Rapid determination of methylated purines in DNA treated with N-methyl-N-nitrosourea using high-performance liquid chromatography.

A method for determining methylated purine bases in [3H]N-methyl-N-nitrosourea (MeNOUr) treated DNA is described. The method combines reversed-phase high performance liquid chromatography (HPLC) of methylated DNA after hydrolysis in dilute acid with the determination of radioactivity in the fractionated eluates. The peaks of the respective methylated purines were indentified by the use of internal standards. The method allows quantitative separation of 3-methyl-adenine (m3Ade), 7-methyl-adenine (m7Ade), 3-methyl-guanine (m3Gua), 7-methyl-guanine (m7Gua) and O6-methyl-guanine (m6Gua) within 20 min. Thus the total time required for determination of methylated purines is limited only by radioactivity measurements in the respective fractions.     

32P-postlabeling analysis of DNA adducts from non-alternant PAH using thin-layer and high performance liquid chromatography

DNA adduct formation in vivo in mouse skin following a single topical application of benzo[a]fluoranthene (BbF), benzo[j]fluoranthene (BjF), benzo[k]fluoranthene (BkF), or indeno[1,2,3-cd]pyrene (IP) was investigated in female CD-1 mice using 32P-postlabeling analysis. Distinct adduct profiles were detected for each of the non-alternant hydrocarbons examined. Two adducts, one major and one minor, were detected using polyethyleneiminecellulose (PEI-cellulose) thin-layer chromatography (TLC) for BbF and BjF while a single major adduct was detected for BkF and IP. The relative extent of binding to mouse skin DNA was in the order BbF greater than BjF greater than BkF greater than IP. 32P-Postlabeled DNA adducts separated by PEI-cellulose TLC were further analyzed by high performance liquid chromatography (HPLC). A single radioactive peak was detected for 32P-labeled DNA adducts of BjF and BkF. Three general areas of radioactivity were detected when 32P-labeled DNA adducts of BbF were separated on HPLC.     

Characteristics of the TEA detector in use with high pressure liquid chromatography

It was found that the response of the TEA detector was highly dependent on the operating temperature of the furnace and on the flow rate of gas that is used to purge the furnace. It was also found that the response of the detector was very highly dependent on the volatility of the nitroso compound and the optimum operating conditions were also very different for low and high volatility compounds. When samples containing equal quantities of NDMA, NPYR, NPRO and NPIC were chromatographed at low furnace temperatures, only the peaks for NPRO and NPIC (low volatility) were observed. When a high furnace temperature was used, only NDMA and NPYR (high volatility) were routinely observed. At intermediate temperatures, all four compounds could usually be observed. Under optimum conditions, the sensitivity was 3 microgram/ml for NPRO and 5 microgram/ml for NPIC.     

32P-postlabelling of 2-hydroxyethylated, ethylated and methylated adducts of 2'-deoxyguanosine 3'-monophosphate

Ethylene oxide, diethyl sulphate and dimethyl sulphate were used to synthesize the corresponding 7-alkylation products of 2'-deoxyguanosine 3'-monophosphate (dGMP). The purified adducts were used as substrates in the 32P-post-labelling reaction with T4 polynucleotide kinase. The kinetics of phosphorylation were studied with 7-(2-hydroxyethyl)-dGMP: most net product was formed by 15 min and only a small increase was seen until 4 h. When different concentrations of the adducts were tested, a complete phosphorylation was noted for 7-methyl-dGMP to the lowest tested amount of 1 fmol. The efficiencies of phosphorylation for 7-ethyl- and 7-hydroxyethyl-dGMP were 1.5 and 0.5% respectively. The proportions phosphorylated were uniform over the concentration range tested. The results demonstrate dramatic differences in the efficiency of phosphorylation between structural analogues, which is probably related to a decreased affinity of the substrate to the enzyme or to an interference in the transfer of the phosphate group on the active site of the enzyme.     

Novel method for DNA methylation analysis using high‐performance liquid chromatography and its clinical application

