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At least 19 recordsLinked to original sources
PubMed1993

HEPES buffer perfusate alters rabbit lung endothelial permeability.

N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) has been shown to cause changes in cultured endothelial cells and smooth muscle function at concentrations from 5 to 25 mM. To determine whether HEPES also affects vascular permeability, the effects of two buffers, HEPES and phosphate, were compared in isolated perfused rabbit lungs. Hemodynamic parameters and vascular protein permeability-surface area products (PS) were measured after perfusion with the buffers. Endothelial permeability was measured for an anionic and a cationic albumin to assess the charge effects of the zwitterion buffer. With HEPES, there were no changes in vascular pressure or resistance but permeability was affected. Cationic albumin permeability increased with 12 mM HEPES (8.7phosphate-->30(12) mM HEPES x ml.min-1.g dry lung-1 x 10(-2)) as did the anionic albumin PS (2.7phosphate-->3.52(12) mM HEPES). The cationic PS returned to baseline (8.1(60) mM HEPES) at 60 mM HEPES, but the anionic PS did not change from the 12 mM HEPES (4.01(60) mM HEPES). In summary, we find that HEPES is not innocuous. Although hemodynamic parameters did not change, endothelial permeability was increased when HEPES was used at normal concentrations. Therefore, HEPES should be used with caution as a physiological buffer in perfused organ systems.

Animals↗
PubMed1976

The effects of hepes buffer on clotting tests, assay of factors V and VIII and on the hydrolysis of esters by thrombin and thrombokinase.

Shorter clotting times were found in the presence of 50 mM Hepes (N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid) buffer than of 50mM Imidazole buffer in one-stage assays of factors V and VIII, in modified APTT and PT tests and in tests of the clotting of human plasma by purified human thrombin. All tests were performed at ionic strength 0.155 in the presence of either Hepes. NaOH or Imidazole. HCl buffer, pH 7.4 at 37 degrees. The faster clotting in the presence of Hepes buffer, therefore, is probably due, at least in part, to acceleration by Hepes of thrombin's enzymatic action on fibrinogen and/or of the polymerization of the fibrin monomers. Hepes may also have effects of other blood clotting reactions. Rates of hydrolysis of TAME or BAME (p-toluenesulfonyl- or benzoyl-L-arginine methyl ester) at pH 7.4 37 degrees by purified human bovine thrombin were essentially the same in 200 mM Hepes as in 250 mM Tris. HCl buffer (rates in Hepes. NaOH or Hepes. KOH buffers were compared with those in Tris. HCl plus NaCl for KCl). However, with purified bovine thrombokinase, rates of TAME hydrolysis in Hepes buffer were accelerated and rates of BAME hydrolysis slightly inhibited. Hepes, therefore, reacts with thrombokinase but whether this accelerates (or inhibits) the rate of converting prothrombin to thrombin remains to be determined. In addition, Hepes has an inhibitory effect on clotting since increasing the concentration of Hepes from 50 mM to 200 mM inhibits clotting in the PT, APTT and bovine thrombin-human plasma tests. Hepes buffer is being added to some plasmas and to some reagents used in clotting tests. It is, therefore, important to realize that its concentration must be monitored closely or erroneous results may be obtained in clotting tests and assays of clotting factors. The clotting times were the same in the presence of 50 mM Tris. HCl as in Imidazole. HCl buffers in APTT tests at three ionic strengths but they differed slightly in plasma-thrombin tests. Depending upon the ionic strength, 17 mM Barbital Sodium. HCl buffer inhibited APTT tests but accelerated plasma-thrombin tests. All the buffers tested, therefore, have individual effects on the clotting tests.

Animals↗
PubMed1997

HEPES inhibits contractile responses of canine basilar artery.

