Design of novel, simple, and inexpensive 3D printing-based miniaturized electrochemical platform containing embedded disposable detector for analytical applications.

This paper describes the development of a novel, simple, and inexpensive electrochemical device containing an integrated and disposable three-electrode system for detection. The base of this platform consists on a PDMS structure containing microchannels which were prototyped using 3D-printed molds. Pencil graphite leads were inserted into these microchannels and utilized as working, counter and reference electrodes in a novel design. Morphological analysis and electrochemical experiments with benchmark redox probes were carried out in order to evaluate the performance and characterize the miniaturized device proposed. Even using inexpensive materials and a simple fabrication protocol, the electrochemical platform developed provided good repeatability and reproducibility over a low cost (ca. $2 per device), acceptable lifetime (ca. 250 voltammetric runs) and extremely reduced consumption of samples and reagents (order of µL). As proof of concept, the analytical feasibility of the platform was investigated through the simultaneous determination of dopamine (DOPA) and acetaminophen (AC). The two analytes showed linear dependence on the concentration range from 1 to 15 µM and the LODs achieved were 0.21 µM for DOPA and 0.29 µM for AC. Moreover, the platform was successfully applied on the determination of DOPA and AC in spiked blood serum and urine samples. The results obtained with the device described here were better than some reports in literature that use more costly electrodic materials and complex modification steps for the detection of the same analytes.

Paracetamol Serum Concentrations in Neonates Treated Enterally for Ductal Closure: A Pilot Study.

We determined serum paracetamol concentrations 4 hours after the eighth dose in infants treated enterally for ductal closure. Serum paracetamol concentrations correlated (P = .0026) with ductal response. No patent ductus arteriosus in a baby with paracetamol levels <20 mg/L closed in response to treatment. Paracetamol levels also correlated (P = .046) with postnatal age.

Simultaneous determination of paracetamol and ciprofloxacin in biological fluid samples using a glassy carbon electrode modified with graphene oxide and nickel oxide nanoparticles.

A simple and highly selective electrochemical method using a glassy carbon electrode (GCE) modified with graphene oxide (GO) and nickel oxide nanoparticles (NiONPs) was developed for the simultaneous determination of paracetamol (PAR) and ciprofloxacin (CIP). The electrochemical characterisation of the modified GCE was performed by cyclic voltammetry and electrochemical impedance spectroscopy. The morphological characterisation of the GO and NiONPs was performed by scanning electron microscopy and transmission electron microscopy. Under optimised conditions, using square wave voltammetry, the simultaneous determination of PAR and CIP using the NiONPs-GO-CTS: EPH/GCE sensor shows a linear concentration range from 0.10 to 2.9µmolL-1 and 0.040-0.97µmolL-1, with detection limits of 6.7 and 6.0 nmol L-1, respectively. The NiONPs-GO-CTS: EPH/GCE sensor showed good reproducibility, repeatability and stability. Furthermore, the proposed method was successfully applied for the simultaneous determination of PAR and CIP in synthetic biological fluid samples.

Serum Acetaminophen Protein Adduct Concentrations in Pediatric Emergency Department Patients.

OBJECTIVES: Acetaminophen toxicity is a common cause of pediatric liver failure. The diagnosis may be limited by the short window of detection of acetaminophen in serum. Recently acetaminophen protein adducts (APAP-CYS) have been used as a biomarker with a longer duration of detection. The objective of this study was to describe the serum concentrations of APAP-CYS in pediatric patients with and without reported therapeutic acetaminophen exposure. METHODS: A cross-sectional study of children age 1 to <12 years presenting to a pediatric emergency department. Subjects were stratified by recent acetaminophen use and had serum APAP-CYS measured using LC/MS. RESULTS: One hundred patients were enrolled. All of the patients whose caregivers denied acetaminophen exposure had nondetectable APAP-CYS. Fifty-two percent of subjects who were reported to have taken acetaminophen in the preceding 2 weeks had detectable serum APAP-CYS. The APAP-CYS concentrations were positively correlated with higher overall dose and more recent ingestion. CONCLUSIONS: APAP-CYS is detectable in the majority of children taking acetaminophen and not detected in the majority of children who are not exposed to acetaminophen.

Bioequivalence and Pharmacokinetic Evaluation Study of Acetaminophen vs. Acetaminophen Plus Caffeine Tablets in Healthy Mexican Volunteers.

