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Facioscapulohumeral dystrophy (FSHD) has a hypomethylation-related epigenetic background and exhibits a different course in male and female patients. The differences between males and females have been linked to the levels of sex hormones. This study is the first to investigate the possible effect of these hormones on methylation status. We hypothesized that the levels of sex-related hormones, estradiol, testosterone, progesterone, and prolactin might be associated with the methylation status of the proximal part of the D4Z4. We also investigated the effect of fT3, folic acid, and vitamin B12 levels. We collected blood from 28 FSHD patients and 28 controls. DNA was extracted from each individual for bisulfite methylation analysis and serum was separated for biochemical analysis of estradiol, testosterone, progesterone, prolactin, fT3, folic acid, and B12 analysis. Methylation analysis was specified to the DR1, 5P regions and the proximal region covering both DR1 and 5P. Methylation levels were compared between FSHD patients and controls. The correlation of methylation levels with estradiol, testosterone, progesterone, prolactin, fT3, folic acid, and B12 was investigated. We found that the 5P region and the proximal region were significantly hypomethylated in FSHD patients compared to the controls, but not the DR1 region. Male patients exhibited a significant reduction in DNA methylation compared to male controls. Older FSHD patients exhibited a notable decrease in fT3 levels and hypomethylation of the 5P region. Analyses of each CpG revealed seven hypomethylated positions that were significantly different from the control group. Two of the positions demonstrated a correlation with progesterone in the control group. With the exception of one position, the methylation levels were inversely correlated with vitamin B12 in FSHD patients. The results of our study indicate that the methylation of the proximal D4Z4 region, particularly at specific positions, may be associated with progesterone. In addition, vitamin B12 may be an indicator of hypomethylation. We suggest that examining position-specific methylations may be a useful approach for the development of epigenetic treatment modalities.
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BACKGROUND: Sphingolipid species in the lung epithelium have a critical role for continuity of membrane structure, vesicular transport, and cell survival. Sphingolipid species were reported to have a role in the inflammatory etiology of cystic fibrosis by previous work. The aim of the study was to investigate the levels of plasma sphingomyelin and ceramide in adult cystic fibrosis (CF) patients and compared with healthy controls. MATERIALS AND METHODS: Blood samples were obtained from CF patients at exacerbation (n = 15), discharge (n = 13) and stable periods (n = 11). Healthy individuals (n = 15) of similar age served as control. Levels of C16-C24 sphingomyelin and C16-C24 ceramide were measured in the plasma by LC-MS/MS. Also, cholesterol and triglyceride levels were determined in plasma samples of the patients at stable period. RESULTS: All measured sphingomyelin and ceramide levels in all periods of CF patients were significantly lower than healthy controls except C16 sphingomyelin level in the stable period. However, plasma Cer and SM levels among exacerbation, discharge, and stable periods of CF were not different. CF patients had significantly lower cholesterol levels compared to healthy individuals. We found significant correlation of cholesterol with C16 sphingomyelin. CONCLUSION: We observed lower plasma Cer and SM levels in adult CF patients at exacerbation, discharge, and stable periods compared to healthy controls. We didn't find any significant difference between patient Cer and SM levels among these three periods. Our limited number of patients might have resulted with this statistical insignificance. However, percentage of SM16 levels were increased at discharge compared to exacerbation levels, while percentage of Cer16 and Cer 20 decreased at stable compared to exacerbation. Inclusion of a larger number of CF patients in such a follow up study may better demonstrate any possible difference between exacerbation, discharge, and stable periods.
