Influence of phloretin and alcohols on barrier defects in the erythrocyte membrane caused by oxidative injury and electroporation

Oxidative damage by diamide, periodate and oxygen-derived reactive species, but also exposure to electroporation induce in the erythrocyte membrane dynamic, presumably fluctuating, defects having the properties of aqueous holes with definable radii and selectivities. These leaks, which can be quantified by measuring tracer fluxes or rates of colloid-osmotic lysis, are here shown to be inhibited by phloretin and a small number of related phenol compounds (phenolphthalein, hydroxyacetophenones, nitrophenol), while a host of other 'membrane-active' agents is not effective in this respect. I50 values range from about 200 microM for phloretin and phenolphthalein to about 10 mM for 4-nitrophenol. Inhibition by phloretin is reversible, not competitive and not related in its extent to the extent of leakiness. In contrast, the enhancement of transbilayer mobility of amphiphilic lipid probes, which invariably goes along with leak formation of the type described, is not affected by phloretin. Aliphatic alcohols (hexanol, butanol) have an amplifying effect on leaks induced by oxidative damage but do not affect leaks induced by electroporation. The alcohol-amplified leaks maintain the properties of aqueous holes as indicated by a low activation energy of leak fluxes. Since both, inhibition and stimulation of leak fluxes do not go along with appreciable changes of the apparent radii of the aqueous holes, changes in the dynamics (opening and closing) of the defects are proposed to underly the effects of phloretin and alkanols. The membrane lipid domain is likely to be the site of the leaks and of their modulation.     

Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. I. Impairment of resealing and formation of aqueous pores in the ghost membrane after modification of SH groups.

Resealed human erythrocyte ghosts prepared by a two-step procedure were shown to have small residual barrier defects with the properties of aqueous pores, such as size discrimination of hydrophilic nonelectrolytes (erythritol to sucrose), indicative of an apparent pore radius of about 0.7 nm, and a low activation energy (about 12-20 kJ/mol (mannitol, sucrose)) of the leak fluxes. As in other cases (Deuticke et al. (1991) Biochim. Biophys. Acta 1067, 111-122) these leak fluxes can be inhibited by phloretin. Treatment of such resealed ghosts with the mild SH oxidizing agent, diamide, induces additional membrane leaks to the same extent and with the same properties as in native erythrocytes (Deuticke et al. (1983) Biochim. Biophys. Acta 731, 196-210), including reversibility of the leak by SH reducing agents, inhibition by phloretin and stimulation by alkanols. In contrast, resealed ghosts prepared either from diamide-treated erythrocytes or by adding diamide to the 'open' membranes prior to reconstitution of high ionic strength and raising the temperature, exhibit a state of greater leakiness. This leakiness is somewhat different in its origin from the former class of leaks, since it can also be produced by N-ethylmaleimide, which is essentially ineffective when added to the membrane in its 'tight' state. The leaks induced in the 'open' state of the membrane, which can be regarded as a consequence of an impaired resealing, are nevertheless reversible by reducing agents added after resealing and are comparable in many, but not all their characteristics to leaks induced in the 'tight' state of the membrane. Resealing in the presence of the isothiocyanostilbenes DIDS or SITS mimicks the leak forming effect of diamide by modifying a small population of SH groups, while amino groups seem not to be involved. The findings indicate and substantiate an important role of the redox state of membrane skeletal protein sulfhydryls in the maintenance and the re-establishment of the barrier function of the erythrocyte membrane.     

Oxidative stress of human erythrocytes by iodate and periodate. Reversible formation of aqueous membrane pores due to SH-group oxidation