OBJECTIVE: The aim of this clinical trial was to establish the bioequivalence of two tablets containing acetaminophen 650 mg (reference) and acetaminophen 650 mg plus caffeine 65 mg (test), administered orally, in fasting conditions in healthy Mexican volunteers. METHODS: Blood samples were taken from 21 male and five female individuals, during a 24-h period, to characterize the pharmacokinetic profile of acetaminophen. Plasma samples were quantified by ultra-performance liquid chromatography, tandem mass spectrometry. Pharmacokinetic metrics (maximum plasma concentration, area under the curve from time zero to the last sampling time, and area under the curve from time zero to infinity) were used to determine the 90 % confidence interval of the test/reference coefficient. RESULTS: The geometric mean values for maximum plasma concentration obtained for the reference and test products were 9.46 ± 34.21 and 9.72 ± 32.38 µg/mL, respectively, whereas for the area under the curve from time zero to the last sampling time the values obtained were 34.93 ± 32.58 and 35.89 ± 31.03 µg h/mL for the reference and test formulations, respectively. The 90 % confidence intervals were within the acceptance range (80-125 %). CONCLUSIONS: The test product was bioequivalent to the reference product. A faster absorption was seen in the test formulation in the Mexican population.

Effects of an Agaricus blazei aqueous extract pretreatment on paracetamol-induced brain and liver injury in rats.

The action of an Agaricus blazei aqueous extract pretreatment on paracetamol injury in rats was examined not only in terms of the classical indicators (e.g., levels of hepatic enzymes in the plasma) but also in terms of functional and metabolic parameters (e.g., gluconeogenesis). Considering solely the classical indicators for tissue damage, the results can be regarded as an indication that the A. blazei extract is able to provide a reasonable degree of protection against the paracetamol injury in both the hepatic and brain tissues. The A. blazei pretreatment largely prevented the increased levels of hepatic enzymes in the plasma (ASP, ALT, LDH, and ALP) and practically normalized the TBARS levels in both liver and brain tissues. With respect to the functional and metabolic parameters of the liver, however, the extract provided little or no protection. This includes morphological signs of inflammation and the especially important functional parameter gluconeogenesis, which was impaired by paracetamol. Considering these results and the long list of extracts and substances that are said to have hepatoprotective effects, it would be useful to incorporate evaluations of functional parameters into the experimental protocols of studies aiming to attribute or refute effective hepatoprotective actions to natural products.

Validação de metodologia analítica e estudo de estabilidade para quantificação sérica de paracetamol / Analytical methodology validation and stability study for serum quantification of acetaminophen

INTRODUÇÃO E OBJETIVO: Paracetamol ou acetaminofeno é atualmente um dos analgésicos-antipiréticos mais utilizados, principalmente em crianças. Porém o fácil acesso ao medicamento e o desconhecimento da população sobre seus efeitos nocivos têm aumentado muito o número de intoxicações por esse medicamento. A análise da concentração sérica de paracetamol confirma o diagnóstico. O resultado não só tem valor de certeza diagnóstica como também avalia o risco de hepatotoxicidade, indicando uso ou não do antídoto específico n-acetilcisteína. O objetivo deste trabalho foi propor um método analítico para quantificação sérica do paracetamol por espectrofotometria visível em 430 nm. MATERIAIS E MÉTODOS: Após desproteinização da amostra, acetaminofeno (n-acetil-p-aminofenol) reage com nitrito de sódio, formando 2,4-nitro-4-acetaminofenol, que assume coloração amarela em meio alcalino. As figuras de mérito linearidade, precisão, exatidão, robustez, recuperação, limites de detecção e qualificação foram avaliadas segundo critérios preconizados pelo International Conference on Harmonisation (ICH) e pela Agência Nacional de Vigilância Sanitária (ANVISA). O estudo de estabilidade foi realizado após ciclos de congelamento/descongelamento, curta duração, longa duração sob refrigeração e em freezer. RESULTADOS: O método se mostrou linear de 20 a 300 mg/l. Os limites de detecção e quantificação foram de respectivamente 3,6 mg/l e 20 mg/l. CONCLUSÃO: O método se mostrou preciso, exato e robusto e apresentou boa recuperação. As amostras-controle foram estáveis nas condições testadas. O método desenvolvido demonstrou possuir todos os parâmetros necessários para ser aplicado na quantificação de paracetamol em amostras de plasma ou soro humano para análise de emergência. Além disso, é uma técnica simples, de rápida execução e baixo custo.

