Disturbance in hemoglobin metabolism and in vivo antimalarial activity of azole antimycotics / Alteración en el metabolismo de la hemoglobina y actividad antimalárica in vivo de azoles antimicóticos

Plasmodium parasites degrade host hemoglobin to obtain free amino acids, essential for protein synthesis. During this event, free toxic heme moieties crystallize spontaneously to produce a non-toxic pigment called hemozoin or ß-hematin. In this context, a group of azole antimycotics, clotrimazole (CTZ), ketoconazole (KTZ) and fluconazole (FCZ), were investigated for their abilities to inhibit ß-hematin synthesis (IßHS) and hemoglobin proteolysis (IHbP) in vitro. The ß-hematin synthesis was recorded by spectrophotometry at 405 nm and the hemoglobin proteolysis was determined by SDS-PAGE 12.5 percent, followed by densitometric analysis. Compounds were also assayed in vivo in a malaria murine model. CTZ and KTZ exhibited the maximal effects inhibiting both biochemical events, showing inhibition of β-hematin synthesis (IC50 values of 12.4 ± 0.9 µM and 14.4 ± 1.4 µM respectively) and inhibition of hemoglobin proteolysis (80.1 ± 2.0 percent and 55.3 ± 3.6 percent, respectively). There is a broad correlation to the in vivo results, especially CTZ, which reduced the parasitemia ( percentP) of infected-mice at 4th day post-infection significantly compared to non-treated controls (12.4 ± 3.0 percent compared to 26.6 ± 3.7 percent, p = 0.014) and prolonged the survival days post-infection. The results indicated that the inhibition of the hemoglobin metabolism by the azole antimycotics could be responsible for their antimalarial effect.

Los parásitos del género Plasmodium degradan la hemoglobina hospedera obteniendo aminoácidos libres para su síntesis proteica. Durante este evento, unidades de hemo libre tóxicas cristalizan espontáneamente formando un pigmento no tóxico denominado ß-hematina. En este trabajo, se investigó la capacidad de un grupo de azoles antimicóticos: clotrimazol (CTZ), ketoconazol (KTZ) y fluconazol (FCZ), en inhibir la síntesis de ß-hematina y la proteólisis de la globina. La síntesis de ß-hematina se registro por espectrofotometría a 405 nm y la proteólisis de la hemoglobina se determino por SDS-PAGE 15 por ciento seguido por análisis densitométrico de las bandas de hemoglobina intactas. Los compuestos fueron también ensayados in vivo en un modelo de malaria murina. CTZ y KTZ inhibieron la síntesis de ß-hematina con CI50 entre 10 y 15 µM y bloquearon la proteólisis de la hemoglobina (80.01 ± 2.04 por ciento y 55.33 ± 3.57 por ciento, respectivamente). En relación directa con los resultados encontrados in vitro, el CTZ redujo la parasitemia de ratones infectados en forma significativa, así como prolongó lo días de sobrevivencia post-infección en comparación con animales controles no tratados. Se sugiere así que la inhibición del metabolismo de la hemoglobina por los antimicóticos azólicos pudiera ser el mecanismo responsable de su actividad antimalárica.

The photodynamic and non-photodynamic actions of porphyrins

Porphyrias are a family of inherited diseases, each associated with a partial defect in one of the enzymes of the heme biosynthetic pathway. In six of the eight porphyrias described, the main clinical manifestation is skin photosensitivity brought about by the action of light on porphyrins, which are deposited in the upper epidermal layer of the skin. Porphyrins absorb light energy intensively in the UV region, and to a lesser extent in the long visible bands, resulting in transitions to excited electronic states. The excited porphyrin may react directly with biological structures (type I reactions) or with molecular oxygen, generating excited singlet oxygen (type II reactions). Besides this well-known photodynamic action of porphyrins, a novel light-independent effect of porphyrins has been described. Irradiation of enzymes in the presence of porphyrins mainly induces type I reactions, although type II reactions could also occur, further increasing the direct non-photodynamic effect of porphyrins on proteins and macromolecules. Conformational changes of protein structure are induced by porphyrins in the dark or under UV light, resulting in reduced enzyme activity and increased proteolytic susceptibility. The effect of porphyrins depends not only on their physico-chemical properties but also on the specific site on the protein on which they act. Porphyrin action alters the functionality of the enzymes of the heme biosynthetic pathway exacerbating the metabolic deficiencies in porphyrias. Light energy absorption by porphyrins results in the generation of oxygen reactive species, overcoming the protective cellular mechanisms and leading to molecular, cell and tissue damage, thus amplifying the porphyric picture

Hemichrone formation during hemoglobin zuricha denaturation

Os espectros de absorçäo obtidos por ressonância eletrônica paramagnética (REP) após oxidaçäo, armazenamento e aqueciemnto de Hb Zurich revelaram numerosos sinais na regiäo de alto campo (g=-) que säo indicativos da formaçäo de hemicromos. Espectros de absorçäo ópticos na regiäo visível obtidos da Hb Zurich oxidada ou de seu precipitado formado espontaneamente mostram aspectos característico da formaçäo de hemicromos. Os sinais de REP aumentam em g =-2) proporcionalmente ao decréscimo em g =-6,37. Além do mais, a relaçäo entre os valores de absorbância em 540 nm (heme) e 280 nm (proteína) näo se alteram durante o aquecimento da Hb Zurich a 50-C e säo similares no precipitado espontâneo desta hemoglobina en na HbA. Estes achados indicam que näo há perda do heme durante os estágios iniciais da desnaturaçäo da Hb Zurich