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DNA methylation is involved in many diseases such as cancer and autoimmunity. We generated recombinant single-chain Fv (scFv) antibodies against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology and a hyperimmunized mouse, and the scFv of most interest were constructed as fusion proteins with green fluorescent protein obtained from Aequorea coerulescens GFP (AcGFP). Using RNA isolated from mouse spleens, we constructed a scFv library consisting of λ light chains. The scFv library was selected against m(5)Cyd-BSA and enriched through four rounds of panning. The scFv library was concentrated about 390-fold and an individual clone was reacted with m(5)Cyd-BSA. Two scFvs with high reactivity for m(5)Cyd-BSA termed 1-2 and 1-12 were produced. Furthermore, methylated DNA-binding activities of the scFvs were confirmed using an indirect immunofluorescence assay. Additionally, N- and C-terminal scFv 1-2 fusion with AcGFP were constructed, and we observed the N-terminal AcGFP exhibited much higher fluorescence intensity than the C-terminal fusions. The AcGFP-scFv 1-2 modified N-terminus of scFv with AcGFP had high fluorescence intensity, but the scFv 1-2-AcGFP modified C-terminus of scFv with AcGFP had low fluorescence intensity. The cross-reactivity of AcGFP-scFv 1-2 was similar to scFv 1-2, and thus, AcGFP-scFv 1-2 could be used in a direct immunofluorescence assay. The scFv fusion proteins may be useful for the detection and quantification of cellular methylated DNA in various specimens.

Thiopurine S-methyltransferase (TPMT) is an enzyme responsible for the detoxification of the widely used thiopurine drugs. TPMT is genetically polymorphic and is associated with large interindividual variations in thiopurine drug toxicity and therapeutic efficacy. In this study, we performed an in-vitro analysis of TPMT variant alleles, namely, TPMT*2, *3A, *3B, *3C, *5, *6, *7, *8, *9, *10, *11, *12, *13, *14, *16, *17, *18, *19, *20, *21, *22, *23, and *24.The wild-type TPMT proteins, TPMT.1 and 23 variants were heterologously expressed in COS-7 cells, and the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of 6-thioguanine S-methylation were determined.The expression levels of TPMT.2, TPMT.3A, TPMT.5, TPMT.12, TPMT.14, and TPMT.22 were considerably lower than that of TPMT.1 (P0.005), and that of TPMT.18 was slightly reduced (P0.05). The kinetic parameters of TPMT.3A, TPMT.3B, TPMT.5, TPMT.14, TPMT.18, TPMT.21, and TPMT.22 could not be accurately established because of no activity in 6-thioguanine S-methylation. The Vmax/Km values of TPMT.2, TPMT.7, TPMT.17, and TPMT.24 were displayed less than 10% of the wild-type.This functional analysis with respect to TPMT variants could provide useful information for individualization of thiopurine drugs therapy.

A novel human cytochrome P450, designated CYP2W1, has recently been identified and is found to be present mainly in tumor cells, particularly in colon cancer cells. In the present study, we report the first systematic investigation of polymorphisms in the human CYP2W1 gene. Based on denaturing high performance liquid chromatography analyses of polymerase chain reaction products, we analyzed nine exons and exon-intron junctions of the gene in DNA samples from 200 Japanese subjects and identified six single nucleotide polymorphisms (SNP). Three of the novel nonsynonymous SNPs were as follows: 173A>C (Glu58Ala) in exon 1 and 5432G>A (Val432Ile) and 5584G>C (Gln482His) in exon 9. Two previously known nonsynonymous SNPs, that is, 2008G>A (Ala181Thr) in exon 4 and 5601C>T (Pro488Leu) in exon 9, were also found. On haplotype analyses, in addition to the wild-type CYP2W1*1A (frequency, 0.295) allele, other alleles, namely, CYP2W1*1B (0.318), CYP2W1*2 (0.005), CYP2W1*3 (0.005), CYP2W1*4 (0.008), CYP2W1*5 (0.003), and CYP2W1*6 (0.368), were also characterized. The most common allele, CYP2W1*6, exhibited the amino acid substitution Pro488Leu. These results were in good agreement with the expected genotype distributions that were calculated using the Hardy-Weinberg equation. The data on variant alleles and comprehensive haplotype structures would be useful for predicting the metabolic phenotypes of CYP2W1 substrates in the Japanese population.

We analyzed all nine exons and exon-intron junctions of the CYP2S1 gene in 200 Japanese individuals and identified the following three novel single nucleotide polymorphisms (SNPs): 4612G>A (Glu147Glu) in exon 3, 5478C>T (Leu230Leu) and 5479T>G (Leu230Arg, CYP2S1*5A) in exon 5. The allele frequencies were 0.013 for 4612G>A, 0.058 for 5478C>T, and 0.003 for 5479T>G. In addition, a known SNP 1324C>G (Pro74Pro) was detected at a frequency of 0.300.

Individualization of drug therapy through genetic testing would maximize the effectiveness of medication and minimize its risks. Recent progress in genetic testing technologies has been remarkable, and they have been applied for the analysis of genetic polymorphisms that regulate drug responses. Clinical application of genetic information to individual health care requires simple and rapid identification of nucleotide changes in clinical settings. We previously reported a novel DNA diagnostic method for detecting single nucleotide polymorphisms (SNPs) using competitive allele-specific short oligonucleotide hybridization (CASSOH) with an immunochromatographic strip. We have developed the method further in order to incorporate an enzyme-linked immunosorbent assay (ELISA) into the final detection step; this enables multiple SNP detection. Special ELISA chips have been fabricated so that disposal of buffer waste is not required and handling procedures are minimized. This method (CASSOH-ELISA) has been successfully applied for the detection of clinically important SNPs in drug metabolism, such as N-acetyltransferase 2, NAT2*6 (590G>A) and NAT*7 (857G>A), and mitochondrial DNA (1555A>G). It would also facilitate point-of-care genetic testing for potentially diverse clinical applications.

