In vitro analysis of human drug glucuronidation and prediction of in vivo metabolic clearance

The glucuronidation of a number of commonly used hepatic uridine diphosphate glucuronosyltransferase drug substrates has been studied in human tissue microsomes. Prediction of in vivo hepatic drug glucuronidation from liver microsomal data yielded a consistent 10-fold under-prediction. Consideration of protein binding was observed to be pivotal when predicting in vivo glucuronidation for acid substrates. Studies using human intestinal microsomes demonstrated the majority of drugs to be extensively glucuronidated such that the intrinsic clearance (CL(int)) of ethinylestradiol (CL(int) = 1.3 microl/min/mg) was twice that obtained using human liver microsomes (CL(int) = 0.7 microl/min/mg). The potential extrahepatic in vivo glucuronidation was calculated for a range of drug substrates from human microsomal data. These results indicate the contribution of intestinal drug glucuronidation to systemic drug clearance to be much less than either hepatic or renal glucuronidation. Therefore, data obtained with intestinal microsomes may be misleading in the assessment of the contribution of this organ to systemic glucuronidation. The use of hepatocytes to assess metabolic stability for drugs predominantly metabolized by glucuronidation was also investigated. Metabolic clearances for a range of drugs obtained using fresh preparations of human hepatocytes predicted accurately hepatic clearance reported in vivo. The use of cryopreserved hepatocytes as an in vitro tool to predict in vivo metabolism was also assessed with an excellent correlation obtained for a number of extensively glucuronidated drugs (R(2) = 0.80, p < 0.001).     

UDP-glucuronosyltransferase (UGT) 2B15 pharmacogenetics: UGT2B15 D85Y genotype and gender are major determinants of oxazepam glucuronidation by human liver

Oxazepam is a commonly used 1,4-benzodiazepine anxiolytic drug that is polymorphically metabolized in humans. However, the molecular basis for this phenomenon is currently unknown. We have previously shown that S-oxazepam glucuronide, the major oxazepam metabolite, is selectively formed by UDP-glucuronosyltransferase (UGT) 2B15, whereas the minor R-oxazepam glucuronide is produced by multiple UGTs other than UGT2B15. Phenotype-genotype studies were conducted using microsomes and DNA prepared from the same set of 54 human livers. Sequencing of the UGT2B15 gene revealed three nonsynonymous polymorphisms, D85Y, T352I, and K523T, with variant allele frequencies of 0.56, 0.02, and 0.40, respectively. D85Y genotype showed a significant effect (p = 0.012) on S-oxazepam glucuronidation with lower median activities in 85Y/Y livers (49 pmol/min/mg protein) compared with 85D/D livers (131 pmol/min/mg), whereas 85D/Y livers were intermediate in activity (65 pmol/min/mg). There was also a significant trend (p = 0.049) for higher S-oxazepam activities in the two 352T/I livers (135 and 210 pmol/min/mg) compared with the remaining 352T/T livers (median, 64 pmol/min/mg). Conversely, K523T genotype had no apparent effect on oxazepam glucuronidation (p > 0.05). Donor gender also significantly influenced S-oxazepam glucuronidation with higher median activities in male (65 pmol/min/mg) compared with female (39 pmol/min/ mg) livers (p = 0.042). R-Oxazepam glucuronidation was not affected by either genotype or gender (p > 0.05). In conclusion, gender and D85Y genotype are identified as major determinants of S-oxazepam glucuronidation by human liver and may explain in part polymorphic oxazepam glucuronidation by human subjects.     

In vitro N-glucuronidation of a novel antiepileptic drug, lamotrigine, by human liver microsomes.