N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) is a commonly-used buffer. This study determined the effect of HEPES on contractility of the dog basilar artery and tested the hypothesis that HEPES inhibits vasoconstriction of isolated arterial segments by generating H2O2. Rings of dog basilar artery with or without endothelium were suspended under isometric tension and contracted with KCl, serotonin, or prostaglandin F2 alpha (PGF2 alpha) in bicarbonate or HEPES buffer. Addition of HEPES, 30 mmol l-1, before or after contraction with KCl, serotonin or PGF2 alpha significantly decreased maximal tension in rings with or without endothelium. Preincubation with HEPES buffer, 10 mmol l-1, significantly decreased maximal contractions to each agonist in rings with endothelium and to KCl and serotonin in rings without endothelium. HEPES, 30 mmol l-1, noncompetitively inhibited concentration-contraction curves to increasing concentrations of each agonist in rings with or without endothelium. Inhibition by HEPES was completely reversible with washing. The inhibitory effects of HEPES on responses to each agonist in rings with endothelium were significantly less in the dark or after coincubation with catalase. Unlike HEPES, effects of H2O2 were endothelium-dependent in that H2O2 caused contractions in rings with endothelium and relaxations in rings without endothelium. 5-(N,N'-dimethyl)-amiloride and 4,4'-diisothiocyanataostilbene-2,2'-disulfonic acid did not affect contractility in this preparation. These results show that HEPES exerts significant inhibitory effects on arterial smooth muscle contractility. The mechanism does not involve endothelium-dependent relaxation, effects on chloride channels or the sodium-hydrogen exchanger or generation of H2O2 by HEPES in the light.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

The critical role of Hepes in SIN-1 cytotoxicity, peroxynitrite versus hydrogen peroxide.

The cytotoxicity of the superoxide anion radical- and nitric oxide-releasing compound SIN-1 to L929 cells was studied in Krebs-Henseleit buffer. pH 7.4, in the presence and absence of Hepes. SIN-1 cytotoxicity was significantly higher in the presence of Hepes than in the absence of Hepes. The available amount of peroxynitrite formed from SIN-1, however, was significantly decreased by Hepes as indicated by decreased oxidation of dihydrorhodamine 123. On the other hand, Hepes largely increased the formation of H2O2 from SIN-1. Catalase protected the L929 cells from SIN-1 cytotoxicity in the buffer with Hepes. In the buffer without Hepes catalase did not have any protective effect. In contrast, tyrosine and tryptophan provided significant protection against SIN-1 cytotoxicity independent of the presence of Hepes. These results demonstrate that the immediate toxic agent formed from SIN-1 decisively depends on the presence of Hepes. In its absence cytotoxicity is most likely mediated by peroxynitrite while in the presence of Hepes, cytotoxicity is conveyed by co-operative action of hydrogen peroxide and reactive nitrogen species.

Animals↗
PubMed1998

Hydrogen peroxide formation by reaction of peroxynitrite with HEPES and related tertiary amines. Implications for a general mechanism.

Organic amine-based buffer compounds such as HEPES (Good's buffers) are commonly applied in experimental systems, including those where the biological effects of peroxynitrite are studied. In such studies 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), a compound that simultaneously releases nitric oxide (.NO) and superoxide (O-2), is often used as a source for peroxynitrite. Whereas in mere phosphate buffer H2O2 formation from 1.5 mM SIN-1 was low ( approximately 15 microM), incubation of SIN-1 with Good's buffer compounds resulted in continuous H2O2 formation. After 2 h of incubation of 1.5 mM SIN-1 with 20 mM HEPES about 190 microM H2O2 were formed. The same amount of H2O2 could be achieved from 1.5 mM SIN-1 by action of superoxide dismutase in the absence of HEPES. The increased H2O2 level, however, could not be related to a superoxide dismutase or to a NO scavenger activity of HEPES. On the other hand, SIN-1-mediated oxidation of both dihydrorhodamine 123 and deoxyribose as well as peroxynitrite-dependent nitration of p-hydroxyphenylacetic acid were strongly inhibited by 20 mM HEPES. Furthermore, the peroxynitrite scavenger tryptophan significantly reduced H2O2 formation from SIN-1-HEPES interactions. These observations suggest that peroxynitrite is the initiator for the enhanced formation of H2O2. Likewise, authentic peroxynitrite (1 mM) also induced the formation of both O-2 and H2O2 upon addition to HEPES (400 mM)-containing solutions in a pH (4.5-7.5)-dependent manner. In accordance with previous reports it was found that at pH >/=5 oxygen is released in the decay of peroxynitrite. As a consequence, peroxynitrite(1 mM)-induced H2O2 formation ( approximately 80 microM at pH 7.5) also occurred under hypoxic conditions. In the presence of bicarbonate/carbon dioxide (20 mM/5%) the production of H2O2 from the reaction of HEPES with peroxynitrite was even further stimulated. Addition of SIN-1 or authentic peroxynitrite to solutions of Good's buffers resulted in the formation of piperazine-derived radical cations as detected by ESR spectroscopy. These findings suggest a mechanism for H2O2 formation in which peroxynitrite (or any strong oxidant derived from it) initially oxidizes the tertiary amine buffer compounds in a one-electron step. Subsequent deprotonation and reaction of the intermediate alpha-amino alkyl radicals with molecular oxygen leads to the formation of O-2, from which H2O2 is produced by dismutation. Hence, HEPES and similar organic buffers should be avoided in studies of oxidative compounds. Furthermore, this mechanism of H2O2 formation must be regarded to be a rather general one for biological systems where sufficiently strong oxidants may interact with various biologically relevant amino-type molecules, such as ATP, creatine, or nucleic acids.

