Diversity and toxicity of the diatom Pseudo-nitzschia Peragallo in the Gulf of Maine, Northwestern Atlantic Ocean.

Multiple species in the toxic marine diatom genus Pseudo-nitzschia have been identified in the Northwestern Atlantic region encompassing the Gulf of Maine (GOM), including the Bay of Fundy (BOF). To gain further knowledge of the taxonomic composition and toxicity of species in this region, Pseudo-nitzschia isolates (n=146) were isolated from samples collected during research cruises that provided broad spatial coverage across the GOM and the southern New England shelf, herein referred to as the GOM region, during 2007-2008. Isolates, and cells in field material collected at 38 stations, were identified using electron microscopy (EM). Eight species (P. americana, P. fraudulenta, P. subpacifica, P. heimii, P. pungens, P. seriata, P. delicatissima and P. turgidula), and a novel form, Pseudo-nitzschia sp. GOM, were identified. Species identity was confirmed by sequencing the large subunit of the ribosomal rDNA (28S) and the internal transcribed spacer 2 (ITS2) for six species (36 isolates). Phylogenetic analyses (including neighbor joining, maximum parsimony, and maximum likelihood estimates and ITS2 secondary structure analysis) and morphometric data supported the placement of P. sp. GOM in a novel clade that includes morphologically and genetically similar isolates from Australia and Spain and is genetically most similar to P. pseudodelicatissima and P. cuspidata. Seven species (46 isolates) were grown in nutrient-replete batch culture and aliquots consisting of cells and growth medium were screened by Biosense ASP ELISA to measure total domoic acid (DA) produced (intracellular + extracellular); P. americana and P. heimii were excluded from all toxin analyses as they did not persist in culture long enough for testing. All 46 isolates screened produced DA in culture and total DA varied among species (e.g., 0.04 to 320 ng ml-1 for P. pungens and P. sp. GOM isolates, respectively) and among isolates of the same species (e.g., 0.24 - 320 ng ml-1 for P. sp. GOM). The 15 most toxic isolates corresponded to P. seriata, P. sp. GOM and P. pungens, and fg DA cell-1 was determined for whole cultures (cells and medium) using ELISA and liquid chromatography (LC) with fluorescence detection (FLD); for seven isolates, toxin levels were also estimated using LC - with mass spectrometry and ultraviolet absorbance detection. Pseudo-nitzschia seriata was the most toxic species (up to 3,500 fg cell-1) and was observed in the GOM region during all cruises (i.e., during the months of April, May, June and October). Pseudo-nitzschia sp. GOM, observed only during September and October 2007, was less toxic (19 - 380 fg cell-1) than P. seriata but more toxic than P. pungens var. pungens (0. 4 fg cell-1). Quantitation of DA indicated that concentrations measured by LC and ELISA were positively and significantly correlated; the lower detection limit of the ELISA permitted quantification of toxicity in isolates that were found to be nontoxic with LC methods. The confirmation of at least seven toxic species and the broad spatial and temporal distribution of toxic Pseudo-nitzschia spp. have significant implications for the regional management of nearshore and offshore shellfisheries resources.

CRYPTIC AND PSEUDO-CRYPTIC DIVERSITY IN DIATOMS-WITH DESCRIPTIONS OF PSEUDO-NITZSCHIA HASLEANA SP. NOV. AND P. FRYXELLIANA SP. NOV.(1).

A high degree of pseudo-cryptic diversity was reported in the well-studied diatom genus Pseudo-nitzschia. Studies off the coast of Washington State revealed the presence of hitherto undescribed diversity of Pseudo-nitzschia. Forty-one clonal strains, representing six different taxa of the P. pseudodelicatissima complex, were studied morphologically using LM and EM, and genetically using genes from three different cellular compartments: the nucleus (D1-D3 of the LSU of rDNA and internal transcribed spacers [ITSs] of rDNA), the mitochondria (cytochrome c oxidase 1), and the plastids (LSU of RUBISCO). Strains in culture at the same time were used in mating studies to study reproductive isolation of species, and selected strains were examined for the production of the neurotoxin domoic acid (DA). Two new species, P. hasleana sp. nov. and P. fryxelliana sp. nov., are described based on morphological and molecular data. In all phylogenetic analyses, P. hasleana appeared as sister taxa to a clade comprising P. calliantha and P. mannii, whereas the position of P. fryxelliana was more uncertain. In the phylogenies of ITS, P. fryxelliana appeared to be most closely related to P. cf. turgidula. Morphologically, P. hasleana differed from most other species of the complex because of a lower density of fibulae, whereas P. fryxelliana had fewer sectors in the poroids and a higher poroid density than most of the other species. P. hasleana did not produce detectable levels of DA; P. fryxelliana was unfortunately not tested. In P. cuspidata, production of DA in offspring cultures varied from higher than the parent cultures to undetectable.

