 21 methanol and 19 ethylene glycol patients died. For methanol ingestion the mean values, for survival compared to death, were 48 (range: 6–138) and 90 (range: 49–159) mOsm/kg water for osmolal gap (p=0.0052), 31 (range: 11–50) and 41 (range: 30–53) mmol/L for anion gap (p=0.0065) and 7.21 (range: 6.60–7.50) and 6.70 (range: 6.34–7.22) for arterial pH (p<0.0001). The area under the ROC curve was highest for arterial pH, 0.94 (95% CI: 0.89–0.99). For ethylene glycol, these were 49 (range: 0–189) and 79 (range: 25–184) mOsm/kg water for osmolal gap (p=0.050), 28 (range: 6–48) and 38 (range: 20–66) mmol/L for anion gap (p=0.0037) and 7.08 (range: 6.46–7.39) and 6.98 (range: 6.50–7.16) for pH (p=0.072), for survival compared to death. The area under the ROC curve was highest for anion gap, 0.73 (95% CI: 0.60–0.87). Conclusion. Post-methanol ingestion a large osmolal gap, anion gap and low pH (<7.22) were associated with increased mortality; and pH has the highest predictive value. Post-ethylene glycol ingestion, both osmolal gap and anion gap were associated with increased mortality.

Prediction of Torsade de Pointes From the QT Interval: Analysis of a Case Series of Amisulpride Overdoses

Electrocardiograms (ECGs) from a case series of 86 amisulpride overdose events in 66 patients were reviewed for abnormal QT intervals and torsade de pointes (TdP). Eight patients exhibited TdP. In this investigative case series, the magnitude of prolongation of the QT interval was a stronger predictor of TdP than the mere presence of a prolongation per se.

A dosing regimen for immediate N-acetylcysteine treatment for acute paracetamol overdose

Context. Current treatment of paracetamol (acetaminophen) poisoning involves initiating a 3-phase N-acetylcysteine (NAC) infusion after comparing a plasma concentration, taken ≥4 h post-overdose, to a nomogram. This may result in dosing errors, a delay in treatment, or possibly more adverse effects – due to the use of a high dose rate for the first infusion when treatment is initiated. Objective. Our aim was to investigate a novel dosing regimen for the immediate administration of NAC on admission at a lower infusion rate. Methods. We used a published population pharmacokinetic model of NAC to simulate a scenario where a patient presents to the hospital 2 h post-overdose. The conventional regimen is commenced 6 h post-overdose when the 4-h plasma paracetamol concentration is available. We investigated an NAC infusion using a lower dosing rate initiated immediately on presentation. We determined a dosing rate that gave an area under the curve (AUC) of the concentration-time curve that was the same or greater than that from the conventional regimen on 90% of occasions. Results. Lower dosing rates of NAC initiated immediately resulted in a similar exposure to NAC. An infusion of 110 mg/kg over the first 5 h (22 mg/kg/h) followed by the last two phases of the conventional regimen, or 200 mg/kg over 9 h (22.6 mg/kg/h) followed by the last phase of the conventional regimen could be used. Conclusion. The novel dosing regimen allowed immediate treatment of a patient using a lower dosing rate. This greatly simplifies the current dosing regimen and may reduce NAC adverse effects while ensuring the same amount of NAC is delivered.

A statistical model for red blood cell survival

A statistical model for the survival time of red blood cells (RBCs) with a continuous distribution of cell lifespans is presented. The underlying distribution of RBC lifespans is derived from a probability density function with a bathtub-shaped hazard curve, and accounts for death of RBCs due to senescence (age-dependent increasing hazard rate) and random destruction (constant hazard), as well as for death due to initial or delayed failures and neocytolysis (equivalent to early red cell mortality). The model yields survival times similar to those of previously published studies of RBC survival and is easily amenable to inclusion of drug effects and haemolytic disorders.

Modeling red blood cell survival data.

Anaemia of chronic kidney disease (CKD) is a common complication in patients with renal impairment, especially in end-stage renal failure. As well as erythropoietin deficiency, decreased red blood cell survival is a contributing factor. However, it remains unclear which mechanism underlies the altered survival of red blood cells (RBCs). In this work a previously developed statistical model for RBC survival was applied to clinical data obtained from 14 patients with CKD undergoing hemodialysis as well as 14 healthy controls using radioactive chromium (51Cr) as random labelling method. A classical two-stage approach and a full population analysis were applied to estimate the key parameters controlling random destruction and senescence in the model. Estimating random destruction was preferred over estimating an accelerated senescence in both approaches and both groups as it provided the better fit to the data. Due to significant nonspecific random loss of the label from the cells that cannot be quantified directly only a relative RBC survival can be obtained from data using 51Cr as labelling method. Nevertheless, RBC survival was found to be significantly reduced in CKD patients compared to the controls with a relative reduction of 20–30% depending on the analysis method used.

The Pharmacokinetics of Methanol in the Presence of Ethanol A Case Study

Background and ObjectiveMethanol is a toxic alcohol that can cause significant morbidity and mortality in overdose, while ethanol is a readily available and effective antidote. Little is known about the pharmacokinetics of methanol in the presence of ethanol and vice versa. This paper explores the influence of methanol and ethanol on the pharmacokinetics of each other along with the effect of continuous venovenous hae-modiafiltration (CVVHD) on alcohol removal.MethodsMultiple plasma, urine and dialysate samples were collected from a 42-year-old male who ingested 166 g of methanol. Methanol and ethanol concentrations in both plasma and urine were assayed and the concentration-time data were modelled using nonlinear mixed-effects modelling software NONMEM® VI. Simulations were performed using the final model parameters in MATLAB® software where a variety of initial doses and ethanol infusions were assessed.ResultsThe final model included a competitive metabolic interaction between methanol and ethanol as well as first-order elimination due to renal, CVVHD and an additional non-renal non-CVVHD mechanism. Simulations from the model show a loading dose of 28.4g/70kg of ethanol results in a target plasma concentration of 1 g/L. Due to the competitive interaction between methanol and ethanol, higher amounts of methanol require lower maintenance doses of ethanol but for longer. CVVHD was shown to increase the dose rate of ethanol required but to decrease the duration of the maintenance phase.ConclusionA detailed understanding of the pharmacokinetics of methanol and ethanol in the presence of each other is required to accurately determine the doses of ethanol required to treat different methanol poisonings.