Metabolism Of Phenacetin Research Articles

The effect of transcatheter arterial chemoembolization on CYP1A2 activity in patients with hepatocellular carcinoma

CYP1A2, a constitutive enzyme expressed in the liver, is among the phase I enzymes responsible for polycyclic aromatic hydrocarbons metabolism. Phenacetin O-de-ethylation is a marker for CYP1A2 activity. This study investigates the metabolism of phenacetin in patients with hepatocellular carcinoma (HCC). The phenacetin test was performed in 56 normal subjects and 92 HCC patients. The test was repeated in HCC patients after treatment with transcatheter arterial chemoembolization (TACE). The recovery of phenacetin's urinary metabolites was studies in 12 normal subjects and 14 patients with HCC. Compared with normal controls, the recovery of phenacetin O-de-ethylated metabolites decreased by 42.5% (P < 0.01) in patients with HCC and diminished further after TACE (P < 0.05). The ratio of plasma total paracetamol to phenacetin was much lower than in normal controls (P < 0.01) and was reduced by 40.7% more after TACE (P < 0.05). The metabolism of phenacetin is impaired in patient with HCC. TACE damages the activity of CYP1A2. The phenacetin test can be used to predict effect of TACE on liver function in HCC patients.

Key Residues Controlling Phenacetin Metabolism by Human Cytochrome P450 2A Enzymes

Cytochrome P450s (P450s) metabolize a large number of diverse substrates with specific regio- and stereospecificity. A number of compounds, including nicotine, cotinine, and aflatoxin B(1), are metabolites of the 94% identical CYP2A13 and CYP2A6 enzymes but at different rates. Phenacetin and 4-aminobiphenyl were identified as substrates of human cytochromes P450 1A2 and 2A13 but not of CYP2A6. The purpose of this study was to identify active site amino acids that are responsible for CYP2A substrate specificity using phenacetin as a structural probe. Ten amino acid residues that differ in the CYP2A13 and CYP2A6 active sites were exchanged between the two enzymes. Phenacetin binding revealed that the six substitution, CYP2A13 S208I, A213S, F300I, A301G, M365V, and G369S decreased phenacetin affinity. Although incorporation of individual CYP2A13 residues into CYP2A6 had little effect on this enzyme's very low levels of phenacetin metabolism, the combination of double, triple, and quadruple substitutions at positions 208, 300, 301, and 369 increasingly endowed CYP2A6 with the ability to metabolize phenacetin. Enzyme kinetics revealed that the CYP2A6 I208S/I300F/G301A/S369G mutant protein O-deethylated phenacetin with a K(m) of 10.3 muM and a k(cat) of 2.9 min(-1), which compare very favorably with those of CYP2A13 (K(m) of 10.7 muM and k(cat) of 3.8 min(-1)). A 2.15 A crystal structure of the mutant CYP2A6 I208S/I300F/G301A/S369G protein with phenacetin in the active site provided a structural rationale for the differences in phenacetin metabolism between CYP2A6 and CYP2A13.

Identification of active site residues essential for the metabolism of phenacetin to acetaminophen in human 2A cytochromes P450

Human cytochromes P450 from the 2A subfamily have differing abilities to metabolize carcinogens and procarcinogens, including the nicotine‐derived 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK), aflatoxin B1, and 4‐aminobiphenyl. Recently, Fukami et al. established that phenacetin, a hepatocarcinogen and known substrate for the CYP1A family, is even more efficiently metabolized by human lung cytochrome P450 2A13 (CYP2A13), while the human liver cytochrome P450 2A6 (CYP2A6) has almost no activity despite being 94% identical in amino acid sequence. To determine the basis for substrate specificity between these two closely related human 2A enzymes, site directed mutagenesis was used to construct eleven CYP2A13 single point mutants with mutations at positions in or near the active site where CYP2A13 and CYP2A6 wild type enzymes differ. Phenacetin metabolism kinetics were determined for the wild type enzymes CYP2A13 and CYP2A6, and eleven CYP2A13 mutants. Two CYP2A13 mutants, A117V and L366I, each increase phenacetin metabolism activity greater than four‐fold. Four amino acid substitutions, S208I, F300I, A301G, and G369S, virtually abolished phenacetin metabolism in CYP2A13. The CYP2A6 enzyme is being modified by substitution of one or more 2A13 active site residues to determine those required to incorporate phenacetin metabolism into CYP2A6.NIH GM76343

