Hepatic Hydroxylation Research Articles

Metabolism and effects on biliary lipid secretion of murocholic acid in the hamster

The metabolism of murocholic acid (MC), a 6β-hydroxylated bile acid, was investigated after intravenous (i.v.), intraduodenal (i.d.) or intragastric (i.g.) administration to bile fistula hamsters. The effects on biliary cholesterol and phospholipid secretion were measured during intravenous infusions of increasing doses of [ 3H]MC. At an infusion rate of 0.1 or 1 μmol · min −1· kg −1, the hepatic uptake was effective. More than 90% of the dose was recovered in bile within 4 h. A bolus injection of 500 μg of [ 3H]MC in the duodenum led to a rapid and efficient biliary secretion of radioactivity. Increasing i.v. infused doses of MC had no effect on bile flow or biliary cholesterol output compared to the controls. Phospholipid secretion was significantly reduced (0.113 μmol · min −1 · kg −1 versus 0.238 μmol · min −1 · kg −1 in controls per μmol · min −1 · kg −1 of excreted bile acids) as MC progressively replaced the endogenous bile acid pool in bile. After i.v. and i.d. administration, MC was secreted in bile as glyco and tauro conjugates without additional hepatic hydroxylation, sulfation or glucuronidation. The i.g. ingestion of MC followed by the faecal analysis of metabolites showed the formation of hyodeoxycholic acid and 3α-OH-6-oxo-5β-cholan-24-oic acid. An equivalent experiment with hyodeoxycholic acid gave MC and the same oxo bile acid. We concluded that MC is metabolized by the hamster liver as an endogenous bile acid, which undergoes intestinal bacterial transformation into a 6-oxo derivative and is then reduced into hyodeoxycholic acid. This process is completely reversible. Progressive replacement of endogenous bile acids in bile by infused MC does not modify choleresis and biliary cholesterol output. In contrast, the phospholipid secretion is decreased. This effect is probably related to the hydrophilic properties of MC.

Age-related changes in biotransformation of azoxymethane and methylazoxymethanolin vitro

1. Age-related changes in hepatic hydroxylation of azoxymethane (AZO) to methylazoxymethanol (MAM), as well as colonic phase I metabolism of MAM by alcohol dehydrogenase (ADH) were examined in young (2-4 months), middle-aged (12-14 months), and old (22-24 months) male Fischer 344 rats. In addition, the possibility that colonic glucuronyltransferase might be involved in the biotransformation of MAM was also investigated. 2. A significant decrease in hepatic conversion of AZO to MAM was found in old vs young rats, concomitant with a decrease in hepatic cytochrome P-450 content, while no age-related difference was found in the colonic metabolism of MAM by ADH. MAM inhibition of colonic 4-methylumbelliferone glucuronyltransferase was non-competitive, suggesting indirectly that colonic glucuronyltransferase is not involved in conjugation of MAM. 3. It is concluded that ageing in the male Fischer 344 rat results in alternations of AZO and MAM biotransformation which indicate that AZO may be less carcinogenic in older rats.

Biomimetic models for monooxygenases.

The microsomal mixed function oxidase system contains the cytochrome P-450 oxidative drug metabolizing family of enzymes. The catalytic cycle of cytochrome P-450 is believed to involve the formation of an active iron-oxygen species which is responsible for oxygen transfer to the substrate. This assumption is supported by the fact that a number of peroxidative agents can replace NADPH, the reductase, and oxygen as co-reactants in most oxidative reactions of microsomal cytochrome P-450. We have found that a mixture of either ferrous or ferric ions with hydrogen peroxide (Fenton and Ruff reagents) can serve as biomimetic models for cytochrome P-450 in hydroxylation, exposidation, sulfoxidation, and N-demethylation of various drugs. The existance of an iron-oxo active species in both Fenton and Ruff type reactions has been postulated and provides reaction cycles similar to those of cytochrome p-450. Other model systems for the hepatic hydroxylation and epoxidation using transition metal complexes with porphyrin are also discussed. The present paper reviews the various biomimetic models of the heme cytochrome P-450 and emphasizes their simulation of hepatic drug metabolism and their potential medical and industrial applications.