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BACKGROUND: The most common cause of intra-abdominal adhesion (IAA) is previous abdominal surgery and mortality. IAA can cause serious complications such as chronic abdominal pain, ileus, and infertility. Approximately 3% of all laparotomies are related to adhesions. IAA reduces the quality of life of the patient, causes morbidity, and increases health expenditures. In this study, we aimed to investigate the preventive effect of fucoxanthin (Fx) on IAA in the intra-abdominal surgical adhesion model that experimentally created in rats. METHODS: This study used 21 Sprague-Dawley rats divided into three groups. After anesthesia, the abdomen was opened, the cecum and right abdominal wall were damaged with a sterile toothbrush until petechiae bleeding was seen. No additional action was taken to the control group. In the sham group, 5 cc saline solution was released into the peritoneum before the abdomen was closed. In the Fx group, 35 mg/kg Fx was instilled intraperitoneally and the abdomen was closed. On the 21st post-operative day, all subjects were anesthetized with standard anesthesia. Macroscopic adhesions were quantitatively evaluated according to the Mazuji classifica-tion. The cecum anterior wall and parietal peritoneum were excised for pathological sampling. A pathologist, unaware of the groups, evaluated inflammation, fibroblastic activity, and vascular proliferation. In addition, serum tumor necrosis factor-alpha (TNF-α) and interleukin-10 levels were measured. RESULTS: No rat was lost during the study period. Congenital adhesion was not observed in any of the subjects at the first laparo-tomy. Adhesion was significantly less macroscopically in the Fx group compared to the control and sham group (p<0.001 and p<0.001). Fibroblastic activity was found to be significantly less in the Fx group compared to the sham and control groups (p<0.001 and p<0.001). Vascular proliferation was found to be significantly less in the Fx group than in the sham and control groups (p<0.001 and p<0.001). The inflammation score was significantly lower in the Fx group compared to the other two groups (p<0.001 and p<0.001). The inflam-mation score in the sham group was lower than the control group and was statistically significant (p<0.001). TNF-α level was found to be statistically significantly lower in the Fx group compared to the sham and control groups (p<0.001 and p<0.001). CONCLUSION: As a result of experimental study, we can say that Fx is effective in preventing IAAs and decreases the level of TNF-α, a pro-inflammatory cytokine.
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Parede Abdominal , Fator de Necrose Tumoral alfa , Parede Abdominal/patologia , Animais , Humanos , Inflamação/patologia , Inflamação/prevenção & controle , Complicações Pós-Operatórias/prevenção & controle , Qualidade de Vida , Ratos , Ratos Sprague-Dawley , Aderências Teciduais/prevenção & controle , XantofilasRESUMO
We investigated plasma sphingomyelin (CerPCho) and ceramide (Cer) levels in pediatric patients with cystic fibrosis (CF) and primary ciliary dyskinesia (PCD). Plasma samples were obtained from CF (n = 19) and PCD (n = 7) patients at exacerbation, discharge, and stable periods. Healthy children (n = 17) of similar age served as control. Levels of 16-24 CerPCho and 16-24 Cer were measured by LC-MS/MS. Concentrations of all CerPCho and Cer species measured at exacerbation were significantly lower in patients with CF than PCD. 16, 18, 24 CerPCho, and 22, 24 Cer in exacerbation; 18, 24 CerPCho, and 18, 20, 22, 24 Cer at discharge; 18, 24 CerPCho and 24 Cer at stable period were significantly lower in CF patients than healthy children (p < 0.001 and p < 0.05). All CerPCho and Cer levels of PCD patients were significantly higher except 24 CerPCho and 24 Cer during exacerbation, 24 CerPCho at discharge, and 18, 22 CerPCho levels at stable period (p < 0.001 and p < 0.05) compared with healthy children. There was no significant difference among exacerbation, discharge, and stable periods in each group for Cer and CerPCho levels. This is the first study measuring plasma Cer and CerPCho levels in PCD and third study in CF patients. The dramatic difference in plasma levels of most CerPCho and Cer species found between two diseases suggest that cilia pathology in PCD and CFTR mutation in CF seem to alter sphingolipid metabolism possibly in opposite directions.
