A enzima delta-aminolevullnato desidratase

Delta-aminolevulinic acid dehydratase (ALA-D, EC 4.2.1.24) is a sulfhydryl-containing enzyme that asymetrically condenses two molecules of delta-aminolevulinic acid (ALA), catalyzing the formation of porphobilinogen, the monopyrrole precursor of ali biological tetrapyrroles (corrins, porphyrins, chlorins). The two ALA molecules have been termed Aside ALA and P-side ALA in reference to their fates as the acetyl and propionyl halves of the product. P-side ALA binds first and forms a Schiffbase with an active-site Iysine. ALA-D is a cytosolic enzyme present in mammals, plants, fungi and bacteria. Bovine enzyme has a molecular mass of 280 000 Da and is composed of eight similar subunits of 35 000 Da, but only four of the subunits form a Schiff-base with the substrate (half-site reactivity). ALA-D from all organisms requires a bivalent metal ion for activity. Although the considerable sequence conservation among ALA-D enzyme from various organisms, there are species-dependent differences in metal ion requirements for enzyme activity. ALA-D is a zinc-dependent enzyme in animals, yeast and some bacteria. Mammalian enzyme bounds 8 zinc ions/octamer. Bovine ALA-D contains two types of Zn2+ binding sites (A and B), each at a stoichiometry of four per octamer. A-metal-ion-binding sites, with a single cysteine residue among its ligands, bind the four zinc ions essential for ALA-D activity (catalytic zinc), which plays a role in A-side ALA binding, in inter-ALA bond formation and in product binding. B-metal-ion-binding sites, with four cysteine residues among its ligands, bind zinc ions refered to as structural, which seems to be involved in the protection of sulfhydryl groups from oxidation. An A-zinc-ion-binding site has been proposed to be present at a number of four per octamer on the enzyme from plants, but has not been demonstrated yet. ALA-D from plants contains two types of magnesium binding sites: four B-metal-ion-binding sites (bind magnesium essential for ALA-D activity) and eight C-metal-ion-binding sites (bind magnesium that activates the enzyme but is not essential for activity). The cysteine-rich sequence of mammalian ALA-D that presumably corresponds to the B-metalion-binding site is replaced by an aspartate-rich sequence in plant ALA-D, probably accounting for the difference on metal-ion requirement (Mg2+ instead of Zn2+ on B-metal-ion-binding site from plant ALA-D). E. coli ALA-D binds eight Zn2+ (presumably four at A-metal-ion-binding site and four at B-metalion-binding site) and eight Mg2+ (presumably at C-metal-ion-binding site) per octamer. Due to its sulfhydrilic nature ALA-D is inhibited by heavy metals such as lead and mercury, serving as a measure of metal intoxication. In addition the inhibition of this enzyme has been implicated with pathological changes observed in some types of porphyrias, hepatorenal tyrosinemia and after lead or mercury exposure. ALA-D inhibition may impair haeme biosynthesis and leads to ALA accumulation, which besides being a potent agonist of y-aminobutiric acid autoreceptors may act as a prooxidant.A delta-aminolevulinato desidratase (ALA-D, E.C. 4.2.1.24) é uma enzima citosólica encontrada em bactérias, vegetais e animais. A reação catalisada pela ALA-D faz parte da rota de biossíntese dos compostos tetrapirrólicos (corrinas, bilinas, clorofilas e hemes). Esta enzima catalisa a condensação assimétrica de duas moléculas de ácido deltaaminolevulínico (ALA), formando porfobilinogênio. Um grupo Ɛ-amino de um resíduo de lisina presente no sítio ativo da enzima forma uma base de Schiff com a primeira molécula de substrato, a qual origina a cadeia lateral P (ácido propiônico) da molécula de porfobilinogênio. A segunda molécula de ALA originará a cadeia lateral A (ácido acético). A enzima de fígado bovino apresenta um peso molecular de 280 000 Da, sendo composta por 8 subunidades iguais, de 35 000 Da cada uma, no entanto apenas metade das subunidades parece estar envolvida na catálise. Independente da fonte, todas as enzimas ALA-D isoladas até o momento requerem um íon metálico divalente para estar ativas. Apesar do grande grau de similaridade existente entre os genes da ALA-D provenientes de diferentes organismos, a enzima requer metais diferentes para ativação, de acordo com a sua fonte (zinco para a enzima de animais, leveduras e algumas bactérias, e magnésio para a enzima de plantas). A enzima de mamíferos liga 8 íons zinco por octâmero. Foi detectada a existência de 2 sítios estruturalmente distintos para ligação do zinco na ALA-D bovina (sítios A e B). Os sítios A seriam compostos por 5 ligantes, entre eles um -SH de um resíduo de cisteína, e estariam envolvidos na ligação das 4 moléculas de zinco essenciais para a completa ativação da ALA-D (referidas como catalíticas), as quais parecem ser importantes para a união da segunda molécula de substrato, formação da primeira ligação entre as duas moléculas de ALA e união do produto. Os sítios B seriam compostos por 4 resíduos de cisteína e estariam envolvidos na união dos 4 íons zinco não essenciais (referidos como estruturais), os quais teriam a função de manter grupos -SH da enzima no estado reduzido. Tem sido proposto que o sítio A estaria presente também na ALA-D de vegetais, num número de 4 por octâmero, no entanto isto ainda não foi demonstrado. Cada octâmero da ALA-D de plantas apresentaria, ainda, 4 sítios B para união de íons magnésio essenciais e 8 sítios C para união de íons maqnesio não essenciais, cuja função é ativar a enzima. Na enzima de plantas a região que corresponde ao sítio B de união do íon metálico parece conter resíduos de aspartato ao invés dos resíduos de cisteína presentes na enzima de origem animal. Isto explicaria porque o sítio B da ALA-D de plantas liga Mg2+ ao invés de Zn2+. A ALA-D de E. Coli possui, aparentemente, 8 sítios para união de zinco (4 sítios A ou α e 4 sítios B ou β) e 8 sítios para união de magnésio (supostamente sítio C) por octâmero. Devido a sua natureza sulfidrílica a ALA-D é inibida por metais pesados, como chumbo e mercúrio, servindo como um índice para avaliar a intoxicação pelo metal. Além disso, as alterações patológicas observadas em alguns tipos de porfiria, na tirosinemia hepatorenal e após exposição a chumbo e mercúrio parecem estar relacionadas à inibição desta enzima. A inibição da ALA-D prejudica a biossíntese do heme e paralelamente provoca um acúmulo de ácido 5-aminolevulínico (seu substrato), que pode atuar como um prooxidante, além ser um potente agonista dos autoreceptores gabaérgicos

