Isotope Dilution Methodology Research Articles

Removal and formation of chlorinated triclosan derivatives in wastewater treatment plants using chlorine and UV disinfection

Triclosan, a common antimicrobial agent, may react during the disinfection of wastewater with free chlorine to form three chlorinated triclosan derivatives (CTDs). This is of concern because the CTDs may be photochemically transformed to tri- and tetra-chlorinated dibenzo- p-dioxins when discharged into natural waters. In this study, wastewater influent, secondary (pre-disinfection) effluent, and final (post-disinfection) effluent samples were collected on two occasions each from two activated sludge wastewater treatment plants, one using chlorine disinfection and one using UV disinfection. Concentrations of triclosan and three CTDs were determined using ultra performance liquid chromatography–triple quadrupole mass spectrometry with isotope dilution methodology. Triclosan and the CTDs were detected in every influent sample at levels ranging from 453 to 4530 and 2 to 98 ng L −1, respectively, though both were efficiently removed from the liquid phase during activated sludge treatment. Triclosan concentrations in the pre-disinfection effluent ranged from 36 to 212 ng L −1, while CTD concentrations were below the limit of quantification (1 ng L −1) for most samples. In the treatment plant that used chlorine disinfection, triclosan concentrations decreased while CTDs were formed during chlorination, as evidenced by CTD levels as high as 22 ng L −1 in the final effluent. No CTDs were detected in the final effluent of the treatment plant that used UV disinfection. The total CTD concentration in the final effluent of the chlorinating treatment plant reached nearly one third of the triclosan concentration, demonstrating that the chlorine disinfection step played a substantial role in the fate of triclosan in this system.

Single‐unit muscle sympathetic nervous activity and its relation to cardiac noradrenaline spillover

Recent work using single-unit sympathetic nerve recording techniques has demonstrated aberrations in the firing pattern of sympathetic nerves in a variety of patient groups. We sought to examine whether nerve firing pattern is associated with increased noradrenaline release. Using single-unit muscle sympathetic nerve recording techniques coupled with direct cardiac catheterisation and noradrenaline isotope dilution methodology we examined the relationship between single-unit firing patterns and cardiac and whole body noradrenaline spillover to plasma. Participants comprised patients with hypertension (n=6), depression (n=7) and panic disorder (n =9) who were drawn from our ongoing studies. The patient groups examined did not differ in their single-unit muscle sympathetic nerve firing characteristics nor in the rate of spillover of noradrenaline to plasma from the heart. The median incidence of multiple spikes per beat was 9%. Patients were stratified according to the firing pattern: low level of incidence (less than 9% incidence of multiple spikes per beat) and high level of incidence (greater than 9% incidence of multiple spikes per beat). High incidence of multiple spikes within a cardiac cycle was associated with higher firing rates (P <0.0001) and increased probability of firing (P <0.0001). Whole body noradrenaline spillover to plasma and (multi-unit) muscle sympathetic nerve activity in subjects with low incidence of multiple spikes was not different to that of those with high incidence of multiple spikes. In those with high incidence of multiple spikes there occurred a parallel activation of the sympathetic outflow to the heart, with cardiac noradrenaline spillover to plasma being two times that of subjects with low nerve firing rates (11.0 ± 1.5 vs. 22.0 ± 4.5 ng min⁻¹, P <0.05). This study indicates that multiple within-burst firing and increased single-unit firing rates of the sympathetic outflow to the skeletal muscle vasculature is associated with high cardiac noradrenaline spillover.