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Ceramidas/sangue , Transtornos da Motilidade Ciliar/sangue , Transtornos da Motilidade Ciliar/genética , Fibrose Cística/sangue , Esfingomielinas/sangue , Adolescente , Estudos de Casos e Controles , Criança , Cromatografia Líquida , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/genética , Mutação , Estudos Prospectivos , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Ischemic preconditioning (IPC) is defined as raising tolerance to subsequent ischemic stress by exposing tissues to sub-lethal ischemia. Although many candidates have been suggested, recent studies have clearly demonstrated that adenosine-mediated ADORA2B receptor (ADORA2BR) activation is the main mechanism involved in IPC. While the tissue-protective role of this mechanism has been demonstrated in different ischemia/reperfusion (I/R) models, its role in flap surgery-derived I/R damage has not to date been investigated. OBJECTIVE: To investigate the role of adenosine and ADORA2BR activation in IPC-mediated tissue protection in an epigastric flap model. METHODS: Animals were divided into five main groups, all of which were then divided into two subgroups depending on whether or not they were exposed to IPC before the I/R procedure, which consisted of 6 hours of ischemia and 6 days of reperfusion. No drugs were administered in Group 1 (the control group). Animals in Group 2 were pretreated with CD73-inhibitor before IPC application or the ischemic period. Animals in Group 3 were pretreated with adenosine. Animals in Group 4 were pretreated with an ADORA2BR antagonist, and those in Group 5 with an ADORA2BR agonist. After 6 days of reperfusion, tissue survival was evaluated via histological and macroscopic analysis. RESULTS: IPC application significantly enhanced CD73 expressions and adenosine concentrations (pâ<â0.01). Flap survivals were increased by IPC in Group 1 (pâ<â0.05). However, CD73 inhibition blocked this increase (Group 2). In Group 3, adenosine improved flap survival even in the absence of IPC (pâ<â0.01). While an ADORA2BR antagonist attenuated the tissue-protective effect of IPC (pâ<â0.01), the ADORA2BR agonist improved flap survival by mimicking IPC in groups 4 and 5. CONCLUSION: These results provide pharmacological evidence for a contribution of CD73 enzyme-dependent adenosine generation and signaling through ADORA2BR to IPC-mediated tissue protection. They also suggest for the first time that ADORA2BR agonists may be used as a potential preventive therapy against I/R injury in flap surgeries.
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Adenosina/metabolismo , Precondicionamento Isquêmico/métodos , Receptor A2B de Adenosina/metabolismo , Traumatismo por Reperfusão/patologia , Retalhos Cirúrgicos/patologia , Sobrevivência de Tecidos/efeitos dos fármacos , Animais , Feminino , Humanos , Ratos , Ratos WistarRESUMO
The 8-hydroxy-2'-deoxyguanosine (8-OHdG) damages are base damages induced by reactive oxygen species. We aimed to investigate the role of Androgen Receptor and NKX3.1 in 8-OHdG formation and repair activation by quantitating the DNA damage using Aklides.NUK system. The data demonstrated that the loss of NKX3.1 resulted in increased oxidative DNA damage and its overexpression contributes to the removal of menadione-induced 8-OHdG damage even under oxidative stress conditions. Moreover, 8-oxoguanine DNA glycosylase-1 (OGG1) expression level positively correlates to NKX3.1 expression. Also in this study, first time a reliable cell-based quantitation method for 8-OHdG damages is reported and used for data collection.
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Dano ao DNA/genética , Proteínas de Homeodomínio/genética , Estresse Oxidativo/genética , Neoplasias da Próstata/genética , Fatores de Transcrição/genética , Linhagem Celular Tumoral , Desoxiguanosina/genética , Genômica/métodos , Humanos , Masculino , Células PC-3 , Receptores Androgênicos/genéticaRESUMO
Limited data are available on the serum levels of different sphingomyelin (CerPCho) and ceramide (CER) species in sickle-cell disease (SCD). This study was aimed at identifying the levels of C16-C24 CerPCho and C16-C24 CER in serum obtained from SCD patients and controls. Circulating levels of neutral sphingomyelinase (N-SMase) activity, ceramide-1-phosphate (C1P), and sphingosine-1-phosphate (S1P) were also determined. Blood was collected from 35 hemoglobin (Hb)A volunteers and 45 homozygous HbSS patients. Serum levels of C16-C24 CerPCho and C16-C24 CER were determined by an optimized multiple reaction monitoring (MRM) method using ultrafast liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Serum activity of N-SMase was assayed by standard kit methods, and C1P and S1P levels were determined by enzyme-linked immunosorbent assay. A significant decrease was observed in the serum levels of C18-C24 CerPCho in patients with SCD compared to controls. No significant difference was found in C16 CerPCho levels between the two groups. Very-long-chain C22-C24 CER were significantly decreased in SCD, while long-chain C16-C20 CER levels showed no significant difference between SCD patients and controls. Significant positive correlation was found between the serum total cholesterol levels and C18-C24 CerPCho and C22-C24 CER in SCD patients. Patients with SCD had significantly elevated serum activity of N-SMase as well as increased circulating levels of C1P and S1P compared to controls. The decrease in serum levels of C18-C24 CerPCho in patients with SCD was accompanied by decreased levels of C22-C24 CER. Future studies are needed to understand the role of decreased CerPCho and CER in the pathophysiology of SCD.
