Speciation analysis of lead in marine animals by using capillary electrophoresis couple online with inductively coupled plasma mass spectrometry.

We herein reported a environment-friendly microwave-assisted extraction used to extract trace lead compounds from marine animals and a ultrasensitive method for the analysis of Pb²âº, trimethyl lead chloride (TML) and triethyl lead chloride (TEL) by using CE-ICP-MS. The extraction method is simple and has a high extracting efficiency. It can be used to completely extract both inorganic lead and organolead in marine animal samples without altering its species. The analytical method has a detection limit as low as 0.012-0.084 ng Pb/mL for Pb²âº, TML, and TEL, and can be used to determine ultratrace Pb²âº, TML, and TEL in marine animals directly without any preconcentration. With the help of above methods, we have successfully determined Pb²âº, TML, and TEL in clam and oyster tissue within 20 min with a RSD (n = 6) < 5% and a recovery of 91-104%. Our results showed that Pb²âº was the main species of lead in clam and oyster, and organolead (TML) was only found in oyster. The proposed method provides a realistic approach for the accurate evaluation of lead pollution in seafood.

Effect of trimethyllead chloride on slowly activating (SV) channels in red beet (Beta vulgaris L.) taproots.

The patch-clamp technique was used to examine the effect of trimethyllead chloride (Met(3)PbCl) on SV channel activity in red beet (Beta vulgaris L.) taproot vacuoles. It was found that in the control bath the macroscopic currents showed the typical slow activation and a strong outward rectification of the steady-state currents. An addition of Met(3)PbCl to the bath solution blocked, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant τ increased several times in the presence of 100 µM trimethyllead chloride at all voltages tested. When single channel properties were analyzed, only little channel activity could be recorded in the presence of 100 µM Met(3)PbCl. Trimethyllead chloride decreased significantly (by about one order of magnitude) the open probability of single channels. The recordings of single channel activity obtained in the presence and absence of Met(3)PbCl showed that organolead only slightly (by ca. 10%) decreased the unitary conductance of single channels. It was also found that Met(3)PbCl diminished significantly the number of SV channel openings, whereas it did not change the opening times of the channels. Taken together, these results suggest that Met(3)PbCl binding site is located outside the channel selectivity filter.

Superstatistics analysis of the ion current distribution function: Met3PbCl influence study.

A novel analysis of ion current time series is proposed. It is shown that higher (second, third and fourth) statistical moments of the ion current probability distribution function (PDF) can yield new information about ion channel properties. The method is illustrated on a two-state model where the PDF of the compound states are given by normal distributions. The proposed method was applied to the analysis of the SV cation channels of vacuolar membrane of Beta vulgaris and the influence of trimethyllead chloride (Met(3)PbCl) on the ion current probability distribution. Ion currents were measured by patch-clamp technique. It was shown that Met(3)PbCl influences the variance of the open-state ion current but does not alter the PDF of the closed-state ion current. Incorporation of higher statistical moments into the standard investigation of ion channel properties is proposed.

Development of a methodology for the simultaneous determination of inorganic and organolead compounds using supercritical fluid extraction followed by gas chromatography-mass spectrometry and its application to environmental matrices.

A method for the extraction of triethyl lead (TEL(+)), trimethyl lead (TML(+)), and Pb(2+) from sand was developed using supercritical modified CO(2)-CH(3)OH extraction and in situ complexation with sodium diethyldithiocarbamate (NaDDTC) using a 2(5) factorial exploratory design is described. The screened variables were (i) pressure (69-193 bar), (ii) temperature (40-150 degrees C), (iii) ligand amount (0-100 mg), (iv) methanol volume (0.0-0.5 mL) and (v) static time (0-45 min). The optimum extraction conditions found were as follow: pressure, 193 bar; temperature, 40 degrees C; amount of NaDDTC, 100 mg; methanol volume, 0.5 mL; static time 45 min; and CO(2) flow rate, 1 mL min(-1). Under these conditions the following recoveries were obtained (TML(+) 97+/-2%, TEL(+) 70+/-5%, and Pb(2+) 100+/-4%). The presence of NaDDTC is not necessary for the extraction of TML(+) and TEL(+), but it is a very significative parameter for Pb(2+). A second experimental design 2(2)+star for temperature and pressure was realized, but the results were not better than those of the first model. SFE extract derivatization was achieved with pentylmagnesium bromide, and target analyte determination was carried out by gas chromatography-mass spectrometry. Detection limits in the full-scan mode were 4, 10, and 39 pg as lead for TMPeL, TEPeL and PbPe(4), respectively. The method was validated with urban dust containing TML(+) (CRM 605. Pb 7.9 +/-1.2 microg kg(-1)) and river sediment containing inorganic lead (GBW08301. Pb 79.0+/-12.0 mg kg(-1)) as reference materials. The proposed method was applied to lead analysis in sand collected from an oil-polluted beach in Chile.