In vitro inhibition of hepatic steroid hydroxylation by tamoxifen, a series of tamoxifen analogues and related compounds

The in vitro inhibition of the cytochrome P-450 (P-450) isozyme specific positional hydroxylation of androst-4-ene-3,17-dione (androstenedione) by the alkylamino containing compounds trans-and cis-tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, SKF 525-A and the non-alkylamino containing compounds tamoxifen metabolite E, and tamoxifen analogue U-23469 was assessed in pooled hepatic microsomes isolated from untreated male rats. P-450 IIA 1-mediated androstenedione 7α-hydroxylation appeared refractory to inhibition, with the lowest I 50s being approximately 200 μM ( cis-and trans-tamoxifen, 4-hydroxytamoxifen). (According to the recently recommended nomenclature for cytochromes P-450 (Nebert DW and González FJ, Ann Reu Biochem 56: 945–993, 1987), rat hepatic cytochromes P-450 UT-A, PB-B, PCN-E and UT-F are encoded by genes IIC 11, IIB 1, IIIA 1 2 and IIA 1, respectively. I 50s toward the P-450 IIC 11-, IIB 1-, and IIIA 1 2 -catalysed reactions, androstenedione 16α-, 16β-and 6β-hydroxylations, respectively, were generally in the range 70–190 μM. However, metabolite E exhibited a rather specific and potent capacity to inhibit androstenedione 16α-hydroxylase activity (I 50 = 18 μM). Since a number of alkylamine compounds have been shown to sequester microsomal P-450 as an inactive metabolite intermediate (MI), the tamoxifen analogues were investigated for their in vitro MI complexation capacity. However, spectral binding studies revealed that the incubation of these compounds with NADPH-fortified microsomal fractions did not result in MI complex formation. In binding experiments conducted with oxidised microsomal fractions it was apparent that most of the tamoxifen analogues are type I ligands of quite high affinity for ferric P-450 ( K s range 10–60 μM). It seems unlikely that MI formation is involved in the observed inhibition of androstenedione hydroxylation by tamoxifen and congeners. Instead, and in contrast to the situation observed with SKF 525-A, it would appear that the inhibitory capacity of the tamoxifen analogues is more closely related to type I binding capacity with ferric P-450. A finding of particular interest is that metabolite E, in which the alkylamino side-chain is absent, elicited a type I interaction of high capacity. The maximal absorbance change of the type I interaction of this compound with microsomal P-450 was about three-fold greater than the other compounds. It therefore appears likely that removal of the tamoxifen side-chain results in quite potent inhibition of P-450 IIC 11-mediated monooxygenase activity and an enhanced binding interaction with the cytochrome.

Pharmacogenetic aspects of biological variability

ABSTRACTIn the field of pharmacogenetics, new insights have been obtained over the past few years with respect to hepatic hydroxylation and acetylation of drugs. Metabolic defects within the particular enzyme systems can be accompanied by multiple consequences, such as altered pharmacokinetics and unexpected side‐effects of drugs.In Caucasian populations, the genetic defect of hydroxylation of debrisoquine occurs in approximately 9% of the population. The mephenytoin‐type drug hydroxylation defect, resulting from another hydroxylation polymorphism, can be demonstrated in 5% and 18% of Caucasian and Asian population samples, respectively. Such interindividual (phenotypic) differences of oxidative drug elimination can be detected after the peroral administration of a single dose of debrisoquine and mephenytoin followed by an 8 h urine collection. In hydroxylation‐deficient subjects, dose‐dependent drug toxicities occur after chronic drug administration of β‐blockers, antidepressants, perhexiline, phenacetine and others.With respect to the acetylation polymorphism, rapid and slow acetylator phenotypes exhibit approximately a 1: 1 distribution in Caucasian populations. That particular phenotype can be determined in plasma or urine after peroral administration of 500 mg sulfadimidine. A new test for the assessment of polymorphic acetylation has recently become available in the form of acetylamino‐formylamino‐methyluracil analysis in urine after peroral administration of caffeine. The clinical consequences of the inherited rapid and slow acetylation capacity of the liver are primarily relevant for slow acetylator phenotypes, because side‐effects of isoniazid, salazopyrine, hydralazine and procainamide occur more frequently in this condition.Pharmacogenetics and ecogenetics are two closely related entities. It might therefore be speculated that pharmacogenetically determined predispositions will also influence the metabolism of as yet unknown endo‐ and xenobiotic substances. Their varying abundances, for

Seasonal variations in aldrin epoxidase (MFO) activity of yellow-legged herring gulls: the relationship to breeding and PCB residues.

