A preliminary evaluation of a novel method to monitor a triple antioxidant combination (vitamins E, C and α-lipoic acid) in diabetic volunteers using in vitro methaemoglobin formation

Eight otherwise healthy diabetic volunteers took a daily antioxidant supplement consisting of vitamin E (200 IU), vitamin C (250 mg) and α-lipoic acid (90 mg) for a period of 6 weeks. Diabetic dapsone hydroxylamine-mediated methaemoglobin formation and resistance to erythrocytic thiol depletion was compared with age and sex-matched non-diabetic subjects. At time zero, methaemoglobin formation in the non-diabetic subjects was greater at all four time points compared with that of the diabetic subjects. Resistance to glutathione depletion was initially greater in non-diabetic compared with diabetic samples. Half-way through the study (3 weeks), there were no differences between the two groups in methaemoglobin formation and thiol depletion in the diabetic samples was now lower than the non-diabetic samples at 10 and 20 min. At 6 weeks, diabetic erythrocytic thiol levels remained greater than those of non-diabetics. HbA(1c) values were significantly reduced in the diabetic subjects at 6 weeks compared with time zero values. At 10 weeks, 4 weeks after the end of supplementation, the diabetic HbA1(c) values significantly increased to the point where they were not significantly different from the time zero values. Total antioxidant status measurement (TAS) indicated that diabetic plasma antioxidant capacity was significantly improved during antioxidant supplementation. Conversion of α-lipoic acid to dihydrolipoic acid (DHLA) in vivo led to potent interference in a standard fructosamine assay kit, negating its use in this study. This report suggests that triple antioxidant therapy in diabetic volunteers attenuates the in vitro experimental oxidative stress of methaemoglobin formation and reduces haemoglobin glycation in vivo.     

Methaemoglobin formation due to nitrite, disulfiram, 4-aminophenol and monoacetyldapsone hydroxylamine in diabetic and non-diabetic human erythrocytes in vitro

Nitrite, monoacetyl dapsone hydroxylamine, 4-aminophenol and disulfiram-mediated methaemoglobin formation was studied in human diabetic and non-diabetic erythrocytes in vitro. Diabetic intact erythrocytes were significantly less sensitive compared with those of non-diabetics to haemoglobin oxidation caused by the hydroxylamine, nitrite and 4-aminophenol, but not disulfiram. In haemolysates, differential sensitivity did occur with disulfiram and was partially retained with 4-aminophenol and nitrite. The differences were lost with 4-aminophenol, nitrite and disulfiram in the presence of haemoglobin purified from the respective erythrocyte types. Diethyl maleate reduced methaemoglobin formation in non-diabetic intact erythrocytes with 4-aminophenol, the hydroxylamine and disulfiram, but not with nitrite. Overall, the differential sensitivity to methaemoglobin formation seen in diabetic compared with non-diabetic erythrocytes, is probably linked to differences in the respective cells' cytosolic anti-oxidant systems.     

Use of in vitro methaemoglobin generation to study antioxidant status in the diabetic erythrocyte

Poor glycaemic control in diabetes and a combination of oxidative, metabolic, and carbonyl stresses are thought to lead to widespread non-enzymatic glycation and eventually to diabetic complications. Diabetic tissues can suffer both restriction in their supply of reducing power and excessive demand for reducing power. This contributes to compromised antioxidant status, particularly in the essential glutathione maintenance system. To study and ultimately correct deficiencies in diabetic glutathione maintenance, an experimental model would be desirable, which would provide in vitro a rapid, convenient, and dynamic reflection of the performance of diabetic GSH antioxidant capacity compared with that of non-diabetics. Xenobiotic-mediated in vitro methaemoglobin formation in erythrocytes drawn from diabetic volunteers is significantly lower than that in erythrocytes of non-diabetics. Aromatic hydroxylamine-mediated methaemoglobin formation is GSH-dependent and is indicative of the ability of an erythrocyte to maintain GSH levels during rapid thiol consumption. Although nitrite forms methaemoglobin through a complex GSH-independent pathway, it also reveals deficiencies in diabetic detoxification and antioxidant performance compared with non-diabetics. Together with efficient glycaemic monitoring, future therapy of diabetes may include trials of different antiglycation agents and antioxidant combinations. Equalization in vitro of diabetic methaemoglobin generation with that of age/sex-matched non-diabetic subjects might provide an early indication of diabetic antioxidant status improvement in these studies.     

