Human Serum Paraoxonase Research Articles

The human paraoxonase‐1 phenotype modifies the effect of statins on paraoxonase activity and lipid parameters

Human serum paraoxonase-1 (PON1) protects lipoproteins against oxidation by hydrolysing lipid peroxides in oxidized low-density lipoprotein, therefore it may protect against atherosclerosis. One of the two common PON1 gene polymorphisms within the PON1 gene is the Q192R, whose prevalence can be estimated by phenotype distribution analysis. The goal of this study was to clarify the role of PON1 phenotypes on the effect of three different statins on paraoxonase activity and lipid parameters. One hundred and sixty-four patients with type IIb hypercholesterolaemia were involved in the study. We examined the effect of 10 mg day(-1) atorvastatin, 10/20 mg day(-1) simvastatin and 80 mg day(-1) extended-release fluvastatin treatment on lipid levels and paraoxonase activity in patients with different PON1 phenotypes. The phenotype distribution of PON1 was determined by the dual substrate method. Three months of statin treatment significantly increased paraoxonase activity in every statin-treated group. In patients with AB+BB phenotype, statin treatment was significantly more effective on paraoxonase activity than in the AA group. Statin treatment more effectively decreased triglyceride levels in the AB+BB group compared with the AA group in the whole study population and in the simvastatin-treated group. Atorvastatin treatment was significantly more effective on apolipoprotein B levels in patients with AB+BB phenotype than in the AA phenotype group. The PON1 phenotype may be a novel predictive factor for the effectiveness of statin treatment on PON1 activity and serum lipid levels; however, different types of statins may exert different effects on these parameters.

Intravenous anesthetics inhibit human paraoxonase-1 (PON1) activity in vitro and in vivo

Objectives Here we evaluated the in vitro and in vivo effects of the intravenous anesthetics, etomidate, propofol, and ketamine, on the activity of human serum paraoxonase (hPON1). Design and methods hPON1 was purified from human serum using simple chromatographic methods, including DEAE–Sephadex anion exchange and Sephadex G-200 gel filtration chromatography. Results The three anesthetics dose-dependently decreased in vitro hPON1 activity. Inhibition mechanisms are: etomidate was noncompetitive, propofol was competitive, and ketamine was uncompetitive. In vivo studies were performed on five patients for each drug. PON1 was significantly inhibited by 0.3 mg/kg etomidate ( p < 0.05), 2 mg/kg propofol ( p < 0.001), and 1 mg/kg ketamine ( p < 0.05) for up to 5 min following intravenous administration. Conclusions Our results showed that anesthetics significantly inhibit hPON1 activity, both in vitro and in vivo, with rank order etomidate > propofol > ketamine in vitro, and propofol > etomidate > ketamine in vivo.

Pitavastatin induces PON1 expression through p44/42 mitogen-activated protein kinase signaling cascade in Huh7 cells

It has been shown that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors have pleiotropic effects and that human serum paraoxonase (PON1) inhibits the oxidative modification of low-density lipoprotein. We investigated the effects of pitavastatin on PON1 gene promoter activity and PON1 protein expression through the activation of mitogen-activated protein (MAP) kinase signaling cascades in cultured Huh7 cells. Both PON1 gene promoter activity and PON1 protein expression were elevated by pitavastatin stimulation. Pitavastatin phosphorylated p44/42 MAP kinase. The effects of pitavastatin on PON1 promoter activity and PON1 protein expression were attenuated by PD98059. The cotransfection of Sp1 expression vector increased PON1 promoter activity, and mithramycin suppressed pitavastatin-enhanced PON1 promoter activity. The latter activity was attenuated by cotransfection with the expression vector of sterol regulatory element-binding protein-2 (SREBP-2) with mutated p44/42 MAP kinase specific phosphorylation sites. Pitavastatin increased the Sp1-PON1 DNA complex and this effect was attenuated by PD98059. These observations suggest that pitavastatin phosphorylates p44/42 MAP kinase and then activates the transcription of PON1 gene and increases the PON1 protein expression in Huh7 cells. Furthermore, we speculate that pitavastatin affects both the phosphorylation of SREBP-2 and the Sp1 binding to PON1 DNA through the activation of p44/42 MAP kinase signaling cascade.

