Species difference and characterization of intestinal esterase on the hydrolizing activity of ester-type drugs

The ability of the esterase from intestine was studied for hydrolysis of ester-type drugs during absorption. The intestinal esterase is present in the absorption sites in the intestine and hydrolyzes to a large extent during the absorption. In a study of the dietary effect on intestinal esterase, the esterase activity increased in rats fed a high-fat diet, decreased in those fasted or fed a fat-free diet, whereas the esterase activity in the rat treated with phenobarbital showed no marked change. Thus the esterase from intestinal mucosa appears to be characteristically quite different from hepatic esterase. The esterase from human intestine was characterized and compared with esterase from rats, mice, rabbits, guinea pigs and dogs. There was a difference in the substrate specificity of the esterase and there were significant species differences in the electrophoretic behavior of the enzyme among the species tested. These results indicate that intestinal esterase from humans differs characteristically from esterases in experimental animals.     

Etomidate and plasma esterase activity in man and experimental animals.

No hydrolysis of etomidate in plasma in vitro was detected in samples from man, horse, cow, sheep, guinea pig or white rabbit. Brown rabbits showed a moderate degree of hydrolysis and it was marked in plasma from Wistar rats. In this species, a single enzyme, an alliesterase, participated in the hydrolysis in plasma. Etomidate did not interfere with the hydrolysis of procaine by plasma pseudocholinesterase in man.     

Species difference in nisoldipine oxidation activity in the small intestine

Species difference in nisoldipine oxidation activities was investigated using small intestinal microsomes of rats, guinea pigs, dogs, monkeys and humans. The oxidation activities were estimated by measuring metabolites formation (BAY o 3199 and BAY r 9425) of nisoldipine. For the preparation of small intestinal microsomes of various animal species, the effect of protease inhibitors was preliminarily investigated. The formation of BAY o 3199 significantly increased in the rat small intestinal microsomes prepared with trypsin inhibitor. Using the trypsin inhibitor-treated small intestinal microsomes of various animals, metabolic intrinsic clearances (CL(int, in vitro)) for BAY o 3199 and BAY r 9425 formations were estimated based on an Eadie-Hofstee plot. The total CL(int,in vitro) estimated by the sum of CL(int, in vitro) for both formations in the small intestines of all species was much lower than that in the liver. There was a marked species difference in the nisoldipine oxidation activities in the small intestines, with the rank order being humans=monkeys>dogs>rats>guinea pigs, versus the following order in the liver: rats>monkeys=guinea pigs>humans>dogs. The formations of both BAY o 3199 and BAY r 9425 in the human intestinal microsomes were inhibited by pretreatment with troleandomycin (TAO) and antiserum against CYP3A4. Similar inhibition profile by TAO was obtained from the monkey intestinal microsomes. These results implied that monkeys would be a good predictor of human small intestinal metabolism for CYP3A4 substrates.     

RP—HPLC法测定正常人血浆中阿司匹林酯酶活性

目的:建立同时测定血浆中阿司匹林和水杨酸浓度的高效液相色谱法,采用水杨酸浓度与阿司匹林和水杨酸浓度之和的比值表示阿司匹林酯酶的活性。方法:色谱条件:采用 Phenomenex C_(18)(4.6 mm×150 mm,5μm)色谱柱,流动相为乙腈-0.072 mmol·L~(-1)磷酸(22:78,v/v),流速1.0 mL·min~(-1),检测波长228 nm,柱温30℃。测定阿司匹林和水杨酸浓度,计算水杨酸浓度与阿司匹林和水杨酸浓度之和的比值,绘制概率分布直方图。结果:100名健康人血浆阿司匹林酯酶活性呈正态性分布;33名健康女性和67名健康男性血浆中阿司匹林酯酶活性均显示呈正态性分布。结论:本方法简便、准确、快速,适合于体外血浆中阿司匹林酯酶活性测定研究。     

Molecular cloning and expression of chicken neuropathy target esterase activity domain

