Influence of deuterium depleted water on freeze-dried tissue isotopic composition and morphofunctional body performance in rats of different generations

The influence of deuterium depleted water on the body of different rat generations was investigated in physiological conditions. As a result of this study it was established that the most significant and rapid reduction in D/H equilibrium was observed in plasma (by 36.2%), and lyophilized kidney tissues (by 15.8%). Less pronounced deuterium decrease was characteristic of liver tissue (9.3%) and heart (8.5%). Stabilization of the isotopic exchange reaction rate was fixed in the blood and tissues of rats, starting from the second generation. At the same time when deuterium depleted water (40 ppm) was used in dietary intake, the change in morphological and functional parameters in laboratory animals associated with the processes of adaptation to the effects of substress isotopic D/H gradient was also noted. The study shows that modification of only drinking water intake regime can’t significantly change the deuterium content in tissues of metabolically active organs, because of the concurrent deuterium receipt in feed substances of plant and animal origin.     

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.     

双标水法：实验室之间的稳定同位素测量对比

双标水(doubly labeled water,DLW)法是研究动物野外能量代谢最准确的方法,其中最关键、最困难的环节是同位素的定量分析。为了检验不同同位素实验室测定动物血样氢氧同位素丰度的准确性及其对估算能量代谢的影响程度,成都地质学院(CCG)和美国加利福尼亚大学洛杉机分校(UCLA)的同位素实验室于1990年5月开展了合作,以成都附近农村的5只山羊为研究对象进行了双标水法的实验。将采集的动物血样分别在两个实验室进行了D和~(13)O含量测定,取得了成功的结果:两个实验室所测得的~(13)O值近于相同,而UCLA同位素实验室测得的~(13)O值略高于CCG的测定值。由此所计算出的能量代谢值却非常接近,其差值平均为6.6％;水循环平均相差1.2％,差异均不显著(P>0.05)。UCLA实验室的质谱仪已为英国和荷兰的同类实验室所校正,这确认了我们的合作测定成果的可信度和有效性,证明CCG和UCLA实验室的测定结果均可以满足双标法同位素定量测定的需要。测定结果是可信的。本文还就两地实验室所测结果产生误差的原因进行了初步分析,对于进行同类研究的其它实验室也有借鉴意义。     

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.     

Comparison of Modeled and Observed Environmental Influences on the Stable Oxygen and Hydrogen Isotope Composition of Leaf Water in Phaseolus vulgaris L

In this paper we describe how a model of stable isotope fractionation processes, originally developed by H. Craig and L. I. Gordon ([1965] in E Tongiorgi, ed, Proceedings of a Conference on Stable Isotopes in Oceanographic Studies and Paleotemperature, Spoleto, Italy, pp 9-130) for evaporation of water from the ocean, can be applied to leaf transpiration. The original model was modified to account for turbulent conditions in the leaf boundary layer. Experiments were conducted to test the factors influencing the stable isotopic composition of leaf water under controlled environment conditions. At steady state, the observed leaf water isotopic composition was enriched above that of stem water with the extent of the enrichment dependent on the leaf-air vapor pressure difference (VPD) and the isotopic composition of atmospheric water vapor (AWV). The higher the VPD, the larger was the observed heavy isotope content of leaf water. At a constant VPD, leaf water was relatively depleted in heavy isotopes when exposed to AWV with a low heavy isotope composition, and leaf water was relatively enriched in heavy isotopes when exposed to AWV with a large heavy isotope composition. However, the observed heavy isotope composition of leaf water was always less than that predicted by the model. The extent of the discrepancy between the modeled and observed leaf water isotopic composition was a strong linear function of the leaf transpiration rate.     

Body composition in mink (Mustela vison) kits during 21-42 days postpartum using estimates of hydrogen isotope dilution and direct carcass analysis

We compared carcass analysis and hydrogen isotope dilution methods to measure total body water (TBW) and body composition in a small altricial carnivore, the mink. Dilution space (D) of mink at 21-42 days of age (n=20), was determined after subcutaneous administration of tritiated water. The same animals were then used to determine TBW and body composition by carcass analysis and to derive predictive empirical relationships between TBW and total body fat, protein and energy. A separate validation set of 27 kits was used to test the accuracy of predicting body composition from TBW. D overestimated TBW by a consistent and predictable 4.1% (R(2)=0.999, P<0.001). Our estimates of fat, protein and energy content, using equations derived from TBW, were not significantly different than those obtained from direct carcass analysis (P>0.980) in either the initial or validation set of mink. TBW was shown to decrease from 81 to 76% and total body protein to increase from 14 to 19% of LBM of the kits from 21 to 42 days of age. Although a rapidly changing hydration state was apparent in neonates, we conclude that when this is taken into account, accurate estimates of body composition can be obtained from hydrogen isotope dilution.     