Effects of oxidised alpha-lipoic acid and alpha-tocopherol on xenobiotic-mediated methaemoglobin formation in diabetic and non-diabetic human erythrocytes in-vitro

The effects of oxidised alpha-lipoic acid and alpha-tocopherol were investigated on a human erythrocytic in vitro model of diabetic metabolic stress. Preincubation of non-diabetic and diabetic erythrocytes with oxidised alpha-lipoic acid or alpha-tocopherol resulted in marked increases in nitrite-mediated methaemoglobin formation. In contrast, oxidised alpha-lipoic acid resulted in considerable reductions in 4-aminophenol-mediated methaemoglobin formation in both diabetic and non-diabetic cells. alpha-Tocopherol showed an increase only in diabetic cells, at one time point. Monoacetyl dapsone hydroxylamine (MADDS-NHOH)-mediated methaemoglobin formation was reduced by oxidised alpha-lipoic acid in non-diabetic and diabetic cells at all three time points, although alpha-tocopherol had no effect with MADDS-NHOH. In diabetic cells only, alpha-tocopherol incubation caused a reduction in GSH levels compared with non-diabetic cells. As the agents showed pro- as well as anti-oxidant effects in this study, further studies are required to demonstrate potential diabetic benefit from alpha-lipoic acid adminstration.     

Effects of the antioxidants dihydrolipoic acid (DHLA) and probucol on xenobiotic-mediated methaemoglobin formation in diabetic and non-diabetic human erythrocytes in vitro

The antioxidant effects of dihydrolipoic acid (DHLA) and probucol were investigated in a human erythrocytic in-vitro model of diabetic oxidative stress, where xenobiotics were used to form methaemoglobin. 4-Aminophenol mediated haemoglobin oxidation in non-diabetic erythrocytes was not affected by the presence of either DHLA or probucol. However, with diabetic cells, there were significant increases (P<0.01) in 4-aminophenol-mediated haemoglobin oxidation in the presence of DHLA. Methaemoglobin formed by nitrite in non-diabetic and diabetic cells was not altered by either DHLA or probucol except at one time point in diabetic cells. In non-diabetic as well as diabetic cells, methaemoglobin formed by MADDS-NHOH was significantly reduced at all three time points in the presence of DHLA (P<0.0001) but unaffected by probucol. In the presence of DHLA only, methaemoglobin formed by the products of rat microsomal oxidation of both 4-aminopropiophenone and benzocaine was markedly reduced for both xenobiotics in diabetic and non-diabetic cells (P<0.0001) compared with cells incubated in the absence of DHLA. There were no significant differences between total cellular thiol levels determined between diabetic and non-diabetic erythrocytes, nor did DHLA or probucol affect resting thiol levels. MADDS-NHOH caused a significant thiol depletion in diabetic cells, which was restored in the presence of DHLA. A further study is required to determine how DHLA attenuates the potent REDOX reactions that occur during hydroxylamine-mediated methaemoglobin formation.     

The methaemoglobin forming and GSH depleting effects of dapsone and monoacetyl dapsone hydroxylamines in human diabetic and non-diabetic erythrocytes in vitro

