A study of the free radical scavenging effects of Piper betle leaf extract in patients with vitiligo.

BACKGROUND: Vitiligo is an idiopathic skin disease manifested by depigmented macules. It is characterised by melanocyte destruction, and redox imbalance is proposed to play a contributory role. AIM: The aim of this study was to analyze the effects of an ethanolic extract of Piper betle leaves on the generation of reactive oxygen species in erythrocytes sourced from vitiligo patients. METHODS: The effect of Piper betle on the generation of reactive oxygen species in erythrocytes was measured by flow cytometry in patients with active and stable vitiligo versus healthy controls, using 5-(and-6)-chloromethyl-2'-7'-dichlorodihydrofluorescein diacetate. RESULTS: The generation of reactive oxygen species in erythrocytes was higher in patients with vitiligo (n = 23) compared to healthy controls (n = 18). The geometrical mean fluorescence channel was 23.05 ± 2.11 in patients versus 17.77 ± 1.79 in controls, P = 0.039. The levels of reactive oxygen species were higher in patients with active vitiligo. Treatment of erythrocytes with Piper betle in concentrations of 0.5 and 1.0 µg/ml significantly decreased the baseline levels of reactive oxygen species by 31.7% in healthy controls, and 47.6% and 44.3% in patients with active vitiligo, respectively. Piper betle effectively scavenged hydrogen peroxide, which was evident by a decrease in the geometrical mean fluorescence channel by 52.4% and 62.9% in healthy controls, and 45.0% and 57.0% in patients with active vitiligo. LIMITATIONS: The study had a small sample size. Future studies should focus on evaluation of the antioxidant role of Piper betle at the lesional site. CONCLUSION: This pilot study indicates that patients with active vitiligo demonstrate enhanced generation of reactive oxygen species in erythrocytes, which was significantly reduced following ex vivo treatment with Piper betle.

Hemolytic drugs aniline and dapsone induce iron release in erythrocytes and increase the free iron pool in spleen and liver.

Incubation of rat erythrocytes with the hydroxylated metabolites of aniline and dapsone (4-4'-diaminodiphenylsulfone), phenylhydroxylamine and dapsone hydroxylamine, respectively, induced marked release of iron and methemoglobin formation. On the contrary, no release of iron nor methemoglobin formation was seen when the erythrocytes were incubated with the parent compounds (aniline and dapsone). The acute intoxication of rats with aniline or dapsone induced a marked increase in the erythrocyte content of free iron and methemoglobin, indicating that the xenobiotics are effective only after biotransformation to toxic metabolites in vivo. Prolonged administration of aniline or dapsone to rats produced continuous release of iron from erythrocytes. Marked iron overload was seen in the spleen and in the liver Kupffer cells, as detected histochemically. The spleen weight in these subchronically treated animals was significantly increased. The free iron pool was markedly increased in the spleen and to a lower extent in the liver. The possible relationships between iron release in erythrocytes, oxidative damage seen in senescent cells, hemolysis, overwhelmed capacity of spleen and liver to keep iron in storage forms and subsequent increase in low molecular weight, catalitically active iron is discussed.

Combination of two mutant alpha spectrin alleles underlies a severe spherocytic hemolytic anemia.

We studied a patient with a severe spherocytic hemolytic anemia without family history of spherocytosis. Analysis of patient's erythrocyte membrane proteins revealed spectrin deficiency and a truncated alpha spectrin protein. We determined that the patient is a compound heterozygote with two mutations in alpha spectrin gene. Mutation in the paternal allele, designated alpha spectrin(PRAGUE), is a transition A to G in the penultimate position of intron 36 that leads to skipping of exon 37, frameshift, and production of the truncated alpha spectrin protein. The maternal allele, designated alpha spectrin(LEPRA), contains transition C-->T in position -99 of intron 30. This mutation enhances an alternative acceptor splice site 70 nucleotides upstream from the regular site. The alternative splicing causes a frameshift and premature termination of translation leading to a significant decrease in alpha spectrin production. The alpha(LEPRA) mutation is linked to a spectrin alphaIIa marker that was found to be associated with recessive or nondominant spectrin-deficient hereditary spherocytosis in approximately 50% of studied families. We conclude that the alpha(LEPRA) mutation combined in trans with the alpha(PRAGUE) mutation underlie the severe hemolytic anemia in the proband. We suggest that allele alpha spectrin(LEPRA) may be frequently involved in pathogenesis of recessive or nondominant spectrin-deficient hereditary spherocytosis.

