Deuterated Form Research Articles

Application of Deuterated A-Tocopherols to the Biokinetics and Bioavailability of Vitamin E

alpha-Tocopherol, a superior chain-breaking, peroxyl radical-trapping antioxidant and the most active component of vitamin E, is elevated in liver tumor cells, contributing to their greater resistance towards lipid peroxidation compared to cells from normal tissues. Also, in regenerating rat liver the level of vitamin E has been found to fluctuate in phase with the rate of cell division. In order to study the biokinetics and mechanisms of the distribution of vitamin E in organs and within tissues of animals, deuterated forms of alpha-tocopherol have been synthesized and their uptake into blood and tissues has been measured by gas chromatography-mass spectrometry. Measurement of the competitive uptake from a mixture of the RRR- and SRR-alpha-tocopherol stereoisomers labelled with different amounts of deuterium shows that the liver exerts a strong preference for secretion of the natural (RRR) stereoisomer into the plasma. It is suggested that a tocopherol-binding protein plays a key role in this process.

Pressure-induced transition of the hydrogen bond in the ferroelectric compounds KH2P04 and KD2P04

THE prototypical ferroelectric compound KH2PO4 contains hydro-gen bonds that exhibit interesting properties. The proton lies in one of two equivalent positions between oxygen atoms of two PO4tetrahedra. Below the ferroelectric transition temperature Tc there exists ordering of the protons in one of these two minima. A large increase in Tc is observed when the proton is substituted by a deuteron. Here we report results which show that in both KH2PO4and its deuterated form KD2PO4 at room temperature (that is, above Tc), the hydrogen-bond length decreases initially with increasing pressure, but then an abrupt and pronounced elongation occurs at a critical pressure Pc, corresponding to 2.7 and 4.2 GPa for KH2PO4 and KD2PO4 respectively. At Pc, the bond lengths are nearly equal to those predicted if the proton (deuteron) were to be located in a single minimum at the midpoint of the two oxygens. The P–O bond lengths in the PO4 tetrahedra also begin to decrease above Pc. These observations suggest that there may be a fairly complex interrelation of the various bond lengths involved in the ferroelectric transition, and reveal a hitherto unsus-pected property of the hydrogen bond.

The role of oxidative biotransformation of halothane in the guinea pig model of halothane-associated hepatotoxicity.

The role of the oxidative pathway of halothane biotransformation in mediating the hepatotoxicity of halothane in the guinea pig was examined by utilizing the deuterated form of halothane (d-halothane), which is resistant to oxidative metabolism. Male outbred Hartley guinea pigs were exposed to either 1% v/v halothane or d-halothane, FIO2 = 0.21, for 4 h. Significant reductions in both oxidative and overall halothane biotransformation were observed with the use of d-halothane as indicated by decreased plasma levels of trifluoroacetic acid and bromide ion, respectively, immediately following exposure. Plasma fluoride ion, indicative of the reductive metabolism of halothane, was significantly increased with the use of d-halothane. These changes in metabolism were accompanied by a reduced hepatotoxic response as indicated by significantly decreased plasma ALT levels 24-96 h following exposure and a significantly lesser incidence of centrilobular necrosis. Thus, the oxidative biotransformation of halothane is implicated as a mechanism of injury in guinea pigs.

Altered retention of derivatives of tele-methylimidazoleacetic acid in acid-hydrolyzed samples of brain measured by capillary gas chromatography and mass spectrometry

Classical chromatographic science holds that chemical substances, even those of similar structure, can be separated according to each compound’s independent interaction with chromatographic media. In gas chromatography (GC), the hallmark of chromatographic separation of a substance is its characteristic retention, often expressed as a retention time, highly reproducible under defined conditions of column composition, size, gas flow, temperature, etc. With capillary GC columns, the range and variability of characteristic retention times are extremely narrow. Significant deviations from established retention times are uncommon when measured under identical chromatographic conditions’,2. Yet it has been observed, for instance, that if a trace constituent elutes after a much larger component3, the latter acts as a transient stationary phase, increasing retention of the former. Absent such phenomena, in capillary GC analysis it is rare1,2*4 to observe selective deviations from well characterized retentions of substances due to component(s) of the injectate under otherwise identical conditions. Under identical GC conditions we observed that the retention times of both derivatized tele-methylimidazoleacetic acid (t-MIAA) and its deuterated form ([2H3]t-MIAA) were consistently and reversibly increased. This occurred in samples prepared from homogenates of rat brains that were hydrolyzed but not in those prepared from aliquots of the same homogenates kept at room temperature or frozen. We present results of a typical experiment, extended briefly to study this effect.

