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Comparison of ³²P-postlabeling and high-resolution GC/MS in quantifying N7-(2-hydroxyethyl)guanine adducts

CHEMICAL RESEARCH IN TOXICOLOGY 1999年第10期12卷 979-984页

作者： Eide, I Zhao, CY Kumar, R Hemminki, K Wu, KY Swenberg, JA Statoil Res Ctr N-7005 Trondheim Norway Karolinska Inst Novum Dept Biosci Ctr Nutr & Toxicol S-14157 Huddinge Sweden Univ N Carolina Dept Environm Sci & Engn Chapel Hill NC 27599 USA

This study compares P-32-postlabeling and high-resolution gas chromatography/mass spectrometry (GC/MS) in the quantification of N7-(2-hydroxyethyl)guanine adducts (7-HEG) in DNA obtained from the same tissue samples of control rats and rats exposed to ethene. The samples were obtained from two independent studies. In one study, male Sprague-Dawley rats were exposed to 300 ppm ethene for 12 h/day for 3 days ("Euro samples"). In the other study, male F-344 rats were exposed to 3000 ppm ethene for 6 h/day for 5 days ("U.S. samples"). DNA from liver and kidney from the European study was isolated in the European laboratory, and DNA from liver and spleen from the U.S. study was isolated in the U.S. laboratory. The DNA samples were coded, divided into two portions, and exchanged between the two laboratories. All DNA samples from both laboratories were analyzed with respect to 7-HEG adducts by P-32-postlabeling and high-resolution GC/MS in the European and U.S. laboratories, respectively. However, the U.S. samples were repurified in the European laboratory before the postlabeling analysis. The data from the Euro and the U.S. samples were therefore treated separately in the regression analysis of the P-32-postlabeling versus GC/MS data. The slope of the regression line for the Euro samples was 1.19 (r = 0.97), implying that the GC/MS data were slightly lower than the postlabeling data (one possible outlier was excluded). The slope of the regression line for the U.S. samples was 0.61 (r = 0.94), implying that the GC/MS data were somewhat higher than the postlabeling data. The main conclusion from this study is that there is very good agreement between the P-32-postlabeling and high-resolution GC/MS methods in quantifying 7-HEG adducts to DNA, particularly when identical DNA samples are analyzed and the RNA content is <2%. The paper also discusses the background levels of adducts, the interorgan distribution, comparison between different strains, and exposure conditions.

关键词： DNA加合物/分析气相色谱-质谱法鸟嘌呤/类似物和衍生物鸟嘌呤/分析肾/化学肝/化学磷放射性同位素/诊断应用大鼠近交F344 大鼠 Sprague-Dawley 回归分析动物男(雄)性大鼠

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Muscle abnormalities in juvenile dermatomyositis patients - P-31 magnetic resonance spectroscopy studies

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ARTHRITIS AND RHEUMATISM 2000年第10期43卷 2359-2367页

作者： Park, JH Niermann, KJ Ryder, NM Nelson, AE Das, A Lawton, AR Hernanz-Schulman, M Olsen, NJ Vanderbilt Univ Dept Med Sch Med Nashville TN 37232 USA

Objective. To characterize metabolic abnormalities in the muscles of children with the juvenile variant of dermatomyositis (JDM) by the use of noninvasive P-31 magnetic resonance spectroscopy (MRS), Methods, Thirteen patients with JDM (ages 4-16 years) were studied. Biochemical status was evaluated with P-31 MRS by determining the concentrations of the high-energy phosphate compounds, ATP and phosphocreatine (PCr), ratios of inorganic phosphate (Pi) to PCr (Pi:PCr ratio), levels of free cytosolic ADP, and phosphorylation potentials (PPs) during rest, exercise, and recovery. Results. Significant metabolic abnormalities were observed in the thigh muscles of 10 severely affected patients during rest, 2 graded levels of exercise, and recovery. Mean ATP and PCr levels in the muscles of JDM patients were 35-40% below the normal control values (P < 0.003). These data, along with elevated Pi:PCr ratios, higher ADP levels, and abnormal values for PPs, indicated defective oxidative phosphorylation in the mitochondria of diseased JDM muscles. MRS findings were normal in 2 additional patients who had improved with prednisone treatment and in 1 patient who had no muscle weakness (amyopathic variant of JDM). Conclusion. JDM patients can be monitored with noninvasive P-31 MRS without sedation: Biochemical defects in energy metabolism are concordant with the weakness and fatigue reported by JDM patients. Quantitative MRS data are useful for evaluating patients and optimizing drug treatment regimens.

