Overexpression

Overexpression merely of p53 decreases SIRT1 abundance and attenuates the effects of metformin on AMPK activation and cellular triglycerides. Overexpression of p53 decreased SIRT1 protein abundance and attenuated metformin-induced AMPK and ACC phosphorylation (Fig. 5, A and B). In addition, treatment with nutlin-3, which increased the abundance of p53 (Fig. 4C), reduced the transcription of SIRT1 as evidenced by reduced SIRT gene expression at both 12 and 16 h (Fig. 5C). Since both AMPK and SIRT1 have lipid-lowering effects in vitro and in vivo (8, 12, 22, 35, 60, 62), we hypothesized that p53 overexpression would attenuate the lipid-lowering effect of metformin. As shown in Fig. 6, the ability of metformin to inhibit triglyceride accumulation was blunted in HepG2 cells in which p53 was overexpressed.

Fig. 5. Overexpression of p53 decreases SIRT1 abundance and diminishes AMPK activation by metformin. Cells infected with adenovirus Ad-��-gal (control) or Ad-p53 for 24 h in 25 mM glucose, followed by 24 h incubation in 25 mM glucose with or without 2 … Fig. 6. Increasing p53 abundance attenuates the effect of metformin on cellular triglycerides. HepG2 cells infected with adenovirus Ad-��-gal (control) or Ad-p53 for 24 h in 25 mM glucose media, followed by 24 h incubation in 25 mM glucose with or without … Metformin effects are partially restored by the SIRT1 activator SRT2183 in cells overexpressing p53. We next tested whether SIRT1 activation in cells overexpressing p53 would restore the effects of metformin on AMPK activity and triglyceride accumulation.

Coincubation with the SIRT1 activator SRT2183 restored some, but not all, of the effects of metformin in these cells, as evidenced by their decreased triglyceride content and increased AMPK phosphorylation (Fig. 7). Although no effect on ACC phosphorylation was observed (Fig. 7A), these results support the notion that p53 overexpression attenuates the effects of metformin in part by decreasing SIRT1 activity. Fig. 7. The effects of metformin are partially restored by the SIRT1 activator SRT2183. A: representative Western blot demonstrating partial restoration of p-AMPK with SRT2183, with no apparent effect on Ac-H3 or Ac-p53. B: triglyceride content in cellular lysates … DISCUSSION In this study, we investigated the interactions between AMPK/SIRT1 signaling and p53 protein abundance in HepG2 cells under conditions of nutrient excess.

The results demonstrate that activation of AMPK and SIRT1 in response to metformin treatment decreases p53 abundance. They also reveal that metformin diminishes oxidative stress and enhances p53 deacetylation, likely contributing to the MDM2-mediated increase in p53 degradation. Conversely, we showed that overexpression of p53 diminishes SIRT1 abundance and prevents metformin-induced activation Anacetrapib of AMPK and reductions in triglyceride content.

The reasons for the failed specific T cell activation after

The reasons for the failed specific T cell activation after http://www.selleckchem.com/products/baricitinib-ly3009104.html the third and fourth vaccination are not clear. However, one may speculate that the vaccine currently has a suboptimal immunogenicity and therefore the primed responses are unable to permanently break the inhibition of the HCV-specific responses. Hence, repeated vaccination may therefore have no beneficial effects. Thus, similar studies using a vaccine with improved immunogenicity merits further investigation. In addition, it is also likely that the strong regulatory effects present in the chronic infection are difficult to overcome with the used vaccine alone. Furthermore, four vaccine doses with this vaccine alone were not sufficient to achieve sustained virological response by itself in patients with chronic HCV.

However, when combined with pegylated IFN and ribavirin treatment or in the future with DAAs,44 therapeutic vaccination possibly will have an influence on the response rates and clearance of HCV. In our small study, 75% of the vaccinated patients, all infected by HCV genotype 1, were cured with the subsequent SOC treatment, which is an encouraging result. However, with the development of highly effective HCV-targeted drugs, the role for a therapeutic vaccine is likely to be small. On the other hand, it is possible that some patient groups in need of alternative treatment options, may still benefit from therapeutic vaccines. Such groups could include patients resistant to DAAs, patients not eligible for DAA treatment, patients exposed to HCV repeatedly (e.g.

