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PubMed2000

pH(i) responses to osmotic cell shrinkage in the presence of open-system buffers.

Changes in plasma volume in vivo cause rapid changes in extracellular pH by altering the plasma bicarbonate concentration at a constant Pco(2) (Garella S, Chang BS, and Kahn SI. Kidney Int 8: 279, 1975). Few studies have examined the possibility that changes in cell volume produce comparable changes in intracellular pH (pH(i)). In the present study, alveolar macrophages were exposed to hyperosmotic medium in the absence or presence of the open-system buffers CO(2)-HCO(3)(-), propionic acid-propionate, or NH(3)-NH(4)(+). In the absence of open-system buffers, exposure to twice-normal osmolarity (2T) produced a slow cellular alkalinization [change in pH(i) (DeltapH(i)) approximately 0.38; exponential time constant (tau) approximately 120 s]. In the presence of 5% CO(2), 2T caused a biphasic pH(i) response: a rapid increase (DeltapH(i) approximately 0.10, tau approximately 15 s) followed by a slower pH(i) increase. Identical rapid pH(i) increases were produced by 2T in the presence of propionic acid (20 mM). Conversely, 2T caused a rapid pH(i) decrease (DeltapH(i) approximately -0.21, tau approximately 10 s) in the presence of NH(3) (20 mM). Thus osmotic cell shrinkage caused rapid pH(i) changes of opposite direction in the presence of a weak acid buffer (contraction alkalosis with CO(2) or propionic acid) vs. a weak base buffer (contraction acidosis with NH(3)). Graded DeltapH(i) were produced by varying extracellular osmolarity in the presence of open-system buffers; osmolarity increases of as little as 5-10% produced significant DeltapH(i). The rapid pH(i) responses to 2T were insensitive to inhibitors of membrane H(+) transport (ethylisopropylamiloride and bafilomycin A(1)). The results are consistent with shrinkage-induced disequilibria in the total cellular buffer system (i.e., intrinsic buffers plus added weak acid-base buffer).

Amiloride↗
PubMed1990

Buffer agents do not reverse intramyocardial acidosis during cardiac resuscitation.

We investigated the effects of carbon dioxide-producing and carbon dioxide-consuming buffers on intramyocardial pH and on cardiac resuscitability. In 29 pigs, intramyocardial pH was continuously measured with a glass electrode advanced into the midmyocardium of the posterior left ventricle through a diaphragmatic window. Ventricular fibrillation (VF) was electrically induced by alternating current applied to the epicardium of the left ventricle. After 3 minutes of VF, precordial compression was begun and continued for an interval of 8 minutes. Sodium bicarbonate (a carbon dioxide-generating buffer), Carbicarb (a carbon dioxide-consuming buffer), and hypertonic sodium chloride (control solution) were infused into the right atrium during cardiac resuscitation. Defibrillation was attempted by transthoracic direct-current shock after 11 minutes of VF. Intramyocardial pH progressively decreased from an average value of 7.26 before VF to 6.87 before infusion of buffers. Systemic circulation and great cardiac vein pH significantly increased after administration of the two buffer agents. However, intramyocardial pH continued to decline to an average of 6.62 after 11 minutes of VF, and this decline was not altered by either buffer solution or by the saline control. As in previous studies, resuscitability was closely related to coronary perfusion pressure at the time of direct-current countershock but not to pH. Accordingly, the rationale of reversing acidosis by the administration of these buffer agents is not supported. Even more important, neither carbon dioxide-consuming nor carbon dioxide-producing buffers altered myocardial acidosis or improved myocardial resuscitability under controlled experimental conditions of cardiac arrest.

Acidosis↗
PubMed1988

Analysis of the buffering systems in dental plaque.