Amines↗
PubMed1995

Simultaneous measurement of pH and membrane potential in rat dorsal vagal motoneurons during normoxia and hypoxia: a comparison in bicarbonate and HEPES buffers.

1. The effects of oxygenated and hypoxic bicarbonate/CO2, 10 and 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered artificial cerebrospinal fluid (ACSF) have been studied in a rat brain slice preparation. Double-barreled pH-selective microelectrodes were used to measure intracellular pH (pHi) and membrane potential in dorsal vagal motoneurons (DVMs) and to measure extracellular pH (pHe) in the dorsal vagal motonucleus. 2. In bicarbonate ACSF, pHi averaged 7.24 +/- 0.05 (mean +/- SE, n = 21) and ranged from 6.86 to 7.79 pH units. pHe averaged 7.13 +/- 0.08 (n = 10). 3. On changing from oxygenated bicarbonate ACSF to either 10 or 25 mM HEPES ACSF, pHi decreased by 0.13-0.15 units, and the membrane depolarized by 10-11 mV. pHe also decreased in 10 mM HEPES ACSF (pHe 6.89 +/- 0.02, n = 8) but not in 25 mM HEPES ACSF (pHe 7.15 +/- 0.06, n = 3). In most neurons changes in pHi preceded changes in membrane potential. 4. In bicarbonate ACSF and in 25 mM HEPES ACSF, there was a significant linear relationship between prehypoxic pHi and the direction and amplitude of the hypoxia-induced membrane potential change (either an hyperpolarization or a depolarization). 5. In 10 mM HEPES ACSF, hypoxia always induced a depolarization; there was no correlation between prehypoxic pHi and the membrane potential response. 6. In bicarbonate ACSF and in 10 and 25 mM HEPES ACSF, hypoxia resulted in intracellular and extracellular acidification. However, the extracellular acidification in hypoxic 10 mM HEPES buffer was most pronounced (pH 6.40 +/- 0.11, n = 8), reflecting a preexisting extracellular acidification in oxygenated 10 mM HEPES buffer. 7. Various hypotheses that could give rise to a relationship between changes in membrane potential and pH are discussed; arguments are presented in favor of the concept that modulation of ion channels by either pHi or pHe, or both, is responsible for the observed correlations.

Animals↗
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↗
PubMed1989

Culture of sheep zygotes in synthetic oviduct fluid medium with different concentrations of sodium bicarbonate and HEPES.

An investigation was conducted of the viability of sheep zygotes in vivo subsequent to culture in synthetic oviduct fluid medium (SOFM) in which sodium bicarbonate was partly or wholly replaced with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid); concentrations of 0, 6.25, 12.5, 18.75 and 25 mM HEPES were studied. The addition of HEPES lowered the pH during the culture period, and was associated with a reduction in the percentage of zygotes that developed to blastocysts (range 60.4 to 85.2%); this reduction was significant (P < 0.05) at concentrations of 18.75 and 25 mM. There was also a significant (P < 0.05) reduction in the percentage of zygotes that commenced hatching (range 47.2 to 74.1%) and a significant (P < 0.05) delay in the time of blastocyst formation (range 4 to 7 d) in medium containing 25 mM HEPES. Viability, as assessed by elongation of the trophoblast after transfer to recipient ewes, was similar for zygotes cultured for 3 d either in medium with 12.5 mM HEPES or in medium without HEPES (90.3 and 93.9%, respectively). Extending the culture period to 5 d was associated with a general decline in viability (P < 0.05), with 67.7% of zygotes cultured with HEPES elongating compared with 45.5% for those cultured without HEPES. This study confirms that sheep zygotes can be successfully cultured in SOFM for up to 3 d without loss of viability. The addition of HEPES provides additional buffering capacity but zygote development may be compromised.