The hepatic mixed function oxidases (MFO) constitute a defense mechanism which enables the organism to make xenobiotics more polar and thus render them more readily excretable. The degree of induction of this system is an expression of its exposure to xenobiotics, but it is also a function of endogenous physiological mechanisms. These two forms of induction may lead to mutual interference: foreign compounds may stimulate hepatic hydroxylation and affect the metabolism of steroid hormones; the later may in turn stimulate the activity of the MFO system favoring the degradation of the xenobiotics. Induction and detoxication processes of endogenous and exogenous compounds have been observed in mammals in laboratory experiments. Relationships between MRO activity, the reproductive cycle and variations in tissue levels of liposoluble xenobiotics, have been reported for marine organisms. In birds, seasonal variations of MFO levels have been observed, but the relationship between these enzyme variations and the levels of contaminants in the animal tissues has never been made clear. The authors aim to clarify this relationship by determining the levels of PCBs residues and aldrin epoxidase activities in Yellow-legged Herring gull (Larus cachinnans) specimens from different areas of Italy collected during two phases of the annual cycle, namelymore » those of reproduction (spring) and of sexual inactivity (autumn). This species was chosen because of its wide distribution, its opportunistic feeding habits and its adaptive capacity in polluted environments.« less

Biliary lipid secretion in the rat. The uncoupling of biliary cholesterol and phospholipid secretion from bile acid secretion by sulfated glycolithocholic acid

Glycolithocholic acid and its sulfated derivative are major metabolites of the secondary bile acid lithocholic acid in man. Both compounds are known to induce cholestasis in experimental animals. We compared the effects of these endogenous hepatotoxins on bile production and biliary lipid composition in rats with chronic biliary drainage. The compounds were administered enterally at relatively low rates (5–50% of the rats' endogenous bile acid secretion in these experiments) to simulate enterohepatic circulation. Both compounds were substantially secreted into bile (more than 90% of dose); sulfated glycolithocholic acid unchanged and glycolithocholic acid after hepatic hydroxylation predominantly in the form of glyco-β-muricholic acid (cf. Kuipers et al. (1986) Am. J. Physiol. 251, G189–G194). Neither glycolithocholic acid nor its sulfated derivative affected the biliary excretion of endogenous bile acids or bile flow in these experiments. In spite of this, phospholipid and cholesterol secretion were significantly reduced by sulfated glycolithocholic acid but were not altered by glycolithocholic acid. Phospholipid and cholesterol secretion rapidly decreased to 25 and 50% of their initial values, respectively, at biliary output rates of sulfated glycolithocholic acid up to 2 μmol/h, and did not further decrease when this output was increased to 6 μmol/h. Small unilamellar liposomes consisting of cholesterol, [ Me- 14C]choline-labeled phosphatidylcholine, phosphatidylserine and [ 3H]cholesteryl oleate in a 5:4: 1:0.1 molar ratio were employed to label intrahepatic lipid pools. Administration of sulfated glycolithocholic acid slightly reduced bile acid synthesis from [ 3H]cholesteryl oleate, but significantly reduced the biliary secretion of [ 14C]phospholipid. Glycolithocholic acid did not affect the hepatic processing of liposomal lipids. It is concluded that sulfated glycolithocholic acid at low doses causes the uncoupling of biliary lipid secretion from that of bile acids, which might represent an initiating event in sulfated glycolithocholic acid hepatotoxicity.

Metabolite involvement in the behavioral effects of bromocriptine in cats

There is a lag phase of 30–60 min before the onset of bromocriptine (BC) action. This delay may be necessary for the formation of active metabolites. The objective was to determine whether the abnormal behavioral effects induced by BC involve active hepatic metabolites. Thus, we studied the effect of an inhibitor of hepatic hydroxylation metabolism (SKF 525A) on the behavior of BC-treated cats. Experiments began after six weeks of habituation and involved i.p. injections of: (1) propylene glycol (drug vehicle); (2) SKF 525A (70 mg/kg); (3) BC (10 mg/kg); and (4) SKF 525A followed 30 min later by BC. Each cat received the four treatments with two weeks elapsing between consecutive experiments. The frequency of 12 behaviors was scored for 60 min after 1 h posttreatment. BC alone induced emergent behavioral changes (hallucinatory-like, limb flicks, abortive grooms) that were not observed following control injections (vehicle and SKF 525A). There was a complete elimination of BC-induced hallucinatory-like behavior/escape by SKF 525A pretreatment. Other emergent behaviors were similarly reduced but persisted in all cats. The large frequency of grooming induced by BC was significantly reduced. SKF 525A pretreatment was correlated with a significant increase in staring and quiet sitting and a failure of BC to increase activities such as rubbing, treading and kneading. But many other BC-induced behaviors showed no changes. The data demonstrated that particular BC-induced changes in cats are antagonized by SKF 525A. The behavioral suppression caused by SKF 525A is compatible with the involvement of active hepatic metabolites from BC. The findings suggest that hepatic metabolites resulting from hydroxylation, rather than hydrolysis, of the BC parent molecule are responsible for the hallucinatory-like behavior/escape.

