DNA Repair Biosensor-Identified DNA Damage Activities of Endophyte Extracts from Garcinia cowa.

Recent developments in chemotherapy focus on target-specific mechanisms, which occur only in cancer cells and minimize the effects on normal cells. DNA damage and repair pathways are a promising target in the treatment of cancer. In order to identify novel compounds targeting DNA repair pathways, two key proteins, 53BP1 and RAD54L, were tagged with fluorescent proteins as indicators for two major double strand break (DSB) repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). The engineered biosensor cells exhibited the same DNA repair properties as the wild type. The biosensor cells were further used to investigate the DNA repair activities of natural biological compounds. An extract from Phyllosticta sp., the endophyte isolated from the medicinal plant Garcinia cowa Roxb. ex Choisy, was tested. The results showed that the crude extract induced DSB, as demonstrated by the increase in the DNA DSB marker γH2AX. The damaged DNA appeared to be repaired through NHEJ, as the 53BP1 focus formation in the treated fraction was higher than in the control group. In conclusion, DNA repair-based biosensors are useful for the preliminary screening of crude extracts and biological compounds for the identification of potential targeted therapeutic drugs.

A double-blinded randomized controlled trial of silymarin for the prevention of antituberculosis drug-induced liver injury.

BACKGROUND: Hepatitis is a common adverse effect of antituberculosis drugs. Silymarin prevented drug-induced hepatoxicity in animals with anti-oxidative mechanisms but its effect in human has been unknown. We aimed to evaluate the efficacy of silymarin for preventing antituberculosis-drug induced liver injury (antiTB-DILI) in patients with tuberculosis. METHODS: A double-blind randomized placebo-controlled trial was performed. Tuberculosis patients were randomly allocated to receive placebo or silymarin. The outcomes of interests were antiTB-DILI and the maximum liver enzymes at week 4. Antioxidative enzymes (i.e., superoxide dismutase (SOD), glutathione and malondialdehyde assays) were assessed. The risks of antiTB-DILI between the two groups were compared. A number need to treat was estimated. RESULTS: A total of 55 out of 70 expected numbers of patients were enrolled. There were 1/27 (3.7%) and 9/28 (32.1%) patients who developed antiTB-DILI in the silymarin and the placebo groups. Risk reduction was 0.28 (0.10, 0.47), i.e., receiving silymarin was 28% at lower risk for antiTB-DILI than placebo. This led to prevention of 28 patients from being antiTB-DILI among 100 treated patients. Median (IQR) of ALT levels at week 4 in the placebo and the silymarin group were 35.0 (15, 415) IU/L and 31.5 (20, 184) IU/L (p = 0.455). The decline of SOD level at week 4 in the silymarin group was less than the placebo group (p < 0.027). CONCLUSIONS: Silymarin reduced the incidence of antiTB-DILI. The benefit of silymarin may be explained from superoxide dismutase restoration. Larger clinical trials are required to confirm the result of our small study [Clinicaltrials.Gov Identifier Nct01800487].

Effect of high dose ergocalciferol in chronic kidney disease patients with 25-hydroxyvitamin D deficiency.

OBJECTIVE: To evaluate 25 hydroxyvitamin D (25-OH-D) deficiency in a cohort ofpredialysis CKD patients and the treatment effect and safety of high dose ergocalciferol supplement in predialysis CKD. MATERIAL AND METHOD: Fifty-six predialysis CKD patients who came for a regular visit at a single hospital with calculated glomerular filtration rate < or =60 mL/min/1.73 m2 were screened for 25-OH-D levels. Forty-four patients with 25-OH-D deficiency were recruited into this prospective observational study that examined the effect of high dose oral ergocalciferol supplementation. After eight weeks, 37 patients completed the follow-up and biochemical parameters were reevaluated and analyzed. RESULTS: The mean 25-OH-D level of 56 patients was 25.6 +/- 8 ng/mL. Forty-four (78.5%) patients had 25-OH-D levels less than 30 ng/mL and four (7.1%) had severe deficiency with the level less than 15 ng/mL. High dose ergocalciferol supplement successively increased 25-OH-D levels in 35 (95%) patients. 25-OH-D levels increased significantly from 22 +/- 4.8 to 34.5 +/- 10.8 ng/mL after eight weeks (p < 0.001). During the study period, there were no changes in serum calcium, phosphate, and PTH. There was no other side effect associated with the treatment. CONCLUSION: 25-OH-D deficiency were found in this cohort of predialysis CKD patients. Ergocalciferol was a safe and effective supplement for the 25-OH-D in predialysis CKD.