W39F, F52Y, S98G, S98A, and S98C mutants of the neocarzinostatin apoprotein (apo-NCS) were newly prepared and investigated their physicochemical properties. The circular dichroism (CD) spectra of F78W, F52Y, S98A, S98G, S98C were superimposable with that of wild type 1R49 protein although the minor spectral change seemed to be in the ellipticity of W39F. The results suggest that position 52, 78, and 98 involving natural chromophore binding do not play a major role in the inducing overall structural changes of the protein. Conversely, the position 39 would be affected slightly. Ethidium bromide (EtdBr) binding to mutants was also evaluated by the monitoring of total fluorescence intensity and fluorescence polarization (FP). The observed dissociation constant in the FP study was 4.4 microM for wild type, 2.2 microM for S98A, 1.3 microM for S98G, 9.7 microM for S98C, respectively. When S98G and F52Y, the calculated maximum change of the total fluorescence intensity was increased, suggesting that the EtdBr binding to S98G or F52Y were slightly improved compared with the wild type. Then, a total of 14 amino acids randomly substituted phage displayed library of apo-NCS was successfully prepared, because substitution of the amino acid structured the chromophore-binding cavity were not change the overall structural features. The phages which bound glycyrrhetic acid conjugated bovine serum albumin were enriched from this library using phage display technique as the pilot experiments. Although more precision investigation still needs, it should be possible to select variants that have new functions not found in nature.

Summary: In this study, the entire coding sequence and the exon-intron junctions of the thiopurine S-methyltransferase (TPMT ) gene from 200 Japanese individuals were screened for mutation. Three novel single nucleotide polymorphisms (SNPs) were identified-106G > A in exon 3 (Gly36Ser, *20 allele), 967A > G in 3'-untranslated region, and -87C > T in intron 8. The allele frequencies were 0.003 for 106G > A, 0.003 for 967A > G, and 0.010 for IVS8 -87C > T. In addition, the three known SNPs, 474 T > C (Ile158Ile), 719A > G (Tyr240Cys, *3C allele), and IVS4 + 35C > T were detected at frequencies of 0.299, 0.010, and 0.421, respectively.

The cytochrome P450 enzymes (GYP) play important roles in the metabolism of many therapeutic agents and environmental compounds. Differences in the activities of these enzymes are thought to be responsible for individual variability in drug response and/or toxicity. Among the GYP enzymes, the isoform CYP4B1 is of particular interest and involved in the metabolism of several protoxins such as 2-aminoanthracene and 2-aminofluorene, all of which are bladder carcinogens. The present study compared the relative expression pattern of CYP4B1 in cDNA panels of human tissues. We developed an assay for CYP4B1 mRNA by performing real-time RT-PCR. Using this method, CYP4B1 mRNA levels were analyzed in human tissues. CYP4B1 mRNA was selectively expressed in lung, bladder, and prostate. These findings suggest that a high expression of CYP4B1 increases the risk of tumor by activation of carcinogenic compounds.

Human 2, 4-dienoyl-CoA reductase (DECR) is an auxiliary enzyme of β-oxidation, participates in the metabolism of unsaturated fatty enoyl-CoA esters having double bonds in both even- and odd-numbered position. DECR gene is approximately 30kb and comprises 10 exons. Four single nucleotide polymorphisms (SNPs) of human DECR have been reported on SNP database, but any SNPs of DECR in the Japanese population have not been determined. To establish a rapid and sensitive means of detecting DECR SNPs, we analyzed those of 200 Japanese individuals using denaturing HPLC (DHPLC). We detected a novel SNP 43441G>A (Ala295Thr) in exon 8. The allele frequency of this polymorphism in the Japanese population appears to be extremely low (0.5%).

Satratoxins, members of the trichothecene mycotoxin family, have been known to be harmful to health. However, the mechanisms underlying the toxicity still remain unclear. The present study is undertaken to elucidate the mechanisms of the satratoxin H-induced cytotoxicity in PC12 cells. Satratoxin H caused cytotoxicity, which was reflected from apoptosis determined by chromatin staining and flow cytometry. Satratoxin H stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Pre-incubation with SB203580, a p38 MAPK inhibitor, or SP600125, a JNK inhibitor, but not PD98059, an ERK inhibitor, reduced satratoxin-induced cytotoxicity. Co-incubation of cells with glutathione, N-acetyl-L-cysteine or glutathione reductase inhibited cytotoxicity and the phosphorylation of p38 MAPK induced by satratoxin H. Our data suggest that satratoxin H-induced apoptosis in PC12 cells is dependent on the activation of p38 MAPK/JNK and the increase in reactive oxygen species.

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%).

Thiazolidinedione, peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, has been used as an anti-diabetic drug and as an useful tool to elucidate multiple PPARgamma functions by in vitro and in vivo studies. We investigated the effects of thiazolidinediones on prostanoid production in lipopolysaccharide-stimulated cells. The high concentrations (>10 microM) of rosiglitazone and pioglitazone significantly increased lipopolysaccharide-stimulated prostanoid production such as thromboxane A2 and prostaglandin E2. However, PPARgamma antagonist could not inhibit them. In PPARgamma-deficient cells, thiazolidinediones increased prostaglandin E2 production. Thiazolidinediones increased arachidonic acid (AA) release from the cell membrane by not stimulating AA releasing process involving several phospholipase A2s but inhibiting AA reuptaking process. The expression of cyclooxygenase-1 and cyclooxygenase-2 were not affected by thiazolidinediones. In this study, we demonstrated that high concentrations of TZDs increased AA release by the inhibition of AA reuptaking process, leading to subsequent increase in the prostanoid production in a PPARgamma-independent manner. This mechanism provides useful information for the elucidation of multiple PPARgamma functions and diabetic drug therapy.

