D2 18O (deuterium oxide) method for CO2 output in small mammals and economic feasibility in man.

A test of the validity of the isotopic steady state relationships of the doubly labeled water (H2O) method has been carried out with D2 18O in small mammals (three chipmunks and one mouse). CO2 outputs calculated just from 1) the rate of water intake and 2) the ratios of the isotopic concentrations in the body water to the intake water agreed satisfactorily with observed values. Moreover, reconstructed energy and material balances agreed reasonably with similar balances reconstructed for an immediately succeeding period on the same animals studied by the previously validated decay procedure. We conclude from an error analysis that by expressing the isotopic specific activities as abundances in excess of the body water of a subject on a given regimen, the decay procedure is economically feasible in the human with available accuracy of isotopic analyses and the present cost of H2 18O. The method therefore appears to be a useful tool ready for application to the field of human energy metabolism.     

Heavy water (D2O)-induced shape changes, movements and F-actin redistribution in human neutrophil granulocytes

Heavy water (D2O) induces characteristic shape changes and a distinct type of movement in human neutrophil granulocytes. In contrast to front-tail polarity as evoked by chemotactic peptides and microtubule-disassembling agents, D2O-based media produce non-polar neutrophils with many small or long surface projections. This phenotype is similar to that elicited by both phorbol myristate acetate and diacylglycerols, but the surface projections are smaller and more densely placed and are often associated with a single large projection. D2O-induced non-polar cells with surface projections perform continuous shape changes without front-tail polarity and without the unidirectional movement and cytoplasmic streaming seen in cells with front-tail polarity. Some of the cells show circus movements of a large projection indicating circular polarity. In neutrophils suspended in D2O, F-actin is shifted to the cell periphery, mainly into the surface projections of activated cells. The D2O-induced effects are reversed in H2O-based medium. D2O is dominant over the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), colchicine and taxol in that the combined action of D2O with any of these agents results in the D2O-induced phenotype. In contrast, cytochalasin B alone and in combination with fMLP induces a considerable decrease of non-polar cells and an increase of spherical cells similar to non-stimulated cells in H2O-based medium. Earlier studies indicated that D2O acts on microtubules. Our results suggest that D2O may act on the microfilament system. Neutrophils suspended in D2O-based medium may represent a useful model to study the relationship between shapes, movements, and particular functions of these cells.     

Polymerization and gelation of fibrinogen in D2O

The solution properties of fibrinogen and the thrombin-induced activation and gelation of fibrinogen in 95% D2O at pH 7.4 were compared to those in H2O under similar conditions. The initial release rates of fibrinopeptides A and B in D2O were slightly slower than those in H2O. However, the values of the Michaelis-Menten parameters Km and V for the release of the two peptides in D2O and H2O in the presence of 0.5 M NaCl were about the same. From turbidity measurements at 450 nm it is obvious that fibrinogen is soluble in a slightly more narrow range of NaCl concentration and that the fibrin gels have a higher degree of lateral aggregation in D2O than in H2O. The variation of fibrinogen concentration, thrombin concentration, pH and ionic a strength have a similar dependence on the final gel structure and clotting time in D2O and H2O. SDS-gel electrophoresis on fibrin samples, which were cross-linked by factor XIII, yielded results where the cross-linking of the gamma-chain appeared to be the same in D2O and H2O. The alpha-chain cross-linking was somewhat faster in D2O than in H2O. When fibrinogen solutions in 95% D2O were incubated at 20 mM CaCl2, a slow gelation of fibrinogen was observed, which was found to be induced by trace amounts of factor XIII. The final gel turbidity appeared to be about the same for this gelation as for that induced by thrombin. The differences in solubility for fibrinogen, kinetics for the enzyme reaction and optical properties for the fibrin gels in D2O and H2O may be explained by differences in electrostatic interactions, hydrogen bonding and hydration of fibrinogen in these two media.     