The metabolism of the antiepileptic drug lamotrigine was characterized in human liver microsomes. For that purpose a high performance liquid chromatography method allowing the separation of lamotrigine glucuronide from the parent compound, and the quantitation of the glucuronide, was developed. The drug undergoes glucuronidation on the 2-nitrogen atom of the triazine ring, leading to a quaternary ammonium-linked glucuronide. This metabolite was positively identified from its hydrolysis by beta-glucuronidase and its associated radioactivity when UDP-[U-14C] glucuronic acid was used as the cosubstrate. Structural confirmation of the glucuronide was finally obtained by high performance liquid chromatography-mass spectrometry, by using a thermospray interface. The reaction proceeded with an apparent Vmax of 0.65 nmol/min/mg and Km of 2.56 mM. The average value of lamotrigine glucuronidation in four human samples of transplantable liver was 0.43 +/- 0.14 nmol/min/mg, thus indicating a large interindividual variation. An interspecies comparison of hepatic lamotrigine glucuronidation (human, rabbit, rat, monkey) revealed that the rate of glucuronidation was low. Of all the species considered, humans glucuronidated the drug to the greatest extent, with a specific activity 2-fold higher than that observed in rabbit liver microsomes. In contrast, the activity was greater than 20 times lower in monkey (0.019 nmol/min/mg) and at the limit of detection in rat liver microsomes. However, in this species, phenobarbital treatment enhanced lamotrigine glucuronidation slightly (0.017 nmol/min/mg). Among the drugs that undergo quaternary ammonium-linked glucuronidation, chlorpromazine, but not imipramine, amitriptyline and cyproheptadine, inhibited the glucuronidation of lamotrigine in vitro (IC50 of 5.0 x 10(-4) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetics of zidovudine in patients with liver cirrhosis

The pharmacokinetics of zidovudine (azidothymidine, AZT) was investigated after oral administration (200 mg) in 14 human immunodeficiency virus seronegative patients with liver cirrhosis. They were divided in three groups according to the severity of the liver disease quantitated by the Child-Pugh score. Plasma and urine concentrations of zidovudine and its glucuronidated metabolite (GAZT) were measured simultaneously by HPLC assay. Findings were compared with those previously measured in six healthy volunteers. As a consequence of a marked drop in oral clearance (10 +/- 4 versus 38 +/- 15 ml/min/kg), zidovudine concentrations, half-life, and mean residence time were increased in patients with cirrhosis. No difference could be established between the three groups. The reason for such a decrease in oral clearance of zidovudine was the reduction in the GAZT formation clearance (236 +/- 73 versus 1540 +/- 540 ml/min); this led to a decrease in the AUC ratio of GAZT and zidovudine (1.3 +/- 0.6 versus 4.6 +/- 0.7), which was directly related to the severity of the cirrhosis. In patients, as in volunteers, formation of GAZT rate limits its elimination. To avoid important cumulation of zidovudine after repeated dosing in patients with acquired immunodeficiency syndrome who have hepatic impairment, a dosage adjustment could be proposed.     

Effect of endotoxin shock on the clearance of lidocaine and indocyanine green in the perfused rat liver

Endotoxin administration and cecal ligation and puncture produce significant hepatocellular dysfunction when studied in vivo. Specific factors that are present in vivo after endotoxin administration and cecal ligation and puncture, such as alterations in liver blood flow, circulating mediators, and hypoxia, can alter hepatic function. In this study, we used an isolated perfused liver to evaluate the effects of in vivo administration of endotoxin on hepatic function using indocyanine green (ICG) as a global marker of function and lidocaine and its metabolite, MEGX, as specific markers of the CYP450 enzyme system. Endotoxin (Escherichia coli; 45 mg/kg i.p.) was administered to rats followed by a 6-h monitoring before preparation of the isolated in situ perfused liver. Livers from control and endotoxin groups received either ICG (control, n = 6; endotoxin, n = 5) or lidocaine (control, n = 8; endotoxin, n = 8). A separate group of rats (n = 6) received cimetidine (an inhibitor of the CYP450 enzyme system) at a dose of 80 mg/kg daily for 3 days. Livers were perfused via the portal vein by using a single-pass system with a balanced salt solution 6 h after receiving either endotoxin or saline or 24 h after receiving the last dose of cimetidine. After a 40-min stabilization period, ICG or lidocaine was infused via the portal vein until steady-state concentrations were reached in the venous outflow. The total hepatic clearance and intrinsic hepatic clearance for ICG and lidocaine were unchanged in the livers obtained from endotoxin-treated rats. This model could adequately detect CYP450 inhibition because cimetidine-treated rats had significantly lower initial MEGX concentrations (0.63 +/- 0.03 mg/L) compared with control (0.77 +/- 0.03 mg/L) and endotoxin-treated (0.74 +/- 0.04 mg/L) rats. Septic livers had significantly higher initial hepatic oxygen consumption (HVO2) than did control livers (45 +/- 3 microL/min/g vs 82 +/- 9 microL/min/g). The HVO2 remained higher in the septic livers and significantly increased throughout the study, which demonstrated that the livers remained viable and functional. These data indicate that there is no detectable hepatocellular dysfunction after endotoxin shock using ICG, lidocaine, and MEGX in the isolated perfused liver; therefore the dysfunction reported from in vivo studies may be reversible when the liver is removed from the shocked environment.     

