Stationary pH Research Articles

IoT based system for real-time monitoring the hydrogen-ion activity in water bodies

The paper presents design and principle of operation of a mobile combined pH meter. Sensor’s network underlying the IoT system for monitoring water bodies hydrogen ions activity in real time was demonstrated. The measuring device was tested on liquids with different pH levels. The obtained measurement results were compared using a stationary pH meter OHAUS Starter 3100.

Quantitative analysis of nine types of virus-like particles in human papilloma virus bulk by size-exclusion chromatography

据统计,5%以上的人类癌症由人乳头瘤病毒(HPV)导致。HPV疫苗的使用,尤其是多价HPV疫苗的使用,可有效预防HPV感染和肿瘤的发生。例如,9价HPV疫苗可有效预防90%以上HPV相关癌前病变。人乳头瘤病毒样颗粒(VLP)是HPV疫苗的唯一抗原。VLP由360份衣壳蛋白L1组成。VLP的含量测定对HPV原液和HPV疫苗的质量评价至关重要。该文发展了一种以体积排阻色谱(SEC)为基础的9种型别人乳头病毒样颗粒的定量方法。实验优化了包括色谱柱类型、色谱柱孔径、流动相离子强度和流动相pH值在内的色谱条件。经过考察,以SHIMSEN Ankylo SEC-300色谱柱(300 mm×7.8 mm, 3 μm)为固定相,以含有300 mmol/L NaCl和50 mmol/L磷酸盐(pH 7.0)的缓冲溶液为流动相时,VLP的色谱峰更窄,从而可获得更高的响应和更好的灵敏度,因此选择该色谱条件用于VLP与基质的分离。优化所得的方法具有较宽的线性范围,良好的重复性(峰面积的相对标准偏差不大于5.0%)和灵敏度(定量限为4.58~15.24 μg/mL)。将方法用于HPV原液中VLP的含量测定,监测VLP的稳定性。结果显示,HPV原液中VLP颗粒不稳定,于4 ℃放置一周后,VLP含量与生产后立即测得的含量相比存在一定程度的降解。此外,方法还可用于疫苗上清液中游离蛋白质的分析,监测铝佐剂对VLP的吸附情况。被测厂家的铝佐剂可较好的吸附VLP,无明显残余蛋白质检出。与传统的蛋白质定量方法相比,如Folin-酚法(Lowry法),该法具有操作简单、自动化程度高、分析通量高等优点,可实现VLP含量的批量化分析。

24-hour multi-pH recording of the postprandial acid pocket and the nocturnal acid distribution at the esophagogastric junction in healthy volunteers.

Postprandial stationary pH monitoring studies have identified the acid pocket. To what extent a similar pool of acid is present in the fasting state or at night remains however unclear. The study was performed in 9 HV without a hiatal hernia. A pH-impedance-pressure catheter was positioned at the Z-line. First, the presence of the acid pocket was monitored under stationary conditions during 2hours after ingestion of a standardized meal. Thereafter, the equipment was connected to an ambulatory monitoring device for 24-hour recording. Under stationary conditions, a postprandial acid pocket was present in 7 of the 9 HV, from 9±7minutes after meal onwards during 47±8minutes. During ambulatory 24-hour monitoring, postprandial acid pockets emerged significantly later, but no differences in duration or position were detected. During nighttime, an acid pool was detected with its proximal border at the level of the cardia, which at later, time points gradually moved to a more distal position. This led to a gradual decrease in nocturnal acid exposure from proximal to distal, a phenomenon that was preceded by a bust of gastric contractions. Nocturnal reflux originated from the cardiac region, and was more acidic in the early compared with late nocturnal period. The acid pocket is present in the postprandial period under both stationary and ambulatory conditions. Of interest, at night, a pool of acid can be demonstrated which is periodically shifted more distally. This pool of acid represents the reservoir from which nocturnal reflux originates.

Designing Stationary Reaction-Diffusion Patterns in pH Self-Activated Systems.

