RESUMEN
In this study, the possibility of enhancing cold stress tolerance of soybean plants (Glycine max L.) by exogenous application of 5-aminolevulinic acid (ALA) was investigated. ALA was added to the Hoagland solution at various concentrations ranging from 0 to 40 µM for 12 h. After ALA treatment, the plants were subjected to cold stress at 4°C for 48 h. ALA at low concentrations (5-10 µM) provided significant protection against cold stress compared to non-ALA-treated plants, enhancing chlorophyll content (Chl) as well as relative water content (RWC). Increase of thiobarbituric acid reactive species (TBARS) levels was also prevented, whereas exposure to higher ALA concentrations (15-40 µM) brought about a dose dependent increase of these species, reaching a maximum of 117% in plants pre-treated with 40 µM ALA compared to controls. ALA pre-treatment also enhanced catalase (CAT) and heme oxygenase-1 (HO-1) activities. These findings indicate that HO-1 acts not only as the rate limiting enzyme in heme catabolism, but also as an antioxidant enzyme. The highest cold tolerance was obtained with 5 µM ALA pre-treatment. Results show that ALA, which is considered as an endogenous plant growth regulator, could be used effectively to protect soybean plants from the damaging effects of cold stress by enhancing the activity of heme proteins, e.g., catalase (CAT) and by promoting heme catabolism leading to the production of the highly antioxidant biliverdin and carbon monoxide, without any adverse effect on the plant growth.
Asunto(s)
Ácido Aminolevulínico/farmacología , Antioxidantes/farmacología , Catalasa/metabolismo , Glycine max/metabolismo , Hemo-Oxigenasa 1/metabolismo , Ácido Aminolevulínico/análisis , Biliverdina/farmacología , Frío , Estrés Oxidativo , Hojas de la Planta/química , Glycine max/efectos de los fármacos , Agua/análisisRESUMEN
OBJETIVO: Este estudo teve como objetivo averiguar a atividade enzimática da N-acetil-β-D-glicosaminidase (NAG) como possível biomarcador precoce de disfunção renal para a exposição ocupacional ao chumbo inorgânico. MATERIAIS E MÉTODOS: Foi selecionado um grupo de 30 pessoas do sexo masculino expostas ao chumbo inorgânico em uma fábrica de baterias localizada no estado do Paraná. Fizeram parte do grupo os funcionários que mostraram valores de chumbo sanguíneo inferiores a 40 mg/dl. O grupo controle foi representado por 15 adultos saudáveis com similaridade em relação à idade e ao gênero do grupo exposto. Foram determinados os níveis de plumbemia, do ácido d-aminolevulínico urinário e a atividade da NAG urinária. RESULTADOS E DISCUSSÃO: Foi evidenciado que a atividade urinária da NAG foi significativamente maior (p < 0,05; teste U de Mann-Whitney) no grupo exposto ao chumbo inorgânico quando comparado ao grupo controle, e houve uma correlação negativa com significância (p < 0,05; correlação de Spearman Rank Order) entre o indicador biológico de exposição plúmbica e a atividade urinária da NAG. CONCLUSÃO: Os resultados demonstraram que o aumento da atividade urinária da NAG pode ser utilizado como um biomarcador precoce da exposição ao chumbo inorgânico.
OBJECTIVE: This study aimed to verify the enzymatic activity of N-acetyl-β-D-glucosaminidase (NAG) as a possible early biomarker of renal dysfunction due to occupational exposure to inorganic lead. MATERIALS AND METHODS: We selected a group of 30 males that had been exposed to inorganic lead in a battery factory in the state of Paraná. This group comprised those employees whose blood lead levels were below 40 mg/dl. The control group consisted of 15 healthy adults of similar age and gender compared with the exposed group. Blood lead concentrations, d-aminolevulinic acid levels and urinary NAG activity were measured. RESULTS AND DISCUSSION: It was shown that urinary NAG activity was significantly higher (p < 0.05, U test of Mann-Whitney) in the exposed group in comparison to the control group, and there was a significant negative correlation (p < 0.05, Spearman Rank Order correlation) between the biological indicator of lead exposure and urinary NAG activity. CONCLUSION: The results showed that the increase of urinary NAG activity may be used as an early biomarker of the exposure to inorganic lead.
