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PubMed1997

Determination of the chemical mechanism of malic enzyme by isotope effects.

Carbon-13 isotope effects have been determined for all four carbons of L-malate as a substrate for chicken liver malic enzyme, using either NADP or acetylpyridine-NADP as the other substrate. The effect of deuteration at C2 of malate was then used to tell whether the chemical mechanism of this oxidative decarboxylation was stepwise, with oxaloacetate as an intermediate, or concerted. With NADP, the 13C isotope effects at C3 and C4 both decrease with deuteration of malate, showing a stepwise mechanism, as previously determined [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114]. With acetylpyridine-NADP, however, the 13C isotope effects at both C3 and C4 increase with deuteration of malate. While the increase at C4 could be explained by a secondary 13C isotope effect on hydride transfer, the increase at C3 proves that the chemical mechanism has changed to a concerted one, presumably because hydride transfer is more rate-limiting and the overall equilibrium constant is more favorable by 2 orders of magnitude. The transition state for this concerted reaction is asynchronous, however, with an intrinsic deuterium isotope effect of approximately 5 and a 13C isotope effect of only 1.010-1.015. Equilibrium 13C isotope effects for conversion of carbons 2, 3, and 4 of malate to pyruvate or CO2 are 1.010, 1.011, and 0.988, respectively. Measured 13C isotope effects at C2 of malate are slightly inverse, but no explanation for this is obvious. With NADP, deuterium isotope effects at C3 of 1.17 and 1.08 for di- and monodeuteration and an increase in the 13C isotope effect at C4 upon dideuteration at C3 are consistent with a stepwise mechanism with the deuterium isotope effect at C3 being only on the decarboxylation step. Smaller deuterium isotope effects of 1.03-1.04 from dideuteration at C3 with acetylpyridine-NADP are consistent with a concerted but asynchronous mechanism where C-C cleavage is not far advanced in the transition state.

Animals↗
PubMed2004

Stable isotopic characterization of active pharmaceutical ingredients.

Stable isotopic characterization or "fingerprinting" of active pharmaceutical ingredients (APIs) is a highly-specific means of defining the provenance of these pharmaceutical materials. The isotopic analysts in this study were provided with 20 blind samples of four APIs (tropicamide, hydrocortisone, quinine HCL, and tryptophan) from one-to-five production batch(es) from one-to-five manufacturer(s). Only the chemical identity of the APIs was initially provided to the isotopic analysts. Depending on the API chemical composition, isotopic ratios of either three or four elements (13C/12C, 15N/14N, 18O/16O, and/or D/H) were measured by either elemental analyzer/isotope ratio mass spectrometry (EA/IRMS: carbon (delta13C) and nitrogen (delta15N)) or by thermal conversion-EA/IRMS (TCEA/IRMS; hydrogen (deltaD) and oxygen (delta15N)); in all cases, the isotopic results are reported in the standard delta-notation which represents part-per-thousand () variations from the isotopic ratios of international standards. The stable isotopic analyses of the four suites of APIs spanned broad ranges in absolute value (deltadelta) and in estimated specificity (a product of dynamic ranges (DR, unitless)--note that these are upper limits of specificity because some of these isotope values may be partially interdependent). The five samples of tropicamide from one production batch and one manufacturer demonstrated the narrowest ranges (deltadelta13C=0.13 ; deltadelta15N=0.52 ; deltadelta18O=0.24 ; deltadeltaD=2.8 ) and the smallest specificity of 1:30.9. By contrast, the five samples of tryptophan that came from five separate manufacturers had some of the widest isotopic ranges observed (deltadelta13C=21.32 ; deltadelta15N=5.26 ; deltadelta18O=22.07 ; deltadeltaD=55.3 ) and had the largest specificity of 1:19.6 x 10(6). The isotopic provenance of the four suites of APIs readily emerged from bivariate plots of selected isotope ratios, particularly deltaD versus delta18O.

Carbon Isotopes↗
PubMed2002

Partitioning and bioaccumulation of cadmium in artificial sediment systems: application of a stable isotope tracer technique.

