Bunitrolol Research Articles

Extrapolating In vitro Metabolic Interactions to Isolated Perfused Liver: Predictions of Metabolic Interactions between R-Bufuralol, Bunitrolol, and Debrisoquine

Drug-drug interactions (DDIs) are a great concern to the selection of new drug candidates. While in vitro screening assays for DDI are a routine procedure in preclinical research, their interpretation and relevance for the in vivo situation still represent a major challenge. The objective of the present study was to develop a novel mechanistic modeling approach to quantitatively predict DDI solely based upon in vitro data. The overall strategy consisted of developing a model of the liver with physiological details on three subcompartments: the sinusoidal space, the space of Disse, and the cellular matrix. The substrate and inhibitor concentrations available to the metabolizing enzyme were modeled with respect to time and were used to relate the in vitro inhibition constant (K(i)) to the in vivo situation. The development of the liver model was supported by experimental studies in a stepwise fashion: (i) characterizing the interactions between the three selected drugs (R-bufuralol (BUF), bunitrolol (BUN), and debrisoquine (DBQ)) in microsomal incubations, (ii) modeling DDI based on binary mixtures model for all the possible pairs of interactions (BUF-BUN, BUF-DBQ, BUN-DBQ) describing a mutual competitive inhibition between the compounds, (iii) incorporating in the binary mixtures model the related constants determined in vitro for the inhibition, metabolism, transport, and partition coefficients of each compound, and (iv) validating the overall liver model for the prediction of the perfusate kinetics of each drug determined in isolated perfused rat liver (IPRL) for the single and paired compounds. Results from microsomal coincubations showed that competitive inhibition was the mechanism of interactions between all three compounds, as expected since those compounds are all substrates of rat CYP2D2. For each drug, the K(i) values estimated were similar to their K(m) values for CYP2D2 indicative of a competition for the same substrate-binding site. Comparison of the performance between the novel liver physiologically based pharmacokinetic (PBPK) model and published empirical models in simulating the perfusate concentration-time profile was based on the area under the curve (AUC) and the shape of the curve of the perfusate time course. The present liver PBPK model was able to quantitatively predict the metabolic interactions determined during the perfusions of mixtures of BUF-DBQ and BUN-DBQ. However, a lower degree of accuracy was obtained for the mixtures of BUF-BUN, potentially due to some interindividual variability in the relative proportion of CYP2D1 and CYP2D2 isoenzymes, both involved in BUF metabolism. Overall, in this metabolic interaction prediction exercise, the PBPK model clearly showed to be the best predictor of perfusate kinetics compared to more empirical models. The present study demonstrated the potential of the mechanistic liver model to enable predictions of metabolic DDI under in vivo condition solely from in vitro information.

Roles of phenylalanine at position 120 and glutamic acid at position 222 in the oxidation of chiral substrates by cytochrome P450 2D6

The roles of Phe-120 and Glu-222 in the oxidation of chiral substrates bunitrolol (BTL) and bufuralol (BF) by CYP2D6 are discussed. Wild-type CYP2D6 (CYP2D6-WT) oxidized BTL to 4-hydroxybunitrolol (4-OH-BTL) with substrate enantioselectivity of (R)-(+)-BTL > (S)-(-)-BTL. The same enzyme converted BF into 1''-hydroxybufuralol with substrate enantioselectivity of (R)-BF >> (S)-BF and metabolite diastereoselectivity of (1''R)-OH < (1''S)-OH. The substitution of Phe-120 by alanine markedly increased the apparent K(m) and V(max) values for enantiomeric BTL 4-hydroxylation by CYP2D6. In contrast, the same substitution caused an increase only in V(max) values of (S)-BF 1''-hydroxylation without changing apparent K(m) values, while kinetic parameters (K(m) and V(max) values) for (R)-BF 1''-hydroxylation remained unchanged. Furthermore, the substitution of Glu-222 as well as Glu-216 by alanine remarkably decreased both the apparent K(m) and V(max) values without changing substrate enantioselectivity or metabolite diastereoselectivity. A computer-assisted simulation study using energy minimization and molecular dynamics techniques indicated that the hydrophobic interaction of an aromatic moiety of the substrate with Phe-120 and the ionic interaction of a basic nitrogen atom of the substrate with Glu-222 in combination with Glu-216 play important roles in the binding of BF and BTL by CYP2D6 and the orientation of these substrates in the active-site cavity. This modeling yielded a convincing explanation for the reversal of substrate enantioselectivity in BTL 4-hydroxylation between CYP2D6-WT and CYP2D6-V374M having methionine in place of Val-374, which supports the validity of this modeling.

