Multi-factorial in vivo stable isotope fractionation: causes, correlations, consequences and applications.

Many physical and chemical processes in living systems are accompanied by isotope fractionation on H, C, N, O and S. Although kinetic or thermodynamic isotope effects are always the basis, their in vivo manifestation is often modulated by secondary influences. These include metabolic branching events or metabolite channeling, metabolite pool sizes, reaction mechanisms, anatomical properties and compartmentation of plants and animals, and climatological or environmental conditions. In the present contribution, the fundamentals of isotope effects and their manifestation under in vivo conditions are outlined. The knowledge about and the understanding of these interferences provide a potent tool for the reconstruction of physiological events in plants and animals, their geographical origin, the history of bulk biomass and the biosynthesis of defined representatives. It allows the use of isotope characteristics of biomass for the elucidation of biochemical pathways and reaction mechanisms and for the reconstruction of climatic, physiological, ecological and environmental conditions during biosynthesis. Thus, it can be used for the origin and authenticity control of food, the study of ecosystems and animal physiology, the reconstruction of present and prehistoric nutrition chains and paleaoclimatological conditions. This is demonstrated by the outline of fundamental and application-orientated examples for all bio-elements. The aim of the review is to inform (advanced) students from various disciplines about the whole potential and the scope of stable isotope characteristics and fractionations and to provide them with a comprehensive introduction to the literature on fundamental aspects and applications.

A stable isotope approach to assessing water loss in fruits and vegetables during storage.

Plant tissue water is the source of oxygen and hydrogen in organic biomatter. Recently, we demonstrated that the stable hydrogen isotope value (δ(2)H) of plant methoxyl groups is a very reliable and easily available archive for the δ(2)H value of this tissue water. Here we show in a model experiment that the δ(2)H values of methoxyl groups remain unchanged after water loss during storage of fruits and vegetables under controlled conditions, while δ(2)H and δ(18)O values of tissue water increase. This enhancement is plant-dependent, and the correlation differs from the meteoric water line. The δ(18)O value is better correlated to the weight decrease of the samples. Therefore, we postulate that the δ(2)H value of methoxyl groups and the δ(18)O value of tissue water are suitable parameters for checking postharvest alterations of tissue water, either addition or loss.

Assessment of enzymatic methods in the δ18O value determination of the L-tyrosine p-hydroxy group for proof of illegal meat and bone meal feeding to cattle.

The δ(18)O value of the p-hydroxy group of L-tyrosine depends on the biosynthesis by plants or animals, respectively. In animal proteins it reflects the diet and is therefore an absolute indicator for illegal feeding with meat and bone meal. The aim of this investigation was to perform the positional (18)O determination on L-tyrosine via a one-step enzymatic degradation. Proteins from plants, herbivores, omnivores, and carnivores were characterized by their δ(13)C, δ(15)N, and δ(18)O values, the latter for normalizing the positional δ(18)O values. Their L-tyrosine was degraded by tyrosine phenol lyase to phenol, analyzed as (2,4,6)-tribromophenol. Degradation by tyrosine decarboxylase yielded tyramine. The δ(18)O values of both analytes corresponded to the trophic levels of their sources but were not identical, probably due to an isotope effect on the tyrosine phenol lyase reaction. Availability of the enzyme, easy control of the reaction, and isolation of the analyte are in favor of tyrosine decarboxylase degradation as a routine method.

Essential methodological improvements in the oxygen isotope ratio analysis of N-containing organic compounds.

The quantitative conversion of organically bound oxygen into CO, a prerequisite for the (18)O/(16)O analysis of organic compounds, is generally performed by high-temperature conversion in the presence of carbon at ∼1450°C. Since this high-temperature procedure demands complicated and expensive equipment, a lower temperature method that could be utilized on standard elemental analyzers was evaluated. By substituting glassy carbon with carbon black, the conversion temperature could be reduced to 1170°C. However, regardless of the temperature, N-containing compounds yielded incorrect results, despite quantitative conversion of the bound oxygen into CO. We believe that the problems were partially caused by interfering gases produced by a secondary decomposition of N- and C-containing polymers formed during the decomposition of the analyte. In order to overcome the interference, we replaced the gas chromatographic (GC) separation of CO and N(2) by reversible CO adsorption, yielding the possibility of collecting and purifying the CO more efficiently. After CO collection, the interfering gases were vented by means of a specific stream diverter, thus preventing them from entering the trap and the mass spectrometer. Simultaneously, a make-up He flow was used to purge the gas-specific trap before the desorption of the CO and its subsequent mass spectrometric analysis. Furthermore, the formation of interfering gases was reduced by the use of polyethylene as an additive for analytes with a N:O ratio greater than 1. These methodological modifications to the thermal conversion of N-containing analytes, depending on their structure or O:N ratio, led to satisfactory results and showed that it was possible to optimize the conditions for their individual oxygen isotope ratio analysis, even at 1170°C. With these methodological modifications, correct and precise δ(18)O results were obtained on N-containing analytes even at 1170°C. Differences from the expected standard values were below ±1‰ with standard deviations of the analysis <0.2‰.

delta(34)S-value measurements in food origin assignments and sulfur isotope fractionations in plants and animals.

