Copper complexing decreases the ability of amyloid beta peptide to cross the BBB and enter brain parenchyma.

The amyloid hypothesis states that amyloid beta protein (Abeta) plays a major causal role in the onset of Alzheimer's disease. Toxicity of Abeta can be modified by metal ions. Two mechanisms by which such Abeta and metal ions could interact are by enhanced oxidative stress or by altered fibrillation. Specifically, Abeta fibrillation is increased by aluminum (Al) and copper (Cu) and Al also increases Abeta uptake into brain. Here, we determined whether chelation with Cu would alter uptake of the human or rat 1-42 form of Abeta (Abeta42) by brain or alter Abeta-induced oxidative stress in an immortalized line of rat brain endothelial cells (RBE4). We found that Cu enhanced cytotoxicity of rat, but not of human Abeta, had no effect on glutathione (GSH) or cysteine (CYS) levels. Cu significantly decreased homocysteine (HCYS) levels when complexed with Abeta. Cu chelation did not alter Abeta uptake into brain or other tissues (except for kidney) or alter clearance from blood or brain in vivo, but did increase efflux in an in vitro model of the blood-brain barrier (BBB). Chelation to Cu also impaired the capillary to brain transport of Abeta, an effect opposite to that previously found for chelation of Abeta to Al. These results show that metal ions have varied effects on Abeta uptake by brain and that Cu could be protective against the neurotoxic effects of circulating Abeta.

Misincorporation of free m-tyrosine into cellular proteins: a potential cytotoxic mechanism for oxidized amino acids.

In vitro studies demonstrate that the hydroxyl radical converts L-phenylalanine into m-tyrosine, an unnatural isomer of L-tyrosine. Quantification of m-tyrosine has been widely used as an index of oxidative damage in tissue proteins. However, the possibility that m-tyrosine might be generated oxidatively from free L-phenylalanine that could subsequently be incorporated into proteins as an L-tyrosine analogue has received little attention. In the present study, we demonstrate that free m-tyrosine is toxic to cultured CHO (Chinese-hamster ovary) cells. We readily detected radiolabelled material in proteins isolated from CHO cells that had been incubated with m-[14C]tyrosine, suggesting that the oxygenated amino acid was taken up and incorporated into cellular proteins. m-Tyrosine was detected by co-elution with authentic material on HPLC and by tandem mass spectrometric analysis in acid hydrolysates of proteins isolated from CHO cells exposed to m-tyrosine, indicating that free m-tyrosine was incorporated intact rather than being metabolized to other products that were subsequently incorporated into proteins. Incorporation of m-tyrosine into cellular proteins was sensitive to inhibition by cycloheximide, suggesting that protein synthesis was involved. Protein synthesis using a cell-free transcription/translation system showed that m-tyrosine was incorporated into proteins in vitro by a mechanism that may involve L-phenylalanine-tRNA synthetase. Collectively, these observations indicate that m-tyrosine is toxic to cells by a pathway that may involve incorporation of the oxidized amino acid into proteins. Thus misincorporation of free oxidized amino acids during protein synthesis may represent an alternative mechanism for oxidative stress and tissue injury during aging and disease.

High performance liquid chromatography analysis of 2-mercaptoethylamine (cysteamine) in biological samples by derivatization with N-(1-pyrenyl) maleimide (NPM) using fluorescence detection.

2-Mercaptoethylamine (cysteamine) is an aminothiol compound used as a drug for the treatment of cystinosis, an autosomal recessive lysosomal storage disorder. Because of cysteamine's important role in clinical settings, its analysis by sensitive techniques has become pivotal. Unfortunately, the available methods are either complex or labor intensive. Therefore, we have developed a new rapid, sensitive, and simple method for determining cysteamine in biological samples (brain, kidney, liver, and plasma), using N-(1-pyrenyl) maleimide (NPM) as the derivatizing agent and reversed-phase high performance liquid chromatography (HPLC) with a fluorescence detection method (lambda(ex)=330 nm, lambda(em)=376 nm). The mobile phase was acetonitrile and water (70:30) with acetic acid and o-phosphoric acid (1 mL/L). The calibration curve for cysteamine in serine borate buffer (SBB) was found to be linear over a range of 0-1200 nM (r(2)=0.9993), and in plasma and liver matrix, the r(2) values were 0.9968 and 0.9965, respectively. The coefficients of the variation for the within-run and between-run precisions ranged from 0.68 to 9.90% and 0.63 to 4.17%, respectively. The percentage of relative recovery ranged from 94.1 to 98.6%.

