Stereoselective metabolism of benalaxyl in liver microsomes from rat and rabbit

Benalaxyl (BX), methyl‐N‐phenylacetyl‐N‐2,6‐xylyl alaninate, is a potent acylanilide fungicide and consist of a pair of enantiomers. The stereoselective metabolism of BX was investigated in rat and rabbit microsomes in vitro. The degradation kinetics and the enantiomer fraction (EF) were determined using normal high‐performance liquid chromatography with diode array detection and a cellulose‐tris‐(3,5‐dimethylphenylcarbamate)‐based chiral stationary phase (CDMPC‐CSP). The t_1/2 of (?)‐R‐BX and (+)‐S‐BX in rat liver microsomes were 22.35 and 10.66 min of rac‐BX and 5.42 and 4.03 of BX enantiomers. However, the t_1/2 of (?)‐R‐BX and (+)‐S‐BX in rabbit liver microsomes were 11.75 and 15.26 min of rac‐BX and 5.66 and 9.63 of BX enantiomers. The consequence was consistent with the stereoselective toxicokinetics of BX in vitro. There was no chiral inversion from the (?)‐R‐BX to (+)‐S‐BX or inversion from (+)‐S‐BX to (?)‐R‐BX in both rabbit and rat microsomes. These results suggested metabolism of BX enantiomers was stereoselective in rat and rabbit liver microsomes. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Stereoselective distribution of hydroxychloroquine in the rabbit following single and multiple oral doses of the racemate and the separate enantiomers

Hydroxychloroquine (HCQ) stereoselective distribution was investigated in rabbits after 20 mg/kg po of racemic-HCQ (rac-HCQ) and 20 mg/kg po of each enantiomer, 97% pure (?)-(R)-HCQ and 99% pure (+)-(S)-HCQ. Concentrations were 4 to 6 times higher in whole blood than in plasma. Melanin did not affect plasma and whole blood levels since concentrations did not differ between pigmented and nonpigmented animals. After single and multiple doses of the separate enantiomers, only 5–10% of the antipode could be measured, in blood or plasma. Therefore, there was no significant interconversion from one enantiomer into the other. Following rac-HCQ, plasma (+)-(S)-levels always surpassed (?)-(R)-ones while in whole blood, (?)-(R)-HCQ concentrations were always the highest. When the enantiomers were administered separately, blood concentrations achieved after (?)-(R)-HCQ were higher, especially after multiple doses. These observations suggest that (?)-(R)-HCQ is preferentially concentrated by cellular components of blood. This enantioselective distribution of HCQ could be secondary to a stereoselective protein binding to plasma proteins, although a more specific binding of (?)-(R)-HCQ to blood cells cannot be ruled out. Since in whole blood (?)-(R)-HCQ is retained in cellular components, metabolism would favour the more available (+)-(S)-enantiomer. © 1994 Wiley-Liss, Inc.     

Chiral HPLC determination and stereoselective pharmacokinetics of tetrahydroberberine enantiomers in rats

Tetrahydroberberine (THB), a racemic mixture of (+)‐ and (?)‐enantiomer, is a biologically active ingredient isolated from a traditional Chinese herb Rhizoma corydalis (yanhusuo). A chiral high performance liquid chromatography method has been developed for the determination of THB enantiomers in rat plasma. The enantioseparation was carried out on a Chiral®‐AD column using methanol:ethanol (80:20, v/v) as the mobile phase at the flow rate 0.4 ml/min. The ultraviolet detection was set at 230 nm. The calibration curves were linear over the range of 0.01–2.5 μg/ml for (+)‐THB and 0.01‐5.0 μg/ml for (?)‐THB, respectively. The lower limit of quantification was 0.01 μg/ml for both (+)‐THB and (?)‐THB. The stereoselective pharmacokinetics of THB enantiomers in rats was studied after oral and intravenous administration at a dose of 50 and 10 mg/kg racemic THB (rac‐THB). The mean plasma levels of (?)‐THB were higher at almost all time points than those of (+)‐THB. (?)‐THB also exhibited greater C_max, and AUC_0–∞, smaller CL and V_d, than its antipode. The (?)/(+)‐enantiomer ratio of AUC_0–∞ after oral and intravenous administration were 2.17 and 1.43, respectively. These results indicated substantial stereoselectivity in the pharmacokinetics of THB enantiomers in rats. Chirality, 2012. © 2012 Wiley Periodicals, Inc.     