Carcinogen metabolizing enzymes in nude mice.

Hepatic and cutaneous microsomal hydroxylating and conjugating enzymes from female and male NMRI nu/nu mice were analyzed, and the response of these enzymes to repeated exposures with 3-methylcholanthrene were studied. Sex differences were observed in basal activities of hydroxylating enzymes. These differences were not the same in the liver as in the skin and were reversed in female and male. Activity ratios of hepatic and cutaneous hydroxylating enzymes were between 40-200. The ratios between hydroxylating and conjugating enzymes were much lower in the skin than in liver. Furthermore the ratios were depending on hydroxylating enzymes. 3-Methylcholanthrene treatment increased both hydroxylating and conjugating enzymes in the liver and in the skin. Again, there were sex differences in the induction pattern, and also the induction in the liver was unrelated to that in the skin. There was no correlation in induction between hydroxylating and conjugating enzymes.

Rat hepatic cholesterol 7α-hydroxylase: Biochemical properties and comparison to constitutive and xenobiotic-inducible cytochrome P-450 enzymes

Cytochrome P-450 Ch7 α (cholesterol 7α-hydroxylase) catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids in mammalian liver. The properties of this cytochrome P-450 ( P-450) form were studied in rat hepatic microsomal preparations in comparison to those of several well characterized constitutive and xenobiotic-inducible rat hepatic P-450s. Administration of the bile acid-sequestering resin cholestyramine [4% ( w w ) in the diet] to male or female rats maintained on a reverse light cycle led to a 10- to 15-fold induction of P-450 Ch7 α activity relative to untreated, standard light cycle controls. By contrast, the levels of four hepatic steroid hormone hydroxylating P-450 enzymes, designated 2a, 2c, 3, and PB-4 [Waxman, D. J. (1984) J. Biol. Chem. 259, 15481–15490] , were not significantly affected by cholestyramine treatment. Antibody inhibition experiments established that P-450 Ch7 α is immunochemically distinct from nine other rat hepatic P-450s, including P-450 3, a highly regio- and stereoselective steroid hormone 7α-hydroxylase. P-450 Ch7 α was shown to be selectively inactivated by micromolar concentrations of the disulfide-containing reagents disulfiram (Antabuse ®) and 2,2′-dithiopyridine. This inactivation was readily reversed upon incubation with 2-mercaptoethylamine, suggesting the presence of a highly reactive thiol group at the active site of P-450 Ch7 α . These findings demonstrate that P-450 Ch7 α corresponds to a unique P-450 enzyme exhibiting inductive, biochemical, immunochemical, and regulatory properties distinct from those of nine well-characterized rat hepatic P-450 forms.

Vitamin D metabolites in generalized scleroderma. Evidence of a normal cutaneous and intestinal supply with vitamin D

Vitamin D metabolites in serum were analysed in 20 patients with generalized scleroderma. The concentration of 1,25-dihydroxyvitamin D was normal, however, significantly lower concentrations (p less than 0.05) were found in 7 patients with cutaneous calcinosis in comparison with 13 patients with no calcinosis. Concentrations of 25-hydroxyvitamin D, 24-25-dihydroxyvitamin D, and vitamin D-binding protein (Gc globulin) were all within the normal range. The 24,25-dihydroxy-vitamin D level correlated with the duration of disease (r = 0.4453, p less than 0.05), and 25-hydroxyvitamin D tended to correlate (r = 0.3016, NS). The study strongly indicates that cutaneous synthesis, intestinal absorption and hepatic hydroxylation of vitamin D are not deficient in scleroderma. A relative but specific decrease in the renal hydroxylation to 1,25-dihydroxyvitamin D, i.e. the active hormone, as the disease progresses and calcinosis occurs, is suspected.