POLYMORPHISMS IN TWO PARAOXONASE GENES- PON1 AND PON2 IN ANGIOGRAPHYCALLY ASSESSED CEREBROVASCULAR STENOSIS

Background. Human serum paraoxonase (PON1) is an esterase coded by pon1 gene on chromosome 7 and linked with two additional PON-like genes (pon2 and pon3). Alterations of enzyme anti- atherogenic activity due to polymorphisms in the pon genes my be one of the risk factors for the development of atherosclerosis. Aim. To examine the distribution of polymorphisms of pon1 and pon2 genes in patients with angiografically assessed severe stenosis of cerebral arteries and matched control subjects. Methods. The blood samples were recruited from 40 healthy individuals and 29 patients with >70 % of arterial carotides stenosis. DNA was extracted and used for amplification of the target regions by polymerase chain reaction (PCR). Amplified products were digested with BspPI (Q192R), Hin 1II (L55M), BsrBI (-108C>T) and DdeI (S311C) restriction enzymes and determinated by restriction fragment length polymorphism (RFLP) procedure. Results. Genotype frequencies of pon1 and pon2 genes polymorphisms founded in cases vs. controls were: 15 (0, 52) QQ, 11 (0, 38) QR, 3 (0, 10) RR vs. 23 (0, 58) QQ, 15 (0, 37) QR, 2 (0, 05) RR for Q192R ; 13 (0, 45) LL, 10 (0, 34) LM, 6 (0, 21) MM vs. 16 (0, 40) LL, 18 (0, 45) LM, 6 (0, 15) MM for L55M ; 6 (0, 21) CC, 12 (0, 41) CT ; 11 (0, 38) TT vs. 8 (0, 20) CC, 24 (0, 60) CT, 8 (0, 20) TT for – 108C>T and 1 (0, 03) CC, 8 (0, 28) CS, 20 (0, 69) SS vs. 0 (0, 00) CC, 22 (0, 55) CS, 18 (0, 45) SS for S311C. Conclusions. The results of our study show no significant differences in genotype or allele frequencies between patients with severe stenosis of cerebral arteries and controls.

Increased methionine sulfoxide content of apoA-I in type 1 diabetes

Cardiovascular disease is a major cause of morbidity and premature mortality in diabetes. HDL plays an important role in limiting vascular damage by removing cholesterol and cholesteryl ester hydroperoxides from oxidized low density lipoprotein and foam cells. Methionine (Met) residues in apolipoprotein A-I (apoA-I), the major apolipoprotein of HDL, reduce peroxides in HDL lipids, forming methionine sulfoxide [Met(O)]. We examined the extent and sites of Met(O) formation in apoA-I of HDL isolated from plasma of healthy control and type 1 diabetic subjects to assess apoA-I exposure to lipid peroxides and the status of oxidative stress in the vascular compartment in diabetes. Three tryptic peptides of apoA-I contain Met residues: Q(84)-M(86)-K(88), W(108)-M(112)-R(116), and L(144)-M(148)-R(149). These peptides and their Met(O) analogs were identified and quantified by mass spectrometry. Relative to controls, Met(O) formation was significantly increased at all three locations (Met(86), Met(112), and Met(148)) in diabetic patients. The increase in Met(O) in the diabetic group did not correlate with other biomarkers of oxidative stress, such as N(epsilon)-malondialdehyde-lysine or N(epsilon)-(carboxymethyl)lysine, in plasma or lipoproteins. The higher Met(O) content in apoA-I from diabetic patients is consistent with increased levels of lipid peroxidation products in plasma in diabetes. Using the methods developed here, future studies can address the relationship between Met(O) in apoA-I and the risk, development, or progression of the vascular complications of diabetes.