Human erythrocytes were exposed to oxidative stress by iodate and periodate. Oxidation causes a time- and concentration-dependent increase in membrane permeability for hydrophilic molecules and ions. The induced leak discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy (Ea = 1-4 kcal.mol-1). These results are reconcilable with the formation of aqueous pores. The pore size was approximated to be between 0.45 and 0.6 nm. This increase in permeability is reversible upon treatment with dithioerythritol. Blocking of membrane thiol groups with N-ethylmaleimide protects the membranes against leak formation. The oxidation causes dithioerythritol-reversible modification of membrane proteins as indicated by the gel electrophoretic behavior. These modifications can also be suppressed by blocking the membrane thiol groups with N-ethylmaleimide. About half of the membrane methionine is oxidized to acid hydrolysis-stable derivatives. A fast saturating increase in diene conjugation was observed in whole cells but not in isolated membranes, with only minor degradation of fatty acid chains. The oxidation of cell membrane lipids as well as oxidation of cell surface carbohydrates are not involved in leak formation. Taken together with earlier data (Deuticke, B., Poser, B., Lütkemeier, P. and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196-210), these findings indicate that formation of disulfide bonds by different oxidative mechanisms results in leaks with similar properties.     

Errata

Human erythrocytes were exposed to oxidative stress by iodate and periodate. Oxidation causes a time- and concentration-dependent increase in membrane permeability for hydrophilic molecules and ions. The induced leak discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy (E_a = 1–4 kcal · mol^?1). These results are reconcilable with the formation of aqeous pores. The pore size was approximated to be between 0.45 and 0.6 nm. This increase in permeability is reversible upon treatment with dithioerythritol. Blocking of membrane thiol groups with N-ethylmaleimide protects the membranes against leak formation. The oxidation causes dithioerythritol-reversible modification of membrane proteins as indicated by the gel electrophoretic behavior. These modifications can also be suppressed by blocking the membrane thiol groups with N-ethylmaleimide. About half of the membrane methionine is oxidized to acid hydrolysis-stable derivatives. A fast saturating increase in diene conjugation was observed in whole cells but not in isolated membranes, with only minor degradation of fatty acid chains. The oxidation of cell membrane lipids as well as oxidation of cell surface carbohydrates are not involved in leak formation. Taken together with earlier data (Deuticke, B., Poster, B., Lütkemeier, P., and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196–210), these findings indicate that formation of disulfide bonds by different oxidative mechanisms results in leaks with similar properties.     

Enhancement of transbilayer mobility of a membrane lipid probe accompanies formation of membrane leaks during photodynamic treatment of erythrocytes

In order to further characterize membrane alterations in human erythrocytes subjected to photodynamic treatment the passive transbilayer mobility of a phospholipid analogue was studied in cells illuminated for various lengths of time in the presence of the photosensitizer, aluminum chlorotetrasulfophthalocyanine. These measurements were combined with the characterization of the membrane leaks for polar solutes occurring under the same conditions with respect to their apparent size, number and ion selectivity. The time-dependent photodynamic enhancement of leaks for K+ as well as choline or erythritol was paralleled by a marked increase of the transbilayer reorientation rate of the amphiphilic lipid probe, palmitoyllysophosphatidylcholine from 0.05% min-1 in native cells to 0.32% min-1 after 60 min illumination. The asymmetric orientation of native phospholipids was not affected by this treatment. The leak permeability proved to be due to the formation of pores with apparent radii of about 0.45 nm after 60 min illumination, and of 0.75 nm after 90 min. The number of pores per cell was calculated to be less than 1, the pores are slightly cation-selective (PK/PCl approximately 3:1). Since photodynamic treatment did not induce lipid peroxidation under the prevailing experimental conditions, protein modification must be the primary cause of both, leak permeability and flip enhancement. Since it is also likely that the leak permeability arises from oxidation of intrinsic membrane proteins, the results raise the interesting possibility that oxidative alteration of intrinsic membrane proteins may lead to enhanced transbilayer mobility of lipids.     

Formation and properties of aqueous leaks induced in human erythrocytes by electrical breakdown