Since Alan Turing's 1952 pioneering work, reaction-diffusion (RD) processes are regarded as prototype mechanisms for pattern formation in living systems. Though suspected in many aspects of morphogenetic development, pure RD patterns have not yet been demonstrated in living organisms. The first observations of an autonomous development of stationary chemical patterns were made in the early 1990s. In this Account, we discuss the recent developments for producing stationary pH RD patterns in open spatial reactors. The theoretical analysis of the early experiments anticipated the possibility of finding Turing patterns in a wide range of oscillatory reactions if one could control the kinetic and diffusional rate of some key species. However, no experimentally effective method to produce stationary Turing patterns was attained before 2009, and the number of systems stagnated at two until then. The two precursor reaction systems benefited from unplanned favorable chemical properties of the RD media. Theoretical studies point out that appropriate diffusion rate differences are necessary to produce stationary patterns since a competition between an effective short distance self-activation and a long distance inhibitory process is required. This differential diffusion would naturally lead to differential exchange rates between the RD system and its feed environment, an aspect somewhat overlooked in theoretical and in primal experimental approaches. Our pattern design method takes this aspect into account. A slower diffusion of a self-activated species (here, protons), produced in the RD part of the spatial reactor, generates the accumulation of this species compared to the other species. This accumulation has to be at least partly compensated by an independent scavenging reaction. The above requirement naturally brought us to focus on two-substrate pH oscillatory reactions. Stationary RD patterns are now well documented in six pH driven reaction systems. Furthermore, the coupling with a pH dependent metal ion complexing agent led to stationary patterns in calcium ion concentration. Our effective semiempirical design method does not require a detailed knowledge of the reaction kinetics; thus it is applicable to a broad spectrum of reactions and even to synthetic biological systems. It is based on simple dynamic arguments and on general topological characteristics of a nonequilibrium phase diagram. We first illustrate our method with numerical simulations, based on a realistic but idealized general model of the two-substrate pH-oscillator reaction family, and provide a refined view of the topology of the resulting phase diagrams. Then, we exemplify its effectiveness by observations made in distinct pH self-activated systems. Analogies and differences between experiments and the model calculations are pointed out. Besides standard hexagonal arrays of spots and parallel stripes, hitherto undocumented dynamic phenomena, such as randomly blinking areas and complex dynamic and stationary filamentous structures, were observed. The main challenge, to find low-mobility complexing agents that would selectively and reversibly bind a species controlling the self-activatory kinetic path of the reaction, was readily overcome in multiple ways by anions of weak acids: not only by polymeric substances but in some cases by a pH color indicator or even smaller molecules, depending on their proton binding affinity.

Design of localized spatiotemporal pH patterns by means of antagonistic chemical gradients

Spatially localized moving and stationary pH patterns are generated in two-side-fed reaction-diffusion systems. The patterns are sandwiched between two quiescent zones and positioned by the antagonistic gradients of the reactants of the self-activatory process. Spatial bistability, spatiotemporal oscillations, and formation of stationary Turing patterns have been predicted by numerical simulations and observed in experiments performed by using different hydrogen ion autocatalytic chemical systems. The formation of stationary patterns due to long-range inhibition is promoted by a large molecular weight hydrogen ion binding polymer.

Analysis of vancomycin and its related impurities by hydrophilic interaction liquid chromatography

The current reversed-phase liquid chromatography as the main analytical method for vancomycin is inappropriate due to its poor polarity selectivity and mass spectrometry compatibility. Hydrophilic interaction chromatography (HILIC) method has been verified to provide the glycopeptide substances with good retention and polarity selectivity, therefore, an HILIC method was developed for the analysis of vancomycin and its related impurities including norvancomycin, desvancosaminyl vancomycin, dedichloro vancomycin and crystalline degradation product (CDP-1). In this work, a systematic evaluation of retention behavior of vancomycin and its related impurities in hydrophilic interaction chromatography was performed and the influences of stationary phase, acetonitrile content, buffer salt and pH of mobile phase were studied. According to the results, Click XIon column was selected as stationary phase and ammonium formate was additive in the mobile phase. Vancomycin and its related impurities were totally separated under the optimization conditions. It is concluded that hydrophilic interaction chromatography may be a new strategy for the separation of vancomycin and its structural analogues.

