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PubMed2006

Effects of multivalent salt addition on effective charge of dilute colloidal solutions.

The effective charge Z* is often invoked to account for the accumulation of counterions near the colloid with intrinsic charge Z. Although the ion concentrations c(i) are not uniform in the solution due to the presence of the charged particle, their chemical potentials are uniform everywhere. Thus, on the basis of ion chemical potential, effective ion concentrations c(i)*, which can be experimentally measured by potentiometry, are defined with the pure salt solution as the reference state. The effective charge associated with the charged particle can then be determined by the global electroneutrality condition. Monte Carlo simulations are performed in a spherical Wigner-Seitz cell to obtain the effective charge of the colloid. In terms of the charge ratio alpha=Z*/Z, the effects of added salt concentration, counterion valency, and particle charge are examined. The effective charge declines with increasing salt concentration and the multivalent salt is much more efficient in reducing the effective charge of the colloidal solution. Moreover, the extent of effective charge reduction is decreased with increasing intrinsic charge for a given concentration of added salt. Those results are qualitatively consistent with experimental observations by electrophoresis.

Journal Article↗

PubMed2000

On the Ca2+ dependence of non-transferrin-bound iron uptake in PC12 cells.

Non-transferrin-bound iron (NTBI) uptake has been reported to follow two pathways, Ca(2+)-dependent and Ca(2+)-independent (Wright, T. L., Brissot, P., Ma, W. L., and Weisiger, R. A. (1986) J. Biol. Chem. 261, 10909-10914; Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1990) J. Biol. Chem. 265, 3139-3145). Studies reporting the two pathways have ignored the weak interactions of Ca(2+) with the chelator nitrilotriacetate (NTA) and the reducing agent ascorbate. These studies used a constant ratio of total Fe(2+) to NTA with and without Ca(2+). We observed Ca(2+) activation of NTBI uptake in PC12 cells with the characteristics reported for other cells upon using 1 mm ascorbate and a constant ratio of total Fe(2+) to NTA with or without Ca(2+). However, Ca(2+) did not affect NTBI uptake in solutions without NTA. We then determined conditional stability constants for NTA binding to Ca(2+) and Fe(2+) by potentiometry under conditions of NTBI uptake experiments (pH, ionic strength, temperature, ascorbate, total Fe(2+), and total Ca(2+) concentrations). In solutions based on these constants and taking Ca(2+) chelation into account, Ca(2+) did not affect NTBI uptake over a range of free Fe(2+) concentrations. Thus, the Ca(2+) activation of NTBI uptake observed using the constant total Fe(2+) to NTA ratio was because of Ca(2+)-NTA chelation rather than an activation of the NTBI transporter itself. It is suggested that the previously reported Ca(2+) dependence of NTBI uptake be re-evaluated.

Animals↗

PubMed2000

Electron transfer and binding of the c-type cytochrome TorC to the trimethylamine N-oxide reductase in Escherichia coli.

Reduction of trimethylamine N-oxide (E'(0(TMAO/TMA)) = +130 mV) in Escherichia coli is carried out by the Tor system, an electron transfer chain encoded by the torCAD operon and made up of the periplasmic terminal reductase TorA and the membrane-anchored pentahemic c-type cytochrome TorC. Although the role of TorA in the reduction of trimethylamine N-oxide (TMAO) has been clearly established, no direct evidence for TorC involvement has been presented. TorC belongs to the NirT/NapC c-type cytochrome family based on homologies of its N-terminal tetrahemic domain (TorC(N)) to the cytochromes of this family, but TorC contains a C-terminal extension (TorC(C)) with an additional heme-binding site. In this study, we show that both domains are required for the anaerobic bacterial growth with TMAO. The intact TorC protein and its two domains, TorC(N) and TorC(C), were produced independently and purified for a biochemical characterization. The reduced form of TorC exhibited visible absorption maxima at 552, 523, and 417 nm. Mediated redox potentiometry of the heme centers of the purified components identified two negative midpoint potentials (-177 and -98 mV) localized in the tetrahemic TorC(N) and one positive midpoint potential (+120 mV) in the monohemic TorC(C). In agreement with these values, the in vitro reconstitution of electron transfer between TorC, TorC(N), or TorC(C) and TorA showed that only TorC and TorC(C) were capable of electron transfer to TorA. Surprisingly, interaction studies revealed that only TorC and TorC(N) strongly bind TorA. Therefore, TorC(C) directly transfers electrons to TorA, whereas TorC(N), which probably receives electrons from the menaquinone pool, is involved in both the electron transfer to TorC(C) and the binding to TorA.

