RESUMO
1. The effect of in vitro glycation on delta-aminolevulinic dehydratase (ALA-D) under several experimental conditions was investigated. When preincubated with 500 mM glucose at 37 degrees C for 20 hr, ALA-D was 80% inactivated and glycated hemoglobin levels were increased more than fourfold. 2. Thiobarbituric acid species were not modified during glycation; therefore ALA-D inactivation cannot be attributed to glucose autoxidation. 3. Acetyl salicylic acid was effective in preventing both hemoglobin glycation and ALA-D inactivation by glucose. 4. A method has been developed for measuring protein glycation in vitro, in a crude preparation of red blood cells, which can also be applied to sugars other than glucose.
Assuntos
Aspirina/farmacologia , Glucose/metabolismo , Sintase do Porfobilinogênio/metabolismo , Metabolismo dos Carboidratos , Carboidratos/farmacologia , Glucose/farmacologia , Hemoglobinas Glicadas/metabolismo , Glicosilação/efeitos dos fármacos , Humanos , Técnicas In Vitro , Peroxidação de Lipídeos/efeitos dos fármacos , Sintase do Porfobilinogênio/antagonistas & inibidores , Temperatura , Fatores de TempoRESUMO
1. The effect of long-term griseofulvin (GRIS) topical administration on some indicators of liver damage was examined. 2. Liver porphyrin accumulation was significant; however, no porhyrin crystals were observed under light microscopy. 3. An earlier onset of hepatopathy was established (3-fold) increase of direct bilirubin values after 7 days of treatment; hepatic injury was confirmed by measuring a 6-fold increase of free bilirubin. 4. Enhanced values of alkaline phosphatase and glutamic oxalacetic transaminase (GOT) confirmed the onset of cholestasis. 5. Topical application of GRIS induced measurable hepatopathy. Nevertheless, we cannot discard the possibility that this hepatopathy could also be attributed in part to a direct reaction to xenobiotics.
Assuntos
Antifúngicos/farmacologia , Griseofulvina/farmacologia , Heme/metabolismo , Fígado/efeitos dos fármacos , Administração Tópica , Animais , Antifúngicos/administração & dosagem , Biomarcadores , Griseofulvina/administração & dosagem , Fígado/enzimologia , Fígado/patologia , Testes de Função Hepática , Masculino , CamundongosRESUMO
Aerobic and anaerobic studies have demonstrated that uroporphyrin I-induced inactivation of delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase was dependent on oxygen and mediated by reactive oxygen species. The mechanism of photoinactivation of those heme-enzymes from human erythrocytes by uroporphyrin I by u.v. light was investigated. Enzymes of the heme pathway were preincubated in the presence of specific scavengers for several reactive oxygen species and then exposed to uroporphyrin I and u.v. light. Upon exposure of the enzymes to the porphyrin under u.v. light, and in an aerobic atmosphere, the percentage of enzyme activities with respect to the corresponding controls were 50.2 +/- 5.1 (SD, n = 6), 25.3 +/- 3.0 (SD, n = 6), 25.9 +/- 2.8 (SD, n = 6) and 49.7 +/- 7.5 (SD, n = 8) for delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase, respectively. The presence of sodium azide, histidine or superoxide dismutase did not protect the enzymes against the effects of uroporphyrin I. However, both cysteine and potassium ferrycyanide prevented the enzyme photoinactivation induced by uroporphyrin I. In the presence of either catalase or GSH, the enzyme photoinactivation was lower. Ethanol, glucose and dimethylsulfoxide had no effect on enzyme activity, while ion chelators had variable effects. This study shows that the type II mechanism is not the predominant reaction mediating the uroporphyrin I effect and enzyme photoinactivation would involve an electron transfer. Hydrogen peroxide and hydroxyl radicals could possibly mediate the uroporphyrin I-induced enzyme photoinactivation.
