Ion exchange chromatographic determination of olpadronate, phosphate, phosphite, chloride and methanesulfonic acid.

A method based on single column ion chromatography with UV detection was developed for purity testing and assay of monosodium olpadronate. The analyte aqueous solution is precipitated with methanol to enhance the impurities/olpadronate molar ratio, thus improving purity determination at trace levels. The resulting solution is injected into a standard chromatographic system with UV detector in indirect mode with a Waters IC Pak HR column using diluted nitric acid as the mobile phase. The method was fully validated according to ICH guidelines for the determination of phosphite, phosphate, chloride and methanesulfonic acid in olpadronate being suitable for purity testing and assay.

Spermine binding to liver mitochondria.

Non-equilibrium binding of spermine to mitochondrial membranes is studied in rat liver mitochondria by applying a new thermodynamic treatment of ligand-receptor interactions (Di Noto, V., Dalla Via, L., Toninello, A. and Vidali, M. (1996) Macromol. Theory Simul. 5, 165-181). The presence on mitochondrial membranes of two spermine binding sites, both with monocoordination, is demonstrated. The calculated binding energy is characteristic for weak interactions. The treatment allows also to evaluate the variations of the molar fraction ratio of spermine bound to sites 1 and 2 as function of total bound spermine. The possible role of the two sites is discussed.

The contribution of endogenous polyamines to the permeability transition of rat liver mitochondria.

Spermine and spermidine are present in rat liver mitochondria at the concentrations of 3.5 and 1.8 nanomoles/mg protein, respectively. Addition of 50 microM Ca2+ and 1 mM Pi to respiring mitochondria induces, concomitantly with mitochondrial swelling and Mg2+ efflux, a consistent release of polyamines, which is either prevented by cyclosporin A or Mg2+. Addition of 0.1 mM spermine to mitochondria deenergized by Ca2+ and phosphate restores, like 1 mM Mg2+, transmembrane potential at the physiological level, while either cyclosporin A or ADP are ineffective. The presence of polyamines in the matrix space should be considered relevant, like Mg2+, in controlling the permeability transition of liver mitochondria.

The alterations in the energy linked properties induced in rat liver mitochondria by acetylsalicylate are prevented by cyclosporin A or Mg2+.

The alterations in rat liver mitochondria induced by acetylsalicylate in the presence of low concentrations of Ca2+ (large amplitude swelling, permeability to 14C]sucrose, collapse of transmembrane potential and effluxes of endogenous Mg2+ and accumulated Ca2+) were fully prevented by either cyclosporin A or Mg2+. Cyclosporin A and Mg2+ were also capable of restoring transmembrane potential upon its decrease induced by acetylsalicylate. The loss of endogenous Mg2+ was the primary effect promoted by acetylsalicylate; the other noxious effects followed. These results indicate that Mg2+ are fundamental components of the mitochondrial permeability barrier and that their loss might be responsible for the membrane transition induced by acetylsalicylate.

Inhibition of mitochondrial permeability transition by polyamines and magnesium: importance of the number and distribution of electric charges.

The protective effects of Mg2+ and various natural and synthetic polyamines on the permeability transition of isolated rat liver mitochondria have been compared. The permeability transition was induced by incubating the mitochondria in a sucrose medium at pH 7.4 in the presence of 100 microM Ca2+ and 1 mM phosphate and was monitored via the release of endogenous Mg2+, sucrose permeation, mitochondria swelling and the fall of transmembrane potential. By all of these parameters (only the traces of delta psi have been reported) spermine fully inhibited the transition at 25 microM concentration, spermidine and caldine at 250 microM and Mg2+ at 500 microM concentration. Both putrescine and dien exhibited only a partial protection even at 2.5 mM concentration. The protective action resulted strictly dependent on the number of the positive charges of each cation. In the case of polyamines this number is also determined by the nature of the methylene carbon chains of each compound.

Spermine effect on the binding of casein kinase I to the rat liver mitochondrial structures.