Changes in the Enzymatic Properties of CYP2D6 by the Substitution of Phenylalanine at Position 120 by Alanine

The functional roles of phenylalanine at position 120 (Phe-120) in the oxidation of bunitrolol (BTL), debrisoquine (DB) and bufuralol (BF) by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system (Saccharomyces cerevisiae AH-22 strain). The substitution of Phe-120 by alanine markedly increased the activities of enantiomeric BTL 4-hydroxylase and DB 4-hydroxylase, whereas it did not remarkably affect BF 1′′-hydroxylase activities. Kinetic studies revealed that the substitution of Phe-120 by alanine increased the Km and Vmax values for enantiomeric BTL 4-hydroxylation, but increased only the Vmax value for DB 4-hydroxylation without changing the Km value. Km and Vmax values for BF 1′′-hydroxylation were similar between the mutant and the wild-type. The dissociation constants of the mutant calculated from the binding spectra for BTL enantiomers were higher than those of the wild-type, suggesting that the substitution of Phe-120 by alanine decreased the affinity of CYP2D6 for BTL enantiomers. These results indicate that Phe-120 has an important role in the oxidation of substrates by CYP2D6.

Alteration in Catalytic Properties of Human CYP2D6 Caused by Substitution of Glycine-42 with Arginine, Lysine and Glutamic Acid

The effects of the substitution of glycine at position 42 with various other amino acid residues on the functions of CYP2D6 were studied using debrisoquine (DB) and bunitrolol (BTL) 4-hydroxylations as indices of drug-metabolizing enzymes. The substitution with hydrophobic amino acid residues such as valine and phenylalanine did not affect the enzymatic properties such as reduced CO-difference spectra, microsomal CYP contents and oxidation activities towards DB and BTL. The substitution of glycine-42 with a polar but noncharged amino acid residue (serine) exhibited a similar reduced CO-different spectrum, but the substitution with a charged basic (lysine and arginine) or acidic (glutamic acid) amino acid residue commonly produced a peak at 420 nm in addition to a Soret peak at 450 nm. Cytochrome P450 contents and microsomal contents of G42S, G42K, G42R and G42E estimated spectrophotometrically and estimated by Western blot analysis, respectively, were lower than those of the wild-type. Kinetic analysis revealed that the substitution of glycine-42 with charged amino acid residues such as lysine, arginine and glutamic acid markedly increased the apparent K(m) values for DB and BTL oxidations without remarkable changes in the V(max) values. The subsitution with noncharged amino acid residues such as serine, valine and phenylalanine did not cause such a marked change in the K(m) values. Efficiencies (V(max)/K(m)) as DB and BTL 4-hydroxylases of CYP2D6 mutant proteins having charged amino acid residues were found to be decreased mainly by increasing their K(m) values. These results indicate that the properties of amino acid residues at position 42 affect the behavior of CYP2D6 proteins such as anchoring into ER membranes, conversion of P450 to P420 and incorporation of heme into apoproteins.

Inactivation of rat cytochrome P450 2D enzyme by a further metabolite of 4-hydroxypropranolol, the major and active metabolite of propranolol.

Repetitive administration of propranolol (PL) in rats decreases the activities of cytochrome P450 (CYP) 2D enzyme(s) in hepatic microsomes. We examined the properties of 4-hydroxypropranolol (4-OH-PL) as an inactivator of rat liver microsomal CYP2D enzyme(s) using bunitrolol (BTL) 4-hydroxylation and PL 5- and 7-hydroxylations as indices of CYP2D enzyme activity. Rat microsomal BTL 4-hydroxylase activity was inhibited by the addition of 4-OH-PL to the incubation medium. The inhibition was greater after preincubation of microsomes with 4-OH-PL in the presence of NADPH than in its absence. The type of inhibition kinetics of BTL 4-hydroxylase by 4-OH-PL was changed from a competitive type to a noncompetitive type by the preincubation. The inhibition of rat liver microsomal PL 5- and 7-hydroxylases by 4-OH-PL was blocked efficiently by co-incubation with quinine, a typical inhibitor of rat CYP2D enzyme(s), or to a lesser extent by BTL. However, quinidine, a diastereomer of quinine, did not significantly protect against the enzyme inactivation. The protective capacities of the substrate and inhibitors reflected their affinities for rat CYP2D enzyme(s). BTL hydroxylase was not affected by either 1,4-naphthoquinone or 1,4-dihydroxynaphthalene which are possible metabolites of 4-OH-PL. These results provide further evidence to support the notion that PL is biotransformed by rat CYP2D enzyme(s) to 4-OH-PL, which is further oxidized to a chemically reactive metabolite in the active site. The inactivation of CYP is likely the result of covalent binding of the reactive species to an amino acid residue of the active site.