Effect of sodium ozagrel on the activity of rat CYP2D6

The aim of the study was to investigate the influence of sodium ozagrel on CYP2D6 (cytochromeP450 2D6) activity. The studies were performed with rat urine and liver microsomes and chemical inhibitors. The metabolism of dextromethorphan (dextrophan/dextromethorphan, dextrophan is a metabolite of dextromethorphan) and phenacetin (paracetamol/phenacetin, paracetamol is a metabolites of phenacetin) was used as probe to measure CYP2D6 and CYP1A2 (cytochromeP450 1A2) activity, respectively, determined by high-performance liquid chromatography (HPLC). The results showed that the metabolism of dextrophan/dextromethorphan in the sodium ozagrel-treated group (37 mg/kg) was higher than that of the control (P<0.05/6) in both in vivo and in vitro studies (r=0.9811). The rate of dextromethorphan metabolism was inhibited by sodium ozagrel and cimetidine in rat liver microsomes prepared from sodium ozagrel-treated rats and control rats group (sodium ozagrel IC(50)=26.5 microM, cimetidine IC(50)=86.3 microM in sodium ozagrel-treated group; sodium ozagrel IC(50)=13.9 microM, cimetidine IC(50)=24.8 microM in control group). The inhibitory effect of sodium ozagrel on CYP2D6 activity was noncompetitive with dextromethorphan with a K(i) of 324.94 microM. Kinetic parameters of the reactions were established by using Lineweaver-Burk with K(m)=0.67 mM and V(max)=2.13 pm/min/mg protein for the sodium ozagrel-treated group and K(m)=0.29 mM, and V(max)=0.91 pm/min/mg protein for the control group, respectively. The expression of CYP2D6 protein in the treated group was higher than that of the control group, as determined by Western blotting. The activity and expression of CYP1A2 did not show obvious differences in the control group and sodium ozagrel treated group. In conclusion, sodium ozagrel metabolism in rats is mediated primarily through CYP2D6, and sodium ozagrel can induce CYP2D6 activity.

Impaired clearance of phenacetin in hepatic cirrhosis and fibrosis

CYP450 1A2 is constitutively expressed in liver. Phenacetin O-de-ethylation is a marker reaction for CYP450 1A2 activity. In this paper, the metabolism of phenacetin has been studied in patients with chronic hepatitis B or cirrhosis secondary to a hepatitis B virus infection. The possibility of using the phenacetin test in the evaluation of liver function in these subjects has also been tested. Phenacetin pharmacokinetics and the recovery of its urinary metabolites were studied in 8 normal subjects, 16 patients with chronic hepatitis B and 12 patients with cirrhosis. The phenacetin test was performed in 18 normal subjects and 52 hepatocellular carcinoma (HCC) patients. The test was repeated in HCC patients after treatment with transcatheter arterial chemoembolization (TACE). Compared with normal controls, phenacetin apparent clearance decreased by 47.0% (p < 0.05) and 78.7% (p < 0.01) in patients with chronic hepatitis B and cirrhosis, respectively. The recovery of phenacetin O-de-ethylated metabolites decreased by 24.6 and 72.4% (p < 0.01). 46 of 52 HCC patients (88.4%) had an abnormal phenacetin test before TACE, where the ratio of plasma total acetaminophen to phenacetin was below the lower limit of the normal control range. The ratio was less than 50% of normal controls in 6 HCC patients who had a deterioration in liver function from Child-Pugh class A to Child-Pugh class B after TACE. The metabolism of phenacetin is impaired in patients with chronic hepatitis B and cirrhosis. The phenacetin test can predict the susceptibility of liver function to TACE in HCC patients.