A new liquid chromatography/mass spectrometry method for 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL) in urine

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic nitrosamine produced upon curing tobacco. It is present in tobacco smoke and undergoes metabolism to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in the lungs. NNAL undergoes further uridine diphosphate glucuronosyltransferase (UGT)-mediated metabolism to give N- and O-glucuronide metabolites, which together with free (non-conjugated) NNAL are then excreted in the urine. The ability to conduct validated analyses of free and conjugated NNAL in human urine is important in order to assess inter-individual differences in lung cancer risk from exposure to cigarette smoke. The use of stable isotope dilution (SID) methodology in combination with liquid chromatography/multiple reaction monitoring/mass spectrometry (LC/MRM-MS) provides the highest bioanalytical specificity possible for such analyses. We describe a novel derivatization procedure, which results in the formation of a pre-ionized N-propyl-NNAL derivative. The increased LC/MS sensitivity arising from this derivative then makes it possible to analyze free NNAL in only 0.25 mL urine. This substantial reduction in urine volume when compared with other methods that have been developed will help preserve the limited amounts of stored urine samples that are available from on-going longitudinal biomarker studies. The new high sensitivity SID LC/MRM-MS assay was employed to determine free and conjugated NNAL concentrations in urine samples from 60 individual disease-free smokers. Effects of inter-individual differences in urinary creatinine clearance on NNAL concentrations were then assessed and three metabolizer phenotypes were identified in the 60 subjects from the ratio of urinary NNAL glucuronides/free NNAL. Poor metabolizers (PMs, 14 subjects) with a ratio of NNAL glucuronides/free NNAL <2 (mean = 1.3), intermediate metabolizers (IMs, 36 subjects) with a ratio between 2 and 5 (mean = 3.4), and extensive metabolizers (EMs, 10 subjects) with a ratio >5 (mean = 11.1).

Analysis of endogenous glutathione‐adducts and their metabolites

The ability to conduct validated analyses of glutathione (GSH)-adducts and their metabolites is critically important in order to establish whether they play a role in cellular biochemical or pathophysiological processes. The use of stable isotope dilution (SID) methodology in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides the highest bioanalytical specificity possible for such analyses. Quantitative studies normally require the high sensitivity that can be obtained by the use of multiple reaction monitoring (MRM)/MS rather than the much less sensitive but more specific full scanning methodology. The method employs a parent ion corresponding to the intact molecule together with a prominent product ion that obtained by collision induced dissociation. Using SID LC-MRM/MS, analytes must have the same relative LC retention time to the heavy isotope internal standard established during the validation procedure, the correct parent ion and the correct product ion. This level of specificity cannot be attained with any other bioanalytical technique employed for biomarker analysis. This review will describe the application of SID LC-MR/MS methodology for the analysis of GSH-adducts and their metabolites. It will also discuss potential future directions for the use of this methodology for rigorous determination of their utility as disease and exposure biomarkers.

Integrated effects of abiotic stresses on inoculant performance, legume growth and symbiotic dependence estimated by 15N dilution

Temperature, water, salinity, sodicity, acidity and nutrient disorders are major abiotic stresses that can affect legume growth or the establishment and function of the legume-Rhizobium symbiosis. We have examined the literature where the application of the 15N isotope dilution methodology permits the effect of individual abiotic stresses to be independently and quantitatively separated into plant growth-mediated and BNF (biological N2 fixation)-mediated components. The response of the symbiosis to a particular stress depends on a host of factors, including legume genotype, cultivar, Rhizobium inoculant, climatic conditions, and the duration, timing and severity of the stress. Published data are analysed in terms of the above variables and their interactions. As a general rule, severe stress inhibits both legume dry matter (DM) and the proportional dependence of the legume on BNF as a source of N. The symbiosis is resilient to low to moderate stress, but there may still be a penalty on legume DM. Gaps in knowledge are identified, and general guidelines on the identification and amelioration of abiotic stresses are provided.