Leaks were induced in human erythrocytes by brief (tau = 1-40 microseconds) discharges of high electric fields (3-20 kV/cm). Leak permeabilities were characterized by measuring (a) net and tracer fluxes of K+ and nonelectrolytes under protection of the cells against colloid-osmotic lysis, or (b) rates of colloid osmotic lysis in various salt solutions. The induced permeabilities are essentially stable for hours at 0-2 degrees C. Leak permeability P increases exponentially with the breakdown voltage ED according to a function of the general type P = bED. The basis b varies with the pulse length. A log-linear presentation reveals a biphasic linear relationship with a break at which the slope (= log b) decreases markedly. Elevated ionic strengths of the suspension medium during the electric discharge enhance leak formation. Leak permeability exhibits an apparent activation energy of 29 +/- 5 kJ/mol, indicative of diffusion through aqueous pathways. Somewhat differing equivalent pore radii emerge from measurements with different probes: 0.6-0.8 nm from tracer fluxes of polyols (Mr = 3600, ED = 4-7 kV/cm) and 0.8-1.9 nm from osmotic protection studies with polyethylene glycols (Mr = 200-3300, ED = 6-10 kV/cm). These numbers and the non-monoexponential increase of leak permeability with the field strength suggest a dual mechanism for the increase of leak permeability: an increase of the number of pores at low breakdown voltage and an additional increase of pore size at higher voltage. Estimated numbers of pores range from 1 to 10 per cell, which suggests dynamic fluctuating structural defects to be involved. The leaks discriminate small monovalent inorganic ions in the sequence of free solution mobility. Organic anions are discriminated according to size and charge. Common properties of these electrically induced defects and of chemically induced leaks (diamide, periodate, t-butylhydroperoxide) in the erythrocyte membrane suggest close similarities in the molecular organization.     

Chemo-mechanical leak formation in human erythrocytes upon exposure to a water-soluble carbodiimide followed by very mild shear stress. I. Basic characteristics of the process

Human erythrocytes treated with low concentrations (1-5 mM) of the carboxyl group-modifying reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) lose their native deformability in parallel with extensive cross-linking of the membrane skeleton. After treatment with higher (5-40 mM) concentrations of the reagent the cells develop a hitherto undescribed property: when subjected to even very low shear stresses (resuspension after packing by centrifugation or viscometric shearing at up to 4 s-1) they become highly leaky to ions, lose their K+ with a half-time of about 5 min and subsequently undergo hemolysis. Lysis is not accompanied by cell fragmentation as occurs with mechanical hemolysis, but is colloid-osmotic, due to the formation of aqueous membrane leaks with an apparent radius of about 3 nm. Leakiness and lysis affect an increasing fraction of the cell population, in relation to (a) the concentration of EDC applied, (b) the shearing intensity, and (c) particularly, the hematocrit during shearing. The physical parameter determining the mechanical component of this 'chemo-mechanical' leak formation is not predominantly the shear stress. Rather, cell-cell interactions of as yet undefined nature seem to be involved. The analysis of chemo-mechanical leak formation may provide interesting insights into the influence of mechanical forces on membranes.     

Peroxidative membrane damage in human erythrocytes induced by a concerted action of iodoacetate, vanadate and ferricyanide

Human erythrocytes incubated without substrate in the presence of iodoacetate (0.2 mM), vanadate (0.5 mM) and ferricyanide (5 mM) form aqueous membrane leaks of equivalent radii of 0.5-0.8 nm leading to complete colloid-osmotic lysis within 180 min. All three components are indispensable for the effect. Inosine but not glucose markedly enhances the rate of hemolysis. These effects are due to oxidative damage, as indicated by concomitant destruction of polyunsaturated fatty acids and suppression of both effects by radical scavengers. Hemoglobin is not oxidized under these conditions. GSH and membrane SH levels remain almost normal, and no crosslinking or irreversible aggregation of membrane proteins is observed. In the absence of O2 no membrane damage can be observed. It is proposed that radical formation originates from reduction of O2 by NADPH, analogous to processes described in microsomal membranes. NADH seems not to be involved, since leak formation occurs in spite of the blockage of NADH formation by iodoacetate. Vanadate and ferricyanide are probably required to amplify the peroxidative reaction sufficiently to overcome the cellular antioxidative capacity.     

Tellurite-induced damage of the erythrocyte membrane. Manifestations and mechanisms.

