Modern geochemical and molecular tools for monitoring in-situ biodegradation of MTBE and TBA

Methyl tert-butyl ether (MTBE) is a major gasoline oxygenate worldwide and a widespread groundwater contaminant. Natural attenuation of MTBE is of practical interest as a cost effective and non-invasive approach to remediation of contaminated sites. The effectiveness of MTBE attenuation can be difficult to demonstrate without verification of the occurrence of in-situ biodegradation. The aim of this paper is to discuss the recent progress in assessing in-situ biodegradation. In particular, compound-specific isotope analysis (CSIA), molecular techniques based on nucleic acids analysis and in-situ application of stable isotope labels will be discussed. Additionally, attenuation of tert-butyl alcohol (TBA) is of particular interest, as this compound tends to occur alongside MTBE introduced from the gasoline or produced by (mainly anaerobic) biodegradation of MTBE.     

Isotope Ratio Measurements of Iron in Blood Samples by Multi-collector ICP-MS to Support Nutritional Investigations in Humans

With the perspective of embarking on a human study using a double iron (Fe) stable isotope tracer protocol to assess iron bioavailability, investigations were conducted on Fe isotope ratios in blood samples using a VG Axiom Multi-collector ICP-MS. The factors affecting the precision and accuracy of Fe isotopic ratios, such as spectral- and matrix-induced interferences and Fe recoveries from sample preparation, have been identified and optimized. Major polyatomic interferences (e.g., Ar-O, Ar-OH, and FeH) were significantly reduced by using an Aridus nebulizer and desolvating system. Isobaric metal (e.g., ⁵⁴Cr⁺ on ⁵⁴Fe⁺ and ⁵⁸Ni⁺ on ⁵⁸Fe⁺) interferences and Ca-oxides and hydroxides were quantitatively removed during chemical purification of blood samples and selective isolation of Fe by anion-exchange resin, after mineralization of the blood samples by microwave digestion. Quantitative recoveries of Fe from different steps of sample preparation were verified using whole blood reference material. Fe isotopic compositions of the samples were corrected for instrumental mass bias by the standard-sample bracketing method using the certified reference standard IRMM-014. External precisions on the order of 0.008–0.05 (% RSD), 0.007–0.015 (% RSD), and 0.03–0.09 (% RSD) were obtained for ⁵⁴Fe/⁵⁶Fe, ⁵⁷Fe/⁵⁶Fe, and ⁵⁸Fe/⁵⁶Fe, respectively, in the blood for three replicate measurements. The level of precision obtained in this work enables the detection of low enrichments of Fe in blood, which is highly desired in nutrition tracer studies.     

Synthesis of a 13C-Labeled Tracer for Stream DOC: Labeling Tulip Poplar Carbon with 13CO2

Ecosystem tracer-level additions would benefit from a stable isotope-labeled source of complex organic molecules. We tested a method to label tree C with ¹³C and create a stable isotope tracer for stream dissolved organic carbon (DOC) using tulip poplar (Liriodendron tulipifera L.) seedlings. In 2000, seedlings were grown with 0.82 moles of ¹³CO₂ to assess the distribution and level of ¹³C enrichment in the tree tissues. In 2001, seedlings were grown with 25 times more ¹³CO₂ to generate tissues with a ¹³C signal strong enough for a ¹³C-DOC stream tracer addition. ¹³C enrichment in the trees varied in each year and by tissue age and type. Tissues formed during labeling (new) were more enriched in ¹³C than tissues established prior to the ¹³CO₂ injection (old). Stems were most enriched in ¹³C in both new and old tissues. A higher percentage of ¹³CO₂ was incorporated into seedlings in 2000 (59% ±1) than 2001 (43% ±0). Percent ¹³C incorporation among tree tissue types paralleled biomass distributions. Although tree C and ¹³C were equally soluble in both years, a greater percentage of tree C went into solution in 2001 (30%) than 2000 (20%). The water-soluble tree C accounted for approximately 12% of the injected ¹³CO₂ and had both humic and polysaccharide components. Results from a whole-stream ¹³C-DOC tracer addition demonstrated that tree C could be sufficiently labeled with ¹³CO₂ to create a stream DOC isotope tracer with some polymeric constituents.     

Palaeoenvironmental reconstruction of dinosaur nesting sites based on a geochemical approach to eggshells and associated palaeosols (Maastrichtian, Provence Basin, France)

