Refinement of a Methodology for Untargeted Detection of Serum Albumin Adducts in Human Populations.

Covalently modified blood proteins (e.g., serum albumin adducts) are increasingly being viewed as potential biomarkers via which the environmental causes of human diseases may be understood. The notion that some (perhaps many) modifications have yet to be discovered has led to the development of untargeted adductomics methods, which attempt to capture entire populations of adducts. One such method is fixed-step selected reaction monitoring (FS-SRM), which analyses distributions of serum albumin adducts via shifts in the mass of a tryptic peptide [Li et al. (2011) Mol. Cell. Proteomics 10, M110.004606]. Working on the basis that FS-SRM might be able to detect biological variation due to environmental factors, we aimed to scale the methodology for use in an epidemiological setting. Development of sample preparation methods led to a batch workflow with increased throughput and provision for quality control. Challenges posed by technical and biological variation were addressed in the processing and interpretation of the data. A pilot study of 20 smokers and 20 never-smokers provided evidence of an effect of smoking on levels of putative serum albumin adducts. Differences between smokers and never-smokers were most apparent in putative adducts with net gains in mass between 105 and 114 Da (relative to unmodified albumin). The findings suggest that our implementation of FS-SRM could be useful for studying other environmental factors with relevance to human health

Medial prefrontal cortex neurochemical metabolites in schizophrenia and schizoaffective disorder: A proton magnetic resonance spectroscopy study

Objectives: The aim of this study was to investigate medial prefrontal cortex neurochemical metabolite levels in patients with schizophrenia and schizoaffective disorder. Method: The subjects comprised 15 patients with schizophrenia (SCH), 15 patients with schizoaffective disorder (SAD), and 15 healthy controls. Levels of N-acetyl aspartate (NAA), choline-containing compounds (Cho), creatine-containing compounds (Cr), and myo-inositol (myo-I) were measured in the medial prefrontal cortex (mPFC) using 1H-MRS. Results: Differences were detected in NAA, Cho, Cr, and myo-I levels among the groups. In schizophrenic patients, the left and right mPFC NAA levels and left mPFC Cho level were found to be lower, compared to the control group. In patients with schizoaffective disorder, left and right mPFC NAA, Cho, Cr, and myo-I levels were found to be lower compared to the control group. No difference was detected in mPFC neurochemical metabolite levels between schizophrenia and schizoaffective disorder patients. Conclusion: In this stud were found that in schizophrenia and schizoaffective disorder, NAA and Cho levels are reduced. Consequently, in these two diseases, neuronal activity and neuronal integrity are impaired in the medial prefrontal region, and there are defects in membrane phospholipid metabolism. In addition, the decreases in Cr and myo-I levels in schizoaffective disorder patients point to abnormalities in energy metabolism and glial cells in the medial prefrontal region

Constitutive expression of bioactivating enzymes in normal human prostate suggests a capability to activate pro-carcinogens to DNA-damaging metabolites

BACKGROUND. The constitutive bioactivating capacity of human prostate may play a role in determining risk of adenocarcinoma developing in this tissue. Expression of candidate enzymes that convert exogenous and/or endogenous agents into reactive DNA-damaging species would suggest the potential to generate initiating events in prostate cancer (CaP). METHODS. Normal prostate tissues from UK-resident Caucasians (n = 10) were collected following either radical retropubic prostatectomy (RRP) or cystaprostatectomy (CyP). An analysis of gene and protein expression of candidate metabolizing enzymes, including cytochrome P450 (CYP) 1A1, CYP1A2, CYP1B1, N-acetyltransferase 1 (NAT1), sulfotransferase (SULT) 1A1, SULT1A3, NAD(P) H: quinone oxidoreductase (NQO1), prostaglandin H synthase 1 (cyclooxygenase 1; COX1), and CYP oxidoreductase (POR) was carried out. Quantitative real-time reverse transcriptase polymerase chain reaction, Western blot, and immunohistochemical analysis were conducted. RESULTS. Except for CYP1A1 and CYP1A2, the metabolizing enzymes examined appeared to be expressed with minimal inter-individual variation (in general, approximately two-to fivefold) in the expression levels. Enzymes such as CYP1B1 and NQO1 that are capable of bioactivating pro-carcinogens to reactive metabolites were readily identifiable in human prostate. Immunohistochemical analysis showed that although some expression is located in the stroma, the majority is localized to epithelial cells lining the glandular elements of the tissue; these are the cells from which CaP might arise. CONCLUSION. Constitutive expression of bioactivating enzymes confers the potential to convert a range of exogenous and/or endogenous agents to reactive species capable of inducing DNA damaging events. These findings suggest an organ capability for pro-carcinogen activation that could play an important role in the etiology of human CaP. Prostate 70: 15861599, 2010. (C) 2010 Wiley-Liss, Inc

Metabolic acidosis is common and associates with disease progression in children with chronic kidney disease

WOS: 000415760400028PubMed ID: 28729033Recent studies in adult chronic kidney disease (CKD) suggest that metabolic acidosis is associated with faster decline in estimated glomerular filtration rate (eGFR). Alkali therapies improve the course of kidney disease. Here we investigated the prevalence and determinants of abnormal serum bicarbonate values and whether metabolic acidosis may be deleterious to children with CKD. Associations between follow-up serum bicarbonate levels categorized as under 18, 18 to under 22, and 22 or more mmol/l and CKD outcomes in 704 children in the Cardiovascular Comorbidity in Children with CKD Study, a prospective cohort of pediatric patients with CKD stages 3-5, were studied. The eGFR and serum bicarbonate were measured every six months. At baseline, the median eGFR was 27 ml/min/1.73m(2) and median serum bicarbonate level 21 mmol/l. During a median follow-up of 3.3 years, the prevalence of metabolic acidosis (serum bicarbonate under 22 mmol/l) was 43%, 60%, and 45% in CKD stages 3, 4, and 5, respectively. In multivariable analysis, the presence of metabolic acidosis as a time-varying covariate was significantly associated with log serum parathyroid hormone through the entire follow-up, but no association with longitudinal growth was found. A total of 211 patients reached the composite endpoint (ESRD or 50% decline in eGFR). In a multivariable Cox model, children with time-varying serum bicarbonate under 18 mmol/l had a significantly higher risk of CKD progression compared to those with a serum bicarbonate of 22 or more mmol/l (adjusted hazard ratio 2.44; 95% confidence interval 1.43-4.15). Thus, metabolic acidosis is a common complication in pediatric patients with CKD and may be a risk factor for secondary hyperparathyroidism and kidney disease progression

Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories

This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp)

Inter-laboratory variation in DNA damage using a standard comet assay protocol

There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories