Anna Pastore

Analysis of glutathione: implication in redox and detoxification

BACKGROUND Glutathione is a ubiquitous thiol-containing tripeptide, which plays a central role in cell biology. It is implicated in the cellular defence against xenobiotics and naturally occurring deleterious compounds, such as free radicals and hydroperoxides. Glutathione status is a highly sensitive indicator of cell functionality and viability. Its levels in human tissues normally range from 0.1 to 10 mM, being most concentrated in liver (up to 10 mM) and in the spleen, kidney, lens, erythrocytes and leukocytes. In humans, GSH depletion is linked to a number of disease states including cancer, neurodegenerative and cardiovascular diseases. The present review proposes an analysis of the current knowledge about the methodologies for measuring glutathione in human biological samples and their feasibility as routine methods in clinical chemistry. Furthermore, it elucidates the fundamental role of glutathione in pathophysiological conditions and its implication in redox and detoxification process. TESTS AVAILABLE Several methods have been optimised in order to identify and quantify glutathione forms in human biological samples. They include spectrophotometric, fluorometric and bioluminometric assays, often applied to HPLC analysis. Recently, a liquid chromatography-mass spectrometry technique for glutathione determination has been developed that, however, suffers from the lack of total automation and the high cost of the equipment. CONCLUSION Glutathione is a critical factor in protecting organisms against toxicity and disease. This review may turn useful for analysing the glutathione homeostasis, whose impairment represents an indicator of tissue oxidative status in human subjects.

COQ2 Nephropathy: A Newly Described Inherited Mitochondriopathy with Primary Renal Involvement

Primary coenzyme Q(10) (CoQ(10)) deficiency includes a group of rare autosomal recessive disorders primarily characterized by neurological and muscular symptoms. Rarely, glomerular involvement has been reported. The COQ2 gene encodes the para-hydroxybenzoate-polyprenyl-transferase enzyme of the CoQ(10) synthesis pathway. We identified two patients with early-onset glomerular lesions that harbored mutations in the COQ2 gene. The first patient presented with steroid-resistant nephrotic syndrome at the age of 18 months as a result of collapsing glomerulopathy, with no extrarenal symptoms. The second patient presented at five days of life with oliguria, had severe extracapillary proliferation on renal biopsy, rapidly developed end-stage renal disease, and died at the age of 6 months after a course complicated by progressive epileptic encephalopathy. Ultrastructural examination of renal specimens from these cases, as well as from two previously reported patients, showed an increased number of dysmorphic mitochondria in glomerular cells. Biochemical analyses demonstrated decreased activities of respiratory chain complexes [II+III] and decreased CoQ(10) concentrations in skeletal muscle and renal cortex. In conclusion, we suggest that inherited COQ2 mutations cause a primary glomerular disease with renal lesions that vary in severity and are not necessarily associated with neurological signs. COQ2 nephropathy should be suspected when electron microscopy shows an increased number of abnormal mitochondria in podocytes and other glomerular cells.

Glutathione in blood of patients with Friedreich's ataxia

Oxidative stress and mitochondrial dysfunction have long been considered to play a role in Friedreichs ataxia, a neurodegenerative disease due to a GAA expansion in a gene coding for a mitochondrial protein (frataxin), implicated in the regulation of iron metabolism. Since glutathione is an important antioxidant whose role has been recently proposed in the pathogenesis of some neurodegenerative diseases, we investigated glutathione metabolism in the blood of 14 patients with Friedreichs ataxia by measuring total, free and protein‐bound glutathione concentrations.

Extracorporeal dialysis in neonatal hyperammonemia: modalities and prognostic indicators.

Abstract. We investigated the prognostic indicators in ten hyperammonemic neonates: four treated by continuous arteriovenous hemodialysis (CAVHD), four with continuous venovenous hemodialysis (CVVHD), and two with hemodialysis (HD). Plasma ammonium levels decreased significantly within the first 24 h irrespective of dialysis modality (from 1419 to 114 µmol/l, median values; P<0.0001). CVVHD achieved the highest ammonium clearance. HD provided highest ammonium extraction but clearance was hampered by severe hemodynamic instability. Five patients had a good outcome (normal at follow-up of 9–59 months), five had poor outcome (four died and one has severe neurological damage). Total coma duration was shorter in patients who had a good outcome (47±11 vs 78±13 h; P=0.02). Remarkably, only coma duration before dialysis determined this difference (22.2±10.1 vs 48.8±11.2 h; P=0.02). In cases with good outcome, coma duration was <33 h, whereas the others exceeded this limit. The prognosis was not related to dialysis modality, rapidity in reducing ammonium levels or to the underlying metabolic defect. In conclusion, results showed CVVHD to be the optimal modality for extracorporeal ammonium detoxification. However, the most relevant indicator for prognosis was coma duration before the start of dialysis. Therefore, major efforts should be made to refer patients quickly to highly specialized centers.

