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PubMed2003

Effect of buffers on the properties of microbicidal hydrogels containing monoglyceride as the active ingredient.

Hydrogel formulations containing the monoglyceride monocaprin have shown potent microbicidal activity against several sexually transmitted viruses and bacteria. It is recommended that formulations for preventing infection in the vagina have a low pH as the HIV virus is inactivated at low pH. The object of the work was to investigate how incorporation of buffers into the hydrogel formulations affects physicochemical properties and microbicidal activity of the active substance. Two series of gels were formulated using carbomer (Carbopol 934) and sodium carboxymethylcellulose (NaCMC) as gel-forming agents. The presence of buffers in the gels caused a lowering in gel viscosity, with carbomer gels being more sensitive to buffer presence than NaCMC gels. To obtain viscosity similar to that of a gel without buffer, the amount of polymer needs to be increased. An increase in the amount of NaCMC by 60-70% is needed to obtain the same viscosity as in gel without buffers; but for carbomer, the amount of polymer needs to be doubled. It appears that the effect of maleate buffer on NaCMC gel formation is greater than that of the citrate/lactate buffer; but for carbopol gels, the effects of the buffer systems tested on gel viscosity were equal. The virucidal activity of NaCMC gel buffered with citrate/lactate buffer against herpes simplex virus type 1 and HIV was not reduced by the presence of buffer. The results show that the presence of buffers in the hydrogel formulations affects gel viscosity, but the virucidal effect of the active compound, monocaprin, is not diminished.

Acrylic Resins↗
PubMed1993

Calcium buffer injections delay cleavage in Xenopus laevis blastomeres.

Microinjection of calcium buffers into the two-cell Xenopus laevis embryo delays cell division in a dose-dependent manner. Four calcium buffers in the BAPTA series with different affinities for calcium were used to distinguish between a localized calcium gradient regulating cleavage and the global calcium concentration regulating this event. DibromoBAPTA (Kd = 1.5 microM) was found to delay cleavage at the lowest intracellular concentration (1.3 mM) of the four buffers tested. The effectiveness of the calcium buffers was dependent upon the buffer dissociation constant but not in a linear fashion. The concentration of buffer required to delay cleavage increased as the buffer's dissociation constant shifted above or below that of the optimum buffer, dibromoBAPTA. This relationship between a calcium buffer's effectiveness at delaying cleavage and its calcium affinity provides support for the hypothesis that a calcium concentration gradient is required for normal cell cycle progression (Speksnijder, J. E., A. L. Miller, M. H. Weisenseel, T.-H. Chen, and L. F. Jaffe. 1989. Proc. Natl. Acad. Sci. USA. 86:6607-6611). DibromoBAPTA was also injected with two different amounts of coinjected calcium to test the possibility that the free calcium concentration of the buffer solution is the important parameter for delaying cleavage. However, we found that changes in buffer concentration have a much stronger effect than changes in the free calcium concentration. This observation supports the hypothesis that BAPTA-type buffers exert their effect by shuttling calcium from regions of high concentration to those of lower concentration, reducing any calcium concentration gradients present in the Xenopus embryo.

Animals↗
PubMed2001

Initial effect of sodium bicarbonate on intracellular pH depends on the extracellular nonbicarbonate buffering capacity.