The utility of stable isotope tracers for investigating the relationship between cadmium (Cd) partitioning in artificial sediment-water systems and Cd accumulation in a benthic detritivore (Asellus racovitzai, Isopoda) was explored. In the laboratory, Cd isotopes were applied to synthetic sediment and isotope concentrations were measured in sediment, overlying water and exposed asellids over a 10-day period. Isotope ratios measured in sediment and water were compared to ratios measured in asellids to determine whether Cd partitioning could predict metal bioaccumulation. Two different parameters which might affect Cd partitioning between the sediment and overlying water compartments were investigated: the chemical form in which Cd was added to systems, and the organic matter content of the sediment. To test the effect of chemical form on Cd partitioning, three isotopes of cadmium were individually applied to formulated sediment in varying combinations of 113Cd(NO3)2, 112Cd-humic acid (HA) 114CdSO4. The results demonstrated that chemical form did not influence partitioning, as the Cd isotope that was applied to sediment in the nitrate form exhibited similar partitioning between sediment and overlying water as the isotope that was applied in the sulfate or HA form. However, Cd isotope concentrations in overlying water were strongly related to the pattern of isotope accumulation in asellids suggesting that overlying water concentrations determined Cd bioaccumulation. In contrast, when the organic matter content of sediment was increased through the addition of Sphagnum peat moss, total Cd concentrations in overlying water and tissue were low, and there was no relationship between Cd-isotope concentrations in tissue and water. These results indicate that Cd accumulation occurred primarily from water, and factors that increase metal partitioning to sediment, such as increased sediment organic matter content, decrease Cd accumulation in asellids. The stable isotope tracer method described herein appears to be a useful technique for investigating the relationship between metal partitioning and bioaccumulation in simple sediment systems, but could also be extended to more complex systems, and used with different metals that have multiple stable isotopes.

Animals↗
PubMed2005

Carbon position-specific isotope analysis of alanine and phenylalanine analogues exhibiting nonideal pyrolytic fragmentation.

Recent advances in gas chromatography combustion-isotope ratio mass spectrometry (GCC-IRMS) has made compound-specific isotope analysis routine, but reports on position-specific isotopic analysis are still scarce. On-line GC-pyrolysis (Py) coupled to GCC-IRMS is reported here for isolation and isotopic characterization of alaninol and phenethylamine, analogues of alanine and phenylalanine, respectively. Ideally, pyrolytic fragments will originate from unique sites within the parent molecule, and isotope ratios for each position within the parent can either be measured directly or calculated from fragment isotope ratios without substantially degrading the analytical precision. Alaninol pyrolysis yielded several fragments, of which CO and CH4 were used for isotope ratio calculations. Isotope labeling experiments showed that CO derived entirely from the C(1) position, while all three positions of alaninol contributed to CH4 (29.0 +/- 0.3% from C(1), 3.6 +/- 0.2% from C(2), and 66.9 +/- 1.1% from C(3)). We demonstrate iterative use of mass balance to calculate isotope ratios from all positions despite the nonideal positional fidelity of CH4. Pyrolysis of phenethylamine generated benzene and toluene fragments. Benzene derived entirely from C(ring), and toluene was proportionately formed from C(3) and C(ring). Relative intramolecular isotope ratios (Deltadelta13C) were calculated directly from delta13C of fragments or indirectly by mass balance. Though the C(3) isotope ratio was calculated from the benzene and toluene fragments, propagation of errors showed that the final precision of the determination was degraded due to the small contribution that C(3) makes to toluene. Samples of each amino acid from four different vendors showed natural variability between sources, especially at the C(1) position of alaninol (range of Deltadelta13C approximately 50 per thousand). The average precision was SD(Deltadelta13C) < 0.20 per thousand for directly measured positions of alaninol and phenethylamine. The precision of indirectly measured positions was poorer (SD(Deltadelta13C) = 0.94 per thousand for alaninol, 6.54 per thousand for phenethylamine) due to propagation of errors. These data demonstrate that GC-Py-GCC-IRMS data can be used to extract high-precision isotope ratios from amino acids despite nonideal positional fidelity in fragments and that natural intramolecular variability in delta13C can be used to distinguish different sources of amino acids.

Carbon Isotopes↗
PubMed2001

Temporal trends in the isotope signature of air-borne sulfur in Central Europe.