The delta(34)S values of biological material, especially food commodities, serve as indicators for origin assignments. However, in the metabolism of higher plants sulfur isotope fractionations must be expected. As a matter of fact, the delta(34)S values of the sulfate- and organic-S, respectively, of Brassicaceae and Allium species vegetables showed differences between 3 and 6 per thousand, and differences in glucosinolates were between 0 and 14 per thousand. delta(34)S-value differences of total-S between individual tissues of the same plant were approximately 3 per thousand. It is believed that these relatively small and variable fractionations are due to the partition of individual S-metabolism steps to different plant compartments, where they may occur independently and quantitatively. The delta(34)S values of herbivore muscle meat and milk relative to the diet and between an animal and its child had trophic shifts of approximately 1.5 per thousand. (34)S enrichments of up to 4 per thousand were observed for hair, hooves, and horn, an isotope fractionation of -5 per thousand between the diet sulfate and cartilage. Therefore, the reported agreements between delta(34)S value of biomass and primary S sources are true for only bulk material and not for individual compounds or tissues.

Local modulation of the redox state of p-nitrothiophenol self-assembled monolayers using the direct mode of scanning electrochemical microscopy.

Local reduction of the terminating nitro groups of a p-nitrothiophenol self-assembled monolayer (SAM) under formation of either hydroxylamine or amino groups is invoked using the direct mode of scanning electrochemical microscopy (SECM). By choosing the appropriate potential and a potential pulse sequence, the reduction of the SAM end groups to the desired oxidation state can be achieved, locally restricted to the area of the sample surface directly underneath the positioned SECM tip. Following the "writing" of redox microstructures within the SAM end groups, the local modification of the redox states is visualized ("reading") by using the feedback mode of SECM. The current at the Pt tip electrode is determined by the electron-transfer rate for reoxidation of the redox mediator at the sample surface. Thus, heterogeneities in the SAM surface, which are caused by local differences in the redox state of the end groups, are distinguishable due to the different electron-transfer rates governed by the redox state of the SAM end groups. To further unequivocally prove the successful local modification of the redox state of the SAM end groups during the writing process, the micropatterned surface is selectively modified with biotin at areas with reduced SAM end groups for further complementary binding of an avidin-enzyme conjugate. Selective post-functionalization with an avidin-alkaline phosphatase conjugate allows visualization of the microstructure using the generator-collector mode of SECM.

Stable hydrogen isotope ratios of lignin methoxyl groups as a paleoclimate proxy and constraint of the geographical origin of wood.

Stable isotope ratios of organic compounds are valuable tools for determining the geographical origin, identity, authenticity or history of samples from a vast range of sources such as sediments, plants and animals, including humans. Hydrogen isotope ratios (delta(2)H values) of methoxyl groups in lignin from wood of trees grown in different geographical areas were measured using compound-specific pyrolysis isotope ratio mass spectrometry analysis. Lignin methoxyl groups were depleted in (2)H relative to both meteoric water and whole wood. A high correlation (r(2) = 0.91) was observed between the delta(2)H values of the methoxyl groups and meteoric water, with a relatively uniform fractionation of -216 +/- 19 per thousand recorded with respect to meteoric water over a range of delta(2)H values from -110 in northern Norway to +20 per thousand in Yemen. Thus, woods from northern latitudes can be clearly distinguished from those from tropical regions. By contrast, the delta(2)H values of bulk wood were only relatively poorly correlated (r(2) = 0.47) with those of meteoric water. Measurement of the delta(2)H values of lignin methoxyl groups is potentially a powerful tool that could be of use not only in the constraint of the geographical origin of lignified material but also in paleoclimate, food authenticity and forensic investigations.

A measuring system for the fast simultaneous isotope ratio and elemental analysis of carbon, hydrogen, nitrogen and sulfur in food commodities and other biological material.