OBJECTIVE: The effect of sodium bicarbonate on intracellular pH under conditions close to those in vivo, with both bicarbonate and nonbicarbonate buffering systems, is unknown. We postulated that this effect depends on the nonbicarbonate buffering capacity because the alkali-induced back-titration of these buffers results in a concentration-dependent release of CO2 in the extracellular space, leading to a decrease in intracellular pH. DESIGN: The study was conducted in two stages. First, human hepatocytes were perfused with pH 7 bicarbonate-buffered medium (5 mM HCO3-, 20 torr Pco2) containing no nonbicarbonate buffer or small amounts (5 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES]) or large amounts (20 mM HEPES) of nonbicarbonate buffer. Second, the changes in intracellular pH of hepatocytes placed in acidotic human blood (pH 7, 5 mM HCO3-, 20 torr Pco2) at three hematocrits (40%, 20%, and 5%) were measured. SETTING: Research laboratory at a medical university. SUBJECTS: Cryopreserved human hepatocytes thawed the day before the experiments. INTERVENTIONS: Sodium bicarbonate was infused for 10 mins to increase the HCO3- concentration from 5 to 30 mM. In the second part, 20 mM sodium bicarbonate was added directly to the blood bathing the cells. MEASUREMENTS AND MAIN RESULTS: The intracellular pH was measured with the pH-sensitive fluorescent dye bis-carboxyethyl carboxy-fluorescein in its esterified form, acetoxy-methyl ester, by using a single-cell imaging technique. Gas analyses were performed before and during the sodium bicarbonate load. Sodium bicarbonate caused a decrease in intracellular pH with all media except the artificial medium containing no HEPES. This decrease was small in media with low nonbicarbonate buffering capacity (5 mM HEPES and 5% hematocrit blood) and large in media with high nonbicarbonate buffering capacity (20 mM HEPES and 40% hematocrit blood). The change in intracellular pH was linked closely to the change in Pco2 caused by the sodium bicarbonate. CONCLUSIONS: The effect of sodium bicarbonate on intracellular pH depends on changes in Pco2 in the medium bathing the cells. The increase in Pco2 is correlated with the extracellular nonbicarbonate buffering capacity because of the release of H+ ions coming from the back-titration of these buffers. We conclude that sodium bicarbonate may exacerbate cell acidosis under buffering conditions close to those in vivo and that the initial changes in cell pH caused by sodium bicarbonate depend on the extracellular nonbicarbonate buffering capacity.

Blood↗
PubMed1995

The effects of warming and buffering on pain of infiltration of lidocaine.

OBJECTIVES: To investigate the effects of warming vs buffering, and warming with buffering, on the pain of lidocaine infiltration. METHODS: A randomized, double-blind clinical trial was conducted using volunteers aged 18 years or more and without an allergy to lidocaine. The study consisted of two parts, each comparing two solutions. The solutions for Part I were warm (38.9 degrees C; 102 degrees F) plain lidocaine and room-temperature buffered lidocaine. Warm buffered lidocaine and room-temperature buffered lidocaine were used for Part II. The subjects received two standardized 0.5-mL intradermal injections, one study solution in each forearm. Immediately after each injection, pain was assessed using a 100-mm visual analog pain scale. Pain scores were analyzed by the sign test, with significance defined as p < 0.05. RESULTS: Part I (n = 10): Nine subjects reported room-temperature buffered lidocaine to be less painful than warm plain lidocaine. Mean pain scores were 28 mm lower for room-temperature buffered lidocaine than they were for warm plain lidocaine (p < 0.01). Part II (n = 24): Eleven subjects found warm buffered lidocaine to be the least painful, 11 reported room-temperature buffered lidocaine to be the least painful, and two reported no difference. A mean pain score difference of 2.1 mm favoring warm buffered lidocaine was not statistically significant. Part II had a power of 80% to detect a 10-mm difference between the two solutions at alpha = 0.05. CONCLUSION: To reduce the pain of lidocane infiltration, buffering is more effective than warming. Warming does not enhance buffering.

Adult↗
PubMed1989

The effect of extracellular weak acids and bases on the intracellular buffering power of snail neurones.

1. Intracellular pH (pHi) was measured in snail neurones using pH-sensitive glass microelectrodes. The influence of externally applied weak acids and bases on the total intracellular buffering power (beta T) was investigated by monitoring the pHi changes caused by the intracellular ionophoretic injection of HCl. 2. In the absence of weak acids or bases a reduction in the extracellular HEPES concentration had no effect on pHi or on beta T. It did, however, reduce slightly the rate of pHi recovery following HCl injection. 3. The presence of CO2 greatly increased beta T. However, as predicted for an open buffer system, the contributions to intracellular buffering by CO2 (beta CO2) decreased as pHi decreased. 4. When added to the superfusate, procaine, 4-aminopyridine, trimethylamine and NH4Cl (1-10 mM) all increased steady-state pHi. Procaine was fastest at increasing pHi and 4-aminopyridine the slowest. All four of these weak bases increased beta T. 5. The intracellular buffering action by these weak bases varied. HCl injection in the presence of procaine usually resulted in steady-state pHi changes with no pHi transients. In the presence of the other three weak bases HCl injections resulted in intracellular acidifications which were followed by pHi recovery-like transients. However, these were not blocked by SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) or by CaCl2 and I thus conclude that these transients were as a result of slow or incomplete intracellular buffering by the weak bases. 6. In many cells there was a good correlation between the measured contributions to intracellular buffering by the weak bases (beta base) and those predicted assuming a simple two-compartment open system. In all cases, as predicted, beta base increased as pHi decreased. 7. I found a clear relationship between the concentration of external buffer (HEPES) and the rate at which weak bases, applied to the superfusate, were able to increase pHi. The greater the extracellular buffer concentration the greater was the speed of intracellular alkalinization. 8. Lowering the extracellular buffer concentration reduced the efficiency of intracellular buffering by weak bases in response to an intracellular acid load. HCl injection in the presence of weak base caused a larger initial intracellular acidification if the extracellular HEPES concentration was reduced. 9. In conclusion, both weak acids and weak bases can make very large, pHi-dependent contributions to intracellular buffering by way of open buffer systems.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Aminopyridine↗
PubMed2001

An estimation of buffer values of human whole blood by titration experiment under the open condition for carbon dioxide gas.

We studied the buffer mechanism of human whole blood by means of a titration experiment under the open condition in which blood is exposed to carbon dioxide gas. Van Slyke proposed the theory of a blood buffer mechanism in 1922 (J Biol Chem 52: 525-570). However, his theory is not applicable to our experiments because it did not take into consideration the effects of changes in volume and the concentration of bicarbonate ion in the buffer as a result of titration. We studied the blood buffer mechanism theoretically by using graphical representation and developed a theory, which we then applied to our experiments. Buffer value, as defined by Van Slyke, is the quantity of the base deltan(OH)/V in the sample without volume change (in gram equivalent per liter) required to change one unit in pH. Based on our blood buffer theory, we obtain the experimental buffer value betaprime prime or minute, which is the reciprocal slope of the dpH-bdV/V (deltan(OH) in the sample with volume change) relation during a titration experiment at a constant carbon dioxide pressure (pCO(2)) with volume change dV and an isotonic NaOH concentration of b = 0.15 M. The true buffer value beta under physiological conditions is estimated to be beta = deltan(OH)/VdpH = betaprime prime or minute (0.15 minus sign [HCO(3)(minus sign)])/0.15. Here, [HCO(3)(minus sign)] is the concentration of bicarbonate ion in blood that can be calculated by using the Henderson-Hasselbalch equation. We determined the agreement of our experimental results with the theoretical results and estimated the quantity m(A) of the buffer component, except for the bicarbonate buffer, and the dissociation constant K(A) of the buffer reaction in a neutral pH range. The true buffer value of whole blood can be estimated by using our theory and the results of precise experiments.

Acid-Base Equilibrium↗
PubMed2003

A VSA-based strategy for placing conservation buffers in agricultural watersheds.

Conservation buffers have the potential to reduce agricultural nonpoint source pollution and improve terrestrial wildlife habitat, landscape biodiversity, flood control, recreation, and aesthetics. Conservation buffers, streamside areas and riparian wetlands are being used or have been proposed to control agricultural nonpoint source pollution. This paper proposes an innovative strategy for placing conservation buffers based on the able source area (VSA) hydrology. VSAs are small, variable but predictable portion of a watershed that regularly contributes to runoff generation. The VSA-based strategy involves the following three steps: first, identifying VSAs in landscapes based on natural characteristics such as hydrology, land use/cover, topography and soils; second, targeting areas within VSAs for conservation buffers; third, refining the size and location of conservation buffers based on other factors such as weather, environmental objectives, available funding and other best management practices. Building conservation buffers in VSAs allows agricultural runoff to more uniformly enter buffers and stay there longer, which increases the buffer's capacity to remove sediments and nutrients. A field-scale example is presented to demonstrate the effectiveness and cost-effectiveness of the within-VSA conservation buffer scenario relative to a typical edge-of-field buffer scenario. The results enhance the understanding of hydrological processes and interactions between agricultural lands and conservation buffers in agricultural landscapes, and provide practical guidance for land resource managers and conservationists who use conservation buffers to improve water quality and amenity values of agricultural landscape.

Agriculture↗
PubMed1999

US National Park Buffer Zones: Historical, Scientific, Social, and Legal Aspects.

