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PubMed1989

Alpha-secondary isotope effects in the lipoxygenase reaction.

Isotope effects for the oxidation of [5,6,8,9,11,12,14,15-3H]arachidonic acid catalyzed by soybean lipoxygenase and by 5-lipoxygenase were measured. This labeling pattern represents substitution at each of the vinylic hydrogens of the substrate. The observed isotope effect for soybean lipoxygenase was 1.16 +/- 0.02 and for 5-lipoxygenase was 1.11 +/- 0.05. These isotope effects are inconsistent with any change in hybridization (sp2 to sp3) at the vinylic carbons prior to or during the rate-determining step and are concluded to be most consistent with the formation of a carbanion-like intermediate or transition state. In contrast, the oxidation of arachidonic acid by Ce(IV), which is thought to proceed via a cation radical intermediate, exhibited at most a small isotope effect (1.02 +/- 0.01). The reduction potential for the cation radical formed from arachidonic acid in this reaction is estimated to be 2.7 V vs NHE by comparison of the rates of oxidation of arachidonic acid and cyclohexene by Ce(IV). This is similar to the potential for the cation radical of 2-butene. No isotope effect (1.00 +/- 0.03) was observed in the 5-lipoxygenase reaction for conversion of the initially formed product 5-hydroperoxyeicosatetraenoic acid to the epoxide leukotriene A4. From this it is concluded that there is little carbon-oxygen bond formation prior to or during the rate-determining step for epoxide formation.

Animals↗
PubMed1990

Glutathione reductase: comparison of steady-state and rapid reaction primary kinetic isotope effects exhibited by the yeast, spinach, and Escherichia coli enzymes.

Kinetic parameters for NADPH and NADH have been determined at pH 8.1 for spinach, yeast, and E. coli glutathione reductases. NADPH exhibited low Km values for all enzymes (3-6 microM), while the Km values for NADH were 100 times higher (approximately 400 microM). Under our experimental conditions, the percentage of maximal velocities with NADH versus those measured with NADPH were 18.4, 3.7, and 0.13% for the spinach, yeast, and E. coli enzymes, respectively. Primary deuterium kinetic isotope effects were independent of GSSG concentration between Km and 15Km levels, supporting a ping-pong kinetic mechanism. For each of the three enzymes, NADPH yielded primary deuterium kinetic isotope effects on Vmax only, while NADH exhibited primary deuterium kinetic isotope effects on both V and V/K. The magnitude of DV/KNADH at pH 8.1 is 4.3 for the spinach enzyme, 2.7 for the yeast enzyme, and 1.6 for the E. coli glutathione reductase. The experimentally determined values of TV/KNADH of 7.4, 4.2, and 2.2 for the spinach, yeast, and E. coli glutathione reductases agree well with those calculated from the corresponding DV/KNADH using the Swain-Schaad expression. This suggests that the intrinsic primary kinetic isotope effect on NADH oxidation is fully expressed. In order to confirm this conclusion, single-turnover experiments have been performed. The measured primary deuterium kinetic isotope effects on the enzyme reduction half-reaction using NADH match those measured in the steady state for each of the three glutathione reductases.(ABSTRACT TRUNCATED AT 250 WORDS)

Deuterium↗
PubMed1990

Studies of the mechanism of the delta 5-3-ketosteroid isomerase reaction by substrate, solvent, and combined kinetic deuterium isotope effects on wild-type and mutant enzymes.