The respective methaemoglobin forming and GSH depleting capabilities of monoacetyl dapsone hydroxylamine (MADDS-NHOH) and dapsone hydroxylamine (DDS-NHOH) were compared in human diabetic and non-diabetic erythrocytes in vitro with a view to select the most potent agent for future oxidative stress and antioxidant evaluation studies. Administration of both metabolites to non-diabetic erythrocytes over the 20min period of the study resulted in significantly more methaemoglobin formation at all four time points compared with the diabetic erythrocytes (P<0.0001). At all four time points, significantly more methaemoglobin was formed in response to MADDS-NHOH in non-diabetic cells compared with the effects of DDS-NHOH on diabetic erythrocytes (P<0.0001). At the 5 and 10min time points, significantly more methaemglobin was formed in non-diabetic cells in the presence of MADDS-NHOH compared with DDS-NHOH (P<0.05). At the 5min time point only, significantly more methaemoglobin was formed in the presence of MADDS-NHOH in diabetic cells compared with that of DDS-NHOH (P<0.01). However, compared with diabetic control GSH levels, the presence of DDS-NHOH caused a significant depletion in GSH at 5, 10 and 20min time points in diabetic cells (P<0.001). In addition, the presence of DDS-NHOH caused a significant reduction in GSH levels in diabetic cells in comparison with those of non-diabetics at the 5, 10 and 20min, (P<0.005). DDS-NHOH was also associated with a significant depletion of GSH levels in diabetic cells compared with those of non-diabetic control erythrocytes (P<0.0001). The presence of MADDS-NHOH in diabetic erythrocytes led to a significant reduction in GSH levels at the 20min time point compared with those of non-diabetics (P<0.001), but there were no significant differences at the 5, 10 and 15min points. Due to its greater GSH-depleting action, DDS-NHOH will be selected for future use in the oxidative stress assessment in diabetic erythrocytes.     

Effects of the antioxidants dihydrolipoic acid (DHLA) and probucol on xenobiotic-mediated methaemoglobin formation in diabetic and non-diabetic human erythrocytes in vitro(1)

The antioxidant effects of dihydrolipoic acid (DHLA) and probucol were investigated in a human erythrocytic in-vitro model of diabetic oxidative stress, where xenobiotics were used to form methaemoglobin. 4-Aminophenol mediated haemoglobin oxidation in non-diabetic erythrocytes was not affected by the presence of either DHLA or probucol. However, with diabetic cells, there were significant increases (P<0.01) in 4-aminophenol-mediated haemoglobin oxidation in the presence of DHLA. Methaemoglobin formed by nitrite in non-diabetic and diabetic cells was not altered by either DHLA or probucol except at one time point in diabetic cells. In non-diabetic as well as diabetic cells, methaemoglobin formed by MADDS-NHOH was significantly reduced at all three time points in the presence of DHLA (P<0.0001) but unaffected by probucol. In the presence of DHLA only, methaemoglobin formed by the products of rat microsomal oxidation of both 4-aminopropiophenone and benzocaine was markedly reduced for both xenobiotics in diabetic and non-diabetic cells (P<0.0001) compared with cells incubated in the absence of DHLA. There were no significant differences between total cellular thiol levels determined between diabetic and non-diabetic erythrocytes, nor did DHLA or probucol affect resting thiol levels. MADDS-NHOH caused a significant thiol depletion in diabetic cells, which was restored in the presence of DHLA. A further study is required to determine how DHLA attenuates the potent REDOX reactions that occur during hydroxylamine-mediated methaemoglobin formation.     

Studies on the differential sensitivity between diabetic and non-diabetic human erythrocytes to monoacetyl dapsone hydroxylamine-mediated methaemoglobin formation in vitro

Methaemoglobin generation by monoacetyl dapsone hydroxylamine in non-diabetic and diabetic erythrocytes was investigated in vitro. Methaemoglobin formation in purified haemoglobin isolated from both types of erythrocytes as well as haemolysates from both diabetic and non-diabetic erythrocytes did not differ. Prior to 18 h incubation with 10 and 20 mM glucose diabetic erythrocytes were significantly less sensitive to monoacetyl dapsone-induced methaemoglobinaemia. After pre-incubation the differential was lost although significant change in glutathione concentrations could not be shown between the two cell types. NADH-diaphorase levels measured in diabetics and non-diabetics did not significantly differ. It is possible that diabetic cells display reduced hydroxylamine-mediated methaemoglobin generation due to differences in glutathione metabolism.     

