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PubMed2003

Stable carbon and nitrogen isotope signatures indicate recovery of marine biota from sewage pollution at Moa Point, New Zealand.

Stable carbon and nitrogen isotopes have been used to assess sewage contamination of a sewage outfall, discharging milli-screened effluent into Moa Point Bay, New Zealand, and monitor the recovery of flora and fauna after the outfall's closure. An initial study characterising the extent of the discharge and the effects on seaweed (Ulva lactuca L.), blue mussels (Mytilus galloprovincialis) and limpets (Cellana denticulata) from the area, showed effects of the sewage discharge on flora and fauna were localised within in the bay. The immediate area surrounding the discharge area was found to contain limited biodiversity, with an abundance of Ulva lactuca, a bright green lettuce-like seaweed, typically found in areas with high nutrient input, limpets and small blue mussels. The nitrogen isotopic signature (delta15N) is shown to be a good tracer of sewage pollution in seaweed and associated grazers (i.e. limpets) as a result of the increased contribution of urea and ammonia to seawater nitrogen derived from the effluent. The carbon isotopic signature (delta13C) is suggested as a more appropriate sewage tracer for mussels, which filter feed the effluent's particulate organic matter from the water. Lower carbon:nitrogen ratios were found in Ulva lactuca sampled from around the outfall region compared to uncontaminated control sites. However carbon:nitrogen ratios do not vary significantly amongst shellfish species. After closure, monitoring continued for 9 months and showed that the carbon and nitrogen isotopic signatures of algae (Ulva lactuca L.) returned to similar control site levels within 3 months. Limpet and blue mussels (Cellana denticulata and Mytilus galloprovincialis) showed slower recovery times than the Ulva lactuca, with detectable levels of the sewage-derived carbon and nitrogen remaining in the animal's tissue for up to 9 months.

Animals↗
PubMed2003

A dual isotope approach to identify denitrification in groundwater at a river-bank infiltration site.

The identification of denitrification in the Torgau sand and gravel aquifer, Germany, was carried out by a dual isotope method of measuring both the delta 15N and delta 18O in NO3-. Samples were prepared by an anion exchange resin method (Silva et al., J. Hydrol. 228 (2000) 22) with a modification to the AgNO3-drying process from a freeze-drying to an oven-drying method. The occurrence of denitrification in the aquifer was confirmed by comparing the reduction of dissolved oxygen, dissolved organic carbon and NO3- concentrations with the dual isotope signatures. High nitrate concentrations were associated with low delta 15N and delta 18O values, and vice versa. The denitrification accords with a Rayleigh equation with calculated enrichment factors of epsilon = -13.62@1000 for delta 15N and epsilon = -9.80@1000 for delta 18O. The slope of the straight-line relationship between the delta 15N and delta 18O data demonstrated that the enrichment of the heavy nitrogen isotope was higher by a factor of 1.3 compared with the heavy oxygen isotope. It is concluded that the identification of this factor is a useful means for confirming denitrification in future groundwater studies.

Environmental Monitoring↗
PubMed2002

Investigating the sources of the labile fraction in sediments from silicate-drained rocks using trace elements, and strontium and lead isotopes.

Elemental concentrations of the trace elements Rb, Sr, Th, Pb, Zn, Cu, Ni, Cd and Sb, as well as Sr and Pb isotopic compositions, were determined on the labile fraction (called acid-extracted matter AEM, Négrel et al., Chem. Geol. 166 (2000) 271-85) of soil and sediment along two small rivers located in the centre of France, one flowing from basalt, the other one on granite-gneiss. Oxide-mineral form in AEM (i.e. Fe-Mn oxides), acts as the main carrier phase. Analysis of the relationships between the trace elements, and lead and strontium isotopes allows the origin of the elements (i.e. natural and anthropogenic) and their history, both in the sediment and soil from the small watersheds to be assessed. Lead-isotope compositions in AEM display large fluctuation in the two watersheds and show a large scatter between natural input (basalt and granite), atmospheric input from gasoline, and input from past mining activities. On the basaltic watershed, Pb and other trace elements like Sb deriving from past mining waste are mainly related to atmospheric origin, while on the granitic terrain Pb originates from mineralization. The Sr-isotope compositions of AEM, water and residues are similar in the watershed draining basalt. On the watershed draining granite, AEM and streamwater shows 87Sr/86Sr ratios similar in the upstream part of the watershed and a divergence between the two ratios appears in the downstream part of the watershed implying that oxides have precipitated upstream and are not re-equilibrated during their transport downstream.

France↗
PubMed2003

Advances in quantitative proteomics via stable isotope tagging and mass spectrometry.

