Determination of free thiols in animal horn derived traditional Chinese medicines / 中国中药杂志

This present study is to detect the content of free thiols(-SH) in the horn derived traditional Chinese medicines( TCMs) from different animals and different regions by using fluorescence derivatization method. TCEP was used as a disulfide bond reducing agent,while SBD-F as a derivatization reagent. Fluorescent spectrophotometry was used to determine the content of-SH,and the maximum excitation wavelength and emission wavelength were set as 375 and 510 nm,respectively. As a result,under the optimized condition,the extraction of Caprae Hircus Cornu showed the highest free-SH concentration,followed by Bovis Grunniens Cornu,Bubali Cornu,and Elaphuri Davidiani Cornu. In the present study,we point out that the-SH-contained components might be the most important material basis in animal horn derived TCMs. With good accurate,sensitive and rapid properties,the present method can provide reference basis for the quality evaluation of animal horn derived TCMs and guides for the investigation on effective material basis.

Investigation of Interaction Mechanism Between Signaling Molecule Hydrogen Sulfide and L-Lactic Dehydrogenase / 分析化学

Molecular mechanisms whereby H2S influences its targets have been of intriguing interest. In this work, L-lactic dehydrogenase ( L-LDH) was used as the protein target, and three kinds of H2S-donor reagents ( NaHS, Na2S, and polysulfide) were chosen. The interactions of these H2S-donor reagents with L-LDH were disclosed by molecular fluorescent assays for real-time monitoring of L-LDH activity. The results of the SDS-PAGE showed that H2S might not interact with L-LDH to form disulfide/trisulfide bonding. Circular dichroism spectra assays revealed that H2S reagents could be likely to react with cysteine thiols to yield sulfurated thiol (-SSH) derivatives in L-LDH, and sulfur-containing PS ( polysulfide) was a stronger protein S-sulfurating agent than the other two sulfides. Matrix assisted laser desorptionionization time-of-flight tandem mass spectrometry ( MALDI-TOF-MS/MS) study showed partial S-sulfuration of the active cysteine sites existed in L-LDH. In conclusion, H2S exerts its biological effects as a gasotransmitter through its reactions with cysteine thiols in proteins by S-sulfuration.

Redox-triggered events in lipid biology: thiol modification and lipidome alteration in ALS / Eventos redox na biologia dos lipídios: modificação de tióis e alterações do lipidoma em ELA

Lipids encompass a wide range of hydrophobic molecules present in cells. The molecular characteristics of lipids determine their cellular localization and biological function. In general, lipids are regarded as essential components of membranes, as energy reservoir and modulators of signaling pathways linked to cellular metabolism and survival, among others. In mammals, a large part of the lipids are esterified to polyunsaturated fatty acids (PUFAs), especially docosahexaenoic (DHA) and arachidonic (ARA) acids, essential for several physiological processes, including normal brain development. However, PUFAs are very susceptible to oxidation by reactive oxygen species (ROS) generated endogenously. Once oxidized, lipids are able to modify thiol groups of peptides and proteins leading to modulation of signaling pathways and cellular redox balance. In the chapter 1, we investigated the mechanisms involved in modification of thiol groups of peptides and protein by autoxidation products derived from PUFAs. Here, we identified several glutathione (GSH) adducts covalently modified by hydroxy-endoperoxides derived from both DHA and ARA. Detailed inspection of MS/MS spectra of GSH-adducts revealed that GSH and hydroxy-endoperoxides are likely bonded through a sulfur-oxygen chemical bond in a reaction which involves a nucleophilic attack by the thiolate anion. Also, we suggest that the efficiency of modification of thiol by hydroxy-endoperoxides are also dependent of the thiol reactivity, as demonstrated by covalent modification of the most reactive cysteine residue (Cys111) of the antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1). Chemical modifications of thiol groups by hydroxy-endoperoxides may modulate protein aggregation and cellular redox status, yieldingGSH adducts capable to modulate inflammation, as reported for the enzymatically generated counterparts. In the chapter 2, we investigated the role of lipids in amyotrophic lateral sclerosis (ALS), since inflammation and oxidative stress in motor neurons are hallmarks of this neurodegenerative disease. Using an untargeted lipidomics approach based on mass spectrometry coupled to liquid chromatography (UHPLC-MS/MS), we investigated the lipid metabolism in motor cortex and spinal cord tissues of a rodent model of ALS. Analysis of the motor cortex showed that the main lipid alterations were age-dependent and linked to metabolism of sphingolipids. In contrast, the major lipid alterations in the spinal cord were found in ALS symptomatic group, being the metabolism of ceramides, cholesteryl esters and cardiolipin the most affected. According to our findings and data reported in the literature, we proposed a mechanism based on neuroprotection that involves accumulation of cholesteryl esters esterified to PUFAs in astrocytes. Collectively, our findings suggest that lipids play a crucial role in modulation of cellular process linked to thiol metabolism and neurodegeneration