Quality by design: a systematic and rapid liquid chromatography and mass spectrometry method for eprosartan mesylate and its related impurities using a superficially porous particle column.

The present work describes the systematic development of a robust, precise, and rapid reversed-phase liquid chromatography method for the simultaneous determination of eprosartan mesylate and its six impurities using quality-by-design principles. The method was developed in two phases, screening and optimization. During the screening phase, the most suitable stationary phase, organic modifier, and pH were identified. The optimization was performed for secondary influential parameters--column temperature, gradient time, and flow rate using eight experiments--to examine multifactorial effects of parameters on the critical resolution and generated design space representing the robust region. A verification experiment was performed within the working design space and the model was found to be accurate. This study also describes other operating features of the column packed with superficially porous particles that allow very fast separations at pressures available in most liquid chromatography instruments. Successful chromatographic separation was achieved in less than 7 min using a fused-core C18 (100 mm × 2.1 mm, 2.6 μm) column with linear gradient elution of 10 mM ammonium formate (pH 3.0) and acetonitrile as the mobile phase. The method was validated for specificity, linearity, accuracy, precision, and robustness in compliance with the International Conference on Harmonization Q2 (R1) guidelines. The impurities were identified by liquid chromatography with mass spectrometry.

Effective Pre-Concentration and Analysis of Heavy Metals Using Ligand Step Gradient Focusing in Combination with Isotachophoresis

A capillary electrophoretic method for the pre-concentration of metals based on selective focusing of metal chelates with subsequent online ITP analysis was developed and verified. The selected ions of heavy metals (Pb2+ and Cd2+) were subjected to pre-concentration from the mixture and analysed. Focusing of the metals was carried out in a ligand step gradient, which was created by the addition of a convenient ligand agent to the regular stationary pH step gradient. The metal ions were continuously dosed into the column, where they were selectively trapped (with the exception of Na+) on the stationary ligand step gradient in the form of non-moving zones of citrate complexes with an effective charge of zero. After a detectable amount of analyte was accumulated, the dosing was stopped and the accumulated zones were mobilized to the analytical column, where they were analysed by the ITP method with conductivity or photometric detection. The electrolyte systems for the dosing (mode IEF), mobilizing (mode MBE), and analytical steps (mode ITP) were developed and tested. The cLOD was decreased by approximately two orders of magnitude using an acceptable dosing time of 25 min.

Focusing of alkali earth metals in ligand step gradient

A capillary electroseparation technique for focusing and selective pre-concentration of metal chelates with subsequent on-line isotachophoresis (ITP) analysis was developed and verified. The ions of alkali earth metals (Mg, Ca, Sr, and Ba) were pre-concentrated from the mixture and analyzed. The focusing of the metals was carried out in a ligand step gradient, which was created by the addition of a convenient ligand agent to the regular stationary pH step gradient. The analytical procedure consisted of three steps. During the first step, the metal ions were electrokinetically continuously dosed into the column where they were selectively trapped on the stationary ligand step gradient in the form of unmoving zones of chelate complexes with effectively zero charge. After a detectable amount of analyte was accumulated, the dosing was stopped. The accumulated zones were mobilized to the analytical column, where they were analyzed by the ITP method with conductivity or photometric detection. The proper electrolyte systems for dosing, mobilizing, and analyzing in isoelectric focusing (IEF), moving boundary electrophoresis (MBE), and ITP modes were consequently developed and put into practice. The trapping selectivity can be regulated by the choice of pH and convenient complexing agents. A mixture of alkali earth metals were used as model analytes. Using a 3000 s dosing time, the proposed method improved the detection limit by 5–29 times in comparison to analysis by ITP with classical injection.

Should we bravo?

Should we bravo?

Inhibition of bicarbonate reabsorption in the rat proximal tubule by activation of luminal P2Y1 receptors.

