Roger W. Giese

Measurement of endogenous estrogens: analytical challenges and recent advances.

Recent developments in the analysis of endogenous estrogens (including both free and conjugated estrogens) are reviewed. Largely due to urging by some cancer researchers, new demands are now being placed on such measurements in terms of sensitivity, throughput, multi-analyte detection and accuracy. Especially high sensitivity is required for detecting estrogens in serum from postmenopausal women, children and men, where concentrations at the low pg/ml level are encountered, and one would prefer to test much less than 1 ml of serum. Aside from throughput, meeting all of these demands may be beyond the reach of immunoassay, the method that has created and continues to dominate this field. Both HPLC and GC versions of mass spectrometry are emerging that have some potential to improve the testing of physiological samples for endogenous estrogens. The following topics are covered in this review: related analyses (e.g. detection of estrogens in environmental samples such as water, where 1-1 samples can be collected to provide ng amounts of estrogens); structure and metabolism of estrogens; biological actions (with an emphasis on their role in cancer); immunoassays; HPLC with electrochemical detection; GC-ECD; and various forms of mass spectrometry.

Laser fluorescence detector for capillary electrophoresis

Abstract A simple, rugged, and relatively inexpensive laser-based fluorometer for detection in capillary electrophoresis is described. This is assembled from commercially available components and requires minimal experience for operation. Yet, the detection performance is comparable to those achieved in laser laboratories with sophisticated layouts. As an example, detection of a 3 pM solution of fluorescein is demonstrated. The design principles of laser fluorometric detection are critically examined, and the special adaptation for the present detection arrangement is discussed.

Mono-2-Ethylhexyl Phthalate Induces Oxidative Stress Responses in Human Placental Cells In Vitro

Di-2-ethylhexyl phthalate (DEHP) is an environmental contaminant commonly used as a plasticizer in polyvinyl chloride products. Exposure to DEHP has been linked to adverse pregnancy outcomes in humans including preterm birth, low birth-weight, and pregnancy loss. Although oxidative stress is linked to the pathology of adverse pregnancy outcomes, effects of DEHP metabolites, including the active metabolite, mono-2-ethylhexyl phthalate (MEHP), on oxidative stress responses in placental cells have not been previously evaluated. The objective of the current study is to identify MEHP-stimulated oxidative stress responses in human placental cells. We treated a human placental cell line, HTR-8/SVneo, with MEHP and then measured reactive oxygen species (ROS) generation using the dichlorofluorescein assay, oxidized thymine with mass-spectrometry, redox-sensitive gene expression with qRT-PCR, and apoptosis using a luminescence assay for caspase 3/7 activity. Treatment of HTR-8 cells with 180μM MEHP increased ROS generation, oxidative DNA damage, and caspase 3/7 activity, and resulted in differential expression of redox-sensitive genes. Notably, 90 and 180μM MEHP significantly induced mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2), an enzyme important for synthesis of prostaglandins implicated in initiation of labor. The results from the present study are the first to demonstrate that MEHP stimulates oxidative stress responses in placental cells. Furthermore, the MEHP concentrations used were within an order of magnitude of the highest concentrations measured previously in human umbilical cord or maternal serum. The findings from the current study warrant future mechanistic studies of oxidative stress, apoptosis, and prostaglandins as molecular mediators of DEHP/MEHP-associated adverse pregnancy outcomes.

Malondialdehyde-Deoxyguanosine Adduct Formation in Workers of Pathology Wards. The Role of Air Formaldehyde Exposure

Formaldehyde is an ubiquitous pollutant to which humans are exposed. Pathologists can experience high formaldehyde exposure levels. Formaldehyde-among other properties-induce oxidative stress and free radicals, which react with DNA and lipids, leading to oxidative damage and lipid peroxidation, respectively. We measured the levels of air-formaldehyde exposure in a group of Italian pathologists and controls. We analyzed the effect of formaldehyde exposure on leukocyte malondialdehyde-deoxyguanosine adducts (M(1)-dG), a biomarker of oxidative stress and lipid peroxidation. We studied the relationship between air-formaldehyde and M(1)-dG adducts. Air-formaldehyde levels were measured by personal air samplers. M(1)-dG adducts were analyzed by a (32)P-postlabeling assay. Reduction room pathologists were significantly exposed to air-formaldehyde with respect to controls and to the pathologists working in other laboratory areas (p < 0.001). A significant difference for M(1)-dG adducts between exposed pathologists and controls was found (p = 0.045). The effect becomes stronger when the evaluation of air-formaldehyde exposure was based on personal samplers (p = 0.018). Increased M(1)dG adduct levels were only found in individuals exposed to air-formaldehyde concentrations higher than 66 microg/m(3). When the exposed workers and controls were subgrouped according to smoking, M(1)-dG tended to increase in all of the subjects, but a significant association between M(1)-dG and air-formaldehyde was only found in nonsmokers (p = 0.009). Air-formaldehyde played a role positive but not significant (r = 0.355, p = 0.075, Pearson correlation) in the formation of M(1)-dG, only in nonsmokers. Working in the reduction rooms and exposure to air-formaldehyde concentrations higher than 66 microg/m(3) are associated with increased levels of M(1)-dG adducts.