The aim of this study was to develop a new methodology that is suitable for DNA methylation diagnostics and to demonstrate its clinical applicability. We developed a new anion‐exchange column for high‐performance liquid chromatography (HPLC) with electrostatic and hydrophobic properties. Both cytosine and thymine, corresponding to methylated and unmethylated cytosine after bisulfite modification, respectively, are captured by electrostatic interaction and then discriminated from each other by their hydrophobic interactions. The DNA methylation levels of synthetic DNA were quantified accurately and reproducibly within 10 minutes without time‐consuming pretreatment of PCR products, and the measured values were unaffected by the distribution of methylated CpG within the synthetic DNA fragments. When the DNA methylation status of the FAM150A gene, a marker of the CpG island methylator phenotype specific to clear cell renal cell carcinoma (ccRCC), was examined in 98 patients with ccRCC, bulk specimens of tumorous tissue including cancer cells showing DNA methylation of the FAM150A gene were easily identifiable by simply viewing the differentiated chromatograms, even when the cancer cell content was low. Sixteen ccRCC showing DNA methylation more frequently exhibited clinicopathological parameters reflecting tumor aggressiveness (ie, a larger diameter, higher histological grade, vascular involvement, renal vein tumor thrombi, infiltrating growth, tumor necrosis, renal pelvis invasion and higher pathological TNM stage), and had significantly lower recurrence‐free and overall survival rates. These data indicate that HPLC analysis using this newly developed anion‐exchange column could be a powerful tool for DNA methylation diagnostics, including prognostication of patients with cancers, in a clinical setting.     

Plasma concentrations of carminomycin and carminomycinol in man,measured by high pressure liquid chromatography

In 9 patients with advanced malignant disease who received carminomycin (CMM) in an i.v. bolus injection (dose 18 mg/m²), curves of plasma concentrations of CMM and carminomycinol (CMMOH), a metabolite, versus time were constructed. For determination of plasma concentrations, high pressure liquid chromatography was used. For CMM and CMMOH the median areas under the curves (AUC's) were 31 (range 4–57) × 10⁻⁸mol/l/hr (measured over 24 hr) and 100 (range 30–158) × 10⁻⁸mol/l/hr (measured over 48 hr) respectively. From the data an accumulation of CMMOH in patients receiving treatments separated by brief intervals can be predicted (half-life time of plasma disappearance for CMMOH was 2 days). Clinical toxicity was lowest in those 3 patients showing the lowest AUC for both CMM and CMMOH.     

368例支气管镜刷检液基细胞学与细胞DNA定量分析的研究

目的:对比支气管镜刷检细胞DNA定量分析与液基细胞学诊断结果，探讨细胞DNA定量分析在支气管镜刷检中的应用价值。方法: 选取同时进行细胞DNA定量分析与液基细胞学诊断的368例患者细胞标本和细胞片，将其检测结果与病理活检进行对比。结果: 细胞DNA定量分析在支气管镜刷检诊断中的灵敏度、特异度分别为90.8%(177/195)和79.8%(138/173)，液基细胞学在支气管镜刷检诊断中的灵敏度、特异度分别为68.1%(124/182)和63.4%(118/186)。细胞DNA定量检测的灵敏度、特异度均高于液基细胞学，二者比较差异显著（P<0.01）。细胞DNA定量分析与病理活检阳性诊断符合率为83.5%。结论:细胞DNA定量分析在支气管镜刷检中有很好的应用价值，同时联合液基细胞学检查能提高恶性肿瘤的检出率，降低漏诊的风险。     

Measurement of incorporation of bromodeoxyuridine into DNA by high performance liquid chromatography using a novel fluorescent labelling technique.

5'-Bromo-2-deoxyuridine (BUdR) is a halogenated pyrimidine analogue that is an efficient radiosensitizer through its incorporation into DNA in place of thymidine. Radiosensitization is proportional to percentage replacement and we present here a novel derivatization technique that specifically labels the thymidine and BUdR with 4-bromomethyl-7-methoxycoumarin (BrMMC) to give the highly fluorescent coumarin derivatives which are quantitated using high performance liquid chromatography (HPLC). This allows for a simple single-stage DNA hydrolysis and sensitive peak detection. Data are presented showing the incorporation with time of BUdR into the DNA of Chinese hamster V79 cells. Attention is also drawn to the care needed in the selection of enzymes required for DNA digestion.     

Investigation of DNA damage response and apoptotic gene methylation pattern in sporadic breast tumors using high throughput quantitative DNA methylation analysis technology

Background-  

Sporadic breast cancer like many other cancers is proposed to be a manifestation of abnormal genetic and epigenetic changes. For the past decade our laboratory has identified genes involved in DNA damage response (DDR), apoptosis and immunesurvelliance pathways to influence sporadic breast cancer risk in north Indian population. Further to enhance our knowledge at the epigenetic level, we performed DNA methylation study involving 17 gene promoter regions belonging to DNA damage response (DDR) and death receptor apoptotic pathway in 162 paired normal and cancerous breast tissues from 81 sporadic breast cancer patients, using a high throughput quantitative DNA methylation analysis technology.     