Os lipídeos abrangem uma ampla gama de moléculas hidrofóbicas presentes nas células. As características moleculares dos lipídios determinam sua localização celular e função biológica. Em geral, os lipídios são considerados componentes essenciais de membranas, reservatórios de energia e moduladores de vias de sinalização ligadas ao metabolismo celular, sobrevivência, entre outros. Em mamíferos, grande parte dos lipídios é esterificada em ácidos graxos poli-insaturados (PUFAs), especialmente os ácidos docosahexaenóico (DHA) e araquidônico (ARA), essenciais para vários processos fisiológicos, incluindo o desenvolvimento normal do cérebro. No entanto, os PUFAs são muito suscetíveis à oxidação por espécies reativas de oxigênio (ROS) geradas endogenamente. Uma vez oxidados, lipídios são capazes de modificar grupos tióis de peptídeos e proteínas, levando à modulação das vias de sinalização e alterando o balanço redox celular. No capítulo 1, foram investigados os mecanismos envolvidos na modificação de grupos tióis de peptídeos e proteínas por produtos de auto-oxidação de PUFAs. Com as análises realizadas foi possível identificar vários adutos de glutationa (GSH) covalentemente modificados por endoperóxidos cíclicos derivados de DHA e ARA. Uma análise detalhada dos espectros de MS/MS dos adutos de GSH revelou que GSH e endoperóxidos cíclicos são provavelmente ligados através de uma ligação química de enxofre-oxigênio, em uma reação que envolve um ataque nucleofílico do ânion tiolato. Além disso, sugerimos que a eficiência da modificação do tiol por endoperóxidos cíclicos também é dependente da reatividade do tiol, como demonstrado pela modificação covalente do resíduo de cisteína mais reativo (Cys111) da enzima antioxidante superóxido dismutase 1(SOD1). Modificações químicas de tióis por endoperóxidos cíclicos podem modular a agregação proteica e o status redox celular, produzindo adutos de GSH capazes de modular a inflamação, como relatado para os conjugados de GSH gerados enzimaticamente. No capítulo 2, nós investigamos o papel dos lipídios na esclerose lateral amiotrófica (ALS), uma vez que a inflamação e o estresse oxidativo nos neurônios motores contribuem para o desenvolvimento desta doença neurodegenerativa. Usando uma abordagem lipidômica não direcionada baseada em espectrometria de massa acoplada à cromatografia líquida (UHPLC-MS/MS), nós investigamos o metabolismo lipídico no córtex motor e na medula espinhal de um modelo de ratos com ALS. A análise do córtex motor mostrou que as principais alterações lipídicas foram dependentes da idade e ligadas ao metabolismo dos esfingolipídios. Em contraste, as principais alterações lipídicas na medula espinhal foram encontradas no grupo sintomático da ALS, sendo o metabolismo de ceramidas, ésteres de colesterol e cardiolipinas os mais afetados. De acordo com os resultados obtidos e dados relatados na literatura, propusemos um mecanismo baseado em neuroproteção que envolve o acúmulo de ésteres de colesterol esterificados em PUFAs em astrócitos. Coletivamente, nossos achados sugerem que os lipídios desempenham um papel crucial na modulação de processos celulares ligado à oxidação de tióis e à neurodegeneração