, injection drug users at risk of HCV re-infection), and patients in resource-poor environments. To be able to cure 100% of HCV-infected patients, it is not unlikely that successful HCV treatment for selected patients will consist of several DAAs used in combination with immune modulatory measures including therapeutic vaccines. In conclusion, the concept of therapeutic DNA vaccine delivered by in vivo EP to treat chronic HCV is safe and should be further investigated in combination with other compounds with different modes of action, and/or by further improving the immunogenicity. Materials and Methods Human subjects. This trial was approved by the Regional Ethical Council in Stockholm, Sweden and by the Swedish Medical Products Agency. The Declaration of Helsinki protocols were followed and all patients gave their written informed consent.

The trial has been registered at http://www.clinicaltrials.gov (http://clinicaltrials.gov/ct2/show/NCT00563173). Twelve patients with verified chronic HCV genotype 1 infections of a duration of >6 months were included in the study. Background data Entinostat of the patients, five females and seven males, with a mean age of 46 years (range 29�C60 years), have been given in Table 1. All were treatment naive and four had genotype 1b, seven had genotype 1a, and one had genotype 1 unspecified (Table 1).

mRNA expression levels presented were calculated relative to the

mRNA expression levels presented were calculated relative to the average of the housekeeping gene cyclophilin and further normalized to the relative expression levels of the respective Carfilzomib clinical controls. Bile collection and assessment of biliary excretion of bile acids, phospholipids, and cholesterol Bile was collected by cannulation of the gallbladder in mice anesthetized by intraperitoneal injection of hypnorm (fentanyl/fluanisone, 1 mg/kg) and diazepam (10 mg/kg). During the bile collection, body temperature was maintained using a humidified incubator. Bile collection was performed for 30 min, and secretion rates were determined gravimetrically. Biliary bile salt, cholesterol, and phospholipid concentrations were determined and the respective biliary excretion rates calculated as described previously (23, 24).

Fecal sterol analysis Mice were individually housed, and feces were collected over a period of 24 h and separated from the bedding. Fecal samples were dried, weighed, and thoroughly ground. Aliquots thereof were used for determination of neutral sterol and bile acid content by gas-liquid chromatography as described (23, 24). HDL kinetics studies HDL kinetics studies were performed essentially as published previously (21). Autologous HDL was prepared from pooled mouse plasma by sequential ultracentrifugation (density 1.063 < d < 1.21). After extensive dialysis against sterile PBS containing 0.01% EDTA, HDL was labeled with 125I-tyramine-cellobiose (TC) and cholesteryl hexadecyl ether (cholesteryl-1,2,-3H; Perkin Elmer Life Sciences) as previously described (25).

For kinetic studies, 0.4 ��Ci of 125I and 0.7 million dpm of the 3H tracer were injected into the tail veins of fasted wild-type mice treated with AdNull or AdhApoE3. Blood samples were drawn by retroorbital bleeding at 5 min and at 1, 3, 6, 11, and 24 h after injection. Plasma decay curves for both tracers were generated by dividing the plasma radioactivity at each time point by the radioactivity at the initial 5-min time point after tracer injection. Fractional catabolic rates (FCRs) were determined from the area under the plasma disappearance curves fitted to a bicompartmental model using the SAAM II program (26). The use of 3H-cholesteryl ether does not affect turnover rates in vivo compared with 3H-cholesteryl ester-labeled HDL (21).

Organ uptake of HDL apolipoproteins (125I) and HDL-CEs (3H-cholesteryl ether) was determined by measuring the counts recovered in each organ expressed as a percentage of the injected dose, which was calculated by multiplying the initial plasma counts (5-min time point) with the estimated plasma volume (3.5% of total body weight). Brefeldin_A Selective uptake into organs was determined by subtracting the percentage of the injected dose of 125I-HDL recovered in each organ from the percentage of the injected dose of 3H-HDL-CE. In vivo RCT studies In vivo RCT studies were performed essentially as published previously (27).

48 ��g/mL (1 61-3 65) vs 0 10 ��g/mL (0 10-0 36), P < 0 001] and

48 ��g/mL (1.61-3.65) vs 0.10 ��g/mL (0.10-0.36), P < 0.001] and POC [2.78 ��g/mL (2.05-5.37) Vismodegib FDA vs 0.38 ��g/mL (0.38-0.41), P < 0.001]. The area under the receiver operating characteristics curve for identifying an elevated PMN count was 0.977 (95%CI: 0.933 to 0.995) for ELISA and 0.982 (95%CI: 0.942 to 0.997) for POC (P = 0.246 vs ELISA). Using the optimal cut-off value for ELISA (0.63 ��g/mL), ascitic calprotectin had 94.8% sensitivity, 89.2% specificity, positive and negative likelihood ratios of 8.76 and 0.06 respectively, positive and negative predictive values of 60.0% and 99.0% respectively, and 90.0% overall accuracy. Using the optimal cut-off value for POC (0.51 ��g/mL), the respective values were 100.0%, 84.7%, 6.53, 0.00, 52.8%, 100% and 87.7%. Correlation between ELISA and POC was excellent (r = 0.