Tissue distribution of bupivacaine enantiomers in sheep

rac-Bupivacaine HCl was infused intravenously to constant arterial blood drug concentrations in sheep using a regimen of 4 mg/min for 15 min followed by 1 mg/min to 24 h. At 24 h, arterial blood was sampled, the animal was killed with a bolus of KCl solution, then rapidly dissected and samples were obtained from heart, brain, lung, kidney, liver, muscle, fat, gut, and rumen. Tissue:blood distribution coefficients for (+)-(R)-bupivacaine exceeded those of (?)-(S)-bupivacaine (P < 0.05) for heart, brain, lung, fat, gut, and rumen by an overall mean of 43%. Blood:plasma distribution coefficients of (?)-(S)-bupivacaine exceeded those of (+)-(R)-bupivacaine by a mean of 29% and this offset the tissue:blood distribution coefficients so that the previously significant enantioselective differences disappeared. It is concluded that although enantioselectivity of bupivacame distribution is shown by the measured tissue:blood distribution coefficients, it is not shown when tissue:plasma water distribution coefficients are calculated, suggesting that there is no intrinsic difference between the bupivacaine enantiomers in tissue affinity. Sheep given fatal intravenous bolus doses of rac-bupivacaine had significantly greater concentrations of (+)-(R)-bupivacaine than (?)-(S)-bupivacaine in brain (P = 0.028) and ventricle (P = 0.036); these could augment the greater myocardial toxicity of this enantiomer found in vitro. © 1993 Wiley-Liss, Inc.     

Analysis of Oxcarbazepine and the 10‐Hydroxycarbazepine Enantiomers in Plasma by LC‐MS/MS: Application in a Pharmacokinetic Study

Oxcarbazepine is a second‐generation antiepileptic drug indicated as monotherapy or adjunctive therapy in the treatment of partial seizures or generalized tonic–clonic seizures in adults and children. It undergoes rapid presystemic reduction with formation of the active metabolite 10‐hydroxycarbazepine (MHD), which has a chiral center at position 10, with the enantiomers (S)‐(+)‐ and R‐(?)‐MHD showing similar antiepileptic effects. This study presents the development and validation of a method of sequential analysis of oxcarbazepine and MHD enantiomers in plasma using liquid chromatography with tandem mass spectrometry (LC‐MS/MS). Aliquots of 100 μL of plasma were extracted with a mixture of methyl tert‐butyl ether: dichloromethane (2:1). The separation of oxcarbazepine and the MHD enantiomers was obtained on a chiral phase Chiralcel OD‐H column, using a mixture of hexane:ethanol:isopropanol (80:15:5, v/v/v) as mobile phase at a flow rate of 1.3 mL/min with a split ratio of 1:5, and quantification was performed by LC‐MS/MS. The limit of quantification was 12.5 ng oxcarbazepine and 31.25 ng of each MHD enantiomer/mL of plasma. The method was applied in the study of kinetic disposition of oxcarbazepine and the MHD enantiomers in the steady state after oral administration of 300 mg/12 h oxcarbazepine in a healthy volunteer. The maximum plasma concentration of oxcarbazepine was 1.2 µg/mL at 0.75 h. The kinetic disposition of MHD is enantioselective, with a higher proportion of the S‐(+)‐MHD enantiomer compared to R‐(?)‐MHD and an AUC^0‐12 _{S‐(+)/R‐(?)} ratio of 5.44. Chirality 25:897–903, 2013. © 2013 Wiley Periodicals, Inc.     