The present study used a stationary microperfusion technique to investigate in vivo the effect of P2Y1 receptor activation on bicarbonate reabsorption in the rat proximal tubule. Proximal tubules were perfused with a bicarbonate Ringer solution before flow was stopped by means of an oil block. The recovery of lumen pH from the initial value (pH 8.0) to stationary values (pH approximately 6.7) was recorded by a H+-sensitive microelectrode inserted downstream of the perfusion pipette and oil block. The stationary pH value and the t(1/2) of pH recovery were used to calculate bicarbonate reabsorption (JHCO3). Both EIPA and bafilomycin A1 caused significant reductions in proximal tubule JHCO3, consistent with the established contributions of Na/H exchange and H+-ATPase to proximal tubule HCO3 reabsorption. The nucleotides ADP and, to a lesser extent, ATP reduced JHCO3 but AMP and UTP were without effect. 2MeSADP, a highly selective agonist of the P2Y1 receptor, reduced JHCO3 in a dose-dependent manner. MRS-2179, a P2Y1 receptor-specific antagonist, abolished the effect of 2MeSADP, whereas theophylline, an antagonist of adenosine (P1) receptors, did not. The inhibitory action of 2MeSADP was blocked by inhibition of protein kinase C and reduced by inhibition of protein kinase A. The effects of EIPA and 2MeSADP were not additive. The data provide functional evidence for P2Y1 receptors in the apical membrane of the rat proximal tubule: receptor activation impairs acidification in this nephron segment.

Dipole Reversal in Bacteriorhodopsin and Separation of Dipole Components

The electrostatics of purple membranes has been analyzed by measurements of the electric dichroism in dc and ac fields in a broad pH range. The dc data are mainly used to characterize the permanent dipole, whereas the ac data serve as control for changes of global structure and of the induced dipole. At pH values from 8 to 3.5, the dc dichroism is negative at low field strengths and turns to positive values at higher field strengths, in qualitative agreement with the orientation function for disks having a permanent dipole moment perpendicular to the plane and an induced dipole moment in the plane. The minimum value of the dichroism, , indicates the permanent dipole and shows a complex dependence on the pH but does not approach zero at any pH between 3.5 and 8, which would be expected for a simple reversal of the permanent dipole. However, the dependence of on pH shows a λ point at pH 4.9, which reflects reversal of the dipole. Fitting of the stationary dichroism for E ≤ 12 kV m-1 to the orientation function shows a decrease of the permanent dipole μ in the pH range 5 to about 50% of the value found at pH 7. In the same pH range, the limit dichroism, , derived from dc data also decreases to about 50% of the value at pH 7, whereas parallel measurements of the ac dichroism show almost constant values. The combined observations indicate reversal of the permanent dipole, changes of disk bending, and the existence of a fluctuating dipole moment, probably resulting from bending fluctuations. Reversal of the dipole moment at pH 4.9 is confirmed by combined pH and field jump experiments. The direction of the dipole is reverted in the same pH range as the direction of stationary pH changes upon illumination, indicating an important function of the dipole for vectorial proton transfer. Comparison of experimental data with simple calculations of the protein dipole from the crystal structure indicates the existence of a large dipole component, which is directed opposite to the protein dipole at pH 7 and is probably due to a nonsymmetric distribution of charges on lipid residues. The results indicate a high degree of compensation in the electric asymmetry, which seems to be necessary for stability of purple membranes.

