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PubMed1983

Preparation of stable isotope-incorporated peptide internal standards for field desorption mass spectrometry quantification of peptides in biologic tissue.

Stable isotopes have been incorporated into two opioid pentapeptides, leucine enkephalin and methionine enkephalin, using chemical- and enzymatic-catalyzed reactions. Oxygen-18 from H218O was the stable isotope incorporated. High-performance liquid chromatography separated the chemical reaction mixture into three fractions: hydrolyzed peptide fraction, 18O-incorporated enkephalin, and 18O-incorporated enkephalin ester. Analysis of individual isotopic species (18O2, 18O16O, 16O2) in the latter two fractions was done with field desorption mass spectrometry. Porcine esterase II was used to hydrolyse enkephalin esters and recover stable isotope. Up to 90% 18O2 was incorporated. Yields of 18O2 species for the overall procedure were for leucine enkephalin, 56%, and for methionine enkephalin, 38%. This scheme represents the first fast and facile preparation of a stable isotope-incorporated peptide internal standard for use in measuring peptides in biologic extracts and is readily extended to any peptide with a free carboxyl group. Fast atom bombardment-collision activated dissociation-linked field (B/E) scan mass spectrometry unambiguously locates the two 18O atoms in the carboxyl group of the peptide and not in a peptide amide bond or tyrosine side chain. The two 18O-labeled peptide internal standards were used to measure, in a structurally unambiguous fashion, endogenous leucine enkephalin and methionine enkephalin in thalamus tissue at the ppb level. A microcomputer was used for data acquisition and reduction.

Animals↗

PubMed1987

Determination of the urinary cortisol production rate using (1,2,3,4-13C)cortisol. Isotope dilution analyses at very small enrichments.

An isotope dilution mass spectrometric method to determine the urinary cortisol production rate (CPR) in babies and children is described. The method uses stable isotopically labelled (1,2,3,4-13C)cortisol. The tracer is intravenously administered to the patient and urine is collected for the following three days. Following extraction, enzymic hydrolysis, purification and isolation by high-performance liquid chromatography (HPLC) the urinary cortisol metabolites tetrahydrocortisone, tetrahydrocortisol, alpha- and beta-cortolone are separately oxidized the common product, 11-oxo-aetiocholanolone. The methyl oxime tert-butyldimethylsilyl ether derivative (MO TBDMS) was analysed by gas chromatography mass spectrometry. Quantification of the isotope enrichment was carried out by unlabelled, and at m/z 348 for labelled 11-oxo-aetiocholanolone. HPLC isolation of the metabolites together with the oxidation step allowed very small isotope enrichments, sometimes down to 0.1% (1:1000), to be reliably measured against a linear calibration graph containing 0 to 1% (13C4) enrichments. The standards for the calibration graph were synthesized from mixtures of labelled (13C4) cortisol and natural cortisol, and the calibration graph was prepared each time samples were measured. The long term instrumental precision of the isotope dilution analyses was 0.91% for a derivatized sample containing a (13C4) enrichment of 0.5% (measured on six different days over seven months). The coefficient of variation of the complete procedure for the four cortisol metabolites was between 1.17 and 2.14%. The clinical applicability of the method is demonstrated by presenting the results of a CPR determination in a patient.

Adolescent↗

PubMedDate not supplied

Drug metabolite identification: stable isotope methods.

This paper focuses on stable isotope labeling of drugs, in combination with mass spectrometry (MS)-based methods, to facilitate the recognition and identification of metabolites and the employment of stable isotope-labeled derivatization reagents (e.g., bis-trimethylsilylacetamide-d18) in the structure elucidation of metabolites from unlabeled drugs via gas-liquid chromatography-MS techniques. In both cases, it is the so-called isotope peak shift that permits generation of data useful for metabolite identification. Furthermore, judicious labeling of a drug permits characterization of drug-related species (metabolites) by MS-based recognition of isotope cluster signatures. Studies using stable isotope-labeled drugs are exemplified by work on aminopyrine and isopropylantipyrine metabolism; examples of the derivatization peak shift approach include those from studies of timolol and cyproheptadine.

