Pharmacokinetic study of acyl-protected hydroxylamine probe, 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine, for in vivo measurements of reactive oxygen species

1-Acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) is a unique probe for in vivo measurements of reactive oxygen species (ROS), because it is hydrolyzed by esterase to a hydroxylamine form (CP-H), which is oxidized to an electron spin resonance-detectable nitroxyl radical (CP) by a reaction with superoxide anion radical, etc. Although a knowledge of pharmacokinetics is essential for the use of ACP in vivo, such information is limited. We investigated the pharmacokinetics of ACP in mice by examining the time course of the tissue distribution of ACP, CP-H, and CP after intravenous or intraperitoneal injection of ACP. Esterase activity for ACP in tissue homogenates was also measured. The concentration of ACP decreased in all tissues obeying a one-compartment model. ACP was hydrolyzed to CP-H in the liver and kidney predominantly, and the first-pass effect of liver on the hydrolysis of ACP was very large. A homogeneous biodistribution of CP-H was obtained 10 min after the injection of ACP regardless of the injection route, and concentrations remained stable over at least 20 min. Because of these pharmacokinetic properties, ACP should be suitable for the imaging of ROS in animals.     

Whole-body kinetic image of a redox probe in mice using Overhauser-enhanced MRI

Overhauser-enhanced MRI (OMRI) enables visualization of free radicals in animals based on dynamic nuclear polarization. Real-time data of tissue redox status gathered from kinetic images of redox-sensitive nitroxyl radical probes using OMRI provided both anatomic and physiological information. Phantom experiments demonstrated the linear correlation between the enhancement factor and the concentration of a membrane-impermeable probe, carboxy-PROXYL (3-carboxy-2,2,5,5-tetramethyl- pyrrolidine-1-oxyl). Whole-body OMRI images illustrated the in vivo kinetics of carboxy-PROXYL for 25 min. Initial distribution was observed in lung, heart, liver, and kidney, but not brain, corresponding to its minimal lipophilicity. Based on these images (pixel size, 1.33 × 1.33 mm; slice thickness, 50mm), a time-concentration curve with low coefficient of variance (<0.21) was created to assess pharmacokinetic behaviors. A biexponential curve showed a distribution phase from 1 to 10 min and an elimination phase from 15 to 25 min. The α rate constant was greater than the β rate constant in ROIs, confirming that its pharmacokinetics obeyed a two-compartment model. As a noninvasive technique, combining OMRI imaging with redox probes to monitor tissue redox status may be useful in acquiring valuable information regarding organ function for preclinical and clinical studies of oxidative diseases.     

^125I标记白眉蝮蛇毒精氨酸酯酶代谢动力学研究

研究白眉蝮蛇(Agkistrodon halys ussuriensis)毒精氨酸酯酶的代谢动力学,为临床应用提供依据。125I标记的精氨酸酯酶对大鼠一侧颈静脉给药,不同时间从另一测颈静脉取血。5h后处死动物,取各组织、尿液、胆汁和粪便,对各样本的放射性进行测定并拟合时间-放射性关系曲线。代谢动力学拟合曲线符合一室模型,其中生物半衰期T1/2为55.9min,K值为0.0124min-1。125I标记的精氨酸酯酶在体内各组织广泛分布,但有血脑屏障存在,肝、肾和尿液中的放射性比其它组织要高很多,主要通过肝脏降解,肾脏排泄。     

125 I标记白眉蝮蛇毒精氨酸酯酶代谢动力学研究

研究白眉蝮蛇(Agkistrodon halys ussuriensis)毒精氨酸酯酶的代谢动力学,为临床应用提供依据。125I标记的精氨酸酯酶对大鼠一侧颈静脉给药,不同时间从另一测颈静脉取血。5h后处死动物,取各组织、尿液、胆汁和粪便,对各样本的放射性进行测定并拟合时间-放射性关系曲线。代谢动力学拟合曲线符合一室模型,其中生物半衰期T1/2为55.9min,K值为0.0124min-1。125I标记的精氨酸酯酶在体内各组织广泛分布,但有血脑屏障存在,肝、肾和尿液中的放射性比其它组织要高很多,主要通过肝脏降解,肾脏排泄。     

