Strong Isotope Effect Research Articles

Human mitochondrial cytochrome P450 27C1 is localized in skin and preferentially desaturates trans-retinol to 3,4-dehydroretinol

Recently, zebrafish and human cytochrome P450 (P450) 27C1 enzymes have been shown to be retinoid 3,4-desaturases. The enzyme is unusual among mammalian P450s in that the predominant oxidation is a desaturation and in that hydroxylation represents only a minor pathway. We show by proteomic analysis that P450 27C1 is localized to human skin, with two proteins of different sizes present, one being a cleavage product of the full-length form. P450 27C1 oxidized all-trans-retinol to 3,4-dehydroretinol, 4-hydroxy (OH) retinol, and 3-OH retinol in a 100:3:2 ratio. Neither 3-OH nor 4-OH retinol was an intermediate in desaturation. No kinetic burst was observed in the steady state; neither the rate of substrate binding nor product release was rate-limiting. Ferric P450 27C1 reduction by adrenodoxin was 3-fold faster in the presence of the substrate and was ∼5-fold faster than the overall turnover. Kinetic isotope effects of 1.5-2.3 (on kcat/Km ) were observed with 3,3-, 4,4-, and 3,3,4,4-deuterated retinol. Deuteration at C-4 produced a 4-fold increase in 3-hydroxylation due to metabolic switching, with no observable effect on 4-hydroxylation. Deuteration at C-3 produced a strong kinetic isotope effect for 3-hydroxylation but not 4-hydroxylation. Analysis of the products of deuterated retinol showed a lack of scrambling of a putative allylic radical at C-3 and C-4. We conclude that the most likely catalytic mechanism begins with abstraction of a hydrogen atom from C-4 (or possibly C-3) initiating the desaturation pathway, followed by a sequential abstraction of a hydrogen atom or proton-coupled electron transfer. Adrenodoxin reduction and hydrogen abstraction both contribute to rate limitation.

Role of molecular effects in divertor plasma recombination

Molecule-Activated Recombination (MAR) effect is re-considered in view of divertor plasma conditions. A strong isotopic effect is demonstrated. In deuterium plasmas, the reaction chain through D2+ formation, usually considered dominant and included in 2D edge plasma models, is negligible. However, in this case the other branch, through D−, usually neglected in modelling, becomes relatively strong. The overall share of MAR in divertor plasma recycling stays within 20%. The operational parameters of the divertor plasmas, such as the peak power loading on the divertor targets or the pressure limit for partial detachment of the divertor plasma, are insensitive to the presence of MAR, although the latter may be important for correct interpretation of the divertor diagnostics.

Dynamical barrier and isotope effects in the simplest substitution reaction via Walden inversion mechanism

Reactions occurring at a carbon atom through the Walden inversion mechanism are one of the most important and useful classes of reactions in chemistry. Here we report an accurate theoretical study of the simplest reaction of that type: the H+CH4 substitution reaction and its isotope analogues. It is found that the reaction threshold versus collision energy is considerably higher than the barrier height. The reaction exhibits a strong normal secondary isotope effect on the cross-sections measured above the reaction threshold, and a small but reverse secondary kinetic isotope effect at room temperature. Detailed analysis reveals that the reaction proceeds along a path with a higher barrier height instead of the minimum-energy path because the umbrella angle of the non-reacting methyl group cannot change synchronously with the other reaction coordinates during the reaction due to insufficient energy transfer from the translational motion to the umbrella mode.

Interface Defect Hydrogen Depassivation and Capacitance-Voltage Hysteresis of Al2O3/InGaAs Gate Stacks.

We investigate the effects of pre- and postatomic layer deposition (ALD) defect passivation with hydrogen on the trap density and reliability of Al2O3/InGaAs gate stacks. Reliability is characterized by capacitance-voltage hysteresis measurements on samples prepared using different fabrication procedures and having different initial trap densities. Despite its beneficial capability to passivate both interface and border traps, a final forming gas (H2/N2) anneal (FGA) step is correlated with a significant hysteresis. This appears to be caused by hydrogen depassivation of defects in the gate stack under bias stress, supported by the observed bias stress-induced increase of interface trap density, and strong hydrogen isotope effects on the measured hysteresis. On the other hand, intentional air exposure of the InGaAs surface prior to Al2O3 ALD increases the initial interface trap density (Dit) but considerably lowers the hysteresis.

Strong Isotope Effects on Effective Interactions and Phase Behavior in Protein Solutions in the Presence of Multivalent Ions.