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by a conservative tautomeric transfer of the 4 beta-proton to the 6 beta-position using Tyr-14 as a general acid and Asp-38 as a general base [Kuliopulos, A., Mildvan, A. S., Shortle, D., & Talalay, P. (1989) Biochemistry 28, 149]. On deuteration of the 4 beta-position (97.0%) of the substrate, kcat(H)/kcat(4 beta-D) is 6.1 in H2O and 6.3 in D2O. The solvent isotope effect, kcat(H2O)/kcat(D2O), is 1.6 for both the 4 beta-H and 4 beta-D substrates. Mutation of Tyr-55 to Phe lowers kcat 4.3-fold; kcat(H)/kcat/4 beta-D) is 5.3 in H2O and 5.9 in D2O, and kcat(H2O)/kcat(D2O) with the 4 beta-H and 4 beta-D substrates is 1.5 and 1.7, respectively, indicating concerted general acid-base catalysis in either the enolization or the ketonization step of both the wild-type and the Tyr-55----Phe (Y55F) mutant enzymes. An additional slow step occurs with the Y55F mutant. Smaller isotope effects on Km are used to estimate individual rate constants in the kinetic schemes of both enzymes. On deuteration of the 4 alpha-position (88.6%) of the substrate, the secondary isotope effect on kcat/Km corrected for composition is 1.11 +/- 0.02 with the wild-type enzyme and 1.12 +/- 0.02 with the Y55F mutant. These effects decrease to 1.06 +/- 0.01 and 1.07 +/- 0.01, respectively, when the 4 beta-position is also deuterated, thereby establishing these to be kinetic (rather than equilibrium) secondary isotope effects and to involve a proton-tunneling contribution. Deuteration of the 6-position of the substrate (92.0%) produces no kinetic isotope effects on kcat/Km with either the wild-type (1.00 +/- 0.01) or the Y55F mutant (1.01 +/- 0.01) enzyme. Since a change in hybridization from sp3 to sp2 occurs at C-4 only during enolization of the substrate and a change in hybridization at C-6 from sp2 to sp3 occurs only during reketonization of the dienol intermediate, enolization of the substrate constitutes the concerted rate-limiting step. Concerted enolization is consistent with the right angle or antarafacial orientations of Tyr-14 and Asp-38 with respect to the enzyme-bound substrate and with the additive effects on kcat of mutation of these catalytic residues [Kuliopulos, A., Talalay, P., & Mildvan, A. S. (1990) Biophys. J. 57, 39a].

Deuterium↗
PubMed1977

Determination of the rate-limiting steps for malic enzyme by the use of isotope effects and other kinetic studies.

Isotope effects have been measured with Mg2+ as the activator and L-malate labeled with deuterium or tritium at carbon 2 as the substrate over the pH range 4-10. Comparison of the nearly pH-independent deuterium-isotope effect on V/Kmalate of 1.5 with the tritium effect of 2.0 by the method of Northrop (Northrop, D.B. (1975), Biochemistry 14, 2644) gives limits on the true effect of deuterium substitution on the bond-breaking step of 5-8 in the forward reaction and 4-6.5 in the reverse direction. Comparison of the deuterium effect on V/K with the 13C-isotope effect of 1.031 reported by Schimerlik et al. (Schimerlik, M.I., Rife, J.E., and Cleland, W.W. (1975), Biochemistry 14, 5347) allows the deduction that at pH 8 reverse hydride transfer is six to eight times faster than decarboxylation, which is thus largely rate limiting for the catalytic reaction. The absence of a deuterium-isotope effect on V at pH 7-8 and comparison of the Ki of pyruvate as an uncompetitive inhibitor of the forward reaction and a substrate for the reverse reaction indicate that at neutral pH the release of TPNH from enzyme-reduced triphosphopyridine nucleotide (E-TPNH) is the rate-limiting step in the forward direction. The observation of a deuterium effect on V that approaches 3 at pH 4 and 10 shows, however, that, at very low and very high pH, hydride transfer may become partly rate limiting. In the reverse reaction, the probable rate-limiting step at pH 7 is the isomerization of E-TPNH, while at pH 8.5 and above V becomes too large to measure and appears infinite. Substitution of Co2+, Ni2+, or low levels of Mn2+ for Mg2+ gives similar deuterium-isotope effects, although the values of V and Kmalate vary considerably with metal. The kinetics of Mn2+ show pronounced negative cooperativity, with Km values of 7 muM and 5 mM for concentration ranges from 4 to 100 muM and over 1 mM.