Targeted chiral lipidomics analysis of bioactive eicosanoid lipids in cellular systems

We have developed a targeted lipidomics approach that makes it possible to directly analyze chiral eicosanoid lipids generated in cellular systems. The eicosanoids, including prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs) and alcohols (HETEs), have been implicated as potent lipid mediators of various biological processes. Enzymatic formations of eicosanoids are regioselective and enantioselective, whereas reactive oxygen species (ROS)-mediated formation proceeds with no stereoselectivity. To distinguish between enzymatic and non-enzymatic pathways of eicosanoid formation, it is necessary to resolve enantiomeric forms as well as regioisomers. High sensitivity is also required to analyze the eicosanoid lipids that are usually present as trace amounts (pM level) in biological fluids. A discovery of liquid chromatography-electron capture atmospheric pressure chemical ionization/mass spectrometry (LCECAPCI/MS) allows us to couple normal phase chiral chromatography without loss of sensitivity. Analytical specificity was obtained by the use of collision-induced dissociation (CID) and tandem MS (MS/MS). With combination of stable isotope dilution methodology, complex mixtures of regioisomeric and enantiomeric eicosanoids have been resolved and quantified in biological samples with high sensitivity and specificity. Targeted chiral lipidomics profiles of bioactive eicosanoid lipids obtained from various cell systems and their biological implications have been discussed.

Short-term heart rate variability and cardiac norepinephrine spillover in patients with depression and panic disorder

Changes in measures of heart rate variability (HRV) have been associated with an increased risk for sudden cardiac death. The mechanisms underlying this association are not known. The objective of this study was to assess the relationship between the amount of norepinephrine (NE) released from the cardiac sympathetic terminals and short-term HRV. The study comprised 8 healthy subjects, 12 patients with major depression, and 7 patients with panic disorder. Cardiac NE spillover was determined using direct coronary sinus blood sampling coupled with an NE isotope dilution methodology. Short-term HRV was quantified using detrended fluctuation analysis, symbolic dynamics, sample entropy, and standard time and frequency domain measures. Neither HRV nor cardiac NE spillover was significantly different between the analyzed groups. None of the standard HRV metrics was significantly correlated with cardiac NE spillover, but there was a moderate correlation between two complexity measures of HRV (symbolic dynamics) and cardiac NE spillover (patterns with 2 like variations, r = -0.37 and P = 0.05; and patterns with no variations: r = 0.34 and P = 0.06). In conclusion, there is no correlation between standard HRV measures and cardiac NE spillover in humans. Short-term complexity of heart rate is only moderately affected by sympathetic neural outflow. Therefore, the predictive value of most HRV measures for sudden cardiac death may predominantly result from their capacity to capture vagally mediated heart rate modulations.

Isotope dilution strategies for absolute quantitative proteomics

The development of mass spectrometry (MS)-based methodologies for high-throughput protein identification has generated a concomitant need for protein quantification. Numerous MS-based relative quantification methodologies have been dedicated to the extensive comparison of multiple proteomes. On the other hand, absolute quantification methodologies, which allow the determination of protein concentrations in biological samples, are generally restricted to defined sets of proteins. Depending on the selected analytical procedure, absolute quantification approaches can provide accurate and precise estimations. These analytical performances are crucial for specific applications such as the evaluation of clinical biomarker candidates. According to bioanalytical guidelines, accurate analytical processes require internal standards and quality controls. Regarding MS-based analysis of small molecules, isotope dilution has been recognized as the reference method for internal standardization. However, protein quantification methodologies which rely on the isotope dilution principle have been implemented in the proteomic field only recently. In these approaches, the sample is spiked with defined amounts of isotope-labeled analogue(s) of specific proteolytic peptide(s) (AQUA and QconCAT strategies) or protein(s) (PSAQ strategy). In this review, we present a critical overview of these isotope dilution methodologies.

