The relative merits of open- and closed-path analysers for measurement of eddy fluxes

Theoretical and practical aspects of measuring eddy fluxes of trace gases using open-and closed-path analysers are presented. Trace gas fluxes measured with an open-path analyser require the concurrent measurement of sensible and latent heat fluxes to correct for density fluctuations in trace gas concentration caused by these fluxes. A closed-path analyser eliminates the corrections due to sensible heat flux, but not for water vapour, provided temperature fluctuations are completely removed without significantly reducing fluctuations in the trace gas mixing ratio. Theory for the design of heat exchangers and for the attenuation of concentration fluctuations during air flow through tubes is used to provide design criteria for closed-path systems. Spectral transfer functions are used to estimate flux losses caused by flow through the sampling tube and gas analyser. Other factors considered include cross-sensitivity of infrared CO₂ analysers to water vapour, and deterioration of system performance caused by contaminants on the walls of sampling tubes. Of two open-path, infrared CO₂ analysers tested, one showed a strong interaction between CO₂ and water vapour, while the other showed little sensitivity to the presence of water vapour, other than caused by dilution. A commercial closed-path CO₂ analyser also showed little cross-sensitivity to water vapour. Compared to results for a clean sampling tube, the spectral bandwidth for water vapour fluctuations decreased significantly after several weeks of sampling. No such deterioration in bandwidth was observed for CO₂. These findings are attributed to differential adsorption/desorption of water vapour by dust or salt on the tubing walls. Rain and dust must be removed from open-path analysers to obtain satisfactory measurements. Careful system design and maintenance is required for both open- and closed-path systems to ensure satisfactory long-term measurement of trace gas fluxes. With these precautions, both approaches will provide satisfactory flux measurements.     

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.     

Rapid, automated analysis of 13C and 18O of CO2 in gas samples by continuous-flow, isotope ratio mass spectrometry.

A rapid automated method for isotopic analysis of 13C and 18O in CO2 has been developed. A variety of gas samples containing CO2 can be swept from serological tubes into a helium carrier flow; impurities are separated on a GC column so that a pure pulse of CO2 in He flows into the mass spectrometer. Isotopic ratio determinations are carried out as the pulse passes through the mass spectrometer, allowing a sample to be measured approximately every 4 min. A double, concentric needle-probe is used to flush the sample from the tube so that 100% sample recovery is achieved, maximizing sensitivity and preventing the possibility of fractionation. The precision of the technique, sigma(m-1), is better than 0.2% (0.0002 atom per cent excess) for 13C and 0.4% (0.83 p.p.m.) for 18O for 10 micromol of CO2 at natural abundance. Samples containing only atmospheric concentrations of CO2 can also be analyzed.     

Natural abundance deuterium and 18-oxygen effects on the precision of the doubly labeled water method

The doubly labeled water method for measuring total energy expenditure is subject to error from natural variations in the background 2H and 18O in body water. There is disagreement as to whether the variations in background abundances of the two stable isotopes covary and what relative doses of 2H and 18O minimize the impact of variation on the precision of the method. We have performed two studies to investigate the amount and covariance of the background variations. These were a study of urine collected weekly from eight subjects who remained in the Madison, WI locale for 6 wk and frequent urine samples from 14 subjects during round-trip travel to a locale > or = 500 miles from Madison, WI. Background variation in excess of analytical error was detected in six of the eight nontravelers, and covariance was demonstrated in four subjects. Background variation was detected in all 14 travelers, and covariance was demonstrated in 11 subjects. The median slopes of the regression lines of delta2H vs. delta18O were 6 and 7, respectively. Modeling indicated that 2H and 18O doses yielding a 6:1 ratio of final enrichments should minimize this error introduced to the doubly labeled water method.     

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.     