Diagnosis of iron deficiency anemia in children of Northeast Brazil.

OBJECTIVE: To diagnose iron deficiency anemia in children. METHODS: The study was conducted with a sample of 301 children aged six to 30 months attending public daycare centers in the city of Recife, Northeast Brazil, in 2004. The diagnoses of anemia were based on a combination of different hematological and biochemical parameters: hemoglobin, mean corpuscular volume, ferritin, C-reactive protein, transferrin saturation and transferrin receptor. The chi-square test and ANOVA were used in the statistical analysis. RESULTS: Of all children studied, 92.4% had anemia (Hb<110 g/L) and 28.9% had moderate/severe anemia (Hb<90 g/L). Lower levels of hemoglobin were found in children aged 6-17 months. Iron deficiency was found in 51.5% of children using ferritin (<12 microg/L) as parameter. Taking into consideration the combination of hemoglobin level, ferritin and transferrin receptor, 58.1% had anemia with iron deficiency, 34.2% had anemia without iron deficiency and 2.3% had iron deficiency without anemia. Mean ferritin concentration was significantly higher in children with high C-reactive protein when compared with those with normal levels (22.1 vs. 14.8 microg/L). CONCLUSIONS: The use of several biochemical and hematological parameters allowed to diagnosing iron deficiency anemia in two thirds of children, suggesting a need to identify other determinants of anemia without iron deficiency.

Diagnosis of iron deficiency anemia in children of Northeast Brazil / Diagnóstico de anemia por deficiencia de hierro en niños del Noreste de Brasil

OBJETIVO: Diagnosticar anemia por deficiência de ferro em crianças. MÉTODOS: O estudo foi desenvolvido com uma amostra de 301 crianças com idade entre seis e 30 meses, usuárias de creches públicas de Recife, PE, em 2004. Para o diagnóstico da anemia utilizou-se a combinação de diferentes parâmetros hematológicos e bioquímicos: hemoglobina, volume corpuscular médio, ferritina, proteína C-reativa, saturação da transferrina e receptor da transferrina. Para a análise estatística empregou-se o teste do qui-quadrado e ANOVA. RESULTADOS: Do total de crianças, 92,4 por cento tinha anemia (Hb < 110g/L) e 28,9 por cento apresentou anemia moderada/grave (Hb<90g/L). Níveis mais baixos de hemoglobina foram observados em crianças de seis a 17 meses. Encontrou-se deficiência de ferro em 51,5 por cento das crianças, utilizando-se a ferritina (< 12µg/L) como parâmetro. Considerando a combinação da concentração de hemoglobina, ferritina e do receptor de transferrina, 58,1 por cento tinha anemia com deficiência de ferro, 34,2 por cento anemia sem déficit de ferro e 2,3 por cento deficiência de ferro sem anemia. A concentração média de ferritina foi significativamente maior em crianças com proteína C-reativa aumentada quando comparada com aqueles com níveis normais (22,1 versus 14,8 µg/L). CONCLUSÕES: A utilização de diversos parâmetros bioquímicos e hematológicos possibilitou diagnosticar anemia por deficiência de ferro em dois terços das crianças, revelando a necessidade de identificar outros determinantes de anemia sem deficiência de ferro.

OBJETIVO: Diagnosticar anemia por deficiência de ferro em crianças. MÉTODOS: O estudo foi desenvolvido com uma amostra de 301 crianças com idade entre seis e 30 meses, usuárias de creches públicas de Recife, PE, em 2004. Para o diagnóstico da anemia utilizou-se a combinação de diferentes parâmetros hematológicos e bioquímicos: hemoglobina, volume corpuscular médio, ferritina, proteína C-reativa, saturação da transferrina e receptor da transferrina. Para a análise estatística empregou-se o teste do qui-quadrado e ANOVA. RESULTADOS: Do total de crianças, 92,4% tinha anemia (Hb < 110g/L) e 28,9% apresentou anemia moderada/grave (Hb<90g/L). Níveis mais baixos de hemoglobina foram observados em crianças de seis a 17 meses. Encontrou-se deficiência de ferro em 51,5% das crianças, utilizando-se a ferritina (< 12µg/L) como parâmetro. Considerando a combinação da concentração de hemoglobina, ferritina e do receptor de transferrina, 58,1% tinha anemia com deficiência de ferro, 34,2% anemia sem déficit de ferro e 2,3% deficiência de ferro sem anemia. A concentração média de ferritina foi significativamente maior em crianças com proteína C-reativa aumentada quando comparada com aqueles com níveis normais (22,1 versus 14,8 µg/L). CONCLUSÕES: A utilização de diversos parâmetros bioquímicos e hematológicos possibilitou diagnosticar anemia por deficiência de ferro em dois terços das crianças, revelando a necessidade de identificar outros determinantes de anemia sem deficiência de ferro.