A high pressure liquid chromatography study on the removal of DNA-bound aflatoxin B1 in rat liver and in vitro

Following intraperitoneal administration of [3H] aflatoxin B1 (AFB1) to young adult male rats, there is rapid uptake of the carcinogen by the liver, the target organ for carcinogenesis, leading to DNA covalent binding. Acid hydrolysis of this DNA shows that after 2h, the major DNA adduct is trans 8,9-dihydro-8-(7-guanyl)-9-hydroxy AFB1 (AFB1-gua). By 24h after AFB1 administration the major DNA adduct is no longer AFB1-gua but a product with the identical retention time on h.p.l.c. to 8,9-dihydro-8-(N5-formyl-2',5',6' triamino-4' oxo-N5-pyrimidyl)-9-hydroxy AFB1 (AFB1-triamino-Py). 48h after carcinogen administration, only a small amount of AFB1-gua remains and the major product is AFB1-triamino-Py. The half-life of removal of AFB1-gua is 22h, while AFB1-triamino-Py is much more persistent. In vitro incubation studies on DNA isolated from rats treated 2h previously with [3H] AFB1 show that at pH 7.4 AFB1-gua is the major product released from the DNA with some release of 8,9-dihydro-8,9-dihydroxy AFB1, (AFB1-diol). If more extensively reacted AFB1-DNA is used than that obtained from in vivo administration, then the rate of AFB1-diol release is enhanced while that of AFB1-gua is reduced. It would appear, therefore, that much of the release of AFB1 from DNA in vivo within the first 24h is probably not through a DNA repair process but through chemical release arising from the positively charged N7-guanine. There is considerable conversion of AFB1-gua to AFB1-triamino-Py on in vitro incubation of DNA as well as AFB1-gua and AFB1-diol release. By 24h approximately 66% of the bound AFB1 is in the form of AFB1-triamino-Py and after 48h the conversion is complete. The complex pattern of AFB1-release from DNA may have important consequences in both the induction of mutations and in tumour initiation.     

高效液相色谱法同时测定水源水中三种微囊藻毒素

目的 建立一种同时测定水源水中三种微囊藻毒素（MC-LR、MC-RR、MC-YR）的高效液相色谱（high performance liquid chromatography,HPLC）方法。 方法 取500 mL水样,0.45 μm滤膜过滤,滤液过C18柱,依次用10 mL超纯水、20 mL 20%甲醇溶液淋洗,15 mL 80% 甲醇溶液（加入0.05%三氟乙酸）洗脱,蒸发浓缩吹干,溶于1 mL超纯水,待测。采用固相萃取柱,流动相为0.1%磷酸水溶液-乙腈（67∶33）,流速1.0  mL/min,柱温45℃,在238 nm波长紫外线检测器检测。结果 MC-LR、MC-RR和MC-YR 在 0.05~1.00 μg/mL范围内线性良好（r=0.9987、0.9992和0.9997）,且峰形较好;回收率均在80%~110%,相对标准偏差（relative standard deviation,RSD）为5.0%~9.6%;MC-LR、MC-RR和MC-YR的方法检出限（limit of detection,LOD）分别为0.028 μg/L、0.037 μg/L和0.056 μg/L。结论 本方法准确度、灵敏度、回收率高,重现性好,可用于水源水中MC-LR、MC-RR、MC-YR同时检测。     

A comparative study of normal and reverse phase high pressure liquid chromatography for analysis of porphyrins accumulated after 5-aminolaevulinic acid treatment of colon adenocarcinoma cells

Primary adenocarcinoma cells of the rectosigmoid colon (WiDr-cells) were treated with 5-aminolevulinic acid (5-ALA). Cellular porphyrins were separated and quantified by high performance liquid chromatography (HPLC), both as free porphyrin acids after an easy extraction method with a subsequent reverse phase technique, and then as porphyrin esters after a more laborious extraction method and subsequent normal phase technique. The porphyrins were detected by means of a fluorescence detector. Analysis by normal phase HPLC indicated that 81% (739 pmol/mg protein) of the total amounts of fluorescing porphyrins accumulated was protoporphyrin IX, while similar analysis by reverse phase HPLC indicated that PpIX constituted 91% (622 pmol/mg protein) of the accumulated porphyrins. In addition to protoporhyrin IX, copro-, hexa-, hepta- and uroporphyrins were observed in extracts from 5-ALA-treated cells by both methods. The discrepancy between the two methods increased with increasing hydrophilicity of the analysed porphyrins, with uroporphyrin estimated to be 6-fold higher (63 vs. 10 pmol/mg protein) by normal than by reverse phase HPLC.     