Asunto(s)
Humanos , Masculino , Adulto , Persona de Mediana Edad , Acetilglucosaminidasa/análisis , Acetilglucosaminidasa/orina , Acetilglucosaminidasa , Enfermedades Profesionales/diagnóstico , Diagnóstico Precoz , Biomarcadores/análisis , Biomarcadores/orina , Enfermedades Renales/diagnóstico , Enfermedades Renales/enzimología , Ácido Aminolevulínico/análisis , Ácido Aminolevulínico , Creatinina/análisis , Intoxicación por Plomo/diagnóstico , Intoxicación por Plomo/sangre , Intoxicación por Plomo/orinaRESUMEN
Liposomes of different compositions have been designed to improve delivery of aminolevulinic acid (ALA) and its esterified derivatives ALA-Hexyl ester (He-ALA) and ALA-Undecanoyl ester (Und-ALA) for its use in photodynamic therapy (PDT). Egg yolk phosphatidyl choline (PC), phosphatidic acid (PA) and phosphatidyl glycerol (PG) were employed in the preparation of the liposomes. Sonicated vesicles composed of PC, PC-PG (80:20) or PC-PA (80:20) containing ALA or derivatives were obtained and purified by a minicolumn centrifugation method. PC liposomes presented encapsulation percentages around 6% for 2 mM ALA, 13% for 2 mM He-ALA and 51% for 2 mM Und-ALA. The addition of PG or PA to the formulation, resulted in an increased entrapment: 19% for 2 mM ALA, 69% for 2 mM He-ALA and 87% for 2 mM Und-ALA in PC-PG liposomes and 21% for 2 mM ALA, 60% for 2 mM He-ALA and 87% for 2 mM Und-ALA in PC-PA liposomes. Higher concentrations of ALA or derivatives resulted in lower percentages of entrapment. The three formulations containing ALA or derivatives were stable up to 1 week upon storage at 4 degrees C. However, upon dilution with medium, ALA leaked from the liposomes, while on the contrary, He-ALA was highly retained, being therefore a good choice for its use in PDT. The stability of Und-ALA upon dilution could not be tested, but Und-ALA proved to have the highest entrapment efficacy.
Asunto(s)
Ácido Aminolevulínico/análogos & derivados , Ácido Aminolevulínico/análisis , Sistemas de Liberación de Medicamentos , Liposomas/química , Ésteres/análisis , Liposomas/aislamiento & purificación , Ácidos Fosfatidicos , Fosfatidilcolinas , Fosfatidilgliceroles , Fotoquimioterapia/métodosRESUMEN
This work evaluated the levels of 5-aminolevulinic acid (ALA) in the liver of rats exposed to different doses of HCB (25,50, and 100mg/kg b.w. for 4 weeks) and correlated them with lipid peroxidation parameters. Levels of ALA were determined by highpressure liquid chromatography after derivatization with acetylacetone and formaldehyde, followed by fluorescence detection. The methodology was carefully validated, nonetheless hepatic levels of ALA in all animals treated or not were below the detection limit of the method (2.27mg of ALA/g liver). On the other hand, lipid peroxidation, evaluated as thiobarbituric acid reactants production and chemiluminescence was found significantly increased in the livers of all treated rats, in comparison with control values (p<0.05). These results suggest that the hepatic oxidative stress observed in animals following HCB treatment may not necessarily be associated with increased levels of ALA in the liver. Another possibility is that increased levels of ALA in the liver, even below the detection limit of the method are sufficient to induce hepatic oxidative stress
Asunto(s)
Ácido Aminolevulínico/análisis , Hexaclorobenceno , Peroxidación de Lípido , Estrés Oxidativo , Cromatografía Liquida/métodos , FluorescenciaRESUMEN
PURPOSE: In topical photodynamic therapy, 5-ALA and its esters are enzymatically converted in the endogenous photosensitizing compounds such as, for example, protoporphyrin IX (PpIX). In order to elucidate in more detail their enzymatic fate, we have determined in vitro the enzymatic degradation of methyl, butyl, hexyl, and octyl-5-ALA ester derivatives in skin homogenate. Furthermore, in vivo porphyrin accumulation was measured in healthy hairless mice skins. METHODS: Hairless mouse skins were homogenized in isotonic phosphate buffer pH 7.4. 5-ALA esters were added, and aliquots were colleted for HPLC-fluorimetric determinations of remaining content of 5-ALA esters. Furthermore, oil-in-water emulsions containing esters were topically applied to mice skin for 6 h, and the amount of accumulated PpIX in the treated areas was determined by quantitative extraction and confocal fluorescence microscopy. RESULTS: The enzymatic degradation of esters follows pseudo first-order kinetics. The octyl ester had the largest rate constant for enzymatic degradation, followed by hexyl-, butyl-, and methyl-ALA. The long-chained 5-ALA esters, butyl-, hexyl-, and octyl ester, induced significantly more porphyrins than 5-ALA and 5-ALA methyl ester as shown by confocal microscopy and quantitative extraction studies. CONCLUSIONS: 5-ALA derivatives differ widely with respect to their enzymatic degradation. The presence of alkyl chains in 5-ALA esters significantly influences the in vitro enzymatic metabolism and the in vivo PpIX formation in healthy hairless mice skins.