Detection and structural characterization of amino polyaromatic hydrocarbon-deoxynucleoside adducts using fast atom bombardment and tandem mass spectrometry

Fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS) are shown to be useful methods for the detection and structural characterization of nanogram amounts of amino polyaromatic hydrocarbon-nucleoside DNA adducts. The positive ion spectra of four aromatic amine guanosine adducts were studied in detail. The FAB spectra of these adducts exhibit an [MH]+ ion and a more abundant aglycon fragment ion, [AH2]+, which results from the loss of the deoxyribose sugar. The sensitivity of the adducts to FAB was enhanced by preparing trimethylsilyl (TMS) ether derivatives. High-quality full-scan spectra could be obtained on less than 70 ng of the derivatized adducts without signal averaging. With a B/E-linked scan of the [MH]+ ion for the TMS2 species, these same adducts could be detected by examination of their metastable ion spectra at levels as low as 4-5 ng (S/N greater than 10). Collision-induced dissociation (CID) of the [MH]+ ion yields the aglycon fragment and an ion, S1, which results from cleavage through the sugar. The CID spectrum of the aglycon [AH2]+ ion is much more useful, providing structural information relating to the base, the polyaromatic hydrocarbon, and, possibly, the site of covalent attachment. Differentiation of isomeric aminophenanthrene-guanine adducts was demonstrated on the basis of the CID spectra of their respective [AH2]+ ions. The use of TMS derivatives also improves the sensitivity of these methods.

Trace detection of modified dna bases via moving-belt liquid chromatography—mass spectrometry using electrophoric derivatization and negative chemica

Electrophoric derivatives of 5-methylcytosine and 5-hydroxymethyluracil nucleobases are determined using high-performance liquid chromatography-mass spectrometry coupled via a moving-belt interface. Standards as well as samples derived from DNA are analysed. As little as 9.9 pg (signal-to-noise ratio 5) and 180 fg (signal-to-noise ratio 10) of the respective nucleobases are detected in the electron-capture negative chemical ionization mode, and linear responses are observed over a moderate dynamic range. In a comparison study, liquid chromatography-electron-capture negative chemical ionization mass spectrometry is found to have a sensitivity comparable to gas chromatography-electron-capture negative chemical ionization mass spectrometry for 5-hydroxymethyluracil. A detection limit of 60 fg (signal-to-noise ratio 5) by gas chromatography-mass spectrometry is only three-fold better than the amount detected by liquid chromatography-mass spectrometry using the same mass spectrometer.

Detritylation with ytterbium triflate

Abstract Ytterbium triflate catalyzes the hydrolysis of tritylamines and trityloxy compounds to the corresponding amines and alcohols under mild conditions in high yields. The reactions are conducted in tetrahydrofuran in the presence of 1 equivalent of water.

Gas chromatography-mass spectrometry characteristics of methylated perfluoroacyl derivatives of cytosine and 5-methylcytosine

Pentafluorobenzoyl and heptafluorobutyryl derivatives of cytosine and pentafluorobenzoyl-5-methylcytosine have been examined by mass spectrometry following permethylation. Electron impact mass spectra were used to confirm the structures of the derivatives. The permethylated pentafluorobenzoyl derivatives of cytosine and 5-methylcytosine are sensitive for quantitative analysis by capillary gas chromatography-mass spectrometry. Under negative ion chemical ionization conditions 1 femtogram of each is detected.

Breast fine-needle aspiration malondialdehyde deoxyguanosine adduct in breast cancer

Abstract This study has analysed the generation of 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine adduct [M1dG], a biomarker of oxidative stress and lipid peroxidation, in breast fine-needle aspirate samples of 22 patients with breast cancer, at different clinical stages, in respect to 13 controls. The multivariate analysis show that M1dG adduct was higher in cases than in controls (Mean Ratio (MR) = 5.26, 95% CI = 3.16–8.77). Increased M1dG was observed in women with a tumour grade 3 and a pathological diameter 2 (MR = 7.61, 95% CI = 3.91–14.80 and MR = 5.75, 95% CI = 3.13–10.59, respectively). A trend with increasing tumour grade and pathological diameter was present (MR = 1.98, 95% CI = 1.57–2.50 and MR = 2.44, 95% CI = 1.71–3.48, respectively). Not significant effects of age and smoking habit were found (MR = 1.58, 95% CI = 0.92–2.72 and MR = 1.68, 95% CI 0.88–3.20, respectively). An increment over the background frequency of M1dG can contribute to breast cancer development. Increasing severity of breast tumour can influence DNA damage level.

Electrophore-labeling and alkylation of standards of nucleic acid pyrimidine bases for analysis by gas chromatography with electron-capture detection

The pyrimidine bases cytosine, uracil and thymine, along with some analogues, are electrophore-labeled either with pentafluorobenzoyl chloride (PFBC), pentafluorophenylsulfonyl chloride (PPSC), or heptafluorobutyric anhydride. Subsequent alkylation is most successful for PFB-cytosine, PPS-uracil, and PPS-thymine. These same alkylated compounds also have the highest aqueous stability and respond most strongly by gas chromatography-electron-capture detection. One of these derivatives, determined to be N4-PFB-1,3-dimethylcytosine by authentic synthesis, and its 5-methyl analogue, can be detected with good precision down to the 100-fg level. Poor reproducibility is encountered at the 10-fg level.