Novel application of the "doubly labeled" water method: measuring CO2 production and the tissue-specific dynamics of lipid and protein in vivo

The partitioning of whole body carbon flux between fat and lean compartments affects body composition. We hypothesized that it is possible to simultaneously determine whole body carbon (energy) balance and the dynamics of lipids and proteins in specific tissues in vivo. Growing C57BL/6J mice fed a high-fat low-carbohydrate diet were injected with a bolus of "doubly labeled" water (i.e., (2)H2O and H2(18)O). The rate of CO2 production was determined from the difference between the elimination rates of 2H and 18O from body water. The rates of synthesis and degradation of triglycerides extracted from epididymal fat pads and of proteins extracted from heart muscle were determined by mathematically modeling the 2H labeling of triglyceride-bound glycerol and protein-bound alanine, respectively. We found that mice were in positive carbon balance (approximately 20% retention per day) and accumulated lipid in epididymal fat pads (approximately 9 micromol triglyceride accumulated per day). This is consistent with the fact that mice were studied during a period of growth. Modeling the 2H labeling of triglycerides revealed a substantial rate of lipid breakdown during this anabolic state (equivalent to approximately 25% of the newly synthesized triglyceride). We found equal rates of protein synthesis and breakdown in heart muscle (approximately 10% of the pool per day), consistent with the fact that the heart muscle mass did not change. In total, these findings demonstrate a novel application of the doubly labeled water method. Utilization of this approach, especially in unique rodent models, should facilitate studies aimed at quantifying the efficacy of interventions that modulate whole body carbon balance and lipid flux while in parallel determining their impact on (cardiac) muscle protein turnover. Last, the simplicity of administering doubly labeled water and collecting samples allows this method to be used in virtually any laboratory setting.     

Hydrogen and Oxygen Stable Isotope Fractionation in Body Fluid Compartments of Dairy Cattle According to Season,Farm, Breed,and Reproductive Stage

Environmental temperature affects water turnover and isotope fractionation by causing water evaporation from the body in mammals. This may lead to rearrangement of the water stable isotope equilibrium in body fluids. We propose an approach to detect possible variations in the isotope ratio in different body fluids on the basis of different homoeothermic adaptations in varying reproductive stages. Three different reproductive stages (pregnant heifer, primiparous lactating cow, and pluriparous lactating cow) of two dairy cattle breeds (Italian Friesian and Modenese) were studied in winter and summer. Blood plasma, urine, faecal water, and milk were sampled and the isotope ratios of H (²H/¹H) and O (¹⁸O/¹⁶O) were determined. Deuterium excess and isotope-fractionation factors were calculated for each passage from plasma to faeces, urine and milk. The effects of the season, reproductive stages and breed on δ²H and δ¹⁸O were significant in all the fluids, with few exceptions. Deuterium excess was affected by season in all the analysed fluids. The correlations between water isotope measurements in bovine body fluids ranged between 0.6936 (urine-milk) and 0.7848 (urine-plasma) for δ²H, and between 0.8705 (urine-milk) and 0.9602 (plasma-milk) for δ¹⁸O. The increase in both isotopic δ values in all body fluids during summer is representative of a condition in which fractionation took place as a consequence of a different ratio between ingested and excreted water, which leads to an increased presence of the heavy isotopes. The different body water turnover between adult lactating cattle and non-lactating heifers was confirmed by the higher isotopic δ for the latter, with a shift in the isotopic equilibrium towards values more distant from those of drinking water.     

Use of the doubly labeled water method for measurement of energy expenditure, total body water, water intake, and metabolizable energy intake in humans and small animals

The basis of the doubly labeled water method is measurement of the differential rates of disappearance of two isotopes of water (H2 18O and either 2H2O or 3H2O, administered at the start of the study) from body water. Published studies indicate that, in its current forms, this technique can be used to provide accurate and reasonably precise information on carbon dioxide production, total body water, and water intake in free-living humans and many small animals. Total energy expenditure can be calculated from carbon dioxide production with little loss of precision. Metabolizable energy intake can also be predicted, as the sum of total energy expenditure plus an estimate for the change in body energy stores during the measurement, but this prediction is unlikely to be accurate and precise unless the subject is in approximate energy balance.     