Bacterial Proteins↗

PubMed2002

Kinetic and structural basis of reactivity of pentaerythritol tetranitrate reductase with NADPH, 2-cyclohexenone, nitroesters, and nitroaromatic explosives.

The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and alpha,beta-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electron-deficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP.

Carbon↗

PubMed2003

Characterization of the cobaltochelatase CbiXL: evidence for a 4Fe-4S center housed within an MXCXXC motif.

CbiX is a cobaltochelatase required for the biosynthesis of vitamin B12 and is found in Archaea as a short form (CbiXS containing 120-145 amino acids) and in some bacteria as a longer version (CbiXL containing 300-350 amino acids). Purification of either recombinant Bacillus megaterium or Synechocystis CbiXL in Escherichia coli, which is facilitated by the presence of a naturally occurring histidine-rich region of the protein, results in the isolation of a dark brown protein solution. The UV/visible spectrum of the protein is consistent with the presence of a redox group, and the lack of definition within the spectrum is suggestive of a 4Fe-4S center. The presence of an iron-sulfur center was confirmed by EPR analysis of the proteins, which produces a pseudoaxial spectrum with g values at 2.04, 1.94, and 1.90. The EPR spectrum was absent at 70 K, an observation that is diagnostic of a 4Fe-4S center. Redox potentiometry coupled with optical spectroscopy allowed the midpoint potential of the redox center to be determined for the CbiXL from both B. megaterium and Synechocystis. Sequence analysis of CbiXL proteins reveals only two conserved cysteine residues within the CbiXL proteins, which are part of an MXCXXC motif. Mutagenesis of the two cysteines leads to loss of both the EPR spectrum and UV/visible spectral features of the Fe-S center in the protein, clearly indicating that these residues are involved in ligating the cofactor to the apoprotein possibly in a butterfly arrangement. The potential physiological role of the iron-sulfur center is discussed.

Amino Acid Motifs↗

PubMed2003

Cytochrome c551 from Starkeya novella: characterization, spectroscopic properties, and phylogeny of a diheme protein of the SoxAX family.

Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES). Previously characterized SoxAX proteins from Rhodovulum sulfidophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit. In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups. Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits. Apparent molecular masses of 27,229 +/- 10.3 Da and 20,258.6 +/- 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure. Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: +133 mV (pH 6.0); +104 mV (pH 7.0); +49 (pH 7.9) and +10 mV (pH 8.7). EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation. The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals. Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S. novella. Phylogenetic analysis of soxA- and soxX-related gene sequences indicates a parallel development of SoxA and SoxX, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.

Alphaproteobacteria↗

PubMed2004

4-cyanopyridine, a versatile spectroscopic probe for cytochrome P450 BM3.

The nitrogenous pi -acceptor ligand 4-cyanopyridine (4CNPy) exhibits reversible ligation to ferrous heme in the flavocytochrome P450 BM3 (Kd=1.8 microm for wild type P450 BM3) via its pyridine ring nitrogen. The reduced P450-4CNPy adduct displays unusual spectral properties that provide a useful spectroscopic handle to probe particular aspects of this P450. 4CNPy is competitively displaced upon substrate binding, allowing a convenient route to the determination of substrate dissociation constants for ferrous P450 highlighting an increase in P450 substrate affinity on heme reduction. For wild type P450 BM3, Kd(red)(laurate)=82.4 microm (cf. Kd(ox)=364 microm). In addition, an unusual spectral feature in the red region of the absorption spectrum of the reduced P450-4CNPy adduct is observed that can be assigned as a metal-to-ligand charge transfer (MLCT). It was discovered that the energy of this MLCT varies linearly with respect to the P450 heme reduction potential. By studying the energy of this MLCT for a series of BM3 active site mutants with differing reduction potential (Em), the relationship EMLCT + (3.53 x = Em 17,005 cm)(-1) was derived. The use of this ligand thus provides a quick and accurate method for predicting the heme reduction potentials of a series of P450 BM3 mutations using visible spectroscopy, without the requirement for redox potentiometry.