Journal Article↗
PubMed2006

Use of a medium buffered with N-hydroxyethylpiperazine-N-ethanesulfonate (HEPES) in intracytoplasmic sperm injection procedures is detrimental to the outcome of in vitro fertilization.

OBJECTIVE: This study was conducted to determine whether N-hydroxyethylpiperazine-N-ethanesulfonate (HEPES)-buffered medium used for the microinjection of sperm into oocytes may be detrimental for the embryo. DESIGN: Controlled randomized study. SETTING: Private IVF center. PATIENT(S): Women (n = 708) undergoing ICSI. INTERVENTION(S): The women were randomized into two study groups: 2,204 oocytes from 357 women were treated using a medium buffered with bicarbonate without HEPES during the ICSI procedure, and 2,168 oocytes from 351 women were treated using a medium buffered with HEPES during the ICSI procedure. MAIN OUTCOME MEASURE(S): Fertilization rate, degeneration rate, triploid rate, cleavage rate, embryo quality, pregnancy rate, implantation rate, and abortion rate. RESULT(S): Oocytes treated with a HEPES-buffered medium showed a statistically significant higher rate of triploid and degenerated oocytes after fertilization with ICSI compared with oocytes treated with a medium without HEPES. The embryos obtained from oocytes microinjected with a HEPES-buffered medium showed a statistically significant higher rate of highly fragmented embryos compared with the controls. Pregnancy rate and implantation rate were statistically significantly lower in the patient group with oocytes treated with the HEPES-buffered medium. The other parameters evaluated did not show any statistically significant differences. CONCLUSION(S): Our study showed that the use of media buffered with HEPES, during the microinjection of sperm into the oocytes, is detrimental for IVF outcome and should be avoided.

Adult↗
PubMed2001

Sustained in vivo activity of recombinant bovine granulocyte colony stimulating factor (rbG-CSF) using HEPES buffer.

The purpose of this study was to develop a long-acting injectable formulation of bG-CSF for veterinary use. However, in order to achieve sustained in vivo activity it was first necessary to stabilize the protein at the injection site. Preformulation studies, as well as literature, suggest that bG-CSF aggregates at neutral pH ranges (i.e., pH 6-8) and at temperatures of approximately 40 degrees C. Therefore, bG-CSF will not retain its activity for an extended period of time at the injection site. During this study we determined that HEPES buffer has a very significant impact on protein stability as well as on biological performance. Recombinant bovine granulocyte colony stimulating factor (rbG-CSF) was formulated in 1 M HEPES buffer for subcutaneous injection into cows. bG-CSF formulated in 1 M HEPES buffer resulted in sustained in vivo activity of bG-CSF compared to the "control" formulation (control formulation: 5% mannitol, 10 mM acetate buffer, 0.004% tween-80, pH 4). White blood cell (WBC) count was used as a marker to evaluate in vivo activity of the formulation. WBC numbers remained above a threshold value for only 24-30 h for the control formula. However, when bG-CSF was formulated in 1 M HEPES, the WBC remained above threshold for 3 days or 72 h. Formulating bG-CSF in 1 M HEPES at pH 7.5 also resulted in greater solution stability. This was surprising since bG-CSF is intrinsically not stable at neutral pH. The effect of 1 M HEPES on the T(M) (temperature at maximum heat flow on calorimetry scan) of bG-CSF was determined by microcalorimetry. In the absence of 1 M HEPES buffer the T(M) was 48 degrees C (onset approximately 40 degrees C), while bG-CSF formulated in 1 M HEPES buffer has a T(M) of 59 degrees C (onset approximately 50 degrees C). Similar organic buffers, such as MOPS, HEPPS, TES, and tricine, also resulted in improved solution stability as well as in sustained in vivo activity. The dramatic effect of these buffers on stability and biological performance of bG-CSF is not well understood. One hypothesis is that the electrostatic interaction between the zwitterionic form of these buffers and bG-CSF provides stabilization against denaturation.

Animals↗
PubMed1992

A change from HCO3(-)-CO2- to hepes-buffered medium modifies membrane properties of rat CA1 pyramidal neurones in vitro.

