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Semisynthetic flavocytochromes, obtained by covalent binding of riboflavin with cytochromes P450 2B4, were able to catalyze the H2O2-mediated reactions of aniline p-hydroxylation, aminopyrine N-demethylation and p-nitroanizole' O-dealkylation. The rates of the flavocytochrome-catalyzed, H2O2-supported reactions far exceeded those of the appropriate NADH-dependent reactions and were comparable with the cytochrome P450 2B4-catalyzed, peroxide-mediated reaction rates. The kinetic parameters (kcat, K(m)) for the peroxide-dependent flavocytochrome P450 2B4 reactions were obtained. Sodium cyanide and SKF-525A, a specific P450 inhibitor, were both shown to inhibit these reactions. The generation of active oxygen species by flavocytochrome 2B4 was registered by chemiluminescence intensity.

Pharmacological therapy, present and future, will undoubtedly continue to play a large role within the overall management of patients with severe head injury. Nevertheless, limited clinical data are available to evaluate the effect of severe head injury on pharmacokinetics. The disruption of the blood-brain barrier secondary to trauma and/or subsequent hyperosmolar therapy can be expected to result in higher than expected brain drug concentrations. Aggressive dietary protein supplementation may result in increased oxidative drug metabolism. These effects may counterbalance inhibitory influences on drug metabolism secondary to cytokine release during the acute phase response. Alterations in protein binding can also be anticipated with the hypoalbuminaemia and increases in alpha 1-acid glycoprotein typically observed in these patients. Based on studies in other patient populations, moderate hypothermia, a treatment strategy in patients with head injury, can decrease drug metabolism. The pharmacokinetics of the following drugs in patients with severe head injury have been studied: phenytoin, pentobarbital (pentobarbitone), thiopental (thiopentone), tirilazad, and the agents used as marker substrates, antipyrine, lorazepam and indocynanine green (ICG). Several studies have documented increase in metabolism over time with phenytoin, pentobarbital, thiopental, antipyrine and lorazepam. Increases in tirilazad clearance were also observed but attributed to concurrent phenytoin therapy. No changes in the pharmacokinetics of ICG were apparent following head injury. With the frequent use of potent inhibitors of drug metabolism (e.g., cimetidine, ciprofloxacin) the potential for drug interaction is high in patients with severe head injury. Additional pharmacokinetic investigations are recommended to optimise pharmacological outcomes in patients with severe head injury.

Histamine H2-receptor antagonistic properties of the anti-ulcer agent T-593, (±)-(E)-1-[2-hydroxy-2-(4-hydroxyphenyI)ethyl]-3-[2[[[5-(methylamino)methyl-2-furyllmethyl]thio]ethyl]-2-(methylsulfonyl)guanidine, were investigated on [14C]aminopyrine accumulation in isolatea canine gastric mucosai cells and compared with those of ranitidine and famotidine. The potency of T-593-inhibition of [14C-aminopyrine accumulation stimulated by 10−4 M histamine, with an IC50 value of 1.85 × 10−6 M, was approximately 5 times greater than that of ranitidine, but half that of famotidine. T-593 did not affect [14C-aminopyrine accumulation stimulated by carbachol or dibutyryl-cAMP. T-593 depressed the maximal response of the histamine concentration-response curve with a dose-related displacement to the right, indicating that the nature of the H2-receptor antagonism of T-593 was insurmountable and included noncompetitive inhibition. The inhibitory efficacy of T-593 was time-dependent and was retained after the cells were washed. The inhibitory potency of (—)-S-T-593, one of the enantiomers, on the [14C]aminopyrine accumulation stimulated by histamine was approximately twice that of racemic T-593 and it also behaved as an insurmountable H2-receptor antagonist. However, the potency of (+)-R-T-593 was markedly weak. These results suggest that T-593 has H2-receptor antagonism that is insurmountable and this agent slowly associates and dissociates with the receptor in isolated canine gastric mucosal cells and that the specific substance causing H2-receptor antagonism is (—)-S-T-593.

New limits have been established to decrease mortality and morbidity rates after liver resection in cirrhotic and non-cirrhotic patients. Various laboratory data and imaging techniques have been used to complement the Child-Pugh score to predict liver failure after hepatectomy and to assess functional hepatic reserve. The greatest experiences are with the aminopyrine breath test and the galactosyl elimination capacity, which are decreased among hepatic failure patients after liver resection. However, absence of these changes do not totally exclude it. The indocyanine green retention test is the most widely used clearance test. Nevertheless, it remains imperfect because it depends both on hepatic blood flow and on the functional capacity of the liver. Nuclear imaging of the asialoglicoprotein receptors with radiolabelled synthetic asialoglicoproteins provides volumetric information as well a functional assessment of the liver. In summary, while liver function is complex, a successful liver test to assess quantitative functional hepatic reserve still needs to be established. The combination of the Child-Pugh score, the presence of ascites, the serum bilirubin levels, the indocyanine green retention (ICG R15) value, and the remnant liver CT volumetry seems to avoid an index of liver failure after hepatic resection. Cases when ICG R15 is above 15% should be combined with portal vein embolization. If there is no possibility to perform an ICG clearance test, it may be replaced with other available, well known dynamic liver function tests.

Epidermal growth factor (EGF) has acute inhibitory and chronic stimulatory effects on gastric acid secretion. Because a cascade of intracellular events culminating in the activation of a family of serine-threonine protein kinases called extracellular signal-regulated protein kinases (ERKs) is known to mediate the actions of EGF, we undertook studies to explore the functional role of the ERKs in gastric acid secretion. ERK2 was immunoprecipitated from cell lysates of highly purified (>95%) gastric canine parietal cells, and its activity was quantified using in-gel kinase assays. Of the primary gastric secretagogues, carbachol was the most potent inducer of ERK2 activity. Gastrin and EGF had weaker stimulatory effects, whereas no induction was noted in response to histamine. The effect of carbachol appeared to be independent of Ca2+signaling. PD-98059, a selective inhibitor of the upstream ERK activator mitogen-activated protein kinase/ERK kinase, dose-dependently inhibited both carbachol- and EGF-stimulated ERK2 activity, with a maximal effect observed between 50 and 100 μM. ERKs activation is required for induction of the early gene c- fos via phosphorylation of the transcription factor Elk-1 which binds to the c- fos serum response element (SRE). Carbachol stimulated a two- to threefold induction of luciferase activity in cultured parietal cells transfected with either a SRE-luciferase reporter plasmid or with a chimeric GAL4-ElkC expression vector and the 5×GAL-luciferase reporter plasmid. To examine the significance of ERK activation in gastric acid secretion, we tested the effect of PD-98059 on carbachol-stimulated uptake of14C-labeled aminopyrine (AP). Acute inhibition of the ERKs by PD-98059 led to a small increase in AP uptake and a complete reversal of the acute inhibitory effect of EGF on AP uptake induced by either carbachol or histamine. In contrast, exposure of the cells to PD-98059 for 16 h led to a reversal of the chronic stimulatory effect of EGF on AP uptake induced by carbachol. Our data led us to conclude that carbachol induces a cascade of events in parietal cells that results in ERK activation. Although the acute effect of the ERKs on gastric acid secretion appears to be inhibitory, the activation of transcription factors and of early gene expression could be responsible for its chronic stimulatory effects.

Background: We have previously shown that eradication of Helicobacter pylori increases acid secretion in H. pylori-associated enlarged fold gastritis. Aim: To investigate whether locally produced interleukin-1β is possibly involved in the inhibition of acid secretion in H. pylori gastritis. Methods: IL-1β release from the gastric body mucosa was determined by short-term culture of biopsy specimens in 13 patients with enlarged fold gastritis (all H. pylori-positive), five H. pylori-positive and 10 H. pylori-negative patients without enlarged folds. The acid-inhibitory effect of locally produced IL-1β was examined by []> 14C]-aminopyrine uptake assay using isolated rabbit gastric glands. Results: IL-1β release was significantly greater in patients with enlarged fold gastritis, significantly correlated with both basal and tetragastrin-stimulated acid outputs in the H. pylori-positive patients (r = −0.591 and r = −0.641, respectively; P

Background. Liver cell proliferation after partial hepatectomy in rats has been thoroughly investigated. Although DNA synthesis and morphologic restoration have been studied in this rat model, the functional capacity of the remnant liver during regeneration has not been elucidated. Methods. We measured the indocyanine green disappearance rate (ICG-k) and serum aminopyrine (CLamp) in rats at various intervals after two-thirds hepatectomy. Morphologic restoration of the liver after hepatectomy was evaluated on the basis of remnant liver weight, proliferating cell nuclear antigen labeling index, and the DNA content of the regenerating liver. Serial changes in ICG-k and CLamp after two-thirds hepatectomy were compared with the degree of morphologic restoration. Results. ICG-k and CLamp were reduced by one third in rats after two-thirds hepatectomy. Although the rate of restoration of remnant liver weight was steady after hepatectomy, ICG-k and CLamp were lowest about 36 hours after hepatectomy. The restoration of ICG-k was comparable to that of liver weight, but the restoration of CLamp was delayed. Conclusions. Functional liver capacity was minimal during parenchymal cell mitosis in the regenerating liver. Functional restoration after two-thirds hepatectomy was delayed in comparison with morphologic restoration in rat.

1. 1. Microsomal cytochrome P-450 and peroxisomal fatty acid oxidation was studied in the kidney of rats 7 days after bile duct ligation (BDL) and a comparative study between kidney and liver was done. 2. 2. Only in the liver did cholestasis decrease the cytocrome P-450 content and the peroxisomal fatty acid (3-oxidation, the catalase activity, and the microsomal metabolism of lauric acid and aminopyrine. 3. 3. In contrast, cholestasis did not influence these activities in the kidney. The microsomal and peroxisomal activities studied responded in a coordinate way to cholestasis. 4. 4. These results could suggest the possibility of a cause-and-effect relationship between microsomal cytochrome P-450 and peroxisomal activity.

Effects of central nervous stimulants on the spino-bulbo-spinal reflex potential were evaluated in anesthetized intact cats, and compared with those on segmental spinal reflex potentials in anesthetized spinal cats. In spinal cats, strychnine augmented polysynaptic reflex potential, picrotoxin inhibited dorsal root reflex potential, aminopyrine potentiated mono- and poly-synaptic reflex potentials but inhibited dorsal root reflex potential, and 4-aminopyridine potentiated all the three types of segmental reflex potentials. A combination of fenbufen, a non-steroidal antiinflammatory agent, and enoxacin, a new quinolone antimicrobial, inhibited all the three types of segmental reflex potentials. In contrast, all these drugs consistently produced an augmentation of the spino-bulbo-spinal reflex potential in anesthetized intact cats. From these findings, we suggest that the spino-bulbo-spinal reflex potential may be used as an electrophysiological parameter for the evaluation of central nervous stimulants.

The oxidation of aminopyrine, an N-alkyl aromatic amine, by the hydroperoxidase activity of lipoxygenase was studied. Aminopyrine gave rise to a purple color in the presence of H2O2 and lipoxygenase, the color being proportional to the aminopyrine radical cation. The H2O2/aminopyrine radical cation molar ratio was 0.5. The overall reaction was considered as an enzymic-chemical second order mechanism with substrate regeneration. From the equations, the apparent constant of the radical cation's decomposition (k'app) was evaluated under different experimental conditions. It was found to be inversely proportional to the proton concentration but unaffected by the concentration of aminopyrine. These results suggest a new comprehensive mechanism for N-demethylation, which takes into account the described presence of both nitrogen- and carbon-centered radicals and the marked effect of pH on the stability of the radical cation.

The formation of reactive oxygen species (ROS) could cause cellular damage and eventually lead to apoptosis and necrosis. The ratio between oxidized glutathione and reduced glutathione (GSSG-to-GSH ratio) has been used as an important in vitro and in vivo biomarker of the redox balance in the cell and consequently of cellular oxidative stress. This paper optimizes a LC-MS/MS method for the simultaneous determination of GSH and GSSG. The proposed method is based on the derivatization of reduced GSH using iodoacetic acid (IAA) in order to prevent its rapid oxidation to GSSG during sample preparation. The optimized analytical method was applied to evaluate the effect of different pharmaceutical agents on GSSG-to-GSH ratio in cryopreserved rat and human hepatocytes in culture. Hepatocyte viabilities were also determined at the same time by using the WST-1 assay as a direct measurement of cell mitochondrial respiration. The results obtained demonstrate that cryopreserved rat and human hepatocytes in culture are reliable in vitro models for the evaluation of cellular oxidative stress. In addition, the GSSG-to-GSH ratio measurements could be a biomarker of hepatotoxicity providing similar results to those of cytotoxicity assay.

This paper reports the establishment of a method for rapid identification 15 effective components of anti common cold medicine (paracetamol, aminophenazone, pseudoephedrine hydrochloride, methylephedrine hydrochloride, caffeine, amantadine hydrochloride, phenazone, guaifenesin, chlorphenamine maleate, dextromethorphen hydrobromide, diphenhydramine hydrochloride, promethazine hydrochloride, propyphenazone, benorilate and diclofenac sodium) with MRM by LC-MS/MS. The samples were extracted by methanol and were separated from a Altantis T3 column within 15 min with a gradient of acetonitrile-ammonium acetate (containing 0.25% glacial acetic acid), a tandem quadrupole mass spectrometer equipped with electrospray ionization source (ESI) was used in positive ion mode, and multiple reaction monitoring (MRM) was performed for qualitative analysis of these compounds. The minimum detectable quantity were 0.33-2.5 microg x kg(-1) of the 15 compounds. The method is simple, accurate and with good reproducibility for rapid identification many components in the same chromatographic condition, and provides a reference for qualitative analysis illegally added chemicals in anti common cold medicine.

Rectal drug delivery is currently at the focus of attention. Surfactants promote drug release from the suppository bases and enhance the formulation properties. The aim of our work was to develop a sample preparation method for HPLC analysis for a suppository base containing 95% hard fat, 2.5% Tween 20 and 2.5% Tween 60. A conventional sample preparation method did not provide successful results as the recovery of the drug failed to fulfil the validation criterion 95-105%. This was caused by the non-ionic surfactants in the suppository base incorporating some of the drug, preventing its release. As guidance for the formulation from an analytical aspect, we suggest a well defined surfactant content based on the turbidimetric determination of the CMC (critical micelle formation concentration) in the applied methanol-water solvent. Our CMC data correlate well with the results of previous studies. As regards the sample preparation procedure, a study was performed of the effects of ionic strength and pH on the drug recovery with the avoidance of degradation of the drug during the procedure. Aminophenazone and paracetamol were used as model drugs. The optimum conditions for drug release from the molten suppository base were found to be 100 mM NaCl, 20-40 mM NaOH and a 30 min ultrasonic treatment of the final sample solution. As these conditions could cause the degradation of the drugs in the solution, this was followed by NMR spectroscopy, and the results indicated that degradation did not take place. The determined CMCs were 0.08 mM for Tween 20, 0.06 mM for Tween 60 and 0.04 mM for a combined Tween 20, Tween 60 system.

Complement receptor type 1 (CR1) is an integral membrane protein of many hematopoietic cells and is found in a soluble form in plasma. Preliminary data have indicated that soluble complement receptor 1 (sCR1) levels in serum were increased in patients with cirrhosis. In this study, sCR1 was measured in patients with various liver diseases with a newly established enzyme-linked immunosorbent assay (ELISA). sCR1 level was elevated in chronic active hepatitis C (24 patients, 62.6 +/- 31 ng/ML; 31 normal controls, 31.4 +/- 7.8 ng/mL, P.001), and in cirrhosis (35 patients, 143.7 +/- 61 ng/mL, P.001). The levels increased transiently in 3 patients who had amanita phalloides intoxication. In 25 patients with advanced cirrhosis (pretransplantation screening), there were significant inverse correlations between sCR1 and both the prothrombin index (rs = -.60, P.002) and the aminopyrine breath test (rs = -.51, P.01). Following liver transplantation, the levels dropped from 166 +/- 70 to 49 +/- 24 ng/mL (P.0001), and serial measurements in the posttransplantation period showed a correlation with liver dysfunction, regardless of etiology. Since CR1 is not produced by hepatocytes, the most likely explanation for the increased level of sCR1 is reduced is reduced catabolism. Thus, sCR1 may be added to the short list of large glycoproteins that accumulate in liver disease.

