Masakazu Oya

Simultaneous determinations of plasma oxytocin and serum placental leucine aminopeptidase (P-LAP) during late pregnancy

1. Plasma oxytocin concentrations were measured by radioimmunoassay in 231 normal pregnant patients. The mean level of the maternal plasma oxytocin increases gradually with advancing pregnancy, culminating in a marked increase during the last two months. 2. Serum placental leucine aminopeptidase (P-LAP) activities and oxytocinase were measured serially in 78 obstetrically normal pregnant patients during late pregnancy. The daily mean P-LAP activity rises progressively during late pregnancy, reaching a relatively high level at 11 days prior to the onset of labor, then fluctuates slightly until the onset of labor. 3. Simultaneous serial measurements of plasma oxytocin levels and serum P-LAP activities in 9 normal pregnant women during late pregnancy show that the onset of labor is preceded by either an increase in the oxytocin level or a decrease in the P-LAP activity.

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.

Characteristics of monoamine oxidase in mitochondria isolated from chick brain, liver, kidney and heart.

Abstract The substrate- and inhibitor-related characteristics of monoamine oxidase (MAO) were studied with mitochondria of chick brain, liver, kidney and heart. The kinetic constants for MAO in these organs were determined, using 5-hydroxytryptamine (5-HT), tyramine and β-phenylethylamine (PEA) as substrates. For all the substrates, the V max values were highest in kidney, followed in decreasing order by brain, liver and heart. For tyramine and PEA, the K m values were lowest in liver, but for 5-HT it was lowest in heart. Inhibition experiments with clorgyline and deprenyl were carried out on mitochondria of the four organs with the three substrates at their K m concentrations. From the plateaus observed of inhibition by clorgyline, it was concluded that 5-HT was oxidized by both types of MAO in mitochondria of all the organs; PEA was fairly specific for type B MAO in brain, liver and kidney, but non-specific in heart. In heart mitochondria, appreciable amounts of the activities toward tyramine and PEA were due to an amine oxidase distinct from mitochondrial MAO; 5-HT, however, was oxidized exclusively by mitochondrial MAO in this organ. The above atypical characteristics in substrate specificity found in chick tissues support the idea that the type A and type B concept cannot be applied uncritically to all tissues from all species.

Simultaneous Determination of Carboxyhemoglobin and Methemoglobin in Victims of Carbon Monoxide Poisoning

Concentrations of methemoglobin (Met-Hb) and carboxyhemoglobin (HbCO) were simultaneously determined in blood samples from the victims of city gas poisoning, fires, and exhaust fumes poisoning. All the samples contained concentrations of HbCO, although the concentrations for victims of city gas poisoning were significantly higher than those for victims of fires or exhaust fumes poisoning. Only negligible amounts of Met-Hb were detected in the samples from cases of city gas poisoning, while 4.9 to 31.6% of Met-Hb were found in all but one (1.8%) of the samples of the other two cases. Heat denaturation was considered to be the main cause of Met-Hb production in cases of fires, and inhalation of oxides of nitrogen in cases of exhaust fumes poisoning.

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.

A practical method for the accurate determination of methemoglobin in blood containing carboxyhemoglobin

Kinetics of the oxidation of carboxyhemoglobin (HbCO) by potassium ferricyanide was studied photometrically in a weakly acid solution. An increase in the absorbance at 630 nm reached a maximum within 10 min when over a 100-fold excess of ferricyanide to hemoglobin iron was used. A slight decrease in the absorbance was observed after completion of the reaction when over a 500-fold excess of the reagent was used. In the presence of 0.4% Sterox SE, the absorbance began to decrease without complete oxidation. From these findings, a simple, rapid and accurate method for the determination of methemoglobin (Met-Hb) in blood was devised. The method was compared with two other methods, using 11 blood samples containing various amounts of HbCO, and proved to be suitable for blood containing elevated HbCO as well as for ordinary blood.

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.

Identification of menstrual blood stains by the electrophoretic pattern of lactate dehydrogenase isozymes

Abstract A new technique is described for the identification of menstrual blood stains by the electrophoretic separation and quantitation of lactate dehydrogenase (LDH) isozymes. In extracts from menstrual blood stains of up to 2-weeks storage, the LDH-4 and LDH-5 fractions (especially the sum of these two) were markedly elevated. No comparable increase was observed in blood stains from other origins. This method is applicable to the examination of blood stains in medicolegal practice.