Specificity of bacterial response to variation of isotopic composition of water

Culture growth of bacteria with different membrane lipid composition was studied in liquid media with different isotope content of water. The concentration of deuterium in water was varied from 0.01 to 90%. It was shown that large concentrations of deuterium cause the inhibition of all cultures except Deinococcus radiodurans, the most stable living cell. Two cultures, Methylobacterium organophilum and Hyphomonas jannaschiana, showed a pronounced activation at a deuterium concentration of 0.01%. At higher deuterium concentrations, the inhibition of culture growth was observed. The strains Renobacter vacuolatum, Arcocella aquatica NO502 and Caulobacter crescentus did not show any significant effect at low concentrations of deuterium.     

Significant Difference in Hydrogen Isotope Composition Between Xylem and Tissue Water in Populus Euphratica

Deuterium depletions between stem water and source water have been observed in coastal halophyte plants and in multiple species under greenhouse conditions. However, the location(s) of the isotope fractionation is not clear yet and it is uncertain whether deuterium fractionation appears in other natural environments. In this study, through two extensive field campaigns utilizing a common dryland riparian tree species Populus euphratica Oliv., we showed that no significant δ¹⁸O differences were found between water source and various plant components, in accord with previous studies. We also found that no deuterium fractionation occurred during P. euphratica water uptake by comparing the deuterium composition (δD) of groundwater and xylem sap. However, remarkable δD differences (up to 26.4‰) between xylem sap and twig water, root water and core water provided direct evidence that deuterium fractionation occurred between xylem sap and root or stem tissue water. This study indicates that deuterium fractionation could be a common phenomenon in drylands, which has important implications in plant water source identification, palaeoclimate reconstruction based on wood cellulose and evapotranspiration partitioning using δD of stem water.     

Evaluation of the deuterium dilution method to estimate body composition in the barnacle goose: accuracy and minimum equilibration time

We examined body composition in barnacle geese (Branta leucopsis) by proximate carcass analysis and by deuterium isotope dilution. We studied the effect of isotope equilibration time on the accuracy of total body water (TBW) estimates and evaluated models to predict fat-free mass (FFM) and fat mass (FM) from different measurements varying in their level of invasiveness. Deuterium enrichment determined at 45, 90, and 180 min after isotope injection did not differ significantly. At all sampling intervals, isotope dilution spaces (TBW(d)) consistently overestimated body water determined by carcass analysis (TBW(c)). However, variance in the deviation from actual TBW was higher at the 45-min sampling interval, whereas variability was the same at 90 and 180 min, indicating that 90 min is sufficient time to allow for adequate equilibration. At 90 min equilibration time, deuterium isotope dilution overestimated TBW(c) by 7.1% +/= 2.6% (P < 0.001, paired t-test, n=20). This overestimate was consistent over the range of TBW studied, and TBW(c) could thus be predicted from TBW(d) (r2=0.976, P<0.001). Variation in TBW(c) and TBW(d) explained, respectively, 99% and 98% of the variation in FFM. FM could be predicted with a relative error of ca. 10% from TBW estimates in combination with body mass (BM). In contrast, BM and external body measurements allowed only poor prediction. Abdominal fat fresh mass was highly correlated to total FM and, if the carcass is available, allows simple means of fat prediction without dissecting the entire specimen.     

