A field portable gas-exchange system for measuring carbon dioxide and water vapour exchange rates of leaves during fumigation with SO2

Abstract A field portable, steady-state gas-exchange system which measures both CO₂ and water vapour exchange of single intact leaves during fumigations with SO₂ is described. Within the leaf cuvette temperature, light, humidity and both CO₂ and SO₂ concentrations are controlled to preset levels. Gas flow and concentrations are controlled by mass flow controllers. Photosynthetic uptake of CO₂ can be determined either by differential depletion or null balance measurement. Water vapour exchange is measured differentially and transpiration and conductance to water vapour determined. Sulphur dioxide is measured directly within the cuvette exhaust gas line by UV-pulse fluorescence. The performance of this system under field conditions is described and the physiological measurements compared with those obtained with other systems.     

High CO2 levels impair alveolar epithelial function independently of pH

Background

In patients with acute respiratory failure, gas exchange is impaired due to the accumulation of fluid in the lung airspaces. This life-threatening syndrome is treated with mechanical ventilation, which is adjusted to maintain gas exchange, but can be associated with the accumulation of carbon dioxide in the lung. Carbon dioxide (CO₂) is a by-product of cellular energy utilization and its elimination is affected via alveolar epithelial cells. Signaling pathways sensitive to changes in CO₂ levels were described in plants and neuronal mammalian cells. However, it has not been fully elucidated whether non-neuronal cells sense and respond to CO₂. The Na,K-ATPase consumes ∼40% of the cellular metabolism to maintain cell homeostasis. Our study examines the effects of increased pCO₂ on the epithelial Na,K-ATPase a major contributor to alveolar fluid reabsorption which is a marker of alveolar epithelial function.

Principal Findings

We found that short-term increases in pCO₂ impaired alveolar fluid reabsorption in rats. Also, we provide evidence that non-excitable, alveolar epithelial cells sense and respond to high levels of CO₂, independently of extracellular and intracellular pH, by inhibiting Na,K-ATPase function, via activation of PKCζ which phosphorylates the Na,K-ATPase, causing it to endocytose from the plasma membrane into intracellular pools.

Conclusions

Our data suggest that alveolar epithelial cells, through which CO₂ is eliminated in mammals, are highly sensitive to hypercapnia. Elevated CO₂ levels impair alveolar epithelial function, independently of pH, which is relevant in patients with lung diseases and altered alveolar gas exchange.     

A Mathematical Model of the Human Respiratory Control System

The respiratory system exhibits the properties of a control system of the regulator type. Equations describing this biological control system have been derived. Transient and steady-state solutions for various CO₂ and O₂ step input disturbances were obtained utilizing a digital computer and are compared with experimental results. The effectiveness of the respiratory system as a regulator is investigated. Further extensions of the model are suggested.     

Cyclic model of respiration applied to asymmetrical ventilation and periodic breathing

A model taking into account the cyclic character of respiration in humans is developed using two classical simplifications: CO₂ is the only respiratory gas involved; and respiration is regulated only by a CO₂ linear controller. The model is used to investigate two important clinical aspects of respiratory disease: asymmetrical ventilation and periodic breathing. We show that asymmetry in ventilation significantly influences the time course of the CO₂ partial pressure in the expired alveolar air at the mouth and the elimination of CO₂ through the lungs. Furthermore, the CO₂ controller delay plays a major role in periodic breathing.     

Non-invasive tracking of peripheral ventilatory response to carbon dioxide

We propose a new method for quantifying the ventilatory sensitivity of the peripheral chemoreceptors to changes in CO₂ through analysis of the natural breath-to-breath variations in ventilation (y) and alveolar Pco₂ (x). This technique is truly non-invasive in that the need for administering CO₂-enriched mixtures is circumvented, and peripheral chemosensitivity is assessed while the respiratory control system operates in its normal eucapnic state, unperturbed by external interventions. The method is based on solution of the inverse problem relating the cross-correlation between alveolar Pco₂ and ventilation changes, and the autocorrelation of changes in alveolar Pco₂. Tests are performed using simulated data generated by a closed-loop respiratory control model. The impulse response of the controller and the convective delay between lungs and chemoreceptors are estimated from the data. Subsequently, the best exponential fit to the estimated impulse response yields values for the effective controller gain (G) and the associated time constant of the response. The estimates of G contain a small contribution from the control chemoreflex gain; however, the relation between changes in G and changes in peripheral gain remains unaltered. The effects of other details in the computation procedure, such as length of data sequence, maximum number of correlation lags and starting lag number, are also investigated.     