Bacterial Proteins↗

PubMed2005

Role of active site residues and solvent in proton transfer and the modulation of flavin reduction potential in bacterial morphinone reductase.

The reactions of several active site mutant forms of bacterial morphinone reductase (MR) with NADH and 2-cyclohexen-1-one as substrates have been studied by stopped-flow and steady-state kinetic methods and redox potentiometry. The enzymes were designed to (i) probe a role for potential proton donors (Tyr-72 and Tyr-356) in the oxidative half-reaction of MR; (ii) assess the function of a highly conserved tryptophan residue (Trp-106) in catalysis; (iii) investigate the role of Thr-32 in modulating the FMN reduction potential and catalysis. The Y72F and Y356F enzymes retained activity in both steady-state and stopped-flow kinetic studies, indicating they do not serve as key proton donors in the oxidative reaction of MR. Taken together with our recently published data (Messiha, H. L., Munro, A. W., Bruce, N. C., Barsukov, I., and Scrutton, N. S. (2005) J. Biol. Chem. 280, 4627-4631) that rule out roles for Cys-191 (corresponding with the proton donor, Tyr-196, in the structurally related OYE1 enzyme) and His-186 as proton donors, we infer solvent is the source of the proton in the oxidative half-reaction of MR. We demonstrate a key role for Thr-32 in modulating the reduction potential of the FMN, which is decreased approximately 50 mV in the T32A mutant MR. This effects a change in rate-limiting step in the catalytic cycle of the T32A enzyme with the oxidizing substrate 2-cyclohexenone. Despite the conservation of Trp-106 throughout the OYE family, we show this residue does not play a major role in catalysis, although affects on substrate and coenzyme binding are observed in a W106F enzyme. Our studies show some similarities, but also major differences, in the catalytic mechanism of MR and OYE1, and emphasize the need for caution in inferring mechanism by structural comparison of highly related enzymes in the absence of solution studies.

Amino Acid Substitution↗

PubMed2005

Redox and spectroscopic properties of the Escherichia coli nitric oxide-detoxifying system involving flavorubredoxin and its NADH-oxidizing redox partner.

Under anaerobic conditions, the flavodiiron NO-reductase from Escherichia coli (flavorubredoxin, FlRd) constitutes one of the major protective enzymes against nitric oxide. The redox and spectroscopic properties of the rubredoxin (Rd), non-heme diiron, and FMN sites of flavorubredoxin were determined, which was complemented by the study of truncated versions of FlRd: one consisting only of its rubredoxin module, and another consisting of its flavodiiron structural core (lacking the Rd domain). The studies here reported were performed by a combination of potentiometry with visible and EPR spectroscopies. Moreover, we present the first direct EPR evidence for the presence of the non-heme diiron site in the flavodiiron proteins family. Also, the redox properties of the FlRd physiological partner, the NADH:flavorubredoxin oxidoreductase (FlRd-Red), were determined. It is further shown that the redox properties of this complex electron transfer system are fine-tuned upon interaction of the two enzymes.

Electromagnetic Fields↗

PubMed1979

Metal ion/buffer interactions. Stability of binary and ternary complexes containing 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) and adenosine 5'-triphosphate (ATP).

The interaction of 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) with the metal ions (M2+) Mg2+, Ca2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I = 0.1 or 1.0 M, KNO3, 25 degrees C). Stability constants of the M(Tris)2+ complexes were determined; those constants which were measured by both methods agreed well. Ternary complexes containing ATP4- as a second ligand were also investigated and it is shown that in the presence of Tris, mixed-ligand complexes of the type M(ATP)(Tris)2- are formed. The values for delta log KM, where delta log KM = log KM(ATP)M(ATP)Tris--log KMM(Tris), are all negative, thus indicating that the interaction of Tris with M(ATP)2- is somewhat less pronounced than with M2+. However, it should be noted that even in mixed-ligand systems complex formation with Tris may still be considerable, hence great reservations should be exercised in employing Tris as a buffer in systems which also contain metal ions. Distributions of the complex species in dependence on pH are shown for several systems, and the structures of the binary M(Tris)2- and the ternary M(ATP)(Tris)2- complexes are discussed. The participation of a Tris-hydroxo group in complex formation is, at least for the M(Tris)2- species, quite evident.

Adenosine Triphosphate↗

PubMed1988

Respiratory nitrate reductase from Paracoccus denitrificans. Evidence for two b-type haems in the gamma subunit and properties of a water-soluble active enzyme containing alpha and beta subunits.