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Anemia Falciforme/sangue , Ceramidas/sangue , Lisofosfolipídeos/sangue , Esfingomielina Fosfodiesterase/sangue , Esfingomielinas/sangue , Esfingosina/análogos & derivados , Adolescente , Estudos de Casos e Controles , Ceramidas/classificação , Criança , HDL-Colesterol/sangue , LDL-Colesterol/sangue , VLDL-Colesterol/sangue , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Esfingomielinas/classificação , Esfingosina/sangue , Espectrometria de Massas em Tandem , Triglicerídeos/sangueRESUMO
20-Hydroxyeicosatetraenoicacid (20-HETE) is an important mediator that regulates vascular tone and blood pressure (BP). Although various experimental animal hypertension models demonstrated that 20-HETE contributes to increased vascular resistance and BP, these effects have not been studied in Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced hypertension model. In this study, we investigated the effects of 20-HETE on the vascular responsiveness and BP in an L-NAME-induced hypertension. Wistar Albino rats were used in this study. Hypertension was induced by the addition of L-NAME to drinking water for 5 weeks. The study was performed in three stages: first, BP changes were monitored in real time in the presence of 20-HETE enzymatic inhibitor, N-hydroxy-N´-(4-butly-2-methylphenyl)-formamidine (HET-0016) for 1 h. Second, vascular responses of the conduit and resistance arteries were investigated in the presence or absence of HET-0016 in the organ bath. Third, BP was monitored weekly in some hypertensive animals treated with HET-0016 and vascular responses were investigated at the end of the experiment. We demonstrated an increase in 20-HETE levels in the resistance arteries of hypertensive animals. 20-HETE inhibition by HET-0016 significantly decreased BP in L-NAME-induced hypertension model. In addition, HET-0016 treatment caused significant improvement in vascular dilator and constrictor responses in the conduit and resistance arteries. This study demonstrates an important role of 20-HETE in increasing BP and altering vascular responsiveness in L-NAME-induced hypertension model, which suggests a possible involvement of 20-HETE in essential hypertension development in humans.
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Amidinas/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensão/fisiopatologia , Resistência Vascular/efeitos dos fármacos , Animais , Inibidores Enzimáticos/farmacologia , Hipertensão/induzido quimicamente , Masculino , NG-Nitroarginina Metil Éster , Ratos , Ratos Wistar , Técnicas de Cultura de Tecidos , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
BACKGROUND: The cardiovascular system is a primary target of stress and stress is the most important etiologic factor in cardiovascular diseases. Stressors increase expressions of atrial natriuretic peptide (ANP) and apelin in cardiac tissue. AIM: The aim of the present study was to investigate whether stress-induced apelin has an effect on the expression of ANP in the right atrium of rat heart. METHODS: The rats were divided into the control, stress and F13A+stress groups. In the stress and F13A+stress groups, the rats were subjected to water immersion and restraint stress (WIRS) for 6h. In the F13A+stress group, apelin receptor antagonist F13A, was injected intravenously immediately before application of WIRS. The plasma samples were obtained for the measurement of corticosterone and atrial natriuretic peptide. The atrial samples were used for immunohistochemistry and western blot analysis. RESULTS: F13A administration prevented the rise of plasma corticosterone and ANP levels induced by WIRS. While WIRS application increased the expressions of apelin, HIF-1α and ANP in atrial tissue, while F13A prevented the stress-induced increase in the expression of HIF-1α and ANP. CONCLUSION: Stress-induced apelin induces ANP expression in atrial tissue and may play a role in cardiovascular homeostasis by increasing ANP expression under WIRS conditions.
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Receptores de Apelina/metabolismo , Apelina/metabolismo , Fator Natriurético Atrial/biossíntese , Miocárdio/metabolismo , Estresse Psicológico/metabolismo , Animais , Homeostase/fisiologia , Ratos , Ratos WistarRESUMO
Previous studies have revealed the activation of neutral sphingomyelinase (N-SMase)/ceramide pathway in hepatic tissue following warm liver ischemia reperfusion (IR) injury. Excessive ceramide accumulation is known to potentiate apoptotic stimuli and a link between apoptosis and endoplasmic reticulum (ER) stress has been established in hepatic IR injury. Thus, this study determined the role of selective N-SMase inhibition on ER stress and apoptotic markers in a rat model of liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reactions monitoring (MRM) method using ultrafast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared with controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. A significant increase was observed in ER stress markers C/EBP-homologous protein (CHOP) and 78 kDa glucose-regulated protein (GRP78) in IR injury, which was not significantly altered by N-SMase inhibition. Inhibition of N-SMase caused a significant reduction in phospho-NF-kB levels, hepatic TUNEL staining, cytosolic cytochrome c, and caspase-3, -8, and -9 activities which were significantly increased in IR injury. Data herein confirm the role of ceramide in increased apoptotic cell death and highlight the protective effect of N-SMase inhibition in down-regulation of apoptotic stimuli responses occurring in hepatic IR injury.