Neuropathy target esterase (NTE) is recognized as the initial target during the process of organophosphate-induced delayed neuropathy (OPIDN). Adult hens are usually used as the animal model for experimental studies of OPIDN. However, the molecular cloning and characteristics of chicken NTE is unknown. On the basis of the predicted chicken NTE gene middle sequence and its 3'-end cDNA sequence, we cloned the gene sequence of chicken NTE activity domain (cNEST). The cloned cNEST gene is 1740 base pairs and encodes 579 amino acids, showing high identity with human and mouse NEST at amino acid level. The serine hydrolase signature motif GXSXG and the patatin domain were found in the cNEST sequence. Over-expression of cNEST tagged with enhanced green fluorescence protein (EGFP) in monkey kidney COS7 cells increased NTE activity significantly. The increased extent is similar to that in over-expression of hNEST cells. Moreover, over-expression of cNEST led to an accumulation of partial cNEST on the cytoplasmic surface of the endoplasmic reticulum. Partial cNEST located in the cytoplasm by comparing the distribution of cNEST and hNEST. After inhibition with different concentrations of mipafox for 60min, the calculated I(50) value was 4.95microM for COS7 cells over-expressing cNEST. These results firstly confirmed that the protein sequence, enzymatic activity, and cellular location of cNEST are very similar to that of hNEST at molecular level. The inhibition curve of mipafox on NTE activity of cNEST in mammalian cells was also reported here.     

Human liver and plasma aspirin esterase

The plasma, in addition to the liver, is a major site of hydrolysis of aspirin. Human plasma and liver aspirin esterase activities in samples from a group of patients varied over a two fold range and there was a significant correlation between individual plasma and liver activities. Human liver aspirin esterase was present in the cytosolic and microsomal fractions. Cytosolic and microsomal enzymes had different activities and apparent affinities for aspirin.     

绿原酸及其同分异构体对人血浆阿司匹林酯酶活性的影响

目的探究绿原酸及其同分异构体对人血浆阿司匹林酯酶活性的影响。方法以高效液相色谱法测定阿司匹林酯酶体外培养体系中水杨酸(SA)和阿司匹林(ASA)的浓度,以CSA/(CASA+CSA)反映阿司匹林酯酶的活性。通过空白对照组和添加单体成分的实验组间阿司匹林酯酶活性的变化来分析各成分对阿司匹林酯酶活性的影响。结果实验组与空白对照组之间差异没有显著性。结论在本实验浓度下,绿原酸及其同分异构体对酯酶影响不明显,为临床同时使用阿司匹林与含有该类化合物的中药或中药制剂提供理论依据。     

Effects of inhaled cholinesterase inhibitors on bronchial tonus and on plasma and erythrocyte acetylcholine esterase activity in rats

Young adult male and female Wistar rats were inhalationally exposed head-only for 1 or 4 h to different anticholinesterase aerosols. The compounds tested were dichlorvos, fenamiphos, methamidophos, parathion, a pyrimidine thiophosphate and the carbamate propoxur. These compounds are direct or indirect inhibitors of cholinesterase activity. Immediately after termination of exposure to the compounds, the rats were anesthetized with barbiturate and subjected to pulmonary function tests. An acetylcholine provocation test was performed to correlate the effect of the cholinesterase inhibition and lung resistance. The results basically revealed that by inhalation exposure bronchoconstriction in the absence of acetylcholine provocation did not occur at toxicologically significant doses of the pesticides. An increase in lung resistance was observed only after provocation. However, measurements of plasma cholinesterase activity proved to be more sensitive than the provocation test. With regard to their diagnostic value, the results of the reported study may be summarized as follows (beginning with the most sensitive parameter): plasma cholinesterase activity depression greater than or equal to acetylcholine-induced bronchoconstriction greater than or equal to cholinergic symptoms greater than erythrocyte cholinesterase activity depression greater than pulmonary resistance without acetylcholine provocation.     

