nitrophenols and phenylacetic-acid

The capacity of human, minipig, and rat skin and liver subcellular fractions to hydrolyze the anesthetic ester procaine was compared with carboxylesterase substrates 4-methylumbelliferyl-acetate, phenylvalerate, and para-nitrophenylacetate and the arylesterase substrate phenylacetate. Rates of procaine hydrolysis by minipig and human skin microsomal and cytosolic fractions were similar, with rat displaying higher activity. Loperamide inhibited procaine hydrolysis by human skin, suggesting involvement of human carboxylesterase hCE2. The esterase activity and inhibition profiles in the skin were similar for minipig and human, whereas rat had a higher capacity to metabolize esters and a different inhibition profile. Minipig and human liver and skin esterase activity was inhibited principally by paraoxon and bis-nitrophenyl phosphate, classical carboxylesterase inhibitors. Rat skin and liver esterase activity was inhibited additionally by phenylmethylsulfonyl fluoride and the arylesterase inhibitor mercuric chloride, indicating a different esterase profile. These results have highlighted the potential of skin to hydrolyze procaine following topical application, which possibly limits its pharmacological effect. Skin from minipig used as an animal model for assessing transdermal drug preparations had similar capacity to hydrolyze esters to human skin.

Topics: Animals; Enzyme Inhibitors; Esterases; Esters; Female; Humans; Hydrolysis; Liver; Loperamide; Male; Microsomes, Liver; Molecular Structure; Nitrophenols; Paraoxon; Pentanoic Acids; Phenylacetates; Phenylmethylsulfonyl Fluoride; Procaine; Rats; Rats, Wistar; Skin; Swine; Swine, Miniature; Umbelliferones

Skin esterases serve an important pharmacological function as they can be utilised for activation of topically applied ester prodrugs. Understanding the nature of these enzymes, with respect to their role and local activity, is essential to defining the efficacy of ester prodrugs. Minipigs are used as models to study the kinetics of absorption of topically applied drugs. Their skin has structural properties very similar to human skin. However, regional distribution differences in esterase activity from site-to-site could influence cross-species extrapolation. Investigation of the regional site variation of minipig skin esterase activity will facilitate standardization of topically applied drug studies. Furthermore, the characterization of regional skin variation, will aid in translation of minipig results to better predictions of human esterase activity. Here we report the variation in rates of hydrolysis by minipig skin taken from different regional sites, using the esterase-selective substrates: phenyl valerate (carboxylesterase), phenyl acetate (arylesterase) and p-nitrophenyl acetate (general esterase). Skin from ears and back of male minipig showed higher activity than female. Skin from minipig ears and the back showed the highest level of esterase activity and was similar to human breast skin used in vitro absorption studies. These results suggest that skin from the minipig back is an appropriate model for preclinical human skin studies, particularly breast skin. This study supports the use of the minipig, with topical application to the back, as a model for the investigation of pharmacokinetics and metabolism of ester prodrugs.

Topics: Animals; Carboxylesterase; Carboxylic Ester Hydrolases; Cytosol; Drug Evaluation, Preclinical; Esterases; Female; Hydrolysis; Kinetics; Liver; Male; Microsomes; Models, Animal; Nitrophenols; Phenylacetates; Reproducibility of Results; Sex Factors; Skin; Substrate Specificity; Swine; Swine, Miniature; Valerates

Paraoxonase is an esterase that hydrolyzes organophosphate compounds. The enzyme is associated with HDL and could protect LDL against peroxidation, which suggests a possible involvement of paraoxonase in the antiatherogenic properties of HDL. Paraoxonase activity has been shown to be low in patients with myocardial infarction, diabetes mellitus, or familial hypercholesterolemia. Because cardiovascular disease is the main cause of death in chronic renal failure, serum paraoxonase activity was measured by spectrophotometry using three synthetic substrates (phenyl acetate, paraoxon, and 4-nitrophenyl acetate) in 305 patients with kidney disease, including 47 patients with non-end-stage chronic renal failure, 104 patients treated with hemodialysis, 22 patients treated with peritoneal dialysis, and 132 renal transplant patients. Patients were compared with two groups of aged-matched control subjects (total number = 195). Especially with 4-nitrophenyl acetate, paraoxonase activity was lower in patients with some degree of renal insufficiency (chronic renal failure [P < 0.05], chronic hemodialysis [P < 10(-4)], chronic peritoneal dialysis [P < 10(-4)]) than in control subjects. In transplant patients, paraoxonase activity was not found to be different from that in control subjects. The decrease of paraoxonase activity and thus the reduction of its antiatherogenic properties in renal failure could be an essential factor of premature vascular aging, especially when dialysis is used. Renal transplantation seems to restore paraoxonase activity.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Arteriosclerosis; Aryldialkylphosphatase; Esterases; Female; Humans; Kidney Failure, Chronic; Kidney Transplantation; Male; Middle Aged; Nitrophenols; Paraoxon; Peritoneal Dialysis; Phenylacetates; Reference Values; Renal Dialysis

