Continuous Flow Tube Research Articles

Kinetics of chloride transport across fish red blood cell membranes

The continuous flow tube method was used to investigate the kinetics of chloride transport, and its potential oxygenation-dependency, in red blood cells (RBCs) from four teleost fish species and man. A significant interspecific variation in Cl- transport kinetics was found. At 15 &deg;C, the rate constant k for unidirectional 36Cl- efflux was significantly lower in RBCs from eel and carp than in RBCs from rainbow trout and Atlantic cod. The values of k of cod RBCs at 15 &deg;C and of human RBCs at 37 &deg;C were not significantly different. The volume and surface area of the RBCs were evaluated and used to calculate the apparent membrane permeability to Cl- (PCl). The magnitude of PCl followed the sequence: eel<carp<trout&frac34;cod. PCl values in trout and cod at 15 &deg;C were similar to human values at 37 &deg;C. An extrapolation of human values to 15 &deg;C revealed that the Cl- shift at this temperature was considerable faster in all four teleosts than in man. This illustrates appropriate adaption of band-3-mediated anion transport to the different temperature regimes encountered by fish and mammals. The Cl- transport kinetics did not differ significantly between oxygenated and deoxygenated RBCs in any of the species examined. The apparent absence of any effect of a change in haemoglobin oxygen-saturation may be related to the presence of a flexible link which results in minimal interaction between the membrane domain (mediating Cl- transport) and the cytoplasmic domain (to which oxygenation-dependent haemoglobin binding occurs) of band 3. In carp, Cl- transport kinetics were not influenced by pH over the extracellular pH (pHe) range 7.6&shy;8.36, which spans the in vivo pHe range. The data are discussed in relation to the rate-limiting role of red blood cell HCO3-/Cl- exchange for CO2 excretion.

The permeability of the human red cell membrane to steroid sex hormones

The release rates of the 3H-labeled steroid sex hormones estrone, estradiol, estriol, progesterone, and testosterone from the human red blood cell and resealed red cell ghosts were studied at 38°C and pH 7.2 by means of the rapid continuous flow tube method which has a time resolution of a few milliseconds. Further, the equilibria between unbound hormone and hormone bound to red cells, resealed red cell ghosts and albumin were studied by partitioning analysis of trace amounts of labeled hormones. The half-times for release from erythrocytes under physiological conditions ranged from 4 ms (testosterone) to 150 ms (estriol). The release from ghosts was significantly faster than from cells preincubated with hormones at unphysiological high concentrations. Affinities of hormone binding to cells and hormone indicate that as much as 15–35% of the total hormone content in whole blood is confined to red cells. The ratio between bound and free hormone in the cell ranged from 5 to 10, and the ratio between cytoplasma-bound and membrane-bound hormone ranged between 3 and 9. The results are compatible with a model of fast transition of hormone through the red cell membrane and intracellular binding of hormone. We suggest that red cells function as carriers of sex hormones in the bloodstream in a manner similar to that of albumin, and that red cells may be responsible for 5–15% of sex hormone delivery to target tissues.

Chloride and bicarbonate transport in chick embryonic red blood cells.

1. Unidirectional efflux of 36Cl- and H14CO3- from erythrocytes of 4- to 16-day-old chick embryos was measured under steady-state conditions at 37 degrees C and pH 7.7. The efflux rates were high, > 3 s-1, and were, therefore, measured by means of the continuous flow tube method. 2. At day 4 of development the range of permeability coefficients for bicarbonate and chloride (PHCO3 and PCl was 1-30 x 10(-4) cm s-1, with average values of respectively 10 x 10(-4) and 8 x 10(-4) cm s-1. However, the results can be divided into two groups, one with PHCO3 and PCl above 12 x 10(-4) cm s-1, and one with values below 5 x 10(-4) cm s-1. The same range of values was also obtained for day 6 erythrocytes, but the overlap is more conspicuous. At day 16, PHCO3 and PCl were respectively 9 x 10(-4) and 6 x 10(-4) cm s-1 (37 degrees C, pH 7.7). In adult chicken red blood cells PHCO3 and PCl were respectively 7 x 10(-4) and 4 x 10(-4) cm s-1, and in human red blood cells the respective values were 5.6 x 10(-4) and 4 x 10(-4) cm s-1. 3. Chloride self-exchange, measured at 0 degrees C, was almost completely inhibited by addition of 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS) at both days 6 and 16 of embryonic development, supporting the finding that the embryonic chick erythrocytes also have a transmembrane anion exchanger similar to that of other red cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of a collocation method for simulation of distributed parameter bioreactors