Stimulation of K-C1 cotransport in rat red cells by a hemolytic anemia-producing metabolite of dapsone.

Dapsone, a sulfone compound used in the treatment of leprosy and, more recently, Pneumocystis carinii pneumonia, produces as a major side effect a hemolytic anemia. This anemia is characterized by oxidation of hemoglobin to methemoglobin and increased splenic uptake of red blood cells. Using a rat model, Grossman and Jollow (J. Pharmacol. Exp. Ther. 244: 118-125, 1988) found that dapsone hydroxylamine (DDS-NOH), a dapsone metabolite, is responsible for its hemolytic effect in vivo. DDS-NOH also promotes hemoglobin binding to SH groups on rat red cell membrane proteins (Budinsky et al., FASEB J. 2: A801, 1988). Since the binding of hemoglobin and other reagents (e.g., N-ethylmaleimide) to membrane SH groups has been associated with increased K transport in red blood cells, we examined the effect of DDS-NOH on K efflux from rat red blood cells in vitro. Cells shrink when exposed to DDS-NOH (100 microM) in media with plasma-like ionic composition. This shrinkage is prevented if extracellular K is raised to 110 mM or if intra- and extracellular Cl are replaced by methylsulfate (MeSO4), suggesting involvement of a K-Cl cotransport pathway. Indeed, 100 microM DDS-NOH produces a 4- to 5-fold increase in K efflux in cells containing Cl but less than a 2-fold increase in cells containing MeSO4. This stimulatory effect is specific for K; Na efflux is slightly inhibited by 100 microM DDS-NOH. The concentrations of DDS-NOH required for half-maximal stimulation of Cl-dependent K efflux (53 microM) is similar to its half-maximal hemolytic concentration in rats (approximately 100 microM). Furthermore, the stimulation of Cl-dependent K efflux by DDS-NOH is greater than 80% reversed by subsequent treatment of the cells with dithiothreitol, suggesting involvement of SH groups. Our results indicate that DDS-NOH exposure stimulates an apparent K-Cl cotransport in rat red blood cells, resulting in cell shrinkage under physiological ionic conditions. Since shrinkage of red blood cells renders them less deformable (Mohandas et al., J. Clin. Invest. 66: 563-573, 1980), this suggests a pathophysiological mechanism whereby DDS-NOH exposure in vivo could promote increased splenic uptake of red blood cells and hemolytic anemia.

Concentrations of adenine nucleotides in erythrocytes of patients with gout.

Gout has long been known as a disease of the high social class or of those with achieved social status. Brooks and Mueller (1966) studied the relationships between behavior and serum uric acid levels among university professors. They indicated that personal characteristics of drive, achievement, and leadership are positively associated with the uric acid levels in plasma. Hansen and Dimitrakondi (1974) reported a marked decrease in ATP levels in psychotic depression and significant correlation between ATP concentration and mood scores. We estimated the adenine nucleotides in erythrocytes of gouty patients.

The haemolytic action of dapsone: the effect on red-cell glycolysis

Some aspects of red-cell matabolism were studied in blood samples taken from patients on long-term dapsone therapy. Glucose consumption by the Embden-Meyerhof pathway (EMP) was not abnormal and adenosine-triphosphate (ATP) levels lay within the normal range and were well maintained during incubation. Hexose monophosphate patchway (HMP) activity was increased above that which would be expected from the age of the red-cell population. Red-cell reduced glutathione (GSH) levels tended to be lower than normal, proportional to the dose od dapsone and GSH levels were unstable on incubation. Red-cell fractionation studies showed that the older cell fraction had lower GSH levels and more Heinz bodies but proportionally greater HMP activity than the younger cell fraction. It is suggested that the low GSH levels are probably due to the binding of GSH to sulphydryl groups in haemoglobin and possibly the red-cell membrane. The increased HMP activity in the older cells many, in part, be a compensatory mechanism.