Metabolite Pools and Metabolic Branching as Factors of in-vivo Isotope Discriminations by Kinetic Isotope Effects

Abstract

Inter- and intra-molecular non-statistical isotope distributions do not only require the existence of a kinetic isotope effect on a defined enzyme catalyzed reaction, but also the prerequisite that this reaction is located at a metabolic branching point. Furthermore a metabolic and isotopic balance demand that the extent of the isotopic shift is reciprocal to the products' yields. On this base the ¹³C-enrichment of L-ascorbic acid in position C-1 and the depletion of glycerol in C-1 are interpreted. The ¹³C-pattern of natural malic acid is discussed as a consequence of isotope effects on the carboxylation of pyruvate and PEP and on the pyruvate dehydrogenase reaction. The patterns of natural products synthezised by transfer of “active acetaldehyde” is proposed to be due to an isotope effect on the thiamine pyrophosphate containing lyase reaction. An isotope effect on the reduction of “active formaldehyde” to “active methyl” and the existence of corresponding pools is responsible for ¹³C-enrichments and depletions of natural products in positions bearing these intermediates. Finally a model for the main nitrogen pools and for isotope discriminations between α-amino, ω-amino-N and amide pools in plants is proposed.     

Hydrogen Isotope Effects in Electrochemical Reductions of Organic Chloro Compounds

Abstract

The kinetic hydrogen isotope effects in electrochemical reductions of CCl₃COOD, CDCl₂COOD, CCl₄, CDCl₃, benzyl chloride, 1-chloronaphthalene and 4-chlorobenzonitrile in deuterated reaction media were determined. The aliphatic chloro compounds are reduced with rather small isotope effects k_H/k_D = 1.2…1.7, as expected if anionic intermediates are formed and protonated. Benzyl chloride is reduced with an apparent k_H/k_D = 2.1, which is probably too high due to radical reactions. Aromatic chloro compounds are reduced with significant participation of a second mechanism via radical intermediates which abstract carbon-bound hydrogen atoms from the components of the reaction system. The resulting isotope effects are k_H/k_D = 1.2 for the part of the reduction which proceeds via anions and k_H/k_D ～ 2.5 for the competing radical reaction.     

Isotope Effects in the Electrochemical Reduction of Oxalic Acid-D2 and Benzoic Acid-D

Abstract

The electrochemical reduction of model carboxylic acid at lead cathodes in deuterated media was studied for potential use in the synthesis of special deuterated compounds.

Oxalic acid-d₂ in D₂O gives good yields of glyoxylic acid-d₂. An unexpected large isotope effect of 5.3 ± 1.7 was found in this reaction, leading to significant depletion of deuterium content in the aldehyde group.

Benzoic acid-d in CD₃OD/diluted D₂SO₄ yields benzyl alcohol-d, 7,7-d₂. The isotope effect of 2.4 ± 1.0 is within the expected range. No deuterium is incorporated into the aromatic nucleus. Essentially unlabelled benzyl alcohol is obtained in CD₃OH/dil. H₂SO₄.     

Oxygen and Carbon Isotope Variations of Carbonates and Silicates from the Carbonatite Massif Tiksheozero (Northern Karelia)

Abstract

Carbon and oxygen isotope compositions have been determined for carbonate minerals from different types of carbonatites (large carbonatite bodies, veins, eruptive breccia), as well as from different temperature classes of carbonatites (according to Samoilov [1]). It could be shown, that only high temperature carbonatites exhibit small variations of δ¹⁸O and δ¹³C falling in the range of “high temperature mantle carbonates”, whereas low temperature calcites and dolomites show wide variations in δ¹⁸O. These changes in both isotope and mineral compositions of low temperature carbonatites are interpreted as a result of repeated interaction with fluids. The oxygen isotope ratios in most of the eruptive breccia have been changed by secondary processes. The primary deep-seated isotope record has been preserved only in samples, in which carbonate occurs as fine-grained calcite groundmass.

Preliminary results on oxygen isotope composition of different silicate minerals (amphibole, magnetite, biotite, albite, apatite) indicate isotope equilibrium for the mineral pair calcite-amphibole with isotope temperatures representing superimposed processes.     

Stable Carbon Isotope Fractionation in Lower Plants from the Schirmacher and Untersee Oases (Central Dronning Maud Land,East Antarctica)

Abstract

Lower plants (algae, lichens, mosses) from the Antarctic continent have been analysed for their stable carbon isotope composition. In contrast to lichens and mosses which exhibit quite normal δ¹³C-values the studied microbial benthos is characterized by an extremely low carbon isotope fractionation (δ¹³C-values up to ?1.4‰. vs. PDB). Limited CO₂ availability and bicarbonate uptake are probably the main factors responsible for this phenomenon.     