An orthogonal collocation method which belongs to the class of functional approximation methods of partial differential equations (PDEs) is applied in two distributed parameter bioreactor models. The models represent microbial growth and substrate consumption in a continuous-flow tube reactor. The growth is described by the Contois model. The points of the spatial discretisation method, the collocation points, are zeros of a Jacobi polynomial. These polynomials form a weighted set of orthogonal functions. The effect of the location and number of the collocation points is studied via simulations. It is concluded that in general relatively good accuracy is obtained already with a low number of points. In the case of the nondiffusional model, the approximation became unstable when the number of points was increased. The approximations of the diffusional model behaved smoothly and more and more accurately when the number of points was increased. Theoretically, the number of points needed for a certain accuracy level can be studied on the basis of the order reduction parameter obtained from the steady-state solution of the original PDE. Simulations for varying values of this parameter confirm the result that the decrease in the absolute value of the parameter lowers the error level of the approximation for the same number of points.

Continuous breathing circuit flow and tracheal tube cuff leak: sources of error during pediatric indirect calorimetry.

To determine whether continuous gas flow in the breathing circuit or an airleak around the tracheal tube cuff will introduce errors into the measurement of oxygen consumption (VO2) with indirect calorimetry. Nonrandomized, controlled trial. Experimental laboratory. Ten healthy, anesthetized mongrel dogs, weighing 8 to 12 kg. Data were recorded at seven levels of flow, from 0 to 12 L/min in excess of minute ventilation, through a continuous breathing circuit. Data were recorded at five levels of tracheal tube cuff leak from 0% to 40% of inspiratory minute volume. VO2 was measured using an indirect calorimeter with constant internal gas flow and calculated from results of blood gas analysis, cooximetry, and thermodilution cardiac output determinations at all levels of continuous breathing circuit flow and cuff leak. BP, heart rate, respiratory rate, arterial and mixed venous blood gases, and body temperature were measured to assess stability of cardiopulmonary function. Continuous breathing circuit flow did not affect the accuracy of indirect calorimetry until the total flow reached a critical value (11.5 L/min) that was slightly below the internal flow constant of the metabolic monitor (12.4 L/min). At higher circuit flows, measured VO2 decreased in a linear fashion, while calculated VO2 remained unchanged. Above the critical flow, the error of indirect calorimetry correlated significantly only with the total circuit flow (r2 = .64), not with the exhaled concentration of CO2 (r2 = .005) or the inspiratory-expiratory oxygen difference (r2 = .004). The continuous flow rate at the critical circuit flow was 66 +/- 15% of the subjects' peak inspiratory flow. Increasing tracheal tube cuff leak produced a progressive decrease in measured VO2 but not in calculated VO2. The difference between measured and calculated VO2 was linearly related to the magnitude of the leak (r2 = .56), and was statistically significantly larger at all levels of cuff leak, when compared with measurements during complete cuff seal. An indirect calorimeter in which measurement of VO2 is based on internal constant flow rather than spirometry can be used to accurately measure VO2 from a continuous-flow breathing circuit, if the total circuit flow is less than the internal flow. This limitation may restrict the use of continuous flow to a level below the subject's peak inspiratory flow. The accuracy of indirect calorimetry cannot be guaranteed for any amount of tracheal tube cuff leak.