Chemical and biophysical mechanisms underlying the thiol-dependent lytic action of tellurite (and selenite) on human erythrocytes were investigated using native and GSH-depleted cells. Exposure of GSH-depleted cells to tellurite alone produces oxidative cross-linking of membrane thiols paralleled by a moderate membrane leakiness comparable in its extent to that induced by other SH-oxidizing agents (diamide, periodate). Exposure to tellurite in presence of endogenous or exogenous GSH produces marked leakiness which stems from the formation of aqueous leaks permeant to ions and nonelectrolytes and sensitive to inhibition by phloretin. Apparent pore radii, derived from exclusion limits for polar non-electrolytes, range from 0.3 to at least 1.3 nm. Leak size increases with increasing exposure time and concentration of the modifier. Leak formation is paralleled by membrane rigidification based on the cross-linking of spectrin. Thiol-dependent leak formation by tellurite in GSH-depleted cells can be sustained not only by exogenous GSH but also by other thiols. Progress of leak formation by tellurite/thiol can not be reliably quenched by procedures such as removal of tellurite from the medium, inhibition of anion transport via band-3 protein, washing of the cells or low temperature. The reaction can, however, be terminated, even in the presence of tellurite, by addition of N-ethylmaleimide, presumably due to the blockage of thiols or thiol-analogous tellurium compounds. N-ethylmaleimide even brings about a partial reversal of leakiness, suggesting the contribution of a reversible and an irreversible component of tellurite damage. Membrane perturbation by tellurite/thiol involves the formation of a membrane permeant tellurium species, possibly HTe-, which is likely to induce progressive damage of membrane proteins by a redox shuttle going along with a formation of elemental tellurium and its reduction by thiols.     

Thyroid hormone status and membrane n-3 fatty acid content influence mitochondrial proton leak

Proton leak, as determined by the relationship between respiration rate and membrane potential, was lower in mitochondria from hypothyroid rats compared to euthyroid controls. Moreover, proton leak rates diminished even more when hypothyroid rats were fed a diet containing 5% of the lipid content as n-3 fatty acids. Similarly, proton leak was lower in euthyroid rats fed the 5% n-3 diet compared to one containing only 1% n-3 fatty acids. Lower proton leaks rates were associated with increased inner mitochondrial membrane levels of n-3 fatty acids and a decrease in the ratio of n-6/n-3 fatty acids. This trend was evident in the phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin phospholipid fractions. These results suggest that a significant portion of the effect of thyroid hormone status on proton leak is due to alterations in membrane fatty acid composition, primarily changes in n-3 content. Both the hypothyroid state and dietary effects appear to be mediated in part by inhibition of the Delta6- and Delta5-desaturase pathways.     

Nucleotide effects on liver and muscle mitochondrial non-phosphorylating respiration and membrane potential

Uncoupling protein-1 homologs are hypothesized to mediate mitochondrial proton leak. To test this hypothesis, we determined the effects of ATP and other nucleotides on liver and skeletal muscle mitochondrial non-phosphorylating respiration (VO(2)), membrane potential, FCCP-stimulated respiratory control ratios, and swelling. Neither ATP nor CTP affected liver or muscle proton leak, but both inhibited the respiratory chain. Unexpectedly, CMP stimulated liver proton leak (EC(50) approximately 4.4+/-0.5 mM). Using CMP chromatography, we identified two proteins (M(r)=31.2 and 32.6 kDa) from liver mitochondria that are similar in size to members of the mitochondrial carrier protein family. We conclude (a) liver and muscle mitochondrial proton leak is insensitive to ATP and CTP, and (b) CMP activates a leak in liver mitochondria. The CMP-inducible leak may be mediated by a 30-32 kDa protein. Based on the high concentrations required, CMP is unlikely to be a physiologically important leak regulator. Nonetheless, our results show that tissues other than brown fat have inducible leaks that may be protein-mediated.     

Leak measurements in spontaneously breathing premature newborns by using the flow-through technique

A new method for measuring and correcting airleaks during lung-function testing in infants has been validated invitro and in vivo by using a flow-through system that measured theinflow and outflow of a face mask. An adjustable leak was quantified byusing suction flow to validate the accuracy of leak measurements. Tovalidate the leak correction, the volume of a pump was measured withdifferent air leaks (0-30%). The method developed was tested in67 infants breathing spontaneously. There was good agreement betweenmeasured and simulated leaks (r = 0.998, P < 0.001; 95% limits ofagreement were 0.3 and 0.1%, respectively). The volume wasgenerally underestimated because of leaks, and the volume error was upto 94% compared with the maximum error of 5% after leak correction.With continuous leak measurements in vivo, there were <4% actualleaks (median 2.6%), and we did not observe any leaks in >7% ofcases. The leak correction improved the accuracy of ventilatorymeasurements. The monitoring of leaks is helpful for airtight placementof the face mask and for prevention of serious measurement errorscaused by leaks.

