A rationally designed oligopeptide shows significant conformational changes upon binding to sulphate ions.

Oligopeptides that interact with oxoanions were developed by rational design methods. The substrate-binding site of the enzyme purine nucleoside phosphorylase served as a model for the design of the ionophores. The amino acids involved in the complexation of oxoanions were linked through flexible spacer residues. These spacers were chosen such that the relative orientation of the interacting amino acids was conserved. Several peptide sequences were preselected based on intermolecular H-bond frequencies. These frequencies were calculated from molecular dynamics trajectories of the corresponding peptide-anion complexes and used to score the binding properties of the peptides. The most promising peptides were prepared using solid phase peptide synthesis. Anion binding of the peptide ionophores was screened using circular dichroism (CD) and confirmed by NMR spectroscopy. CD measurements performed in methanol revealed a significant conformational change of a linear undecapeptide upon binding to sulphate ions. Two-dimensional-NMR experiments confirmed that a conformation with high helical content is formed in the presence of sulphate ions. These conformational changes induced by the anion stimulate the development of new transduction mechanisms in chemical sensors.

Long-term excessive magnesium supplementation is deleterious whereas suboptimal supply is beneficial for bones in rats.

The long-term effects of a suboptimal magnesium supply inducing a marginal or moderate deficiency or of an excessive magnesium supplementation corresponding to a basal diet with a high pharmacological intake were investigated in 36 growing Sprague-Dawley female rats. The rats were randomly divided in three groups and received a purified diet with 7 g calcium, 5 g phosphorus and either 0.2, 0.5 or 2 g magnesium per kg diet for 7 months. At the end of the trial, plasma and erythrocyte total magnesium concentrations were significantly lower in the magnesium-deficient group than in the respective control group. Serum concentrations of 1,25-dihydroxyvitamin D, PTH and IGF-I and the length of the right humeri were not affected by the dietary treatment. The volumes corrected for body weight, the medio-lateral diameters and the ratios dry weight/length of the right humeri, and the dry weight corrected for body weight of the left tibiae and of the right humeri were significantly smaller in the magnesium-supplemented group than in the two other groups. The magnesium contents of the left tibiae and of the first lumbar vertebrae were significantly lower in the magnesium-deficient group than in the two other groups. In the right femora, dual energy X-ray absorptiometry revealed significantly smaller areas in the proximal part and significantly smaller mineral contents in the second proximal quarter in the magnesium-supplemented group compared with the two other groups. Peripheral quantitative computer tomography of the right humeri revealed in the cortex significantly larger values for the relative area, mineral content, mineral density and thickness in the magnesium-deficient group compared with the control group. The maximum point of the load-deformation curve was significantly reduced in the fifth lumbar vertebrae and in the proximal femoral metaphyses of the magnesium-supplemented group. These results indicate that the long-term suboptimal magnesium supply improved some of the parameters indicators of bone health whereas the long-term supplementation was deleterious.

Development of new dyes for use in integrated optical sensors.

New chromoionophores have been developed, focused on NIR applications so that optode membranes may be used in monolithically integrated optical sensors. The wavelength of maximum absorbance has been estimated for a new model compound by the Pariser-Parr-Pople (PPP) method. Several cyanine type dyes have been tested as membrane chromoionophores. Membrane composition has been altered to overcome solubility problems. In this way, simple pH-sensitive optode membranes have been produced.

Postparturient hypocalcemia of dairy cows: a model for the study of the interdependence of Ca, Pi, and Mg homeostasis.

Disorders of calcium, phosphorus and magnesium homeostasis in ruminants provide natural models for the study of the physiology and pathophysiology of these minerals. The knowledge that can be acquired with a better understanding of the pathogenesis of these diseases could give useful clues in the puzzle of human osteoporosis. In the present study, the case of parturient paresis of dairy cows is reexamined with a newly developed technique for the measurements of serum ionized magnesium concentrations (Mg2+). The concentrations of total magnesium (Mgtot), ionized calcium (Ca2+), total calcium (Catot), and inorganic phosphate (Pi) were also determined in the sera of seventeen 3- to 16-year-old Brown Swiss and crossed Simmental/Red Holstein cows during the periparturient period. In each animal, a transient increase of Mg2+ and Mgtot serum concentrations was observed in association with the transient decrease after parturition of Ca2+, Catot and Pi serum concentrations. On average, throughout the study, serum Mg2+ concentrations were 68.5% of those of Mgtot whereas serum Ca2+ concentrations were 52% of those of Catot. The possible mechanisms involved in the transient increase of Mg2+ and Mgtot serum concentrations are discussed and the relevance of this data for osteoporosis is outlined.

Parathyroid hormone-related protein and calcium homeostasis during the periparturient period of dairy cows.