In vitro inhibition of the paraoxonase from human serum with sulfonamide

This study was conducted to determine the in vitro effects of sulfonamide on human serum paraoxonase (PON1) activity. The enzyme was purified by two-step using ammonium sulfate precipitation and sepharose-4B-L-tyrosine-1-napthylamine hydrophobic interaction chromatography. Sulfonamide was an effective inhibitor on purified human serum PON1 activity for phenylacetate and paraoxon as substrates with IC50 values of 0.22 and 0.81 mM, respectively. The kinetics of interaction of sulfonamide with the purified enzyme indicated a different inhibition pattern for two substrates. Sulfonamide showed a non-competitive inhibition with Ki of 0.0037 ± 0.0009 mM for phenylacetate and competitive inhibition with Ki of 0.0057 ± 0.0002 mM for paraoxon.

Purification of Human Serum Paraoxonase and Effect of Acetylsalicylic Acid on Paraoxonase Activity

Paraoxonase was purified from human serum using sepharose-4B-l-tyrosine-1-napthylamine affinity chromatography. This enzyme was purified 1797-fold. SDS-polyacrylamide electrophoresis of the enzyme indicates a single protein staining band with an apparent Mr of 43 kDa. The kinetic properties of the purified enzyme were determined. The V.max and Km are 231.5 μ/mg protein (EU) and 3.96 mM using substrate paraoxon respectively. The effect of acetylsalicylic acid on purified human serum paraoxonase has been investigated in vitro.. The inhibition and activation effects of acetylsalicylic acid on serum paraoxonase activity were measured spectrophotometrically using paraoxon as the substrate. The phenotypic distribution of paraoxonase was determined using the dual substrate paraoxon and phenyl acetate as substrates. It was observed that acetylsalicylic acid inhibited human serum paraoxonase activity. In addition, an in vivo. study was performed for acetylsalicylic acid in Sprague-Dawley rats. It was demonstrated that paraoxonase in the serum of Sprague-Dawley rat was activated, but activity in liver and heart was significantly inhibited.

Interethnic Variability of Plasma Paraoxonase (PON1) Activity towards Organophosphates and PON1 Polymorphisms among Asian Populations━A Short Review

Organophosphate (OP) poisoning is a progressively worrying phenomenon as worldwide pesticide production and consumption has doubled. On average, WHO estimates that 3% of agricultural workers in developing Asian countries suffer an episode of pesticide poisoning every year. Furthermore, the threat of OP usage in terrorism is existent, as seen by the subway tragedy in Tokyo in 1995 where sarin was used. Despite these alarming facts, there is currently no global system to track poisonings related to pesticide use. Human serum paraoxonase (PON1) is the enzyme that hydrolyses OP compounds. Serum PON1 levels and activity vary widely among different ethnic populations. Two commonly studied polymorphisms of PON1 are PON1Q192R and PON1L55M. PON1R192 hydrolyses paraoxon faster than PON1Q192 but hydrolyses diazoxon, sarin and soman eight times slower, and vice versa. PON1M55 has lower plasma levels of PON1 than PON1L55. As the prevalence of the different alleles and genotypic distribution vary between the Asian populations we studied, we propose the necessity to study PON1 polymorphisms and its role in OP toxicity in Asian populations. This would help safeguard the proper care of agricultural workers who might be affected by OP poisoning, and alert relevant anti biological terrorism agencies on possible risks involved in the event of an OP attack and provide effective counter measures.

A Gas Chromatographic-Mass Spectrometric Approach to Examining Stereoselective Interaction of Human Plasma Proteins with Soman*