Causes of hypocitraturia in recurrent calcium stone formers: focusing on urinary potassium excretion.

BACKGROUND: Multiple factors associated with hypocitraturia have been identified. However, limited studies addressing the causal relationship to hypocitraturia are available. We therefore conducted this study to determine factors associated with hypocitraturia and show their causal relationship in recurrent calcium stone formers. METHODS: Dietary review and 24-hour urine samples were obtained from all recurrent calcium stone formers referred for metabolic workup in the stone clinic. One month of oral potassium chloride supplementation was prescribed to stone formers to determine the causal relationship between urinary potassium and citrate levels. RESULTS: Eighty-three subjects, 44 men and 39 women, were recruited to participate in this study. Hypocitraturia (citrate < 300 mg/d [<1.43 mmol/d]) was found in 50.6% of subjects. Four independent urinary variables associated with hypocitraturia were identified, including potassium level, net gastrointestinal alkaline absorption, calcium level, and titratable acid. Urinary potassium level was the strongest predictor of urinary citrate level. Hypocitraturic subjects also had lower fruit intake compared with subjects with high urinary citrate levels. Potassium chloride supplementation to a subgroup of this population (n = 58) resulted in a significant increase in urinary citrate excretion (350.73 +/- 27.25 versus 304.15 +/- 30.00 mg/d [1.67 +/- 0.13 versus 1.45 +/- 0.14 mmol/d]; P < 0.02), but no alteration in fractional excretion of citrate (19.7% +/- 2.7% versus 23.1% +/- 2.4%; P > 0.05). CONCLUSION: Hypocitraturia was found to be a common risk factor associated with recurrent calcium stone formation and low urinary potassium level, low alkaline absorption, low urinary calcium level, and high titratable acid excretion. Hypocitraturia is predominantly of dietary origin. Estimation of fruit intake should be included in the metabolic evaluation for recurrent calcium stone formation.

Schedule of taking calcium supplement and the risk of nephrolithiasis.

BACKGROUND: Variation in the timing of calcium supplement may affect gastrointestinal absorption of both calcium and oxalate differently and may associate with variable risk of calcium oxalate nephrolithiasis. There are few human studies addressing specifically the appropriate time for taking calcium supplement. Therefore, this study was performed to compare calcium bioavailability and the risk of calcium oxalate stone formation for calcium supplement taken with meal vs. taken at bedtime. METHODS: Thirty-two healthy male navy privates, 22.7 +/- 1.9 years old (mean +/- SD), who had normal renal function (serum creatinine less than 150 umol/L) and no history of renal stone, participated in the study. The subjects were randomly allocated into two groups of 16 each. Group A took 1 g of calcium carbonate with meal, 3 times/day; and group B took 3 g/day of calcium carbonate at bedtime. After taking the regimens for 1 week, followed by 1 month of washout period, crossover between both groups was done. The diet was controlled throughout the study. Twenty-four-hour urine collections for the determination of urinary constituents were obtained at baseline and after taking both regimens of calcium supplement. Activity product for calcium oxalate was determined to assess the risk of calcium oxalate stone formation. RESULTS: Urinary excretions of calcium were significantly elevated above the baseline values when taking calcium supplement both with meal (3.48 +/- 2.13 mmol/day vs. 5.17 +/- 2.61 mmol/day, P < 0.05) and at bedtime (3.09 +/- 1.70 mmol/day vs. 5.08 +/- 2.34 mmol/day, P < 0.05). There was no difference between the two regimens in the urinary calcium excretions. The urinary oxalate was decreased significantly when the subjects took calcium supplement with meal compared with the corresponding baseline value (0.13 +/- 0.05 vs. 0.17 +/- 0.07 mmol/d, P= 0.01). In contrast, there was no alteration in urinary oxalate when calcium supplement was taken at bedtime compared to the baseline values (0.15 +/- 0.05 mmol/day vs. 0.15 +/- 0.06 mmol/day, P= 0.9). Compared with the corresponding baseline values, there was no significant increase in the activity product for calcium oxalate when taking calcium with meal (0.54 +/- 0.25 vs. 0.57 +/- 0.22, P= 0.54), but it was increased significantly when calcium supplement was taken at bedtime (0.47 +/- 0.21 vs. 0.72 +/- 0.27, P < 0.01). CONCLUSION: Calcium supplement should be taken with meal in order to avoid increasing the risk of calcium oxalate nephrolithiasis.