Animals↗

PubMed1989

One-day test using stable isotopes to measure true fractional calcium absorption.

True calcium absorption can only be measured by the time-consuming and expensive metabolic balance method and is not predicted well by measuring the fractional absorption (FA) of radiocalcium from a fixed calcium carrier. We describe here a 1 day method for measuring the actual fraction of calcium absorbed from the habitual diet (true fractional calcium absorption, TFCA) using stable isotopes of calcium. Oral and intravenous isotopes were administered with each of the three daily meals, and then the ratio of the two isotopes in the urine was measured by mass spectroscopy. In 12 subjects, TFCA determined from stable Ca correlated well with TFCA measured by the balance method (r = 0.71, P less than 0.01), and the mean values were not different (0.26 and 0.26). In contrast, no significant correlation was found between FA and TFCA. The weak relationship between FA and TFCA underscores the importance of tracing dietary calcium rather than a fixed calcium carrier in tests of calcium absorption. Using the new method, TFCA was inversely related to dietary calcium (r = -0.45, P less than 0.05), demonstrating that it could detect physiological changes in calcium absorption. Thus, this test has two important advantages: (1) it provides a simple way to measure TFCA and true fractional calcium absorption (the product of TFCA and dietary calcium), the physiologically relevant variables, and (2) because there is no radiation exposure, the test can be used in pregnant women and children, the isotopes can be prepared in advance, and several isotopes can be used simultaneously.

Administration, Oral↗

PubMed1996

Solvent isotope effects on the refolding kinetics of hen egg-white lysozyme.

Solvent isotope effects have been observed on the in vitro refolding kinetics of a protein, hen lysozyme. The rates of two distinct phases of refolding resolved by intrinsic fluorescence have been found to be altered, to differing extents, in D2O compared with H2O, and experiments have been conducted aimed at assessing the contributions to these effects from various possible sources. The rates were found to be essentially independent of whether backbone amide nitrogens were protiated or deuterated, indicating that making and breaking of their hydrogen bonding interactions is not associated with a substantial isotope effect. Neither were the rates significantly affected by adding moderate concentrations of sucrose or glycerol to the refolding buffer, suggesting that viscosity differences between H2O and D2O are also unlikely to explain the isotope effects. The data suggest that different factors, acting in opposing directions, may be dominant under different conditions. Thus, the isotope effect on the rate-determining step was found to be qualitatively reversed on going to low pH, suggesting that one component is probably associated with changes in the environments of carboxylate groups in forming the folding transition state. This term disappears at low pH as these groups are protonated and an opposing effect then dominates. It was not possible to identify this other effect on the basis of the present data, but a dependence of the hydrophobic interaction on solvent isotopic composition is a likely candidate.

Animals↗

PubMed1993

Isotope effects in peptide group hydrogen exchange.

Kinetic and equilibrium isotope effects in peptide group hydrogen exchange reactions were evaluated. Unlike many other reactions, kinetic isotope effects in amide hydrogen exchange are small because exchange pathways are not limited by bond-breaking steps. Rate constants for the acid-catalyzed exchange of peptide group NH, ND, and NT in H2O are essentially identical, but a solvent isotope effect doubles the rate in D2O. Rate constants for base-catalyzed exchange in H2O decrease slowly in the order NH > ND > NT. The alkaline rate constant in D2O is very close to that in H2O when account is taken of the glass electrode pH artifact and the difference in solvent ionization constant. Small equilibrium isotope effects lead to an excess equilibrium accumulation of the heavier isotopes by the peptide group. Results obtained are expressed in terms of rate constants for the random coil polypeptide, poly-DL-alanine, to provide reference rates for protein hydrogen exchange studies as described in Bai et al. [preceding paper in this issued].

Hydrogen↗

PubMed2004

Is the isotopic composition of nitrous oxide an indicator for its origin from nitrification or denitrification? A theoretical approach from referred data and microbiological and enzyme kinetic aspects.