Effects of lipoic acid and dihydrolipoic acid on 4-aminophenol-mediated erythrocytic toxicity in vitro

The effects of the antioxidant lipoic acid and its reduced form, dihydrolipoic acid (DHLA), were studied on the process of the erythrocytic toxicity of 4-aminophenol in human erythrocytes in vitro. 4-Aminophenol alone caused a stepwise increase in methaemoglobin formation, along with a commensurate decrease in total thiols. At 10 min., in the presence of lipoic acid alone and the thiol depletor 1-chloro-2,4-dinitrobenzene (CDNB) alone, 4-aminophenol-mediated methaemoglobin formation was significantly increased, whilst thiol levels were significantly reduced compared with the 4-aminophenol alone. At 10 min., with DHLA and CDNB alone, 4-aminophenol was associated with significantly increased methaemoglobin formation. However, thiol levels were not significantly different in the presence of DHLA compared with 4-aminophenol alone, although thiol levels were different compared with control (4-aminophenol alone) in the incubations with CDNB alone. At 15 min., only CDNB/4-aminophenol methaemoglobin formation differed from control, whilst thiol levels were significantly lower in the presence of CDNB alone compared with 4-aminophenol alone. Lipoic acid enhanced the toxicity of 4-aminophenol in terms of increased methaemoglobin formation coupled with increased thiol depletion, whilst DHLA showed increased 4-aminophenol-mediated methaemoglobin formation without thiol depletion. Lipoic acid, and to a lesser extent its reduced derivative DHLA, acted as a prooxidant in the presence of 4-aminophenol, enhancing the oxidative stress effects of the amine in human erythrocytes.     

The Effect of Acetylation and Deacetylation on the Disposition of Dapsone and Monoacetyl Dapsone Hydroxylamines in Human Erythrocytes In-vitro†

The fates of both dapsone and monoacetyl hydroxylamine have been studied in terms of acetylation and deacetylation within the human erythrocyte in-vitro. A comparison between the two metabolites showed equipotency in methaemoglobin generation at 15 min, although the monoacetyl derivative was the more rapid haemoglobin oxidizer. Within the erythrocytes, both dapsone and monoacetyl hydroxylamines were found to undergo acetylation, deacetylation and diacetylation. Of the inhibitors of acetylation studied, folate caused an increase in methaemoglobin formation associated with both metabolites, which led to a rise in both acetylated and non-acetylated amine formation. Amethopterin was associated with a rise in hydroxylamine mediated methaemoglobin formation which coincided with a fall in acetylated products. It is possible that the hydroxylamines undergo erythrocytic processes of acetylation and deacetylation before methaemoglobin-mediated reduction to their respective amines.     

Monitoring diabetic antioxidant status: a role for in vitro methaemoglobin formation

Diabetes leads to premature organ and system failure and considerably shortens lifespan. Careful control of glucose levels may not be enough to prevent the onset of complications in most diabetics. Compared with non-diabetics, diabetic tissues must not only resist a much greater long-term threat from hyperglycaemia-mediated reactive species but also defend themselves with compromised antioxidant systems. Although antioxidant therapy is a logical step in the prevention of oxidant and carbonyl stresses in the face of intermittent hyperglycaemia, this approach is not yet universally accepted to be effective in preventing complications. Although there are many biochemical indices of oxidant stress, piecemeal elevations of individual markers may not necessarily reflect true diabetic cellular antioxidant status. A dynamic process such as in vitro methaemoglobin generation may provide an opportunity to compare the response of a diabetic erythrocyte with that of a non-diabetic before and after corrective antioxidant therapy. Due to compromised cellular antioxidant capacity, diabetic cells generate less methaemoglobin in the presence of aromatic amine hydroxylamines, 4-aminophenol and nitrite compared with non-diabetics. Agents such as dihydrolipoic acid have been shown to correct methaemoglobin formation-mediated thiol deficits during in vitro studies. It is hoped that the progress of antioxidant supplementation studies in diabetics can be monitored with the aid of in vitro methaemoglobin generation using agents such as hydroxylamines, 4-aminophenol and nitrite. The most appropriate antioxidants and dosages can thus be recommended to diabetics worldwide to attenuate the development of complications.     