Summary: We sequenced all exons and exon-intron junctions of the CYP2B6 gene from 200 Japanese individuals. We found three novel single nucleotide polymorphisms (SNPs) (1375A>G, 1427G>A and 1454A>T) causing amino acid substitutions (Met 459 Val, Gly 476 Asp and Gln 485 Leu in exon 9), respectively. The detected SNP was as follows: 1) SNP, 031226Hiratsuka01; GENE NAME, CYP2B6 ; ACCESSION NUMBER, AC023172; LENGTH, 25 base; 5′-CAGAACTTCTCC A/G TGGCCAGCCCCG-3′. 2) SNP, 031226Hiratsuka02; GENE NAME, CYP2B6 ; ACCESSION NUMBER, AC023172; LENGTH, 25 base; 5′-CCCAGGAGTGTG G/A TGTGGGCAAAAT-3′. 3) SNP, 031226Hiratsuka03; GENE NAME, CYP2B6 ; ACCESSION NUMBER, AC023172; LENGTH, 25 base; 5′-CCCCAACATACC A/T GATCCGCTTCCT-3′.

Aims The goal of this study was to determine the frequencies of important allelic variants in the TPMT, NAT2, GST, SULT1A1 and MDR-1 genes in the Egyptian population and compare them with the frequencies in other ethnic populations. Methods Genotyping was carried out in a total of 200 unrelated Egyptian subjects. TPMT*2 was detected using an allele-specific polymerase chain reaction (PCR) assay. TPMT*3C and NAT2 variants (*5,*6 and *7) were detected using an allele-specific real-time PCR assay. Detection of GSTM1 and GSTT1 null alleles was performed simultaneously using a multiplex PCR assay. Finally, a PCR-restriction fragment length polymorphism assay was applied for the determination of TPMT*3A (*3B), SULT1A1*2 and MDR-1 (3435T) variants. Results Genotyping of TPMT revealed frequencies of 0.003 and 0.013 for TPMT*3A and TPMT*3C, respectively. No TPMT*2 or *3B was detected in the analysed samples. The frequencies of specific NAT2 alleles were 0.215, 0.497, 0.260 and 0.028 for *4 (wild-type), *5 (341C), *6 (590A) and *7 (857A), respectively. GSTM1 and GSTT1 null alleles were detected in 55.5% and 29.5% of the subjects, respectively. SULT1A1*2 was detected at a frequency of 0.135. Finally, the frequencies of the wild-type allele (3435C) and the 3435T variant in the MDR-1 gene were found to be 0.6 and 0.4, respectively. Conclusions We found that Egyptians resemble other Caucasians with regard to allelic frequencies of the tested variants of NAT2, GST and MDR-1. By contrast, this Egyptian population more closely resemble Africans with respect to the TPMT*3C allele, and shows a distinctly different frequency with regard to the SULT1A1*2 variant. The predominance of the slow acetylator genotype in the present study (60.50%) could not confirm a previously reported higher frequency of the slow acetylator phenotype in Egyptians (92.00%), indicating the possibility of the presence of other mutations not detectable as T341C, G590A and G857A. The purpose of our future studies is to investigate for new polymorphisms, which could be relatively unique to the Egyptian population.

Aims The goal of this study was to determine the frequencies of CYP1A2*1C, *1D, *1E and *1F variants in the Egyptian population and compare frequencies with other populations. Methods Genotyping was performed in a total of 212 unrelated Egyptian subjects using polymerase chain reaction-restriction fragment length polymorphism assay. Results The frequencies of CYP1A2*1C, *1D, *1E and *1F variants in the Egyptian population were 0.07, 0.40, 0.03 and 0.68, respectively. The Egyptians have a lower frequency of CYP1A2*1C, and CYP1A2*1E than the Japanese (0.07 vs 0.21 and 0.03 vs 0.08, respectively), while the frequencies of CYP1A2*1D and CYP1A2*1F did not differ significantly between the two groups. CYP1A2*1F (0.68) frequency in Egyptians was identical to that observed in Caucasians (0.68 among 236 German individuals). Conclusions The present study is the first to describe the frequencies of four known allelic variants of CYP1A2 among the Egyptian population. CYP1A2*1C and *1E occurred at frequencies significantly lower than that in Japanese, while similar frequencies were observed for CYP1A2*1D and *1F. The CYP1A2*1F frequency appeared to be identical to that of Caucasians. This does not exclude the possibility of the presence of new mutations relatively specific to the Egyptian population that have not been identified.

We developed a determination method for human urinary 12-hydroxyeicosatetraenoic acid (12-HETE) using LC–MS–MS. This method, which includes simple extraction and detection in the SRM mode, allows precise and accurate determination of 12-HETE. There was a significant sex difference in urinary 12-HETE levels. Chiral analysis of 12-HETE using LC–MS–MS with column-switching technique revealed that the major enantiomer was 12(S)-HETE. Furthermore, the urinary level in patients with diabetes mellitus (DM) was analyzed. The present in vivo findings indicate that there could be difference in production of 12(S)-HETE between genders and 12(S)-HETE may play a role in the pathogenesis of DM.

