Metabolic isotope effect

Gaebler, O.H., Vitti, T.G. and Vukmirovich, R. 1966 Isotope effects in metabolism of N and from unlabeled dietary proteins. CanadianJournal of Biochemistry 44 1249-1257. 

Although the radioactive isotope H has been extensively used for studies on the uptake of xenobiotics into whole cells, the intrusion of exchange reactions and the large isotope effect renders this isotope rather less straightforward for metabolic studies. Both deuterium H-labeled substrates, and oxygen and OH2 have, however, been extensively used in metabolic studies, since essentially pure labeled compounds are readily available and mass spectrometer facilities have become an essential part of structural determination. 

Mizutani, T. Yamamoto, K. Tajima, K. Isotope effects on the metabolism and pulmonary toxicity of butylated hydroxytoluene in mice by deuteration of the 4-methyl group. Toxicol. Appl. Pharm. 1983, 69, 283-290. 

The authors have also synthesized134 fatty acids labelled with deuterium and carbon-11 in order to investigate if kinetic isotope effects related to fatty acid metabolism can be observed in vivo by pet133,135-137. In vitro, the large kinetic deuterium isotope effects are observed in the oxidation of deuteriated aliphatic carboxylic acids with alkaline permanganate and manganate135-139. 

Reinhold and Bruni studied the metabolism of 7,9-dideuterioellipticine (17) in rats and found that deuterium originally at position 9 was completely lost during the mammalian hydroxylation process (147). Proton and carbon-13 NMR and mass spectral analyses confirmed the complete elimination of deuterium at position 9, thus ruling out the occurrence of an NIH shift mechanism in the hydroxylation of ellipticine. An oxygen-insertion process was rationalized to account for the mechanism of aromatic hydroxylation in rats since this would not be expected to display the NIH shift but should demonstrate an isotope effect. It was... 

For a more detailed and extensive exposition of the use of deuterium isotope effects, including other experimental designs, in drug metabolism studies the interested reader is referred to a recent review article and the references therein (7). 

Nelson SD, Trager WF. The use of deuterium isotope effects to probe the active site properties, mechanism of cytochrome P450-catalyzed reactions, and mechanisms of metabolically dependent toxicity. Drug Metab Dispos 2003 31(12) 1481—1498. 

Atkins, W.M. and Sligar, S.G. (1988) Deuterium isotope effects in norcamphor metabolism by cytochrome P-450cam kinetic evidence for the two-electron reduction of a high-valent iron-oxo intermediate. Biochemistry, 27 (5), 1610-1616. 

Carbon kinetic isotope effects on enzyme-catalyzed decarboxylations are among the most intensively studied enzyme reactions. This is because of the central role that carbon dioxide plays in plant metabolism and also because precise kinetic measurements are relatively easy to obtain since the carbon dioxide liberated in the reaction can be immediately analyzed using isotope ratio mass spectrometry. 

Microbial reduction of nitrate to N2, known as denitrification, is similar. It is kinetically inhibited in the absence of bacteria and is known to induce a kinetic isotope effect (Blackmer and Bremner 1977 Kohl and Shearer 1978 Mariotti et al. 1981 Bryan et al. 1983 Htibner 1986 Mariotti et al. 1988). W N shifts ranging from 6.5%o to 20%o have been observed experimentally. As with sulfate, microbial fractionations appear to depend on the metabolic states of the microbes. 

White RD, Gandolfi AJ, Bowden GT, et al. 1983. Deuterium isotope effect on the metabolism and toxicity of 1,2-dibromoethane. Toxicol AppI Pharmacol 69 170-178. 

Kinetic isotope effects during microbial processes. Micro-organisms have long been known to fractionate isotopes during their sulfur metabolism, particularly during dissimilatory sulfate reduction, which produces the largest fractionations in the sulfur cycle... 

The natural cycles of the bioelements carbon, oxygen, hydrogen, nitrogen and sulphur) are subjected to various discrimination effects, such as thermodynamic isotope effects during water evaporation and condensation or isotope equilibration between water and CO2. On the other hand, the processes of photosynthesis and secondary plant metabolism are characterised by kinetic isotope effects, caused by defined enzyme-catalysed reactions [46]. 

Deuterium isotope effects in the metabolism of drugs and xenobiotics Implications for drug design, 14, 1... 

The production of the antigens was reduced in rats in which a deutero analogue of halothane was given and the metabolism of which would be reduced by an isotope effect. The antigens could be formed in vitro, but only under aerobic conditions and not under anaerobic conditions, implicating the oxidative pathway rather than the reductive (Fig. 7.77). 

During inhalation exposure of human volunteers to low levels of toluene (200-300 mg/mty, approximately 50% of the inhaled toluene was absorbed (Lbf et al., 1993). Such studies at low toluene exposure are complicated by the presence of toluene from other sources, in blood or in urine (Pierce et al., 1996). If the deuterated [ Hgjtoluene is used for exposure, this problem is avoided [but an isotope effect may reduce the rate of the metabolism of deuterated toluene compared to normal toluene, possibly by 30-50%]. When toluene is administered orally, it is virtually completely absorbed from the gastrointestinal tract (Baelum et al., 1993). 

In order to determine intrinsic isotope effects of benzylic hydroxylations, the metabolism of different deuterated toluenes was investigated in detail with rat liver microsomes and compared with the chemical radical chlorination of... 

The formation of 3-halophenols in the metabolism of chlorobenzene, bromobenzene, and fluorobenzene215 cannot be explained on the basis of arene oxides as intermediates. These metabolites may represent examples of a direct hydroxylation of the ring. Besides, the magnitude of the isotopic effects observed during the metabolic formation of such meta-substituted phenols... 

Deuterium isotope effect study of the metabolism of testosterone by CYP2C11... 

If bond breaking occurs during the rate-determining step of the reaction, then the overall reaction will be slowed by the replacement of hydrogen with deuterium. This is an example of a kinetic isotope effect. The use of deuterium as a bioisostere to manipulate rates of metabolism is a fairly new idea in drug discovery. If SD-254 successfully reaches the market, additional deuterated drugs will almost certainly be advanced into clinical trials. 

As the glucose is metabolized, the metabolic derivatives become enriched with the isotopic carbon label and are detected using advanced technologies such as mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Importantly, incorporation of isotopes into molecules can alter reaction mechanisms including the rate of a reaction-this is termed isotopic substitution and forms the basis of the kinetic isotope effect. 

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