Cytochrome P450 1A2 is a major determinant of lidocaine metabolism in vivo: effects of liver function

OBJECTIVES: This study was designed (1) to evaluate the effect of a cytochrome P450 (CYP) 1A2 inhibitor, fluvoxamine, on the pharmacokinetics of intravenous lidocaine and its 2 pharmacologically active metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), to confirm recent in vitro results indicating that CYP1A2 is the main isoform responsible for lidocaine biotransformation and (2) to assess whether liver function has any influence on the fluvoxamine-lidocaine interaction. METHODS: The study was carried out in 10 healthy volunteers and 20 patients with cirrhosis, 10 with mild (Child grade A) and 10 with severe (Child grade C) liver dysfunction, according to a randomized, double-blind, 2-phase, crossover design. In one phase all participants received placebo for 6 days; in the other phase they received 50 mg fluvoxamine for 2 days and 100 mg fluvoxamine for the next 4 days. On day 6, a 1-mg/kg lidocaine dose was administered intravenously 2 hours after the last dose of fluvoxamine or placebo. Plasma concentrations of lidocaine, MEGX, GX, and fluvoxamine were measured up to 12 hours after lidocaine injection. RESULTS: The effects of fluvoxamine coadministration were dependent on liver function. Lidocaine clearance was decreased on average by 60% (from 12.1 mL/min.kg to 4.85 mL/min.kg, P <.001) in healthy subjects and by 44% (from 9.83 mL/min.kg to 5.06 mL/min.kg, P <.001) in patients with mild liver dysfunction, with proportional increases in terminal half-lives, whereas virtually no effect was produced in patients with severe liver dysfunction (4.21 mL/min.kg versus 3.65 mL/min.kg, P >.05). Analogous effects were observed on MEGX and GX formation kinetics, which were drastically impaired in healthy subjects and patients with mild liver cirrhosis but virtually unaffected in patients with severe cirrhosis. CONCLUSION: CYP1A2 is the enzyme principally responsible for the metabolic disposition of lidocaine in subjects with normal liver function. The extent of fluvoxamine-lidocaine interaction decreases as liver function worsens, most likely because of the concomitant decrease in the hepatic level of CYP1A2. These observations indicate that results obtained in healthy subjects cannot be extended a priori to patients with liver dysfunction, but the clinical consequences of inhibition of drug metabolism must also be assessed in such patients.     

Hepatic clearances of antipyrine, indocyanine green, and galactose in normal subjects and in patients with chronic liver diseases

Blood clearance of antipyrine, indocyanine green, and galactose were measured to evaluate the alterations of effective hepatic blood flow and hepatic intrinsic clearances in chronic liver diseases. Galactose blood clearance, which may be taken as effective hepatic blood flow, decreased by approximately 30% in patients with cirrhosis (12.49 +/- 0.76 ml/min/kg; mean +/- SE; n = 17) compared with normal subjects (18.17 +/- 1.03 ml/min/kg; n = 5). In patients with cirrhosis, intrinsic clearances of antipyrine (0.178 +/- 0.014 ml/min/kg; n = 17) and indocyanine green (6.19 +/- 1.38 ml/min/kg; n = 7) showed 61% and 85% reduction, respectively, compared with those of normal subjects (0.462 +/- 0.048 ml/min/kg; n = 5; 41.72 +/- 7.75 ml/min/kg; n = 5). Considering that indocyanine green and antipyrine are eliminated by different hepatic mechanism, these mechanisms may not be equally sensitive to decrements in hepatic function. In addition, fractional reductions of intrinsic clearances for these compounds are thus much greater than that of effective hepatic blood flow.     