Perinatal exposure to methadone and the development of hepatic microsomal testosterone hydroxylation and methadone N-demethylation in the rat

Perinatal exposure to methadone and the development of hepatic microsomal testosterone hydroxylation and methadone N-demethylation in the rat

Hyperphenylalaninaemia caused by defects in biopterin metabolism.

The hepatic phenylalanine hydroxylating system consists of three essential components, phenylalanine hydroxylase, dihydropteridine reductase and the non-protein coenzyme, tetrahydrobiopterin. The reductase and the pterin coenzyme are also essential components of the tyrosine and tryptophan hydroxylating systems. Recent studies have shown that there are three distinct forms of phenylketonuria or hyperphenylalaninaemia, each caused by the lack of one of these essential components. The variant forms of the disease that are caused by the lack of dihydropteridine reductase or tetrahydrobiopterin are characterized by severe neurological deterioration, impaired functioning of tyrosine and tryptophan hydroxylases and the resultant deficiency of tyrosine- and tryptophan-derived monoamine neurotransmitters in brain.

Stereoselectivity of hepatic phenytoin hydroxylation: An in vivo probe for the heterogeneity of the cyt P-450 system in various drug hydroxylation phenotypes in man

Stereoselectivity of hepatic phenytoin hydroxylation: An in vivo probe for the heterogeneity of the cyt P-450 system in various drug hydroxylation phenotypes in man

The Disorder of Vitamin D Metabolism in Patients with Liver Cirrhosis

Liver cirrhosis (LC) is often associated with osteomalacia and osteoporosis. Since it has been shown that serum levels of 25 hydroxy vitamin D (25-OH-D) are reduced in LC, defective hepatic hydroxylation of vitamin D has been postulated to be responsible for the low serum 25-OH-D levels and skeletal demineralization. This study was designed, therefore, to determine serum 25-OH-D and 1 alpha, 25-(OH)2-D levels in patients with LC. Further, the response of serum 1 alpha, 25-(OH)2-D to a single oral dose of 1 alpha-OH-D3 (2 micrograms) was investigated. In 5 patients with severe decompensated LC and 3 patients with compensated LC, serum 25-OH-D and 1 alpha, 25-(OH)2-D levels were respectively measured by the modified method of Belsey and by that of Eisman. Serum 25-OH-D in patients with compensated and decompensated LC was significantly higher than that in normals. Serum levels of 1 alpha, 25-(OH)2-D in patients with decompensated LC were significantly lower than those in patients with compensated LC and normals. After a single oral administration of 1 alpha-OH-D3 at a dose of 2 micrograms, the 1 alpha, 25-(OH)2-D rose in each patient within 6h, reaching the maximum levels at 12h. The percent increase over the basal value in decompensated LC was similar to that in compensated LC.(ABSTRACT TRUNCATED AT 250 WORDS)

Parabolic Response of Hepatic Microsomal Hydroxylating System and Lipids to Graded Doses of Ascorbic Acid in Guinea Pigs on Low and High α-Tocopherol Intake

The influence of marginal ascorbic acid (AA) deficiency and excessive AA consumption on hepatic microsomal cytochrome P-450, aniline hydroxylase and O-demethylase, serum and liver cholesterol, serum triglycerides and liver α-tocopherol was investigated in guinea pigs with low and high vitamin E intake. Male guinea pigs were fed 9 weeks a scorbutogenic diet low in vitamin E (6 mg α-tocopherol per kilogram diet), supplemented with four levels of AA: A, deficient, 1 mg AA per animal per day per os; B, control, 500 mg/kg diet; C, control, 5000 mg/kg diet; D, excess, 20,000 mg/kg diet. The response of the microsomal cytochrome P-450 and aniline hydroxylase, serum and liver cholesterol and serum triglycerides to increasing AA doses showed a parabolic pattern. Deficiency (A): high cholesterol in serum and liver, hypertriglyceridemia, low cytochrome P-450 and aniline hydroxylase in liver microsomes; control groups (B and C): decrease of serum and liver cholesterol and serum triglycerides, increase of cytochrome P-450 and aniline hydroxylase; excess (D): return of all parameters to values not significantly different from the AA-deficient group. When guinea pigs were fed the same diets with four levels of AA plus 10 mg α-tocopherol acetate per animal orally five times a week, the response of the microsomal hydroxylating system and serum triglycerides to the graded AA doses again proved to be parabolic.

