2-Fluoro-beta-alanine, a previously unrecognized substrate for bile acid coenzyme A:amino acid:N-acyltransferase from human liver

Our laboratory has demonstrated recently that conjugates of 2-fluoro-beta-alanine (FBAL) and bile acids are the major biliary metabolites of 5-fluorouracil (FUra) in cancer patients. Bile acids are normally conjugated with glycine or taurine, and therefore the identification of the FBAL-bile acid conjugates suggested that FBAL may also be a substrate for the bile acid conjugating enzyme, bile acid CoA:amino acid:N-acyltransferase. Enzyme activity detected using glycine and taurine as substrates was purified 8-fold from human liver cytosol using a DEAE-cellulose column. This preparation when tested for its activity towards beta-alanine and FBAL using cholyl CoA as the bile acid substrate only catalyzed the formation of FBAL-cholate. beta-Alanine was not a substrate. Confirmation of FBAL-cholate as the enzymatic product was demonstrated by (1) coelution of the product of this reaction on HPLC with authentic FBAL-cholate, (2) specific hydrolysis of this product by cholylglycine hydrolase, and (3) molecular weight of the product (497) being identical to that of the authentic FBAL-cholate. Kinetic experiments demonstrated that the enzyme had an affinity for FBAL (Km 1.45 mM) comparable to taurine (Km 1.32 mM), but greater than glycine (Km 6.45 mM). Formation of FBAL-cholate was inhibited competitively by taurine (Ki 1.27 mM) and glycine (Ki 4.47 mM), suggesting that a single enzyme is responsible for conjugation of glycine, taurine and FBAL with bile acids. These data indicate that the formation of the FBAL-bile acid conjugates in patients receiving FUra results from high affinity of the bile acid conjugating enzyme for FBAL.     

Genetic polymorphism of CYP2D6 in the Japanese population

The frequencies of CYP2D6 mutations in a Japanese population were investigated. Individuals were classified into three groups: control individuals, cancer patients and Parkinsonians. Genotyping for CYP2D6*3, CYP2D6*4 and CYP2D6*18 was carried out using the polymerase chain reaction, and that for CYP2D6*5 was also carried out using XbaI restriction fragment length polymorphism. The frequencies of the CYP2D6*3, CYP2D6*4, CYP2D6*5 and CYP2D6*18 mutant alleles were 0%, 0.77%, 4.10% and 0.53% in more than 256 Japanese control individuals, respectively. Based on these data, the population frequency of the CYP2D6 poor metabolizer phenotype was estimated to be 0.29%. The distribution of the four mutated alleles was not significantly different between control individuals and cancer patients or Parkinsonians.     

A novel single nucleotide polymorphism of the human methylenetetrahydrofolate reductase gene in Japanese individuals

The genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) have been associated with increased toxicity of methotrexate (MTX), a folic acid antagonist that is widely used to treat cancer and immunosuppressive disorders such as rheumatoid arthritis. In this study, we analyzed all the exons and exon/intron junctions of the MTHFR gene from 200 Japanese individuals. We detected a novel single nucleotide polymorphism (SNP) 148C>T (Arg46Trp) in exon 1. The allele frequency of this polymorphism in the Japanese population appears to be extremely low (0.25%).     

Novel single nucleotide polymorphism of the CYP2A13 gene in Japanese individuals

Cytochrome P450 2A13 (CYP2A13) is a human CYP enzyme that is selectively expressed in the respiratory tract. It plays an active role in the metabolic activation of a tobacco-specific procarcinogen. In this study, the entire coding sequence and the exon-intron junctions of the CYP2A13 gene obtained from 395 Japanese individuals were screened for genetic polymorphisms. Eight genetic polymorphisms were found, of which seven gave rise to known variant alleles: CYP2A13*2, CYP2A13*3, CYP2A13*4, CYP2A13*6, and CYP2A13*7. We identified a novel single nucleotide polymorphism (SNP), 5792T>C, in exon 7 that caused an amino acid substitution (Ile331Thr). One of the 395 individuals included in the study was heterozygous for the variant allele, and therefore, the frequency of the allele in the study population was 0.13%.     

