RESUMEN
Under aerobic or anaerobic conditions, tyrosinase undergoes a process of irreversible inactivation induced by its physiological substrate L-dopa. Under aerobic conditions, this inactivation occurs through a process of suicide inactivation involving the form oxy-tyrosinase. Under anaerobic conditions, both the met- and deoxy-tyrosinase forms undergo irreversible inactivation. Suicide inactivation in aerobic conditions is slower than the irreversible inactivation under anaerobic conditions. The enzyme has less affinity for the isomer D-dopa than for L-dopa but the velocity of inactivation is the same. We propose mechanisms to explain these processes.
Asunto(s)
Dihidroxifenilalanina/química , Monofenol Monooxigenasa/química , Agaricales/enzimología , Catálisis , Dominio Catalítico , Catecol Oxidasa/química , Cinética , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Modelos Químicos , Oxígeno/química , Unión Proteica , Espectrofotometría/métodos , Factores de TiempoRESUMEN
There is controversy in the literature concerning the action of tetrahydropterines on the enzyme tyrosinase and on melanogenesis in general. In this study, we demonstrate that tetrahydropterines can inhibit melanogenesis in several ways: i) by non-enzymatic inhibition involving purely chemical reactions reducing o-dopaquinone to L-dopa, ii) by acting as substrates which compete with L-tyr and L-dopa, since they are substrates of tyrosinase; and iii) by irreversibly inhibiting the enzymatic forms met-tyrosinase and deoxy-tyrosinase in anaerobic conditions. Three tetrahydropterines have been kinetically characterised as tyrosinase substrates: 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin, 6-methyl-5,6,7,8-tetrahydropterine and 6,7-(R,S)-dimethyl-5,6,7,8-tetrahydropterine. A kinetic reaction mechanism is proposed to explain the oxidation of these compounds by tyrosinase.
Asunto(s)
Melaninas/biosíntesis , Monofenol Monooxigenasa/antagonistas & inhibidores , Pterinas/farmacología , Agaricales/enzimología , Unión Competitiva , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Cinética , Levodopa/metabolismo , Monofenol Monooxigenasa/metabolismo , Oxidación-Reducción , Pterinas/química , Especificidad por Sustrato , Tirosina/metabolismoAsunto(s)
Muerte , Trasplante de Hígado , Donantes de Tejidos , Recolección de Tejidos y Órganos/métodos , Obtención de Tejidos y Órganos , Isquemia Encefálica/complicaciones , Carcinoma Hepatocelular/cirugía , Hemorragia Cerebral/complicaciones , Femenino , Glucosa , Paro Cardíaco/diagnóstico , Paro Cardíaco/etiología , Humanos , Cirrosis Hepática Alcohólica/cirugía , Neoplasias Hepáticas/cirugía , Masculino , Manitol , Persona de Mediana Edad , Preservación de Órganos/métodos , Soluciones Preservantes de Órganos , Cloruro de Potasio , Guías de Práctica Clínica como Asunto , Procaína , Donantes de Tejidos/provisión & distribución , Obtención de Tejidos y Órganos/normas , Resultado del TratamientoAsunto(s)
Analgésicos Opioides/uso terapéutico , Procedimientos Quirúrgicos Orales , Dolor Postoperatorio/prevención & control , Piperidinas/uso terapéutico , Cuidados Posoperatorios , Analgésicos Opioides/efectos adversos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Piperidinas/efectos adversos , Remifentanilo , Factores de TiempoRESUMEN
OBJECTIVE: To compare the measurements of total resistance and dynamic elastance determined by different techniques of respiratory mechanics analysis based on the time or frequency domains. DESIGN: Prospective study. SETTING: A 12-bed medical and surgical intensive care unit in a 700-bed university hospital. PATIENTS: Eighteen sedoparalyzed patients who needed controlled mechanical ventilation for acute or chronic acute respiratory failure. MEASUREMENTS: The total resistance and dynamic elastance in the time domain were determined by the occlusion technique and by multiple linear regression. The Fourier analysis was used to study the impedance of the respiratory system for elastance and resistance values in the frequency domain. RESULTS: The ANOVA analysis of the elastance variable showed no statistical differences (Ef: 41.4+/-19.0 cmH(2)O/l, Emlr: 40.8+/-17.2 cmH(2)O/l Edyn,occ: 39.5+/-14.0 cmH(2)O/l; ns) and the correlation was very good (r=0.8-0.9). The total resistances were less with multiple linear regression (13.5+/-9.3 cmH(2)O/l per s, p<0.05) than Rmax (17.0+/-11.9 cmH(2)O/l per s) or Rf (17.6+/-10.2 cmH(2)O/l per s). There were no differences between Rmax and Rf (p=0.7) and the correlation between resistances was 0.7-0.9. The agreement analysis for variables without statistical differences showed the following limits: Edyn,occ-f: -17 to 13 cmH(2)O/l; Edyn,occ-mlr: -12 to 9 cmH(2)O/l; Emlr-f: -6 to 8 cmH(2)O/l; Rmax-f: -18 to 19 cmH(2)O/l per s. This last range was related to the autoPEEP level (r=0.9). CONCLUSION: The wide agreement limits show that respiratory mechanics analysis is very dependent on the measurement technique used, particularly for resistance, perhaps due to the higher dependence on frequency.