A patogênese das porfirias agudas

Acute porphyria is a disorder characterized by neurological dysfunctions such as autonomic neuropathy, peripheral neuropathy and encephalopathy. Such dysfunctions arise from an enzymatic defect in the heme biosynthetic pathway leading to decreased heme biosynthesis and accumulation of the heme precursors 5-aminolevulinic acid (ALA) and porphobilinogen. ALA accumulation seems to be responsible for the reduced plasma melatonin levels observed in porphyria patients. This could be related to the intermittent and cyclical nature of porphyria attacks, as well as to psychological alterations observed in the prodromal phase (insomnia, depression, emotional variations). Moreover, it has been proposed that ALA-induced DNA oxidation may explain the higher incidence of primary liver carcinoma in porphyria patients who have experienced a series of acute crises when compared to asymptomatic carriers. On the other hand, it has been suggested that peripheral neuropathy may be related to heme depletion, leading to a dysfunction of nervous system heme proteins. In addition, there is evidence supporting the notion that a depletion of hepatic heme may increase tryptophan plasma levels leading to enhanced serotonin levels in the central nervous system. Such alteration could be the cause of the nausea, abdominal pain and psychomotor disturbances presented by patients.As porfirias agudas são causadas por uma deficiência na via de biossíntese do heme, que provoca ataques caracterizados por disfunções neuroviscerais (neuropatiaautonômica, neuropatia periférica e encefalopatia) e produção excessiva dos precursores porfirínicos, ácido 5-aminolevulínico e porfobilinogênio. O acúmulo de ácido 5- aminolevulínico parece estar envolvido na redução dos níveis de melatonina no plasma dos pacientes, o que poderia estar relacionado com o caráter intermitente e cíclico das crises de porfiria, bem como com algumas alterações psicológicas observadas na fase prodrômica (insônia, depressão, alterações emocionais). Também tem sido sugerido que efeitos oxidantes do ácido 5-aminolevulínico no DNA poderiam explicar a maior suscetibilidade de pacientes que sofreram vários ataques de porfiria ao desenvolvimento de carcinomas hepáticos. No entanto, a neuropatia periférica apresentada pelos pacientes parece não estar relacionada com os aumentos na produção de ácido 5-aminolevulínico. Sugere-se que ela possa estar relacionada a uma depleção de heme que poderia prejudicar o funcionamento de hemoproteínas no sistema nervoso. Além disso, existem evidências convincentes de que a depleção do heme hepático nos pacientes pode provocar um aumento nos níveis de triptofano circulantes, com possíveis conseqüências no sistema nervoso central, tais como aumento nos níveis de serotonina, provocando náuseas, dores abdominais e distúrbios psicomotores e psiquiátricos