Literature data on the isotopic composition of nitrous oxide indicate a general predominance of the alpha-15N-isotopomer and a parallel 18O-enrichment in N2O from nitrification and denitrification, respectively. As the kinetic isotope effects on any individual reactions of the two processes lead to depletions of the heavy isotopes of nitrogen and oxygen in the products, the observed enrichments could mainly be caused by enzymatic reduction of NO, provided it occurs via a symmetric intermediate like hyponitrite; infrared data are in favour of large differences between the binding constants of the isotopomers and isotopologues of this compound. As a matter of fact one of the mechanisms discussed for the nitric oxide reductase from certain microorganisms implies the parallel binding of two NO molecules and the formation of a symmetrical intermediate, while that of the enzyme from other microorganisms reduces NO in a sequential mechanism. In addition, isotope effects on the reduction of N2O to N2 must contribute to the observed isotope characteristics of N2O, especially in context with denitrification. Therefore, the known enzymatic reaction pathways suggest that the alpha-15N-isotopomer preference and the 18O-signature of the produced N2O is not essentially characteristic for its origin from nitrification or denitrification, respectively, but rather from the involved population of microorganisms and the type of their nitric oxide reductases. This has to be confirmed experimentally.

Bacteria↗

PubMed2004

Automated interpretation of mass spectra of complex mixtures by matching of isotope peak distributions.

Mass spectrometry is now firmly established as a powerful technique for the identification and characterization of proteins when used in conjunction with sequence databases. Various approaches involving stable-isotope labeling have been developed for quantitative comparisons between paired samples in proteomic expression analysis by mass spectrometry. However, interpretation of such mass spectra is far from being fully automated, mainly due to the difficulty of analyzing complex patterns resulting from the overlap of multiple peaks arising from the assortment of natural isotopes. In order to facilitate the interpretation of a complex mass spectrum of such a mixture, such as an MS spectrum of a stable-isotope-enriched ion species, we report on the development of a software application, 'Matching' (web accessible), that enables the automatic matching of theoretical isotope envelopes to multiple ion peaks in a raw spectrum. It is particularly useful for resolving the relative abundances of narrow-split paired peaks caused by enrichment with a stable isotope, such as 18O, 13C, 2H, or 15N.

Algorithms↗

PubMed2004

Isotope-coded N-terminal sulfonation of peptides allows quantitative proteomic analysis with increased de novo peptide sequencing capability.

Recently various methods for the N-terminal sulfonation of peptides have been developed for the mass spectrometric analyses of proteomic samples to facilitate de novo sequencing of the peptides produced. This paper describes the isotope-coded N-terminal sulfonation (ICenS) of peptides; this procedure allows both de novo peptide sequencing and quantitative proteomics to be studied simultaneously. As N-terminal sulfonation reagents, 13C-labeled 4-sulfophenyl[13C6]isothiocyanate (13C-SPITC) and unlabeled 4-sulfophenyl isothiocyanate (12C-SPITC) were synthesized. The experimental and reference peptide mixtures were derivatized independently using 13C-SPITC and 12C-SPITC and then combined to generate an isotopically labeled peptide mixture in which each isotopic pair differs in mass by 6 Da. Capillary reverse-phase liquid chromatography/tandem mass spectrometry experiments on the resulting peptide mixtures revealed several immediate advantages of ICenS in addition to the de novo sequencing capability of N-terminal sulfonation, namely, differentiation between N-terminal sulfonated peptides and unmodified peptides in mass spectra, differentiation between N- and C-terminal fragments in tandem mass spectra of multiply protonated peptides by comparing fragmentations of the isotopic pairs, and relative peptide quantification between proteome samples. We demonstrate that the combination of N-terminal sulfonation and isotope coding in the mass spectrometric analysis of proteomic samples is a viable method that overcomes many problems associated with current N-terminal sulfonation methods.

Amino Acid Sequence↗

PubMed2005

delta34S measurements on organic materials by continuous flow isotope ratio mass spectrometry.

