The opsonising activity of a pentraxin-like protein isolated from snapper (Pagrus auratus, Sparidae) serum.

In mammals the pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP) are important components of the immune response. Although pentraxins have been isolated from a number of fish species few studies detail their functional immunological role. In this paper we report the establishment of a flow cytometry based assay to measure the phagocytic activity of isolated snapper head kidney leukocytes (HKLs). This assay was then used to examine the ability of a pentraxin-like protein isolated from the serum of snapper (P. auratus) (Sn-PLP) to act as an opsonin. Incubation of snapper head kidney leukocytes (HKL) with FITC-labelled beads resulted in uptake of these particles by approximately 35% of HKLs. Incubation of beads with Sn-PLP enhanced phagocytosis by snapper HKLs in a dose-dependant manner. Enhanced phagocytosis could be inhibited by addition of a rabbit anti-Sn-PLP antibody suggesting that Sn-PLP may act as a ligand for a HKL cell surface receptor. This study provides further evidence toward a functional role for pentraxins in the host defence repertoire of fish.

Isolation and partial characterization of a pentraxin-like protein with complement-fixing activity from snapper (Pagrus auratus, Sparidae) serum.

Pentraxin-like molecules have been isolated from a number of fish species. However, little is known about the function of these proteins in the teleosts. In this study we report the isolation and characterization of a pentraxin-like molecule from the serum of snapper (Pagrus auratus) that has the ability to activate complement. This pentraxin-like protein was isolated from serum by calcium-dependent binding to agarose. SDS-PAGE analysis demonstrated an oligomeric protein of approximately 200k Da consisting of non-covalently bound subunits of 26 and 23 kDa. Protein sequencing revealed significant (50%) sequence identity with pentraxins from both Atlantic salmon (S. salar) and rainbow trout (O. mykiss). However, polyclonal antibodies raised against snapper pentraxin did not recognise salmon or trout pentraxin in Western blot analysis. Following LPS injection, snapper pentraxin levels increased 2-fold before gradually returning to basal levels. Most significantly, the isolated pentraxin initiated complement-mediated lysis of ligand-coated sheep erythrocytes in a dose-dependent fashion. In view of the similarity between the known fish pentraxins, and their similarity to mammalian serum amyloid P-components we conclude that the isolated protein may be a snapper pentraxin homologue.

Administration of a commercial immunostimulant preparation, EcoActiva as a feed supplement enhances macrophage respiratory burst and the growth rate of snapper (Pagrus auratus, Sparidae (Bloch and Schneider)) in winter.

This study investigated the effects of prolonged administration of a commercial beta-glucan based immunostimulant preparation, EcoActiva, in the form of a feed supplement, on non-specific immune parameters and the growth rate of snapper (Pagrus auratus). Fish held at a temperature representing either summer or winter conditions, were sampled periodically and assayed for head kidney macrophage activity via in vitro superoxide production, and classical and alternative complement activity. Fish were also weighed monthly and the growth rate determined. Fish fed on a diet supplemented with EcoActiva and held at the winter temperature had a significant enhancement of macrophage superoxide anion production upon stimulation with phorbol myristate acetate (PMA), and this increased activity was maintained throughout the trial. Macrophage activity in fish fed the supplemented diet and held at the summer temperature was also increased. However, EcoActiva failed to increase either classical or alternate complement activity. Most significantly EcoActiva resulted in an increase in growth rates of the fish held at the winter temperature as compared to the control fish, although no difference was seen between the groups held at the summer temperature. These results suggest that routine incorporation of beta-glucan preparations like EcoActivaduring winter may enhance macrophage function and growth rates at a time of increased disease susceptibility and little or no growth.

Simultaneous determination of oleuropein and hydroxytyrosol in rat plasma using liquid chromatography with fluorescence detection.

Oleuropein, the main glycoside present in olives, and hydroxytyrosol, the principal degradation product of oleuropein present in olive oil, have been linked to reduction of coronary heart disease and certain cancers. In the present study a direct and sensitive reversed-phase high-performance liquid chromatographic assay was developed for simultaneous quantification of both oleuropein and hydroxytyrosol. The plasma protein was precipitated with acetonitrile, samples were then centrifuged and supernatants were dried, and reconstituted with water prior to injection. The chromatographic analysis was carried out using a phenyl column and an isocratic elution of acidified water and acetonitrile with fluorescence detection at 281 and 316 nm for excitation and emission, respectively. The calibration curve was linear and limits of quantification were 30 ng/ml and 3 microg/ml for hydroxytyrosol and oleuropein, respectively. The method has been successfully applied to monitor oleuropein and hydroxytyrosol plasma levels in the rat.

Structural characterization of the metabolites of hydroxytyrosol, the principal phenolic component in olive oil, in rats.

