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PubMed1997

Determination of urea kinetics by isotope dilution with [13C]urea and gas chromatography-isotope ratio mass spectrometry (GC-IRMS) analysis.

1. Stable urea isotopes can be used to study urea kinetics in humans. The use of stable urea isotopes for studying urea kinetic parameters in humans on a large scale is hampered by the high costs of the labelled material. We devised a urea dilution for measurement of the distribution volume, production rate and clearance of urea in healthy subjects and renal failure patients using the inexpensive single labelled [13C]urea isotope with subsequent analysis by headspace chromatography-isotope ratio MS (GC-IRMS) of the [13C]urea enrichment. 2. The method involves measurement of the molar percentage excess of [13C]urea in plasma samples taken over a 4 h period after an intravenous bolus injection of [13C]urea. During the sample processing procedure, the plasma samples together with calibration samples containing a known molar percentage excess of [13C]urea are acidified with phosphoric acid to remove endogenous CO2, and are subsequently incubated with urease to convert the urea present in the plasma samples into CO2. The 13C enrichment of the generated CO2 is analysed by means of GC-IRMS. This method allows measurement of the molar percentage excess of [13C]urea to an accuracy of 0.02%. 3. Reproducibility studies showed that the sample processing procedure [within-run coefficient of variation (CV) < 2.8% and between-run CV < 8.8%] and the GC-IRMS analysis (within-day CV < 1.3% and between-day CV < 1.3%) could be repeated with good reproducibility. 4. In clinical urea kinetic studies in a healthy subject and in a renal failure patient without residual renal function, reproducible values of the distribution volume, production rate and clearance of urea were determined using minimal amounts of [13C]urea (25-50 mg). 5. Because only low [13C]urea enrichments are needed in this urea dilution method using GC-IRMS analysis, the costs of urea kinetic studies are reduced considerably, especially in patients with renal failure.

Adult↗

PubMedDate not supplied

Isotope ratio and isotope dilution analysis of lead in wine by inductively coupled plasma-mass spectrometry.

The lead content of 12 wine samples was measured by inductively coupled plasma-mass spectrometry using the methods of standard addition and isotopic dilution analysis. An additional wine sample was analysed by external calibration, standard addition and isotopic dilution analysis. The lead content of the wine samples was in the range 30-150 ng ml-1 and good agreement between the different techniques was observed. Analysis of lead isotope ratios revealed significant differences between Australian and European wines, reflecting the different isotopic composition of Australian lead.

Australia↗

PubMed2006

Assessment of the amount of body water in the Red Knot (Calidris canutus): an evaluation of the principle of isotope dilution with 2H, (17)O, and (18)O as measured with laser spectrometry and isotope ratio mass spectrometry.

We have used the isotope dilution technique to study changes in the body composition of a migratory shorebird species (Red Knot, Calidris canutus) through an assessment of the amount of body water in it. Birds were quantitatively injected with a dose of water with elevated concentrations of 2H, (17)O, and (18)O. Thereafter, blood samples were taken and distilled. The resulting water samples were analysed using an isotope ratio mass spectrometry (for 2H and (18)O only) and a stable isotope ratio infrared laser spectrometry (2H, (17)O, and (18)O) to yield estimates of the amount of body water in the birds, which in turn could be correlated to the amount of body fat. Here, we validate laser spectrometry against mass spectrometry and show that all three isotopes may be used for body water determinations. This opens the way to the extension of the doubly labelled water method, used for the determination of energy expenditure, to a triply labelled water method, incorporating an evaporative water loss correction on a subject-by-subject basis or, alternatively, the reduction of the analytical errors by statistically combining the (17)O and (18)O measurements.

Animals↗

PubMed1984

Solvent isotope effects on the glucokinase reaction. Negative co-operativity and a large inverse isotope effect in 2H2O.

The solvent isotope effects on the reaction catalysed by rat-liver glucokinase have been studied. At low concentrations of glucose and high concentrations of MgATP2- there is an inverse solvent isotope effect of 3.5. At high glucose concentrations there is a normal solvent isotope effect of 1.3. In 1H2O there is positive co-operativity with respect to glucose [ Storer , A.C. and Cornish - Bowden , A. (1976) Biochem. J. 159, 7-14], but this is changed to negative co-operativity in 2H2O. The half-saturation points for both glucose and MgATP2- are decreased in 2H2O compared with those in 1H2O. Explanations of these effects in terms of the mnemonical model proposed by Storer and Cornish - Bowden [Biochem. J. 65, 61-69 (1977)] were considered in computer simulation. Two interpretations could account for the results, either a decrease in the rate of interconversion of the two forms of free enzyme postulated in the model, or an increase in the affinity for glucose of the enzyme form with the lower affinity in 1H2O. The results of a proton-inventory analysis were consistent with either of these interpretations. The solvent isotope effects thus provide additional evidence for the mnemonical model as an explanation of glucokinase co-operativity.