     

Escherichia coli alpha-haemolysin induces focal leaks in colonic epithelium: a novel mechanism of bacterial translocation

Extraintestinal pathogenic Escherichia coli (ExPEC) are usually harmless colonizer of the intestinal microflora. However, they are capable to translocate and cause life-threatening disease. Translocation of ExPEC isolates was quantified in colonic monolayers. Transepithelial resistance (R(t)) was monitored and local changes in conductivity analysed with conductance scanning. Confocal microscopy visualized the translocation route. Corroboratory experiments were performed on native rat colon. One translocating strain E. coli O4 was identified. This translocation process was associated with an R(t) decrease (36 +/- 1% of initial resistance) beginning only 2 h after inoculation. The sites of translocation were small defects in epithelial integrity (focal leaks) exhibiting highly increased local ion permeability. Translocation was enhanced by preincubation of monolayers with tumour necrosis factor-alpha or interleukin-13. Mutant strains lacking alpha-haemolysin lost the ability to induce focal leaks, while this effect could be restored by re-introducing the haemolysin determinant. Filtrate of a laboratory strain carrying the alpha-haemolysin operon was sufficient for focal leak induction. In native rat colon, E. coli O4 decreased R(t) and immunohistology demonstrated focal leaks resembling those in cell monolayers. E. coli alpha-haemolysin is able to induce focal leaks in colonic cell cultures as well as in native colon. This process represents a novel route of bacterial translocation facilitated by pro-inflammatory cytokines.     

Membrane and intracellular modes of sugar-dependent increments in red cell stability.

Sugar-dependent increments in red cell stability under osmotic stress can be ascribed to changes either in the membrane or in the intracellular matrix. These two possible modes of action have been tested and characterized. Rheological investigation of membrane-free haemoglobin solutions has shown that D-glucose, but not D-fructose, promotes the formation of a visco-plastic gel structure. Gel strength is a function of glucose concentration, haemoglobin concentration and temperature. The ability of various sugars to promote gel formation correlates with their solution properties. The existence of gel structure reduces K+ and haemoglobin leak from red cells whose membranes were partially destroyed by gamma-radiation. Reduced osmotic swelling in the presence of glucose is also due to gel formation since the glucose effect is lost in resealed red cell ghosts. D-Fructose does not protect red cells against radiation damage; its mode of action in increasing red cell stability under osmotic stress is a membrane effect. Cell sizing using the Coulter Counter has shown that fructose, but not glucose, can increase the maximal volume at lysis. At 50 mM, D-fructose expands the red cell ghost volume by 11.2%; this represents a 7.2% increase in membrane area. Ghost expansion by fructose is fructose concentration dependent (0-100 mM) and is insensitive to temperature variation (0-37 degrees C).     

The biochemistry of virus-induced cell fusion. Changes in membrane integrity

1. Phospholipids prelabelled with [(14)C]acetate, [(32)P]phosphate, [(3)H]- or [(14)C]-choline or [(3)H]inositol are not significantly degraded during fusion of Lettrée cells mediated by Sendai virus, nor are carbohydrates prelabelled with [(3)H]fucose, [(14)C]galactose or [(3)H]glucosamine. Less than 1nmol of lysophosphatidylcholine/10(7) cells is formed during fusion. Diethyl p-nitrophenyl phosphate, which inhibits phospholipase A by more than 95% has no effect on fusion. It is concluded that none of the events leading to cell fusion is accompanied by significant turnover of phospholipids or other membrane components. 2. Intracellular K(+) leaks out during virally mediated cell fusion; the loss is not as extensive as that of intracellularly accumulated choline or deoxyglucose. Movement of Ca(2+) into or out of cells could not be detected. 3. At concentrations of Lettrée cells insufficient to be agglutinated by virus, intracellularly accumulated choline and deoxyglucose leak out. Agglutination caused by concanavalin A does not result in leakage of intracellular metabolites. 4. P815Y cells, which agglutinate but do not fuse in the presence of virus, show leakage of intracellularly accumulated metabolites. The extent of leakage does not alter during the G(1) and S periods of the cell cycle. 5. Leakage is inhibited by Ca(2+), but is unaffected by EDTA. 6. It is concluded that the interaction of Sendai virus with mammalian cells causes a weakening of membrane integrity so that intracellular metabolites leak out. Such destabilization may facilitate viral entry and is therefore an interesting system for further biochemical studies.     