The high-throughput identification and accurate quantification of proteins are essential components of proteomic strategies for studying cellular functions and processes. Techniques that are largely based on stable isotope protein or peptide labeling and automated tandem mass spectrometry are increasingly being applied in quantitative proteomic studies. Over the past year, significant progress has been made toward improving and diversifying these technologies with respect to the methods for stable isotope labeling, process automation and data processing and analysis. Advances in stable isotope protein labeling and recent biological studies that used stable isotope based quantitative proteomics techniques are reviewed.

Affinity Labels↗
PubMed2005

Temporal host-parasite relationships of the wild rabbit, Oryctolagus cuniculus (L.) as revealed by stable isotope analyses.

Natural abundances of the stable isotopes, 15N/14N (delta15N) and 13C/12C (delta13C), were used to study temporal host-parasite relationships of the wild rabbit, Oryctolagus cuniculus (L.). During the 12-month sampling period, temporal isotopic shifts in delta15N were noted for dietary vegetation, host rabbit faeces and fur, but not for muscle or stomach contents. delta15N varied temporally for the parasitic cestode species, Mosgovoyia pectinata but not for Cittotaenia denticulata. Similarly, intestinal parasitic nematodes had apparent species-specific delta15N patterns. Only rabbit fur and intestinal parasitic nematodes did not exhibit temporal shifts in delta13C. Overall, host faeces and stomach contents were isotopically indistinct as a likely consequence of coprophagy. Relative to their host, parasitic nematodes were 15N-enriched, consistent with an increase in trophic level status. Conversely, cestodes were 15N-depleted. Isotopically, each parasite reflected a species-specific relationship with their rabbit host. This technique could be utilized to integrate parasites into food-web studies.

Animals↗
PubMed1991

Interfacial catalysis by phospholipase A2: evaluation of the interfacial rate constants by steady-state isotope effect studies.

The kinetics of hydrolysis of phospholipid vesicles by phospholipase A2 (PLA2) in the scooting mode can be described by the Michaelis-Menten formalism for the action of the enzyme in the interface (E*). E* + S in equilibrium E*S in equilibrium E*P in equilibrium E* + Products The values of the interfacial rate constants cannot be obtained by classical methods because the concentration of the substrate within the lipid bilayer is not easily manipulated. In the present study, carbonyl-carbon heavy atom isotope effects for the hydrolysis of phospholipids have been measured in both vesicles and in mixed micelles in which the phospholipid was present in the nonionic detergent Triton X-100. A large [14C]carbonyl carbon isotope effect of 1.12 +/- 0.02 was measured for the cobra venom PLA2-catalyzed hydrolysis of dipalmitoylphosphatidylcholine in Triton X-100. In contrast, no isotope effect (1.01 +/- 0.01) was measured for the action of the porcine pancreatic and cobra venom enzymes on vesicles of dimyristoylphosphatidylmethanol in the scooting mode. In a second experiment, the hydrolysis of vesicles was carried out in oxygen-18 enriched water. Analysis of the released fatty acid product by mass spectrometry showed that it contained only a single oxygen-18. All of these results were used to estimate both the forward and reverse commitments to catalysis. The lack of doubly labeled fatty acid demonstrated that the product is released from the E*P complex faster than the reverse of the esterolysis step. The small isotope effect in vesicles demonstrated that the E*S complex goes on to products faster than substrate is released from the enzyme. The relevance of these results to an understanding of substrate specificity and inhibition of PLA2 is discussed. In addition, the conditions placed on the values of the rate constants obtained in the present study together with results obtained in the other studies described in this series of papers have led to the evaluation of most of the interfacial rate constants for the hydrolysis of phospholipid vesicles by PLA2.

Animals↗
PubMed2001

Characterization of the transition-state structure of the reaction of kanamycin nucleotidyltransferase by heavy-atom kinetic isotope effects.

The transition-state structure for the reaction catalyzed by kanamycin nucleotidyltransferase has been determined from kinetic isotope effects. The primary (18)O isotope effects at pH 5.7 (close to the optimum pH) and at pH 7.7 (away from the optimum pH) are respectively 1.016 +/- 0.003 and 1.014 +/- 0.002. Secondary (18)O isotope effects of 1.0033 +/- 0.0004 and 1.0024 +/- 0.0002 for both nonbridge oxygen atoms were measured respectively at pH 5.7 and 7.7. These isotope effects are consistent with a concerted reaction with a slightly associative transition-state structure.

Adenosine Monophosphate↗
PubMed1985

Carbon isotope effect on dehydration of bicarbonate ion catalyzed by carbonic anhydrase.