QT interval variability and cardiac norepinephrine spillover in patients with depression and panic disorder

Suggestions were made that increased myocardial sympathetic activity is reflected by elevated QT variability (dynamic changes in QT interval duration). However, the relationship between QT variability and the amount of norepinephrine released from the cardiac sympathetic terminals is unknown. We thus attempted to assess this relationship. The study was performed in 17 subjects (12 with major depressive disorder and 5 with panic disorder). Cardiac norepinephrine spillover (measured by direct catheter technique coupled with norepinephrine isotope dilution methodology) was assessed before and 4 mo after treatment with selective serotonin reuptake inhibitor (SSRI) antidepressants. The distribution of the cardiac norepinephrine spillover was bimodal, with the majority of patients having values of < or =10 ng/min. There was a positive correlation between cardiac norepinephrine spillover and corrected QT interval (r = 0.7, P = 0.03) but not with any of the QT variability measures. However, in a subgroup of five patients who had high levels of cardiac norepinephrine spillover (>20 ng/min) a tendency for a strong positive correlation with variance of QT intervals (r = 0.9, P = 0.08) was observed. There were significant correlations between the severity of depression and QT variability indexes normalized to the heart rate [QTVi and QT interval/R-R interval (QT/RR) coherence] and between the severity of anxiety and the QT/RR residual and regression coefficient, respectively. Treatment with SSRI antidepressants substantially reduced depression score but did not affect any of the QT variability indexes. We conclude that in depression/panic disorder patients with near-normal cardiac norepinephrine levels QT variability is not correlated with cardiac norepinephrine spillover and is not affected by treatment with SSRI.

Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats

Human milk contains prebiotic oligosaccharides, which stimulate the growth of intestinal bifidobacteria and lactobacilli. It is unclear whether the prebiotic capacity of human milk contributes to the larger bile salt pool size and the more efficient fat absorption in infants fed human milk compared with formula. We determined the effect of prebiotic oligosaccharides on bile salt metabolism in rats. Rats were fed a control diet or an isocaloric diet containing a mixture of galactooligosaccharides (GOS), long-chain fructooligosaccharides (lcFOS), and acidified oligosaccharides (AOS) for 3 wk. We determined synthesis rate, pool size, and fractional turnover rate (FTR) of the primary bile salt cholate by using stable isotope dilution methodology. We quantified bile flow and biliary bile salt secretion rates through bile cannulation. Prebiotic intervention resulted in significant changes in fecal and colonic flora: the proportion of lactobacilli increased 344% (P < 0.01) in colon content and 139% (P < 0.01) in feces compared with the control group. The number of bifidobacteria also increased 366% (P < 0.01) in colon content and 282% in feces after the prebiotic treatment. Furthermore, pH in both colon and feces decreased significantly with 1.0 and 0.5 pH point, respectively. However, despite this alteration of intestinal bacterial flora, no significant effect on relevant parameters of bile salt metabolism and cholate kinetics was found. The present data in rats do not support the hypothesis that prebiotics naturally present in human milk contribute to a larger bile salt pool size or altered bile salt pool kinetics.