Light-induced generation of membrane potential in intact chloroplasts suspended in H2O or D2O media

The membrane potential changes induced by short flashes and continuous light were investigated in isolated chloroplasts of Peperomia metallica suspended in H2O- or D2O media. The potential generated in H2O-suspended chloroplasts by a single turnover flash is approximately two times lower than the maximal level of potential induced by continuous light. The photoelectric response of D2O-suspended chloroplasts differs from that of H2O-suspended chloroplasts by an increased amplitude and a prolonged phase of the potential rise. Te dark decay of the potential proceeds 2-3 times slower in the D2O-suspended chloroplasts as compared to the H2O-suspended chloroplasts. The magnitude of the flash-induced potential is somewhat lower for the chloroplasts in D2O than for the chloroplasts in the H2O medium. The results obtained suggest that the substitution of H2O for D2O results in a decrease of the ionic conductance and an increase of stability of thylakoid membranes. It was shown that the rise of electrical potential under continuous illumination proceeds in two stages. The difference kinetics of membrane potential changes are observed under conditions of separate activity of two systems of photosynthesis.     

Stability of globular proteins in H2O and D2O

In several experimental techniques D2O rather then H2O is often used as a solvent for proteins. Concerning the influence of the solvent on the stability of the proteins, contradicting results have been reported in literature. In this paper the influence of H2O-D2O solvent substitution on the stability of globular protein structure is determined in a systematic way. The differential scanning calorimetry technique is applied to allow for a thermodynamic analysis of two types of globular proteins: hen's egg lysozyme (LSZ) with relatively strong internal cohesion ("hard" globular protein) and bovine serum albumin (BSA), which is known for its conformational adaptability ("soft" globular protein). Both proteins tend to be more stable in D2O compared to H2O. We explain the increase of protein stability in D2O by the observation that D2O is a poorer solvent for nonpolar amino acids than H2O, implying that the hydrophobic effect is larger in D2O. In case of BSA the transitions between different isomeric forms, at low pH values the Nm and F forms, and at higher pH values Nm and B, were observed by the presence of a supplementary peak in the DSC thermogram. It appears that the pH-range for which the Nm form is the preferred one is wider in D2O than in H2O.     

Investigation of the electron transfer reactions and redox characteristics of photoactive bacteriochlorophyll in Rhodobacter sphaeroides reaction centers modified by D2O and cryoprotectants

The effects of D2O, glycerol and dimethyl sulfoxide (DMSO) on redox potential Em of bacteriochlorophyll of a special P2 or [P(M)P(L)] pair, the rate of energy migration from bacteriopheophytin H(M) to [P(M)P(L)], electron transfer from [P(M)P(L)] to bacteriopheophytin H(L) and then to quinone Q(A) in reaction centers (RC) of Rhodobacter sphaeroides were studied. The H2O --> D2O substitution did not change Em of the special pair, whereas addition of 70% glycerol or 35% DMSO (v/v) increased the values of Em by 30 and 45 mV, respectively. Rate constants of energy migration km(H(M)* (km)--> P2), charge separation ke([P(M)P(L)] *H(L) (ke)--> [P(M)P(L)] +H(L)-), electron transfer to quinone kQ did not change after the glycerol addition, whereas isotopic substitution and addition of DMSO caused a 2-3-fold increase in km, ke, and kQ values. Theoretical analysis of the redox center potential dependence on dielectric permeability epsilon, swelling of the protein globule in a solvent, and on changes in the charge distribution (charge shifts) in the protein interior near the redox center was carried out. It has been shown that the H2O replacement with DMSO can result in the Em increase by tens of mV. No correlation was found between the Em values and the rate of charge separation upon isotopic substitution and addition of cryoprotectants. The effect of epsilon of the medium on the rate of electron transport due to changes of energy of intermolecular interaction between the donor and acceptor molecules was estimated.     