The results indicated here, together with those previously reported, show that spermine, ubiquitous polyamine, while promoting the transmembrane translocation of casein kinase II (CKII) across the outer membrane to more internal compartments of rat liver mitochondria, promotes the binding of casein kinase I (CKI) to the external surface of outer mitochondrial membrane but inhibits its spontaneously occurring binding to more internal structures.

Effect of spermine on mitochondrial glutathione release.

Spermine prevents glutathione release induced in rat liver mitochondria by the combined addition of Ca2+ and phosphate. Spermine also inhibits mitochondrial swelling, membrane potential decrease, oxygen uptake and [14C] sucrose entry stimulated by the above reported agent. Mitochondrial swelling is completely prevented by 25 microM spermine while higher concentrations (100 microM) are required for the full inhibition of glutathione release. Therefore, polyamines decrease the mitochondrial inner membrane permeability and, by preventing mitochondrial glutathione loss, also act as protective agents against oxidative stress.

Effects of palmitoyl CoA and palmitoyl carnitine on the membrane potential and Mg2+ content of rat heart mitochondria.

Palmitoyl CoA and palmitoyl carnitine added to rat heart mitochondria in amounts above 20 and 50 nmoles/mg protein, respectively, induced a fall in transmembrane potential and loss of endogenous Mg2+. The dissipation of membrane potential by low concentrations of palmitoyl CoA in the presence of Ca2+, but not that of high concentrations of palmitoyl CoA alone, was prevented by either ruthenium red, Cyclosporin A or Mg2+, but reversed only by Mg2+. The fall of membrane potential induced by palmitoyl carnitine was not prevented by any of these factors. It is suggested that the action of both palmitoyl CoA and palmitoyl carnitine at high concentrations is due to a non specific disruption of membrane architecture, while that of low concentrations of palmitoyl CoA in the presence of Ca2+ is associated specifically with energy dissipation due to Ca2+ cycling.

Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter.

We present evidence that polyamine uptake into rat liver mitochondria is mediated by a specific polyamine uniporter. Polyamine transport is not mediated by the ornithine, lysine, or Ca2+ transporters of mitochondria. Polyamine transport is a saturable process, with apparent Km values of 0.13 mM for spermine, 0.26 mM for spermidine, and 1 mM for putrescine. These substrates are mutually competitive inhibitors, indicating a common transport system. Polyamine transport is strictly dependent on membrane potential and insensitive to medium pH, showing that these polycations are transported electrophoretically. Spermine, spermidine, and putrescine are taken up by rat liver mitochondria at rates that increase with increasing valence of the transported species. The activation enthalpies for transport were 24, 32, and 59 kJ/mol for putrescine, spermidine, and spermine, respectively. These values, which amount to about 12 kJ/mol per charge transferred, may be compared to a value of 76 kJ/mol observed for monovalent tetraethylammonium cation. Flux-voltage analysis is consistent with the hypothesis that the mitochondrial polyamine transporter catalyzes transport via a channel mechanism.

Bidirectional transport of spermine in rat liver mitochondria.

Further study of the mitochondrial transport of spermine (Toninello et al. (1988) J. Biol. Chem. 263, 19407) shows that, after loading rat liver mitochondria with [14C]spermine and [32P]phosphate, these components are released together into the surrounding medium by adding mersalyl or N-ethylmaleimide. On later addition of dithioerythritol, both are recaptured, but if acetate or nigericin are added instead, only spermine re-enters and there is continued export of phosphate. This bidirectional transport of spermine in and out mitochondria is driven, respectively, by membrane potential and pH gradient at constant protonmotive force. Results using [14C]spermine or [32P]phosphate, in conjunction with the their unlabelled isomers and with or without carbonyl cyanide/p-trifuloromethoxyphenylhydrazone (FCCP) present suggest that there is a continuous energy-dependent efflux-influx cycling of spermine and phosphate.

Electrophoretic polyamine transport in rat liver mitochondria.