Tissue-like cultures of rat hepatocytes in study of phase I and phase II drug metabolism

To investigate phase I and phase II enzyme activities in drug metabolism with tissue-like cultures of rat hepatocytes. The gel entrapment and spheroid culture of hepatocytes were used as tissue-like cultures and the monolayer culture was used as a control. The metabolism of phenacetin and 7-hydroxycoumarin (7-HC) was evaluated as the activities of phase I and phase II enzymes after incubated in medium for a period of time. The metabolites were assayed by HPLC. The hepatocytes were exposed to beta-naphthoflavone (BNF, 50 micromol x L(-1)) before the phase I and phase II enzyme activities were analyzed. In monolayer culture, phase I parameters decreased quickly and did not detected at d 5, and the phase II enzyme activities were not detected at d 7. In other two models of tissue-like cultures, the activities of phase I and phase II enzyme maintained at 32%-50% of the initial value at d 7. Paracetamol formation rates in spheroid culture maintained at 96% of that at d 1. The phase I enzyme activities of the spheroid culture were maintained from d 1 to d 3 at a level of 2.7-3.9-fold higher than the monolayer culture. After exposure to BNF the activities on phase I enzyme increased by about 2.5-fold (P <0.05) in all three culture models, while the increase in phase II enzyme was not significant. The gel entrapment culture and spheroid culture are superior to the monolayer culture in maintenance of drug metabolic enzyme activities.

In vitro metabolism and inductive or inhibitive effect of DL111 on rat cytochrome P4501A enzyme

In vitro metabolism and the inductive or inhibitive effect of DL111, a non-hormonal early pregnancy-terminating agent, toward cytochrome P450 (CYP) enzymes in rat liver microsomes were studied. In vitro metabolism of DL111 was performed in different rat liver microsomes (pretreated with phenobarbital (PB), dexamethasone (Dex), β-naphthoflavone (BNF), DL111, respectively) and the catalytic abilities of these microsomes for DL111 were compared with control group. DL111 was well metabolized in microsomes pretreated with β-naphthoflavone and itself. The K m and V max was 41.76±3.26 μM and 15.34±1.03 nmol min −1 mg −1 protein for β-naphthoflavone group, 48.17±6.06 μM and 17.54±1.79 nmol min −1 mg −1 protein for DL111 group, 77.81±4.73 μM and 3.087±0.202 nmol min −1 mg −1 protein for control group, respectively. The rats were pretreated intraperitoneally with the same daily dose of DL111 for different days. The DL111-pretreated microsomal enzymatic activities were evaluated by measuring the metabolic abilities for specific substrates of various enzymes. The results showed that DL111 had the same inductive function as β-naphthoflavone (the specific inducer of CYP1A) toward rat liver microsomes. The inhibitive effect of DL111 on CYP1A was investigated by coincubating DL111 with the specific substrates of CYP1A—ethoxyresorufin or phenacetin in the microsome induced by β-naphthoflavone, and the inhibitive level was compared with fluvoxamine (Flu), the specific inhibitor of CYP1A. DL111 inhibited significantly the metabolism of phenacetin and ethoxyresorufin with the inhibition constant ( K i) 6.836±0.10 and 1.222±0.230 μM, respectively and its inhibition potential on CYP1A was higher than fluvoxamine.

Use of a cocktail of probe substrates for drug-metabolizing enzymes for the assessment of the metabolic capacity of hepatocyte preparations

1. A cocktail of the following probe substrates for human drug-metabolizing enzymes was used to characterize hepatocyte preparations: phenacetin (for CYP1A2), diclofenac (CYP2C9), diazepam (CYP2C19), bufuralol (CYP2D6), midazolam (CYP3A4/5) and 7-hydroxycoumarin (for glucuronidation and sulphation).2. The cocktail was incubated with cryopreserved human, dog or minipig hepatocytes or with freshly prepared rat hepatocytes. Sample analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an Open Access environment that allowed less experienced MS operators to login, submit and analyse sample sets using predefined settings without the immediate attendance of an experienced analyst. Intrinsic clearances (CLint) were calculated from the disappearance of the compounds from the incubations.3. Initially, the cocktail used for human, rat and dog hepatocyte incubations contained 7-ethoxycoumarin instead of 7-hydroxycoumarin. However, 7-ethoxycoumarin had an inhibitory effect on the metabolism of phenacetin.4. The highest CLint estimated with human and dog hepatocytes was observed for 7-hydroxycoumarin. For rat and minipig hepatocytes, the highest CLint was observed for bufuralol. In incubations with dog and minipig hepatocytes, the lowest CLint was seen with diclofenac, whereas for human and rat hepatocytes, the lowest value was observed with diazepam and phenacetin, respectively.5. When the cocktail was incubated together with human hepatocytes and 1 μM ketoconazole, the CLint of midazolam was decreased to about 7.5% of the control value, whereas the metabolism of the other cocktail compounds was virtually unaffected by this CYP3A inhibitor.6. It is suggested that a cocktail of specific human probe substrates for drug-metabolizing enzymes can be used routinely for the determination of the metabolic capacity of hepatocyte preparations in order to ensure the quality and reproducibility of experiments. Moreover, a cocktail of specific probe substrates can also be a useful tool for studies on enzyme inhibition.