Trialkyllead compounds induce the opening of the MTP pore in rat liver mitochondria.

The interactions of the tributyl, triethyl and trimethyllead compounds with energized mitochondria have been investigated in this paper. It has been shown that the (alkyl)(3)Pb-Cl compounds induce swelling in mitochondria suspended in a sucrose medium. The phenomenon is more marked the higher the lipophilicity and occurs in the following order: (Bu)(3)Pb>(Et)(3)Pb>(Me)(3)Pb. As swelling is inhibited by cyclosporine, this suggests that the swelling is due to the opening of a trans-membrane pore (MTP pore) in the mitochondria. As this pore can be responsible for the inhibition of the ATP synthesis, and, consequently for cell death, the opening of the pore could be one of the reasons for the toxicity of the (alkyl)(3)Pb-X compounds.

Emissions of organo-metal compounds via the leachate and gas pathway from two differently pre-treated municipal waste materials -- A landfill reactor study.

Due to their broad industrial production and use as PVC-stabilisers, agro-chemicals and anti-fouling agents, organo-metal compounds are widely distributed throughout the terrestrial and marine biogeosphere. Here, we focused on the emission dynamics of various organo-metal compounds (e.g., di,- tri-, tetra-methyl tin, di-methyl mercury, tetra-methyl lead) from two different kinds of pre-treated mass waste, namely mechanically-biologically pre-treated municipal solid waste (MBP MSW) and municipal waste incineration ash (MWIA). In landfill simulation reactors, the emission of the organo-metal compounds via the leachate and gas pathway was observed over a period of 5 months simulating different environmental conditions (anaerobic with underlying soil layer/aerated/anaerobic). Both waste materials differ significantly in their initial amounts of organo-metal compounds and their environmental behaviour with regard to the accumulation and depletion rates within the solid material during incubation. For tri-methyl tin, the highest release rates in leachates were found in the incineration ash treatments, where anaerobic conditions in combination with underlying soil material significantly promoted its formation. Concerning the gas pathway, anaerobic conditions considerably favour the emission of organo-metal compounds (tetra-methyl tin, di-methyl mercury, tetra-methyl lead) in both the MBP material and especially in the incineration ash.

Vibrational spectra and ab initio analysis of tert-butyl, trimethylsilyl, trimethylgermyl, trimethylstannyl, and trimethylplumbyl derivatives of 3,3-dimethylcyclopropene IX. 3,3-Dimethyl-1-(trimethylplumbyl)cyclopropene.

The geometrical parameters and quantum mechanical force fields (QMFF's) of 3,3-dimethyl-1-(trimethylplumbyl)cyclopropene (I), 3,3-dimethyl-1-(t-butyl)cyclopropene (II), 3,3-dimethyl-1-(trimethylsilyl)cyclopropene (III), 3,3-dimethyl-1-(trimethylgermyl)cyclopropene (IV), and 3,3-dimethyl-1-(trimethylstannyl)cyclopropene (V) were calculated at the pseudopotential (HF/SDDAll) level. Analysis of the optimised geometrical parameters was performed. The set of scale factors for correction of the pseudopotential QMFF of III was determined using its earlier well-characterised vibrational spectrum. Transferral of the set of scale factors obtained for III to the QMFF's of I, II, IV and V was followed by calculation of the fundamental vibrational frequencies. Analysis of the results for these molecules revealed some peculiarities in the vibrational frequencies obtained at the pseudopotential level.