It is generally believed that histamine-stimulated gastric acid secretion involves a transient elevation of intracellular Ca2+ and the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) cascade through phosphorylation, whose actions ultimately effect the fusion of H(+)-K(+)-adenosinetriphosphatase (ATPase)-containing vesicles to the apical plasma membrane of parietal cells. To dissect the signaling events underlying gastric acid secretion, we have developed a permeabilized gastric gland model using Staphylococcus alpha-toxin. The advantage of this model is its ability to retain cytosolic components that are required for the secretory machinery. Here we show that acid secretion in alpha-toxin-permeabilized glands is a cAMP-dependent process, reaching a maximal stimulation at 100 microM cAMP. The cAMP-elicited acid secretion, as monitored by the accumulation of the weak base aminopyrine (AP), required functional mitochondria or exogenously supplied ATP. Maximal stimulation elicited by cAMP for AP uptake by permeabilized glands was 51-85% of intact glands. Moreover, secretory activity was potentiated by 0.1 mM ATP. The recruitment of H(+)-K(+)-ATPase-rich tubulovesicles into the apical plasma membrane was measured using biochemical and morphological assays, thus validating the cell activation processes in response to cAMP. From this permeabilized model, [gamma-32P]ATP was used to directly phosphorylate target proteins. A number of proteins whose phosphorylation-dephosphorylation is specifically modulated by cAMP were found. These studies establish the first permeabilized gland model in which the resting-to-secreting transition can be triggered and show that cAMP-mediated phosphorylation is correlated with secretory activity.

Albumin and fibrinogen synthesis rates were measured in 15 subjects with different clinical stages of postviral cirrhosis and compared with galactose elimination capacity and aminopyrin breath test. Forty-three mg per kg body weight [ 2 H 5 ring]phenylalanine with an isotopic enrichment of 10 atom% were intravenously injected. [ 2 H 5 ring]phenylalanine enrichments in the plasma-free phenylalanine and the albumin and fibrinogen isolates were measured by gas chromatography-mass spectrometry. Fractional synthesis rates of albumin were normal in Child A cirrhosis (7.6 ± 2.2%d), but were lower in both Child B (3.5 ± 0.8%d) and C (4.5 ± 2.8%d). Absolute rates of albumin synthesis were (103 ± 30 mg/kg/d) in the child A group and substantially lower in the Child B (50 ± 3 mg/kg/d) and C (36 ± 20 mg/kg/d) group. The average fractional synthesis rate of fibrinogen was 16.7 ± 7.5%d and the absolute synthesis rate 11.6 ± 6.4 mg/kg/d. The values of the galactose elimination capacity and the aminopyrin breath test were below the normal range in all patients, gradually decreasing with an increase in the severity of the clinical stage of cirrhosis. Albumin synthesis rates significantly correlated with the Child scores, the galactose elimination capacity, and the aminopyrin breath test, whereas fibrinogen synthesis rates showed no such correlations. (Hepatology 1996 Jul;24(1):53-9)

Isolated rat parietal cells were used to investigate the role of intracellular Ca 2+ in the action of cAMP-dependent secretagogues and cross talk between cAMP- and Ca 2+ -dependent stimulatory pathways. Aminopyrine accumulation (an index of acid produced and trapped by the parietal cells), cytosolic free Ca 2+ , morphological transformation and cell viability were used to investigate parietal cell function and stimulation. The increase of cytosolic free Ca 2+ promoted by gastrin, or carbachol, was abolished by the intracellular Ca 2+ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′ -tetraacetic acid (BAPTA, 10 μM). Also, the morphological transformations induced by dibutyryladenosine 3′:5′-cyclic monophosphate (DBcAMP), gastrin, and S p-adenosine-cyclic-3′,5′-monophosphothioate ( S p-cAMPS) were completely abolished by BAPTA (10 μM). In aminopyrine accumulation the action of 1 mM DBcAMP was dose-dependently reduced by BAPTA. The Ca 2+ ionophore A23187 alone, in the range of 1 pM to 1 μM, had no effect but it dose-dependently potentiated the action of 1 mM DBcAMP in aminopyrine accumulation. The inhibitory actions of BAPTA on DBcAMP- and histamine-stimulated aminopyrine accumulation were dose-dependently reversed by A23187. Histamine-stimulated protein kinase activity and viability parameters as cellular lactate dehydrogenase (LDH) and trypan blue exclusion were not changed by BAPTA. These results indicated that in isolated parietal cells: (1) the action of cAMP-dependent secretagogues in aminopyrine accumulation and morphological transformation are dependent on cytosolic free Ca 2+ ; (2) Ca 2+ -induced morphological transformation is essential for aminopyrine accumulation; (3) a threshold level of one second messenger is required for stimulation of aminopyrine accumulation by the other second messenger.

BACKGROUND & AIMS: Epidermal growth factor (EGF) inhibits secretagogue- stimulated gastric acid secretion via an EGF receptor located on parietal cells. The aim of this study was to examine whether this growth factor inhibited carbachol-stimulated acid secretion through a protein kinase C-dependent mechanism. METHODS: The effect of EGF on carbachol-stimulated aminopyrine uptake, inositol trisphosphate formation, and intracellular Ca2+ ([Ca2+]i) in purified cultured parietal cells was studied. The ability of protein kinase A and C inhibitors to alter the inhibitory action of EGF was assessed. EGF- mediated translocation and activation of protein kinase C in parietal cells were determined. RESULTS: EGF dose dependently inhibited carbachol-stimulated aminopyrine uptake in a pertussis toxin- insensitive, genistein (tyrosine kinase inhibitor)--sensitive manner, with a maximal inhibitory effect (37.5% +/- 6.8%) achieved at 10(-7) mol/L. EGF did not significantly inhibit carbachol-stimulated inositol trisphosphate formation and did not alter the initial transient increase or sustained plateau in [Ca2+]i stimulated by this secretagogue. The protein kinase C inhibitors H-7 and staurosporine dose dependently reversed the inhibitory action of EGF, whereas H-89 (protein kinase A inhibitor) failed to alter the effect of EGF. EGF pretreatment increased the translocation of alpha and beta 1 isoforms of protein kinase C and stimulated kinase activity in parietal cells. EGF did not down-regulate the parietal cell muscarinic receptor. CONCLUSIONS: The inhibitory action of EGF on carbachol-stimulated parietal cell activity seems to involve protein kinase C. (Gastroenterology 1996 Feb;110(2):469-77)

Many drugs, industrial pollutants, and other xenobiotics are known to be oxidized by peroxidases to potentially harmful free-radical intermediates. We have examined the possibility that certain compounds, acting as efficient peroxidase substrates, may stimulate the formation of reactive free radicals by acting as mediators of electron transfer reactions (redox mediators). To explore this hypothesis, we have investigated the interaction of two well-known peroxidase substrates, chlorpromazine and aminopyrine. As shown by ESR and UV-visible spectroscopy, chlorpromazine radical was able to oxidize aminopyrine to aminopyrine cation radical. The rate constant for this rapid, pH-dependent, reaction was estimated to be 1 x 10(7) M-1 s-1 at pH 4.5. Transient-state and steady-state kinetic studies both showed that rate constants for chlorpromazine oxidation to its cation radical by horseradish peroxidase (HRP) were about 100-fold greater than for the corresponding HRP-catalyzed oxidation of aminopyrine to its cation radical. When both aminopyrine and chlorpromazine were present with HRP and H2O2, aminopyrine cation radical formation was stimulated 100-fold. Concomitantly, the accumulation of chlorpromazine cation radical was completely inhibited in the presence of aminopyrine. Similar results were obtained when lactoperoxidase, myeloperoxidase, or the myeloperoxidase mimic HOCl were substituted for HRP. These data suggest that chlorpromazine can act as a redox mediator for peroxidase-catalyzed oxidation of aminopyrine and other chemicals. We suggest that some peroxidase substrates, acting as redox mediators, may stimulate the production of toxic free-radical intermediates from various drugs and other xenobiotics. As such, this may have implications for a number of adverse effects caused by these xenobiotic chemicals.