Determination of the antihypertensive drug 1-[2-ethoxy-2-(3′-pyridyl)ethyl]-4-(2′-methoxyphenyl)piperazine (IP/66) in rat and human plasma by high-performance liquid chromatography and isotope dilution mass spectrometry

In connection with pharmacokinetic studies on the antihypertensive drug 1-[2-ethoxy-2-(3′-pyridyl)ethyl]-4-(2′-methoxyphenyl)piperazine (IP/66) (I), appropriate high-performance liquid chromatographic (HPLC) and gas chromatographic—mass spectrometric isotope dilution (GC—MS—ID) methods for its determination in rat and human plasma, respectively, were developed. In both techniques, deproteinized and basified plasma samples were extracted and purified by adsorption on an Extrelut-1 column, then the drug was eluted with dichloromethane. Quantitative HPLC analysis was performed on a C 8 reversed-phase column. The mobile phase was phosphate buffer (0.02 M, pH 2.8)—acetonitrile (65:35), with UV detection at 208 nm. The internal standard was 1-[2-butoxy-2-(3′-pyridyl)ethyl]-4-(2′-methoxyphenyl)piperazine, a homologue of I. The inter-assay coefficient of variation (C.V.) was 9.9% for a drug level of 2 μg/ml. Quantitative GC—MS—ID analysis was performed with a DB-17 fused-silica capillary column using the selected-ion monitoring technique. The deuterated form of I, 1-[2-ethoxy-2-(3′-pyridyl)ethyl]-4-2′-trideuteromethoxyphenyl)-piperazine, utilized as internal standard, was synthesized. The inter-assay C.V. was 7.36% for a drug level of 1 ng/ml.

On the structural evidence for a direct proton tunnelling effect in the KH2PO4-type transition

High-resolution neutron diffraction results for KH2PO4 (KDP) and its deuterated form (DKDP) have been used to re-examine evidence that the large increase in the phase transition temperature, Tc, on deuteration can be explained simply by the increase in the phase transition temperature, Tc, on deuteration can be explained simply by the increase in the O...O hydrogen-bond length, 2R, without invoking proton tunnelling effects. It is shown that deuteration at constant 2R still gives rise to an increase in Tc by about 40 K. This may represent the direct proton tunnelling effect on Tc. However, there are other geometric changes on deuteration at constant 2R, and the effect of these needs to be assessed before any more definite conclusion can be reached.

Diode laser spectroscopy of the ν4 band of NH 2OH and the ν5 band of NHDOD

Doppler-limited spectroscopy of the ν 4 band (OH bend) of hydroxylamine NH 2OH has been carried out using a diode laser spectrometer in the range 1320 to 1400 cm −1. Hydroxylamine is so close to being a symmetric rotor that in the v 4 = 1 excited state the rovibrational effects cause the b and c molecular axes to be interchanged relative to their orientation in the ground state. The recognition of this fact allowed earlier work on the ν 5 band ( 1) to be corrected. The ν 4 band is perturbed by Coriolis-type interactions with at least two other modes. These are (i) a mainly c-type interaction with the combination band ( ν 6 + ν 9) and (ii) an a-type interaction with what is probably the very weak fundamental ν 8. Effective unperturbed parameters are calculated for the ν 4 band. Accurate constants have been obtained for the ν 5 band (NHD-wagging motion) of the partially deuterated form of hydroxylamine, NHDOD. These are the first gas-phase infrared data on any deuterated form of hydroxylamine.