Reversible deformation-dependent erythrocyte cation leak. Extreme sensitivity conferred by minimal peroxidation.

To determine the threshold at which red blood cells (RBC) begin to manifest deformation-dependent leakiness to monovalent cations, we examined net passive potassium leak during elliptical deformation. Normal RBC did not begin to leak appreciable amounts of potassium until shear stress reached 204 dyn/cm2, at which point they had attained greater than 96% of their maximal deformation. In striking contrast, RBC that had undergone minimal, physiologically relevant degrees of peroxidative damage induced by t-butylhydroperoxide began to leak potassium at only 59 dyn/cm2 when they had reached only 63% of their maximal deformation. The cation leak identified in this manner is not prelytic, and it is fully reversible. Therefore, these data may be relevant to abnormal cation leaks that develop in sickle red cells that have membranes damaged by auto-oxidative stress and that manifest an exuberant but reversible leakiness to monovalent cation during sickling-induced deformation of the cell membrane.     

Erythrocyte ion transport and membrane lipid composition in young and adult rats with NO-deficient hypertension

The aim of our study was to search for abnormalities of sodium and potassium transport in erythrocytes of male Wistar rats subjected to chronic L-NAME treatment (40 mg/kg/day) for 4 weeks either from weaning (4-week-old) or in adulthood (12-week-old). Sodium content, Na(+),K(+)-pump and Na(+),K(+)-cotransport activity, cation leaks as well as membrane cholesterol and phospholipid contents were determined in fresh erythrocytes. Chronic inhibition of NO synthase elicited similar blood pressure rise in both age groups which did not differ in the degree of NO synthase inhibition. No significant ion transport abnormalities were disclosed in erythrocytes of young NO-deficient rats, whereas erythrocyte Na(+) content, outward Na(+),K(+)-cotransport and inward Na(+) leak were significantly reduced in adult hypertensive animals compared to age-matched controls. It should be noted that the erythrocytes of adult control rats were characterized by higher activity of Na(+),K(+)-pump and Na(+),K(+)-cotransport, increased Na(+) and Rb(+) leaks and elevated membrane cholesterol content compared to those of young normotensive controls. Increased Na(+) leak and elevated membrane cholesterol content but reduced membrane phospholipid content were revealed in erythrocytes of adult hypertensive rats when compared to young hypertensive rats. It can be concluded that young and adult Wistar rats did not differ in the extent of NO synthase inhibition and blood pressure rise elicited by chronic L-NAME treatment. Our results exclude the important participation of classical sodium transport abnormalities in the pathogenesis of this NO-deficient form of experimental hypertension.     

Top-down control analysis of the effect of temperature on ectotherm oxidative phosphorylation

Top-down control and elasticity analysis was conducted on mitochondria isolated from the midgut of the tobacco hornworm (Manduca sexta) to assess how temperature affects oxidative phosphorylation in a eurythermic ectotherm. Oxygen consumption and protonmotive force (measured as membrane potential in the presence of nigericin) were monitored at 15, 25, and 35 degrees C. State 4 respiration displayed a Q(10) of 2.4-2.7 when measured over two temperature ranges (15-25 degrees C and 25-35 degrees C). In state 3, the Q(10)s for respiration were 2.0 and 1.7 for the lower and higher temperature ranges, respectively. The kinetic responses (oxygen consumption) of the substrate oxidation system, proton leak, and phosphorylation system increased as temperature rose, although the proton leak and substrate oxidation system showed the greatest thermal sensitivity. Whereas there were temperature-induced changes in the activities of the oxidative phosphorylation subsystems, there was no change in the state 4 membrane potential and little change in the state 3 membrane potential. Top-down control analysis revealed that control over respiration did not change with temperature. In state 4, control of respiration was shared nearly equally by the proton leak and the substrate oxidation system, whereas in state 3 the substrate oxidation system exerted over 90% of the control over respiration. The proton leak and phosphorylation system account for <10% of the temperature-induced change in the state 3 respiration rate. Therefore, when the temperature is changed, the state 3 respiration rate is altered primarily because of temperature's effect on the substrate oxidation system.     