In vivo effect of diallyl sulfide and cimetidine on phenacetin metabolism and bioavailability in rat.

Numerous cytochrome P450 inhibitors have been described as effective modulators of cytochrome P450 isoforms activity in vitro. Their inhibitory efficiency may be considerably modified after in vivo application. The aim of this study was to examine the effect of oral administration of diallyl sulfide--a cytochrome P450 2E1 inhibitor and cimetidine--a cytochrome P450 2C6 and 2C11 inhibitor on rat serum concentration of phenacetin and its metabolite acetaminophen. Both inhibitors increased area under the curve (AUC(0-4 h)) for phenacetin by 50%. Only cimetidine reduced AUC(0-4 h) for acetaminophen indicating inhibition of O-deethylation activity. Quinidine--a cytochrome P450 2D subfamily and P-glycoprotein inhibitor did not change significantly phenacetin bioavailability. These results suggest that diallyl sulfide inhibits the deacetylation pathway catalysed by arylamine N-acetyl transferase. Beside cytochrome P450 1A2 other cytochrome P450 isoforms (2A6 and/or 2C11) are involved in phenacetin O-deethylation in rat.

Directly-coupled HPLC-NMR spectroscopic studies of metabolism and futile deacetylation of phenacetin in the rat

The metabolism and futile deacetylation of phenacetin has been investigated in the rat via 1H NMR spectroscopic analysis of urine. Animals were dosed with either phenacetin or phenacetin–C 2H 3 and urine samples were collected for −24–0 (pre-dosing), 0–8, 8–24, and 24–48 h post-dosing. Drug metabolites of the two compounds were concentrated from the urine using solid-phase extraction prior to the use of directly-coupled HPLC- 1H NMR spectroscopy for separation and identification. Following dosing of phenacetin, the metabolites identified were paracetamol glucuronide, paracetamol and N-hydroxyparacetamol, whilst paracetamol and N-hydroxyparacetamol sulphate were identified following dosing of phenacetin–C 2H 3. Quantitatively the percentage futile deacetylation of phenacetin–C 2H 3 metabolites was found to be 32% in both paracetamol and N-hydroxyparacetamol sulphate. This study further indicated the importance of futile deacetylation in simple analgesics and the value of directly-coupled HPLC-NMR spectroscopy for the study of this process.

Inhibitory monoclonal antibodies to human cytochrome P450 1A2: analysis of phenacetin O-deethylation in human liver.

Human cytochrome P450 1A2 metabolizes a large number of common drugs and engages in carcinogen metabolism and activation. Baculovirus-expressed 1A2 was used to immunize mice producing hybridomas yielding monoclonal antibodies (MAbs). Three of 2050 clones assayed yielded the MAbs, MAb 26-7-5, MAb 951-5-1, MAb 1812-2-4, which were specific for 1A2 as assessed by enzyme-linked immunosorbent assay and immunoblots. The three MAbs inhibited 1A2-catalysed metabolism of phenacetin, 7-ethoxycoumarin, chlorzoxazone and phenanthrene by more than 85%. The MAbs were highly specific to 1A2 and did not inhibit 11 other human P450s. The phenancetin O-deethylation activity varied from 0.44-2.49 nmol/min/nmol P450 in eight human liver microsomes samples. MAb 26-7-5 inhibited 1A2-dependent phenacetin O-deethylation in these samples by 64-84% indicating the amount of 1A2 contribution to this reaction and in addition a role for other P450s in the O-deethylation. Independent analysis of recombinant human P450s showed that 1A1, 1A2, 2A6 and 2C19 exhibited phenacetin O-deethylation activity, with 1A1 and 1A2 being the most active followed by 2C19 and 2A6. Eight other P450s were inactive towards phenacetin O-deethylation. The role of different P450 in eight liver samples was analysed with specific individual inhibitory MAbs. Inhibitory antibodies to 1A2, 2C8/9/18/19, 2A6, 2D6, 2E1, and 1A1 were combinatorially added to the microsomes. The O-deethylation activity was inhibited by antibodies to 1A2 (64-84%), to 2C19 (4.6-20%) and to 2A6 (0-8.8%). The total activity inhibited by antibodies to P450 2E1, 2D6 and 1A1 was less than 4.5%, indicating a minor role for these P450s in phenancetin metabolism in human liver microsomes. Thus, 1A2, 2C 9 and 2A6 are the dominant P450s for phenacetin O-deethylation. These studies demonstrate the use of inhibitory MAbs to P450s for a simple and precise assessment of the quantitative role of each P450 in the metabolism of substrates, including drugs, carcinogens, mutagens, environmental chemicals and endobiotics.