The kinetics of release of 4-nitrophenol were followed by stopped-flow spectrophotometry with two 4-nitrophenyl ester substrates of penicillin G acylase from Kluyvera citrophila. With the ester of acetic acid, but not of propionic acid, there was a pre-steady-state exponential phase, the kinetics of which were inhibited by phenylacetic acid (a product of hydrolysis of specific substrates) to the extent predicted from Ki values. This was interpreted as deriving from rapid formation (73 mM-1.s-1) and slow hydrolysis (0.76 s-1) of an acetyl derivative of the side chain of the catalytic-centre residue Ser-290. With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of the wild-type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were 11.1 mM-1.s-1 and 0.051 s-1 respectively. The ratio of these rate constants was three times that for the wild-type enzyme, suggesting that the mutant is less impaired in the rate of formation of an acetyl-enzyme than in its subsequent hydrolysis.

Topics: Acylation; Catalysis; Enterobacteriaceae; Hydrolysis; Kinetics; Mutation; Nitrophenols; Penicillin Amidase; Penicillin G; Phenylacetates; Phenylpropionates; Spectrophotometry

In contrast with the general thought that penicillin G acylases (PGAs) were only able to hydrolyse amides or esters of higly hydrophobic acids, we have demonstrated that the PGA from Kluyvera citrophila catalysed the hydrolysis of 4-nitrophenyl esters of acetic, propionic, butyric and valeric acids. Values of kcat. and kcat./Km were greatest for the first compound and less than values for benzylpenicillin by factors of 30 and 7, respectively. 4-Nitrophenyl acetate was hydrolysed faster than 2-nitrophenyl acetate but slower than phenyl acetate. The pH dependence of the reaction was similar to that of benzylpenicillin. Several experiments showed that hydrolysis of 4-nitrophenyl acetate was not catalysed by contaminating esterase activity. The implications for the structural basis of substrate binding are discussed. These substrates open, for the first time, a way to investigate the kinetic parameters of PGA at the presteady-state and provides a new perspective about the role of PGA in nature.

Topics: Esters; Kinetics; Kluyveromyces; Nitrophenols; Penicillin Amidase; Penicillin G; Phenylacetates; Recombinant Proteins; Substrate Specificity

The hydrolysis of paraoxon (POX), phenylacetate (PA) and beta-naphthylacetate (BNA) was studied in human serum. Based upon correlations between enzyme activities, upon reversible inhibition by EDTA and upon progressive inhibition by iso-OMPA, tabun, eserine and bis-4 nitrophenylphosphate, the following conclusions were drawn about the number and specificity of enzymes involved in the hydrolysis. Two paraxonases hydrolyse paraoxon: one sensitive and the other insensitive to EDTA. The EDTA-sensitive paraoxonase also hydrolysed BNA. The EDTA-insensitive hydrolysis of BNA and PA was attributed to a serine esterase. The EDTA-sensitive hydrolysis of PA is probably due to more than one enzyme, which might be an arylesterase and a carboxylesterase.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aryldialkylphosphatase; Cholinesterase Inhibitors; Cholinesterases; Edetic Acid; Esterases; Female; Humans; Hydrolysis; Kinetics; Male; Middle Aged; Naphthaleneacetic Acids; Nitrophenols; Organophosphates; Organophosphorus Compounds; Paraoxon; Phenylacetates; Phosphoric Monoester Hydrolases; Physostigmine; Sensitivity and Specificity; Substrate Specificity; Tetraisopropylpyrophosphamide; Thiocholine

A new method for phenotyping human serum arylesterase (EC 3.1.1.2) is described and evaluated. The aromatic esters, phenyl acetate and 4-nitrophenyl acetate, were compared as substrates for spectrophotometric measurement of arylesterase activity. A method for arylesterase phenotyping, based upon inhibition of the enzymatic hydrolysis of 4-nitrophenyl acetate by phenyl acetate, was developed. The method was applied to serum samples from 158 blood donors and showed a distinct separation of the three phenotypes defined by a reference method based on the ratio of paraoxonase activity to arylesterase activity using paraoxon and phenyl acetate as substrates. The method was adapted to a Cobas-Fara centrifugal analyser.

Topics: Alleles; Blood Donors; Carboxylic Ester Hydrolases; Humans; Hydrolysis; Isoenzymes; Kinetics; Nitrophenols; Phenotype; Phenylacetates; Substrate Specificity

Topics: Barbiturates; Chlorides; Chlortetracycline; Hypnosis; Hypnotics and Sedatives; Nitrophenols; Phenylacetates; Uncoupling Agents