Separation of Hydrogen Isotopes

Isotopes of hydrogen are separated by reacting a normal alkane, its deuterated form, atomic bromine, a deactivating gas, and a isotopically selective vibrational sensitizer gas selected from the class consisting of carbon dioxide, carbon monoxide, nitrogen, and mixtures thereof and separating the products which are enriched in deuterium.

Refractive indices of 14 nonlinear crystals isomorphic to KH_2PO_4

Refractive indices for the crystals KDP, ADP, RDP, KDA, ADA, RDA, CDA, KDP:Rb, and most of their deuterated forms were measured from 404 to 1064 nm with ±3 × 10−5 accuracy. Numerical fits to standard and modified versions of the double-resonance Sellmeier formula were made, permitting interpolation of the index data consistent with the measurement accuracy. Samples of KDP from different crystal growers had slightly different (~3 × 10−4) refractive indices, which correlated with deviations in the lattice parameters.

Raman amide bands of type-II beta-turns in cyclo-(VPGVG)3 and poly-(VPGVG), and implications for protein secondary-structure analysis.

AbstractThe cyclopentadecapeptide, c(VPGVG)3, a model structure for protein type‐II β‐turns [W. J. Cook et al. (1980) J. Am. Chem. Soc. 102, 5502–5505], has been investigated by laser Raman spectroscopy. Data obtained from both normal and deuterated crystals identify amide I, III, I′, and III′ bands characteristic of the β(II)turn. The structurally related polypentapeptide poly(VPGVG) in normal and deuterated forms has also been investigated, and exhibits the same Raman amide bands as c(VPGVG)3. The coacervate of poly(VPGVG), obtained by heating the solution to 40°C, likewise exhibits a Raman spectrum very similar to that of the c(VPGVG)3 crystal. Raman spectra thus indicate closely similar secondary structures for crystalline c(VPGVG)3 and aqueous poly(VPGVG), and provide an empirical basis for interpreting the conformation sensitive amide bands of globular proteins in terms of β(II)turn structures that may be present. An important conclusion from the present findings is that the amide I Raman profile of a protein may not be sufficient in general to distinguish turns from helix and sheet secondary structures, since the major amide I peaks of the β(II)turn at 1676 and 1652 cm−1 overlap, respectively, with amide I profiles generated by β‐sheet and α‐helix conformations.

Spectroscopic studies of metal complexes containing π-delocalized sulfur ligands. The pre-resonance Raman spectra of the antitumor agent 2-formylpyridine thiosemicarbazone and its Cu(II) and Zn(II) complexes

The pre-resonance Raman spectra of 2-formylpyridine thiosemicarbazone have been measured at three pH values corresponding to the fully protonated (H 2FPT +), half protonated (HFPT) and deprotonated (FPT −) forms of the ligand. Assignments of the vibrations coupled with the π→π * transition have been made by comparison with the spectrum of the deuterated form (DFPT). The pre-resonance Raman spectra of the Zn(II) and Cu(II) complexes, [ZnFPT] +, [CuFPT] + and [CuHFPT] 2+, have also been measured. The spectral pattern of the Cu(II) complexes shows resonance enhancement of vibrations coupled with the π→π *, as well as with the ligand to metal charge transfer transitions. In addition, it is consistent with coordination through thiolate sulfur in [CuFPT] + and thione sulfur in [CuHFPT] 2+.

Effects of acute and chronic phenelzine on regional monoamine metabolism in rats and its potentiation by deuterium substitution