The carbon-13 kinetic isotope effect on the dehydration of HCO3- by bovine carbonic anhydrase has been measured. To accomplish this, bicarbonate was added to a buffer solution at pH 8 containing carbonic anhydrase under conditions where purging of the product CO2 from the solution is rapid. Measurement of the isotopic composition of the purged CO2 as a function of the concentration of carbonic anhydrase permits calculation of the isotope effect on the enzymic reaction. The isotope effect on the dehydration is k12/k13 = 1.0101 +/- 0.0004. This effect is most consistent with a ping-pong mechanism for carbonic anhydrase action, in which proton transfer to or from the enzyme occurs in a step separate from the dehydration step. Substrate and product dissociation steps are at least 2-3-fold faster than the hydration/dehydration step.

Animals↗
PubMed1985

Positional isotope exchange and kinetic experiments with Escherichia coli guanosine-5'-monophosphate synthetase.

The kinetic mechanism of Escherichia coli guanosine-5'-monophosphate synthetase has been determined by utilizing initial velocity kinetic patterns and positional isotope exchange experiments. The initial velocity patterns of MgATP, XMP, and either NH3 or glutamine (as nitrogen source) were consistent with the ordered addition of MgATP followed by XMP and then NH3. The enzyme catalyzes the exchange of 18O from the beta-nonbridge positions of [beta,beta,beta gamma,gamma,gamma,gamma-18O6]ATP into the alpha beta-bridge position only in the presence of XMP and Mg2+. The exchange reaction did not require NH3. The isotope exchange reaction increased as the XMP concentration increased and then decreased at saturating levels of XMP. These results also support the ordered addition of MgATP followed by XMP. GMP synthetase catalyzes the hydrolysis of ATP to AMP and PPi along with an ATP/PPi exchange reaction in the absence of NH3. These data taken together support a mechanism in which the initial step in the enzymatic reaction involves formation of an adenyl-XMP intermediate. Psicofuranine, an irreversible inhibitor of the enzyme, acts by preventing the release or further reaction of adenyl-XMP with H2O or NH3 but does not suppress the isotope exchange or ATP/PPi exchange reactions. GMP synthetase has also been shown to require a free divalent cation for full activity. When Ca2+ replaces Mg2+ in the reaction, the positional isotope exchange reaction is enhanced but the reaction with NH3 to form GMP is greatly suppressed.

Adenosine Triphosphate↗
PubMed1987

Determination of the energetics of the UDP-glucose pyrophosphorylase reaction by positional isotope exchange inhibition.

A method has been developed for obtaining qualitative information about enzyme-catalyzed reactions by measuring the inhibitory effects of added substrates on positional isotope exchange rates. It has been demonstrated for ordered kinetic mechanisms that an increase in the concentration of the second substrate to add to the enzyme will result in a linear increase in the ratio of the chemical and positional isotope exchange rates. The slopes and intercepts from these plots can be used to determine the partitioning ratios of binary and ternary enzyme complexes. The method has been applied to the reaction catalyzed by UDP-glucose pyrophosphorylase. A positional isotope exchange reaction was measured within oxygen- 18-labeled UTP as a function of variable glucose 1-phosphate concentration in the forward reaction. In the reverse reaction, a positional isotope exchange reaction was measured within oxygen- 18-labeled UDP-glucose as a function of increasing pyrophosphate concentration. The results have been interpreted to indicate that the interconversion of the ternary central complexes is fast relative to product dissociation in either direction. In the forward direction, the release of UDP-glucose is slower than the release of pyrophosphate. The release of glucose 1-phosphate is slower than the release of UTP in the reverse reaction.

Isotope Labeling↗
PubMed1988

Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase.

Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the bridge position of pyrophosphate to a nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. The maximum rates of isotope exchange at equilibrium for the [14C]fructose 1,6-bisphosphate in equilibrium fructose 6-phosphate, [32P]Pi in equilibrium MgPPi, and Mg[32P]PPi in equilibrium fructose 1,6-bisphosphate exchange reactions increasing all four possible substrate-product pairs in constant ratio are identical, consistent with a rapid equilibrium mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate (F6P)/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate (FBP) pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi, in agreement with initial velocity studies [Bertagnolli, B.L., & Cook, P.F. (1984) Biochemistry 23, 4101]. Neither back-exchange by [32P]Pi nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction.

Carbon Radioisotopes↗
PubMed2005

Tracking sources of unsaturated zone and groundwater nitrate contamination using nitrogen and oxygen stable isotopes at the Hanford site, Washington.

