Influence of deuterium depleted water on freeze-dried tissue isotopic composition and morphofunctional body performance in rats of different generations

The influence of deuterium depleted water on the body of different rat generations was investigated in physiological conditions. As a result of this study it was established that the most significant and rapid reduction in D/H equilibrium was observed in plasma (by 36.2%), and lyophilized kidney tissues (by 15.8%). Less pronounced deuterium decrease was characteristic of liver tissue (9.3%) and heart (8.5%). Stabilization of the isotopic exchange reaction rate was fixed in the blood and tissues of rats, starting from the second generation. At the same time when deuterium depleted water (40 ppm) was used in dietary intake, the change in morphological and functional parameters in laboratory animals associated with the processes of adaptation to the effects of substress isotopic D/H gradient was also noted. The study shows that modification of only drinking water intake regime can’t significantly change the deuterium content in tissues of metabolically active organs, because of the concurrent deuterium receipt in feed substances of plant and animal origin.     

Influence of deuterium depleted water on the organism of laboratory animals in various functional conditions of nonspecific protective systems

The influence of deuterium depleted water with the modified isotope composition on an organism of animals is studied in physiological conditions and development of chronic endogenous intoxication of hepatorenal genesis. The influence of this water on isotope composition of plasma and tissues (the liver and kidneys) in laboratory animals is shown. The impact of this water on biochemical indicators (AST, ALT, alkaline phosphatase, creatinine, bilirubin) and dynamics in body weight of laboratory animals within 42 days is established. As a result of studies the possibility of the preventive use of deuterium depleted water for correction of metabolic processes is shown in various conditions of the functional system of the body’s detoxification.     

Intramolecular deuterium distributions reveal disequilibrium of chloroplast phosphoglucose isomerase

D, deuterium
δ D(NMR), chemical shift axis in a deuterium NMR spectrum
F6P, fructose-6-phosphate
G6P, glucose-6-phosphate
IRMS, isotope ratio mass spectrometry
NMR, nuclear magnetic resonance
PGI, phosphoglucose isomerase

Intramolecular deuterium distributions of the carbon-bound hydrogens of glucose were measured using deuterium nuclear magnetic resonance. Glucose isolated from leaf starch of common bean ( Phaseolus vulgaris cv. Linden) or spinach ( Spinacia oleracea cv. Giant nobel) was depleted in deuterium in the C(2) position, compared with glucose isolated from leaf sucrose or bean endosperm starch. In beans, the depletion of C(2) was independent of the light intensity during growth (150 or 700 μ mol photons s^–1 m^–2). The ratio of glucose-6-phosphate to fructose-6-phosphate ([G6P]/[F6P]) in bean chloroplasts was 0·9 in high light, indicating that the phosphoglucose isomerase reaction was not in equilibrium ([G6P]/[F6P]) ≈ 3). This implies that the kinetic isotope effect of phosphoglucose isomerase depleted deuterium in the C(2) position of G6P. Because the depletion was the same, the chloroplastic ([G6P]/[F6P]) ratio was in disequilibrium irrespective of the light intensity. If the ([G6P]/[F6P]) ratio was in equilibrium, a large chloroplastic pool of G6P would be unavailable for regeneration of ribulose-1,5-bisphospate. We argue that chloroplast phosphoglucose isomerase activity is regulated to avoid this. The deuterium depletion of C(2) explains the known low overall deuterium abundance of leaf starch. This example shows that measurements of intramolecular deuterium distributions can be essential to understand overall deuterium abundances of plant material.     

Isotopic effects of low concentration of deuterium in water on biological systems

Isotopic effects of deuterium in water are studied in a broad range of concentrations on a number of biological objects of different organization levels. The results obtained show that biological objects are sensitive to variations of isotope composition in water. A decrease or increase in deuterium concentrations in water may cause activation or inhibition of biological functions. The values of biological isotopic effects of low deuterium concentration may even be higher than those of high deuterium concentration. No regularity in response for all the objects studied failed to find out in a range of deuterium concentration in water from 4 ppm to 1%.     