A Mathematical Model of the Controlled Plant of the Réspiratory System

Ability to predict the dynamic response of oxygen, carbon dioxide tensions, and pH in blood and tissues to abrupt changes in ventilation is important in the mathematical modeling of the respiratory system. In this study, the controlled plant (the amount and distribution of O₂ and CO₂) of the respiratory system is modeled. Although the body tissues are divided into a finite number of “compartments” (three tissue groups), in contrast to earlier models, the blood and tissue gas tensions within each compartment are considered to be continuously distributed in time and in one spatial coordinate. The mass conservation equations for oxygen and carbon dioxide involved in the blood-tissue gas exchange are described by a set of partial differential equations which take into account convection of O₂ and CO₂ caused by the flow of blood as well as diffusion due to local tension gradients. Nonlinear algebraic equations for the dissociation curves, which take into account the Haldane and Bohr effects in blood, are used to obtain the relationships between concentrations and partial pressures. Time-variable delays caused by the arterial and venous transport of the respiratory gases are also included. The model so constructed successfully reproduced actual O₂ and CO₂ tensions in arterial blood, and in muscle venous and mixed venous blood when ventilation was abruptly changed.     

Respiratory control in bullfrogs: Cutaneous versus pulmonary response to selective CO2 exposure

Summary Bullfrogs,Rana catesbeiana, were exposed to high ambientP _Co2 (15–22 Torr) at 20°C through either their skin or their lungs. The objective was to evaluate the effectiveness of the gas exchange surface not exposed to high CO₂ to excrete the excess CO₂ load. Frogs exposed to high CO₂ through their skin increased pulmonary ventilation and controlled arterialP _CO2, close to the normal value. In contrast, frogs that breathed CO₂ did not increase their skin CO₂ conductance, and arterialP _CO2, increased significantly. These results support the concept that the lungs are the primary effector mechanism for respiratory control in the bullforg while the skin is a passive, poorly controlled avenue for CO₂ loss. They also reveal that the lungs of this anuran are affective eliminators of CO₂.     

Nonequilibrium Steady-State Differences in Partial Pressure of CO2 and in Concentration of Weak Acids and Bases between Blood and Tissue

Several investigators have demonstrated that under conditions where little or no gas exchange occurs across the alveolar capillary membrane the PCO₂ is higher in the alveolus than in the mixed venous blood, whereas there are no PO₂ differences. Gurtner et al. have explained the ΔPCO₂ by a model in which H⁺ dissociation of proteins due to an electrical field caused by a negatively charged capillary wall (Wien effect) sets up an intracapillary PCO₂ difference between wall and bulk phase which is maintained by blood flow. The model is not specific for CO₂ and predicts that weak acids should be concentrated in a manner similar to CO₂ whereas weak bases should be relatively excluded from the alveolar space. Measurements of the steady-state distribution of the uncharged forms of the weak acids 5,5-dimethyloxyazoladinedione (DMO) and barbital and of the weak base tris(hydroxymethyl)aminomethane (THAM) between mixed venous blood and a fluid-filled lobe of lung were made in living dogs. The results agree fairly well with the predicted values.     

