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.

<AbstractText>Plasma protein biomarkers could be an efficient alternative for population-based screening for early detection of colorectal cancer (CRC). The objective of this study was to evaluate and validate plasma proteins individually and as a signature for early detection of CRC.</AbstractText><p><div><b>METHODS</b></div>In a three-stage design, proteins were measured firstly by liquid chromatography/multiple reaction monitoring-mass spectrometry (LC/MRM-MS) and later by proximity extension assay (PEA) in a discovery set consisting of 96 newly diagnosed CRC cases and 94 controls free of neoplasms at screening colonoscopy. Two algorithms (one for each measurement method) were derived by Lasso regression and .6<em>3</em>2+ bootstrap based on 11 proteins that were included in both the LC/MRM-MS and PEA measurements. Additionally, another algorithm was constructed from the same eleven biomarkers plus amphireglin, the most promising protein marker in the PEA measurements that had not been available from the LC/MRM-MS measurements. Lastly the three prediction signatures were validated with PEA in independent samples of participants of screening colonoscopy (CRC (<i>n</i> = 56), advanced adenoma (<i>n</i> = 101), and participants free of neoplasm (<i>n</i> = 102)).</p><AbstractText>The same four proteins were included in all three prediction signatures; mannan binding lectin serine protease 1, osteopontin, <em>serum</em> <em>paraoxonase</em> <em>lactonase</em> <em>3</em> and transferrin receptor protein 1, and the third prediction signature additionally included amphiregulin. In the independent validation set from a true screening setting, the five-marker blood-based signature including AREG presented areas under the curves of 0.82 (95% CI, 0.74-0.89), 0.86 (95% CI, 0.77-0.92) and 0.76 (95% CI, 0.64-0.86) for all, early and late stages CRC, respectively.</AbstractText><AbstractText>Two different measurement methods consistently identified four protein markers and an algorithm additionally including amphiregulin, a marker measured by PEA only, showed promising performance for detecting early stage CRC in an independent validation in a true screening setting. These proteins may be potential candidates for blood-based tests for early detection of CRC.</AbstractText>

Recent studies suggest that <em>paraoxonase</em>-1 (PON1), complexed with high-density lipoproteins, is the major <em>lactonase</em> in the circulation. Using 5-hydroxy eicosatetraenoate δ-lactone (5-HETEL) as the substrate, we observed <em>lactonase</em> activity in <em>serum</em> from Pon1-/- mice. However, 6-12 carbon fatty acid γ- and δ-lactones were not hydrolyzed in <em>serum</em> from Pon1-/- mice. <em>Serum</em> from both wild-type and Pon1-/- mice contained a <em>lactonase</em> activity towards 5-HETEL and <em>3</em>-oxo-dodecanoyl-homoserine lactone that was resistant to inactivation by EDTA. This <em>lactonase</em> activity was sensitive to the serine esterase inhibitor phenyl methyl sulfonyl fluoride and co-eluted with carboxylesterase activity by size-exclusion chromatography. Analysis of <em>serum</em> from the Es1e mouse strain, which has a deficiency in the carboxylesterase, ES-1, proved that this activity was due to ES-1. PON1 activity predominated at early time points (<em>3</em>0 s), whereas both PON1 and ES-1 contributed equally at later time points (15 min). When both PON1 and ES-1 were inhibited, 5-HETEL was stable in mouse <em>serum</em>. Thus, while long-chain fatty acid lactones are substrates for PON1, they can be hydrolyzed by ES-1 at neutral pH. In contrast, medium-chain length fatty acid lactones are stable in mouse <em>serum</em> in the absence of PON1, suggesting that PON1 plays a specific role in the metabolism of these compounds.

OBJECTIVE

To study the relationship between the lactonase activities and status of paraoxonase 1 (PON1) and its association with the PON1 genetic polymorphisms in women with polycystic ovarian syndrome (PCOS).

METHODS

A case-control study.

METHODS

A total of 455 PCOS patients and 441 control women were included in this study. The lactonase activities and concentrations of PON1 were assayed using 5-thiobutyl butyrolactone (TBBL) and 7-O-diethylphosphoryl-3-cyano-4-methyl-7-hydroxycoumarin (DEPCyMC) respectively. A normalized lactonase activity (NLA) was estimated based on the ratio of TBBLase:DEPCyMCase activity. The PON1 genotypes, serum malondialdehyde (MDA) levels and total antioxidant capacity were analyzed.