Regioselective deuterium labeling of estrone and catechol estrogen metabolites

Increased exposure to estrogens and estrogen metabolites is linked with increased rates of breast, ovarian and other human cancers. Metabolism of estrogen can led to formation of electrophilic o-quinones capable of binding to DNA. In order to gain insight into the mechanism of estrogen-induced DNA damage, estrone and catechol estrogens derived from estrone, have been regioselectively labeled with deuterium at the 1-position. Estrone-1-d, estrone-1,2,4-d₃, 4-hydroxyestrone-1-d and 2-hydroxyestrone-1-d have been synthesized with or without deuteriums at the 16-position. The key labeling step involves deuterated trifluoroacetic acid exchange catalyzed by t-butyl alcohol. This economical, straightforward labeling technique makes available a range of estrone compounds containing deuterium at the 1-position.     

Changes in the functional activity of mitochondria isolated from the liver of rat that passed the preadaptation to ultra-low deuterium concentration

The incubation in deuterium-depleted medium of mitochondria isolated from the liver of rats that consumed drinking diet with depleted deuterium (46 ppm) revealed a higher (by 35%) generation of hydrogen peroxide in comparison with the mitochondria (isolated from the liver of rats that consumed drinking diet with 152 ppm deuterium) incubated in medium that contained 152 ppm deuterium. Succinate addition to the reaction system led to an increase in the production of hydrogen peroxide in isolated mitochondria by 44–81%, whereas the difference in the generation of H₂O₂ between the organelles incubated in mediums 46 and 152 ppm was reduced by 14%. The revealed change in the functional activity of mitochondria suggests the ability of the organism to adapt to the deuterium-depleted drinking diet, which is probably due to the formation of the D/H isotope transmembrane gradient.     

A comparative study of purple membranes partially rehydrated with water and deuterium oxide

The photoelectric signals of dried oriented purple membrane samples were studied at various hydration degrees (humidities between 0.036–0.13 gH₂0/gbR) in water and deuterium oxide. A modified photocycle was found both in water and deuterium oxide at very low humidities, as obtained previously in the case of water. The dependence of the lifetime on temperature and hydration degree, for the LM and MbR transitions, was calculated by using an exponential decomposition of the electric signals. The Eyring parameters were calculated from the temperature dependence, in order to obtain comparative information concerning the isotope effect following deuteration. The activation enthalpies and entropies for the L decay showed an abrupt change at a water content of about 0.06 gH₂0/gbR, but the isotope effect was present only at humidities below this value. In the case of the M decay, an isotope effect was found at all humidities, the values of Eyring parameters being smaller in deuterium oxide. The activation entropies have negative values in the case of strongly dehydrated samples, both in water and deuterium oxide. Correspondence to: G. Váró     

Use of deuterium labelling from deuterium oxide to demonstrate carotenoid transformations in photosynthetic bacteria

Carotenoid biosynthesis in many purple photosynthetic bacteria of the Rhodospirillaceae is inhibited by nicotine, and biosynthetic intermediates accumulate. If the inhibitor is removed and the bacteria are then incubated in buffered 99.6% deuterium oxide, deuterium is incorporated specifically into the C-2 position in both cyclic and acyclic carotenoids that are then formed from the previously accumulated hydrocarbon precursors. The deuterated molecular species can be detected and assayed by mass spectrometry. By use of this procedure, direct proof has been obtained for the conversion of lycopene into β-carotene and rhodopin in Rhodomicrobium vannielii, of neurosporene into spheroidene in Rhodopseudomonas sphaeroides and of spheroidene into hydroxyspheroidene in Rps. gelatinosa. The results confirm the operation of the biosynthetic pathways postulated for these organisms, and prove that formation of the acyclic 1-hydroxy-1,2-dihydro end-group characteristic of the carotenoids of photosynthetic bacteria occurs by addition of water to the C-1,2 double band.     

Biogeochemistry of the stable isotopes of hydrogen and carbon in salt marsh biota

Deuterium to hydrogen ratios of 14 plant species from a salt marsh and lagoon were 55‰ depleted in deuterium relative to the environmental water. Carbon tetrachloride-extractable material from these plants was another 92‰ depleted in deuterium. This gave a fractionation factor from water to CCl₄ extract of 1.147. This over-all fractionation was remarkably constant for all species analyzed. Plants also discriminate against ¹³C, particularly in the lipid fraction. Data suggest that different mechanisms for carbon fixation result in different fractionations of the carbon isotopes. Herbivore tissues reflected the isotopic ratios of plants ingested. Apparently different metabolic processes are responsible for the different degrees of fractionation observed for hydrogen and carbon isotopes.     