Asunto(s)
Enfermedad Crítica , Interpretación Estadística de Datos , Rendimiento Pulmonar , Análisis Numérico Asistido por Computador , Insuficiencia Respiratoria/diagnóstico , Insuficiencia Respiratoria/fisiopatología , Mecánica Respiratoria , Procesamiento de Señales Asistido por Computador , Enfermedad Aguda , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Sesgo , Enfermedad Crónica , Análisis de Fourier , Humanos , Modelos Lineales , Persona de Mediana Edad , Respiración de Presión Positiva Intrínseca/etiología , Estudios Prospectivos , Respiración Artificial/efectos adversos , Insuficiencia Respiratoria/terapia , Índice de Severidad de la EnfermedadRESUMEN
Captopril and mesna are molecules with a free thiol group, used as active ingredients due to their hypotensor and mucolytic properties, respectively. These compounds cross the hematoencephalic barrier and, due to the reactivity of their thiol group, can form adducts with the o-quinones formed during the oxidation of mono- and o-diphenols. Polyphenol oxidase from plants and fungi can be used as a tool for generating o-quinones in their action on o-diphenols and facilitate the formation of adducts in the presence of captopril or mesna. The spectrophotometric characterization of these adducts is useful from several points of view. Here, using the end-point method, which involves the exhaustion of oxygen in the medium, we determined the molar absorptivity of the adducts of different o-diphenols with captopril and mesna. Besides the analytical interest of this approach, we also use it to make a kinetic characterization of polyphenol oxidase as it acts on o-diphenolic substrates that produce unstable o-quinones.
Asunto(s)
Captopril/química , Mesna/química , Fenoles/química , Catecol Oxidasa/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Neurotransmisores/química , Oxidación-Reducción , Fenoles/metabolismo , Polifenoles , Quinonas/química , Espectrofotometría , Compuestos de Sulfhidrilo/químicaRESUMEN
The oxidation of green tea catechins by polyphenol oxidase/O2 and peroxidase/H2O2 gives rise to o-quinones and semiquinones, respectively, which inestability, until now, have hindered the kinetic characterization of enzymatic oxidation of the catechins. To overcome this problem, ascorbic acid (AH2) was used as a coupled reagent, either measuring the disappearance of AH2 or using a chronometric method in which the time necessary for a fixed quantity of AH2 to be consumed was measured. In this way, it was possible to determine the kinetic constants characterizing the action of polyphenol oxidase and peroxidase toward these substrates. From the results obtained, (-) epicatechin was seen to be the best substrate for both enzymes with the OH group of the C ring in the cis position with respect to the B ring. The next best was (+) catechin with the OH group of the C ring in the trans position with respect to the B ring. Epigallocatechin, which should be in first place because of the presence of three vecinal hydroxyls in its structure (B ring), is not because of the steric hindrance resulting from the hydroxyl in the cis position in the C ring. The epicatechin gallate and epigallocatechin gallate are very poor substrates due to the presence of sterified gallic acid in the OH group of the C ring. In addition, the production of H2O2 in the auto-oxidation of the catechins by O2 was seen to be very low for (-) epicatechin and (+) catechin. However, its production from the o-quinones generated by oxidation with periodate was greater, underlining the importance of the evolution of the o-quinones in this process. When the [substrate] 0/[IO4 (-)] 0 ratio = 1 or >>1, H2O2 formation increases in cases of (-) epicatechin and (+) catechin and practically is not affected in cases involving epicatechin gallate, epigallocatechin, or epigallocatechin gallate. Moreover, the antioxidant power is greater for the gallates of green tea, probably because of the greater number of hydroxyl groups in its structure capable of sequestering and neutralizing free radicals. Therefore, we kinetically characterized the action of polyphenol oxidase and peroxidase on green tea catechins. Furthermore, the formation of H2O2 during the auto-oxidation of these compounds and during the evolution of their o-quinones is studied.
Asunto(s)
Catequina/química , Catequina/metabolismo , Té/enzimología , Ácido Ascórbico , Catecol Oxidasa/metabolismo , Indicadores y Reactivos , Cinética , Oxidación-Reducción , Peroxidasa/metabolismo , Té/químicaRESUMEN
From the kinetic study carried out in part I of this series (preceding article) an analysis quantifying the relative contribution to the global process of the uni- and bimolecular routes has been carried out. This analysis suggests a way to predict the time course of the relative contribution as well as the effect on this relative weight of the initial zymogen, inhibitor and activating enzyme concentrations.