Pharmacokinetics and Tissue Distribution of Metronidazole in the Newborn Infant

Metronidazole pharmacokinetics and tissue distribution were studied in 11 infants varying in gestational age from 28 to 40 weeks. Elimination half-life was inversely related to gestational age, and ranged from 22.5 to 109 hours. Hepatic hydroxylation of metronidazole was not evident in infants less than 35 weeks' gestation, unless they had been exposed prenatally to betamethasone. A dosage schedule of 15 mg/kg intravenously as an initial single dose is proposed, and will provide adequate therapeutic levels for 48 hours in the preterm infant and for 24 hours in the term infant. Subsequently a dose of 7.5 mg/kg/12 hours is suggested for the first week of life.

Enhancement of hepatic microsomal aniline hydroxylation by Habu snake (Trimeresurus flavoviridis) venom fractions.

Enhancement of aniline hydroxylation by Habu snake venom was studied in vitro. The fraction which was isolated from Habu venom through column chromatography caused the enhancement of aniline hydroxylation of hepatic microsomes obtained from untreated rats. This fraction was heat stable and free from phospholipase activity. Enhancement of aniline hydroxylation was not clear in crude Habu venom or the unheated fraction, but appeared after heating of the fraction. This fraction enhanced aniline hydroxylation at low concentrations, followed by inhibition at high concentrations, but inhibited aminopyrine N-demethylation dose-dependently. Aniline hydroxylation was enhanced by the heated fraction at more than 1 mM aniline, and the enhancement was rather decreased at more than 10 mM aniline. The degree of the enhancement by the heated fraction increased with increasing pH.

Effect of inhibitors on ω‐and (ω‐1)‐hydroxylation of lauric acid by frog liver microsomes

To investigate the involvement of different cytochrome P-450 monooxygenases in fatty acid hydroxylation in frog liver microsomes, the effect of various inhibitors of cytochrome P-450 monooxygenases on the omega- and (omega-1)-hydroxylation of laurate was examined. The omega/omega-1-hydroxylation ratios were changed significantly by various levels of carbon monoxide (CO) inhibition; the formation of omega-hydroxylaurate was more sharply inhibited by various levels of CO than was the formation of (omega-1)-hydroxylaurate. On the contrary, metyrapone inhibited only the formation of (omega-1)-hydroxylaurate and stimulated the formation of omega-hydroxylaurate, 7,8-Benzoflavone as well as CO was more inhibitory to the omega-hydroxylation of laurate. At low concentrations of KCN (0.2 and 0.1 mM), the (omega-1)-hydroxylase activity was stimulated, but both the omega- and (omega-1)-hydroxylase activities were inhibited at the higher concentrations (5-10 mM). The effect of drugs and hydroxylaurate isomers on the omega- and (omega-1)-hydroxylation was also examined. Aminopyrine showed a stimulative effect on omega-hydroxylase activity and no effect on the (omega-1)-hydroxylase activity, while p-nitroanisole inhibited the (omega-1)-hydroxylase activity and showed almost no effect on the omega-hydroxylase activity. 12-Hydroxylaurate inhibited both the omega- and (omega-1)-hydroxylase activities, but the omega-hydroxylase activity was inhibited to a much greater extent. 11-Hydroxylaurate had no effect on either hydroxylation. These findings strongly support the hypothesis that different cytochrome P-450 species are involved in the hepatic microsomal hydroxylation of laurate at omega- and (omega-1)-positions in the frog.

Assessment of quinidine gluconate for nonlinear kinetics following chronic dosing.

Two different chronic dosing regimens of quinidine gluconate were administered to each of four healthy volunteers in a pilot study to evaluate quinidine for nonlinear pharmacokinetics. Analysis of plasma quinidine levels following the last dose indicates that disproportionate increases in steady-state plasma concentrations can occur in some subjects as the daily dose increases. Measurement of 2'-oxoquinidinone (2'-OXO) and 3-hydroxyquinidine (3-OH) metabolites revealed that the formation of 2'-OXO is proportional to the availability of quinidine base. Hydroxylation was a more variable process. Rate-limited hydroxylation was documented in one subject, and an apparent increase in hepatic microsomal enzyme-mediated hydroxylation was shown in a second subject who ingested large amounts of caffeine daily. By using a highly selective high-performance liquid chromatography assay technique, the total body clearance of quinidine was found to be greater than previously published data. Our results suggest that some individuals may exhibit dose-dependent elimination of quinidine and that the variability in quinidine's pharmacokinetics is related in part to its hydroxylation. Future studies must use highly specific quinidine assays and control for variables that may influence this route of metabolism.