RESULTS

The lactonase activities and levels of PON1 were higher in PCOS patients than in the control women. However, the NLA did not significantly differ between groups. The -108C→T variation of the PON1 gene showed decreased lactonase activities and levels of PON1 in a genotype-dependent manner (CC>CT>TT); the 192Q→R variation of the PON1 gene showed increased PON1 lactonase activities and NLA; and the 55L→M variation of the PON1 gene showed decreased lactonase activities and levels of PON1 but an increased NLA. A multivariable regression analysis showed that the -108C/T, 192Q/R, and 55L/M variations of the PON1 gene, serum apolipoprotein A1, and MDA levels were significant predictors of PON1 lactonase activity, PON1 level, and NLA.

CONCLUSIONS

The serum lactonase activities and concentrations of PON1 are increased in PCOS patients. The increased oxidative stress and the -108C/T, 192Q/R, and 55L/M genetic polymorphisms of PON1 may be associated with these changes.

Changes in <em>paraoxonase</em> 1 (PON1) activities have been observed in a variety of diseases involving oxidative stress, such as CVD. However, its role in obesity has not been fully established. In the present study, we aimed (1) to genotype sixteen PON1 SNP, (2) to measure <em>serum</em> PON1 activities and (<em>3</em>) to correlate these findings with the incidence of childhood obesity and related traits. We conducted a case-control study of 189 normal-weight and 179 obese prepubertal children, and we measured four different PON1 activities: <em>lactonase</em>; <em>paraoxonase</em>; arylesterase; diazoxonase. Although none of these activities was significantly different between the obese and normal-weight children, <em>lactonase</em> activity was found to be positively correlated with HDL-cholesterol and ApoA1 levels and negatively correlated with myeloperoxidase and fatty acid-binding protein 4 levels. Among the sixteen genotyped PON1 SNP, only the intronic SNP rs854566 exhibited a significant association with obesity (OR 0·61, 95 % CI 0·41, 0·91; P= 0·016). This genetic variant was also associated with increased diazoxonase, <em>lactonase</em> and arylesterase activities and decreased <em>paraoxonase</em> activity. Other genetic variants exhibited different association patterns with <em>serum</em> activities based on their location within the PON1 gene, and SNP that were located within the promoter were strongly associated with <em>lactonase</em>, arylesterase and diazoxonase activities. The functional variant Q192R exhibited the greatest effect on <em>paraoxonase</em> activity (P= 5·88 × 10(-42)). In conclusion, SNP rs854566 was negatively associated with childhood obesity and with increased <em>serum</em> PON1 activities in prepubertal children. We determined that <em>lactonase</em> is a reliable indicator of PON1 activities and should be included in future studies of PON1 function.

BACKGROUND

High-density lipoprotein (HDL) particles are protective against atherosclerosis. However, HDL function is impaired in metabolic syndrome (MetS) due to low-grade inflammation and dyslipidemia. Foods containing polyphenols, such as grapes, may prevent HDL dysfunction via antioxidant or anti-inflammatory effects. We evaluated the effects of grape powder ingestion on measures of HDL function in adults with MetS.

METHODS

Twenty adults (age: <em>3</em>2-70 years; body mass index: 25.<em>3</em>-45.4 kg/m2) consumed either 60 grams/day of freeze-dried grape powder (GRAPE) or a placebo for 4 weeks, separated by a <em>3</em>-week washout period, in a randomized, double-blind crossover study. The primary outcome was <em>serum</em> <em>paraoxonase</em>-1 (PON1) arylesterase activity, a measure of HDL antioxidant function. Secondary outcomes included PON1 <em>lactonase</em> activity, plasma lipids, metabolic markers, cholesterol efflux capacity, and other HDL functional markers.

RESULTS

After 4 weeks, GRAPE did not alter the <em>serum</em> PON1 activity or other markers of HDL function compared with placebo. Measures of HDL function were positively correlated with each other and inversely with measures of insulin resistance and inflammation. GRAPE intake led to a significant reduction in fasting plasma triglycerides compared with placebo (P = 0.0<em>3</em>2). No other significant effects of GRAPE were observed for other plasma lipids, anthropometrics, or metabolic measures.

