Thasinas Dissayabutra

Long interspersed nuclear element-1 hypomethylation and oxidative stress: correlation and bladder cancer diagnostic potential.

Although, increased oxidative stress and hypomethylation of long interspersed nuclear element-1 (LINE-1) associate with bladder cancer (BCa) development, the relationship between these alterations is unknown. We evaluated the oxidative stress and hypomethylation of the LINE-1 in 61 BCa patients and 45 normal individuals. To measure the methylation levels and to differentiate the LINE-1 loci into hypermethylated, partially methylated and hypomethylated, peripheral blood cells, urinary exfoliated cells and cancerous tissues were evaluated by combined bisulfite restriction analysis PCR. The urinary total antioxidant status (TAS) and plasma protein carbonyl content were determined. The LINE-1 methylation levels and patterns, especially hypomethylated loci, in the blood and urine cells of the BCa patients were different from the levels and patterns in the healthy controls. The urinary TAS was decreased, whereas the plasma protein carbonyl content was increased in the BCa patients relative to the controls. A positive correlation between the methylation of LINE-1 in the blood-derived DNA and urinary TAS was found in both the BCa and control groups. The urinary hypomethylated LINE-1 loci and the plasma protein carbonyl content provided the best diagnostic potential for BCa prediction. Based on post-diagnostic samples, the combination test improved the diagnostic power to a sensitivity of 96% and a specificity of 96%. In conclusion, decreased LINE-1 methylation is associated with increased oxidative stress both in healthy and BCa subjects across the various tissue types, implying a dose-response association. Increases in the LINE-1 hypomethylation levels and the number of hypomethylated loci in both the blood- and urine-derived cells and increase in the oxidative stress were found in the BCa patients. The combination test of the urinary hypomethylated LINE-1 loci and the plasma protein carbonyl content may be useful for BCa screening and monitoring of treatment.

Oxidative Stress Induces Hypomethylation of LINE-1 and Hypermethylation of the RUNX3 Promoter in a Bladder Cancer Cell Line

Increased oxidative stress and changes in DNA methylation are frequently detected in bladder cancer patients. We previously demonstrated a relationship between increased oxidative stress and hypomethylation of the transposable long-interspersed nuclear element-1 (LINE-1). Promoter hypermethylation of a tumor suppressor gene, runt-related transcription factor 3 (RUNX3), may also be associated with bladder cancer genesis. In this study, we investigated changes of DNA methylation in LINE-1 and RUNX3 promoter in a bladder cancer cell (UM-UC-3) under oxidative stress conditions, stimulated by challenge with H2O2 for 72 h. Cells were pretreated with an antioxidant, tocopheryl acetate for 1 h to attenuate oxidative stress. Methylation levels of LINE-1 and RUNX3 promoter were measured by combined bisulfite restriction analysis PCR and methylation-specific PCR, respectively. Levels of LINE-1 methylation were significantly decreased in H2O2-treated cells, and reestablished after pretreated with tocopheryl acetate. Methylation of RUNX3 promoter was significantly increased in cells exposed to H2O2. In tocopheryl acetate pretreated cells, it was markedly decreased. In conclusion, hypomethylation of LINE-1 and hypermethylation of RUNX3 promoter in bladder cancer cell line was experimentally induced by reactive oxygen species (ROS). The present findings support the hypothesis that oxidative stress promotes urothelial cell carcinogenesis through modulation of DNA methylation. Our data also imply that mechanistic pathways of ROS-induced alteration of DNA methylation in a repetitive DNA element and a gene promoter might differ.

The Impact of Macro-and Micronutrients on Predicting Outcomes of Critically Ill Patients Requiring Continuous Renal Replacement Therapy

Critically ill patients with acute kidney injury (AKI) who receive renal replacement therapy (RRT) have very high mortality rate. During RRT, there are markedly loss of macro- and micronutrients which may cause malnutrition and result in impaired renal recovery and patient survival. We aimed to examine the predictive role of macro- and micronutrients on survival and renal outcomes in critically ill patients undergoing continuous RRT (CRRT). This prospective observational study enrolled critically ill patients requiring CRRT at Intensive Care Unit of King Chulalongkorn Memorial Hospital from November 2012 until November 2013. The serum, urine, and effluent fluid were serially collected on the first three days to calculate protein metabolism including dietary protein intake (DPI), nitrogen balance, and normalized protein catabolic rate (nPCR). Serum zinc, selenium, and copper were measured for micronutrients analysis on the first three days of CRRT. Survivor was defined as being alive on day 28 after initiation of CRRT.Dialysis status on day 28 was also determined. Of the 70 critically ill patients requiring CRRT, 27 patients (37.5%) survived on day 28. The DPI and serum albumin of survivors were significantly higher than non-survivors (0.8± 0.2 vs 0.5 ±0.3g/kg/day, p = 0.001, and 3.2±0.5 vs 2.9±0.5 g/dL, p = 0.03, respectively) while other markers were comparable. The DPI alone predicted patient survival with area under the curve (AUC) of 0.69. A combined clinical model predicted survival with AUC of 0.78. When adjusted for differences in albumin level, clinical severity score (APACHEII and SOFA score), and serum creatinine at initiation of CRRT, DPI still independently predicted survival (odds ratio 4.62, p = 0.009). The serum levels of micronutrients in both groups were comparable and unaltered following CRRT. Regarding renal outcome, patients in the dialysis independent group had higher serum albumin levels than the dialysis dependent group, p = 0.01. In conclusion, in critically ill patients requiring CRRT, DPI is a good predictor of patient survival while serum albumin is a good prognosticator of renal outcome.