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%.     

Dietary and physiological controls on the hydrogen and oxygen isotope ratios of hair from mid‐20th century indigenous populations

A semimechanistic model has recently been proposed to explain observed correlations between the H and O isotopic composition of hair from modern residents of the USA and the isotopic composition of drinking water, but the applicability of this model to hair from non‐USA and preglobalization populations is unknown. Here we test the model against data from hair samples collected during the 1930s–1950s from populations of five continents. Although C and N isotopes confirm that the samples represent a much larger range of dietary “space” than the modern USA residents, the model is able to reproduce the observed δ²H and δ¹⁸O values given reasonable adjustments to 2 model parameters: the fraction of dietary intake derived from locally produced foods and the fraction of keratin H fixed during the in vivo synthesis of amino acids. The model is most sensitive to the local dietary intake, which appears to constitute between 60% and 80% of diet among the groups sampled. The isotopic data are consistent with a trophic‐level effect on protein H isotopes, which we suggest primarily reflects mixing of ²H‐enriched water and ²H‐depleted food H in the body rather than fractionation during biosynthesis. Samples from Inuit groups suggest that humans with marine‐dominated diets can be identified on the basis of coupled δ²H and δ¹⁸O values of hair. These results indicate a dual role for H and O isotopic measurements of keratin, including both biological (diet, physiology) and environmental (geographic movement, paleoclimate) reconstruction. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Contributions of evaporation, isotopic non-steady state transpiration and atmospheric mixing on the delta18O of water vapour in Pacific Northwest coniferous forests

Changes in the ²H and ¹⁸O of atmospheric water vapour provide information for integrating aspects of gas exchange within forest canopies. In this study, we show that diurnal fluctuations in the oxygen isotope ratio (δ¹⁸O) as high as 4‰ were observed for water vapour (δ¹⁸O_vp) above and within an old‐growth coniferous forest in the Pacific Northwest region of the United States. Values of δ¹⁸O_vp decreased in the morning, reached a minimum at midday, and recovered to early‐morning values in the late afternoon, creating a nearly symmetrical diurnal pattern for two consecutive summer days. A mass balance budget was derived and assessed for the ¹⁸O of canopy water vapour over a 2‐d period by considering the ¹⁸O‐isoflux of canopy transpiration, soil evaporation and the air entering the canopy column. The budget was used to address two questions: (1) do δ¹⁸O values of canopy water vapour reflect the biospheric influence, or are such signals swamped by atmospheric mixing? and (2) what mechanisms drive temporal variations of δ¹⁸O_vp? Model calculations show that the entry of air into the canopy column resulted in an isotopically depleted ¹⁸O‐isoflux in the morning of day 1, causing values of δ¹⁸O_vp to decrease. An isotopically enriched ¹⁸O‐isoflux resulting from transpiration then offset this decreased δ¹⁸O_vp later during the day. Contributions of ¹⁸O‐isoflux from soil evaporation were relatively small on day 1 but were more significant on day 2, despite the small H₂¹⁶O fluxes. From measurements of leaf water volume and sapflux, we determined the turnover time of leaf water in the needles of Douglas‐fir trees as ≈ 11 h at midday. Such an extended turnover time suggests that transpiration may not have occurred at the commonly assumed isotopic steady state. We tested a non‐steady state model for predicting δ¹⁸O of leaf water. Our model calculations show that assuming isotopic steady state increased isoflux of transpiration. The impact of this increase on the modelled δ ¹⁸O_vp was clearly detectable, suggesting the importance of considering isotopic non‐steady state of transpiration in studies of forest ¹⁸O water balance.     