Assuntos
Hemeproteínas/efeitos da radiação , Liases/efeitos da radiação , Uroporfirinas/farmacologia , Amônia-Liases/efeitos da radiação , Elétrons , Sequestradores de Radicais Livres , Humanos , Peróxido de Hidrogênio/sangue , Radical Hidroxila , Hidroximetilbilano Sintase/efeitos da radiação , Oxigênio/sangue , Sintase do Porfobilinogênio/efeitos da radiação , Superóxidos/sangue , Uroporfirinogênio Descarboxilase/efeitos da radiaçãoRESUMO
The correlation between blood lead level (BLL), delta-aminolevulinate dehydratase (ALA-D) activity, and other common biochemical parameters used to assess a plumbism diagnosis have been carefully analyzed, with the aim of correctly interpreting the data handled in the laboratory. No correlation was observed between BLL and free erythrocyte porphyrins. In the case of ALA-D or Zn-reactivated ALA-D despite the direct correlation with BLL, the curve follows a potential or a logarithmic line, which is not the best to calculate BLL. The so-called Zn-ALA-D-reactivation index (iZn) has been defined as the ratio between the activity of Zn-reactivated ALA-D and the activity of ALA-D. The plot of BLL against the iZn revealed a very good linear relationship which allows an estimate of BLL with reasonable accuracy within a very wide range.
Assuntos
Intoxicação por Chumbo/diagnóstico , Chumbo/sangue , Sintase do Porfobilinogênio/sangue , Zinco/sangue , Adulto , Alanina/sangue , Alanina/urina , Sítios de Ligação , Criança , Pré-Escolar , Eritrócitos/metabolismo , Feminino , Hemoglobinas/metabolismo , Humanos , Chumbo/análise , Masculino , Porfobilinogênio/sangue , Porfobilinogênio/urina , Sintase do Porfobilinogênio/análise , Porfirinas/sangue , Porfirinas/urina , Padrões de Referência , Reprodutibilidade dos Testes , Espectrofotometria Atômica , Zinco/análiseRESUMO
A frequent coexistence of diabetes and porphyria disease has been reported. Under normal conditions, porphyrin biosynthesis is well regulated to only form the amount of heme required for the synthesis of the various hemoproteins. The activity of some heme enzymes and rhodanese in streptozotocin (STZ) induced diabetic mice and in allylisopropylacetamide (AIA) induced experimental acute porphyria mice has been examined. The role of alpha-tocopherol (alpha-T), reported to prevent protein glycation in vitro, has also been investigated. AIA induced hepatic delta-aminolevulinic acid synthetase (ALA-S) activity in control animals but was ineffective in the diabetic group. alpha-Tocopherol did not modify ALA-S activity in either group. delta-Aminolevulinic acid dehydratase (ALA-D) and deaminase activities were significantly diminished both in liver and blood of diabetic animals. alpha-Tocopherol prevented inhibition of ALA-D, deaminase and blood rhodanese activities in diabetic animals but alpha-tocopherol by itself did not affect the basal levels of the enzymes studied. The potential use of alpha-tocopherol to prevent late complications of diabetes, including the onset of a porphyria like syndrome is considered.
Assuntos
Alilisopropilacetamida/toxicidade , Diabetes Mellitus Experimental/enzimologia , Porfiria Aguda Intermitente/enzimologia , Vitamina E/farmacologia , 5-Aminolevulinato Sintetase/sangue , 5-Aminolevulinato Sintetase/metabolismo , Animais , Glicemia/análise , Glicemia/efeitos dos fármacos , Heme/metabolismo , Masculino , Camundongos , Nucleosídeo Desaminases/sangue , Nucleosídeo Desaminases/metabolismo , Sintase do Porfobilinogênio/sangue , Sintase do Porfobilinogênio/metabolismo , Porfiria Aguda Intermitente/induzido quimicamente , Estreptozocina/toxicidade , Tiossulfato Sulfurtransferase/metabolismoRESUMO
1. Heme regulation before the appearance of hyperplastic nodules was investigated in mice models of hepatocarcinogenesis. 2. With this aim 5-aminolaevulinate synthetase (ALA-S), microsomal heme-oxygenase (MHO), mitochondrial and cytoplasmic rhodanese activities were examined throughout a period of 35 days in animals exposed to dietary p-dimethylaminoazobenzene (DAB). 3. ALA-S activity was significantly diminished (50%) on day 14, then showing a sharply rising profile from day 28 onwards, and reaching 350% on day 35. 4. A similar profile was observed for mitochondrial rhodanese activity. 5. Changes in MHO and cytoplasmic rhodanese activities were almost the opposite to those observed for ALA-S. 6. The distinctive alteration in mitochondrial and cytoplasmic rhodanese would suggest that it plays a subtle role in ALA-S regulation during carcinogenesis initiation through a mechanism that appears to involve subcellular localization controls perhaps by means of the breakage of cystine trisulphide postulated to act as an ALA-S activator. 7. Taking into account the present results, we suggest a probable mechanism for the onset of hepatocarcinogenesis that includes a primary activating liver status, provoking biochemical aberration leading to the stage of initiation of hepatocarcinogenesis involving the whole organ.