Monitoring in Situ Liver Metabolism in Rats Using Microdialysis. Comparison of Microdialysis Mass-Transport Model Predictions to Experimental Metabolite Generation Data

□ The generation of metabolites from two model compounds, phenacetin and acetaminophen, included in the perfusion fluid of a microdialysis probe implanted into rat liver was studied. When 60μM phenacetin was included in the perfusion fluid using a flow rate of 1.0μL/min, acetaminophen and acetaminophen sulfate were recovered at concentrations that ranged between 0.4 and 1.6μM. Acetaminophen sulfate ([AS]gain) diffused back into the microdialysis probe on a micromolar percentage basis of 8.9±2.4% (n=3) when acetaminophen was passed through the probe at a concentration between 11 and 12μM. When 220–240μM acetaminophen was passed through the probe, the percentage of acetaminophen sulfate recovered was 4.8±1.4% (n=3) (P<0.1 compared to the 11μM group). No acetaminophen glucuronide was detected in the dialysate samples. A mathematical model that describes mass transport in microdialysis sampling was used to predict the concentration of metabolite that could be recovered into the dialysate after the loss of a substrate compound that undergoes metabolism. The model predicts a metabolite recovery of 23.6% using estimates for phenacetin metabolism and 21.5% using estimates for acetaminophen metabolism. The results presented here indicate that microdialysis has potential to be used to study local in situ metabolism and with further refinements of the microdialysis mass-transport model may be used to estimate in vivo metabolic formation rates.

CHARACTERIZATION OF RAT HEPATIC CYTOCHROME P-450 ACTIVITIES FOLLOWING INHALATION EXPOSURE TO p-CHLOROBENZOTRIFLUORIDE

p-Chlorobenzotrifluoride (PCBTF), an intermediate in the synthesis of some herbicides, is currently being evaluated as an industrial solvent. The neurotoxicity of PCBTF was evaluated in a 90-day inhalation study in male and female Sprague-Dawley rats exposed for 6 h/day to target concentrations of 0, 10, 50, and 250 ppm PCBTF. Following exposure for 90 days, the livers were weighed, frozen, and microsomal fractions prepared to assess cytochrome P-450 (CYP450) content and activities. A significant increase in liver weight and CYP450 content in female rats was observed in the high exposure group (250 ppm), while the increase in males was not found to be statistically significant. 7-Ethyoxyresorufin O -deethylase (EROD), a measure of CYP1A1 activity, and acetanilide and phenacetin metabolism, a measure of CYP1A2 activity, were increased about twofold in both males and females at the high exposure level (250 ppm). Pentoxyresorufin O dealkylase (PROD) and benzyloxyresorufin O -dealkylase (BROD), a measure of CYP2B activity, were increased approximately fivefold in males at 250 ppm; however, the increase in females at 250 ppm was only twofold. Relatively small changes were observed in chlorzoxazone hydroxylation (CYP2E1) in the male high exposure group, while there were no differences in the females. Conversely, nifedipine oxidation (CYP3A1/3A2) showed a small increase in the females at 250 ppm and no change in males. Western blots of protein levels correlated generally well with increases in enzyme activity (control vs. high exposure level) for CYP1A2, CYP2B, and CYP2E1. The relatively modest induction of a wide range of hepatic CYP450 activities was limited to rats exposed to the 250 ppm dose of PCBTF, supporting the NOEL of 50 ppm. In addition, the results suggest that there are gender differences associated with the induction of CYP450 activities.