Herbal extracts differentially inhibit oxidative effects caused by the biotransformation of nifurtimox, nitrofurantoin and acetaminophen on rat liver microsomes / Extractos herbales inhiben diferencialmente los efectos oxidativos causados por la biotransformación de nifurtimox, nitrofurantoína y acetaminofeno en microsomas hepáticos de rata]

Inflammation is a cellular defensive mechanism associated to oxidative stress. The administration of nitrofurantoin, nifurtimox and acetaminophen generates oxidative stress by their biotransformation through CYP450 system. The main adverse effect described for the first two drugs is gastrointestinal inflammation and that of the last, hepatitis. Therefore, standardised dry extracts from Rosmarinus officinalis, Buddleja globosa Hope, Cynara scolymus L., Echinacea purpurea and Hedera helix were tested to evaluate their capacity to decrease drug-induced oxidative stress. For that, rat liver microsomes were incubated with drugs in the presence of NADPH (specific CYP450 system cofactor) to test oxidative damage on microsomal lipids, thiols, and GST activity. All drugs tested induced oxidation of microsomal lipids and thiols, and inhibition of GST activity. Herbal extracts prevented these phenomena in different extension. These results show that antioxidant phytodrugs previously evaluated could alleviate drugs adverse effects associated to oxidative stress.

Inflamación es un mecanismo de defensa el cual está asociado a estrés oxidativo. La administración de nitrofurantoína, nifurtimox y paracetamol genera estrés oxidativo al metabolizarse a través del sistema CYP450. El principal efecto adverso de los dos primeros fármacos es inflamación gastrointestinal y del tercero, hepatitis. Por lo tanto, utilizamos diversos extractos herbales para disminuir el estrés oxidativo inducido por estos fármacos. Para esto se incubaron microsomas hepáticos de rata con dichos fármacos en presencia de NADPH (cofactor específico del sistema CYP450) y se evaluó el daño oxidativo generado sobre los lípidos, los tioles y la actividad GST microsómica. Todos los fármacos indujeron oxidación de los lípidos y los tioles microsómicos e inhibieron la actividad GST. Los extractos herbales previnieron estos fenómenos oxidativos en diferente extensión. Estos resultados indican que fitofármacos antioxidantes previamente evaluados, podrían aliviar los efectos adversos asociados a estrés oxidativo de los fármacos.

Oxidative stress parameters as biomarkers of risk factor for diabetic foot among the patients with type 2 diabetes

The aim of this study was to determine whether plasma levels of carbonylated proteins, total antioxidant capacity (TAC) and reduced protein thiols could be suitable biomarkers of risk factors for diabetic foot. Individuals with type 2 diabetes with normal protective sensation (normal foot group) vs. loss of protective sensation and/or signs of peripheral arterial disease and/or foot deformities and/or history of ulcers and/or neuropathic fractures and/or amputation (diabetic foot group) were compared. The diabetic foot group showed higher carbonylated protein levels (P = 0.0457) and lower levels of TAC (P = 0.0148) and reduced protein thiols (P = 0.0088), compared with the normal foot group. In general, several other parameters of risk of diabetes complication (blood levels of glycated hemoglobin, glucose and cholesterol, duration of diabetes, body mass index and waist circumference) showed a tendency of higher values in the diabetic foot group. The results suggest that the plasma levels of carbonylated proteins, TAC and reduced protein thiols could furnish information about the risk of diabetic foot, considering that the changes in these biomarkers were associated with the loss of sensitivity and foot ulcerations.

