Biomedical subjects

M Mizugaki

Publications and source records attributed to M Mizugaki.

At least 37 records · Page 2Linked to original sources

PubMed2000

Troglitazone has a reducing effect on thromboxane production.

Effects of troglitazone, an antidiabetic thiazolidinedione that enhances insulin sensitivity, on thromboxane (TX) production were assessed in human erythroleukemia (HEL) cells and human platelets. Measurement of TX was performed by using the gas chromatography/selected ion monitoring (GC/SIM) method. We found that troglitazone reduced the TX production from HEL cells and human platelets. Furthermore, troglitazone also reduced arachidonic acid (AA)-induced TX production from HEL cell and thrombin-induced TX release from platelets. In addition, we compared the effect of troglitazone with that of alpha-tocopherol and BRL 49653. Other thiazolidinedione compound BRL 49653 had effects similar to troglitazone, but alpha-tocopherol had no effect on TX production. Our findings suggest that the thiazolidinedione group had an antithrombotic effect and was beneficial in preventing vascular complications often observed in diabetes mellitus.

Arachidonic Acid↗

PubMed2000

Epigallocatechin gallate increase the prostacyclin production of bovine aortic endothelial cells.

We describe the effect of (-) epigallocatechin gallate (EGCg), one of catechins known in tea, on the prostacyclin (PGI) production by bovine aortic endothelial cells. The amounts of 6-keto-PGF(1alpha) and Delta(17)-6-keto-PGF(1alpha), stable metabolites of PGI(2) and PGI(3), released in culture medium were measured using gas chromatography/selected ion monitoring (GC/SIM). The prostacyclin production of endothelial cells was increased by EGCg in a dose- and time-dependent manner. The effect by EGCg was stronger than any other catechins (catechin, epicatechin, epigallocatechin, and epicatechin gallate). When endothelial cells incubated with EGCg and arachidonic acid (AA) or eicosapentaenoic acid (EPA), PGI(2), and PGI(3) production were increased greater than those incubated with AA or EPA alone. Furthermore, gallic acid, that also has a pyrogallol structure, increased PGI(2) production. These observations indicate that catechins increase the prostacyclin production and that the pyrogallol structure is significant to this function.

Animals↗

PubMed2000

Determination of urinary 8-epi-prostaglandin F(2alpha) using liquid chromatography-tandem mass spectrometry: increased excretion in diabetics.

Liquid chromatography-tandem mass spectrometry (LC/MS-MS) was applied to the quantitative analysis of urinary 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)) level. 8-Epi-PGF(2alpha) and its internal standard, [(2)H(4)]-8-epi-PGF(2alpha), were extracted from urine by using a solid phase extraction cartridge and loaded to LC/MS-MS in selected reaction monitoring (SRM) mode. The standard curve showed good linearity in the range of 40 pg to 10 ng (r = 0. 997). The accuracy of the added 8-epi-PGF(2alpha) ranged from 96.8 to 104.9% with a mean +/- SD of 99.5+/-2.5%. The average level +/- SD of urinary 8-epi-PGF(2alpha) in 13 healthy volunteers (five women and eight men, 31+/-7.4 years old) was 429.4+/-149.6 pg/mg creatinine. The level of seven patients with noninsulin dependent diabetes mellitus (two women and five men, 40+/-13.6 years old), 630.9+/-275.6 pg/mg creatinine, was statistically higher than that of healthy volunteers (P<0.05). This finding suggested that diabetics are in a highly oxidative condition. This simple and rapid LC/MS-MS method can be used to elucidate the pathophysiological feature of diabetes or for monitoring the curative effect.

Adult↗

PubMed2000

Difference in urinary LTE4 and 11-dehydro-TXB2 excretion in asthmatic patients.