Animals↗
PubMed1976

Studies on the intramolecular and intermolecular kinetic isotope effects in pyruvate carboxylase catalysis.

A deuterium kinetic isotope effect of 2.1 was observed when [2H3]pyruvate was used as the substrate for pyruvate carboxylase. The effect is on Vmax/Km alone and disappears at infinite substrate concentration. This is interpreted to mean that the slowest step in the overall catalysis is in the half-reaction involving the carboxylation of enzymebiotin by ATP and HCO3-. A tritium intramolecular isotope effect of 4.8 and an intermolecular effect of 1.2 were also observed. The former was interpreted as the isotope effect on the "effective kcat", while the latter the one on V max/Km. With these data, the rate constant for binding of pyruvate was estimated to be 4.5 X 10(6) M-1 min-1, and the deuterium kinetic isotope effect on the catalytic step to be 3.1. Relative values for various rate constants were also obtained. Fluoropyruvate was also shown to be a substrate, reacting six times slower. A deuterium kinetic isotope effect of 1.5 was observed, which remained even at infinite substrate concentration. This is interpreted to mean that the slowest step in the overall catalysis is now the carboxylation of fluoropyruvate.

Deuterium↗
PubMed1998

Evaluation of cytochrome P450 mechanism and kinetics using kinetic deuterium isotope effects.

In this paper two hypotheses are tested: (i) the active oxygen species is similar in energetics for all cytochrome P450 (CYP) enzymes and (ii) linear free-energy relationships can be used to evaluate the mechanism of the reaction of these enzymes. A series of intramolecular isotope effects were determined and compared for CYPs 1A2, 2B1, 2C9, 2E1, and P450cam. The results indicate that the isotope effects are very similar for each of these isoforms of P450 and that the first hypothesis is likely to be true. Attempts to establish a linear free-energy relationship were only moderately successful: log Vmax = 0.11sigma+p + 1.73; r2 = 0.588. It was determined, through the use of intermolecular isotope effects, that the rates of hydrogen atom abstraction are masked. Thus, the second hypothesis is found to be false. This is likely to be a general result for CYP reactions, and linear free-energy relationships can only be used to determine the mechanism under very special circumstances. In all cases, the rate-limiting step should be evaluated with isotope effect experiments before any mechanistic conclusions can be drawn. If the intermolecular isotope effects are found to be masked, no mechanistic conclusion can be drawn from the linear free-energy relationship study.

Binding, Competitive↗
PubMed2003

"Nonavailable" soil cadmium is bioavailable to snails: evidence from isotopic dilution experiments.

Isotopic dilution techniques were initially used to evaluate the bioavailability of trace metals contained in soils to plants, i.e., the phytoavailability. Here, we use for the first time an isotopic technique to evaluate the zooavailability of cadmium (Cd), i.e., the bioavailability to an animal organism. A terrestrial invertebrate, the snail Helix aspersa, was exposed for 14 days to a polluted soil that was spiked with 109Cd. Isotopic composition of snail tissues was then determined, allowing the computing of the L value, which can be considered as a biological estimate of the bioavailable Cd pool in the soil. It showed that the bioavailable pool of Cd to H. aspersa represented 58% of the total soil Cd. The Evalue, a chemical estimate of the soil Cd labile pool, was calculated with short-term isotopic exchange kinetics experiments and a time-dependent model describing the isotopic exchange over time. The E(14 days) value indicated that the labile soil Cd pool represented 49.6% of the total soil Cd. The L value was significantly higher than the E value, showing that snails accessed the nonlabile soil Cd pool, generally considered as nonbioavailable. The nonlabile pool contributed for 16% to the total Cd accumulated by snails. These results showed that the uptake of Cd in the nonlabile pool by soil organisms could be important and should be considered in risk assessment procedures for metal polluted soils.

Animals↗
PubMed2004

Identifying the sources of subsurface contamination at the Hanford Site in Washington using high-precision uranium isotopic measurements.