/ This review will trace the evolution of beyond boundary/buffer zone thinking and policy responses in the US National Park Service (NPS); address buffer zone science, benefits, and limitations; examine pertinent legal and social concerns; highlight some agency attempts to create buffer zone-like areas; and propose highlights of a protected area strategy, with buffer zones and corridors as one component. Some findings follow. The need to expand national parks to accommodate large ungulate movement began in the late 1800s, but the recognition that such land was also needed to thwart human impacts such as poaching surfaced in the 1930s. External park buffer zone recommendations by 1930s park scientists were not implemented, and other related adopted policy forgotten, supporting the belief that great insight can be discovered in forgotten institutional history. Buffer zones can remedy some impacts but not others, but their benefits are multiple and underappreciated. The science of buffer zones is very immature and deserves more attention. A present primary obstacle to creating park buffer zones and connecting corridors is a social climate opposing federal initiatives that may intrude on the rights of private landowners. Some proactive NPS bufferlike activity examples are reviewed, but there were none where permanent, complete, effective nonlegislated park buffer zones, derived from nonfederal property, circumscribed large natural area parks. The need for buffer zones and corridors may be a symptom of inadequate regional planning. Options to create buffer zones from private and federal land are outlined. A comprehensive, overall protected area strategy must include more than just buffer zones, with highlights provided. Because optimal regional planning for US national parks is now thwarted by land-use politics, American society must soon decide what is most crucial to future well-being. KEY WORDS: Buffer zone; Reserve; Boundary; Policy; Planning

Journal Article↗
PubMed2006

Retention of ionisable compounds on high-performance liquid chromatography XVII. Estimation of the pH variation of aqueous buffers with the change of the methanol fraction of the mobile phase.

The use of methanol-aqueous buffer mobile phases in HPLC is a common election when performing chromatographic separations of ionisable analytes. The addition of methanol to the aqueous buffer to prepare such a mobile phase changes the buffer capacity and the pH of the solution. In the present work, the variation of these buffer properties is studied for acetic acid-acetate, phosphoric acid-dihydrogenphosphate-hydrogenphosphate, citric acid-dihydrogencitrate-hydrogencitrate-citrate, and ammonium-ammonia buffers. It is well established that the pH change of the buffers depends on the initial concentration and aqueous pH of the buffer, on the percentage of methanol added, and on the particular buffer used. The proposed equations allow the pH estimation of methanol-water buffered mobile phases up to 80% in volume of organic modifier from initial aqueous buffer pH and buffer concentration (before adding methanol) between 0.001 and 0.01 mol L(-1). From both the estimated pH values of the mobile phase and the estimated pKa of the ionisable analytes, it is possible to predict the degree of ionisation of the analytes and therefore, the interpretation of acid-base analytes behaviour in a particular methanol-water buffered mobile phase.

Chromatography, High Pressure Liquid↗
PubMed1990

IgE receptor-mediated phosphatidylinositol hydrolysis and exocytosis from rat basophilic leukemia cells are independent of extracellular Ca2+ in a hypotonic buffer containing a high concentration of K+.

In isotonic buffer, IgE receptor-mediated exocytosis from rat basophilic leukemia cells is dependent on extracellular Ca2+, with half-maximal degranulation requiring 0.4 mM Ca2+. No significant exocytosis occurs in the absence of extracellular Ca2+. This absolute requirement for Ca2+ is eliminated by suspending the cells in a hypotonic buffer containing 60 to 80 mM K+; Na+ cannot substitute for K+. Optimal Ca2(+)-independent exocytosis occurs in a buffer containing 20 mM dipotassium Pipes, pH 7.1, 40 mM KCl, 5 mM glucose, 7 mM Mg acetate, 0.1% BSA, and 1 mM EGTA. The cells maintain this Ca2(+)-independent exocytosis even if they are preincubated with 1 mM EGTA for 40 min at 37 degrees C before triggering. Exocytosis is eliminated as isotonicity is approached by adding sucrose, NaCl, KCl, or potassium glutamate to the buffer. Quin 2 fluorescence measurements reveal only a very small rise in [Ca2+]i when the cells are triggered in hypotonic buffer in the absence of extracellular Ca2+ and the presence of 1 mM EGTA. In isotonic buffer, degranulation does not occur under conditions that lead to such a small rise in [Ca2+]i. Sustained IgE receptor-mediated phosphatidylinositol hydrolysis, which is also Ca2+ dependent in isotonic buffer, becomes independent of Ca2+ in the hypotonic buffer. In fact, the rate of phosphatidylinositol hydrolysis in hypotonic buffer in the absence of Ca2+ (and presence of 1 mM EGTA) is twice that observed in isotonic buffer in the presence of 1 mM Ca2+. These data show that in hypotonic buffer, the requirement of IgE receptor-mediated PI hydrolysis for extracellular Ca2+ is eliminated, and degranulation proceeds with a [Ca2+]i of 0.1 microM, the baseline level of [Ca2+]i found in resting cells. These results are consistent with the hypothesis that, in isotonic buffer, the Ca2+ requirement for mast cell degranulation is for the generation of second messengers via hydrolysis of membrane phosphatidylinositols.