D2O induced alterations of mitosis in PtK1 cells

Deuterium oxide (D2O) was applied to PtK1 cells to assess its effect on mammalian mitosis. Cells exposed to culture medium containing up to 50% D2O were able to enter and complete mitosis, but the duration of mitosis was increased proportionally to the concentration of D2O applied. Cells exposed to 50% D2O showed increases of more than 300% for the interval between nuclear envelope breakdown and anaphase onset, and approximately 65% for the interval between anaphase onset and initial furrowing. At a concentration of 80%, D2O acted as an inhibitor of mitosis; after 8 h exposure to this concentration, cultures showed an increase in the proportion of mulinucleate cells and an absence of mitotic figures. When applied early in anaphase, 80% D2O effectively slowed chromosome separation, prolonging anaphase for more than 60 min. Normal chromosome motion was restored when medium containing D2O was replaced with control medium. Mitotic chromosomes remained condensed throughout prolonged anaphase intervals. Immunofluoresence examination of spindles stained using a monoclonal anti-tubulin revealed no pronounced increase in microtubule polymerization after exposure of cells to 20-80% D2O.     

Steady-state nitrogen isotope effects of N2 and N2O production in Paracoccus denitrificans

Nitrogen stable-isotope compositions (delta15N) can help track denitrification and N2O production in the environment, as can knowledge of the isotopic discrimination, or isotope effect, inherent to denitrification. However, the isotope effects associated with denitrification as a function of dissolved-oxygen concentration and their influence on the isotopic composition of N2O are not known. We developed a simple steady-state reactor to allow the measurement of denitrification isotope effects in Paracoccus denitrificans. With [dO2] between 0 and 1.2 microM, the N stable-isotope effects of NO3- and N2O reduction were constant at 28.6 per thousand +/- 1.9 per thousand and 12.9 per thousand +/- 2.6 per thousand, respectively (mean +/- standard error, n = 5). This estimate of the isotope effect of N2O reduction is the first in an axenic denitrifying culture and places the delta15N of denitrification-produced N2O midway between those of the nitrogenous oxide substrates and the product N2 in steady-state systems. Application of both isotope effects to N2O cycling studies is discussed.     

Effects of deuterium oxide on the rate and dissociation constants for saxitoxin and tetrodotoxin action. Voltage-clamp studies on frog myelinated nerve

The actions of tetrodotoxin (TTX) and saxitoxin (STX) in normal water and in deuterium oxide (D2O) have been studied in frog myelinated nerve. Substitution of D2O for H2O in normal Ringer's solution has no effect on the potency of TTX in blocking action potentials but increases the potency of STX by approximately 50%. Under voltage clamp, the steady-state inhibition of sodium currents by 1 nM STX is doubled in D2O as a result of a halving of the rate of dissociation of STX from the sodium channel; the rate of block by STX is not measurably changed by D2O. Neither steady-state inhibition nor the on- or off-rate constants of TTX are changed by D2O substitution. The isotopic effects on STX binding are observed less than 10 min after the toxin has been added to D2O, thus eliminating the possibility that slow-exchange (t 1/2 greater than 10 h) hydrogen-binding sites on STX are involved. The results are consistent with a hypothesis that attributes receptor-toxin stabilization to isotopic changes of hydrogen bonding; this interpretation suggests that hydrogen bonds contribute more to the binding of STX than to that of TTX at the sodium channel.     