INTRODUCTION AND OBJECTIVE: Acetaminophen or paracetamol is currently one of the most used analgesic-antipyretic agents, mainly with children. However, both the easy access to this medicine and the population's unawareness of its toxic effects have contributed to a rise in the number of intoxications caused by this drug. Assessment of serum acetaminophen confirms the diagnosis. Not only does the result have diagnostic reliability but it also evaluates the risk of hepatotoxicity, indicating or not the administration of the specific antidote n-acetylcysteine. The aim of this study is to present an analytical method to the assessment of serum acetaminophen by spectrophotometric detection at 430 nm. MATERIALS AND METHODS: After sample deproteinization, acetaminophen (n-acetyl-p-aminophenol) reacts with sodium nitrite forming 2.4-nitro-4-acetaminophenol, which becomes yellowish in alkaline medium. For method validation, linearity, precision, accuracy, robustness, recovery and detection limits were evaluated according to ICH and ANVISA criteria. The stability study was carried out after freezing/defreezing cycles, short-time duration, long-time duration under refrigeration and long-time duration under freezing. RESULTS: The method showed to be linear from 20 to 300 mg/l. The detection and quantification limits were 3.6 mg/l and 20 mg/l, respectively. CONCLUSION: The method was precise, accurate and robust and showed good recovery. The control-samples were stable in all tested conditions. The method developed presented all the necessary parameters to be applied in acetaminophen quantification in plasma samples or human serum for emergency analyzes. Furthermore, it is a simple, time and cost-effective technique.

Evaluation of acetaminophen P-glycoprotein-mediated salivary secretion by rat submandibular glands.

The constant ratio between saliva and plasma acetaminophen concentrations (S/P) during the elimination phase is assumed to result from the equilibrium established among the free-drug concentrations in the arterial blood, venous blood and saliva. Salivary secretion of acetaminophen is assumed to result from a passive diffusion of the drug to saliva from the blood that supplies the salivary glands. However, the constant S/P ratio during acetaminophen disposition and the finding that P-glycoprotein (P-gp), a protein recognized to pump substrates out of the cell, is expressed in duct cells of the submandibular glands questions the mechanisms involved in acetaminophen salivary secretion. Thus, we intended to evaluate the existence of a P-glycoprotein-mediated transport of acetaminophen in rat submandibular glands. Acetaminophen (30 mg/kg, i.v.) pharmacokinetics was assessed in controls and in rats pre-treated with erythromycin (100 mg/kg) as a P-glycoprotein inhibitor. Acetaminophen pharmacokinetic parameters were calculated from saliva and plasma levels considering a non-compartmental analysis. Mean plasma and salivary profiles of control and pre-treated animals were almost superimposable. No difference could be found in S/P ratios in control and erythromycin pre-treated animals (P > 0.05). Moreover, no statistical difference could be found in the kinetic parameters calculated from saliva or plasma drug level (P > 0.05). These observations indicate that acetaminophen salivary secretion in rat submandibular glands is not related to P-glycoprotein-mediated transport under the experimental conditions of the present work.

¿Contribuye el TNF-alfa a la fisiopatolog'ia de las hepatopatias? / Does TNF-alpha contribute to liver disease physiopathology?

The aim of the present paper is to establish the possible role of serum TNF in the pathophysiology of three experimental models of liver injury: paracetamol intoxication, cholestasis followed by paracetamol intoxication and cholestasis. We concluded that under our experimental conditions the serum TNF-alpha levels were not responsible for the inflammatory phenomena described in our previous paper as apopt.

¿Contribuye el TNF-alfa a la fisiopatologia de las hepatopatias? / Does TNF-alpha contribute to liver disease physiopathology?

The aim of the present paper is to establish the possible role of serum TNF in the pathophysiology of three experimental models of liver injury: paracetamol intoxication, cholestasis followed by paracetamol intoxication and cholestasis. We concluded that under our experimental conditions the serum TNF-alpha levels were not responsible for the inflammatory phenomena described in our previous paper as apopt. (Au)

Drug glucuronidation and hepatic lipid microsomal membrane profile in cholestatic rats followed paracetamol intoxication.

