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PubMed2006

Lactic acid buffering, nonmetabolic CO2 and exercise hyperventilation: a critical reappraisal.

It has been suggested that hyperventilation and the disproportionate increase in VCO2 versus VO2 above the ventilatory threshold (V(TH)) in ramp exercise are due to the production of nonmetabolic CO2 in muscle because of lactic acid buffering by plasma bicarbonate entering the cell in exchange with lactate [Wasserman, K., 1982. Dyspnea on exertion. Is it the heart or the lungs? JAMA 248, 2039-2043]. According to this model, plasma standard bicarbonate concentration decreases in a approximately 1:1 ratio with the increase in plasma lactate concentration, 1 mmol of CO2 is generated above that produced by aerobic metabolism for each mmol of lactic acid buffered, and nonmetabolic CO2 produced in the muscle is partly responsible for hyperventilation because of the resulting increase in the CO2 flow to the lungs. The present report shows that this model is not consistent with experimental data: (1) bicarbonate is not the main buffer in the muscle; (2) the decrease in standard bicarbonate concentration is not the mirror image of the increase in lactate concentration; (3) buffering by bicarbonate does not increase CO2 production in muscle (no nonmetabolic CO2 is produced in tissues); (4) the CO2 flow to the lungs, which should not be confused with VCO2 at the mouth, does not increase at a faster rate above than below V(TH). The disproportionate increase in VCO2 at the mouth above V(TH) is due to hyperventilation (not the reverse) and to the low plasma pH which both reduce the pool of bicarbonate readily available in the body.

Acid-Base Equilibrium↗
PubMed2002

Further evidence for distinct reactive intermediates from nitroxyl and peroxynitrite: effects of buffer composition on the chemistry of Angeli's salt and synthetic peroxynitrite.

The nitroxyl (HNO) donor Angeli's salt (Na(2)N(2)O(3); AS) is cytotoxic in vitro, inducing double strand DNA breaks and base oxidation, yet may have pharmacological application in the treatment of cardiovascular disease. The chemical profiles of AS and synthetic peroxynitrite (ONOO(-)) in aerobic solution were recently compared, and AS was found to form a distinct reactive intermediate. However, similarities in the chemical behavior of the reactive nitrogen oxide species (RNOS) were apparent under certain conditions. Buffer composition was found to have a significant and unexpected impact on the observed chemistry of RNOS, and varied buffer conditions were utilized to further distinguish the chemical profiles elicited by the RNOS donors AS and synthetic ONOO(-). Addition of HEPES to the assay buffer significantly quenched oxidation of dihydrorhodamine (DHR), hydroxylation of benzoic acid (BA), and DNA damage by both AS and ONOO(-), and oxidation and nitration of hydroxyphenylacetic acid by ONOO(-). Additionally, H(2)O(2) was produced in a concentration-dependent manner from the interaction of HEPES with both the donor intermediates. Interestingly, clonogenic survival was not affected by HEPES, indicating that H(2)O(2) is not a contributing factor to in vitro cytotoxicity of AS. Variation in RNOS reactivity was dramatic with significantly higher relative affinity for the AS intermediate toward DHR, BA, DNA, and HEPES and increased production of H(2)O(2). Further, AS reacted to a significantly greater extent with the unprotonated amine form of HEPES while the interaction of ONOO(-) with HEPES was pH-independent. Addition of bicarbonate only altered ONOO(-) chemistry. This study emphasizes the importance of buffer composition on chemical outcome and thus on interpretation and provides further evidence that ONOO(-) is not an intermediate formed between the reaction of O(2) and HNO produced by AS.

Animals↗
PubMed1999

Individual variations of pH, buffer capacity, and concentrations of calcium and phosphate in unstimulated whole saliva.