CHRACTERIZATION OF CYTOCHROME P4502D6 VARIANTS EXPRESSED IN YEAST CELLS

To evaluate CYP2D6 mutants as drug-metabolizing enzymes, we expressed four single amino acid-substituted mutants (P34S, G42R, R296C and S486T) and two double amino acid-substituted mutants (P34S/S486T and R296C/S486T) in yeast cells. The profiles of P34S, G42R and P34S/S486T in microsomal expression levels and oxidation capacities towards debrisoquine (DB) and bunitrolol (BTL) were much different from those of the wild type, while the behavior of R296C, S486T and R296C/S486T was similar to the wild type in these indices. CYP contents in microsomal and whole cell fractions, which were measured spectrophotometrically and immunochemically, indicated that a part of P34S protein was located in microsomal membranes whereas most of P34S protein was in cytosolic fraction. Enzyme assay revealed that P34S showed much lower activities in DB and BTL 4-hydroxylations than the wild type in terms of nmol/min/mg protein, but similar activities to the wild type in terms of nmol/min/nmol CYP, indicating that P34S, which can anchor to endoplasmic reticulum membrane and hold a prosthetic heme group, retains normal functions. However, G42R did not have normal functions even in terms of nmol/min/nmol CYP; i.e., its Km values for DB and BTL oxidations were much higher than those of the wild type. The results indicate that similar to the substitution of Pro-34 to Ser, that of Gly-42 to Arg results in the decrease in the enzymatic function but in a manner different from P34S.

Contribution of P-glycoprotein to bunitrolol efflux across blood-brain barrier.

In this study, we investigated the mechanism of the blood-brain barrier (BBB) transport of bunitrolol (BTL), as a model of beta-blocker, in vivo and in vitro. In order to define the contribution of P-glycoprotein (P-gp) to the active efflux of BTL from brain to blood, we examined the in vivo brain distribution of BTL in mdr1a(-/-) mice with a disrupted mdr1a gene. After intravenous administration of BTL to mdr1a(-/-) mice, the brain concentration and Kp value of BTL were significantly increased as compared with those in mdr1a(+/+) mice. Next, the contribution of the mdr1a P-gp to in vitro uptake of BTL was compared in LV500 cells and L cells (mouse mdr1a-expressing cells and host cells, respectively). The intracellular accumulations of [3H]vinblastine and BTL by LV500 cells were lower than those by L cells, but were significantly increased by verapamil, a P-gp inhibitor. Furthermore, the BTL uptake by KB-VJ300 cells, which express human P-gp, was also significantly lower than that by KB host cells, and was increased by verapamil. The steady-state uptake of BTL by LLC-GA5-COL300 cells, expressing human P-gp, was significantly increased in the presence of 20 microM cyclosporin A (another P-gp inhibitor), which had no effect in the LLC-PK1 host cells. On the other hand, the steady-state intracellular accumulation of BTL by MBEC4 cells, which express mdr1b P-gp instead of mdr1a P-gp, was not significantly changed in the presence of verapamil. This finding suggested that BTL is not a good substrate for mdr1b P-gp. In conclusion, our results suggest that BTL is transported from brain to blood by mdr1a P-gp in mice and by MDR1 in humans, and this presumably accounts for the low brain distribution of BTL.

Enantioselectivity of bunitrolol 4-hydroxylation is reversed by the change of an amino acid residue from valine to methionine at position 374 of cytochrome P450-2D6.