The isotope ratio of each of the light elements preserves individual information on the origin and history of organic natural compounds. Therefore, a multi-element isotope ratio analysis is the most efficient means for the origin and authenticity assignment of food, and also for the solution of various problems in ecology, archaeology and criminology. Due to the extraordinary relative abundances of the elements hydrogen, carbon, nitrogen and sulfur in some biological material and to the need for individual sample preparations for H and S, their isotope ratio determination currently requires at least three independent procedures and approximately 1 h of work. We present here a system for the integrated elemental and isotope ratio analysis of all four elements in one sample within 20 min. The system consists of an elemental analyser coupled to an isotope ratio mass spectrometer with an inlet system for four reference gases (N(2), CO(2), H(2) and SO(2)). The combustion gases are separated by reversible adsorption and determined by a thermoconductivity detector; H(2)O is reduced to H(2). The analyser is able to combust samples with up to 100 mg of organic material, sufficient to analyse samples with even unusual elemental ratios, in one run. A comparison of the isotope ratios of samples of water, fruit juices, cheese and ethanol from wine, analysed by the four-element analyser and by classical methods and systems, respectively, yielded excellent agreements. The sensitivity of the device for the isotope ratio measurement of C and N corresponds to that of other systems. It is less by a factor of four for H and by a factor of two for S, and the error ranges are identical to those of other systems.

The prediction of isotopic patterns in phenylpropanoids from their precursors and the mechanism of the NIH-shift: basis of the isotopic characteristics of natural aromatic compounds.

The theoretical 2H-distribution in the aromatic ring of phenylpropanoids can be predicted from that of their precursors--erythrose-4-phosphate, phosphoenolpyruvate and NADPH--and by invoking the mechanism of the NIH-shift and implied deuterium isotope effects. For each position in the non-oxygenated ring, the predicted natural 2H-abundance is in excellent agreement with experimental data obtained from quantitative 2H NMR-measurements on natural compounds, especially concerning the relative 2H-abundances p > o > or = m. For the p-hydroxylated derivatives, the experimentally determined 2H-abundance sequence order m > o can also be deduced, assuming an anisotropic migration (intramolecular isotope effect) of the p-hydrogen atom to the two differently 2H-substituted m-positions during the NIH-shift (intramolecular hydrogen transfer) and an in vivo deuterium kinetic isotope effect of approximately 1.20 on the final hydrogen elimination from the proposed ketodiene intermediate. The predicted 2H-distribution pattern of methyl salicylate 10, a representative of an o-hydroxylated natural compound, is in excellent agreement with that reported from 2H NMR analyses. However, for salicyl alcohol, minor differences between the theoretical and experimentally determined pattern are found that cannot yet be satisfactorily explained. On the other hand, a very good agreement is found between the theoretical and experimental pattern of coumarin, provided a deuterium kinetic isotope effect of approximately 1.30 is assumed for the elimination of the H-atoms from the ketodiene intermediate. The secondary m-hydroxylation of p-coumaric acid in the biosynthesis of vanillin seems to proceed without large isotope effects. Parallel differences are also observed for the 18O-kinetic isotope effects on the corresponding monooxygenase-catalysed reactions. The results demonstrate convincingly that the mechanisms of these general reactions of the phenylpropanoid biosynthetic pathway are identical and follow general principles. Small observed differences between the 2H-patterns of individual natural aromatic compounds originating from the same hydroxylation type can therefore be assigned to differences of the patterns of the precursors, the extent and the orientation of the hydrogen migration, and the kinetic isotope effect on the final hydrogen elimination. The evidence for the existence of general systematic rules governing isotopic patterns in the shikimic acid pathway and its subsequent reactions is further supported by the recently reported 13C-distribution pattern of vanillin, which is also in agreement with that predicted from the precursors. Hence, it is apparent that the systematics of the isotope patterns of phenylpropanoids are in line with the generally accepted biosynthetic reactions in the shikimic acid pathway and that this knowledge can strengthen their value as an essential support for the distinction of natural and synthetic aromatic compounds.

Isotope characteristics of vegetables and wheat from conventional and organic production.

Multielement isotope ratio analysis was checked for its suitability as a means for the discrimination between agricultural products from integrated/conventional or organic production, respectively. Differences were mainly found for delta15N-values. Paprika and tomatoes from organic production in greenhouses showed delta15N-values above+7 per thousand, whereas corresponding products from conventional cultivation had delta15N-values near 0 per thousand. Lettuce, onions, cabbage and Chinese cabbage from field production had delta15N-values in the range of+5 to+6 and+5.5 to+7.5 per thousand, respectively (conventional and organic production); these overlapping differences do not permit a reliable discrimination. The same is true for wheat, showing average delta15N-values of+2.3+/-1.0 and+3.6+/-1.6 per thousand, respectively. The unexpected relative high 15N-enrichments of vegetables from integrated production are discussed as originating, at least partially, from 15N-enrichment in the soil by NH3 evaporation and denitrification.

Biosynthesis of gallic acid in Rhus typhina: discrimination between alternative pathways from natural oxygen isotope abundance.

