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PubMed1982

Stable isotopes of lithium: in vivo differential distribution between plasma and cerebrospinal fluid.

Stable and radioactive isotopes of an element have been utilized to study a variety of complex phenomena including metabolic pathways and enzyme reactions. It is generally assumed that isotopes of an element are not treated in a differential manner. Nevertheless we have demonstrated that the two stable isotopes of lithium (Li), one of mass 6 and the other with a mass of 7, are differentially taken up by red blood cells with 6-8% higher quantities of 6-lithium (6Li) being accumulated. We report here on the in vivo pharmacokinetics of Li isotopes. Nineteen adult cats were given a single dose of either 6LiCl by gastric intubation (1 meq/kg) and 2 weeks later the treatment was crossed. Plasma concentrations of the two Li isotopes were determined. In another series of studies 19 adult cats were given either 6LiCl or 7LiCl by intubation, cerebrospinal fluid (CSF) and blood samples were collected simultaneously and isotropic Li concentrations of the spinal fluid and plasma were determined. The average plasma concentration of 6Li was higher than 7-lithium (7Li) between 2 and 9 hr postadministration. The elimination curve for both isotopes had an initial fast and later slow component. For the slow component 6Li had an average half-life of 12.9 and 7Li 15.9 hr. Comparison of the CSF/plasma ratio for 6Li and for 7Li indicated that the ratios were higher for 6Li than 7Li 2-6 hr after administration. These data suggest that factors in addition to concentration gradients influence the transport of Li isotopes across the blood-brain barrier. These studies demonstrate the acute pharmacokinetics of the two Li isotopes are not the same in compartmental distribution and rate of disappearance from plasma. It is conceivable 6Li may have a greater toxic or therapeutic effect than 7Li in the treatment of manic-depressive illness.

Administration, Oral↗
PubMed1998

Magnesium isotopic abundance measurement in humans: comparison of two mass spectrometric methods.

Pharmacokinetic studies using stable isotopes of magnesium as tracers need to determine the isotopic abundance in biological media by means of mass spectrometry. Of mass spectrometric techniques, electronic impact-mass spectrometry (EI-MS) and inductively coupled plasma-mass spectrometry (ICP-MS) can be used. We have measured the isotopic abundance in plasma and urinary samples and compared the precision and accuracy of these two methods. Graphical representations showed that mean differences were close to 0, there was no obvious relationship between the difference and the mean, and no systematic bias was evidenced at low or high isotopic abundance. This was shown for isotopic abundance of either 25 Mg and 26 Mg. EI-MS is able to measure magnesium isotopic abundance with an intra-day precision between 0.14 and 0.45 per cent and with an inter-day precision between 0.20 and 1.23 per cent. ICP-MS exhibited an intra-day precision between 0.01 and 0.06 per cent and an inter-day precision between 0.01 and 0.15 per cent. Our results showed that, despite the similar isotopic abundances in biological samples obtained with the two methods, a large difference in precision clearly favours ICP-MS in studies of magnesium behaviour using stable isotopes.

Biological Availability↗
PubMed2003

Tracing the pathways of neotropical migratory shorebirds using stable isotopes: a pilot study.

We evaluated the potential use of stable isotopes to establish linkages between the wintering grounds and the breeding grounds of the Pectoral Sandpiper (Calidris melanotos), the White-rumped Sandpiper (Calidris fuscicollis), the Baird's Sandpiper (Calidris bairdii), and other Neotropical migratory shorebird species (e.g., Tringa spp.). These species molt their flight feathers on the wintering grounds and hence their flight feathers carry chemical signatures that are characteristic of their winter habitat. The objective of our pilot study was to assess the feasibility of identifying the winter origin of individual birds by: (1) collecting shorebird flight feathers from several widely separated Argentine sites and analyzing these for a suite of stable isotopes; and 2) analyzing the deuterium and 18O isotope data that were available from precipitation measurement stations in Argentina. Isotopic ratios (delta13C, delta15N and delta34S) of flight feathers were significantly different among three widely separated sites in Argentina during January 2001. In terms of relative importance in separating the sites, delta34S was most important, followed by delta15N, and then delta13C. In the complete discriminant analysis, the classification function correctly predicted group membership in 85% of the cases (jackknifed classification matrix). In a stepwise analysis delta13C was dropped from the solution, and site membership was correctly predicted in 92% of cases (jackknifed matrix). Analysis of precipitation data showed that both deltaD and delta18O were significantly related to both latitude and longitude on a countrywide scale (p < 0.001). Other variables, month, altitude, explained little additional variation in these isotope ratios. Several issues were identified that will likely constrain the degree of accuracy one can expect in predicting the geographic origin of birds from Argentina. There was unexplained variation in isotope ratios within and among the different wing feathers from individual birds. Such variation may indicate that birds are not faithful to a local site during their winter stay in Argentina. There was significant interannual variation in the deltaD and delta18O of precipitation. Hence, specific locations may not have a constant signature for some isotopes. Moreover, the fractionation that occurs in wetlands due to evaporation significantly skews local deltaD and delta18O values, which may undermine the strong large-scale gradients seen in the precipitation data. We are continuing the research with universities in Argentina with a focus on expanding the breadth of feather collection and attempting to resolve the identified issues.