Fluorescence and Photosynthetic Activity of Chloroplasts in Acid and Alkaline Zones of Chara corallina

Continuous profiles of local pH near the cell surface of Chara corallinawere recorded during uniform longitudinal movement of an internodal cell relative to a stationary pH microelectrode. Under illumination, the pH profile consisted of alternating acid and alkaline bands with a pH difference of up to 3 pH units. After darkening, the bands disappeared and pH became uniformly distributed along the cell length. Chlorophyll fluorescence of chloroplasts was measured by microfluorometry at different locations within one cell, and significant differences were observed in close relation to light-dependent pH banding. The chlorophyll fluorescence yield was lower in zones of low external pH than in alkaline zones both under actinic and saturating light. The fluorescence parameters Fand F" m and the quantum yield of photosystem II (PSII) displayed variations along the cell length in accordance with pH changes in unstirred layers of the medium. The results show that PSII photochemical efficiency and the rate of noncyclic electron transport are higher in the chloroplasts of acid zones (zones of H+extrusion from the cell) than in alkaline zones. The dependence of photosynthetic electron transport on local pH near the cell surface may result from different contents of CO2in acid and alkaline regions. The acid zones are enriched with CO2that readily permeates through the membrane providing the substrate for the Calvin cycle. Conversely, a poorly permeating form, HCO–3is predominant in alkaline zones, which may restrict the dark reactions and photosynthetic electron flow.

Capillary electrophoresis of DNA in the 20–500 bp range: recent developments

The present mini-review summarizes recent developments in the field of DNA separations by capillary zone electrophoresis (CZE), as developed by our group. Separation of antisense oligonucleotides in sieving liquid polymers in isoelectric buffers is first discussed. It is shown that the use of isoelectric buffers (notably His) permits very high voltage gradients (up to 1000 V/cm) with much reduced transit times and increased resolution of all truncated and failed sequences. Oligonucleotides can also be analyzed by zone electrophoresis against a stationary pH gradient (typically a pH 6.5–10 range): if injected at the alkaline end, the sample components experience stacking and zone sharpening due to modulation of charge as the oligonucleotides move along the pH gradient. Oligonucleotides having the same length, but differing by one single nucleotide in the chain, can be separated in free solution (i.e., in the absence of a sieving matrix) at strongly acidic pH values (pH 3.0–3.3) where charge differences due to base protonation are maximized. By working in free solution, it has also been possible to measure accurately the free mobility of DNAs, shown to reach a constant value of 3.75±0.04·10−4 cm2 V−1 s−1 at 25°C and in Tris–acetate–EDTA buffer, pH 8.3, above a critical length of ca. 400 bp. However, when double-stranded, rather than single-stranded, DNA is analyzed in isoelectric His buffer, some peculiar phenomena are observed: improved resolution for smaller DNA fragments (up to ca. 150 bp) and a rapid deterioration of resolution above this critical length. Direct binding of His to the DNA helix is hypothesized, via a bidentate salt bridge of the two charged amino groups of His on the negatively charged oxygen of the phosphate group. Upon extensive binding, occupying every available phosphate site, π–π interactions could occur among the stacks of bound His residues, thus further stabilizing the complex.

Low-tech electrophoresis, small but beautiful, and effective: electrophoretic titration curves of proteins.

Migration across a stationary pH gradient results in the electrophoretic titration of a protein's dissociable groups. From the resulting curves, some properties of the protein may be derived, including overall amino acid composition and type of mutation between polymorphic variants, as well as range of stability or, for enzymes, of catalytic activity. Analysis with this technique is a stringent purity criterion; other applications allow the study of interacting systems and the planning of chromatographic fractionations based on differences in surface charge.

Recent advances in capillary zone electrophoresis of DNA

The present minireview summarizes recent developments in the field of DNA separations by capillary zone electrophoresis (CZE), as developed by our group. Separation of antisense oligonucleotides in sieving liquid polymers in isoelectric buffers is discussed first. It is shown that the use of isoelectric buffers permits very high voltage gradients (up to 1000 V cm −1) with much reduced transit times and increased resolution of all truncated and failed sequences. Oligonucleotides can also be analysed by zone electrophoresis against a stationary pH gradient (typically a pH 6.5–10 range): if injected at the alkaline end, the sample components experience stacking and zone sharpening due to modulation of charge as the oligonucleotides move along the pH gradient. Oligonucleotides having the same length, but differing by one single nucleotide in the chain, can be separated in free solution (i.e. in the absence of a sieving matrix) at strongly acidic pH values (pH 3.0–3.3) where charge differences due to base protonation are maximized. By working in free solution, it has also been possible to measure accurately the free mobility of DNAs, shown to reach a constant value of 3.75±0.04·10 −4 cm 2 V −1 s −1 at 25°C and in Tris–acetate–EDTA buffer, pH 8.3, above a critical length of ca. 400 base pairs. Finally, detection of point mutations in human genomic DNA is proven to be feasible in nonisocratic CZE, by running temperature-programmed CZE. The temperature gradient is activated within the capillary lumen by voltage ramps during the run, by exploiting joule effects. This technique has been proven to work for all point mutants, from low-, to intermediate-, to high-melters and has been applied to a number of point mutants in cystic fibrosis and thalassemia.