In the mid-1990s, a groundwater plume of uranium (U) was detected in monitoring wells in the B-BX-BY Waste Management Area at the Hanford Site in Washington. This area has been used since the late 1940s to store high-level radioactive waste and other products of U fuel-rod processing. Using multiple-collector ICP source magnetic sector mass spectrometry, high-precision uranium isotopic analyses were conducted of samples of vadose zone contamination and of groundwater. The isotope ratios 236U/238U, 234U/238U, and 238U/235U are used to distinguish contaminant sources. On the basis of the isotopic data, the source of the groundwater contamination appears to be related to a 1951 overflow event at tank BX-102 that spilled high-level U waste into the vadose zone. The U isotopic variation of the groundwater plume is a result of mixing between contaminant U from this spill and natural background U. Vadose zone U contamination at tank B-110 likely predates the recorded tank leak and can be ruled out as a significant source of groundwater contamination, based on the U isotopic composition. The locus of vadose zone contamination is displaced from the initial locus of groundwater contamination, indicating that lateral migration in the vadose zone was at least 8 times greater than vertical migration. The time evolution of the groundwater plume suggests an average U migration rate of approximately 0.7-0.8 m/day showing slight retardation relative to a groundwater flow of approximately 1 m/day.

Environmental Monitoring↗
PubMed2005

Enrichment of stable carbon and hydrogen isotopes during anaerobic biodegradation of MTBE: microcosm and field evidence.

The conventional approach to evaluate biodegradation of organic contaminants in groundwater is to demonstrate an increase in the concentration of transformation products. This approach is problematic for MTBE from gasoline spills because the primary transformation product (TBA) can also be a component of gasoline. Compound-specific stable isotope analysis may provide a useful alternative to conventional practice. Changes in the delta13C and deltaD of MTBE during biodegradation of MTBE in an anaerobic enrichment culture were compared to the delta13C and deltaD of MTBE in groundwater at nine gasoline spill-sites. The stable isotopes of hydrogen and carbon were extensively fractionated during anaerobic biodegradation of MTBE. The stable isotope enrichment factor for carbon (epsilonC) in the enrichment cultures was -13 (-14.1 to -11.9 at 95% confidence level), and the hydrogen enrichment factor (epsilonH) was -16 (-21 to -11 at 95% confidence level). The isotope enrichment factors for carbon and hydrogen during anaerobic biodegradation indicate that the first reaction is enzymatic hydrolysis of the O-Cmethyl bond. The ratio of epsilonH to epsilonC was consistent between the enrichment culture and the field site that provided the inoculum, and with the other eight sites, suggesting a common degradation pathway. Compound-specific isotope evidence is discussed in terms of its utility for monitoring in situ biodegradation, in particular, for measuring how much MTBE was degraded. For the studied field sites, significant biodegradation of the original mass of MTBE is suggested, in some cases exceeding 90%.

Accidents↗
PubMed2002

Using equilibrium isotope effects to detect intramolecular OH/OH hydrogen bonds: structural and solvent effects.

A comparative (1)H NMR study of partially deuterated 1,3- and 1,4-diols has demonstrated that intramolecular hydrogen bonds of different geometry can give rise to equilibrium isotope shifts of opposite sign in hydrogen-bond-accepting solvents such as DMSO-d(6), acetone-d(6), and THF-d(8). The sign inversion is interpreted in terms of the ability of solvent molecules to form competitive intermolecular hydrogen bonds with the diol and in terms of the limiting chemical shifts for the interior and exterior hydroxyl groups. Deuterium is shown to prefer the intermolecular solvent hydrogen bond by 10.9 +/- 0.5 cal/mol for 1,4-diol 3 dissolved in DMSO-d(6) at room temperature. Pyridine-d(5) is shown to be capable of amplifying positive (downfield) isotope shifts measured in DMSO-d(6), in some cases by as much as a factor of 3. Its use is demonstrated for the assignment of the syn or anti relative configuration of 2,4-pentanediol and for the amplification of isotope shifts used to detect intramolecular hydrogen bonds in alpha- and beta-cyclodextrin. Studies in apolar solvents such as CD(2)Cl(2) and benzene-d(6) reveal that the isotope shift is negative (upfield) for all hydrogen bond geometries studied. Larger isotope shifts are measured in benzene-d(6), and a rationale for this amplification is presented. The use of apolar solvents is particularly useful for assigning the syn or anti configuration of 2,4-pentanediol.