Stereoselective cyclooxygenase inhibition in cellular models by the enantiomers of ketoprofen

The pharmacological activity of rac-ketoprofen and its enantiomers was investigated in vitro using different cellular models. The effect of these compounds on arachidonic acid metabolism was assessed by measuring the inhibition of prostanoid generation under the action of several agonists. Thus, we have evaluated the inhibition of (1) thromboxane B₂ synthesis in rabbit platelets and human polymorphonuclear leukocytes (PMNs), (2) prostaglandin E₂ synthesis in three cultured cells, namely human umbilical vein endothelial cells (HUVEC), human keratinocytes, and mouse macrophage-like P388D1 cells. The IC₅₀ values found for (+)-(S)-ketoprofen were in the range between 0.1 nM and 0.8 μM, being slightly lower in all models than those found for rac-ketoprofen (0.4 nM–3 μM). On the other hand, (?)-(R)-ketoprofen showed inhibition of cyclooxygenase only at concentrations two or three orders of magnitude higher than those required for the (+)-(S) enantiomer. These results, obtained with cell types of relevance for inflammatory processes and with compounds of high optical purity, demonstrate that the prostanoid biosynthesis inhibition caused by the drug rac-ketoprofen is exclusively due to its dextrorotatory enantiomer. © 1993 Wiley-Liss, Inc.     

Stereoselective Determination of Metoprolol and its Metabolite α‐Hydroxymetoprolol in Plasma by LC‐MS/MS: Application to Pharmacokinetics during Pregnancy

Metoprolol is available for clinical use as a racemic mixture. The S‐(?)‐metoprolol enantiomer is the one expressing higher activity in the blockade of the β₁‐adrenergic receptor. The α‐hydroxymetoprolol metabolite also has activity in the blockade of the β₁‐adrenergic receptor. The present study describes the development and validation of a stereoselective method for sequential analysis of metoprolol and of α‐hydroxymetoprolol in plasma using high‐performance liquid chromatography with tandem mass spectrometry (LC‐MS/MS). 1‐ml aliquots of plasma were extracted with dichloromethane : diisopropyl ether (1:1, v/v). Metoprolol enantiomers and α‐hydroxymetoprolol isomers were separated on a Chiralpak AD column (Daicel Chemical Industries, New York, NY, USA) and quantitated by LC‐MS/MS. The limit of quantitation obtained was 0.2 ng of each metoprolol enantiomer/ml plasma and 0.1 ng/ml of each α‐hydroxymetoprolol isomer/ml plasma. The method was applied to the study of kinetic disposition of metoprolol in plasma samples collected up to 24 h after the administration of a single oral dose of 100‐mg metoprolol tartrate to a hypertensive parturient with a gestational age of 42 weeks. The clinical study showed that the metoprolol pharmakokinetics is enantioselective, with the observation of higher area under the curve (AUC)^0?∞ values for S‐(?)‐metoprolol (AUC_S‐(?)/AUC_R‐(+) = 1.81) and the favoring of the formation of the new chiral center 1′R of α‐hydroxymetoprolol (AUC^0?∞_1′R/1′S = 2.78). Chirality, 25:1–7, 2013. © 2012 Wiley Periodicals, Inc.     

Enantioselective Toxic Effects and Degradation of Myclobutanil Enantiomers in Scenedesmus obliquus

Research on the enantioselective environmental behavior of chiral pesticides has been a hot spot of environmental chemistry recently. In this study, the acute toxicity of myclobutanil enantiomers was investigated with the aquatic algae Scendesmus obliquus. After exposure for 96 h, the EC₅₀ values for (?)‐myclobutanil, rac‐myclobutanil and (+)‐myclobutanil were 3.951, 2.760, and 2.128 mg/L, respectively. The photosynthetic pigment (chlorophyll a, chlorophyll b, and carotenoids) and antioxidant enzyme activities catalase (CAT) were determined to evaluate the different toxic effects when S. obliquus were exposed to 1.5, 5 and 15 mg/L of rac‐myclobutanil, (?)‐myclobutanil, and (+)‐myclobutanil for 96 h, respectively. In addition, the degradation of myclobutanil enantiomers in S. obliquus was also studied. Myclobutanil in the medium inoculated with algae degraded faster than in the uninoculated medium. The degradation of (?)‐myclobutanil was faster than that of (+)‐myclobutanil at a concentration of 3 mg/L. On the basis of these data, the acute toxicity and toxic effects of myclobutanil against S. obliquus were concluded to be enantioselective, and such enantiomeric differences should be taken into consideration in pesticide risk assessment. Chirality 25:858–864, 2013. © 2013 Wiley Periodicals, Inc.     

