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We developed methods for the fluorometric assay of 3-hydroxykynurenine and 3-hydroxy-anthranilic acid, which are suspected as carcinogens in bladder cancer. It was shown that the urinary excretion of 3-hydroxyanthranilic acid increased in patients with bladder cancer. We also developed methods for the fluorometric assay of glucuronide and sulfate of 3-hydroxyanthranilic acid and showed that the excretion of these conjugated forms was minor in humans. The distribution of 3-hydroxykynurenine was studied and data obtained suggested that it has an affinity for the pancreas. We then developed methods for determination of the related compounds of tryptophan. Fluorescence reaction with UV radiation was applied to the determinations of kynurenic acid, kynurenine, quinolinic acid, nicotinic acid, nicotinamide, N1-methyl-nicotinamide, isatin, xanthrenic acid, and melatonin in the serum or urine. Furthermore, the fluorescence reaction with UV radiation was applied to some drugs, e.g., indomethacin, isoniazid, naldixic acid, nicorandil, and disodium cromoglycate. The relationship was investigated between the tumor promoter, 12-tetradecanoyl-phobol-13-acetate (TPA), and delayed hypersensitivity in mice. The foot pad reaction (FPR) in mice was suppressed by the application of TPA following the application of 7,12-dimethylbenz[alpha]-anthracene (DMBA), a tumor initiator, in BALB/c mice, while the FPR was suppressed by the application of TPA alone in C3H/He mice. CD8+ and CD4+ T cells, which suppress the FPR, were induced in BALB/c and C3H/He mice, respectively. These T cells produced soluble factors that inhibited the FPR in mice.

Changes in the expressions of interleukin-1 beta (IL-1 beta) mRNA in the rat brain were investigated during inflammatory or non-inflammatory stress. We utilized competitive reverse transcription PCR (RT-PCR) to quantitate precisely the minute amount of IL-1 beta mRNA in small tissues which were micro-punched out from discrete sites in the brain. Intraperitoneal injection of lipopolysaccharide (100 micrograms/kg) caused a widespread and high elevation of IL-1 beta mRNA in the hypothalamus, hippocampus and cerebral cortex. Immobilization stress significantly increased IL-1 beta mRNA only in the hypothalamus, when examined in the brain block samples, which reached a peak 30 minutes after the start of stress. However, examination of the small tissues punched out from discrete sites in the brain revealed a profound elevation of IL-1 beta mRNA in the CA1 region of the hippocampus and in all the hypothalamic nuclei examined 30 minutes after the start of immobilization stress, but not in the CA3 region and the dentate gyrus or cerebral cortex. The method shown in this report proved to be useful and may be applied to quantify the low amount of mRNA in the small areas in the brain.

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).

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.

In tryptophan metabolites, 3-hydroxykynurenine and 3-hydroxyanthranilic acid have been reported to show a carcinogenic action to mice bladder and the relation of the metabolites to human bladder cancer has been discussed. We developed methods for the fluorometric assay of these compounds and showed that the excretion of 3-hydroxyanthranilic acid increased in the patients with bladder cancer. We also devised methods for the fluorometric assay of glucuronide and sulfate of 3-hydroxyanthranilic acid and showed that the minor excretion of these conjugated forms was shown in humans. The distribution of these compounds was also studied and the obtained data suggests that 3-hydroxykynurenine has affinity for the pancreas. We then developed methods for the determination of other metabolites of tryptophan. A fluorescence reaction with UV irradiation was found and applied to the determination. This method is the most sensitive to kynurenic acid but can be applied to kynurenine, nicotinamide and quinolinic acid. Furthermore, this methods also applied to the determination of some medicines, e.g. indomethacin, isoniazid, nalidixic acid, nicorandil and disodium cromoglicate in the serum or urine. We further devised other methods for the determination of xanthurenic acid and 5-hydroxyindoles.

The purpose of this study was 1) to evaluate the usefulness of myocardial microdialysis method in the beating canine heart for the measurements of myocardial interstitial adenosine, inosine and hypoxanthine concentrations and 2) to determine their changes during myocardial ischemia. Microdialysis probes modified for the beating myocardium were implanted in the left ventricular myocardium of pentobarbital-anesthetized dogs. The implanted microdialysis probes were perfused with saline solution containing low molecular heparin in order to prevent thrombotic obstruction of the dialysis membrane. The concentrations of adenosine and its metabolites, inosine and hypoxanthine, in the effluent dialysate were determined using high-performance liquid chromatography (HPLC). Dialysate concentrations of adenosine and its metabolites were considerably high immediately after the insertion of microdialysis probes. However, the concentrations gradually declined and stabilized by 150 min. Occlusion of the left anterior descending artery for 30 min caused a marked increase in dialysate adenosine and inosine levels, and a small increase in hypoxanthine level in the ischemic myocardium. These results suggest that the myocardial microdialysis method is very useful for the in vivo monitoring of interstitial levels of adenosine and its metabolites in the beating canine heart.