Alcohols↗
PubMed2004

Characterization of fluxional hydrogen-bonded complexes of acetic acid and acetate by NMR: geometries and isotope and solvent effects.

1H, (2)H, and (13)C NMR spectra of enriched CH(3)(13)COOH acid without and in the presence of tetra-n-butylammonium acetate have been measured around 110 K using a liquefied Freon mixture CDF(3)/CDF(2)Cl as a solvent, as a function of the deuterium fraction in the mobile proton sites. For comparison, spectra were also taken of the adduct CH(3)(13)COOH.SbCl(5) 1 and of CH(2)Cl(13)COOH under similar conditions, as well as of CH(3)(13)COOH and CH(3)(13)COO(-) dissolved in H(2)O and D(2)O at low and high pH at 298 K. The low temperatures employed allowed us to detect several well-known and novel hydrogen-bonded complexes in the slow hydrogen bond exchange regime and to determine chemical shifts and coupling constants as well as H/D isotope effects on chemical shifts from the fine structure of the corresponding signals. The measurements show that self-association of both carboxylic acids in Freon solution gives rise exclusively to the formation of cyclic dimers 2 and 3 exhibiting a rapid degenerate double proton transfer. For the first time, a two-bond coupling of the type (2)J(CH(3)COOH) between a hydrogen-bonded proton and the carboxylic carbon has been observed, which is slightly smaller than half of the value observed for 1. In addition, the (1)H and (2)H chemical shifts of the HH, HD, and the DD isotopologues of 2 and 3 have been determined as well as the corresponding HH/HD/DD isotope effects on the (13)C chemical shifts. Similar "primary", "vicinal", and "secondary" isotope effects were observed for the novel 2:1 complex "dihydrogen triacetate" 5 between acetic acid and acetate. Another novel species is the 3:1 complex "trihydrogen tetraacetate" 6, which was also characterized by a complex degenerate combined hydrogen bond- and proton-transfer process. For comparison, the results obtained previously for hydrogen diacetate 4 and hydrogen maleate 7 are discussed. Using an improved (1)H chemical shift-hydrogen bond geometry correlation, the chemical shift data are converted into hydrogen bond geometries. They indicate cooperative hydrogen bonds in the cyclic dimers; i.e., widening of a given hydrogen bond by H/D substitution also widens the other coupled hydrogen bond. By contrast, the hydrogen bonds in 5 are anticooperative. The measurements show that ionization shifts the (13)C signal of the carboxyl group to low field when the group is immersed in water, but to high field when it is embedded in a polar aprotic environment. This finding allows us to understand the unusual ionization shift of aspartate groups in the HIV-pepstatin complex observed by Smith, R.; Brereton, I. M.; Chai, R. Y.; Kent, S. B. H. Nature Struct. Biol. 1996, 3, 946. It is demonstrated that the Freon solvents used in this study are better environments for model studies of amino acid interactions than aqueous or protic environments. Finally, a novel correlation of the hydrogen bond geometries with the H/D isotope effects on the (13)C chemical shifts of carboxylic acid groups is proposed, which allows one to estimate the hydrogen bond geometries and protonation states of these groups. It is shown that absence of such an isotope effect is not only compatible with an isolated carboxylate group but also with the presence of a short and strong hydrogen bond.

Journal Article↗
PubMed2005

Intrinsic isotope effects on benzylic hydroxylation by the aromatic amino acid hydroxylases: evidence for hydrogen tunneling, coupled motion, and similar reactivities.