Enantioselective Pharmacokinetics of Doxazosin and Pharmacokinetic Interaction Between the Isomers in Rats

In this study, the stereoselective pharmacokinetics of doxazosin enantiomers and their pharmacokinetic interaction were studied in rats. Enantiomer concentrations in plasma were measured using chiral high‐pressure liquid chromatography (HPLC) with fluorescence detection after oral or intravenous administration of (–)‐(R)‐doxazosin 3.0 mg/kg, (+)‐(S)‐doxazosin 3.0 mg/kg, and rac‐doxazosin 6.0 mg/kg. AUC values of (+)‐(S)‐doxazosin were always larger than those of (–)‐(R)‐doxazosin, regardless of oral or intravenous administration. The maximum plasma concentration (C_max) value of (–)‐(R)‐doxazosin after oral administration was significantly higher when given alone (110.5 ± 46.4 ng/mL) versus in racemate (53.2 ± 19.7 ng/mL), whereas the C_max value of (+)‐(S)‐doxazosin did not change significantly. The area under the curve (AUC) and C_max values for (+)‐(S)‐doxazosin after intravenous administration were significantly lower, and its Cl value significantly higher, when given alone versus in racemate. We speculate that (–)‐(R)‐doxazosin increases (+)‐(S)‐doxazosin exposure probably by inhibiting the elimination of (+)‐(S)‐doxazosin, and the enantiomers may be competitively absorbed from the gastrointestinal tract. In conclusion, doxazosin pharmacokinetics are substantially stereospecific and enantiomer–enantiomer interaction occurs after rac‐administration. Chirality 27:738–744, 2015. © 2015 Wiley Periodicals, Inc.     

Enzymatic and chromatographic chiral discrimination of racemic (thio)glycidyl esters: Part I. A chemoenzymatic approach to both enantiomers of thioglycidyl esters

Both hitherto unknown (+)-(R)- and (?)-(S)-thioglycidyl esters, (R)-( 2 ) and (S)-( 2 ), have been synthesized with different high enantiomeric excesses (ee) by two routes from the corresponding rac-glycidyl esters rac-( 1 ). The first includes a porcine pancreatic lipase (PPL)-mediated kinetic resolution of these esters followed by sulfuration with practically complete inversion to the (+)-(R)-enantiomer (+)-(R)-( 2 ) (36–86% ee). (?)-(S)-Thioglycidyl esters (?)-(S)-( 2 ) are obtained by the reverse reaction sequence (43–80% ee). In the latter case the hydrolysis rate is lower than that of analogous glycidyl esters. Moreover, the dependence of enantiomeric excess on the size of the acyl-group is of the opposite tendency. Therefore, in both cases suitable selection of the acid residue gives rise to maximum enantioselectivity. The irreversible lipase-catalyzed acylation of rac-glycidol and rac-thioglycidol, however, was found to be a less suitable alternative. The enantiomeric excess of recovered homochiral esters was determined by chiral chromatography using modified cellulose stationary phases (OB, OD). © 1993 Wiley-Liss, Inc.     

Crystallographic studies for the chiral recognition of the novel chiral stationary phase derived from (+)‐(R)‐18‐crown‐6 tetracarboxylic acid

