O(6)-Alkylguanine-DNA-alkyltransferase (ATase) is an important modulator of alkylating agent-induced toxicity and carcinogenicity, but those factors which influence the expression of this repair protein in human tissues are poorly characterised. In this study, we have determined ATase levels in macroscopically normal and tumour tissues from 76 individuals with benign or malignant colorectal disease. All tissue samples had detectable ATase activity, with values ranging from 35 to 451 fmol/mg protein. ATase activity in normal rectal tissue was significantly higher than that in normal tissue from the sigmoid colon (148 +/- 76 vs. 100 +/- 40 fmol/mg protein, p = 0.01), whereas ATase levels within different regions of the colon (proximal vs. sigmoid colon) were similar. In normal tissue, inter-individual variation in ATase activity was 4-fold in the colon and 6-fold in the rectum, whereas in tumour tissue the corresponding figures were approx. 13.0- and 7-fold, respectively. There was no detectable difference in normal tissue ATase activity between individuals with benign or malignant disease of the colon. Normal and tumour tissue ATase activities were strongly correlated in the sigmoid colon (r = 0.80) and rectum (r = 0.59) but not the caecum (r = -0.03). In a multivariate analysis, ATase activity in normal colon tissue increased with age (p = 0.01) and current smoking (p = 0.06), whereas tumour ATase activity increased only with use of anti-histamines (p = 0.05). In rectal tumour tissue, activity decreased with age (p = 0.05) and use of anti-muscarinic medications (p = 0.01): in normal rectal tissue, no modulating factors were identified.
Male and female BK-TO mice were infected with different numbers of Schistosoma mansoni cercariae under standard environmental conditions. Promutagenic methylation damage (O6-methyldeoxyguanosine; O6-MedG) was detected in liver DNA, but not in kidney, spleen or bladder DNA of infected animals. It was shown that levels of hepatic O6-MedG increased with increasing intensities of schistosomal infection. Possible mechanisms of action are discussed. These include the activating effects of schistosomes and their products on murine macrophages and subsequent endogenous formation of N-nitroso compounds by the activated macrophages.
Background: Current sampling for total homocysteine (tHcy) is problematic, requiring plasma separation within 15 min. The aim of this study was to develop a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the measurement of tHcy in plasma and dried blood spots and to determine whether the dried blood spot concentration could be used to predict plasma concentrations of tHcy. Methods: LC-MS/MS methodology was optimized to measure tHcy in plasma and dried blood spots. Fifty blood samples collected from heart transplant patients were used to form dried blood spots and for plasma analysis. Plasma tHcy was also measured using the Abbott IMx 1 method and values were compared to the tHcy concentrations determined in plasma and dried blood spots using LC-MS/MS methodology. Results: The plasma tHcy LC-MS/MS results compared well with the IMx values: LC-MS/MS=1·18(IMx)-0·44 ( r 2 =0·915). The within-batch precision ( n =10) of the plasma LC-MS/MS method was < 2·0% at 14·6 and 37·7 µmol/L, respectively; the between-batch precision ( n=10) was 5·0 and 8·0%, respectively, at these concentrations. The method was found to be sensitive down to 1 µmol/L and linear up to at least 100 µmol/L. Dried blood spot LC-MS/MS results were considerably lower than the plasma IMx values ( P < 0·0001): dried blood spot LC-MS/MS=0·33IMx+1·77 ( r 2 =0·682). The within-batch precision ( n=20) of the dried blood spot LC-MS/MS method was 7·3% and 4·7% at concentrations of 4·0 and 7·9 µmol/L, respectively; the between-batch precision was 12·6% and 7·9% at concentrations of 5·1 and 8·0 µmol/L, respectively. To assess whether dried blood spots are suitable as a screening test to predict plasma tHcy concentrations, arbitary cut-off levels were compared. If it is assumed that a plasma tHcy concentration of >15 µmol/L is raised, a dried blood spot result of >6·8 µmol/L has a sensitivity and specificity in detecting a raised plasma tHcy of 83·3% and 96·2%, respectively, and a positive and negative predictive value of 95% and 86%, respectively, with an efficiency of 90%. Use of a dried blood spot cut-off concentration of 6·2 µmol/L for predicting high plasma tHcy concentrations (above 15 µmol/L) has a sensitivity and specificity of 95·8% and 73·1%, respectively, positive and negative predictive values of 76% and 95%, respectively, and an efficiency of 84%. Conclusions: We have developed a precise and accurate LC-MS/MS method for measuring plasma tHcy concentrations, which uses a small volume of plasma and is suitable for routine use. A satisfactory LC-MS/MS method for the measurement of tHcy in dried blood spots was also developed; this method might be useful in routine screening for raised plasma concentrations of tHcy.
A rapid LC-MS/MS method for the analysis of the immunosuppressant cyclosporin A (CsA) in whole blood is presented. The method has good precision (intra-assay CV<5%, inter-assay CV<10%) and the accuracy is demonstrated by the analysis of UK NEQAS (n=27) and patient (n=180) samples. Calibration is linear to at least 5000µg/L and the superior characteristics of the assay will allow for the analysis of CsA at peak or trough concentrations in a routine environment.
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