Effect of deuterium oxide on gastrin release from rat antral mucosal fragments

The effects of the microtubule stabilizing agent, deuterium oxide, on in vitro rat antral gastrin release were examined under basal conditions and during stimulation with isobutyl methylxanthine and bombesin plus isobutyl methylxanthine. Basal gastrin release from antral mucosal fragments was unaffected by increasing media concentration of deuterium oxide (12.5 to 75% v/v) during 1 h incubations. Gastrin release stimulated by isobutyl methylxanthine (0.1 mM), a potent inhibitor of phosphodiesterase activity, was inhibited completely by 12.5% deuterium oxide. Bombesin (1 × 10^?8 M) in the presence of IBMX (0.1 mM) stimulated gastrin release (29.7 ± 1.9% of total gastrin). This was significantly greater than gastrin released under control conditions and with IBMX alone: 12.0 ± 1.1 (P < 0.001) and 20.2 ± 2.6% of total gastrin (P < 0.02), respectively. Partial inhibition of bombesin-IBMX stimulated gastrin release was achieved with 12.5% and 25% deuterium oxide and stimulation of gastrin release was inhibited completely by 50% deuterium oxide. In contrast to these results, gastrin release stimulated by the calcium ionophore A23187 was not inhibited by 50% deuterium oxide. Additional studies were performed to assess reversibility of the effects of deuterium oxide on stimulated gastrin release. Antral tissue exposed to initial culture medium containing deuterium oxide (50%) and bombesin-IBMX for 60 min was exchanged for medium without deuterium oxide. Restimulation of antral tissue during the second hour of culture resulted in gastrin release that was comparable to that observed in cultures not exposed to deuterium oxide during the first hour of culture. Reversibility of the effects of deuterium oxide suggest that a functional alteration in microtubular function is restored by removal of heavy water from the culture medium. Results of these experiments indicate that deuterium oxide is capable of inhibiting gastrin release stimulated by the peptide hormone bombesin and by the phosphodiesterase inhibitor isobutyl methylxanthine. Furthermore, these results suggest that increased levels of intracellular calcium achieved by the action of ionophore A23187 prevent microtubular stabilization by deuterium oxide.     

Adaptation and growth response of Arabidopsis thaliana to deuterium

Summary Arabidopsis seeds were sown aseptically on mineral media containing between 0 and 90% of heavy water (D₂O). Initially, a D₂O level of over 50% was lethal for the plants. However, after culture for six successive generations on 50% D₂O, plants were capable of growing marginally on media containing up to 70% D₂O, but not higher. With increasing concentration, deuterium progressively delays germination, slows growth, reduces survival, results in bleaching of the leaves and delays flowering. Pollen fertility is not affected measurably but seed set is reduced with increasing levels of deuteration so that at 70% D₂O few seeds were obtained. The viability of the seeds harvested from plants grown on deuterated media is low. No chlorophyll or morphological mutants were observed among a large number of plant progenies. Seeds from plants cultured on D₂O media for several generations grow normally on proteated media in the very first generation.     

Uptake of deuterium into proteins of fertilized and unfertilized Arbacia eggs suspended in heavy water

When fertilized and unfertilized eggs of Arbacia punctulata are suspended in heavy water, deuterium is incorporated into stable positions in the egg proteins. The rate of incorporation of the isotope is considerably greater in fertilized than in unfertilized eggs, and is accelerated at the time of formation of the blastula. The result of calculation of the maximum deuterium concentration which would be reached on complete turnover indicates that at least one out of every ten stably bound hydrogen atoms of the egg proteins is a deuterium atom. This has been interpreted as evidence that at the time of formation of the sea urchin blastula and in the period of development which follows, synthesis and breakdown are simultaneous processes leading to the redistribution of amino acids among the egg proteins.     

Raman spectroscopy and deuterium exchange of the filamentous phage fd

The filamentous phage fd has been investigated using the techniques of Raman spectroscopy and deuterium exchange. Despite the rather uniform secondary structure of the fd phage coat protein, which is predominantly alpha-helix, the deuterium exchange is complex. A substantial fraction of the helical peptides exchange deuterium by 8 h at room temperature, yet another substantial fraction does not exchange following an additional 5 months at 4 degrees C. Heating the phage to 70 degrees C for several hours leads to additional deuterium exchange compared to samples soaked for 5 months in heavy water. We suggest that the wide variation in peptide exchange rates may be related to the phage protein quaternary structure, which has been shown to be a double layer of tightly packed helices. The accomplishment of enhanced exchange by reaction at high temperature combined with digital difference spectroscopic methods has enabled us to define the structure of the amide III and III' bands. The complexity of these bands is unexpected for a simple helical protein, but we suggest that the complexity arises at least in part from end-effects that become important in short alpha-helices.     

The bioactivation of 1,2-dibromoethane in rat hepatocytes: deuterium isotope effect

The metabolism and genotoxicity of 1,2-dibromoethane (EDB) and its deuterium substituted analog ( d4EDB ) were studied in isolated rat hepatocytes. There was a marked isotope effect on the metabolism of EDB by hepatocytes. This was due to decreased microsomal oxidation of d4EDB . Cytosolic metabolism of EDB, as measured by bromide ion release, was unaffected by deuterium substitution. The genotoxicity of the two analogs was assessed by assaying for the presence of EDB induced single-strand breaks in DNA. As measured by the alkaline elution technique, both compounds caused DNA single-strand breaks when incubated at a concentration of 0.1 mM with hepatocytes. No difference in the degree of DNA damage could be demonstrated between hepatocytes incubated with EDB or d4EDB . These data suggest that the GSH transferase mediated metabolism of EDB is responsible for the genotoxic effects of EDB observed in hepatocytes.     

Probing conserved helical modules of portal complexes by mass spectrometry-based hydrogen/deuterium exchange

The Double-stranded DNA bacteriophage P22 has a ring-shaped dodecameric complex composed of the 84 kDa portal protein subunit that forms the central channel of the phage DNA packaging motor. The overall morphology of the P22 portal complex is similar to that of the portal complexes of Phi29, SPP1, T3, T7 phages and herpes simplex virus. Secondary structure prediction of P22 portal protein and its threading onto the crystal structure of the Phi29 portal complexes suggested that the P22 portal protein complex shares conserved helical modules that were found in the dodecameric interfaces of the Phi29 portal complex. To identify the amino acids involved in intersubunit contacts in the P22 portal ring complexes and validate the threading model, we performed comparative hydrogen/deuterium exchange analysis of monomeric and in vitro assembled portal proteins of P22 and the dodecameric Phi29 portal. Hydrogen/deuterium exchange experiments provided evidence of intersubunit interactions in the P22 portal complex similar to those in the Phi29 portal that map to the regions predicted to be conserved helical modules.     

The nature of bile salt micelles as studied by deuterium NMR

Deuterium NMR of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid was studied. Molecular sizes obtained from deuterium spin-lattice relaxation time (T₁) data of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid in methanol and in water are in accordance with monomeric and tetrameric structures in the two media, respectively. The deuterium T₁ and intensity of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid in aqueous solution at pH 8.0–8.8 were studied as functions of NaCl and lecithin concentrations. The results indicated that tetramers are in equilibrium with larger aggregates when secondary micelles are formed in the precense of NaCl, and that 3α, 12α-dihydroxy-7,7-dideutero-5β-cholanoic acid forms mixed micelles with lecithin with a molecular ratio of 2 : 3.     

Selective deuterium labeling of steroidal estrogens

Refluxing estrone ($\text{1}$) and equilenin ($\text{8}$) in methanol-OD under basic conditions places a deuterium atom at position 4 and 16, and for estradiol at position 4. The location of the label in ring A is confirmed by nmr examination of the aromatic protons. Milder procedures that can be used for labeling the $\text{ortho}$ and $\text{para}$ positions of phenol and the cresols were not successful among steroids. Using palladized charcoal and deuterium, the benzylic hydrogens of estrone can be exchanged. Following the same procedure, but using hydrogen for a back exchange, one deuterium atom remains at position 6 as shown by C¹³ nmr spectroscopy.     

