Takatoshi Matsumoto

Trace levels of pyrroloquinoline quinone in human and rat samples detected by gas chromatography/mass spectrometry

A detailed procedure for the assay of free pyrroloquinoline quinone (PQQ) in human and rat samples by gas chromatography/mass spectrometry (GC/MS) has been established with stable-isotopic PQQ as internal standard. PQQ was extracted from the samples, after addition of the internal standard, with butanol under acid conditions and with Sep-Pak C18 cartridges. After derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ by selected ion monitoring, respectively. Trace amounts of free PQQ were detected in eight organs, plasma and urine of the human, and in three organs of the rat. The PQQ level was highest in the human spleen (5.9 +/- 3.4 ng/g tissue, followed by the pancreas and lung, and it was below detection limits for human brain and heart. Trace levels of PQQ were also found in rat small intestine, liver and testis. Our data are far below those measured by the redox cycling method of Gallops group for human plasma, adrenal and urine.

A sensitive fluorometric assay for serum monoamine oxidase with kynuramine as substrate

The detailed procedure of a new sensitive fluorometric assay for human serum monoamine oxidase (MAO) with kynuramine as substrate is described. The data on its reproducibility, stability, correlation with another method, and serum MAO levels for 150 healthy subjects and 205 patients with various diseases, are presented to support the usefulness of this method. Since our method is much simpler and more sensitive than other methods conventionally used, we can recommend it for routine clinical investigation.

Oxidation of phenylethanolamine and octopamine by type A and type B monoamine oxidase effect of substrate concentration

Abstract Phenylethanolamine (PEOA) and octopamine (OA) were characterized as substrates for type A and type B monoamine oxidase (MAO) at various substrate concentrations, using rat brain mitochondria. The experiments on sensitivity to clorgyline and deprenyl showed that the inhibition patterns with PEOA as substrate differed markedly at different substrate concentrations: at 12.5 μM, PEOA acted as a specific substrate for type B MAO, but at 125 and 1250 μM it became a common substrate for both types of MAO. However, when OA was used as substrate, there were only slight or no differences in the inhibition patterns among the various concentrations tested; OA was found to be a common substrate for both types of MAO. Benzylamine was also examined for comparison and confirmed to be highly specific for type B MAO over a wide concentration range of the substrate. Kinetic analyses were carried out for PEOA and OA. High and low affinities for MAO were identified for PEOA: K m values were 22.7 and 465 μM, and V max values were 6.90 and 19.2 nmoles/mg of protein/30 min respectively. Pretreatment of the enzyme with 10 −6 M clorgyline resulted in the disappearance of the low affinity component, and pretreatment with 10 −6 M deprenyl resulted in the disappearance of the high affinity component. Therefore, the high affinity corresponded to that for type B MAO and the low one to that for type A MAO. For OA, however, the double reciprocal plots were linear with a single affinity component showing K m and V max values of 455 μM and 90.9 nmoles/mg of protein/ 30 min respectively. From the present study, it can be concluded that, when sensitivity of MAO to clorgyline or deprenyl is studied, it is necessary to check the effect of substrate concentration for each substrate and enzyme preparation, suspecting the different affinities of the substrate for type A and type B MAO.

3-(p-hydroxyphenyl)propionic acid as a new fluorogenic reagent for amine oxidase assays.

A detailed procedure of a new and extremely sensitive fluorometric assay for amine oxidases is presented. Hydrogen peroxide, produced by the oxidase reaction, reacted with 3-(p-hydroxyphenyl)propionic acid in the presence of peroxidase to yield a fluorescent compound by which enzyme activity could be determined. The enzyme reaction was terminated by NaOH solution, which increased the fluorescence intensity three- to fivefold. The detection limit thus obtained was as little as 0.02 nmol. The alkalinization also contributed to stopping the enzyme reaction and to the clarification of assay mixtures containing turbid enzyme preparations.

Acetylputrescine deacetylase from Micrococcus luteus K-11

Abstract An acetylputrescine deacetylase was induced in Micrococcus luteus K-11, and was partially purified and characterized briefly. The enzyme was most active toward acetylputrescine, followed by N 8 -acetylspermidine and acetylcadaverine, but was inactive toward N 1 -acetylspermidine and N 1 -acetylspermine. The K m value for acetylputrescine was 0.321 mM. It was almost unaffected by -SH blocking agents but was inhibited by metal ions such as Cu 2+ and Ni 2+ . Its molecular weight estimated by Sephacryl S-200 column chromatography was 115000.