Sulfur (S) isotope ratios of thoroughly dried organic samples were measured by direct thermal decomposition in an elemental analyzer coupled to an isotope ratio mass spectrometer in continuous flow mode (EA-CF-IRMS). For organic samples of up to 13 mg weight and with total S contents of more than 10 microg, the reproducibility of the delta34S(organic) values was +/-0.4 per thousand or better. However, the delta34S values of organic samples measured directly by online EA-CF-IRMS analysis were between 0.3 and 2.9 per thousand higher than those determined on BaSO4 precipitates produced by Parr Bomb oxidation from the same sample material. Our results suggest that structural oxygen in organic samples influences the oxygen isotope ratios of the SO2 produced from organic samples. Consequently, SO2 generated from organic samples appears to have different 18O/16O ratios than SO2 generated from BaSO4 precipitates and inorganic reference materials, resulting in a deviation from the true delta34S values because of 32S16O18O contributions to mass 66. It was shown that both the amount of structural oxygen in the organic sample, and the difference of the oxygen isotope ratios between organic samples and tank O2, influenced the magnitude of the observed deviation from the true delta34S value after direct EA-CF-IRMS analysis of organic samples. Suggestions are made to correct the difference between measured delta34S(organic) and true delta34S values in order to obtain not only reproducible, but also accurate S isotope ratios for organic materials by EA-CF-IRMS.

Mass Spectrometry↗

PubMed2006

(13)C/(12)C isotope labelling to study leaf carbon respiration and allocation in twigs of field-grown beech trees.

In situ (13)C/(12)C isotopic labelling was conducted in field-grown beech (Fagus sylvatica) twigs to study carbon respiration and allocation. This was achieved with a portable gas-exchange open system coupled to an external chamber. This method allowed us to subject leafy twigs to CO(2) with a constant carbon isotope composition (delta(13)C of -51.2 per thousand) in an open system in the field. The labelling was done during the whole light period at two different dates (in June 2002 and October 2003). The delta(13)C values of respiratory metabolites and CO(2) that is subsequently respired during the night were measured. It was found that night-respired CO(2) is not completely labelled (only ca. 58% and 27% of new carbon is found in respired CO(2) immediately after the labelling in June 2002 and October 2003, respectively) and the labelling level progressively disappeared during the next day. It is concluded that the carbon respired by beech leaves after illumination was supplied by a mixture of carbon sources in which current carbohydrates were not the only contributors. In addition, as has been found in herbaceous plants, isotopic data before labelling showed that carbon isotope discrimination favoring the (13)C isotope occurred during the night respiration of beech leaves.

Carbon↗

PubMed1997

Kinetics of enzymes with iso-mechanisms: solvent isotope effects.

Kinetic isotope effects on enzymatic reactions which employ general acid or general base catalytic mechanisms may arise during reprotonations of free enzyme. These effects reveal kinetically significant isomerizations of the free enzyme, or iso-mechanisms. The effects are expressed kinetically at high concentrations of substrate, on Vmax or Kcat, but only thermodynamically at low substrate, on Vmax/K(m). The effects are also expressed on the noncompetitive inhibition constant of product inhibition, Kiip, because this parameter is dependent upon the steady-state concentration of the product form of free enzyme. A normal isotope effect on isomerization will decrease Vmax and Kiip, but not necessarily to the same degree. Which is greater will depend upon how rate-limiting the isomerization is to a complete turnover. Together they are related to the full effect on isomerization, DKiso, by their product: DKiso = DVmax DKiip. Moreover, precisely how rate-limiting the isomerization is to a turnover can be shown to be numerically equal to (DVmax - 1)/(DKiipDVmax - 1), which surprisingly, holds whether there are other isotope effects present or not. The new relationships applied to published data on bovine carbonic anhydrase II reveal an intrinsic solvent isotope effect of DK = 9 +/- 4, and an iso step that is less than 80% rate-limiting. Applied to porcine pepsin, a significant DV is accompanied by excessive standard error on DKiip, precluding the calculation of a definitive intrinsic solvent isotope effect.

Carbonic Anhydrases↗

PubMed2000

Development of a stable isotope approach for the inductively coupled plasma-mass spectrometry determination of oxidized metallothionein in biological materials.