Genetic polymorphisms of Glycine N-acyltransferase (GLYAT) in a French Caucasian population

1. In humans, the glycine N-acyltransferase enzyme (GLYAT) is thought to be important in the detoxification of endogenous and xenobiotic compounds which contain a carboxylic acid group, such as benzoic, isovaleric, or acetylsalicylic acids.

2. The aim of this work was to report a comprehensive investigation of GLYAT genetic polymorphisms in DNA samples from 55 subjects of French Caucasian origin, using polymerase chain reaction–single-strand conformation polymorphism and sequencing strategies.

3. Seven different polymorphisms of the GLYAT gene were identified, including two polymorphisms in the 5′ flanking region of the gene (g.?8457C>T and g.?8010A>G), two polymorphisms in intron 5 (g.13931A>G and g.13944C>T) and three missense mutations in exon 2 (g.49T>A; p.Ser17Thr), exon 5 (g.13886A>G; p.Asn156Ser) and exon 6 (g.14435C>T; p.Arg199Cys). In addition to the wild-type allele GLYAT*1 (2.7%), four novel alleles were identified: GLYAT*2A (75.5%), *2B (4.5%), *3 (16.4%) and *4 (0.9%), and five different genotypes.

4. Localisation of the p.Ser17Thr and p.Arg199Cys missense mutations in predicted secondary structures suggest that these variants might have a potential role on the GLYAT protein activity. These results could be helpful in investigating the potential association of GLYAT variants with an incidence of reduced efficiency in xenobiotic carboxylic acids detoxification in humans.

     

Urinary bile acid and bile alcohol excretion does not reflect the genetic polymorphism of debrisoquine hydroxylation

Excretion of the major urinary bile alcohol 27-nor-5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24,25- pentol , and of cholic, chenodeoxycholic, deoxycholic and lithocholic acid was measured in 24 h urine collections of 10 extensive and seven poor metabolizers of debrisoquine. There was no significant difference of the excretion of these cholesterol metabolites between the two groups, indicating that cholesterol hydroxylation to bile alcohols and bile acids is probably not controlled by the same genes responsible for the 'debrisoquine-type' hydroxylation polymorphism.     

Genetic polymorphism of CYP2C19 and lansoprazole pharmacokinetics in Japanese subjects

Objective: We investigated whether interindividual differences in the pharmacokinetic disposition of lansoprazole are attributed to the genetic polymorphism of CYP2C19 which occurred by two mutations, CYP2C19m1 and CYP2C19m2, in 20 Japanese subjects. Methods: Polymerase chain reaction (PCR) restriction fragment length polymorphism procedures were used to detect the CYP2C19m1 mutation in exon 5 and the CYP2C19m2 mutation in exon 4 using SmaI and BamHI, respectively. Results: Ten subjects were homozygous (wt/wt subjects) for the wt allele in both exon 5 and exon 4, four subjects were heterozygous (wt/m1) for the CYP2C19m1 mutation, and two subjects were heterozygous (wt/m2) for the CYP2C19m2. The remaining four subjects had both mutated alleles in CYP2C19 genes, i.e., two were homozygous (m1/m1) for the defect in exon 5 and two were heterozygous (m1/m2) for the two defects in exons 5 and 4. The subjects in group 1 (wt/wt, wt/m1 and wt/m2) were the extensive metabolizers (EMs) for 5-hydroxylation of lansoprazole and were in the range of hydroxylation indexes from 3.83 to 19.8, whereas the subjects in group 2 (m1/m1 and m1/m2) were the poor metabolizers (PMs) and the indexes were in the range of 38.5 to 47.6. In group 2, AUC, t_1/2 and CL/f of lansoprazole were significantly greater, longer, and lower, respectively, than those in group 1. Conclusion: The hydroxylation of lansoprazole to 5-hydroxylansoprazole was apparently impaired in the subjects with the genetic defects of CYP2C19 (m1/m1 or m1/m2). Received: 12 November 1996 / Accepted in revised form: 18 February 1997     