Stable isotopes (δD and δ13C) are geographic indicators of natal origins of monarch butterflies in eastern North America

Wing membranes of laboratory and field-reared monarch butterflies (Danaus plexippus) were analyzed for their stable-hydrogen (δD) and carbon (δ¹³C) isotope ratios to determine whether this technique could be used to identify their natal origins. We hypothesized that the hydrogen isotopic composition of monarch butterfly wing keratin would reflect the hydrogen isotope patterns of rainfall in areas of natal origin where wings were formed. Monarchs were reared in the laboratory on milkweed plants (Asclepias sp.) grown with water of known deuterium content, and, with the assistance of volunteers, on native milkweeds throughout eastern North America. The results show that the stable hydrogen isotopic composition of monarch butterflies is highly correlated with the isotopic composition of the milkweed host plants, which in turn corresponds closely with the long-term geographic patterns of deuterium in rainfall. Stable-carbon isotope values in milkweed host plants were similarly correlated with those values in monarch butterflies and showed a general pattern of enrichment along a southwest to northeast gradient bisecting the Great Lakes. These findings indicate that natal origins of migratory and wintering monarchs in Mexico can be inferred from the combined δD and δ¹³C isotopic signatures in their wings. This relationship establishes that analysis of hydrogen and carbon isotopes can be used to answer questions concerning the biology of migratory monarch butterflies and provides a new approach to tracking similar migratory movements of other organisms. Received: 1 July 1998 / Accepted: 11 November 1998     

Reproducibility of gas chromatography-mass spectrometry measurements of 2H labeling of water: application for measuring body composition in mice

Deuterium-labeled water (2H2O) has emerged as a novel isotope tracer. Following the administration of 2H2O, it is possible to study the dynamics of carbohydrate, protein, lipid, and DNA and to determine body composition. Those studies require reliable measurements of the 2H labeling of water. Although simple gas chromatography-mass spectrometry (GC-MS) methods have been developed for measuring the 2H enrichment of biological fluids, investigators have not reported on the intra- and/or interdaily variability of the measurements. We have experimentally examined the reproducibility of one GC-MS method for measuring the 2H labeling of water. Briefly, hydrogen (deuterium) atoms in water were exchanged with those bound to acetone, and the 2H labeling of acetone was then determined under electron impact ionization. We found that the coefficient of variation is generally less than 0.5% when water is labeled between 0 and 2.8 mole percentage excess 2H. We demonstrated that this highly reproducible result allows one to use 2H2O and the "acetone method" to measure physiological parameters such as body composition in mice.     

Interspecies differences in the empty body chemical composition of domestic animals

Domestication of animals has resulted in phenotypic changes by means of natural and human-directed selection. Body composition is important for farm animals because it reflects the status of energy reserves. Thus, there is the possibility that farm animals as providers of food have been more affected by human-directed selection for body composition than laboratory animals. In this study, an analysis was conducted to determine what similarities and differences in body composition occur between farm and laboratory animals using literature data obtained from seven comparative slaughter studies (n = 136 observations). Farm animals from four species (cattle, goats, pigs and sheep) were all castrated males, whereas laboratory animals from three species (dogs, mice and rats) comprised males and/or females. All animals were fed ad libitum. The allometric equation, Y = aX^b, was used to determine the influence of species on the accretion rates of chemical components (Y, kg) relative to the growth of the empty body, fat-free empty body or protein weights (X, kg). There were differences between farm and laboratory animals in terms of the allometric growth coefficients for chemical components relative to the empty BW and fat-free empty BW (P < 0.01); farm animals had more rapid accretion rates of fat (P < 0.01) but laboratory animals had more rapid accretion rates of protein, water and ash (P < 0.01). In contrast, there was no difference in terms of the allometric growth coefficients for protein and water within farm animals (P > 0.2). The allometric growth coefficients for ash weight relative to protein weight for six species except sheep were not different from a value of 1 (P > 0.1), whereas that of sheep was smaller than 1 (P < 0.01). When compared at the same fat content of the empty body, the rate of change in water content (%) per unit change in fat content (%) was not different (P > 0.05) across farm animal species and similar ash-to-protein ratios were obtained except for dogs. The fraction of empty body energy gain retained as fat increased in a curvilinear manner, and there was little variation among farm animals at the same fat content of the empty body. These findings may provide the opportunity to develop a general model to predict empty body composition across farm animal species. In contrast, there were considerable differences of chemical body composition between farm and laboratory animals.     

Isotopic effects of low concentration of deuterium in water on biological systems

Isotopic effects of deuterium in water are studied in a broad range of concentrations on a number of biological objects of different organization levels. The results obtained show that biological objects are sensitive to variations of isotope composition in water. A decrease or increase in deuterium concentrations in water may cause activation or inhibition of biological functions. The values of biological isotopic effects of low deuterium concentration may even be higher than those of high deuterium concentration. No regularity in response for all the objects studied failed to find out in a range of deuterium concentration in water from 4 ppm to 1%.     