Naturally occurring polyamines (spermidine, putrescine, cadaverine), as the well studied spermine, are transported into rat liver mitochondrial matrix provided that mitochondria are energized and the electrical membrane potential has a value of about 180 mV. This condition is achieved by the presence of inorganic phosphate, or acetate, or nigericin in the incubation medium. Valinomycin plus K(+) almost completely blocks polyamine transport.The obtained results clearly show that all naturally occurring polyamines are transported by an electrophoretic mechanism in responce to a high negative inner electrical potential.The distribution ratio of polyamines across the mitochondrial membrane is far from the thermodynamic equilibrium by many orders of magnitude. This result might suggest the existence of a different pathway for polyamine efflux.

Transport and action of spermine in rat heart mitochondria.

At concentrations of 0.5-1.0 mM, spermine fully prevents the fall of membrane potential induced in rat heart mitochondria either by aging at room temperature or by the addition of palmitoyl CoA. Spermine also prevents the inhibitory action of palmitoyl CoA on adenylate translocase activity. When added to heart mitochondria de-energized by the same damaging conditions (aging or addition of palmitoyl CoA) spermine restores both membrane potential (provided that ATP is also added) and the activity of adenylate translocase. A part of added spermine is immediately bound to anionic sites on mitochondrial membranes, another part is slowly transported into heart mitochondria. Whereas binding is an energy independent process, transport is driven by the transmembrane potential. Spermine penetrates the mitochondrial matrix at significant rates only at high membrane potential, such as that produced either by phosphate transport or addition of nigericin.

On the mechanism of spermine transport in liver mitochondria.

Spermine penetrates the mitochondrial matrix at significant rates which increase sharply and non-ohmically with membrane potential. In this respect, spermine uptake is qualitatively similar to that of other cations whose electrophoretic transport has been studied in mitochondria. At 200 mV and 1 mM spermine, the observed rate of spermine uptake was about 7 nmol x mg-1 x min-1, and the rate constant was about 8 times greater than that of tetraethylammonium cation. These rates are remarkably rapid considering that spermine is largely tetravalent at the pH of the experiment. The fluxes of spermine and tetraethylammonium are log-linear with membrane potential. The slope of the tetraethylammonium plot is consistent with leakage of this ion across a sharp Eyring barrier located in the middle of the membrane. The slope of the spermine plot is half that predicted by such a leak pathway, raising the possibility that spermine may cross the inner membrane by means of a channel. Whatever its mechanism of penetration, if comparable rates of uptake obtain in vivo and if spermine is not metabolized within the mitochondrial matrix, then a separate efflux mechanism would appear to be required to prevent unlimited spermine loading.

Protective action of methylglyoxal bis (guanylhydrazone) on the mitochondrial membrane.

At low concentrations (0.5-1.0 mM) methylglyoxal bis (guanylhydrazone) (MGBG) exhibited a clearcut protection of rat liver mitochondria against the deenergizing action of either Ca2+, or oxidizing agents (butylhydroperoxide and oxaloacetate). Such a protection resulted from the prevention of transmembrane potential decay, discharge of accumulated Ca2+, release of mitochondrial Mg2+, adenine nucleotides and pyridine nucleotides and mitochondrial swelling. At high concentrations (5-10 mM) MGBG induced functional alterations of mitochondria (decrease of transmembrane potential, lower capability to accumulate and to retain Ca2+) which can be reversed by resuspension of mitochondria in a MGBG free medium. These reversible mitochondrial alterations by high MGBG concentrations are interpreted as a consequence of an aggregation and coprecipitation of suspended mitochondria.

Transport and function of L-carnitine and L-propionylcarnitine: relevance to some cardiomyopathies and cardiac ischemia.

