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Multiphoton fluorescence lifetime microscopy has revolutionized studies of pathophysiological and xenobiotic dynamics, enabling the spatial and temporal quantification of these processes in intact organs in vivo. We have previously used multiphoton fluorescence lifetime microscopy to characterise the morphology and amplitude weighted mean fluorescence lifetime of the endogenous fluorescent metabolic cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) of mouse livers in vivo following induction of various disease states. Here, we extend the characterisation of liver disease models by using nonlinear regression to estimate the unbound, bound fluorescence lifetimes for NAD(P)H, flavin adenine dinucleotide (FAD), along with metabolic ratios and examine the impact of using multiple segmentation methods. We found that NAD(P)H amplitude ratio, and fluorescence lifetime redox ratio can be used as discriminators of diseased liver from normal liver. The redox ratio provided a sensitive measure of the changes in hepatic fibrosis and biliary fibrosis. Hepatocellular carcinoma was associated with an increase in spatial heterogeneity and redox ratio coupled with a decrease in mean fluorescence lifetime. We conclude that multiphoton fluorescence lifetime microscopy parameters and metabolic ratios provided insights into the in vivo redox state of diseased compared to normal liver that were not apparent from a global, mean fluorescence lifetime measurement alone.

CASE REPORT: We report a fatal case of a 37 year old gentleman who ingested a MCPA/bromoxynil co-formulation herbicide. Although clinically well on initial examination, our patient declined dramatically over his 18 h admission with increasing CO2 production, hyperthermia and metabolic derangement to eventually die from cardiac asystole 20 h post ingestion. Two hours after ingestion the MCPA concentration was 83.9 μg/mL and bromoxynil concentration was 137 μg/mL. DISCUSSION: The patients' mechanism of death appeared to be uncoupling of oxidative phosphorylation, excess CO2 production and hyperthermia. There is limited knowledge on the acute toxicity of these herbicides, in particular bromoxynil, and this case highlights the relentless progression of severe toxicity in humans. The collaboration was supported by an NHMRC Program Grant (1055176). Geoff Isbister is supported by an NHMRC Senior Research Fellowship ID 1061041 and Mike Roberts is supported by an NHMRC Senior Principal Research Fellowship ID 1002611.

Simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods have been developed and validated for quantification of paraquat (PQ) in plasma and urine. Plasma and urine sample preparation were carried out by one-step protein precipitation using cold acetonitrile (-20 to -10 ?C). After centrifugation, an aliquot of 10 L of supernatant was injected into a KinetexTM hydrophilic interaction chromatography (HILIC) column with a KrudKatcherTM Ultra in-line filter. The chromatographic separation was achieved using the mobile phase mixture of 250 mM ammonium formate (with 0.8% aqueous formic acid) in water and acetonitrile at a flow rate of 0.3 mL/min. Detection was performed using an API2000 triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear over the concentration range of 10–5000 ng/mL, with an LLOQ of 10 ng/mL. The inter- and intra-day precision (% R.S.D.) were

Background. Herbicides are commonly ingested for self-harm, but relatively little has been published on poisoning with herbicides other than paraquat and glyphosate. We report here a case series of patients with acute exposure to a combination herbicide (brand name Tiller Gold or Whip Super) containing the selective phenoxy herbicide fenoxaprop-P-ethyl, the sulfonylurea herbicide ethoxysulfuron and the safener isoxadifen ethyl. Method. Clinical data on all patients presenting with Tiller Gold or Whip Super poisoning to two General Hospitals in Sri Lanka from 2002-2008 were collected prospectively until discharge. Results. Eighty-six patients with a history of Tiller Gold or Whip Super ingestion were included. The main clinical features were an epigastric burning sensation and vomiting; however, most of those who vomited had received gastric lavage or forced emesis. Eight patients had a reduced level of consciousness on admission (Glasgow coma scale 9-14) that resolved without intervention over several hours. Only symptomatic and supportive care was required. The median hospital stay was 1 day (IQR: 1-2) and the case fatality was zero (95% confidence interval: 0-4.2%). This low case fatality compared favorably with the case fatality of other common herbicides in our cohort: paraquat > 40%, propanil > 10%, 4-chloro-2-methylphenoxyacetic acid > 5%, and glyphosate > 2%. Conclusion. This combination herbicide product appears to be safe in patients with acute self-poisoning, particularly in comparison with other herbicides, and causing few clinical features.