1. Intracellular recordings were obtained from CA1 pyramidal neurones in rat hippocampal slices. Perfusion with a HCO3(-)-CO2-free, HEPES-buffered medium at pH 7.4 produced a wide variety of reversible effects on neuronal excitability, compared to responses obtained under standard (21 mM-HCO3-, 5% CO2, pH 7.4) conditions. 2. Introduction of HCO3(-)-CO2-free medium most commonly elicited, within 5-20 min, a fall in resting membrane potential (Vm), a rise in threshold for Na(+)-dependent action potential generation, and a reduction in input resistance. Anomalous inward rectification in the hyperpolarizing direction and subthreshold inward rectification were commonly reduced in HEPES-buffered medium. More prolonged exposure (> or = 25 min) to HCO3(-)-CO2-free medium produced, on occasion, Na+ spike inactivation. 3. The amplitudes of the fast and medium after-hyperpolarizations (AHPs) following a single depolarizing current-evoked action potential were attenuated during perfusion with HEPES-buffered medium at pH 7.4, as was the composite AHP following a train of action potentials. 4. Perfusion with HEPES-buffered medium at pH 7.4 reduced the degree of spike frequency adaptation and abolished depolarizing current-evoked burst-firing behaviour when this was present under standard conditions. 5. In tetrodotoxin (TTX)- and tetraethylammonium (TEA)-poisoned neurones, perfusion with HCO3(-)-CO2-free medium at pH 7.4 slightly raised the threshold for activation of Ca(2+)-dependent potentials and slightly reduced their duration, compared to responses obtained in HCO3(-)-CO2-buffered medium at the same pH. The AHP following the Ca2+ spike was, however, markedly attenuated. 6. Perfusion with a low-pH HCO3(-)-CO2-buffered medium (7 mM-HCO3-, 5% CO2, pH 6.9) produced changes qualitatively similar to those observed during perfusion with HEPES-buffered medium at pH 7.4. Raising the pH of the HEPES-buffered medium to 7.8 or 7.9 reversed inconsistently and then only in part the changes noted on the transition from a HCO3(-)-CO2- to a HEPES-buffered medium at the same pH (7.4). 7. The effects noted are unlikely to be due to a direct action of HEPES itself on neuronal membrane conductances. Rather, I suggest that they are likely to be caused by intracellular acidosis consequent upon the omission of HCO3- and CO2 from the extracellular medium.

Action Potentials↗
PubMed1996

Electrochemical evidence of surfactant activity of the hepes pH buffer which may have implications on trace metal availability to cultures in vitro.

Hydrogen ion buffers are required for many different types of in vitro biological and chemical studies, but they may not be inert enough, thus interfering with the system under study. N-Hydroxyethylpiperazine-N'-ethanesulfonic acid (Hepes), a zwitterionic buffer in the 7.0-8.0 pH range, has been used in different biological studies. In this work, surfactant activity of Hepes and the buffer's capability to bind copper(II) were investigated through different electrochemical techniques: ion-selective electrode potentiometry, potentiometric stripping analysis, differential pulse anodic stripping voltammetry, normal pulse anodic stripping voltammetry, and alternating current polarography. Hepes concentrations between 0.1 (0.24 g liter-1) and 25 mmol liter-1 (6.0 g liter-1) were studied at pH 8.0 (and at pH 6.5 for comparison). At pH 8.0 Hepes displayed surfactant activity, particularly when magnitude of Hepes > 10 mmol liter-1 (2.4 g liter-1). The ability of Hepes to bind copper(II) was detected when Hepes was in large excess (magnitude of Hepes/magnitude of Cu between 10,000 and 2000). Eventual implications in the bioavailability of copper(II) are also discussed.

Cells, Cultured↗
PubMed1987

Enhanced release and synthesis of lipoprotein lipase in rat heart cell cultures exposed to high concentrations of Hepes.