Changes in Thiol Content, Glutathione and Hydrogen Peroxide Levels as a Measure of Oxidative Stress in Periplaneta Americana Treated with Metarhizium Anisopliae Spp.

Periplaneta americana (American cockroach) treated with conidia of entomopathogenic fugal isolates of Metarhizium anisopliae revealed a decrease in the thiol content and an increment in the levels of oxidized glutathione as well as hydrogen peroxide (H2O2). T-SH levels decreased to 48% and 50% at 24 hours and 48 hours post treatment respectively at LC90 of M20 isolate. Low virulent isolate M48, on the other hand, recorded a decrease of 76% and 78% in the T-SH levels at 24 and 48 hours post treatment respectively with LC30 treatment. Remarkable increase of 200% in the levels of H2O2 recorded at LC90 of M20 and 102% increase at LC30 of M19 was remarkable and focuses on the extent of oxidative stress induced by the fungal infection. Dynamics in the levels of GSH, GSSG, H2O2 and thiols in the insects treated with different fungal isolates in a time and dose dependant manner reveals oxidative stress induced by the fungal infection and the information would facilitate to explore the antioxidant defense system in augmented resistance or susceptibility of cockroach against entomopathogenic fungal conidia.

Effect of (+)-catechin hydrate on oxidative stress induced by high sucrose and high fat diet in male Wistar rats.

Increased lipid peroxidation and reduced glutathione levels in liver of rats fed high sucrose high fat (HSHF) diet were normalized by concomitant administration of (+)-catechin hydrate. Plasma non-enzymatic antioxidants viz. α-tocopherol, ascorbic acid and total thiols decrease were also significantly less in rats administered with (+)-catechin hydrate concomitantly with HSHF diet. Thus the present results indicate that (+)-catechin hydrate has antioxidant activity and is effective in reducing oxidative stress. The study is of clinical importance as oxidative stress is known to be the cause of many clinical manifestations viz. cancer, Parkinson’s disease, atherosclerosis, heart failure, myocardial infarction and many other diseases.

Effectiveness of pranayama on the levels of serum protein thiols and glutathione in breast cancer patients undergoing radiation therapy: A randomized controlled trial.

The effect of Pranayama on the levels of protein thiols and glutathione was studied among breast cancer patients receiving radiation therapy. 160 patients were randomised into experimental and control group using block randomisation. The experimental group received fractionated radiation for five days a week and performed Pranayama (Nadishodhana, Brahmari and Sheethali) for 30 minutes twice daily for five days a week. The control group received only radiation. Blood samples were collected from both the groups at the end of six weeks of radiation therapy and analysed for the levels of serum protein thiols and glutathione. An independent sample ‘t’ test showed a significant difference in the level of serum protein thiols between the two groups (t = 4.43 p 0.001). A Mann- Whitney U test showed a significant difference (z = –3.07 p 0.002) in the level of glutathione as well. These Pranayama techniques improve the antioxidant status of breast cancer patients receiving radiation therapy.

Evaluation of oxidative protein damage in patients with hypercholesterolemia.