Non-invasive technique for examining the respiratory proprioceptor system in man

Our technique enables non-invasive experiments to be conducted on the proprioceptor part of respiratory control, while eliminating misleading responses due to interaction with the chemoreceptor system; interaction was prevented by stabilizing arterial P_O2 and P_CO2 with the aid of an optimal regulator based on a mini-computer which controlled the inspired gas mixture. The proprioceptor system in a human was disturbed by applying positive pressure pulses at the mouth, responses were derived from continuous air-flow measurement. The classical inflation inhibiting reflex and an effect akin to Head's paradoxical reflex were demonstrated.     

Membrane oxygenators: current developments in design and application

Cardiopulmonary bypass (CPB) procedures require a blood-gas exchanger (oxygenator) to temporarily replace the respiratory function of the lungs. In the past the majority of CPB procedures have been carried out with bubble oxygenators which effect gas exchange by dispersion of bubbles into the blood. Membrane oxygenators, on the other hand, utilize a hydrophobic gas permeable membrane between the blood and gas phases.Bubble oxygenators are being superseded by membrane types for CPB due to improvements in membrane technology and mass transfer efficiency. These advances are reviewed in this paper and are illustrated by reference to the gas exchange and operating characteristics of a number of clinical oxygenators designed for adult CPB.Membrane oxygenatorsare also being used for long-term support in the treatment of acute respiratory failure. Operated in a partial bypass circuit, the oxygenator may have to function for several days or weeks. In one particular treatment method, the rate of spontaneous breathing is controlled by the partial or total removal of the metabolic CO₂ production by the membrane oxygenator. For this method, known as extracorporeal CO₂ removal (ECCO₂R), the oxygenator must be optimized for CO₂ transfer at low blood flow rates. The suitability of clinical oxygenators for ECCO₂R is discussed in terms of gas exchange and functionality over a prolonged operation.     

C3 photosynthesis in silico

A computer model comprising light reactions, electron–proton transport, enzymatic reactions, and regulatory functions of C₃ photosynthesis has been developed as a system of differential budget equations for intermediate compounds. The emphasis is on electron transport through PSII and PSI and on the modeling of Chl fluorescence and 810 nm absorptance signals. Non-photochemical quenching of PSII excitation is controlled by lumenal pH. Alternative electron transport is modeled as the Mehler type O₂ reduction plus the malate-oxaloacetate shuttle based on the chloroplast malate dehydrogenase. Carbon reduction enzymes are redox-controlled by the ferredoxin–thioredoxin system, sucrose synthesis is controlled by the fructose 2,6-bisphosphate inhibition of cytosolic FBPase, and starch synthesis is controlled by ADP-glucose pyrophosphorylase. Photorespiratory glycolate pathway is included in an integrated way, sufficient to reproduce steady-state rates of photorespiration. Rate-equations are designed on principles of multisubstrate-multiproduct enzyme kinetics. The parameters of the model were adopted from literature or were estimated from fitting the photosynthetic rate and pool sizes to experimental data. The model provided good simulations for steady-state photosynthesis, Chl fluorescence, and 810 nm transmittance signals under varying light, CO₂ and O₂ concentrations, as well as for the transients of post-illumination CO₂ uptake, Chl fluorescence induction and the 810 nm signal. The modeling shows that the present understanding of photosynthesis incorporated in the model is basically correct, but still insufficient to reproduce the dark-light induction of photosynthesis, the time kinetics of non-photochemical quenching, ‘photosynthetic control’ of plastoquinone oxidation, cyclic electron flow around PSI, oscillations in photosynthesis. The model may find application for predicting the results of gene transformations, the analysis of kinetic experimental data, the training of students.     

Optimization behavior of brainstem respiratory neurons

A recent model of respiratory control suggested that the steady-state respiratory responses to CO₂ and exercise may be governed by an optimal control law in the brainstem respiratory neurons. It was not certain, however, whether such complex optimization behavior could be accomplished by a realistic biological neural network. To test this hypothesis, we developed a hybrid computer-neural model in which the dynamics of the lung, brain and other tissue compartments were simulated on a digital computer. Mimicking the controller was a human subject who pedalled on a bicycle with varying speed (analog of ventilatory output) with a view to minimize an analog signal of the total cost of breathing (chemical and mechanical) which was computed interactively and displayed on an oscilloscope. In this manner, the visuomotor cortex served as a proxy (homolog) of the brainstem respiratory neurons in the model. Results in 4 subjects showed a linear steady-state ventilatory CO₂ response to arterial PCO₂ during simulated CO₂ inhalation and a nearly isocapnic steady-state response during simulated exercise. Thus, neural optimization is a plausible mechanism for respiratory control during exercise and can be achieved by a neural network with cognitive computational ability without the need for an exercise stimulus.     