In this article, we have studied the influence of the isotopic composition of the solvent (H2O or D2O) on the effective interactions and the phase behavior of the globular protein bovine serum albumin in solution with two trivalent salts (LaCl3 and YCl3). Protein solutions with both salts exhibit a reentrant condensation phase behavior. The condensed regime (regime II) in between two salt concentration boundaries (c* < cs < c**) is significantly broadened by replacing H2O with D2O. Within regime II, liquid-liquid phase separation (LLPS) occurs. The samples that undergo LLPS have a lower critical solution temperature (LCST). The value of LCST decreases significantly with increasing solvent fraction of D2O. The effective protein-protein interactions characterized by small-angle X-ray scattering demonstrate that although changing the solvent has negligible effects below c*, where the interactions are dominated by electrostatic repulsion, an enhanced effective attraction is observed in D2O above c*, consistent with the phase behavior observed. As the LCST-LLPS is an entropy-driven phase transition, the results of this study emphasize the role of entropy in solvent isotope effects.

A strong developmental isotope effect in Caenorhabditis elegans induced by 5,5-deuterated lysine.

Effects exerted by heavy isotope substitution in biopolymers on the functioning of whole organisms have not been investigated. We report on the decrease of permissive temperature of nematodes fed with bacteria containing 5,5-D2-lysine. We synthesized 5,5-dideuterolysine and, taking advantage of lysine being an essential amino acid, showed that C. elegans with modified lysine poorly develop from larvae into fertile adult hermaphrodites. This effect occurs only at high temperature within the permissible range for C. elegans (25°C) and completely vanishes at 15°C. The only known metabolic involvement of C5 in lysine is in post-translational modification through lysyl hydoxylases. Indeed, siRNA experiments showed that deficiency of let-268/plod lysyl-hydroxylase/glycosydase further amplifies the isotope effect making it apparent even at 20°C, whereas control siRNAs as well as another lysyl-hydroxylase (psr-1/jmjdD) siRNA do not. We report for the first time that a site-specific deuteration may strongly affect the development of the whole animal organism especially under the conditions of deficiency of the corresponding enzyme. These findings provide the basis for our ongoing efforts to employ isotope effects for fine tuning of metabolic pathways to mitigate pathological processes.

Proton Conductivity in Phosphoric Acid: The Role of Quantum Effects.

Phosphoric acid has one of the highest intrinsic proton conductivities of any known liquids, and the mechanism of this exceptional conductivity remains a puzzle. Our detailed experimental studies discovered a strong isotope effect in the conductivity of phosphoric acids caused by (i)a strong isotope shift of the glass transition temperature and (ii)a significant reduction of the energy barrier by zero-point quantum fluctuations. These results suggest that the high conductivity in phosphoric acids is caused by a very efficient proton transfer mechanism, which is strongly assisted by quantum effects.

Interplay between crystal and magnetic structures in YFe2(HαD1−α)4.2 compounds studied by neutron diffraction

We report a detailed magnetic structure investigation of YFe2(HαD1−α)4.2 (α=0, 0.64, 1) compounds presenting a strong (H,D) isotope effect by neutron diffraction and Mössbauer spectroscopy analysis. They crystallize in the same monoclinic structure (Pc space group) with 8 inequivalent Fe sites having different H(D) environment. At low temperature, the compounds are ferromagnetic (FM) and show an easy magnetization axis perpendicular to the b axis and only slightly tilted away from the c axis. Upon heating, they display a first order transition from a ferromagnetic towards an antiferromagnetic (AFM) structure at TM0 which is sensitive to the H/D isotope nature. The AFM cell is described by doubling the crystal cell along the monoclinic b axis. It presents an unusual coexistence of non magnetic Fe layer sandwiched by two thicker ferromagnetic Fe layers which are antiparallel to each other. This FM-AFM transition is driven by the loss of ordered moment on one Fe site (Fe7) through an itinerant electron metamagnetic (IEM) behaviour. The key role of the Fe7 position is assigned to both its hydrogen rich atomic environment and its geometric position. Above TM0 a field induced metamagnetic transition is observed from the AFM towards the FM structure accompanied by a cell volume increase. Both thermal and magnetic field dependence of the magnetic structure are found strongly related to the anisotropic cell distortion induced by (H,D) order in interstitial sites.

Dual Carbon-Bromine Stable Isotope Analysis Allows Distinguishing Transformation Pathways of Ethylene Dibromide.