胃癌DNA甲基化差异片段的筛选与分析

目的筛选和分析胃癌和正常胃组织间DNA甲基化差异片段。方法通过甲基化敏感性代表性差异分析法(methylation-sensitive-representationaldifferenceanalysis,MS-RDA),筛选胃癌和正常胃组织间DNA甲基化差异片段,经克隆、测序后进行生物信息学分析。结果获得3个甲基化差异片段CRS1308、CRS1309、CRS1310序列。其中CRS1309和CRS1310已被GenBank收录,登陆号分别为AY887106和AY887107。CRS1309序列与LOC440683基因、LOC440887基因的3'端、DRD5基因均有很高的相似性;CRS1310序列与1999年MinoruToyota在人类结肠癌中分离出的核糖体RNA上的甲基化差异性CpG岛有很高的相似性。结论胃癌和正常胃组织间DNA甲基化存在差异,LOC440683、LOC440887、DRD5基因的异常甲基化可能与胃癌的发生相关,胃癌中CRS1310序列所在核糖体RNA上的CpG岛易发生甲基化。     

Large-scale quantitative clinical proteomics by label-free liquid chromatography and mass spectrometry

We previously reported the development of an integrated proteome platform, namely 2-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL), for quantitative comparison of large peptide datasets generated by nano-flow liquid chromatography (LC) and mass spectrometry (MS). The key technology of 2DICAL was the precise adjustment of the retention time of LC by dynamic programming. In order to apply 2DICAL to clinical studies that require comparison of a large number of patient samples we further refined the calculation algorithm and increased the accuracy and speed of the peptide peak alignment using a greedy algorithm, which had been used for fast DNA sequence alignment. The peptide peaks of each sample with the same m/z were extracted every 1 m/z and displayed with along the horizontal axis. Here we report a precise comparison of more than 150 000 typtic peptide ion peaks derived from 70 serum samples (40 patients with uterine endometrial cancer and 30 controls). The levels of 49 MS peaks were found to differ significantly between cancer patients and controls ( P <  0.01, Welch's t -test and interquartile range [IQR] of >40), and the differential expression and identification of selected three proteins was validated by immunoblotting. 2DICAL was is highly advantageous for large-scale clinical proteomics because of its simple procedure, high throughput, and quantification accuracy. ( Cancer Sci 2009; 100: 514–519)     

Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis

This report describes the development and validation of quantitative microsatellite analysis (QuMA) for rapid measurement of relative DNA sequence copy number. In QuMA, the copy number of a test locus relative to a pooled reference is assessed using quantitative, real-time PCR amplification of loci carrying simple sequence repeats. Use of simple sequence repeats is advantageous because of the large numbers that are mapped precisely. In addition, all markers are informative because QuMA does not require that they be polymorphic. The utility of QuMA is demonstrated in assessment of the extent of deletions of chromosome 2 in leukemias arising in radiation-sensitive inbred SJL mice and in analysis of the association of increased copy number of the putative oncogene ZNF217 with reduced survival duration in ovarian cancer patients.     

Plasma pharmacokinetics of morphine and morphine-6-glucuronide using high performance liquid chromatography and colorimetric detection]

This pilot study was undertaken in a group of 7 patients receiving morphine either by oral route under a controlled release form (Moscontin tablets), or by subcutaneous route with continuous infusion. Complete pharmacokinetics over 24 h were carried out with blood samples taken every hour. The measurements of morphine and of its metabolite, morphine-6-glucuronide (M6G) were performed by high-performance liquid chromatography (HPLC) with coulometric detection, using nalorphine (NAL) as an internal standard. The morphinics were extracted on a Bond Elut C18 column according to a double liquid-solid extraction. The extract was chromatographed by ion-pairing on a mu Bondapak C18 column, 10 microns (300 x 3.9 mm). The minimal detectable concentrations were respectively 1 and 5 ng/ml for M and M6G. When Moscontin was given at dosages < 1 mg/kg/d, the areas under the curve over 24 h (AUC 24 h) of M were rather close to those of M6G (ratio 1.1 +/- 0.1). However, with dosages > 1 mg/kg/d, a difference appeared and gradually rose to a M/M6G ratio of 1.3 +/- 0.04. With subcutaneous infusion, the plasma levels of M6G were from 2 to 17-fold lower than those of M.     