Ellagic acid and its methyl-derivatives inhibit a newly found nitratase activity.

We have recently shown that low density lipoprotein (LDL) was able to denitrate albumin-bound 3-NO(2)-Tyr residues and to concomitantly release NO(3)(-) through a Ca(2+)-dependent process that has been ascribed to a specific protein structure. A lipophilic food component (gamma-tocopherol), which is easily loaded into LDL has been found to totally inhibit denitrating activity. We presently found that ellagic acid (EA) and its methylated derivatives, 4,4'O-methyl- and 3,3'O-methyl-ellagic acids (MeEA1 and MeEA2, respectively), amphipathic phenolic components of certain fruits and beverages, were also able to inhibit this activity, with a total inhibition for EA and a 60% inhibition for MeEA1 and MeEA2. EA exhibited the highest affinity for protein plasma, whereas a higher affinity of MeEA1 and MeEA2 (with MeEA1 > MeEA2) than EA was found for lipoprotein fractions, suggesting that the inhibition-driving property is protein affinity. As a result of this nitratase-inhibition property EA and its natural metabolite MeEA2 may have a beneficial role in special physiopathological conditions.

Analysis of trace levels of domoic acid in seawater and plankton by liquid chromatography without derivatization, using UV or mass spectrometry detection.

Quantitation of trace levels of domoic acid (DA) in seawater samples usually requires labour-intensive protocols involving chemical derivatization with 9-fluorenylmethylchloroformate and liquid chromatography with fluorescence detection (FMOC-LC-FLD). Procedures based on LC-MS have been published, but time-consuming and costly solid-phase extraction pre-concentration steps are required to achieve suitable detection limits. This paper describes an alternative, simple and inexpensive LC method with ultraviolet detection (LC-UVD) for the routine analysis of trace levels of DA in seawater without the use of sample pre-concentration or derivatization steps. Qualitative confirmation of DA identity in dubious samples can be achieved by mass spectrometry (LC-MS) using the same chromatographic conditions. Addition of an ion-pairing/acidifying agent (0.15% trifluoroacetic acid) to sample extracts and the use of a gradient elution permitted the direct analysis of large sample volumes (100 microl), resulting in both high selectivity and sensitivity (limit of detection=42 pg ml(-1) by LC-UVD and 15 pg ml(-1) by LC-MS). Same-day precision varied between 0.4 and 5%, depending on the detection method and DA concentration. Mean recoveries of spiked DA in seawater by LC-UVD were 98.8% at 0.1-10 ng ml(-1) and 99.8% at 50-1000 ng ml(-1). LC-UVD exhibited strong correlation with FMOC-LC-FLD during inter-laboratory analysis of Pseudo-nitzschia multiseries cultures containing 60-2000 ng DA ml(-1) (r(2)>0.99), but more variable results were obtained by LC-MS (r(2)=0.85). This new technique was used to confirm the presence of trace DA levels in low-toxicity Pseudo-nitzschia spp. isolates (0.2-1.6 ng ml(-1)) and in whole-water field samples (0.3-5.8 ng ml(-1)), even in the absence of detectable Pseudo-nitzschia spp. cells in the water column.

Salicylate enables cochlear arachidonic-acid-sensitive NMDA receptor responses.