Acute oxygen supplementation restores markers of hepatocyte energy status and hypoxia in cirrhotic rats

The oxygen limitation hypothesis states that hepatocyte hypoxia is the mechanism determining metabolic restriction in the cirrhotic liver. Therefore we studied markers of hepatocyte energy state and cellular hypoxia in livers of normal and cirrhotic rats before and after oxygen supplementation. Rats with carbon tetrachloride-induced cirrhosis and procedural control rats were exposed to either room air or a hyperoxic gas mixture for 1 h immediately before freeze clamping and perchloric acid extraction of liver tissue. Extracts were assessed by (31)P NMR and enzymatic assays. Livers from cirrhotic rats breathing room air showed a reduced ratio of ATP/ADP, an increased ratio of inorganic phosphate/ATP, and a trend toward an increased ratio of lactate/pyruvate compared with procedural control livers (ATP/ADP 1.73 +/- 0.35 versus 2.68 +/- 0.61, P <.05; P(i)/ATP 2.74 +/- 0.48 versus 1.56 +/- 0.26, P <.05; lactate/pyruvate 29.3 +/- 6.4 versus 22.5 +/- 7.4, P =.18). After supplementation with oxygen for 1 h, these ratios in cirrhotic livers approached control values. A variety of other metabolic markers affected by cirrhosis showed variable trends toward normal in response to oxygen supplementation, whereas minor trends toward an increase in ATP levels in control animals suggest the possibility of marginal oxygen limitation in normal livers. The data are consistent with the hypothesis that hepatocytes in cirrhotic livers have normal metabolic capacity but are constrained by a deficit in oxygen supply. Interventions aimed at increasing oxygen supply to the liver may have both short- and long-term therapeutic value in the management of cirrhosis.     

Differential effect of chronic renal failure on the pharmacokinetics of lidocaine in patients receiving and not receiving hemodialysis

BACKGROUND AND OBJECTIVES: The effect of chronic renal failure (CRF) on the pharmacokinetics of lidocaine, a drug cleared almost exclusively by hepatic metabolism, has thus far only been evaluated in patients undergoing regular hemodialysis. This study had 2 objectives: (1) to investigate the effect of CRF on the pharmacokinetics of lidocaine in both patients undergoing hemodialysis and patients not undergoing hemodialysis and (2) to test the effects of plasma from the patients examined and of lidocaine metabolites possibly accumulated in vivo on lidocaine biotransformation in vitro. METHODS: In a clinical investigation we studied the kinetics of lidocaine and its metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), after intravenous injection of 1 mg/kg lidocaine in 15 healthy volunteers (creatinine clearance [CL(cr)] >80 mL/min x 1.73 m(-2)), 10 subjects with moderate renal insufficiency (CL(cr) between 30 and 60 mL/min x 1.73 m(-2)), 10 subjects with severe renal insufficiency (CL(cr) <30 mL/min x 1.73 m(-2)), and 10 functionally anephric patients undergoing long-term hemodialysis. In experiments in vitro we determined the effects of plasma and GX on the formation rate of the primary lidocaine metabolite, MEGX, by use of human liver microsomes. RESULTS: In patients not undergoing hemodialysis, lidocaine kinetic parameters were altered in proportion to the degree of renal function impairment, but only in patients with severe renal insufficiency were differences statistically significant: clearance was about half that of control subjects (mean +/- SD, 6.01 +/- 2.54 mL/min x kg versus 11.87 +/- 2.97 mL/min x kg; P < .001), and half-life was approximately doubled (4.55 +/- 1.71 hours versus 2.24 +/- 0.55 hours, P < .001). No such alterations were observed in patients undergoing regular hemodialysis, whose values were similar to those of the control group. The steady-state volume of distribution and MEGX levels were independent of renal function, whereas GX levels were more than double those of control subjects (P < .05) in all CRF groups. No inhibitory effect of plasma was observed, for any of the subjects examined, on lidocaine biotransformation in vitro. GX was found to be a competitive inhibitor, but its apparent inhibition constant value (52 +/- 6 micromol/L) was 2 orders of magnitude higher than its concentrations in vivo. CONCLUSIONS: Our in vivo findings have both clinical and methodologic implications: (1) Lidocaine dose adjustment may be required in patients with severe renal insufficiency who are not receiving hemodialysis. (2) Results of studies evaluating the effect of CRF on metabolic drug disposition are not of general validity, unless both patients undergoing hemodialysis and patients not undergoing hemodialysis have been examined. Our in vitro observations exclude that impairment of lidocaine disposition is the result of direct inhibition of metabolizing enzymes by accumulated metabolites or uremic toxins. Alternative mechanisms, suggested by the results of recent in vitro studies, are discussed.     