Asunto(s)
Ácido Aminolevulínico/química , Ácido Aminolevulínico/metabolismo , Protoporfirinas/metabolismo , Piel/metabolismo , Ácido Aminolevulínico/análisis , Animales , Ésteres , Masculino , Ratones , Ratones Pelados , Fármacos Fotosensibilizantes/metabolismo , Protoporfirinas/análisis , Piel/químicaRESUMEN
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 profirias 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 aaumento na concentração de ácido 5-aminolevulínico no plasma e no líquor, acompanhado de um aumento na excreção urinária deste composto...
Asunto(s)
Humanos , Adulto , Ácido Aminolevulínico/análisis , ADN , Mutación/genética , Porfobilinógeno/análisis , Porfiria Intermitente Aguda , Porfirinas , Sangre , Heces , Manejo de Especímenes , OrinaRESUMEN
The quenchings of pyrene fluorescence by 5-aminolevulinic acid (5-ALA) in dodecyl sulfate (SDS) micelles and stratum corneum lipids liposomes (SCLLs) were studied in order to verify the capacity of incorporation of 5-ALA in these systems. Static and dynamic quenching constants based on the simultaneous determination of fluorescence intensity quenching and fluorescence decay measurements were determined. 5-ALA was incorporated at crescent concentrations in SDS micelles and SCLLs containing the fluorescent probe (pyrene, 1,15 x '10 POT.menos 5 M') and the rate constants of quenching (kq) of pyrene were determined. 5-ALA was able to quench the fluorescence of the probe in both systems studied. A greater extent...
Asunto(s)
Ácido Aminolevulínico/análisis , Ácido Aminolevulínico/uso terapéutico , Fluorescencia , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/terapia , Fotoquimioterapia , Pirenos , Colorantes Fluorescentes , Micelas , Espectrometría de FluorescenciaRESUMEN
A simple and sensitive method for determining 5-aminolevulinic acid (ALA) in biological samples is described. ALA is derivatized with o-phthaldehyde to give a compound with favorable properties for high-performance liquid chromatography with electrochemical detection. The method does not require extensive pretreatment of the samples and its detection limit is in the range of 1 pmol/20 microl injection. This method was applied to the determination of plasma ALA from normal and lead-exposed subjects, where 0.26+/-0.08 microM (n=30) and 2.6+/-0.75 microM (n=30), respectively were found. We also determined ALA in rat tissues, namely liver and brain. and the uptake of ALA by cultured fibroblasts and hepatocytes to illustrate the diversified applicability of the method.
Asunto(s)
Ácido Aminolevulínico/análisis , Contaminantes Ocupacionales del Aire/toxicidad , Ácido Aminolevulínico/sangre , Animales , Encéfalo/metabolismo , Células CHO , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cricetinae , Electroquímica , Humanos , Plomo/toxicidad , Hígado/citología , Hígado/metabolismo , Masculino , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , o-FtalaldehídoRESUMEN
The heme precursor 5-aminolevulinic acid (ALA), acting as a prooxidant, has been proposed to underlie the clinical manifestations of various porphyric disorders. Accordingly, ALA-generated oxyradicals where shown to cause oxidative lesions in biomolecules and isolated cell organelles and to release iron from ferritin. In rats, administered ALA triggered oxidative stress in liver, brain and red muscles. We now study the correlation between the plasma antioxidant capacity and tissue oxidative damage, after acute (one and two doses) and prolonged (eight doses) ALA treatment of rats (one dose of ALA = 40 mg/kg body weight). The in situ spontaneous chemiluminescence intensity increased 5-fold in brain, 50% in liver and 4-fold in soleus muscle upon two dose-treatment, indicating tissue response to oxidative injury by ALA. Chemiluminescence reached the highest intensity after one or two doses of ALA and decreased after eight doses in all tissues. The plasma trapping capacity, evaluated by the luminol/2-amidinopropane system, gave a parallel response: maximum values after two doses and decreased values after prolonged treatment. After eight doses, the ALA concentration was found to be 3-fold above the normal value in plasma, 48% higher in liver and 38% higher in total brain. These data indicate that the plasma antioxidant system responds to ALA treatment and is correlated with tissue chemiluminescence. In vitro studies showed that ALA does not interfere with the antioxidant plasma capacity, neither promoting oxidation of plasma elements nor binding to plasma proteins.