EPI-743 reverses the progression of the pediatric mitochondrial disease—Genetically defined Leigh Syndrome

BACKGROUND Genetically defined Leigh syndrome is a rare, fatal inherited neurodegenerative disorder that predominantly affects children. No treatment is available. EPI-743 is a novel small molecule developed for the treatment of Leigh syndrome and other inherited mitochondrial diseases. In compassionate use cases and in an FDA Expanded Access protocol, children with Leigh syndrome treated with EPI-743 demonstrated objective signs of neurologic and neuromuscular improvement. To confirm these initial findings, a phase 2A open label trial of EPI-743 for children with genetically-confirmed Leigh syndrome was conducted and herein we report the results. METHODS A single arm clinical trial was performed in children with genetically defined Leigh syndrome. Subjects were treated for 6 months with EPI-743 three times daily and all were eligible for a treatment extension phase. The primary objective of the trial was to arrest disease progression as assessed by neuromuscular and quality of life metrics. Results were compared to the reported natural history of the disease. RESULTS Ten consecutive children, ages 1-13 years, were enrolled; they possessed seven different genetic defects. All children exhibited reversal of disease progression regardless of genetic determinant or disease severity. The primary endpoints--Newcastle Pediatric Mitochondrial Disease Scale, the Gross Motor Function Measure, and PedsQL Neuromuscular Module--demonstrated statistically significant improvement (p<0.05). In addition, all children had an improvement of one class on the Movement Disorder-Childhood Rating Scale. No significant drug-related adverse events were recorded. CONCLUSIONS In comparison to the natural history of Leigh syndrome, EPI-743 improves clinical outcomes in children with genetically confirmed Leigh syndrome.

Nitrosylation of Human Glutathione Transferase P1-1 with Dinitrosyl Diglutathionyl Iron Complex in Vitro and in Vivo

We have recently shown that dinitrosyl diglutathionyl iron complex, a possible in vivo nitric oxide (NO) donor, binds with extraordinary affinity to one of the active sites of human glutathione transferase (GST) P1-1 and triggers negative cooperativity in the neighboring subunit of the dimer. This strong interaction has also been observed in the human Mu, Alpha, and Theta GST classes, suggesting a common mechanism by which GSTs may act as intracellular NO carriers or scavengers. We present here the crystal structure of GST P1-1 in complex with the dinitrosyl diglutathionyl iron ligand at high resolution. In this complex the active site Tyr-7 coordinates to the iron atom through its phenolate group by displacing one of the GSH ligands. The crucial importance of this catalytic residue in binding the nitric oxide donor is demonstrated by site-directed mutagenesis of this residue with His, Cys, or Phe residues. The relative binding affinity for the complex is strongly reduced in all three mutants by about 3 orders of magnitude with respect to the wild type. Electron paramagnetic resonance spectroscopy studies on intact Escherichia coli cells expressing the recombinant GST P1-1 enzyme indicate that bacterial cells, in response to NO treatment, are able to form the dinitrosyl diglutathionyl iron complex using intracellular iron and GSH. We hypothesize the complex is stabilized in vivo through binding to GST P1-1.

Antioxidant enzymes in blood of patients with Friedreich's ataxia

Background and Aims: Increased generation of reactive oxygen species and mitochondrial dysfunction may underlie the pathophysiology of Friedreichs ataxia, the most common inherited ataxia, due to GAA expansion in a gene coding for a mitochondrial protein (frataxin), implicated in the regulation of iron metabolism. Because iron overload would cause oxidative stress in Friedreichs ataxia, we investigated the enzyme antioxidant system in the blood of 14 patients by determining superoxide dismutase, glutathione peroxidase, and glutathione trasferase catalytic activities. We also studied the glutathione S-transferase genotype polymorphism in order to evaluate its possible influence on enzyme activity. Methods: Blood samples were obtained from 14 unrelated patients with Friedreichs ataxia and 21 age matched healthy subjects. Antioxidant enzyme determinations were spectrophotometrically assayed using specific substrates; the glutathione S-transferase genotype polymorphism was analysed by endonuclease restriction mapping of exon 5 and 6 amplification products. Results: There was a significant elevation of the superoxide dismutase/glutathione peroxidase activity ratio (0.037 (0.01) v 0.025 (0.008) of controls) and an 83% rise of glutathione transferase specific activity (0.22 (0.1) v 0.12 (0.03) nmol/min/mg protein) in blood of patients with Friedreichs ataxia than in the controls. The genotype polymorphism of glutathione S-transferase enzyme did not show any relevant differences when compared to that of healthy subjects. Conclusions: Data show an impairment in vivo of antioxidant enzymes in patients with Friedreichs ataxia and provide evidence of an increased sensitivity to oxidative stress, supporting a consistent role of free radical cytotoxicity in the pathophysiology of the disease.

