Human resource management, employee attitudes and business performance in Post Office Ltd

Whilst a consistent link between the adoption of human resource management (HRM) practices by organisations and their performance has been confirmed by numerous studies, there is a need for greater understanding of why such effects occur. Recently, the attention of researchers has shifted towards understanding the so-called ‘black box’ linking HRM and business performance. This study focuses on this area of research by testing processes through which HRM may affect performance, in particular the process of HR implementation, mediation mechanisms, and fit with internal and external boundary conditions. This research was based on a sample of 136 Post Office branches in the UK and investigated the role of HR implementation, employee attitudes and competitive environment. The study revealed that HR implementation, a climate for service, job satisfaction and effective organisational commitment predicted independent measures of economic and service performance in branches. Employee attitudes moderated the relationship between implemented HRM and service performance, and both job satisfaction and commitment were found to mediate relationships between a climate for service and service performance. Finally, relative levels of competition faced by branches moderated the relationship between employee attitudes and sales. The findings demonstrate how the process of HR implementation, interactions with employee attitudes and moderation by external competition all influence the impact of HR systems on service and economic performance outcomes. These results illustrate the need for greater attention to processes of internal and external fit within HRM research in order to develop theory relating to why HR systems affect performance.

Development of a neurotoxicity test-system, using human post-mitotic, astrocytic and neuronal cell lines in co-culture

Astrocytes are essential for neuronal function and survival, so both cell types were included in a human neurotoxicity test-system to assess the protective effects of astrocytes on neurons, compared with a culture of neurons alone. The human NT2.D1 cell line was differentiated to form either a co-culture of post-mitotic NT2.N neuronal (TUJ1, NF68 and NSE positive) and NT2.A astrocytic (GFAP positive) cells (∼2:1 NT2.A:NT2.N), or an NT2.N mono-culture. Cultures were exposed to human toxins, for 4 h at sub-cytotoxic concentrations, in order to compare levels of compromised cell function and thus evidence of an astrocytic protective effect. Functional endpoints examined included assays for cellular energy (ATP) and glutathione (GSH) levels, generation of hydrogen peroxide (H2O2) and caspase-3 activation. Generally, the NT2.N/A co-culture was more resistant to toxicity, maintaining superior ATP and GSH levels and sustaining smaller significant increases in H2O2 levels compared with neurons alone. However, the pure neuronal culture showed a significantly lower level of caspase activation. These data suggest that besides their support for neurons through maintenance of ATP and GSH and control of H2O2 levels, following exposure to some substances, astrocytes may promote an apoptotic mode of cell death. Thus, it appears the use of astrocytes in an in vitro predictive neurotoxicity test-system may be more relevant to human CNS structure and function than neuronal cells alone. © 2007 Elsevier Ltd. All rights reserved.

The debate on human automatism in mid-Victorian England

During the 1830s, Marshall Hall carried out innumerable experiments on a great variety of animals to establish the concept of a ‘reflex arc’. In France F.L.Goltz showed that decerebrate frogs were still capable of complex behaviours. Thomas Laycock in England and Ivan Sechenov in Russia sought to apply the reflex idea to the brain. This paper follows the debate in the periodical literature of mid-Victorian England and discusses the contributions of WB Carpenter, Herbert Spencer, TH Huxley, W Clifford and others. The previous outing of this issue in the post-Cartesian seventeenth century had been largely suppressed by ecclesiastical authority. In the nineteenth century ecclesiastical power had waned, at least in England, and the debate could take a more open form. As neurophysiology and behavioural science developed, with the widespread acceptance of Darwinian evolution, it became more and more difficult to deny that brain and mind were part of the natural world and subject to the usual laws of cause and effect. This, of course, had powerful implications for the human self-image and for jurisprudence. These implications are still with us and the work of neurophysiologists such as Benjamin Libet have only reinforced them. Should humans be regarded as ‘automata’ and, if so, what becomes of ‘free will’, ‘responsibility’, and the rule of law? The Victorian debate is still useful and relevant.