Currently, many millions of people treated for various ailments receive high doses of salicylate. Consequently, understanding the mechanisms by which salicylate induces tinnitus is an important issue for the research community. Behavioral testing in rats have shown that tinnitus induced by salicylate or mefenamate (both cyclooxygenase blockers) are mediated by cochlear NMDA receptors. Here we report that the synapses between the sensory inner hair cells and the dendrites of the cochlear spiral ganglion neurons express NMDA receptors. Patch-clamp recordings and two-photon calcium imaging demonstrated that salicylate and arachidonate (a substrate of cyclooxygenase) enabled the calcium flux and the neural excitatory effects of NMDA on cochlear spiral ganglion neurons. Salicylate also increased the arachidonate content of the whole cochlea in vivo. Single-unit recordings of auditory nerve fibers in adult guinea pig confirmed the neural excitatory effect of salicylate and the blockade of this effect by NMDA antagonist. These results suggest that salicylate inhibits cochlear cyclooxygenase, which increased levels of arachidonate. The increased levels of arachidonate then act on NMDA receptors to enable NMDA responses to glutamate that inner hair cells spontaneously release. This new pharmacological profile of salicylate provides a molecular mechanism for the generation of tinnitus at the periphery of the auditory system.

First evidence for an LDL- and HDL-associated nitratase activity that denitrates albumin-bound nitrotyrosine--physiological consequences.

In the systemic circulation, LDL occurs in the form of a weakly nitrated LDL-albumin complex (LAC). The question here is whether LAC (or HDL) is able to denitrate the albumin-bound 3-NO(2)-tyrosine (3NT). Nitrated albumin was incubated in the presence of lipoprotein fraction (LPF) to be tested, with or without Ca(2+). After precipitation and centrifugation, supernatants (SNs) and protein pellets (PP) were collected. HCl proteolysis was carried out with deuterated 3NT as an internal standard, and amino acids were derivatized for GC-MS analysis, whereas SNs were used for NO(2) (-)/NO(3) (-)-fluorimetric assays. A loss of 3NT, higher with albumin-low LDL than with albumin-rich LDL or HDL, was found in PP only in the presence of Ca(2+). gamma-Tocopherol loading of LPF inhibited 3NT loss. 3NT loss was found for the first time to be stoichiometrically equivalent to NO(3) (-), proving that the 3NT loss must be ascribed to a 3NT-denitrating nitratase activity. 3NT loss and NO(3) (-) production that clearly cannot be attributed to PON-1 were impaired by D-penicillamine and phenylacetate, inhibitor, and substrate of PON-1, respectively, leading to speculate on the active site. Finally, nitratase activity and albumin contribute to beneficially convert peroxynitrite (ONOO(-)) into nonbioactive NO(3) (-). But, in inflammatory conditions, xanthine oxidoreductase is expressed leading to detrimentally reduce O(2) and NO(3) (-) into O(2) (*) (-) and NO(*) that may interact, reconstituting the ONOO(-) pool. The real consequence of nitratase activity and the physiological significance of nitration/denitration processes remain to be explored.

Peroxynitrite mild nitration of albumin and LDL-albumin complex naturally present in plasma and tyrosine nitration rate-albumin impairs LDL nitration.

In blood, peroxynitrite (ONOO- ) and CO2 react to form NO2* and CO3* through the short-lived adduct ONOOCO2-, leading to protein-bound tyrosine nitration. ONOO(-) -modified LDL is atherogenic. Oxidatively modified LDL generally appears in the vessel wall surrounded by antioxidants. Human serum albumin (HSA) is one of them, partly associated to LDL as a LDL-albumin complex (LAC). The purpose was to study the effect of a mild nitration on LAC and whether albumin may interfere with LDL nitration. To do so, SIN-1 was used as ONOO- generator in the presence or absence of 25 mM HCO3-. The human serum albumin (HSA)-bound tyrosine nitration rate was found to be 4.4 x 10(-3) mol/mol in the presence of HCO3-. HSA decreased the LAC-tyrosine nitration rate from 2.5 x 10(-3) to 0.6 x 10(-3) mol/mol. It was concluded that HSA impaired the apoB-bound tyrosine nitration. These findings raise the question of the patho-physiological significance of these nitrations and their interactions which may potentially prevent both atheromatous plaque formation and endothelium dysfunction in particular and appear to be beneficial in terms of atherogenic risk.

Regional Brazilian diet-induced pre-natal malnutrition in rats is correlated with the proliferation of cultured vascular smooth muscle cells.