Genetic polymorphisms of glycine N-acyltransferase (GLYAT) in a French Caucasian population

In humans, the glycine N-acyltransferase enzyme (GLYAT) is thought to be important in the detoxification of endogenous and xenobiotic compounds which contain a carboxylic acid group, such as benzoic, isovaleric, or acetylsalicylic acids. The aim of this work was to report a comprehensive investigation of GLYAT genetic polymorphisms in DNA samples from 55 subjects of French Caucasian origin, using polymerase chain reaction-single-strand conformation polymorphism and sequencing strategies. Seven different polymorphisms of the GLYAT gene were identified, including two polymorphisms in the 5' flanking region of the gene (g.-8457C>T and g.-8010A>G), two polymorphisms in intron 5 (g.13931A>G and g.13944C>T) and three missense mutations in exon 2 (g.49T>A; p.Ser17Thr), exon 5 (g.13886A>G; p.Asn156Ser) and exon 6 (g.14435C>T; p.Arg199Cys). In addition to the wild-type allele GLYAT*1 (2.7%), four novel alleles were identified: GLYAT*2A (75.5%), *2B (4.5%), *3 (16.4%) and *4 (0.9%), and five different genotypes. Localisation of the p.Ser17Thr and p.Arg199Cys missense mutations in predicted secondary structures suggest that these variants might have a potential role on the GLYAT protein activity. These results could be helpful in investigating the potential association of GLYAT variants with an incidence of reduced efficiency in xenobiotic carboxylic acids detoxification in humans.     

育龄女性基因多态性对补充叶酸降低HCY的影响

目的阐明MTHFRC677T、A1298C和MTRRA66G多态性在河南汉族育龄女性中的分布特征及其对补充叶酸降低HCY的影响。方法选取2011年7月至2013年4月在郑州大学第三附属医院进行优生咨询的育龄女性425名。荧光定量PCR检测MTHFRC677T、A1298C和MTRRA66G的基因多态性;化学发光法测定血叶酸浓度;循环酶法测定血HCY浓度。结果①河南地区汉族育龄女性中MTHFR677T、1298C和MTRR66G等位基因频率分别为62．1％、13．1％、23．7％。②补充叶酸前MTHFRC677T基因型叶酸浓度Tr〈CT〈CC,TT基因型HCY浓度远高于CC和CT基因型（P〈0．001）。400μg／d补充后HCY浓度迅速下降（TT〉CT〉CC）,但6．3％CT、16．9％TT基因型HCY浓度没有降低至正常范围。结论河南地区汉族育龄女性具有其他省份不同的遗传特征,MTHFRC677T各基因型对补充叶酸降低HCY的反应不同。     

Genetic polymorphism of UDP-glucuronosyltransferase 2B7 (UGT2B7) at amino acid 268: ethnic diversity of alleles and potential clinical significance

UGT2B7 catalyses the glucuronidation of a diverse range of drugs, environmental chemicals and endogenous compounds. Hence, coding region polymorphisms of UGT2B7 are potentially of pharmacological, toxicological and physiological significance. Two variant UGT2B7 cDNAs encoding enzymes with either His or Tyr at residue 268 have been isolated. The variants, referred to as UGT2B7*1 and UGT2B7*2, respectively, arise from a C to T transversion at nucleotide 802 of the UGT2B7 coding region. Analysis of genomic DNA from 91 unrelated Caucasians and 84 unrelated Japanese demonstrated the presence of the variant alleles encoding UGT2B7*1 and UGT2B7*2 in both populations. However, while there was an approximately equal distribution of subjects homozygous for each allele in the Caucasian population, subjects homozygous for the UGT2B7*1 allele were over 10-fold more prevalent than UGT2B7*2 homozygotes in Japanese. The frequencies of the UGT2B7*1 and UGT2B7*2 alleles were 0.511 and 0.489, respectively, in Caucasians, and 0.732 and 0.268, respectively, in Japanese. The 95% confidence intervals for the two alleles did not overlap between Caucasians and Japanese. Rates of microsomal androsterone, menthol and morphine (3-position) glucuronidation were determined for genotyped livers from Caucasian donors. Statistically significant inter-genotypic differences were not apparent for any of the three substrates. Although the UGT2B7 polymorphism characterized here is probably not associated with altered enzyme activity, the results highlight the need to consider ethnic variability in assessing the consequences of UGT polymorphisms.     