Methyl ester of prostaglandin D2 as a delivery system of prostaglandin D2 into brain

We examined the permeability of the blood-brain barrier to a methyl ester of prostaglandin D2 and the brain uptake was assessed by radioactivity measurements and radioimmunoassay. When the methyl ester (1 mg/kg) was administered intravenously into mice, it was rapidly taken up by the brain (189 ng/g brain at 30 s) and disappeared from the brain with a half-life of 9 s, whereas it was hardly detectable in the blood. The methyl ester transported into the brain was hydrolyzed to prostaglandin D2 and the time course of prostaglandin D2 levels showed an accumulation phase with a peak at 30 s. The total amount of prostaglandin D2 and its methyl ester was 279 ng/g brain at 30 s after injection, corresponding to 0.5% of the administered dose and being 6-times higher than that after prostaglandin D2 injection. The advantage of the methyl ester over prostaglandin D2 for brain uptake was observed at doses higher than 0.2 mg/kg where the methyl ester which escaped from hydrolysis in the blood was taken up more effectively than prostaglandin D2. In in vitro experiments, the esterase activity on the methyl ester was shown to be 20-times greater in the plasma than in the brain homogenate. These results indicate that the esterification of prostaglandin D2 may serve as a good system for the delivery of prostaglandin D2 into the brain.     

A novel ascorbic acid-resistant nitroxide in fat emulsion is an efficient brain imaging probe for in vivo EPR imaging of mouse

The loss of paramagnetism of nitroxide radicals due to reductant reactions in biological systems, places a fundamental time constraint on their application as an imaging probe in in vivo EPR imaging studies. However, in vitro studies of the newly synthesized tetraethyl-substituted piperidine nitroxide radical demonstrated high resistivity to paramagnetic reduction when exposed to ascorbic acid, a common reduction agent in biological systems. In this work we investigated the use of these nitroxides as an imaging probe in EPR imaging of small rodents. 2,2,6,6-Tetraethyl-piperidine nitroxide (TEEPONE) is not highly soluble in aqueous media, thus a lipid-based emulsion system of lecithin was used to solubilize TEEPONE. The obtained solution was homogenous and with low viscosity, allowing smooth intravenous injection into mice tail vein. Acquired three dimensional (3D) EPR images of mouse head clearly showed TEEPONE distributed in all tissues including brain tissues, with an average measurable signal half-life of more than 80 min, thus demonstrating high resistivity to reduction due to ascorbic acid in in vivo animal studies, and the potential for use of this compound in in vivo studies of animal model systems.     

A novel ascorbic acid-resistant nitroxide in fat emulsion is an efficient brain imaging probe for in vivo EPR imaging of mouse

Abstract

The loss of paramagnetism of nitroxide radicals due to reductant reactions in biological systems, places a fundamental time constraint on their application as an imaging probe in in vivo EPR imaging studies. However, in vitro studies of the newly synthesized tetraethyl-substituted piperidine nitroxide radical demonstrated high resistivity to paramagnetic reduction when exposed to ascorbic acid, a common reduction agent in biological systems. In this work we investigated the use of these nitroxides as an imaging probe in EPR imaging of small rodents. 2,2,6,6-Tetraethyl-piperidine nitroxide (TEEPONE) is not highly soluble in aqueous media, thus a lipid-based emulsion system of lecithin was used to solubilize TEEPONE. The obtained solution was homogenous and with low viscosity, allowing smooth intravenous injection into mice tail vein. Acquired three dimensional (3D) EPR images of mouse head clearly showed TEEPONE distributed in all tissues including brain tissues, with an average measurable signal half-life of more than 80 min, thus demonstrating high resistivity to reduction due to ascorbic acid in in vivo animal studies, and the potential for use of this compound in in vivo studies of animal model systems.     

ESR measurement of radical clearance in lung of whole mouse.

Clearance of the nitroxide radicals, hydroxy-TEMPO and carboxy-PROXYL, in whole-mouse lung was directly measured by in vivo ESR. After injecting a nitroxide radical, distribution of the nitroxide radical all over the lung was confirmed by ESR imaging. The ESR signal of hydroxy-TEMPO was reduced in the lung and the clearance obeyed first-order kinetics, whereas the signal of carboxy-PROXYL remained constant. Comparison of the clearance rates of live and dead mice indicated the presence of 2 different clearance systems in the lung: loss of its paramagnetism in the lung, and transfer from alveolar to the blood circulation system.     