OBJECTIVES: Pre-natal malnutrition induces hypertension and insulin resistance, pathologies commonly linked to atherosclerotic disease. The proliferation of vascular smooth muscle cells (SMCs) is important during development of the atherosclerotic plaque. In this work, we investigated whether the serum of pre-natal malnourished Wistar rats could alter the proliferation of aortic and renal artery SMCs in culture. Malnutrition was induced by feeding a basic regional diet available in a rural area of Pernambuco State, Brazil. This diet was rich in carbohydrates and deficient in proteins, lipids, vitamins and minerals, including sodium chloride. METHODS AND RESULTS: Serum was obtained from the blood of 90-day-old control and pre-natal undernourished rats. SMCs from control Wistar rats at the 6th passage were allowed to adhere to plates in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal calf serum (10%). Subsequently, the SMCs were maintained in DMEM supplemented with rat serum (10%). The number of cells was counted on the 3rd, 6th and 8th days of culture into rat serum. [3H]-thymidine incorporation into SMCs was evaluated after 20 h or 6 days of incubation. The birth weight of male and female undernourished offspring was 25% (p<0.05) and 46% (p<0.05) lower, respectively, than their corresponding control groups. On the 8th day of culture, the number of aortic SMCs in the serum of undernourished male and female rats, as well as renal artery SMCs in the serum of undernourished female rats, was higher than in the serum of control rats. The [3H]-thymidine incorporation was higher in aortic SMCs incubated for 6 days in the serum of undernourished male and female rats. At confluence, the density of aortic SMCs was higher than that of renal artery SMCs. CONCLUSIONS: Pre-natal malnutrition produces serum with altered properties that can affect the proliferation of SMCs and may contribute to atherosclerotic disease.

Fluorescence spectroscopy for monitoring deterioration of extra virgin olive oil during heating.

The potential of fluorescence spectroscopy for characterizing the deterioration of extra virgin olive oil (EVOO) during heating was investigated. Two commercial EVOO were analysed by HPLC to determine changes in EVOO vitamin E and polyphenols as a result of heating at 170 degrees C for 3 h. This thermal oxidation of EVOO caused an exponential decrease in hydroxytyrosol and vitamin E (R(2)=0.90 and 0.93, respectively) whereas the tyrosol content was relatively stable. At the same time, amounts of preformed hydroperoxides (ROOH), analysed by an indirect colorimetric method, decreased exponentially during the heating process (R(2)=0.94), as a result of their degradation into secondary peroxidation products. Fluorescence excitation spectra with emission at 330 and 450 nm were recorded to monitor polyphenols and vitamin E evolution and ROOH degradation, respectively. Partial least-squares calibration models were built to predict these indicators of EVOO quality from oil fluorescence spectra. A global approach was then proposed to monitor the heat charge from the overall fluorescence fingerprint. Different data pretreatment methods were tested. This study indicates that fluorescence spectroscopy is a promising, rapid, and cost-effective approach for evaluating the quality of heat-treated EVOO, and is an alternative to time-consuming conventional analyses. In future work, calibration models will be developed using a wide range of EVOO samples.

Potential anti-atherogenic cell action of the naturally occurring 4-O-methyl derivative of gallic acid on Ang II-treated macrophages.

We have recently established that the blood concentrations of gallic acid (GA), a polyphenolic component naturally found in food, and its O-methyl derivatives are very low (practically < or = 1 microM) in physiological (postprandial) condition. Using acellular oxidant systems and macrophage-differentiated promonocytes (MDPs) THP-1, we show here that the direct and indirect (through depressing effect on the superoxide cell production) antioxidant properties of these components were not effective at these concentrations. In contrast, 4-O-methyl GA was the most efficient component to depress AT1R and CD36 mRNA expression in Ang II-treated MDPs, suggesting a strong inhibition of Ang II-triggered pro-atherogenic mechanisms of foam cell formation.

Red-wine beneficial long-term effect on lipids but not on antioxidant characteristics in plasma in a study comparing three types of wine--description of two O-methylated derivatives of gallic acid in humans.

The purpose of this double clinical study was (1) to evaluate the effect of one single intake (300 ml) of red wine (RW) on the plasma antioxidant capacity (pAOC) and plasma phenolics over the 24-h time period following the intake, and (2) to compare the long-term effects of daily intakes (250 ml/d) of RW, white wine (WW) and Champagne (CH) on the plasma and LDL characteristics of healthy subjects. In the first part, blood samples were collected just before and after wine consumption. In the second part, subjects received the 3 types of wine successively, only at the mealtime, over 3-week periods separated by a 3-week wash out. Blood samples were drawn in fasting condition before and after each 3-week wine consumption period. The peak of pAOC was at 3-4 h following the single intake of RW, that of catechin was at 4 h (0.13 micromol/l) and that of gallic acid and caffeic acid was earlier (< or = 1.5 and 0.3 micromol/l, respectively). In plasma, the major form of gallic acid was 4-O-methylated, but a minor form (the 3-O-methyl derivative) appeared. In the long term study, no wine was able to change LDL oxidizability, but some other parameters were modified specifically: RW decreased pAOC (without changing TBARS and uric acid plasma levels), LDL lipids and total cholesterol (TC), and increased plasma apoA1, whereas CH increased plasma vitamin A. The beneficial effect of RW seems to mainly be explained by its action on lipid and lipoprotein constants, and not by its antioxidant one.