Chemical Reactions of Small Gas Phase Metal Clusters

The study of gas phase chemical reactions of neutral metal clusters with simple molecules has become feasible due to the development of the pulsed fast flow reactor and the low pressure continuous flow tube reactor. In both devices a laser vaporization cluster source is coupled to the flow tube reactor. An inert carrier gas enable both cluster formation and a continuously variable concentration of the reagent. Upon exiting the reactor the product composition is frozen in an adiabatic expansion. Clusters and reaction products are formed into a molecular beam and detected by pulsed laser ionization and time-of-flight mass spectrometry. This contribution focuses mainly on the performance of the continuous flow tube reactor. This device offers the unique advantage that a quantitative analysis of the reactions becomes principally possible, because the clusters can be exposed to controlled concentrations of reagent for a determinable time interval at a controlled temperature. It is possible to distinguish between kinetically and thermodynamically controlled reactions. One of the major problems in the data analysis is to relate the detected distribution of cluster ions to the original distribution of the neutrals. Fragmentation of the clusters, differences in ionization efficiencies, and desorption of adsorbates can distort the initialmore » distribution. A rate equation analysis allows the authors to relate the observed ion intensities to the abundance distribution of the neutral clusters. The reaction of iron clusters with D{sub 2}O is discussed as an application.« less

Glucose transport in human red cell membranes. Dependence of age, ATP, and insulin

Glucose self-exchange flux ( J ex) and net efflux ( J net) in human red cells and ghosts were studied at 25°C and pH 7.2 by means of the combined use of the Millipore-Swinnex filtering method and the continuous flow tube method to show the dependence of time of storage after aspiration, ATP and insulin. In fresh cells (RBC), ghosts (G), ghosts with 2 mM ATP (G+), and cells stored at 4°C > 60 days (OC) both J ex and J net follow simple Michaelis-Menten kinetics where J = J max · C i · (K 1 2 + C i ) −1 . J ex max and J net max (nmol · cm −2 · s −1), respectively, was: (RBC) 0.27 and 0.19, (G) 0.24 and 0.27, (G +) 0.23 and 0.24, (OC) 0.23 and 0.20. K 1 2 , ex and K 1 2 , net (mM), respectively, was: (RBC) 7.5 and 1.3, (G) 4.8 and 14.2, (G +) 11.6 and 6.8, (OC) 3.8 and 9.0. In ghosts, the ATP-dependent fraction of the permeability shows a hyperbolic dependence on glucose concentrations lower than 80 mM. Insulin up to 1 μM had effect on neither J ex nor J net in RBC. Based on reported values of cytochalasin B binding sites the turnover rate per site in RBC appears to be as high as in maximally insulin-stimulated fat cells. Our results suggest that the number of transport sites remains constant, independent of age, ATP and insulin.

Diffusional solute flux during osmotic water flow across the human red cell membrane.

The effect of solvent drag on the unidirectional efflux of labeled water, urea, and chloride from human red cells was studied by means of the continuous flow tube method under conditions of osmotic equilibrium and net volume flow. Solvent (water) flow out of cells was created by mixing cells equilibrated in 100 mM salt solution with a 200-mM or 250-mM salt solution, while flow of water into cells was obtained by equilibrating the cells in the higher concentration and mixing them with the 100-mM solution. Control experiments constitute measurements of efflux of [14C]ethanol in normal cells and 3H2O in cells treated with p-chloromercuribenzosulfonate under the conditions described above. In both instances, the solute is known to penetrate the membrane through nonporous pathways. As anticipated, the tracer flux of neither urea nor chloride showed any dependence on net solvent flow, regardless of the direction. If one assumes the recently reported reflection coefficient for urea of 0.7, the urea tracer flux should change by at least 24% under volume flow conditions. Since such changes would be easily detected with our method, we conclude that the pathways for water, for urea, and for chloride are functionally separated.

Urea permeability of human red cells.

The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.

Kinetics of glucose transport in human erythrocytes.