873, P < 0.001). The mean �� SD of the difference was -0.11 �� 0.48 ��g/mL with limits of agreement of + 0.8 ��g/mL (95%CI: 0.69 to 0.98) and -1.1 ��g/mL (95%CI: -1.19 to -0.91). CONCLUSION: Ascitic calprotectin reliably predicts PMN count > 250/��L, which may prove useful in the diagnosis of SBP, especially with a readily available bedside testing device. Keywords: Calprotectin, Ascites, Liver cirrhosis, Spontaneous bacterial peritonitis, Polymorphonuclear cells INTRODUCTION Liver cirrhosis is the clinical end-stage of different entities of chronic liver disease when patients suffer from substantial mortality and morbidity, both of which are positively correlated with disease severity[1,2].

Ascites is the most common complication, and around 60% of patients with compensated cirrhosis develop ascites within 10 years of disease onset[3]. Spontaneous bacterial peritonitis (SBP) is an important cause of morbidity and mortality in cirrhotic patients with ascites. SBP is estimated to affect 10%-30% of cirrhotic patients hospitalised with ascites, and mortality in this group approaches 30%[4,5]. Many of these patients are asymptomatic, and it is therefore recommended that all patients with ascites undergo paracentesis at the time of admission to confirm the SBP status[5]. Although SBP is less prevalent in an outpatient setting, it is reasonable to also evaluate the ascitic fluid of outpatients because of the high mortality associated with SBP. The diagnosis of SBP is based upon the polymorphonuclear (PMN) leukocyte cell count exceeding 250/��L in ascitic fluid[6,7].

Currently, differential cell count is usually performed by a manual method using light microscopy and counting chambers. However, Cilengitide the diagnosis is often delayed when laboratory personnel are not readily available or in the private practice setting where specimens are sent to an offsite laboratory. This is a major drawback, as rapid diagnosis of SBP and immediate initiation of antibiotic treatment is of paramount importance.

Indicative of its role in oligomerization, acetyl-CoA inhibits co

Indicative of its role in oligomerization, acetyl-CoA inhibits cold inactivation of Acot12 (24) and facilitates reactivation of the enzyme upon warming (17). Our finding especially that acetyl-CoA promotes Acot12 tetramer formation is in accordance with these observations. Although temperature per se did not influence oligomerization of Them1, its dimerization was effected by the presence of a fatty acyl-CoA. Collectively, these findings demonstrate that the substrates of Acot12 and Them1 mediate oligomerization. ATP promotes tetramerization of Acot12 (25) and its activation (18). Here we showed that it also induced dimerization and increased activity for Them1. Like Acot12 (18), ATP hydrolysis was not required for the activation of Them1, as evidenced by similar effects of ATP and ATP-��-S on oligomerization and enzymatic activity.

ADP induced Them1 to dimerize yet decreased its activity. This is in keeping with the observation that ADP promotes tetramerization of Acot12 while at the same inactivating the enzyme (26). The effects of ATP and ADP on Acot12 were attributed to the potential presence of a nucleotide binding site in proximity to the enzyme active site (26). Because nucleotide effects on Acot12 activity were consistent with simple competitive displacements, it was concluded that ATP and ADP more likely induce conformational changes that lead to different catalytic activities and substrate affinities. Consistent with this possibility for Them1, Lineweaver-Burk plots were indicative of a mixed mechanism of inhibition.

The mechanism by which CoASH inhibits Acot12 (18) and Them1 is not known but may be due to the presence of ADP within its molecular structure. With the exception of Acot12, medium- and long-chain acyl-CoAs are generally the preferred substrates of type II Acots (1), an observation that extends to the more recently characterized Them4 (22) and Them5/Acot15 (23). Although previously suggested to hydrolyze medium- and long-chain fatty acyl-CoAs (9), in the current study Them1 cleaved a range of substrates, albeit demonstrating a relative preference for medium- to long-chain fatty acyl-CoAs. The steady-state kinetic constants for fatty acyl-CoA hydrolysis for Them1 fall in similar ranges as those of Them2 (8), Them4 (22), and Them5 (23).