关键词：成本及成本分析皮肌炎/代谢能量代谢运动(Exercise) 磁共振成像肌骨骼/病理学肌骨骼/放射性核素显像磷放射性同位素/诊断应用磷酰化休息工作青少年儿童儿童学龄前女(雌)性人类男(雄)性

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NONFLUORESCENT MALIGNANT-MELANOMA OF CHOROID DIAGNOSED WITH RADIOACTIVE PHOSPHORUS UPTAKE TEST

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AMERICAN JOURNAL OF OPHTHALMOLOGY 1975年第4期79卷 634-640页

作者： SHIELDS, JA ANNESLEY, WH TOTINO, JA WILLS EYE HOSP RETINA SERVONCOL UNITPHILADELPHIAPA 19130

A 57 yr old woman presented with a small malignant melanoma of the choroid in the macular area of the right eye. The lesion failed to produce a visual field defect or to show fluorescence with angiography, but the radioactive phosphorus uptake (32P) test was positive. Histologically, the retinal pigment epithelium was intact over the tumor and the sensory retina was normal. This unusual finding was the most likely explanation for the normal visual field. The intact retinal pigment epithelium over this non fluorescent melanoma suggests that the destruction of the retinal pigment epithelium occurring with most choroidal melanomas may partially explain why these tumors usually demonstrate fluorescence. This case emphasizes the great accuracy of the 32P test for diagnosing small malignant tumors of the choroid, even before other popular diagnostic modalities indicate the malignancy of the lesion.

关键词：脉络膜/病理学脉络膜肿瘤/诊断脉络膜肿瘤/病理学荧光素血管造影术荧光黑色素瘤/诊断黑色素瘤/病理学检眼镜检查磷放射性同位素/诊断应用显微摄影术视网膜/病理学超声检查女(雌)性人类中年人

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Marked impairment of protein tyrosine phosphatase 1B activity in adipose tissue of obese subjects with and without type 2 diabetes mellitus

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JOURNAL OF LABORATORY AND CLINICAL MEDICINE 1999年第2期134卷 115-123页

作者： Cheung, A Kusari, J Jansen, D Bandyopadhyay, D Kusari, A Bryer-Ash, M Univ Tennessee Coll Med Dept Med Memphis TN USA Vet Adm Med Ctr Res Serv Memphis TN 38104 USA Tulane Univ Sch Med Dept Physiol New Orleans LA 70112 USA Tulane Univ Sch Med Dept Surg New Orleans LA 70112 USA

Protein tyrosine phosphatases (PTPs) are required for the dephosphorylation of the insulin receptor (IR) and its initial cellular substrates, and it has recently been reported that PTP-1B may play a role in the pathogenesis of insulin resistance in obesity and type 2 diabetes mellitus (DM), We therefore determined the amount and activity of PTP-1B in abdominal adipose tissue obtained from lean nondiabetic subjects (lean control (LC)), obese nondiabetic subjects (obese control (OC)), and subjects with both type 2 DM (DM2) and obesity (obese diabetic (OD)), PTP-1B protein levels were 3-fold higher in OC than in LC (1444 +/- 195 U vs 500 +/- 146 U (mean +/- SEM), P < .015), while OD exhibited a 5.5-fold increase (2728 +/- 286 U, P < .01), PTP activity was assayed by measuring the dephosphorylating activity toward a phosphorus 32-labeled synthetic dodecapeptide, In contrast to the increased PTP-1B protein levels, PTP-1B activity per unit of PTP-1B protein was markedly reduced, by 71% and 88% in OC and On, respectively. Non-PTP-1B tyrosine phosphatase activity was comparable in all three groups. Similar results were obtained when PTP-1B activity was measured against intact human IR, A significant correlation was found between body mass index (BMI) and PTP-1B level (r = 0.672, P < .02), whereas BMI and PTP-1B activity per unit of PTP-1B showed a strong inverse correlation (r = -0.801, P < .002). These data suggest that the insulin resistance of obesity and DM2 is characterized by the increased expression of a catalytically impaired PTP-1B in adipose tissue and that impaired PTP-1B activity may be pathogenic for insulin resistance in these conditions.