Suppression and summation in contrast gain control for human vision

Over the last ten years our understanding of early spatial vision has improved enormously. The long-standing model of probability summation amongst multiple independent mechanisms with static output nonlinearities responsible for masking is obsolete. It has been replaced by a much more complex network of additive, suppressive, and facilitatory interactions and nonlinearities across eyes, area, spatial frequency, and orientation that extend well beyond the classical recep-tive field (CRF). A review of a substantial body of psychophysical work performed by ourselves (20 papers), and others, leads us to the following tentative account of the processing path for signal contrast. The first suppression stage is monocular, isotropic, non-adaptable, accelerates with RMS contrast, most potent for low spatial and high temporal frequencies, and extends slightly beyond the CRF. Second and third stages of suppression are difficult to disentangle but are possibly pre- and post-binocular summation, and involve components that are scale invariant, isotropic, anisotropic, chromatic, achromatic, adaptable, interocular, substantially larger than the CRF, and saturated by contrast. The monocular excitatory pathways begin with half-wave rectification, followed by a preliminary stage of half-binocular summation, a square-law transducer, full binocular summation, pooling over phase, cross-mechanism facilitatory interactions, additive noise, linear summation over area, and a slightly uncertain decision-maker. The purpose of each of these interactions is far from clear, but the system benefits from area and binocular summation of weak contrast signals as well as area and ocularity invariances above threshold (a herd of zebras doesn't change its contrast when it increases in number or when you close one eye). One of many remaining challenges is to determine the stage or stages of spatial tuning in the excitatory pathway.

The role of tissue transglutaminase in 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity in differentiated human SH-SY5Y neuroblastoma cells

Tissue transglutaminase (TG2) can induce post-translational modification of proteins, resulting in protein cross-linking or incorporation of polyamines into substrates, and can also function as a signal transducing G protein. The role of TG2 in the formation of insoluble cross-links has led to its implication in some neurodegenerative conditions. Exposure of pre-differentiated SH-SY5Y cells to the Parkinsonian neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+) resulted in significant dose-dependent reductions in TG2 protein levels, measured by probing Western blots with a TG2-specific antibody. Transglutaminase (TG) transamidating activity, on the other hand, monitored by incorporation of a polyamine pseudo-substrate into cellular proteins, was increased. Inhibitors of TG (putrescine) and TG2 (R283) exacerbated MPP+ toxicity, suggesting that activation of TG2 may promote a survival response in this toxicity paradigm.

Effects of glutathione, N-acetyl-cysteine, α-lipoic acid and dihydrolipoic acid on the cytotoxicity of a 2-pyridylcarboxamidrazone antimycobacterial agent in human mononuclear leucocytes in vitro

A series of antioxidants was used to explore the cytotoxicity of one particularly toxic antimycobacterial 2-pyridylcarboxamidrazone anti-tuberculosis agent against human mononuclear leucocytes (MNL), in comparison with isoniazid (INH) to aid future compound design. INH caused a significant reduction of nearly 40% in cell recovery compared with control (P < 0.0001), although the co-incubation with either glutathione (GSH, 1 mM) or (NAC, 1 mM) showed abolition of INH toxicity. In contrast, the addition of GSH or NAC 1 h after INH failed to protect the cells from INH toxicity (P < 0.0001). The 2-pyridyl-carboxamidrazone 'Compound 1' caused a 50% reduction in cell recovery compared with control (P < 0.001), although this was abolished by the presence of either GSH or NAC. A 1 h post incubation with either NAC or GSH after Compound 1 addition failed to protect the cells from toxicity (P < 0.001). Co-administration of lipoic acid (LA) abolished Compound 1-mediated toxicity, although again, this effect did not occur after LA addition 1 h post incubation with Compound 1 (P < 0.001). However, co-administration of dihydrolipoic acid (DHLA) prevented Compound 1-mediated cell death when incubated with the compound and also after 1 h of Compound 1 alone. Pre-treatment with GSH, then removal of the antioxidant resulted in abolition of Compound 1 toxicity (vehicle control, 63.6 ± 16.7 versus Compound 1 alone 26.1 ± 13.6% versus GSH pre-treatment, 65.7 ± 7.3%). In a cell-free incubation, NMR analysis revealed that GSH does not react with Compound 1, indicating that this agent is not likely to directly deplete membrane thiols. Compound 1's MNL toxicity is more likely to be linked with changes in cell membrane conformation, which may induce consequent thiol depletion that is reversible by exogenous thiols. © 2004 Elsevier B.V. All rights reserved.