Biochemical and clinical effects of Whey protein supplementation in Parkinson's disease: A pilot study

BACKGROUND Parkinsons disease (PD) is an oxidative stress-mediated degenerative disorder. Elevated plasma homocysteine (Hcy) is frequently found in the levodopa-treated PD patients, is associated with disease progression and is a marker of oxidative stress. Whey protein is a rich source of cysteine, and branched-chain amino acids (BCAA). It has been shown that supplementation with Whey protein increases glutathione synthesis and muscle strength. OBJECTIVES AND METHODS In this study, we conducted a placebo-controlled, double-blind study (NCT01662414) to investigate the effects of undenatured Whey protein isolate supplementation for 6months on plasma glutathione, plasma amino acids, and plasma Hcy in PD patients. Clinical outcome assessments included the unified Parkinsons disease rating scale (UPDRS) and striatal L-3,4-dihydroxy-6-(18)F-fluorophenylalanine (FDOPA) uptake were determined before and after supplementation. 15 patients received Whey protein, and 17 received Soy protein, served as a control group. RESULTS Significant increases in plasma concentration of reduced glutathione and the ratio of reduced to oxidized glutathione were found in the Whey-supplemented patients but not in a control group. This was associated with a significant decrease of plasma levels of Hcy. The plasma levels of total glutathione were not significantly changed in either group. Plasma BCAA and essential amino acids (EAA) were significantly increased in the Whey-supplemented group only. The UPDRS and striatal FDOPA uptake in PD patients were not significantly ameliorated in either group. However, significant negative correlation was observed between the UPDRS and plasma BCAA and EAA in the pre-supplemented PD patients. CONCLUSION This study is the first to report that Whey protein supplementation significantly increases plasma reduced glutathione, the reduced to oxidized glutathione ratio, BCAAs and EAAs in patients with PD, together with a concomitant significant reduction of plasma Hcy. However, there were no significant changes in clinical outcomes. Long-term, large randomized clinical studies are needed to explore the benefits of Whey protein supplementation in the management of PD patients.

Combination of vitamin E and vitamin C alleviates renal function in hyperoxaluric rats via antioxidant activity

Hyperoxaluria and oxidative stress are risk factors in calcium oxalate (CaOx) stone formation. Supplement with antioxidant could be effective in prevention of recurrent stone formation. The present study aims to evaluate the protective effects of vitamin E and vitamin C in hyperoxaluric rat. The experiment was performed in rats for 21 days. Rats were divided into 5 groups as follows: control (group 1, n=8), hyperoxaluric rats (group 2, n=8), hyperoxaluric rats with vitamin E supplement (group 3, n=7), hyperoxaluric rats with vitamin C supplement (group 4, n=7) and hyperoxaluric rats with vitamin E and C supplement (group 5, n=7). Hyperoxaluria was induced by feeding hydroxyl L-proline (HLP) 2% w/v dissolved in drinking water. Intraperitoneal 200 mg/kg of vitamin E was given in groups 3 and 5 on days 1, 6, 11 and 16, while 500 mg of vitamin C was injected intravenously in groups 4 and 5 on days 1 and 11. Renal functions and oxidative status were measured. The urinary oxalate excretion was increased in HLP supplement rats, while glomerular filtration rate, proximal water and sodium reabsorption were significantly lower in group 2 compared with a control (P<0.05). Giving antioxidants significantly lower urinary calcium oxalate crystals (P<0.05). Hyperoxaluric rats had higher plasma malondialdehyde (PMDA) and lower urinary total antioxidant status (UTAS), which were alleviated by vitamin E and/or vitamin C supplement. In conclusion, giving combination of vitamin E and vitamin C exerts a protective role against HLP-induced oxalate nephropathy.