Enhanced intraarticular free radical reactions in adjuvant arthritis rats

One of the reasons of rheumatoid arthritis (RA) development is widely recognized the relation of free radical reactions in tissue injuries. The aim of this study was to evaluate the location where in vivo free radical reactions was enhanced in adjuvant arthritis (AA) model rats using in vivo electron spin resonance (ESR)/nitroxyl spin probe technique. The signal decay after intravenous injection of spin probe was enhanced in AA than that in control and suppressed by the pre-treatment of dexamethasone (DXT). Interestingly, the decay in joint cavity occurred prior to paw swelling of AA and suppressed by a simultaneous injection of free radical scavengers, indicating that the enhancement of free radical reactions in joint cavity of AA rats. This technique would be useful tool to determine the location of the enhanced free radical reactions and evaluate the activity of antioxidant medicine with non-invasive real-time measurement.     

Partial purification and characterization of sialate O-acetylesterase from bovine brain

From bovine brain an esterase was purified 2,600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8-6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e.g. alpha-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-4-O-acetylneuraminic acid. Other primary acetyl esters such as 6-O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, however, is a substrate for this esterase. Whereas N-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. N-Acetylated sialic acids are better substrates than the analogous N-glycoloyl derivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced intraarticular free radical reactions in adjuvant arthritis rats

One of the reasons of rheumatoid arthritis (RA) development is widely recognized the relation of free radical reactions in tissue injuries. The aim of this study was to evaluate the location where in vivo free radical reactions was enhanced in adjuvant arthritis (AA) model rats using in vivo electron spin resonance (ESR)/nitroxyl spin probe technique. The signal decay after intravenous injection of spin probe was enhanced in AA than that in control and suppressed by the pre-treatment of dexamethasone (DXT). Interestingly, the decay in joint cavity occurred prior to paw swelling of AA and suppressed by a simultaneous injection of free radical scavengers, indicating that the enhancement of free radical reactions in joint cavity of AA rats. This technique would be useful tool to determine the location of the enhanced free radical reactions and evaluate the activity of antioxidant medicine with non-invasive real-time measurement.     

Development of separable electron spin resonance-computed tomography imaging for multiple radical species: an application to .OH and .NO

A method of separable ESR-CT (electron spin resonance-computed tomography) imaging for multiple radical species was developed and applied to imaging of .OH and .NO. The algorithm was improved by combining filtered back-projection with a modified algebraic reconstruction technique to enhance accuracy and shorten calculation time. With this algorithm, spectral-spatial images of the phantom consisting of 3-carbamoyl-2,2,5,5,-tetramethylpyrrolidine-N-oxyl and 2-phenyl-4,4,5,5,-tetramethylimidazoline-3-oxide-1-oxyl could be obtained in different directions by rotating the spatial axis. The spatial function of individual radicals was extracted by each of the two methods from each spectral-spatial image. The separative 2D images of each radical were individually constructed using the spatial function obtained with the two methods. By comparing the separative images with the phantom sample, the algorithm for separable ESR-CT imaging was established. This ESR-CT technique was combined with L-band ESR spectroscopy and applied to the separative imaging of .OH and .NO, which were spin trapped with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) and Fe(2+)-N-methyl-D-glucamine dithiocarbamate complex, respectively. The ESR signal of DMPO-OH decreased gradually during data acquisition, and the decrease was calibrated by extrapolating the signal intensity to the beginning of data sampling. Both the position and size of the individual images for .OH and .NO were in very good agreement with the findings for the sample.     