The uridin-diphosphoglucuronyl-transferase (UDP-GT) is a membrane-bound enzyme responsible for glucuronidation of endogenous and exogenous compounds. This work established the UDP-GT activity and its lipid membrane microenvironment in two experimental models: acute paracetamol intoxication, and cholestasis followed by acute paracetamol intoxication. Cholestasis was performed by bile duct ligation. After 7 days animals were injected with paracetamol (BDL-APAP group). Sham-operated rats were injected at day 7 with paracetamol (APAP group). Cholestatic and sham-operated rats injected with vehicle (BDL and control groups). UDP-GT activity was measured by a kinetic method for different substrates. Microsomal membrane phospholipid composition, cholesterol content and ultrastructure were determined. BDL-APAP group showed an increment in the UDP-GT activity except for chloramphenicol, morphine and paracetamol if compared to controls and to BDL group. The same increment was observed when BDL-APAP was compared to APAP except for chloramphenicol and lorazepam. Between BDL and APAP groups similar levels of activity were detected except for paracetamol. Microsomal phospholipid profile: phosphatidylcholine showed the lowest content in the BDL group, with a significant recovery in the BDL-APAP and APAP groups. Phosphatidylserine was markedly decreased in the APAP group compared to the rest and phosphatidylinositol was decreased in all the groups if compared to control values. An increment of phosphatidylethanolamine was seen in the APAP and BDL-APAP groups if compared to BDL and control values. A significant increment of microsomal cholesterol content was seen in BDL. Under these conditions, a different lipid microenvironment is produced, resulting in an increment of the enzyme activity for a variety of substrates.

Predicting concentrations in children presenting with acetaminophen overdose.

OBJECTIVE: To predict serum concentrations to evaluate and improve guidelines for the treatment of children (1 to 5 years) with accidental ingestion of acetaminophen elixir. METHODS: Acetaminophen concentrations for 1000 children were simulated with pharmacokinetic parameters and their expected variability. The distribution of concentrations arising from a 300 mg/kg dose at different age groups was predicted. These predictions were validated by comparison with concentrations obtained at 4 hours from 121 children with accidental ingestion of acetaminophen elixir. RESULTS: No child who presented with overdose had a concentration in the probable risk area of the Rumack-Matthew toxicity nomogram. Enteral charcoal administered 98 minutes (SD 44) after ingestion had no effect on serum concentrations. The simulation predicted that an acetaminophen dose of 300 mg/kg would result in concentrations of 32 to 208 mg/L (95% CI) at 4 hours after ingestion. The maximum concentration occurred before 2 hours in 95% of simulated children. CONCLUSION: Children (1 to 5 years) with reported ingestion of >250 mg/kg acetaminophen elixir should have serum concentrations measured at 2 hours after ingestion rather than at the 4-hour time point recommended in adults. This can be expected to speed discharge and reduce anxiety. The use of enteral charcoal is unlikely to enhance acetaminophen elimination, unless it is given within an hour of acetaminophen ingestion.

Gastric emptying in rats with acetaminophen-induced hepatitis

The objective of this work was to study the gastric emptying (GE) of liquids in fasted and sucrose-fed rats with toxic hepatitis induced by acetaminophen. The GE of three test meals (saline, glucose and mayonnaise) was evaluated in Wistar rats. For each meal, the animals were divided into two groups (N = 24 each). Group I was fed a sucrose diet throughout the experiment (66 h) while group II was fasted. Forty-two hours after the start of the experiment, each group was divided into two subgroups (N = 12 each). Subgroup A received a placebo and subgroup B was given acetaminophen (1 g/kg). Twenty-four hours later, the GE of the three test meals was assessed and blood samples were collected to measure the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and acetaminophen. In group IB, the mean AST and ALT values were 515 and 263 IU/l, respectively, while for group IIB they were 4014 and 2472 IU/l, respectively. The mean serum acetaminophen levels were higher in group IIB (120 µg/ml) than in group IB (87 µg/ml). The gastric retention values were significantly higher in group IIB than in group IIA for all three test meals: saline, 51 vs 35 percent; glucose, 52 vs 38 percent and mayonnaise, 51 vs 29 percent(median values). The correlation between gastric retention and AST levels was significant (P<0.05) for group IIB for the three test meals: r = 0.73, 0.67 and 0.68 for saline, glucose and mayonnaise, respectively. We conclude that GE is altered in rats with hepatic lesions induced by acetaminophen, and that these alterations may be related to the liver cell necrosis caused by the drug

Gastric emptying in rats with acetaminophen-induced hepatitis.