Effects of protein and carbohydrate content of diet on drug conjugation

Eight healthy subjects were fed a high-protein-low-carbohydrate diet and, after a 3-day washout period, an isocaloric low-protein-high-carbohydrate diet. They received acetaminophen and oxazepam, drugs metabolized primarily by conjugation, on days 11 and 13, respectively, of each diet. Changing the diets of subjects from the high-protein-low-carbohydrate diet to the low-protein-high-carbohydrate diet resulted in a 14% increase in urinary recovery of acetaminophen glucuronide and a 32% increase in urinary recovery of oxazepam glucuronide (p less than 0.05). The increases in glucuronidation were at the expense of other pathways of metabolism, and there were no significant changes in the metabolic clearance rates of acetaminophen and oxazepam. Mean renal clearances of acetaminophen glucuronide, acetaminophen sulfate, and oxazepam glucuronide decreased 45%, 32%, and 54%, respectively (p less than 0.05), when the subjects were switched to the low-protein-high-carbohydrate diet.     

Effects of parenteral nutrition on hepatic elimination of lidocaine: a study using the isolated perfused rat liver

The effects of acute and a short-term (7 days) parenteral nutrition (PN, consisting of 24.2% dextrose and 5.2% amino acids) on hepatic lidocaine elimination were studied using an isolated perfused rat liver preparation. In the acute study, lidocaine was coinfused with PN solution or normal saline at 0.123 ml/min for 80 min. There was no significant difference in hepatic lidocaine elimination between control and PN-treated groups (six per group). In the post-PN study, only lidocaine was infused for 80 min. Lidocaine hepatic clearance and extraction ratio were reduced by 29.8 and 31.1% (P less than .05), respectively, compared to what occurred in livers isolated from control animals with free access to standard laboratory rat-chow and water (six per group). Mass balance at steady state showed that recovery was higher for lidocaine (46.58 +/- 7.28% vs. 25.56 +/- 5.59%) and lower for N-(N,N-diethylglycyl)-2-hydroxymethyl-6-methylaniline (0.21 +/- 0.01% vs. 2.75 +/- 0.58%) in the PN group compared to chow-fed controls. Recoveries of other lidocaine metabolites: N-(N-ethylglycy)-2,6-xylidine, 3-hydroxylidocaine, 3-hydroxy-N-(N-ethylyglycyl)-2,6-xylidine and N-(N-ethylglycyl)-2-hydroxymethyl-6-methylaniline were similar in the PN- and chow-fed group. These findings suggest that acute PN infusion has no effect on lidocaine metabolism, and that the impaired hepatic lidocaine elimination after 7-days of PN occurs as a result of selective inhibition of hepatic enzyme activities such as those involved in aryl methyl hydroxylation and in other unidentified metabolic pathway(s).     

Zidovudine, trimethoprim, and dapsone pharmacokinetic interactions in patients with human immunodeficiency virus infection.

Zidovudine is widely prescribed for the treatment of human immunodeficiency virus (HIV) infection. Trimethoprim and dapsone are commonly used in the management of Pneumocystis carinii pneumonia in HIV-infected patients. To examine the pharmacokinetic interactions among these drugs, eight HIV-infected patients (26 to 43 years old) with a mean CD4 count of 524.4 +/- 405.7 cells per mm3 received zidovudine (200 mg), trimethoprim (200 mg), and dapsone (100 mg) as single agents and in two- and three-drug combinations. Blood and urine samples were collected at a specified time and analyzed for zidovudine, zidovudine-glucuronide, trimethoprim, dapsone, and monoacetyl-dapsone concentrations under single-dose and steady-state conditions. Zidovudine did not influence the pharmacokinetic disposition of dapsone or trimethoprim. Dapsone had no effect on the pharmacokinetic disposition of zidovudine. Trimethoprim significantly decreased the renal clearance of zidovudine by 58% (5.0 +/- 1.8 versus 2.1 +/- 0.5 ml/min/kg of body weight [P < 0.05]). There was a concurrent 54% decrease in the mean urinary recovery of zidovudine (11.7 +/- 3.5 versus 5.4 +/- 3.0 [P < 0.05]), and the metabolic ratio was decreased by 78% (0.32 +/- 0.4 versus 0.07 +/- 0.05 [P < 0.05]). The mean area under the concentration-time curve from 0 to 6 h of the zidovudine-glucuronide/ zidovudine ratio was unchanged. We conclude that zidovudine, trimethoprim, and dapsone can be coadministered to patients with AIDS without significant pharmacokinetic interaction. However, in AIDS patients with liver impairment and impaired glucuronidation, doses of zidovudine may need to be decreased.     