CONCLUSIONS

Grape powder consumption did not impact HDL function in this cohort of adults with MetS. However, it was shown to improve fasting triglycerides, a risk factor for cardiovascular disease.

BACKGROUND

Paraoxonase 1 (PON1) is a high- density lipoprotein (HDL)-associated enzyme, displaying esterase and lactonase activity. The PON1 is involved in a variety of inflammatory diseases, metabolizing toxic oxidized lipids and detoxifying of organophosphorus insecticide compounds and nerve agents.

OBJECTIVE

The aim of this study was to investigate the effects of methanolic date seed extract (DSE) on paraoxonase and arylesterase activities in hypercholesterolemic rats.

METHODS

Experiments were conducted in two groups of normal and hypercholesterolemic rats and continued for four weeks. Two weeks after receiving the normal and hypercholesterolemic diet, different dosages of DSE were administered during the last two weeks of the treatment. Blood samples were taken from animals before administration of DSE (at day 14) and at the end of the experimental period (at day 28). Paraoxonase and arylesterase activities of PON1 enzyme were assayed by kit using paraoxone and phenylacetate as the substrates. Relative changes in serum paraoxonase and arylesterase activities and total antioxidant capacity (TAOC) were compared between the two groups during this interval.

RESULTS

Administration of DSE significantly increased serum paraoxonase and arylesterase activities in treated hypercholesterolemic groups compared to untreated ones. There was a significant difference in the TAOC of serum between the normal diet and hypercholesterolemic groups. However, DSE did not change the TAOC in hypercholesterolemic groups significantly.

CONCLUSIONS

DSE increases serum paraoxonase and arylesterase activities. These beneficial effects may be subjected to the presence of natural antioxidants such as phenolic compounds in the date seed. Despite this, DSE did not increase TAOC in treated hypercholesterolemic groups compared to the untreated ones based on ABTS (2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid) radical reduction assay. This indicates that the hypercholesterolemic diet, apart from DSE and atorvastatin effects, may be responsible for the serum TAOC reduction. However, it is concluded that DSE may be useful in decreasing the symptoms of diseases resulting from the low activity of paraoxonase.

OBJECTIVE

To derive a plasma biomarker protein panel from a list of 141 candidate proteins which can differentiate transient ischaemic attack (TIA)/minor stroke from non-cerebrovascular (mimic) conditions in emergency department (ED) settings.

METHODS

Prospective clinical study (#NCT0<em>3</em>050099) with up to three timed blood draws no more than <em>3</em>6 h following symptom onset. Plasma samples analysed by multiple reaction monitoring-mass spectrometry (MRM-MS).

METHODS

Totally 545 participants suspected of TIA enrolled in the EDs of two urban medical centres.

RESULTS

90-day, neurologist-adjudicated diagnosis of TIA informed by clinical and radiological investigations.

RESULTS

The final protein panel consists of 16 proteins whose patterns show differential abundance between TIA and mimic patients. Nine of the proteins were significant univariate predictors of TIA [odds ratio (95% confidence interval)]: L-selectin [0.726 (0.596-0.88<em>3</em>)]; Insulin-like growth factor-binding protein <em>3</em> [0.727 (0.594-0.889)]; Coagulation factor X [0.740 (0.60<em>3</em>-0.908)]; <em>Serum</em> <em>paraoxonase</em>/<em>lactonase</em> <em>3</em> [0.76<em>3</em> (0.6<em>3</em>0-0.924)]; Thrombospondin-1 [1.<em>3</em>1<em>3</em> (1.081-1.595)]; Hyaluronan-binding protein 2 [0.776 (0.6<em>3</em>7-0.945)]; Heparin cofactor 2 [0.775 (0.6<em>3</em>4-0.947)]; Apolipoprotein B-100 [1.249 (1.0<em>3</em>7-1.50<em>3</em>)]; and von Willebrand factor [1.256 (1.0<em>3</em>4-1.527)]. The scientific plausibility of the panel proteins is discussed.

CONCLUSIONS

Our panel has the potential to assist ED physicians in distinguishing TIA from mimic patients.

OBJECTIVE

Obesity has a great impact on cardiovascular morbidity and mortality, the treatment of this pathological state is important given the significant health consequences. Lifestyle and behaviour changes play a significant role in weight management. The purpose of this study was to investigate the impact of an intensive multidisciplinary lifestyle intervention on well-known atherogenic factors in a group of overweight and obese subjects.