Effects of N-acetylcysteine amide (NACA), a novel thiol antioxidant against glutamate-induced cytotoxicity in neuronal cell line PC12.

Oxidative stress plays an important role in neuronal cell death associated with many different neurodegenerative conditions such as cerebral ischemia and Parkinson's disease. Elevated levels of glutamate are thought to be responsible for CNS disorders through various mechanisms causing oxidative stress induced by a nonreceptor-mediated oxidative pathway which blocks cystine uptake and results in depletion of intracellular glutathione (GSH). The newly designed amide form of N-acetylcysteine (NAC), N-acetylcysteine amide (NACA), was assessed for its ability to protect PC12 cells against oxidative toxicity induced by glutamate. NACA was shown to protect PC12 cells from glutamate (Glu) toxicity, as evaluated by LDH and MTS assays. NACA prevented glutamate-induced intracellular GSH loss. In addition, NACA restored GSH synthesis in a Glu (10 mM) plus buthionine-sulfoximine (BSO) (0.2 mM)-treated group, indicating that the intracellular GSH increase is independent of gamma-GSC (gamma-glutamylcysteinyl synthetase). The increase in levels of reactive oxygen species (ROS) induced by glutamate was significantly decreased by NACA. Measurement of malondialdehyde (MDA) showed that NACA reduced glutamate-induced elevations in levels of lipid peroxidation by-products. These results demonstrate that NACA can protect PC12 cells against glutamate cytotoxicity by inhibiting lipid peroxidation, and scavenging ROS, thus preserving intracellular GSH.

Liquid chromatography analysis of N-(2-mercaptopropionyl)-glycine in biological samples by ThioGlo 3 derivatization.

N-(2-Mercaptopropionyl)-glycine (MPG) is a synthetic aminothiol antioxidant that is used in the treatment of cystinuria, rheumatoid arthritis, liver and skin disorders. Recent studies have shown that MPG can function as a chelating, cardioprotecting and a radioprotecting agent. Several other studies have shown that it may also act as a free radical scavenger because of its thiol group. Thiol-containing compounds have been detected in biological samples by various analytical methods such as spectrophotometric and colorimetric methods. However, these methods require several milliliters of a sample, time-consuming procedures and complicated derivatization steps, as well as having high detection limits. The present study describes a rapid, sensitive and relatively simple method for detecting MPG in biological tissues by using reverse-phase HPLC. With ThioGlo 3 [3H-Naphto[2,1-b] pyran, 9-acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl) phenyl-3-oxo-)] as the reagent, highly fluorescent derivatives of thiols can be obtained that are suitable for HPLC. MPG is derivatized with ThioGlo 3 and is then detected flourimetrically by reverse phase HPLC using a C18 column as the stationary phase. Acetonitrile: Water (75:25) with acetic acid and phosphoric acid (1 mL/L) is used as the mobile phase (excitation wavelength, 365 nm; emission wavelength, 445 nm). The calibration curve for MPG is linear over a range of 10-2500 nM (r=0.999) and the coefficients of the variation of within-run and between-run precision were found to be 0.3 and 2.1%, respectively. The detection limit was 5.07 nM per 20 microL injection volume. Quantitative relative recovery of MPG in the biological samples (plasma, lung, liver, kidney and brain) ranged from 90+/-5.3 to 106.7+/-9.3 %. Based on these results, we have concluded that this method is suitable for determining MPG in biological samples.