关键词：脂肪组织/酶学印迹法蛋白质细胞系糖尿病 2型/酶学酶激活/生理学成纤维细胞/细胞学水解作用神经组织蛋白质类/分析神经组织蛋白质类/代谢肥胖症/酶学磷放射性同位素/诊断应用磷酰化沉淀素试验成年人老年人动物女(雌)性人类男(雄)性中年人大鼠

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Determination of in vitro- and in vivo-formed DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline by capillary liquid chromatography/microelectrospray mass spectrometry

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CHEMICAL RESEARCH IN TOXICOLOGY 1999年第10期12卷 1019-1027页

作者： Gangl, ET Turesky, RJ Vouros, P Northeastern Univ Dept Chem Boston MA 02115 USA Northeastern Univ Barnett Inst Boston MA 02115 USA Nestec Ltd Nestle Res Ctr CH-1000 Lausanne Switzerland

Capillary liquid chromatography/microelectrospray mass spectrometry has been applied to the detection of deoxyribonucleoside adducts of the food-derived mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)from in vitro and in vivo sources. Constant neutral loss (CNL) and selective reaction monitoring (SRM) techniques with a triple-quadrupole mass spectrometer enabled sensitive and specific detection of IQ adducts in vitro and in animals. Detection of 1 adduct in 10(4) unmodified bases is achieved using CNL scanning detection, while the lower detection limits using SRM approach 1 adduct in 10(7) unmodified bases using 300 mu g of DNA. The DNA adducts N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-C8-IQ) and 5-(deoxyguanosin-N-2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-N-2-IQ) were detected in kidney tissues of chronically treated cynomolgus monkeys at levels and in proportions consistent with previously published P-32-postlabeling data [Turesky, R. J., et al. (1996) Chem. Res, Toxicol. 9, 403-408]. Thus, capillary tandem LC/MS is a highly sensitive technique, which can be used to screen for DNA adducts in vivo.

关键词：色谱法高压液相色谱法液相 DNA/化学 DNA加合物/分析同位素标记肾/化学成束猴质谱分析法诱变剂/化学磷放射性同位素/诊断应用喹啉类/化学动物

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Sphingosine 1-phosphate as a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells

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BLOOD 2000年第10期96卷 3431-3438页

作者： Yatomi, Y Ohmori, T Ge, RL Kazama, F Okamoto, H Sano, T Satoh, K Kume, S Tigyi, G Igarashi, Y Ozaki, Y Yamanashi Med Univ Dept Lab Med Yamanashi 4093898 Japan Hokkaido Univ Grad Sch Pharmaceut Sci Dept Biomembrane & Biofunct Chem Sapporo Hokkaido Japan Univ Tennessee Ctr Hlth Sci Dept Physiol Memphis TN 38163 USA

The serum-borne lysophospholipid mediators sphingosine 1-phosphate (Sph-1-P) and lysophosphatidic acid (LPA) have been shown to be released from activated platelets and to act on endothelial cells. In this study, we employed the repeated lipid extraction (under alkaline and acidic conditions), capable of detecting Sph-1-P, LPA, and possibly structurally similar lysophospholipids, whereby a marked formation of [P-32]Sph-1-P, but not [P-32]LPA, was observed In [P-32]orthophosphate-labeled platelets. Platelet Sph-1-P release, possibly mediated by protein kinase C, was greatly enhanced in the presence of albumin, which formed a complex with Sph-1-P. This finding suggests that platelet Sph-1-P may become accessible to depletion by albumin when its transbilayer movement (flipping) across the plasma membrane is enhanced by protein kinase C. Although human umbilical vein endothelial cells expressed receptors for both Sph-1-P and LPA, Sph-1-P acted much more potently than LPA on the cells in terms of intracellular Ca++ mobilization, cytoskeletal reorganization, and migration. The results suggest that Sph-1-P, rather than LPA, is a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells, under the conditions in which critical platelet-endothelial interactions (including thrombosis, angiogenesis, and atherosclerosis) occur. Furthermore, albumin-bound Sph-1-P may account for at least some of the serum biological activities on endothelial cells, which have-been ascribed to the effects of albumin-bound LPA, based on the similarities between LPA and serum effects. (C) 2000 by The American Society of Hematology.