In various parts of the Northern hemisphere air-borne S exhibits a seasonality, with isotopically light (i.e., 32S-rich) sulfur predominating in the warm summer months. Such seasonality has been reported from the United States, Canada, Japan, and China. Elevated biological emissions of isotopically light S in summer, a temperature-dependent isotope fractionation accompanying the oxidation of SO2, and heavy rains in winter bringing 34S-rich marine S have been suggested as the controlling mechanisms. In the atmosphere of Central Europe, one of the most severely polluted regions of the world, we have found an opposite seasonal trend: Isotopically light SO2-S predominates in the cold winter months, whereas isotopically heavy SO2-S is typical of the summer. The low delta34S values of air-borne SO2 in winter are influenced by low-delta34S emissions from local coal-burning power plants. The coal contains isotopically light S (mean delta34S of 1.6/1000). Higher demand for electricity during the heating season leads to higher anthropogenic S emission rates in winter. On a yearly basis, atmospheric sulfate S in Central Europe is isotopically heavier than atmospheric SO2-S by 4/1000. Atmospheric oxidation of SO2 is accompanied by an isotope fractionation resulting in 34S-enriched sulfate. In addition to the seasonality in air-borne delta34S(SO2), we report also an interannual trend of 1/1000 yr(-1) toward isotopically light sulfate S in atmospheric deposition. This interannual trend cannot be explained by a change in pollution sources accompanying the present massive environmental cleanup. To investigate the role of biological S emissions from the soil of heavily polluted ecosystems, we conducted a series of laboratory experiments using repacked soil columns and 34S-enriched precipitation under summer and winter temperatures. These experiments indicate that, under summer temperatures, the 34S-labeled precipitation is largely captured by the upper organic-rich soil horizons, a high proportion (53-74%) of S input is revolatilized, and the biologically reemitted S is isotopically light. Under winter temperatures more precipitation S is leached to the bottom of the soil columns. Our experiments have shown that biological emissions in Central Europe can be sizable. Yet, they cannot be singled out in the overall SO2 isotope pattern in the atmosphere. The main reason is continuous, variable (0-4/1000), open-system depletion in 34S in the residual SO2 during the isotopically selective SO2-to-SO4(2-) conversion.

Air Pollutants↗
PubMed2003

Low turnover rates of carbon isotopes in tissues of two nectar-feeding bat species.

Stable isotopes of carbon are commonly used to characterize dietary preferences in animals. Because turnover rates of carbon isotopes are related to metabolic rate, we wanted to determine the rates at which carbon isotopes are exchanged in tissues of two species of nectar-feeding bats (Leptonycteris curasoae and Glossophaga soricina), both of which have relatively high mass-specific metabolic rates. To test the hypothesis that isotope turnover is higher in nectar-feeding bats, because of their high mass-specific metabolic rates, than in other eutherian mammals, we conducted diet-switching experiments and chose three target tissues (hair, wing membrane and blood) to evaluate the isotopic turnover rates. We made the following predictions: (1) isotopic composition should change towards higher delta(13)C-values due to the turnover of carbon isotopes of C(3) origin with those of C(4)/CAM origin; (2) the turnover rates of carbon isotopes would differ between the three types of tissues in the following order of decreasing turnover rates: blood>wing membrane>hair; and (3) turnover rates of nectar-feeding bats should exceed those reported for other small mammals because of the high mass-specific metabolic rate of nectar-feeding bats. Compared to the initial diet, target tissues were enriched in heavy carbon isotopes by 2.8 per thousand in L. curasoae and by 2.6 per thousand in G. soricina. After changing the diet from C(3) to C(4)/CAM origin we found an increase in abundance of (13)C in blood and wing membrane in all experimental subjects. The estimated half life of carbon isotope turnover ranged from 100 to 134 days and did not differ significantly between blood and wing membrane, nor did it differ between the two species. The low turnover rate in wing membrane may reflect its specific composition and the relatively low temperature of this tissue, and long-lived erythrocytes in bat blood may be responsible for the low turnover rate of carbon isotopes in blood. The turnover rate of stable carbon isotopes in hair was low in L. curasoae and undetectable in G. soricina, which may be explained by the seasonal growth of the hair in these two species. Because both species are small (10 and 25 g, respectively) and nectar-feeding bats have higher mass-specific metabolic rates than bats in temperate regions or similar sized terrestrial mammals, our findings of low turnover rates were unexpected.

Analysis of Variance↗
PubMed1998

Isotopic ratio analysis in residential lead-based paint and associated surficial dust.