Chiral discrimination observed in high‐performance liquid chromatography (HPLC) with the novel chiral stationary phase (CSP‐18C6I) derived from (+)‐(R)‐18‐crown‐6 tetracarboxylic acid [(+)‐18C6H₄] was investigated by X‐ray crystallographic analysis of the complex composed of the R‐enantiomer of 1‐(1‐naphthyl)ethylamine (1‐NEA) and (+)‐18C6H₄. Mixtures of 1‐NEA (the R‐ or S‐enantiomer) and (+)‐18C6H₄ were dissolved in methanol‐water (1:1) solution and allowed to stand for crystallization. The R‐enantiomer crystallized with (+)‐18C6H₄ as a co‐crystal, although the S‐enantiomer did not. This result was in good agreement with the enantiomer elution order of 1‐NEA in CSP‐18C6I. The apparent binding constants (K_a) of the enantiomers to the (+)‐18C6H₄ obtained from ¹H‐NMR experiments also supported the above‐mentioned result. The X‐ray crystal structure of the 1:1 complex of the R‐enantiomer and (+)‐18C6H₄ indicated the four sets of hydrogen bond association between the naphthylethylammonium cation and oxygen of polyether ring or carbonyl group of (+)‐18C6H₄. Chirality 11:173–178, 1999. © 1999 Wiley‐Liss, Inc.     

Field response of the northern spruce bark beetle Ips duplicatus (Sahlberg) (Coleoptera: Curculionidae,Scolytinae) to different combinations of synthetic pheromone with (−)‐α‐pinene and (+)‐limonene

相似文献   

Enantioselective toxic effects of hexaconazole enantiomers against Scenedesmus obliquus

Enantioselectivity in ecotoxicity of chiral pesticides in the aquatic environment has been a subject of growing interest. In this study, the toxicological impacts of hexaconazole enantiomers were investigated with freshwater algae Scenedesmus obliquus. After 96 h of exposure, the EC₅₀ values for rac‐hexaconazole, (+)‐hexaconazole, and (?)‐hexaconazole were 0.178, 0.355, and 0.065 mg l^?1, respectively. Therefore, the acute toxicities of hexaconazole enantiomers were enantioselective. In addition, the different toxic effects were evaluated when S. obliquus were exposed to 0.2, 0.5, and 1.0 mg l^?1 of rac‐hexaconazole, (+)‐hexaconazole, and (?)‐hexaconazole during 96 h, respectively. The chlorophyll a and chlorophyll b contents of S. obliquus treated by (?)‐hexaconazole were lower than those exposed to (+)‐hexaconazole, whereas the malondialdehyde contents of S. obliquus treated by (?)‐form were higher than those exposed to (+)‐form at higher concentrations. In general, catalase activities were significantly upregulated by exposure to (?)‐enantiomer than (+)‐enantiomer at all three concentrations. However, superoxide dismutase activities exposed to (?)‐hexaconazole were lower than that exposed to (+)‐hexaconazole at 0.2 mg l^?1 and 0.5 mg l^?1. On the basis of these data, the acute toxicity and toxic effects of hexaconazole against S. obliquus were enantioselective, and such enantiomeric differences must be taken into consideration in pesticide risk assessment. Chirality 24:610–614, 2012. © 2012 Wiley Periodicals, Inc.     

Asymmetric epoxidation of cinnamyl alcohol with optically active titanium complexes

A family of titanium(IV) alkoxo compounds [{Ti(O‐i‐Pr)₂(OR)₂}₂] 1–4 prepared by alcohol exchange of Ti(O‐i‐Pr)₄ and a chiral higher‐boiling alcohol [ROH = 1,2:3,4‐di‐O‐isopropylidene‐α‐d ‐galactopyranose, 1,2:5,6‐di‐O‐isopropylidene‐α‐d ‐glucofuranose, (1R,2S,5R)‐(?)‐menthol, (1S‐endo)‐(?)‐borneol, (1S,2R,5S)‐(+)‐menthol, and (+)‐borneol] has been tested to evaluate their catalytic activity and stereoselectivity in the asymmetric epoxidation of cinnamyl alcohol. © 2005 Wiley‐Liss, Inc. Chirality     

Influence of N‐hexane inhalation on the enantioselective pharmacokinetics and metabolism of verapamil in rats