We examined which cytochrome P-450 (P-450) species other than CYP1A participates in the oxidative metabolism of theophylline (TP) in mouse hepatic microsomes. Among the three metabolic pathways of TP, only 8-hydroxylation was selectively enhanced by acetone, a potent inducer of CYP2E. We assumed that two P-450 populations with different metabolic ability were involved in this metabolic process, and kinetic analyses revealed that the enhancement was due to the induction of a high-capacity P-450 population. The 8-hydroxylation at a substrate concentration, where most of the total activity was attributed to the catalysis of the high-capacity phase, was markedly impaired by CYP2E inhibitors such as 4-methylpyrazole and aminoacetonitrile, whereas the N-demethylations were little affected by these agents. The activity of TP 8-hydroxylation was significantly correlated with that of p-nitrophenol hydroxylation, a probe for CYP2E, in untreated microsomes. The activities of these oxidative reactions were modified to a similar degree by known enzyme inhibitors with a range of inhibitory potencies and affinity for P-450 isoforms. On the other hand, a relationship between TP N-demethylations and p-nitrophenol hydroxylation was not apparent, but there was a behavioral similarity between the two types of N-demethylations. The results indicated that TP 8-hydroxylation, which accounts for a large portion of TP oxidations, involves CYP2E, and that its N-demethylations are mediated by a common or closely similar P-450 species distinct from CYP2E.

In this work, the thermal behaviour of three active substances (phenazone, aminophenazone, phenylbutazone) was studied by drawing up the TG/DTG/DTA curves in air/nitrogen atmosphere at 10 °C min −1 heating rate. The information on the thermal-induced events was corroborated with the IR spectra of the solid samples (pharmaceutical compounds and the remaining chars after heating treatment), respectively with the ones obtained by evolved gases analysis (EGA). The data on a possible drug–excipient interaction were obtained from the thermoanalytical study of mixtures of these active compounds with talc, magnesium stearate, starch and microcrystalline cellulose. No changes were observed by TG/DTG/DTA curves of mixtures in comparison with the pure compound. Even if the three active substances contain the same heterocyclic ring, having similar molecular structures, their thermal behaviour is not similar. According to thermal and evolved gas analysis, it was proved that formation of CO 2 does not involve atmospheric oxygen. By stoichiometric means, the molecular breakdown of aminophenazone can generate only carbon monoxide, which undergoes disproportionation, generating CO 2 .

In the present study we compared various CCK(B) receptor antagonists and tried to detect a difference in biological activity between the C-terminal octapeptides of cholecystokinin (CCK-8) and [Leu11]gastrin-(5-17) in isolated rabbit gastric glands. Binding experiments showed that different CCK(B)/gastrin receptor agonists bound with high affinity and that antagonists inhibited this binding in accordance with a CCK(B)/gastrin pharmacological profile. [Leu11]gastrin-(5-17), CCK-8 and cionin were found to induce [14C]aminopyrine accumulation to 25% above the basal level. Under the same experimental conditions, histamine induced a response twice as great as the response obtained with [Leu11]gastrin-(5-17) or CCK-8. [Leu11]gastrin-(5-17) (10(-7) M), CCK-8 (10(-8) M) and cionin (10(-8) M) appeared to be full agonists. CCK(B)/gastrin receptor antagonists including L-365,260 (3R-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin++ +-3-yl)-N-(3-methylphenyl) urea), L-364,718 (3S-(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin++ +-3-yl)-1H-indole-2-carboximide) (a selective CCK(A) receptor antagonist), PD-135,158 (4([2-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2-[[[1.7.7-trimethyl-bicyclo[2. 2.1]hept-2-yl)oxy]carbonyl]amino]propyl]amino]-1-phenylethyl] amino-4-oxo-[1S-1alpha.2beta[S*(S*)]4alpha]]-butano nate N-methyl-D-glucamine) (bicyclo system 1S-endo), YM-022 ((R)-1-[2,3-dihydro-1-(2'-methylphenacyl)-2-oxo-5-phenyl-1H-1,4-++ +benzodiazepin-3-yl]-3-(3-methylphenyl)urea) and JMV-180 (Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-O-CH2-CH2-C6H5) exhibited the same profile for inhibition of [Leu11]gastrin-(5-17) or CCK-8-induced [14C]aminopyrine accumulation in rabbit gastric glands. These results suggested that [Leu11]gastrin-(5-17) and CCK-8 induced [14C]aminopyrine accumulation by the same mechanism. [Leu11]gastrin-(5-17)- or CCK-8-induced [14C]aminopyrine accumulation was inhibited by about 40% by the histamine H2 receptor blocker cimetidine. These results are consistent with there being cooperativity between [Leu11]gastrin-(5-17) (or CCK-8) and histamine in the acid secretory pathway. Similarly, the CCK(B)/gastrin receptor antagonists were tested against histamine-induced [14C]aminopyrine accumulation and surprisingly, only compound L-365,260 appeared active and even more potent than cimetidine.

Lauric acid hydroxylation and aminopyrine N-demethylation were studied in kidney and liver microsomes from rats treated with fish oil. Different doses of fish oil containing 20% eicosapentaenoic acid and 10% docosahexaenoic acid were provided daily to the rats for seven days. In all the groups studied, the lauric acid metabolism was higher in kidney microsomes and the aminopyrine metabolism in the liver microsomes. Although no effect on the renal cytochrome P-450 concentration was detectable, all four fish oil doses increased the hepatic concentration of cytochrome P-450 by a mean 27%. The higher fish oil doses used increased the renal and hepatic microsomal metabolism of aminopyrine. The lauric acid metabolism was increased by fish oil only in the liver. Fish oil, a known inducer of fatty acid peroxisomal beta-oxidation, also induced microsomal activity. These results show that liver and kidney respond in different ways to dietary factors such as fish oil. In addition, our study would suggest that fish oil increased the activity of two different families of liver cytochrome P-450. The activity of kidney lauric acid 11- and 12-hydroxylation, however, was not modulated by fish oil.

Epidermal growth factor (EGF) acutely inhibits acid secretion; however, prolonged administration of EGF has been reported to increase acid production. We undertook these studies to examine whether the physiological effects of EGF on acid secretion are mediated by regulation of gastric H+,K+-ATPase, the principle enzyme responsible for acid secretion. EGF in concentrations equivalent to those in plasma increased H+,K(+)-ATPase alpha-subunit mRNA levels. Using H+,K(+)-ATPase-luciferase constructs transfected into primary cultured parietal cells, a significant step up in EGF inducibility was observed between bases -162 and -156 (5'-GACATGG-3') relative to the cap site. This EGF response element (ERE) conferred EGF inducibility when linked to homologous and heterologous promoters. The ERE is homologous to the 3' half-site of the c-fos serum response element to which rNFIL-6, rE12, and SRE-ZBP bind. Electrophoretic mobility shift assays using an ERE probe and parietal cell nuclear extracts revealed a specific DNA-protein complex, the formation of which was changed by neither E12 and NFIL-6 consensus oligonucleotides nor antibodies for NFIL-6, SRE-ZBP, and E12. Our studies indicate that EGF induces gastric H+,K(+)-ATPase alpha-subunit gene expression via an interaction between a specific ERE and a novel transcriptional factor and that this may be a physiologic mechanism by which EGF regulates acid secretion.