The present study investigated dual carbon-bromine isotope fractionation of the common groundwater contaminant ethylene dibromide (EDB) during chemical and biological transformations, including aerobic and anaerobic biodegradation, alkaline hydrolysis, Fenton-like degradation, debromination by Zn(0) and reduced corrinoids. Significantly different correlation of carbon and bromine isotope fractionation (ΛC/Br) was observed not only for the processes following different transformation pathways, but also for abiotic and biotic processes with, the presumed, same formal chemical degradation mechanism. The studied processes resulted in a wide range of ΛC/Br values: ΛC/Br = 30.1 was observed for hydrolysis of EDB in alkaline solution; ΛC/Br between 4.2 and 5.3 were determined for dibromoelimination pathway with reduced corrinoids and Zn(0) particles; EDB biodegradation by Ancylobacter aquaticus and Sulfurospirillum multivorans resulted in ΛC/Br = 10.7 and 2.4, respectively; Fenton-like degradation resulted in carbon isotope fractionation only, leading to ΛC/Br ∞. Calculated carbon apparent kinetic isotope effects ((13)C-AKIE) fell with 1.005 to 1.035 within expected ranges according to the theoretical KIE, however, biotic transformations resulted in weaker carbon isotope effects than respective abiotic transformations. Relatively large bromine isotope effects with (81)Br-AKIE of 1.0012-1.002 and 1.0021-1.004 were observed for nucleophilic substitution and dibromoelimination, respectively, and reveal so far underestimated strong bromine isotope effects.

Photodegradation of polycyclic aromatic hydrocarbons in soils under a climate change base scenario

The photodegradation of polycyclic aromatic hydrocarbons (PAHs) in two typical Mediterranean soils, either coarse- or fine-textured, was here investigated. Soil samples, spiked with the 16 US EPA priority PAHs, were incubated in a climate chamber at stable conditions of temperature (20 °C) and light (9.6 W m−2) for 28 days, simulating a climate change base scenario. PAH concentrations in soils were analyzed throughout the experiment, and correlated with data obtained by means of Microtox® ecotoxicity test. Photodegradation was found to be dependent on exposure time, molecular weight of each hydrocarbon, and soil texture. Fine-textured soil was able to enhance sorption, being PAHs more photodegraded than in coarse-textured soil. According to the EC50 values reported by Microtox®, a higher detoxification was observed in fine-textured soil, being correlated with the outcomes of the analytical study. Significant photodegradation rates were detected for a number of PAHs, namely phenanthrene, anthracene, benzo(a)pyrene, and indeno(123-cd)pyrene. Benzo(a)pyrene, commonly used as an indicator for PAH pollution, was completely removed after 7 days of light exposure. In addition to the PAH chemical analysis and the ecotoxicity tests, a hydrogen isotope analysis of benzo(a)pyrene was also carried out. The degradation of this specific compound was associated to a high enrichment in 2H, obtaining a maximum δ2H isotopic shift of +232‰. This strong isotopic effect observed in benzo(a)pyrene suggests that compound-specific isotope analysis (CSIA) may be a powerful tool to monitor in situ degradation of PAHs. Moreover, hydrogen isotopes of benzo(a)pyrene evidenced a degradation process of unknown origin occurring in the darkness.

A closer examination of the coupling between ionic hydrogen bond (IHB) stretching and flanking group motions in (CH3OH)2H(+): the strong isotope effects.

The intermode coupling between shared proton (O-H(+)-O) fundamental stretching and flanking modes in (CH3OH)2H(+) was revisited in the following contexts: (1) evaluation of Hamiltonian matrix elements represented in a "pure state" (PS) basis and (2) tuning of coupling strengths using H/D isotopic substitution. We considered four experimentally accessible isotopologues for this study. These are: (CH3OH)2H(+), (CD3OH)2H(+), (CH3OD)2D(+), and (CD3OD)2D(+). Potential energy surfaces (PESs), as well as dipole moment surfaces (DMSs), were constructed at the MP2/aug-cc-pVDZ level. Multidimensional vibrational calculations were conducted by solving a reduced dimensional Schrödinger equation using a discrete variable representation (DVR). We found that vibrational states in (CH3OH)2H(+) and (CD3OH)2H(+) are much more heavily mixed than those in (CH3OD)2D(+) and (CD3OD)2D(+). Furthermore, each isotopologue chooses to strongly couple between out-of-phase in-plane CH3 rocking and its out-of-plane counterpart. Lastly, the interaction between O-O stretching and O-H(+)-O stretching was explored. We found that between the first overtone of O-O stretching and its combination tone with O-H(+)-O fundamental stretching, only the second couples with O-H(+)-O fundamental stretching. We hope that our isotopologue calculations would motivate experimentalists to measure them in the future.