Animals↗
PubMed1999

pK-matched running buffers for gel electrophoresis.

Electrophoresis through agarose and polyacrylamide-type gels is the standard method to separate, identify, and purify nucleic acids. Properties of electrophoresis buffers such as pH, ionic strength, and composition affect performance. The buffers in use contain a weak acid or weak base buffered by a compound with a dissimilar pK. Herein, three pK-matched buffers were developed, each containing two effective buffering components: one weak base and one weak acid which have similar pKa at 25 degrees C (within 0.3 pK units): (i) Ethanolamine/Capso, pH 9.6; (ii) triethanolamine/Tricine, pH 7.9; and (iii) Bis-Tris/Aces, pH 6.7. On agarose gels, the buffers in various concentrations were tested for separation of double-stranded DNA fragments with various DNA markers, agarose gel concentrations, and field strengths. Mobility was inversely proportional to the logarithm of molecular weight. The buffers provided high resolution without smearing at more dilute concentration than is possible with standard TAE (Tris/acetate, pH 8.0) or TBE (Tris/borate, pH 8.3) buffers. The buffers were also tested in 7 M urea denaturing LongRanger sequencing gels and in nondenaturing polyacrylamide SSCP gels. The pK-matched buffers provide good separation and high resolution, at a broad range of potential pH values. In comparison to TAE and TBE, pK-matched buffers provide higher voltage and current stability, lower working concentration, more concentrated stock solutions (up to 200x), and lower current per unit voltage, resulting in less heat generation.

Buffers↗
PubMed1995

Comparison of the buffer capacity of endocytotic vesicles, lysosomes and cytoplasm in cells derived from the proximal tubule of the kidney (opossum kidney cells).

During transepithelial acid-base transport cells of the proximal tubule of the kidney have to maintain a relative constant intracellular pH. Herein cellular buffer capacity plays an important role. We measured vesicular buffer capacity in proximal tubule-derived opossum kidney cells and compared it with cytoplasmic buffer capacity to determine the possible importance of vesicular buffer capacity for cellular pH homeostasis. Under HCO(3-)-free conditions endocytotic vesicular buffer capacity was 43 +/- 4 mmol.l-1. delta pH-unit-1 (n = 7) and exceeded cytoplasmic buffer capacity (19 +/- 3 mmol.l-1.delta pH-unit-1; n = 7) significantly. Lysosomal buffer capacity was 19 +/- 6 mmol.l-1.delta pH-unit-1; (n = 5). Inhibition of vesicular H(+)-ATPase using bafilomycin A1 led to a dramatic increase of vesicular pH but to a decrease of cytoplasmic pH indicating the importance of organellar buffer systems. We estimated that endocytotic buffer capacity accounts for approximately 23% of cellular buffer capacity under our experimental condition and thus, impairment of endosomal acidification may affect cytoplasmic pH indeed. From our results we conclude that endocytotic vesicles have a large buffer capacity and might play a role in cellular pH homeostasis.

Ammonium Chloride↗
PubMed1983

Effects of NaOH-PIPES buffer used in aldehyde fixative on alkaline phosphatase activity in rat hepatocytes.

Effects of NaOH-PIPES buffer used as a vehicle for aldehyde fixative on alkaline phosphatase (ALPase) activity demonstrated cyto- and biochemically were compared with those of routinely used cacodylate buffer. The reaction products showing ALPase activity demonstrated ultracytochemically were confined to the bile canalicular membranes when cacodylate buffer (0.1 M) was used. However, when PIPES buffer (0.03 M or 0.1 M) was used, the activity was observed on whole membranes of hepatocytes. The activities of the sinusoidal, lateral and bile canalicular membranes were completely suppressed by an addition of 2.5 mM levamisole. Moreover, the same results were obtained when HEPES or low concentration of cacodylate buffer (0.01 M) was used. Biochemical estimation revealed that much higher activity was retained when PIPES or HEPES buffer was used as compared with that when cacodylate buffer was used. Maximum preservation of ALPase activity was obtained when PIPES buffer was used. Cacodylate buffer showed an inhibitory effect on the hepatic ALPase activity in proportion to the buffer concentration. In conclusion, PIPES buffer preserves the alkaline phosphatase activity much better and is a better vehicle for the aldehyde fixatives in alkaline phosphatase cytochemistry.