Saúde e nutrição de crianças de áreas urbanas da Zona da Mata Meridional de Pernambuco: resultados preliminares de um estudo de coorte / Child health and nutrition in urban areas in the South of Pernambuco: preliminary results of a cohort study

OBJETIVOS: apresentar as características socioeconômicas e demográficas, o perfil nutricional de crianças ao nascer e aos 12 meses de vida, além dos dados longitudinais sobre aleitamento, diarréia e situação vacinal durante o primeiro ano de vida. MÉTODOS: uma amostra de 652 recém-nascidos foi recrutada de setembro de 1997 a agosto de 1998 e acompanhada durante os primeiros 18 meses de vida. Essas crianças residiam nas áreas urbanas de quatro municípios da zona da mata meridional de Pernambuco. A coleta de dados foi realizada através de visitas domiciliares. RESULTADOS: cerca de 60 por cento das famílias tinham uma renda per capita ú ® salário mínimo e 41 por cento das mães referiam menos de quatro anos de escolaridade. A mediana do aleitamento materno exclusivo e total foi de 0 dias e 94 dias, respectivamente. A incidência da diarréia foi de dois episódios/criança/ano nos primeiros 12 meses de vida e a prevalência de déficit peso/idade e comprimento/idade (<-2 escores z) aos 12 meses foram de 6,8 por cento e 11 por cento, respectivamente. Apenas 66 por cento das crianças aos 12 meses tinham completado o esquema vacinal. CONCLUSÕES: o desenvolvimento desta pesquisa prospectiva contribuirá para uma maior compreensão dos problemas de saúde e nutrição da população infantil e para o adequado planejamento de intervenções na área.

Grape and grape seed extract capacities at protecting LDL against oxidation generated by Cu2+, AAPH or SIN-1 and at decreasing superoxide THP-1 cell production. A comparison to other extracts or compounds.

A large body of evidence supports the key role of oxidized low-density lipoprotein in atherosclerosis. The aim of this study was to compare the capacity of natural polyphenols (PP) from Vitis vinifera and Olea europea at protecting LDL against oxidation brought about by Cu2+, oxygen-centered radical-generating AAPH, or peroxynitrite-generating SIN-1 in vitro systems, or at impairing superoxide production in promonocyte cells (THP-1) conveniently differentiated into adherent macrophages. PP were either from the whole grape (fraction A) containing mainly procyanidins, (epi)-catechin and anthocyanins, or from grape seed extracts (fractions B and C) consisting of tannins and procyanidin oligomers with a higher content in B than in C, or from a grape skin extract (fraction D) consisting mainly of anthocyanins, or from a hydrosoluble olive mill wastewater PP extract (fraction E) containing hydroxytyrosol and oleuropein. Chlorogenic acid (F) and catechin (G) were taken as archetypes of PP preventing oxidation partly as copper scavenger and as radical scavenger only, respectively. All grape fractions were efficient towards Cu2+ system (equally or more efficient than F), whereas they were rather poorly efficient towards AAPH and SIN-1 (less efficient than G but as efficient as F). Among the PP fractions, B was the most effective at protecting LDL in the SIN-1 system and at impairing THP-1 superoxide production. Taken together, these data suggest that the PP fraction from grape seed rich in procyanidins achieves the best compromise between the direct and indirect (i.e. cell-mediated) types of action in protecting LDL against oxidation, strengthening the need for improving the knowledge of its bioavailability in humans.

Synergistic antioxidative properties of phenolics from natural origin toward low-density lipoproteins depend on the oxidation system.