Asunto(s)
Ácido Aminolevulínico/farmacología , Antioxidantes/metabolismo , Estrés Oxidativo , Amidinas/química , Amidinas/metabolismo , Ácido Aminolevulínico/análisis , Ácido Aminolevulínico/sangre , Animales , Ácido Ascórbico/química , Ácido Ascórbico/metabolismo , Proteínas Sanguíneas/efectos de los fármacos , Proteínas Sanguíneas/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Cromanos/química , Cromanos/metabolismo , Glutatión/química , Glutatión/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Mediciones Luminiscentes , Luminol/química , Luminol/metabolismo , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Oxidación-Reducción , Plasma/efectos de los fármacos , Plasma/metabolismo , Ratas , Ratas Wistar , Compuestos de Sulfhidrilo/análisis , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Ácido Úrico/química , Ácido Úrico/metabolismoRESUMEN
O Saturnismo e a PAI representam porfirias relacionadas ao acúmulo de ALA, como resultado da inibiçäo de várias enzimas da via biossintética do heme pelo chumbo e defeito genético na biossíntese da PBG-desminase, respectivamente. Bechara et al (1993 - artigo de revisäo) propöem a auto-oxidaçäo da forma enólica do ALA catalisada por complexos de ferro, gerando espécies reativas de oxigênio e o radical ALA, como processo disparador dos sintomas observados nessas patologias. O efeito deletério destas espécies sobre importantes macromoléculas (proteínas, DNA) e estruturas celulares (mitocôndria, retículo endoplasmático) foi evidenciado em vários estudos in vivo e in vitro. O objetivo desta tese é estudar o envolvimento de ALA como espécie pro-oxidante no saturnismo e no processo de envelhecimento. Para tanto, foi necessário o desenvolvimento de um método de dosagem do ALA, pois os métodos até entäo descritos na literatura embora dotados de sensibilidade adequada, eram extremamente trabalhosos e lentos devido ao elevado número de etapas envolvidas na preparaçäo da amostra. O método aqui descrito utiliza a cromatografia em fase reversa em HPLC acoplado a um detector eletroquímico e a derivatizaçäo pré-coluna do ALA com omicron-ftalaldeído (OPA) a um produto indólico. De posse de um método simples e seletivo para a quantificaçäo do ALA, o passo seguinte foi analisar a correlaçäo dos níveis do ALA em plasma com os de chumbo eritrocitário, protoporfirina IX eritrocitária, superóxido dismutase eritrocitária, metehemoglobina (sangue) e quimioluminescência (urina) em amostras coletadas de indivíduos expostos ocupacionalmente ao chumbo. Os resultados obtidos indicaram um grau de correlaçäo acentuado entre vários dos parâmetros testados reforçando o papel do ALA como espécie pró-oxidante de relevância na etiologia do saturnismo. Além disso, foram determinados os níveis de ALA em plasma, fígado e cérebro de ratos caracterizados como adultos jovens (6-8 meses) e velhos (24 meses) e sua correlaçäo com parâmetros indicativos do status oxidativo destes animais. Foi observado um aumento significativo do ALA nos ratos velhos tanto em plasma como em fígado, sendo que o mesmo apresentou uma correlaçäo positiva em fígado e cérebro com ferro näo hemínico, carbonil proteína e "thiobarbituric acid reactive substances" (TBARS). Esses dados nos permitem inferir ao ALA um papel pró-oxidante no processo de envelhecimento
Asunto(s)
Animales , Ratas , Adulto , Anciano , Ácido Aminolevulínico/análisis , Ácido Aminolevulínico/farmacología , Envejecimiento/efectos de los fármacos , Plomo/sangre , Plomo/farmacología , Oxidantes , Porfirias , Cromatografía , Enfermedades ProfesionalesRESUMEN
delta-Aminolevulinic acid (ALA), a heme precursor accumulated in acute intermittent porphyria and saturnism, undergoes autoxidation leading to ammonium ion and probably the corresponding alpha-ketoaldehyde. This reaction is accelerated by addition of oxyhemoglobin (oxyHb) and other iron complexes. OxyHb is concomitantly oxidized to metHb; the apparent second-order rate constant of oxyHb/ALA coupled oxidation is ca. 10 M-1 min-1.1H NMR and uv spectral studies suggest that ALA undergoes enolization before consuming the dissolved oxygen. Spin-trapping experiments demonstrate formation of both the hydroxyl radical and a substrate-derived carbon-centered radical during ALA oxidation. Generation of active oxygen species by ALA might be related to the neuropathy associated to some acquired and inherited porphyrinpathies.