Assuntos
Heme/biossíntese , Neoplasias Hepáticas Experimentais/metabolismo , 5-Aminolevulinato Sintetase/metabolismo , Animais , Citoplasma/enzimologia , Heme Oxigenase (Desciclizante)/metabolismo , Cinética , Fígado/enzimologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Masculino , Camundongos , Microssomos Hepáticos/enzimologia , Mitocôndrias Hepáticas/enzimologia , Tiossulfato Sulfurtransferase/metabolismo , p-DimetilaminoazobenzenoRESUMO
1. Basal levels and allyl-isopropylacetamide (AIA) or veronal induced levels of delta-amino-levulinate synthetase (ALA-S), cytoplasmic and mitochondrial rhodanese were determined in tumor (T) and liver of both normal mice (NM) and T-bearing mice (TBM). 2. Rhodanese tumoral mitochondrial levels were higher than the hepatic normal mitochondrial fraction, while the cytoplasmic activity was nearly equal in all sources. 3. In neither case was the activity of tumoral ALA-S and rhodanese altered by any of the porphyrinogenic drugs. 4. Mitochondrial and cytoplasmic rhodanese activity was also measured in tumor and liver of TBM at different intervals after transplantation. We concluded that the behaviour of rhodanese is a property inherent to the tissue and not one attained with time.
Assuntos
5-Aminolevulinato Sintetase/metabolismo , Neoplasias Mamárias Experimentais/enzimologia , Tiossulfato Sulfurtransferase/metabolismo , 5-Aminolevulinato Sintetase/biossíntese , Alilisopropilacetamida/farmacologia , Animais , Barbital/farmacologia , Citoplasma/enzimologia , Indução Enzimática/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/enzimologia , Tiossulfato Sulfurtransferase/biossínteseRESUMO
1. URO-D was investigated in crude extracts from mouse mammary carcinoma, normal mouse (NM) liver and tumor-bearing mouse (TBM) liver. 2. URO-D from TBM liver and tumor appears to be more sensitive to increasing concentrations of UROgen than the NM liver enzyme. 3. In tumor the rate-limiting step seems to be the decarboxylation of the first carboxyl group, but this was not so clear for the NM and the TBM liver URO-D. 4. URO-D activity was enhanced when incubated at higher temperatures in the presence of its substrate, suggesting that UROgen might afford some protection of the enzyme against heat inactivation. 5. The optimum pH for all three sources is around 7.0.
Assuntos
Neoplasias Mamárias Experimentais/enzimologia , Uroporfirinogênio Descarboxilase/metabolismo , Animais , Concentração de Íons de Hidrogênio , Cinética , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade por Substrato , Temperatura , UroporfirinogêniosRESUMO
1. delta-Aminolevulinic acid synthetase (ALA-S), rhodanese and microsomal heme oxygenase (MHO), were quantitated in Cl4C induced regenerating mouse liver. 2. Maximal hepatomegalia was observed at 48 hr after i.p. injection of a single dose of the toxin. 3. ALA-S activity decreased on day 2, and then significantly increased (50%) between days 3 and 7, returning afterwards to control values. 4. Cytoplasmic rhodanese, as well as MHO activities, exhibited a clear correlation as compared with the ALA-S activity profile. 5. Porphyrin biosynthesis from precursor delta-aminolevulinic acid (ALA) was significantly increased even after 15 days of intoxication. 6. Present results would indicate that Cl4C is acting in a dual fashion.