Adenosine Triphosphate↗

PubMed1981

Stable isotope fractionation by Clostridium pasteurianum. 4. Sulfur isotope fractionation during enzymatic S3O6(2-), S2O3(2-), and SO3(2-) reductions.

Cell-free extracts from Clostridium pasteurianum grown on SO3(2-) utilize H2 to reduce S3O6(2-), S2O3(2-), SO3(2-) to H2S at a much faster rate than extracts from SO4(2-)-grown cells. This further supports the concept of an inducible dissimilatory type SO3(2-) reductive pathway in this organism. 35S dilution experiments further support the concept that S3O6(2-) and S2O3(2-) are pathway intermediates. The inducible SO3(2-) reductase is ferredoxin linked and the kinetics of the reduction and the sulfur isotope fractionation of the product can be altered by altering the growth conditions. The attending sulfur isotope fractionations are similar to those observed during the chemical decomposition of these compounds. In the case of S2O3(2-), 35S labelling experiments verified the conclusions derived from the stable isotope fractionation data concerning the relative reduction rates of the sulfane and sulfonate sulfurs. The reduction rates were also affected by enzyme concentration. The integrity of the whole cell is a necessary requirement for the large inverse isotope effects previously reported.

Clostridium↗

PubMed2005

Stable isotope dilution-based accurate comparative quantification of nitrogen-containing metabolites in Arabidopsis thaliana T87 cells using in vivo (15)N-isotope enrichment.

Stable isotope dilution-based comparative quantification of nitrogen-containing metabolites for highly sensitive and selective metabolomics was developed using liquid chromatography/mass spectrometry (LC/MS) and (15)N-isotope enrichment. We produced metabolically stable isotope-labeled Arabidopsis T87 cells by culturing with (15)N-labeled medium. We found that the growth of cells maintained in (15)N-labeled medium is very similar to the growth in normal medium, as evidenced by cell morphology, doubling time, and measurement of chlorophyll and carotenoid contents. Complete incorporation of (15)N in folate, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) in T87 cells was accomplished after culturing for 21 d. Accurate comparative quantification of folate, SAM, and SAH was established by means of LC/MS using the isotopomers of the target metabolites as internal standards. The within- and between-run assay coefficients of variation for the folate, SAM, and SAH levels were all less than 8.5%. Stable isotope labeling by nitrogen source in Arabidopsis T87 cell culture provided simple, inexpensive, and accurate amino acid profiling. This interesting new protocol is valuable for the study of dynamic changes in N-compound pools in cultured cells.

Arabidopsis↗

PubMed1992

15N-leucine and 15N-isoleucine isotope dilution techniques versus the 15N-isotope dilution technique for determining the recovery of endogenous protein and amino acids in digesta collected from the distal ileum in pigs.

Twelve gilts, with an average initial BW of 38 kg, were fitted with simple T-cannulas at the distal ileum and two catheters in the external jugular veins. The animals were fed twice daily (0800 and 2000) 700 g of one of four diets containing either soybean meal, canola meal, wheat, or barley as the sole protein source. 15N-leucine was infused continuously for 9 d at a rate of 40 mg/kg of BW daily via one of the jugular catheters. Blood samples were taken during feeding. Ileal digesta samples were collected continuously for 24 h on d 7 and 9 of the infusion period. 15N-enrichment excess in leucine and isoleucine in the trichloroacetic acid (TCA)-soluble fraction of blood and in digesta were measured using single-ion monitoring gas chromatography-mass spectrometry. Assuming that the 15N-enrichment excess in leucine and isoleucine in the TCA-soluble fraction of the blood is similar to that in endogenous protein secreted into the digestive tract and that the amino acid composition of endogenous protein is constant, the contribution of endogenous to total protein in ileal digesta was calculated using the 15N-isotope dilution technique for leucine and isoleucine, respectively. These contributions were much smaller for the 15N-leucine than for the 15N-isoleucine isotope dilution technique: 7.1 to 11.0 vs 21.8 to 24.9 g of protein/kg of DM intake. The values obtained with the 15N-isoleucine isotope dilution technique were close to those obtained with the 15N-isotope dilution technique as described in a previous publication.

Amino Acids↗

PubMed1987

Simultaneous determination of radioactive and stable isotopes of cobalt in environmental samples by the isotopic exchange method.

