D2 18O (deuterium oxide) method for CO2 output in small mammals and economic feasibility in man.

A test of the validity of the isotopic steady state relationships of the doubly labeled water (H2O) method has been carried out with D2 18O in small mammals (three chipmunks and one mouse). CO2 outputs calculated just from 1) the rate of water intake and 2) the ratios of the isotopic concentrations in the body water to the intake water agreed satisfactorily with observed values. Moreover, reconstructed energy and material balances agreed reasonably with similar balances reconstructed for an immediately succeeding period on the same animals studied by the previously validated decay procedure. We conclude from an error analysis that by expressing the isotopic specific activities as abundances in excess of the body water of a subject on a given regimen, the decay procedure is economically feasible in the human with available accuracy of isotopic analyses and the present cost of H2 18O. The method therefore appears to be a useful tool ready for application to the field of human energy metabolism.     

Reliability in estimates of body composition of birds: oxygen-18 versus deuterium dilution

Body composition in birds was evaluated indirectly by 18O and 2H dilution. Body composition was determined by whole-body chemical analysis of eight adult roosters (Gallus gallus). In vivo measurements of total body water (TBW) were carried out using doubly labeled water (2H2 18O). Estimated dilution spaces using both the plateau and intercept approaches were compared with the results obtained by carcass lyophilization. Both 18O and 2H slightly overestimated TBW compared with the results obtained by lyophilization, by 2.2%+/-1.9% and 5.7%+/-0.2%, respectively; both differences were statistically significant (P<0.01). The difference between these isotope estimations was significant (P<0.001). However, isotope dilution spaces and TBW were highly correlated. There was a strong inverse relationship between total body fat and TBW percentages (r2=0.98, P<0.0001). The relation between TBW and body protein was significant. Water content in lean body mass (72.8%) obtained in our study was very close to that reported in mammals, demonstrating no fundamental difference in tissue water content between birds and mammals. Estimated body fat and protein values from isotopic dilution did not significantly differ from values obtained by direct chemical analysis (P>0.05), except for body fat in the Pace and Rathbun approach (Table 3). Although estimation of TBW and body composition by isotope dilution is time consuming and expensive, deuterium offers a reliable and low-cost alternative compared with 18O. The advantage of in vivo estimation of TBW with isotopic dilution in combination with the regression approach is that it permits repeated measurements of body composition on the same birds under laboratory and free-living conditions.     

Measurement of energy expenditure in humans by doubly labeled water method

The utility of the doubly labeled water method for the determination of energy expenditure and water output was investigated in humans. Approximately 10 g of 18O and 0.5 g of 2H as water was orally administered to four healthy adults. Total body water was determined from the isotope dilution, and the ensuing 18O and 2H disappearance rates from body water were determined for 13 days by mass spectrometric isotope ratio analysis of the urinary water. During this period, subjects were maintained on a measured diet to determine energy and water intake. The energy expenditure from the doubly labeled water method differed from dietary intake plus change in body composition by an average of 2%, with a coefficient of variation of 6%. The water outputs determined by the two methods differed by 1%, with a coefficient of variation of 7%. The doubly labeled water method is noninvasive, and the subjects could maintain their daily activities without restriction.     

Facile oxygen exchanges of phosphoenolpyruvate and preparation of [18O]phosphoenolpyruvate

Phosphoenolpyruvate when heated in acidic solution exchanges its phosphoryl and carboxyl oxygens rapidly and its enolic oxygen much more slowly with oxygens from water. The incorporation of 18O into phosphoenolpyruvate was measured by gas chromatography-mass spectrometry and phosphorus-31 nuclear magnetic resonance after heating in H218O at 98 degrees C. The rates of exchange of all six oxygens of phosphoenolpyruvate with water increase with increasing acidity, and the phosphoryl oxygens exchange more rapidly than the carboxyl oxygens. The rate of exchange of each oxygen of the phosphoryl group is 16-fold greater than the hydrolysis rate at 1 N HCl. This provides a simple and useful method for the synthesis of [18O]phosphoenolpyruvate highly enriched in its phosphoryl-group oxygens. An enrichment of 89% was obtained with a 50% yield. The [18O]-phosphoenolpyruvate showed a binomial distribution of 18O in the phosphoryl-group oxygens. The exchange may be explained by the reversible formation of a transient cyclic phosphate and, for exchange of the enolic oxygen, a transient acyl phosphate. Preparation of [18O]phosphoenolypyruvate from [18O]Pi by a chemical synthesis from beta-chlorolactate was not satisfactory because of drastic loss of 18O during the procedures used. Some loss of 18O also occurred during an enzymic synthesis with KCNO, [18O]Pi, carbamate kinase, and pyruvate kinase.     