Isoniazid is a rare overdose that causes seizures and there is limited evidence to guide treatment. We report a 20-year-old female migrant who presented with recurrent seizures after ingesting 25 g of isoniazid. She was treated with activated charcoal, repeated doses of midazolam for the seizures, and given multiple doses of pyridoxine (14 mg), limited by availability. She was admitted to intensive care, and 5.5 hours post-ingestion, she was commenced on continuous veno-venous hemodiafiltration (CVVHDF). She was extubated after 24 hours and CVVHDF was ceased 6 hours later (30 hours post-overdose). Her renal function remained normal and her initial lactate was the highest at 2.3. She made a full recovery. Five plasma samples were collected before, during, and after CVVHDF, and isoniazid was quantified with liquid chromatography-tandem mass spectrometry. A pharmacokinetic analysis of time-isoniazid concentration data was fitted to a two-compartment model with first-order input (with fixed ka ) with the effect of CVVHDF modeled as a time-dependent covariate. This suggested that there was initially good clearance with CVVHDF (4 times endogenous clearance), which rapidly declined within hours.

Stress is known to modulate sensitisation to repeated psychostimulant exposure. However, there is no direct evidence linking glucocorticoids and sensitisation achieved by repeated administration of the NMDA receptor antagonist MK-801. We tested the hypothesis that co-administration of RU486, a glucocorticoid receptor (GR) antagonist, prior to repeated daily MK-801 injections would block the expression of locomotor sensitisation due to its dual effects on corticosterone and dopamine. We employed a repeated MK-801 administration locomotor sensitisation paradigm in male Sprague Dawley rats. RU486 or a dimethyl sulfoxide (DMSO) vehicle was co-administered with MK-801 or saline during the induction phase. Subsequent to withdrawal, rats were challenged with MK-801 alone to test for the expression of sensitisation. In a separate cohort of rats, plasma corticosterone levels were quantified from blood samples taken on the 1st, 4th and 7th day of induction and at expression. One day after challenge, nucleus accumbens tissue levels of dopamine and its metabolites DOPAC and HVA were measured. During the induction phase, RU486 progressively enhanced locomotor sensitisation to MK-801. RU486 and MK-801 both showed stimulatory effects on corticosterone levels and this was further augmented when given in combination. Contrary to our hypothesis, RU486 did not block the expression of locomotor sensitisation to MK-801 and actually increased levels of dopamine, DOPAC and HVA in nucleus accumbens tissue. Our results showed that RU486 has augmentative rather than inhibitory effects on MK-801-induced sensitisation. This study indicates a divergent role for glucocorticoids in sensitisation to MK-801 compared to sensitisation with other psychostimulants.

To introduce better antibiotics for the treatment of some infectious diseases in sheep and to expand the range of antibiotics available for veterinary medicine, pharmacokinetics of two antibiotics marbofloxacin (MBX) and trovafloxacin (TVX) were investigated in sheep after intramuscular injection. Variable and irregular plasma concentration-time profiles were observed for TVX but not for MBX. To understand the mechanism of this phenomenon, intravenous studies were performed for both drugs and data were analyzed using a population approach. Deconvolution was then performed using various approaches to obtain absorption profiles of both drugs in sheep after intramuscular injection. The Loo-Riegelman and staircase deconvolution function methods were found to provide more reliable estimates of absorption rate than the Spath-spline and B-spline constraining break points deconvolution methods. The absorption profiles resulting from deconvolution indicated a zero-order absorption process for TVX and a first-order process for MBX. Precipitation of TVX at the injection site was suspected to cause the pseudo zero-order absorption. This hypothesis was supported by the observation of crystalline deposits of TVX in sheep meat after direct injection, using reflectance confocal microscopy. Refereed/Peer-reviewed