The enantioselectivity of 4-hydroxylation of bunitrolol (BTL), a beta-adrenoceptor blocking drug, was studied in microsomes from human liver, human hepatoma (Hep G2) cells expressing CYP2D6, and lymphoblastoid cells expressing CYP2D6. Kinetics in human liver microsomes showed that the Vmax value for (+)-BTL was 2.1-fold that of (-)-BTL, and that the Km value for (+)-BTL was lower than that for the (-)-antipode, resulting in the intrinsic clearance (Vmax/Km) of (+)-BTL being 2.1-fold over its (-)-antipode. CYP2D6 (CYP2D6-met) expressed in Hep G2 cells had a methionine residue at position 373 of the amino acid sequence and a rat-type N-terminal peptide (MELLNGTGLWSM) instead of the human-type (MGLEALVPLAVIV), and showed enantioselectivity of [(+)-BTL < (-)-BTL] for the rate of BTL 4-hydroxylation. In contrast, enantioselectivity [(+)-BTL > (-)-BTL] for Hep G2-CYP2D6 (CYP2D6-val) with a human-type N-terminal peptide that had a valine residue at 374, which corresponds to the methionine of the CYP2D6-met variant, was the same as that for human liver microsomes. We further confirmed that CYP2D6-met and CYP2D6-val expressed in human lymphoblastoid cells, both of which have methionine and valine, respectively, at position 374 and a human-type N-terminal peptide, exhibited the same enantioselectivities as those obtained from CYP2D6-met and CYP2D6-val expressed in the Hep G2 cell system. These results indicate that the amino acid at 374 of CYP2D6 is one of the key factors influencing the enantioselectivity of BTL 4-hydroxylation.

CHARACTERIZATION OF OXIDATION OF OPTICALLY ACTIVE SUBSTRATES BY RAT AND HUMAN CYP2D ENZYMES EXPRESSED IN SACCHAROMYCES CEREVISIAE

To validate the usefulness of recombinant CYP enzymes in metabolic studies of optically active drugs, enantiomer selectivity was examined for oxidation of optically active R-blokers by rat CYP2D enzymes (CYP2D1, -2D2, -2D3 and -2D4) and human CYP2D6 expressed in yeast cells, and was compared with the profile of liver microsomes (Ms). In bunitrolol (BTL) 4hydroxylation rate, selectivity of [(+)-BTL S(-)-PL] in a low PL concentration range. CYP2D2 having the highest PL oxidation activities among the rat CYP2D enzymes showed selectivity of [R(+)PL S(-)-PL] in a low PL concentration range, whereas recombinant CYP2D6 did not show clear selectivity. PL oxidation by human CYP2D6 showed biphasic kinetics. Kinetics of racemic bufuralol (BUF) were monophasic for rat CYP2D2 and human CYP2D6 but biphasic for rat CYP2D4. Considering these results, different enantioselectivity between liver Ms and recombinant CYP2D enzymes and biphasic kinetics for recombinant CYP enzymes may be caused by different microenvironments surrounding CYP enzymes between liver and yeast cells.

Characterization of hepatic cytochrome P450 isozyme composition in the transgenic rat expressing low level human growth hormone

1. The present authors have previously developed a transgenic rat carrying a chimeric gene of the mouse whey acidic protein promoter and the structural portion of human growth hormone (GH) gene. Among this (hGH-TG) rat, a line (low GH rat) missing a male-specific pulsatile GH secretary pattern due to suppression of endogenous GH secretion and having a continuous low GH (hGH and rat GH) level in the peripheral circulation was identified. The latter rat was also characterized as having severe obesity with age. This strain (low Gh rat) was used to correlate the sex-specific secretory pattern of GH with the sex-specific expression of cytochrome P450 (CYP) in rat. 2. Comparisons were made between the low GH rat and the non-transgenic rat as to the expression of liver microsomal CYP isozymes. The following enzyme activities were assessed: testosterone (T) hydroxylation and oxidation; ethoxyresorufin O-dealkylation (EROD); bunitrolol (BTL) 4-hydroxylation and T5 alpha-reduction. Protein expression of CYP1A, CYP2C11, CYP2D, CYP2E1, CYP3A2 and CYP4A1 were also assessed by Western blot analysis. 3. Enzyme activities and protein expression of CYP2C11 (T16 alpha and 2alpha-hydroxylase and 17-oxidase activities) and CYP3A2 (T6beta and 2beta-hydroxylase activities) levels, which are known to be higher in the male than in the female rat, were significantly lower in the adult male low GH rat than in the control male rat. In contrast, CYP2A1 (T7 alpha hydroxylase) and T5-alpha-reductase activities, which are known to be specifically elevated in the female, were significantly higher in the adult male low GH rat than in the control male rat. Thus, the loss of male-specific secretory pattern of GH results in feminization of the pattern of expression of CYP and T5 alpha-reductase activity in the liver. 4. In contrast to other GH-deficient models so far studied, an increase in CYP4A1 and a decrease in CYP2E1 protein expression were observed in the low GH rat. These trends are consistent with the characteristic phenotype of obesity in the transgenic rat because CYP4A1 and CYP2E1 enhance fatty acid excretion and glyconeogenesis from fatty acids respectively.

