Evaluation of 6,6′-dithionicotinic acid as alternative chromogen in a modified Ellman method—comparison in various species

For the diagnosis and therapy monitoring of intoxications with organophosphorus compounds, the determination of acetylcholinesterase (AChE) activity in whole blood is crucial. Usually, this testing is done with the colorimetric Ellman test using 412?nm wavelength and 5,5′-dithiobis-nitrobenzoic acid (DTNB) as chromogen. However, it has been described that the assay sensitivity is impaired by the Soret band of hemoglobin.

In order to enable more sensitive determination of AChE activity in human, porcine, rat, and guinea pig whole blood dilutions, we tested the alternative chromogen 6,6′-dithionicotinic acid (DTNA) with an optimal absorption wavelength of 340?nm and compared it with a modified Ellman assay using a wavelength of 436?nm.

DTNB resulted in a higher background absorption compared with DTNA in human and porcine blood samples, although the saturation of sulfhydryl groups was delayed with DTNA in all species. A slightly higher whole blood AChE activity was recorded in DTNB samples.

In conclusion, the results of the present study with human, porcine, guinea pig, and rat whole blood do not provide evidence that using DTNA for the spectrophotometric determination of AChE activity in whole blood is superior to a modified Ellman assay using DTNB.     

总谷胱甘肽的测定方法

本文介绍了测定谷胱甘肽总含量的循环法。原理是,在NADPH和GR维持谷胱甘肽总量不变的条件下,GSH和D7NB反应,生成TNB的速率与样品中总谷胱甘肽量呈正比。由于采用了γ-GT抑制剂和快速测定,克服了人血浆中谷胱甘肽含量极低,离体后消退极快,不易准确测定的困难。本法灵敏度可达0.1μM左右,回收率为93～106％。     

Protection of rats against 3-butene-1,2-diol-induced hepatotoxicity and hypoglycemia by N-acetyl-l-cysteine

3-Butene-1,2-diol (BDD), an allylic alcohol and major metabolite of 1,3-butadiene, has previously been shown to cause hepatotoxicity and hypoglycemia in male Sprague-Dawley rats, but the mechanisms of toxicity were unclear. In this study, rats were administered BDD (250 mg/kg) or saline, ip, and serum insulin levels, hepatic lactate levels, and hepatic cellular and mitochondrial GSH, GSSG, ATP, and ADP levels were measured 1 or 4 h after treatment. The results show that serum insulin levels were not causing the hypoglycemia and that the hypoglycemia was not caused by an enhancement of the metabolism of pyruvate to lactate because hepatic lactate levels were either similar (1 h) or lower (4 h) than controls. However, both hepatic cellular and mitochondrial GSH and GSSG levels were severely depleted 1 and 4 h after treatment and the mitochondrial ATP/ADP ratio was also lowered 4 h after treatment relative to controls. Because these results suggested a role for hepatic cellular and mitochondrial GSH in BDD toxicity, additional rats were administered N-acetyl-l-cysteine (NAC; 200 mg/kg) 15 min after BDD administration. NAC treatment partially prevented depletion of hepatic cellular and mitochondrial GSH and preserved the mitochondrial ATP/ADP ratio. NAC also prevented the severe depletion of serum glucose concentration and the elevation of serum alanine aminotransferase activity after BDD treatment without affecting the plasma concentration of BDD. Thus, depletion of hepatic cellular and mitochondrial GSH followed by the decrease in the mitochondrial ATP/ADP ratio was likely contributing to the mechanisms of hepatotoxicity and hypoglycemia in the rat.     

Immunochemical and biochemical analyses of myosin light chains from normal and denervated cardiac muscle

Rabbit antisera were prepared against the combined low molecular weight subunits (the light chains) of canine cardiac myosin. The antisera reacted specifically with the light chains and with purified myosin. There were no immunochemical reactions with the heavy myosin chains. The antisera were then used to test preparations of light chains isolated from hearts maintained in a denervated condition for 12, 4, or 2 weeks prior to killing the animals. Denervation was accomplished by mediastinal neural ablation. The light chains prepared from the denervated hearts all reacted with the antisera and showed immunological identity with the light chains from control muscle. The specific adenosine triphosphatase activity of myosin prepared from hearts denervated for 12, 4, or 2 weeks exhibited a marked deviation from control activity. We concluded that, although cardiac denervation did not affect the immunological properties of myosin light chains, the biological activity of cardiac myosin was greatly reduced after 2 weeks of denervation and recovered somewhat after 4 weeks.     