Deuterium kinetic isotope effects for hydroxylation of the methyl group of 4-methylphenylalanine have been used as a probe of the relative reactivities of the hydroxylating intermediates in the aromatic amino acid hydroxylases phenylalanine, tyrosine, and tryptophan hydroxylase. When there are three deuterium atoms in the methyl group, all three enzymes exhibit an intrinsic isotope effect of about 13. The temperature dependence of the isotope effect is consistent with moderate tunneling, with the extent of tunneling identical for all three enzymes. In the case of phenylalanine hydroxylase, the presence of the regulatory domain has no effect on the values. The intrinsic primary and secondary isotope effects were determined using 4-methylphenylalanine containing one or two deuterium atoms in the methyl group. With one deuterium atom, the intrinsic primary and secondary effects have average values of 10 and 1.1, respectively. With two deuterium atoms, the primary effects decrease to 7.4 and the secondary effect increases to 1.3, consistent with coupled motion of the primary and secondary hydrogens. The results with all three enzymes are consistent with a hydrogen abstraction mechanism. The similarities of the isotope effects and extent of tunneling establish that the reactivities of the hydroxylating intermediates in the three enzymes are essentially identical.

Benzene↗
PubMed2001

Authentication of vegetable oils by bulk and molecular carbon isotope analyses with emphasis on olive oil and pumpkin seed oil.

The authenticity of vegetable oils consumed in Slovenia and Croatia was investigated by carbon isotope analysis of the individual fatty acids by the use of gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS), and through carbon isotope analysis of the bulk oil. The fatty acids from samples of olive, pumpkin, sunflower, maize, rape, soybean, and sesame oils were separated by alkaline hydrolysis and derivatized to methyl esters for chemical characterization by capillary gas chromatography/mass spectrometry (GC/MS) prior to isotopic analysis. Enrichment in heavy carbon isotope ((13)C) of the bulk oil and of the individual fatty acids are related to (1) a thermally induced degradation during processing (deodorization, steam washing, or bleaching), (2) hydrolytic rancidity (lipolysis) and oxidative rancidity of the vegetable oils during storage, and (3) the potential blend with refined oil or other vegetable oils. The impurity or admixture of different oils may be assessed from the delta(13)C(16:0) vs. delta(13)C(18:1) covariations. The fatty acid compositions of Slovenian and Croatian olive oils are compared with those from the most important Mediterranean producer countries (Spain, Italy, Greece, and France).

Croatia↗
PubMed1999

Stable isotope characterization of milk components and whey ethanol.

A multi-isotopic study of several components of milk has been carried out on commercial samples and on milk produced in feeding experiments involving different kinds of diets originating from C(3) or C(4) photosynthetic metabolisms and exhibiting a relatively wide range of isotope ratios. The dispersion of the carbon, hydrogen, and nitrogen isotope parameters of dried matter and of the lactose, protein, and lipid components has been estimated. In addition, the carbohydrates were represented by the site specific isotope ratios (SNIF-NMR) of ethanol resulting from standardized fermentation of lactose. The rates of response of the isotopic parameters to changes in the feeding materials is slower for the minor components, proteins, and lipids than for lactose and ethanol. For similar diets, the nonexchangeable sites of lactose and the methyl site of ethanol, in particular, are relatively enriched in deuterium in the case of polygastric animals, cow, goat, and ewe, as compared to the monogastric species, sow and mare, and woman. From an analytical point of view, the carbon and hydrogen parameters of ethanol provide efficient criteria for identifying a whey origin with respect to other agricultural and fossil sources.

Animals↗
PubMed1997

13C and (15)N Kinetic Isotope Effects on the Decarboxylation of 3-Carboxybenzisoxazole. Theory vs Experiment.

Nitrogen and carbon kinetic isotope effects were measured on the decarboxylation of 3-carboxybenzisoxazole at room temperature. The nitrogen isotope effect in acetone is 1.0312 +/- 0.0006. The carbon isotope effect shows no dependence on the solvent polarity: 1.0448 +/- 0.0007 in 1,4-dioxane, 1.0445 +/- 0.0001 in acetonitrile, 1.0472 +/- 0.0013 in DMF, and 1.0418 +/- 0.0027 in water. These isotope effects were modeled theoretically at the semiempirical (AM1, PM3, SAM1) and ab initio (up to B3LYP/6-31++G) levels. The comparison of the theoretical and experimental results leads to the conclusion that none of the theory levels employed is capable of quantitatively predicting these isotope effects.