Verapamil (VER) is commercialized as a racemic mixture of the (+)‐(R)‐VER and (?)‐(S)‐VER enantiomers. VER is biotransformed into norverapamil (NOR) and other metabolites through CYP‐dependent pathways. N‐hexane is a solvent that can alter the metabolism of CYP‐dependent drugs. The present study investigated the influence of n‐hexane (nose‐only inhalation exposure chamber at concentrations of 88, 176, and 352 mg/m³) on the kinetic disposition of the (+)‐(R)‐VER, (?)‐(S)‐VER, (R)‐NOR and (S)‐NOR in rats treated with a single dose of racemic VER (10 mg/kg). VER and NOR enantiomers in rat plasma was analyzed by LC‐MS/MS (m/z = 441.3 > 165.5 for the NOR and m/z 455.3 > 165.5 for the VER enantiomers) using a Chiralpak® AD column. Pharmacokinetic analysis was performed using a monocompartmental model. The pharmacokinetics of VER was enantioselective in control rats, with higher plasma proportions of the (?)‐(S)‐VER eutomer (AUC^0?∞ = 250.8 vs. 120.4 ng/ml/h; P ≤ 0.05, Wilcoxon test). The (S)‐NOR metabolite was also found to accumulate in plasma of control animals, with an S/R AUC^0?∞ ratio of 1.5. The pharmacokinetic parameters AUC^0?∞, Cl/F, Vd/F, and t_1/2 obtained for VER and NOR enantiomers were not altered by nose‐only exposure to n‐hexane at concentrations of 88, 176, or 352 mg/m³ (P > 0.05, Kruskal‐Wallis test). However, the verapamil kinetic disposition was not enantioselective for the animals exposed to n‐hexane at concentrations equal to or higher than the TLV‐TWA. This finding is relevant considering that the (?)‐(S)‐VER eutomer is 10–20 times more potent than R‐(+)‐VER in terms of its chronotropic effect on atrioventricular conduction in rats and humans. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Microbial Resolution of (±)-1 and 2-Decalols

By microorganisms or esterase they produce, (±)-1 and 2-decalyl acetates were asymmetrically hydrolyzed to (?)-1-(R)-trans,cis-1-decalol (IIa), (+)-1-(S)-cis,cis-1-decalol (IIIb), (+)-1-(R)-cis,trans-1-decalol (IVa) and (+)-1-(S)-trans,trans-2-decalol (VIIb), (?)-cis,cis-2-decalol (IXb) with the acetates of their antipodes, whereas the axial acetates of (±)-decalols were scarecely hydrolyzed.     

Distribution of enantiomers of volatile organic compounds in selected fruit distillates

The enantiomer ratios of chiral volatile organic compounds in fruit distillates were determined by multidimensional gas chromatography using solid‐phase microextraction (SPME) as a sample treatment procedure. Linalool and its oxides, limonene, α‐terpineol, and nerolidol, were present at the highest concentration levels, while significantly lower amounts of β‐citronellol and lactones were found in the studied samples. However, almost all terpenoids mainly occur as a racemic or near‐racemic mixture; enantiomer distribution of some chiral organic compounds in fruit distillates correlated to a botanical origin. In particular, a significant enantiomeric excess of (R)‐linalool and (S)‐α‐terpineol was found only for pear brandy, and likewise the dominance (R)‐limonene and the second eluted enantiomer of nerolidol for Sorbus domestica and strawberry, respectively. The distribution of γ‐lactones stereoisomers was more nonspecific, with a general excess of the R‐enantiomer.     

Studies of Enantiomeric Degradation of the Triazole Fungicide Hexaconazole in Tomato,Cucumber, and Field Soil by Chiral Liquid Chromatography–Tandem Mass Spectrometry

Chiral pesticide enantiomers often show different bioactivity and toxicity; however, this property is usually ignored when evaluating their environmental and public health risks. Hexaconazole is a chiral fungicide used on a variety of crops for the control of many fungal diseases. This use provides opportunities for the pollution of food and soil. In this study, a sensitive and convenient chiral liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) method was developed and validated for measuring hexaconazole enantiomers in tomato, cucumber, and soil. Separation was by a reversed‐phase Chiralcel OD‐RH column, under isocratic conditions using a mixture of acetonitrile‐2 mM ammonium acetate in water (60/40, v/v) as the mobile phase at a flow rate of 0.4 mL/min. Parameters including the matrix effect, linearity, precision, accuracy and stability were undertaken. Then the proposed method was successfully applied to investigate the possible enantioselective degradation of rac‐hexaconazole in plants (tomato and cucumber) and soil under field conditions. The degradation of the two enantiomers of hexaconazole proved to be enantioselective and dependent on the media: The (+)‐enantiomer showed a faster degradation in plants, while the (?)‐enantiomer dissipated faster than the (+)‐form in field soil, resulting in relative enrichment of the opposite enantiomer. The results of this work demonstrate that both the environmental media and environmental conditions influenced the direction and rate of enantioselective degradation of hexaconazole. Chirality 25:160–169, 2013. © 2013 Wiley Periodicals, Inc.     