Formation of the prebiotic molecule NH2CHO on astronomical amorphous solid water surfaces: accurate tunneling rate calculations.

Investigating how formamide forms in the interstellar medium is a hot topic in astrochemistry, which can contribute to our understanding of the origin of life on Earth. We have constructed a QM/MM model to simulate the hydrogenation of isocyanic acid on amorphous solid water surfaces to form formamide. The binding energy of HNCO on the ASW surface varies significantly between different binding sites, we found values between ∼0 and 100 kJ mol-1. The barrier for the hydrogenation reaction is almost independent of the binding energy, though. We calculated tunneling rate constants of H + HNCO → NH2CO at temperatures down to 103 K combining QM/MM with instanton theory. Tunneling dominates the reaction at such low temperatures. The tunneling reaction is hardly accelerated by the amorphous solid water surface compared to the gas phase for this system, even though the activation energy of the surface reaction is lower than the one of the gas-phase reaction. Both the height and width of the barrier affect the tunneling rate in practice. Strong kinetic isotope effects were observed by comparing to rate constants of D + HNCO → NHDCO. At 103 K we found a KIE of 231 on the surface and 146 in the gas phase. Furthermore, we investigated the gas-phase reaction NH2 + H2CO → NH2CHO + H and found it unlikely to occur at cryogenic temperatures. The data of our tunneling rate constants are expected to significantly influence astrochemical models.

Evidence That Compound I Is the Active Species in Both the Hydroxylase and Lyase Steps by Which P450scc Converts Cholesterol to Pregnenolone: EPR/ENDOR/Cryoreduction/Annealing Studies.

Cytochrome P450scc (CYP 11A1) catalyzes the conversion of cholesterol (Ch) to pregnenolone, the precursor to steroid hormones. This process proceeds via three sequential monooxygenation reactions: two hydroxylations of Ch first form 22(R)-hydroxycholesterol (HC) and then 20α,22(R)-dihydroxycholesterol (DHC); a lyase reaction then cleaves the C20-C22 bond to form pregnenolone. Recent cryoreduction/annealing studies that employed electron paramagnetic resonance (EPR)/electron nuclear double resonance (ENDOR) spectroscopy [Davydov, R., et al. (2012) J. Am. Chem. Soc. 134, 17149] showed that compound I (Cpd I) is the active intermediate in the first step, hydroxylation of Ch. Herein, we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the second and third steps of the enzymatic process, as conducted by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc complexes with HC and DHC. This procedure is validated by showing that the cryoreduced ternary complexes of oxy-cytochrome P450scc with HC and DHC are catalytically competent and during annealing generate DHC and pregnenolone, respectively. Cryoreduction of the oxy-P450scc-HC ternary complex trapped at 77K produces the superoxo-ferrous P450scc intermediate along with a minor fraction of ferric hydroperoxo intermediates. The superoxo-ferrous intermediate converts into a ferric-hydroperoxo species after annealing at 145 K. During subsequent annealing at 170-180 K, the ferric-hydroperoxo intermediate converts to the primary product complex with the large solvent kinetic isotope effect that indicates Cpd I is being formed, and (1)H ENDOR measurements of the primary product formed in D2O demonstrate that Cpd I is the active species. They show that the primary product contains Fe(III) coordinated to the 20-O(1)H of DHC with the (1)H derived from substrate, the signature of the Cpd I reaction. Hydroperoxo ferric intermediates are the primary species formed during cryoreduction of the oxy-P450scc-DHC ternary complex, and they decay at 185 K with a strong solvent kinetic isotope effect to form low-spin ferric P450scc. Together, these observations indicated that Cpd I also is the active intermediate in the C20,22 lyase final step. In combination with our previous results, this study thus indicates that Cpd I is the active species in each of the three sequential monooxygenation reactions by which P450scc catalytically converts Ch to pregnenolone.