A liquid chromatographic-tandem mass spectrometric (LC/MS-MS) method was developed for the simultaneous quantification of prostaglandin (PG) E(2), PGF(2alpha), 6-keto-PGF(lalpha) and thromboxane (TX) B(2). These eicosanoids and their deuterium derivatives, using as internal standards, were extracted by solid-phase extraction and analyzed using LC/MS-MS in the selected reaction-monitoring (SRM) mode. A good linear response over the range of 10 pg to 10 ng for each eicosanoid was demonstrated. The accuracy of added eicosanoids ranged from 94.1 to 106.6% and coefficients of variation ranged from 0.62 to 7.8%. Furthermore, we applied this method for the determination of eicosanoids in the human synovial cell-cultured medium, stimulated by lipopolysaccharide (LPS). LPS produced each eicosanoid and they increased in a time-dependent manner. The production levels after 24 h stimulation were 6-keto-PGF(1alpha)PGE(2)TXB(2)PGF(2alpha). This simultaneous quantification method is so useful to clarify the function of synovial cells in rheumatoid arthritis (RA).

We have developed a simultaneous quantification method for prostaglandin (PG) E(2), PGD(2), PGF(2 alpha), 8-epi-PGF(2 alpha), 6-keto-PGF(1 alpha) and thromboxane (TX) B(2). Using [3,3,4,4-(2)H(4)]PGE(2), [3,3,4,4-(2)H(4)]PGD(2), [3,3,4,4-(2)H(4)]8-epi-PGF(2 alpha), [3,3,4,4-(2)H(4)]PGF(2 alpha), [3,3,4,4-(2)H(4)]6-keto-PGF(1 alpha) and [18,18,19,19-(2)H(4)]TXB(2) as internal standards (I.S.), the eicosanoids and their I.S. were simultaneously extracted by solid-phase extraction from cell-cultured medium, derivatized to methyl ester/methoxim/tert.-butyldimethylsilyl ether derivatives and analyzed using gas chromatography-mass spectrometry in the selected ion monitoring mode. The accuracy for the added eicosanoids ranged from 92 to 113%, and coefficients of variation ranged from 0.1 to 12.2%. Increased eicosanoids in RAW264.7 and U937 cells stimulated by lipopolysaccharide were suppressed by NS-398 and indometacin. This simultaneous quantification method can be applied routinely for assaying eicosanoids in vitro.

Aims The goal of this study was to determine the frequencies of important allelic variants of CYP2C9, CYP2C19, CYP2E1 and DPYD in the Egyptian population and compare them with the frequencies in other ethnic populations.

Neocarzinostatin (NCS) is the first discovered anti-tumor antibiotic having an enediyne-containing chromophore and an apoprotein with a 1:1 complex. An artificial gene library for NCS apoprotein (apo-NCS) production in Escherichia coli was designed and constructed on a phage-display vector, pJuFo. The recombinant phages expressing pre-apo-NCS protein were enriched with a mouse anti-apo-NCS monoclonal antibody, 1C7D4. The apo-NCS gene (encsA) for E. coli was successfully cloned, and then re-cloned into the pRSET A vector. After the his-tagged apo-NCS protein had been purified and cleaved with enterokinase, the binding properties of the recombinant protein as to ethidium bromide (EtBr) were studied by monitoring of total fluorescence intensity and fluorescence polarization with a BEACON 2000 system and GraphPad Prism software. A dissociation constant of 4.4 +/- 0.3 microM was obtained for recombinant apo-NCS in the fluorescence polarization study. This suggests that fluorescence polarization monitoring with EtBr as a chromophore mimic may be a simplified method for the characterization of recombinant apo-NCS binding to the NCS chromophore. When Phe78 on apo-NCS was substituted with Trp78 by site-directed mutagenesis using a two stage megaprimer polymerase chain reaction, the association of the apo-NCS mutant and EtBr observed on fluorescence polarization analysis was of the same degree as in the case of the wild type, although the calculated maximum change (DeltaIT(max)) in total fluorescence intensity decreased from 113.9 to 31.3. It was suggested that an environmental change of the bound EtBr molecule on F78W might have dramatically occurred as compared with in the case of wild type apo-NCS. This combination of monitoring of fluorescence polarization and total fluorescence intensity will be applicable for determination and prediction of the ligand state bound or associated with the target protein. The histone-specific proteolytic activity was also re-investigated using this recombinant apo-NCS preparation, and calf thymus histone H1, H2A, H2B, H3, and H4. The recombinant apo-NCS does not act as a histone protease because a noticeable difference was not observed between the incubation mixtures with and without apo-NCS under our experimental conditions.

We have generated mutant mice for epidermal-type fatty acid binding protein by the gene targeting technique and examined the phenotype in detail. Despite a lack in the expression of epidermal-type fatty acid binding protein mRNA and its protein in the skin and other tissues of the mutant mice, the animals appeared normal in gross and histologic examination. Northern blot analysis of other fatty acid binding proteins revealed a distinct elevated gene expression of heart-type fatty acid binding protein in the skin of the homozygous mice. In analyses of the skin, no differences were observed in contents of major fatty acids, electron microscopic appearance as well as inflammatory responses in ear skin between the mutant and wild-type mice. Basal transepidermal water loss of homozygous mice was lower than that of the wild mice. When acetone was applied to the skin for disruption of the water permeability barrier, recovery in transepidermal water loss was delayed, although maximum transepidermal water loss upon acetone treatment was similar between homozygous and wild-type mice in terms of size and time course. The molecular mechanism by which epidermal-type fatty acid binding protein contributes to the water barrier function of the skin remains to be elucidated.

