The paraoxonase gene family contains at least three members: PON1, PON2, and PON3. The physiological roles of the corresponding gene products are still uncertain. Until recently, only the serum paraoxonase/arylesterase (PON1) had been purified and characterized. Here we report the purification, cloning, and characterization of rabbit serum PON3. PON3 is a 40-kDa protein associated with the high density lipoprotein fraction of serum. In contrast to PON1, PON3 has very limited arylesterase and no paraoxonase activities but rapidly hydrolyzes lactones such as statin prodrugs (e.g. lovastatin). These differences facilitated the complete separation of PON3 from PON1 during purification. PON3 hydrolyzes aromatic lactones and 5- or 6-member ring lactones with aliphatic substituents but not simple lactones or those with polar substituents. We cloned PON3 from total rabbit liver RNA and expressed it in mammalian 293T/17 cells. The recombinant PON3 has the same apparent molecular mass and substrate specificity as the enzyme purified from serum. Rabbit serum PON3 is more efficient than rabbit PON1 in protecting low density lipoprotein from copper-induced oxidation. This is the first report that identifies a second PON enzyme in mammalian serum and the first to describe an enzymatic activity for PON3.

The <em>paraoxonase</em> (PON) family contains three genes (PON1/2/<em>3</em>) that are believed to be involved in the protection against oxidative stress. PON1 and PON<em>3</em> are circulating in <em>serum</em> attached to high-density lipoprotein fraction (HDL), whereas PON2 is ubiquitously expressed. The intestine is the second major organ that synthesizes lipoproteins; therefore, we examined PON mRNA expression and protein levels in gastrointestinal biopsies from humans, from C57BL6 mice, and from Caco-2 cells, a colon carcinoma-derived cell line that exhibits properties of intestinal epithelium at differentiation. PON 1/2/<em>3</em> mRNA and proteins were present in human biopsies with variable expression among different gastrointestinal segments. Only PON2 and PON<em>3</em> were present in mice. All PON mRNA, proteins, and enzymatic activities were present in Caco-2 cells. Oxidation of CaCo-2 cells with ferrum ascorbate had no significant effect on PON mRNA expression, but it increased <em>paraoxonase</em> and <em>lactonase</em> activity, whereas statinase activity was decreased. We showed polarized secretion of PON1 (basolateral) and PON2 (apical) into Caco-2 culture medium, raising the possibility that intestine is capable of producing and releasing PON1 and PON<em>3</em> to the circulation, whereas PON2 is released at the brush-border membrane to intestinal lumen where it may perform another yet unclear function.

<em>Serum</em> <em>paraoxonases</em> (PONs) are detoxifying <em>lactonases</em> that were first identified in mammals. Three mammalian families are known, PON1, 2, and <em>3</em> that reside primarily in the liver. They catalyze essentially the same reaction, lactone hydrolysis, but differ in their substrate specificity. Although some members are highly specific, others have a broad specificity profile. The evolutionary origins and substrate specificities of PONs therefore remain poorly understood. Here, we report a newly identified family of bacterial PONs, and the reconstruction of the ancestor of the three families of mammalian PONs. Both the mammalian ancestor and the characterized bacterial PONX_OCCAL were found to efficiently hydrolyze N-acyl homoserine lactones that mediate quorum sensing in many bacteria, including pathogenic ones. The mammalian PONs may therefore relate to a newly identified family of bacterial, PON-like "quorum-quenching" <em>lactonases</em>. The appearance of PONs in metazoa is likely to relate to innate immunity rather than detoxification. Unlike the bacterial PON, the mammalian ancestor also hydrolyzes, with low efficiency, lactones other than homoserine lactones, thus preceding the detoxifying functions that diverged later in two of the three mammalian families. The bifunctionality of the mammalian ancestor and the trade-off between the quorum-quenching and detoxifying <em>lactonase</em> activities explain the broad and overlapping specificities of some mammalian PONs versus the singular specificity of others.

OBJECTIVE

We investigated the analytical performance of a new assay of the lactonase activity of paraoxonase-1 and its efficacy in the assessment of liver damage.

METHODS

Serum lactonase activity was determined by the hydrolysis of 5-thiobutyl butyrolactone in 633 healthy individuals and 369 patients with chronic liver disease. Paraoxonase-1, 2, and 3 gene polymorphisms were analyzed by the MassArray method.

RESULTS

Linearity was up to 10 U/L. Detection limit was 0.12 U/L. Imprecision was < or = 17.7%. Lactonase values in our normal population were 5.99 (3.29-13.61) U/L. Lactonase activity showed a lower influence of genetic polymorphisms than the classical assay using paraoxon. Both measurements showed a similar efficiency in testing for liver dysfunction.

CONCLUSIONS

We report a reliable assay using a non-toxic substrate for the measurement of serum lactonase activity. The influence of genetic variability is low. The assay could be a useful addition to tests evaluating liver impairment.