An estimation of CO<Subscript>2</Subscript> fixation capacity in mangrove forest using two methods of CO<Subscript>2</Subscript> gas exchange and growth curve analysis

In many coastal areas of South-East Asia, attempts have been made to revive coastal ecosystem by initiating projects that encourage planting of mangrove trees. Compared to the terrestrial trees, mangrove trees possess a higher carbon fixation capacity. It becomes a very significant option for clean development mechanism (CDM) program. However, a reliable method to estimate CO₂ fixation capacity of mangrove trees has not been established. Acknowledging the above fact, we decided to set up an estimation method for the CDM program, using gas exchange analysis to estimate mangrove productivity, we put into consideration the net CO₂ fixation of reforested Kandelia candel (5-, 10-, and 15-year-old stand). This was estimated by gas exchange analysis and growth curve analysis. In growth curve analysis, we drew a growth curve of a single stand using data of above- and below-ground biomass. In the gas exchange analysis, we calculated CO₂ fixation capacity by (1) measuring respiration rate of each organ of stand and calculating respiratory CO₂ emission from above- to below-ground biomass. (2) Measuring the single-leaf photosynthetic rate in response to light intensity and calculating the photosynthetic CO₂ absorption. (3) We also developed a model for the diurnal changes in temperature, and monthly averages based on one-day estimation of CO₂ absorption and emission, which we corrected by this model in order to estimate the net CO₂ fixation capacity in response to temperature. Comparing the biomass accumulation of the two methods constructed for the same forest, the above-ground biomass accumulation of 10-year-old forest (34.3 ton ha⁻¹ yr⁻¹) estimated by gas exchange analysis was closely compared to those of growth curve analysis (26.6 ton ha⁻¹ yr⁻¹), suggesting that the gas exchange analysis was capable of estimating mangrove productivity. The validity of the estimated CO₂ fixation capacity by the gas exchange analysis and the growth curve analysis was also discussed.     

Effect of Mechanical Loading on Ventilatory Response to CO2 and CO2 Excretion

The ventilatory response to CO₂ (S) and respiratory exchange ratio have been measured in 10 healthy subjects breathing naturally and through added resistive loads. The changes in these values produced by the added loads were shown to be correlated with the unloaded CO₂ responsiveness. The results indicated that poorly responsive individuals had a greater depression of ventilatory response to CO₂ and were more liable to retain CO₂.These observations raise the possibility that the constitutional CO₂ responsiveness of an individual influences the alveolar ventilation achieved in the presence of airways obstruction. The propensity to develop respiratory failure may thus be conditioned by the premorbid CO₂ responsiveness.     

Effect of exercise-induced hyperventilation on airway resistance and cycling endurance