METHODS

A total of 44 people with overweight/obesity underwent a lifestyle intervention based on nutritional education, psychological support and a <em>3</em>-month exercise training program with a frequency of twice a week. Several anthropometric and biochemical parameters were measured before and after the lifestyle intervention.

RESULTS

Lifestyle intervention led to a significant reduction in metabolic profile including body mass index (BMI), waist circumference, systolic and diastolic blood pressure, plasma glucose, and plasma triglycerides. These reductions were also accompanied by a significant increase in maximal oxygen consumption and muscle strength. Furthermore, paraoxonase and lactonase activities and the concentration of Apoliproteins A1 (APO A1) were significantly increased and the serum levels of oxLDL reduced without any changes in the circulating levels of LDL and HDL.

CONCLUSIONS

In conclusion, our study suggests that an intensive lifestyle intervention in obese subjects promotes a series of beneficial antiatherogenic changes which included increased enzyme activites of paraoxonase and lactonase, concentration of Apoliproteins A1 and decreased circulating levels of oxLDL.

High-density lipoprotein (HDL) plays an important role in preventing atherosclerosis. The antioxidant effect of HDL is mostly associated with <em>paraoxonase</em> 1 (PON1) activity. Increasing PON1 activity using nutrients might improve HDL function and quality and thus, decrease atherosclerotic risk. We previously isolated and identified a novel active compound, lyso-DGTS (C20:5,0) from Nannochloropsis sp. ethanol extract. In the present study, its effect on PON1 activities was examined and the mechanism by which the compound affects PON1 activity was explored. Lyso-DGTS elevated recombinant PON1 (rePON1) <em>lactonase</em> and esterase activities in a dose- and time-responsive manner, and further stabilized and preserved rePON1 <em>lactonase</em> activity. Incubation of lyso-DGTS with human <em>serum</em> for 4 h at <em>3</em>7 °C also increased PON1 <em>lactonase</em> activity in a dose-responsive manner. Using tryptophan-fluorescence-quenching assay, lyso-DGTS was found to interact with rePON1 spontaneously with negative free energy (ΔG = -22.87 kJ mol-1 at 25 °C). Thermodynamic parameters and molecular modeling calculations showed that the main interaction of lyso-DGTS with the enzyme is through a hydrogen bond with supporting van der Waals interactions. Furthermore, lyso-DGTS significantly increased rePON1 influx into macrophages and prevented lipid accumulation in macrophages stimulated with oxidized low-density lipid dose-dependently. In vivo supplementation of lyso-DGTS to the circulation of mice fed a high-fat diet via osmotic mini-pumps implanted subcutaneously significantly increased <em>serum</em> PON1 <em>lactonase</em> activity and decreased <em>serum</em> glucose concentrations to the level of mice fed a normal diet. Our findings suggest a beneficial effect of lyso-DGTS on increasing PON1 activity and thus, improving HDL quality and atherosclerotic risk factors. © 2018 BioFactors, 44(<em>3</em>):299-<em>3</em>10, 2018.

BACKGROUND

<em>Paraoxonase</em>-<em>3</em> (PON<em>3</em>), as a high density lipoprotein (HDL)-associated <em>lactonase</em>, is capable of preventing the oxidative modification of low density lipoprotein (LDL). PON<em>3</em> activity in follicular fluid (FF) is three times more than its activity in <em>serum</em>. However, the detailed role of PON<em>3</em> in women's fertility remains unknown. The aim of this study was to investigate the correlation between PON<em>3</em> activity in the FF of women undergoing assisted reproductive technique (ART), in vitro fertilization (IVF), or intra-cytoplasmic sperm injection (ICSI).

METHODS

This cross-sectional study consisted of 50 women from couples with male factor infertility (MFI) or with female factor infertility (FFI). The FF samples were obtained during the ART intervention. PON<em>3</em> activity, HDL cholesterol (HDL C), total antioxidant status (TAS) and the level of malondialdehyde (MDA) were determined. The morphology of the embryo was determined using embryo cell number (ECN) and embryo fragmentation score (EFS). In addition, fertilization rate (FR) was used an oocyte fertilization index.