Objectives: The routine detection of polymorphisms affecting drug sensitivity in patients before treatment is important in the identification of drug responders or nonresponders, and patients at increased risk of drug toxicity. Here, we present an assay for the simultaneous and rapid genotyping of five polymorphisms influencing drug sensitivity. Design and methods: We used a hybridization probe assay on the LightCycler to detect five single nucleotide polymorphisms (SNPs): INPP1 (973C>A), ADRB2 (R16G and Q27E), HTR2A (102T>C), and mtDNA (1555A>G). Two fluorescent labeled hybridization probes were designed for the simultaneous detection of the five SNPs and detection of the variant alleles was performed by melting curve analysis. Results: All five SNPs were detected with a single thermocycle protocol within 40 min. The genotypes determined in this assay were identical to those obtained with conventional PCR and restriction fragment length polymorphism analysis. Conclusions: To our knowledge, we report here for the first time a method for simultaneous detection of five SNPs, on a single thermocycle protocol by the LightCycler. This method is rapid, highly sensitive, and high-throughput, and is thus suitable for routine clinical use and large-scale epidemiologic studies.

In order to clarify the binding site of drugs on human serum albumin (HSA), competition studies were carried out using ELISA for glycyrrhetic acid (GA), which is mainly bound to the diazepam site on HSA.The release of GA from HSA was detected as a displacement by the various drugs [bezafibrate (B), delapril HCI, diclofenac Na (D), doxifluridine, famotidine, ibuprofen (I), loxoprofen Na (L-1) and its metabolite (loxoprofen Na trans-OH (L-2)), pravastatin Na]. The percentages of GA release by the addition of I, D, L-1, L-2, which belong to nonsteroidal anti-inflammatory drugs, and B were 96.0%, 86.5%, 25.4%, 25.6% and 12.6% respectively. In addition, the percentages of GA release by addition of the other 4 drugs ranged between 3. 2 and 4.1%. These results showed that I and D were strongly bound to the diazepam site, while the binding of L-1, L-2 and B was weak. Thus, L-1, L-2 and B may predominantly bind to other site (s) on HSA.Therefore, ELISA for GA was suggested to be helpful for studying the binding site (diazepam binding site) of the drugs on HSA.

To investigate the association between NAT2 genotypes and the incidence of isoniazid (INH)-induced adverse reactions, in the hope of identifying a pharmacogenetic approach that could be useful in the prediction and prevention of adverse reactions in Japanese patients, we retrospectively studied the genotypes of NAT2 in 102 Japanese patients treated with INH (without rifampicin co-administration). The subjects were classified into three groups according to their genotypes: rapid-type, intermediate-type, and slow-type. The clinical conditions of the patients were followed-up in order to evaluate the development of any adverse drug reactions (ADRs) and correlate them with patient genotypes. Six out of the 102 patients (5.9%) developed various ADRs following INH treatment. These reactions included nausea/vomiting, fever, visual impairment, and peripheral neuritis. We found a statistically significant difference between the incidence of ADRs and NAT2 genotype. The incidence of ADRs was significantly higher in the slow type than in the other two types, as 5 out of the 6 ADR patients were of the slow-type, and the other one was of the intermediate-type, while no patients of the rapid-type developed any ADRs. The results indicated that the genes coding for slow acetylation were associated with the incidence of serious ADRs following INH treatment. Our findings suggest that determination of NAT2 genotype might be clinically useful in the evaluation of patients at high risk of developing ADRs induced by INH.

Thromboxane and leukotrienes have been implicated in inflammation. However, the production level of these eicosanoids in patients with rheumatoid arthritis is still unclarified. In the present study, endogenous synthesis of thromboxane and cysteinyl leukotrienes in patients was investigated. The production of eicosanoids in patients is assessed by measuring stable urinary metabolites,11-dehydro thromboxane B2 and leukotriene E4, using gas chromatography/selected ion monitoring and liquid chromatography/tandem mass spectrometry. The level of urinary thromboxane in patients was significantly higher than that in healthy volunteers (P0.05). Furthermore, we investigated the effects of administered drugs on the production of these eicosanoids. The urinary thromboxane level of the untreated group (1630 +/- 613 pg/mg creatinine) was much higher than that of healthy volunteers (342 +/- 263 pg/mg creatinine). The level in the group receiving NSAID alone was similar to that in healthy volunteers, and the group receiving steroid alone showed slightly lower thromboxane levels than the untreated group. On the other hand, the leukotriene E4 level in patients (280 +/- 360 pg/mg creatinine) was also significantly higher than that in healthy volunteers (59 +/- 54 pg/mg creatinine, P0.05). In particular, the group receiving methotrexate (904 +/- 685 pg/mg creatinine) had higher leukotriene levels than not only healthy volunteers but also other medicated groups. These findings demonstrated that endogenous thromboxane and leukotriene production in patients with rheumatoid arthritis are enhanced, and the effects of medication on the production of these eicosanoids differed in thromboxane and leukotriene E4.

G We investigated possible motor effects of 7-nitroindazole (7-NI), an neuronal nitric oxide synthase (nNOS) inhibitor, and N -nitro-L- arginine methyl ester ( L-NAME), an non-selective NOS inhibitor in mice using catalepsy and pole tests in comparison with dopamine D2 receptor antagonist, haloperidol. We also studied the change in dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) contents of these compounds. The administration of 7-NI and L-NAME (40-160 mg / kg, s.c.) dose-dependently induced motor deficit in both catalepsy and pole tests. The motor deficit induced by 7-NI was more pronounced than the one produced by L-NAME. In contrast, haloperidol showed a marked motor deficit in mice. Haloperidol showed a marked motor deficit as compared with 7-NI and L-NAME. For dopamine, DOPAC and HVA contents, haloperidol exhibited a significant decrease in dopamine content and a significant increase in DOPAC and HVA content in the striatum. In contrast, 7-NI showed a significant increase in the striatal dopamine content. However, 7-NI had no significant change in the striatal DOPAC and HVA contents. On the other hand, no significant change in the striatal dopamine, DOPAC and HVA contents was observed in L-NAME-treated mice. The present study also showed that the motor deficit induced by 7-NI or L-NAME was significantly attenuated by the treatment with L-arginine. These results demonstrate that NOS inhibitors as well as dopamine D receptor antagonist haloperidol can induce motor deficit in mice. The present study also suggests that 2 the mechanism in the motor deficit caused by NOS inhibitors may be different from that in the motor deficit induced by haloperidol. Furthermore, our findings suggest that nNOS may play some role in control of motor behavior. © 2001 Elsevier Science B.V. All rights reserved.