Journal Article↗
PubMed2005

Tautomerism of sterically hindered schiff bases. Deuterium isotope effects on 13C chemical shifts.

A series of sterically hindered o-hydroxy Schiff bases derived from o-hydroxyaceto- and benzophenones with very short intramolecular hydrogen bonds were described qualitative and quantitatively by deuterium isotope effects on (13)C chemical shift, (n)DeltaC(XD), (n)DeltaF(XD), (1)J(N,H) coupling constants, deltaNCH(3) chemical shifts and UV spectra. All the investigated compounds are found to be tautomeric. The tautomeric character is described by the signs of the deuterium isotope effects on the (13)C chemical shifts. For the 3-nitro-5-chloro derivatives at low temperature, the equilibrium is shifted almost fully toward the proton transferred form in CD(2)Cl(2). Intrinsic deuterium isotope effects on chemical shifts of these compounds as well as (1)J(N,H) coupling constants suggest that a zwitterionic resonance form is dominant for the proton transferred form. Structures, (1)H, (19)F, and (13)C chemical shifts, and deuterium isotope effects on (13)C chemical shifts are calculated by ab initio methods. The potential energy functions and the total deuterium isotope effects are calculated, and they are shown to correspond well with the experimental findings.

Journal Article↗
PubMed2003

Contaminant source apportionment by PIMMS lead isotope analysis and SEM-image analysis.

By combining scanning electron microscopy (SEM) image analysis and laser ablation plasma ionisation multi-collector mass spectrometry (LA-PIMMS), high precision lead isotope analyses can be obtained from individual metal-rich particles. Soils from Wolverhampton and Nottingham were sampled on the basis of high Pb concentrations or brownfield location. Pressed powder pellets of each were rastered by LA-PIMMS to obtain a bulk Pb-isotope signature. The results plot along an apparent mixing line between the major sources of lead contamination in the UK, that is UK ore deposits and alkyl-lead from petrol additives (Australian ore). Two particularly lead-rich soils were chosen to investigate the lead distribution and isotope variability between size and density fractions. The fine-grained and low-density fractions contained most of the lead and have Pb-isotope ratios comparable with the bulk soils. By contrast, the small, lead-enriched denser fractions contained only a minor proportion of the total lead but Pb-isotope signatures indicating relative enrichment in one or other of the end-members from the mixing line. Further characterisation of individual Pb-rich grains is in progress.

Environmental Monitoring↗
PubMed1984

The response of tumour cells to radiation and cytotoxic drugs--a comparison of clonogenic and isotope uptake assays.

We have carried out a series of experiments to compare the response to radiation and drugs of cells disaggregated from solid tumours as assayed by clonogenic survival and by an isotope incorporation method. This latter assay consisted of measuring the 24 h uptake of tritium labelled thymidine into cells plated in liquid medium upon a layer of semi-solid agar. The isotope was administered 4 days after plating. For cells from the RIF-1 mouse tumour, good agreement was seen between response to radiation, adriamycin, vincristine and CCNU as measured by the two assays. The two curves for radiation response, for example, showed similar shoulders and subsequent exponential regions. For cells from xenografts of the NCI-H69 human small cell lung cancer line, the response to radiation was dose-related for both assays, but the curve for clonogenic assay was about twice as steep as that for isotope uptake. For a range of five cytotoxic drugs, good agreement was seen between the two assays over the first 1 1/2 decades of response but with a tendency for the isotope uptake curve to plateau with further increasing drug dose. It appears that, at least for these two well-defined experimental tumour systems, the isotope uptake assay can provide a rapid quantitative assessment of cellular drug and radiation sensitivity comparable to that provided by clonogenic assay but in a much shorter period of time.

Animals↗