The isotopic exchange method is studied for the simultaneous determination of radioactive and stable isotopes of cobalt in environmental samples. Radioactive cobalt is isotopically exchanged with Co-CyDTA in HC1 solution of the sample. The solvent extraction method is used for the separation of Co2+ and Co-CyDTA species from each other. The amounts of radioactive and stable cobalt isotopes are calculated with activities of Co and Co-CyDTA at the exchange equilibrium. The methods for eliminating interfering ions are discussed. The results are in good concordance with those obtained by the conventional analytical methods.

Cobalt Isotopes↗

PubMed1994

Measurement of blood volume with an enriched stable isotope of chromium (53Cr) and isotope dilution by combined gas chromatography-mass spectrometry.

We used an established isotope dilution gas chromatography-mass spectrometry method to measure blood volumes with an enriched stable isotope of chromium. The results were compared with those of the conventional method, which involves radioactive 51Cr. The two methods were compared simultaneously in two male subjects of different sizes, and essentially identical volumes were obtained. The isotope dilution method is insensitive to contamination with natural (unenriched) chromium and can be used to measure all of the stable isotopes of chromium. The method has potential applications in simultaneous erythrocyte survival studies. The absence of radioactivity makes possible an accurate means of studying blood volume changes in children and during pregnancy.

Blood Volume Determination↗

PubMed1993

Performance of human mass balance/metabolite identification studies using stable isotope (13C, 15N) labeling and continuous-flow isotope-ratio mass spectrometry as an alternative to radioactive labeling methods.

Stable isotope labeling in therapeutic and subtherapeutic quantities of drug (15N2(13)C-phenobarbital) can be quantitated in biological matrices (urine) and high performance liquid chromatography (HPLC) peaks from urine using continuous-flow isotope-ratio mass spectrometry (CF-IRMS). Standard curves for 15N2(13)C-phenobarbital were reproducible and linear (R2 > 0.985) over the ranges of 3-100 micrograms/ml for whole urine (15N2 or 13C labeling) and 0.1-8.0 micrograms/mL for HPLC peaks derived from urine (15N2 labeling). The lower limit of quantitation values for urine drug concentration was 0.46-2.62 micrograms/mL in whole urine and 0.10-0.70 micrograms/mL in HPLC peaks. Validation samples quantitated with these standard curves yielded close to expected values. These data suggest stable isotope labeling and CF-IRMS may be used as an alternative to 14C labeling and radioactivity counting methods in mass balance/metabolite identification and other biomedical studies.

Carbon Isotopes↗

PubMed1999

Memory effects in combining isotope ratio monitoring with isotope dilution mass spectrometry.

Combining isotope ratio monitoring with isotope dilution techniques provides very accurate results in the quantitative analysis of volatile organic chemical compounds by gas chromatography/mass spectrometry (GC/MS). However, this method requires that spikes highly enriched in (13)C be used. This may lead to memory effects which will be investigated in more detail. They occur when the component of the mixture to be investigated exhibits an isotope ratio which is different from that of the component eluted earlier from the column during the chromatographic separation process. A residue of this component, which is shown in the gas chromatogram as tailing, falsifies the result of the isotope ratio measurement. This also leads to false amount-of-substance measurement results. Memory effects can be avoided by using spikes of low (13)C content, by adjusting the composition of the reference solution to that of the sample, or by ensuring effective sample preparation, thus separating disturbing mixture components prior to the measurement. Copyright 1999 John Wiley & Sons, Ltd.

Journal Article↗

PubMed2001

Theoretical evaluation of error in the isotopic analysis of carbon and oxygen as CO (2)(+): considerations for determining two different isotopic abundance ratios simultaneously.

The computations involved in the CO(2)(+) ion beam method of determining simultaneously a pair of constituent elemental isotopic abundance ratios P and Q (viz. (13)C/(12)C and (17)O/(16)O, or (13)C/(12)C and (18)O/(16)O, or (17)O/(16)O and (18)O/(16)O) are worked out, and the possible implications of their involvement as an analytical step are evaluated theoretically. It is shown, as an immediate consequence, that accurate measurements of the required isotopic CO(2)(+) abundance ratios (R(j) and R(k)) do not necessarily mean that the results (P; Q) are equally accurate. It is demonstrated that, and also explained why, the results can be far more inaccurate, or even in some cases more accurate, than the (R(j);R(k)) values themselves. It is clarified how the errors of analysis (delta(P) and delta(Q)) are actually governed, and elaborated further by evaluating their variations as a function of different possible parameters which control their magnitudes. The investigations thus help to predict the required analytical conditions for accurate isotopic analysis of carbon and/or oxygen samples of any origin as CO(2)(+). The considerations for the case of natural samples predict that, while it should be generally possible to simultaneously determine the isotopic abundance ratios of (13)C/(12)C and (18)O/(16)O with an accuracy better than the measurements themselves, the determination of either the ((13)C/(12)C and (17)O/(16)O) ratios or the ((17)O/(16)O and (18)O/(16)O) ratios, with an accuracy as good as that of the measurements, would be extremely difficult and may, in practice, be impossible.