A semi-micro method was used for investigation of the buffering properties of whole plaque, plaque fluid, and washed plaque bacteria. Artifacts associated with titration of samples containing live bacteria were noted and their effects estimated. All three sample types showed minimal buffering in the region of neutrality, with much stronger buffering in the regions pH 4-5.5 and pH 8-9. For the range pH 4-7, almost 90% of the total buffer capacity of plaque appeared to be accounted for by macromolecules of bacterial cell walls and plaque matrix. Extracellular buffers in plaque fluid removable by centrifugation contributed up to 11%. These buffers (probably soluble proteins, peptides, organic acids, and phosphate) are, potentially at least, capable of exchange with saliva. In vitro, bicarbonate (dissolved in the extracellular fluid) contributed only 2-5% of total buffering; there was no evidence of formation of carbamino compounds. However, in vivo, salivary bicarbonate may be important as a continually replenished source of additional buffering.

Adult↗
PubMed2004

BIOKID: randomized controlled trial comparing bicarbonate and lactate buffer in biocompatible peritoneal dialysis solutions in children [ISRCTN81137991].

BACKGROUND: Peritoneal dialysis (PD) is the preferred dialysis modality in children. Its major drawback is the limited technique survival due to infections and progressive ultrafiltration failure. Conventional PD solutions exert marked acute and chronic toxicity to local tissues. Prolonged exposure is associated with severe histopathological alterations including vasculopathy, neoangiogenesis, submesothelial fibrosis and a gradual loss of the mesothelial cell layer. Recently, more biocompatible PD solutions containing reduced amounts of toxic glucose degradation products (GDPs) and buffered at neutral pH have been introduced into clinical practice. These solutions contain lactate, bicarbonate or a combination of both as buffer substance. Increasing evidence from clinical trials in adults and children suggests that the new PD fluids may allow for better long-term preservation of peritoneal morphology and function. However, the relative importance of the buffer in neutral-pH, low-GDP fluids is still unclear. In vitro, lactate is cytotoxic and vasoactive at the concentrations used in PD fluids. The BIOKID trial is designed to clarify the clinical significance of the buffer choice in biocompatible PD fluids. METHODS/DESIGN: The objective of the study is to test the hypothesis that bicarbonate based PD solutions may allow for a better preservation of peritoneal transport characteristics in children than solutions containing lactate buffer. Secondary objectives are to assess any impact of the buffer system on acid-base status, peritoneal tissue integrity and the incidence and severity of peritonitis. After a run-in period of 2 months during which a targeted cohort of 60 patients is treated with a conventional, lactate buffered, acidic, GDP containing PD fluid, patients will be stratified according to residual renal function and type of phosphate binding medication and randomized to receive either the lactate-containing Balance solution or the bicarbonate-buffered Bicavera solution for a period of 10 months. Patients will be monitored by monthly physical and laboratory examinations. Peritoneal equilibration tests, 24-h dialysate and urine collections will be performed 4 times. Peritoneal biopsies will be obtained on occasion of intraabdominal surgery. Changes in small solute transport rates, markers of peritoneal tissue turnover in the effluent, acid-base status and peritonitis rates and severity will be analyzed.

Adolescent↗
PubMed1999

Physiological HEPES buffer proposed as a calibrator for pH measurement in human blood.

N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid, known as HEPES buffer, with pK in the physiological range was studied for use as an alternative to conventional phosphate buffer for the calibration of pH in modern clinical analyzers. In different series of aqueous equimolar HEPES buffer, pH was measured at 37 degrees C with a capillary glass electrode standardized previously using phosphate, and variations due to changes in total HEPES buffer concentration (0.025 to 0.320 mol/l), and NaCl (0 to 0.250 mol/l) were monitored. For 0.05 equimolar HEPES buffer without NaCl, the pH of 7.362+/-0.003 (n = 15) obtained coincided well with the reference pH (7.364) from the National Institute of Standards and Technology (NIST). In particular, in the preferred 0.05 equimolar HEPES buffer/0.110 mol/l NaCl, which is isotonic to human plasma (0.160 mol/l), and termed physiological HEPES buffer (PHB), the pH of 7.346+/-0.003 (n = 84) can be related to the calculated corresponding reference pH from NIST without liquid junction (7.374), and is also compatible with the pH measured in normal arterial blood, pH = 7.403+/-0.003 (n = 20). Hence, in the two-point calibration of clinical analyzers, PHB, which is defined operationally with respect to the glass electrode and to phosphate buffer, may be useful as a calibrator in the range of buffer adjustment control to meet the correct values for pH when measuring in blood. Whereas Na-HEPES salt is hygroscopic and does not meet the declared purity grade (> 99%), pure HEPES acid is non-hygroscopic and conforms to the manufacturer's purity grade (> or = 99%). Therefore, for easy preparation of PHB, HEPES acid is the preferred starting material.