Effect of bolus fluid intake on energy expenditure values as determined by the doubly labeled water method.

The doubly labeled water (DLW, 2H(2)18O) method is a highly accurate method for measuring energy expenditure (EE). A possible source of error is bolus fluid intake before body water sampling. If there is bolus fluid intake immediately before body water sampling, the saliva may reflect the ingested water disproportionately, because the ingested water may not have had time to mix fully with the body water pool. To ascertain the magnitude of this problem, EE was measured over a 5-day period by the DLW method. Six subjects were dosed with 2H2(18)O. After the reference salivas for the two-point determination were obtained, subjects drank water (700-1,000 ml), and serial saliva samples were collected for the next 3 h. Expressing the postbolus saliva enrichments as a percentage of the prebolus value, we found 1) a minimum in the saliva isotopic enrichments were reached at approximately 30 min with the minimum for 2H (95.48 +/- 0.43%) being significantly lower than the minimum for 18O (97.55 +/- 0.44, P less than 0.05) and 2) EE values calculated using the postbolus isotopic enrichments are appreciably higher (19.9 +/- 7.5%) than the prebolus reference values. In conclusion, it is not advisable to collect saliva samples for DLW measurements within approximately 1 h of bolus fluid intake.     

A rapid analytical technique for the determination of energy expenditure by the doubly labelled water method

The doubly labelled water method involves the administration of water enriched in 2H and 18O followed by determination of the turnover rates of these isotopes. Since 18O is eliminated from the body as both CO2 and water, while 2H leaves only as water, the difference between the two turnover rates provides a measure of CO2 production and hence energy expenditure. Isotopic analysis by conventional stable isotope ratio analysis (SIRA) is labour intensive and time consuming, as it requires off-line conversion of water samples to gases (H2 and CO2) followed by sequential analysis for each of the two isotopes using the mass spectrometer. Lack of suitable automated instrumentation with the ability to process large numbers of samples has prevented routine application of the method. We describe here an automated technique in which body water samples (urine, saliva, breath water or milk) are analysed simultaneously for 2H and 18O. The single bench system comprises two mass spectrometer analysers, one for measuring 2H from H2 gas, the other for measuring 18O from the water vapour (masses 18, 20). Both analysers share a common heated inlet system into which microlitre quantities of the body fluids are injected from an autosampler (102 samples). The water vapour flows both directly to one analyser for 18O measurement and into a uranium reduction furnace for conversion to H2, prior to 2H measurement by the second analyser. Both analysers also share vacuum and electronic components, enabling savings in both space and cost. In this paper we present results illustrating performance characteristics and procedures for routine application to human subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen and oxygen isotope ratios in body water and hair: modeling isotope dynamics in nonhuman primates

The stable isotopic composition of drinking water, diet, and atmospheric oxygen influence the isotopic composition of body water ((2)H/(1)H, (18)O/(16)O expressed as δ(2) H and δ(18)O). In turn, body water influences the isotopic composition of organic matter in tissues, such as hair and teeth, which are often used to reconstruct historical dietary and movement patterns of animals and humans. Here, we used a nonhuman primate system (Macaca fascicularis) to test the robustness of two different mechanistic stable isotope models: a model to predict the δ(2)H and δ(18)O values of body water and a second model to predict the δ(2)H and δ(18)O values of hair. In contrast to previous human-based studies, use of nonhuman primates fed controlled diets allowed us to further constrain model parameter values and evaluate model predictions. Both models reliably predicted the δ(2)H and δ(18)O values of body water and of hair. Moreover, the isotope data allowed us to better quantify values for two critical variables in the models: the δ(2)H and δ(18)O values of gut water and the (18)O isotope fractionation associated with a carbonyl oxygen-water interaction in the gut (α(ow)). Our modeling efforts indicated that better predictions for body water and hair isotope values were achieved by making the isotopic composition of gut water approached that of body water. Additionally, the value of α(ow) was 1.0164, in close agreement with the only other previously measured observation (microbial spore cell walls), suggesting robustness of this fractionation factor across different biological systems.     