Carnitine, an essential cofactor in fatty acid oxidation, plays a central role in myocardial metabolism. Interpretation of the biochemical features of disturbed myocardial function, particularly in ischemia, may be facilitated by understanding carnitine biosynthesis, transport and function. Biosynthesis: In man, deoxycarnitine, the immediate precursor of carnitine, is synthesized in all tissues, whereas the last step, the conversion of deoxycarnitine into carnitine may only take place in liver, kidney and brain (Figs. 1 and 2). Deoxycarnitine formed by organs like muscle or heart is released into the plasma, taken up by liver and kidney, converted into carnitine which is secreted into the bloodstream to be taken up by heart or muscle (Fig. 2). Carnitine transport and cellular function: The myocardial uptake of carnitine against a large concentration gradient (Table 1) occurs in an 1:1 exchange-diffusion process. Under physiological conditions, intracellular deoxycarnitine is exported and extracellular carnitine is imported. According to this model, myocardial carnitine deficiency may be due either to a functional alteration of the sarcolemmal carnitine carrier or to a deficient synthesis of deoxycarnitine. D-carnitine, acetylcarnitine and long-chain acylcarnitine esters are also transported by the carrier at different rates. This might account for the release of endogenous acylcarnitines accumulated in anoxic or ischemic conditions, contributing to the cardioprotective effect of carnitine by reduction in intracellular long-chain acyl-coenzyme A.(ABSTRACT TRUNCATED AT 250 WORDS)

Action of spermine on phosphate transport in liver mitochondria.

Spermine, at concentrations similar to those normally present in the cytosol of liver cells, facilitates the transport of phosphate into mitochondria and thus its accumulation within the matrix space. Both mersalyl and N-ethylmaleimide (NEM) inhibit phosphate influx either in the absence or in the presence of spermine. These inhibitors also inhibit, but only partially, the efflux from mitochondria of phosphate generated within the matrix space by the hydrolysis of ATP induced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or the valinomycin-K+ system. The inhibition of phosphate efflux by both mersalyl and NEM is almost completely removed, unlike that of phosphate influx, by spermine. The possibility that spermine may induce phosphate efflux by damaging mitochondrial membranes and consequently inducing an unspecific permeability to phosphate is excluded by the full restoration of transmembrane potential once FCCP has been removed by albumin. Since spermine does not react with either thiol groups or thiol group reagents, the simplest explanation of the reported results is that the pathway of phosphate efflux is distinct from that of phosphate influx.

Uptake of spermine by rat liver mitochondria and its influence on the transport of phosphate.

Spermine, a polyamine present in the mammalian cells at rather high concentration, has, among other actions, a remarkable stabilizing effect on mitochondria, functions which have generally been attributed to the capability of this and other polyamines to bind to membrane anionic sites. In the present paper evidence is provided that at physiological concentrations spermine may also be transported into rat liver mitochondrial matrix space, provided that mitochondria are energized and inorganic phosphate is simultaneously transported. The close dependence of spermine transport is also demonstrated by the concurrent efflux of spermine and inorganic phosphate when mitochondria preloaded with the two ionic species are deenergized either with uncouplers or respiratory chain inhibitors. Furthermore, Mersalyl, the known inhibitor of phosphate transport, prevents both spermine uptake and release. Mg2+ inhibits the transport of spermine conceivably by competing for the some binding sites on the mitochondrial membrane. The physiological significance of these results is discussed.

On the mechanism of citrate and isocitrate protective action on rat liver mitochondria.

Both citrate and isocitrate prevent the damage (efflux of endogenous Mg2+ and pyridine nucleotides, decay of delta psi and release of accumulated Ca2+) induced in rat liver mitochondria by Ca2+ and phosphate fluxes. Addition of fluorocitrate suppresses the action of isocitrate, but not that of citrate. The same results have been obtained with mitochondria isolated from animals treated with fluoroacetate. It is suggested that citrate directly and isocitrate by prior conversion into citrate exert the protective action by chelating and retaining Mg2+ within the mitochondria.

On the mechanism by which Mg2+ and adenine nucleotides restore membrane potential in rat liver mitochondria deenergized by Ca2+ and phosphate.

The presence of ATP or ADP in the incubation medium prevents the collapse of membrane potential induced by external Ca2+ and phosphate. The same adenine nucleotides are unable to restore collapsed membrane potential unless Mg2+ are also added. Bongkrekate is also able to prevent the effects of external Ca2+ and phosphate and when added after membrane potential has collapsed strongly potentiates the restorative action of ATP or ADP. Atractyloside has an opposite effect.