Isotopic signatures of N2O produced by ammonia-oxidizing archaea from soils

N₂O gas is involved in global warming and ozone depletion. The major sources of N₂O are soil microbial processes. Anthropogenic inputs into the nitrogen cycle have exacerbated these microbial processes, including nitrification. Ammonia-oxidizing archaea (AOA) are major members of the pool of soil ammonia-oxidizing microorganisms. This study investigated the isotopic signatures of N₂O produced by soil AOA and associated N₂O production processes. All five AOA strains (I.1a, I.1a-associated and I.1b clades of Thaumarchaeota) from soil produced N₂O and their yields were comparable to those of ammonia-oxidizing bacteria (AOB). The levels of site preference (SP), δ¹⁵N^bulk and δ¹⁸O -N₂O of soil AOA strains were 13–30%, −13 to −35% and 22–36%, respectively, and strains MY1–3 and other soil AOA strains had distinct isotopic signatures. A ¹⁵N-NH₄⁺-labeling experiment indicated that N₂O originated from two different production pathways (that is, ammonia oxidation and nitrifier denitrification), which suggests that the isotopic signatures of N₂O from AOA may be attributable to the relative contributions of these two processes. The highest N₂O production yield and lowest site preference of acidophilic strain CS may be related to enhanced nitrifier denitrification for detoxifying nitrite. Previously, it was not possible to detect N₂O from soil AOA because of similarities between its isotopic signatures and those from AOB. Given the predominance of AOA over AOB in most soils, a significant proportion of the total N₂O emissions from soil nitrification may be attributable to AOA.     

D2O as a modifier of ionic specificity of Na, K-ATPase

Effect of heavy water D2O on the rate of hydrolysis of ATP and pNPP by Na,K-ATPase was studied. Heavy water of high concentration inhibits the rate of ATPase reaction in all the studied ratios of the ions Na/K at constant ionic strength 150 mM. Activation of the enzyme was observed in the solution with low concentration of heavy water (less than 5%). The value of isotope effects depended on the ratio between sodium and potassium ion concentrations in the medium. At low temperature no activation of the enzyme with heavy water in low concentration was observed. Substitution of usual water for the heavy one was accompanied by a decrease of apparent constants of enzyme activation with sodium and potassium ions. During pNPP hydrolysis with Na,K-ATPase an increase of reaction rate in the medium with heavy water was observed. Substitution of potassium ions by cesium resulted in an increase of isotope effects during ATP and pNPP hydrolysis. Analysis of isotope effects in terms of the molecular model of sodium pump proposed permits a conclusion that the isotope effects of heavy water are explained by its influence as a solvent, the binding centres of potassium and sodium ions are localized in different regions of the enzyme differing in physico-chemical properties. The structure of sodium centres is controlled by hydrogen bonds, and of potassium ones--by hydrophobic interactions; the transport of ions by the enzyme is accompanied by dehydration of ions.     

Hydrogen isotope fractionation during H2/CO2 acetogenesis: hydrogen utilization efficiency and the origin of lipid-bound hydrogen

Hydrogen metabolism was studied in the anaerobic bacterium, Sporomusa sp. strain DMG 58, by measuring natural abundance levels of deuterium in H₂, H₂O, and individual fatty acids during acetogenic growth on H₂/CO₂. Four cultures were grown, each in medium with a distinct hydrogen‐isotopic composition (δD‐H₂O). The δD value of H₂ was quantified in the residual gas exiting the growth chambers and found to decrease concurrently with net H₂ consumption, indicating rapid isotope exchange between H₂ and H₂O. An isotopic mass balance was used to constrain the efficiency with which H₂ was activated by the cell and the reducing equivalents catabolized, which we term the H₂ utilization efficiency. Results indicate that H₂ utilization efficiency in these cultures is less than 20% during the growth phase, and less than 2% after the growth phase. The gross rate of cellular H₂ activation was similar in the growth phase and afterward. Biomass harvested at the end of each experiment was used to analyse the D/H of individual membrane lipids. Values of δD were highly correlated between lipids and water (δD‐lipids = 0.59 × δD‐water – 381‰; R² = 0.995), indicating the source of lipid hydrogen is in isotopic equilibrium with water. Results are consistent with two possibilities: (i) water is the sole source of hydrogen to lipids, and the fractionation during biosynthesis is significantly larger than previously observed (α = 0.59), or (ii) hydrogen from H₂ is incorporated into lipids, but only after reaching isotopic equilibrium with H₂O. Fatty acids were strongly depleted in deuterium relative to all other organisms studied thus far, and such large depletions may prove useful as biomarkers for studying H₂ cycling in anoxic environments as well as in the geological record.     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the rumen ciliate Dasytricha ruminantium