In order to evaluate the risk of development of dental caries and/or of formation of dental calculus, salivary variables have often been used, but not with particular success. A reason for the apparent lack of association could be that the individual temporal variation of a characteristic was so substantial relative to the overall variation that it is not possible to characterize an individual by a single salivary measurement. The aim here was to examine the individual variation of pH, buffer capacity, and concentrations of calcium and phosphate and to compare it with the overall variation of the characteristics in order to shed light on the above problem. Eight weekly samples of up to 4 ml of unstimulated whole saliva were collected from 11 dental students before tooth brushing on their arrival at 8 a.m. in the dental school. Calcium was determined by atomic absorption spectroscopy, phosphate colorimetrically, and pH electrometrically. The buffer capacity was assessed by titration of the saliva sample from the pH initially observed to pH 3. It was found that within each individual the concentration of calcium and of phosphate, pH, the hydroxyapatite ion product and the buffer capacity varied considerably over the 7 weeks. The individual range frequently covered more than a third of the total range. Further, within each of the variables, single individuals could be found whose samples covered 60% or more of the overall range, whilst others covered less than 10% of the range. It was therefore concluded that, although collected at the same time of the day, pH, buffer capacity and concentrations of calcium and phosphate in unstimulated whole saliva in the single individual vary so much that characterization of individuals and of their saliva based on a single salivary analysis is unreliable and hazardous.

Buffers↗
PubMed2000

Buffer effects on electric signals of light-excited bacteriorhodopsin.

Buffers change the electric signals of light-excited bacteriorhodopsin molecules in purple membrane if their concentration and the pH of the low-salt solution are properly selected. "Positive" buffers produce a positive component, and "negative" buffers a negative component in addition to the signals due to proton pumping. Measurement of the buffer effects in the presence of glycyl-glycine or bis-tris propane revealed an increase of approximately 2 and a change of sign and a decrease to approximately -0.5 in the translocated charge in these cases, respectively. These factors do not depend on temperature. The Arrhenius parameters established from the evaluation of the kinetics indicate activation enthalpies of 35-40 kJ/mol and negative activation entropies for the additional signals. These values agree with those found by surface-bound pH-sensitive probes in the search of the timing of proton release and uptake. The electric signals were also measured in the case of D(2)O solutions with similar results, except for the increased lifetimes. We offer a unified explanation for the data obtained with surface-bound probes and electric signals based on the clusters at extracellular and cytoplasmic sites of bacteriorhodopsin participating in proton release and uptake.

Bacteriorhodopsins↗
PubMed1990

Intracellular diffusion in the presence of mobile buffers. Application to proton movement in muscle.

Junge and McLaughlin (1987) derived an expression for the apparent diffusion constant of protons in the presence of both mobile and immobile buffers. Their derivation applies only to cases in which the values of pH are considerably greater than the largest pK of the individual buffers, a condition that is not expected to hold in skeletal muscle or many other cell types. Here we show that, if the pH gradients are small, the same expression for the apparent diffusion constant of protons can be derived without such constraints on the values of the pK's. The derivation is general and can be used to estimate the apparent diffusion constant of any substance that diffuses in the presence of both mobile and immobile buffers. The apparent diffusion constant of protons is estimated to be 1-2 x 10(-6) cm2/s at 18 degrees C inside intact frog twitch muscle fibers. It may be smaller inside cut fibers, owing to a reduction in the concentration of mobile myoplasmic buffers, so that in this preparation a pH gradient, if established within a sarcomere following action potential stimulation, could last 10 ms or longer after stimulation ceased.

Action Potentials↗
PubMed1999

Diffusion barriers limit the effect of mobile calcium buffers on exocytosis of large dense cored vesicles.