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Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Fígado/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Esfingomielina Fosfodiesterase/genética , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/patologia , Caspases/biossíntese , Ceramidas/metabolismo , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glicoesfingolipídeos/metabolismo , Proteínas de Choque Térmico/biossíntese , Humanos , Fígado/lesões , Fígado/patologia , Ratos , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Fator de Transcrição CHOP/biossínteseRESUMO
Oxidative stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS)-2 have been shown in the pathogenesis of liver ischemia-reperfusion (IR) injury. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression therefore this study determined the role of selective N-SMase inhibition on nitrative and oxidative stress markers following liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Nitrative and oxidative stress markers were determined by evaluating NOS2 expression, protein nitration, nitrite/nitrate levels, 4-hydroxynonenal (HNE) formation, protein carbonyl levels and xanthine oxidase/xanthine dehydrogenase (XO/XDH) activity. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reaction monitoring method using ultra-fast liquid chromatography coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared to controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. NOS2 expression, nitrite/nitrate levels and protein nitration were significantly greater in IR injury and decreased with N-SMase inhibition. Treatment with a selective N-SMase inhibitor significantly decreased HNE formation, protein carbonyl levels and the hepatic conversion of XO. Data confirm the role of nitrative and oxidative injury in IR and highlight the protective effect of selective N-SMase inhibition. Future studies evaluating agents blocking N-SMase activity can facilitate the development of treatment strategies to alleviate oxidative injury in liver I/R injury.
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Fígado/enzimologia , Fígado/patologia , Traumatismo por Reperfusão/enzimologia , Esfingomielina Fosfodiesterase/metabolismo , Alanina Transaminase/sangue , Compostos de Anilina/uso terapêutico , Animais , Compostos de Benzilideno/uso terapêutico , Ceramidas/metabolismo , Dimetil Sulfóxido/uso terapêutico , Imuno-Histoquímica , Fígado/efeitos dos fármacos , Masculino , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Ratos Wistar , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia , Esfingomielina Fosfodiesterase/genética , Esfingomielinas/metabolismo , Espectrometria de Massas em TandemRESUMO
This study aimed to determine circulating levels of polyunsaturated fatty acids (PUFAs), secretory phospholipase A2 (sPLA2), lipoprotein lipase (LPL) and measure circulating protein levels of angiopoietin-like protein 3 (ANGPTL3), ANGPTL4, cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in patients with acne vulgaris. Serum from 21 control subjects and 31 acne vulgaris patients were evaluated for levels of arachidonic acid (AA, C20:4n- 6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3). PUFA levels were determined by an optimized multiple reaction monitoring (MRM) method using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods Serum EPA levels were significantly decreased while AA/EPA and DGLA/EPA ratio were significantly increased in acne vulgaris patients compared to controls. Serum levels of AA, DGLA and DHA showed no significant difference while activity of sPLA2 and LPL were significantly increased in acne vulgaris compared to controls. Results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly decreased serum EPA levels and increased activity of sPLA2, AA/EPA and DGLA/EPA ratio. Increased LPL activity in the serum of acne vulgaris patients can be protective through its anti-dyslipidemic actions. This is the first study reporting altered EPA levels and increased sPLA2 activity in acne vulgaris and supports the use of omega-3 fatty acids as adjuvant treatment for acne patients.