Journal Article↗

PubMed2005

Investigation of the feeding effect on the 13C/12C isotope ratio of the hormones in bovine urine using gas chromatography/combustion isotope ratio mass spectrometry.

The effect of the feeding on the 13C/12C isotope ratio of four endogenous steroid hormones testosterone (T), epi-testosterone (epi-T), dehydroepiandrosterone (DHEA) and etiocholanolone (ETIO) in bovine urine was investigated. An analytical method to determine the accurate isotope ratio was developed including an extensive clean up followed by enrichment of the analytes in two steps of HPLC fractionation. Feeding experiments with four young animals were performed using C3 and C4 plants (grass, maize silage, hay, etc.) over a time period of about 280 days. One cattle was used as a control animal with no change of its diet over the full period. The detection of the 13C/12C isotope ratio of the acetylated extracts was performed by gas chromatography/combustion isotope ratio mass spectrometry. After the first change of the feeding from C4 to C3 plants significant changes of the delta 13C % values were observed from the -19 to -23% level to the -24 to -32% level for etiocholanolone and epi-testosterone in urine of three animals, whereas the DHEA values remained under the level of the two metabolites. Testosterone could not be detected with GC-C-IRMS due to its low concentration in young animals. After the second change of the diet from C3 to C4 plants (after 222 days), the measured delta 13C % values have been stabilised at the original level. The results show that in case of the feeding with only C3 plants the endogenous delta values of -32% can be reached. In this case the contribution of exogenous material with a delta value of -32% could not be detected independently of the concentration. If the diet contains C4 plants the difference or the ratio of the delta 13C % values becomes the determinant in the discriminatory power. For validation of the method a human and a cattle were treated with testosterone and the delta 13C % values were measured in incurred human and cattle urine.

Animal Feed↗

PubMed1982

Kinetic isotope effects in the oxidation of isotopically labeled NAD(P)H by bacterial flavoprotein monooxygenases.

Three bacterial flavoprotein monooxygenases, p-hydroxybenzoate hydroxylase, orcinol hydroxylase, and salicylate hydroxylase, have been examined for steady-state kinetic isotope effects with (4R)-[4-2H]NAD(P)H and (4R)-[4-3H]NAD(P)H. The observed isotope selections are for deuterium, DV = 1.8-3.5 and D(V/K) = 1.7-5.1, and for tritium, T(V/K) = 5-19. For both orcinol hydroxylase and p-hydroxybenzoate hydroxylase, reduction of enzyme-bound FAD by (4R)-[4-2H]NAD(P)H in pre-steady-state assays reveals intrinsic deuterium isotope effects of 10 +/- 2 on this redox step. These values are at the upper end of substrate deuterium effects seen in enzymatic reactions. Suppression of approximately 83% of the intrinsic isotope effects in the overall reaction rate (e.g., kH/kD = 10 down to DV = 2.5) corroborates earlier kinetic data on p-hydroxybenzoate hydroxylase [Husain, M., & Massey. V. (1979) J. Biol. Chem. 254, 6657] and suggests that these bacterial phenolic monooxygenases balance out internal transition states such that no single barrier is fully rate limiting.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed2005

Activities of Pu and Am isotopes and isotopic ratios in a soil contaminated by weapons-grade plutonium.

An accident and fire at the former McGuire Air Force Base and Boeing Michigan Aeronautical Research Center (BOMARC) site in New Jersey resulted in dispersion of weapons-grade plutonium in particulate form to the local environment. Soil samples collected at the BOMARC site were measured for their activities and isotopic ratios of Pu and Am isotopes by radioanalytical techniques. The activities of the Pu and Am isotopes in the BOMARC soil were markedly higher than fallout levels, and they decreased nearly exponentially with increasing particle size of the soil. The measured (241)Am activity was compared to calculated values based on decay of (241)Pu. The activity ratios of (238)Pu/(239,240)Pu, (241)Pu/(239,240)Pu, and (241)Am/(239,240)Pu observed in the BOMARC soil were much lower than those attributed to nuclear reprocessing plants and Chernobyl fallout. From the activity ratios of (241)Pu/(239,240)Pu and (241)Am/(239,240)Pu, the origin of the Pu isotopes was identified as weapons-grade and the time since production of the material was estimated. Furthermore, the atomic ratio of (240)Pu/(239)Pu in the BOMARC soil was remarkably lower than the fallout value influenced by nuclear weapons testing and the Chernobyl accident. The atomic ratio of (240)Pu/(239)Pu was very close to the value of the weapons-grade Pu detected from the Thule accident in Greenland. This work demonstrates the utility of radioanalytical measurements and decay calculations for defining characteristics of the source term and discriminating multiple processes that contribute to a source. Such an approach would also be needed to respond to a terrorist event involving an improvised nuclear device or radiological dispersal device.