Induction of the mitochondrial permeability transition by selenium compounds mediated by oxidation of the protein thiol groups and generation of the superoxide

The cancer chemopreventive effect of selenium compounds cannot be fully explained by the role of selenium as a component of antioxidant enzymes, suggesting that other mechanisms, such as thiol oxidation or free radical generation, also underlie this effect. The toxicities of six different selenium compounds (selenite, selenate, selenocystine, selenocystamine, selenodioxide, and selenomethionine) have now been compared in HepG2 human hepatoma cells and isolated rat liver mitochondria. Selenite, selenocystine, and selenodioxide induced apoptosis in HepG2 cells and mediated oxidation of protein thiol groups in both HepG2 cells and isolated mitochondria. Selenocystamine oxidized protein thiol groups in isolated mitochondria and crude extracts of HepG2 cells but not in intact HepG2 cells, suggesting that this compound is not able to cross the cell membrane. The selenium compounds capable of oxidizing thiol groups also induced the mitochondrial permeability transition (MPT) in isolated mitochondria. Furthermore, they generated the superoxide (O(2) .-) on reaction with glutathione in the presence of mitochondria, and an O(2) .-) scavenger inhibited their induction of the MPT. These results suggest that the pro-apoptotic action of selenium compounds is mediated by both thiol oxidation and the generation of O(2) .-), both of which contribute to opening of the MPT pore.     

beta-Amyloid aggregation induced by human acetylcholinesterase: inhibition studies

The aggregation of beta-amyloid (1-40) (Abeta) induced by human recombinant acetylcholinesterase (HuAChE) was studied by means of circular dichroism (CD) and by thioflavin T fluorescence spectroscopy. Abeta was incubated alone and with HuAChE. The kinetic of fibrils formation was followed for 48 hr. The increasing beta-conformation content induced by HuAChE, preliminary to the formation of Abeta fibrils, was determined by circular dichroism. This phenomenon was found to be related to the thioflavin T emission of fluorescence at 490 nm. Incubation experiments were performed in the presence of known AChE inhibitors (physostigmine, edrophonium, decamethonium, propidium) and drugs used for Alzheimer's disease (AD) (tacrine, donepezil), to test their capability of preventing the HuAChE-induced Abeta aggregation. The non-competitive or mixed mode of AChE inhibition was confirmed to be an essential feature. At 100 microM propidium, decamethonium, donepezil and physostigmine were found to inhibit the HuAChE-induced Abeta aggregation by 82, 25, 22 and 30%, respectively.     

DTNB inhibits calcium response of rat brain cortical slices to anoxia of various duration

The effects of DTNB on changes in intracellular Ca²⁺ content in slices of rat cortex induced by 2- or 10-min anoxia were studied fluorometrically. DTNB (200 M) prevented excessive intracellular Ca²⁺ accumulation during and after 10-min anoxia and moderate increase in Ca²⁺ content induced by 2-min anoxia. Our results suggest that redox sites of NMDA receptors participates in both pathogenic and adaptive Ca²⁺-mediated processes activated by anoxia.     

Alteration in the redox state of plasma in heart-transplant patients with moderate hyperhomocysteinemia

Hyperhomocysteinemia has recently been suggested to contribute to the progression of the so-called chronic rejection or cardiac allograft vasculopathy (CAV) in heart-transplant patients in which the major determinant of the increase in homocysteine (Hcy) was the progressive decline of renal function. The exact mechanisms of tissue injury by Hcy is unknown, but some aspects of its toxicity have been related to its capacity for altering the redox state of plasma and forming protein adducts by intermediate lactone. To study the relationships between Hcy levels and variations in the redox state governed by thiols, plasma levels of Hcy, cysteine, glutathione, cysteinylglycine, and corresponding disulfides and protein-mixed disulfides were evaluated in subjects with moderate hyperhomocysteinemia represented by heart-transplant patients with (HTRF) and without (HT) renal failure, as well as patients with renal failure of different origin (RF), and compared with those of a control group (C) of normal subjects matched for age and sex. Plasma levels of Hcy and the corresponding protein mixed disulfides increased progressively in HTs, RFs, and HTRFs with respect to control. These changes were correlated with cysteine variations (as cystine and protein-mixed disulfides) but not with glutathione or cysteinylglycine that varied only as disulfides with a similar tendency. Moreover, an alteration in the plasma redox was evidenced by the decrease in thiol/disulfide ratios of cysteine, Hcy, and cysteinylglycine. In all groups, cysteine was directly correlated with Hcy but not with glutathione or cysteinylglycine, which in turn were correlated each other. Therefore levels of plasma cysteine were more linked to Hcy than to metabolism of glutathione. The clinical meaning of cysteine changes remains undefined and requires further study.