Assessment of renal function by the stable oxygen and hydrogen isotopes in human blood plasma

Water (H(2)O) is the most abundant and important molecule of life. Natural water contains small amount of heavy isotopes. Previously, few animal model studies have shown that the isotopic composition of body water could play important roles in physiology and pathophysiology. Here we study the stable isotopic ratios of hydrogen (δ(2)H) and oxygen (δ(18)O) in human blood plasma. The stable isotopic ratio is defined and determined by δ(sample) = [(R(sample)/R(STD))-1] * 1000, where R is the molar ratio of rare to abundant, for example, (18)O/(16)O. We observe that the δ(2)H and the δ(18)O in human blood plasma are associated with the human renal functions. The water isotope ratios of the δ(2)H and δ(18)O in human blood plasma of the control subjects are comparable to those of the diabetes subjects (with healthy kidney), but are statistically higher than those of the end stage renal disease subjects (p<0.001 for both ANOVA and Student's t-test). In addition, our data indicate the existence of the biological homeostasis of water isotopes in all subjects, except the end stage renal disease subjects under the haemodialysis treatment. Furthermore, the unexpected water contents (δ(2)H and δ(18)O) in blood plasma of body water may shed light on a novel assessment of renal functions.     

Gas chromatography-mass spectrometry assay of the (18)o enrichment of water as trimethyl phosphate

We have developed an assay for determining the 18O enrichment of water in biological fluids. Urine, plasma, or whole blood is reacted with phosphorous pentachloride to yield phosphoric acid. Derivatization of phosphoric acid with diazomethane generates trimethyl phosphate. The enrichment of trimethyl phosphate is nearly four times that of water and is assayed using gas chromatography-mass spectrometry (electron impact ionization). Yang et al. (1998, Anal. Biochem. 258, 315-321) assayed the 2H enrichment of body water after exchange with acetone, by gas chromatography-mass spectrometry. The combination of our 18O method and the 2H method of Yang et al. allows one to measure energy expenditure via "doubly labeled" water (2H(2)O + H(2)18O), using small samples of body fluids. These techniques were used to measure energy expenditure in mice, in which the 18O enrichment of body water can be monitored down to 0.025%.     

Cooperative binding of oxygen to the water-splitting enzyme in the filamentous cyanobacterium Oscillatoria chalybea

In the filamentous cyanobacterium Oscillatoria chalybea photolysis of water does not take place in the complete absence of oxygen. A catalytic oxygen partial pressure of 15x10(-6) Torr has to be present for effective water splitting to occur. By means of mass spectrometry we measured the photosynthetic oxygen evolution in the presence of H(2)(18)O in dependence on the oxygen partial pressure of the atmosphere and analysed the liberations of (16)O(2), (16)O(18)O and (18)O(2) simultaneously. The observed dependences of the light-induced oxygen evolution on bound oxygen yield sigmoidal curves. Hill coefficient values of 3.0, 3.1 and 3.2, respectively, suggest that the binding is cooperative and that four molecules of oxygen have to be bound per chain to the oxygen evolving complex. Oxygen seems to prime the water-splitting reaction by redox steering of the S-state system, putting it in the dark into the condition from which water splitting can start. It appears that in O. chalybea an interaction of oxygen with S(0) and S(1) leads to S(2) and S(3), thus yielding the typical oxygen evolution pattern in which even after extensive dark adaptation substantial amounts of Y(1) and Y(2) are found. The interacting oxygen is apparently reduced to hydrogen peroxide. Mass spectrometry permits to distinguish this highly specific oxygen requirement from the interaction of bulk atmospheric oxygen with the oxygen evolving complex of the cyanobacterium. This interaction leads to the formation H(2)O(2) which is decomposed under O(2) evolution in the light. The dependence on oxygen-partial pressure and temperature is analysed. Structural peculiarities of the cyanobacterial reaction centre of photosystem II referring to the extrinsic peptides might play a role.     