Blood Chemical Analysis↗
PubMed1999

Effect of fumaric acid and dietary buffering capacity on ileal and fecal amino acid digestibilities in early-weaned pigs.

Experiments were conducted to determine the effect of fumaric acid supplementation and buffering capacity of the diet on ileal and fecal digestibilities of CP, GE, and amino acids in early-weaned pigs. In two experiments, 12 pigs each were weaned at 14 d of age and fitted with a simple T-cannula at the distal ileum between 15 and 17 d of age. In both experiments, the pigs were fed four diets, based on wheat and soybean meal without (control) or with 1, 2, or 3% fumaric acid according to a balanced two-period change-over design. In Exp. 2, the buffering capacity of the diets was increased by supplementation of 3% sodium bicarbonate. The pigs were fed three times daily: equal amounts at 8-h intervals. The diets were supplied at a rate of 5% (wt/wt) of body weight. The inclusion of fumaric acid to the diet with a low buffering capacity increased (P < .05) the ileal digestibilities of CP, GE, and the majority of amino acids. The highest responses were found at an inclusion level of 2% fumaric acid. The improvements in apparent ileal amino acid digestibilities ranged from 4.9 to 12.8 percentage units. Supplementation of fumaric acid to a diet with a high buffering capacity led only to numerical increases in ileal digestibilities of CP, GE, and amino acids. In both studies, fumaric acid supplementation had no effect (P > .05) on the fecal digestibilities of CP, GE, and amino acids, except histidine. A high buffering capacity of the diet decreased the ileal amino acid digestibilities by 1 to 10 percentage units compared with diets with the low buffering capacities. In both experiments, ileal and fecal digestibilities were higher (P < .05) in Period 2 (on d 24 after weaning) than in Period 1 (on d 11 after weaning). A positive correlation (P < .05) between ileal digestibilities and fumaric acid supplementation to the diets with the low buffering capacities was observed in Period 1 (on d 11 after weaning), but not in Period 2 (on d 24 after weaning). No relationship (P > .05) between ileal digestibilities and fumaric acid supplementation to the diets with a high buffering capacity could be established. In conclusion, supplementation of fumaric acid to starter diets during the first 3 to 4 wk after weaning increases the ileal digestibilities of GE, CP, and amino acids.

Amino Acids↗
PubMed1989

A high pH discontinuous buffer system for resolution of isozymes in starch gel electrophoresis.

A Tris-citrate pH 9.5 gel/borate pH 8.2 electrode discontinuous buffer system for starch gel electrophoresis of proteins was developed to resolve iso- and allozymes of aspartate aminotransferase in frogs (Hyla crucifer). This buffer system also enhanced resolution of NADP-dependent malate dehydrogenase and the L-lactate dehydrogenase-A locus in this species. It provided good resolution of NAD-dependent malate dehydrogenase in esocid fishes, and esterases, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, and S-aconitate hydratase in ambystomatid salamanders. Variation suppressed by other buffers was revealed by this buffer for some enzyme encoding loci, while at other loci, this buffer suppressed electromorph variability. The concentration of tris(hydroxymethyl)aminomethane in gels made with this buffer was much higher than in pH 8.7 "Poulik" gels, but running characteristics of the two gel types were similar. Gels made with this new buffer were less prone to splitting and "warping" than Poulik gels, and were easier to handle. When screening a given taxon for enzyme variability, tests using multiple buffers are essential to maximize the amount of electrophoretically detectable variation.