Effects of lipoic acid and dihydrolipoic acid on total erythrocytic thiols under conditions of restricted glucose in vitro

The effects of lipoic acid and dihydrolipoic acid were explored on total thiol maintenance in diabetic and non-diabetic human erythrocytes in vitro over 22 hr in a 37 degrees C incubation system with no added glucose. Over 18-22.5 hr after treatment in both non-diabetic and diabetic cells, lipoic acid (1 mM) was associated with greater loss of cellular thiols than dihydrolipoic acid (1 mM), compared to respective control values. At 0.1 mM, in non-diabetic cells, although lipoic acid-treated cells' thiol levels were significantly lower than control, there was no significant difference between dihydrolipoic acid-treated cells and control cells regarding thiol levels. In addition, at 0.1 mM, dihydrolipoic acid-treated diabetic cells showed a reduction in thiol levels compared to control. At 0.01 mM, lipoic acid-treated cells had significantly lower measured thiol levels compared with diabetic cells exposed to dihydrolipoic acid, whereas in non-diabetic cells, dihydrolipoic acid-treated erythrocytic thiol levels were significantly greater than those treated with lipoic acid, although there were no other significant differences between the groups. At 22.5 hr, control values of methaemoglobin rose to 6.4 +/- 1.1% in diabetic cells and 3.6 +/- 2.1% in non-diabetic cells. Lipoic acid (1 mM) showed greater methaemoglobin formation in diabetic rather than non-diabetic cells (13.6 +/- 1.5% versus 11.6 +/- 1.5%), whereas dihydrolipoic acid-treated diabetic and non-diabetic cells were less potent in methaemoglobin generation (8.5 +/- 2.4% and 8.4 +/- 1.4%, respectively). These studies suggest that in certain circumstances such as hypoglycaemia, lipoic acid administration may actually be detrimental to cellular oxidant protection status.     

Effects of dihydrolipoic acid (DHLA), α-lipoic acid. N-acetyl cysteine and ascorbate on xenobiotic-mediated methaemoglobin formation in human erythrocytes in vitro

α-Lipoic acid, dihydrolipoic acid (DHLA), N-acetyl cysteine and ascorbate were compared with methylene blue for their ability to attenuate and/or reduce methaemoglobin formation induced by sodium nitrite, 4-aminophenol and dapsone hydroxylamine in human erythrocytes. Neither α-lipoic acid, DHLA, N-acetyl cysteine nor ascorbate had any significant effects on methaemoglobin formed by nitrite, either from pre-treatment, simultaneous addition or post 30 min addition of the agents up to the 60 min time point, although N-acetyl cysteine did reduce methaemoglobin formation at 120 min (P<0.05). In all three treatment groups at 30, 60 and 120 min, there were no significant effects mediated by DHLA or N-acetyl cysteine on 4-aminophenol (1 mM)-mediated haemoglobin oxidation. Ascorbate caused marked significant reductions in 4-aminophenol methaemoglobin in all treatment groups at 30–120 min except at 30 min in the simultaneous addition group (P<0.0001). Neither α-lipoic acid, nor N-acetyl cysteine showed any effects on hydroxylamine-mediated methaemoglobin formation at 30 and 60 in all treatment groups. In contrast, DHLA significantly reduced hydroxylamine-mediated methaemoglobin formation at all three time points after pre-incubation and simultaneous addition (P<0.001), while ascorbate was ineffective. Compared with methylene blue, which was effective in reducing methaemoglobin formation by all three toxins (P<0.01), ascorbate was only highly effective against 4-aminophenol mediated methaemoglobin, whilst the DHLA-mediated attenuation of dapsone hydroxylamine-mediated methaemoglobin formation indicates a possible clinical application in high-dose dapsone therapy.     