The objective of this work was to study the gastric emptying (GE) of liquids in fasted and sucrose-fed rats with toxic hepatitis induced by acetaminophen. The GE of three test meals (saline, glucose and mayonnaise) was evaluated in Wistar rats. For each meal, the animals were divided into two groups (N = 24 each). Group I was fed a sucrose diet throughout the experiment (66 h) while group II was fasted. Forty-two hours after the start of the experiment, each group was divided into two subgroups (N = 12 each). Subgroup A received a placebo and subgroup B was given acetaminophen (1 g/kg). Twenty-four hours later, the GE of the three test meals was assessed and blood samples were collected to measure the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and acetaminophen. In group IB, the mean AST and ALT values were 515 and 263 IU/l, respectively, while for group IIB they were 4014 and 2472 IU/l, respectively. The mean serum acetaminophen levels were higher in group IIB (120 micrograms/ml) than in group IB (87 micrograms/ml). The gastric retention values were significantly higher in group IIB than in group IIA for all three test meals: saline, 51 vs 35%; glucose, 52 vs 38% and mayonnaise, 51 vs 29% (median values). The correlation between gastric retention and AST levels was significant (P < 0.05) for group IIB for the three test meals: r = 0.73, 0.67 and 0.68 for saline, glucose and mayonnaise, respectively. We conclude that GE is altered in rats with hepatic lesions induced by acetaminophen, and that these alterations may be related to the liver cell necrosis caused by the drug.

Fulminant hepatitis associated with centrilobular hepatic necrosis in young children.

OBJECTIVE: To describe fulminant hepatic failure (FHF) in children in the United States with clinical and histopathologic features distinctly different from those typical of FHF. PATIENTS: Seven young children were seen in early 1994 with encephalopathy, coagulopathy, and elevated aminotransferase levels. Liver failure was preceded by a prodromal viral illness that resulted in a period of fasting without dehydration. Unlike the majority of children with FHF, these patients had serum bilirubin levels < 171 mumol/L (10 mg/dl). All children had received therapeutic doses of acetaminophen during the prodromal illness. HISTOPATHOLOGIC FINDINGS: Histologic findings included zonal necrosis of hepatocytes in a centrilobular distribution, which is characteristic of toxic liver injury but is atypical for viral hepatitis and sporadic non-A non-B hepatitis. OUTCOME: Six patients recovered spontaneously, and one died of complications of liver failure and fungal sepsis. The cause of this disorder remains unknown, but we postulate a viral or environmental insult that preferentially damages zone 3 hepatocytes. The potential for this injury may have been augmented by ingestion of therapeutic doses of acetaminophen while patients were in a fasted state. The prognosis was good compared with typical FHF in children and correlated with the degree of liver necrosis on histologic examination.

N-acetylcysteine stimulates hepatic glycogen deposition in the rat.

The action of N-acetylcysteine on hepatic glycogen deposition was investigated. Emphasis was also given to the protective action of this compound against hepatic glycogen depletion by paracetamol. Rats fasted for 24 hours were injected intraperitoneally with (1) vehicle, (2) paracetamol (500 mg/kg), (3) N-acetylcysteine (1200 mg/kg) and (4) paracetamol plus N-acetylcysteine. The rats were refed immediately after the drug injections. Paracetamol inhibited glycogen deposition in the 12 hours following injection. The plasma levels of paracetamol were in the range that inhibits energy metabolism in isolated mitochondria and in the isolated perfused liver. N-Acetylcysteine increased the rate of glycogen deposition either in the presence or in the absence of paracetamol. The latter effect may be responsible, partly at least, for the protective action of N-acetylcysteine against glycogen depletion caused by toxic doses of paracetamol.

Daño hepático por acetaminofeno: caso clínico / Hepatic damage due to acetaminophen: a clinical case

Se presenta el caso de una paciente de 27 años de edad, con el antecedente de ingesta excesiva de fármacos en forma voluntaria. Por sospecha de corresponder a una dosis alta de acetaminofeno, se evalúa con niveles plasmáticos de la droga (que confirman la ingesta) enzimas hepáticas y pruebas de coagulación, mostrando evolución rápida y progresiva al daño hepático agudo. No se obtuvo restos de fármacos del lavado gástrico efectuando al ingreso y no se pudo contar con el antídoto específico (n-acetilcisteína). La paciente evolucionó inicialmente soporosa, y luego con sensibilidad en hipocondrio derecho e ictericia. Se usó fitonodiona IM y plasma fresco congelado para corregir las alteraciones de coagulación, hidratación parenteral, ranitidina EV, dieta cero por boca. Se observó paulatina recuperación. Se revisa el mecanismo de daño hepático del acetaminofeno y se comentan las posibilidades terapéuticas