Three hydroxylations incorporating molecular oxygen in the aerobic biosynthesis of ubiquinone in Escherichia coli.

The biosynthetic origin of the oxygen atoms of ubiquinone 8 from aerobically grown Escherichia coli was studied by 18O labeling. An apparatus was developed which allowed the growth of cells under a defined atmosphere. Mass spectral analysis of ubiquinone 8 from cells grown under highly enriched 18O2 showed that three oxygen atoms of the quinone are derived from molecular oxygen. It was established that the molecular oxygen is incorporated into the two methoxyl groups (at C-5 and C-6) and one of the carbonyl positions of the ubiquinone molecule by demonstrating that only one of the incorporated oxygens will exchange with water under acidic conditions that specifically catalyze the exchange of carbonyl, but not methoxyl, oxygens. That the C-4 carbonyl oxygen is derived from molecular oxygen was shown by the incorporation of three atoms of 18O2 into ubiquinone 8 biosynthesized from added 4-hydroxybenzoic acid. Comparison of ubiquinone 8 and menaquinone 8 from E. coli grown under 18O2 confirmed that the labeled carbonyl oxygen of the [18O2]ubiquinone 8 is incorporated biosynthetically and not by chemical exchange in the cell. It is concluded that the three hydroxylation reactions involved in the pathway for the aerobic biosynthesis of ubiquinone are all catalyzed by monooxygenases. The implications of this study for the anaerobic biosynthesis of ubiquinone 8 in E coli are discussed.     

Oxidation of reduced cytochrome c oxidase with 18O2. A search for mu-oxo-bridged metal species in the oxidized enzyme

We have measured the increase in 18O content of water produced from single turnover oxidations of anerobically reduced cytochrome c oxidase with 18O2 in order to test the hypothesis that a reduced atom of oxygen, originating from dioxygen, remains bound to oxidized cytochrome c oxidase in the form of a mu-oxo-bridge between two metal components when a single turnover occurs. When water samples produced by oxidizing the reduced enzyme with 18O2 were compared to natural abundance control samples obtained by oxidizing with 16O2, all of the 18O2 reduced in a single turnover could be accounted for in the form of additional H218O produced. We conclude that neither atom of the dioxygen reduced is incorporated into the enzyme as a bridge which is stable in the absence of oxidoreductive reactions on the time scale of several minutes.     

A new measurement technique reveals temporal variation in delta18O of leaf-respired CO2

The oxygen isotope composition of CO(2) respired by Ricinus communis leaves (delta(18)O(R)) was measured under non-steady-state conditions with a temporal resolution of 3 min using a tunable diode laser (TDL) absorption spectrometer coupled to a portable gas exchange system. The SD of delta(18)O measurement by the TDL was +/- 0.2 per thousand and close to that of traditional mass spectrometers. Further, delta(18)O(R) values at isotopic steady state were comparable to those obtained using traditional flask sampling and mass spectrometric techniques for R. communis grown and measured in similar environmental conditions. As well as higher temporal resolution, the online TDL method described here has a number of advantages over mass spectrometric techniques. At isotopic steady state among plants grown at high light, the "one-way flux" model was required to accurately predict delta(18)O(R). A comparison of measurements and the model suggests that plants grown under low-light conditions have either a lower proportion of chloroplast CO(2) that isotopically equilibrates with chloroplast water, or more enriched delta(18)O of CO(2) in the chloroplast that has not equilibrated with local water. The high temporal resolution of isotopic measurements allowed the first measurements of delta(18)O(R) when stomatal conductance was rapidly changing. Under non-steady-state conditions, delta(18)O(R) varied between 50 and 220 per thousand for leaves of plants grown under different light and water environments, and varied by as much as 100 per thousand within 10 min for a single leaf. Stomatal conductance ranged from 0.001 to 1.586 mol m(-2) s(-1), and had an important influence on delta(18)O(R) under non-steady-state conditions not only via effects on leaf water H(2) (18)O enrichment, but also via effects on the rate of the one-way fluxes of CO(2) into and out of the leaf.     