The rate of unidirectional D-[14C]glucose efflux from human red blood cells was determined at self-exchange and net-efflux conditions by means of the Millipore-Swinnex filtering technique and the rapid continuous flow tube technique with which initial rates can be measured within fractions of a second. Determinations at 38, 25 and 10 degrees C of the concentration dependence of glucose self-exchange flux and net efflux showed that both self exchange and net efflux followed simple Michaelis-Menten kinetics at all temperatures. At 38 degrees C the maximal self-exchange flux and the maximal net efflux were identical (6 X 10(-10) mol/cm2.sec). The cellular glucose concentration for half-maximal flux (K1/2) was 6.7 mM for self exchange and 8.2 mM for net efflux. By lowering temperature the maximal glucose self-exchange flux progressively exceeded the maximal net efflux, and was about three times larger at 10 degrees C. K1/2 for self exchange increased to 12.6 mM at 10 degrees C, while K1/2 for net efflux decreased to 4.4 mM. At 38 degrees C the glucose permeability at self exchange at a constant extracellular glucose concentration of 40 mM showed a bell-shaped pH dependence between pH 6 and pH 9. A maximum was found at pH 7.2, whereas the apparent permeability coefficient was halved both at pH 6 and pH 9. The temperature dependence of glucose transport was determined between 47 and 0 degrees C at a cellular glucose concentration of 100 mM which ensured greater than 85% saturation of the glucose transport system within the temperature range. The Arrhenius activation energy of glucose transport was not constant. By lowering the temperature, the activation energy increased gradually for net efflux from 55 kJ/mole between 38 and 47 degrees C to 151 kJ/mole between 0 and 10 degrees C. The temperature dependence of self-exchange flux showed a more pronounced change around 10 degrees C. The Arrhenius activation energy was found to be 61 kJ/mole above and 120 kJ/mole below 10 degrees C.

Permeability of human red cells to a homologous series of aliphatic alcohols. Limitations of the continuous flow-tube method.

Human red cell permeability to the homologous series of methanol, ethanol, n-propanol, n-butanol, and n-hexanol was determined in tracer efflux experiments by the continuous flow tube method, whose time resolution is 2-3 ms. Control experiments showed that unstirred layers in the cell suspension were less than 2 X 10(-4) cm, and that permeabilities less than or equal to 10(-2) cm s-1 can be determined with the method. Alcohol permeability varied with the chain length (25 degrees C): Pmeth 3.7 X 10(-3) cm s-1, Peth 2.1 X 10(-3) cm s-1, Pprop 6.5 X 10(-3) cm s-1, Pbut less than or equal to 61 X 10(-3) cm s-1, Phex 8.7 X 10(-3) cm s-1. The permeability for methanol, ethanol, and n-propanol was concentration independent (1-500 mM). The permeability to n-butanol and n-hexanol, however, increased above the upper limit of determination at alcohol concentrations of 100 and 25 mM, respectively. The activation energies for the permeability to methanol, n-propanol, and n-hexanol were similar, 50-63 kJ mol-1. Methanol permeability was not reduced by p-chloromercuribenzene sulfonate (PCMBS), thiourea, or phloretin, which inhibit transport of water or hydrophilic nonelectrolytes. It is concluded (a) that all the alcohols predominantly permeate the membrane lipid bilayer structure; (b) that both the distribution coefficient and the diffusion coefficient of the alcohols within the membrane determine the permeability, and (c) that the relative importance of the two factors varies with changes in the chain length.

Diffusional water permeability of human erythrocytes and their ghosts.

The diffusional water permeability of human red cells and ghosts was determined by measuring the rate of tracer efflux by means of an improved version of the continuous flow tube method, having a time resolution of 2-3 ms. At 25 degrees C, the permeability was 2.4 x 10(3) and 2.9 x 10(3) cm s-1 for red cells and ghosts, respectively. Permeability was affected by neither a change in pH from 5.5 to 9.5, nor by osmolality up to 3.3 osmol. Manganous ions at an extracellular concentration of 19 mM did not change diffusional water permeability, as recently suggested by NMR measurements. A "ground" permeability of 1 x 10(3) cm s-1 was obtained by inhibition with 1 mM of either p- chloromercuribenzoate (PCMB) or p-chloromercuribenzene sulfonate (PCMBS). Inhibition increased temperature dependence of water permeability for red cells and ghosts from 21 to 30 kJ mol-1 to 60 kJ mol-1. Although diffusional water permeability is about one order of magnitude lower than osmotic permeability, inhibition with PCMB and PCMBS, temperature dependence both before and after inhibition, and independence of osmolality showed that diffusional water permeability has qualitative features similar to those reported for osmotic permeability, which indicates that the same properties of the membrane determine both types of transport. It is suggested that the PCMB(S)-sensitive permeability above the ground permeability takes place through the intermediate phase between integral membrane proteins and their surrounding lipids.