OBJECTIVES: This investigation assessed the contribution of lead in lead-based paint (7 samples) to lead-laden dust (8 samples) in a single suburban vacant residence using isotopic ratio analysis. METHODS: Interior/exterior lead-based paint surface concentration was measured by X-ray fluorescence while dust and scrapings were analyzed chemically for total lead content and by mass spectrometry for the associated isotopic ratios. RESULTS: Four out of 5 comparisons of paint (7 samples) and dust (8 samples) for a given location did not match isotopically. In the one location where the isotopic ratio of the paint and dust samples matched closely, some portions of the paint were not intact. One explanation for the isotopic ratio match is that the dust sample may have actually been contaminated with paint flecks. This explanation appears likely since the isotopic ratio for the lead in the dust and paint sample were not in the modern average range of US environmental lead, strongly indicating a local point source of the lead in this dust sample, namely the paint at this location. Lead dust samples whose isotopic ratio lies in the modern average range for US environmental lead cannot be correlated to the paint which is beneath them, since the isotopic ratio of lead in the dust may actually be a composite of many sources of lead over time, as suggested by an isotopic ratio in the modern average range. CONCLUSIONS: From the samples from this one house, the data dispute the contention that intact lead-based paint chalks and creates lead-contaminated dust on its surface. While leaded household dust may contribute to children's lead exposure, intact paint need not contribute to surficial lead-laden dust. Isotopic ratio measurements can be useful for point-source determination by virtue of sample match and by placement of the ratio on the spectrum of isotopic ratio values for lead. Point-source assessment based on isotopic ratio was either strengthened or weakened by placement outside or within the average range for US environmental lead, respectively.

Air Pollutants↗
PubMed2005

Simultaneous mass bias and fractionation corrections utilizing isotopic solid standards and laser ablation ICPMS.

Homogeneous incorporation of analytes of known isotopic abundance into sol-gel-derived standards that mimic important mineral systems has the potential to contribute significantly to providing solid standard benchmarks for a range of applications. This preliminary study reports on the synthesis of solid glass standards produced via the sol-gel method and their doping with Standard Reference Material (SRM) 981 Common Lead Isotopic Standard and SRM 982 Equal-Atom Lead Isotopic Standard. Custom isotopic materials were also prepared using mixtures of the two isotopic SRMs. Particles from these solid samples were then introduced into an inductively coupled plasma mass spectrometer via laser ablation to determine whether materials of suitable homogeneity could be developed as isotopic reference materials. Preliminary results for Pb isotope ratios show that these solid isotopic reference standards are capable of correcting for instrumental mass bias and laser ablation-induced bias due to fractionation simultaneously. Correction factors generated from the quotient of the certified and measured Pb isotopic ratios in sol-gel disks spiked with SRMs 981 and 982 were successfully applied to produce accurate isotope ratios using comparative control/unknown checks. These correction factors were also used to assign Pb isotopic ratios in NIST SRM 612 Trace Elements in Glass that were in excellent agreement with published measurements, suggesting that tunable matrix sol-gel disks can serve as adequate control matrixes for evaluation of isotope ratios in glass samples.

Journal Article↗
PubMed1991

Comparison of Modeled and Observed Environmental Influences on the Stable Oxygen and Hydrogen Isotope Composition of Leaf Water in Phaseolus vulgaris L.

In this paper we describe how a model of stable isotope fractionation processes, originally developed by H. Craig and L. I. Gordon ([1965] in E Tongiorgi, ed, Proceedings of a Conference on Stable Isotopes in Oceanographic Studies and Paleotemperature, Spoleto, Italy, pp 9-130) for evaporation of water from the ocean, can be applied to leaf transpiration. The original model was modified to account for turbulent conditions in the leaf boundary layer. Experiments were conducted to test the factors influencing the stable isotopic composition of leaf water under controlled environment conditions. At steady state, the observed leaf water isotopic composition was enriched above that of stem water with the extent of the enrichment dependent on the leaf-air vapor pressure difference (VPD) and the isotopic composition of atmospheric water vapor (AWV). The higher the VPD, the larger was the observed heavy isotope content of leaf water. At a constant VPD, leaf water was relatively depleted in heavy isotopes when exposed to AWV with a low heavy isotope composition, and leaf water was relatively enriched in heavy isotopes when exposed to AWV with a large heavy isotope composition. However, the observed heavy isotope composition of leaf water was always less than that predicted by the model. The extent of the discrepancy between the modeled and observed leaf water isotopic composition was a strong linear function of the leaf transpiration rate.