Recent development of flameless atomic absorption spectrophotometry has made possible more precise and sensitive determinations of metals including lead. This means that a sample size of microliter order of blood has come to give a sufficient analytic signal. Blood lead which has usually been determined using venous blood was determined in the present study using a small amount of capillary blood. The method developed is of lead determination in the capillary blood sample from earlobe by means of flameless atomic absorption spectrophotometry equipped with an automated microsampling system or autosampler. Thus, 70 microliters of heparinized whole blood sample from earlobe gave a satisfactory result in the lead analysis. The procedure is as follows. 1) Blood obtained from the earlobe is collected in a heparinized capillary tube. 2) The blood is diluted in the ratio of 1 : 9 with Triton 5000 X solution for complete hemolysis of erythrocytes, by which the matrix-bound lead is released and a better distribution of samples is made possible in the graphite tube. 3) Finally the sample is analyzed by a flameless atomic absorption spectrophotometer with an autosampler. Making sure the analytical method to be accurate and reliable for blood lead determination, the authors compared capillary blood lead levels (Pb-Bc) with venous ones (Pb-Bv) from the same subjects in the same sampling session. It was found that the correlation between Pb-Bc and Pb-Bv was highly significant (n = 144, r = +0.998, y = 0.97 x+0.32, p less than 0.001) and that they were almost of the same level. It was concluded that the method developed may be recommended for the routine clinical use.

The determination of small amount of chlorine in the atmosphere is carried on ordinarily by omicron-toluidine method at present. However, this method is not recommended because the issued colour is unstable and omicron-toluidine itself belongs in the first group member of the Ordinance on Prevention of Particular Chemical Hazards Substances. Authors recommend 4-nitroaniline method, reported by J. Gabbay etc., because this method is stable and more practical. Although the colour that issued from the reaction of chlorine with 4-nitroaniline is affected by the co-existence of reducing gas, e.g. SO2, H2S or organic gas, but this effect may be removed the co-existence of reducing gas, e.g. SO2, H2S or organic gas, but this effect may be removed by introducing the sample gas into the layer of H2SO4-52Cr2O7 mixture immersed in glassfiber filter for micro-analysis. In this paper, influence of pH of the issued colour, stability, stream velocity of samplegas and effect of reducing substance are discussed.

Concentrations of cefotiam (CTM) in the exudate after abdominal operation were studied. The exudates were collected by 6 mm paper discs and the antibiotic concentrations were determined by the paper disc method using Proteus rettgeri ATCC 9250 as test organism. Comparison between agar well and paper disc assay for CTM in ascites specimens showed good correlation. Two grams of CTM was intravenously given to the patients after abdominal operations. The concentration of CTM in the exudate for 6 hours after injection was 0.4--14.2 micrograms/ml and this value exceeded the antibacterial level of CTM. It is, therefore, concluded that CTM will be effective for the postoperative prophylaxis.

The purpose of our study was to examine the ischemia induced enzymatic changes of decaylation-reacylation cycle of membrane phospholipids in dog brain. In this study, we developed new modified method for assay of phospholipase A1, A2 and lysophospholipase which is simpler and needs only a smaller amount of materials. For the first report, we introduced this new method and demonstrated some properties of phospholipase A1, A2 and lysophospholipase in dog brain. Crude enzyme solution for assays of phospholipase A1, A2 and lysophospholipase was gained from extraction of frozen brain with aceton, butanol and saline. The level of phosphorus in the enzyme extract was determined and only those extracts which had a level of phosphorus within a certain range were used. The substrates for assays were L-alpha-[beta-palmitoyl-1-14C] phosphatidylcholine, dipalmitoyl for phospholipase A1 and A2 and L-lysophosphatidylcholine-1-[1-14C] palmitoyl for lysophospolipase respectively. Each radioactive substrates was diluted with cold carrier lipid to give the proper specific activity. Reaction system including substrate, buffer [pH 7.0] and enzyme extract was incubated for 10 hours at 38 degrees C. But for the assay of phospholipase A1 and A2, enzyme solution was pre-incubated at 70 degrees C for 5 minutes. In our new method, reaction mixture was directly separated by TLC without extracting lipids. Enzyme activities were calculated from radio thin-layer chromatograms. Furthermore, we made a comparison between our method and the former one. The value of each enzyme activity was slightly higher in our method than in the former one. However, it was revealed that the results were reproducible in both methods.