Effect of ursodeoxycholic acid administration on bile acid composition in hamster bile

The modification in the composition of bile acids in hamster by the administration of high dose of ursodeoxycholic acid (UDCA) was investigated. Male Golden Syrian hamsters were divided into five groups: a control group, two groups that received 0.5 g of UDCA per 100 g of standard diet during 30 and 60 days and another two groups that received 1 g of UDCA per 100 g of standard diet during 30 and 60 days. After ether anaesthesia the gallbladder was removed and bile was immediately aspirated. Bile acids were determined by high performance liquid chromatography (HPLC). Taurolithocholic (TLCA) and glycolithocholic acids (GLCA) increased significantly in all treated groups. The glyco/tauro ratio of 0.69 in controls became more than 1 in treated animals except in the case of lithocholic acid (LCA) conjugates which remained less than 1. UDCA derivatives increased proportionally to the administered dose and the cholic/cheno ratio diminished significantly. A moderate increase of 3- and 7-keto derivatives of chenodeoxycholic acid (CDCA) was observed in all treated groups but the above mentioned increment was especially evident in 3-keto derivatives. A high percentage of UDCA administered in the hamster was likely transformed to CDCA and the glyco conjugates of the bile acids were the predominant species except for the LCA derivatives.     

Interaction of 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T) with the acyl-CoA: amino acid N-acyltransferase enzymes of bovine liver mitochondria

The amino acid conjugation of the phenoxyherbicides 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T) by animals was examined at the level of the enzymes catalyzing the reactions. The phenoxyherbicides were not substrates for the bile acid conjugating system but were substrates for the mitochondrial xenobiotic conjugating system. The two mitochondrial xenobiotic-CoA: amino acid N-acyltransferases (benzoyltransferase and phenylacetyltransferase) were separated and tested for activity towards 2,4-D-CoA and 2,4,5-T-CoA. The phenylacetyltransferase showed activity towards phenylacetyl-CoA, phenoxyacetyl-CoA and 2,4-D-CoA, but not 2,4,5-T-CoA. Benzoyltransferase conjugated both 2,4-D-CoA and 2,4,5-T-CoA. The overall rates of conjugation of the phenoxyherbicides were slow relative to the standard substrates with both enzymes. This slow rate was found to be due in both cases to a relatively high Km for glycine, and a very slow catalytic rate constant. Both enzymes did, however, have a very high affinity for 2,4-D-CoA and 2,4,5-T-CoA so these compounds proved to be potent alternate substrate inhibitors of both enzymes. The data show that the inefficient in vivo conjugation of the phenoxyherbicides relative to structurally similar compounds can be understood in terms of the kinetic properties of the mitochondrial N-acyltransferases. Further, the potential for the interference of the phenoxyherbicides with the conjugation of other compounds is revealed.     

Review article: bile acid therapy

Over the past 3 years there has been a renewed interest in bile acid therapy not only because of the promising results obtained by combining this therapy with extracorporeal shock-wave lithotripsy for rapid gallstone dissolution, but also because of its novel use as a treatment for primary biliary cirrhosis. This article reviews the use of bile acids for both these indications.     

Genetic polymorphism of Japanese cultivated Rheum species in the internal transcribed spacer region of nuclear ribosomal DNA

Journal of Natural Medicines - In order to develop new domestic production of Rhei Rhizoma (RR) from Rheum specimens cultivated in the Sugadaira Medicinal Plant Cultivation Test Field (SMPCF), the...     