Use of deuterated water for measurement of short-term cholesterol synthesis in humans

Previous methods for measurement of cholesterol synthesis de novo in humans have either required extended measurement periods or been indirect. Recently, a technique based on the rate of incorporation of deuterium from D2O into the plasma cholesterol pool has been developed. Following oral ingestion of D2O, deuterium enrichment over time in free plasma cholesterol after combustion and reduction was determined using isotope ratio mass spectrometry. This methodology enabled direct measurement of plasma cholesterol synthesis over intervals as short as 4 h. The technique has been used to demonstrate changes in synthetic rate in response to feeding conditions and genetic influences. Fasting over 36 h resulted in markedly reduced deuterium uptake into cholesterol in healthy males. Diurnal variations in synthetic rate have also been identified, with elevated synthesis observed during nocturnal periods in both fed and fasted subjects. In addition, the influence of apolipoprotein E phenotype on cholesterol synthesis has been shown using this technique. Individuals carrying the apoprotein epsilon 2 allele demonstrated lower synthesis compared with those possessing the epsilon 4 allele. Thus, the deuterium incorporation technique for measuring cholesterol synthesis demonstrates potential as a valuable stable isotope method for human nutrition studies.     

Deuterium Isotope Effect in γ-Aminobutyric Acid Transamination: Determination of Rate-Limiting Step

The rate of transamination of gamma-aminobutryic acid (GABA) catalyzed by hog brain gamma-aminobutyrate aminotransferase was substantially reduced when the hydrogen at the gamma-carbon position was replaced by deuterium. The deuterium isotope effect of this reaction has been substantiated by fluorometric, radiometric, and mass spectrometric procedures and assessed kinetically. The ratios of Vmax of the nonlabeled substrate/Vmax of the deuterated substrate obtained under different conditions ranged from 6 to 7. This indicates that the cleavage of the hydrogen from the gamma-carbon is the rate-determining step in GABA transamination. Similar isotope effects have also been shown to occur in the peripheral system in vivo.     

Deuterium oxide dilution accurately predicts water intake in sheep and goats

The aim of this study was to test whether the deuterium oxide dilution technique accurately predicts water intake in sheep and goats. Two other issues were also studied: (i) a comparison of water intake in sheep and goats and (ii) an assessment of whether observations of drinking behaviour can accurately measure the water intake. In this study, eight dry Boer goats and eight dry German Black Head Mutton ewes were kept under controlled stable conditions. Animals had access to hay and water ad libitum. Diurnal drinking behaviour was recorded by video. Individual daily water intake was measured and estimated for 2 weeks by re-weighing water buckets and from water kinetics using the deuterium oxide dilution technique, respectively. In addition, dry matter intakes were directly measured and were significantly higher in sheep than in goats. The average daily water consumption by drinking differed significantly between the two species, with higher intakes in sheep than in goats. Total body water expressed as a percentage of body mass did not differ between species. Measurement methods of total water intake (TWI) using deuterium oxide dilution and re-weighing water buckets did not differ significantly in both species (P = 0.926). Results obtained for measured and estimated TWI confirm that the isotope dilution technique gives reliable results for estimates of water intake in sheep and goats. The higher amount of water intake in sheep was also reflected by their drinking behaviour. Sheep spent approximately 0.3% per 24-h drinking, while Boer goats spent only 0.1%. However, measured and estimated TWIs were only moderately correlated to the daily time spent drinking. The lower water intake found in Boer goats confirms a superior water management capacity compared with Black Head Mutton sheep even under temperate conditions.     

The effect of external gamma-irradiation on the distribution of deuterium oxide in the rat body

External gamma-irradiation (7.74 and 15.48 x 10(-2) C/kg) does not influence the distribution of deuterium oxide within a rat body: it is distributed uniformly among the organs and tissues as it is observed in nonirradiated animals. The effect of external irradiation favors the retention of deuterium oxide within the organs and tissues. The delayed excretion of deuterium oxide from the body can enhance the biological effect. This should be taken into account in standardizing the combined radiation effects.     

