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PubMed2005

The use of isotope effects to determine enzyme mechanisms.

Isotope effects are one of the most powerful kinetic tools for determining enzyme mechanisms. There are three methods of measurement. First, one can compare reciprocal plots with labeled and unlabeled substrates. The ratio of the slopes is the isotope effect on V/K, and the ratio of the vertical intercepts is the isotope effect on V(max). This is the only way to determine V(max) isotope effects, but is limited to isotope effects of 5% or greater. The second method is internal competition, where the labeled and unlabeled substrates are present at the same time and the change in their ratio in residual substrate or in product is used to calculate an isotope effect, which is that on V/K of the labeled reactant. This is the method used for tritium or (14)C, or with the natural abundances of (13)C, (15)N, or (18)O. The third method involves perturbations from equilibrium when a labeled substrate and corresponding unlabeled product are present at chemical equilibrium. This also gives just an isotope effect on V/K for the labeled reactant. The chemistry is typically not fully rate limiting, so that the isotope effect on V/K is given by: (x)(V/K)=((x)k+c(f)+c(r)(x)K(eq))/(1+c(f)+c(r)) where x defines the isotope (D, T, 13, 15, 18 for deuterium, tritium, (13)C, (15)N, or (18)O), and (x)(V/K), (x)k, and (x)K(eq) are the observed isotope effect, the intrinsic one on the chemical step, and the isotope effect on the equilibrium constant, respectively. The constants c(f) and c(r) are commitments in forward and reverse directions, and are the ratio of the rate constant for the chemical reaction and the net rate constant for release from the enzyme of the varied substrate (direct comparison) or labeled substrate (internal competition and equilibrium perturbation) for c(f), or the first product released or the one involved in the perturbation for c(r). The intrinsic isotope effect, (x)k, can be estimated by comparing deuterium and tritium isotope effects on V/K, or by comparing the deuterium isotope effect with (13)C ones with deuterated and undeuterated substrates. Adding a secondary deuterium isotope effect and its effect on the (13)C one can give an exact solution for all intrinsic isotope effects and commitments. The effect of deuteration on a (13)C isotope effect allows one to tell if the two isotope effects are on the same or different steps. Applications of these methods to several enzyme systems will be presented.

Aspartate Carbamoyltransferase↗
PubMed1999

[Stable oxygen and carbon isotopes in biological studies].

Biogenic chemical elements in the Earth biosphere are presented both by light and heavy stable isotopes (16O and 18O, 12C and 13C). Because of inequality of these isotopes the light ones dominate in the exchange processes between the animate and inanimate nature and this leads to changes in the ratios of isotope modifications in the environment. A comparatively constant isotopic composition of the Earth atmosphere is primarily maintained by water. It is the heavy carbon isotope that inputs to the most isotope-heavy compounds such as marine carbonates within the atmosphere--hydrosphere system. As in nature, biogenic isotopes fractionate inside hermetically sealed habitats with the regenerative life-support systems; however, the mechanism of relative stability of the isotope composition is unknown. The problem of providing the best isotopic composition of biogenic chemical elements aboard manned space vehicles was first raised by Yu.Ye.Sinyak and A.I. Grigoriev in 1996. The review contains the data on the levels of oxygen isotopes and photosynthetically generated oxygen in water pools in different regions of the Earth. discussion of the ratios of the oxygen and carbon isotopes in the atmospheric carbon dioxide, and the range of isotopes variations and presumed "sites" of isotopes fractionating in cellular structures of autotrophs. The wealth of literary findings effectuates the conclusion that artificial environments for humans should reproduce the natural relationships and variations of biogenic isotopes. Yet, what biogenic isotopes ratios are desirable aboard manned space vehicles can be determined in extensive investigations of the effects of various modifications of stable isotopes on humans with due account for the unfavourable factors of space flight.

Biology↗
PubMed1998

Isotope ratio determination in boron analysis.