Species differences in avian serum B esterases revealed by chromatofocusing and possible relationships of esterase activity to pesticide toxicity

Serum cholinesterase (BChE) and carboxylesterase (CbE) activities were investigated in ten species of birds. Multiple forms of serum BChE and CbE were also separated by chromatofocusing. Higher CbE activity and a wider range of CbE and BChE forms were present in the sera of omnivorous/herbivorous birds than carnivores. Omnivores/herbivores studied were the starling, house sparrow, tree sparrow, pigeon, partridge and magpie. Serum CbE activities of these species ranged from 0.46 to 2.93 mumol/min/mL with 2-6 forms separated by chromatofocusing. 0-6 forms of BChE were separated by the same method. The serum CbE activities of the little owl, tawny owl, barn owl and razorbill ranged from 0.19 to 0.58 mumoles/min/mL with 0-2 forms separated by chromatofocusing. No ChE forms were present within the pH gradient. These results may be significant in contributing to the understanding of the selective toxicity of organophosphorus and carbamate pesticides.     

Species difference in biliary excretion of methylmercury

Species difference in the biliary excretion of methylmercury was studied in male rats, mice, rabbits and guinea pigs. The rates of mercury excretion (% dose/2 hr) into the bile of the rats, mice, rabbits and guinea pigs during the 2 hr from 2 to 4 hr after the administration of methylmercury were 0.61, 0.091, 0.036 and 0.019, respectively. These results suggest that biliary excretion and enterohepatic circulation of methylmercury in the latter three species may not influence the fate of this compound as significantly as in rats. Most of the methylmercury excreted into the bile of rats was bound to glutathione (GSH). In the mouse bile, 40% of the methylmercury was bound to GSH and the rest was found in a fraction eluted at the void volume of the Sephadex G-15 column. However, in the case of the rabbits and guinea pigs, methylmercury-GSH was scarcely detectable in the bile and almost all of the methylmercury was eluted at the void volume of the column.     

Species difference in temperature dependence of cardiac (Na+ + K+)-ATPase activity

Cardiac (Na+ + K+)-ATPase from several species of animals were comparatively studied with respect to their molecular form and temperature dependence. The molecular weight of the catalytic subunit varied a little among species, but the difference did not correlate with the sensitivity of the enzyme to ouabain inhibition. Analysis of Arrhenius plots of the activities of the enzymes showed that enzymes showing break points of 24-25 degrees were relatively insensitive to ouabain inhibition whereas those enzymes with break points of 29-31 degrees were much more sensitive to the glycoside. This suggests that there is a difference in the interaction of cardiac (Na+ + K+)-ATPase with lipids between the ouabain-sensitive and -insensitive animals.     

NTE酯酶活力域真核表达载体的构建及其表达

目的构建人神经病变靶标酯酶（NTE）酯酶活力域（NESP）真核表达载体，并在细胞中表达。方法利用含有特定酶切位点的引物，通过PCR扩增出其酯酶活力域，经T载体克隆测序正确后，双酶切回收cDNA片段定向插入到pcDNA3．1（＋）中，通过酶切鉴定其真核表达载体的构建。然后将其转染到真核细胞中，通过NTE活力测定，检测其表达效果。结果经PCR获得了NTE的1．6kb酯酶活力域的cDNA片段，T载体克隆后测序证实完全正确，然后克隆到真核表达载体中获得了NTE酯酶活力域真核表达载体pcDNA—NEST。瞬时转染到COS7和SH-SY5Y细胞中，NTE的活力与对照细胞相比显著提高。结论成功构建了NEST真核表达载体，并在哺乳动物细胞中表达。     

Epoxide hydrolase activity in human skin.

1 Epoxide hydrolase (EH) activity was measured in biopsied skin (n = 42) using 7-[H3]-styrene oxide as substrate, and separation of the products by high performance liquid chromatography. 2 EH activity (mean +/- s.d.) was present in separated epidermis (139 +/- 105 pmol glycol formed mg-1 min-1) and dermis (165 +/- 120 pmol glycol formed mg-1 microsomal protein min-1). 3 Whole skin EH activity (mean +/- s.d.) varied widely (433 +/- 254 pmol glycol formed mg-1 microsomal protein min-1) 4 No significant difference in EH activity was observed in skin from breast, penis and leg. 5 Skin EH activity does not appear to contribute significantly to the systemic metabolism of epoxide, but may be important in determining the effects of epoxides formed within the epidermis.     