In vivo mechanistic studies on the metabolic activation of 2-phenylpropionic acid in rat

Two alternative metabolic pathways, acyl glucuronidation and acyl-CoA formation, are implicated in the generation of reactive acylating metabolites of carboxylic acids. Here, we describe studies that determine the relative importance of these two pathways in the metabolic activation of a model substrate, 2-phenylpropionic acid (2-PPA), in vivo in rats. Male Sprague-Dawley rats were pretreated with and without (-)-borneol (320 mg/kg i.p.), an inhibitor of acyl glucuronidation, or trimethylacetic acid (TMA, 500 mg/kg i.p.), an inhibitor of acyl-CoA formation, before receiving 2-PPA (racemic, 130 mg/kg). After administration of 2-PPA, livers were collected over a 2-h period and analyzed for 2-PPA acyl glucuronidation and 2-PPA-CoA formation by high-performance liquid chromatography. Covalent binding was measured by scintillation counting of washed liver protein precipitates. Results showed that pretreatment with TMA led to a 49% decrease in covalent binding of 2-PPA to liver proteins, when a 64% decrease in the exposure of 2-PPA-CoA was observed. Conversely, 95% inhibition of acyl glucuronidation by (-)-borneol, led to a 23% decrease in covalent binding to protein. These results suggest that metabolic activation by 2-PPA-CoA formation contributes to covalent adduct formation to protein in vivo to a greater extent than metabolic activation by acyl glucuronidation for this model substrate.     

Determinants of hepatic function in liver cirrhosis in the rat. Multivariate analysis.

We investigated the determinants of hepatic clearance functions in a rat model of liver cirrhosis induced by phenobarbital/CCl4. Aminopyrine N-demethylation (ABT), galactose elimination (GBT), and serum bile acids (SBA) were determined in vivo. The livers were then characterized hemodynamically: intrahepatic shunting (IHS) was determined by microspheres and sinusoidal capillarization by measuring the extravascular albumin space (EVA) by a multiple indicator dilution technique. The intrinsic clearance was determined by assaying the activity of the rate-limiting enzymes in vitro. Hepatocellular volume (HCV) was measured by morphometry. ABT and SBA, but not GBT, differentiated cirrhotic from normal liver. IHS ranged from normal to 10%; all cirrhotic livers showed evidence of sinusoidal capillarization (reduced EVA). The cirrhotic livers showed a bimodal distribution of HCV, HCV being decreased in 50% of the cirrhotic livers. Multivariate analysis showed EVA and portal flow to be the main determinants of microsomal (ABT) and cytosolic (GBT) clearance function; SBA, by contrast, were determined solely by IHS. We conclude that sinusoidal capillarization is the main determinant of hepatic clearance, while serum bile acids reflect intrahepatic shunting. These findings emphasize the importance of alterations of hepatic nutritional flow to explain reduced clearance function in cirrhosis of the liver.     

Hepatic artery flow and propranolol metabolism in perfused cirrhotic rat liver.