Metabolic and Isotopic Correlations between D-Glucose,L-Ascorbic Acid and L-Tartaric Acid

Abstract

L-Ascorbic acid in fruit juices is enriched in ¹³C by 4.8 δ-units relative to its precursor glucose, and this enrichment is mainly located in position C-1. On the other hand, biotechnologically produced L-ascorbic acid preserves the pattern as expected from glucose. In grapes L-tartaric acid is synthesized from glucose via L-ascorbic acid and it is one of its main products. It preserves C-atoms 1 to 4 from glucose and the measured δ¹³C-value is identical to that calculated from this precursor.

The findings are interpreted as overlapping influences of isotope discriminations after metabolic branching and relative turnover rates leading to isotopic balances. The results can also be used to distinguish between identical products by different synthetic processes.     

Platinized Magnesium as Reducing Agent for Hydrogen Isotope Analysis

Abstract

Platinized magnesium has recently been proposed as a new reducing agent for the conversion of small quantities of water to hydrogen in a flame-sealed borosilicate glass tube at 400°C for isotopic analysis. The reagent, Mg-Pt, in contrast to zinc can be prepared in every laboratory by coating a magnesium granulate with a thin layer of platinum by reaction with H₂PtCl₆·6H₂O dissolved in acetone-ether mixture. Excellent reproducibility of the isotope ratios in hydrogen gas prepared from water samples has been obtained using 4 μl of water and 120 mg of the reagent.     

A Modified Technique for the Preparation of SO2 from Sulphates and Sulphides for Sulphur Isotope Analyses

Abstract

A modified technique for the conversion of sulphates and sulphides to SO₂ with the mixture of V₂O₅—SiO₂ for sulphur isotopic analyses is described. This technique is more suitable for routine analysis of large number of samples. Modification of the reaction vessel and using manifold inlet system allows to analyse up to 24 samples every day. The modified technique assures the complete yield of SO₂, consistent oxygen isotope composition of the SO₂ gas and reproducibility of δ³⁴S measurements being within 0.10‰. It is observed, however, oxygen in SO₂ produced from sulphides differs in δ¹⁸O with respect to that produced from sulphates.     

Helium and Argon Isotope Abundances in Rocks of Lavozero Alkaline Massif

Abstract

He and Ar isotope abundances in alkaline rocks of the Lovozero layered ore-bearing complex are used to discuss the origin of hydrogen-hydrocarbon gases (HHG).

Three procedures to extract - crushing, milling and melting - and the chemical analysis of these gases have been described, and then the isotope analysis of the ratios of ³He/⁴He and ⁴⁰Ar/³⁶Ar is related. It is suggested, that (⁴He/⁴⁰Ar)_rad ratios and ⁴He concentrations decrease and ³He/⁴He increase with the distance from the ore zone, the former ratio being the most contrast indicator of ore mineralization. A direct correlation has been revealed between helium isotopic ratios and methane concentrations.

The results of measurements confirm the idea on the crustal origin of HHG.     

Microcombustion of ng Amounts of Carbon in Non-Volatile Materials for isotope Ratio Evaluation

Abstract

A novel microcombustion technique for carbon isotopic analysis of nanogram amounts of carbon in non-volatile materials based on isotope ratio monitoring (irm) mass spectrometry is described. Liquid or solid samples placed in a quartz sleeve are combusted at 1000°C in a continuous stream of helium and oxygen. CO₂ removed from the carrier gas stream by cryogenic trapping is transferred onto a GC column. Following GC separation, the CO₂ is transferred via an open split to the ion source of a gas isotope ratio mass spectrometer. Reproducibility for samples >25 nmol carbon is <1‰. Problems associated with blanks from various sources and with reproducible deposition of small sample amounts led to variable accuracy, which was dependent on the compound class being analysed. Minimum sample size was in the range from 5 to 10 nmol carbon. Measurements of dissolved organic carbon (DOC) of groundwater from Germany yielded consistent values of δ¹³C = -28.8‰.     

Isotope Anomalies of Carbon,Hydrogen and Nitrogen in Peat from the Area of the Tunguska Cosmic Body Explosion (1908)

Abstract

Peat profiles from the area of the Tunguska explosion epicentre indicate significant carbon and hydrogen isotopic effects which are clearly associated with the zone of the 1908 “catastrophe”, and which cannot be attributed to any known terrestrial processes. We explain them with the presence of extraterrestrial matter similar to carbonaceous chondrites or, more probably, to cometary matter. Initial data on nitrogen content and its isotope composition are consistent with the assumption of acid rainfall following the passage and explosion of the Tunguska cosmic body, as is known to have occurred during the Cretaceous-Tertiary boundary.     