Bronchoconstrictor cysteinyl leukotrienes (LT) and thromboxane (TX) A2 have been implicated in the pathogenesis of asthma. Determination of urinary leukotriene E4 (LTE4) and 11-dehydro-TXB2 levels are often used to assess cysteinyl LT and TXA2 production in humans. To define the potential role in the pathogenesis of asthma, we investigated the urinary LTE4 and 11-dehydro-TXB2 levels. LTE4 and 11-dehydro-TXB2 levels were determined using liquid chromatography/tandem mass spectrometry (LC/MS) and gas chromatography/mass spectrometry (GC/MS), respectively. Urinary LTE4 levels in asthmatic patients (192 +/- 122 pg/mg creatinine, n = 14) were significantly higher (P < 0.005) than those in healthy volunteers (55 +/- 16 pg/mg creatinine, n = 13), but no significant difference in 11-dehydro-TXB2 levels was observed. A significant inverse correlation (r = -0.821, P < 0.005) was found between urinary LTE4 levels and the forced expiratory volume in 1 s (FEV1) but no significant correlation was observed between urinary 11-dehydro-TXB2 levels and FEV1. The present findings suggest that cysteinyl LTs play a more important role in the pathogenesis of asthma than TXA2.

Adult↗

PubMed2000

Changes of the thromboxane A2/prostacyclin balance in the urine of patients with renal diseases analyzed by gas chromatography/selected ion monitoring.

The thromboxane A2/prostacyclin (TX/PGI) ratios were measured in patients with renal diseases to elucidate the relationship between the ratios and the pathological changes of the diseases. Urinary stable metabolites of thromboxane A2 and prostacyclin, 11-dehydro-thromboxane B2 and 2,3-dinor-6-keto-prostaglandin F1alpha, respectively, were converted to 1-methyl ester-propylamide-9,12,15-tris-dimethylisopropylsilyl ether derivative and 1-methyl ester-6-methoxime-9,12,15-tris-dimethylisopropylsilyl ether derivative, respectively, and applied to a gas chromatography/selected ion monitoring. The TX/PGI ratios of 10 outpatients and 6 inpatients with chronic glomerulonephritis were higher than those of 13 healthy volunteers. In an inpatient with systemic lupus erythematoides, the TX/PGI ratios were gradually lowered to the normal level with the therapies. Furthermore, the ratios seemed to change in advance of the changes of the levels of urinary protein and hematuria. These observations suggested that the TX/PGI ratio was a useful index to assess the pathological condition of renal diseases and the effects of treatment.

Adult↗

PubMed2000

Temporal changes of dopaminergic and glutamatergic receptors in 6-hydroxydopamine-treated rat brain.

Quantitative receptor autoradiography was used to examine the sequential patterns of changes in dopaminergic and glutamatergic receptors in the brain of rats lesioned with 6-hydroxydopamine. The animals were unilaterally lesioned in the medial forebrain bundle and the brains were analyzed at 1, 2, 4 and 8 weeks of postlesion. Degeneration of the nigrostriatal pathway caused a significant increase in dopamine D(2) receptors in the ipsilateral striatum from 1 to 8 weeks of postlesion. In the ipsilateral substantia nigra (SN), a significant decrease in dopamine D(2) receptors was also observed from 1 to 8 weeks of postlesion. On the other hand, dopamine D(1) receptors were increased in the ipsilateral ventromedial striatum from 2 to 4 weeks of postlesion. In the ipsilateral SN, a transient increase in dopamine D(1) receptors was observed only 1 week after lesioning. However, other regions in both ipsilateral and contralateral sides showed no significant change in dopamine D(1) and D(2) receptors during postlesion except for a transient change in a few regions. N-Methyl-D-aspartate (NMDA) receptors showed no significant changes in all brain regions studied during the postlesion. In contrast, a transient increase in excitatory amino acid transport sites was observed only in the frontal cortex and ventromedial striatum of the ipsilateral side at 2 weeks of postlesion. However, glycine receptors showed a significant change in any brain areas of both ipsilateral and contralateral sides after lesioning. The change in the brain areas of contralateral side was more pronounced than that of ipsilateral side for glycine receptors. In addition, dopamine uptake sites showed a severe damage in the ipsilateral striatum from 1 to 8 weeks after lesioning. In the contralateral side, in contrast, no significant change in dopamine uptake sites was found in the striatum during the postlesion. These results indicate that unilateral injection of 6-hydroxydopamine in the medial forebrain bundle can cause a significant increase in dopamine D(1) and D(2) receptors in the striatum. The increase in dopamine D(2) receptors was more pronounced than that in dopamine D(1) receptors in the striatum after 6-hydroxydopamine treatment. In contrast, dopamine uptake sites showed a severe damage in the striatum during the postlesion. Furthermore, our results support the existence of dopamine D(2) receptors on the neurons of SN, but not dopamine D(1) receptors. For glutamatergic receptor system, the present study suggests that the changes in glycine receptors may be more susceptible to degeneration of nigrostriatal pathway than NMDA receptors and excitatory amino acid transport sites. Thus, our findings are of interest in relation of degeneration of the nigrostriatal pathway that occurs in Parkinson's disease