The organophosphorus (OP) nerve agent soman (GD) contains two chiral centers (a carbon and a phosphorus atom), resulting in four stereoisomers (C+P+, C-P+, C+P-, and C-P-); the P- isomers exhibit a mammalian toxicity that is approximately 1000-fold greater than that of the P+ isomers. The capacity to assess the binding or hydrolysis of each of the four stereoisomers is an important tool in the development of enzymes with the potential to protect against GD intoxication. Using a gas chromatography-mass spectrometry-based approach, we have examined the capacity of plasma-derived human serum albumin, plasma-purified human butyrylcholinesterase, goat milk-derived recombinant human butyrylcholinesterase, and recombinant human paraoxonase 1 to interact with each of the four stereoisomers of GD in vitro at pH 7.4 and 25 degrees C. Under these experimental conditions, the butyrylcholinesterase samples were found to bind GD with a relative preference for the more toxic stereoisomers (C-P- > C+P- > C-P+ > C+P+), while human serum albumin and paraoxonase 1 interacted with GD with a relative preference for the less toxic isomers (C-P+/C+P+ > C+P-/C-P-). The results indicate that these human proteins exhibit distinct stereoselective interactions with GD. The approach described presents a means to rapidly assess substrate stereospecificity, supporting future efforts to develop more effective OP bioscavenger proteins.

Association between Human Paraoxonase 1 Activity and Intima-Media Thickness in Subjects under 55 Years of Age with Carotid Artery Disease

Background: Human serum paraoxonase (PON1) protects lipoproteins against oxidation by hydrolyzing lipid peroxides in oxidized low-density lipoprotein (oxLDL); therefore, it may protect against atherosclerosis. PON1 activity and polymorphisms have been inconsistently associated with carotid artery disease. The goal of this study was to clarify the role of PON1 activity and phenotype on carotid artery disease and its correlation with some inflammatory and immune markers in subjects under 55 years with early-onset carotid atherosclerosis. Methods: Sixty patients with occlusive carotid artery disease and 30 healthy controls were enrolled. Intima-media thickness (IMT) was measured by high-resolution ultrasound of both common carotid arteries. Anti-oxLDL antibody levels were determined by ELISA. Results: In the whole study population we found a negative correlation between PON1 activity and IMT (r = –0.27, p = 0.011), and between salt-stimulated PON1 activity and IMT (r = –0.24, p = 0.02). Both PON1 activity and salt-stimulated PON1 activity negatively correlated with anti-oxLDL levels (r = –0.28, p = 0.008; r = –0.26, p = 0.01). PON1 activity was lower in patients compared to controls; however, the difference was not significant.PON1 phenotype distribution of patients and controls did not differ significantly. Conclusion: The importance of PON1 activity as a predictive risk factor for early-onset occlusive carotid artery disease should be assessed in future studies.

Simple procedures for purification and stabilization of human serum paraoxonase-1

Human paraoxonases-1 is one of the most important detoxifying enzymes. In this study using simple chromatographic procedures human paraoxonases-1 was purified from human pooled plasma. The enzyme was purified using DEAE Sephadex an anion exchanger and G-200 a gel filtration chromatographic media. Results showed a single band of approximately 43 KD proteins in SDS–PAGE, corresponding to the human PON1. Using paraoxon as the substrate the activity was related to the concentration of calcium and sodium ions (K m = 1.2 ± 0.2 mM). Phenyl acetate hydrolyzing activity was independent of sodium and calcium ions (K m = 0.78 ± 0.08 mM). Keeping at 25 °C for 20 days 75% of the enzyme original activity was restored in 20% (v/v) glycerol. EDTA and zinc chloride both inhibited the enzyme activity. In conclusion the applied procedures can be used for large scale purification. It would greatly facilitate their structural and functional characterization and permit examination of their weak, yet potentially most biologically relevant activities, in the complete absence of other serum proteins.