The nitrogen and oxygen isotopic compositions of nitrate in pore water extracts from unsaturated zone (UZ) core samples and groundwater samples indicate at least four potential sources of nitrate in groundwaters at the U.S. DOE Hanford Site in south-central Washington. Natural sources of nitrate identified include microbially produced nitrate from the soil column (delta15N of 4 - 8 per thousand, delta18O of -9 to 2 per thousand) and nitrate in buried caliche layers (delta15N of 0-8 per thousand, delta 18O of -6to 42 per thousand). Isotopically distinctindustrial sources of nitrate include nitric acid in low-level disposal waters (delta15N approximately per thousand, delta 18O approximately 23%o) per thousandnd co-contaminant nitrate in high-level radioactive waste from plutonium processing (6'5delta1of 8-33 % o, per thousand18delta oO -9 to 7%0). per thousandThe isotopic compositions of nitrate from 97 groundwater wells with concentrations up to 1290 mg/L NO3- have been analyzed. Stable isotope analyses from this study site, which has natural and industrial nitrate sources, provide a tool to distinguish nitrate sources in an unconfined aquiferwhere concentrations alone do not. These data indicate that the most common sources of high nitrate concentrations in groundwater at Hanford are nitric acid and natural nitrate flushed out of the UZ during disposal of low-level wastewater. Nitrate associated with high-level radioactive UZ contamination does not appear to be a major source of groundwater nitrate at this time.

Bacteria↗
PubMed2006

Sedimented organic nitrogen isotopes in freshwater wetlands record long-term changes in watershed nitrogen source and land use.

Although historic land use is widely recognized as an important determinant of watershed N cycling, efforts to examine land use legacy effects are limited by incomplete historical data. This research evaluates N isotopes of sedimented organic matter (delta15N(org)), in a palynological context, as a long-term proxy of changes in N source to wetland biota. N and S isotope measurements of organic sediments, fossil plant fragments, and living plants are used to explore isotope stratigraphies of wetland sediment cores. Processes potentially contributing to isotope stratigraphies are investigated including the following: a change in N source, diagenesis, and denitrification. We document the delta15N(org) stratigraphy of a core from the Smithsonian Environmental Research Center, MD, U.S.A. spans approximately 350 years, during which time delta15N(org) increases from +2 per thousand to +7 per thousand. Reconstructed population density and wastewater inputs to the watershed suggest that the increase in delta15N reflects changing land use from forested conditions to increasing nutrient inputs from human waste. Our results illustrate the importance of hydrologic connectivity in delivering waste-derived N in a watershed characterized by relatively low human population density. These results also demonstrate how this approach can expand the temporal horizon over which we can assess human impacts to watershed N dynamics.

Agriculture↗
PubMed2004

Nature of vibrational coupling in helical peptides: an isotopic labeling study.

Infrared (IR) and vibrational circular dichroism (VCD) spectra were measured for a series of isotopically ((13)C on two or more amide Cdouble bond]O) labeled, 25 residue, alpha-helical peptides of the sequence Ac-(AAAAK)(4)AAAAY-NH(2) that were also studied in the previous paper. Theoretical IR and VCD simulations were performed for correspondingly isotopically labeled Ac-A(24)-NHCH(3) constrained to an alpha-helical conformation by use of property tensor transfer from density functional theory (DFT) calculations on Ac-A(10)-NHCH(3). The simulations predicted and experiments confirmed that the vibrational coupling constants between i, i + 1 and i, i + 2 residues differ in sign, thus leading to a reversal of the (13)C VCD pattern and explaining the large shift in the (13)C amide I frequency as reported in the previous paper. The sign of the coupling constant remained consistent for larger label separation (with the exception of i, i + 4) and for more labels with uniform separation. Such effects confirm that the isotopically labeled group vibrations are essentially only coupled to each other and are effectively uncoupled from those of the unlabeled groups. This development confirms the utility of isotopic labels for site-specific structural studies with vibrational spectra. Observed spectral effects cannot be explained by considering only transition dipole coupling (TDC) between amide oscillators, particularly for smaller label separations, but the TDC and ab initio predicted couplings roughly converge at large separation.

Alanine↗
PubMed2002

Detection and quantification of 5-chlorocytosine in DNA by stable isotope dilution and gas chromatography/negative ion chemical ionization/mass spectrometry.