Strong Isotope Effect in the Vibrational Response of the Hydration Shells of Hydrophobic Ions

We have studied the properties of water molecules at the surface of salt solutions containing hydrophobic anions like triflate (CF3SO3–), ethanesulfonate (C2H5SO3–), and butanesulfonate (C4H9SO3–) using vibrational sum-frequency generation (VSFG) spectroscopy. The VSFG spectra reveal a surprisingly strong isotope effect in the intra- and intermolecular mixing of the water molecules contained in the hydration shells of the hydrophobic anions. The O–H stretch vibrations of H2O molecules in the hydration shell are strongly mixed, whereas the O–D stretch vibrations of hydrating D2O molecules are decoupled. This isotope effect is not observed for other ions like perchlorate (ClO4–), and can be explained from the structure of the hydration shells of the hydrophobic ions.

Effect of electron-nuclei interaction on internuclear motions in slow ion-atom collisions

The electron-nuclei interaction affects the internuclear motion in slow ion-atom collisions, which in turn affects theoretical results for the cross sections of various collision processes. The results are especially sensitive to the details of the internuclear dynamics in the presence of a strong isotope effect on the cross sections, as is the case, e.g., for the charge transfer in low-energy collisions of ${\text{He}}^{2+}$ with H, D, and T. By considering this system as an example, we show that internuclear trajectories defined by the Born-Oppenheimer (BO) potential in the entrance collision channel, which effectively accounts for the electron-nuclei interaction, are in much better agreement with trajectories obtained in the ab initio electron-nuclear dynamics approach [R. Cabrera-Trujillo et al., Phys. Rev. A 83, 012715 (2011)] than the corresponding Coulomb trajectories. We also show that the use of the BO trajectory instead of the Coulomb trajectory in the calculations of the charge-transfer cross sections within the adiabatic approach improves the agreement of the results with ab initio calculations.

Metal hydride differential scanning calorimetry as an approach to compositional determination of mixtures of hydrogen isotopologues and helium

Gaseous mixtures of diatomic hydrogen isotopologues and helium are often encountered in the nuclear energy industry and in analytical chemistry. Compositions of stored mixtures can vary due to interactions with storage and handling materials. When tritium is present, it decays to form ions and helium-3, both of which can lead to further compositional variation. Monitoring of composition is typically achieved by mass spectrometry, a method that is bulky and energy-intensive. Mass spectrometers disperse sample material through vacuum pumps, which is especially troublesome if tritium is present. Our ultimate goal is to create a compact, fast, low-power sensor that can determine composition with minimal gas consumption and waste generation, as a complement to mass spectrometry that can be instantiated more widely. We propose calorimetry of metal hydrides as an approach to this, due to the strong isotope effect on gas absorption, and demonstrate the sensitivity of measured heat flow to atomic composition of the gas. Peak shifts are discernible when mole fractions change by at least 1%. A mass flow restriction results in a unique dependence of the measurement on helium concentration. A mathematical model is presented as a first step toward prediction of the peak shapes and positions. The model includes a useful method to compute estimates of phase diagrams for palladium in the presence of arbitrary mixtures of hydrogen isotopologues. We expect that this approach can be used to deduce unknown atomic compositions from measured calorimetric data over a useful range of partial pressures of each component.

Quasiclassical-Trajectory Investigation on the Isotopic Effect of H(D)+LiF→H(D)F+Li (v = 0–4, j = 0) Reaction **Supported by the Jilin University under Grant No 419080106440, the Chinese National Fusion Project for the International Thermonuclear Experimental Reactor under Grant No 2010GB104003, and the National Natural Science Foundation of China under Grant No 10974069.

A quasi-classical trajectory (QCT) method is employed to investigate the scalar properties and vector correlations of H+LiF→HF+Li and D+LiF→DF+Li reactions. The collision energy (Ecol = 4–25 kcal/mol) and vibrational excitation effects (v = 0–4) are studied by using the Aguado–Paniagua2-potential energy surface (AP2-PES) [J. Chem. Phys. 107 (1997) 10085]. The reaction probability, cross section and rate constant are calculated, which demonstrate obvious energy and vibrational excitation dependences in the probability, cross section, and a high-temperature region of the rate constant. In addition, two product angular distributions P(θr) and P(φr) are calculated to facilitate a deeper insight into vector correlations. The H+LiF→HF+Li and D+LiF→DF+Li reactions reveal strong isotopic effects. Moreover, these scalar and vector results of both the reactions show sensitive behaviors to the changes of vibrational levels and the collision energy.

Hydrogenation of CO2 to methanol and CO on Cu/ZnO/Al2O3: Is there a common intermediate or not?