Paraoxonase Phenotype Distribution in a Healthy Iranian Population

Human serum paraoxonase (PON1) is a high-density lipoprotein-associated esterase that protects against organophosphate neurotoxicity, and is proposed to play a role in lipid metabolism and the onset of cardiovascular disease. In the present study, paraoxonase activities and phenotype distribution in serum of 132 healthy Iranian individuals aged 17-68 years were assessed using dual substrate method. In the study population, a wide interindividual variability (up to 15-fold) of paraoxonase activity was found. The mean of basal, salt-stimulated paraoxonase and arylesterase activities were 81.8 +/- 57 U/ml, 153.1 +/- 117.5 U/ml and 80.7 +/- 12.8 kU/l, respectively. The ratio of salt-stimulated paraoxonase activity to arylesterase activity was used for definition of phenotypes. Based on the observed ratios, three distinct phenotypes AA (low activity), AB (intermediate activity) and BB (high activity) were determined. The PON1 ratio varied from 0.5 to 6.8. The paraoxonase phenotype frequencies were approximately 48% (AA), 41% (AB) and 11% (BB). In this work, serum triglycerides had significant positive correlation (r = 0.334, P < 0.05) with paraoxonase activity, whereas high-density lipoprotein did not. No significant decrease in paraoxonase activity by smoking was observed. Age and sex had no influences on PON1 activities. In conclusion, the distribution of paraoxonase phenotypes in this Iranian population was trimodal and comparable to that of Caucasians from North America; however, overall enzyme activity was lower than that reported for Caucasians.

Human serum paraoxonase concentration predicts cardiovascular mortality in hemodialysis patients

Human serum paraoxonase (PON1) is associated with high-density lipoprotein, and inhibits oxidative modification of low-density lipoprotein. Therefore, PON1 is supposed to contribute to the prevention of atherosclerosis. We and other investigators have shown that the enzymatic activities and concentrations of PON1 were decreased in maintenance hemodialysis (HD) patients. However, the effect of PON1 status on the long-term outcome of HD patients has not been reported. In this study, we examined the association between baseline PON 1 status and cardiovascular mortality in an observation study of an outpatient HD population. The relation between baseline cardiovascular risk factors and clinical events was investigated, during 6 years of follow-up, in 81 HD patients (50 males and 31 females) whose enzymatic activities, concentrations and genetic polymorphisms of PON1 had been determined in a previous study. During follow-up for 6 years, we recorded 42 deaths, including 24 fatal cardiovascular events. In univariate analyses, baseline PON1 concentration was associated with not only cardiovascular mortality (p < 0.005), but also all-cause mortality (p < 0.001) during the period of follow-up, as were age, preexisting cardiovascular disease (CVD) and hemoglobin concentration. In a multivariate Cox regression analysis, PON1 concentration retained significant associations with cardiovascular mortality (p < 0.05) and all-cause mortality (p < 0.005) even after correction of known risk factors for CVD or mortality in HD patients. Using Kaplan-Meier survival curves, we assessed the association between low and high concentrations of PON1 divided according to the median value (7.52 U/ml). Significantly increased cardiovascular mortality (log rank 6.125, p = 0.01) and all-cause mortality (log rank 7.113, p < 0.01) were detected in the patients with low PON1 concentrations. These data suggest that low PON 1 concentration may be an independent predictor of cardiovascular mortality in maintenance HD patients.

Direct detection of stereospecific soman hydrolysis by wild‐type human serum paraoxonase

Human serum paraoxonase 1 (HuPON1; EC 3.1.8.1) is a calcium-dependent six-fold beta-propeller enzyme that has been shown to hydrolyze an array of substrates, including organophosphorus (OP) chemical warfare nerve agents. Although recent efforts utilizing site-directed mutagenesis have demonstrated specific residues (such as Phe222 and His115) to be important in determining the specificity of OP substrate binding and hydrolysis, little effort has focused on the substrate stereospecificity of the enzyme; different stereoisomers of OPs can differ in their toxicity by several orders of magnitude. For example, the C+/-P- isomers of the chemical warfare agent soman (GD) are known to be more toxic by three orders of magnitude. In this study, the catalytic activity of HuPON1 towards each of the four chiral isomers of GD was measured simultaneously via chiral GC/MS. The catalytic efficiency (k(cat)/K(m)) of the wild-type enzyme for the various stereoisomers was determined by a simultaneous solution of hydrolysis kinetics for each isomer. Derived k(cat)/K(m) values ranged from 625 to 4130 mm(-1).min(-1), with isomers being hydrolyzed in the order of preference C+P+ > C-P+ > C+P- > C-P-. The results indicate that HuPON1 hydrolysis of GD is stereoselective; substrate stereospecificity should be considered in future efforts to enhance the OPase activity of this and other candidate bioscavenger enzymes.