Animals↗
PubMed1983

Calculation of substrate turnover rate in stable isotope tracer studies.

The determination of substrate turnover rate with stable isotope-labeled compounds has advantages of being safe and applicable in the study of children and pregnant women. Currently, a majority of these studies has been performed with primed constant-rate infusion technique. The isotope enrichment of the substrate in the plasma is measured by gas chromatography-mass spectrometry (GCMS). The turnover rate is then calculated from steady-state kinetics. A number of different equations have been used by various investigators for this purpose. Based on theoretical consideration and experimental data, it is concluded that the equation P = [(1/E) - 1] I or P = (y/x) I should be used for a correct turnover rate calculation, where P is turnover rate in mumol X kg-1, E the isotope enrichment, I the infusion rate in mumol X kg-1 X min-1, and y/x the mole ratio of tracee to tracers. A GCMS standard curve constructed from isotope enrichment versus isotope peak abundance should be used for the former equation, whereas a standard curve constructed from mole ratio (x/y) versus isotope ratio should be used for the latter equation. Interchange of standard curves or use of other equations will produce erroneous turnover rate. This is especially significant when a low enriched isotope-labeled compound is used as a tracer.

Blood Glucose↗
PubMed2006

Flushing time and storage effects on the accuracy and precision of carbon and oxygen isotope ratios of sample using the Gasbench II technique.

Continuous-flow isotope ratio mass spectrometry interfaced with a Gasbench II is used for automated and faster analyses of delta(13)C and delta(18)O in water, carbonate, and air samples that are accurate and highly precise. Prior to online chemistry and measurement using the Gasbench technique, rubber septa-capped glass vials are routinely flushed to remove air. Due to the small amounts of sample gas required for isotope analyses using current techniques, care should be taken to properly flush these vials to avoid contamination of sample gas with air. Our results indicate that isotopic composition of sample CO(2) gas remains constant when 10 mL vials are flushed (rate of 100 mL/min) for > or =600 s, whereas for vials flushed <600 s, the isotopic composition becomes substantially lighter with decreasing time of flushing, which affects the accuracy of analyses. This largely depends on the isotopic composition (and volume) of air that still remains after flushing. This effect is more pronounced on delta(18)O than on delta(13)C of sample CO(2) gas because there is very little carbon in the air. After 24 h storage in vials with punctured septa, both delta(13)C and delta(18)O of CO(2) become isotopically heavier compared with first day analyses, suggesting time-dependent changes in isotopic composition. The magnitude of shift depends on the concentration and the isotopic composition of CO(2) in laboratory air as well as on fractionation due to outflow of sample gas or inflow of air via punctured septa. Contamination of sample gas with air can be observed as a secondary peak on chromatograms that precedes sample peaks, and the intensity of these peaks depends on the amount of air. Such peaks are always present with short flushing times. For accuracy and better precision, irrespective of the magnitude of the secondary peaks, the analyses should be discarded if these appear in the chromatograms.

Journal Article↗
PubMedDate not supplied

Tools and procedures for quantitative microbeam isotope ratio imaging by secondary ion mass spectrometry.