Variability of the stable isotopic (δ¹³C and δ¹⁸O) and trace element (Mg, Sr, Fe, and Mn) content from dinosaur eggshells from the Provence Basin has been addressed by considering the stratigraphic distribution of the eggshells, the palaeoenvironments, and the chemical content of associated sediments. Preferentially, in situ eggshells were analysed. Samples were collected from two sections, one belonging to the floodplain (Roques Hautes) and the other to the channel belt (Rousset-Bréguières), as determined from sedimentological criteria. On a regional scale, this combined analysis provided new insights into palaeohydrology, vegetation cover and dinosaur behaviour. Different sources of drinking water were identified: (1) the main stream waters with significant salinity levels, (2) lateral feeders with water composition close to that of precipitation. Water composition is reflected in the eggshell trace element contents: a high Sr content for the eggshells from the channel belt (mean 1200 ppm) contrasting with the very low values for the floodplain (mean 250 ppm). During the first part of the Maastrichtian, evaporation rates were quite significant, as seen from the 1.5‰ difference between the eggshell δ¹⁸O (drinking water) and the palaeosol carbonate nodules (precipitation). This contrast diminished and became nearly insignificant in the second half of the Maastrichtian. Vegetation was composed of C₃ plants and two ecosystems are reconstructed: a riparian forest extending along the channel belt and open vegetation in the floodplain. The contrasted isotopic and trace element content of the eggshells was a crucial factor in identifying animal migration from one site to another. Migration was quite limited, suggesting that the animals settled for some time at the places where they laid their eggs. Intraspecific variations among modern birds are well correlated with the environment and may be greater than interspecies variations, which limits the potential of geochemical analysis to characterise dinosaur species.     

A Liquid Chromatography–Mass Spectrometry Method to Measure Stable Isotopic Tracer Enrichments of Glycerol and Glucose in Human Serum

Stable isotopes are commonly used as tracers for the measurement of glycerol and glucose kinetics in metabolic studies. Traditionally, the analysis of these isotopes has been performed using gas chromatography-mass spectrometry, which requires that the analytes first be derivatized. The derivatization process adds considerable complexity to the method. Liquid chromatography-mass spectrometry (LCMS) can measure many metabolites directly with limited sample preparation. We present a novel analytical method for the measurement of [1,1,2,3,3-(2)H(5)]glycerol (d(5)-glycerol) and [6,6-(2)H(2)]glucose (d(2)-glucose) isotopic tracer enrichments in human serum in a single run by LCMS. After a simple extraction step, the sample is separated isocratically by HPLC, and the isotopes are measured using positive electrospray ionization with selected ion monitoring of the sodium-adduct ions. The method is linear over a wide range of d(2)-glucose and d(5)-glycerol enrichments. The within-day standard deviation of measurement of serum samples was 0.05 mole% excess (MPE) for d(2)-glucose and 0.25 MPE for d(5)-glycerol. The variation of tracer enrichment among days was about double that measured within 1 day.     

Parallel labeling experiments validate Clostridium acetobutylicum metabolic network model for 13C metabolic flux analysis

In this work, we provide new insights into the metabolism of Clostridium acetobutylicum ATCC 824 obtained using a systematic approach for quantifying fluxes based on parallel labeling experiments and ¹³C-metabolic flux analysis (¹³C-MFA). Here, cells were grown in parallel cultures with [1-¹³C]glucose and [U-¹³C]glucose as tracers and ¹³C-MFA was used to quantify intracellular metabolic fluxes. Several metabolic network models were compared: an initial model based on current knowledge, and extended network models that included additional reactions that improved the fits of experimental data. While the initial network model did not produce a statistically acceptable fit of ¹³C-labeling data, an extended network model with five additional reactions was able to fit all data with 292 redundant measurements. The model was subsequently trimmed to produce a minimal network model of C. acetobutylicum for ¹³C-MFA, which could still reproduce all of the experimental data. The flux results provided valuable new insights into the metabolism of C. acetobutylicum. First, we found that TCA cycle was effectively incomplete, as there was no measurable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, and malate and oxaloacetate. Second, an active pathway was identified from pyruvate to fumarate via aspartate. Third, we found that isoleucine was produced exclusively through the citramalate synthase pathway in C. acetobutylicum and that CAC3174 was likely responsible for citramalate synthase activity. These model predictions were confirmed in several follow-up tracer experiments. The validated metabolic network model established in this study can be used in future investigations for unbiased ¹³C-flux measurements in C. acetobutylicum.     

Present status of sentinel lymph node biopsy in cervical cancer

Cervical cancer is the fourth most common cancer in women, and seventh overall. This disease represents a medical, economic and social burden. In early FIGO stage patients (IA, IB1 and IIA1), nodal involvement is the most important prognostic factor. Imaging evaluation of nodal metastasis is of limited value. In order to determine lymph node involvement, allow loco-regional control of the disease, define the need for adjuvant radiotherapy and improve survival, standard surgery for early disease is radical hysterectomy with systematic pelvic lymphadenectomy. However, this surgical treatment has risks and complications: longer operative time, larger blood loss, neurovascular or ureteral injury, lower-limb lymphedema, symptomatic lymphocysts, hydronephrosis. A method that allows to define the presence of regional metastasis with less morbidity and equal or greater precision is particularly relevant. The use of the sentinel lymph node biopsy is intended to reach that purpose. The present study reviews recent literature on the role of sentinel lymph node biopsy in cervical cancer, analyzing its indications and contraindications, injection and detection techniques, tracers used, surgical and pathological approaches and its applicability in up-to-date clinical practice.