The effects of ruminal concentrations of CO2 and O2 on glucose-stimulated and endogenous fermentation of the rumen isotrichid ciliate Dasytricha ruminantium were investigated. Principal metabolic products were lactic, butyric and acetic acids, H2 and CO2. Traces of propionic acid were also detected; formic acid present in the incubation supernatants was found to be a fermentation product of the bacteria closely associated with this rumen ciliate. 13C NMR spectroscopy revealed alanine as a minor product of glucose fermentation by D. ruminantium. Glucose uptake and metabolite formation rates were influenced by the headspace gas composition during the protozoal incubations. The uptake of exogenously supplied D-glucose was most rapid in the presence of O2 concentrations typical of those detected in situ (i.e. 1-3 microM). A typical ruminal gas composition (high CO2, low O2) led to increased butyrate and acetate formation compared to results obtained using O2-free N2. At a partial pressure of 66 kPa CO2 in N2, increased cytosolic flux to butyrate was observed. At low O2 concentrations (1-3 microM dissolved in the protozoal suspension) in the absence of CO2, increased acetate and CO2 formation were observed and D. ruminantium utilized lactate in the absence of extracellular glucose. The presence of both O2 and CO2 in the incubation headspaces resulted in partial inhibition of H2 production by D. ruminantium. Results suggest that at the O2 and CO2 concentrations that prevail in situ, the contribution made by D. ruminantium to the formation of ruminal volatile fatty acids is greater than previously reported, as earlier measurements were made under anaerobic conditions.     

Preparative cell electrophoresis with D2O as a stabilizing agent.

The method of ascending preparative cell electrophoresis in a nonstabilized vertical column can be greatly improved by layering the cells on a starting cushion of buffer prepared in D2O and by stabilizing the liquid column above it with a D2O gradient. D2O/H2O mixtures have no apparent biochemical effects on the cells, and their physiochemical effects are short-lived and reversible. It was possible, by this method, to separate a mixture of glutaraldehyde-fixed erythrocytes of three species (rabbit, horse and chicken) into three distinct zones after 15 minutes of electrophoresis. It was also possible to obtain an enriched human lymphocyte fraction containing 96% T-cells, after 1 hour of electrophoresis.     

D2O inhibition of interphase thymocyte death]

Using the method of flow cytometry and biochemical analysis it was shown that D2O, an agent that stabilizes microtubules, prevented the internucleosome fragmentation of DNA in thymocytes exposed to gamma radiation and dexamethasone in vitro. It was also found that D2O is ineffective with respect to Ca2+/Mg2(+)-dependent nuclease. The transfer of irradiated cells from a medium containing 90% of D2O to a normal one caused rapid DNA degradation; the fragmentation process ceased with the irradiated cells being transferred from H2O to heavy water. The results obtained permit us to assume that the disturbance of microtubules is not a trigger mechanism of DNA degradation by apoptosis, but is some intermediate stage of cell death preceding the chromatin fragmentation proper.     

A new measurement technique reveals temporal variation in delta18O of leaf-respired CO2