Background Synthesis of cysteinyl leukotrienes (LTs) is known to play a part in the pathogenesis of inflammatory diseases. Objectives To define the involvement of cysteinyl LTs in atopic dermatitis (AD). Methods Synthesis of cysteinyl LTs was assessed in patients with AD and healthy volunteers by measuring urinary LTE4, a useful index of systemic cysteinyl LT synthesis, using liquid chromatography/tandem mass spectrometry. Results Mean ± SD urinary LTE4 levels in patients with AD (125 ± 69 pg mg−1 creatinine, n = 20) were significantly higher (P

Queuosine is a modified nucleoside located at the first position of the tRNA anticodon, which is synthesized by tRNA-guanine transglycosylase (TGT). Although the levels of queuosine in cancer cells have been reported to be lower than those in normal cells, the expression levels of TGT remain to be determined. We determined the expression levels of a subunit of TGT (TGT60KD). Contrary of our expectations, the results revealed higher levels of expression of TGT60KD than that in normal cells, and the level of queuosine in the tRNA fraction corresponded with that of TGT60KD expression. These results suggest the possibilities that the expression levels of TGT60KD regulate TGT activity and the levels of queuosine, and that TGT60KD plays significant roles in carcinogenesis. To our knowledge, this is a first report of increased expression levels of TGT60KD in human cancer cells.

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.

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

We studied sequential changes in muscarinic cholinergic receptors, high-affinity choline uptake sites and dopamine D2 receptors in the brain after 6-hydroxydopamine lesions of the medial forebrain bundle in rats. The animals were unilaterally lesioned in the medial forebrain bundle and the brains were analyzed at 1, 2, 4 and 8 weeks postlesion. [3H]Quinuclidinylbenzilate (QNB), [3H]hemicholinum-3 (HC-3) and [3H]raclopride were used to label muscarinic cholinergic receptors, high-affinity choline uptake sites and dopamine D2 receptors, respectively. The degeneration of nigrostriatal pathway produced a transient decrease in [3H]QNB binding in the parietal cortex of both ipsilateral and contralateral sides at 2 and 8 weeks postlesion. [3H]QNB binding also showed a mild but insignificant decrease in the ipsilateral striatum throughout the postlesion periods. No significant change was observed in the substantia nigra (SN) of both ipsilateral and contralateral sides throughout the postlesion periods. In contrast, [3H]HC-3 binding showed no significant change in the parietal cortex of both ipsilateral and contralateral sides during the postlesion. However, [3H]HC-3 binding was upregulated in the ipsilateral dorsolateral striatum throughout the postlesion periods. The ventromedial striatum also showed a significant increase in [3H]HC-3 binding at 1 week and 2 weeks postlesion. On the other hand, no significant change in [3H]raclopride binding was found in the parietal cortex of both ipsilateral and contralateral sides during the postlesion. [3H]Raclopride binding showed a conspicuous increase in the ipsilateral striatum (35-52% of the sham-operated values in the lateral part and 39-54% in the medial part) throughout the postlesion periods. In the contralateral side, a mild increase in [3H]raclopride binding was also found in the striatum (10-15% of the sham-operated values in the lateral part and 22% in the medial part) after lesioning. However, a significant decline in [3H]raclopride binding was observed in the ipsilateral SN and ventral tegmental area during the postlesion. The present study indicates that 6-hydroxydopamine injection of medial forebrain bundle in rats can cause functional changes in high-affinity choline uptake site in the striatum, as compared with muscarinic cholinergic receptors. Furthermore, our studies demonstrate an upregulation in dopamine D2 receptors in the striatum and a decrease in the receptors in the SN and ventral tegmental area after the 6-hydroxydopamine injection. Thus, these findings provide further support for neurodegeneration of the nigrostriatal pathway that occurs in Parkinson's disease.

A case involving a suicidal ingestion of Aconitum tubers is presented. A 40-year-old woman in Hokkaido, Japan ingested ground aconite and died of aconite intoxication about 4 h after ingestion. The Aconitum alkaloids were quantitated using gas chromatography-selected ion monitoring from extracts of the body fluids and organs. The blood and urine concentrations of jesaconitine, the main alkaloid of the aconite in this case, were 69.1 ng/mL and 237.8 ng/mL, respectively. Higher values of the alkaloid were demonstrated in the kidneys, the liver, and in the bile rather than other organs or serum, suggesting the alkaloids were eliminated by the liver and kidneys. In the gastrointestinal tract, the highest value of jesaconitine (471.3 ng/g) was in the ileal contents. These findings show that Aconitum alkaloids were found in the liver and kidneys in much higher concentrations than in serum and suggest that they were eliminated not only via urine but also in feces. Feces may be useful to detect Aconitum alkaloid if other biological samples are not available.