1. The b-type haem centres of the three (alpha, beta and gamma) subunit nitrate reductase from Paracoccus denitrificans have been analysed by redox potentiometry. Two components were identified with mid-point potentials +95 mV and +210 mV. 2. Washing, in the absence of Mg2+ ions, of cytoplasmic membrane vesicles from P. denitrificans promoted selective release of nitrate reductase activity. The released enzyme was purified by chromatography and shown to contain alpha and beta, but not gamma polypeptides. A haem spectrum was absent, consistent with the lack of the gamma subunit. The alpha and beta polypeptides of the water-soluble nitrate reductase had molecular masses that were identical to those of the detergent-purified enzyme and also of the nitrate reductase in cytoplasmic membranes. This observation, together with the failure of protease inhibitors to prevent release from the membrane, indicates that the release is not related to limited proteolysis of the alpha and/or beta polypeptides. The relative molecular mass of the water-soluble alpha beta enzyme was estimated to be approximately 200,000. 3. The water-soluble nitrate reductase was released from intact inverted cytoplasmic membrane vesicles as judged by loss of NADH-NO3- reductase activity and retention by the vesicles after washing of uncoupler-sensitive NADH-oxidase activity. These observations show that alpha and beta polypeptides, and therefore the active site for nitrate reduction, are located on the cytoplasmic side of the membrane. 4. Attempts to reverse the nitrate reductase activity of the enzyme, using nitrate as reductant plus ferricyanide or chlorate as tested oxidants, were unsuccessful. The implications for the mechanism of the enzyme are discussed.

Cytoplasm↗

PubMed1992

EPR studies of cytochrome aa3 from Sulfolobus acidocaldarius. Evidence for a binuclear center in archaebacterial terminal oxidase.

The purified cytochrome aa3-type oxidase from Sulfolobus acidocaldarius (DSM 639) consists of a single subunit, containing one low-spin and one high-spin A-type hemes and copper [Anemüller, S. and Schäfer, G. (1990) Eur. J. Biochem. 191, 297-305]. The enzyme metal centers were investigated by electron paramagnetic resonance spectroscopy (EPR), coupled to redox potentiometry. The low-spin heme EPR signal has the following g-values: gz = 3.02, gy = 2.23 and gx = 1.45 and the high-spin heme exhibits an almost axial spectrum (gy = 6.03 and gx = 5.97, E/D < 0.002). In the enzyme as isolated the low-spin resonance corresponds to 95 +/- 10% of the enzyme concentration, while the high-spin signal accounts for only 40 +/- 5%. However, taking into account the redox potential dependence of the high-spin heme signal, this value also rises to 95 +/- 10%. The high-spin heme signal of the Sulfolobus enzyme shows spectral characteristics distinct from those of the Paracoccus denitrificans one: it shows a smaller rhombicity (gy = 6.1 and gx = 5.9, E/D = 0.004 for the P. denitrificans enzyme) and it is easier to saturate, having a half saturation power of 148 mW compared to 360 mW for the P. denitrificans protein, both at 10 K. The EPR spectrum of an extensively dialyzed and active enzyme sample containing only one copper atom/enzyme molecule does not display CuA-like resonances, indicating that this enzyme contains only a CUB-type center. The EPR-redox titration of the high-spin heme signal, which is assigned to cytochrome a3, gives a bell shaped curve, which was simulated by a non-interactive two step redox process, with reduction potentials of 200 +/- 10 mV and 370 +/- 10 mV at pH = 7.4. The decrease of the signal amplitude at high redox potentials is proposed to be due to oxidation of a CUB(I) center, which in the CUB(II) state is tightly spin-coupled to the heme a3 center. The reduction potential of the low-spin resonance was determined using the same model as 305 +/- 10 mV at pH = 7.4 by EPR redox titration. Addition of azide to the enzyme affects only the high-spin heme signal, consistent with the assignment of this resonance to heme a3. The results are discussed in the context of the redox center composition of quinol and cytochrome c oxidases.

Electron Spin Resonance Spectroscopy↗

PubMed1994

Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha.