NMR spectroscopic studies on the metabolism and futile deacetylation of phenacetin in the rat

1. 1H-NMR spectroscopy of urine was used to determine the % deacetylation and re-acetylation of 2H-labelled (in the acetyl) phenacetin metabolites in the rat. 2. Male Sprague- Dawley rats were each dosed with either phenacetin or phenacetin C2H8 at 50 mg kg−1. The total urinary recoveries for phenacetin and phenacetin-C2H8 were 47.6 ± 16.7 and 50.1 ± 16.2% respectively (not significantly different, p > 0.05). Paracetamol sulphate and glucuronide are the major urinary metabolites of both protio and deuteriophenace tin. 3. The futile deacetylationgiven by the urinary recovery of protio-acetyl metabolitesof phenacetin-C2H8 was 29.6 ± 0.9% for paracetamol sulphate and 36.6 ± 3.1% for paracetamol glucuronide. These observations demonstrate a high level of futile deacetylation in the paracetamol conjugates formed by metabolism of phenacetin-C2H8 and this may indicate a high metabolic flux through the nephrotoxic intermediate 4-aminophenol. 4. The level of futile deacetylation for phenacetin was significantly higher than that found previously in studies of labelled paracetamol in rat or man, and may be important in understanding the higher nephrotoxicity of phenacetin as compared with paracetamol.

Intermolecular interactions of antimicrobial fluoroquinolones with purified rat liver CYP1A2 studied by proton nuclear magnetic resonance spectroscopy.

1. Binding and inhibition of antimicrobial fluoroquinolones towards liver CYP1A2 purified from 3-methylcholanthrene-treated rats were investigated using proton nuclear magnetic resonance (nmr) and phenacetin metabolism. 2. The proton nmr longitudinal relaxation rate study indicated that the paramagnetic effects of the haem iron of CYP1A2 were observed in protons of enoxacin with a 1,8-naphthyridine skeleton and its 4'-nitrogen atom on the 7-piperazine ring probably participated in specific binding to the haem iron. These data suggest a facile accessibility and strong binding of enoxacin to the active site of the enzyme. On the contrary, the binding region of norfloxacin with a quinoline skeleton could not be specified, and an 8-fluorinated derivative (AT-3970) had much lower paramagnetic effects and no specific binding region. 3. In a reconstituted CYP1A2 system, enoxacin exhibited the most potent inhibition of phenacetin O-deethylation. The metabolism was less inhibited by norfloxacin, and AT-3970 had a weak inhibitory activity. 4. The binding ability of the fluoroquinolones to the CYP1A2 active site is likely to determine their inhibitory activity against phenacetin metabolism.

Role of the hepatic artery in the metabolism of phenacetin and acetaminophen: An intravital microscopic and multiple-indicator dilution study in perfused rat liver,

Role of the hepatic artery in the metabolism of phenacetin and acetaminophen: An intravital microscopic and multiple-indicator dilution study in perfused rat liver,

Role of the Hepatic Artery in the Metabolism of Phenacetin and Acetaminophen: An Intravital Microscopic and Multiple–Indicator Dilution Study in Perfused Rat Liver

We studied the pattern of intermixing of the hepatic arterial and portal venous flows in a perfused rat liver preparation under constant flow (12 ml/min) with intravital epifluorescent microscopy; changes in the steady state extraction ratio of carbon 14-labeled phenacetin and tritiated acetaminophen, probes metabolized primarily in perivenous and periportal regions of the rat liver, respectively; and the spaces accessed by noneliminated reference indicators introduced as a bolus into the hepatic artery and portal vein at different hepatic arterial/portal venous flow regimens of 0:12, 2:10 and 4:8. The sinusoidal velocities for the hepatic arterial- and portal venous (hepatic arterial/portal venous flow at 4:8)-infused fluorescein isothiocyanate-erythrocytes (100 microliters/min) were 327 +/- 78 and 301 +/- 63 microns/sec, respectively, and the velocity for the solely portal venous-perfused liver (12 ml/min) was 347 +/- 74 microns/sec; the flow-weighted sinusoidal velocity was highly correlated to the sinusoidal volume for the dually perfused rat liver. Small but significant decreases in the extraction ratio of [14C]phenacetin (from 0.989 to 0.984 and 0.980) and tritiated acetaminophen (from 0.631 to 0.607 to 0.563), delivered simultaneously into the hepatic artery and portal vein, were observed with an increment of hepatic arterial flow within the same liver preparation; oxygen consumption rate also fell slightly, in parallel fashion. When a multiple-indicator dilution dose containing chromium 51-labeled RBCs, iodine 125-labeled albumin and tritiated water or [14C]urea was injected into the hepatic artery (which accesses both the peribiliary capillary plexus [nonsinusoidal] and the sinusoidal bed) and portal vein (which enters only the sinusoids) at 10-min intervals within each steady state, the blood volume, total albumin space, albumin Disse space, total water and parenchymal cellular water spaces were unchanged after portal venous injection for all hepatic arterial/portal venous flow ratios, suggesting that the arterial flow is ineffective in perturbing average sinusoidal flow dynamics. However, slightly larger total water spaces were obtained with hepatic arterial injection. This excess water space was almost completely accounted for by the "nonsinusoidal" extravascular space associated with the peribiliary capillary plexus; it averaged 0.03 ml/gm and was independent of flow. The anomaly, a reduced flow-weighted sinusoidal velocity for the dually perfused liver, an unchanged diameter of the terminal hepatic venule (32 microns) among the hepatic arterial/portal venous flow ratios and the reduction in the extraction ratio of the drug probes and oxygen consumption rates suggest that some of the arterial flow must have entered the sinusoids somewhat downstream.