A simple and rapid high performance liquid chromatographic method was developed, validated and applied for the simultaneous determination of marbofloxacin (MBX) and trovafloxacin (TVX) in sheep plasma. Samples were extracted with 20% perchloric acid and MBX and TVX were separated on a C18 column using a gradient mobile phase system consisting of 17.5 mM of NaH2PO4 and 1.5 mM of tetrabutylammonium hydroxide aqueous solution, pH 3 (A) and 50% acetonitrile and 50% methanol (B), with UV detection at 293 and 270 nm. The retention times of MBX and TVX were 4.9 and 6.6 min respectively. The detection and quantification limits for MBX and TVX were 2 ng/mL and 10 ng/mL respectively for both compounds. The calibration curves were linear over a concentration range of 10-50,000 ng/mL for both antibiotics. The linearity, precision, accuracy, recovery and stability of the assay were evaluated from spiked sheep plasma. The method was successfully applied to sheep plasma samples obtained from MBX and TVX pharmacokinetic studies. Refereed/Peer-reviewed

Carbofuran (CFN), carbosulfan (CSN) and fenobucarb (FBC) are carbamate pesticides that are widely used in gardening and agriculture for the control of insects. Human poisoning due to occupational or self-poisoning exposures is also reported, so assays are required to quantify the plasma concentration of these insecticides. An LC–MS/MS method was developed and validated for the simultaneous quantification of these three carbamate insecticides in the plasma of patients with acute intentional self-poisoning. Plasma samples were pretreated by acetonitrile for protein precipitation. Chromatography was carried out on a Luna C18(2) analytical column with gradient elution using a mobile phase containing acetonitrile and water with 10 mM ammonium acetate. Mass spectrometric analysis was performed by an Applied Biosystems MDS Sciex API 2000 triple quadrupole mass spectrometer coupled with electrospray ionization (ESI) source in the positive ion mode. The total run time was 7 min. The assay was validated over a concentration range from 10 to 1000 ng/ml for CSN and FBC and 20–2000 ng/ml for CFN. The precision and accuracy for both intra- and inter-day determination of all analytes were acceptable (

Surgical site infections are common, so effective antibiotic concentrations at the sites of infection, i.e., in the interstitial fluid (ISF), are required. The aim of this study was to evaluate contemporary perioperative prophylactic dosing of cefazolin by determining plasma and subcutaneous ISF concentrations in patients undergoing elective/semielective abdominal aortic aneurysm (AAA) open repair surgery. This was a prospective pharmacokinetic study in a tertiary referral hospital. Cefazolin (2 g) was administered as a 3-min slow bolus 30 min prior to incision in 12 enrolled patients undergoing elective/semielective AAA open repair surgery. Serial blood, urine, and ISF (via microdialysis) samples were collected and analyzed using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Cardiac output was determined using pulse waveform contours with Vigileo. The recruited patients had a median (interquartile range) age of 70 (66 to 76) years and weight of 88 (81 to 95) kg. The median (interquartile range) terminal volume of distribution was 0.14 (0.11 to 0.15) liter/kg, total clearance was 0.05 (0.03 to 0.06) liter/h, and minimum observed unbound concentration was 5.7 (5.4 to 8.1) mg/liter. The penetration of unbound drug from plasma to ISF was 85% (78% to 106%). We found correlations present, albeit weak, between cefazolin clearance and cardiac output ( r 2 = 0.11) and urinary creatinine clearance ( r 2 = 0.12). In conclusion, we found that a single 2-g dose of cefazolin administered 30 min before incision provides plasma and ISF concentrations in excess of the likely MICs for susceptible pathogens in patients undergoing AAA open repair surgery.