Phenelzine is a monoamine oxidase inhibitor with antidepressant properties. The present study investigated effects of acute (1-2 mg kg-1 4 h s.c.) and chronic (0.25-2 mg kg-1 day-1 Alzet miniosmotic pumps, 13 days s.c.) administration of phenelzine on regional monoamine metabolism in rats. The effects of these phenelzine treatments were compared with those of equivalent doses of a deuterated form of the drug (phenelzine-d4). The following brain regions and compounds were assessed using high performance liquid chromatography with electrochemical detection: Striatum: dopamine, DOPAC, HVA, 5-HT, 5-HIAA; hypothalamus: dopamine, 5-HT, 5-HIAA, noradrenaline; hippocampus: 5-HT, 5-HIAA, noradrenaline; frontal cortex: dopamine, noradrenaline, 5-HT, 5-HIAA. Acute drug administration increased levels of dopamine, 5-HT and noradrenaline with the exception of dopamine in the hypothalamus and frontal cortex and 5-HT in the hypothalamus. DOPAC, HVA and 5-HIAA levels were decreased. After chronic administration amine levels increased with the exception of dopamine administration amine levels increased with the exception of dopamine in the hypothalamus. The respective acid metabolites were also decreased. These effects of phenelzine were markedly potentiated by deuterium which was substituted for hydrogen in the side chain. The potentiation of these effects was enhanced with chronic administration, differences between phenelzine and phenelzine-d4 effects being more marked at lower doses.

The harmonic force field and vibrational spectra of pyrrole

The complete harmonic vibrational force field of pyrrole has been calculated by the ab initio gradient method at the Hartree-Fock level using the 4–21 basis set. The force field was then scaled with a set of six factors transferred from benzene, and the vibrational spectrum of pyrrole was calculated. This a priori prediction, made with no reference to observations on pyrrole, agreed with the known experimental fundamental frequencies with a mean deviation of 12 cm-1 for in-plane modes and 20 cm-1 for out-of-plane modes except for the NH wagging and NH stretch. A new set of ten scale factors was next obtained by direct fitting of the computed force field to the observed pyrrole spectrum, producing the best force field obtainable by combined use of the theoretical and experimental information. This force field reproduced the entire pyrrole spectrum with mean deviations of 4·2 cm-1 (in-plane) and 5·9 cm-1 (out-of-plane). The spectra of three deuterated forms of pyrrole were also computed. Infrared absorption intensities were calculated and proved very useful in examining assignments of the two ring torsional modes and the CH stretching modes.

A study of the vibrational behavior of imidazole by the ab initio gradient method

The complete harmonic vibrational force field of imidazole has been computed at the Hartree—Fock level using the 4–21 basis set of Gaussian orbitals. The harmonic constants were scaled by using scale factors previously derived by fitting the computed force field of benzene to the observed benzene vibrational spectrum. The resulting scaled force field was then used to predict the vibrational spectrum of imidazole. The NH wagging and NH stretching frequencies, which have no counterpart in benzene, were poorly predicted, but the mean deviation between experiment and prediction was only 9 cm −1 for the other in-plane (A′) vibrations and 27 cm −1 for the out-of-plane (A″) vibrations. In order to fit the NH modes and to obtain a better fit for the out-of-plane vibrations, a new set of scale factors, some of which turned out to be identical with those from benzene, was derived by fitting the computed spectrum of imidazole to the observed spectrum. This new set of scale factors was used to predict the vibrational spectra of several deuterated forms of imidazole (−1D, −3D, and −4D). The imidazole scale factors are presumably more accurate in scaling computed spectra of other 5-membered heterocyclic ring compounds. Dipole moment derivatives were also calculated and used to predict infrared intensities which are compared with experimental values. A few uncertainties in the experimental assignment of the imidazole spectrum can be clarified.

Quantitation of vitamin B 6 in biological samples by isotope dilution mass spectrometry

Methods have been developed for the simultaneous quantitative analysis of vitamin B 6 forms in biological samples by isotope dilution mass spectrometry using deuterated forms of pyridoxine, pyridoxal, pyridoxamine, and pyridoxic acid. The biological fluid or tissue sample was homogenized and then treated with a cocktail containing appropriate amounts of each deuterated vitamer, as well as the deuterated, phosphorylated vitamer forms. The individual vitamers were isolated from the homogenate by a complex high-performance liquid chromatographic procedure that provided separate fractions for each of the six vitamers found in biological samples. Aldehydic B 6 vitamers were reduced to the alcohol form prior to acetylation and analysis by gas chromatography/mass spectrometry ( GC MS ). The three resulting vitamers were analyzed by electron ionization GC MS using a silicone capillary column. The methods have been applied to analysis of vitamin B 6 in liver, milk, urine, and feces at levels as low as 0.02 nmol/ml.