Hydroxytyrosol is quantitatively and qualitatively the principal phenolic antioxidant in olive oil. Recently it was shown that hydroxytyrosol and five metabolites were excreted in urine when hydroxytyrosol was dosed intravenously or orally in an olive oil solution to rats. The conclusive identification of three metabolites of hydroxytyrosol by MS/MS as a monosulfate conjugate, a 3-O-glucuronide conjugate, and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid) has been established in this investigation. The structural configurations of the glucuronide conjugate and 4-hydroxy-3-methoxyphenylacetic acid were confirmed by (1)H NMR. The radical scavenging potencies of homovanillic acid, homovanillic alcohol, hydroxytyrosol, and the metabolites were examined with the radical 2,2-diphenyl-1-picrylhydrazyl. These studies showed them to be potent antioxidants with SC(50) values of 14.8 and 11.4 microM for homovanillic acid and homovanillic alcohol, respectively. The 3-O-glucuronide conjugate was more potent than hydroxytyrosol, with an SC(50) of 2.3 in comparison to 11.0 microM, and the monosulfate conjugate was almost devoid of radical scavenging activity.

Comparison of radical scavenging effect, inhibition of microsomal oxygen free radical generation, and serum lipoprotein oxidation of several natural antioxidants.

Typical components of the Mediterranean diet, such as olive oil and red wine, contain high concentrations of complex phenols, which have been suggested to have an important antioxidant role. The aim of the present work was to determine the inhibitory potency of compounds such as oleuropein, hydroxytyrosol, and other structurally related compounds, such as gallic acid, toward reactive oxygen species generation and free radical scavenging ability. The potency of these compounds was also examined with respect to protecting in vitro low-density lipoprotein oxidation. These studies indicate that complex phenols, such as hydroxytyrosol, and gallic acid both inhibit free radical generation and act as free radical scavengers. The use of three different approaches to determine antioxidant potency demonstrates that activity in one test does not necessarily correlate with activity in another. It was also demonstrated that the presence of two phenolic groups is not always associated with antioxidant activity.

Inhibition of CYP3A-mediated oxidation in human hepatic microsomes by the dietary derived complex phenol, gallic acid.

Plant polyphenols, such as gallic acid, have been reported to have a range of biological activities including antimutagenic effects. Previously, we reported that gallic acid (3,4,5-trihydroxybenzoic acid), an agent found in wine and tea, inhibits androstenedione 6beta-hydroxylase activity (Ki 70 microm), a cytochrome P450 (CYP3A) marker in human liver microsomes. The pre-incubation of gallic acid (100 microM) with human liver microsomes in the absence of NADPH, as compared with the presence of NADPH, before assay of androstenedione 6beta-hydroxylase activity significantly increased the inhibitory effects of the gallic acid (0.03 +/- 0.03 nmol (mg microsomal protein)(-1) min(-1) compared with 0.20 +/- 0.06 nmol (mg microsomal protein) (-1) min(-1) (P < 0.05)). The antioxidant ascorbic acid and the radical scavenger glutathione prevented this observed increase in inhibition. Removal of gallic acid-derived products from the incubation completely restored CYP3A activity. In contrast, the activities of CYP1A and CYP2E, and non-CYP mediated reductive microsomal 17beta-hydroxysteroid dehydrogenase activity were refractory to inhibition by gallic acid.

Major phenolic compounds in olive oil: metabolism and health effects.

It has been postulated that the components in olive oil in the Mediterranean diet, a diet which is largely vegetarian in nature, can contribute to the lower incidence of coronary heart disease and prostate and colon cancers. The Mediterranean diet includes the consumption of large amounts of olive oil. Olive oil is a source of at least 30 phenolic compounds. The major phenolic compounds in olive oil are oleuropein, hydroxytyrosol and tyrosol. Recently there has been a surge in the number of publications that has investigated their biological properties. The phenolic compounds present in olive oil are strong antioxidants and radical scavengers. Olive "waste water" also possesses compounds which are strong antioxidant and radical scavengers. Typically, hydroxytyrosol is a superior antioxidant and radical scavenger to oleuropein and tyrosol. Hydroxytyrosol and oleuropein have antimicrobial activity against ATTC bacterial strains and clinical bacterial strains. Recent syntheses of labeled and unlabelled hydroxytyrosol coupled with superior analytical techniques have enabled its absorption and metabolism to be studied. It has recently been found that hydroxytyosol is renally excreted unchanged and as the following metabolites as its glucuronide conjugate, sulfate conjugate, homovanillic acid, homovanillic alcohol, 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylacetaldehyde. Studies with tyrosol have shown that it is excreted unchanged and as its conjugates. This review summarizes the antioxidant abilities; the scavenging abilities and the biological fates of hydroxytyrosol, oleuropein and tyrosol which have been published in recent years.