Purification of plasmid DNA for use in human gene therapy

Two key issues defined the focus of this research in manufacturing plasmid DNA for use In human gene therapy. First, the processing of E.coli bacterial cells to effect the separation of therapeutic plasmid DNA from cellular debris and adventitious material. Second, the affinity purification of the plasmid DNA in a Simple one-stage process. The need arises when considering the concerns that have been recently voiced by the FDA concerning the scalability and reproducibility of the current manufacturing processes in meeting the quality criteria of purity, potency, efficacy, and safety for a recombinant drug substance for use in humans. To develop a preliminary purification procedure, an EFD cross-flow micro-filtration module was assessed for its ability to effect the 20-fold concentration, 6-time diafiltration, and final clarification of the plasmid DNA from the subsequent cell lysate that is derived from a 1 liter E.coli bacterial cell culture. Historically, the employment of cross-flow filtration modules within procedures for harvesting cells from bacterial cultures have failed to reach the required standards dictated by existing continuous centrifuge technologies, frequently resulting in the rapid blinding of the membrane with bacterial cells that substantially reduces the permeate flux. By challenging the EFD module, containing six helical wound tubular membranes promoting centrifugal instabilities known as Dean vortices, with distilled water between the Dean number's of 187Dn and 818Dn,and the transmembrane pressures (TMP) of 0 to 5 psi. The data demonstrated that the fluid dynamics significantly influenced the permeation rate, displaying a maximum at 227Dn (312 Imh) and minimum at 818Dn (130 Imh) for a transmembrane pressure of 1 psi. Numerical studies indicated that the initial increase and subsequent decrease resulted from a competition between the centrifugal and viscous forces that create the Dean vortices. At Dean numbers between 187Dn and 227Dn , the forces combine constructively to increase the apparent strength and influence of the Dean vortices. However, as the Dean number in increases above 227 On the centrifugal force dominates the viscous forces, compressing the Dean vortices into the membrane walls and reducing their influence on the radial transmembrane pressure i.e. the permeate flux reduced. When investigating the action of the Dean vortices in controlling tile fouling rate of E.coli bacterial cells, it was demonstrated that the optimum cross-flow rate at which to effect the concentration of a bacterial cell culture was 579Dn and 3 psi TMP, processing in excess of 400 Imh for 20 minutes (i.e., concentrating a 1L culture to 50 ml in 10 minutes at an average of 450 Imh). The data demonstrated that there was a conflict between the Dean number at which the shear rate could control the cell fouling, and the Dean number at which tile optimum flux enhancement was found. Hence, the internal geometry of the EFD module was shown to sub-optimal for this application. At 579Dn and 3 psi TMP, the 6-fold diafiltration was shown to occupy 3.6 minutes of process time, processing at an average flux of 400 Imh. Again, at 579Dn and 3 psi TMP the clarification of the plasmid from tile resulting freeze-thaw cell lysate was achieved at 120 Iml1, passing 83% (2,5 mg) of the plasmid DNA (6,3 ng μ-1 10.8 mg of genomic DNA (∼23,00 Obp, 36 ng μ-1 ), and 7.2 mg of cellular proteins (5-100 kDa, 21.4 ngμ-1 ) into the post-EFD process stream. Hence the EFD module was shown to be effective, achieving the desired objectives in approximately 25 minutes. On the basis of its ability to intercalate into low molecular weight dsDNA present in dilute cell lysates, and be electrophoresed through agarose, the fluorophore PicoGreen was selected for the development of a suitable dsDNA assay. It was assesseel for its accuracy, and reliability, In determining the concentration and identity of DNA present in samples that were eleclrophoresed through agarose gels. The signal emitted by intercalated PicoGreen was shown to be constant and linear, and that the mobility of the PicaGreen-DNA complex was not affected by the intercalation. Concerning the secondary purification procedure, various anion-exchange membranes were assessed for their ability to capture plasmid DNA from the post-EFD process stream. For a commercially available Sartorius Sartobind Q15 membrane, the reduction in the equilibriumbinding capacity for  ctDNA in buffer of increasing ionic demonstrated that DNA was being.adsorbed by electrostatic  interactions only. However, the problems associated with fluid distribution across the membrane demonstrated that the membrane housing was the predominant cause of the .erratic breakthrough curves. Consequently, this would need to be rectified before such a membrane could be integrated into the current system, or indeed be scaled beyond laboratory scale. However, when challenged with the process material, the data showed that considerable quantities of protein (1150 μg) were adsorbed preferentially to the plasmid DNA (44 μg). This was also shown for derived Pall Gelman UltraBind US450 membranes that had been functionalised by varying molecular weight poly-L~lysine and polyethyleneimine ligands. Hence the anion-exchange membranes were shown to be ineffective in capturing plasmid DNA from the process stream. Finally, work was performed to integrate a sequence-specific DNA·binding protein into a single-stage DNA chromatography, isolating plasmid DNA from E.coli cells whilst minimising the contamination from genomic DNA and cellular protein. Preliminary work demonstrated that the fusion protein was capable of isolating pUC19 DNA into which the recognition sequence for the fusion-protein had been inserted (pTS DNA) when in the presence of the conditioned process material. Althougth the pTS recognition sequence differs from native pUC19 sequences by only 2 bp, the fusion protein was shown to act as a highly selective affinity ligand for pTS DNA alone. Subsequently, the scale of the process was scaled 25-fold and positioned directly following the EFD system. In conclusion, the integration of the EFD micro-filtration system and zinc-finger affinity purification technique resulted in the capture of approximately 1 mg of plasmid DNA was purified from 1L of E.coli  culture in a simple two stage process, resulting in the complete removal of genomic DNA and 96.7% of cellular protein in less than 1 hour of process time.