The oxygen isotope composition of CO(2) respired by Ricinus communis leaves (delta(18)O(R)) was measured under non-steady-state conditions with a temporal resolution of 3 min using a tunable diode laser (TDL) absorption spectrometer coupled to a portable gas exchange system. The SD of delta(18)O measurement by the TDL was +/- 0.2 per thousand and close to that of traditional mass spectrometers. Further, delta(18)O(R) values at isotopic steady state were comparable to those obtained using traditional flask sampling and mass spectrometric techniques for R. communis grown and measured in similar environmental conditions. As well as higher temporal resolution, the online TDL method described here has a number of advantages over mass spectrometric techniques. At isotopic steady state among plants grown at high light, the "one-way flux" model was required to accurately predict delta(18)O(R). A comparison of measurements and the model suggests that plants grown under low-light conditions have either a lower proportion of chloroplast CO(2) that isotopically equilibrates with chloroplast water, or more enriched delta(18)O of CO(2) in the chloroplast that has not equilibrated with local water. The high temporal resolution of isotopic measurements allowed the first measurements of delta(18)O(R) when stomatal conductance was rapidly changing. Under non-steady-state conditions, delta(18)O(R) varied between 50 and 220 per thousand for leaves of plants grown under different light and water environments, and varied by as much as 100 per thousand within 10 min for a single leaf. Stomatal conductance ranged from 0.001 to 1.586 mol m(-2) s(-1), and had an important influence on delta(18)O(R) under non-steady-state conditions not only via effects on leaf water H(2) (18)O enrichment, but also via effects on the rate of the one-way fluxes of CO(2) into and out of the leaf.     

Stable isotope fractionation by Clostridium pasteurianum. 1. 34S/32S: inverse isotope effects during SO4-2- and SO3-2- reduction.

During growth on minimal salts--sucrose media supplemented with various concentrations (10-4-10-2 M) of sodium sulfate, Clostridium pasteurianum grew at a normal rate and only evolved sulfide in late stages of growth on 10-2 M SO4-2-. The evolved sulfide was slightly enriched in 34S as compared to the medium sulfur. On the other hand, sulfide was evolved during growth on all concentrations of sulfite tested. Large normal and inverse isotopic effects were observed in the evolved sulfide during SO3-2- reductions. In contrast, the intracellular sulfur showed much smaller fractionations. The complexity of the isotopic patterns suggests that a dissimilatory sulfite reductase system may be induced by high concentrations of sulfite.     

Altered O2-/H2O2 production ratio by in vitro and in vivo primed human neutrophils

Human neutrophils were primed by exudation or pretreatment with a synthetic diacylglycerol (diC10), the Ca2+ ionophore ionomycin or lipopolysaccharide (LPS). Compared to control cells, these primed cells showed a significantly decreased O2-/H2O2 ratio when stimulated with formylmethionyl-leucyl-phenylalanine (FMLP). This shift indicates a comparative (and net) increased H2O2 detection in the extracellular medium and can not be explained by a dose-dependent impairment in either O2- or H2O2 detecting capacity. An altered H2O2 degenerating capacity was not observed in the primed cells. We propose that priming enhances the capacity to divalently reduce oxygen and thereby directly produce H2O2.     

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.     

The effects of H2O2 on electromechanical activity of the ileum longitudinal muscle

The effects of H2O2 on electrical and mechanical activity of the longitudinal layer from the guinea-pig ileum were studied using sucrose-gap technique and the influence of H2O2 on ionic current was investigated in single smooth muscle cells by the patch-clamp method. In most of the preparations tested, the spontaneous activity observed was composed of slow waves with superimposed action potentials (APs). Both were resistant to tetrodotoxin and atropine. H2O2 (1 mmol/l) evoked sustained 3-5 mV membrane depolarisation, doubled the amplitude of the slow waves and increased their frequency, augmented the APs and reduced their splitting. These changes were accompanied with significant contraction, which had an amplitude comparable to that of the tonic component of 50 mmol/l K+-induced contraction. Calcium-free solution caused membrane depolarisation, reduction of the slow wave amplitude and frequency, disappearance of APs and decreased the mechanical tension of the preparations. Application of H2O2 (1 mmol/l) into the zero-calcium bath solution recovered the APs, which was accompanied by a low amplitude contraction. H2O2 (up to 1 mmol/l) increased the L-type calcium current (I(Ca)) both under conventional whole-cell patch-clamp configuration and under amphotericin-perforated patches by 16 +/- 3%. These data demonstrated that contractile response of the ileum longitudinal smooth muscle preparation evoked by H2O2 was mainly due to the enhanced electrical activity.