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Acne Vulgar/sangue , Ácido Eicosapentaenoico/sangue , Acne Vulgar/enzimologia , Adolescente , Adulto , Proteína 3 Semelhante a Angiopoietina , Proteína 4 Semelhante a Angiopoietina/sangue , Proteínas Semelhantes a Angiopoietina/sangue , Criança , Ciclo-Oxigenase 2/sangue , Dinoprostona/sangue , Feminino , Humanos , Inflamação/sangue , Lipase Lipoproteica/sangue , Masculino , Adulto JovemRESUMO
RATIONALE: Urinary liver fatty acid binding protein (L-FABP) has been evaluated as a promising early biomarker of renal ischemia in human kidney transplant patients. The use of L-FABP in clinical practice requires that this biomarker be associated with an analytical method that combines specificity, accuracy and robustness. This study aimed to evaluate an optimized multiple reaction monitoring (MRM) method using ultrafast liquid chromatography coupled with tandem mass spectrometry to measure urinary L-FABP levels in renal transplant recipients. METHODS: Purified recombinant human L-FABP tryptic standard was analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS/MS) and liquid chromatography (LC)/MS/MS to select for peptides that provided specificity and adequate response in developing an MRM method for urinary L-FABP quantification. Human urine samples collected from kidney transplant recipients were isolated, concentrated, precipitated and trypsin digested before mass spectrometric analysis of L-FABP. L-FABP levels were also measured in urine samples by enzyme immunoassay. RESULTS: The tryptic peptide ion MH(+) of (50) FTITAGSK(57) (m/z 824) provided an adequate signal and was used for quantification of L-FABP under conditions employed for LC/MS/MS analysis. MALDI-TOF-MS/MS spectra obtained by collision-induced dissociation of the parent MH(+) ion (50) FTITAGSK(57) resulted in a y3 product ion that was used for quantitative analysis by the MRM method. Urinary L-FABP content measured by both ELISA and LC/MS/MS after transplantation was significantly higher compared to before transplantation levels. The Spearman correlation coefficient between the two methods was statistically significant. Intra-day and inter-day coefficients of variation provided good repeatability and reproducibility for validation of LC/MS/MS analysis. CONCLUSIONS: LC/MS/MS quantification of L-FABP may provide a new reference method to determine changes in this potential biomarker in human kidney transplant patients.
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Proteínas de Ligação a Ácido Graxo/urina , Sequência de Aminoácidos , Cromatografia Líquida/métodos , Proteínas de Ligação a Ácido Graxo/análise , Feminino , Humanos , Nefropatias/urina , Transplante de Rim , Masculino , Peptídeos/análise , Peptídeos/urina , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Detection of 8-OHdG-base damage has been a big challenge for decades, though different analytical methods are developed. The recent approaches that are used for quantitating either the total amount of base damage or the amount of base damage per cell from different sources of samples are not automated. We have developed a method for automated damage detection from a single cell and applied it to 8-OHdG quantitation.
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Dano ao DNA/efeitos dos fármacos , Desoxiguanosina/análogos & derivados , Análise de Célula Única/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , 8-Hidroxi-2'-Desoxiguanosina , Dano ao DNA/genética , Reparo do DNA/genética , Desoxiguanosina/química , Desoxiguanosina/isolamento & purificação , Humanos , Estresse Oxidativo/genéticaRESUMO
Hydrogen sulfide (H2S) is produced enzymatically by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), as well as other enzymes in mammalian tissues. These discoveries have led to the crowning of H2S as yet another toxic gas that serves as a gasotransmitter like NO and CO. H2S is thought to exert its biological effects through its reaction with cysteine thiols in proteins, yielding sulfurated thiol (-SSH) derivatives. One of the first proteins shown to be modified by H2S was glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [1] where the S-sulfuration of the active site cysteine (Cys 152) resulted in ~7-fold increase in the activity of the enzyme. In the present study we have attempted to reproduce this result with no success. GAPDH in its reduced, or hydrogen peroxide, or glutathione disulfide, or nitrosonium oxidized forms was reacted with sulfide or polysulfides. Sulfide had no effect on reduced GAPDH activity, while polysulfides inhibited GAPDH to ~42% of control. S-sulfuration of GAPDH occurred at Cys 247 after sulfide treatment, Cys 156 and Cys 247 after polysulfide treatment. No evidence of S-sulfuration at active site Cys 152 was discovered. Both sulfide and polysulfide was able to restore the activity