Development of a post-mitotic, neuronal-astrocytic cell system, for the prediction of acute neurotoxicity in humans

The human NT2.D1 cell line was differentiated to form both a 1:2 co-culture of post-mitotic NT2 neuronal and NT2 astrocytic (NT2.N/A) cells and a pure NT2.N culture. The respective sensitivities to several test chemicals of the NT2.N/A, the NT2.N, and the NT2.D1 cells were evaluated and compared with the CCF-STTG1 astrocytoma cell line, using a combination of basal cytotoxicity and biochemical endpoints. Using the MTT assay, the basal cytotoxicity data estimated the comparative toxicities of the test chemicals (chronic neurotoxin 2,5-hexanedione, cytotoxins 2,3- and 3,4-hexanedione and acute neurotoxins tributyltin- and trimethyltin- chloride) and also provided the non-cytotoxic concentration-range for each compound. Biochemical endpoints examined over the non-cytotoxic range included assays for ATP levels, oxidative status (H2O2 and GSH levels) and caspase-3 levels as an indicator of apoptosis. although the endpoints did not demonstrate the known neurotoxicants to be consistently more toxic to the cell systems with the greatest number of neuronal properties, the NT2 astrocytes appeared to contribute positively to NT2 neuronal health following exposure to all the test chemicals. The NT2.N/A co-culture generally maintained superior ATP and GSH levels and reduced H2O2 levels in comparison with the NT2.N mono-culture. In addition, the pure NT2.N culture showed a significantly lower level of caspase-3 activation compared with the co-culture, suggesting NT2 astrocytes may be important in modulating the mode of cell death following toxic insult. Overall, these studies provide evidence that an in vitro integrated population of post-mitotic human neurons and astrocytes may offer significant relevance to the human in vivo heterogeneous nervous system, when initially screening compounds for acute neurotoxic potential.