Background: Hypercholesterolemia is highly prevalent in Indian population and known to contribute towards increased mortality and morbidity related to cardiovascular and cerebrovascular disorders. An antioxidant defence system consisting of enzymatic and non-enzymatic compounds prevents oxidative damage of lipoproteins in the plasma. When the activity of this system decreases or the reactive oxygen species (ROS) production increases, oxidative stress may occur.The –SH group (reduced thiols) bound to proteins (protein thiols) play a major role in maintaining the antioxidant status of the body. Protein thiols acts as major extracellular antioxidant, they react with reactive oxygen species (ROS) and prevent LDL oxidation. Such thiols have been studied in different disease conditions and found to be decreased compared to healthy control samples. Reduced concentration of protein thiol found to have positive correlation with increase serum level of LDL cholesterol. In the current work we have measured the level of serum protein thiols along with lipid profile in newly diagnosed hyperlipidemic patients and we tried to establish the relationship between serum protein thiols and lipid profile parameters. Objective: To study the level of protein thiols as a potent antioxidant in patient with an increased level of cholesterol. Materials: After obtaining prior consent, blood (2 ml) was taken using aseptic precautions from hypercholesterolemic patients (n = 25) and age and sex matched healthy controls (n = 25) in plain vacutainers. Serum protein thiols were measured by spectrophotometric method using 5, 5′ dithio-bis (2-nitrobenzoic acid) (DTNB). Triglyceride levels were measured by Cobas 6000 using a GPO Trinder method and HDL levels by Cobas 6000 using a direct- homogenous method. LDL levels were calculated. Results: There was a significant decrease in the levels of protein thiols p< 0.001 in hypercholesterolemic patients when compared to healthy controls and a corresponding correlatable increase in the level of LDL cholesterol due to oxidative damage. Conclusion: There may be a role for protein thiols as a biomarker in pathophysiology of cardiovascular and cerebrovascular disorders in patients with hyperlipidemia.

Indicators of oxidative stress in thyroid cancer.

Ferric reducing antioxidant power (FRAP), myeloperoxidase (MPO) activity and the levels of protein thiols and carbonyls were estimated in the blood samples of thyroid cancer patients (n = 20) before and after thyroidectomy, as well as in healthy controls (n = 10) to study the extent of damage caused by tumor tissue proliferation-induced oxidative stress and to ascertain that oxidative stress levels drop, when there was no proliferation. A significant decrease (p<0.001) in the levels of serum protein thiols and FRAP as well as a significant increase (p<0.001) in the levels of protein carbonyls and MPO activity in the blood of thyroid cancer patients before surgery was observed as compared to healthy controls. All the parameters studied also showed a significant difference (p<0.001) in their respective levels in thyroid cancer patients, pre- and post-thyroidectomy. These findings present the role of oxidative stress as a pathological implication of thyroid cancer.

Assessment of free radical-mediated damage in head and neck squamous cell carcinoma patients and after treatment with radiotherapy.

Reactive oxygen species (ROS) produced as a part of cellular metabolism can interact with biological macromolecules such as DNA, proteins and lipids and interfere with their normal functions, leading to the loss of cellular viability. ROS have been implicated in many pathophysiological conditions including cancer. In the present study, the damage caused by ROS and the effect of radiation in head and neck squamous cell carcinoma (HNSCC) patients were assessed in the erythrocytes by analyzing the superoxide dismutase (SOD) and catalase (CAT) activities, and levels of total thiols (T-SH) and malondialdehyde (MDA, a marker for lipid peroxidation). Blood samples were collected before the start of treatment and after the completion of radiotherapy. Both SOD and CAT activities were decreased in untreated patients, but elevated in patients after treatment. The T-SH levels were also depleted in untreated HNSCC patients, but elevated non-significantly after radiation therapy (p>0.05). The levels of MDA showed a significant increase in both untreated patients and after radiation therapy when compared with normal subjects (p<0.05). Thus, the present study indicated that the free radical-mediated damage was aggravated in untreated HNSCC patients, but the levels of antioxidants returned to baseline or nearly so after the treatment with radiation therapy.

Determinación de tioles totales y tioles solubles en ácido en el pez colossoma macropomum (cuvier, 1818) expuesto a cadmio / Total and Soluble Thiols in Acid Determination on the Freshwater Fish Colossoma macropomum (Cuvier, 1818) Exposed to Cadmium