Animals↗

PubMed2000

Sequential changes of [H]forskolin, [H]cyclohexyladenosine and [H]PN200-110 binding sites in the brain of 6-hydroxydopamine-lesioned rats.

Receptor autoradiographic technique was studied to investigate sequential changes in adenylyl cyclase, adenosine A1 receptors and L-type calcium channels in the striatum and substantia nigra 1-8 weeks after unilateral 6-hydroxydopamine injection of the medial forebrain bundle in rats. [3H]Forskolin, [3H]cyclohexyladenosine (CHA) and [3H]PN200-110 were used to label adenylyl cyclase, adenosine A1 receptors and L-type calcium channels, respectively. The degeneration of the nigrostriatal pathway caused a significant increase in [3H]forskolin binding in the striatum of both the ipsilateral and contralateral sides from 2 to 4 weeks post-lesion. The ipsilateral substantia nigra showed a transient increase in [3H]forskolin binding 4 weeks post-lesion. In contrast, [3H]CHA binding showed no significant change in most brain areas after lesioning. On the other hand, a conspicuous decrease in [3H]PN200-110 binding was observed in the dorsolateral striatum of ipsilateral side 4 weeks post-lesion. Thereafter, the striatum of both the ipsilateral and contralateral sides showed a significant decrease in [3H]PN200-110 binding 8 weeks post-lesion. These results demonstrate that unilateral 6-hydroxydopamine into the medial forebrain bundle of rats can experimentally cause a significant increase in adenylyl cyclase binding sites in the striatum and substantia nigra, whereas no conspicuous change in adenosine A1 receptors is observed in these areas during post-lesion. In contrast, L-type calcium channels were progressively damaged in the striatum after unilateral 6-hydroxydopamine treatment. These findings suggest that adenylyl cyclase and calcium system may contribute to the degeneration processes of the dopaminergic neurones.

Adenosine↗

PubMed2000

Rapid detection of CYP2C18 genotypes by real-time fluorescence polymerase chain reaction.