Past adult lead exposure is linked to neurodegeneration measured by brain MRI.

OBJECTIVE: To determine whether cumulative lead dose in former organolead workers was associated with MRI measures of white matter lesions (WML) and global and structure-specific brain volumes. METHODS: MRIs, tibia lead, and other measures were obtained from 532 former organolead workers with a mean age of 56 years and a mean of 18 years since last occupational exposure to lead. Cumulative lead dose was measured by tibia lead, obtained by X-ray fluorescence, and expressed as microg lead per gram of bone mineral (microg Pb/g). WML were evaluated using the Cardiovascular Health Study grading scale. A total of 21 global and specific brain regions were evaluated. RESULTS: A total of 36% of individuals had WML grade of 1 to 7 (0 to 9 scale). Increasing peak tibia lead was associated with increasing WML grade (p = 0.004). The adjusted OR for a 1 microg Pb/g increase in tibia lead was 1.042 (95% CI = 1.021, 1.063) for a CHS grade of 5+ (> or = 5 vs < 5). In linear regression, the coefficient for tibia lead was negative for associations with all structures. Higher tibia lead was significantly related to smaller total brain volume, frontal and total gray matter volume, and parietal white matter volume. Of nine smaller specific regions of interest, higher tibia lead was associated with smaller volumes for the cingulate gyrus and insula. CONCLUSIONS: These data suggest that cumulative lead dose is associated with persistent brain lesions, and may explain previous findings of a progressive decline in cognitive function.

Simultaneous multi-species determination of trimethyllead, monomethylmercury and three butyltin compounds by species-specific isotope dilution GC-ICP-MS in biological samples.

An accurate and sensitive multi-species species-specific isotope dilution GC-ICP-MS method was developed for the simultaneous determination of trimethyllead (Me3Pb+), monomethylmercury (MeHg+) and the three butyltin species Bu3Sn+, Bu2Sn2+, and BuSn3+ in biological samples. The method was validated by three biological reference materials (CRM 477, mussel tissue certified for butyltins; CRM 463, tuna fish certified for MeHg+; DORM 2, dogfish muscle certified for MeHg+). Under certain conditions, and with minor modifications of the sample pretreatment procedure, this method could also be transferred to environmental samples such as sediments, as demonstrated by analyzing sediment reference material BCR 646 (freshwater sediment, certified for butyltins). The detection limits of the multi-species GC-ICP-IDMS method for biological samples were 1.4 ng g(-1) for MeHg+, 0.06 ng g(-1) for Me3Pb+, 0.3 ng g(-1) for BuSn3+ and Bu3Sn+, and 1.2 ng g(-1) for Bu2Sn2+. Because of the high relevance of these heavy metal alkyl species to the quality assurance of seafood, the method was also applied to corresponding samples purchased from a supermarket. The methylated lead fraction in these samples, correlated to total lead, varied over a broad range (from 0.01% to 7.6%). On the other hand, the MeHg+ fraction was much higher, normally in the range of 80-100%. Considering that we may expect tighter legislative limitations on MeHg+ levels in seafood in the future, we found the highest methylmercury contents (up to 10.6 microg g(-1)) in two shark samples, an animal which is at the end of the marine food chain, whereas MeHg+ contents of less than 0.2 microg g(-1) were found in most other seafood samples; these results correlate with the idea that MeHg+ is usually of biological origin in the marine environment. The concentration of butyltins and the fraction of the total tin content that is from butyltins strongly depend on possible contamination, due to the exclusively anthropogenic character of these compounds. A broad variation in the butylated tin fraction (in the range of <0.3-49%) was therefore observed in different seafood samples. Corresponding isotope-labeled spike compounds (except for trimethyllead) are commercially available for all of these compounds, and since these can be used in the multi-species species-specific GC-ICP-IDMS method developed here, this technique shows great potential for routine analysis in the future.

Species-specific GC/ICP-IDMS for trimethyllead determinations in biological and environmental samples.