RESULTS

Of 50 women, 20 women belonged to FFI group and the remaining <em>3</em>0 women belonged to MFI group. PON<em>3</em> activity in FF of women in FFI group was significantly lower (p<0.05) in comparison with corresponding value in MFI group. The value of PON<em>3</em> activity/MDA in the FFI group was lower than that in MFI group. Moreover, MDA level in the FF of FFI group was significantly higher (p<0.05) than its concentration in MFI group. Meanwhile, no significant difference was found in HDL-C concentration and TAS of both groups. No significant correlation was observed between the ECN and FF biochemical parameters. There was also a negative correlation between FR and MDA (r=-0.42, p=0.02), whereas a positive relation between FR with PON<em>3</em> activity (r=0.59, p=0.004), HDL-C (r=0.<em>3</em>5, p=0.04) and PON<em>3</em>/MDA (r=0.59, p=0.001).

CONCLUSIONS

According to the results of this study, PON<em>3</em> activity level as a key component of antioxidant system in FF may directly be associated with the success rate of ART and fertilization rate in women.

Blood-based protein biomarkers are increasingly being explored as supplementary or efficient alternatives for population-based screening of colorectal cancer (CRC). The objective of the current study was to compare the diagnostic potential of proteins measured with different proteomic technologies. The concentrations of protein biomarkers were measured using proximity extension assays (PEAs), liquid chromatography/multiple reaction monitoring-mass spectrometry (LC/MRM-MS) and quantibody microarrays (QMAs) in plasma samples of 56 CRC patients and 99 participants free of neoplasms. In another approach, proteins were measured in <em>serum</em> samples of <em>3</em>0 CRC cases and <em>3</em>0 participants free of neoplasm using immunome full-length functional protein arrays (IpAs). From all the measurements, 9, 6, <em>3</em>5 and 14 protein biomarkers overlapped for comparative evaluation of (a) PEA and LC/MRM-MS, (b) PEA and QMA, (c) PEA and IpA, and (d) LC/MRM-MS and IpA measurements, respectively. Correlation analysis was performed, along with calculation of the area under the curve (AUC) for assessing the diagnostic potential of each biomarker. DeLong's test was performed to assess the differences in AUC. Evaluation of the nine biomarkers measured with PEA and LC/MRM-MS displayed correlation coefficients >+0.6, similar AUCs and DeLong's <i>p</i>-values indicating no differences in AUCs for biomarkers like insulin-like growth factor binding protein 2 (IGFBP2), matrix metalloproteinase 9 (MMP9) and <em>serum</em> <em>paraoxonase</em> <em>lactonase</em> <em>3</em> (PON<em>3</em>). Comparing six proteins measured with PEA and QMA showed good correlation and similar diagnostic performance for only one protein, growth differentiation factor 15 (GDF15). The comparison of <em>3</em>5 proteins measured with IpA and PEA and 14 proteins analyzed with IpA and LC/MRM-MS revealed poor concordance and comparatively better AUCs when measured with PEA and LC/MRM-MS. The comparison of different proteomic technologies suggests the superior performance of novel technologies like PEA and LC/MRM-MS over the assessed array-based technologies in blood-protein-based early detection of CRC.

Keywords: LC/MRM-MS; biomarkers; colorectal cancer; diagnosis; microarray; plasma proteins; proximity extension assays.

In our study, we aimed to evaluate the effects of <i>Moringa oleifera</i> leaves extract on rat <em>paraoxonase</em> 1 (rPON1) and catalase (rCAT) activities in alloxan-induced diabetic rats. Our study included three groups; group C (control, n = 5); group D (diabetic, n = 5); and group DM (<i>M. oleifera</i> extract-supplemented diabetic rats, n = 5). Daily oral administration of <i>M. oleifera</i> extract at 200 mg/kg doses produced an increase in endogenous antioxidants. <em>Serum</em> rPON1 (<em>lactonase</em>) and liver cytosol catalase activities were determined by a spectrophotometric assay using progress curve analysis. We found a decrease in the Vm value of rPON1 in diabetic rats, but dihydrocoumarin (DHC) affinity (Km) was slightly increased. The value of Vm for the DM group was found to be reduced approximately by a factor of <em>3</em> compared with those obtained for group C, whereas Km was largely changed (96 times). Catalase activity was significantly higher in the DM group. These data suggest that the activation of rPON1 and rCAT activities by <i>M. oleifera</i> extracts may be mediated via the effect of the specific flavonoids on the enzyme structure. In addition, through molecular blind docking analysis, rPON1 was found to have two binding sites for flavonoids. In contrast, flavonoids bound at four sites in rCAT. In conclusion, the data suggest that compounds from <i>M. oleifera</i> leaves extract were able to influence the catalytic activities of both enzymes to compensate for the changes provoked by diabetes in rats.