of glutathione disulfide oxidized GAPDH, but not to control untreated levels. Treatment of glutathione disulfide oxidized GAPDH with polysulfide also produced S-sulfuration of Cys 156. Treatment of a C156S mutant of GAPDH with sulfide and polysulfide resulted in S-sulfuration of Cys 152, which also caused a decrease and not an increase in enzymatic activity. Computational chemistry shows S-sulfuration of Cys 156 may affect the position of catalytic Cys 152, raising its pKa by 0.5, which may affect the nucleophilicity of Cys 152. The current study raises significant questions about the reported ability of H2S to activate GAPDH by the sulfuration of its active site thiol, and indicates that polysulfide is a stronger protein S-sulfurating agent than sulfide.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Ativação Enzimática , Gliceraldeído-3-Fosfato Desidrogenases/química , Humanos , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Técnicas In Vitro , Espectrometria de Massas , Modelos Moleculares , Oxirredução , Sulfetos/metabolismo , Sulfetos/farmacologiaRESUMO
The aim of the present study was to assess omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) in liver tissue and evaluate changes in the n6-associated inflammatory pathway following liver ischemia/reperfusion (IR) injury. Male Wistar rats which were allowed free access to standard rat chow were included in the study. Blood vessels supplying the median and left lateral hepatic lobes were occluded with an arterial clamp for 60 min, followed by 60 min of reperfusion. Levels of arachidonic acid (AA, C20:4n6), dihomogammalinolenic acid (DGLA, C20:3n6), eicosapentaenoic acid (EPA, C20:5n3) and docosahexaenoic acid (DHA, C22:6n3) in liver tissue were determined by an optimized multiple reaction monitoring method using ultra fastliquid chromatography coupled with tandem mass spectrometry. Phospholipase A2 (PLA2), cyclooxygenase (COX) and prostaglandin E2 (PGE2) were measured in tissue samples to evaluate changes in the n6 inflammatory pathway. Total histopathological score of cellular damage were significantly increased following hepatic IR injury. n3 and n6 PUFA levels were significantly increased in postischemic liver tissue compared to those in nonischemic controls. No significant difference was observed in the AA/DHA and AA/EPA ratio in postischemic liver tissues compared with that in the control. Tissue activity of PLA2 and COX as well as PGE2 levels were significantly increased in postischemic liver tissues compared to those in nonischemic controls. The results of the present study suggested that increased hydrolysis of fatty acids via PLA2 triggers the activity of COX and leads to increased PGE2 levels. Future studies evaluating agents which block the formation of eicosanoids derived from n6 PUFAs may facilitate the development and application of treatment strategies in liver injury following IR.
Assuntos
Ácidos Graxos Ômega-3/análise , Ácidos Graxos Insaturados/análise , Alanina Transaminase/sangue , Animais , Dinoprostona/análise , Isquemia/metabolismo , Isquemia/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
BACKGROUND: Sodium metabisulfite is commonly used as preservative in foods but can oxidize to sulfite radicals initiating molecular oxidation. Ghrelin is a peptide hormone primarily produced in the stomach and has anti-inflammatory effects in many organs. This study aimed to assess endogenous omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) in rat peripheral organs following sodium metabisulfite treatment and determine the possible effect of ghrelin on changes in n-6 inflammatory pathway. METHODS: Male Wistar rats included in the study were allowed free access to standard rat chow. Sodium metabisulfite was given by gastric gavage and ghrelin was administered intraperitoneally for 5 weeks. Levels of arachidonic acid (AA, C20:4n-6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) in liver, heart and kidney tissues were determined by an optimized multiple reaction monitoring (MRM) method using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Cyclooxygenase (COX) and prostaglandin E2 (PGE2) were measured in tissue samples to evaluate changes in n-6 inflammatory pathway. RESULTS: Omega-6 PUFA levels, AA/DHA and AA/EPA ratio were significantly increased in liver tissue following sodium metabisulfite treatment compared to controls. No significant change was observed in heart and kidney PUFA levels. Tissue activity of COX and PGE2 levels were also significantly increased in liver tissue of sodium metabisulfite treated rats compared to controls. Ghrelin treatment decreased n-6 PUFA levels and reduced COX and PGE2 levels in liver tissue of sodium metabisulfite treated rats. CONCLUSION: Current results suggest that ghrelin exerts anti-inflammatory action through modulation of n-6 PUFA levels in hepatic tissue.