A new enzymatic assay for total diamines and polyamines in urine of cancer patients

SummaryA detailed procedure of a new photometric assay for total diamines and polyamines in human urine using soybean seedling amine oxidase (SSAO) as an enzyme reagent is described. It is based on the unique substrate specificity of SSAO that the enzyme is active toward all diamines and polyamines. The amines were purified from urine by cation-exchange chromatography and incubated with SSAO. Hydrogen peroxide formed in the oxidase reaction was measured photometrically by coupling 4-aminoantipyrine with phenol in the presence of peroxidase. For its simplicity and sensitivity, our method seems useful for routine clinical investigation. The data obtained from normal subjects and patients of various cancers are presented to validate the present method.

Oxidation of β-phenylethylamine by both types of monoamine oxidase: Effects of substrate concentration and PH

Abstract β-Phenylethylamine (PEA) was characterized as substrate for both type A and type B monoamine oxidase (MAO) in rat brain mitochondria at different substrate concentrations and at different pHs of the reaction media. The experiments on sensitivity to clorygline and deprenyl showed that the inhibition patterns with PEA as substrate differed markedly at different substrate concentrations: at 10 μM, PEA acted as a specific substrate for type B MAO, but at 50–1000 μM it became a common substrate for both types of MAO. The inhibition patterns were also affected markedly by a small change in pH of the reaction medium, especially when PEA concentrations were 50 and 100 μM: the change in pH from 7.2 to 7.8 resulted in the incresse in the proportion of type A MAO by 20–30 per cent. To investigate the mechanisms of such changes in substrate specificity of PEA, kinetic analyses were carried out at pH 7.2 and 7.8 with the uninhibited, the clorgyline-treated (type B) and the deprenyl-treated (type A) enzyme. The Lineweaver-Burk plots for the uninhibited MAO showed strong substrate inhibition for both pHs, which is more marked at pH 7.8 than at pH 7.2. Pretreatment of the enzyme with 10 −7 M clorgyline resulted in generally similar K m values for PEA to those of the uninhibited enzyme, and the substrate inhibition at pH 7.8 was also stronger than that at pH 7.2. After pretreatment with 10 −7 M deprenyl, the K m values were higher and the V max values were lower than those of the uninhibited or the clorgyline-treated enzyme; there was no or only slight substrate inhibition in these curves. These results suggest that the remarkable changes in substrate specificity observed at different PEA concentrations and at different pHs may be due to the strong substrate inhibition of type B MAO.

Oxidation of synephrine by type A and type B monoamine oxidase.

Synephrine (SP) was found to be a substrate for monoamine oxidase (MAO) in rat brain mitochondria, showing the Km and Vmax values of 250 μM and 32.6 nmoles/mg of protein/30 min respectively. The inhibition studies showed that the SP oxidation was carried out by both type A and type B MAO and a major part of the activity was due to type A MAO.

Percutaneous Bowel Drainage for Jaundice due to Afferent Loop Obstruction Following Pancreatoduodenectomy: Report of a Case

A case of jaundice due to an obstruction of the afferent loop following a pancreatoduodenectomy is presented. The dilated loop of the jejunum was drained percutaneously with a 12-F gastrostomy tube. Localized peritonitis around the puncture site was managed conservatively and the obstructive jaundice improved. The treatment strategy for this type of jaundice is discussed.

Metabolism of acetylpolyamines by monoamine oxidase, diamine oxidase and polyamine oxidase

N1-Monoacetylspermine, N1,N12-diacetylspermine and N1-monoacetylspermidine were found to be good substrates for rat liver polyamine oxidase, but not for rat liver mitochondrial monoamine oxidase. N8-Monoacetylspermidine, monoacetylcadaverine, monoacetylputrescine and monoacetyl-1,3-diaminopropane were oxidized by the monoamine oxidase when the substrate concentration was 10.0 mM, but not by the polyamine oxidase. All the acetylpolyamines except N1,N12-diacetylspermine were also oxidized by hog kidney diamine oxidase although their affinities for the oxidase appeared low. The present data suggest that acetylpolyamines are not easily metabolized in vivo by either monoamine oxidase or diamine oxidase in mammalian tissues although N1-monoacetylspermine, N1,N12-diacetylspermine and N1-monoacetylspermidine are attacked by polyamine oxidase.