32P-postlabelling with high-performance liquid chromatography for analysis of abundant DNA adducts in human tissues

Abundant complex DNA adducts can be detected in human tissues by a combined 32P-postlabelling and high-performance liquid chromatography (HPLC) method. The HPLC profiles reveal a panorama of nuclease P1-resistant human adducts, which are not among the known human DNA adducts and are suspected of being endogenous. Lipid peroxidation-induced DNA adducts and I-compounds are two possible candidates for these adducts. Therefore, we performed two experiments: one was to identify chromatographically the lipid peroxidation-induced adducts among other human adducts with two acrolein- and crotonaldehyde-derived propano adduct standards (Acr-dG3 and Cro-dG1&2) and a structurally unknown adduct (Cro-DNA) derived from crotonaldehyde-treated DNA; and the other was to analyse the adducts in breast tissue from patients with breast cancer and from controls and to compare their behaviour with that of I-compounds in cancerous tissues. In the first experiment, Acr-dG3 and Cro-dG1 were detected in three human lung tissues, at levels ranging from 3.4 to 8.9 (x 10(-8)) and from not detectable to 2.9 (x 10(-8)), respectively. Acr-dG3 and Cro-DNA were detected in three human colon tissues, at levels of 0.2-0.4 (x 10(-8)) and 1.2-3.4 (x 10(-8)), respectively. In the second experiment, adjacent and tumorous breast tissues from 15 patients with breast cancer (of an average age of 33.4 years) and normal breast tissue from 18 controls (of an average age of 57.3) were analysed for the abundant complex adducts. The total adduct levels in the adjacent and tumorous tissues were lower than in the normal tissues (with medians of 8.0, 11.8 and 13.3 (x 10(-7)), respectively). Significant differences in the adduct levels between adjacent or tumorous tissues and normal tissues were observed in three HPLC peaks, and age was significantly associated with three peaks. These results are consistent with our speculation that the abundant adducts are comprised of lipid peroxidation-induced adducts and human homologues of I-compounds.     

Quantitative analysis of N-(guanin-8-yl)-N-acetyl-2-aminofluorene and N-(guanin-8-yl)-2-aminofluorene in modified DNA by hydrolysis in trifluoroacetic acid and high pressure liquid chromatography

N-acetyl-2-aminofluorene (AAF)- and 2-aminofluorene (AF)-modified DNA was hydrolyzed in dry trifluoroacetic acid (TFA). The hydrolysate was analyzed by high-pressure liquid chromatography. Using this procedure N-(guanin-8-yl)-[G-3H]2-aminofluorene was released quantitatively from DNA, modified by the reaction with [G-3H]N-hydroxy-2-aminofluorene. From DNA that had been reacted with [G-3H]N-acetoxy-N-acetyl-2-aminofluorene, 70% of the total bound radioactivity was isolated as N-guanin-8-yl)-[G-3H]-N-acetyl-2-aminofluorene. Thirty eight percent of the DNA bound radioactivity after i.p. injection of [G-3H]N-acetyl-2-aminofluorene followed by isolation of the rat liver DNA and subsequent hydrolysis in trifluoroacetic acid was identified as N-(guanin-8-yl)-[G-3H]-N-acetyl-2-aminofluorene. In this DNA the corresponding deacetylated compound N-(guanin-8-yl)-[G-3H]2-aminofluorene could not be detected.     

Mitochondrial DNA copy number and cancer risks: A comprehensive Mendelian randomization analysis

Mitochondrial DNA plays a critical role in the pathophysiology of cancer. However, the associations between mitochondrial DNA copy number (mtDNA-CN) and cancer risk are controversial. Mendelian randomization (MR) analyses were performed using three independent instrumental variables (IVs) to explore potential associations between mtDNA-CN and 20 types of cancer. The three sets of IVs were primarily obtained from participants in the UK Biobank and the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium using different methods. The outcome data of cancers were investigated using summary statistics from the FinnGen cohort. The potential causal associations were evaluated using the MR-Egger regression, weighted median, inverse-variance weighted (IVW), and weighted mode methods. The robustness of IVW estimates was validated using leave-one-out sensitivity analysis. Additionally, a meta-analysis was conducted to pool results from three sets of IVs. The results revealed that genetically predicted mtDNA-CN was not associated with cancer risk (odds ratio = 1.02; 95% confidence interval: 0.95–1.10). Subgroup analyses indicated no causal association between mtDNA-CN and breast, lung, prostate, skin, colorectal, gastric, liver, cervical uteri, esophageal, thyroid, bladder, pancreas, kidney, corpus uteri, ovary, brain, larynx, and anus cancers. It was observed that mtDNA-CN was associated with lip, oral cavity, and testis cancers. However, these results should be interpreted with caution because a small number of patients with lip and oral cavity or testis cancers were included. The comprehensive MR analysis demonstrated that mtDNA-CN is not a suitable biomarker for tumor risk assessment.