The purpose of the present study was to investigate the effect of exercise induced hyperventilation and hypocapnia on airway resistance (R _aw), and to try to answer the question whether a reduction of R _aw is a mechanism contributing to the increase of endurance time associated with a reduction of exercise induced hyperventilation as for example has been observed after respiratory training. Eight healthy volunteers of both sexes participated in the study. Cycling endurance tests (CET) at 223 (SD 47) W, i.e. at 74 (SD 5)% of the subject's peak exercise intensity, breathing endurance tests and body plethysmograph measurements of pre- and postexercise R _aw were carried out before and after a 4-week period of respiratory training. In one of the two CET before the respiratory training CO₂ was added to the inspired air to keep its end-tidal concentration at 5.4% to avoid hyperventilatory hypocapnia (CO₂-test); the other test was the control. The pre-exercise values of specific expiratory R _aw were 8.1 (SD 2.8), 6.8 (SD 2.6) and 8.0 (SD 2.1) cm H₂O · s and the postexercise values were 8.5 (SD 2.6), 7.4 (SD 1.9) and 8.0 (SD 2.7) cm H₂O · s for control CET, CO₂-CET and CET after respiratory training, respectively, all differences between these tests being nonsignificant. The respiratory training significantly increased the respiratory endurance time during breathing of 70% of maximal voluntary ventilation from 5.8 (SD 2.9) min to 26.7 (SD 12.5) min. Mean values of the cycling endurance time (t _cend) were 22.7 (SD 6.5) min in the control, 19.4 (SD 5.4) min in the CO₂-test and 18.4 (SD 6.0) min after respiratory training. Mean values of ventilation ( _E) during the last 3␣min of CET were 123 (SD 35.8) l · min⁻¹ in the control, 133.5 (SD 35.1) l · min⁻¹ in the CO₂-test and 130.9 (SD 29.1) l · min⁻¹ after respiratory training. In fact, six subjects ventilated more and cycled for a shorter time, whereas two subjects ventilated less and cycled for a longer time after the respiratory training than in the control CET. In general, the subjects cycled longer the lower the _E, if all three CET are compared. It is concluded that R _aw measured immediately after exercise is independent of exercise-induced hyperventilation and hypocapnia and is probably not involved in limiting t _cend, and that t _cend at a given exercise intensity is shorter when _E is higher, no matter whether the higher _E occurs before or after respiratory training or after CO₂ inhalation. Accepted: 11 September 1996     

Simultaneous flue gas bioremediation and reduction of microalgal biomass production costs

A flue gas originating from a municipal waste incinerator was used as a source of CO₂ for the cultivation of the microalga Chlorella vulgaris, in order to decrease the biomass production costs and to bioremediate CO₂ simultaneously. The utilization of the flue gas containing 10–13% (v/v) CO₂ and 8–10% (v/v) O₂ for the photobioreactor agitation and CO₂ supply was proven to be convenient. The growth rate of algal cultures on the flue gas was even higher when compared with the control culture supplied by a mixture of pure CO₂ and air (11% (v/v) CO₂). Correspondingly, the CO₂ fixation rate was also higher when using the flue gas (4.4 g CO₂ l⁻¹ 24 h⁻¹) than using the control gas (3.0 g CO₂ l⁻¹ 24 h⁻¹). The toxicological analysis of the biomass produced using untreated flue gas showed only a slight excess of mercury while all the other compounds (other heavy metals, polycyclic aromatic hydrocarbons, polychlorinated dibenzodioxins and dibenzofurans, and polychlorinated biphenyls) were below the limits required by the European Union foodstuff legislation. Fortunately, extending the flue gas treatment prior to the cultivation unit by a simple granulated activated carbon column led to an efficient absorption of gaseous mercury and to the algal biomass composition compliant with all the foodstuff legislation requirements.     

Compliance of the respiratory system in newborn and adult rats after gestation in hypoxia

We hypothesized that hypoxia during gestation modifies the compliance of the respiratory system of newborn and adult rats. Pregnant rats were placed in a hypobaric chamber at an inspired oxygen pressure of 86 mmHg (equivalent to 12% O₂ in normobaria) from day 4 of gestation until day 2 post-partum. Three- day-old rat pups were smaller than controls, with higher hematocrit; the lungs were also small, with less protein and DNA content. The pressure (x-axis)-volume (y-axis) curve of the respiratory system was displaced to the right of the control curve, and the compliance of the respiratory system, measured on the inflation or deflation limb of the pressure-volume curve, was decreased by ∼20–25%, depending upon the normalization procedure (per body mass or per dry lung weight). In 50-day-old rats exposed to hypoxia during gestation, body weight, hematocrit, lung mass and DNA content were normal; the compliance of the respiratory system, measured at ventilation frequencies between 20 cpm and 100 cpm, was higher than in controls by ∼20%. It is concluded that the effects of prenatal hypoxia on the compliance of the respiratory system can vary with age. In the rat the process of alveolar formation initiates postnatally. Hence, in the newborn the effects of the prenatal hypoxia on the compliance of the respiratory system are likely to be dominated by the hypoxic pulmonary hypoplasia and hypertension, which decrease the compliance of the respiratory system. In the adult, the effects of the decreased alveolar formation are the prevailing ones, increasing the compliance of the respiratory system. Accepted: 3 January 2000     