Americium↗

PubMed2004

Lead isotopic compositions of environmental certified reference materials for an inter-laboratory comparison of lead isotope analysis.

Lead isotope ratios, viz. 207Pb/206Pb and 208Pb/206Pb, of the commercially available certified reference materials (CRMs) issued in Japan are presented with an objective to provide a data set, which will be useful for the quality assurance of analytical procedures, instrumental performance and method validation of the laboratories involved in environmental lead isotope ratio analysis. The analytical method used in the present study was inductively coupled plasma quadrupole mass spectrometry (ICPQMS) preceded by acid digestion and with/without chemical separation of lead from the matrix. The precision of the measurements in terms of the relative standard deviation (RSD) of triplicated analyses was 0.19% and 0.14%, for 207Pb/20Pb and 208Pb/206Pb, respectively. The trueness of lead isotope ratio measurements of the present study was tested with a few CRMs, which have been analyzed by other analytical methods and reported in various literature. The lead isotopic ratios of 18 environmental matrix CRMs (including 6 CRMs analyzed for our method validation) are presented and the distribution of their ratios is briefly discussed.

Journal Article↗

PubMed1994

Clinically feasible stable isotope technique at a reasonable price: analysis of 13CO2/12CO2-abundance in breath samples with a new isotope selective-nondispersive infrared spectrometer.

Up to now, stable isotope analysis of carbon dioxide in breath samples is carried out with sensitive but very expensive and complex isotope ratio mass spectrometry (IRMS). Aiming at a more widespread application of breath tests in gastroenterological diagnostic routine, we tested a newly developed isotope selective non-dispersive infrared spectrometer (NDIRS) in comparison to IRMS. 13C-urea breath tests were performed in 63 patients as the routine screening method for Helicobacter pylori infection. Breath samples at baseline and (15) 30 min after administration of the test solution containing 13C-urea were analysed both by NDIRS and conventional IRMS. The correlation between the delta values of both devices was linear and in good agreement (r = 0.96; p < 0.0001; Y = 1.01 X -0.94). Comparing the delta over baseline-values, the correlation was Y = 1.11 X -0.36 (r = 0.98; p < 0.0001). Referring to the diagnosis of Helicobacter pylori infection with IRMS we calculated a sensitivity of 95.0% and an unchanged specificity (100%) for NDIR analysis. In conclusion, NDIRS appears a promising, easy to operate, and low cost potential alternative to conventional IRMS thus encouraging further detailed investigation and more widespread application of the noninvasive stable isotope technique in breath tests for gastrointestinal function testing.

Adolescent↗

PubMed1998

Performance of human mass balance studies with stable isotope-labeled drug and continuous flow-isotope ratio mass spectrometry: a progress report.

We propose performing human mass balance studies by administering stable isotope labeled (13C or 15N) drug and quantitating excess (above background) 13C or 15N in urine, serum, and feces by continuous flow-isotope ratio mass spectrometry (CF-IRMS). Theoretical calculations and empirical data (dynamic range, linearity, sensitivity, precision, accuracy) are presented to establish that commercially available CF-IRMS instruments can quantitate stable isotope labeled (one or two 15N or 13C labels) drug concentrations of 1.0 microg/mL or greater in urine, serum (15N), or feces. More than two 13C labels may be necessary to quantitate 1.0 microg/mL of drug in serum. Three volunteers received 650 mg of 15N13C2-acetaminophen, and urine was collected for 72 hours. Percent of administered label recovered in urine from the three subjects was 97.4, 78.9, and 95.4 for 13C and 90.3, 77.0, and 90.6 for 15N. Fecal recovery of label for one subject was 0.9% (13C2) and 1.1% (15N). Serum pharmacokinetic values obtained by counting 13C or 15N in one subject were as expected for acetaminophen. This method appears to be promising, and further validation is ongoing.

Acetaminophen↗