Fast exocytosis in melanotropic cells, activated by calcium entry through voltage-gated calcium channels, is very sensitive to mobile calcium buffers (complete block at 800 microM ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA)). This indicates that calcium diffuses a substantial distance from the channel to the vesicle. Surprisingly, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), having a similar KD for calcium as EGTA but a approximately 100 times faster binding rate, blocked exocytosis only twice as effectively as EGTA. Using computer simulations, we demonstrate that this result cannot be explained by free diffusion and buffer binding rates. We hypothesized that local saturation of calcium buffers is involved. A diffusion barrier for both calcium and buffer molecules, located 50-300 nm from the membrane and reducing diffusion 1000 to 10,000 times, generated similar calcium concentrations for specific concentrations of EGTA and BAPTA. With such barriers, calcium rise phase kinetics upon short step depolarizations (2-20 ms) were faster for EGTA than for BAPTA, implying that short depolarizations should allow exocytosis with 50 microM EGTA but not with 25 microM BAPTA. This prediction was confirmed experimentally with capacitance measurements. Coupling exocytosis to calcium dynamics in the model, we found that a barrier with a approximately 3000 times reduced diffusion at approximately 130 nm beneath the membrane best explains the experimentally observed effects of EGTA and BAPTA on block and kinetics of release.

Animals↗
PubMed2000

Mitochondria buffer non-toxic calcium loads and release calcium through the mitochondrial permeability transition pore and sodium/calcium exchanger in rat basal forebrain neurons.

Mitochondria participate in intracellular Ca2+ buffering and signalling. They are also major mediators of cell death. Toxic Ca2+ accumulation in mitochondria is widely believed to initiate cell death in many cell types by opening the permeability transition pore (PTP). In non-neuronal cells, the PTP has been implicated as a Ca2+ release mechanism in physiological Ca2+ signalling. In neurons, Ca2+ release from mitochondria has been attributed primarily to mitochondrial Na+/Ca2+ exchange. Using fura-2 ratiometric microfluorimetry in acutely dissociated rat basal forebrain neurons, we show that mitochondria are able to buffer non-toxic Ca2+ loads arising from caffeine-sensitive internal stores or from extracellular influx through voltage gated channels. We also show that these non-toxic Ca2+ loads are reversibly released from mitochondria through the PTP and the Na+/Ca2+ exchanger. Evoked Ca2+ transients have characteristic peak and shoulder features mediated by mitochondrial buffering and release. Depolarizing mitochondria with carbonyl cyanide m-chlorophenylhydrazone (CCCP, 5 microM) causes release of mitochondrial Ca2+ and prevents Ca2+ uptake. In CCCP, the magnitudes of evoked Ca2+ transients are increased, and the peak and shoulder features are eliminated. The PTP antagonist, cyclosporin A, (CSA, 2 microM) and the Na+/Ca2+ exchange blocker, clonazepam, (CLO, 20 microM) reversibly inhibited both the shoulder features of evoked Ca2+ transients and Ca2+ transients associated with CCCP application. We suggest that central neuronal mitochondria buffer and release Ca2+ through the PTP and Na+/Ca2+ exchanger during physiological Ca2+ signalling. We also suggest that CLO blocks both the PTP and the mitochondrial Na+/Ca2+ exchanger.

Animals↗
PubMed1985

Phosphate buffer and salt medium concentrations affect the inactivation of T4 phage by platinum(II) complexes.

The initial rate of inactivation of T4 phage by solutions of [Pt(NH3)2Cl2], [PtenCl2] and [Pten(H2O)2] (NO3)2 at fixed values of pH is strongly reduced by phosphate buffer, slightly reduced by acetate buffer and apparently not influenced by bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-Tris) buffer and HEPES buffer. The phosphate abolishes the antiphage activity of the platinum complexes probably by some sort of complex formation. This together with dimerization reactions qualitatively explains the tailing off of the phage inactivation rate. High concentrations of NaNO3 as the salt medium give increased phage inactivation rates, which are also strongly pH-dependent.

Buffers↗
PubMed1978

Use of a sulfate: hydrogen sulfate buffer to preserve urine for steroid analysis.