When characterized using a short- and a long-chain fatty acyl-CoA substrate, THEM1a and THEM1b yielded similar kinetic constants as Them1, suggesting that the additional Carfilzomib 13 aa at the C terminus of THEM1a (9) serves a function separate from the enzymatic activity of the protein. The intracellular concentrations of fatty acyl-CoAs remain an important unresolved issue in understanding the biologic functions of Acots. Total intracellular concentrations of long-chain fatty acyl-CoAs range from 5 ��M in neutrophils to 164 ��M in liver (27), with estimates of 30�C90 ��M in cytosol, 0.2�C3.1 mM in mitochondria, and 0.4 mM in peroxisomes (2).

Expression levels relative to HAd were determined using the ����C

Expression levels relative to HAd were determined using the ����Ct method. Histology and IHC Tissue sections (5 ��m) were cut from formalin fixed paraffin embedded (FFPE) blocks. A section for each specimen upon was stained with hematoxylin and eosin (H&E) to confirm the histological features. Unless stated otherwise, all incubations were done at room temperature. Sections were deparaffinized in xylene and hydrated in decreasing concentrations of ethanol. The tissue sections were then subjected to epitope retrieval by steaming in 10 mM sodium citrate buffer, pH 6.0. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol. Blocking of non-specific sites was achieved by incubation with 5% bovine serum albumin for one hour.

Specimens were then incubated overnight at 4��C with a 1:200 dilution of anti-CDO1 antibody (Sigma-Aldrich). The sections were incubated for one hour with a 1:1,000 dilution of biotinylated anti-rabbit IgG (Jackson ImmunoResearch) followed by a 30-minute incubation with a 1:1,000 dilution of HRP-conjugated streptavidin (Jackson ImmunoResearch). Immunocomplexes were visualized with 3,3-diaminobenzidine tetrahydrochloride (Covance, England). Incubations omitting the primary antibody were used as negative controls for each sample. Quantitation of CDO1 IHC Images from three to five fields of each CDO1 antibody-stained tumor specimen and of its matched control (no CDO1 antibody) were acquired using Nikon Eclipse E400 together with NIS Elements software. To minimize variation, identical exposure conditions were applied to each specimen and its corresponding control.

The CDO1 signal intensity was calculated by NIS Elements for each field of a given tumor sample. The CDO1 expression level was calculated by subtracting the average intensity of all fields of the control slide from the average intensity of all fields of the CDO1-stained slide. DNA extraction and analysis of promoter hypermethylation Genomic DNA was extracted from tumors and from hMSCs before and after each adipogenic induction cycle using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer��s protocol. The optional RNase A treatment was performed. The concentration and purity of DNA was determined by Nanodrop spectrophotometer. A total of 1 ��g of genomic DNA was bisulfite-converted and purified using the EZ Methylation-Direct Kit (Zymo Research).

In all, 9 ng of converted DNA was used as the template in a 25 ��L methylation-specific quantitative PCR reaction.27 Briefly, Cilengitide the reaction contained 1��TaqMan? Universal Master Mix (Life Technologies), 0.2 ��M fluorescent probe, and 1.2 pmol each primer (sequences in Supplementary Table 1 from Ref. 24). The thermal cycling protocol was as follows: 95��C (10 minutes) followed by 50 cycles of 95��C (15 seconds), 60��C (1 minute), and 72��C (15 seconds). Amplification of the unmethylated ��-actin promoter was used as the reference gene (sequences in Supplementary Table 1).

(a) ��You shouldn’t hurt or interfere with me, and I shouldn’t hu

(a) ��You shouldn’t hurt or interfere with me, and I shouldn’t hurt or interfere with you�� [17, page 148]. (b) ��You scratch my back and I scratch yours.�� (c) ��You help me today and I will help you tomorrow in return��.(2) Concrete Materialistic and Individualistic Perspective ��The contents of the exchange or the deal are often concrete or materialistic things selleck chemical such as money or food, or things which are perceived as good to serve one’s own needs or interests such as praise from authorities. The perspective of judgment is individualistic; self-interests precede group or others’ interests.It should be noted that things that are too general or abstract such as basic rights of human beings are seldom considered or valued in the exchange or deal.