Urinary stone risk factors in the descendants of patients with kidney stone disease

BackgroundEvidence has indicated that immediate family members of nephrolithiasis patients had high opportunity to develop stones. However, they are usually not regarded to be at risk, since it is unclear if there are any lithogenic abnormalities found in non-stone-forming nephrolithiasis relatives. Our aim was to investigate urinary metabolic abnormalities in the children of nephrolithiasis patients, compared with the general population.MethodsThe 24-h urinary metabolic profile was studied for 28 calcium oxalate nephrolithiasis patients (NL) and 46 of their descendants (ND), as well as 40 non-stone-forming volunteers (V) and 34 of their descendants (VD).ResultsThere was no difference between age, gender, and serum creatinine between NL vs. V (parental groups) and ND vs. VD (descendant groups). High urinary oxalate in nephrolithiasis and urinary calcium in their descendants was detected. In addition, an elevated urinary excretion rate of calcium, phosphate, protein, and albumin, along with low citrate excretion and high urinary supersaturation was observed in both the nephrolithiasis patients and their descendants. Approximate 17.8–24.4% of the nephrolithiasis descendants had a urinary supersaturation higher than the nephrolithiasis level, but none was found in VD group. The level of urinary supersaturation index was correlated with urinary protein and albumin excretion in nephrolithiasis family.ConclusionIt was demonstrated that nephrolithiasis offspring carried several urinary metabolic risks predisposing to stone formation which are similar to their parents, and about one in every five nephrolithiasis children had nephrolithiasis level urinary supersaturation.

Lime powder regimen supplement alleviates urinary metabolic abnormalities in urolithiasis patients: LPR prevent urolithiasis recurrence

Urolithiasis has high recurrent rate after surgical removal within 3 years. Potassium citrate compound is used to prevent stone recurrence but it has intolerable gastrointestinal adverse effects. We conducted a phase 2 clinical study of lime power regimen (LPR), a limeade‐based supplement containing potassium and citrate for 6 months period of treatment, to evaluate its effects on biochemical and clinical aspects of recurrent urolithiasis.

rs11567842 SNP in SLC13A2 gene associates with hypocitraturia in Thai patients with nephrolithiasis

Hypocitraturia is a profound risk for kidney stone formation and recurrence. Sodium-dicarboxylate cotransporter-1 (NaDC-1) is a main transporter responsible for citrate reabsorption in renal proximal tubules. To investigate an association of sodium-dicarboxylate cotransporter-1 (NaDC-1) polymorphism with hypocitraturia in Thai patients with nephrolithiasis (NL). Exonic SNPs in NaDC-1 were screened in peripheral blood DNA of 13 NL patients. The rs11567842 (A/G) variant was found and further genotyped in 145 NL patients and 115 non-stone forming controls. NL patients had significantly lower level of urinary citrate than the controls. Based on logistic regression, hypocitraturia was significantly associated with urinary stone formation (adjusted OR 8.34, 95% CI 4.63–15.04). Significant association of urinary citrate level with rs11567842 genotype was found only in the NL group. NL patients with GG genotype had significantly higher urinary citrate than those with AA and AG genotypes. GG carrying patients had significantly reduced risk for hypocitraturia (adjusted OR 0.15; 95% CI 0.05–0.48, AA as reference). In selected 15 calcium oxalate stone patients, AA carriers had significantly higher intrarenal NaDC-1 mRNA than GG and AG carriers. Homozygous GG of rs11567842 SNP in NaDC-1 gene was a protective genotype for hypocitraturia in kidney stone patients. The findings suggested that patients with AA genotypes were more susceptible to hypocitraturia than those with GG, hence carrying a higher risk for kidney stone recurrence.

Changes of Klotho protein and Klotho mRNA expression in a hydroxy-L-proline induced hyperoxaluric rat model

Klotho protein is recognized as having a renoprotective effect and is used as a biomarker for kidney injury. We investigated the level of Klotho protein in hyperoxaluria-induced kidney injury and the effects of vitamin E (Vit E) and vitamin C (Vit C) supplementation. Hyperoxaluria was induced by feeding 2% (w/v) Hydroxy-L-proline (HLP) in the drinking water for 21 days. Rats were divided into 5 groups; control (Group 1, n=7), HLP treated rats that received nothing else (Group 2, n=7), Vit E (Group 3, n=6), Vit C (Group 4, n=6) and both Vit E and Vit C (Group 5, n=7). Vit E (200 mg/kg) was injected on days 1, 6, 11 and 16, while Vit C (500 mg/kg) was given intravenously on days 1 and 11. The Klotho protein levels and oxidative status were measured. The expression level of kidney Klotho protein expression was significantly reduced by HLP-treatment, while the mRNA expression was higher (P<0.05), the plasma and kidney malondialdehyde and kidney superoxide dismutase activities were increased, and the kidney reduced glutathione and urinary total antioxidant status were decreased (P<0.05). All of these changes were ameliorated by administration of Vit E, Vit C or especially the co-administration of both. In conclusion, HLP-induced hyperoxaluria reduced the kidney Klotho protein level, which could be restored by Vit E and/or Vit C.