1. HCI, commonly used to preserve urine for steroid analysis, cannot safely be supplied to out of town patients. 2. An equimolar mixture of Na2SO4 and NaHSO4 is a safe alternative which may be used to protect specimens en route to the laboratory. 3. Addition of bisulfate buffer to urine (20 g/l) reduced the pH to 2.0-3.5 (31 specimens; 24 hr urine volumes, 560-3320 ml). 4. Little change in pH was observed when buffered urine (12 specimens) was kept at 30 degrees for 14 days (initial pH 2.62 +/- 0.38 (mean +/- standard deviation); PH after 14 days, 2.59 +/- 0.48). 5. Analysis of the creatinine, 17-oxosteroids, 17-oxogenicsteroids, estrogens, pregnanediol, androsterone + etiocholanolone and estriol in urine samples acidified to pH 2-3 with HCl, and in the same samples acidified with buffer, gave results which were indistinguishable from each other. 6. Almost no change in steroid and creatinine concentrations was observed in samples of buffered urine kept for 10 days at room temperature (20-26 degrees).

Buffers↗
PubMed1998

Quasi-linear pH gradients for chromatofocusing using simple buffer mixtures: local equilibrium theory and experimental verification.

Chromatofocusing utilizes internally generate, retained pH gradients to focus proteins into narrow chromatographic bands. One of the characteristics of current chromatofocusing methods is that they use expensive polyampholyte buffers containing large numbers of ill-defined components in order to generate linear or quasi-linear pH gradients. In addition to being costly to use, polyampholyte buffers also tend to associate with proteins and often yield irreproducible gradient shapes. In order to avoid the various difficulties associated with the use of polyampholyte buffers, this study investigates the use of mixtures of simple buffering species to generate quasi-linear pH gradients on a weak-base ion-exchange column packing. The ability of these gradients to separate protein mixtures was also investigated. To optimize the conditions used, a computer simulation method using a local equilibrium model developed that predicts the shape of the pH gradient. Several experiments were performed that demonstrate the usefulness of the method and the accuracy of the model.

Acid-Base Equilibrium↗
PubMed1985

Intracellular pH and contraction of isolated rabbit and cat papillary muscle: effect of superfusate buffering.

The influence of external buffering on surface pH (pHs), intracellular pH (pHi) and developed twitch tension was investigated in rabbit and cat papillary muscle. pHs and pHi were measured using single and double-barreled microelectrodes respectively. In 20 mM HEPES buffered solution, steady state pHi is close to that in control CO2/HCO-3 (25 mM HCO-3, 5% CO2) solution. pHs and developed tension also do not differ greatly from their control values. Decreasing the HEPES concentration to 5 mM, at constant external pH, lowers pHs considerably. The surface acidosis is associated with a small intracellular acidification; steady state pHi in 5 mM HEPES is always more acid than that in control CO2/HCO-3. A significant decrease in developed tension is also seen in 5 mM HEPES. Alteration of the superfusion velocity influences pHs only slightly. Stimulation of the muscle at high frequency is shown to increase surface acidification, the extent of which is dependent on the buffer concentration. The conclusion from the present experiments is that in papillary muscle external buffering influences intracellular pH and contraction via its effect on pHs.

Acid-Base Equilibrium↗
PubMed2003

Relationship between stability of folding intermediates and amyloid formation for the yeast prion Ure2p: a quantitative analysis of the effects of pH and buffer system.

The dimeric yeast protein Ure2 shows prion-like behaviour in vivo and forms amyloid fibrils in vitro. A dimeric intermediate is populated transiently during refolding and is apparently stabilized at lower pH, conditions suggested to favour Ure2 fibril formation. Here we present a quantitative analysis of the effect of pH on the thermodynamic stability of Ure2 in Tris and phosphate buffers over a 100-fold protein concentration range. We find that equilibrium denaturation is best described by a three-state model via a dimeric intermediate, even under conditions where the transition appears two-state by multiple structural probes. The free energy for complete unfolding and dissociation of Ure2 is up to 50 kcal mol(-1). Of this, at least 20 kcal mol(-1) is contributed by inter-subunit interactions. Hence the native dimer and dimeric intermediate are significantly more stable than either of their monomeric counterparts. The previously observed kinetic unfolding intermediate is suggested to represent the dissociated native-like monomer. The native state is stabilized with respect to the dimeric intermediate at higher pH and in Tris buffer, without significantly affecting the dissociation equilibrium. The effects of pH, buffer, protein concentration and temperature on the kinetics of amyloid formation were quantified by monitoring thioflavin T fluorescence. The lag time decreases with increasing protein concentration and fibril formation shows pseudo-first order kinetics, consistent with a nucleated assembly mechanism. In Tris buffer the lag time is increased, suggesting that stabilization of the native state disfavours amyloid nucleation.