An accurate and sensitive species-specific isotope dilution GC/ICPMS method was developed for the determination of trimethyllead (Me3Pb+) in biological and environmental samples. A trimethyllead spike was synthesized from 206Pb-enriched metallic lead by reaction of lead halide with methyllithium and subsequent formation of trimethyllead iodide. The isotopic composition of the spike solution was determined by GC/ICPMS after derivatization with tetraethylborate, and its concentration was determined by reverse isotope dilution analysis. The species-specific GC/ICP-IDMS method was validated by reference material CRM 605 (urban dust) certified for Me3Pb+. The method was also applied to determine the Me3Pb+ content in six biological reference materials (DORM 2, CRM 278, CRM 422, CRM 463, CRM 477, MURST-ISS-A2) and one sediment reference material (CRM 580) for which no certified values of this species exist. The Me3Pb+ concentrations in the biological reference materials vary in the range of 0.3-17 ng g(-1) (as Pb) except for the Antarctic Krill (MURST-ISS-A2), where the concentration was less than the detection limit of 0.09 ng g(-1), which was also found for the sediment. Up to 20% of total lead was methylated in the biological reference materials, whereas much higher methylation fractions were found for mercury. The method was also applied to seafood samples purchased from a supermarket with Me3Pb+ concentrations in the limited range of 0.3-0.7 ng g(-1). On the contrary, the portion of methylated lead in these samples varied over more than 2 orders of magnitude from 0.02 to 7.5%.

Degradation of alkyllead compounds to inorganic lead in contaminated soil.

In glass columns with sandy soil from a former antiknocking agents factory hydrophobic tetraalkyllead was transformed in oxygen-saturated water to inorganic lead. Up to 324 mg l(-1) trialkyllead, but only very little dialkyllead accumulated. After 740 days 49.1+/-6.7% of the organic lead was converted to inorganic lead. Conversion of hydrocarbons was 39.6+/-5.1%. To reduce toxicity of high trialkyllead concentrations the water of soil columns was replaced by tap water after 450d. Trialkyllead in the new water increased again to more than 150 mg l(-1). If the alkyllead-containing water from these columns was diluted to concentrations of alkyllead compounds that were found in the groundwater after air injection (total alkyllead<10 mg l(-1)) and used as a source of alkyllead compounds in columns with non-contaminated sandy soil, elimination of tetra-, tri- and dialkyllead compounds followed first-order kinetics. In the soil 85.8-93.6% of the alkyllead dissappeared in only 170 days with 51% being converted to inorganic lead. This makes in situ remediation reasonable.

Metal cation-methyl interactions in CB11Me12(-) salts of Me3Ge+, Me3Sn+, and Me3Pb+.

Oxidation of Me(6)M(2) (M = Ge, Sn) and Me(4)Pb with the CB(11)Me(12)(*) radical in alkane solvents produced the insoluble salts Me(3)M(+)CB(11)Me(12)(-), characterized by CP-MAS NMR and EXAFS. The cations interact with methyl groups of CB(11)Me(12)(-) with coordination strength increasing from Pb to Ge. Density functional theory (DFT) calculations for the isolated ion pairs, Me(3)M(+)CB(11)Me(12)(-) (M = Ge, Sn), revealed three isomers with the cation above methyl 2, 7, or 12, and not above a BB edge or a BBB triangle. The interaction has a considerable covalent component, with the cation attempting to perform a backside S(E)2 substitution on the methyl carbon. In a fourth less favorable isomer the cation is near methyl 1, inclined toward methyl 2, and interacts with hydrogens. DFT atomic charge distributions and plots of the electrostatic potential on the surface of spheres centered at the CB(11)H(12)(-) and CB(11)Me(12)(-) icosahedra display the effects of uneven charge distribution within the anion and contradict the common belief that the negative charge of the cage anion is concentrated primarily on the cage boron atoms 7-12; in CB(11)Me(12)(-), roughly half is on the cage carbon and the rest on methyls 7-12.

Maternal and umbilical cord blood lead levels: an Istanbul study.