Journal Article↗
PubMed2005

The effect of growth rate on tissue-diet isotopic spacing in rapidly growing animals. An experimental study with Atlantic salmon (Salmo salar).

The difference in isotopic composition between a consumer's tissues and that of its diet is a critical aspect of the use of stable isotope analyses in ecological and palaeoecological studies. In a controlled feeding experiment with the Atlantic salmon, Salmo salar, we demonstrate for the first time that the value of tissue-diet isotope spacing in nitrogen in a growing animal is not constant, but varies inversely with growth rate. The value of tissue-diet isotopic spacing in N reflects N use efficiency. Thus, in salmon, growth rate is accompanied by, or requires, increased N use efficiency. The total range in tissue-diet isotopic spacing in N seen in the experimental population of 25 fish was 1 per thousand, approximately 50% of the total trophic shift. Mean equilibrium tissue-diet isotopic spacing (+/-standard deviation) in salmon averaged 2.3 per thousand (+/-0.3 per thousand) and 0.0 per thousand (+/-0.3 per thousand) for N in muscle and liver, respectively, and 2.1 per thousand (+/-0.1 per thousand) and 1.6 per thousand (+/-0.3 per thousand) for C in muscle and liver, respectively. Feeding with a mixed dietary source (wheat and fish-meal origin) resulted in tissue-diet isotopic fractionation in both C and N due to the differential digestibility of food components with distinct isotopic composition. The rate of change in isotopic composition of S. salar tissues was dominated by growth, but the estimated contribution of metabolic turnover to change in tissue N was relatively high for an ectothermic animal at ca. 20-40%. The estimated half-life for metabolic turnover of the tissue N pool was ca. 4 months in both muscle and liver tissue. This is the first study to demonstrate a direct relationship between tissue-diet isotopic spacing in N and growth rate and adds to the growing list of factors known to influence the level of isotopic separation between a consumer's tissue and that of its diet.

Animal Feed↗
PubMed2003

Effects of elemental composition on the incorporation of dietary nitrogen and carbon isotopic signatures in an omnivorous songbird.

The use of stable isotopes to infer diet requires quantifying the relationship between diet and tissues and, in particular, knowing of how quickly isotopes turnover in different tissues and how isotopic concentrations of different food components change (discriminate) when incorporated into consumer tissues. We used feeding trials with wild-caught yellow-rumped warblers (Dendroica coronata) to determine delta15N and delta13C turnover rates for blood, delta15N and delta13C diet-tissue discrimination factors, and diet-tissue relationships for blood and feathers. After 3 weeks on a common diet, 36 warblers were assigned to one of four diets differing in the relative proportion of fruit and insects. Plasma half-life estimates ranged from 0.4 to 0.7 days for delta13C and from 0.5 to 1.7 days for delta15N . Half-life did not differ among diets. Whole blood half-life for delta13C ranged from 3.9 to 6.1 days. Yellow-rumped warbler tissues were enriched relative to diet by 1.7-3.6% for nitrogen isotopes and by -1.2 to 4.3% for carbon isotopes, depending on tissue and diet. Consistent with previous studies, feathers were the most enriched and whole blood and plasma were the least enriched or, in the case of carbon, slightly depleted relative to diet. In general, tissues were more enriched relative to diet for birds on diets with high percentages of insects. For all tissues, carbon and nitrogen isotope discrimination factors increased with carbon and nitrogen concentrations of diets. The isotopic signature of plasma increased linearly with the sum of the isotopic signature of the diet and the discrimination factor. Because the isotopic signature of tissues depends on both elemental concentration and isotopic signature of the diet, attempts to reconstruct diet from stable isotope signatures require use of mixing models that incorporate elemental concentration.

Animals↗
PubMed2004

Subtle modification of isotope ratio proteomics; an integrated strategy for expression proteomics.