The oxygen limitation theory states that capillarization of the sinusoidal endothelium in cirrhosis impairs hepatocellular oxygen uptake manifesting as a reduction in oxygen-dependent enzyme activity including phase 1 drug metabolism. The hepatic artery supplies highly oxygenated blood to the liver. Therefore, we tested whether augmentation of hepatic arterial blood flow could improve hepatic oxygenation and function in cirrhosis. Rats were treated with carbon tetrachloride and phenobarbitone to induce hepatic cirrhosis or fibrosis. We used a bivascular rat liver perfusion model to examine the effects of increased hepatic artery flow on propranolol clearance and oxygen consumption. Each liver was perfused at three hepatic artery flow rates, 1 to 3, 4 to 6, and 7 to 9 ml/min with a constant portal venous flow of 7 to 9 ml/min. Increasing the hepatic artery flow led to improvement in propranolol clearance in control (n = 7, P <.001), fibrotic (n = 8, P <.001), and cirrhotic (n = 6, P <.001) livers. Intrinsic clearance of propranolol increased only in the cirrhotic livers (P =.01), indicating an improvement in enzyme activity. Regression analysis indicated that this improvement was mediated by change in oxygen delivery alone (P =.001). The results confirm that propranolol metabolizing enzyme activity in cirrhosis can be improved by increasing oxygen delivery by increasing hepatic arterial blood flow. These findings suggest that increasing hepatic arterial blood flow may be an important therapeutic strategy for improving global liver function in cirrhosis.     

Increased estrogen sulfation of estradiol 17beta-D-glucuronide in metastatic tumor rat livers

Changes in the disposition of estradiol 17beta-d-glucuronide (E(2)17G), a substrate of the organic anion-transporting polypeptide family (Oatp) and multidrug resistance-associated protein 2 (Mrp2), were examined in livers of male Wag/Rij rats that were injected with CC531 cells intraportally to induce metastatic tumors (n = 5) or with phosphate-buffered saline for sham-operated controls (n = 4). Multiple indicator dilution, single-pass liver perfusions revealed extremely high influx clearances of [(3)H]E(2)17G (>190 ml/min) in both groups. In recirculating liver perfusions, [(3)H]E(2)17G decayed monoexponentially in the reservoir perfusate, and the total (9.19 +/- 1.33 versus 8.18 +/- 0.94 ml/min) and biliary (4.94 +/- 1.07 versus 4.60 +/- 0.86 ml/min) clearances were similar in both groups (P > 0.05). The metabolic clearance of E(2)17G was higher in the tumor group (4.60 +/- 0.64 versus 3.23 +/- 0.23 ml/min, P < 0.05). E(2)3S17G, the 3-sulfate metabolite, whose identity was confirmed by mass spectrometry, appeared only in bile and not perfusate. Liver microsomal incubations of E(2)3(35)S17G and [(3)H]estrone sulfate revealed similar sulfatase activities between the tumor and sham livers, albeit the activities were much lower for E(2)3(35)S17G. Oatp1a1 and Oatp1b2 protein expression in liver membrane fragments was reduced by 42% and 38%, respectively, whereas that of cytosolic estrogen sulfotransferase (Sult1e1) was significantly increased (41%) with tumor (P < 0.05). All of the observations were captured by modeling. From modeling, we showed that reduction of the high influx clearance (546 to 283 ml/min) failed to lower the total clearance of E(2)17G, whereas up-regulation of Sult1e1 increased the E(2)17G sulfation clearance (2.56 to 3.69 ml/min) in livers with metastatic tumors.     

Lorazepam and oxazepam kinetics in women on low-dose oral contraceptives

Women on low-dose estrogen oral contraceptives (OC) and drug-free control women matched for age, weight, and cigarette smoking habits, received single 2-mg IV doses of lorazepam or single 30-mg oral doses of oxazepam, two benzodiazepines metabolized by glucuronide conjugation. Kinetics were determined from multiple plasma concentrations measured during 48 hr after dosing. Mean kinetic variables for lorazepam in control and OC groups (n = 15 in each group) were: volume of distribution (Vd), 1.33 and 1.45 l/kg; elimination t1/2, 13.1 and 12.2 hr; total clearance, 1.25 and 1.50 ml/min/kg; free fraction in plasma, 10.3% and 10.3% unbound. For oxazepam, kinetic variables in the two groups (n = 14 and 17) were: Vd, 1.05 and 1.19 l/kg; t1/2, 7.6 and 7.2 hr; total clearance, 1.60 and 2.03 ml/min/kg; free fraction, 4.6% and 4.9% unbound. None of these differences were significant. Thus, metabolic clearance by glucuronidation of lorazepam and oxazepam is not significantly affected by OC, in contrast with the highly significant reduction in clearance of the oxidized benzodiazepine diazepam.     