Using an approach in line with that of a previous report, we assessed the antioxidant activity of several natural, polyphenol- or tocotrienol-rich mixtures: extracts from Elaesis Guineensis oil (A) and Vitis vinifera (B), a Coffea robusta powder (C), and extracts from Olea europea mill wastewaters (D), Solanum melongena (E), and Lycopersicon esculentum (F). The copper- and 2-2'-azobis(2-amidinopropane) hydrochloride (AAPH)-oxidation systems were used in the presence of low-density lipoprotein. For comparison, antioxidant activities of chlorogenic acid and catechin, as archetypes of molecules highly efficient with the copper- and the AAPH-oxidation system, respectively, were assessed. The aim was to establish the occurrence of synergistic antioxidant actions among some of these natural mixtures. On a molar basis, the highest specific antioxidant activities (SAA) were found for B, chlorogenic acid, and C in the copper system, and for A, catechin, and B in the AAPH system. On a mass basis, the highest SAA were found, respectively, for chlorogenic acid, B, and catechin, and for catechin, chlorogenic acid, and B. These results show that large discrepancies take place in the evaluations between the two systems. B and C exhibited a synergistic antioxidant efficiency, in the presence or absence of A, but only with the copper system. This was also true for the two types of A+B+C mixture that were tested. It is thought that this association might provide an ideal combination, incorporating both the radical scavenger and the transition-metal ion chelation properties of B and C.

Cord blood essential fatty acid and alpha-tocopherol in full-term newborns in a Northeast Brazil area.

Malnutrition of children during the first two years of life constitutes a public health concern in Brazil, particularly in the Northeast. Most of the nutrition data are concerned with protein-energy malnutrition and hypovitaminosis A. The purpose of this cross-sectional study was to assess the essential fatty acid (EFA) status, which is crucial in physical and mental development, and that of vitamin E which prevents against the oxidative loss of EFA physiological properties, in 81 full-term newborns. Blood samples were obtained from the residual blood of the umbilical cord (UC) at delivery. Fatty acid composition of UC plasma did not show any sign of EFA deficiency. The levels of docosahexanoic (DHA) and arachidonic acid (AA) appeared to be quite similar to those obtained in European populations. UC plasma vitamin E content was 6.31 +/- 1.99 mumol/L whereas the lipid-normalized vitamin E was 2.36 mumol/mmol of lipids. An interesting point was that newborns with vitamin E inferior to the median value (5.80 mumol/L) revealed significantly lower contents of linoleic acid and DHA in UC than newborns superior to the median value. Together with the absolute or normalized plasma level of vitamin E, this supports the observation that one quarter of the community's newborns is deficient in vitamin E.

New insight on the relationship between LDL composition, associated proteins, oxidative resistance and preparation procedure.

Oxidized low density lipoprotein (LDL) plays an important role in atherogenesis. It is generally thought that LDL is mainly oxidized in the intima of vessel walls, surrounded by hydrophilic antioxidants and proteins such as albumin. The aim of this study was to investigate the possible interrelationships between oxidation resistance of LDL and its protein and lipid moieties. Proteins and to a lesser extent lipids, appeared to be the major determinants in the LDL Cu2+-oxidation resistance, which in turn depend on the ultracentrifugation (UC) procedure used. Comparing high speed/short time (HS/ST, 4 h), high speed/long time (HS/LT, 6-16h) and low speed/long time (LS/LT, 24h) conditions of UC, HS with the shortest time (4h) led to prepare LDL (named LDL.HS-4 h) with higher total protein and triglyceride contents, unchanged total cholesterol, phospholipids and Vitamin E, and higher Cu2+-oxidation resistance. Among proteins, only albumin allows to explain changes. PAF acetyl hydrolase appeared to be unaffected, whereas its pro-oxidant role was established and found only in the absence of albumin. In contrast the pro-oxidant role of caeruloplasmin took place regardless of the albumin content of LDL. The antioxidant effect of albumin (the oxidation lag time was doubled for 20mol/mol albumin per LDL) is assumed to be due to its capacity at decreasing LDL affinity for Cu2+. Interestingly, the LDL.HS-4 h albumin content mirrored the intrinsic characteristics of LDL in the plasma and was not affected by added free albumin. Moreover, it has been verified that in 121 healthy subjects albumin was the best resistance predictor of the Cu2+-oxidation of LDL.HS-4 h, with a multiple regression equation: lag time (min) = 62.1 + 0.67(HSA/apoB) + 0.02(TG/apoB)-0.01(TC/apoB); r = 0.54, P < 0.0001. Accounted for by lag time, the oxidation resistance did not correlate with alpha-tocopherol and ubiquinol contents of LDL. The mean albumin content was about 10mol/mol, and highly variable (0-58 mol/mol) with subjects. The LDL.HS-4h may account for the status of LDL in its natural environment more adequately than LDL resulting from other conditions of UC.