Assuntos
Ácidos Graxos Ômega-6/biossíntese , Grelina/farmacologia , Inflamação/metabolismo , Fígado/efeitos dos fármacos , Sulfitos/farmacologia , Ácido 8,11,14-Eicosatrienoico/análise , Animais , Ácido Araquidônico/análise , Dinoprostona/análise , Ácidos Docosa-Hexaenoicos/análise , Ácido Eicosapentaenoico/análise , Ácidos Graxos Ômega-3/análise , Ácidos Graxos Ômega-3/biossíntese , Ácidos Graxos Ômega-6/análise , Rim/química , Fígado/metabolismo , Masculino , Miocárdio/química , Prostaglandina-Endoperóxido Sintases/análise , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização por Electrospray , Sulfitos/antagonistas & inibidoresRESUMO
The aim of this study was to determine circulating omega-6, omega-3 polyunsaturated fatty acids and prostaglandin E2 (PGE2) levels in steady state sickle cell disease (SCD) patients. Blood was collected from healthy hemoglobin volunteers and steady state homozygous HbSS patients who had not received blood transfusions in the last 3 months. Plasma levels of arachidonic acid (AA, C20:4n-6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) were determined by an optimized multiple reaction monitoring method using ultrafast liquid chromatography coupled with tandem mass spectrometry. PGE2 was measured in serum samples by enzyme immunoassay. Plasma AA and DGLA were significantly increased while EPA and DHA were significantly decreased in SCD plasma compared to control. Serum PGE2 levels, AA/DHA and AA/EPA ratio was significantly higher in SCD patients when compared to control group. The significant increase in PGE2 levels, AA/EPA and AA/DHA ratio confirms the presence of a proinflammatory state in SCD patients.
Assuntos
Anemia Falciforme/patologia , Dinoprostona/sangue , Ácidos Graxos Ômega-3/sangue , Ácidos Graxos Ômega-6/sangue , Plasma/química , Adolescente , Adulto , Criança , Pré-Escolar , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
BACKGROUND: This study aimed to determine early postoperative changes of plasma polyunsaturated fatty acids (PUFAs) following laparoscopic sleeve gastrectomy (LSG). METHODS: Ten obese patients (mean BMI: 51.10 ± 11.59 kg/m²) underwent LSG and eleven normal weight control patients (mean BMI: 24.37 ± 2.33 kg/m²) underwent laparoscopic abdominal surgery. Fasting blood samples were collected prior to surgery, at day 1 after surgery and after postoperation oral feeding. Plasma levels of arachidonic acid (AA, C20:4n6), dihomo-gamma-linolenic acid (DGLA, C20:3n6), eicosapentaenoic acid (EPA, C20:5n3) and docosahexaenoic acid (DHA, C22:6n3) were determined by an optimized multiple reaction monitoring (MRM) method using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Prostaglandin E2 (PGE2) was measured in serum samples by enzyme immunoassay. RESULTS: A significant decrease was observed in insulin and HOMA IR levels in sleeve gastrectomy patients after postoperation oral feeding compared to preoperation. Plasma AA levels and AA/EPA ratio were significantly increased in sleeve gastrectomy patients after postoperation oral feeding compared to postoperation day 1. Serum PGE2 levels and AA/DHA ratio was significantly higher in sleeve gastrectomy patients at preoperation, postoperation day 1 and after postoperation oral feeding when compared to control group patients. CONCLUSION: Increased peripheral insulin sensitivity associated with LSG may play a role in the significant increase of plasma AA levels in sleeve gastrectomy patients following postoperation oral feeding. The significant increase in PGE2 levels and AA/DHA ratio in sleeve gastrectomy group patients also confirms the presence of a proinflammatory state in obesity.
Assuntos
Ácidos Graxos Insaturados/sangue , Gastrectomia , Ácido 8,11,14-Eicosatrienoico/sangue , Ácido Araquidônico/sangue , Dinoprostona/sangue , Ácidos Docosa-Hexaenoicos/sangue , Ácido Eicosapentaenoico/sangue , Humanos , Insulina/sangue , Laparoscopia , Obesidade/sangue , Projetos Piloto , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
This study aimed to determine the role of selective neutral sphingomyelinase (N-SMase) inhibition on arachidonic acid (AA) mediated inflammation following liver ischemia-reperfusion (IR) injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Levels of AA in liver tissue were determined by multiple reaction monitoring (MRM) using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Phospholipase A2 (PLA2), cyclooxygenase (COX) and prostaglandin E2 (PGE2) were measured in liver tissue. Arachidonic acid levels, activity of PLA2, COX and PGE2 levels were significantly increased in postischemic liver tissue compared to nonischemic controls. N-SMase inhibition significantly decreased COX activity and PGE2 levels in postischemic liver. Future studies evaluating agents blocking N-SMase activity can facilitate the development of treatment strategies to alleviate inflammation in liver I/R injury.