Porfirias agudas: aspectos laboratoriais

Porphyrias are disorders characterized by identical neurological disfunctions arising from an enzymatic defect in the heme biosynthetic pathway. The acute porphyrias, namely 5-aminolevulinic acid dehydratase porphyria, acute intermittent porphyria, hereditary coproporphyria and variegate porphyria are caused respectively by deficiencies in the enzymes 5aminolevulinic acid dehydratase, porphobilinogen deaminase, coproporphyrinogen decarboxilase and protoporphyrinogen oxidase. Acute porphyrias are characterized by an increase of plasma and liquor 5aminolevulinic acid levels, followed by increased urinary excretion of this compound. Increases in the urinary excretion of porphyrins or other porphyrin precursors may be observed depending on the type of acute porphyria. Diagnosis of acute porphyria cases rests on the measurement of porphyrins or porphyrin precursors in urine, blood and faeces. Since the clinical manifestations are generally triggered by ambiental or acquired factors, screenings for gene carriers among relatives of AIP are important to identify latent cases. The diagnosis of gene carriers was usually based on the determination of the activity of blood heme biosynthetic enzymes and more recently on DNA analysis.As porfirias são causadas por deficiência parcial de uma das enzimas da via de biossíntese do heme, caracterizando-se por disfunções neuroviscerais bastante semelhantes. As porfirias agudas são decorrentes da deficiência das enzimas delta-aminolevulinato desidratase (ALAD), porfobilinogênio desaminase, coproporfirinogênio oxidase ou protoporfirinogênio oxidase, que provocam, respectivamente, porfiria por deficiência da ALAD, porfiria aguda intermitente, coproporfiria hereditária e porfiria variegada. Todas as porfirias agudas caracterizam-se por um aumento na concentração de ácido 5-aminolevulínico no plasma e no líquor, acompanhado de um aumento na excreção urinária deste composto. Dependendo do tipo de porfiria aguda que acomete o paciente, podem ser observados ainda aumentos na excreção urinária de outros precursores porfirínicos e mesmo de porfirinas. Os casos de porfirias agudas podem ser detectados pela dosagem de porfirinas e seus precursores na urina, sangue e fezes. A triagem das famílias portadoras é importante para detectar casos latentes, pois a expressão clínica da doença geralmente está ligada a fatores ambientais ou adquiridos que provocam os ataques agudos. A identificação de portadores pode ser realizada através da determinação da atividade das enzimas da via de biossíntese do heme no sangue e mais recentemente através da análise do DNA

Antioxidant activity of blackberry (Rubus sp.) genotypes from the Southern Region of Brazil

The antioxidant activity and bioactive compounds of different blackberry fruit genotypes from the major Brazilian producer region (three cultivars and four selections) were evaluated and compared to the Cherokee cultivar. Phenolic and anthocyanic extracts were obtained and evaluated for each fruit genotype. The phenolic extracts of selections 02/96 and 07/001 presented higher antioxidant activity than those of cultivars in most assays. This activity was partially correlated to the higher amount of total phenolics in these samples. Thus, the phenolic compounds are probably the major responsible for the antioxidant activity in the diphenyl-2-picrylhydrazyl radical scavenging assay (DPPH), ferric reducing antioxidant power (FRAP) assay and thiobarbituric acid reactive substances (TBARS) assay. Quercetin seems to be responsible for the antioxidant activity of blackberry phenolic extracts in the β-carotene bleaching assay. Concerning anthocyanic extracts, the selection 02/96 and Cherokee cultivar from harvest 2007 had higher antioxidant activity than the other genotypes in most assays. Anthocyanins appear to be the major responsible for the antioxidant activity of anthocyanic extracts in the DPPH and FRAP assays, although ascorbic acid also contributed to the DPPH antioxidant activity. Selection 02/96 appears to have higher antioxidant activity than the commercial cultivars cultivated in the southern Brazil and appears to be promising for nutritional and health purposes