Amyloid↗
PubMed1988

The H+/OH- flux localizes around the channel mouth in buffered solution.

The effects of buffering agents present in the solution on the fluxes of H+/OH- through a single channel were theoretically examined under physiological biochemical conditions, i.e. where the concentration of the buffer is in the millimolar range and the pH is around neutral. The solution of the diffusion equation for particles of finite lifetime showed that the convergence permeability can be defined as well as in the case of infinite lifetime, i.e. under non-buffered conditions. The presence of buffers resulted in localization of the H+/OH- fluxes around the channel mouth and enhanced leveling of the ionic concentrations. These results present the basis for arguments on vesicle kinetics and on local chemiosmotic models.

Animals↗
PubMed1992

Impact of site release vaginal pH buffer cream on introital colonization by gram-negative bacilli.

Colonization of the vaginal introitus by gram-negative bacilli is a critical event in the pathogenesis of bacteriuria and is promoted by an abnormal pH elevation. To determine whether a pH reduction might inhibit colonization we conducted a placebo controlled, crossover study of a site release vaginal pH buffer cream. A total of 12 premenopausal women with a history of urinary tract infection participated in the 12-week investigation. The mean introital pH during application of the placebo and buffer was 4.87 and 4.61, respectively (p less than 0.01). However, the percentage of positive introital cultures during application of the placebo and buffer (40.3 and 35.3, respectively) and the mean density of colonization (23,600 and 22,700 bacteria per ml., respectively) were not significantly different. The percentage of positive introital cultures and the mean colonization density were also not different during application of the placebo and buffer when the patients were retrospectively stratified as light and heavy colonizers. These data suggest that alteration of the introital pH within a range of 4.5 to 5.0 does not influence the growth of uropathogens on the vaginal mucosa.

Buffers↗
PubMed1993

Proteases of polymorphonuclear neutrophils: influence of different salts and buffers on pericellular lysis.

Polymorphonuclear neutrophils (PMN) contain neutral proteases which are cytochemically detectable on blood smears. The activity of these proteases can be visualized by pericellular lysis, i.e. through a disk like degradation of plasma and erythrocytes around centrally located neutrophils. This degradation is inhibited by protease inhibitors in a concentration-dependent manner. Striking differences in halo formation were found depending on the type of buffer and salt used and change of temperature. A halo formation was obtained in Tris-HCl buffer, pH 7.5 without salt, at room temperature (22 degrees C), and with short incubation periods (30 min). Borate buffer, pH 8.5, showed a comparable halo formation only at 37 degrees C, after addition of salts, and after longer incubation times (180 min). When different alkali chlorides were used, halo size increased with molecular mass. Optimum halo size was achieved at a concentration of 250 mM. Addition of MgCl2 and CaCl2 (250 mM) resulted in a small halo formation at 22 degrees C, but inhibited halo formation at 37 degrees C. The present study shows that the pericellular lysis caused around neutrophils by the release of proteases is affected by a complex interaction of salts, temperature, buffers and thermodynamic aspects, all of which should be evaluated carefully in these kinds of experiments.

Buffers↗
PubMed1996

Risk of aspirin-associated major upper-gastrointestinal bleeding with enteric-coated or buffered product.