In man, CYP2C19, a liver enzyme, plays an important role in the metabolism of severaldrugs. Mutation of the CYP2C19 gene results in a poor metaboliser phenotype. S-Mephenytoin hydroxylation genetic polymorphism is due to two mutations of theCYP2C19 gene, namely CYP2C19*2, located in exon 5, and CYP2C19*3, located inexon 4. CYP2C18 is also polymorphically expressed. The mutant alleles of this enzyme areCYP2C18m1, located in exon 2 and CYP2C18m2, located in the 5 0 -flanking region. Wehave developed an allele-specific TaqMan polymerase chain reaction (PCR) assay withwhich to detect CYP2C18 mutant alleles. This assay combines hybridization of theTaqMan probe and allele-specific amplification primers to the target DNA. The TaqManprobe is labelled with 6-carboxyfluorescein at the 5 0 end and 6-carboxytetramethylrhoda-mine together with a phosphate at the 3 0 end. Genotypes are separated according to thedifferent threshold cycles of the wild type and mutant primers.We applied this procedure to DNA extracted from the blood or saliva of 144 healthyJapanese volunteers. The wt=wt, wt=m1, wt=m2, m1=m1, m1=m2 and m2=m2 genotypes ofthe CYP2C18 alleles detected by the assay were consistent with the results obtained fromrestriction enzyme cleavage. In accordance with a previous report, the genotypes ofCYP2C18m1 and CYP2C18m2 coincided with those of CYP2C19*3 and CYP2C19*2,respectively. Therefore, detection of CYP2C18 mutant alleles also allows that of CYP2C19mutant alleles. Among 19 poor metabolisers, eight showed the homozygousCYP2C19*2=CYP2C19*2, two the homozygous CYP2C19*3=CYP2C19*3 and nine thecompound heterozygous CYP2C19*2=CYP2C19*3 genotype.We found the allele-specific TaqMan PCR assay rapid, simple and cost-effective, as wellas suitable for high-throughput applications in a routine laboratory. This assay allows thefast and reliable detection of inherited disorders that might influence diagnosis andtreatment.Human CYP2C is a major subfamily of enzymes inthe liver, of which four members have been iden-tified (CYP2C8, CYP2C9, CYP2C18 andCYP2C19; Goldstein & de Morais 1994). Of these,CYP2C19 plays the most important role in themetabolism of several drugs, such as omeprazole,lansoprazole, diazepam and proguanil (Goldstein dMinoletti et al 1999) and diazepam (Jung et al1997). CYP2C18 was also recently reported to behighly expressed in the human epidermis (Zaphiro-poulos 1997).The poor metaboliser phenotype is associatedwith CYP2C19*2 and, less commonly, CYP2C19*3genetic mutations. A single G to A transition causesboth mutations. The CYP2C19*2 mutation is inexon 5 and that of CYP2C19*3 is in exon 4 (deMorais et al 1994a, b). Poor metabolisers have twodefective alleles, whereas those who are eitherhomozygous or heterozygous for CYP2C19*1 are

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.

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.

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.

The in vitro effect of C-AGP (pure α1-acid glycoprotein from the ascitic fluid of cancer patients) on NK cell cytotoxicity was tested using normal healthy human PBMC. C-AGP had no inhibitory effect on basal NK cell activity. C-AGP selectively suppressed the augmentation of NK cell activity by rIFNαA and rIFNγ, but C-AGP did not prevent the NK activation by rIL-2. NK cells in PBMC treated with C-AGP for 12 h and then washed just once, to remove the C-AGP, fully recovered the ability to respond to rIFNαA. However, after the treatment of PBMC with C-AGP for 5 or 6 days, NK cells failed to respond to rIFNαA, in spite of washing to remove C-AGP from the cultures. Monocytes were necessary for the suppressive effect of C-AGP on rIFNαA activation of NK cells. Indomethacin restored the ability of NK cells to respond to rIFNαA in C-AGP-treated PBMC. These results suggest that monocytes are able to selectively suppress the response of NK cells to IFNs in the presence of, or following treatment with C-AGP.

We established microdetermination methods of prostaglandin (PG) metabolites by GC-selected ion monitoring (GC-SIM) and applied them to the clinical investigations. At first the microdetermination of delta 17-6-keto-PGF1 alpha, a hydrolyzed metabolite of PGI2, is described. An authentic delta 17-6-keto-PGF1 alpha was prepared from eicosapentaenoic acid (EPA) incubated with a homogenate from the bovine aortic intima. [18O] delta 17-6-Keto-PGF1 alpha was synthesized to obtain an internal standard for GC-SIM of delta 17-6-keto-PGF1 alpha. A good linear response over the range of 10 pg-5 ng was demonstrated. Chromatographic conditions using a MP-65HT column presented nearly baseline separation of delta 17-6-keto-PGF1 alpha and 6-keto-PGF1 alpha. Furthermore, a monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate delta 17-6-keto-PGF1 alpha in the human blood sera. Using the prepared immunoaffinity columns of this antibody, delta 17-6-keto-PGF1 alpha was clearly detected in the human blood sera by GC/MS analysis. We were able to detect delta 17-6-keto-PGF1 alpha of the amount ranging from 6 to 26 pg/ml in the human blood plasma. The present method can be applied to the determination of delta 17-6-keto-PGF1 alpha in the human urine and plasma. Diabetes mellitus induces platelet alterations such as hyperaggregation. Variations in PG production seem to be related to this phenomenon but the changes in PG levels remain unclear. So we microanalyzed the 11-dehydrothromboxane B2 (TXB2) and 2,3-dinor-6-keto-PGF1 alpha, which were stable metabolites of TXA2 and PGI2, in the urine and investigated the relationship between the thromboxane/prostacyclin (TX/PGI) ratio and diabetes mellitus. The TX/PGI ratio in the urine of diabetics was higher than that of healthy volunteers. In murine, the TX/PGI ratio of STZ-induced mice was also higher than that of non-induced mice. The ratio of db/db mice also increased with the progress of diabetes mellitus. Furthermore, we investigated the relationship between the retinal vein occlusion (RVO), a thrombotic disease in which the retinal vein is blocked by blood aggregations, and the TX/PGI ratio. The TX/PGI level in patients with the RVO, who were not combine diabetes, was significantly higher than that in healthy volunteers. One of the causes of the RVO may be due to the variation of thromboxane production. This GC-SIM method can be used to determine the TX/PGI ratio in the urine.