EFEITO DA INGESTA DIÁRIA DE LEITE NAS CONCENTRAÇÕES DE COMPOSTOS ORGANOCLORADOS NO TECIDO ADIPOSO

Os níveis dos pesticidas organoclorados p,p-DDE, γ-HCH, HCB e Aldrin foram determinados em amostras de tecido adiposo de residentes em Córdoba (Espanha) e relacionados com dados referentes a ingesta diária de leite das doadoras. O consumo médio de leite por parte das mulheres interrogadas foi de aproximadamente 350 mL ao dia. Para relacionar os níveis dos resíduos presentes nas amostras de tecido adiposo ao volume de leite consumido, diariamente, pelas doadoras foi realizado o teste de correlação linear simples. Os resultados obtidos mostraram a inexistência de significação (p>0,05) em relação aos valores detectados nas amostras e a quantidade de leite consumida por essas mulheres. EFFECT OF DAILY MILK INTAKE IN THE CONCENTRATIONS OF ORGANOCHLORINE COMPOUNDS IN ADIPOSE TISSUE Abstract The levels of organochlorine pesticides p,p-DDE, γ-HCH, HCB and Aldrin were determined in samples of adipose tissue from residents in Córdoba (Spain) and related to data on the daily milk intake by the donors. The average milk intake by women interrogated was around 350 mL per day. The levels of residues found in adipose tissue samples were correlated to the average daily milk intake by the donors using a simple linear correlation test. The results obtained showed inexistence of signification (p>0,05) in relation to the values detected in samples and the quantification of consumed milk by this women

Structure–Biological Activity Relationships of Extra-Virgin Olive Oil Phenolic Compounds: Health Properties and Bioavailability

Extra-virgin olive oil is regarded as functional food since epidemiological studies and multidisciplinary research have reported convincing evidence that its intake affects beneficially one or more target functions in the body, improves health, and reduces the risk of disease. Its health properties have been related to the major and minor fractions of extra-virgin olive oil. Among olive oil chemical composition, the phenolic fraction has received considerable attention due to its bioactivity in different chronic diseases. The bioactivity of the phenolic compounds could be related to different properties such as antioxidant and anti-inflammatory, although the molecular mechanism of these compounds in relation to many diseases could have different cellular targets. The aim of this review is focused on the extra-virgin olive oil phenolic fraction with particular emphasis on (a) biosynthesis, chemical structure, and influence factors on the final extra-virgin olive oil phenolic composition; (b) structure–antioxidant activity relationships and other molecular mechanisms in relation to many diseases; (c) bioavailability and controlled delivery strategies; (d) alternative sources of olive biophenols. To achieve this goal, a comprehensive review was developed, with particular emphasis on in vitro and in vivo assays as well as clinical trials. This report provides an overview of extra-virgin olive oil phenolic compounds as a tool for functional food, nutraceutical, and pharmaceutical applications.Junta de Andalucía B-AGR-466-UGR1

Caracterização fisico-química e capacidade antioxidante de pitangas (Eugenia uniflora L.)