In this work we demonstrate the use of secondary ion mass spectrometry (SIMS) combined with the Lispix image processing program (Bright 1995) to generate quantitative isotope ratio images from a test sample of a calcium-aluminum rich inclusion from the Allende meteorite that is known to contain discrete mineral grains with perturbed Mg isotopic ratios. Using 19.5 keV impact O- primary ion bombardment and detection of positive secondary ions, microbeam imaging SIMS has allowed us to identify, from the isotope ratio images, enrichments in the 26Mg/24Mg isotope ratio of approximately 5-15% in selected mineral grains. Using custom image processing software, each isotopic ratio image is corrected on an individual pixel basis for a number of factors including detector dead-time, mass bias effects, and isobaric interferences. We have developed procedures for correlating the isotopic images with polarized optical microscopy so that targeted mineral grains could be identified for further SIMS analysis. Finally, additional image processing tools have been developed to allow for pixel-by-pixel evaluation of the influence of detector dead-time and count rate errors on the isotopic ratio images and for correlation of the isotopic images with elemental distribution maps.

Journal Article↗
PubMed2004

Deuterium stable isotope ratios as tracers of water resource use: an experimental test with rock doves.

Naturally-occurring deuterium stable isotope ratios can potentially be used to trace water resource use by animals, but estimating the contribution of isotopically distinct water sources requires the accurate prediction of isotopic discrimination factors between water inputs and an animal's body water pool. We examined the feasibility of using estimates of water fluxes between a bird and its environment with a mass-balance model for the deuterium stable isotope ratio of avian body water (deltaDbody) to predict isotopic discrimination factors. Apparent fractionation and thus discrimination factors were predicted to vary with the proportion of an animal's total water losses than could be attributed to evaporative processes. To test our ability to predict isotopic discrimination, we manipulated water intake and evaporative water loss in rock doves (Columba livia) by providing them with fresh water or 0.15 M NaCl solution in thermoneutral or hot environments. After we switched the birds from drinking water with deltaD=-95 per thousand VSMOW (Vienna Standard Mean Ocean Water) to enriched drinking water with deltaD=+52 per thousand VSMOW, steady-state deltaDbody was approached asymptotically. The equilibrium deltaDbody was enriched by 10-50 per thousand relative to water inputs. After isotopic equilibrium was reached, the degree of enrichment was positively related (r2=0.34) to the fraction of total water loss that occurred by evaporation (revap/rH2O)supporting the major prediction of the model. The variation we observed in discrimination factors suggests that the apparent fractionation of deuterium will be difficult to predict accurately under natural conditions. Our results show that accurate estimates of the contribution of different water sources to a bird's body water pool require large deuterium isotopic differences between the sources.

Animals↗
PubMed2005

The US national isotope program: current status and strategy for future success.

Since their introduction in the 1940s, peaceful use of stable isotopes and radioisotopes in the United States has expanded continuously. Today, new isotopes for diagnostic and therapeutic uses are not being developed, critical isotopes for national security are in short supply, and demand for isotopes critical to homeland security exceeds supply. While commercial suppliers, both domestic and foreign, can only meet specific needs, the nation needs a consistent, reliable supply of radioactive and stable isotopes for research, medical, security, and space power applications. The national isotope infrastructure, defined as both facilities and trained staff at national laboratories and universities, is in danger of being lost due to chronic underfunding. With the specific recommendations given herein, the US Department of Energy may realign and refocus its Isotope Program to provide a framework for a successful National Isotope Program.

Government Agencies↗
PubMed2005

Size-fractionated thorium isotopes (228Th, 230Th, 232Th) in surface waters in the Jiulong River estuary, China.