CHANGE IN SUBSTRATE ENANTIO AND REGIOSELECTIVITY FOR VARIOUS HETEROLOGOUS CYP2D6 EXPRESSION SYSTEMS

Enantio and regioselectivity of adrenoceptor blocking agents [propranolol (PL) and bunitrolol (BTL)] was examined for CYP2D6 expressed in various heterologous expression systems. In human liver microsomes, propranolol was oxidized at 4 and 5-position of the naphthalene ring and at the side chain to form 4-hydroxypropranolol, 5-hydroxypropranolols and N-desisopropyl-propranolol, respectively. BTL was oxidized to 4-hydroxybunitrolol by human liver microsomes. At a lower concentration range of PL or BTL, the ring hydroxylation of the substrates is mainly mediated by CYP2D6 in human liver. As for the enantioselectivity, CYP2D6 expressed in Sf9 insect cells showed enantioselectivity of R(+) S(-)]. CYP2D6 expressed in Escherichia coli also gave the same results. This was proved to be caused by the change of an amino acid residue at position 374 of CYP2D6 from valine as wildtype to methionine as allelic variant. As for the regioselectivity, CYP2D6 expressed in lymphoblastoid cells had PL 4- and 5-hydroxylase, and BTL 4-hydroxylase activities, but did not show PL N-desisopropylase activity, on the one hand. CYP2D6 expressed in E. coli had PL 4- and 5-hydroxylase, and N-desalkylase activities but did not exhibit BTL 4-hydroxylase activity, on the other. These results suggest that different environment surrounding the CYP2D6 proteins expressed in heterologous cells or N-truncation of the enzyme to yield high efficiency in expression in E. coli. affects the properties of CYP2D6 proteins.

SUBSTRATE SPECIFICITIES OF CYP 2D SABFAMILY

The Dark Agouti (DA) rat has been proposed as a poor metabolizer model for the human debrisoquine 4-hydroxylase polymorphism. Earlier studies suggested that the poor metabolizer phenotype in the DA rat is due to the absence of the expression of CYP2D1 mRNA. Although CYP2D 1 catalyzes debrisoquine 4-hydroxylation, we found that another P450 of CYP2D subfamily, presumably CYP2D2, which we have purified from SD rat livers by chasing bunitrolol (BTL) 4-hydroxylase activity as an indicator also has the ability to catalyze debrisoquine 4-hydroxylation. CYP 2D2 has previously been described as inactive species. Thus we obtained CYP2D1 and 2D2 cDNA from Dr. F. J. Gonzalez of NIH and using baculovirus expression system, these two isozymes were expressed in Sf9 insect cells. We found that both isozymes can catalyze debrisoquine 4-hydroxylation, but CYP 2D2 has about 10 fold higher activity than CYP 2D1. In addition, CYP 2D2 was able to catalyze BTL 4-hydroxylation and propranolol 4-, 5-, and 7-hydroxylations while CYP 2D1 was not able to catalyze BTL 4-hydroxylation and propranolol 7-hydroxylation. These results indicate that CYP isozyme responsible for PM phenotype in DA rats is not CYP2D1 as previously indicated but CYP2D2. Levels of expression of CYP proteins were assessed by Western blotting using isozyme specific peptide antibodies. The level of expression of CYP2D 1 in DA rats was about the same level as that in SD rats, but the level of expression of CYP2D2 was markedly lower in DA rats than that in SD rats. The levels of CYP2D 1 and 2D2 mRNAs were examined by Northern blotting to find that levels of both mRNAs were markedly lower in DA rats than those in SD rats. Although the discrepancy between expression levels of CYP 2D1 proteins and its mRNA remains unresolved, it is clear that the reduced level of expression of CYP2D2 is responsible for the PM phenotype of DA rats.