Current mean lead concentrations in umbilical cords and in maternal blood in Istanbul were 1.69 +/- 0.91 (standard deviation) microg/dl and 2.37 +/- 0.89 microg/dl, respectively. These levels were much lower than values reported in previous years. Umbilical cord blood lead levels, which correlated significantly with mothers' blood lead levels, were approximately 70% of the latter. The drastic decrease in blood lead levels likely reflects the reduction in use of tetraalkyl lead in gasoline subsequent to 1989.

Mass-spectrometry assisted heavy-atom derivative screening of human Fc gamma RIII crystals.

A heavy-atom screening method aided by mass spectrometry is described here. Using mass spectrometry, several heavy-atom compounds have been screened in order to obtain potential phasing derivatives for the crystals of a human immunoglobulin Fc receptor, Fc gamma RIII. Of these, HgCl(2), trimethyllead acetate (TMLA), KAu(CN)(2), K(2)PtCl(4) and PbAc(2) reacted with Fc gamma RIII in solution, whereas KAuCl(4), ethylmercuric thiosalicylate (EMTS) and Na(2)WO(4) did not. To validate the mass-spectrometry results, these heavy-atom compounds were also used to soak crystals of Fc gamma RIII and crystallographic data were collected after soaking. The calculated R(iso) indicated that HgCl(2), TMLA, K(2)PtCl(4) and PbAc(2) were likely to form derivatives, whereas KAu(CN)(2) and Na(2)WO(4) were not. The anomalous difference Patterson maps calculated for the HgCl(2) and TMLA derivative data sets were of good quality and can readily be interpreted by hand. In general, the number of binding sites obtained from the crystallographic phase refinement of the derivatives agrees with those obtained from the mass spectrometry, suggesting that mass spectrometry can be applied for rapid searching of suitable heavy-atom derivatives for X-ray crystallography.

Speciation of ionic alkyllead compounds in human urine by gas chromatography-mass spectrometry after butylation through a Grignard reaction.

One analytical procedure for the determination of ionic alkyllead in human urine has been studied. The system consists of the extraction of Me3Pb+, Et3Pb+ and Pb2+ at pH 9.0 with diethyldithiocarbamate to an organic phase. Then, the ionic compounds are butylated with BuMgCl and the final organic solution is analyzed by GC-MS-SIM. The elimination of both foam and gels in the extraction step and the general procedure for the urine are discussed. The recovery of compounds ranges from 105.1% for Me3Pb+ to 97.2% for Et3Pb+ using hexane as extracting agent and detection limits are 18.4 pg/ml of Me3Pb+ and 19.2 pg/ml of Et3Pb+ in urine. The speciation of ionic alkylleads in the urine of a petrol station worker showed a value of 27.9 pg/ml of Me3Pb+ in urine and Et3Pb+ was below the detection limit.

Interactions of trialkyllead compounds with rat liver mitochondria.

The interactions of two trialkyllead (TAL) compounds, (trimethyl)Pb-Cl and (tributyl)Pb-Cl, with mitochodria from rat liver have been studied. A stimulation of the respiratory rate induced by the trialkyllead compounds added at low doses was observed which was not dependent on the presence of chloride in the medium. In contrast with the major current view, we propose that trialkyllead compounds behave as uncouplers of the oxidative phosphorylation and not (or not only) as Cl-/OH- exchangers. In fact the present results suggest that the TAL compounds enter the mitochondria as (alkyl)3Pb+ cations and are extruded as electroneutral (alkyl)3 Pb-OH compounds, the overall result being the transport of a proton through the membrane as in the case of classical uncouplers. The uncoupling effect could explain the toxicity of the compounds as a result of the decrease in the energy level of the cell. Furthermore, such a mechanism, in which the uptake of TAL compounds is supposed to be driven by a negative potential, could explain their preferential toxicity for neuronal cells, which maintain a higher negative-inside potential than most other cell types.

Voltage-activated calcium channel currents of rat dorsal root ganglion cells are reduced by trimethyl lead.

