The analysis of acetaminophen (paracetamol) and seven metabolites in rat, pig and human plasma by U(H)PLC–MS

A U(H)PLC–MS/MS method is described for the analysis of acetaminophen and its sulphate, glucuronide, glutathione, cysteinyl and N-acetylcysteinyl metabolites in plasma using stable isotope-labeled internal standards. P-Aminophenol glucuronide and 3-methoxyacetaminophen were monitored and semi-quantified using external standards. The assay takes 7.5 min/sample, requires only 5 μl of plasma and involves minimal sample preparation. The method was validated for rat plasma and cross validated for human and pig plasma and mouse serum. LOQ in plasma for these analytes were 0.44 μg/ml (APAP-C), 0.58 μg/ml (APAP-SG), 0.84 μg/ml (APAP-NAC), 2.75 μg/ml (APAP-S), 3.00 μg/ml (APAP-G) and 16 μg/ml (APAP). Application of the method is illustrated by the analysis of plasma following oral administration of APAP to male Han Wistar rats

The analysis of acetaminophen (paracetamol) and seven metabolites in rat, pig and human plasma by U(H)PLC–MS

A U(H)PLC–MS/MS method is described for the analysis of acetaminophen and its sulphate, glucuronide, glutathione, cysteinyl and N-acetylcysteinyl metabolites in plasma using stable isotope-labeled internal standards. P-Aminophenol glucuronide and 3-methoxyacetaminophen were monitored and semi-quantified using external standards. The assay takes 7.5 min/sample, requires only 5 μl of plasma and involves minimal sample preparation. The method was validated for rat plasma and cross validated for human and pig plasma and mouse serum. LOQ in plasma for these analytes were 0.44 μg/ml (APAP-C), 0.58 μg/ml (APAP-SG), 0.84 μg/ml (APAP-NAC), 2.75 μg/ml (APAP-S), 3.00 μg/ml (APAP-G) and 16 μg/ml (APAP). Application of the method is illustrated by the analysis of plasma following oral administration of APAP to male Han Wistar rats

Metabolism and Effects on Endogenous Metabolism of Paracetamol (Acetaminophen) in a Porcine Model of Liver Failure

The metabolic fate, toxicity and effects on endogenous metabolism of paracetamol (acetaminophen, APAP) in 22 female Landrace cross large white pigs were evaluated in a model of acute liver failure (ALF). Anaesthetized pigs were initially dosed at 250 mg/kg via an oroduodenal tube with APAP serum concentrations maintained above 300 mg/L using maintenance doses of 0.5-4g/h until ALF. Studies were undertaken to determine both the metabolic fate of APAP and its effects on the endogenous metabolic phenotype of ALF in using 1H NMR spectroscopy. Increased concentrations of citrate combined with pre-ALF increases in circulating lactate, pyruvate and alanine in plasma suggest mitochondrial dysfunction and a switch in hepatic energy metabolism to glycolysis in response to APAP treatment. A specific liquid chromatography-tandem mass spectrometry assay was used to quantify APAP and metabolites. The major circulating and urinary metabolite of APAP was the phenolic glucuronide (APAP-G), followed by p-aminophenol glucuronide (PAP-G) formed from N-deacetylated APAP. The PAP produced by N-deacetylation was the likely cause of the methaemoglobinemia and kidney toxicity observed in this, and previous, studies in the pig. The phenolic sulfate of APAP, and the glutathione-derived metabolites of the drug were only found as minor components (with the cysteinyl conjugate detected but not the mercapturate). Given its low sulfation, combined with significant capacity for N-deacetylation the pig may represent a poor translational model for toxicology studies for compounds undergoing significant metabolism by sulfation, or which contain amide bonds which when hydrolysed to unmask an aniline lead to toxicity. However, the pig may provide a useful model where extensive amide hydrolysis is seen for drugs or environmental chemicals in humans, but not in e.g., the rat and dog which are the pre-clinical species normally employed for safety assessment

MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure

Objective: Acute liver failure (ALF) is characterised by overwhelming hepatocyte death and liver inflammation with massive infiltration of myeloid cells in necrotic areas. The mechanisms underlying resolution of acute hepatic inflammation are largely unknown. Here, we aimed to investigate the impact of Mer tyrosine kinase (MerTK) during ALF and also examine how the microenvironmental mediator, secretory leucocyte protease inhibitor (SLPI), governs this response. Design: Flow cytometry, immunohistochemistry, confocal imaging and gene expression analyses determined the phenotype, functional/transcriptomic profile and tissue topography of MerTK+ monocytes/macrophages in ALF, healthy and disease controls. The temporal evolution of macrophage MerTK expression and its impact on resolution was examined in APAP-induced acute liver injury using wild-type (WT) and Mer-deficient (Mer−/−) mice. SLPI effects on hepatic myeloid cells were determined in vitro and in vivo using APAP-treated WT mice. Results: We demonstrate a significant expansion of resolution-like MerTK+HLA-DRhigh cells in circulatory and tissue compartments of patients with ALF. Compared with WT mice which show an increase of MerTK+MHCIIhigh macrophages during the resolution phase in ALF, APAP-treated Mer−/− mice exhibit persistent liver injury and inflammation, characterised by a decreased proportion of resident Kupffer cells and increased number of neutrophils. Both in vitro and in APAP-treated mice, SLPI reprogrammes myeloid cells towards resolution responses through induction of a MerTK+HLA-DRhigh phenotype which promotes neutrophil apoptosis and their subsequent clearance. Conclusions: We identify a hepatoprotective, MerTK+, macrophage phenotype that evolves during the resolution phase following ALF and represents a novel immunotherapeutic target to promote resolution responses following acute liver injury

MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure.

OBJECTIVE: Acute liver failure (ALF) is characterised by overwhelming hepatocyte death and liver inflammation with massive infiltration of myeloid cells in necrotic areas. The mechanisms underlying resolution of acute hepatic inflammation are largely unknown. Here, we aimed to investigate the impact of Mer tyrosine kinase (MerTK) during ALF and also examine how the microenvironmental mediator, secretory leucocyte protease inhibitor (SLPI), governs this response. DESIGN: Flow cytometry, immunohistochemistry, confocal imaging and gene expression analyses determined the phenotype, functional/transcriptomic profile and tissue topography of MerTK+ monocytes/macrophages in ALF, healthy and disease controls. The temporal evolution of macrophage MerTK expression and its impact on resolution was examined in APAP-induced acute liver injury using wild-type (WT) and Mer-deficient (Mer-/-) mice. SLPI effects on hepatic myeloid cells were determined in vitro and in vivo using APAP-treated WT mice. RESULTS: We demonstrate a significant expansion of resolution-like MerTK+HLA-DRhigh cells in circulatory and tissue compartments of patients with ALF. Compared with WT mice which show an increase of MerTK+MHCIIhigh macrophages during the resolution phase in ALF, APAP-treated Mer-/- mice exhibit persistent liver injury and inflammation, characterised by a decreased proportion of resident Kupffer cells and increased number of neutrophils. Both in vitro and in APAP-treated mice, SLPI reprogrammes myeloid cells towards resolution responses through induction of a MerTK+HLA-DRhigh phenotype which promotes neutrophil apoptosis and their subsequent clearance. CONCLUSIONS: We identify a hepatoprotective, MerTK+, macrophage phenotype that evolves during the resolution phase following ALF and represents a novel immunotherapeutic target to promote resolution responses following acute liver injury

Hole Expansion of Aluminum Alloys for the Automotive Industry

The introduction of Aluminum alloys in the automotive industry due to their high strength-to-weight ratio has brought with it a number of technical hurdles which require overcoming in order that their full potential may be realized. One of the issues that require addressing is that of edge cracking, a phenomenon which is particularly difficult to predict. This is often observed during the initial drawing operation in a traditional automotive stamping plant. A useful measure of a materials susceptibility to edge cracking is the Hole Expansion Ratio. Currently there is a standard (ISO 16630) which provides for holes to be expanded by a conical punch, where the original hole is introduced via punching. This reflects the traditional processing route within the automotive industry. Investigations have been conducted using both conical and flat-topped punches, as well as using drilling and reaming, and CNC machining to introduce the initial hole for comparison with the standard punched route in order to understand the effect of different processes on the susceptibility to edge cracking. The hole expansion ratio for a number of aluminum alloys, both 5xxx and 6xxx series, has been determined. Regression analyses of hole expansion ratios against material thickness and UTS have been conducted, and a relationship has been established for the different punch and hole types. Consideration is also given to combining a materials hole expansion ratio with its Forming Limit Curve (FLC), with a focus on tool design and tool buy-off being presented

Experimental Evaluation of Springback in Aluminium Alloys Using Optical Measurement and Numerical Analysis

The aim of this paper is to establish an efficient and effective means of understanding the springback behaviour of a 5xxx-O and a 6xxx-T61 series aluminium alloy. The tooling used for investigating the springback is a small U-channel stamping draw die using two die radii of 8 or 12 mm and a punch radius of 12 mm. The samples were drawn to depths of 50 or 75 mm with a minimum of 5 samples being drawn for each condition. The U-channel samples were then evaluated using an optical scanning technique and the springback values were calculated using a numerical analysis as developed by one of the authors. The comparison of the sidewall springback, sidewall curl and flange springback under different conditions is conducted and the effect of the draw depth and die radius on the final springback is shown. Statistical analyses of both the drawing condition and the scanning technique are conducted to understand the effect of process variation on the results obtained. The 6xxx series shows higher springback values when compared to 5xxx series alloy. An increase in drawing depth suggests higher springback for both the alloys