The periplasmic nitrate reductase of Thiosphaera pantotropha has been purified from a mutant strain (M-6) that overproduces the enzyme activity under anaerobic growth conditions. The enzyme is a complex of a 93-kDa polypeptide and a 16-kDa nitrate-oxidizable cytochrome c552. The complex contains molybdenum; a fluorescent compound with spectral features of a pterin derivative can be extracted. In contrast to the dissimilatory membrane-bound nitrate reductases, the periplasmic nitrate reductase shows high specificity for nitrate as a substrate and is insensitive to inhibition by azide. The 93-kDa subunit exhibits immunological cross-reactivity with the catalytic subunit of Rhodobacter capsulatus N22DNAR+ periplasmic nitrate reductase. Mass spectrometric comparisons of holo-cytochrome c552 and apo-cytochrome c552 demonstrated that the polypeptide bound two haem groups. Mediated redox potentiometry of the cytochrome indicated that the haem groups have reduction potentials (pH = 7.0) of approximately -15 mV and + 80 mV. The functional significance of these potentials is discussed in relation to the proposed physiological role of the enzyme as a redox valve.

Cell Membrane↗

PubMed2004

Thermodynamic and kinetic analysis of the isolated FAD domain of rat neuronal nitric oxide synthase altered in the region of the FAD shielding residue Phe1395.

In rat neuronal nitric oxide synthase, Phe1395 is positioned over the FAD isoalloxazine ring. This is replaced by Trp676 in human cytochrome P450 reductase, a tryptophan in related diflavin reductases (e.g. methionine synthase reductase and novel reductase 1), and tyrosine in plant ferredoxin-NADP(+) reductase. Trp676 in human cytochrome P450 reductase is conformationally mobile, and plays a key role in enzyme reduction. Mutagenesis of Trp676 to alanine results in a functional NADH-dependent reductase. Herein, we describe studies of rat neuronal nitric oxide synthase FAD domains, in which the aromatic shielding residue Phe1395 is replaced by tryptophan, alanine and serine. In steady-state assays the F1395A and F1395S domains have a greater preference for NADH compared with F1395W and wild-type. Stopped-flow studies indicate flavin reduction by NADH is significantly faster with F1395S and F1395A domains, suggesting that this contributes to altered preference in coenzyme specificity. Unlike cytochrome P450 reductase, the switch in coenzyme specificity is not attributed to differential binding of NADPH and NADH, but probably results from improved geometry for hydride transfer in the F1395S- and F1395A-NADH complexes. Potentiometry indicates that the substitutions do not significantly perturb thermodynamic properties of the FAD, although considerable changes in electronic absorption properties are observed in oxidized F1395A and F1395S, consistent with changes in hydrophobicity of the flavin environment. In wild-type and F1395W FAD domains, prolonged incubation with NADPH results in development of the neutral blue semiquinone FAD species. This reaction is suppressed in the mutant FAD domains lacking the shielding aromatic residue.

Amino Acid Sequence↗

PubMed1993

Solubility, stability and ionization behaviour of famotidine.

The pKa of famotidine was determined at 23 +/- 0.2 degrees C to be 6.76, 6.98 and 6.89 by a spectrophotometric, solubility, and partitioning method, respectively. The pH-solubility profile of famotidine indicated an intrinsic solubility of 2.7 mM at 23 degrees C. Degradation of famotidine followed pseudo-first-order kinetics over a pH range of 1-11 at 37 +/- 0.2 degrees C and at an ionic strength (mu) of 0.5. The pH-rate profile was accounted for by the specific acid and base catalysed reactions as well as water-catalysed decomposition of both protonated and free famotidine. A pKa of 6.60, determined by potentiometry at 37 +/- 0.5 degrees C (mu = 0.5), was used in the kinetic calculations. Maximum stability occurred at pH 6.3. Undissociated acetic acid, dihydrogen phosphate ion and glycine anion were found to contribute to the general acid and base catalyses. pH-dependency of the apparent octanol-water partition coefficient of famotidine indicated a partition coefficient of 0.23 for free famotidine at 23 +/- 0.2 degrees C.

Buffers↗

PubMed1996

Determining the adequacy of sodium balance in hemodialysis using a kinetic model.