Role of Cl- in electrogenic H+ secretion by cortical distal tubule.

The presence of an electrogenic H+-ATPase has been described in the late distal tubule, a segment which contains intercalated cells. The present paper studies the electrogenicity of this transport mechanism, which has been demonstrated in turtle bladder and in cortical collecting duct. Transepithelial PD (Vt) was measured by means of Ling-Gerard microelectrodes in late distal tubule of rat renal cortex during in vivo microperfusion. The tubules were perfused with electrolyte solutions to which 2 x 10(-7) M bafilomycin or 4.6 x 10(-8) M concanamycin were added. No significant increase in lumen-negative Vt upon perfusion with these inhibitors as compared to control, was observed as well as when 10(-3) m amiloride, 10(-5) M benzamil or 3 mM Ba2+ were perfused alone or in combination. The effect of an inhibition of electrogenic H+ secretion, i.e., increase in lumen-negative Vt by 2-4 mV, was observed only when Cl- channels were blocked by 10(-5) M 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). This blocker also reduced the rate of bicarbonate reabsorption in this segment from 1.21 +/- 0.14 (n = 8) to 0.62 +/- 0.03 (8) nmol.cm-2.sec-1 as determined by stationary microperfusion and pH measurement by ion-exchange resin microelectrodes. These results indicate that: (i) the participation of the vacuolar H+ ATPase in the establishment of cortical late distal tubule Vt is minor in physiological conditions, but can be demonstrated after blocking Cl- channels, thus suggesting a shunting effect of this anion; and, (ii) the rate of H+ secretion in this segment is reduced by a Cl- channel blocker, supporting coupling of H+-ATPase with Cl- transport.

Regulation of nephron acidification by corticosteroids.

The present paper reviews work from our laboratories evaluating the importance of adrenal cortical hormones in acidification by proximal and cortical distal tubules. Proximal acidification was determined by stationary microperfusion, and measurement of bicarbonate reabsorption using luminal pH determination was performed with H(+)-ionsensitive microelectrodes. Rats were adrenalectomized (ADX) 48 h before the experiments, and corticosteroids (aldosterone (A), corticosterone (B), and 18-OH corticosterone (18-OH-B)) were injected intramuscularly 100 and 40 min before the experiments. In ADX rats stationary pH increased significantly to 7.03 as compared to sham-operated rats (6.78). Bicarbonate reabsorption decreased from 2.65 +/- 0.18 in sham-operated rats to 0.50 +/- 0.07 nmol cm-2 s-1 after ADX. The administration of the three hormones stimulated proximal tubule acidification, reaching, however, only 47.2% of the sham values in aldosterone-treated rats. Distal nephron acidification was studied by measuring urine minus blood pCO2 differences (U-B pCO2) in bicarbonate-loaded rats treated as above. This pCO2 difference is used as a measure of the distal nephron ability to secrete H+ ions into an alkaline urine. U-B pCO2 decreased significantly from 39.9 +/- 1.26 to 11.9 +/- 1.99 mmHg in ADX rats. When corticosteroids were given to ADX rats before the experiment, U-B pCO2 increased significantly, but reached control levels only when aldosterone (two 3-microgram doses per rat) plus corticosterone (220 micrograms) were given together. In order to control for the effect of aldosterone on distal transepithelial potential difference one group of rats was treated with amiloride, which blocks distal sodium channels. Amiloride-treated rats still showed a significant reduction in U-B pCO2 after ADX. Only corticosterone and 18-OH-B but not aldosterone increased U-B pCO2 back to the levels of sham-operated rats. These results show that corticosteroids stimulate renal tubule acidification both in proximal and distal nephrons and provide some clues about the mechanism of action of these steroids.