Role of the intestinal bile acid transporters in bile acid and drug disposition

Membrane transporters expressed by the hepatocyte and enterocyte play critical roles in maintaining the enterohepatic circulation of bile acids, an effective recycling and conservation mechanism that largely restricts these potentially cytotoxic detergents to the intestinal and hepatobiliary compartments. In doing so, the hepatic and enterocyte transport systems ensure a continuous supply of bile acids to be used repeatedly during the digestion of multiple meals throughout the day. Absorption of bile acids from the intestinal lumen and export into the portal circulation is mediated by a series of transporters expressed on the enterocyte apical and basolateral membranes. The ileal apical sodium-dependent bile acid cotransporter (abbreviated ASBT; gene symbol, SLC10A2) is responsible for the initial uptake of bile acids across the enterocyte brush border membrane. The bile acids are then efficiently shuttled across the cell and exported across the basolateral membrane by the heteromeric Organic Solute Transporter, OSTα-OSTβ. This chapter briefly reviews the tissue expression, physiology, genetics, pathophysiology, and transport properties of the ASBT and OSTα-OSTβ. In addition, the chapter discusses the relationship between the intestinal bile acid transporters and drug metabolism, including development of ASBT inhibitors as novel hypocholesterolemic or hepatoprotective agents, prodrug targeting of the ASBT to increase oral bioavailability, and involvement of the intestinal bile acid transporters in drug absorption and drug-drug interactions.     

Linuron and carbaryl differentially impair baseline amino acid and bile salt olfactory responses in three salmonids

For salmon, amino acid and bile salt detection form the basis for important behaviors including predator evasion and conspecific recognition, respectively. For this reason, decreases in olfactory sensory neuron responses to the amino acid l-serine and the bile salt taurocholic acid (TChA) have been used in studies as indicators of acute olfactory pesticide toxicity to environmental contaminants such as metals and pesticides. In this study, we first compare baseline responses to these two odorant classes across three salmonids, and then explore how two currently used pesticides alter these responses. We found baseline differences in electro-olfactogram (EOG) responses and their sensitivity to pesticide exposure between rainbow trout, coho and sockeye salmon. For example, rainbow trout had lower baseline EOGs than either coho or sockeye (e.g. 10(-5)M TChA EOGs of 1.34+/-0.17 versus 2.57+/-0.46 and 2.72+/-0.43 mV, respectively). At 15 min after exposure to 10 microg/L of the herbicide linuron, rainbow l-serine-evoked EOGs were 49.6% of control versus 78.5 and 69.8% for sockeye and coho, indicating rainbow were more sensitive to linuron. In contrast, at 30 min of exposure to 100 microg/L carbaryl, l-serine-evoked EOGs of sockeye were 49.7% of control versus 60.3 and 62.3% for rainbow and coho, suggesting sockeye were more sensitive to carbaryl. In all species the l-serine-evoked EOGs did not return to baseline by 15 min after 100 microg/L carbaryl exposure, suggesting persisting impairment of amino acid detection. The TChA-evoked EOGs were less affected by carbaryl exposure (i.e. EOGs were 83.3, 84.9 and 66.0% of control 15 min after exposure) and not affected at all by 100 microg/L linuron exposure. Species-specific differences in pesticide sensitivity may limit extrapolation of toxicity across salmonids while the generally greater sensitivity of amino acid olfaction may lead to selective impairment of behaviors such as predator evasion.     

Mesophase formation during cholesterol gallstone dissolution in human bile: effect of bile acid composition

Duodenal bile obtained from patients with gallstones who were acutely infused with chenodeoxycholic acid, ursodeoxycholic acid, or cholic acid were examined for the propensity toward the formation of a liquid crystalline mesomorphic phase when cholesterol gallstones were incubated in these bile acids. Bile taken from patients infused with ursodeoxycholic acid was found to be enriched in ursodeoxycholic acid; mesophase formation was detected in these samples but not in bile from patients infused with chenodeoxycholic acid or cholic acid.