The importance of sodium balance avoiding intradialytic cardiovascular instability and interdialytic hypertension and pulmonary edema is well known. An early analytical single-pool kinetic model created to evaluate sodium balance in hemodialysis, using flame photometry to determine plasma and dialysate sodium concentrations, has been shown to have a level of imprecision of +/- 2.8 mEq/l in predicting end-dialysis sodium plasma water concentrations (NaPWt). The ionometric determination of sodium concentrations seems to be more accurate and refers to the activity of the sodium capable of crossing dialysis membranes. On the basis of the theoretical premises of the model mentioned above, we developed a computerized single-pool kinetic model which makes it possible to calculate the ionized dialysate sodium activity (NaDI) required to reach a pre-established target of end-dialysis blood sodium activity (NaBI). Thirty-seven non-diabetic and anuric patients undergoing regular thrice-weekly hemodialysis were given their usual dialysis treatment, with NaDI at the usual value for each patient (range 137-147 mEq/l) and kept constant throughout dialysis. At the beginning and end of the session, NaDI and NaBI were measured in quadruplicate by means of a Nova-1 device (Direct Potentiometry, Pabisch Instruments). The validity of this kinetic model was tested by considering the difference between predicted and observed (P-O) NaBI at the end of dialysis [(t)]. P-O NaBI(t) was -0.37 +/- 0.42 mEq/l, which was statistically different from 0 (p < 0.001). When P-O NaBI(t) was plotted against ONaBI(t), it was more negative at the higher values of ONaBI(t). P-O intradialytic sodium removal (Nag) was -12.5 +/- 17.8 mEq/session, which was also statistically different from 0 (p < 0.001). The imprecision of this kinetic model was less than 0.84 mEq/l, as estimated by doubling the SD of P-O NaBI(t) (0.42 = 0.84 mEq/l). Although the reasons for its inaccuracy especially at higher ONaBI(t) values remain to be clarified, these data are the expression of a satisfactory clinical model.

Aged↗

PubMed1988

Sodium measurements in the presence of paraproteins by four direct ISE methods and flame photometry compared.

Sodium was determined by flame photometry and by direct potentiometry using four different systems (Ektachem DT60, Corning 614, Orion 1020 and Nova-1) in serum samples from patients with normal proteins and paraproteins. Flame photometric and direct potentiometric differences (ISE-flame), showed the expected relationship with total protein whether or not the sample contained paraprotein, an exception being the Ektachem DT60 on normal protein. With the exception of the Ektachem DT60 with samples with normal protein levels, correlation between different ISE-flame estimates was good. Differences between direct potentiometric measurements are probably more attributable to efforts made by manufacturers to relate them to flame photometric measurements in 'normal samples', than to the volume occupied by increased protein content. The apparent lack of influence of protein on ISE-flame for the Ektachem DT60 was shown to relate to the original external reference electrode salt bridge used. A second study showed that changing this fluid to an equi-transferrant high salt content caesium chloride based reference fluid partially unmasked the protein effect.

Electrodes↗

PubMed2001

Antioxidant capacity of phenolic and related compounds: correlation among electrochemical, visible spectroscopy methods and structure-antioxidant activity.

We investigated the antioxidant activity of phenylpropionic acids--caffeic (CAF), ferulic (FER), para-coumaric (COU) and cinnamic (CIN)--and phenolic acids and related compounds--gallic (GAL), methyl gallate (meGAL), vanillic (VAN) and gentisic (GEN)--using visible spectroscopy, inhibition of nitroblue tetrazolium (NBT) reduction, and electrochemical methods including cyclic voltammetry and potentiometry. In the spectroscopic assays, only CAF, GAL and meGAL were able to inhibit NBT reduction. The same compounds showed the lowest oxidation potentials (Epa) and the highest redox potentials deltaE) in the cyclic voltammetric and potentiometric studies, respectively. In addition, it was observed that the greater the number of hydroxyls linked to the aromatic ring, the greater was the antioxidant activity of the analysed compounds. The correlations of Spermann--used to compare the methods between themselves and the methods with the relationship structure-antioxidant activity--were r = -0.9762 for the cyclic voltammetric-potentiometric methods. r = 0.8333 for the inhibition of NBT reduction-potentiometric methods and r = -0.8095 for the inhibition of NBT reduction-cyclic voltammetric methods. The correlations for cyclic voltammetric, potentiometric and inhibition of NBT reduction methods-number of hydroxyls linked to the aromatic ring were r = -0.9636, 0.9636 and 0.9142, respectively. These findings indicate that the electrochemical methods together with spectroscopic studies are a good tool to evaluate the antioxidant activity of substances.

Antioxidants↗