Aldehydes↗
PubMed1981

Role of luminal buffers in renal tubular acidification.

The acidification of kinetics of artificial solutions containing buffers of different permeancy were studied in rat proximal tubules by means of stationary microperfusion techniques. Luminal pH changes were measured by antimony microelectrodes and used to calculate net rates of acidification and the approach to steady-state pH levels. For most buffer species, tracer efflux out of the lumen was compared with changes in buffer concentration as derived from calculations based on the Henderson Hasselbalch equation. Steady-state luminal pH was similar for most buffer systems studied. However, secretory hydrogen ion fluxes into the lumen were significantly higher for permeant than for less permeant buffers. The most likely explanation is that permeant buffers behave as "open" systems maintaining constant low diffusible acid levels in the lumen, whereas impermeant buffers behave as "closed" systems in which non-ionized acid levels are maintained at higher levels. A behavior consistent with this thesis was directly demonstrated for glycodiazine and, to a lesser degree, for DMO. In contrast, phosphate and creatinine behave like buffers in a "closed" system. Characteristics of proximal tubular acidification, of buffer reabsorption, and the effect thereupon of carbonic anhydrase inhibitors are satisfactorily explained by an essential role of (1) hydrogen ion secretion, (2) pK differences, and (3) different permeance of the non-ionized buffer species. However, specific transport mechanisms may, in addition, also contribute to differences in transepithelial buffer movement.

Animals↗
PubMed2004

Comparison of the effect of different pH buffering techniques on the toxicity of copper and zinc to Daphnia magna and Pseudokirchneriella subcapitata.

During the time-course of ecotoxicity tests with algae and chronic (reproductive) toxicity tests with daphnids, in which algae are present as a food source, pH can dramatically increase due to photosynthetic activity. As pH changes can significantly affect metal speciation and thus its bioavailability, it may be necessary to buffer the pH of the exposure medium. One class of buffers (Good's N-subtituted aminosulfonic acids) are increasingly being used in biological and chemical applications, including ecotoxicity testing. However, the potential effect of these buffers on metal toxicity has, so far, scarcely been examined. In this study we investigated if MOPS (3-N morpholino propane sulfonic acid) affected the toxicity of copper and zinc to two standard test organisms: the cladoceran Daphnia magna and the green alga Pseudokirchneriella subcapitata. First, we demonstrate that up to a concentration of 750 mg l(-1) (which proved to be sufficient for pH buffering) MOPS did not affect 21-day net reproduction of D. magna or the 72-h population growth of P. subcapitata. Second, we conducted bioassays in copper and zinc spiked standard media for the pH range 6-8. For D. magna the possible effect of 750 mg l(-1) MOPS on acute copper and zinc toxicity was investigated by performing parallel 48-h toxicity tests in NaHCO3 and MOPS buffered test media. Seventy-two hour growth inhibition assays with P. subcapitata were performed in parallel in MOPS and NaHCO3 buffered test media and in test media with daily manual pH adjustment with HCl. For daphnids no significant differences in copper and zinc toxicity were observed between MOPS or NaHCO3 buffered test media. For algae no significant differences in metal toxicity were observed between MOPS and HCl buffered media, but in test media buffered with NaHCO3 an increased copper and zinc toxicity was observed as a consequence of pH increases during the test. Clearly, the results of this study demonstrate the importance of pH buffering in metal toxicity testing and the suitability of the MOPS buffer for that purpose.

Animals↗
PubMed1995

Assessment of the antielastase activities in bronchoalveolar lavage fluid: effect of assay buffer ionic strength.