Thorium isotopes (228Th, 230Th, 232Th and 234Th) are useful tracers for studying particle dynamics and trace element scavenging in marine environments. In this study, surface waters were collected along a salinity gradient from the Jiulong River estuary, China, for determination of activity concentrations of 228Th, 230Th and 232Th in different size fractions, namely, the >53 microm, 10-53 microm, 2-10 microm, 0.4-2 microm, 10 kDa-0.4 microm and the <10 kDa fractions. Our results indicated that the activity concentrations of 228Th, 230Th and 232Th in the Jiulong River estuarine waters were significantly higher than most of the previously reported values in coastal and oceanic seawaters, suggesting a higher lithogenic U and Th contribution from the Jiulong River Basin. When normalized to the particulate mass concentration, the activity concentrations of the three thorium isotopes decreased with increasing particle size, demonstrating the important role of surface areas of particles in controlling the scavenging of thorium from the water column. The partitioning of three thorium isotopes showed a common characteristic, i.e., the >53 microm fraction had the least share (0-1%), while the 10-53 microm fraction had the largest share of Th isotopes. The average value of the 230Th/232Th activity ratio (230Th/232Th)(A.R.) increased from 0.8 in the >53 microm fraction to 3.7 in the 10 kDa-0.4 microm fraction, indicating that the radiogenic Th isotopes are preferentially scavenged by the small size particles. (230Th/232Th)(A.R.) in the <10 kDa and 10 kDa-0.4 microm fractions were similar, however, suggesting a similar chemical composition and/or equilibrium partitioning between the low molecular weight and colloidal Th. It was very interesting to note that the geochemical behaviors of the three Th isotopes were different from each other. Dissolved 228Th had the highest concentration in the mid-salinity region, showing a non-conservative behavior with additional input. In contrast, dissolved 232Th showed a concave profile, indicating a net removal of 232Th during the mixing of fresh water with seawater. The behavior of Th isotopes in the <10 kDa fraction followed those in the dissolved phases. The difference in geochemical behaviors among three Th isotopes was ascribed to their different sources in the estuary.

China↗
PubMed1997

Identification of historical lead sources in roof dusts and recent lake sediments from an industrialized area: indications from lead isotopes.

X-ray fluorescence and stable lead (Pb) isotopic analyses have been undertaken on dusts, known from microscopic investigation to contain significant quantities of industrially- and urban-derived particulate matter, present in the roof cavities of houses in the Illawarra region (N.S.W., Australia), with the objective of examining the historic record of Pb pollution. All investigated houses contained in excess of 250 micrograms g-1 Pb, with dwellings close to a copper smelter, in a large industrial complex including a major steelworks, containing higher (> 2500 micrograms g-1) Pb concentrations. The isotopic composition in the dusts, expressed here as 206Pb/204Pb, is relatively constant at 17.0, irrespective of dwelling age or distance from the industrial complex. Contamination of the dusts by Pb sourced from paint cannot explain the isotopic uniformity of the dust samples. Isotopic modelling indicates that the dusts contain Pb derived from the copper smelter, gasoline-air Pb and a minor contribution from coal-utilising sources. Lead loading was also investigated in the adjacent lagoon, which acts as a natural sink for particulate matter in the Illawarra region. Isotopic data and modelling indicate that one natural and four anthropogenic sources contribute to the Pb burden of this lagoon. The natural source consists of Permian rocks cropping out in the catchment area which have a 206Pb/204Pb of approximately 18.7. The suggested anthropogenic sources are an old disbanded base-metal (Pb) smelter (206Pb/204Pb approximately 16.2-16.3), the copper smelter (206Pb/204Pb approximately 17.9), gasoline-air derived Pb (206Pb/204Pb approximately 16.4-16.5) and industries utilising coal, for example the recently closed thermal coal-fired power station (206Pb/204Pb approximately 18.9). The relative contributions of the base-metal (mainly lead) smelter and gasoline-air Pb in the sediment can only be partly assessed due to the isotopic similarity of these sources. Likewise the natural background and coal source (e.g. power station) contributions can only be estimated from historical data. Age estimations for sediment cores, using 137Cs, provide some control on these assessments. Near surface sediments in the lagoon have a relatively constant 206Pb/204Pb of 17.6-17.7, irrespective of sample location. Isotopic calculations, together with records of particulate matter pollution emissions, indicate a link between the Pb in roof dusts (206Pb/204Pb approximately 17.0) and Pb contamination of the near surface (upper 20 cm) lagoonal sediments via a homogeneous, non-unique source of lead whose isotopic composition closely matches that of the dusts. Over the last 5 decades, atmospheric fallout of Pb-bearing particulate matter appears to have been the dominant pathway for addition of Pb to the lagoon and dwellings in the Illawarra region.

Australia↗
PubMed2006

Chromatographic purification for the determination of dissolved silicon isotopic compositions in natural waters by high-resolution multicollector inductively coupled plasma mass spectrometry.