The use of isotope dilution analysis (IDA) with inductively coupled plasma-mass spectrometry (ICP-MS) for the determination of oxidized metallothionein (MT) by a Cd-saturation method is investigated. The method developed here is a modification of an earlier methodology which used a radioactive Cd isotope ((109)Cd). While retaining the many advantages of this previous approach, the procedure presented here uses stable isotope ratio measurements ((114)Cd/(111)Cd) for the determination of MT. Experimental parameters governing the instrumental precision and accuracy for isotope ratio measurements of Cd by ICP-MS were characterized. Systematic errors, including mass bias, detector dead time, and spectroscopic interferences, could be easily corrected. The isotope dilution ICP-MS method was validated by the determination of very low levels of cadmium in biological certified reference materials (NIST SRM 2670 freeze-dried urine, IAEA H-8 horse kidney, and BCR TP-25 lichens). Finally, the IDA procedure was evaluated for the determination of oxidized MT by a Cd-saturation method previously developed using radioactive (109)Cd. The final procedure was applied to the quantification of MT in Long-Evans Cinnamon rat liver cytosol samples and the results were compared with data obtained for the same samples using the reference (109)Cd methodology. A good agreement between the analytical values obtained by both methods was observed.

Acetonitriles↗

PubMed2001

Uses of isotope effects in the study of enzymes.

There have been few recent additions to the technical methods employed in the study of isotope effects, notable exceptions being the use of high pressure as an experimental variable and the measurement of heavy-atom isotope effects on maximal velocities using continuous-flow techniques. Most of the innovations are in the realm of new experimental designs that allow the asking of new questions. These designs include the use of isotope effects to: determine kinetic mechanisms, distinguish between changes in enzymatic activity and loss of active enzyme, distinguish between reactant-state origins and transition-state origins and quantify hydrogen tunneling, separate and quantify multiple origins of solvent isotope effects, distinguish between concerted and stepwise chemical mechanisms, characterize bond order changes in ligand binding, distinguish different pathways of inhibitor binding, and estimate intrinsic isotope effects.

Biochemistry↗

PubMed1980

Are sulfur isotope ratios sufficient to determine the antiquity of sulfate reduction?

Sulfur isotope fractionation values have been measured in sedimentary sulfides of varying ages, The 'Antiquity and evolutionary status of bacterial sulfate reduction...' has been inferred from these measurements by Schidlowski (1979). However, under experimental conditions, the isotope values vary widely due to inadequately controlled variables. Thus the direct extrapolation of sulfur isotope fractionation values measured in the laboratory to those measured in sedimentary rocks is unwarranted. New sulfur transforming microbes have been described and recent measurements indicate that inorganic processes affect sulfur isotope fractionation values. This information is summarized here; at present sulfur isotope fractionation values are insufficient to determine the antiquity of sulfate reduction.

Desulfovibrio↗

PubMed2003

Compound-specific stable isotope analysis of organic contaminants in natural environments: a critical review of the state of the art, prospects, and future challenges.

Compound-specific stable isotope analysis (CSIA) using gas chromatography-isotope ratio mass spectrometry (GC/IRMS) has developed into a mature analytical method in many application areas over the last decade. This is in particular true for carbon isotope analysis, whereas measurements of the other elements amenable to CSIA (hydrogen, nitrogen, oxygen) are much less routine. In environmental sciences, successful applications to date include (i) the allocation of contaminant sources on a local, regional, and global scale, (ii) the identification and quantification of (bio)transformation reactions on scales ranging from batch experiments to contaminated field sites, and (iii) the characterization of elementary reaction mechanisms that govern product formation. These three application areas are discussed in detail. The investigated spectrum of compounds comprises mainly n-alkanes, monoaromatics such as benzene and toluene, methyl tert-butyl ether (MTBE), polycyclic aromatic hydrocarbons (PAHs), and chlorinated hydrocarbons such as tetrachloromethane, trichloroethylene, and polychlorinated biphenyls (PCBs). Future research directions are primarily set by the state of the art in analytical instrumentation and method development. Approaches to utilize HPLC separation in CSIA, the enhancement of sensitivity of CSIA to allow field investigations in the microg L(-1) range, and the development of methods for CSIA of other elements are reviewed. Furthermore, an alternative scheme to evaluate isotope data is outlined that would enable estimates of position-specific kinetic isotope effects and, thus, allow one to extract mechanistic chemical and biochemical information.