Rate coefficients and kinetic isotope effects of the abstraction reaction of H atoms from methylsilane

ABSTRACT

Thermal rate constants for chemical reactions using improved canonical variational transition state theory (ICVT) with small-curvature tunnelling (SCT) contributions in a temperature range 180–2000 K are reported. The general procedure is used with high-quality ab initio computations and semi-classical reaction probabilities along the minimum energy path (MEP). The approach is based on a vibrational adiabatic reaction path and is applied to the multiple-channel hydrogen abstraction reaction H + SiH₃CH₃ → products and its isotopically substituted variants. All the degrees of freedom are optimised and harmonic vibrational frequencies and zero-point energies are calculated at the MP2 level with the cc-pVTZ basis set. Single-point energies are calculated at a higher level of theory; CCSD(T)-F12a/VTZ-F12. ICVT/SCT rate constants show that the quantum tunnelling contributions at low temperatures are relatively important and the H-abstraction channel from SiH₃ group of SiH₃CH₃ is the major pathway. The total rate constants are given by the following expression: k_tot(ICVT/SCT) = 2.29 10^?18 T^2.42 exp(?350.9/T) cm³ molec^?1 s^?1. These calculated rates are in agreement with the available experiments. The ICVT/SCT method is further exploited to predict primary and secondary kinetic isotope effects, respectively).     

Chemical and Isotope Investigations in Dissolved Gases of a Meromictic Residual Lake

Abstract

Lake Hufeisen near Halle/Saale has been formed in an old mining pit. Its deepest part is trench shaped and filled with water of sodium chloride content. Due to the density difference strong mixing processes in this part of the water body are prevented all over the year (meromictic lake). Therefore anoxic conditions have been established and the organic matter of the lake sediments is converted to carbon dioxide and methane. Over a period of more than one year gas samples were collected from different water depths. The investigations of the chemical composition (mainly nitrogen, carbon dioxide and methane) and the carbon isotope ratios show different relationships for individual species. The results are discussed with respect to the behaviour of the gases in the water column and to the stability of the density stratification in the saline bottom water.     

Metabolite Pools and Metabolic Branching as Factors of in-vivo Isotope Discriminations by Kinetic Isotope Effects

Abstract Inter- and intra-molecular non-statistical isotope distributions do not only require the existence of a kinetic isotope effect on a defined enzyme catalyzed reaction, but also the prerequisite that this reaction is located at a metabolic branching point. Furthermore a metabolic and isotopic balance demand that the extent of the isotopic shift is reciprocal to the products' yields. On this base the (13)C-enrichment of L-ascorbic acid in position C-1 and the depletion of glycerol in C-1 are interpreted. The (13)C-pattern of natural malic acid is discussed as a consequence of isotope effects on the carboxylation of pyruvate and PEP and on the pyruvate dehydrogenase reaction. The patterns of natural products synthezised by transfer of "active acetaldehyde" is proposed to be due to an isotope effect on the thiamine pyrophosphate containing lyase reaction. An isotope effect on the reduction of "active formaldehyde" to "active methyl" and the existence of corresponding pools is responsible for (13)C-enrichments and depletions of natural products in positions bearing these intermediates. Finally a model for the main nitrogen pools and for isotope discriminations between α-amino, ω-amino-N and amide pools in plants is proposed.     

Fractionation of Carbon Isotopes During Carbohydrate Fermentation in Ruminants

Abstract

In the forestomaches of ruminants the carbohydrates of the diet are transformed by microbiotic fermentation via pyruvate into short chain-fatty acids (acetate, propionate, and with less quantity butyrate). Since carbohydrates, i.e. glucose, cannot penetrate the rumen epithelium, metabolically needed glucose must be resynthesized intermediary mainly from propionate by gluconeogenesis.

Lactose in milk of lactating cows is formed up to 98% from glucose of that origin [1]. Glucose absorption is possible in the small intestine, but contrary to monogastric animals, this is without quantitative relevance.

Therefore, an experimentally found higher abundance of ¹³C in lactose of about 3‰, compared with milk-fat [2], is not explainable only by passing on the well known isotopic differences between carbohydrates and fat in the diet. According to the conclusion of De NIRO and EPSTEIN [3], who identified the decarboxylation of pyruvate to be the relevant step of ¹³C depletion in fat in cultures of Escheria coli, we assume an isotopic shift between propionate and acetate produced by the ruminal microorganisms using pyruvate as the last common precursor of both fatty acids.

In vitro-investigation of ¹³C/¹²C - ratios in CO₂ produced during the decarboxylation of pyruvate in fresh rumen-fluid, confirmed the responsibility of that enzyme-reaction for isotope fractionation also in the multiple-step-system of ruminal carbohydrate conversion.     