Stereoselectivity in bunitrolol 4-hydroxylation in liver microsomes from marmosets and Japanese monkeys.

The stereoselectivity in 4-hydroxylation of bunitrolol (BTL), a beta-adrenoreceptor blocking agent, was examined in liver microsomes from monkeys (marmosets and Japanese monkeys) and compared with the results of human liver microsomes. The formation of (+)-4-OH-BTL from (+)-BTL from (+)-BTL was significantly higher than that of (-)-4-OH-BTL from (-)-BTL in the liver microsomal fractions from the two kinds of monkeys. The 4-OH-BTL-forming activity from racemic BTL was significantly lower than from enantiomeric BTL, indicating a possible metabolic interaction between BTL enantiomers. The in vitro profiles observed in the monkeys were very similar to those in humans, but the stereoselectivity in BTL metabolism [(+)-BTL > (-)-BTL] in the primates was found to be reverse to that in rats [S. Narimatsu et al., Anal. Biochem., 222, 256-261 (1994)]. The 4-OH-BTL-forming activity from BTL enantiomers was significantly suppressed by quinidine and quinine, while the former was more potent than the latter, and also by alpha-naphthoflavone. Furthermore, the activity was also suppressed by antisera against rat cytochromes P450-2D2 and -1A2 in concentration-dependent manners. However, kinetics showed that enantiomeric BTL 4-hydroxylation was monophasic in liver microsomes from marmosets of both genders and from male Japanese monkeys. These results suggest that cytochrome P450-2D and -1A enzymes with similar Km values are involved in BTL 4-hydroxylation in monkey liver microsomes.

Development of a High-Performance Liquid Chromatographic Method for the Analysis of Enatiomer/Enantiomer Interaction in Oxidative Metabolism of Bunitrolol in Rat Liver Microsomes

A high-performance liquid chromatographic method for the assay of enantiomeric 4-hydroxybunitrolol (4-OH-BTL) formed from racemic bunitrolol (BTL) in rat liver microsomes was developed. Racemic bunitrolol was incubated with rat fiver microsomes fortified with an NADPH-generating system. Metabolites extracted with ethyl acetate were converted to acetyl derivatives with acetic anhydride in pyridine. The derivatives of 4-OH-BTL were well separated by the liquid chromatography equipped with a chiral column. Using this method, the metabolic interaction of BTL enantiomers was examined, The 4-OH-BTL-forming activities from enantiomeric BTL were higher than those from racemic BTL in rat liver microsomes, while the formation of ratios of 4-OH-BTL enantiomer to its antipode were the same under the two conditions. The Ki values obtained from kinetic studies using each BTL enantiomer as an inhibitor of its antipode were almost the same (ca 0.9 μM), which were close to their Michaelis constants (Km values). Oxidative activities of enantiomeric and racemic BTL were almost equally inhibited by debrisoquine and quinidine, a typical substrate and a selective inhibitor of the CYP2D subfamily, respectively. These results indicate that a BTL enantiomer is a mutual metabolic inhibitor of its antipode and BTL enantiomers compete for the same CYP2D isozyme in rat liver microsomes.

Radioimmunoassay for bunitrolol

A specific, sensitive, and simple radioimmunoassay was developed for bunitrolol (BNL), a beta adrenoceptor antagonist. Antisera against BNL were produced by conjugating both dl- and l-BNL-hemiglutarate to bovine serum albumin and immunizing rabbits with the two immunogens. The antiserum against dl-BNL recognized both d- and 1-BNL to the same degree, whereas the antiserum against l-BNL did not show any specificity to the 1-isomer and rather bound well with the d-isomer. Both antisera did not appreciably cross-react with the major metabolites of BNL, 4-hydroxy BNL and o-nitrilophenoxylactic acid. [3H]BNL and the antiserum against dl-BNL were used to develop a radioimmunoassay. The antibody bound [3H]BNL was separated from the free by the ammonium sulfate method. The radioimmunoassay is able to determine as little as 100 pg of BNL directly from plasma without the need for extraction. This procedure was then applied to determine plasma concentrations of BNL in rats administered with 10 mg/kg of dl-BNL, p.o. BNL was rapidly absorbed from the gastrointestinal tract. Plasma concentra-Tion-time curves for dl-BNL were found to fit well with two-compartment model.