The influence of energy-transfer inhibitors on proton permeability and photophosphorylation in normal and preilluminated Rhodospirillum rubrum chromatophores.

(1) Chromatophores were preilluminated in the presence of phenazine methosulphate or diaminodurene, and without phosphorylation substrates; next they were transferred to fresh medium and assayed for light-induced proton uptake, light-induced 9-aminoacridin fluorescence quenching, and photophosphorylation. (2) Preillumination in the presence of phenazine methosulphate or diaminodurene causes an inhibition of the photophosphorylation rate. The presence of ADP + MgCl2 + phosphate, or ADP + MgCl2 + arsenate during preillumination provides full protection against this effect. (3) Preilluminated chromatophores are leaky for protons. The leak is expressed as an accelerated dark decay, and a diminished extent of succinate-supported, light-induced proton uptake. The extent of light-induced 9-aminoacridin fluorescence quenching is also diminished. (4) The proton leak can be closed by oligomycin and by dicyclohexyl carbodiimide (at concentrations similar to those used to inhibit photophosphorylation), but not by aurovertin. Closure of the proton leak results in partial restoration of the photophosphorylation rate. (5) The inhibition of phosphorylation by oligomycin or dicyclohexyl carbodiimide is time-dependent. In untreated chromatophores, the time-dependence is determined by the extent of membrane energization. In preilluminated chromatophores, the time-dependence is determined in addition by the extent to which the proton leaks have been closed. The reasons for this are briefly discussed.     

Barium swallows after free jejunal transfer: should they be performed routinely?

Fistula formation after free jejunal transfer for pharyngoesophageal reconstruction is a serious complication with potentially critical consequences. Barium swallow is used postoperatively to check for anastomotic competence before feeding but has been unreliable as a predictor of leak at our institution. The objective of this study was to evaluate the role of routine postoperative barium swallow in 41 consecutive jejunal transfers. Thirty-nine patients who underwent 41 consecutive free jejunal transfers had a routine barium swallow performed between postoperative days 12 and 17. Radiologic findings and clinical outcome were evaluated and correlated. All barium swallows were reviewed by a single experienced radiologist in a blinded fashion. One total and one partial flap failure necessitated a second free jejunal transfer. Pharyngocutaneous fistulae developed after nine free jejunal transfers, of which the barium swallow was normal in four (44 percent) and showed a leak in five (56 percent). In the 32 free jejunal transfers with no clinical leaks, 6 (19 percent) had radiologic leakage of contrast. Thus, barium swallow was normal in 30 patients and showed leakage in 11 patients. Normal barium swallow correlated with uncomplicated clinical course in 26 of 30 cases. In the remaining four cases (13 percent), however, a delayed fistula developed, which was secondary to flap necrosis in one case (negative predictive value 87 percent). On the other hand, radiologic leaks corroborated clinical fistula in 5 of 11 cases (45 percent), whereas no fistula developed in 6 cases (positive predictive value 46 percent). Of the five patients with clinical fistulae, four had early leaks (within 1 week), and the barium swallow did not provide additional information. The fifth patient developed a delayed leak 2 weeks after the barium swallow. Review of these barium swallows at the time of this study reversed the initial report of leakage in three patients, improving the predictive value to 63 percent. These patients had an uncomplicated clinical course. The positive predictive value of clinical assessment alone was 63 percent. We conclude that barium studies following free jejunal transfers can be difficult to interpret, but an experienced radiologist can improve their accuracy. A normal barium swallow, however, does not ensure an uneventful clinical course. Similarly, radiologic leaks do not imply a clinical complication of fistula. Clinical judgment should therefore be exercised in initiating oral intake after free jejunal transfer. Barium swallow should be used only as an adjunct to aid in patient management.