Inhibitors of sterol synthesis. Oleate ester of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one as a substrate for pancreatic cholesterol esterase

5 alpha-Cholest-8(14)-en-3 beta-yl-15-one oleate (15-ketosteryl oleate), the oleate ester of a compound with the capacity to lower serum cholesterol, was effectively hydrolyzed by partially purified porcine pancreatic cholesterol esterase with an apparent Km of 0.28 +/- 0.01 mM and a Vmax of 0.62 +/- 0.01 mumol/min per mg protein compared to an apparent Km of 0.19 +/- 0.02 mM and a Vmax of 0.37 +/- 0.02 mumol/min per mg protein for cholesteryl oleate. The 15-ketosteryl oleate was also hydrolyzed by highly purified rat pancreatic cholesterol esterase with an apparent Km of 0.20 +/- 0.01 mM and a Vmax of 86.7 +/- 3.0 mumol/min per mg protein compared to an apparent Km of 0.43 +/- 0.01 mM and a Vmax of 119.8 +/- 2.6 mumol/min per mg protein for cholesteryl oleate. 15-Ketosteryl oleate is, therefore, a good substrate for pancreatic cholesterol esterase from either source. The 15-ketosterol is a weak competitive inhibitor of partially purified porcine pancreatic cholesterol esterase when cholesteryl oleate is the substrate.     

Ganglioside participation in the regulation of free-radical reactions in brain membranes]

The effect of monosialoganglioside GM1 on induced free radical reactions in the synaptosomal and myelin membranes induced by Fe(2+)-H2O2 system was studied. The formation of free radicals was determined by measuring luminol-dependent chemoluminescence. It was found that preincubation of the membranes with GM1 (10(-11)-10(-6) M) and 12 or 12-palmitate, 13-acetate phorbol ester (10(-7)-10(-6) M) or alpha-tocopherol (10(-6) M) results in the decrease of chemiluminescent response. The inhibiting effect of alpha-tocopherol (but not of other compound tested) takes place without any preincubation as well. When the effect of GM1 was studied over a wide range of GM1 concentrations, a biphasic kinetics was observed, the highest per cent of inhibition of chemoluminescence being found at 10(-8) M. The data obtained provide evidence that the inhibition of free radical reactions in the brain membranes by nanomolar concentration of GM1 is not due to its interaction with lipid radicals. The results suggested that the inhibiting effect of GM1 is mediated through signal transduction system.     

Hydrolysis of dolichyl esters by rat liver lysosomes

Dolichyl ester hydrolase activity is broadly distributed among the organs of the rat. The highest activity was found in spleen, brain, lung, and thyroid tissues, whereas this activity is very low in stomach and intestine. The esterase involved is localized to the lumen of lysosomes and, to some extent, in the plasma membranes. Hydrolysis occurs with both alpha-saturated and alpha-unsaturated polyisoprenes esterified with different fatty acids, but the rate of hydrolysis is strongly dependent on the nature of the substrate. The enzyme involved is inhibited by divalent cations, EDTA and EGTA and also by one of the products, dolichol. The esterase is activated by 3-[(3-cholamidopropyl) dimethylammonio]-1-propranesulfonic acid and taurodeoxycholate and inhibited by Triton X-100. Dolichyl esterase activity is completely inhibited by alpha- and beta-naphthyl acetate, phenylmethylsulfonyl fluoride, and beta-chloromethylmercurisulfate. These inhibitors, as well as the pH optimum for dolichyl ester hydrolysis, clearly differentiate the enzyme involved from cholesteryl esterase and triglyceride lipase. Microsomal phospholipase A hydrolyzes dolichyl esters at a slow rate only. In vivo labeling experiments with [3H]mevalonate demonstrated that newly synthesized dolichol is transported in esterified form to the lysosomes, where this lipid is slowly hydrolyzed by the esterase. The possibility is raised that the role of the fatty acyl moiety may be to target dolichol to its final location in the cell.     

Apomorphine: sterotyped behavior and regional distribution in rat brain.

The distribution of apomorphine following subcutaneous injection of 20 mg/kg (as the hydrochloride) was measured spectrofluorometrically in specific regions of rat brain. Measurable concentrations were found in the brain within a few minutes of injection, the drug was still detectable for at least 60 min in all regions, and maximum concentration was observed 20 min after injection. Stereotyped behavior, characteristic of apomorphine action, followed a time course parallel to accumulation of the drug in the brain.     