Effects of calcium supplements on the risk of renal stone formation in a population with low oxalate intake.

It has been speculated that calcium supplement in subjects with low oxalate intake might increase the risk of calcium stone formation due to an increase in calcium absorption without a significant reduction in oxalate absorption. There have been no human studies addressing specifically the effects of taking calcium supplements in populations whose dietary oxalate is low. This study was conducted to determine the effects of calcium supplements on the risk of calcium stone formation in a population with low oxalate intake. Thirty-two healthy male navy privates, 22.7 +/- 1.9 (mean +/- SD) years old, who had oxalate intake of less than 1 mmol/day, a serum creatinine of less than 150 micromol/l, and no history of renal stones, participated in the study. Dietary oxalate was controlled to be under 1 mmol/day throughout the study. Twenty-four hour urine collections for the determination of urinary constituents were obtained at baseline and after taking calcium supplements. Detection of calcium oxalate was performed to assess the risk of calcium oxalate stone formation. The urinary excretion of calcium was significantly elevated above baseline values while taking the calcium supplements (3.48 +/- 2.13 vs 5.17 +/- 2.61 mmol/d, p < 0.05) and urinary oxalate was significantly decreased when the subjects took calcium supplements compared to the corresponding baseline value (0.13 +/- 0.05 vs 0.17 +/- 0.07 mmol/d, p = 0.01). Urinary citrate was significantly elevated when the subjects took calcium supplements compared to the baseline (0.83 +/- 0.57 vs 0.64 +/- 0.39 mmol/d, p = 0.03). There was no significant alteration in the activity products of calcium oxalate while taking the calcium supplements (0.54 +/- 0.25 vs 0.57 +/- 0.22, p = 0.54). The effect of calcium supplements with meals, for the reduction of the risk of calcium stone formation, was unchanged, even in a population whose oxalate intake is rather low. Taking calcium supplements resulted in a reduction in urinary oxalates and an elevation in urinary citrates. Both alterations in urinary constituents counterbalanced the elevation in urinary calcium which resulted from the calcium supplements.

Risk of calcium oxalate nephrolithiasis in postmenopausal women supplemented with calcium or combined calcium and estrogen.