BACKGROUND: Aspirin products are known to cause irritation and injury to the gastric mucosa. The belief that enteric-coated and buffered varieties are less likely to occasion major upper-gastrointestinal bleeding (UGIB) than plain aspirin was tested in data from a multicentre case-control study. METHODS: 550 incident cases of UGIB admitted to hospital with melaena or haematemesis and confirmed by endoscopy, and 1202 controls identified from population census lists, were interviewed about use of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) during the 7 days before the onset of bleeding (cases) or interview (controls). Relative risks of UGIB for each type of aspirin used regularly (at least every other day) were calculated overall, and according to dose, by multiple logistic regression, with control for age, sex, marital status, date, education, cigarette smoking, alcohol use, and use of NSAIDs. FINDINGS: The relative risks of UGIB for plain, enteric-coated, and buffered aspirin at average daily doses of 325 mg or less were 2.6, 2.7, and 3.1, respectively. At doses greater than 325 mg, the relative risk was 5.8 for plain and 7.0 for buffered aspirin; there were insufficient data to evaluate enteric-coated aspirin at this dose level. There were no important differences in risk attributable to the three aspirin forms according to bleeding site (gastric vs duodenal), or when users of NSAIDs were excluded. INTERPRETATION: Use of low doses of enteric-coated or buffered aspirin carries a three-fold increase in the risk of major UGIB. The assumption that these formulations are less harmful than plain aspirin may be mistaken.

Adolescent↗
PubMed2003

Short-term fluoride and cations release from polyacid-modified composites in a distilled water, and an acidic lactate buffer.

Fluoride and various cations release from three commercial compomers (Dyract, Dyract AP and Compoglass-F) and a resin-modified glass ionomer (Fuji-II LC) as a control were measured up to 7 days in distilled water and 0.01M lactate buffer solution with pH 4.1. The surface morphological change before and after the release experiment was observed with a scanning electron microscopy. Fluoride, aluminum and strontium ions were released from Dyract, Dyract AP and Fuji-II LC much more in the lactate buffer than in the distilled water. With compoglass-F containing barium instead of strontium, barium was released in the same way. X-ray diffraction analysis confirmed that fluoride release from Dyract and Dyract AP was derived mainly from SrF(2) contained in Dyract and Dyract AP. However, fluoride release from Compoglass-F was derived from glassy phase though it contained much fluoride as YbF(3). Cumulative release amount of each species versus square root of time plot showed good linearity, indicating that the dissolution was controlled by the diffusion mechanism. The surface characteristics of disks for each material, which were immersed in the lactate buffer, were quite different from the surface before and after immersion in deionized water, especially with Dyract. After immersion in the lactate buffer, many voids which were left after dissolution of the filler particle, were seen obviously on the surface of Dyract.

Aluminum↗
PubMed2003

Effects of calcium buffers and calbindin-D28k upon histamine-induced calcium oscillations and calcium waves in HeLa cells.

The effects of the artificial Ca(2+) buffers EGTA and BAPTA upon histamine-induced Ca(2+) oscillations and calcium waves were studied in HeLa cells. These events were also examined in HeLa cell lines transfected with the intracellular calcium-binding protein calbindin-D28k (CaBP; HeLa-CaBP) or the pCINeo vector alone (HeLa-pCINeo). High concentrations of the Ca(2+) indicators fluo-3 and fura-2 significantly influenced the oscillatory pattern of intracellular Ca(2+) in HeLa-pCINeo cells exposed to 1 microM histamine. Loading cells with low concentrations of the cell-permeant esters of the artificial Ca(2+)-buffers EGTA or BAPTA, resulted in fewer cells with a distinct "baseline" oscillatory pattern, and loading with higher concentrations of BAPTA almost completely abolished them. In HeLa-CaBP cells, stimulation with 1 microM histamine resulted in individual Ca(2+) spikes that had a flattened profile when compared to control cells; peak [Ca(2+)](i) was lowered, the rate of increase in [Ca(2+)](i) was slower and transients were prolonged. When compared to HeLa-pCINeo cells, loading with EGTA or BAPTA, or transfection of CaBP, significantly reduced the propagation velocity (by up to 60%) of Ca(2+) waves induced by exposure to 100 microM histamine. We conclude that intracellular Ca(2+) buffering exerts a significant influence on global Ca(2+) responses in HeLa cells and the propagation of Ca(2+) waves that underlie them. The relative effectiveness of different Ca(2+) buffers, including CaBP, appears to be particularly dependent upon the rapidity of their binding kinetics, with BAPTA being the most effective.

Aniline Compounds↗