In vitro hydrolysis of prostaglandin F2α, esters by serum or plasma of different animal species

The methyl ester of prostaglandin F_2α was hydrolyzed by undiluted human serum with a t1/2 of about 5 min, and the ethyl ester was hydrolyzed at one third of this rate. The 2′-propyl and 3′-pentyl esters were de-esterified at a rapid initial rate and at a slower second rate beginning after 10 min incubation. Alterations at the carbon-15 position of prostaglandin F_2α such as 15(S)-15-methyl or the 15-acetate or 15-hexanoate resulted in a reduction in the rate of hydrolysis of the primary esters at carbon-1.Species variation in serum esterase was very large, with rat serum showing activity more than 500 times that of human serum. Rates of hydrolysis in monkey serum were lower than that of human, and activity in the peripheral blood of the dog was extremely low. Plasma esterase activity in the mesenteric blood of the dog was several times higher than that found in the plasma from the peripheral circulation.     

Effects of caffeic acid phenethyl ester and alpha-tocopherol on reperfusion injury in rat brain

Oxygen-derived free radicals have been implicated in the pathogenesis of cerebral injury after ischaemia-reperfusion. Caffeic acid phenethyl ester (CAPE), an active component of propolis extract, exhibits antioxidant properties. The purpose of the present study was to investigate the effects of ischaemia and subsequent reperfusion on rat brain and to investigate the effects of two free radical scavengers, CAPE and alpha-tocopherol, on this in vivo model of cerebral injury. Ischaemia was induced by bilateral occlusion of the carotid arteries for 20 min and reperfusion was achieved by releasing the occlusion to restore the circulation for 20 min. Control rats underwent a sham operation. CAPE at 10 micromol kg(-1) or alpha-tocopherol at 25 micromol kg(-1) was administered intraperitoneally before reperfusion. Reperfusion led to significant increase in the activity of xanthine oxidase and higher malondialdehyde levels in the brain. Acute administration of both CAPE and alpha-tocopherol suppressed ischaemia-reperfusion-induced cerebral lipid peroxidation and injury, but CAPE seems to offer a better therapeutic advantage over alpha-tocopherol.     

Esterases of developing human brain

Abstract The free and bound nonspecific esterases occurring in, respectively, the saline and triton X-100 extracts of adult and developing human brain were studied by starch gel electrophoresis (zymograms). Zymograms of the free esterase fraction visualized with NA as substrate were qualitatively similar at 5-12 days of age to the electrophoretic patterns observed in adult material. In both adult and developing brain, zymograms of bound esterase resembled those of the free enzyme, the major difference being the presence in the former of a slow, broad, anodic zone of diethyl-p-nitrophenyl phosphate-inhibited enzyme. Esterases characteristic of adult white matter and having preferential affinity for alpha-naphthylpropionate, alpha-naphthyl butyrate and alpha-naphthyl valerate were not identified in infant brain until about 4 months postnatal age. A far-moving, anodic enzyme was distinctly evident in zymograms of brains of less than 1 month of age. This enzyme hydrolysed NP, NB, and NV more actively than alpha-naphthyl acetate. It was present in the adult brain but, in contrast to the infant, was no longer electrophoretically-separable from another enzyme which had greater affinity for NA and had previously been designated the A₁₀ band. Quantitative assays demonstrated that the bound esterase increased in cerebral and cerebellar cortex during development. In contrast, the proportion of free to bound esterase showed little change in white matter. Acetylcholinesterase and butyrylcholinesterase zymograms became identical to adult patterns from 1 to 4 months of age. Thiolacetic acid esterase was present at 38 weeks gestation. Some functional and anatomical correlations were attempted in explanation of the biochemical observations.     

A rapid spectrophotometric method for the determination of esterase activity

We have developed a spectrophotometric assay method which continuously records esterase activity at 510 nm by monitoring absorbance changes due to the formation of a diazo dye complex. In our method, α-naphthyl ester substrates are hydrolyzed by enzymatic action to α-naphthol which couples to Fast Blue RR salt (a diazonium salt) forming a diazo dye complex. Our method is unique in directly monitoring the formation of the diazo dye complex without extracting the color of the complex as in other methods that use naphthyl esters and diazo coupling of reaction products. The method appears to be limited to α-naphthyl ester substrates, however, since β-naphthyl esters did not give a linear change in absorbance in the enzymatic reactions tested. With this assay method, one can use a single substrate both to determine esterase units quantitatively in solution and to detect esterase staining activity on gel electrophoresis.