While attempting to optimize conditions for synthesis of lipoprotein lipase by cultured heart cells, we encountered an unexpected rise in enzyme activity when media were supplemented inadvertently with 100 mM Hepes buffer (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid). This finding was further investigated and optimal results were obtained at pH 7.0-7.2. The increase in lipoprotein lipase activity was time dependent; after 3-6 h there was a rise in medium activity but cellular activity increased only after 24 h. The increased enzyme activity was defined as lipoprotein lipase by inhibition with antiserum to rat adipose tissue lipoprotein lipase. A 72-h exposure to Hepes resulted in a 30% increase in the incorporation of [35S]methionine into cellular proteins and a 2-fold increase into heparin-releasable proteins. Using heparin Sepharose chromatography and stepwise elution, a lipoprotein lipase enriched fraction was recovered with 2 M NaCl. The amount of [35S]methionine and [3H]galactose incorporated into protein of this fraction derived from Hepes-treated cells was 2-6-fold that of controls. A 4-fold increase in cellular lipoprotein lipase mass in Hepes-treated cells was shown by immunoblotting. Results obtained with Hepes-conditioned medium suggest the presence of cell-derived compounds that enhance release and subsequent synthesis of lipoprotein lipase. The effect of Hepes-conditioned medium on lipoprotein lipase resembled to some extent that of the addition of heparin. Therefore, it appears that when Hepes is first added to the culture medium, it might promote a release of heparan sulfate or related compounds, possibly by virtue of its negatively charged sulfonic acid residue. The accumulated heparan sulfate could then promote a sustained release of lipoprotein lipase into the culture medium which in turn leads to increased enzyme synthesis.

Animals↗
PubMed1990

Inhibition of an outwardly rectifying anion channel by HEPES and related buffers.

The effect of pH buffers and related compounds on the conductance of an outwardly rectifying anion channel has been studied using the patch-clamp technique. Single-channel current-voltage relationships were determined in solutions buffered by trace amounts of bicarbonate and in solutions containing N-substituted taurines (HEPES, MES, BES, TES) and glycines (glycylglycine, bicine and tricine), Tris and bis-Tris at millimolar concentrations. HEPES (pK alpha = 7.55) reduced the conductance of the channel when present on either side of the membrane. Significant inhibition was observed with 0.6 mM HEPES on the cytoplasmic side (HEPESi) and this effect increased with [HEPESi] so that conductance at the reversal potential was diminished approximately 25% with 10 mM HEPEsi and approximately 70% at very high [HEPESi]. HEPESi block was relieved by applying positive voltage but positive currents were not consistent with a Woodhull-type blocking scheme in that calculated dissociation constants and electrical distances depended on HEPES concentration. Results obtained by varying total HEPESi concentration at constant [HEPES-] and vice versa suggest both the anionic and zwitterionic (protonated) forms of HEPES inhibit. Structure-activity studies with related compounds indicate the sulfonate group and heterocyclic aliphatic groups are both required for inhibition from the cytoplasmic side. TES (pK alpha = 7.54), substituted glycine buffers (pK alpha = 8.1-8.4) and bis-Tris (pK alpha = 6.46) had no measurable effect on conductance and appear suitable for use with this channel.

Buffers↗
PubMed2004

Oxidative DNA damage induced by HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer in the presence of Au(III).

Oxidative DNA damage was investigated by free radicals generated from HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer, which is widely used in biochemical or biological studies, in the presence of Au(III). The effect of free radicals on the DNA damage was ascertained by gel electrophoresis, electron spin resonance (ESR) spectroscopy and circular dichroism (CD) spectroscopy. ESR results indicated the generation of nitrogen-centered cationic free radicals from the HEPES in the presence of Au(III) which cause the DNA damage. No ESR spectra were observed for phosphate, tris(hydroxymethyl)aminomethane (Tris-HCl) and acetate buffers in the presence of Au(III) or for HEPES buffer in the presence of other metal ions such as Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Pd(II) or [Au(III)(TMPyP)](5+) and [Pd(II)(TMPyP)](4+), where [H(2)(TMPyP)](4+) denotes tetrakis(1-methylpyridium-4-yl)porphyrin. Consequently, no DNA damage was observed for these buffer agents (e.g., phosphate, Tris-HCl or acetate) in the presence of Au(III) or for HEPES in the presence of other metal ions or the metalloporphyrins mentioned above. No detectable inhibitory effect on the DNA damage was observed by using the typical scavengers of reactive oxygen species (ROS) ()OH, O(2)(-) and H(2)O(2). This non-inhibitory effect indicated that no reactive oxygen species were generated during the incubation of DNA with HEPES and Au(III). The drastic change in CD spectra from positive ellipticity to negative ellipticity approximately at 270 nm with increasing concentration of Au(III) also indicated the significant damage of DNA. Only HEPES or Au(III) itself did not damage DNA. A mechanism for the damaging of DNA is proposed.