Benzylidenemalonaldehydes: A redox reaction during the attempted cycloaddition on nitrosobenzene

Reaction of p-chlorobenzylidenemalonaldehyde with nitrosobenzene represents a reduction-oxidation process leading to 2-formyl-3-anilino-3-(4-chlorophenyl)acrylic acid (III). The structure of the product, including two intramolecular hydrogen bonds, has been proved by analysis of the 1H and 13C NMR spectra of the acid III, its deuterated form and the methyl ester obtained by reaction with diazomethane.

A mass spectrometer study of the reaction of erbium with the residual gases in a vacuum coating unit and with heavy water

A quadrupole gas analyser was used to study the gases inside a conventional diffusion-pumped vacuum evaporator prior to and following the evaporation of erbium. Studies have also been done on the effects of admitting heavy water (D 2O) to the system. It has been found that erbium at the evaporation source reduces H 2O and D 2O to produce H 2 and D 2 respectively and that CO in the vacuum is converted to CH 4 in the presence of H 2O. Various deuterated forms of CH 4 occur in the presence of H 2O and D 2O. An explanation is given of why vacuum-deposited films of erbium are frequently contaminated by ErH 2 in the presence of water even though Er 2O 3 is thermodynamically much more stable than ErH 2.

ChemInform Abstract: INTRAMOLECULAR HYDROGEN BONDING. 2. ENORMOUS DEUTERIUM ISOTOPE EFFECT ON THE PHOSPHORESCENCE OF 6‐HYDROXYBENZANTHRONE

The laser-excited fluorescence and phosphorescence spectra of the title compound and its hydroxy-deuterated analogue have been measured in an n-hexane Shpol'skii matrix at 10 K. The deuterium substitution has only a minor effect on the fluorescence intensity and the fluorescence and phosphorescence vibronic patterns but leads to a greater than order of magnitude increase in the phosphorescence quantum yield. The phosphorescence lifetimes have been measured to be 19 ms for the normal isotopic species and 300 ms for the deuterated form. This probably represents the largest deuterium isotope effect yet observed in aromatic molecule photophysics.

Simultaneous quantitation of krebs cycle and related acids by mass fragmentography

Methods are described for simultaneous quantitation of Krebs cycle and related acids by gas chromatography—mass spectrometry using deuterium-labelled acids and n-butyl-d9-esters of the organic acids as internal standards. Using sulphuric acid as esterification catalyst, only lactic, succinic, fumaric, malic, maleic and citric acids were found to be stable to hydrogen exchange and could be used as reference standards in the deuterated form. In contrast, pyruvic, oxalacetic, α-ketoglutaric and malonic acids were found to exchange their deuterium readily and could not be employed for this purpose. All the acids could be quantitated using n-butyl-d9-esters of reference organic acids as internal standards, following separate preparation of the n-butyl derivatives of the unknown acids. The method is suitable for routine analysis of organic acids at the picogram level in perchloric acid extracts of tissues.

ChemInform Abstract: THE ABNORMAL ISOTOPIC EFFECT IN THE INFRARED SPECTRA OF THE HYDROGEN BOND IN 2‐THIAPYRIDONE

Abstract The abnormal IR spectral properties of the hydrogen bond in 2-thiapyridone have been investigated together with the complex effect of deuteration on the NH⋯S hydrogen bonds. The hydrogen-bond spectra of the deuterated form of 2-thiapyridone have been remeasured in order to confirm the results of some recent investigations. The spectra have also been theoretically constructed based on the strong coupling model of the vibrational spectra of the hydrogen-bonded dimers. In addition, a dependence of the resonance interaction energy of the dimeric νNH vibrations on the normal coordinates has been assumed thus describing the low-energy vibrations of the hydrogen bridges of the dimer.