Ambystoma↗
PubMed1997

Effect of dietary forage concentration and buffer addition on duodenal flow of trans-C18:1 fatty acids and milk fat production in dairy cows.

Milk fat depression in cows fed high grain diets has been shown to be related to increased trans-C18:1 fatty acids in milk. Trans-C18:1 fatty acids are produced as a result of incomplete biohydrogenation of dietary polyunsaturated fatty acids in the rumen. The objective of this study was to determine the effects of varying amounts of dietary concentrate and buffer addition on duodenal flow, apparent absorption, and incorporation of trans-C18:1 fatty acids into milk fat. Four ruminally and duodenally cannulated multiparous Holstein cows in midlactation were fed diets consisting of 60% (high) or 25% (low) forage with or without buffer (1.5% NaHCO3 and 0.5% MgO). Treatments were arranged in a 2 x 2 factorial within a 4 x 4 Latin square design. The diet containing low forage and no buffer increased the flow of trans-C18:1 fatty acids to the duodenum compared with the effects of other diets (120 vs. 57 to 66 g/d). Ruminal pH was lower for cows fed the low forage diets. The addition of buffer increased ruminal pH by 0.19 and 0.02 units for cows fed the low forage and high forage diets, respectively. Cows fed the diet containing low forage and no buffer produced milk with a lower fat percentage. The addition of buffer to the low forage diet partially corrected milk fat depression. Milk trans-C18:1 fatty acids were higher for cows fed the low forage diet without buffer than for cows fed the other diets (5.8% vs. 3.0%). Altered ruminal function resulting from low ruminal pH for cows fed the low forage diet and no buffer may play a role in the increased production of trans-C18:1 fatty acids in the rumen.

Animal Feed↗
PubMedDate not supplied

[Mathematical model of a generalized calcium buffer in cardiac muscle cells].

The problem of reduction of the earlier model for the calcium buffer system of cardiomyocytes was solved. A simpler model of generalized buffer, was obtained, which reproduces the behavior of the initial system in typical situations. To identify the parameters of the equations of the generalized buffer, recursive algorithms of filtration were developed. The model consists of two equations, which describe separately the fast and slow components of the buffer system. It is shown that the error of the generalized buffer model relative to the initial model of the calcium buffer system does not exceed 10% in a wide range of Ca2+ transients. The introduction of the generalized buffer into the model of mechanochemical coupling provides a more realistic description of a decrease in free calcium concentration. The new model adequately describes all mechanical events simulated by the initial model. In terms of the model, the effect of the slow component of the generalized buffer in a single contraction-relaxation cycle was studied. It was shown that the presence of the slow component leads to a marked decrease in the amplitude of contraction and increase in relaxation time.

Buffers↗
PubMed1999

Isocitrate as calcium ion activity buffer in coagulation assays.

BACKGROUND: Ca(2+) activity close to the physiological concentration of 1.3 mmol/L is essential in blood coagulation. Is this also true for the performance of global diagnostic coagulation assays? We searched for compounds that would buffer Ca(2+) activity at approximately 1.3 mmol/L without disturbing coagulation reactions and investigated whether such Ca(2+) buffering improves diagnostic efficacy in global diagnostic coagulation tests. METHODS: Buffering was investigated by mixing CaCl(2) and 11 candidate compounds and determining Ca(2+) activity. The best candidates were added to mixtures of plasma and thromboplastin to detect interference with coagulation reactions. The best of these candidates, isocitrate, was used to modify an activated partial thromboplastin time (APTT), buffering final Ca(2+) activity to approximately 1.3 mmol/L. Plasma samples from 22 healthy individuals and 120 patients were analyzed with original and modified APTT to determine whether diagnostic efficacy was improved. RESULTS: Two suitable Ca(2+) buffers, citrate and isocitrate, were found. Isocitrate was preferred as being less coagulation inhibitory, a better Ca(2+) buffer, and possibly a better anticoagulant. The isocitrate-modified APTT showed a final Ca(2+) activity of 1.60 +/- 0.07 mmol/L, compared with 2.73 +/- 0.20 mmol/L for the original APTT. The means and SDs for the healthy individuals were determined for both procedures, and the values were used to express patient deviation from normality (difference from mean divided by SD). The deviation was greater for the modified APTT; 4.3 +/- 5.7, compared with 3.6 +/- 5.0 (P <0.005) for the original APTT. CONCLUSIONS: Isocitrate can be used to buffer Ca(2+) activity at physiological concentrations and can serve as an anticoagulant. APTT with isocitrate-buffered Ca(2+) activity shows signs of improved diagnostic efficacy.