Nature of hydrogen transfer in soybean lipoxygenase 1: separation of primary and secondary isotope effects

Previous measurements of the kinetics of oxidation of linoleic acid by soybean lipoxygenase 1 have indicated very large deuterium isotope effects, but have not been able to distinguish the primary isotope effect from the alpha-secondary effect. To address this question, singly deuterated linoleic acid was prepared, and enantiomerically resolved using the enzyme itself. Noncompetitive measurements of the primary deuterium isotope effect give a value of ca. 40 which is temperature-independent. The enthalpy of activation is low and isotope-independent, and there is a large isotope effect on the Arrhenius prefactor. A very large apparent secondary isotope effect (ca. 2.1) is measured with deuterium in the primary position, but a greatly reduced value (1.1) is observed with protium in the primary position. Mutagenesis of the active site leads to a significant reduction in k(cat) and perturbed isotope effects, in particular, a secondary effect of 5.6 when deuterium is in the primary position. The anomalous secondary isotope effects are shown to arise from imperfect stereoselectivity of hydrogen abstraction which, for the mutant, is attributed to a combination of inverse substrate binding and increased flexibility at the reactive carbon. After correction, a very large primary (76-84) and small secondary (1.1-1.2) kinetic isotope effects are calculated for both mutant and wild-type enzymes. The weight of the evidence is taken to favor hydrogen tunneling as the primary mechanism of hydrogen transfer.     

Human erythrocyte glutathione reductase: chemical mechanism and structure of the transition state for hydride transfer

Kinetic parameters and primary deuterium kinetic isotope effects for NADH and five pyridine nucleotide substrates have been determined at pH 8.1 for human erythrocyte glutathione reductase. DV/KNADH and DV are equal to 1.4 and are pH independent below pH 8.1, but DV decreases to 1.0 at high pH as a group exhibiting a pK of 8.6 is deprotonated. This result suggests that as His-467' is deprotonated, the rate of the isotopically insensitive oxidative half-reaction is specifically decreased and becomes rate-limiting. For all substrates, equivalent V and V/K primary deuterium kinetic isotope effects are observed at pH values below 8.1. The primary deuterium kinetic isotope effect on V, but not V/K, is sensitive to solvent isotopic composition. The primary tritium kinetic isotope effects agree well with the corresponding value calculated from the primary deuterium kinetic isotope effects by using the Swain-Schaad relationship. This suggests that the primary deuterium kinetic isotope effects observed in these steady-state experiments are the intrinsic primary deuterium kinetic isotope effects for hydride transfer. The magnitude of the primary deuterium kinetic isotope effect is dependent on the redox potential of the pyridine nucleotide substrate used, varying from approximately 1.4 for NADH and -320 mV reductants to 2.7 for thioNADH to 4.2-4.8 for 3-acetylpyridine adenine dinucleotide (3APADH). The alpha-secondary tritium kinetic isotope effects also increase as the redox potential of the pyridine nucleotide substrate becomes more positive. Together, these data indicate that the transition state for hydride transfer is very early for NADH and becomes later for thioNADH and 3APADH, as predicted by Hammond's postulate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactation in the tammar wallaby (Macropus eugenii). I. Milk consumption and the algebraic description of the lactation curve

The milk intake of suckling tammar wallabies ( Macropus eugenii ) was estimated between 213 and 339 days of age using a double-isotope procedure in which tritiated water (TOH) was injected into the dams and deuterium oxide (D₂O) injected into the offspring. Recycling of isotopes between mothers and offspring was shown not to be a significant source of error. Estimates of body water (isotope dilution space) in the mothers or offspring, as a proportion of their body weight, were similar to values obtained with other macropodids and with placental mammals (0.73–0.75). Total water inflow in the offspring increased throughout the period, but the proportion of this contributed by milk fell in sigmoid fashion from 95.6% at 213 d to 3.7% at 340 d. Milk intakes reached a peak of about 86 ml/d, which was low compared with placental species. Before the peak, intakes were more closely related to body weight than age. Peak milk intake corresponded with the period of emergence from the pouch. Milk intakes were also in excellent agreement with published data obtained earlier in lactation, in animals drawn from the same population. These data were therefore combined to provide estimates of intake for the whole of lactation in the tammar wallaby, and algebraic models fitted to describe the pattern of milk intake in relation to body weight or age. With body weight, the pattern of milk intake was well described by a rising linear phase to a peak intake of about 86 ml/d near 1110 g body weight, followed by a gradual exponential decline. When related to age, the pattern was well described by a rising logistic phase (peak at 251 d) followed by a sharp exponential decline. It is shown that this latter pattern is markedly similar to the shape of the functions describing the energy requirements for the sum of pregnancy and lactation in a placental species, the sheep.