Cloning and characterization of α-L-arabinofuranosidase and bifunctional α-L-arabinopyranosidase/β-D-galactopyranosidase from Bifidobacterium longum H-1

Aims: This study focused on the cloning, expression and characterization of recombinant α‐l ‐arabinosidases from Bifidobacterium longum H‐1. Methods and Results: α‐l ‐Arabinofuranosidase (AfuB‐H1) and bifunctional α‐l ‐arabinopyranosidase/β‐d ‐galactosidase (Apy‐H1) from B. longum H‐1 were identified by Southern blotting, and their recombinant enzymes were overexpressed in Escherichia coli BL21 (DE3). Recombinant AfuB‐H1 (rAfuB‐H1) was purified by single‐step Ni²⁺‐affinity column chromatography, whereas recombinant Apy‐H1 (rApy‐H1) was purified by serial Q‐HP and Ni²⁺‐affinity column chromatography. Enzymatic properties and substrate specificities of the two enzymes were assessed, and their kinetic constants were calculated. According to the results, rAfuB‐H1 hydrolysed p‐nitrophenyl‐α‐l ‐arabinofuranoside (pNP‐αL‐Af) and ginsenoside Rc, but did not hydrolyse p‐nitrophenyl‐α‐l ‐arabinopyranoside (pNP‐αL‐Ap). On the other hand, rApy‐H1 hydrolysed pNP‐αL‐Ap, p‐nitrophenyl‐β‐d ‐galactopyranoside (pNP‐βD‐Ga) and ginsenoside Rb2. Conclusions: Ginsenoside‐metabolizing bifidobacterial rAfuB‐H1 and rApy‐H1 were successfully cloned, expressed, and characterized. rAfuB‐H1 specifically recognized the α‐l ‐arabinofuranoside, whereas rApy‐H1 had dual functions, that is, it could hydrolyse both β‐d ‐galactopyranoside and α‐l ‐arabinopyranoside. Significance and Impact of the Study: These findings suggest that the biochemical properties and substrate specificities of these recombinant enzymes differ from those of previously identified α‐l ‐arabinosidases from Bifidobacterium breve K‐110 and Clostridium cellulovorans.     

Stereoselective urinary excretion of bupivacaine and its metabolites during epidural infusion

A sensitive and efficient chiral assay for bupivacaine and its three principal metabolites desbutylbupivacaine, 4′‐hydroxybupivacaine, and 3′‐hydroxybupivacaine has been applied to urine from five male patients receiving postoperative epidural infusions of rac‐bupivacaine fentanyl over 60–120 hr. The fraction of the dose of bupivacaine (total dose 840–2093 mg) accounted for in urine was 75 ± 6%. The rate of excretion of bupivacaine enantiomers approximated a steady state after ∼30 hr with values of 1.27 ± 0.26 and 0.76 ± 0.13 mg hr⁻¹ for (R)‐ and (S)‐enantiomers, respectively. The fraction of the dose of bupivacaine enantiomer excreted unchanged in the urine (fe) varied from 14.3% to 39.1% for (+)‐(R)‐bupivacaine and 9.2% to 14.0% for (−)‐(S)‐bupivacaine in the five patients. The rate of excretion of all metabolites also reached a steady state after ∼30 hr and the relative amounts of metabolites excreted into urine (fm) suggest bupivacaine is subject to regioselective and stereoselective clearance, which may vary from patient to patient. Chirality 11:50–55, 1999. © 1999 Wiley‐Liss, Inc.