Using the conventional whole-cell patch-clamp recording technique with cultured neurones of rat dorsal root ganglions (DRG), we analysed the effects of trimethyl lead (TML) on voltage-activated calcium channel currents. TML reduces voltage-activated calcium channel currents in a dose-dependent manner, with a threshold concentration below 0.5 microM and a total reduction of the current ( > or =80% of the control current) at concentrations above 50 microM. Half of the current is abolished at TML concentrations between 1 and 5 microM. The action is irreversible and is not voltage dependent. After application of TML the current decreases with each activation of the channel until a steady state is reached after 8-12 min, when the channel was activated every 10 s. The channel had to be in the open state for TML to act. TML is a potent compound for reducing voltage activated calcium channel currents. These effects of TML must be taken into account in explaining the neurotoxic effects of this organic metal compound.

Organotypic spinal cord culture in serum-free fibrin gel: a new approach to study three-dimensional neurite outgrowth and of neurotoxicity testing: Effects of modulating the actin and tubulin dynamics and protein kinase activities.

Spinal cord explants from embryonic day seven (E7) chicken embryos were cultured without serum and in the presence of aprotinine, in a three-dimensional fibrin matrix. These conditions promote a robust, radial, unfasciculated outgrowth of neurites that are tipped by elaborate growth cones. Routinely after 5 days, the neurite outgrowth intensity (NOI) was determined by measuring the optical density of the immunostained neurites (image analysis program OPTIMAS version 5.2) within defined areas, extending radially for up to 3 mm from the explant border. A dose-dependent inhibition of NOI was determined for the cytoskeleton-affecting drugs nocodazole (half maximal inhibition ([I50]), 0.02 microM), taxol ([I50], 0.016 microM), cytochalasin D ([I50], 0.006 microM), and tetramethyl lead ([I50], 0.05 microM). Likewise, NOI was decreased in a dose-dependent fashion by ML-9 and RO-31-8220, inhibitors of myosin-light chain kinase and protein kinase C (PKC), respectively. The addition of 1,2-dioctanoyl-s,n-glycerol, a potent activator of PKC, led, at 5 microM, to an increase and at 30 and 60 microM to a decrease in NOI. The described system provides a rapid, reproducible, and quantitative assay for the effects of exogenous factors on the mode and intensity of neurite outgrowth.

Trimethyl lead neurotoxicity in the rat: changes in glial fibrillary acidic protein (GFAP).

The literature on the toxicology of lead provides little evidence of the neurotoxicity of organic lead compounds. Toxicant-induced changes in the concentration of glial fibrillary acidic protein (GFAP) in the brain may help clarify at which stage of neurotoxicity astrocytes are affected and whether GFAP may provide an index of toxicity. Male F344 rats (> 42 days old) were exposed to 0 (control), 8 or 16 ppm lead as trimethyl lead (TMPb) in drinking water for up to 14 days. Weight Gain was significantly reduced in both exposed groups. Control rats had the expected brain regional pattern of GFAP concentration with the highest in the hippocampus and cerebellum and lowest in the cerebral cortex. The hippocampus was the region very sensitive to TMPb, with increased GFAP in rats exposed to 8 and 16 ppm TMPb with decreases in GFAP in rats exposed to 8 and 16 ppm TMPb for 14 days. There was a significant time-response in rats exposed to 8 ppm TMPb with decreases in GFAP on day 7 and increases on day 14. A hypothesis concerning this biphasic change in GFAP concentrations is discussed. The results indicate that GFAP may be used to indicate the role of the astrocyte in the neurotoxicity of TMPb. GFAP concentration, as biomarker of TMPb effect, was as sensitive to TMPb as body weight and thus may provide a marker of neurotoxicity.

Trimethyllead acetate: a first-choice heavy atom derivative for protein crystallography.

The three-dimensional conformation of a protein provides a wealth of biochemical information and with the advent of cloning techniques that allow the preparation of proteins almost at will, a renewed interest has arisen in the crystallographic determination of protein structures. As in any research technique, however, there are often many difficulties encountered in an X-ray crystallographic investigation. One of these is the "phase problem." Although in recent years there has been considerable progress in the development of techniques for phase determination, including the use of molecular replacement and multiple wavelength measurements, the multiple isomorphous replacement method is still the most successful method for obtaining a three-dimensional structure. Here we report the use of trimethyllead acetate as a heavy atom compound of first choice in the preparation of an isomorphous heavy atom derivative.