In man, CYP2C19, a liver enzyme, plays an important role in the metabolism of several drugs. Mutation of the CYP2C19 gene results in a poor metaboliser phenotype. S-Mephenytoin hydroxylation genetic polymorphism is due to two mutations of the CYP2C19 gene, namely CYP2C19*2, located in exon 5, and CYP2C19*3, located in exon 4. CYP2C18 is also polymorphically expressed. The mutant alleles of this enzyme are CYP2C18m1, located in exon 2 and CYP2C18m2, located in the 5'-flanking region. We have developed an allele-specific TaqMan polymerase chain reaction (PCR) assay with which to detect CYP2C18 mutant alleles. This assay combines hybridization of the TaqMan probe and allele-specific amplification primers to the target DNA. The TaqMan probe is labelled with 6-carboxyfluorescein at the 5' end and 6-carboxytetramethylrhodamine together with a phosphate at the 3' end. Genotypes are separated according to the different threshold cycles of the wild type and mutant primers. We applied this procedure to DNA extracted from the blood or saliva of 144 healthy Japanese volunteers. The wt/wt, wt/m1, wt/m2, m1/m1, m1/m2 and m2/m2 genotypes of the CYP2C18 alleles detected by the assay were consistent with the results obtained from restriction enzyme cleavage. In accordance with a previous report, the genotypes of CYP2C18m1 and CYP2C18m2 coincided with those of CYP2C19*3 and CYP2C19*2, respectively. Therefore, detection of CYP2C18 mutant alleles also allows that of CYP2C19 mutant alleles. Among 19 poor metabolisers, eight showed the homozygous CYP2C19*2/CYP2C19*2, two the homozygous CYP2C19*3/CYP2C19*3 and nine the compound heterozygous CYP2C19*2/CYP2C19*3 genotype. We found the allele-specific TaqMan PCR assay rapid, simple and cost-effective, as well as suitable for high-throughput applications in a routine laboratory. This assay allows the fast and reliable detection of inherited disorders that might influence diagnosis and treatment.

Aryl Hydrocarbon Hydroxylases↗

PubMed2000

Detection assay of rare variants of the thiopurine methyltransferase gene by PCR-RFLP using a mismatch primer in a Japanese population.

Thiopurine methyltransferase (TPMT) catalyzes the metabolism of important drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. The identification and frequency distributions of several variant TPMT alleles (TPMT*2--*8) have been described recently in many ethnic groups. We have recently demonstrated that TPMT*3C is the most common allele in Japanese subjects; however, it remains to be elucidated whether TPMT*4--*8 variants also exist in Japanese subjects. To detect polymorphisms in the TPMT gene (TPMT*4--*8), we have developed a mismatch polymerase chain reaction and restriction fragment length polymorphism method and conducted a population study of Japanese subjects. Genotyping of these variant forms was carried out in 192 Japanese healthy volunteers. The TPMT*4, TPMT*5, TPMT*6, TPMT*7, and TPMT*8 variants were not detected in any of the samples analyzed. This study provides the first analysis of the TPMT*4--*8 variants in a sample of the Japanese population and indicates that TPMT*4--*8 variants do not occur or are rare alleles in this population.

Alleles↗

PubMed2000

High throughput detection of drug-metabolizing enzyme polymorphisms by allele-specific fluorogenic 5' nuclease chain reaction assay.

We have developed an allele-specific fluorogenic 5' nuclease chain reaction assay for detecting polymorphisms in the following human drug-metabolizing enzyme genes: CYP2C9 (CYP2C9*2 and *3), CYP2C19 (CYP2C19*2 and *3), CYP2D6 (CYP2D6*4, *10, *14, *18, and *21(C8)), N-acetyltransferase 2 (NAT2*5B, *6A, and *7B), thiopurine methyltransferase (TPMT*3C), and aldehyde dehydrogenase2 (ALDH2*2). This method is a marriage of two emerging technologies, the use of allele-specific amplification primers for target DNA and hybridization of the TaqMan probe. The TaqMan probe is labeled with both a fluorescent reporter dye and a quencher dye. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. All assays are performed using a single thermocycling protocol. This genotyping method is rapid and highly sensitive and yields a high throughput. It could be applied toward automated large-scale genotyping.

Aldehyde Dehydrogenase↗

PubMed2000

Determination of serum concentrations of glycyrrhizin in humans by semi-micro high-performance liquid chromatography after administration of a therapeutic dose.

A simple and sensitive semi-micro high-performance liquid chromatography (HPLC) was established for determining the serum levels of glycyrrhizin (GL) in humans. Butyl p-hydroxybenzoate was used as the internal standard and serum was deproteinized by methanol. The samples were separated on a Capcell Pak C18 UG120 column (150 x 1.5 mm i.d.; particle size, 5 microm). The detection limit of GL in serum was 100 ng/ml, which enables determination of serum levels of GL after administration of a therapeutic dose. The time-course study suggested that the elimination rate of GL differed between subjects for the same administered dose, although the sample was too small to allow a meaningful comment. In clinical practice, GL is used for its antiviral and anti-inflammatory effects. Excessive administration of GL can induce pseudoaldosteronism; however the optimal GL concentration in serum remains to be determined. The determination method reported here is expected to aid in the safe and efficient use of the drug in clinical practice.