Accuracy and Biological Variation of Human Serum Paraoxonase 1 Activity and Polymorphism (Q192R) by Kinetic Enzyme Assay

Paraoxonase 1 (PON1) phenotype is a better predictor of atherosclerosis risk than are PON1 genetic polymorphisms alone. Larger studies are required to determine the role of PON1 and there is a need for standardized PON1 assays between laboratories. We have adapted 5 enzyme kinetic assays for high-throughput automated analysis of PON1 activity. Using different substrates and reaction conditions, we measured PON1 activity and used activity ratios to identify the PON1 Q192R genetic polymorphisms and assessed the accuracy of the genotype assignments in 79 adult study participants by comparing them with genotypes determined by AlwI restriction enzyme digestion of a 176-bp PCR amplification product from genomic DNA. Imprecision was determined using pooled serum and purified enzyme preparations. Biological variability was estimated by analysis of serial samples from 17 individuals. Variability parameters were compared with total cholesterol as a point of reference to a recognized biomarker of coronary heart disease risk. Salt stimulation and inhibition ratios were 97.4% and 94.7% correct in assigning Q192R genotype, respectively. Analytical imprecision (CV) was 1.0%-3.0% for phenylacetate and paraoxon substrate assays and 3.0%-8.0% for the para-nitrophenylacetate substrate assays. Combination of the 2 ratios into a double ratio resulted in 100% correct genotype classification. The described methods for measurement of PON1 activity and accurate genotype assignment are rapid and have potential to facilitate the efficient investigation of PON1 status in clinical and epidemiological studies.

Human Serum Paraoxonase Activity Decreases After Vertical Banded Gastroplasty*

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Human serum paraoxonase gene polymorphisms, Q192R and L55M, are not associated with the risk of cerebral infarction in Chinese Han population

Objectives: It has been reported that human serum paraoxonase (PON1) gene is associated with coronary heart disease (CHD) and diabetes mellitus (DM). However, little is known about the role of PON1 gene polymorphism in cerebral infarction (CI). For this, we have investigated the relationship between PON1 gene polymorphisms, Q192R and L55M, and CI in Chinese Han population.Methods: The PON1 genotypes, Q192R and L55M, from 153 CI patients and 153 healthy individuals, were determined by polymerase chain reaction (PCR) and restriction enzyme digestion. Their allele frequencies were then determined. The association of the PON1 gene polymorphism with the risk of CI was analysed by statistical analysis software.Results: The frequencies of PON1-Q192R genotypes in CI and control group are 13.7 and 9.8% (QQ), 51.6 and 53.6% (QR), 34.6 and 36.6% (RR) respectively. There is no significant difference in PON1-Q192R genotype (p=0.566) and allele frequencies (p=0.505) between CI patients and controls. The frequencies of PON1-55 genotypes in the CI and control group are 96.7 and 93.5% (LL), 3.3 and 6.5% (LM) respectively. No MM genotype was found in both CI and control group. No significant difference in genotype (p=0.289) and allele (p=0.296) distribution between CI patients and controls was observed.Discussion: Our results suggest that the human serum paraoxonase polymorphisms, Q192R and L55M, are not associated with the risk of cerebral infarction in Chinese Han population.

Novel purification strategy for human PON1 and inhibition of the activity by cephalosporin and aminoglikozide derived antibiotics

