Enantioselective determination of metoprolol acidic metabolite in plasma and urine using liquid chromatography chiral columns: applications to pharmacokinetics

Enantioselective separations on chiral stationary phases with or without derivatization were developed and compared for the HPLC analysis of (+)-(R)- and (-)-(S)-metoprolol acidic metabolite in human plasma and urine. The enantiomers were analysed in plasma and urine without derivatization on a Chiralcel OD-R column, and in urine after derivatization using methanol in acidic medium on a Chiralcel OD-H column. The quantitation limits were 17 ng of each enantiomer/ml plasma and 0.5 microgram of each enantiomer/ml urine using both methods. The confident limits show that the methods are compatible with pharmacokinetic investigations of the enantioselective metabolism of metoprolol. The methods were employed in a metabolism study of racemic metoprolol administered to a patient phenotyped as an extensive metabolizer of debrisoquine. The enantiomeric ratio (+)-(R)/(-)-(S)-acid metabolite was 1.1 for plasma and 1.2 for urine. Clearances were 0.41 and 0.25 l/h/kg, respectively, for the (+)-(R)- and (-)-(S)-enantiomers. The correlation coefficients between the urine concentrations of the acid metabolite enantiomers obtained by the two methods were >0.99. The two methods demonstrated interchangeable application to pharmacokinetics.     

Genomic divergence of zebu and taurine cattle identified through high-density SNP genotyping

Background

Natural selection has molded evolution across all taxa. At an arguable date of around 330,000 years ago there were already at least two different types of cattle that became ancestors of nearly all modern cattle, the Bos taurus taurus more adapted to temperate climates and the tropically adapted Bos taurus indicus. After domestication, human selection exponentially intensified these differences. To better understand the genetic differences between these subspecies and detect genomic regions potentially under divergent selection, animals from the International Bovine HapMap Experiment were genotyped for over 770,000 SNP across the genome and compared using smoothed F_ST. The taurine sample was represented by ten breeds and the contrasting zebu cohort by three breeds.

Results

Each cattle group evidenced similar numbers of polymorphic markers well distributed across the genome. Principal components analyses and unsupervised clustering confirmed the well-characterized main division of domestic cattle. The top 1% smoothed F_ST, potentially associated to positive selection, contained 48 genomic regions across 17 chromosomes. Nearly half of the top F_ST signals (n = 22) were previously detected using a lower density SNP assay. Amongst the strongest signals were the BTA7:~50 Mb and BTA14:~25 Mb; both regions harboring candidate genes and different patterns of linkage disequilibrium that potentially represent intrinsic differences between cattle types. The bottom 1% of the smoothed F_ST values, potentially associated to balancing selection, included 24 regions across 13 chromosomes. These regions often overlap with copy number variants, including the highly variable region at BTA23:~24 Mb that harbors a large number of MHC genes. Under these regions, 318 unique Ensembl genes are annotated with a significant overrepresentation of immune related pathways.

Conclusions

Genomic regions that are potentially linked to purifying or balancing selection processes in domestic cattle were identified. These regions are of particular interest to understand the natural and human selective pressures to which these subspecies were exposed to and how the genetic background of these populations evolved in response to environmental challenges and human manipulation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-876) contains supplementary material, which is available to authorized users.     

Assessing signatures of selection through variation in linkage disequilibrium between taurine and indicine cattle

Background

Signatures of selection are regions in the genome that have been preferentially increased in frequency and fixed in a population because of their functional importance in specific processes. These regions can be detected because of their lower genetic variability and specific regional linkage disequilibrium (LD) patterns.

Methods

By comparing the differences in regional LD variation between dairy and beef cattle types, and between indicine and taurine subspecies, we aim at finding signatures of selection for production and adaptation in cattle breeds. The VarLD method was applied to compare the LD variation in the autosomal genome between breeds, including Angus and Brown Swiss, representing taurine breeds, and Nelore and Gir, representing indicine breeds. Genomic regions containing the top 0.01 and 0.1 percentile of signals were characterized using the UMD3.1 Bos taurus genome assembly to identify genes in those regions and compared with previously reported selection signatures and regions with copy number variation.