Evolutionary Conserved Role of c-Jun-N-Terminal Kinase in CO2-Induced Epithelial Dysfunction

Elevated CO₂ levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO₂ signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO₂ levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO₂ levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO₂-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO₂ signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO₂ signaling in mammals, diptera and nematodes.     

Evaluating the efficiency of enzyme accelerated CO2 capture: chemical kinetics modelling for interpreting measurement results

In this paper, the efficiency of the carbonic anhydrase (CA) enzyme in accelerating the hydration of CO₂ is evaluated using a measurement system which consists of a vessel in which a gaseous flow of mixtures of nitrogen and CO₂ is bubbled into water or water solutions containing a known quantity of CA enzyme. The pH value of the solution and the CO₂ concentration at the measurement system gas exhaust are continuously monitored. The measured CO₂ level allows for assessing the quantity of CO₂, which, subtracted from the gaseous phase, is dissolved into the liquid phase and/or hydrated to bicarbonate. The measurement procedure consists of inducing a transient and observing and modelling the different kinetics involved in the steady-state recovery with and without CA. The main contribution of this work is exploiting dynamical system theory and chemical kinetics modelling for interpreting measurement results for characterising the activity of CA enzymes. The data for model fitting are obtained from a standard bioreactor, in principle equal to standard two-phase bioreactors described in the literature, in which two different techniques can be used to move the process itself away from the steady-state, inducing transients.     

A field portable system for the measurement of gas exchange of leaves under natural and controlled conditions: examples with field-grown Eucalyptus pauciflora Sieb. ex Spreng. ssp. pauciflora, E. behriana F. Muell. and Pinus radiata R. Don.

Abstract A field portable system is described which measures the response of gas exchange of one leaf to changes in environmental parameters under controlled conditions, and which simultaneously measures the gas exchange of another leaf as the climatic parameters vary naturally. The system consists of two independently operating cuvettes. It enables detailed studies of photosynthesis and stomata/transpiration of leaves attached to the plant in their natural position. It provides control of temperature, humidity, CO₂ and oxygen concentration (or, alternatively, of other gases) as well as of light. Infrared gas analyzers for CO₂ and H₂O are used which allow similar time constants for the measurement of the two gases. Examples of a diurnal course of gas exchange of a leaf in its natural exposition and of experiments with steady-state responses of gas exchange are presented. In Eucalyptus pauciflora Sieb. ex Spreng. ssp. pauciflora, a set of response curves of CO, assimilation (A) to CO₂, as measured at various leaf temperatures and light levels, shows carboxylation efficiency to be light saturated at the lower photon irradiances the lower the leaf temperature is. Carboxylation efficiency is maximal at 25°C. At ambient CO, partial pressure stomata open in a way that CO₂ assimilation occurs at a rate found within the curvature region of the CO₂ response function of A. The light-independent CO² compensation point as a function of temperature is presented. Applying a combined heat/low humidity pulse (15 or 60 min) on leaves of Eucalyptus behriana F. Muell. or Pinus radiata R. Don, respectively, leads to a lower level of intercellular carbon dioxide partial pressure (C_i) during the decline in A and leaf conductance to water vapour (g). A lower C_i level is maintained during recovery of A and g, A almost reaching the pre-pulse level but not g. The existence of an after-effect indicates that the response to the combined high temperature/low humidity pulse is a multi-step process.