Assuntos
Heme/metabolismo , Regeneração Hepática/fisiologia , Fígado/metabolismo , 5-Aminolevulinato Sintetase/metabolismo , Ácido Aminolevulínico/metabolismo , Animais , Tetracloreto de Carbono/farmacologia , Sistema Enzimático do Citocromo P-450/deficiência , Heme Oxigenase (Desciclizante)/metabolismo , Cinética , Fígado/efeitos dos fármacos , Fígado/enzimologia , Regeneração Hepática/efeitos dos fármacos , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Porfirinas/biossíntese , Tiossulfato Sulfurtransferase/metabolismoRESUMO
1. A clear biphasic response of the enzyme activities as a function of intoxication time due to the topical cutaneous griseofulvin treatment was observed. 2. The initial acute induction of ALA-S activity would be due to depletion of free heme in the regulatory pool caused by cytochrome P 450 destruction. 3. The second induction peak, would be due to less heme formation, secondary to the ferrochelatase inhibition, as expected for the erythropoietic protoporphyria model. 4. The biphasic response of hepatic ALA-D and PBGase activities would be related to ALA-S activity changes and the subsequent augmented available substrates. 5. Endogenous liver porphyrin distribution in cytosolic, mitochondrial and nuclear fractions was investigated. 6. The in vitro biosynthesis of porphyrins confirmed both the biphasic model and the hepatic porphyrins subcellular distribution. 7. Two mechanisms to explain the action of griseofulvin at shorter and longer times of intoxication are proposed.
Assuntos
Griseofulvina/farmacologia , Heme/metabolismo , 5-Aminolevulinato Sintetase/metabolismo , Amônia-Liases/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Citosol/efeitos dos fármacos , Citosol/enzimologia , Técnicas In Vitro , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Sintase do Porfobilinogênio/metabolismo , Porfirinas/metabolismo , Proteínas/metabolismoRESUMO
1. Porphobilinogenase (PBGase) and hydroxymethylbilane synthetase (HMB-S) were investigated in crude extracts from mouse mammary carcinoma, normal mouse liver and tumor bearing mouse liver. 2. A Michaelis-Menten kinetics for both enzymes in either source was observed. Km values of 87 to 108 microM, Vmax of 1.57-1.83 nmol porphyrins/2 hr and a Hill coefficient of n = 1 were obtained for PBGase and Km values of 13 to 19 microM and Vmax of 2.6-4.8 were obtained for HMB-S. 3. Porphyrin synthesis was linear up to 180 min of incubation in all cases for PBGase and HMB-S, and greatly increased with higher incubation temperature being maximal at 60 degrees C and nil at 70 degrees C, optimal temperature was 37 degrees C for either enzyme in either source. 4. Uroporphyrinogen III synthetase was heat inactivated while HMB-S was a heat stable enzyme. Optimum pH was 8.2 for PBGase and HMB-S in either tissue.
Assuntos
Amônia-Liases/metabolismo , Hidroximetilbilano Sintase/metabolismo , Neoplasias Mamárias Experimentais/enzimologia , Animais , Concentração de Íons de Hidrogênio , Cinética , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Porfirinas/biossíntese , TemperaturaRESUMO
1. Aminolevulinate dehydratase (ALA-D) was studied in crude extract from mouse mammary carcinoma, normal mouse liver and tumour bearing mouse liver. 2. A Michaelis-Menten behaviour and Km values between 0.24 and 0.31 mM were obtained for the enzyme in either source. 3. In all three tissues there was a linear relationship between porphobilinogen formation and incubation time, up to 120 min, ALA-D was thermostable and optimum pH was at 6.8. 4. There seems to be no structural alterations in tumoural ALA-D as compared with the enzyme from liver of both normal and tumour bearing mice.
Assuntos
Neoplasias Mamárias Experimentais/enzimologia , Sintase do Porfobilinogênio/metabolismo , Animais , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Porfobilinogênio/metabolismoRESUMO
1. Basal levels and allyl-isopropylacetamide (AIA) or veronal induced levels of delta-aminolevulinate synthetase (ALA-S), cytochrome P-450 (cyt P-450) and cytochrome oxidase were determined in tumor (T) and liver of both normal mice (NM) and T bearing mice (TBM). 2. Basal levels of ALA-S were nearly the same in either source. The amount of cyt P-450 was lower in TBM liver than in NM liver, and no detectable in T. While the basal activity of cytochrome oxidase in TBM liver and T were higher than those of NM liver. 3. In AIA intoxicated animals there was a lower induction of ALA-S in liver of TBM than in NM liver. There was no induction in T ALA-S. The loss of cyt P-450 was less in TBM liver when compared with NM liver. 4. The induction level of cyt P-450 after veronal administration was nearly the same in liver of both TBM and NM. 5. We conclude that lower induction of liver ALA-S activity in TBM liver is due to correspondingly lower drug metabolism ability of TBM liver. Otherwise our results suggest that the control mechanism operating in T and probably in its original tissue are different from those described for normal liver.