Blood Chemical Analysis↗
PubMed2006

Evaluation of the buffering capacity of powdered cow's, goat's and soy milk and non-prescription antacids in the treatment of non-ulcer dyspepsia.

BACKGROUND: Non-ulcer dyspepsia (NUD) is the term most commonly used to describe a heterogeneous and often ill-defined group of dyspepsia patients whose symptoms of upper abdominal pain, discomfort or nausea persist in the absence of identifiable cause. Treatment choice commonly includes over the counter medicines and home remedies, e.g. milk. OBJECTIVE: To determine the relative buffering capacity of goat's, cow's and soy milk, non-prescription antacid drugs and combinations thereof. METHODS: The buffering capacities of 25 ml aliquots of each of the powdered milk products, the antacids alone and the combination of antacid and milk were determined. Statistical analysis was used to determine any significant differences in buffering capacity. RESULTS: When the antacids were examined alone, significant differences in buffering capacity were observed. When powdered milk products were examined alone, cow's milk had a significantly higher buffering capacity than either goat's or soy milk. There was no significant difference between goat's and soy milk. In the combination of cow's milk with each of the antacids, brand A and B had a similar buffering capacity, significantly higher than that observed with brand C. CONCLUSIONS: The combination with best observed buffering capacity was brand A with cow's milk, and the weakest buffering capacity was observed with brand C with soy milk. The results obtained can be attributed to the chemical constituents of the antacids and the milk products.

Acid-Base Equilibrium↗
PubMed1991

Pharmacokinetic study of a new oral buffered acetylsalicylic acid (ASA) formulation in comparison with plain ASA in healthy volunteers.

A single-blind, randomized, crossover pharmacokinetic study was carried out to investigate the bioavailability of a new oral buffered 325 mg acetylsalicylic acid (ASA) formulation (ASPIRINA 03) in comparison with a 325 mg plain tablet. Twelve healthy volunteers of both sexes, aged between 20 and 37 years, received buffered or plain ASA on two separate occasions with a wash-out interval of at least two weeks. ASA and salicylic acid (SA) plasma levels were determined by a chromatographic method. The results showed no difference between the area under concentration time curve (AUC0-infinity) ASA values of both formulations (p = 0.19), and buffered ASA relative bioavailability was 102.49% (= bioequivalence). A significant difference was found between the AUC0-30 min ASA values: 90.5 micrograms. min/ml with buffered and 67.7 micrograms. min/ml with the plain tablet (p less than 0.05). The buffered ASA time of maximum concentration was shorter (28 +/- 8 min) than the plain one (38 +/- 19 min, p less than 0.05). The plasma concentrations and pharmacokinetic parameters of SA were not significantly different after the administration of the two ASA formulations. The plain ASA tablet had a significantly lower (p less than 0.05) dissolution rate than buffered ASA tablet. Moreover, the buffered ASA tablet significantly (p less than 0.01) increased the pH by 0.5 units. In conclusion, the bioavailability of the new oral buffered ASA was equivalent to that of plain ASA, but the plasma concentration peak was reached in a shorter time.

Administration, Oral↗
PubMed1994

Which bicarbonate concentration is adequate to lactate-buffered substitution fluids in maintenance hemofiltration?