Monoethylglycinexylidide formation kinetics: a novel approach to assessment of liver function

A novel quantitative liver function test is described which is based on monoethylglycinexylidide (MEGX) formation after lidocaine bolus injection. Following the administration of small single doses of lidocaine hydrochloride (1 mg/kg), monoethylglycinexylidide serum concentration-time curves were determined by a novel highly sensitive fluorescence polarisation immunoassay (FPIA) in healthy volunteers, liver donors and patients with liver cirrhosis. The FPIA allowed rapid and reliable monoethylglycinexylidide determinations in serum and urine (between-days coefficient of variation: less than 10.3%, recovery: 80-113%). Monoethylglycinexylidide concentrations measured by FPIA in 32 serum samples from patients correlated well those determined by HPLC. The monoethylglycinexylidide concentration in serum determined 15 min after a lidocaine bolus injection proved to be a highly sensitive and specific indicator of hepatic dysfunction. Average monoethylglycinexylidide concentrations in serum obtained 15 min after lidocaine injection were substantially lower in patients with liver cirrhosis than in healthy volunteers. The average monoethylglycinexylidide concentrations in serum were also substantially lower in liver donors with ballooning or fatty changes of hepatocytes than in donors without relevant alterations of liver histology. By means of monoethylglycinexylidide formation in the liver donors, primary function of the transplanted liver was correctly predicted in 32/37 cases and initial non-function in 4/6 cases.     

Right heart failure impairs hepatic elimination of p-nitrophenol without inducing changes in content or latency of hepatic UDP-glucuronosyltransferases

Congestive heart failure has been shown to affect oxidative drug metabolism, however, there has been little study of its effects on drug conjugation. Using the isolated perfused livers from rats with right ventricular failure (RVF) due to pulmonary artery constriction, we studied the effects of RVF on hepatic elimination of p-nitrophenol (PNP) under controlled flow and oxygen delivery conditions. Hepatic clearance of the drug was found to be significantly impaired in RVF as compared with the sham group (0.80 +/- 0.23 versus 1.28 +/- 0.26 ml/min/g of liver). The impairment of PNP clearance in RVF occurred in parallel with significant reduction in metabolic formation clearance of p-nitrophenyl-beta-D-glucuronide; the major metabolite of PNP (0.51 +/- 0.12 versus 1.03 +/- 0.26 ml/min/g of liver). The intrinsic drug-glucuronidation capacity of livers was evaluated by measuring the microsomal content and activity of the UDP-glucuronosyltransferase(s) (UDP-GT) toward p-nitrophenol. There was no significant difference between sham and the RVF groups in either the content or the activity of the UDP-GT. The latency of the UDP-GT enzymes in microsomes was measured and was found to be similar between the two groups. The results of this study show that RVF impairs hepatic elimination of PNP and that this appears to be independent of changes in hepatic perfusion and oxygenation or alterations in hepatic content, activity, and latency of the UDP-GT.     

Pharmacokinetics of intravenous cefetamet and oral cefetamet pivoxil in patients with hepatic cirrhosis.

The pharmacokinetics of orally administered cefetamet pivoxil and intravenously administered cefetamet were studied in 12 healthy subjects and 12 patients with hepatic cirrhosis without ascites. Cirrhosis had no detectable effect on the pharmacokinetics of cefetamet and on the bioavailability of cefetamet pivoxil. After intravenous cefetamet in control versus cirrhotic subjects, respectively, the following mean +/- standard deviation values were observed: total body clearance, 128 +/- 10.2 versus 123 +/- 28.8 ml/min; steady-state volume of distribution, 23.2 +/- 2.2 versus 22.7 +/- 4.6 liters; half-life, 2.42 +/- 0.21 versus 2.35 +/- 0.41 h. Renal and nonrenal clearances of cefetamet were similar in both groups, as were the mean residence times and areas under the plasma concentration-time curve. For oral cefetamet pivoxil, no differences were detected in the mean values of the percentage of dose absorbed: 44.6 +/- 9.1 versus 50.1 +/- 12.9. The rate of appearance of cefetamet in the plasma also was not affected by cirrhosis: similar mean values were found for the mean residence time and the maximum concentration in plasma and its time of occurrence.