A diet high in cholesterol and deficient in vitamin E induces lipid peroxidation but does not enhance antioxidant enzyme expression in rat liver.

Expression of antioxidant enzymes (AOE), an important mechanism in the protection against oxidative stress, could be modified by the redox status of the cells. The aim of this project was to evaluate the role of vitamin E deficiency in association with a high-cholesterol diet in the hepatic lipid peroxidation and the expression of AOE. Two groups of 6 male rats were fed with a high-cholesterol or a high-cholesterol vitamin E-deficient diet. All animals were sacrificed at 72 days of treatment. Liver lipid peroxidation index (Malondialdehyde; MDA) and hepatic AOE were evaluated. Total liver RNA was extracted, and the steady state messenger RNA (mRNA) levels of glutathion peroxydase, manganese superoxide dismutase, Cu/Zn superoxide dismutase and catalase were examined by northern blot. After 72 days on the diet, a significant increase in the lipid peroxidation index was observed in the vitamin E deficient group (MDA : 4.45 +/- 0.29 nmol/mg protein versus 3.65 +/- 0.1 nmol/mg protein in vitamin E normal group). Despite this oxidative stress, the activities and mRNA levels of liver AOE were not significantly different in the 2 groups. These preliminary results show that chronic vitamin E deficiency associated with high cholesterol diet is able to increase lipid peroxidation without modulation of AOE expression and activity in the liver. This suggests that beneficial effects of dietary vitamin E are due to a plasma antioxidant effect or a cell mediated action, rather than to a specific modulation of cellular enzymes.

Convective and diffusive losses of vitamin C during haemodiafiltration session: a contributive factor to oxidative stress in haemodialysis patients.

BACKGROUND: Enhanced oxidative stress in haemodialysis (HD) patients may be considered as a risk factor for accelerated atherosclerosis. Reduced antioxidant defences include impairment in enzyme activities and decreased plasma levels of hydrophilic vitamin C (vit C), and cellular levels of lipophilic vitamin E (vit E). METHODS: We investigated plasma levels of vit C in 19 patients undergoing regular haemodiafiltration (HDF) (mean age 62+/-7 years) and in 1846 healthy elderly subjects (HS) (mean age 69+/-5 years). The contribution of convection and diffusion was determined using paired filtration dialysis (PFD), a modified HDF technique which physically separates convective from diffusive fluxes. Blood samples were collected before and after the HDF session; in addition at 60 min of HDF, samples were drawn from arterial lines (AL) and venous lines (VL), dialysate (D) and ultrafiltrate (UF). Blood levels of total vit C were determined using an HPLC fluorescence method. Markers of oxidative stress were also assessed in both populations as follows: levels of malondialdehyde (MDA) were determined by fluorometric assay, measurements of advanced oxidation protein products (AOPP) and glutathione peroxidase (GSH-Px) activity were performed by spectrophotometric assay, and plasma vit E content was obtained by an HPLC procedure. RESULTS: A significant reduction in plasma vit C level was observed in HDF patients when compared with HS (1.6+/-1.4 microg/ml in HDF vs 6.6+/-3.7 microg/ml in HS; P<0.01). The HDF session was associated with a dramatic reduction in vit C levels (1.87+/-1.57 microg/ml before HDF and 0.98+/-0.68 microg/ml after HDF); at 60 min of HDF, concentrations were as follows: AL=1.35+/-1.27 microg/ml; VL=0.37+/-0.31 microg/ml, D=0.40+/-0.34 microg/ml, UF=1.24+/-1.18 microg/ml; corresponding to a diffusive flux of 271 microg/min and a convective flux of 126 microg/min. Total loss of vit C could be assessed at 66 mg/session (8--230 mg/session). According to this loss of vit C, presence of an oxidative stress was demonstrated in HD population as shown by a significant increase in MDA (1.66+/-0.27 microM in HD vs 0.89+/-0.25 microM in HS; P<0.01) and AOPP (77.5+/-29.3 microM in HD vs 23.5+/-13.2 microM in HS; P<0.01) levels, and a decrease in GSH-Px activity (259.2+/-106.3 U/l in HD vs 661.2+/-92.2 U/l in HS; P<0.01). No change in plasma vit E between both populations (30.7+/-9.1 microM in HD vs 35.3+/-7.34 microM in HS) was observed. CONCLUSIONS: These results suggest that HDF with highly permeable membranes is associated with a significant loss of vit C. Diffusive transport is responsible for two-thirds whereas convective phenomenon accounts for only one-third of this loss.