Human plasma paraoxonase (HuPON1): an anti-atherogenic enzyme with organophosphate hydrolase activity

Human plasma paraoxonase (PON1) is a calcium-dependent organophosphate-hydrolase. In plasma, PON1 is bound to high-density lipoproteins (HDL). PON1 prevents the oxidation of LDL and scavenges oxidized phospholipids, thus protecting from atherogenesis. Improving the prophylaxis and treatment of organophosphate (OP) poisoning is a public health concern that also interests the civilian safety and the military. In this context, engineering and formulation of enzymes able to scavenge or hydrolyze OPs, such as PON1, are widely studied. Determination of the PON1 three-dimensional structure is a key step toward improvement of the enzyme functional properties.     

Age dependency of esterase activity in rat and human keratinocytes

The age and species dependent characteristics of cutaneous esterase activity were examined in cultured keratinocytes of neonatal and adult humans and of rats at the age of 1, 3, 10, and 50 d. The existence of esterases was characterized using fluorescein-5-isothiocyanate diacetate under a confocal laser scanning microscope. In vitro hydrolysis of ethyl nicotinate (EN), an esterified prodrug of nicotinic acid, was investigated in homogenate of cultured keratinocytes, and the Michaelis-Menten parameters (V(max) and K(m)) of EN were evaluated. Together with development and growth of rats and humans, V(max) and V(max)/K(m) increased drastically, suggesting that esterases in keratinocytes develop markedly during the growth process. The affinity parameter, K(m), was almost the same among the ages in each species. These findings in cultured keratinocytes corresponded with our previous report using dissected skin specimens. Species differences in V(max), V(max)/K(m) and K(m) were also observed, and these parameters of EN hydrolysis in rats were significantly higher than that in humans. In conclusion, cultured keratinocytes can be an advantageous method with which to estimate cutaneous activation of ester prodrugs in humans and during the growth process.     

Age-related changes in esterase activity in rabbit eyes

Thus far, the influence of age on the bioavailability of topically applied ophthalmic drugs has been studied by considering age-dependent changes in membrane permeability and protein binding. The specific objective of this research was to determine variation of ocular esterase activity with age and pigmentation of the rabbit. This was achieved by monitoring the fluorescence intensity as a function of time upon incubating ocular tissue homogenates of albino and pigmented rabbits of various age groups with α-naphthyl acetate, both in the presence and absence of esterase inhibitors. In general, esterase activity was the highest in the iris-ciliary body followed by the cornea and then the aqueous humor. Of the age groups studied, peak ocular esterase activity was reached at 6 weeks in the albino rabbit, and beyond 6 weeks in the pigmented rabbit. In the 6-week-old group the albino rabbit exceeded the pigmented rabbit in ocular esterase activity, whereas in the 12-week-old group it was the pigmented rabbit that exceeded the albino rabbit in ocular esterase activity. The conclusion was that in establishing a dosage regimen, age-dependent changes in ocular esterase activity must be considered in conjunction with age-dependent changes in the other factors that also help determine the amount of active drug ultimately reaching its receptor.     

Studies on the alpha-glucoside hydrolase inhibitor, adiposin. III. alpha Glucoside hydrolase inhibitory activity and antibacterial activity in vitro

Adiposin-1 and -2 exhibited potent inhibitory activities against alpha-amylase, human salivary alpha-amylase and disaccharidases isolated from porcine small intestine. The effect of adiposin-1 and -2 on hydrolysis of glucoamylase was non-competitive. Adiposin showed antimicrobial activities against some Gram-positive bacteria, Gram-negative bacteria belonging to Enterobacteriaceae, Some anaerobic bacteria and some phytopathogenic fungi, and showed a synergistic effect on the antibacterial activity with some maltooligosaccharides. However, these antibacterial activities were suppressed by addition of various other saccharides.