Spontaneous Hydrolysis and Dehydration of Dehydroascorbic Acid in Aqueous Solution

The interaction of water with dehydroascorbic acid was examined by incubating dehydroascorbic acid and ascorbic acid in¹⁸O-labeled water for various amounts of time and then oxidizing the products with hydrogen peroxide or reducing the products with mercaptoethanol, with analysis by gas chromatography mass spectrometry. Based on mass changes, dehydroascorbic acid readily exchanged three oxygen atoms with H₂¹⁸O. When mercaptoethanol was used to reduce dehydroascorbic acid (which had been incubated in H₂¹⁸O) to ascorbic acid, the newly formed ascorbic acid also contained three labeled oxygen atoms. However, ascorbic acid incubated in H₂¹⁸O for the same amount of time under identical conditions exchanged only two labeled oxygen atoms. Electron impact mass spectrometry of derivatized ascorbic acid created a decarboxylation product which had only two labeled oxygen atoms, regardless if 3-oxygen-labeled or 2-oxygen-labeled ascorbic acid was the parent compound, isolating the extra oxygen addition to carbon 1. These data suggest that dehydroascorbic acid spontaneously hydrolyzes and dehydrates in aqueous solution and that the hydrolytic-hydroxyl oxygen is accepted by carbon 1. Ascorbic acid, on the other hand, does not show this same tendency to hydrolyze.     

Body composition assessment in extreme obesity and after massive weight loss induced by gastric bypass surgery

Body composition methods were examined in 20 women [body mass index (BMI) 48.7 +/- 8.8 kg/m(2)] before and after weight loss [-44.8 +/- 14.6 (SD) kg] after gastric bypass (GBP) surgery. The reference method, a three-compartment (3C) model using body density by air displacement plethysmography and total body water (TBW) by H(2)18O dilution (3C-H(2)18O), showed a decrease in percent body fat (%BF) from 51.4 to 34.6%. Fat-free mass hydration was significantly higher than the reference value (0.738) in extreme obesity (0.756; P < 0.001) but not after weight reduction (0.747; P = 0.16). %BF by H(2)18O dilution and air displacement plethysmography differed significantly from %BF by 3C-H(2)18O in extreme obesity (P < 0.05) and 3C models using (2)H(2)O or bioelectrical impedance analysis (BIA) to determine TBW improved mean %BF estimates over most other methods at both time points. BIA results varied with the equation used, but BIA better predicted %BF than did BMI at both time points. All methods except BIA using the Segal equation were comparable to the reference method for determining changes over time. A simple 3C model utilizing air displacement plethysmography and BIA is useful for clinical evaluation in this population.     

Baboons, Water, and the Ecology of Oxygen Stable Isotopes in an Arid Hybrid Zone

Abstract Baboons regularly drink surface waters derived from atmospheric precipitation, or meteoric water. As a result, the oxygen isotope (δ(18)O) composition of their tissues is expected to reflect that of local meteoric waters. Animal proxies of the oxygen isotope composition of meteoric water have practical applications as paleoenvironmental recorders because they can be used to infer aridity and temperature in historic and fossil systems. To explore this premise, we measured the δ(18)O values of hair from two baboon species, Papio anubis and Papio hamadryas, inhabiting Awash National Park, Ethiopia. The hybridizing taxa differ in their ranging behavior and physiological response to heat. Papio hamadryas ranges more widely in the arid thornbush and is inferred to ingest a greater proportion of leaf water that is enriched in (18)O as a result of evaporative fractionation. It is also better able to conserve body water, which reduces its dependence on meteoric waters depleted in (18)O. Taken together, these factors would predict relatively higher δ(18)O values in the hair (δ(18)O(hair)) of P. hamadryas. We found that the δ(18)O(hair) values of P. hamadryas were higher than those of P. anubis, yet the magnitude of the difference was marginal. We attribute this result to a common source of drinking water, the Awash River, and the longer drinking bouts of P. hamadryas. Our findings suggest that differences in δ(18)O values among populations of Papio (modern or ancient) reflect different sources of drinking water (which might have ecological significance) and, further, that Papio has practical value as a paleoenvironmental recorder.     