Plasma and milk concentrations of parathyroid hormone-related protein (PTHrP) at various stages of pregnancy and lactation were determined in thirty-nine 3- to 16-year-old Brown Swiss and Red Holstein x Simmental dairy cows originating from 4 herds. Eighteen of the cows were separated into 2 groups: low-parity (LP, n = 8) cows if they were in their first or second pregnancy and high-parity (HP, n = 10) cows if they were in their third or greater pregnancy. Blood samples were collected from each cow on 1 occasion, 15 to 5 days before calving, and blood and milk samples were collected daily during 6 days after calving. Serum total and ionized calcium (Ca(tot) and Ca2+, respectively) and milk Ca(tot) concentrations were also quantified. A transient postpartum decrease of serum Ca(tot) and Ca2+ concentrations was observed, whereas milk Ca(tot) concentration was constant. Plasma concentration of PTHrP was detected in 11 of 21 cows by use of an immunoradiometric assay (range, 0.45 to 1.82 pmol/L). Daily mean (+/- SD) colostrum and milk PTHrP concentrations ranged from 3.25 (+/- 3.23) to 4.69 (+/- 1.36) nmol/L in LP cows and 2.74 (+/- 0.5) to 5.95 (+/- 0.33) nmol/L in HP cows. In all cows of the HP group and most cows of the LP group, milk PTHrP concentration was highest in the day-1 sample. Milk PTHrP concentration correlated positively with milk Ca(tot) concentration in HP cows (r = 0.5959, P < 0.0001). In contrast, there was a negative relation between milk PTHrP and milk Ca(tot) concentrations in LP cows (r = -0.3285, P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

The concentration of ionized magnesium in serum during the periparturient period of non-paretic dairy cows.

Ion-selective electrodes have recently been designed for determining the ionized concentration of magnesium (Mg2+) in serum. This development may allow new insights into some metabolic diseases of cattle. For this report, the concentrations of Mg2+, total magnesium (Mgtot), ionized calcium (Ca2+), total calcium (Catot), and inorganic phosphate (P(i)) were determined in sera from seventeen 3- to 16-year-old Brown Swiss and crossed Simmental/Red Holstein cows during the periparturient period. In each animal, a transient increase of Mg2+ and Mgtot serum concentrations was observed in association with the transient decrease in serum concentrations of Ca2+, Catot and P(i) after parturition. On average, throughout the study, the serum Mg2+ concentrations were 68.5% of those of Mgtot, whereas the serum Ca2+ concentrations were 52% of those of Catot. The possible mechanisms involved in the transient increase of Mg2+ and Mgtot serum concentrations are discussed.

Nitrite-selective microelectrodes.

The development of nitrite-selective liquid membrane microelectrodes based on a synthetic charged ionophore is described. The addition of potassium tetrakis(4-chlorophenyl)borate and poly(vinyl chloride) to the membrane phase is essential to lower the ohmic resistance and to prolong the lifetime of the microelectrodes. The detection limits for sodium nitrite solutions without and with an ion background of 0.1M chloride are 10(-5.1) and 10(-4.2)M, respectively. The comparison with macroelectrodes shows that the miniaturization reduces, to some extent, the selectivity and the slopes of the EMF response functions.

Novel membraneless amperometric peroxide biosensor based on a tetrathiafulvalene-p-tetracyanoquinodimethane electrode.

Amperometric biosensors for the detection of hydrogen peroxide are prepared by adsorbing peroxidase (POD, EC 1.11.1.7, lipophilized with caprylic aldehyde) to TTF-TCNQ/silicone oil paste electrodes. This is the first time a reductase is coupled to an organic conducting salt electrode. At -50 mV vs Ag/AgCl and pH 6.0, the current vs concentration function can be described by the enzyme kinetic Michaelis-Menten formalism. Stable signals are obtained within 10 s. The detection limit is typically in the low nanomolar range for H2O2. The enzyme stability under storage, standby, and various operation conditions is discussed.

Characterization procedure for ion-selective electrode assays of magnesium activity in aqueous solutions of physiological composition.

A magnesium ion-selective liquid membrane electrode based on a synthetic neutral carrier is presented. The selectivity for Mg2+ over Na+, K+, and H+ is sufficient for assays in the physiological range, but a chemometric correction of approximately 10% is still necessary for the Ca2+ interference. We optimized the membrane composition especially with respect to selectivity and accuracy by performing a new characterization procedure in which we simulated the variety of blood serum samples with 10 aqueous solutions. After a three-point calibration, these solutions were measured alternately with the middlerange calibrator for 3 min. The magnesium activity was evaluated after correcting for the calcium interference according to the Nikolsky equation. The selectivity coefficient, KpotMgCa, was itself fitted to give the minimum deviations from the assigned magnesium activities. Electrodes with an optimized membrane composition show an average deviation from theoretical activity values of about 1.6% with logKpotMgCa = -0.8.