Isolated guinea pig parietal cells, the function of which is similar to that of human parietal cells, were used in this study. The accumulation of 14C-aminopyrine (14C-AP) was measured to study the inhibitory mechanism of leminoprazole in cells. Stimulation by 10 microM histamine, 0.1 mM carbachol, 1 microM gastrin or 1 mM db-cAMP brought about satisfactory incorporation of 14C-AP, and leminoprazole concentration-dependently inhibited acid secretion induced by these stimulants. At 10(-5) M, almost 100% inhibition was observed. The IC50 values of leminoprazole obtained from its inhibitory action on histamine, carbachol, gastrin and db-cAMP-stimulated acid secretion were 4.0 x 10(-7) M, 3.5 x 10(-7) M, 2.5 x 10(-7) M and 5.6 x 10(-7) M, respectively. Thus the extent of inhibition was the same for the responses to all the secretagogues. These results indicate that the site of action of leminoprazole is intracellular and distal from cAMP (intracellular second messenger), but not at the receptor sites. The results also strongly suggest that the inhibitory action of leminoprazole on H+,K(+)-ATPase may contribute to the inhibitory effect of this drug on gastric acid secretion.

We tested sonicates of Helicobacter pylori, H. mustelae, and H. felis for inhibition of acid secretion in rabbit and ferret isolated gastric glands. Three H. pylori strains, two of three H. mustelae strains, and two H. felis strains significantly inhibited acid secretion in rabbit cells by 95.2-93.3%, 55.9% and 96.4%, and 83.4-96%, respectively. All Helicobacter strains examined inhibited acid secretion by ferret cells by 65.3-76.8%, 89.1-97.6%, and 85.8-92.8%. H. pylori inhibited acid secretion after stimulation with histamine and isobutylmethylxanthine or with 8-bromo-cyclic adenosine monophosphate (P0.05 for all tests). These findings demonstrate that acid inhibition is a property common to the three Helicobacter species tested. It occurs independently of the mammalian origin of the parietal cell, and it does not involve blockade of histamine-2 receptors.

NADPH-cytochrome P-450 oxidoreductase (EC 1.6.2.4) was purified from the microsomal fraction of tobacco (Nicotiana tabacum) BY2 cells by chromatography on two anion-exchange columns and 2',5' ADP-Sepharose 4B column. The purified enzyme showed a single protein band with a molecular weight of 79 kDa on SDS-PAGE and exhibited a typical flavoprotein redox spectrum, indicating the presence of an equimolar quantity of FAD and FMN. This enzyme followed Michaelis-Menten Kinetics with Km values of 24 microM for NADPH and 16 microM for cytochrome c. An in vitro reconstituted system of the purified reductase with a partially purified tobacco cytochrome P-450 preparation showed the cinnamic acid 4-hydroxylase activity at the rate of 14 pmol min-1 nmol-1 P-450 protein and with a purified rabbit P-4502C14 catalyzed N-demethylation of aminopyrine at the rate of 6 pmol min-1 nmol-1 P-450 protein. Polyclonal antibodies raised against the purified reductase reacted with tobacco reductase but not with yeast reductase on Western blot analysis. Anti-yeast reductase antibodies did not react with the tobacco reductase. This result indicate that the tobacco reductase was immunochemically different from the yeast reductase. The anti-tobacco reductase antibodies totally inhibited the tobacco reductase activity, but not the yeast reductase. Also, Western blot analyses using the anti-tobacco reductase antibodies revealed that leaves, roots and shoots of Nicotiana tabacum plants contained an equal amount of the reductase protein. From these results, it was suggested that there are different antibody binding sites, which certainly participate in enzyme activity, between tobacco and yeast reductase.

The relationship among impaired selenium status, lipid peroxidation, and liver function was examined in 19 hospitalized patients with severe alcoholic cirrhosis. Plasma selenium was found to be significantly lower (mean +/- SD: 54 +/- 13 micrograms/L) than in healthy controls (83 +/- 11 micrograms/L) and plasma malondialdehyde, assessed as thiobarbituric acid reactants, which reflects lipid peroxidation, was increased (2.0 +/- 1.2 mumol/L vs < 1.2 mumol/L in controls). The mean 14C aminopyrine breath test, an indicator of liver function, was lower than normal (2.7 +/- 1.9 vs 6.3 +/- 0.9% in controls) and found to be significantly correlated with plasma selenium (r = 0.59, p < 0.05). A prospective, randomized selenium supplementation trial was conducted in a group of 16 patients who received either daily 100 micrograms selenium as enriched yeast during 4 mo or a placebo. Among the 10 patients who completed the study, plasma selenium significantly increased in the supplemented group (n = 4; before: 58 +/- 10 micrograms/L, and after 101 +/- 12 micrograms/L, p < 0.01) contrary to the placebo group (n = 6, before: 47 +/- 10 micrograms/L, after: 57 +/- 9 micrograms/L, n.s.). 14C aminopyrine breath test improved in three out of four selenium-supplemented patients and in three out of six placebo patients, but the small number of patients did not allow statistical evaluation. These results demonstrate that low selenium status in alcoholic cirrhosis is correlated to liver function and could be improved by supplementation.

Background In 2007, sorafenib was the first drug able to improve overall survival in patients with advanced hepatocellular carcinoma. Aim In 2005 we designed a phase II study to assess safety and efficacy of sunitinib. Methods This is a single arm, open-label, single-centre phase II trial. Eligibility criteria were advanced hepatocellular carcinoma; no prior chemotherapy, performance status 0–1; and Child ≤ B8. The treatment schedule was 50 mg each day orally, 4 weeks on, 2 weeks off. Results Between 10/2007 and 10/2010, 34 patients were enrolled. A significant worsening of liver functional reserve after sunitinib was observed. Grade 3/4 adverse effects occurred in 80% of patients and included fatigue (47%), nausea (15%), liver failure (15%), encephalopathy (12%) and upper gastrointestinal bleeding (12%). Six patients (18%) died within 60 days of enrolment. A partial response was observed in 4 patients (12%). Median time to tumour progression was 2.8 months and median overall survival was 5.8 months. Conclusion A dose of 50 mg/d induces a high rate of severe adverse events. Toxicity remains a key concern also at the dose of 37.5 mg/d. However, sunitinib is able to induce a prolonged response in some patients. Positron Emission Tomography/Computed Tomography scans may select good responders.

Acute respiratory infections are the second leading cause of morbidity in children under 18 years. Several drugs have been used with variable efficacy and safety, trying to reduce the associated symptoms and improve quality of life.To evaluate the efficacy and safety of buphenine, aminophenazone and diphenylpyraline hydrochloride when compared with placebo for the control of symptoms associated with common cold in children 6-24 months of age.Randomized clinical trial, double blind, placebo controlled, in 100 children24 months of any gender, with symptoms associated to common cold. They received the drug under study vs. placebo for seven days. Both groups received acetaminophen. The change on common cold related symptoms were evaluated. Statistic analysis was made with STATA 11.0 for Mac.Fifty-three children were randomized to study drug and forty-seven to placebo. Age of children in each group was similar (12.2 +/- 5.8 months vs. 12.7 +/- 5.8 months, p NS). There were significant differences between groups in relation to rhinorrea and sneezing resolution, with better results in Flumil group and no adverse events observed.The results in this study indicates that Flumil is a safe and effective drug for control of symptoms present in the common cold in children aged 6-24 months.

In order to evaluate the pharmacokinetics of metamizol in the presence of morphine in arthritic rats, after subcutaneous administration of the drugs, an easy, rapid, sensitive and selective analytical method was proposed and validated. The four main metamizol metabolites (4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoantipyrine) were extracted from plasma samples (50–100 μl) by a single solid-phase extraction method prior to reverse-phase high performance liquid chromatography with diode-array detection. Standard calibration graphs for all metabolites were linear within a range of 1–100 μg/ml ( r 2 ≥ 0.99). The intra-day coefficients of variation (CV) were in the range of 1.3–8.4% and the inter-day CV ranged from 1.5 to 8.4%. The intra-day assay accuracy was in the range of 0.6–9.6% and the inter-day assay accuracy ranged from 0.9 to 7.5% of relative error. The lower limit of quantification was 1 μg/ml for all metabolites using a plasma sample of 100 μl. Plasma samples were stable at least for 4 weeks at −20 °C. This method was found to be suitable for studying metamizol metabolites pharmacokinetics in arthritic rats, after simultaneous administration of metamizol and morphine, in single dose.