A procedure is described for accurate Si isotope ratio measurements by multicollector inductively coupled plasma mass spectrometry (MC-ICPMS). Dissolved silicon was preconcentrated and separated from other elements present in natural surface waters using anion-exchange chromatography. The optimized procedure provides virtually complete elimination of major inorganic constituents while maintaining Si recovery in excess of 97%. High-resolution capabilities of MC-ICPMS used in this study allow interference-free measurements of 28Si and 29Si isotopes using conventional solution nebulization sample introduction without aerosol desolvation. Owing to the magnitude of polyatomic ion contributions in the region of mass 30, mostly from 14N16O+, measurements of the 30Si isotope can be affected by tailing of the interference signals, making exact matching of analyte and nitric acid concentrations in all measurement solutions mandatory. Isotope abundance ratio measurements were performed using the bracketing standards approach and on-line correction for mass-bias variations using an internal standard (Mg). Uncertainties, expressed as 95% confidence intervals, for replication of the entire procedure are better than +/-0.18/1000 for delta29Si and +/-0.5/1000 for delta30Si. For the first time with MC-ICPMS, the quality of Si isotope abundance ratio measurements could be verified using a three-isotope plot. All samples studied were isotopically heavier than the IRMM-018 Si isotopic reference material.

Journal Article↗
PubMed2006

Determination of the S isotope composition of methanesulfonic acid.

Sulfur (S) isotopes have been used to apportion the amount of biogenic and anthropogenic sulfate in remote environments, an important parameter that is used to model the global radiation budget. A key assumption in the apportionment calculations is that there is little isotope selectivity as reduced compounds such as dimethyl sulfide (DMS) are oxidized. This paper describes a method to determine, for the first time, the S isotope composition of methanesulfonic acid (MSA), the product of DMS oxidation. The isotope composition of MSA was measured directly by EA-IRMS and was used as an isotope reference for the method. Synthetic mixtures approximating the conditions expected for aerosol MSA samples were prepared to test this method. First, MSA solutions were measured alone and then in combination with MSA and SO4(2-). In synthetic mixtures, SO4(2-) was separated from MSA by precipitating it as BaSO4 prior to preparation of MSA for isotope analysis. The delta 34S value for MSA solutions was -2.6 per thousand (SD +/- 0.4 per thousand), which is not different from the delta 34S obtained from MSA filtrate after precipitating SO4(2-) from the mixture (-2.7 +/- 0.3 per thousand). However, these values are offset from direct EA-IRMS analysis of MSA used as the isotope reference by -1.1 +/- 0.2 per thousand, and this must be accounted for in reporting MSA measurements. The S isotope measurements using this method approach a limiting value above 300 microg of MSA. This is approximately equal to the MSA found in 20,000 m3 of air, assuming ambient concentrations of approximately 15 ng m(-3). Three samples of MSA from the Pacific Ocean measured using this technique have an average delta 34S value of +17.4 +/- 0.7 per thousand.

Journal Article↗
PubMed1983

Enzymes of the beta-ketoadipate pathway in Pseudomonas putida: primary and secondary kinetic and equilibrium deuterium isotope effects upon the interconversion of (+)-muconolactone to cis,cis-muconate catalyzed by cis,cis-muconate cycloisomerase.

Primary and secondary kinetic and secondary equilibrium deuterium isotope effect studies on the cis,cis-muconate cycloisomerase catalyzed interconversion of cis,-cis-muconate (CCM) and (+)-muconolactone (ML) have been performed. The primary and solvent kinetic deuterium isotope effects upon Vmax for the reactions of (+)-[5R-2H]muconolactone in water (HOH) and (+)-muconolactone in deuterium oxide (DOD) to form cis,cis-muconate are about 2.5-2.6 with the heavier isotopic reactions being the slower ones. The secondary equilibrium isotope effect for the formation cis,-cis-[2,3,4,5-2H4] muconate from (+)-[2,3,4,5S-2H4]muconolactone is 1.32 for KH/KD = [( cis,cis-muconate]/[(+)-muconolactone])/ [(+)-[2,3,4,5S-2H4]muconolactone]) and agrees well with the measured value of 1.45 on the basis of the fumarase reaction [Cook, P. F., Blanchard, J. S., & Cleland, W. W. (1980) Biochemistry 19, 4853-4858]. The secondary kinetic deuterium isotope effect determined by the equilibrium perturbation method [Cleland, W. W. (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M. H., & Northrop, D. B., Eds.) pp 153-175, University Park Press, Baltimore, MD] for the conversion of cis,cis-[2,3,4,5-2H4] muconate to (+)-[2,3,4,5S-2H4]muconolactone is 0.66, expressed as (VmaxCCM(H)/KmCCM(H]/(VmaxCCM(D)/KmCCM(D]. From the equilibrium and kinetic secondary deuterium isotope effects, the calculated value for the kinetic secondary deuterium isotope effect for the reverse reaction, (VmaxML(H)/KmML(H]/(VmaxML(D)/KmML(D], is about 0.96.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipates↗
PubMed1985

Secondary isotope effects and structure-reactivity correlations in the dopamine beta-monooxygenase reaction: evidence for a chemical mechanism.