Use of minor modification of isotope ratio to code samples for expression proteomics is being investigated. Alteration of (13)C abundance to approximately 2% yields a measurable effect on peptide isotopic distribution and inferred isotope ratio. Elevation of (13)C abundance to 4% leads to extension of isotopic distribution and background peaks across every unit of the mass range. Assessment of isotope ratio measurement variability suggests substantial contributions from natural measurement variability. A better understanding of this variable will allow assessment of the contribution of sequence dependence. Both variables must be understood before meaningful mixing experiments for relative expression proteomics are performed. Subtle modification of isotope ratio ( approximately 1-2% increase in (13)C) had no effect upon either the ability of data-dependent acquisition software or database searching software to trigger tandem mass spectrometry or match MSMS data to peptide sequences. More severe modification of isotope ratio caused a significant drop in performance of both functionalities. Development of software for deconvolution of isotope ratio concomitant with protein identification using LC-MSMS, or any other proteomics strategy, is underway (Isosolv). The identified peptide sequence is then be used to provide elemental composition for accurate isotope ratio decoding and the potential to control for specific amino acid biases should these prove significant. It is suggested that subtle modification of isotope ratio proteomics (SMIRP) offers a convenient approach to in vivo isotope coding of plants and might ultimately be extended to mammals including humans.

Carbon Isotopes↗
PubMed1999

Documenting the diet in ancient human populations through stable isotope analysis of hair.

Fundamental to the understanding of human history is the ability to make interpretations based on artefacts and other remains which are used to gather information about an ancient population. Sequestered in the organic matrices of these remains can be information, for example, concerning incidence of disease, genetic defects and diet. Stable isotopic compositions, especially those made on isolates of collagen from bones, have been used to help suggest principal dietary components. A significant problem in the use of collagen is its long-term stability, and the possibility of isotopic alteration during early diagenesis, or through contaminating condensation reactions. In this study, we suggest that a commonly overlooked material, human hair, may represent an ideal material to be used in addressing human diets of ancient civilizations. Through the analysis of the amino-acid composition of modern hair, as well as samples that were subjected to radiation (thus simulating ageing of the hair) and hair from humans that is up to 5200 years old, we have observed little in the way of chemical change. The principal amino acids observed in all of these samples are essentially identical in relative abundances and content. Dominating the compositions are serine, glutamic acid, threonine, glycine and leucine, respectively accounting for approximately 15%, 17%, 10%, 8% and 8% of the total hydrolysable amino acids. Even minor components (for example, alanine, valine, isoleucine) show similar constancy between the samples of different ages. This constancy clearly indicates minimal alteration of the amino-acid composition of the hair. Further, it would indicate that hair is well preserved and is amenable to isotopic analysis as a tool for distinguishing sources of nutrition. Based on this observation, we have isotopically characterized modern individuals for whom the diet has been documented. Both stable nitrogen and carbon isotope compositions were assessed, and together provide an indication of trophic status, and principal type (C3 or C4) of vegetation consumed. True vegans have nitrogen isotope compositions of about 7/1000 whereas humans consuming larger amounts of meat, eggs, or milk are more enriched in the heavy nitrogen isotope. We have also analysed large cross-sections of modern humans from North America and Europe to provide an indication of the variability seen in a population (the supermarket diet). There is a wide diversity in both carbon and nitrogen isotope values based at least partially on the levels of seafood, corn-fed beef and grains in the diets. Following analysis of the ancient hair, we have observed similar trends in certain ancient populations. For example, the Coptics of Egypt (1000 BP) and Chinchorro of Chile (5000-800 BP) have diets of similar diversity to those observed in the modern group but were isotopically influenced by local nutritional sources. In other ancient hair (Egyptian Late Middle Kingdom mummies, ca. 4000 BP), we have observed a much more uniform isotopic signature, indicating a more constant diet. We have also recognized a primary vegetarian component in the diet of the Neolithic Ice Man of the Oetztaler Alps (5200 BP). In certain cases, it appears that sulphur isotopes may help to further constrain dietary interpretations, owing to the good preservation and sulphur content of hair. It appears that analysis of the often-overlooked hair in archaeological sites may represent a significant new approach for understanding ancient human communities.

Amino Acids↗
PubMed2000

Isotope recycling in lactating dogs (Canis familiaris).