We investigated the metabolic and hemodynamic effects of a lactate- and a bicarbonate-buffered (bicarbonate concentration 31.4 mmol/l, type I) hemofiltration substitution fluid in a prospective crossover study of 3 weeks each in 11 patients on maintenance hemofiltration. The lactate-buffered hemofiltration (lactate concentration 34-44.5 mmol/l) lead to hyperlactatemia in all patients without signs of overt lactic acidosis but showed a better control of acid-base balance (pH, base excess, standard bicarbonate) than the type I bicarbonate-buffered fluid (p < 0.01). In 6 patients a higher concentration of bicarbonate- (39.7 mmol/l, type II) buffered fluid was tested. The parameters of acid-base balance showed a better control during type II than during type I bicarbonate hemofiltration and were similar to the lactate-buffered phase. Plasma lactate levels between type I and type II bicarbonate hemofiltration were not different. Also in the steady state phase of the treatment (days 7-9 [week 3]) parameters of acid-base balance rose more to normal values during type II than during lactate-buffered hemofiltration. Hemodynamic parameters showed no differences between the three types of buffers used. Furthermore, also the type II bicarbonate fluid was well tolerated. Bicarbonate in a higher concentration (39.7 mmol/l) proved to be a safe and practical alternative to lactate-buffered hemofiltration.

Acid-Base Equilibrium↗
PubMed1994

Effects of alkaline buffers on cytoplasmic pH in lymphocytes.

OBJECTIVE: To study experimentally possible adverse effects of bicarbonate on cytoplasmic pH. DESIGN: Open, randomized control trial of white blood cells from human volunteers. SETTING: Experimental laboratory in a large university hospital. SUBJECTS: Lymphocytes prepared from human blood. INTERVENTIONS: The fluorescent intracellular probe 2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was used to study the influence on cytoplasmic pH after the addition of different alkaline buffers to lymphocytes with normal as well as decreased initial intracellular pH. MEASUREMENTS AND MAIN RESULTS: In normal lymphocytes, sodium bicarbonate caused a marked, dose-dependent acidification of the cytoplasm followed by a slow, often unpredictable increase. Ringer's acetate solution decreased the intracellular pH dose-dependently this acidification effect continued throughout the measurements. In contrast, trometamol (tris) and Carbicarb both caused a pronounced dose-dependent and lasting alkalinization of the cytoplasm. Tris buffer mixture (Tribonate) produced a slight initial dose-dependent acidification, followed by a slow increase in cytoplasmic pH to values above those recorded during control measurements. Lymphocytes that were preincubated in acetate showed similar results after addition of tris buffer mixture or sodium bicarbonate. Lymphocytes with intracellular acidosis due to preincubation in an acid buffer demonstrated a more pronounced and dose-dependent decrease of cytoplasmic pH immediately after addition of the bicarbonate-containing buffers (sodium bicarbonate and tris buffer mixture). The decrease was only partly compensated for over the next 10 mins. Only the buffers that were not producing CO2 could fully compensate for the severe extra- and intracellular acidosis imposed on the lymphocytes by preincubation in an acid medium. CONCLUSION: Use of bicarbonate-containing buffers often results in an initial decrease of cytoplasmic pH.

Buffers↗
PubMed1996

Radiation-induced cell killing is highly dependent upon buffer treatment (filtration compared to autoclaving) due to metal-catalyzed formation of hypochlorite: a cautionary note.

Buffer solutions used in experiments in radiation biology may be sterilized by either autoclaving or filtration. We show here that for phosphate-buffered saline such differences in buffer treatment may result in widely differing dose-effect curves for cell killing. The temperature-dependent transformation of monophosphate ions into di- or polyphosphate evidently proceeds to an appreciable extent upon autoclaving the buffers at 120 degrees C for 10 to 20 min. This increases the capability of the buffer to chelate spurious metal contaminations and, as a consequence, to reduce the amount of cytotoxic hypochlorite being produced. Depending on conditions of buffer treatment we have observed dose modification factors for the colony-forming ability of yeast cells up to the order of 3. Thus effects due to buffer treatment might easily outweigh the effect which the experiment was originally designed to determine. We strongly advise, therefore, that results of parallel sets of experiments in which different methods of buffer sterilization have been used should not be compared directly.