The chemical mechanism of hydroxylation, catalyzed by dopamine beta-monooxygenase, has been explored with a combination of secondary kinetic isotope effects and structure-reactivity correlations. Measurement of primary and secondary isotope effects on Vmax/Km under conditions where the intrinsic primary hydrogen isotope effect is known allows calculation of the corresponding intrinsic secondary isotope effect. By this method we have obtained an alpha-deuterium isotope effect, Dk alpha = 1.19 +/- 0.06, with dopamine as substrate. The beta-deuterium isotope effect is indistinguishable from one. The large magnitude of Dk alpha, together with our previous determination of a near maximal primary deuterium isotope effect of 9.4-11, clearly indicates the occurrence of a stepwise process for C-H bond cleavage and C-O bond formation and hence the presence of a substrate-derived intermediate. To probe the nature of this intermediate, a structure-reactivity study was performed by using a series of para-substituted phenylethylamines. Deuterium isotope effects on Vmax and Vmax/Km parameters were determined for all of the substrates, allowing calculation of the rate constants for C-H bond cleavage and product dissociation and dissociation constants for amine and O2 loss from the enzyme-substrate ternary complex. Multiple regression analysis yielded an electronic effect of p = -1.5 for the C-H bond cleavage step, eliminating the possibility of a carbanion intermediate. A negative p value is consistent with formation of either a radical or a carbocation; however, a significantly better correlation is obtained with sigma p rather than sigma p+, implying formation of a radical intermediate via a polarized transition state. Additional effects determined from the regression analyses include steric effects on rate constants for substrate hydroxylation and product release and on KDamine, consistent with a sterically restricted binding site, and a positive electronic effect of p = 1.4 on product dissociation, ascribed to a loss of product from an enzyme-bound Cu(II)-alkoxide complex. These results lead us to propose a mechanism in which O-O homolysis [from a putative Cu(II)-OOH species] and C-H homolysis (from substrate) occur in a concerted fashion, circumventing the formation of a discrete, high energy oxygen species such as hydroxyl radical. The substrate and peroxide-derived radical intermediates thus formed undergo a recombination, kinetically limited by displacement of an intervening water molecule, to give the postulated Cu(II)-alkoxide product complex.

Copper↗
PubMed1988

Glutathione reductase: solvent equilibrium and kinetic isotope effects.

Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D2O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456.

Glutathione↗
PubMed1996

Beta-secondary and solvent deuterium kinetic isotope effects on beta-lactamase catalysis.

Beta-Secondary and solvent deuterium kinetic isotope effects have been determined for the steady-state kinetic parameters V/K and V for turnover of a depsipeptide substrate, m-[[(phenylacetyl)glycyl]-oxy]benzoic acid, and of a beta-lactam substrate, penicillanic acid, by three typical class A beta-lactamases and a class C beta-lactamase. The isotope effects on alkaline hydrolysis of these substrates have been used as a frame of reference. The effect of the transition state conformation of the substrates in determining the beta-secondary isotope effects has been explicitly considered. The inverse beta-secondary isotope effects on both V/K and V for the class A enzymes with both substrates indicate transition states where the carbonyl group of the scissile bond has become tetrahedral and therefore reflect typical acyl-transfer transition states. The solvent isotope effects indicate that enzyme deacylation (as reflected in V for the Staphylococcus aureus PC1 beta-lactamase) may be a classical general-base-catalyzed hydrolysis but that there is little proton motion in the enzyme acylation transition state (as revealed by V/K) for the TEM beta-lactamase and Bacillus cereus beta-lactamase I. These results provide kinetic support for the conjecture made on structural grounds that class A beta-lactamases employ an asymmetric double-displacement mechanism. The isotope effects on V/K for the class C beta-lactamase of Enterobacter cloacae P99 suggest an acyl-transfer transition state for the penicillin, although, as for the class A enzymes, without significant proton motion. On the other hand, the V/K transition state for depsipeptide does not seem to involve covalent chemistry. Suggestive of this conclusion are the measured beta-secondary isotope effect of 1,002 +/- 0.012 and the inverse solvent isotope effect. These results provide an example of a significant difference between the kinetics of turnover of a beta-lactam and a depsipeptide by a beta-lactamase. The V transition state for both substrates with the P99 beta-lactamase probably involves acyl-transfer (deacylation) where the conformation of the acyl-enzyme is closely restricted. The conformations of acyl-enzymes of the PC1 and P99 beta-lactamases correlate to the (different) dispositions of general base catalysts at their active sites.