Novel magnesium ion-selective microelectrodes based on a neutral carrier.

Two types of Mg(2+)-selective microelectrodes based on a synthetic neutral carrier and containing poly-(vinyl chloride) (PVC) are described. The first is applicable to intracellular Mg2+ assays and suffers no interference from alkali and all other alkaline-earth metal cations. The second can be used for determining intra- as well as extracellular Mg2+ activities, but Ca2+ and especially lipophilic cations (e.g. acetylcholine) may interfere. Cyclohexanone is used for dissolving the PVC-containing membrane phases thus avoiding problems encountered with the much more volatile tetrahydrofuran. The procedure gives a higher output of working microelectrodes and can also be applied with other membrane phases.

[Chemical sensors in medical use: ion-selective electrodes, limitations and potentials]. / Chemische Sensoren im medizinischen Einsatz: Ionenselektive Elektroden, Grenzen und Chancen.

The electrolyte concentrations of potassium and sodium, for example, are routinely analyzed in diluted serum and plasma. In contrast, electrolyte activities are quantified by ISEs directly in undiluted whole blood and plasma and promise to be highly informative. Nevertheless, some drawbacks, such as the interaction between the biological matrix and the ISE surface, limit the accuracy of measurements. A symmetrical measuring arrangement allows one to localize and quantify the known sources of errors. These are minimized by adequate techniques. The membrane composition for sodium selective electrodes is optimized in particular. The interpretation of ISE values as compared to FAES (flame atomic emission spectroscopy) is closely related to the water concentration of the samples. Comparisons between ISE and FAES results obviously allow one to evaluate the water concentration of samples, as is shown by a linear regression plot.

Characterization of sodium-selective optode membranes based on neutral ionophores and assay of sodium in plasma.

Plasticized poly(vinyl chloride) membranes that incorporate a highly lipophilic sodium-selective neutral ionophore (ETH 4120) and novel H(+)-selective chromo-ionophores, lipophilic isologs of Nile Blue, are used in the competitive binding of Na+ and H+ for a reversible, optical determination of sodium activities in buffered solutions at different selected pH values. These optode membranes are used to assay total sodium concentrations in pH-buffered human blood plasma.

Lifetime of neutral-carrier-based liquid membranes in aqueous samples and blood and the lipophilicity of membrane components.

On the basis of previously reported correlations between the lipophilicity of membrane components, their partition coefficient between the membrane and the sample, and the lifetime of corresponding neutral-carrier-based sensors, the lipophilicities of ionophores and plasticizers in analytically relevant ion-selective electrodes, ISFETs, and optodes are analyzed and reported. Equations for the estimation of the lifetimes of liquid membranes in continuous-flow systems are presented, and the experimental determination of the lipophilicity values by thin-layer chromatography (TLC) is described. The required lipophilicities for the lifetimes of liquid membranes over 30 24-h days for different applications in aqueous solutions as well as in blood are presented. A comparison of the experimental results of lifetime measurements with calculated theoretical values is given. The experimental results of the determination of the lipophilicity by TLC are compared with the lipophilicities estimated on the basis of Hansch parameters.

Neutral ionophore-based selective electrode for assaying the activity of magnesium in undiluted blood serum.

The concentration of free, ionized magnesium in undiluted blood serum can be determined potentiometrically with a new magnesium-selective carrier in polymeric membrane electrodes (by weight 66% plasticizer, approximately 33% polyvinyl chloride and approximately 1% ionophore). However, discrimination of calcium by the new ionophore-added membrane is not sufficient to keep calcium interference to less than 1%. To correct for the interference by calcium, we determine the calcium activity of the serum sample with a calcium-selective electrode and calibrate the magnesium-selective electrode with standard solutions containing calcium in the concentrations found. In this way we determined the free ionized magnesium in five plasma samples from five different persons and found it to be within the range expected for magnesium in undiluted serum.

A self-consistent set of reference values for 23 clinical chemical analytes.

Heparinized plasma of 528 blood donors was subjected to the 23 most frequently ordered chemical and enzymatic tests. The central fraction of the distribution of all results for each test was estimated. Out of the 528 donors a reference population has been selected. Because of the lack of other criteria, the result for any test of a blood donor was selected as a value belonging to the reference population if the results for the other 22 analytes of this particular donor lay within their own central fraction. On this basis an iterative procedure for the selection was programmed, considering the interaction between tests. The procedure was stopped when the reference limits for all 23 tests were converging. Fractions from 0.90 to 0.98 were applied to results for men and women donors separately. The elimination procedure and the criteria to select the best fitted fraction are discussed. The derived reference intervals are designated a "self-consistent set of reference values."