Buffers↗
PubMed1997

Linearized buffered Ca2+ diffusion in microdomains and its implications for calculation of [Ca2+] at the mouth of a calcium channel.

Immobile and mobile calcium buffers shape the calcium signal close to a channel by reducing and localizing the transient calcium increase to physiological compartments. In this paper, we focus on the impact of mobile buffers in shaping steady-state calcium gradients in the vicinity of an open channel, i.e. within its "calcium microdomain." We present a linear approximation of the combined reaction-diffusion problem, which can be solved explicitly and accounts for an arbitrary number of calcium buffers, either endogenous or added exogenously. It is valid for small saturation levels of the present buffers and shows that within a few hundred nanometers from the channel, standing calcium gradients develop in hundreds of microseconds after channel opening. It is shown that every buffer can be assigned a uniquely defined length-constant as a measure of its capability to buffer calcium close to the channel. The length-constant clarifies intuitively the significance of buffer binding and unbinding kinetics for understanding local calcium signals. Hence, we examine the parameters shaping these steady-state gradients. The model can be used to check the expected influence of single channel calcium microdomains on physiological processes such as excitation-secretion coupling or excitation-contraction coupling and to explore the differential effect of kinetic buffer parameters on the shape of these microdomains.

Adenosine Triphosphate↗
PubMed2000

Acid-base control for biocatalysis in organic media: new solid-state proton/cation buffers and an indicator

Although great care is generally taken to buffer aqueous enzyme reactions, active control of acid-base conditions for biocatalysis in low-water media is rarely considered. Here we describe a new class of solid-state acid-base buffers suitable for use in organic media. The buffers, composed of a zwitterion and its sodium salt, are able to set and maintain the ionisation state of an enzyme by the exchange of H+ and Na+ ions. Surprisingly, equilibrium is established between the different solid components quickly enough to provide a practical means of controlling acid-base conditions during biocatalysed reactions. We developed an organosoluble chromoionophore indicator to screen the behaviour of possible buffer pairs and quantify their relative H+/Na- exchange potential. The transesterification activity of an immobilised protease, subtilisin Carlsberg, was measured in toluene in the presence of a range of buffers. The large observed difference in rates showed good correlation with that expected from the measured exchange potentials. The maximum water activities accessible without formation of hydrates or solutions of the buffers are reported here. The indicator was also used to monitor, for the first time in situ, changes in the acid-base conditions of an enzyme-catalysed transesterification reaction in toluene. We found that even very minor amounts of an acidic by-product of hydrolysis were leading to protonation of the enzyme, resulting in rapid loss of activity. Addition of solid-state buffer was able to prevent this process, shortening reaction times and improving yields. Solid-state buffers offer a general and inexpensive way of precisely controlling acid-base conditions in organic solvents and thus also have potential applications outside of biocatalysis.

Journal Article↗
PubMed1986

Membrane alterations in cerebral cortex when using PIPES buffer.

When used for vascular perfusion of brain, 0.1 M PIPES-buffered 3% glutaraldehyde resulted in the formation of expanded, vesicle-filled cell processes limited by multiple membrane layers. These structures, termed multivesicular myelin figures and interpreted as artefacts, were most common in layer 2 of the cerebral cortex. When cacodylate or phosphate buffer was used instead of PIPES buffer in the primary fixative, such structures were not seen. The use of a more concentrated initial aldehyde fixative, PIPES-buffered, markedly reduced the size and numbers of these artefacts when compared to PIPES-buffered 3% glutaraldehyde only. Slowing the initial perfusion rate increased the size and frequency of occurrence of multivesicular myelin figures with PIPES buffer when compared to optimum perfusions. Prolonged initial exposure to PIPES buffer by using it to wash out the blood and then perfusing with fixative 5 min later did not increase the number or size of multivesicular myelin figures but did reduce the multivesicular nature of the artefacts. We suggest that the non-toxic nature of PIPES buffer allowed the formation of these membranous artefacts, while phosphate and cacodylate interfered with the cellular activity during the process of fixation.

Alkanesulfonates↗