(3) Ignoring Others’ Positive Claims and Welfare ��Since people at this stage are holding a concrete individualistic perspective, the positive claims or welfare of others are in general not their concern or responsibility unless such claims and welfare are part of the exchange or deal. In other words, ��one has a right to ignore the positive claims or welfare of others as long as one does not directly violate their freedom or injure them�� [17, page 215].4.3. Stage 3: Primary Group Affection and ConformityPrimary group refers to family, gang, group of friends or intimates, club, school, party, organization, company and so forth. Generally speaking, members of a primary group share common interests, philosophy, ideology, and in some cases properties.

Kohlberg’s [17, 22] Stage 3: ��Mutual Interpersonal Expectations, Relationships, and Interpersonal Conformity,�� and Chinese Cardinal Relationships and family affection are the major bases for elaborating the characteristics of this stage. 4.3.1. Altruism and Human Relationships The study of altruism in terms of kin selection and group selection in sociobiology is a good example of primary group altruism. Sociobiology, as defined by Wilson [24], is ��the systematic study of the biological basis of all social behavior�� [24, page 4]. Its central theoretical problem is ��how can altruism, which by definition reduced personal fitness, possibly evolve by natural selection�� [4, page 3]? The answer provided by sociobiologists is kinship. As mentioned before, the evolution of altruism involves mainly group selection and kin selection. People at this stage are willing to perform altruistic acts towards ingroup members at great sacrifice; however, they are much less willing to do so for outgroup members. The Chinese emphasize family integrity, group intimacy, and loyalty. Children are taught to be affective Dacomitinib and altruistic to their primary group members [25].

The existence dimension refers to an individual perception of his

The existence dimension refers to an individual perception of his or her life. Life has meaning under all circumstances. Frankl contended that our main motivation for living is the will to find a life meaning. We have the freedom to find meaning in what we do and in what we experience. The existence dimension of the purpose in life includes whether life best is perceived to be enthusiastic versus boring, exciting versus monotonous, or new versus unchanged. Youth researchers can use the scale to examine the concept of life meaning among early adolescents. A norm table can be designed to examine the trend of the purpose in life among early adolescents as well as for cross-cultural comparison. The EPIL provides an avenue for unique and down-to-earth application of measuring life purposes.

The existence domain of PIL is more relevant to adolescents. Other domains such as death and retirement are not entirely relevant to early adolescents [15, 24]. The EPIL provides a practical approach to measure early adolescents’ purposes in life.The current study has several limitations. First, the research findings are based on the perceptions of early adolescents in Hong Kong. There is a need to replicate the current study in adolescents with different ethnicities and contexts. Second, the respondents are from convenience sampling and not from sampling, although the sample size is large. The application of the findings to other adolescent populations should be interpreted with caution because of questionable generalizability. Third, items specifically related to early adolescents’ purposes in life are not included.

One example is the inclusion of the importance of the academic achievement, which is demonstrated to be one of the utmost concerns among adolescents [29]. Fourth, most existing studies adopt the PIL, which differs from the EPIL. Thus, EPIL scores cannot be compared with past PIL scores directly. Despite these limitations, the current study is the first to validate the EPIL for early adolescents. The measure can be used as outcome indicators in positive youth development programs in Chinese contexts. In fact, in the Project P.A.T.H.S., measures derived from the PIL were used to assess the existential well-being of Chinese adolescents in Hong Kong [30�C34].

Medicinal plant remedies are an integral part of the history and culture of people in developing countries where they are widely used to cover basic health care needs and their use is also becoming part of the integrative healthcare system of developed nations as complementary and alternative medicines [1]. Plant remedies are favoured as a cheaper Cilengitide and readily available alternative form of treatment and Borris [2] estimated that between 250000 and 500000 plant species exist on earth thereby representing a biologically and chemically diverse resource.

Inclusion criteria for controls were age between 40 and 65 years

Inclusion criteria for controls were age between 40 and 65 years and signing of the informed consent for study participation. Exclusion criteria for controls were the same as described above and the presence of ED.2.2. Clinical and Laboratory ParametersThe severity of ED was determined selleck chem Volasertib by application of the IIEF test. The weight, height, and abdominal circumference of the subjects were measured, and their body mass index (BMI, kg/m2) was calculated. Systolic and diastolic blood pressure (BP) was measured after a 5min rest and again 10min later; the mean value was recorded.