Drs. Otte and Whooley Reply

Back to table of contents Previous article Next article Letters to the EditorFull AccessDrs. Otte and Whooley ReplyCHRISTIAN OTTE M.D.MARY A. WHOOLEY M.D.,CHRISTIAN OTTE M.D.Search for more papers by this authorMARY A. WHOOLEY M.D.Search for more papers by this author,Published Online:1 Jan 2008https://doi.org/10.1176/appi.ajp.2007.07091377rAboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail To The Editor: We appreciate the thoughtful comments by Mr. Bayles et al. on our recent article. We agree that sympathetic nervous activation may contribute to the development of left ventricular hypertrophy and that this could be a potential mechanism by which s allele carriers of the 5-HTTLPR might be at increased risk for worse cardiovascular outcome. However, in our cohort there were no differences between s allele carriers and l/l homozygote subjects with regard to left ventricular hypertrophy, defined as a left ventricular mass index >90 g/m 2 by echocardiography (202/383 [54%] of s allele carriers vs. 99/174 [59%] of l/l homozygote subjects). Further, both groups did not differ in functionally relevant variables, such as left ventricular ejection fraction (s allele carriers: mean=62% [SD=10] vs. l/l homozygote subjects: mean=61% [SD=10]) and wall motion score (s allele carriers: mean=1.2 [SD=36] vs. l/l homozygote subjects: mean=1.2 [SD=33]), an index of ischemia. This suggests that greater 24-hour urinary norepinephrine in s allele carriers did not contribute to left ventricular hypertrophy in this study. However, as Dr. Bayles et al. correctly point out, human sympathetic nervous system activity is regionalized, with the outflow to some organs, such as the heart, sometimes being preferentially activated. Indeed, 24-hour urinary norepinephrine as a broad measure of overall sympathetic nervous system activity might lack the sensitivity to detect associations between sympathetic nervous activity and regional processes, such as the development of left ventricular hypertrophy. Given the large number of participants in our epidemiological cohort study, we were unable to use more localized measures of norepinephrine, such as cardiac catheterization techniques coupled with norepinephrine isotope dilution methodology. However, we agree that it would be an interesting next step to examine the association between HTTLPR and bimodal sympathetic activation in depressed patients (1). Certainly, much more work has to be done to elucidate the mechanisms by which 5-HTTLPR, depression, stress, and catecholamine secretion are linked and how they might affect outcome in cardiovascular disease. Hamburg, GermanyThe authors’ disclosures accompany the original article.This letter (doi: 10.1176/appi.ajp.2007.07091377r) was accepted for publication in September 2007. FiguresReferencesCited byDetailsCited ByNone Volume 165Issue 1 January, 2008Pages 137-138THE AMERICAN JOURNAL OF PSYCHIATRY January 2008 Volume 165 Number 1 Metrics PDF download History Published online 1 January 2008 Published in print 1 January 2008

Assay of the Set of All Sudan Azodye (I, II, III, IV, and Para-Red) Contaminating Agents by Liquid Chromatography−Tandem Mass Spectrometry and Isotope Dilution Methodology

A high-throughput mass spectrometric method is presented for the simultaneous detection of Sudan I, II, III, IV, and Para-Red azodyes in foodstuff. The method is based on the use of deuterium-labeled internal standards and atmospheric pressure chemical ionization (APCI) in a triple-quadrupole instrument. The gas-phase breakdown pattern of each labeled and unlabeled analogue displays the naphthoic moiety as a common fragment. The search for the parents of this common species (parent ion scans) allows, by flow injection and in a single run, the evaluation of the presence of each polluting species spiked in typical foodstuffs. A detailed assay of each azodye was performed by LC-APCI and isotope dilution method, through the multiple reaction monitoring approach, using deuterium-labeled internal standards. Sudan dyes can be quantified above the threshold of 10 ppb except for Sudan Para-Red, for which the limit of quantification was 20 ppb, likely due to the different ionization efficiency.

Sympathetic activity in major depressive disorder: identifying those at increased cardiac risk?

Evidence exists linking major depressive disorder (MDD) with clinical cardiovascular events. The importance of the sympathetic nervous system in the generation of cardiac risk in other contexts is established. To examine the importance of the sympathetic nervous system in the generation of cardiac risk in patients with major depressive disorder (MDD). Studies were performed in 39 patients meeting the Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV) criteria for MDD and in 76 healthy subjects. Treatment for patients consisted of selective serotonin reuptake inhibition (SSRI) for 12 weeks. Whole body and cardiac sympathetic activity were examined using noradrenaline isotope dilution methodology and sympathetic nerve recording techniques. Measurement of the extraction of infused tritiated noradrenaline by the heart, and estimation of cardiac dihydroxyphenylglycol production provided direct quantification of neuronal noradrenaline reuptake. Sympathetic activity, particularly in the heart and for the whole body, in patients with MDD followed a bimodal distribution. Elevated values were observed in patients with co-morbid panic disorder (P = 0.006). Consistent with a defect in noradrenaline reuptake, the cardiac extraction of tritiated noradrenaline (0.80 +/- 0.01 versus 0.56 +/- 0.04%, P < 0.001) and cardiac dihydroxyphenylglycol overflow (109 +/- 8 versus 73 +/- 11, P = 0.01) were reduced in patients with MDD. SSRI therapy abolished the excessive sympathetic activation, with whole body noradrenaline spillover falling from 518 +/- 83 to 290 +/- 41 ng/min (P = 0.008). We have identified a subset of patients with MDD in whom sympathetic nervous activity is extraordinarily high, including in the sympathetic outflow to the heart. Treatment with an SSRI may reduce sympathetic activity in a manner likely to reduce cardiac risk.