Traditionally, boron (B) isotope ratios have been determined using thermal ionization mass spectrometry (TIMS) and, to some extent, secondary ion mass spectrometry (SIMS). Both TIMS and SIMS use a high-resolution mass analyzer, but differ in analyte ionization methods. TIMS uses electrons from a hot filament, whereas SIMS employs an energetic primary ion beam of Ga+, Cs+, or O- for analyte ionization. TIMS can be used in negative or positive ion modes with high sensitivity and precision of B isotope ratio determination. However, isobaric interferences may be a problem, if the sample is not well purified and/or memory of the previous sample is not removed. Time-consuming sample preparation, analyte (B) purification, and sample determination processes limit the applications of TIMS for routine analyses. SIMS can determine B and its isotope ratio in intact solid samples without destroying them, but has poorer resolution and sensitivity than TIMS, and is difficult to standardize for biological samples. Development of plasma-source mass spectrometry (MS) enabled the determination of B concentration and isotope ratio without requiring sample purification. Commonly used plasma-source MS uses an Ar inductively coupled plasma (ICP) as an ionization device interfaced to a low-resolution quadrupole mass analyzer. The quadrupole ICP-MS is less precise than TIMS and SIMS, but is a popular method for B isotope ratio determination because of its speed and convenience. B determination by ICP-MS suffers no spectroscopic interferences. However, sample matrices, memory effects, and some instrument parameters may affect the accuracy and precision of B isotope ratio determination if adequate precautions are not taken. New generations of plasma-source MS instruments using high-resolution mass analyzers provide better sensitivity and precision than the currently used quadrupole ICP-MS. Because of the convenience and high sample throughput, the high-resolution ICP-MS is expected to be the method of choice for B isotope ratio determination. The current state of instrumental capabilities is adequate for B isotope determination. However, precision and accuracy are primarily limited by sample preparation, introduction, and analytical methodology, including 1. Analyte loss and isotope fractionation during sample preparation. 2. The precision of B isotope determination in small samples, especially those containing low concentrations. 3. Difficult matrices. 4. Memory effects. Sample preparation by alkali fusion allows rapid and complete decomposition of hard-to-digest samples, but high-salt environments of the fused materials require extensive sample purification for B ratio determination. The alternative wet-ashing sample decomposition with HF also results in B loss and isotopic fractionation owing to the high volatility of BF3. Open-vessel dry- or wet-ashing methods usually do not work well for animal samples, and are also prone to B loss and contamination. Closed-vessel microwave digestion overcomes these problems, but the digests of biological materials have high C contents, which cause spectral interference on 11B and affect 11B/10B ratios. Exchange separation/preconcentration of B using exchange (cation or anion exchange, B-specific resin, e.g., Amberlite IRA-743) tend to cause B isotope fractionation, and C eluting from these resin columns may interfere with B isotope ratio determination. Memory effects of B that occur during sample determination may cause serious errors in B isotope ratio determination, especially when samples varying in B concentrations and/or isotope composition are analyzed together. Although the utilization of high-resolution plasma-source MS will undoubtedly improve analytical precision, it is the sample preparation, sample introduction, and analytical methodology that represent the primary limitation to accurate and precise B isotope ratio determination.

Boron↗
PubMed2005

A preliminary multi-stable-isotopic evaluation of three synthetic pathways of Topiramate.

As a preliminary study of the utility of the natural stable-isotopic differentiation of batch samples produced by different synthetic pathways, multi-stable-isotopic analyses (delta(13)C, delta(15)N, delta(18)O, deltaD) of 53 samples of the antiepileptic drug, Topiramate, produced by three different synthetic pathways (designated "A," "B," "C") were performed. From the outset, we note that there are two fundamental variables that determine the stable-isotopic composition of materials-the stable-isotopic composition of the reagents and starting intermediates, and the isotope fractionation that occurs during manufacture of the product. In this study, the stable-isotopic composition of the raw materials was not controlled and we report here data obtained for a suite of samples that was produced by three synthetic pathways. Graphical examination of these data reveals marked data clustering by synthetic pathway, though in some cases with some overlapping values within standard errors. In general, the isotopic composition of Topiramate from the A and B pathways is distinct from the C pathway. The isotopic data from the A and B pathways typically abut each other, sometimes partially overlapping. The deuterium/hydrogen- (deltaD) and oxygen (delta(18)O) isotopic compositions are each significantly linearly related with the paired carbon (delta(13)C) isotopic composition indicating possible isotopic end-members for the raw materials of the present sample suite. Given that H and O typically derive from meteoric water, the linear correlations with delta(13)C indicate that a mixture of carbon sources (viz., perhaps terrestrial C3 photosynthetic organic carbon and marine C3 organic carbon) were used in the production of the batches tested. If the H and O analyzed were derived from meteoric water, then an elementary comparison of the span of the deltaD (DeltadeltaD = 54.6 +/- 2.1 per thousand) and of the delta(18)O (Deltadelta(18)O = 4.71 +/- 0.26 per thousand) values in the Topiramate samples to that of the global isotopic gradients indicates that the water retained in the samples spanned from as much as 11 degrees of latitude (or, approximately 760 statute miles North-to-South). The present isotope results (delta(13)C, delta(15)N, delta(18)O, deltaD) form an initial database against which future samples can be compared to infer specific synthetic pathways. It is clear that to perform a rigorous test of the variables controlling the stable-isotopic composition of pharmaceutical materials that both the stable-isotopic composition of the starting materials and synthetic isotope fractionation must be controlled in future studies.

Fructose↗