Serum triglycerides (mg/dL), HDL-C (mg/dL), LDL-C (mg/dL), total cholesterol (mg/dL), glycaemia (mg/dL), insulin (uUI/mL), glycated haemoglobin (%) albumin (mg/dL), SHBG (nmol/L), total testosterone (ng/mL), free testosterone (ng/mL), bioavailable testosterone (ng/mL), FSH (mUI/mL), LH (mUI/mL), prolactin (ng/mL), progesterone (ng/mL), oestradiol (pg/mL), D-dimer (mg/L), fibrinogen (mg/dL), erythrocyte sedimentation rate (ESR, mm/h), and C-reactive protein (CRP, mg/dL) were studied in samples drawn between 8 and 9 a.m. after a rest period of ��30min. The homeostasis model assessment of insulin resistance (HOMA-IR index, ��U/mg) was also calculated (fasting insulin �� fasting glucose/22.5).Data were also gathered on age, alcohol consumption (>40g/day), smoking (>5cigarettes/day), sedentarism (physical exercise <30min/day), and drug intake (antihypertensives, diuretics, hypocholesterolemics or oral antidiabetics). The prevalence of metabolic syndrome was calculated according to ATP-III criteria.

MS was defined by the presence of three of the following: abdominal circumference >102cm; hypertriglyceridaemia >150mg/dL, HDL-cholesterol <40mg/dL, blood pressure >130/85mmHg, or glycaemia >110mg/dL. Fasting blood samples were obtained between 8 and 9 a.m. after a rest period of ��30min by venipuncture of the patients’ large antecubital veins without stasis and after a 12-hour fasting period. 2.3. Statistical AnalysisUnpaired, two-sided Student’s t tests were applied to compare the mean values of quantitative variables, the Kolmogorov-Smirnov test to examine the normality of their distribution, and the Levene test to study the variance. The Mann-Whitney U test was used if the variables were not normally distributed. Qualitative variables were analysed with chi-square test or Fisher’s exact test if at least one cell had an expected count <5.

Correlations among variables were studied by using the Pearson coefficient or Spearman’s methods if the variables were nonnormally distributed. Binary logistic regression models (the Wald method) were used to obtain estimated adjusted ORs and their 95% confidence intervals (CIs) to measure the association between ED and metabolic syndrome AV-951 criteria.

Then, 5mL of the diluted sample was used to determine the arsenic

Then, 5mL of the diluted sample was used to determine the arsenic (V) and another 5mL aliquot of sample was treated with 1mL each of concentrated nitric acid and H2O2 for the determination http://www.selleckchem.com/products/Pazopanib-Hydrochloride.html of total arsenic [As(III) + As(V)].2.3.2. Soil Samples The soil samples were collected from the agricultural field and soil sludge samples from the pond bed where painted clay idols were immersed. Both samples were collected from the site and stored in polyethylene bags. The soil samples were air dried, and known weight (100g) of sample was placed in a 250mL beaker and extracted four times with 5mL portions of concentrated hydrochloric acid each time. The combined extract was boiled for about 30min, then the solution was cooled and diluted to 50mL with distilled water.

5mL aliquot of diluted sample was used for As(V) determination by the proposed method. Another aliquot of 5mL was treated with 1mL each of concentrated nitric acid and H2O2 solution to determine total arsenic.2.3.3. Vegetable Samples The spinach and tomato leaves were collected from local market. They were dried in sun light and grinded into fine powder. 100g of finely powdered and sieved sample was placed in a beaker. 10mL each of nitric acid and sulfuric acids were added and heated to 100��C for 20min, in fume hood. The solutions were cooled, treated with 10mL of perchloric acid, and heated again in fume hood for 5min, until the dense fumes of sulphur dioxide disappear completely. Then, solutions were cooled and 1mL of HCl was added to remove any heavy metal ions present in the sample.

The filtered solutions were diluted to 100mL using distilled water. Then, 5mL aliquots of diluted samples were used for the estimation of As(V) content as well as total arsenic after treating the sample aliquot with 1mL each of concentrated nitric acid and hydrogen peroxide.2.3.4. Biological Samples Urine Sample ��Urine samples were collected in sterilized glass containers from male individuals, and 10mL of sample was diluted to five times. The diluted samples were deproteinated by treating with 2mL of trichloroacetic acid (30%), and the residue has been removed by centrifugation. The filtrate was treated with 5mL each of concentrated nitric acid and H2O2 to oxidize any As(III) present to As(V) in the sample. Then, the solution was diluted to 100mL and fivemL aliquots of diluted samples were subjected for the analysis of arsenic content.Nail and Hair Samples ��Hair and Nail samples were collected from adults and washed thoroughly with distilled water followed by acetone and finally Batimastat dried in an oven at 100��C. About 0.2g of dried samples were placed in 250mL beakers separately and, 12mL of concentrated HNO3 followed by 2mL of HClO4 were added.