Acylation↗
PubMed2001

Isotopic homogeneity of iron in the early solar nebula.

The chemical and isotopic homogeneity of the early solar nebula, and the processes producing fractionation during its evolution, are central issues of cosmochemistry. Studies of the relative abundance variations of three or more isotopes of an element can in principle determine if the initial reservoir of material was a homogeneous mixture or if it contained several distinct sources of precursor material. For example, widespread anomalies observed in the oxygen isotopes of meteorites have been interpreted as resulting from the mixing of a solid phase that was enriched in 16O with a gas phase in which 16O was depleted, or as an isotopic 'memory' of Galactic evolution. In either case, these anomalies are regarded as strong evidence that the early solar nebula was not initially homogeneous. Here we present measurements of the relative abundances of three iron isotopes in meteoritic and terrestrial samples. We show that significant variations of iron isotopes exist in both terrestrial and extraterrestrial materials. But when plotted in a three-isotope diagram, all of the data for these Solar System materials fall on a single mass-fractionation line, showing that homogenization of iron isotopes occurred in the solar nebula before both planetesimal accretion and chondrule formation.

Journal Article↗
PubMed1998

Solvent kinetic isotope effects of human placental alkaline phosphatase in reverse micelles.

Human placental alkaline phosphatase was embedded in a reverse micellar system prepared by dissolving the surfactant sodium bis(2-ethylhexyl) sulphosuccinate (Aerosol-OT) in 2,2, 4-trimethylpentane. This microemulsion system provides a convenient instrumental tool to study the possible kinetic properties of the membranous enzyme in an immobilized form. The pL (pH/p2H) dependence of hydrolysis of 4-nitrophenyl phosphate has been examined over a pL range of 8.5-12.5 in both aqueous and reverse micellar systems. Profiles of log V versus pL were Ha-bell shaped in the acidic region but reached a plateau in the basic region in which two pKa values of 9.01-9.71 and 9.86-10.48, respectively, were observed in reverse micelles. However, only one pKa value of 9.78-10.27 in aqueous solution was detected. Profiles of log V/K versus pL were bell-shaped in the acidic region. However, they were wave-shaped in the basic region in which a residue of pKa 9.10-9.44 in aqueous solution and 8.07-8.78 in reverse micelles must be dehydronated for the reaction to reach an optimum. The V/K value shifted to a lower value upon dehydronation of a pKa value of 9.80-10.62 in aqueous solution and 11.23-12.17 in reverse micelles. Solvent kinetic isotope effects were measured at three pL values. At pL 9.5, the observed isotope effect was a product of equilibrium isotope effect and a kinetic isotope effect; at pL 10.4, the log V/K value was identical in water and deuterium. The deuterium kinetic isotope effect on V/K was 1.14 in an aqueous solution and 1.16 in reverse micelles. At pL 11.0 at which the log V values reached a plateau in either solvent system, the deuterium kinetic isotope effect on V was 2.08 in an aqueous solution and 0.62 in reverse micelles. Results from a proton inventory experiment suggested that a hydron transfer step is involved in the transition state of the catalytic reaction. The isotopic fractionation factor (pi) for deuterium for the transition state (piT) increased when the pH of the solution was raised. At pL 11.0, the piT was 1.07 in reverse micelles, which corresponds to the inverse-isotope effect of the reaction in this solvent system. Normal viscosity effects on kcat and kcat/Km were observed in aqueous solution, corresponding to a diffusional controlled physical step as the rate-limiting step. We propose that the rate-limiting step of the hydrolytic reaction changes from phosphate releasing in aqueous solution to a covalent phosphorylation or dephosphorylation step in reverse micelles.

Alkaline Phosphatase↗