Isotope recycling in lactating dogs (Canis familiaris)

Isotope-based techniques for the measurement of water turnover, energy expenditure, and milk intake often assume that there is no recycling of isotopes once they have left the labeled animal. In experiments involving lactating females or their suckling offspring, there are several possible routes of isotope recycling. These include the consumption of labeled milk by offspring, the ingestion of labeled excreta, and the rebreathing of exhaled labeled CO(2) or water vapor by both mother and offspring. Isotope recycling might be especially important during lactation because the offspring are in close contact with each other and their mother for prolonged periods. We show here in 24- to 30-day-old domestic dog Canis familiaris puppies that there was no detectable transfer of (18)O or (2)H from labeled to unlabeled pups in two litters (16 pups, 8 labeled, 8 unlabeled) that were weaned early and independent of their mother. However, there was a significant transfer of both isotopes from labeled to unlabeled pups and from labeled pups to their mothers in nine equivalent nursing litters of the same age (27 labeled, 26 unlabeled pups). The increases in enrichment of isotopes in unlabeled offspring were greater than the increases in enrichment of the mothers. This indicates that maternal ingestion of offspring excreta and subsequent transfer of isotope in milk is not the sole pathway of recycling. Additional routes must also be important, such as exchange of isotope between pups on saliva-coated nipples and perhaps direct ingestion of excreta by unweaned young. Recycling is unlikely to be an important factor when determining maternal metabolic rate during peak lactation in domestic dogs. However, experiments that are designed to assess the energy demands of pups and isotope-based estimates of water turnover in offspring may need to take into account any effects of isotope recycling. In a theoretical example, removing the effects of recycling increased the measured energy expenditure in pups by up to 7% and increased the calculated elimination rates of both isotopes by up to 11.1% in (18)oxygen and 10.9% in (2)hydrogen.     

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.     

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 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.     

Soil water uptake by trees using water stable isotopes (δ2H and δ18O)−a method test regarding soil moisture,texture and carbonate

Aims

Stable isotopes of oxygen and hydrogen are often used to determine plant water uptake depths. We investigated whether and to what extend soil moisture, clay content, and soil calcium carbonate influences the water isotopic composition.

Methods

In the laboratory, dried soil samples varying in clay content were rewetted with different amounts of water of known isotopic composition. Further, we removed soil carbonate from a subset of samples prior to rewetting. Water was extracted from samples via cryogenic vacuum extraction and analysed by mass spectrometry.

Results

The isotopic composition of extracted soil water was similarly depleted in both ¹⁸O and ²H with decreasing soil moisture and increasing clay and carbonate content. Soil carbonate changed the δ¹⁸O composition while δ²H was not affected.

Conclusions

Our results indicate that soil carbonate can cause artifacts for ¹⁸O isotopic composition of soil water. At low soil moisture and high carbonate content this could lead to conflicting results for δ¹⁸O and δ²H in plant water uptake studies.     

Accumulation of deuterium oxide in body fluids after ingestion of D2O-labeled beverages

A simple low-cost procedure was developed to compare the temporal profiles of deuterium oxide (D2O) accumulation in body fluids after ingestion of D2O-labeled solutions. D2O concentration was measured in plasma and saliva samples taken at various intervals after ingestion of 20 ml of D2O mixed with five solutions differing in carbohydrate and electrolyte concentrations. An infrared spectrometer was used to measure D2O in purified samples obtained after a 48-h incubation period during which the water (D2O and H2O) in the sample was equilibrated with an equal volume of distilled water in a sealed diffusion dish. The procedure yields 100% recoveries of 60-500 ppm D2O with an average precision of 5%. When compared with values for distilled water, D2O accumulation in serial samples of plasma and saliva was slower for ingested solutions containing 40 and 15% glucose and faster for hypotonic saline and a 6% carbohydrate-electrolyte solution. These differences appear to reflect known differences in gastric emptying and intestinal absorption of these beverages. Therefore this technique may provide a useful index of the rate of water uptake from ingested beverages into the body fluids.     