关键词：生物转运血小板/代谢钙信号/药物作用细胞运动/药物作用色谱法薄层细胞骨架/药物作用内皮血管/细胞学内皮血管/代谢胎血/细胞学胎血/代谢溶血磷脂素类/分离和提纯溶血磷脂素类/代谢磷酸盐类/诊断应用磷酸盐类/代谢磷放射性同位素/诊断应用 RNA 信使/代谢受体细胞表面/遗传学受体 G-蛋白偶联受体溶血磷脂酸受体溶血磷脂血清白蛋白/药理学鞘氨醇/类似物和衍生物鞘氨醇/代谢人类

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Development of a ³²P-postlabeling method for the detection of 1,N²-propanodeoxyguanosine adducts of 2-hexenal in vivo

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CHEMICAL RESEARCH IN TOXICOLOGY 1999年第4期12卷 335-340页

作者： Schuler, D Budiawan Eder, E Univ Wurzburg Dept Toxicol D-97078 Wurzburg Germany

2-Hexenal is an alpha,beta-unsaturated carbonyl compound which forms cyclic 1,N-2-propano adducts in vitro. The adduct formation in vivo was not reported by others to date. Because this type of adduct is considered promutagenic (2-hexenal is actually mutagenic and genotoxic) and humans are permanently exposed to this compound via vegetarian food, 2-hexenal may play a role in carcinogenicity. To improve the cancer risk assessment, we developed a new P-32-postlabeling technique for this compound and optimized the different steps of the postlabeling procedure. The results of the postlabeling methods are shown. A labeling efficiency of 35%, a recovery of 10% for the synthesized standards, and a detection Limit of three 2-hexenal adducts per 10(8) nucleotides was achieved. After gavage of 500 mg/kg of body weight to male Fischer 344 rats, the respective DNA adducts were detected in rat liver DNA, With this study, we demonstrate in vivo adduct formation of 2-hexenal for the first time. Highest adduct levels were found 2 days after gavage, and after 4 days, the level was even higher than after 1 day. No adducts were detected 8 h after gavage. The respective adducts could not be found as a background in tissues of untreated rats or in calf thymus DNA at the limit of detection.

关键词：醛类/代谢色谱法薄层 DNA加合物/分析脱氧鸟苷/代谢氢离子浓度磷放射性同位素/诊断应用大鼠近交F344 动物男(雄)性大鼠

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皮瓣血运重建中的32P观察及其临床应用

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中华外科杂志 1984年第11期22卷 683-684页

作者：曹子明北京部队总医院

皮管、皮瓣形成后,何时为再手术的安全时机?如何确定其血运是否建立完善?直接关系着手术的成败。Hynes用Atropine法和郁血测验法荧光法和测量皮瓣温度法来观察末稍循环。目前,成形外科则以将皮管夹压阻断法来观察和确定血运恢复情况。... 详细信息

皮管、皮瓣形成后,何时为再手术的安全时机?如何确定其血运是否建立完善?直接关系着手术的成败。Hynes用Atropine法和郁血测验法荧光法和测量皮瓣温度法来观察末稍循环。目前,成形外科则以将皮管夹压阻断法来观察和确定血运恢复情况。上述方法有的会带来全身药物反应,有的结果不太可靠。我们用32P测定组织移植后的血运建立情况,并以正常组织的32P半减时间的平均数值为依据,确定其血运是否建立完善和再手术

关键词：磷放射性同位素/诊断应用外科皮瓣

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