H/D exchange experiments on a Cu/ZnO/Al2O3 catalyst have shown that methanol synthesis and RWGS display a strong thermodynamic isotope effect, which is attributed to differences in the zero-point energy of hydrogenated vs. deuterated species. The effect is larger for methanol synthesis and substantially increases the equilibrium yield in deuterated syngas. In the kinetic regime of CO2 hydrogenation, an inverse kinetic isotope effect of H/D substitution was observed, which is stronger for methanol synthesis than for CO formation suggesting that the two reactions do not share a common intermediate. Similar observations were also made on other catalysts such as Cu/MgO, Cu/SiO2, and Pd/SiO2. In contrast to CO2 hydrogenation, the CO hydrogenation on Cu/ZnO/Al2O3 did not show such a strong kinetic isotope effect indicating that methanol formation from CO2 does not proceed via consecutive reverse water gas shift and CO hydrogenation steps. The inverse KIE is consistent with formate hydrogenation being the rate-determining step of methanol synthesis from CO2. Differences in the extent of product inhibition by water, observed for methanol synthesis and reverse water gas shift indicate that the two reactions proceed on different surface sites in a parallel manner. The consequences for catalyst design for effective methanol synthesis from CO2 are discussed.

Strong Isotope Effects on Melting Dynamics and Ice Crystallisation Processes in Cryo Vitrification Solutions

The nucleation and growth of crystalline ice during cooling, and further crystallization processes during re-warming are considered to be key processes determining the success of low temperature storage of biological objects, as used in medical, agricultural and nature conservation applications. To avoid these problems a method, termed vitrification, is being developed to inhibit ice formation by use of high concentration of cryoprotectants and ultra-rapid cooling, but this is only successful across a limited number of biological objects and in small volume applications. This study explores physical processes of ice crystal formation in a model cryoprotective solution used previously in trials on vitrification of complex biological systems, to improve our understanding of the process and identify limiting biophysical factors. Here we present results of neutron scattering experiments which show that even if ice crystal formation has been suppressed during quench cooling, the water molecules, mobilised during warming, can crystallise as detectable ice. The crystallisation happens right after melting of the glass phase formed during quench cooling, whilst the sample is still transiting deep cryogenic temperatures. We also observe strong water isotope effects on ice crystallisation processes in the cryoprotectant mixture. In the neutron scattering experiment with a fully protiated water component, we observe ready crystallisation occurring just after the glass melting transition. On the contrary with a fully deuteriated water component, the process of crystallisation is either completely or substantially supressed. This behaviour might be explained by nuclear quantum effects in water. The strong isotope effect, observed here, may play an important role in development of new cryopreservation strategies.

Isotopic disproportionation during hydrogen isotopic analysis of nitrogen-bearing organic compounds.

High-precision hydrogen isotope ratio analysis of nitrogen-bearing organic materials using high-temperature conversion (HTC) techniques has proven troublesome in the past. Formation of reaction products other than molecular hydrogen (H(2)) has been suspected as a possible cause of incomplete H(2) yield and hydrogen isotopic fractionation. The classical HTC reactor setup and a modified version including elemental chromium, both operated at temperatures in excess of 1400 °C, have been compared using a selection of nitrogen-bearing organic compounds, including caffeine. A focus of the experiments was to avoid or suppress hydrogen cyanide (HCN) formation and to reach quantitative H(2) yields. The technique also was optimized to provide acceptable sample throughput. The classical HTC reaction of a number of selected compounds exhibited H(2) yields from 60 to 90 %. Yields close to 100 % were measured for the experiments with the chromium-enhanced reactor. The δ(2)H values also were substantially different between the two types of experiments. For the majority of the compounds studied, a highly significant relationship was observed between the amount of missing H(2) and the number of nitrogen atoms in the molecules, suggesting the pyrolytic formation of HCN as a byproduct. A similar linear relationship was found between the amount of missing H(2) and the observed hydrogen isotopic result, reflecting isotopic fractionation. The classical HTC technique to produce H(2) from organic materials using high temperatures in the presence of glassy carbon is not suitable for nitrogen-bearing compounds. Adding chromium to the reaction zone improves the yield to 100 % in most cases. The initial formation of HCN is accompanied by a strong hydrogen isotope effect, with the observed hydrogen isotope results on H(2) being substantially shifted to more negative δ(2)H values. The reaction can be understood as an initial disproportionation leading to H(2) and HCN with the HCN-hydrogen systematically enriched in (2)H by more than 50 ‰. In the reaction of HCN with chromium, H(2) and chromium-containing solid residues are formed quantitatively.