Potentiation of lead-induced cell death in PC12 cells by glutamate: protection by N-acetylcysteine amide (NACA), a novel thiol antioxidant.

Oxidative stress has been implicated as an important factor in many neurological diseases. Oxidative toxicity in a number of these conditions is induced by excessive glutamate release and subsequent glutamatergic neuronal stimulation. This, in turn, causes increased generation of reactive oxygen species (ROS), oxidative stress, excitotoxicity, and neuronal damage. Recent studies indicate that the glutamatergic neurotransmitter system is involved in lead-induced neurotoxicity. Therefore, this study aimed to (1) investigate the potential effects of glutamate on lead-induced PC12 cell death and (2) elucidate whether the novel thiol antioxidant N-acetylcysteine amide (NACA) had any protective abilities against such cytotoxicity. Our results suggest that glutamate (1 mM) potentiates lead-induced cytotoxicity by increased generation of ROS, decreased proliferation (MTS), decreased glutathione (GSH) levels, and depletion of cellular adenosine-triphosphate (ATP). Consistent with its ability to decrease ATP levels and induce cell death, lead also increased caspase-3 activity, an effect potentiated by glutamate. Exposure to glutamate and lead elevated the cellular malondialdehyde (MDA) levels and phospholipase-A(2) (PLA(2)) activity and diminished the glutamine synthetase (GS) activity. NACA protected PC12 cells from the cytotoxic effects of glutamate plus lead, as evaluated by MTS assay. NACA reduced the decrease in the cellular ATP levels and restored the intracellular GSH levels. The increased levels of ROS and MDA in glutamate-lead treated cells were significantly decreased by NACA. In conclusion, our data showed that glutamate potentiated the effects of lead-induced PC12 cell death by a mechanism involving mitochondrial dysfunction (ATP depletion) and oxidative stress. NACA had a protective role against the combined toxic effects of glutamate and lead by inhibiting lipid peroxidation and scavenging ROS, thus preserving intracellular GSH.

High performance liquid chromatography analysis of MESNA (2-mercaptoethane sulfonate) in biological samples using fluorescence detection.

MESNA is the sodium salt of 2-mercaptoethane sulfonate, a thiol-containing drug. It is an antioxidant used particularly in renal protection. Several studies have proved that MESNA has beneficial effects in ischemic acute renal failure where it scavenges reactive oxygen species (ROS), due to the presence of the thiol group. It also reduces the size of urinary bladder cancer. MESNA was proved to be effective in preventing hemorrhagic cystitis induced by high doses of several chemotherapeutic regimens such as cyclophosphamide and ifosfamide. It has been shown that MESNA functions as an uroprotective substance in drug-induced experimental bladder cancer models. Moreover, recent studies have suggested that it is also effective in reducing intestinal inflammation in colitis. Because of the increased level of interest in using MESNA for treating various disorders, a new and sensitive method was needed to understand the pharmacokinetics of this drug. Accordingly, we developed a new method for determining free MESNA in biological samples by using ThioGlo-3 [3H-Naphto [2,1-b] pyran, 9-acetoxy-2-(4-(2,5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) phenyl-3-oxo)] as the derivatizing agent. MESNA was detected fluorimetrically by reverse-phase HPLC using acetonitrile:water (75:25) along with acetic acid and phosphoric acid (1 mL/L each) as the mobile phase. The detection limit was 1.64 nm per 20 microL injection volume, with a linearity (r = 0.999) in the calibration curve extending over a range 2.5-2500 nm. The coefficients of variation for within-run and between-run precision were 0.43 and 3.31%, respectively. The relative recoveries in the biological samples were in the range 87 +/- 6 to 93 +/- 2.4%. The concentrations of MESNA in the biological samples (lungs, liver, kidney and brain) were determined. The highest concentration of MESNA was found in plasma. Of all the tissues, the kidney was found to have the highest concentration while the liver had the lowest concentration.