En el presente estudio se evaluó la distribución de tioles totales y de tioles solubles en ácido en diferentes tejidos del pez dulceacuícola Colossoma macropomum en condiciones de cultivo, y aclimatados en el laboratorio durante 15 y 30 días, y el efecto del cadmio sobre estos parámetros en organismos expuestos durante 21 días y depurados 15 días después de la exposición al metal. La distribución de tioles totales (TT) y de tioles solubles en ácido (TSA) varió con las condiciones ambientales en las cuales se encontraban los organismos, observándose que en peces en cultivo, los mayores valores de TT se presentan en el plasma>músculo>branquias, en peces aclimatados a condiciones de laboratorio (15 días), se observó la siguiente distribución: branquias>músculo>hígado>plasma>riñón y en peces aclimatados 30 días fue branquias>riñón>hígado>músculo>plasma. Para los TSA, la distribución fue la siguiente: organismos provenientes de condiciones de cultivo: músculo>plasma>hígado>branquias. En los peces con 15 días de aclimatación: hígado>branquias>músculo; el riñón y el plasma presentaron valores cercano a cero. En peces con 30 días de aclimatación fue branquias>músculo>hígado>riñón>plasma. La exposición crónica a cadmio produjo un aumento significativo de la concentración de TT y de TSA en el plasma. Los organismos depurados 15 días disminuyeron la concentración de TSA en plasma no así la de TT. La determinación de tioles, podría considerarse un importante parámetro auxiliar, en el diagnóstico y monitoreo de peces expuestos a ambientes contaminados por cadmio.

In this study was evaluated the distribution of total thiols (TT) and acid soluble thiols (AST) in tissues of the freshwater fish Colossoma macropomum providing of cultivation and acclimated in laboratory by 15 and 30 days, as well, was evaluated the effect of cadmium on these parameters in Cd-exposure fishes during 21 days and in Cd-depuration fishes (15 days). TT and AST showed variations with environmental conditions; In Farmer fishes were, TT: plasm>muscle>gills; acclimated fishes (15 days): gills>muscle>liver>plasm>kidney and acclimated fishes (30 days): gills>kidney>liver>muscle>plasm. The distribution of AST was: farmer fishes: muscle>plasm>liver>gills. Acclimated fishes (15 days): liver>gills>muscle; the kidney and the plasm presented near values to zero, and acclimated fishes (30 days) was gills>muscle>liver>kidney>plasm. Cadmium exposition produced a significant increase in the TT and TSA concentration of plasm. Cd-depured fishes showed a decrease in plasm TSA concentration but not in plasm TT concentration. Thiol assays could be considered an important parameter in the diagnosis and monitor of fishes environmental Cd exposed.

Progress on Fluorescent Probes for Thiols / 分析化学

Thiols, which are components of many proteins and simple molecules, play an important role in the cellular antioxidant defense system. The quantitative determination of thiols is important in biochemistry and clinical chemistry. Fluorescent probes, which have its apparent advantages in sensitivity and, most importantly, in imaging thiols in vivo, even in single living cells, appear to be particularly attractive. In this review, we classify the fluorescent probes based on their different reaction mechanisms with thiols and summarize the recent progresses of thiols fluorescent probes with fifty-one

Differential Effect of gamma-radiation-induced Heme Oxygenase-1 Activity in Female and Male C57BL/6 Mice

Ionizing radiation produces reactive oxygen species, which exert diverse biological effects on cells and animals. We investigated alterations of heme oxygenase (HO) and non-protein thiols (NPSH), which are known as two major anti-oxidant enzymes, in female and male C57BL/6 mice in the lung, liver, and brain after whole-body gamma-irradiation with 10 Gy (1-7 days) as well as in the lung after whole-thorax gamma-irradiation (WTI) with 12.5 Gy (1-26 weeks). Most significant alteration of HO activity was observed in the liver, which elevated 250% in males. NPSH level in female liver was increased on the 5th-7th days but decreased in males on the 3rd day. In the lung, the elevation of HO activity in both sexes and the pattern of NPSH change were similar to that of the liver. On the other hand, the increase of HO activity on the 16th week and the decrease of NPSH level on the 2nd week were observed only in male lung after WTI. This study shows that the liver is the most sensitive tissue to gamma-irradiation-induced alterations of HO activity in both female and male mice. In addition, there exists significant differential effect of gamma-irradiation on anti-oxidant system in female and male mice.