Contrast matching and discrimation in human vision

The aim of this work was to investigate human contrast perception at various contrast levels ranging from detection threshold to suprathreshold levels by using psychophysical techniques. The work consists of two major parts. The first part deals with contrast matching, and the second part deals with contrast discrimination. Contrast matching technique was used to determine when the perceived contrasts of different stimuli were equal. The effects of spatial frequency, stimulus area, image complexity and chromatic contrast on contrast detection thresholds and matches were studied. These factors influenced detection thresholds and perceived contrast at low contrast levels. However, at suprathreshold contrast levels perceived contrast became directly proportional to the physical contrast of the stimulus and almost independent of factors affecting detection thresholds. Contrast discrimination was studied by measuring contrast increment thresholds which indicate the smallest detectable contrast difference. The effects of stimulus area, external spatial image noise and retinal illuminance were studied. The above factors affected contrast detection thresholds and increment thresholds measured at low contrast levels. At high contrast levels, contrast increment thresholds became very similar so that the effect of these factors decreased. Human contrast perception was modelled by regarding the visual system as a simple image processing system. A visual signal is first low-pass filtered by the ocular optics. This is followed by spatial high-pass filtering by the neural visual pathways, and addition of internal neural noise. Detection is mediated by a local matched filter which is a weighted replica of the stimulus whose sampling efficiency decreases with increasing stimulus area and complexity. According to the model, the signals to be compared in a contrast matching task are first transferred through the early image processing stages mentioned above. Then they are filtered by a restoring transfer function which compensates for the low-level filtering and limited spatial integration at high contrast levels. Perceived contrasts of the stimuli are equal when the restored responses to the stimuli are equal. According to the model, the signals to be discriminated in a contrast discrimination task first go through the early image processing stages, after which signal dependent noise is added to the matched filter responses. The decision made by the human brain is based on the comparison between the responses of the matched filters to the stimuli, and the accuracy of the decision is limited by pre- and post-filter noises. The model for human contrast perception could accurately describe the results of contrast matching and discrimination in various conditions.

Development of visual evoked responses to tritan,red-green and luminance stimuli in human infants

The principal aim of this work was to investigate the development of the S-cone colour-opponent pathway in human infants aged 4 weeks to 6 months. This was achieved by recording transient visual evoked responses to pattern-onset stimuli along a tritanopic confusion axis (tritan stimuli) at and around the adult isoluminant match. For comparison, visual evoked responses to red-green and luminance-modulated stimuli were recorded from the same infants at the same ages. Evoked responses were also recorded from colour-normal adults for comparison with those of the infants. The transient VEP allowed observation of response morphology as luminance differences were introduced to the chromatic stimuli. In this way, an estimate of isoluminance was possible in infants. Estimated isoluminant points for a group of six infants aged 6 to 10 weeks closely approximated the adult isoluminant match. This finding has implications for the use of photometric isoluminance in infant work, and suggests that photopic spectral sensitivity is similar in infants and adults. Abnormalities of the visual evoked responses to tritan, red-green and luminance-modulated stimuli in an infant with cystic fibrosis are reported. The results suggest abnormal function of the retino-striate visual pathway in this infant, and it is argued that these may be secondary to his illness, although data from more infants with cystic fibrosis are needed to clarify this further. A group of nine healthy infants demonstrated evoked responses to tritan stimuli by 4 to 10 weeks and to red-green stimuli by 6 to 11 weeks post-term age. Responses to luminance-modulated stimuli were present in all nine infants at the earliest age tested, namely 4 weeks post-term. The slightly earlier age of onset of evoked responses to tritan stimuli than for red-green may be explained by the relatively lower cone contrast afforded by red-green stimuli. Latency of the evoked response to both types of chromatic stimuli and to luminance-modulated stimuli decreased with age at a similar rate, suggesting that the visual pathways transmitting luminance and chromatic information mature at similar rates in young infants.

Measurement of HNE-protein adducts in human plasma and serum by ELISA-Comparison of two primary antibodies

There is increasing evidence that non-enzymatic post-translational protein modifications might play key roles in various diseases. These protein modifications can be caused by free radicals generated during oxidative stress or by their products generated during lipid peroxidation. 4-Hydroxynonenal (HNE), a major biomarker of oxidative stress and lipid peroxidation, has been recognized as important molecule in pathology as well as in physiology of living organisms. Therefore, its detection and quantification can be considered as valuable tool for evaluating various pathophysiological conditions.The HNE-protein adduct ELISA is a method to detect HNE bound to proteins, which is considered as the most likely form of HNE occurrence in living systems. Since the earlier described ELISA has been validated for cell lysates and the antibody used for detection of HNE-protein adducts is non-commercial, the aim of this work was to adapt the ELISA to a commercial antibody and to apply it in the analysis of human plasma samples.After modification and validation of the protocol for both antibodies, samples of two groups were analyzed: apparently healthy obese (n=62) and non-obese controls (n=15). Although the detected absolute values of HNE-protein adducts were different, depending on the antibody used, both ELISA methods showed significantly higher values of HNE-protein adducts in the obese group. © 2013 The Authors.