This study was carried out to obtain more information about the physicochemical properties, composition, and antioxidant activity of pitanga fruits (Eugenia uniflora L.), particularly fruits from the State of Rio Grande do Sul, Brazil. Pitanga with different flesh colors (purple, red, and orange) from tree selections cultivated at Embrapa Clima Temperado (RS-Brazil) were analyzed. Only slight differences were observed in the quality parameters and in the proximate and fatty acid compositions among the fruits studied. The extracts from purple-fleshed pitanga had the highest total phenolic and anthocyanin contents along with the highest antioxidant capacity. The antioxidant capacity (DPPH and FRAP assays) of methanolic pitanga extracts was highly correlated with the total phenolic content, but in ethanolic extracts, the anthocyanin content was correlated only with the FRAP antioxidant capacity. Orange fleshed pitanga had higher β-cryptoxanthin and β-carotene levels than those of the red fruit, which had higher lycopene content. The results indicate that the purple-fleshed pitanga, cultivated in Rio Grande do Sul, is a rich source of phenolic compounds and has high antioxidant capacity. The red and orange-fleshed pitanga, on the other hand, are rich sources of carotenoids.Este estudo foi realizado para obter mais informações sobre as propriedades físico-químicas, composição e atividade antioxidante de frutos de pitanga (Eugenia uniflora L.), especialmente os do Rio Grande do Sul (Brasil). Foram comparadas pitangas com diferentes colorações de polpa (roxa, vermelha e laranja) de seleções cultivadas na Embrapa Clima Temperado (RS-Brasil). Foram observadas pequenas diferenças nos parâmetros de qualidade e na composição centesimal e de ácidos graxos entre as frutas com diferentes colorações de polpa. Os extratos de pitanga roxa apresentaram maiores conteúdos totais de fenólicos e de antocianinas, bem como, a maior capacidade antioxidante. A capacidade antioxidante (valores de DPPH e FRAP) dos extratos metanólicos de pitanga apresentou alta correlação com o conteúdo de fenólicos totais, mas nos extratos etanólicos, o conteúdo de antocianinas correlacionou-se apenas com a capacidade antioxidante avaliada pelo método de FRAP. A pitanga de cor laranja apresentou maiores teores de β-criptoxantina e β-caroteno, enquanto que a de cor vermelha continha alto teor de licopeno. Os resultados indicam que a pitanga de cor roxa, cultivada no Rio Grande do Sul, é uma fonte rica de compostos fenólicos e possui alta capacidade antioxidante. As de cor vermelha e laranja, por outro lado, são fontes ricas de carotenoides.147154Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Optimization and validation of an enzymatic method to quantify glucose in potato tubers

The processing industry requires potato tubers with low levels of reduced sugars, glucose and fructose, which is responsible for the whiteness of the chip color. The objective of this research was to optimize and validate a simple, inexpensive, and precise enzymatic method to quantify glucose in potato tubers. This method will be useful in breeding programs to select clones for processing purposes with low levels of glucose. The validation procedure followed the recommendations described in INMETRO document DOQ-CGCRE-008 in 2003. The method was linear between 1.25 to 40µg of glucose in the sample. The quantification limit was 0.319mg g-1 of potato fresh weight. The linear working interval of the method started at the quantification limit and ended at 1.32mg g-1 of potato fresh weight. The average recovery in the extraction was 99.0%. The proposed method was used to determine glucose level in samples of Macaca tubers, which ranged from 1.4 to 2.9mg g-1 potato fresh weight

Replacement of pork meal by plant protein sources in Hungarian carp diets

O objetivo deste trabalho foi avaliar o crescimento e a qualidade de carcaça de carpa-húngara alimentada com dietas em que houve substituição da farinha de carne suína por farelos de soja e canola, bem como determinar parâmetros bioquímicos do metabolismo dos peixes e a qualidade sensorial do filé. Cada um dos farelos contribuiu com 50% da proteína na mistura. Cinco dietas foram avaliadas, com níveis de substituição (0, 25, 50, 75 e 100%) da proteína da farinha de carne suína pela mistura das fontes vegetais. A inclusão de fontes proteicas vegetais nas dietas reduziu o crescimento, a deposição de gordura corporal e no filé, e o colesterol total dos peixes. A cor e o sabor dos filés não foram afetados pela inclusão das fontes proteicas vegetais. A dieta à base de farinha de carne suína é mais eficiente para o crescimento da carpa-húngara, e proporciona maior deposição de proteína no peixe inteiro e no filé.The objective of this work was to evaluate growth and carcass composition of Hungarian carp fed with diets in which pork meat meal was replaced by a combination of canola and soybean meals, as well as to determine fish metabolism biochemical parameters and the sensorial quality of the fillet. Each plant meal contributed with 50% of the dietary protein of the mixture. Five diets were tested, with replacement levels of 0, 25, 50, 75 and 100% of pork meal by plant protein sources. The inclusion of the plant-protein meal in the diet results in lower overall growth, lower body and fillet lipid deposition and lower total cholesterol of the fish. Color and flavor of the fillets were not affected by inclusion of plant protein sources. A pork meat meal based diet is more efficient for Hungarian carp growth, and provides higher whole fish and fillet protein deposition