Results

For all comparisons, the top 0.01 and 0.1 percentile included 26 and 165 signals and 17 and 125 genes, respectively, including TECRL, BT.23182 or FPPS, CAST, MYOM1, UVRAG and DNAJA1.

Conclusions

The VarLD method is a powerful tool to identify differences in linkage disequilibrium between cattle populations and putative signatures of selection with potential adaptive and productive importance.     

Stereoselective analysis of metoprolol and its metabolites in rat plasma with application to oxidative metabolism

We investigated the stereoselective kinetic disposition and metabolism of metoprolol (MET) in rats. The racemic MET (15 mg/kg) was given by oral gavage and blood samples were collected from 0 to 10h (n=6 at each time point). The enantiomeric concentrations of MET and its metabolites alpha-hydroxymetoprolol (alpha-OHM) and O-demethylmetoprolol (ODM) were determined by HPLC using a Chiralpak AD chiral column and fluorescence detection. The pharmacokinetic parameters of unchanged MET and the formation of ODM did not show to be stereoselective. In contrast, the AUC (ng h/mL) of alpha-hydroxymetoprolol isomers were higher to I'R [638.2(525.2-706.2) for 1'R2R and 659.6(580.4-698.1) for 1'R,2S, mean, (95%CI)] than to I'S products [58.3(47.4-66.1) for 1'S,2R and 57.1(44.7-67.9) for 1'S,2S, mean, (95%CI)]. We conclude that the kinetic disposition of unchanged MET and the formation of ODM are not enantioselective in rats but the metabolism of alpha-OHM yields predominantly the 1'R-product.     

Impact of temperature on Na2Sr(PO4)F:Eu3+ phosphor

In this article, we report the synthesis of Na₂Sr_1‐x(PO₄)F:Eu_x phosphor via a combustion method. The influence of different annealing temperatures on the photoluminescence properties was investigated. The phosphor was excited at both 254 and 393 nm. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors emit strong orange and red color at 593 and 612 nm, respectively, under both excitation wavelengths. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors annealed at 1050°C showed stronger emission intensity compared with 600, 900 and 1200°C. Moreover, Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphor was found to be more intense when compared with commercial Y₂O₃:Eu³⁺ phosphor. Copyright © 2013 John Wiley & Sons, Ltd.     

Influence of quinidine,cimetidine, and ketoconazole on the enantioselective pharmacokinetics and metabolism of metoprolol in rats

Metoprolol is a β‐blocker and its racemic mixture is used for the treatment of hypertension. In the present study we investigated the influence of CYP2D and CYP3A on the stereoselective metabolism of metoprolol in rats. Male Wistar rats (n = 6 per group) received racemic metoprolol (15 mg/kg) orally, with or without pretreatment with the CYP inhibitor ketoconazole (50 mg/kg), cimetidine (150 mg/kg), or quinidine (80 mg/kg). Blood samples were collected up to 48 h after metoprolol administration. The plasma concentrations of the stereoisomers of metoprolol, O‐demethylmetoprolol (ODM), α‐hydroxymetoprolol (OHM) (Chiralpak® AD column), and metoprolol acidic metabolite (AODM) (Chiralcel® OD‐R column) were determined by HPLC using fluorescence detection (λ_exc = 229 nm; λ_em = 298 nm). CYP3A inhibition by ketoconazole reduced the plasma concentrations of ODM and AODM and favored the formation of OHM. CYP2D and CYP3A inhibition by cimetidine reduced the plasma concentrations of OHM and AODM and favored the formation of ODM. The inhibition of CYP2D by quinidine reduced the plasma concentrations of OHM and favored the formation of ODM. In conclusion, the results suggest that CYP3A is involved in the formation of ODM and CYP2D is involved in the formation of AODM. Chirality 2009. © 2009 Wiley‐Liss, Inc.     