Evaluating the potential and limitations of double-spiking species-specific isotope dilution analysis for the accurate quantification of mercury species in different environmental matrices

A new double-spiking approach, based on a multiple-spiking numerical methodology, has been developed and applied for the accurate quantification of inorganic mercury (IHg) and methylmercury (MeHg) by GC-ICPMS in different environmental matrices such as water, sediments and a wide range of biological tissues. For this purpose, two enriched mercury species (201MeHg and 199IHg) were added to the samples before sample preparation in order to quantify the extents of the methylation and demethylation processes, and thereby correct the final species concentrations. A critical evaluation of the applicability of this methodology was performed for each type of matrix, highlighting its main advantages and limitations when correcting for the conversion reactions of the species throughout the whole sample preparation procedure. The double-spike isotope dilution (DSIDA) methodology was evaluated by comparing it with conventional species specific isotope dilution (IDA) when analysing both certified reference materials and environmental samples (water, biotissues and sediment). The results demonstrate that this methodology is able to provide both accurate and precise results for IHg and MeHg when their relative concentrations are not too different (ratio MeHg/IHg > 0.05), a condition that holds for most natural waters and biotissues. Significant limitations on the accurate and precise determination of the demethylation factor are however observed, especially for real sediment samples in which the relative concentrations of the species are substantially different (ratio MeHg/IHg < 0.05). A determination of the sources of uncertainty in the methylation/demethylation factors has demonstrated that the accurate and precise measurement of the isotope ratios in the species involved in the transformations is crucial when quantifying the extents of these reactions. Although the double-spike methodology is established as a reference approach that permits the correction of most analytical biases and the accurate quantification of Hg species, some limitations have been identified for the first time in this work.

The role of arbuscular mycorrhiza in legume symbiotic performance

Legumes may respond to non-rhizobial inoculants such as arbuscular mycorrhizal (AM) fungi either through an effect on plant growth or, in addition, through an effect on the function of the legume– Rhizobium symbiosis. We have examined the literature where the application of 15N isotope dilution methodology permits the effect of indigenous AM and AM inoculants to be quantitatively separated into plant-growth-mediated and biological N 2 fixation (BNF)-mediated components. These studies clearly demonstrate the beneficial effects that both indigenous and inoculated AM have on legume growth, N uptake and the proportional dependence of the legume on atmospheric N 2. While the published data allow an assessment of various biological, edaphic and environmental factors that affect the response of various legumes to AM inoculation, they also highlight the paucity of quantitative field data and the lack of understanding of the interaction of legume genotype with AM species with respect to legume symbiotic performance.

Semicarbazide in Canadian bakery products

Levels of semicarbazide were determined by liquid chromatography-tandem mass spectrometry using isotope dilution (13C15N2-semicarbazide) methodology, and they were measured, after hydrolysis in 0.125 M hydrochloric acid and derivatization with 2-nitrobenzaldehyde, as a sum of free and bound semicarbazide. Levels of semicarbazide in 11 bakery products, which were sampled at three time intervals from the same source, varied from not detected (<1 ng g−1) to 560 ng g−1. In some instances, concentrations of semicarbazide varied between batches of the same product, at times more than tenfold, suggesting that the addition of azodicarbonamide to the same product is not standardized in many baking establishments.