BACKGROUND: Recent studies showed that postmenopausal women lost less bone mass when supplemented with calcium or estrogen therapy. However, the safety of the treatments in terms of the risk of calcium oxalate stone formation is unknown. We therefore conducted this study to determine the alteration in calcium oxalate supersaturation after calcium supplement or after combined calcium and estrogen therapy in postmenopausal osteoporotic women. METHODS: Fifty-six postmenopausal women were enrolled in this study. All subjects were more than 10 years postmenopausal with vertebral or femoral osteoporosis by bone mineral density criteria. They were randomly allocated to receive either 625 mg of calcium carbonate (250 mg of elemental calcium) at the end of a meal three times a day (group A, n=26) or calcium carbonate in the same manner plus 0.625 mg/day of conjugated equine estrogen and 5 mg medrogestone acetate from day 1-12 each month (group B, n=30). The age (mean +/- S.E.M.) was 66.3 +/- 1.2 and 65.1 +/- 1.1 years, weight 54.1 +/- 1.2 and 55.3 +/- 2.1 kg, in group A and group B, respectively. Urine specimens (24-h) were collected at baseline and 3 months after treatment for the determination of calcium oxalate saturation by using Tiselius's index (AP(CaOx)) and calcium/citrate ratio. RESULTS: After 3 months of treatment, there was no significant alteration from baseline for urinary excretion of calcium, citrate and oxalate. Urinary phosphate excretion was significantly reduced (6.3 +/- 0.7 vs. 5.1 +/- 0.7 mmol/day for group A and 8.2 +/- 0.9 vs. 5.8 +/- 0.7 mmol/day for group B, P<0.05), whereas net alkaline absorption was significantly elevated (10.1 +/- 3.6 vs. 20.1 +/- 4.4 meq/day for group A and 4.8 +/- 3.2 vs. 19.9 +/- 3.6 meq/day for group B, P<0.05). Calcium/citrate ratio and AP(CaOx) determined at baseline were not different from the corresponding values after treatment in both groups; calcium/citrate: 10.1 +/- 3.1 vs. 10.1 +/- 2.5 for group A and 9.3 +/- 1.8 vs. 11.9 +/- 2.5 for group B and AP(CaOx): 1.1 +/- 0.1 vs. 1.3 +/- 0.2 for group A and 1.2 +/- 0.2 vs. 1.1 +/- 0.1 for group B. There were eight and nine patients with high AP(CaOx), or >2, at baseline and after treatment, respectively. CONCLUSIONS: Calcium supplement with a meal or combined calcium supplement and estrogen therapy is not associated with a significant increased risk of calcium oxalate stone formation in the majority of postmenopausal osteoporotic patients. Determination of urinary saturation for calcium oxalate after calcium and estrogen supplements, especially at the initial phase of treatment, may be helpful in the avoidance of nephrolithiasis.

Dosage of potassium citrate in the correction of urinary abnormalities in pediatric distal renal tubular acidosis patients.

Potassium citrate is an alkaline agent that has been recommended for the prevention of nephrolithiasis in distal renal tubular acidosis (RTA). Information on the effectiveness and the optimal dose of potassium citrate in the correction of urinary abnormalities in pediatric distal RTA is limited, however. We conducted this study to determine the effectiveness and the optimal dose of potassium citrate for the correction of urinary abnormalities and the prevention of nephrolithiasis in children with distal RTA. Eight pediatric distal RTA patients participated in this study. The mean +/- SEM age was 9.7 +/- 1.2 years, and mean body weight was 29.1 +/- 4.7 kg. After initial evaluation, all patients were treated with increasing dosages of potassium citrate starting from 2 mEq/kg/d in three divided doses. The dosage was increased progressively in a stepwise fashion every 2 months from 2 mEq/kg/d to 3 mEq/kg/d, then to 4 mEq/kg/d. Blood and 8-hour overnight urine samples were obtained at baseline and every 2 months before increasing the dosage of potassium citrate. Urinary saturations for calcium oxalate and calcium phosphate were estimated by using Tiselius's indices. The basal urinary calcium-to-creatinine, phosphate-to-creatinine, and calcium-to-citrate ratios and urinary saturation for calcium oxalate and calcium phosphate were elevated significantly, whereas citrate-to-creatinine ratio was reduced significantly in distal RTA patients. These ratios were normalized gradually with the increasing dosage of potassium citrate. All the aforementioned abnormalities were normalized only after the dosage of potassium citrate was raised to 4 mEq/kg/d. The elevation in urinary saturation of calcium phosphate could not be normalized throughout the study, however. These results suggest that 4 mEq/kg/d of potassium citrate supplement can correct successfully most of the urinary abnormalities and the elevated urinary saturation for calcium oxalate but not for calcium phosphate in children with distal RTA. Monitoring of urinary calcium-to-creatinine ratio or citrate-to-creatinine ratio is valuable to ensure adequate potassium citrate supplementation in this group of patients.