Reduction of enantioselectivity in the kinetic disposition and metabolism of verapamil in rats exposed to toluene

Toluene and verapamil are subject to extensive oxidative metabolism mediated by CYP enzymes, and their interaction can be stereoselective. In the present study we investigated the influence of toluene inhalation on the enantioselective kinetic disposition of verapamil and its metabolite, norverapamil, in rats. Male Wistar rats (n = 6 per group) received a single dose of racemic verapamil (10 mg/kg) orally at the fifth day of nose-only toluene or air (control group) inhalation for 6 h/day (25, 50, and 100 ppm). Serial blood samples were collected from the tail up to 6 h after verapamil administration. The plasma concentrations of verapamil and norverapamil enantiomers were analyzed by LC-MS/MS by using a Chiralpak AD column. Toluene inhalation did not influence the kinetic disposition of verapamil or norverapamil enantiomers (p > 0.05, Kruskal-Wallis test) in rats. The pharmacokinetics of verapamil was enantioselective in the control group, with a higher plasma proportion of the S-verapamil (AUC 250.8 versus 120.4 ng x h x mL(-1); p < or = 0.05, Wilcoxon test) and S-norverapamil (AUC 72.3 versus 52.3 ng x h x mL(-1); p < or = 0.05, Wilcoxon test). Nose-only exposure to toluene at 25, 50, or 100 ppm resulted in a lack of enantioselectivity for both verapamil and norverapamil. The study demonstrates the importance of the application of enantioselective methods in studies on the interaction between solvents and chiral drugs.     

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.     

Symmetric cyanine dyes for detecting nucleic acids

A novel approach to the design of sensitive fluorescent probes for nucleic acids detection is proposed. Suitable modifications of tri- and pentamethine cyanine dyes in the polymethine chain and/or in the heterocyclic residues can result in a significant decrease in unbound dye fluorescence intensity and an increase in dye emission intensity in the presence of DNA compared to the unsubstituted dye. The sharp enhancement in the fluorescence intensity upon dye interaction with double-stranded DNA permits the application of the modified tri- and pentamethine dyes as fluorescent probes in double-stranded DNA detection in homogeneous assays.     

Low levels of taurine introgression in the current Brazilian Nelore and Gir indicine cattle populations

Background

Nelore and Gir are the two most important indicine cattle breeds for production of beef and milk in Brazil. Historical records state that these breeds were introduced in Brazil from the Indian subcontinent, crossed to local taurine cattle in order to quickly increase the population size, and then backcrossed to the original breeds to recover indicine adaptive and productive traits. Previous investigations based on sparse DNA markers detected taurine admixture in these breeds. High-density genome-wide analyses can provide high-resolution information on the genetic composition of current Nelore and Gir populations, estimate more precisely the levels and nature of taurine introgression, and shed light on their history and the strategies that were used to expand these breeds.

Results

We used the high-density Illumina BovineHD BeadChip with more than 777 K single nucleotide polymorphisms (SNPs) that were reduced to 697 115 after quality control filtering to investigate the structure of Nelore and Gir populations and seven other worldwide populations for comparison. Multidimensional scaling and model-based ancestry estimation clearly separated the indicine, European taurine and African taurine ancestries. The average level of taurine introgression in the autosomal genome of Nelore and Gir breeds was less than 1% but was 9% for the Brahman breed. Analyses based on the mitochondrial SNPs present in the Illumina BovineHD BeadChip did not clearly differentiate taurine and indicine haplotype groupings.

Conclusions

The low level of taurine ancestry observed for both Nelore and Gir breeds confirms the historical records of crossbreeding and supports a strong directional selection against taurine haplotypes via backcrossing. Random sampling in production herds across the country and subsequent genotyping would be useful for a more complete view of the admixture levels in the commercial Nelore and Gir populations.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0109-5) contains supplementary material, which is available to authorized users.