The biosynthetic pathway of gallic acid in leaves of Rhus typhina is studied by oxygen isotope ratio mass spectrometry at natural oxygen isotope abundance. The observed delta18O-values of gallic acid indicate an 18O-enrichment of the phenolic oxygen atoms of more than 30 per thousand above that of the leaf water. This enrichment implies biogenetical equivalence with oxygen atoms of carbohydrates but not with oxygen atoms introduced by monooxygenase activation of molecular oxygen. It can be concluded that all phenolic oxygen atoms of gallic acid are retained from the carbohydrate-derived precursor 5-dehydroshikimate. This supports that gallic acid is synthesized entirely or predominantly by dehydrogenation of 5-dehydroshikimate.

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.

Fundamentals and systematics of the non-statistical distributions of isotopes in natural compounds.

The intermolecular and intramolecular non-statistical distribution of the isotopes of the bio-elements in natural compounds must obviously be controlled by logical principles. However, a critical review of the available isotope patterns of natural compounds indicates that a previously discussed general thermodynamic order and its mechanistic foundation cannot satisfactorily explain all experimental data. In the present contribution it is shown that a partial thermodynamic order can eventually be attained for defined positions and compounds under steady-state conditions of metabolism. However, as biological systems are generally open and irreversible, many other in vivo isotope discriminations are dominated by kinetic isotope effects, even in context with reversible reactions. On the other hand, kinetic isotope effects can only become effective in vivo in combination with metabolic branching and the implied isotope shifts of the products are balanced by their relative yields. In vivo, the influences of thermodynamic and kinetic isotope effects are modulated by interferences of the actual metabolic conditions, such as the nature and kind of precursors, alternative metabolic pathways, metabolite pools and fluxes, and by reaction mechanisms. This is demonstrated by giving examples for the isotopes of hydrogen, carbon, nitrogen, oxygen and sulfur, while simultaneously the particularities of the individual elements are elaborated. The resulting general theory of the origin of non-statistical isotope distributions in biological systems permits the interpretation and prediction of isotope patterns and provides the scientific basis for the elucidation of biosyntheses and origin assignments of natural compounds.

The in vivo nitrogen isotope discrimination among organic plant compounds.

The bulk delta 15 N-value of plant (leaf) biomass is determined by that of the inorganic primary nitrogen sources NO(3)(-), NH(4)(+) and N(2), and by isotope discriminations on their uptake or assimilation. NH(4)(+) from these is transferred into "organic N" mainly by the glutamine synthetase reaction. The involved kinetic nitrogen isotope effect does not become manifest, because the turnover is quantitative. From the product glutamine any further conversion proceeds in a "closed system", where kinetic isotope effects become only efficient in connection with metabolic branching. The central and most important corresponding process is the GOGAT-reaction, involved in the de novo nitrogen binding and in recycling processes like the phenylpropanoid biosynthesis and photorespiration. The reaction yields relatively 15N-depleted glutamate and remaining glutamine, source of 15N-enriched amide-N in heteroaromatic compounds. Glutamate provides nitrogen for all amino acids and some other compounds with different 15N-abundances. An isotope equilibration is not connected to transamination; the relative delta 15 N-value of individual amino acids is determined by their metabolic tasks. Relative to the bulk delta 15 N-value of the plant cell, proteins are generally 15N-enriched, secondary products like chlorophyll, lipids, amino sugars and alkaloids are depleted in 15N. Global delta 15 N-values and 15N-patterns of compounds with several N-atoms can be calculated from those of their precursors and isotope discriminations in their biosyntheses.

Microbial biosensor array with transport mutants of Escherichia coli K12 for the simultaneous determination of mono-and disaccharides.

An automated flow-injection system with an integrated biosensor array using bacterial cells for the selective and simultaneous determination various mono- and disaccharides is described. The selectivity of the individually addressable sensors of the array was achieved by the combination of the metabolic response, measured as the O(2) consumption, of bacterial mutants of Escherichia coli K12 lacking different transport systems for individual carbohydrates. Kappa-carrageenan was used as immobilization matrix for entrapment of the bacterial cells in front of 6 individually addressable working electrodes of a screen-printed sensor array. The local consumption of molecular oxygen caused by the metabolic activity of the immobilized cells was amperometrically determined at the underlying screen-printed gold electrodes at a working potential of -600 mV vs. Ag/AgCl. Addition of mono- or disaccharides for which functional transport systems exist in the used transport mutant strains of E. coli K12 leads to an enhanced metabolic activity of the immobilized bacterial cells and to a concomitant depletion of oxygen at the electrode. Parallel determination of fructose, glucose, and sucrose was performed demonstrating the high selectivity of the proposed analytical system.