Immunoaffinity extraction for the high-performance liquid chromatographic determination of equilin and its metabolites in plasma has been achieved. The antibody raised against an equilin 3-O-carboxymethyl ether-bovine serum albumin conjugate was characterized as having a high affinity for equilin and equilenin. One mL of the immunoaffinity adsorbent prepared by immobilization of an antibody was capable of retaining up to 1 microgram of equilin and equilenin, to 100 ng of other metabolites including 2-methoxylated and 17 beta-reduced compounds, and to 0.3 micrograms of glucuronic acid conjugates at C-3. The adsorbates were recovered qualitatively by elution with 90% aqueous (v/v) methanol without any interfering peaks on the chromatogram.

Chromatography, Affinity↗

PubMedDate not supplied

Separation and detection of bile acid 3-glucuronides in human urine by liquid chromatography/electrospray ionization-mass spectrometry.

A method for the separation and detection of bile acid 3-glucuronides by liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) has been developed. On the ESI mode, glucuronides were characterized by an intense pseudo-molecular ion [M-h]- with a doubly charged ion [M-2H]2-, and the ratio of these negative ions were markedly influenced by an acidic component of salt added to a mobile phase, according to a pKa value of an acidic moiety at C-24. Bile acid 3-glucuronides in human urine were extracted with a SepPak C18 cartridge, followed by purification on lipophylic ion exchange gel, piperidinohydroxypropyl Sephadex LH-20. Subsequent resolution into unconjugated, glycine and taurine conjugated bile acid 3-glucuronides was attained by LC on a Develosil ODS-HG-5 column using 20 mM ammonium acetate (pH 7.0)-methanol (10:12, v/v) as a mobile phase, where 3-glucuronides were monitored with a characteristic negative ion [M-H]-. The newly developed method was applied to the quantitatiation of bile acid 3-glucuronides in human urine with satisfactory sensitivity and reliability.

Bile Acids and Salts↗

PubMedDate not supplied

Separatory determination of diastereomeric ibuprofen glucuronides in human urine by liquid chromatography/electrospray ionization-mass spectrometry.

A method for the separatory determination of diastereomeric isomers of glucuronic acid conjugates of ibuprofen having a carboxyl group at the chiral center by liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) has been developed. The authentic specimens of acyl glucuronides of R(-)- and S(+)-ibuprofen were chemically synthesized by the Mitsunobu reaction. In the ESI mode, the glucuronides were characterized by an abundant quasi-molecular ion [M-H]-, and the formation of the negative ion was markedly influenced by a drift voltage. The resolution of diastereomeric isomers was achieved on a Develosil ODS-HG-5 column with 20 mM ammonium acetate (pH 5.0):acetonitrile (5:2, v/v) as a mobile phase where diastereomers were monitored with a corresponding quasi-molecular ion. After oral administration of racemic ibuprofen, a preferential excretion of (S)-ibuprofen glucuronide into the urine was observed.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMedDate not supplied

Preparation of glycine-conjugated bile acids and their gas/liquid chromatographic analysis on an aluminum-clad flexible fused silica capillary column.

This paper describes a method for the direct gas/liquid chromatographic (GC) analysis of 46 glycine-conjugated bile acids, which differ from one another in the number, position and configuration of the hydroxyl groups at positions C-2, C-3, C-4, C-6, C-7 and/or C-12. Free bile acids were converted quantitatively on a micro scale to ethyl ester-trimethylsilyl (Et-TMS) and methyl ester-dimethylethylsilyl (Me-DMES) ether derivatives of the corresponding glycine conjugates. The Et-TMS and Me-DMES ethers of the glycine conjugates were chromatographed on an aluminum-clad flexible fused silica capillary column coated with a thin film (0.1 micron) of chemically bonded and cross-linked methylpolysiloxane. Relative retention time (RRT) and methylene unit (MU) values were determined for the 46 compounds and their GC behaviour was discussed. The derivatization procedure and the retention data would be useful for the direct GC identification of unknown glycine-conjugated bile acid mixtures extracted from biological samples.

Bile Acids and Salts↗

PubMedDate not supplied

Study of three intragenic polymorphisms in the Machado-Joseph disease gene (MJD1) in relation to genetic instability of the (CAG)n tract.

Intergenerational instability is one of the most important features of the disease-associated trinucleotide expansions, leading to variation in size of the repeat among and within families, which manifests as variable age at onset and severity, and is probably the basis for the occurrence of anticipation. Several factors are known to affect the degree of instability, namely the type of repeated sequence, its initial size, the presence or absence of interruptions in the repetitive tract and the gender of the transmitting parent. A recent study demonstrated the effect of an intragenic polymorphism (C987GG/G987GG) in the Machado-Joseph disease causative gene, immediately downstream of the CAG repeat, on the intergenerational instability of the expanded repeat. Surprisingly, there was an effect not only of the specific allele in cis to the disease chromosome, but also of the allele on the normal chromosome, suggesting the existence of an interaction between the normal and expanded alleles that affects the fidelity of replication of the (CAG)n tract. This effect could be a direct effect of the polymorphism studied or, alternatively, this polymorphism could be in disequilibrium with some other flanking sequence which affects the instability of the repetitive (CAG)n tract. In order to confirm the previous results in a different population and to distinguish between a direct and indirect effect of the CGG/GGG polymorphism, we typed 70 parent-progeny pairs for which the variation in the (CAG)n length in the MJD1 gene was known, for three intragenic polymorphisms: C987GG/G987GG and two additional, newly described ones, TAA1118/TAC1118 and A669TG/G669TG. We also typed a control population of 125 individuals for the A669TG/G669TG, C987GG/G987GG and TAA1118/TAC1118 polymorphisms, in an attempt to identify any association between haplotype and (CAG)n length in normal chromosomes, suggestive of an instability-predisposing effect of the repeat-flanking sequences, which could have led to the origin of the MJD mutation in the human population. We confirmed the effect of the C987GG/G987GG polymorphism on intergenerational instability when present in trans. Our results suggest that this effect is restricted to a small region of the gene, immediately downstream of the CAG repeat, which includes this particular nucleotide substitution and the stop codon of the MJD1 cDNA, and is not a more widespread chromosomal effect. The lack of a significant association of any specific intragenic haplotype with larger CAG repeats in normal chromosomes, together with the absence of an effect of the intragenic haplotype in cis on the intergenerational instability of the expanded (CAG)n in MJD families does not indicate the existence of an instability-predisposing haplotype.