The aims of this study were to evaluate the predictive performances of all previously derived limited sampling strategies (LSSs) for total prednisolone, and to derive LSSs for free prednisolone in an independent cohort of adult kidney transplant recipients.Total and free prednisolone area under the concentration-time curve profiles from 0 to 12 hours post-dose (AUC(0-12)) were collected from 20 subjects. All previously published total prednisolone LSSs were identified from the literature. Free prednisolone LSSs were developed using multiple linear regression analyses. AUC predicted by each of the LSSs was compared with AUC(0-12). Median percentage prediction error (MPPE) and median absolute percentage prediction error (MAPE) were calculated to evaluate bias and imprecision.Total dose-adjusted prednisolone exposure varied 5-fold among study participants, while free dose-adjusted prednisolone exposure varied 3-fold. Correlation (r²) between total and free prednisolone AUC(0-12) was 0.79 (p = 0.0001) for the entire study cohort. This correlation was poorer in those early compared with late post-transplant (r² = 0.42 (p = 0.04) versus r² = 0.59 (p = 0.009) respectively). Ten previously published LSSs for total prednisolone and 15 derived LSSs for free prednisolone performed with acceptable levels of bias and imprecision (15%). Of the free prednisolone LSSs, an equation incorporating 0.25-, 2- and 4-h concentrations showed the highest predictive power (AUC₀-₁₂ = -17.20 + 1.18 × C₀.₂₅ + 2.75 × C₂ + 4.45 × C₄; MPPE = 0.1%, MAPE = 4.6%).Wide between-subject variability in drug exposure suggests a role for TDM. LSSs can accurately estimate both total and free prednisolone AUC(0-12). However, given the poor correlation observed between the two parameters, our data suggest that free prednisolone LSSs may be preferable.

Self-poisoning with herbicides is an important reason for hospital admission and death in Asia. Although some herbicides have a well-described toxicity profile in humans, many of the newer compounds rely on extrapolation from animal results as no published literature on clinical outcomes of human self-poisoning has been described. One example of these compounds is bispyribac, a selective herbicide used in rice and wheat cultivation that is marketed in two containers, one containing bispyribac 400 g/L with a solvent and the other the surfactant, polyethylene glycol. We present the first case series of acute human self-poisoning with an herbicide product containing bispyribac.Clinical data for all patients who presented with acute poisoning from a bispyribac-containing herbicide (Nominee) to two general hospitals in Sri Lanka from June 2002 to January 2009 were collected prospectively. Admission and serial blood samples were collected from consenting patients to confirm exposure and to study the toxicokinetics of bispyribac, respectively.One hundred ten patients with a history of bispyribac ingestion presented after a median time of 4 h post-ingestion. There were three deaths at 15, 6, and 5 h post-ingestion because of asystolic cardiac arrest. All three patients had reduced Glasgow Coma Score (GCS) (3, 12, and 13, respectively) of whom the former two had co-ingested ethanol and developed tonic-clonic seizures. Admission blood sample was obtained from the former two of these patients but bispyribac was detected in only one of these patients. The other patient presented 2.5 h post-ingestion with a GCS of 12 but bispyribac was not detected. Excluding the patient with undetectable bispyribac, a conservative estimate of the case fatality ratio at 1.81% (95% confidence interval 0.32-5.8) can be made. The majority of the remaining patients had self-limiting upper gastrointestinal symptoms and eight patients had an abnormal GCS on presentation to hospital. The overall median hospital stay was 3 days. Bispyribac was not detectable on admission in 21 patients; in the remaining patients, the median plasma concentration was 50.55 microg/mL (interquartile range 1.28-116.5; n=32). The peak concentration was noted around 3 h post-ingestion and plasma bispyribac concentration did not predict the severity of poisoning.The majority of patients developed self-resolving symptoms and were successfully managed in rural general hospitals without transfer to larger tertiary hospitals. Patients who died developed significant poisoning within 6 h and plasma bispyribac concentrations did not appear to predict mortality. The lack of correlation between bispyribac outcomes and the available plasma concentrations may be because of exposure to nonbispyribac components or other undefined factors. Clinical outcomes from acute self-poisoning with bispyribac-containing herbicides appear to be relatively more favorable than other commonly used herbicides.