Proton transfer within the active-site cavity of carbonic anhydrase III

The maximal turnover rate of CO2 hydration catalyzed by the carbonic anhydrases is limited by proton transfer steps from the zinc-bound water to solution, steps that regenerate the catalytically active zinc-bound hydroxide. Catalysis of CO2 hydration by wild-type human carbonic anhydrase III (HCA III) (k(cat) = 2 ms (-1)) is the least efficient among the carbonic anhydrases in its class, in part because it lacks an efficient proton shuttle residue. We have used site-directed mutagenesis to test positions within the active-site cavity of HCA III for their ability to carry out proton transfer by replacing various residues with histidine. Catalysis by wild-type HCA III and these six variants was determined from the initial velocity of hydration of CO2 measured by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and H2O at chemical equilibrium by mass spectrometry. The results show that histidine at three positions (Lys64His, Arg67His and Phe131His) have the capacity to transfer protons during catalysis, enhancing maximal velocity of CO2 hydration and 18O exchange from 4- to 15-fold compared with wild-type HCA III. Histidine residues at the other three positions (Trp5His, Tyr7His, Phe20His) showed no firm evidence for proton transfer. These results are discussed in terms of the stereochemistry of the active-site cavity and possible proton transfer pathways.     

Impact of 2H and 18O Pool Size Determinations on the Calculation of Total Energy Expenditure

Measurement of total energy expenditure using [²H,¹⁸O]water requires both accurate and precise determination of the rates of disappearance of ²H and ¹⁸O from body water over time and determination of the ²H and ¹⁸O pool sizes. However, the impact of the isotopic determination of body water upon the determination of energy expenditure is often overlooked. For measurement of total body water per se, the delay after administration before sampling body fluids becomes important, and saliva sampling can be used to resolve the timing of early samples for body water determination. For energy expenditure measurement per se, linear regression can be used to define the initial dilution. Because the hydrogen tracer dilutes into a pool significantly larger than body water pool per se due to the presence of labile hydrogens, a correction to the isotope pool size must be applied. The theoretical calculations of the exchangeable hydrogen pool presented here suggest that the hydrogen pool size is <3% greater than the body water pool and data are provided to support this idea. Finally, the two approaches used to define the body water pool space contribution to the calculation of energy expenditure using ²H₂¹⁸O are reviewed. Using a pool size based upon the average of the two pool spaces limits the effect of pool size error in the calculation of energy expenditure.     

Internal conductance to CO(2) diffusion and C(18)OO discrimination in C(3) leaves

(18)O discrimination in CO(2) stems from the oxygen exchange between (18)O-enriched water and CO(2) in the chloroplast, a process catalyzed by carbonic anhydrase (CA). A proportion of this (18)O-labeled CO(2) escapes back to the atmosphere, resulting in an effective discrimination against C(18)OO during photosynthesis (Delta(18)O). By constraining the delta(18)O of chloroplast water (delta(e)) by analysis of transpired water and the extent of CO(2)-H(2)O isotopic equilibrium (theta(eq)) by measurements of CA activity (theta(eq) = 0.75-1.0 for tobacco, soybean, and oak), we could apply measured Delta(18)O in a leaf cuvette attached to a mass spectrometer to derive the CO(2) concentration at the physical limit of CA activity, i.e. the chloroplast surface (c(cs)). From the CO(2) drawdown sequence between stomatal cavities from gas exchange (c(i)), from Delta(18)O (c(cs)), and at Rubisco sites from Delta(13)C (c(c)), the internal CO(2) conductance (g(i)) was partitioned into cell wall (g(w)) and chloroplast (g(ch)) components. The results indicated that g(ch) is variable (0.42-1.13 mol m(-2) s(-1)) and proportional to CA activity. We suggest that the influence of CA activity on the CO(2) assimilation rate should be important mainly in plants with low internal conductances.