Determination of 18O by prompt nuclear reaction analysis: application for measurement of microsamples

A method is described for the routine determination of 18O concentrations in microsamples of biological fluids. The method utilizes the prompt nuclear reaction 18O(p, alpha o)15N, and 846-keV protons from a 3-MeV Van de Graaff Accelerator are focused on approximately 2,000-A-thick Ta2O5 targets prepared by anodic oxidation from 50-microliter samples of water distilled from blood or other biological fluids. The broad cross section of the resonance peak for this nuclear reaction (47 keV) ensures high yields, especially at small reaction angles, and the high-energy alpha particles produced by the reaction (4 MeV) are readily separated from scattered protons by the use of an aluminized Mylar foil of suitable thickness. Background levels of 18O (0.204 atom%) can be detected with run times of approximately 5-8 min, and the sensitivity of the method is of the order of 0.05 atom %. Experimental error due to sample preparation was found to be 1.7%, and counting errors were close to theoretical limits so that total error was of the order of 2.5%. Duplicate samples were analyzed by use of the 18O(p, alpha o)15N reaction at Lucas Heights, Australia, and the 18O(p,n)18F reaction by the method of Wood et al. (Anal. Chem. 47: 646-650, 1975) at the University of California, Los Angeles, and the agreement was excellent (y = 1.0123x - 0.0123, r = 0.991, P less than 0.001). The theoretical limitations and the general applicability of the method in biological studies designed to estimate the rate of metabolism of free-ranging animals are discussed.     

Oxygen isotope enrichment of organic matter in Ricinus communis during the diel course and as affected by assimilate transport

The oxygen isotope composition in leaf water and organic compounds in different plant tissues is useful for assessing the physiological performance of plants in their environment, but more information is needed on Delta(18)O variation during a diel course. Here, we assessed Delta(18)O of the organic matter in leaves, phloem and xylem in stem segments, and fine roots of Ricinus communis during a full diel cycle. Enrichment of newly assimilated organic matter in equilibrium with leaf water was calculated by applying a nonsteady-state evaporative enrichment model. During the light period, Delta(18)O of the water soluble organic matter pool in leaves and phloem could be explained by a 27 per thousand enrichment compared with leaf water enrichment. Leaf water enrichment influenced Delta(18)O of phloem organic matter during the night via daytime starch synthesis and night-time starch remobilization. Phloem transport did not affect Delta(18)O of phloem organic matter. Diel variation in Delta(18)O in organic matter pools can be modeled, and oxygen isotopic information is not biased during transport through the plant. These findings will aid field studies that characterize environmental influences on plant water balance using Delta(18)O in phloem organic matter or tree rings.     

Effects of environmental parameters, leaf physiological properties and leaf water relations on leaf water delta18O enrichment in different Eucalyptus species

Stable oxygen isotope ratios (delta18O) have become a valuable tool in the plant and ecosystem sciences. The interpretation of delta18O values in plant material is, however, still complicated owing to the complex interactions among factors that influence leaf water enrichment. This study investigated the interplay among environmental parameters, leaf physiological properties and leaf water relations as drivers of the isotopic enrichment of leaf water across 17 Eucalyptus species growing in a common garden. We observed large differences in maximum daily leaf water delta18O across the 17 species. By fitting different leaf water models to these empirical data, we determined that differences in leaf water delta18O across species are largely explained by variation in the Péclet effect across species. Our analyses also revealed that species-specific differences in transpiration do not explain the observed differences in delta18O while the unconstrained fitting parameter 'effective path length' (L) was highly correlated with delta18O. None of the leaf morphological or leaf water related parameters we quantified in this study correlated with the L values we determined even though L was typically interpreted as a leaf morphological/anatomical property. A sensitivity analysis supported the importance of L for explaining the variability in leaf water delta18O across different species. Our investigation highlighted the importance of future studies to quantify the leaf properties that influence L. Obtaining such information will significantly improve our understanding of what ultimately determines the delta18O values of leaf water across different plant species.