Characterization of tissue transglutaminase in human osteoblast-like cells

Tissue transglutaminase (tTG) is a calcium-dependent and guanosine 5'-triphosphate (GTP) binding enzyme, which catalyzes the post-translational modification of proteins by forming intermolecular ε(ϒ-glutamyl)lysine cross-links. In this study, human osteoblasts (HOBs) isolated from femoral head trabecular bone and two osteosarcoma cell lines (HOS and MG-63) were studied for their expression and localization of tTG. Quantitative evaluation of transglutaminase (TG) activity determined using the [1,414C]-putrescine incorporation assay showed that the enzyme was active in all cell types. However, there was a significantly higher activity in the cell homogenates of MG-63 cells as compared with HOB and HOS cells (p <0.001). There was no significant difference between the activity of the enzyme in HOB and HOS cells. All three cell types also have a small amount of active TG on their surface as determined by the incorporation of biotinylated cadaverine into fibronectin. Cell surface-related tTG was further shown by preincubation of cells with tTG antibody, which led to inhibition of cell attachment. Western blot analysis clearly indicated that the active TG was tTG and immunocytochemistry showed it be situated in the cytosol of the cells. In situ extracellular enzyme activity also was shown by the cell-mediated incorporation of fluorescein cadaverine into extracellular matrix (ECM) proteins. These results clearly showed that MG-63 cells have high extracellular activity, which colocalized with the ECM protein fibronectin and could be inhibited by the competitive primary amine substrate putrescine. The contribution of tTG to cell surface/matrix interactions and to the stabilization of the ECM of osteoblast cells therefore could by an important factor in the cascade of events leading to bone differentiation and mineralization.

Adaptive responses of the embryo to maternal diet and consequences for post-implantation development

Maternal periconceptional (PC) nutrition, coupled with maternal physiological condition, can impact on reproductive performance and potential across mammalian species. Oocyte quality and embryo development are affected adversely by either nutrient restriction or excess. Moreover, the quality of maternal PC nutrition can have lasting effects through fetal development and postnatally into adulthood. Chronic disease, notably cardiovascular and metabolic disease, and abnormal behaviour have been identified in adult offspring in small and large animal models of PC nutrient restriction. These long-term effects associate with compensatory responses that begin from the time of early embryo development. This review assesses the field of PC nutrition in vivo on short- and long-term developmental consequences in rodent and ruminant models and considers the implications for human health. © IETS 2012.

Glutaredoxin-1 up-regulation induces soluble vascular endothelial growth factor receptor 1, attenuating post-ischemia limb revascularization

Glutaredoxin-1 (Glrx) is a cytosolic enzyme that regulates diverse cellular function by removal of GSH adducts from S-glutathionylated proteins including signaling molecules and transcription factors. Glrx is up-regulated during inflammation and diabetes. Glrx overexpression inhibits VEGF-induced endothelial cell (EC) migration. The aim was to investigate the role of up-regulated Glrx in EC angiogenic capacities and in vivo revascularization in the setting of hind limb ischemia. Glrx overexpressing EC from Glrx transgenic mice (TG) showed impaired migration and network formation and secreted higher level of soluble VEGF receptor 1 (sFlt), an antagonizing factor to VEGF. After hind limb ischemia surgery Glrx TG mice demonstrated impaired blood flow recovery, associated with lower capillary density and poorer limb motor function compared to wild type littermates. There were also higher levels of anti-angiogenic sFlt expression in the muscle and plasma of Glrx TG mice after surgery. Non-canonical Wnt5a is known to induce sFlt. Wnt5a was highly expressed in ischemic muscles and EC from Glrx TG mice, and exogenous Wnt5a induced sFlt expression and inhibited network formation in human microvascular EC. Adenoviral Glrx-induced sFlt in EC was inhibited by a competitive Wnt5a inhibitor. Furthermore, Glrx overexpression removed GSH adducts on p65 in ischemic muscle and EC, and enhanced nuclear factor kappa B (NF-kB) activity which was responsible for Wnt5a-sFlt induction. Taken together, up-regulated Glrx induces sFlt in EC via NF-kB -dependent Wnt5a, resulting in attenuated revascularization in hind limb ischemia. The Glrx-induced sFlt may be a part of mechanism of redox regulated VEGF signaling.

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. © 2003 Elsevier B.V. All rights reserved.