The structurally characterized lower rim 1,3-di{4-antipyrine}amide conjugate of calix[4]arene (L) exhibits high selectivity toward Hg(2+) among other biologically important metal ions, viz., Na(+), K(+), Ca(2+), Mg(2+), Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Ag(+) as studied by fluorescence, absorption, and ESI MS. L acts as a sensor for Hg(2+) by switch-off fluorescence and exhibits a lowest detectable concentration of 1.87 ± 0.1 ppm. The complex formed between L and Hg(2+) is found to be 1:1 on the basis of absorption and fluorescence titrations and was confirmed by ESI MS. The coordination features of the mercury complex of L were derived on the basis of DFT computations and found that the Hg(2+) is bound through an N(2)O(2) extending from both the arms to result in a distorted octahedral geometry with two vacant sites. The nanostructural features such as shape and size obtained using AFM and TEM distinguishes L from its Hg(2+) complex and were different from those of the simple mercuric perchlorate. L is also suited to sense pyrimidine bases by fluorescence quenching with a minimum detection limit of 1.15 ± 0.1 ppm in the case of cytosine. The nature of interaction of pyrimidine bases with L has been further studied by DFT computational calculations and found to have interactions through a hydrogen bonding and NH-π interaction between the host and the guest.

Hepatic arteriovenous malformations (HAVMs) in hereditary hemorrhagic telangiectasia (HHT) have long been considered to have scarce clinical significance in most cases. Nevertheless, data are lacking regarding the influence of HAVMs on the liver first-pass effect on drugs in HHT patients. To gain insight into the effect of HAVMs on hepatic drug clearance by means of two specific (13)C-labeled probes, namely the (13)C-methacetin and (13)C-aminopyrine, 46 HHT patients and 44-matched healthy controls were enrolled. The liver first-pass effect was studied by the (13)C-based breath test using methacetin and aminopyrine. The methacetin breath test showed statistically significant reduced metabolism rates (p0.0001) in HHT when compared with controls, both in patients with and without CT-detectable HAVMs, and when expressed both as cumulative (13)C-percentage dose per hour and as (13)C-percentage peak after 15 min. In contrast, no significant difference was found between HHT and controls regarding aminopyrin metabolism rates. In HHT, (13)C%-methacetin breath test values are significantly lower than those found in normal subjects, probably due to the effect of hepatic shunts. A reduced perfusion and an impaired hepatic metabolism might affect hepatic drug clearance in HHT. Therefore, an appropriate dosage adjustments should be considered when high-hepatic-metabolism drugs are administered to HHT patients.

Indan derivatives, namely, 5-(5',6'-dichloroindan-1'-yl)-tetrazole (12a) and 5-(5',6'-dichloroindan-1'-yl)- methyltetrazole (12b), were synthesized conveniently from 5,6-dichloroindan-1-carboxylic acid (9a) and 5,6- dichloroindan-1-acetic acid (9b), respectively, as potential analgesic and anti-inflammatory agents. The analgesic and anti-inflammatory properties of 9a, 9b, 12a and 12b were evaluated by the acetic acid induced writhing in Swiss albino mice and the carrageenan-induced rat paw edema models, respectively. Compounds 9a and 12a exhibited significant analgesic activity with the doses of 50 and 100 mg/kg body weight, comparable to that of the positive controls, phenylbutazone, indomethacin and aminopyrine. The anti-inflammatory potencies of 9a and 12a were also comparable to that of the positive control, phenylbutazone. Compounds 9b and 12b showed analgesic and anti-inflammatory activities, but were weaker than that of compounds 9a and 12a.

Impaired hepatic function is a major contributory factor to the high incidence of postoperative morbidity and mortality in patients with malignant biliary obstruction. Dynamic hepatic function tests such as indocyanine green (ICG) retention and aminopyrine breath tests were evaluated in such patients to define whether they were clinically useful for prediction of postoperative morbidity and mortality. Forty-four patients with malignant biliary obstruction undergoing surgery for relief of obstructive jaundice were recruited into the study. Indocyanine green retention and aminopyrine breath tests were carried out in all patients pre-operatively and repeated in 36 patients postoperatively. The ICG retention was abnormal in all patients before surgery and there was significant improvement 2 weeks after surgery (32.8 +/- 2.5% vs 18.3 +/- 2.8%, P = 0.001). The change in ICG retention levels correlated with the serum bilirubin levels but the pre-operative ICG retention value could not predict postoperative morbidity and mortality. The aminopyrine breath test was abnormal in all but one patient. It correlated with pre-operative prothrombin time of the patients before surgery but it did not improve significantly after surgery and was not predictive of postoperative outcome. It is concluded that both ICG retention and aminopyrine breath tests have limited clinical value in the pre-operative evaluation of patients with malignant biliary obstruction.

We investigated the effects of extracellular magnesium on stimulus-secretion coupling in isolated rat parietal cells. A high concentration of extracellular magnesium (10mM) decreased basal and carbachol-stimulated calcium levels, whereas low magnesium levels (0.2mM) had the opposite effect. The calcium-triggered acid secretion was influenced in the same manner. Basal and carbachol-stimulated acid secretion could be enhanced by incubation in a buffer of low magnesium concentration, whereas a high magnesium concentration totally suppressed the carbachol-induced acid secretion. These results demonstrate that magnesium plays a modulative role in calcium-dependent stimulus-secretion coupling in rat parietal cells.

BACKGROUND Rapid assessment of hepatic function early after reperfusion of the liver graft is of great importance, because it may allow for prompt detection of incipient hepatic graft failure. The current study was undertaken to determine whether the continuous recording of neuromuscular transmission could be used as an on-line assessment of hepatic function during liver transplantation when a muscle relaxant with high hepatic uptake is used. METHODS We quantified and compared the effect of liver exclusion and graft reperfusion on the level of vecuronium-induced neuromuscular blockade in nine pigs studied twice within 3 days. During the 1st day (control session), an intravenous infusion of vecuronium was administered to maintain a constant 90-95% twitch depression during 180 min. The twitch response was then allowed to recover spontaneously to 75% of its prerelaxant value. Neuromuscular transmission was continuously measured on the right anterior leg using an acceleration transducer. During the same time period, the metabolic rate of 14C-labeled aminopyrine (a well-established quantitative test of the liver microsomal function) was determined by measuring the excretion of 14CO2 in expired air after administration of an intravenous bolus of 14C-labeled aminopyrine. Two days later, the pigs underwent a hepatic autotransplantation, during which vecuronium was administered to maintain a constant 90-95% twitch depression. After reperfusion of the liver graft, the vecuronium infusion rate was maintained at its anhepatic level, and the recovery index of the neuromuscular blockade (the time from 25% to 75% recovery of twitch height) was calculated. The aminopyrine breath test was performed during the last 30 min of the anhepatic phase, and during 3 h after reperfusion of the liver graft. RESULTS During control studies, the mean infusion rate of vecuronium was 1.30 +/- 0.33 mg.kg-1.h-1 and the recovery index was 3.4 +/- 0.5 min. During liver dissection, the infusion rate of vecuronium was similar to the control value (1.18 +/- 0.16 mg.kg-1.h-1), then considerably decreased to 0.05 +/- 0.03 mg.kg-1.h-1 during the anhepatic phase. After reperfusion of the liver graft, the recovery index was markedly prolonged to 35.5 +/- 15.8 min, indicating a prolongation of the recovery of neuromuscular blockade by a factor of 10.4. Excretion of 14CO2 was equal to zero during the anhepatic phase and then increased to 0.19 +/- 0.11% during the 1st h after reperfusion of the liver graft, an excretion rate corresponding to 11.2% of control conditions. The relationship between individual changes in the recovery index of the neuromuscular blockade and 14CO2 excretion in expired air after reperfusion of the liver graft showed a strong significant correlation (r2 = 0.71). CONCLUSIONS These results indicate that, compared with the control studies, there is a similar decrease in the recovery rate of vecuronium-induced neuromuscular blockade and in the metabolic rate of 14C-labeled aminopyrine during the progressive recovery of hepatic function immediately after unclamping of the liver vessels. Metabolism of 14C-labeled aminopyrine increased progressively during the reperfusion phase. Therefore, recording of neuromuscular transmission during liver transplantation could serve as a continuous and easy to perform assessment of liver graft function provided that a muscle relaxant with a high hepatic uptake is used for neuromuscular blockade.