<em>Serum</em> <em>paraoxonase</em> (PON1) is well recognized for its ability to hydrolyze arylesters, toxic oxon metabolites of organophosphate insecticides and nerve agents. PON1 is a member of gene family including also PON2 and PON<em>3</em>; however, the later two enzymes have very limited arylesterase and practically no organophosphatase activity. We have established that all three PONs are <em>lactonases</em>/lactonyzing enzymes with overlapping, but also distinct substrate specificity. Dihydrocoumarin (DHC), long chain fatty acid lactones and acylhomoserine lactones (AHLs) are hydrolyzed by all three PONs and likely represent their natural substrates. The <em>3</em>D structure of PON1 is a six-bladed beta-propeller containing two Ca(2+) ions necessary for the enzyme stability and enzymatic activity. Senescence marker protein (SMP<em>3</em>0), another putative six-bladed beta-propeller, hydrolyzes DFP, sarin and soman in the presence of Mg(2+) or Mn(2+). More recently, SMP<em>3</em>0 was characterized as a glucono<em>lactonase</em> with a role in vitamin C metabolism. Bacterial phosphotriesterases (PTEs) are members of the amidohydrolase superfamily and differ in their structure from the eukaryotic organophosphatases; PTEs are (beta/alpha)(8) barrels with an active site containing two transition metal ions such as Co(2+), Mn(2+) or Zn(2+). PTE from Pseudomonas diminuta hydrolyzes paraoxon extremely efficiently; this enzyme was shown to hydrolyze also DHC and other lactones. At least <em>3</em> more bacterial <em>lactonases</em>, dubbed PTE-like <em>lactonases</em> (or PLL), have been identified to possess both <em>lactonase</em> and organophosphatase activities. Lactones are natural compounds, many of them with high biological activity, while organophosphates are human-made chemicals introduced in the 20th century. Thus, it is plausible that <em>lactonase</em> is the primary activity for which the enzymes discussed here evolved for, while the organophosphatase activity arose as a promiscuous activity during their evolution. Laboratory (directed) evolution studies provided mechanisms for their catalytic versatility and demonstrated experimentally the evolvability of promiscuous enzyme functions.

OBJECTIVE

To evaluate and validate <em>3</em> spectrophotometric assays for measuring <em>serum</em> activity of <em>paraoxonase</em> type-1 (PON1), an enzyme associated with high-density lipoproteins, in dogs.

METHODS

22 healthy adult dogs and 10 dogs with eccentrocytosis.

METHODS

2 methods were adapted for use in 96-well microplates with phenyl acetate and 5-thiobutyl butyrolactonase as substrates, and 1 was adapted for use in an automated analyzer with p-nitrophenyl acetate as substrate. Blood samples were collected from all dogs, serum was harvested, and serum PON1 activity was measured with each method.

RESULTS

Imprecision was low for all <em>3</em> methods, with the exception of interassay imprecision for 5-thiobutyl butyrolactonase, and results were linear across serial sample dilutions. The <em>3</em> methods were able to detect low PON1 activity when EDTA was used for blood sample collection, yielded lower PON1 values in sick dogs with eccentrocytosis than in healthy dogs, and yielded highly correlated results.

CONCLUSIONS

The methods described here may allow a wider use of PON1 activity as a biomarker of oxidative stress in dogs in clinical and research settings. Results of each method were robust and precise (with the exception of the interassay values for the lactonase method), and the methods were easy to set up in a laboratory.

<em>Serum</em> <em>paraoxonase</em> (PON1) is a high-density lipoprotein (HDL)-associated esterase/<em>lactonase</em> implicated to play a role in protection against atherosclerosis. However, the exact mechanism(s) and substrates for PON1 are still uncertain. In this article, we review some of the evidence for PON1's antioxidant activity, as well as our efforts to identify the actual substrates and products for this activity. We originally reported that PON1 had phospholipase activity toward oxidized phosphatidylcholine (J. Biol. Chem. 276:2447<em>3</em>-24481; 2001). Subsequently, Marathe et al. (J. Biol. Chem. 278:<em>3</em>9<em>3</em>7-<em>3</em>947; 200<em>3</em>) reported that this activity was due to a contaminating lipase. However, that article did not replicate the conditions used in our previous study. To address this controversy, we purified <em>serum</em> PON1 by a modified method that separates the <em>paraoxonase</em> activity from an activity detectable as platelet-activating factor acetyl hydrolase (PAF-AH) (Teiber et al., J. Lipid. Res. 2004; Epub ahead of print, PMID 15<em>3</em>42686) and reexamined the oxidation of phosphatidylcholine by peroxynitrite using <em>3</em>-morpholinosydnonimine as a peroxynitrite generator and apolipoprotein AI-phosphatidylcholine- PON1 complexes. The phosphatidylcholines were studied by electrospray ionization tandem mass spectrometry. PON1 preparations free of PAF-AH activity showed no phospholipase activity when reconstituted into apolipoprotein AI-phosphatidylcholine complexes. We conclude that PON1 does not affect the accumulation of phosphatidylcholine oxidation products. Further, we have no evidence that PON1 has an intrinsic phospholipase A2 activity toward oxidized phospholipids.