Common mutation in methylenetetrahydrofolate reductase. Correlation with homocysteine and other risk factors for vascular disease

A common mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene results in elevated homocysteine levels and, presumably, in increased atherosclerotic risk. We evaluated serum homocysteine levels, MTHFR genotype, and a panel of variables in a sample of 155 middle-aged Italian subjects (mean age 38.1 years). Biometrical, hematological, and biochemical variables (including serum folate and vitamin B12) and lifestyle characteristics were investigated. MTHFR genotype was studied by polymerase chain reaction. The frequency of the genotype Val/Val (homozygosity for the mutant allele) was 16.13%. The Val/Val genotype was associated with increased levels of homocysteine; no differences among genotypes were seen in individuals with folate or vitamin B12 levels at or above the median values. In multivariate analysis, MTHFR genotype was an independent predictor of homocysteine levels in both biochemical and non biochemical regression models. Sex and diastolic blood pressure emerged as non biochemical variables independently associated with homocysteine. Apart from cofactors, uric acid was the only biochemical variable independently associated with homocysteine, particularly in subjects with Val/Val genotype. The observed parallel increases in homocysteine and uric acid levels in subjects with thermolabile MTHFR warrant further investigation.

Treatment of doxorubicin-resistant MCF7/Dx cells with nitric oxide causes histone glutathionylation and reversal of drug resistance

Acquired drug resistance was found to be suppressed in the doxorubicin-resistant breast cancer cell line MCF7/Dx after pre-treatment with GSNO (nitrosoglutathione). The effect was accompanied by enhanced protein glutathionylation and accumulation of doxorubicin in the nucleus. Among the glutathionylated proteins, we identified three members of the histone family; this is, to our knowledge, the first time that histone glutathionylation has been reported. Formation of the potential NO donor dinitrosyl-diglutathionyl-iron complex, bound to GSTP1-1 (glutathione transferase P1-1), was observed in both MCF7/Dx cells and drug-sensitive MCF7 cells to a similar extent. In contrast, histone glutathionylation was found to be markedly increased in the resistant MCF7/Dx cells, which also showed a 14-fold higher amount of GSTP1-1 and increased glutathione concentration compared with MCF7 cells. These results suggest that the increased cytotoxic effect of combined doxorubicin and GSNO treatment involves the glutathionylation of histones through a mechanism that requires high glutathione levels and increased expression of GSTP1-1. Owing to the critical role of histones in the regulation of gene expression, the implication of this finding may go beyond the phenomenon of doxorubicin resistance.

Protein Glutathionylation in Cardiovascular Diseases

The perturbation of thiol-disulfide homeostasis is an important consequence of many diseases, with redox signals implicated in several physio-pathological processes. A prevalent form of cysteine modification is the reversible formation of protein mixed disulfides with glutathione (S-glutathionylation). The abundance of glutathione in cells and the ready conversion of sulfenic acids to S-glutathione mixed disulfides supports the reversible protein S-glutathionylation as a common feature of redox signal transduction, able to regulate the activities of several redox sensitive proteins. In particular, protein S-glutathionylation is emerging as a critical signaling mechanism in cardiovascular diseases, because it regulates numerous physiological processes involved in cardiovascular homeostasis, including myocyte contraction, oxidative phosphorylation, protein synthesis, vasodilation, glycolytic metabolism and response to insulin. Thus, perturbations in protein glutathionylation status may contribute to the etiology of many cardiovascular diseases, such as myocardial infarction, cardiac hypertrophy and atherosclerosis. Various reports show the importance of oxidative cysteine modifications in modulating cardiovascular function. In this review, we illustrate tools and strategies to monitor protein S-glutathionylation and describe the proteins so far identified as glutathionylated in myocardial contraction, hypertrophy and inflammation.