Bioactivation of benzocaine to a methaemoglobin-forming metabolite by rat and human microsomes in vitro

Benzocaine-mediated methaemoglobin-generation was compared with that of dapsone in vitro. Direct incubation of benzocaine with washed human erythrocytes alone at up to 15 mM did not result in significant methaemoglobin formation (0.4 ± 0.1%). With rat microsomes, dapsone-dependent methaemoglobin formation was almost two-fold that of benzocaine at 30 min (56.5 ± 0.7% vs 31.6 ± 2.4% P < 0.005)). Benzocaine-mediated methaemoglobin formation was significantly reduced in the presence of DDC (diethyldithiocarbamate) at the 10 (P < 0.005) and 20 (P < 0.025) min time points. At 30 min, cimetidine reduced benzocaine-mediated methaemoglobin from 34.4 ± 8.7% to less than 3% (P < 0.005). The methaemoglobin forming capacity of dapsone was significantly inhibited at all three time points by both DDC (P < 0.005) and cimetidine (P < 0.005). Incubation of benzocaine with microsomes from five human livers showed that each liver produced methaemoglobin-forming metabolites. No inhibitory effect was seen with DDC, although cimetidine caused a significant reduction (32.8 ± 12.4% overall) in benzocaine-mediated methaemoglobin formation in the four livers tested.     

Resistance to glutathione depletion in diabetic and non-diabetic human erythrocytes in-vitro

We have investigated the resistance of erythrocytes from diabetics and non-diabetics to glutathione depletion caused by p-benzoquinone, 1-chloro-2,4-dinitrobenzene (CDNB), diethyl maleate and 4-aminophenol. Incubation of erythrocytes with 4-aminophenol (2 mM) caused a precipitous reduction (>80%) in cellular glutathione levels although there was no significant difference between 4-aminophenol-mediated glutathione depletion in the diabetic and non-diabetic cells. p-Benzoquinone and CDNB were both associated with a less severe initial reduction in glutathione levels (>50% at 30 min) although p-benzoquinone caused greater depletion (P < 0.001) at 4.5 h (21.1 +/- 3.1%, non-diabetic; 20.0 +/- 1.0%, diabetic) compared with CDNB (49.2 +/- 2.2%, non-diabetic; 51.3 +/- 1.1% diabetic). Although there was no significant difference between the two types of cell in terms of level of depletion, administration of diethyl maleate caused a significant reduction in glutathione levels at 30 min (P < 0.0005), 3.5 h (P < 0.05) and 4.5 h (P < 0.05) in erythrocytes from diabetic man compared with those from non-diabetic man. Co-administration of buthionine sulphoximine (20 mM) and 4-aminophenol (1 mM) also led to a significant reduction in glutathione levels in diabetic cells at 30 min (P < 0.05), 3.5 h (P < 0.02) and 4.5 h (P < 0.007) compared with those in non-diabetic cells. The observations that diabetic red cells' resistance to depletion was similar to that of nondiabetic cells for three of the four depletors, and that the combination of 4-aminophenol and buthionine sulphoximine-mediated inhibition of glutathione synthesis was required to illustrate differences suggests that diabetic complications might be a result of the long-term effect of small deficiencies in oxidative self-defence mechanisms such as glutathione.     

Begrenzende Faktoren der Methämoglobinbildung durch Phenylhydroxylamin in Hämolysaten verschiedener Tierarten

Summary 1. Addition of ATP, G-6-P, G-6-PDH and NADP to haemolysates of erythroyctes from pigs, cows, and sheep increased the formation of methaemoglobin through phenylhydroxylamine by various degrees, according to the animal source used. The results revealed limiting factors for the formation of methaemoglobin in haemolysates. These factors are compared with the limiting factors found in intact erythrocytes.2. Phosphorylation of glucose limits NADP reduction and, consequently, the methaemoglobin formation in haemolysates of erythrocytes from the three animals. The individual limiting factors are as follows: in sheep erythrocytes, the activity of hexokinase; in pig erythrocytes, the glucose utilisation; in cow erythrocytes, the ATP concentration.3. When the hexokinase reaction is circumvented by the addition of G-6-P, the activity of NADPH-diaphorase limits the formation of methaemoglobin in the haemolysates of cow erythrocytes. However, the formation of methaemoglobin in haemolysates of pigs and cows can be increased by the addition of NADP before the activity of diaphorase becomes the limiting factor.4. After adding a complete NADPH regenerating system, the velocities of methaemoglobin formation in haemolysates from the three animals were found to correlate with the corresponding disphorase activities.