Capillary zone electrophoresis of oligonucleotides in isoelectric buffers and against a stationary pH gradient.

Capillary zone electrophoresis of oligonucleotides in a background electrolyte of two different types of stationary buffers is proposed: single, isoelectric amphoteres and focused carrier ampholytes. In the first case, two zwitterionic molecules are evaluated: lysine and histidine. Although the former has a five times higher buffering power (beta) at the pI (9.74) than the latter (pI 7.47), due to the favorable delta pK value (1.6 vs. 3) and thus should be the preferred species, a new parameter for evaluating the performance of isoelectric buffers is proposed: the beta/lambda ratio, i.e., the ratio between the buffering power and its conductivity. Ideal buffers are those with the highest beta/lambda ratio, since this allows delivering very high voltage gradients with minimal Joule effects. Since the pI of Lys is situated in a pH region (9.74) where bulk water begins to conduct, whereas His has a pI close to neutrality, the beta/lambda ratio is more favorable for His than for Lys. In the second case (zone electrophoresis of oligonucleotides against a preformed pH gradient), it is shown that migration against a pH 6.5-10 Pharmalyte carrier ampholyte pH gradient offers a unique analyte resolution. This is possibly due to two effects: (i) When injected at the alkaline extreme (ca. pH 10) of the pH gradient, the oligonucleotide zones undergo a stacking effect, with consequent zone sharpening, due to modulation of their free mobility via protonation of the -OH group (enolate ion) in the hetero aromatic rings of G and T, which undergo a lactam-lactim transition. (ii) As the zones migrate down the pH gradient, they transit through a pH 6.5-8.5 zone where, for Pharmalytes, the beta/lambda ratio reaches a maximum and is constant as well. This last condition allows high voltage gradients (typically 1000 V/cm, even in 75 microm capillaries) to be delivered, thus greatly reducing the analysis time and maintaining peak sharpness, due to limited diffusion.

Regulation of rat proximal tubule Na/H exchange by protein kinase C.

The effect of protein kinase C (PKC) activation on fluid and bicarbonate transport in renal tubules has been discussed controversially. Stimulation and inhibition have been shown to depend on factors such as experimental model and exposure time to the mediator of enzyme activation. We studied the role of PKC activation by phorbol-12-myristate-13-acetate (PMA) and by 1,2-dioctanoyl glycerol (DOG) in proximal bicarbonate reabsorption (JHCO3-) by 'in vivo' stationary microperfusion and ion-exchange resin microelectrode determination of luminal pH. Both PMA (10(-8) mol/l) and DOG (10(-3) mol/l) added to lumen or to peritubular capillaries reduced the net JHCO3- significantly. When added to lumen, the inhibition was 44 and 32%, respectively. This reduction did not involve changes in lumen stationary pH, but was mediated by a marked increase in the halftime of luminal bicarbonate disappearance; from 4.22 +/- 0.23 to 6.27 +/- 0.51 s with PMA and from 3.90 +/- 0.25 to 6.33 +/- 0.48 s with DOG. This effect was intensified by 10(-6) mol/l okadaic acid, a phosphatase inhibitor (inhibition of JHCO3- increased to 61%), and reduced by 30% by 10(-6) mol/l H7, an inhibitor of PKC. H89, a protein kinase A inhibitor, did not affect the inhibitory action of PMA. Our data suggest that PKC activation reduces the rate of H ion secretion (bicarbonate reabsorption) in convoluted segments of rat renal proximal tubules and that phosphorylation of the Na+/H+ exchanger by this kinase is the cause of the reduction in net secretion of H ions.