Alleles↗

PubMedDate not supplied

Genetic variation in ABCB1 influences paclitaxel pharmacokinetics in Japanese patients with ovarian cancer.

Paclitaxel, an antineoplastic agent used for the treatment of ovarian cancer, is metabolized by cytochrome P450 (CYP)3A4 and CYP2C8 and is excreted from cells by ATP-binding cassette (ABCB1) (multi-drug resistance [MDR1], P-glycoprotein). Expression of these proteins is regulated by pregnane X receptor (PXR). Although there are common genetic polymorphisms in the genes encoding these proteins, their effect on the clinical efficacy of paclitaxel is unclear. We therefore examined the relationship of the paclitaxel pharmacokinetics in 13 patients with ovarian cancer to polymorphisms in CYP2C8, CYP3A5, ABCB1, and PXR. We found high interindividual variability in the plasma concentrations of two metabolites, 6alpha-hydroxypaclitaxel and p-3'-hydroxypaclitaxel. All the patients were genotyped as CYP2C8*1/*1. Neither the CYP3A5 A6986G (CYP3A5*3) nor the PXR C-25385T alleles were associated with altered plasma concentrations of paclitaxel and its metabolites. ABCB1 T-129C, T1236C, and G2677(A,T), however, was associated with lower area under the plasma concentration-time curve (AUC) of paclitaxel. We also observed a significant correlation between the AUC (r=-0.721) or the total clearance of paclitaxel (CL(tot)) (r= 0.673) and the ABCB1 mutant allele dosage in each patient. Taken together, our findings suggest that interindividual variability in paclitaxel pharmacokinetics could be predicted by ABCB1 genotyping.

ATP Binding Cassette Transporter, Subfamily B↗

PubMedDate not supplied

Brain natriuretic peptide is a sensitive indicator of impaired left-ventricular function in elderly patients with cardiovascular disease.

We studied the relation between the plasma concentration of brain natriuretic peptide (BNP) and echocardiographic findings to determine the sensitivity of BNP as an indicator of left-ventricular dysfunction in elderly patients with various cardiovascular diseases. The plasma concentration of BNP was positively correlated with left-ventricular end-diastolic and end-systolic dimensions (LVEDD and LVESD, respectively) and inversely correlated with the left-ventricular ejection fraction (LVEF) in patients with prior myocardial infarction, dilated cardiomyopathy, and valvular heart disease. The plasma concentration of BNP decreased significantly in association with an increase in LVEF and decreases in LVEDD and LVESD in patients with congestive heart failure following therapy. These observations indicate that the plasma concentration of BNP is a sensitive marker of impaired left-ventricular function in elderly patients with various cardiovascular diseases and may be useful for evaluating the improvement in left-ventricular function and the efficacy of therapy in patients with congestive heart failure.

Aged↗

PubMedDate not supplied

Three cases of bronchial asthma after middle ages associated with pets.

Three cases of bronchial asthma after middle ages associated with pets were presented. Case 1 was a 39-year-old man who was admitted due to exacerbation of asthma after having fed a cat and a dog for 2 years. IgE-RAST was positive for cat fur and house dust. Inhalation test of cat hair antigen was positive. His symptoms improved by avoidance of the cat. Case 2 was 54-year-old woman suffering from asthma for 6 years after having fed a cat. IgE-RAST was positive for dog hair and cat fur. Symptoms did not improve by any treatment because she could not give up pets. Case 3 was a 45-year-old woman suffering from asthma attacks for two months. She had kept a dog for 1 year. IgE-RAST was positive for dog hair. Her symptoms disappeared by avoidance of the dog. Patients with pet allergy have increased in recent years because of the pet boom, but it is not so common to have asthma attacks in aged people. Bronchial asthma caused by allergens of pets easily improves by avoidance of them and it is important to establish early diagnosis of this disorder.

Adult↗

PubMedDate not supplied

Slowly progressive dementia and multiple cerebral cortical infarctions following mitral valve replacement.

We describe the clinicopathological findings of a woman, 83 years of age at the time of death, who demonstrated dementia and numerous cerebral infarctions. She had a history of repeated mitral valve replacements 15 and 13 years prior to death and showed a dull responsive state 1 month after the second operation. Thereafter, dementia manifested and slowly progressed. Brain computed tomography revealed cortical atrophy and ventricular dilatation. Histological examination revealed a large number of minute foci of infarction in the cerebral cortex. Such lesions may have developed in association with the valve replacement and resulted in progressive dementia.

Aged↗