Isotope-based techniques for the measurement of water turnover, energy expenditure, and milk intake often assume that there is no recycling of isotopes once they have left the labeled animal. In experiments involving lactating females or their suckling offspring, there are several possible routes of isotope recycling. These include the consumption of labeled milk by offspring, the ingestion of labeled excreta, and the rebreathing of exhaled labeled CO(2) or water vapor by both mother and offspring. Isotope recycling might be especially important during lactation because the offspring are in close contact with each other and their mother for prolonged periods. We show here in 24- to 30-day-old domestic dog Canis familiaris puppies that there was no detectable transfer of (18)O or (2)H from labeled to unlabeled pups in two litters (16 pups, 8 labeled, 8 unlabeled) that were weaned early and independent of their mother. However, there was a significant transfer of both isotopes from labeled to unlabeled pups and from labeled pups to their mothers in nine equivalent nursing litters of the same age (27 labeled, 26 unlabeled pups). The increases in enrichment of isotopes in unlabeled offspring were greater than the increases in enrichment of the mothers. This indicates that maternal ingestion of offspring excreta and subsequent transfer of isotope in milk is not the sole pathway of recycling. Additional routes must also be important, such as exchange of isotope between pups on saliva-coated nipples and perhaps direct ingestion of excreta by unweaned young. Recycling is unlikely to be an important factor when determining maternal metabolic rate during peak lactation in domestic dogs. However, experiments that are designed to assess the energy demands of pups and isotope-based estimates of water turnover in offspring may need to take into account any effects of isotope recycling. In a theoretical example, removing the effects of recycling increased the measured energy expenditure in pups by up to 7% and increased the calculated elimination rates of both isotopes by up to 11.1% in (18)oxygen and 10.9% in (2)hydrogen.

Animals↗
PubMed1993

Use of nitrogen-15 kinetic isotope effects to elucidate details of the chemical mechanism of human immunodeficiency virus 1 protease.

We have used 15N kinetic isotope effects of the HIV-1 protease-catalyzed peptidolysis of Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 to characterize the chemical mechanism of this enzyme. In addition, the multiple isotope effects have been determined by measuring the 15N kinetic isotope effects in both H2O and D2O. The isotope effects, measured on values of V/K, were determined by the incorporation of a radiolabel (tritium and 14C in peptides bearing the heavy and light isotopes, respectively) at a position remote from the isotopically labeled scissile peptide bond, such that the isotope effect was determined by measurement of the change in the 14C/3H ratio in recovered substrates at various fractions of reaction. At pH = 6.0 (37 degrees C), the nitrogen isotope effects were slightly, but significantly, inverse in both solvents: 15(V/K)H2O = 0.995 +/- 0.002, and 15(V/K)D2O = 0.992 +/- 0.003. The observation of an inverse nitrogen kinetic isotope effect implies that bonding to the nitrogen atom is becoming stiffened in a reaction transition state, and since this inverse isotope effect is enhanced in D2O, this isotope effect likely arises from protonation of the proline nitrogen atom.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

Conformational equilibrium isotope effects in glucose by (13)C NMR spectroscopy and computational studies.

Anomeric equilibrium isotope effects for dissolved sugars are required preludes to understanding isotope effects for these molecules bound to enzymes. This paper presents a full molecule study of the alpha- and beta-anomeric forms of D-glucopyranose in water using deuterium conformational equilibrium isotope effects (CEIE). Using 1D (13)C NMR, we have found deuterium isotope effects of 1.043 +/- 0.004, 1.027 +/- 0.005, 1.027 +/- 0.004, 1.001 +/- 0.003, 1.036 +/- 0.004, and 0.998 +/- 0.004 on the equilibrium constant, (H/D)K(beta/alpha), in [1-(2)H]-, [2-(2)H]-, [3-(2)H]-, [4-(2)H]-, [5-(2)H]-, and [6,6'-(2)H(2)]-labeled sugars, respectively. A computational study of the anomeric equilibrium in glucose using semiempirical and ab initio methods yields values that correlate well with experiment. Natural bond orbital (NBO) analysis of glucose and dihedral rotational equilibrium isotope effects in 2-propanol strongly imply a hyperconjugative mechanism for the isotope effects at H1 and H2. We conclude that the isotope effect at H1 is due to n(p) --> sigma* hyperconjugative transfer from O5 to the axial C1--H1 bond in beta-glucose, while this transfer makes no contribution to the isotope effect at H5. The isotope effect at H2 is due to rotational restriction of OH2 at 160 degrees in the alpha form and 60 degrees in the beta-sugar, with concomitant differences in n --> sigma* hyperconjugative transfer from O2 to CH2. The isotope effects on H3 and H5 result primarily from syn-diaxial steric repulsion between these and the axial anomeric hydroxyl oxygen in alpha-glucose. Therefore, intramolecular effects play an important role in isotopic perturbation of the anomeric equilibrium. The possible role of intermolecular effects is discussed in the context of recent molecular dynamics studies on aqueous glucose.