The aim of this work was to study the action of methotrexate on the cytochrome P-450 hepatic monooxygenases and to follow the evolution of the metabolic processes with time, after oral and intra-peritoneal administration of these drugs to rats. In order to perform this study, we used a new technique the [13C]-aminopyrine breath test (ABT). At the end of the in vivo study, the rats were sacrificed and the cytochrome P-450 concentration as well as microsomal enzymatic activities of aminopyrine N-demethylase, 7-ethoxyresorufin desalkylase, 7-ethoxycoumarin desalkylase, 7-pentoxyresorufin desalkylase were determined. A histological study of the liver was also carried out. Finally, haematological and biochemical parameters were also determined. In this study, 3 series of 6 rats were used: a control group, a group receiving 70 mg/kg of methotrexate by intra-peritoneal route, and a group of rats treated orally with 1 mg/kg of methotrexate every 2 days. For each observation, the variation of the ABT scores and of the cytochrome P-450 amounts as well as the microsomal aminopyrine N-demethylase activity were in good agreement.

Rabbit gastric glands were treated with α-toxin to test for permeabilization of basolateral membrane and retention of functional activity of parietal cells. Treatment with up to 400 U α-toxin/mL resulted in a dose-dependent increase in permeabilization, as judged by nuclear uptake of trypan blue (960 daltons), while causing relatively little loss of cytoplasmic macromolecules in the size range of lactate dehydrogenase (134 000 daltons). In the presence of cAMP and ATP, α-toxin-permeabilized resting gastric glands were stimulated to accumulate aminopyrine by ~10-fold over glands incubated without added nucleotides. Aminopyrine accumulation in stimulated permeabilized glands was inhibited by specific H+,K+-ATPase inhibitors, omeprazole and SCH-28080, and by the selective inhibitor of protein kinase A, H-89 (IC50 = 7.17 ± 2.05 μM; n = 4). Aminopyrine accumulation in the α-toxin-treated glands was dependent on both exogenous ATP and cAMP; however, when no exogenous ATP was present, cAMP-activated aminopyrine accumulation reached ~50% of maximum, and at levels of ATP > 0.05 mM, maximal aminopyrine accumulation occurred without exogenous cAMP. In the presence of ATP alone, aminopyrine accumulation in permeabilized glands achieved 61.1 ± 3.2% (n = 10; range, 50–70%) of the values measured on paired samples of intact glands stimulated with histamine plus isobutylmethylxanthine. These results demonstrate the functional responsiveness of α-toxin-permeabilized resting gastric glands. The participation of a protein kinase A dependent pathway during activation of permeabilized parietal cell is proposed.Key words: aminopyrine accumulation, H+,K+-ATPase, H-89, protein kinase A, oxidative phosphorylation, digitonin.

Cholesterol values measured with the Reflotron dry chemistry (Boehringer Mannheim, Mannheim, Germany) and enzymatic (CHOD-PAP) routine method in 4150 venous blood samples from 580 infants aged 7 months, 1778 children aged 13 months to 19 years, and 1792 adults aged 18-63 years showed good correlation between the two assay systems (r > 0.92). However, the mean cholesterol concentrations were 4.5%, 3.7% and 2.3% lower in infants, children and adults, respectively, if measured with the Reflotron analyser as compared with values obtained with the CHOD-PAP method. These differences in the values were greater when cholesterol values were low (significance for the slope of regression line, p < 0.001). Values were below the detection limit of the Reflotron assay (2.59 mmol l-1) in 104 (4.4%) and 4 (0.2%) of all paediatric and adult samples, respectively. When measured with the CHOD-PAP method, 75 (72%) and 3 (75%) of these same samples showed values which exceeded the 2.59 mmol l-1 detection limit. We conclude that Reflotron dry chemistry analyser is precise in fast measurement of serum cholesterol both in all paediatric subjects and adults. A particular problem in paediatric age groups is that about 4% of children have values below the detection limit of the Reflotron analyser.

This study was conducted to evaluate in vivo the hepatotoxic effects of CCL 4 administration to rats using 13 C breath tests: aminopyrine breath test (ABT) was used to monitor CCL 4 -induced cytochrome P450 inactivation, and galactose breath test (GBT) to quantitatively measure the CCl 4 -induced decrease of liver function. The ABT results showed profound aminopyrine demethylation inhibition lasting for three days and complete recovery at day 7, while GBT results were decreased only one day after CCl 4 . The protection induced by a first CCl 4 dose against a second one paralleled cytochrome P450 inactivation: a second CCl 4 dose given three days after the first one induced no GBT decrease and a mild increase of serum transaminase activities. On the other hand, the second dose administered 7 days after the first one produced a GBT decrease similar to the one observed after the first one. These results should be taken into consideration to determine the optimal CCl 4 dosing schedule in the rat CCl 4 -induced cirrhosis model.

The methanol extract of Enhydra fluctuans, given orally at the dose of 250 and 500 mg/kg, was evaluated for its analgesic activity using the acetic acid induced writhing and the tail-flick methods. The extract showed promising activity in both tests.

Background: The prediction of fibrosis is an essential part of the assessment and management of patients with chronic liver disease. Non-invasive tests (NITs) have a number of advantages over the traditional standard of fibrosis assessment by liver biopsy, including safety, cost-effectiveness, and widespread accessibility. Objectives: The aim of this study was to determine the accuracy of certain biomarkers and transient elastography (TE) alone or in combination to predict the stage of liver fibrosis in chronic hepatitis C (CHC). Also, we examined whether the combination of certain biomarkers and TE could increase the diagnostic accuracy of liver fibrosis assessment. Patients and Method: A total of 446 patients who were previously diagnosed with CHC were included in the study. In the study group, 6 blood-based scores (APRI, Forns, Fib-4, Hepascore, FibroTest, and Fibrometer) were calculated, and TE was performed to validate the stage of fibrosis, compared with liver biopsy (LB) as the standard. Results: Significant fibrosis (F ≥ 2) was predicted with an AUROC of 0.727, 0.680, 0.714, 0.778, 0.688, 0.797, and 0.751 for the APRI, Forns, Fib-4, FibroTest, Hepascore, and Fibrometer scores and TE (Fibroscan), respectively. Severe fibrosis (F ≥ 3) was predicted, with AUROCs ranging between 0.705 and 0.811 for Hepascore and Fibrometer, respectively. Of the biomarkers, Fibrometer had the highest AUROC value in predicting both significant and severe fibrosis. The combination of APRI or FIB-4 with Fibrometer increased the diagnostic accuracy for significant fibrosis (from 69.07 to 82.27 for APRI, P = 0.001 and from 57.74 to 81.33, P = 0.001 for Fib-4). Combining APRI or Fib-4 with TE also increased the diagnostic accuracy (from 69.07 to 80.70%, P = 0.001 for APRI and from 57.74 to 81.33%, P = 0.001 for Fib-4) for significant fibrosis. The association that included Fibrotest was also reliable for the improvement of diagnostic accuracy. These combinations were more accurate or the assessment of severe fibrosis. Conclusions: The synchronous association between a simple, inexpensive score and a complex but expensive score or TE increases the diagnostic accuracy of non-invasive methods for the assessment of liver fibrosis stage.