A method for the quantitation of diesterditerpene-type Aconitum alkaloids and their hydrolysis products by gas chromatography-selected ion monitoring was applied to a clinical case study. A 45-year-old male attempted suicide by oral intake of Aconitum alkaloids, which are highly intoxicant extracts of Aconitum tubers. It was estimated that he had ingested approximately 11 mg of diesterditerpene-type alkaloids but was saved by intensive gastric irrigation. Mesaconitine, aconitine, hypaconitine, and their hydrolysis products were detected in the serum on the first day only. On the other hand, some alkaloids were still detectable in the urine even six days after intoxication. Aconitum alkaloids are biotransformed, and their hydrolysis products are excreted time-dependently to the urine. The urine was a useful material to identify the toxicants in the case of aconite intoxication.

Although the role of an adhesion molecule such as P-selectin may be important in the pathogenesis of stroke, temporal, spatial, and cellular profiles of the expression of such a protein has not been fully studied in the case of the middle cerebral artery (MCA) occlusion and reperfusion in rat brain. Change in expression of immunoreactive P-selectin was examined in rat brain after transient MCA occlusion (MCAO) in comparison to that of permanent occlusion with an anti-P-selectin monoclonal antibody. Western blot analyses were performed to ensure the selective detection of immunoreactive P-selectin protein with the monoclonal antibody using brain homogenates before and after MCAO. Temporal, spacial, and cellular changes of P-selectin expressions were evaluated with rat brain sections at 2, 8h, 1 and 3 days of permanent MCAO, and at 2, 8h, 1, 3 and 7 days of reperfusion after 1 h of transient MCAO. Western blot showed a single band with a molecular weight of 140 kDa for both cases with permanent occlusion and reperfusion. P-selectin immunoreactivity was not normally present in rat brain sections. However, it was expressed mainly in the post-capillary venules of the cerebral cortex and caudate in the MCA territory with a peak at 2-8h after permanent occlusion and at 8h to 1 day after the reperfusion. The expression was diminished by 1 day of permanent occlusion and 3 days of reperfusion. The maximum staining in the case of permanent MCAO was stronger than the case with reperfusion. However, spacial distribution of the staining was similar in the cerebral cortex and caudate between the cases with permanent of transient MCAO. These results suggest a different temporal but similar spacial and cellular expression of P-selection immunoreactivity between permanent occlusion and reperfusion of MCA in rat brain.

We describe variations of 11-dehydrothromboxane B 2 (11-dehydro-TXB 2 ) levels in human urine samples. Retinal vein occlusion (RVO) is a thrombotic disease in which the retinal vein is blocked by blood aggregations. We considered the possibility that 11-dehydro-TXB 2 plays an important role in the formation of RVO. Thus, we determined the 11-dehydro-TXB 2 levels in patients with RVO using gas chromatography/selected ion monitoring (GC/SIM) and compared them with those of healthy volunteers. The thromboxane levels in patients with RVO, who did not also have diabetes, were significantly higher than those in healthy volunteers. One cause of RVO may be the variation of thromboxane production. Furthermore, this GC/SIM method can be applied to the prevention and treatment of not only RVO, but also of general thrombosis.

We devised a simple and effective purification for the microdetermination of thromboxane B 3 (TXB 3 ), a hydrolysis product of TXA 3 . [ 18 O 2 ]TXB 3 was synthesized by the repeated base-catalyzed hydrolysis of methyl ester derivatives in [ 18 O]water, to obtain an internal standard (IS) for the gas chromatography/selected ion monitoring (GC/SIM) of TXB 3 . The methyl ester (ME)-methoxime (MO)-dimethylisopropylsilyl (DMIPS) ether derivative was prepared, then GC/SIM was carried out by monitoring the ion at m/z 668 for TXB 3 and that at m/z 672 for IS. A good linear response over the range of 10 pg ∼ 10 ng was demonstrated. We were able to detect the levels of TXB 3 in the medium of human erythroleukemia (HEL) cell cultured with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). This method can be used to determine 3-series thromboxane in biological samples.

This paper describes a newly developed method using gas chromatography-selected ion monitoring (GC-SIM) for the simultaneous determination of diester-diterpene type Aconitum alkaloids and their hydrolysis products in human serum. When the alkaloids were converted into their TMS derivatives, they produced a well defined peak on selected ion recording (SIR). Good linear response over the range of 100 pg to 7.5 ng was demonstrated for each alkaloid. When the alkaloids were spiked into human serum, good quantitative recovery was observed.

Ascorbic acid (AA) accumulates in vitreous at a concentration several times higher than in plasma. It has been suggested that AA may serve as an antioxidant that protects ocular tissues from free radical attack. There are many reports about the concentration of AA in ocular tissues. However, AA in adult human vitreous humor has not been determined. We measured concentrations of AA from pathologic human vitreous samples and compared the results.AA was measured by high-performance liquid chromatography (HPLC) with UV detection. Human vitreous humor was collected from patients undergoing pars plana vitrectomy.AA was quantified in vitreous humor of proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), macular hole (MH), idiopathic premacular fibrosis (PMF), and Terson syndrome (Terson). The concentrations of AA were 120.9 +/- 36.3 micrograms/ml (mean +/- SD), 129.8 +/- 36.6, 311.5 +/- 126.7, 446.9 +/- 154.2 and 406.0 +/- 22.0, respectively. There was no significant difference between the PDR and the PVR groups (unpaired t-test). Patients with PDR and PVR showed significantly lower concentrations of AA than those with MH, PMF, and Terson (p0.01).These findings suggest that increased oxidative stress may be produced in the ocular tissues of eyes with PDR and PVR, and AA appears to be consumed (oxidized) in performing its protective role.