OBJECTIVE: To evaluate the effect of assay buffer ionic strength on assessment of the antielastase activities in bronchoalveolar lavage fluid. METHOD: An improved assay protocol in which elastase (in Tris-HCI buffer) is added to increasing volumes of test samples (made up to equal volume with phosphate-buffered saline) was used. RESULTS: The percent NE activity inhibited by BALF decreased with increasing NaCl concentration of the buffer. Inhibition of pancreatic elastase (PE) was not affected. One hundred percent inhibition of NE by pure AAT and SLPI standards occurred at molar ratios of 0.91 +/- 0.03 for AAT-to-NE and 0.83 +/- 0.02 for SLPI-to-NE when assayed in buffer with < or = 0.15 mol NaCl/L, compared to ratios of 0.99 +/- 0.02 and 1.06 +/- 0.02, respectively, for assays in buffers with 0.50-1.00 mol NaCl/L (p < 0.05 for AAT-to-NE; p < 0.02 for SLPI-to-NE). The AAT-to-PE molar ratio at 100% inhibition of PE was not affected. Assays in buffer with < or = 0.15 mol NaCl/L indicated that 86.9 +/- 4.1% of AAT and 100.9 +/- 4.9% of SLPI in BALF were active against NE, while assays in buffer with 0.50-1.00 mol NaCl/L showed that 84.4 +/- 3.5% of AAT and 81.6 +/- 5.9% of SLPI present were active. AAT inhibited NE and PE equally only in buffer with 0.50-1.00 mol NaCl/L. CONCLUSIONS: The results of assays of BALF antielastase activities depend on the assay buffer NaCl concentration, which may account for the conflicting reports in the literature. The buffer with 0.50-1.00 mol NaCl/L appear to be optimal for valid quantitation of anti-NE activities in BALF.

Adsorption↗
PubMed1988

Determination of buffering capacity of rat myocardium during ischemia.

To determine the buffering capacity of ischemic rat myocardium, lactate production was altered by glycogen depletion prior to total global ischemia. Lactate production was monitored by 1H-NMR spectroscopy in perfused rat hearts and determined by enzymatic assay of freeze-clamped tissue extracts. Intracellular pH was measured by 31P-NMR spectroscopy. The relationship between total lactate produced and pH varied considerably, depending on the final pH reached. At pH greater than 6.4 this relationship is linear with a total buffering capacity (delta lactate/delta pH) of 25 mumol H+/g wet weight per pH unit. At lower pH values (pH less than 6.4), the total buffering capacity increases progressively. Since ischemia is invariably accompanied by ATP and phosphocreatine (PCr) hydrolysis, the proton production/consumption during high-energy phosphate hydrolysis must be considered when evaluating the intrinsic buffering capacity of the myocardium against proton loads produced by lactate production from glucose and glycogen. Schemes are presented which allow an estimation of the contribution of ATP and PCr hydrolysis and the buffering by the CO2/HCO3- system during ischemia. At pH greater than 6.4, the majority (about 60%) of buffering is due to hydrolysis of adenosine triphosphate, phosphocreatine in the heart, and neutralization of sodium bicarbonate in the perfusate. At pH less than 6.4 an increasing proportion of cardiac buffering is from intrinsic cardiac buffers, most likely from intracellular proteins. After correction for these contributions to the observed total cardiac buffering capacity, the intrinsic buffering capacity of the myocardium can be accounted for by a high capacity (170 mumol/g wet weight) but low pKa (5.2) buffering system.

Adenosine Triphosphate↗
PubMed2006

Do sperm contribute to the buffering capacity of steelhead trout (Oncorhynchus mykiss) semen?

The motility of salmonid sperm is pH-sensitive and the buffering capacity of the seminal plasma is low. The objective of the present study was to determine the extent to which sperm contribute to the buffering capacity of whole steelhead (Oncorhynchus mykiss) semen. To determine the buffering capacity, semen and seminal plasma samples were titrated with HCl and pH measurements taken at 1-2 min. The buffering capacity of semen was not different from that of seminal plasma over the pH range 7.5 to 8.5 and was approximately 15% to 20% less over the range 6.0 to 7.0. Comparable results were obtained for the semen and seminal plasma of the chinook salmon (Oncorhynchus tshawytscha). To assess whether the intracellular environment could influence the buffering capacity, the effects of cell disruption with n-butanol and Triton X-100 (TX-100) were determined. Over the pH range 7.5 to 8.5, the presence of n-butanol or TX-100 resulted in a doubling of the buffering capacity of the semen; TX-100, but not n-butanol, increased semen buffering capacity over the pH range 6.0 to 7.0. To determine whether the sperm's intracellular compartment might contribute to the buffering capacity over a longer duration, semen and seminal plasma samples were acidified with HCl and the pH measured over several hours. These data suggest that intact sperm contribute no more than about 25% to the buffering capacity of whole semen. The buffering capacity of steelhead semen and seminal plasma were comparably and modestly temperature sensitive. The results suggest that the sperm may contribute to the buffering capacity of the semen over a physiological pH range, however, if so, the effect is relatively small.

Animals↗