Background & Aims: Although numerous non-invasive tests are currently available to explore liver function and disease activity in patients with HCV-related chronic diseases, none of these indicate the likelihood of disease progression in the individual patient. We aimed at assessing the prognostic ability of C-13(2)-aminopyrine breath test (C-13-ABT) in the prediction of liver fibrosis progression in patients with HCV chronic hepatitis who prospectively entered a long-term follow-up. Methods: Fifty patients with HCV-related chronic disease who underwent paired liver biopsy (at baseline and after a mean period of 86 months) were included in the study. C-13-ABT was carried out at baseline and every 3 years. Histological progression was defined as increase of at least 2 fibrosis units according to Ishak score. Results: Fourteen patients progressed of at least 2 fibrosis units during the follow-up. These patients were more frequently infected with a HCV-1b genotype and had, at baseline, a significantly older age, higher BMI, AST levels, and AST to platelet ratio index (APRI). C-13-ABT was altered in 57% of cases at baseline and in 100% of the cases at 3-year follow-up. In the univariate analysis, age (p = 0.005), BMI (p = 0.006), platelet count (p = 0.03), AST (p = 0.012) and ALT (p = 0.04) levels, APRI (p = 0.03), and baseline C-13-ABT results (p

This study determined the within-subject and between-subject variability of different ways of expressing the results of the (13)C-aminopyrine breath test ((13)C-ABT) and the effect of shortening the test duration. The (13)C-ABT was conducted on three separate occasions in 10 healthy volunteers and on a single occasion in 22 patients with established liver cirrhosis. The within-subject variability of cumulative percentage dose recovered (cPDR), using measured CO(2) production rate (VCO(2)), in the reference group over three trials was 15% over 120 min. Higher within-subject variability in cPDR would have been evident if the test was terminated at either 30 or 60 min. Substitution of predicted VCO(2) to calculate cPDR yielded comparable values at all time points. Significant differences between cirrhotics and reference group were evident after just 10 min using PDR/h, cPDR or enrichment (all P0.05). The ABT demonstrates clinically acceptable reproducibility. Shortening of the duration may make the test more acceptable clinically, but it is associated with increasing imprecision.

The purpose of this study was to identify both the possibilities of in vitro generation of 1-phenyl-2,3-dimethyl-4-dimethylamino-5-pyrazolone (aminophenazone) radical cation (abbreviated APH*+) and the mechanisms of interaction with different antioxidants.The chromophore radical cation APH*+ was obtained by oxidizing APH with ammonium persulfate. The stages of the oxidation reaction and the production of the intermediate compounds were studied by spectrophotometrical and potentiometric titration. As standard antioxidant there was used a catechin solution in distilled water with concentrations ranging between 50-250 microg/mL, in weak alkaline environment.The oxidation of APH with ammonium persulfate developed in four stages, involving the production of some intermediate compounds with different colorations. A mechanism for this reaction has been proposed. The radical cation forms to an APH - ammonium persulfate molar ratio of 2 : 1. In the presence of an antioxidant (catechin) the radical cation APH*+ forms to a molar ratio APH-ammonium persulfate of 1:1, in weak alkaline environment. It has an absorbtion maxima at lambda = 540 nm. The molar extinction coefficient of APH*+ at lambda = 540 nm has been calculated. Its absorbance was proportional with catechin concentration in the range 50-250 microg/mL (r2 = 0.9988).The APH-ammonium persulfate system can be used for spectrophotometric determination of total polyphenols in different systems.

Aminopyrine is metabolized by cytochrome P450 (CYP) in the liver. The investigators evaluated influences of different PPIs on CYP activity as assessed by the [(13)C]-aminopyrine breath test ([(13)C]-ABT). Subjects were 15 healthy volunteers with different CYP2C19 status (5 rapid metabolizers [RMs], 5 intermediate metabolizers [IMs], and 5 poor metabolizers [PMs]). Breath samples were collected before and every 15 to 30 minutes for 3 hours after oral ingestion of [(13)C]-aminopyrine 100 mg on day 8 of each of the following regimens: control; omeprazole 20 mg and 80 mg, lansoprazole 30 mg, and rabeprazole 20 mg. Changes in carbon isotope ratios in carbon dioxide ((13)CO(2)/(12)CO(2)) in breath samples were measured by infrared spectrometry and expressed as delta-over-baseline (DOB) ratios (‰). Mean areas under the curve of DOB from 0 to 3 h (AUC(0-3h) of DOB) were significantly decreased by omeprazole 20 mg and lansoprazole 30 mg but not by rabeprazole 20 mg. Conversely, higher PPI dose (ie, omeprazole 80 mg) seemed to further decrease AUC(0-3h) of DOB in RMs but increased it in PMs. Omeprazole and lansoprazole at the standard doses inhibit CYP activity but rabeprazole does not, whereas high-dose omeprazole seems to induce CYPs.

To understand the molecular basis of sequential N-dealkylation by cytochrome P450 2B enzymes, we studied the binding of amidopyrine (AP) as well as the metabolites of this reaction, desmethylamidopyrine (DMAP) and aminoantipyrine (AAP), using the X-ray crystal structure of rabbit P450 2B4 and two nuclear magnetic resonance (NMR) techniques: saturation transfer difference (STD) spectroscopy and longitudinal (T(1)) relaxation NMR. Results of STD NMR of AP and its metabolites bound to P450 2B4 were similar, suggesting that they occupy similar niches within the enzyme's active site. The model-dependent relaxation rates (R(M)) determined from T(1) relaxation NMR of AP and DMAP suggest that the N-linked methyl is closest to the heme. To determine the orientation(s) of AP and its metabolites within the P450 2B4 active site, we used distances calculated from the relaxation rates to constrain the metabolites to the X-ray crystal structure of P450 2B4. Simulated annealing of the complex revealed that the metabolites do indeed occupy similar hydrophobic pockets within the active site, while the N-linked methyls are free to rotate between two binding modes. From these bound structures, a model of N-demethylation in which the N-linked methyl functional groups rotate between catalytic and noncatalytic positions was developed. This study is the first to provide a structural model of a drug and its metabolites complexed to a cytochrome P450 based on NMR and to provide a structural mechanism for how a drug can undergo sequential oxidations without unbinding. The rotation of the amide functional group might represent a common structural mechanism for N-dealkylation reactions for other drugs such as the local anesthetic lidocaine.

Background Platelet function of patients with subarachnoid hemorrhage (SAH) may play an important part in both rebleeding and delayed cerebral ischemia, but little is known about aggregation pathways during the acute phase of stroke. Analgesics are used regularly in the first days after bleeding, and some can potentially inhibit the cyclooxygenase (COX) enzyme. We examined the platelet function of patients with SAH in order to describe their basal situation and determine whether the administration of intravenous nonsteroidal antiinflammatory drugs (NSAIDs) affected platelet aggregation. Methods Arachidonic acid (AA)–induced aggregation and the platelet function analyzer (PFA)-100 test with collagen/epinephrine cartridges were used to study a group of SAH patients that was treated with dexketoprofen and dipyrone and to compare them to patients that had received no analgesia. Results Ninety-six consecutive SAH patients prospectively enrolled in platelet studies. Twenty-seven patients were taking NSAIDs (10 on dexketoprofen and 17 on dipyrone), and there were 15 cases in the control group. AA-induced aggregation was 10% ± 3.2% for NSAIDs (mean ± standard error), specifically 17.2% ± 7% for dexketoprofen and 5.7% ± 1% for dipyrone. Aggregation in the control group was 72.4% ± 6% ( P = .001). Both analgesics slowed the platelet plug formation during the PFA-100 test, with closure times of 237.2 ± 25 seconds for dexketoprofen and 198.4 ± 22 seconds for dipyrone and 138.1 ± 21 seconds in controls ( P = .02). Conclusions The administration of COX-inhibiting analgesics leads to an hypoaggregability state in the first days of SAH. Further insight into their impact on complications such as rebleeding and delayed cerebral ischemia is needed in order to optimize the headache treatment of SAH.