Adult↗

PubMed2000

[Drug Compliance Scale. I. Development of the Drug Compliance Scale].

The failure of patients to comply with treatment regimens recommended by their physicians is a significant clinical problem. Researches on the assessment of compliance have, however, been precluded by methodological difficulties such as lack of adequate measures. The purpose of this study was to develop a self-administered questionnaire to evaluate drug compliance. First, questionnaire containing a 52-items complied by two doctors, a pharmacist and a nurse, was tested on 81 outpatients, all volunteers, attending the departments of psychosomatic medicine and internal medicine. Four items were temporarily removed for later analysis because they directly inquired about drug compliance (drug compliance items). The other 48 items were analyzed and three factors consisting of 26 items were further studied: expectation on taking medicine, rejection to taking medicine and seeking knowledge of drugs. Chronback's alpha coefficients representing internal consistency of the three factors were sufficiently high (ranging from .75 to .84). Furthermore, we preformed a simplified pill count to validate the 4 drug compliance items. There was a weak to moderate correlation between the result of pill count and each of 4 drug compliance items. A new self-administered questionnaire of 30 items was thus developed and named the Drug Compliance Scale.

Drug Administration Schedule↗

PubMed2000

[Drug Compliance Scale. II. Psychological factors affecting drug compliance in the department of psychosomatic medicine].

The purpose of this study was to investigate psychological factors affecting drug compliance in the department of psychosomatic medicine. Seventy-four outpatients were asked to answer a battery of self-administered questionnaire including the Drug Compliance Scale (DCS) that we had recently developed and other questionnaire evaluating psychological and vegetative symptoms, self-efficacy and attributional styles on the promotion of health and personality closely related to interpersonal relationships. Results of path analysis indicated that attributional styles and self-efficacy mainly affected three factors of DCS such as expectation on taking medicine, rejection to taking medicine and seeking knowledge of drugs, through which they influenced drug compliance, and also indicated that personality and self-efficacy mainly affected the stability of mood state, suggesting a further influence on drug compliance.

Adolescent↗

PubMed2000

[Microdetermination of eicosanoids using mass spectrometry].

Prostaglandins (PGs), thromboxanes (TXs) and leukotrienes (LTs), named eicosanoids, are derived from polyunsaturated fatty acids such as arachidonic and eicosapentaenoic acids. Eicosanoids have various physiological actions, and they are closely associated with various pathological conditions. Quantitative analysis of eicosanoids production, therefore, may be useful as an index of pathological states and medical therapeutic effects. Quantitative methods require high sensitivity and selectivity because of the low concentration, short half-life time of their active compounds and coexistence of structurally similar interfering compounds. From this view point, gas chromatography/mass spectrometry (GC/MS) is one of the useful techniques for determination of eicosanoids. Using GC/MS, we have developed determination methods for various eicosanoids and applied them to clinical studies. Although GC/MS is highly sensitive and selective, it often requires tedious and time-consuming derivatization and purification procedures. Liquid chromatography/mass spectrometry (LC/MS), on the other hand, is a simple and rapid determination method. LC/MS is sufficiently applicable to determination of prostanoids, and we previously established a new determination method for LTE4. In the present paper, I describe an advanced determination method for 11-dehydro TXB2, a stable urinary metabolite of TXA2, using liquid chromatography/tandem mass spectrometry (LC/MS-MS).

Chromatography, Liquid↗

PubMed1999

Determination of leukotriene E4 in human urine using liquid chromatography-tandem mass spectrometry.