Condensed Phase Isotope Effects

It is well known that the condensed phase physical properties of molecular isotopic isomers are not the same. These differences in the vapor pressure, molar volume, surface tension, etc., are purely quantal phenomena. The effects are most exterme for isotopic substitution at hydrogen isotopes. Of the various physical properties which have been investigated the isotope effect on vapour pressure (VPIE) shows the largest ralative effects. In face, for hydrogen-deuterium substitution they range all the way from about inverse 2% per deuterium atom for nonpolar hydrocarbons (by inverse we mean that the molecule substituted with the heavier isotope has the higher vapor pressure) to as much as 20% normal (P_H > P_D ) per deuterium atom for molecules which are strongle associated in the condensed phase. Such effects are about one order of magnitude larger than isotope effects which are observed in most systems on molar volumes, surface tension, etc. (Thus, for example, benxene at 20°C shows a rather small VPIE of 0.4% per atom D 3 but the molar volume effect is only 0.05% per D and the surface tension effect 0.03% [4].) This it would appear is the reason a majority of recent studies have been directed toward the VPIE. The emphasis in this paper reflects that trend and the balance of the discussion will deal almost exclusively with the vapor pressure isotope effect.     

Determination of 13CO2/12CO2 Ratio by IRMS and NDIRS

Abstract

Breath tests using ¹³C-labelled substrates require the measurement of ¹³CO₂/¹²CO₂ ratio in breath gas samples. Next to isotope ratio mass spectrometry (IRMS), which is very sensitive but also complex and expensive, alternatively isotope selective nondispersive infrared spectrometry (NDIRS) can be used to determine the ¹³CO₂/¹²CO₂ ratio in expired breath. In this study we compared NDIRS- with IRMS-results to investigate whether the less expensive and very simply to operate NDIRS works as reliable as IRMS. For this purpose we applicated 1-¹³C-Phenylalanine to patients with advanced liver cirrhosis and healthy volunteers and took duplicated breath samples for IRMS and NDIRS at selected time points. Our data show a good correlation between these two methods for a small number of samples as required for simple breath tests. Longer series, where repeated measurements are required on the NDIRS instrument lead to a decreasing correlation. This indicates the superiority of IRMS concerning ¹³CO₂-kinetics over longer time periods.     

Preparation of Naturally 13C-Rich Free Fatty Acids (FFA) from Corn Oil and their Application in Metabolic Studies of Lactating Cows

Abstract

When the ¹³C/¹²C ratios in lactose and milk fat of normally fed healthy cows are compared to ¹³C/¹²C in feed a metabolic depletion of ¹³C in the milk constituents becomes obvious.

Due to a carbon isotope effect in the decarboxylation step of pyruvate to Acetyl-Co-A resulting in a ¹³C depleted acetate, depot fat and milk fat are especially reduced in ¹³C relative to ¹²C.

Under pathophysiological conditions after longer fasting periods or in ketosis, long chain fatty acids from depot fat are metabolized and their ¹³C depleted carbon atoms enter the citric acid cycle via Acetyl-Co-A. Then the intermediary products of this cycle become ¹³C depleted too and with a drain on the oxalacetate pool by gluconeogenesis ¹³C depleted carbon atoms are channeled into lactose.

This hypothesis is apt to explain a ¹³C/¹²C decrease in lactose of ketotic cows relative to healthy controls. It is supported by the observation that ¹³C-rich fatty acids from corn oil dosed orally to a ketotic cow increase the ¹³C/¹²C ratios in lactose. So a crossing-over of carbon atoms from long chain fatty acids into glucose and lactose seems highly probable.     

The Selective Flux of Sulfur and Implications for Magmatic Sulfur Isotope Fractionation

The selective flux of sulfur during magma emplacement is proposed to explain some abnormal δ³⁴S data from granitic and basaltic rocks. It is assumed that on the one hand a quasi-equilibrium isotope fractionation exists between sulfate and sulfide during magma cooling, and on the other hand a non-equilibrium fractionation occurs between the fluxed sulfur and the magmatic sulfur. The results show that at high fO₂, ³⁴S is preferably enriched in sulfate with decreasing temperature, without a corresponding depletion of sulfide in ³⁴S. The δ³⁴S value of solidified rocks is then significantly shifted in the positive direction due to the selective degassing or assimilation. Conversely, at low fO₂, ³⁴S is preferably depleted in sulfide as temperature declines, while a corresponding ³⁴S-enrichment in sulfate does not occur. As a consequence, δ³⁴S value of the rocks is driver in the negative direction.