Carbon Isotopes↗
PubMed2001

Microbial influences on local carbon isotopic ratios and their preservation in carbonate.

Analysis of the carbon isotopic composition of carbonates is a valuable tool for studying microbial processes and looking for evidence of life. Microbial fixation of CO2 and conversion of organic carbon to CO2 can produce measurable delta 13C shifts in a microbial mat environment. Diffusion modeling demonstrates that substantial isotopic shifts can develop within the mat and in the diffusion boundary layer in the fluid when CO2 fixation is rapid and prolonged for several hours. Carbonates that precipitate during rapid CO2 fixation can preserve these microbially produced isotopic shifts. However, continued precipitation during intervals when respiration dominates or after the cessation of active microbial growth commonly dilutes autotrophic isotopic signatures. Thus, preserved isotopic signatures rarely reflect the magnitude of isotopic shifts within the mat. Interpretation of observed isotopic shifts in microbial mat carbonate depends on fully characterizing ambient delta 13C and eliminating other origins for isotopic shifts. The carbon isotopic composition of reservoirs can vary substantially, both on Earth and on other planets. Characterizing the reservoir composition and any changes through time is critical to evaluating microbially induced shifts. In addition, careful evaluation of non-microbial causes for shifts in isotopic composition is essential for a reliable interpretation. Complicating processes include recrystallization, calcite precipitation over extended periods of time, variable precipitation rates and water chemistry, and mixing of carbonates having different isotopic signatures.

Carbon Dioxide↗
PubMed2002

The carbon isotope composition of ancient CO2 based on higher-plant organic matter.

Carbon isotope ratios in higher-plant organic matter (delta(13)C(plant)) have been shown in several studies to be closely related to the carbon isotope composition of the ocean-atmosphere carbon reservoir, and, in particular, the isotopic composition of CO(2). These studies have primarily been focused on geological intervals in which major perturbations occur in the oceanic carbon reservoir, as documented in organic carbon and carbonates phases (e.g. Permian-Triassic and Triassic-Jurassic boundary, Early Toarcian, Early Aptian, Cenomanian-Turonian boundary, Palaeocene-Eocene Thermal Maximum (PETM)). All of these events, excluding the Cenomanian-Turonian boundary, record negative carbon isotope excursions, and many authors have postulated that the cause of such excursions is the massive release of continental-margin marine gas-hydrate reservoirs (clathrates). Methane has a very negative carbon isotope composition (delta(13)C, ca. 60 per thousand ) in comparison with higher-plant and marine organic matter, and carbonate. The residence time of methane in the ocean-atmosphere reservoir is short (ca. 10 yr) and is rapidly oxidized to CO(2), causing the isotopic composition of CO(2) to become more negative from its assumed background value (delta(13)C, ca. -7 per thousand ). However, to date, only the Early Toarcian, Early Aptian and PETM are well-constrained chronometric sequences that could attribute clathrate release as a viable cause to create such rapid negative delta(13)C excursions. Notwithstanding this, the isotopic analysis of higher-plant organic matter (e.g. charcoal, wood, leaves, pollen) has the ability to (i) record the isotopic composition of palaeoatmospheric CO(2) in the geological record, (ii) correlate marine and non-marine stratigraphic successions, and (iii) confirm that oceanic carbon perturbations are not purely oceanographic in their extent and affect the entire ocean-atmosphere system. A case study from the Isle of Wight, UK, indicates that the carbon isotope composition of palaeoatmospheric CO(2) during the Mid-Cretaceous had a background value of 3 per thousand, but fluctuated rapidly to more positive (ca. +0.5 per thousand ) and negative values (ca. 10 per thousand ) during carbon cycle perturbations (e.g. carbon burial events, carbonate platform drowning, large igneous province formation). Hence, fluctuations in the carbon isotope composition of palaeoatmospheric CO(2) would compromise our use of palaeo-CO(2) proxies that are dependent on constant carbon isotope ratios of CO(2).

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