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method was developed for the quantitation of urinary leukotriene E4 (LTE4). LTE4 and its internal standard were extracted by solid-phase extraction and analysed using LC-MS-MS in the selected reaction monitoring (SRM) mode. A good linear response over the range of 10 pg to 10 ng was demonstrated. The accuracy of added LTE4 ranged from 97.0% to 108.0% with a mean and SD of 100.6+/-2.4%. We detected LTE4 (63.1+/-18.7 pg/mg creatinine, n=10) in healthy human urine. This method can be used to determine LTE4 in biological samples.

Adult↗

PubMed1999

Anti-P-selectin antibody attenuates rat brain ischemic injury.

We examined a protective effect of anti-P-selectin monoclonal antibody against rat ischemic brain injury with 24 h of middle cerebral artery occlusion (MCAO). Anti-rat P-selectin monoclonal antibody, was intravenously injected at a dose of 1 mg/kg at 5 min before MCAO. Control animals received the same volume of vehicle. MCAO was accomplished by an insertion of nylon thread with silicone coating for 24 h. Application of anti-P-selectin antibody significantly reduced infarct size and brain water content at 24 h of MCAO. Although leukocyte infiltration was not normally detected, it became remarkably evident at 1 day of MCAO. However, treatment with ARP 2-4 significantly reduced the number of leukocytes. These results demonstrated that administration of monoclonal antibody against P-selectin attenuated infarct size and brain edema, which was associated with reduction of leukocyte infiltration.

Animals↗

PubMed1999

Application of a recombinant Fab fragment from a phage display library for sensitive detection of a target antigen by an inhibition ELISA system.

We have found that the recombinant Fab (rFab) produced by phage display system was detectable for a target antigen more sensitive than the parental monoclonal antibody (MoAb). The Fab phage display library was constructed from hybridoma cells producing APU-6 MoAb specific for a modified nucleoside, pseudouridine that have been studied as a urinary marker for malignancy. Fab-displayed phage clones were screened by a direct ELISA, and the single positive clone was finally obtained. Although the reaction pattern of rFab against pseudouridine and uridine was almost identical to that of MoAb, detection sensitivity of rFab was approximately 30 times higher than that of MoAb. Since the sensitivity of rFab was almost identical to that of Fab fragment prepared by papain digestion of MoAb, the increased sensitivity is considered to be the nature of Fab fragment. The sensitivity of established assay system was sufficient for quantitative determination of serum pseudouridine levels in healthy individuals and cancer patients. This procedure may be applicable for improvement of detection sensitivity of a MoAb-based inhibition ELISA system for drugs or low molecular weight compounds.

Amino Acid Sequence↗

PubMed1999

Rapid detection of CYP2C9*3 alleles by real-time fluorescence PCR based on SYBR Green.

CYP2C9 catalyzes the metabolism of important drugs such as phenytoin, S-warfarin, tolbutamide, losartan, and nonsteroidal anti-inflammatory drugs. A functional polymorphism of the CYP2C9 gene has been described. The single-base mutation of A1061C (Ile359Leu) in the CYP2C9 gene termed CYP2C9*3 was found at a frequency of about 2.1% in Japanese. We developed a rapid mutation analysis method for detecting the CYP2C9*1 genotype. This method is a marriage of two emerging technologies: allele-specific amplification primers for target DNA and a new double-stranded DNA-selective fluorescent dye, SYBR Green. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. We applied this procedure to DNA extracted from the blood of healthy Japanese volunteers. The CYP2C9 wild-type CYP2C9*1/CYP2C9*1 and heterozygous CYP2C9*1/CYP2C9*3 genotypes of the CYP2C9 alleles detected by the assay were consistent with the results obtained from restriction enzyme cleavage. No genotype of CYP2C9*3/CYP2C9*3 was found in these samples. Using plasmid DNA containing a point mutation of CYP2C9*3 as template, the assay separated the three genotypes. We conclude that this simple, rapid, and inexpensive procedure is applicable to routine high-throughput assays.

Alleles↗