Protein aggregation. The effect of deuterium oxide on large protein aggregates of C-phycocyanin

The amount of 11s aggregate in phycocyanin, normally stimulated by hydrophobic forces, is dramatically increased by the presence of deuterium oxide. Proteins in which hydrophobic forces are not proposed as a mechanism for aggregation are unaffected by deuterium oxide. These observations are consistent with the lower critical micelle concentration reported for ionic detergents in deuterium oxide. Phycocyanin samples containing a majority of material sedimenting faster than 11s were also investigated in the presence of deuterium oxide with the following findings: the most rapidly sedimenting species in water buffer is 24s; in deuterium oxide more than 10% of the protein sediments at 67s and substantial amounts of other species with sedimentation coefficients larger than 24s are present. These large quantities of species sedimenting faster than 24s are found in deuterium oxide buffers from pD5.5 to 7.0. Sucrose-density-gradient studies in deuterium oxide at pD6.0 confirm the presence of large amounts of more rapidly sedimenting species. Spectrophotometric studies on fractions from the sucrose-density-gradient experiments indicate with the presence of higher aggregates a red shift of the visible-absorption maximum and an enhancement of the E(620)/E(280) ratio. Fluorescence-emission studies show a greater relative fluorescence efficiency for these higher aggregates and are consistent with the suggested enhancement of higher aggregates in deuterium oxide. The existence of phycocyanin aggregates of such a large size is suggested to be of importance in vivo, with phycocyanin playing a role as a structural protein.     

Measurement of gluconeogenesis using glucose fragments and mass spectrometry after ingestion of deuterium oxide.

We report a new method to measure the fraction of glucose derived from gluconeogenesis using gas chromatography-mass spectrometry and positive chemical ionization. After ingestion of deuterium oxide by subjects, glucose derived from gluconeogenesis is labeled with deuterium. Our calculations of gluconeogenesis are based on measurements of the average enrichment of deuterium on carbon 1, 3, 4, 5, and 6 of glucose and the deuterium enrichment in body water. In a sample from an adult volunteer after ingestion of deuterium oxide, fractional gluconeogenesis using the "average deuterium enrichment method" was 48.3 +/- 0.5% (mean +/- SD) and that with the C-5 hexamethylenetetramine (HMT) method by Landau et al. (Landau BR, Wahren J, Chandramouli V, Schumann WC, Ekberg K, Kalhan SC; J Clin Invest 98: 378-385, 1996) was 46.9 +/- 5.4%. The coefficient of variation of 10 replicate analyses using the new method was 1.0% compared with 11.5% for the C-5 HMT method. In samples derived from an infant receiving total parenteral nutrition, fractional gluconeogenesis was 13.3 +/- 0.3% using the new method and 13.7 +/- 0.8% using the C-5 HMT method. Fractional gluconeogenesis measured in six adult volunteers after 66 h of continuous fasting was 83.7 +/- 2.3% using the new method and 84.2 +/- 5.0% using the C-5 HMT method. In conclusion, the average deuterium enrichment method is simple, highly reproducible, and cost effective. Furthermore, it requires only small blood sample volumes. With the use of an additional tracer, glucose rate of appearance can also be measured during the same analysis. Thus the new method makes measurements of gluconeogenesis available and affordable to large numbers of investigators under conditions of low and high fractional gluconeogenesis ( approximately 10 to approximately 90) in all subject populations.     

Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry.

C. glutamicum meso-diaminopimelate dehydrogenase is an enzyme of the L-lysine biosynthetic pathway in bacteria. The binding of NADPH and diaminopimelate to the recombinant, overexpressed enzyme has been analyzed using hydrogen/deuterium exchange and electrospray ionization/mass spectrometry. NADPH binding reduces the extent of deuterium exchange, as does the binding of diaminopimelate. Pepsin digestion of the deuterated enzyme and enzyme-substrate complexes coupled with liquid chromatography/mass spectrometry have allowed the identification of eight peptides whose deuterium exchange slows considerably upon the binding of the substrates. These peptides represent regions known or thought to bind NADPH and diaminopimelate. One of these peptides is located at the interdomain hinge region and is proposed to be exchangeable in the "open," catalytically inactive, conformation but nonexchangeable in the "closed," catalytically active conformation formed after NADPH and diaminopimelate binding and domain closure. Furthermore, the dimerization region has been localized by this method, and this study provides an example of detecting protein-protein interface regions using hydrogen/deuterium exchange and electrospray ionization.