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Stipendiat der Alexander von Humboldt-Stiftung 1965.     

The interaction of phosphine with haemoglobin and erythrocytes.

1. Phosphine progressively converts oxyhaemoglobin to methaemoglobin and hemichrome species, with the product formed being time- and concentration-dependent. 2. The reaction of phosphine with oxyhaemoglobin leads to the formation of phosphite and phosphate. 3. Incubation of rat erythrocytes with various concentrations of phosphine results in the progressive uptake of phosphine by the erythrocytes in a temperature-dependent first-order process. 4. Uptake of phosphine by erythrocytes causes crenation, but conversion of oxyhaemoglobin to methaemoglobin and hemichrome could not be demonstrated.     

Studies on the mechanism of toxicity of the mycotoxin sporidesmin. 2--Evidence for intracellular generation of superoxide radical from sporidesmin

Formation of hydrogen peroxide, the dismutation product of superoxide radical, has been demonstrated in erythrocytes incubated with the mycotoxin sporidesmin. Erythrocytic thiols, both non-protein and protein-bound, were depleted in the presence of sporidesmin, whilst haemoglobin was oxidized to methaemoglobin. Irreversible haemoglobin oxidation also occurred in these cells, shown by the formation of Heinz bodies; purified haemoglobin likewise suffered oxidative damage when incubated with sporidesmin in the presence of glutathione. Sporidesmin has previously been shown to generate superoxide radical in vitro; the erythrocytic changes induced by the mycotoxin, which are characteristically produced by compounds which generate 'active oxygen' species, suggest that it is also capable of generating this radical intracellularly.     

The interaction of phosphine with haemoglobin and erythrocytes

1. Phosphine progressively converts oxyhaemoglobin to methaemoglobin and hemichrome species, with the product formed being time- and concentration-dependent.

2. The reaction of phosphine with oxyhaemoglobin leads to the formation of phosphite and phosphate.

3. Incubation of rat erythrocytes with various concentrations of phosphine results in the progressive uptake of phosphine by the erythrocytes in a temperature-dependent first-order process.

4. Uptake of phosphine by erythrocytes causes crenation, but conversion of oxyhaemoglobin to methaemoglobin and hemichrome could not be demonstrated.     

Studies on the toxicity and efficacy of some ester analogues of dapsone in vitro using rat and human tissues

The toxicity and efficacy of a series of 13 anti-tubercular sulphone esters has been evaluated using human and rat tissues. The toxicity studies involved comparison of the esters' ability to generate rat microsomally mediated NADPH-dependent methaemoglobin with that of dapsone. All the compounds formed significantly less methaemoglobin in the 1 compartment studies compared with dapsone itself. The ethyl, propyl, 3-methyl-butyl cyclopentyl esters and the carboxy parent derivative all yielded less than 5% of the methaemoglobin generated by dapsone. The 3-nitro benzoic acid ethyl and propyl esters generated 30 and 25% of dapsone's methaemoglobin formation. A similar effect was seen in the 2 compartment system, except for the butyl ester, which yielded similar haemoglobin oxidation to dapsone. The low toxicity ethyl and propyl esters, were also low in toxicity using human liver microsomes, producing less than 30% of the dapsone mediated methaemoglobin. All the compounds except the benzoic acid parent were superior to dapsone in terms of suppression of human neutrophil respiratory burst using a lucigenin-based chemiluminescence assay. The most potent derivatives were the phenyl, propyl and 3-nitro benzoic acid ethyl esters, which were between two- and threefold more potent compared with dapsone in arresting the respiratory burst. Overall, the ethyl ester showed the best combination of low toxicity in the rat and human microsomal systems and its IC(50) was approximately 40% lower than that of dapsone in neutrophil respiratory burst inhibition. These compounds indicate some promise for future development in their superior anti-inflammatory capability and lower toxicity compared with the parent sulphone, dapsone.