Omeprazole Does Not Enhance the Metabolism of Phenacetin, a Marker of CYP1A2 Activity, in Healthy Volunteers

Omeprazole has been reported to increase cytochrome P450IA2 (CYP1A2) activity in vitro, but whether this effect also occurs in vivo is controversial. To clarify this issue, the effect of omeprazole (20 mg/day for 8 days) on the kinetics and metabolism of phenacetin, an in vivo marker of CYP1A2 activity, was examined in 10 healthy volunteers. The pharmacokinetic parameters of phenacetin and metabolically derived paracetamol on the 8th day of omeprazole administration were very similar to those observed in a control session in the absence of omeprazole administration, the only significant difference being a higher peak plasma phenacetin concentration during omeprazole treatment. It is concluded that at the dosage used omeprazole does not increase the rate of oxidative and conjugative reactions involved in the metabolism of phenacetin and paracetamol respectively. These data are consistent with the hypothesis that omeprazole is generally devoid of inducing effects on CYP1A2 activity in vivo, at least in a Caucasian population with a low prevalence of the omeprazole-mephenytoin poor metabolizer phenotype.

Effect of an experimental malaria infection on the metabolism of phenacetin in the rat isolated perfused liver.

1. The effect of infection with the rodent malaria parasite Plasmodium berghei on the metabolism of phenacetin has been investigated in a rat isolated perfused liver preparation. 2. A bolus dose of phenacetin (10 mg) was introduced into the perfusate reservoir of both control (n = 4) and malaria-infected (n = 4) liver preparations, and samples of bile and perfusate were collected (0-4 h) for hplc analysis of phenacetin, paracetamol and its phase II metabolites. 3. Whereas malaria had no effect on the hepatic clearance of phenacetin (control: 0.64 +/- 0.15 versus malaria: 0.66 +/- 0.14 ml min-1), there was a significant reduction in the hepatic clearance of generated paracetamol (control: 1.22 +/- 0.15 versus malaria: 0.41 +/- 0.08 ml min-1) and the total recovery in bile and perfusate of paracetamol glucuronide (control: 1.18 +/- 0.44 versus malaria: 0.29 +/- 0.20 mg). There was no significant change during malaria infection in the total recovery of either phenacetin (control: 1.30 +/- 0.73 versus malaria: 0.79 +/- 0.36 mg) or paracetamol sulphate (control: 0.81 +/- 0.25 versus malaria: 0.74 +/- 0.16 mg),

Metabolism of phenacetin in V79 Chinese hamster cell cultures expressing rat liver cytochrome P4501A2 compared to isolated rat hepatocytes

In a genetically engineered V79 cell line (XEMd-MZ) expressing rat cytochrome P4501A2 the activity of phenacetin- O-deethylase was determined and compared with freshly isolated rat hepatocytes. In the V79 cells the apparent K m , was 0.99 μM (N = 4), compared to the high affinity K m (0.23 μM, N = 4) found in freshly isolated rat hepatocytes, where as the V max found in the XEMd-MZ cells (14.90 pmol/min/10 6 cells) corresponded to the V max for the high affinity site of the hepatocytes (18.2 pmol/min/10 −6 cells). The metabolic capacity of phenacetin was quantitatively comparable to that of freshly isolated hepatocytes on a cell to cell basis.