Assuntos
5-Aminolevulinato Sintetase/metabolismo , Alilisopropilacetamida/farmacologia , Barbital/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Heme/biossíntese , Fígado/enzimologia , Neoplasias Mamárias Experimentais/enzimologia , Animais , Indução Enzimática/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
1. The effect of colchicine, vincristine and griseofulvin (GRIS) on the porphyrinogenic action of 2-allyl-2-isopropylacetamide (AIA) and veronal was studied in vivo and using the in vitro experimental model of tissue explant cultures. 2. Complete prevention by colchicine was found in liver and heart explant from animals treated with AIA and veronal. 3. Vincristine, GRIS and colchicine reversed AIA induction in liver explants, however reversal was partial or nil in skin and heart explants depending on the antimitotic and the tissue. 4. The usefulness of the combination of the in vivo experimental model and the in vitro explant tissue culture model, for this kind of studies is emphasized.
Assuntos
Colchicina/farmacologia , Heme/metabolismo , Porfirinas/biossíntese , 5-Aminolevulinato Sintetase/antagonistas & inibidores , 5-Aminolevulinato Sintetase/biossíntese , Alilisopropilacetamida/farmacologia , Animais , Indução Enzimática/efeitos dos fármacos , Griseofulvina/farmacologia , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Masculino , Camundongos , Vincristina/farmacologiaRESUMO
1. Porphyrin biosynthesis from delta-aminolevulinic acid (ALA) was investigated using the technique of tissue explant cultures, in both human breast cancer and its original normal tissue. 2. The activity of ALA-dehydratase, porphobilinogenase and uroporphyrinogen decarboxylase was directly determined in both tumor and normal mammary tissues. 3. Porphyrin synthesis capacity of human breast carcinoma was 20-fold enhanced, as compared with normal tissue, at least between the stages of porphobilinogen and coproporphyrinogen formation. 4. The activity of the three enzymes examined was always lower in normal tissue than in tumoral tissue. 5. Present findings show that porphyrin biosynthesis is increased in breast cancer tissue.
Assuntos
Neoplasias da Mama/metabolismo , Heme/biossíntese , Adulto , Idoso , Amônia-Liases/metabolismo , Mama/enzimologia , Mama/metabolismo , Neoplasias da Mama/enzimologia , Técnicas de Cultura , Feminino , Heme/metabolismo , Humanos , Pessoa de Meia-Idade , Sintase do Porfobilinogênio/metabolismo , Uroporfirinogênio Descarboxilase/metabolismoRESUMO
We recorded fluorescence excitation and emission spectra of uro- and coproporphyrin under different experimental conditions, to see how these conditions influence quantifications based on measurement of fluorescence intensity. We found that, for bands alpha and beta of the emission spectra and the main peak of the excitation spectra, fluorescence depends on pH and is minimal near pH 5 and near pH 7-7.5 for copro- and uroporphyrin, respectively. For band gamma of the emission spectra there was a constant decrease of fluorescence with increasing alkalinity of the solution. The intensity of porphyrin fluorescence also depends on ionic strength, reaching sharp maxima at 0.1 mol/L (for uroporphyrin) and 1 mol/L (for coproporphyrin). The organic mixture ethyl acetate:acetic acid (4:1 by vol), commonly used to extract porphyrins from biological samples, markedly diminishes the fluorescence of both porphyrins as compared with the same concentration of each porphyrin in aqueous acidic solvent. Furthermore, when we measured different ratios of uro:copro mixture at three distinct pHs and buffers, we found that at pH 10.5 (in carbonate buffer) the measured units of fluorescence depend only on total porphyrin concentration and not on the composition of the mixture.