Keywords: Moringa oleifera leaves extract; progress kinetics and blind docking; rat liver catalase; serum paraoxonase 1.

OBJECTIVE

To validate our previously developed 16 plasma-protein biomarker panel to differentiate between transient ischaemic attack (TIA) and non-cerebrovascular emergency department (ED) patients.

METHODS

Two consecutive cohorts of ED patients prospectively enrolled at two urban medical centers into the second phase of SpecTRA study (training, cohort 2A, n = 575; test, cohort 2B, n = 528). Plasma samples were analyzed using liquid chromatography/multiple reaction monitoring-mass spectrometry. Logistic regression models which fit cohort 2A were validated on cohort 2B.

RESULTS

Three of the panel proteins failed quality control and were removed from the panel. During validation, panel models did not outperform a simple motor/speech (M/S) deficit variable. Post-hoc analyses suggested the measured behaviour of L-selectin and coagulation factor V contributed to poor model performance. Removal of these proteins increased the external performance of a model containing the panel and the M/S variable.

CONCLUSIONS

Univariate analyses suggest insulin-like growth factor-binding protein <em>3</em> and <em>serum</em> <em>paraoxonase</em>/<em>lactonase</em> <em>3</em> are reliable and reproducible biomarkers for TIA status. Logistic regression models indicated L-selectin, apolipoprotein B-100, coagulation factor IX, and thrombospondin-1 to be significant multivariate predictors of TIA. We discuss multivariate feature subset analyses as an exploratory technique to better understand a panel's full predictive potential.

<p><div><b>Background</b></div><em>Paraoxonase</em> 1 (PON1) is a calcium-dependent multifunctional enzyme that binds to high-density lipoproteins. The physiological function of PON1 is related to its <em>lactonase</em> activity. However, this activity has not been analyzed in women with gestational diabetes mellitus (GDM). The present study investigated the <em>lactonase</em> activities and status of PON1 and their association with <i>PON1</i> genetic variants and oxidative stress indices in Chinese women with GDM.</p><p><div><b>Methods</b></div>This is a case-control study of <em>3</em>47 women with GDM and 288 women with uncomplicated pregnancies. PON1 levels and <em>lactonase</em> activities were analyzed using 7-O-diethylphosphoryl-<em>3</em>-cyano-4-methyl-7-hydroxycoumarin (DEPCyMC) and 5-thiobutyl butyrolactone (TBBL), respectively. A normalized <em>lactonase</em> activity (NLA) was estimated based on the ratio of TBBLase to DEPCyMCase activity. <em>Serum</em> malondialdehyde (MDA), total oxidant status (TOS), total antioxidant capacity (TAC) levels, and <i>PON1</i> genetic variants and oxidative stress indices in Chinese women with GDM.</p><p><div><b>Results</b></div>PON1 <em>lactonase</em> activity and levels of TOS, TAC, and MDA were higher in the GDM women compared with the control women. The <i>PON1 -108C→T</i> genetic variation decreased the levels and <em>lactonase</em> activities of PON1 in a genotype-dependent manner in the patient and control groups. GDM patients with the <i>PON1 -108TT</i> genotype displayed lower NLA than those with the <i>-108CC</i> or <i>-108CT</i> genotype. GDM patients with the <i>RR</i> genotype of <i>PON1 192Q/R</i> polymorphism had significantly lower PON1 <em>lactonase</em> activities and NLA and tended to have decreased PON1 levels compared with those with the <i>QQ</i> or <i>QR</i> genotype. Multivariable regression analysis revealed that the <i>PON1 -108C/T</i> or <i>192Q/R</i> variations, apolipoprotein (apo) A1, apoB, TAC, MDA, or age was significant predictors of the levels, <em>lactonase</em> activities, or NLA of PON1.</p><p><div><b>Conclusions</b></div>The <em>lactonase</em> activities of PON1 are increased in women with GDM. <i>PON1</i> genetic variants, increased oxidative stress, and abnormalities in lipoproteins may be associated with these changes.<i>PON1</i> genetic variants and oxidative stress indices in Chinese women with GDM.</p>