Human serum paraoxonase (PON1, EC 3.1.8.1.) is a high-density lipid (HDL)-associated, calcium-dependent enzyme; its physiological substrates are not known. In this study, a new purification strategy for human PON1 enzyme was developed using two-step procedures, namely ammonium sulfate precipitation and sepharose-4B- l-tyrosine-1-napthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43 kDa. Overall purification rate of our method was found 227-fold. The V max and K m of the purified enzyme were determined 227.27 EU and 4.16 mM, respectively. The in vitro effects of commonly used antibiotics, namely gentamycin sulfate and cefazolin sodium was also investigated on the purified human serum PON1 enzyme and human liver PON1 enzyme from human hepatoma cell (HepG2). Gentamycin sulfate and cefazolin sodium caused a dose- and time-dependent decrease on PON1 activity in HepG2 cells. Moreover, gentamycin sulfate and cefazolin sodium were effective inhibitors on purified human serum PON1 activity with IC 50 of 0.887 and 0.0084 values, respectively. The kinetics of interaction of gentamycin sulfate and cefazolin sodium with the purified human serum PON1 indicated a different inhibition pattern. Cefazolin sodium showed a competitive inhibition with K i of 0.012 ± 0.00065 mM. However, Gentamycin sulfate was inhibited in non-competitive manner with K i of 0.026 ± 0.015. In order to determine the inhibition statue of these drugs on a living system, the effects of same antibiotics on PON1 enzyme activity of mouse serum PON1 and liver PON1 were investigated in vivo. Gentamycin sulfate (3.2 mg/kg) and cefazolin sodium (106.25 mg/kg) leads to the significant decrease in mouse serum PON1 after 2, 4, 6 h and 2, 4 h drug administration, respectively. Cefazolin sodium did not exhibit any inhibition effect for the liver PON1, in vivo.

Effects of Some Antibiotics on Paraoxonase from Human Serum in Vitro and from Mouse Serum and Liver in Vivo

Paraoxonase (PON1, EC 3.1.8.1) is an esterase protein which plays multifunctional role in metabolism. Therefore, in this study the effects of commonly used antibiotics, namely sodium ampicillin, ciprofloxacin, rifamycin SV and clindamycin phosphate, on human PON1 were investigated in vitro and in vivo. Human serum paraoxonase (PON1) was separately purified by ammonium sulfate precipitation and hydrophobic interaction chromatography. The in vitro effects of the antibiotics in purifying human serum paraoxonase was determined using paraoxon as a substrate, and the IC50 values of these drugs exhibiting inhibition effects were found from graphs of hydratase activity % by plotting the concentration of the drugs. It was determined that sodium ampicillin, ciprofloxacin, and clindamycin phosphate were effective inhibitors on human serum PON1, and the inhibition kinetics of interaction of sodium ampicillin, ciprofloxacin, and clindamycin phosphate with the human serum PON1 was also determined, with the Ki of sodium ampicillin, ciprofloxacin, and clindamycin phosphate being 0.00714+/-0.00068, 6.5x10(-6)+/-4.59x10(-7), 0.0291+/-0.0077 mM, respectively. The in vivo effects of the antibiotics on paraoxonase enzyme activity in mouse serum and liver PON1 were also investigated. Mouse liver PON1 activity showed a statistically significant change at 2, 4 and 6 h of drug application in vivo. Sodium ampicillin and clindamycin phosphate exhibited about 80% mouse liver PON1 at 2 or 4 h (p: 0.034, 0.003 and 0.021, respectively). In addition, ciprofloxacin and rifamycin SV only showed inhibition at 4 h incubation. Sodium ampicillin (17.12 mg/kg) lead to a significant decrease in mouse serum PON1 after 4 h drug administration. Ciprofloxacin (3.2 mg/kg), rifamycin SV (3.56 mg/kg) and clindamycin phosphate (2.143 mg/kg) did not exhibit any inhibition effect for the mouse serum PON1, in vivo.

Amphenicol and macrolide derived antibiotics inhibit paraoxonase enzyme activity in human serum and human hepatoma cells (HepG2) in vitro

Human serum paraoxonase (hPON1) was separately purified by ammonium sulfate precipitation and hydrophobic interaction chromatography. The in vitro effects of commonly used antibiotics, namely clarithromycin and chloramphenicol, on purified human serum paraoxonase enzyme activity (serum hPON1) and human hepatoma (HepG2) cell paraoxonase enzyme activity (liver hPON1) were determined. Serum hPON1 and liver hPON1 were determined using paraoxon as a substrate and IC(50) values of these drugs exhibiting inhibition effects were found from graphs of hydratase activity (%) by plotting concentration of the drugs. We determined that chloramphenicol and clarithromycin were effective inhibitors of serum hPON1.