Hypochlorous acid (HOCl) is generated from activated phagocytes during infections and inflammation. One of the major products of HOCl reaction with DNA was 5-chlorocytosine (5Cl-Cyt). In this report, a gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) assay with stable isotope dilution was developed for detection and quantification of 5Cl-Cyt in DNA. During hydrolysis of DNA, 5Cl-Cyt undergoes spontaneous deamination quantitatively forming 5-chlorouracil (5Cl-Ura). The stable isotope of 5Cl-Ura with six mass units higher than the normal 5Cl-Ura was synthesized and used as internal standard of the assay. The adduct-enriched fraction of DNA hydrolysate was derivatized with pentafluorobenzyl bromide before GC/NICI/MS analysis with selected ion monitoring at [M - 181](-) fragments of bispentafluorobenzylated 5Cl-Ura and its isotope analogue. The limit of detection was 20 amol (S/N = 8) of bispentafluorobenzylated 5Cl-Ura injected on column with selective ion monitoring mode and the limit of quantification for the entire assay was 14 fmol of 5Cl-Cyt. Analysis of hypochlorous acid-treated calf thymus DNA by both GC/NICI/MS and HPLC/UV detection provided similar adduct levels and thus verified this new GC/NICI/MS assay. Using this highly specific and ultrasensitive GC/NICI/MS method, the levels of 5Cl-Cyt in untreated calf thymus DNA and human placental DNA were determined as 0.6 and 6.6 adducts per 10(7) normal cytosine, respectively. Peroxynitrite also contributed to 5Cl-Cyt formation in DNA. Level of 5Cl-Cyt in DNA treated with peroxynitrite in the presence of chloride was higher than that without addition of chloride. Thus, quantification of 5Cl-Cyt in DNA by this isotope dilution GC/NICI/MS assay may facilitate research on the role of DNA chlorination in carcinogenesis and in cancer development.

Animals↗
PubMed1998

In vivo measurement of ureagenesis with stable isotopes.

We have utilized stable isotopes to measure in vivo rates of ureagenesis. In one testing procedure, 15NH4Cl was administered orally to controls and to heterozygotes for ornithine transcarbamylase deficiency. Controls produced [15N]urea at a rate that was greater than that of symptomatic carriers, but indistinguishable from that of asymptomatic carriers. In contrast, both symptomatic and asymptomatic heterozygotes produced [5-15N]glutamine more rapidly than the controls. Ureagenesis could also be measured by administering sodium [1-13C]acetate to a healthy adult and measuring subsequent formation of [13C]urea. The latter approach involves the use of isotope ratio mass spectrometry to determine isotopic abundance. This technique is much more sensitive than gas chromatography-mass spectrometry for the measurement of isotopic label, a consideration that makes the method more suitable for the study of subjects in whom ureagenesis is severely compromised, for example the human male neonate with a near complete deficiency of ornithine transcarbamylase.

Adult↗
PubMed1998

Nitrogen in solar energetic particles: isotopically distinct from solar wind.

Stepwise etching of lunar soil ilmenite grains reveals that the 15N/14N ratio of implanted nitrogen decreases with increasing implantation depth within the ilmenite grains, i.e., with increasing energy of implantation. These results show that N derived from solar energetic particles, NSEP, is enriched in the light isotope, 14N, relative to solar-wind nitrogen, NSW. This is in striking contrast to the neon isotopic record: NeSEP is depleted in the light isotope, 20Ne, relative to NeSW. These data suggest either distinct signatures in the respective solar source regions, or fractionation in the acceleration mechanism(s). However, the observed opposite fractionation trends for light N and Ne isotopes do not agree with model predictions.

Extraterrestrial Environment↗
PubMed1981

Kinetic equivalence of stable-isotope-labeled and unlabeled phenytoin.

Stable isotope labeling (SIL) of a drug results in a higher molecular weight than that of the unlabeled drug. SIL tracer doses can be quantitated separately from unlabeled drug by gas chromatography-mass spectrometry (GC-MS) without exposing the patient to radiation. The higher molecular weight of SIL drug could cause a higher energy of activation for (and slowing of) metabolic reactions ("isotope effect"). To evaluate possible isotope effect, three dogs and three men were infused with a mixture containing equal amounts of SIL (2-13C-1,3-15N2) and unlabeled phenytoin (PHT). Plasma and urine were collected at regular intervals. Concentrations of SIL and unlabeled PHT and HPPH (the major metabolite of PHT) were determined by GC-MS. Within each subject there was no trend for concentrations of SIL PHT or HPPH to be higher or lower than concentrations of their unlabeled analogs (greater than 0.20 to 0.90). There was no difference in the distribution and elimination half-lifes (t 1/2s), volume of distribution, volume of central compartment, or clearance of the two forms of PHT. Thus, no isotope effect was found.

Animals↗