Buffers↗
PubMed2001

HEPES prevents edema in rat brain slices.

Brain slices gain water when maintained in bicarbonate-buffered artificial cerebro-spinal fluid (ACSF) at 35 degrees C. We previously showed that this edema is linked to glutamate receptor activation and oxidative stress. An additional factor that may contribute to swelling is acidosis, which arises from high CO2 tension in brain slices. To examine the role of acidosis in slice edema, we added N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) to osmotically balanced ACSF (HEPES-ACSF), thereby increasing buffering capacity beyond that provided by bicarbonate/CO2. Water gain was markedly inhibited in HEPES-ACSF. After 3 h incubation in HEPES-ACSF at 35 degrees C, water gain was limited to that of fresh slices after 1 h recovery in ACSF at room temperature. The effect of HEPES in decreasing slice water gain was concentration dependent from 0.3 to 20 mM. The inhibition of water gain by HEPES suggests that tissue acidosis is a contributing factor in brain slice edema.

Acidosis↗
PubMed1994

Effects of in-vivo administration of taurine and HEPES on the inflammatory response in rats.

The effect of in-vivo administration of N-2-hydroxyethylpiperazine-N'-2- ethane sulphonic acid (HEPES) and taurine on rat paw oedema and reactive oxidant production was examined. Carrageenan-induced paw oedema was attenuated following intraperitoneal injection of HEPES. Chemiluminescence production by isolated peripheral blood mononuclear cells (PBMC) was reduced in HEPES-treated rats. Taurine-treated rats did not exhibit attenuation of paw oedema using subcutaneous or intraperitoneal administration but intracerebroventricular administration produced a significant reduction at a dosage of 4.0 mumol. No reduction in chemiluminescence production was observed by PBMC using subcutaneous or intraperitoneal administration of taurine, but intracerebroventricular administration produced a significant reduction at a dosage of both 0.4 and 4.0 mumol. Intravenous injection of [14C]HEPES or [3H]taurine demonstrated rapid clearance with a significantly longer half-life of HEPES compared with taurine. These results support previous reports of anti-inflammatory activity of taurine when administered centrally. The lack of anti-inflammatory effect when taurine was administered subcutaneously or intraperitoneally may be a consequence of rapid distribution or clearance. The greater anti-inflammatory effects of HEPES compared with taurine may be due to its slower distribution or clearance in-vivo.

Animals↗
PubMed1992

Disparate efficacy of tobramycin on Ca(2+)-, Mg(2+)-, and HEPES-treated Pseudomonas aeruginosa biofilms.

Mucoid exopolysaccharide (MEP) obtained from Pseudomonas aeruginosa 579 was suspended in 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) pH 7.2 containing 0.1-10.0 mM of CaCl2.2H2O or MgCl2.4H2O. MEP treated with HEPES or < 5.0 mM of the Ca2+ or Mg2+ salts remained soluble and bound tobramycin in an equilibrium dialysis bioassay. MEP treated with 5.0 or 10.0 mM of the Ca2+ or Mg2+ salts did not bind tobramycin. Five and 10 mM Ca(2+)-treated MEP precipitated but Mg(2+)-treated MEP did not. Pseudomonas aeruginosa 579 biofilms formed using a defined growth medium having < 1 mM Ca2+ or Mg2+ were treated for 1 h with 10 mM HEPES +/- 5.0 mM CaCl2.2H2O or MgCl2.4H2O, prior to an 8-h exposure to HEPES, or the defined growth medium, +/- 125 micrograms/mL of tobramycin. The tobramycin kill kinetics for the HEPES-, Mg(2+)-, and Ca(2+)-treated biofilms were similar and gradual from T = 0-6 h. The viability of the HEPES- and Mg(2+)-treated populations declined sharply (from 6 to 8 h). Bacteria dispersed from the MEP in control biofilms at 0 and 8 h did not grow in the presence of 7.81 micrograms/mL of tobramycin. Thus, binding of tobramycin of P. aeruginosa 579 MEP may not be as influential to the impediment of tobramycin diffusion as is the steric hindrance imposed by the Ca2+ condensation of the polymer.

Binding Sites↗