Hydrolysable tannin polyphenols in pomegranate and phenolic acids in date fruit and seeds are potent antioxidants and anti-atherogenic agents, and thus, in the present study we investigated the possible benefits of combining them in vivo in atherosclerotic apolipoprotein E KO (E(0)) mice, compared with the individual fruit. In vitro studies revealed that the date seed extract contains more polyphenols than Amari or Hallawi date extracts, and possesses a most impressive free radical scavenging capacity. Similarly, pomegranate juice (PJ), punicalagin, punicalain, gallic acid, and urolithins A and B are very potent antioxidants. E(0) mice consumed 0.5 μmol gallic acid equivalents (GAE) per mouse per day of PJ, Hallawi extract, date seed extract, or a combination for <em>3</em> weeks. Consumption of the combination was the most potent treatment, as it decreased <em>serum</em> cholesterol and triglyceride levels, and increased <em>serum</em> <em>paraoxonase</em> 1 (PON1) activity. Consumption of the combination also significantly reduced mouse peritoneal macrophage (MPM) oxidative stress, MPM cholesterol content, and MPM LDL uptake. Finally, the lipid peroxide content in the aortas of the mice significantly decreased, and the PON <em>lactonase</em> activity of the aortas increased after treatment with the combination. We thus conclude that consumption of pomegranate, together with date fruit and date seeds, has the most beneficial anti-atherogenic effects on E(0) mice <em>serum</em>, macrophages, and aortas, probably due to their unique and varied structures.

BACKGROUND

<em>Serum</em> <em>paraoxonase</em> (PON1) exerts antiatherogenic effects. Novel PON1 enzymatic tests have been recently developed: 5-thiobutyl butyrolactone (TBBL) estimates PON1 <em>lactonase</em> activity, whereas 7-O-diethylphosphoryl-<em>3</em>-cyano-4-methyl-7-hydroxycoumarin (DEPCyMC) is considered a surrogate marker of PON1 concentration. The TBBL to DEPCyMC ratio provides the normalized <em>lactonase</em> activity (NLA), which may reflect the degree of PON1 <em>lactonase</em> catalytic stimulation. The aim of this study was to evaluate for the first time TBBLase and DEPCyMCase activity in patients with coronary artery disease (CAD).

METHODS

An angiography-based case-control study was conducted, including <em>3</em>00 sex- and age-matched subjects [100 CAD-free, 100 CAD without myocardial infarction (MI) and 100 CAD with MI].

RESULTS

A low DEPCyMCase activity (lowest vs. highest tertile: OR 2.96, 95% CI 1.18-7.4<em>3</em>) and a high NLA (highest vs. lowest tertile: OR <em>3</em>.25, 95% CI 1.28-8.26) were both associated with CAD, independent of classical atherosclerosis risk factors, lipid-lowering therapy and PON1 genotype. Total TBBLase activity was, however, not different in CAD compared to CAD-free subjects.

CONCLUSIONS

Novel PON1 activity assays may be associated with CAD. In this study, CAD patients had low DEPCyMCase activity, a possible marker of low PON1 concentration, but showed a high stimulation of PON1 lactonase activity.

Esophageal adenocarcinoma (EAC) is thought to develop from asymptomatic Barrett's esophagus (BE) with a low annual rate of conversion. Current endoscopy surveillance of BE patients is probably not cost-effective. Previously, we discovered <em>serum</em> glycoprotein biomarker candidates which could discriminate BE patients from EAC. Here, we aimed to validate candidate <em>serum</em> glycoprotein biomarkers in independent cohorts, and to develop a biomarker candidate panel for BE surveillance. <em>Serum</em> glycoprotein biomarker candidates were measured in <em>3</em>01 <em>serum</em> samples collected from Australia (4 states) and the United States (1 clinic) using previously established lectin magnetic bead array (LeMBA) coupled multiple reaction monitoring mass spectrometry (MRM-MS) tier <em>3</em> assay. The area under receiver operating characteristic curve (AUROC) was calculated as a measure of discrimination, and multivariate recursive partitioning was used to formulate a multi-marker panel for BE surveillance. Complement C9 (C9), gelsolin (GSN), <em>serum</em> <em>paraoxonase</em>/arylesterase 1 (PON1) and <em>serum</em> <em>paraoxonase</em>/<em>lactonase</em> <em>3</em> (PON<em>3</em>) were validated as diagnostic glycoprotein biomarkers in lectin pull-down samples for EAC across both cohorts. A panel of 10 <em>serum</em> glycoprotein biomarker candidates discriminated BE patients not requiring intervention (BE± low grade dysplasia) from those requiring intervention (BE with high grade dysplasia (BE-HGD) or EAC) with an AUROC value of 0.9<em>3</em>. Tissue expression of C9 was found to be induced in BE, dysplastic BE and EAC. In longitudinal samples from subjects that have progressed toward EAC, levels of <em>serum</em> C9 were significantly (p < 0.05) increased with disease progression in EPHA (erythroagglutinin from Phaseolus vulgaris) and NPL (Narcissus pseudonarcissus lectin) pull-down samples. The results confirm alteration of complement pathway glycoproteins during BE-EAC pathogenesis. Further prospective clinical validation of the confirmed biomarker candidates in a large cohort is warranted, prior to development of a first-line BE surveillance blood test.