Chromatography, High Pressure Liquid↗

PubMed2006

High-precision measurement of mercury isotope ratios in sediments using cold-vapor generation multi-collector inductively coupled plasma mass spectrometry.

An on-line Hg reduction technique using stannous chloride as the reductant was applied for accurate and precise mercury isotope ratio determinations by multi-collector (MC)-ICP/MS. Special attention has been paid to ensure optimal conditions (such as acquisition time and mercury concentration) allowing precision measurements good enough to be able to significantly detect the anticipated small differences in Hg isotope ratios in nature. Typically, internal precision was better than 0.002% (1 RSE) on all Hg ratios investigated as long as approximately 20 ng of Hg was measured with a 10-min acquisition time. Introducing higher amounts of mercury (50 ng Hg) improved the internal precision to <0.001%. Instrumental mass bias was corrected using (205)Tl/(203)Tl correction coupled to a standard-sample bracketing approach. The large number of data acquired allowed us to validate the consistency of our measurements over a one-year period. On average, the short-term uncertainty determined by repeated runs of NIST SRM 1641d Hg standard during a single day was <0.006% (1 RSD) for all isotope pairs investigated ((202)Hg/(198)Hg, (202)Hg/(199)Hg, (202)Hg/(200)Hg, and (202)Hg/(201)Hg). The precision fell to <0.01% if the long-term reproducibility, taken over 11 months (over 100 measurements), was considered. The extent of fractionation has been investigated in a series of sediments subject to various Hg sources from different locations worldwide. The ratio (202)Hg/(198)Hg expressed as delta values (per mil deviations relative to NIST SRM 1641d Hg standard solution) displayed differences from +0.74 to -4.00 per thousand. The magnitude of the Hg fractionation per amu was constant within one type of sample and did not exceed 1.00 per thousand. Considering all results (the reproducibility of Hg standard solutions, reference sediment samples, and the examination of natural samples), the analytical error of our delta values for the overall method was within +/-0.28 per thousand (1 SD), which was an order of magnitude lower than the extent of fractionation (4.74 per thousand) observed in sediments. This study confirmed that analytical techniques have reached a level of long-term precision and accuracy that is sufficiently sensitive to detect even small differences in Hg isotope ratios that occur within one type of samples (e.g., between different sediments) and so far have unequivocally shown that Hg isotope ratios in sediments vary within approximately 5 per thousand.

Chemistry Techniques, Analytical↗

PubMed2005

The effects of salinity, crassulacean acid metabolism and plant age on the carbon isotope composition of Mesembryanthemum crystallinum L., a halophytic C(3)-CAM species.

The carbon isotope composition of the halophyte Mesembryanthemum crystallinum L. (Aizoaceae) changes when plants are exposed to environmental stress and when they shift from C(3) to crassulacean acid metabolism (CAM). We examined the coupling between carbon isotope composition and photosynthetic pathway by subjecting plants of different ages to salinity and humidity treatments. Whole shoot delta(13)C values became less negative in plants that were exposed to 400 mM NaCl in the hydroponic solution. The isotopic change had two components: a direct NaCl effect that was greatest in plants still operating in the C(3) mode and decreased proportionally with increasing levels of dark fixation, and a second component related to the degree of CAM expression. Ignoring the presumably diffusion-related NaCl effect on carbon isotope ratios results in an overestimation of nocturnal CO(2) gain in comparison to an isotope versus nocturnal CO(2) gain calibration established previously for C(3) and CAM species grown under well-watered conditions. It is widely taken for granted that the shift to CAM in M. crystallinum is partially under developmental control and that CAM is inevitably expressed in mature plants. Plants, cultivated under non-saline conditions and high relative humidity (RH) for up to 63 days, maintained diel CO(2) gas-exchange patterns and delta(13)C values typical of C(3) plants. However, a weak CAM gas-exchange pattern and an increase in delta(13)C value were observed in non-salt-treated plants grown at reduced RH. These observations are consistent with environmental control rather than developmental control of the induction of CAM in mature M. crystallinum under non-saline conditions.

Carbon↗