Single‐unit analysis of sympathetic nervous discharges in patients with panic disorder

Patients with panic disorder are at increased cardiac risk. While the mechanisms responsible remain unknown, activation of the sympathetic nervous system may be implicated. Using isotope dilution methodology, investigations of whole-body and regional sympathetic nervous activity have failed to show any differences between patients with panic disorder and healthy subjects. Using direct recording of single unit efferent sympathetic vasoconstrictor nerve activity by microneurography we examined sympathetic nervous function in patients with panic disorder more precisely than previously reported. The activity of multiunit and single unit vasoconstrictor sympathetic nerves was recorded at rest at the level of the peroneal nerve in 10 patients diagnosed with panic disorder and in nine matched healthy volunteers. Multiunit sympathetic activity was not different between the two groups (26+/-3 bursts min-1 in patients with panic disorder and 28+/-3 bursts min-1 in controls). The firing frequency of single unit vasoconstrictor neurones was also similar between the two groups (0.38+/-0.09 versus 0.22+/-0.03 Hz). However, the probability of firing during a sympathetic burst was higher in patients with panic disorder compared with healthy controls (45+/-5%versus 32+/-3%, P<0.05). When only the neural bursts during which the vasoconstrictor neurone was active were considered, we found that in patients with panic disorder the neurones tended to fire more often in a 'multiple spike' pattern than in the controls (i.e. the probability of the neurone firing twice was 25+/-3% in patients with panic disorder compared with 14+/-3% in controls). Quantification from single vasoconstrictor unit recording provides evidence of a disturbed sympathetic firing pattern in patients with panic disorder.

The use of enriched 111Cd as tracer to study de novo cadmium accumulation and quantitative speciation in Anguilla anguilla tissues

In order to determine de novo incorporation of cadmium into fish liver and kidney, the stable isotope 111Cd was used as tracer and the European eel (Anguilla anguilla) as model organism. The exposure of animals to 111Cd (100 ng L−1) gave rise to the in vivo dilution of the natural previously existing Cd in the selected tissues and therefore a change of Cd isotope ratios can be measured. The measurement of this new 114Cd/111Cd ratio by ICP-MS, and the subsequent application of mathematical calculations based on the isotope dilution methodology, allowed us to quantitatively discriminate between the previously accumulated natural Cd and the isotopically enriched Cd (de novo incorporated). This was observed for total cadmium in liver and kidney and also for speciated Cd (e.g., that bound to different cytosolic fractions, such as the Metallothionein fraction (MT fraction). In addition, the quantitative results observed pointed out that in liver natural Cd decreased, while kidney received an appreciable amount of endogenous cadmium, supporting the idea that a redistribution of Cd between liver and kidney in fish occurs, as has previously been described in mammals. Moreover, Cd speciation analysis provided the real cadmium balance (natural Cd versus enriched Cd) in the MT fraction, demonstrating quantitatively the Cd mobilization in hepatic and renal MT fractions. On the other hand, since MTs bind Cd, they were capable of in vivo incorporation of the enriched and stable isotope 111Cd during the period of exposure. The separation of the 111Cd labelled MTs was accomplished by anion-exchange (AE) chromatography, which was coupled on-line with ICP-MS detection to investigate the in vivo ability of individual liver MT isoforms for binding cadmium. The 114Cd/111Cd ratio, measured in three individual MT isoforms, was comparatively lower in the predominant MT-2a isoform, which seems to act in the preferential incorporation of the new incoming Cd. On the other hand, the determination of Cd in separated MT isoforms (by an on-line post-column isotope dilution technique) in conjunction with the calculated Cd isotope ratios has allowed for the first time the determination of both recently (exogenous) and previously (endogenous) Cd bound to individual MT isoforms. The results shown here could open the way to the study of other metalloproteins metals preferences, where radioactive techniques are inadequate for metal speciation analysis.

Development of the metabolic syndrome along the female biological life-path

Development of the metabolic syndrome along the female biological life-path

Is obstructive sleep apnea the cause of sympathetic nervous activation in human obesity?

based on findings in experimental models of obesity, the sympathetic nervous system has commonly been assumed to have a determining role in obesity development through its influence on the regulation of energy expenditure. An earlier hypothesis was that sympathetic nervous activity was low in