Aluminum speciation studies in biological fluids. Part 6. Quantitative investigation of aluminum(III)-tartrate complex equilibria and their potential implications for aluminum metabolism and toxicity

Recent epidemiological studies have confirmed the existence of a correlation between aluminum level in low-silica drinking water and prevalence of Alzheimer's disease. Also, oral aluminum-based phosphate binders and antacids may induce acute aluminum toxicity. Whatever the source of the metal ingested, its bioavailability is a function of the chemical forms under which it occurs in the gastrointestinal tract, i.e. of the ligands with which the Al3+ ion may associate. Dietary acids in particular can favor the bioavailability of aluminum in different ways: by increasing its solubility, by complexing it into neutral species, and/or by acting indirectly on its absorption process. Among these, tartaric acid is commonly found in fruits and in industrial foods and drinks, and may therefore be ingested together with environmental or/and therapeutic aluminum. The present work examines its potential influence on aluminum bioavailability. Firstly, Al(III)-tartrate complex formation constants have been determined under physiological conditions (37 degrees C, 0.15 M NaCl). Then these constants have been used to simulate the influence of tartrate on aluminum speciation in different gastrointestinal situations in which phosphate was also taken into account. Under normal conditions of aluminum contamination, tartrate is expected to keep the metal soluble throughout the whole pH range of the small intestine, which is likely to enhance its bioavailability. Even at low concentrations, tartrate also gives rise to two neutral complexes that span over the 1.5-7.5 pH interval, a phenomenon that is aggravated by increased aluminum levels as may result from aluminum hydroxide therapy. The co-occurrence of dietary phosphate reduces the fraction of aluminum neutralized by tartrate under normal conditions, but this effect quickly decreases with increasing aluminum doses. Even the therapeutic use of aluminum phosphate is not expected to be totally safe in the presence of tartaric acid. As plasma simulations show that no aluminum mobilization can be expected from tartrate that could enhance aluminum excretion, avoiding ingestion of tartaric acid during any form of aluminum-based therapy appears advisable.     

Aluminum speciation studies in biological fluids. Part 9. A quantitative investigation of aluminum(III)-glutamate complex equilibria and their potential implications for aluminum metabolism and toxicity

As a nonessential element, aluminum is likely to be toxic both at low usual dietary levels in the long run (chronic toxicity) and at high therapeutic levels in shorter periods of time (acute toxicity). In both situations, aluminum toxicity is a direct function of aluminum bioavailability, which is itself dependent on Al(3+) solubility and charge neutralization. Dietary acids, by their intrinsic acidity and coordinating capacity, can extend the pH range, thus the section of the gastrointestinal tract, within which the Al(3+) ion remains soluble, and also help Al(3+) diffusion across the intestinal epithelium through the formation of neutral complex species. The present work examines the impact of glutamic acid, an essential amino acid also widely used in industrial food and drinks, on aluminum speciation in the gastrointestinal tract and blood plasma. Complex formation between the Al(3+) ion and glutamate has first been investigated through potentiometric titrations, complex stoichiometries being then checked by ESI mass spectrometry and NMR measurements. A series of mono- and polynuclear species has been characterized, whose influence on aluminum distribution in vivo has been assessed by computer simulation. The capacity of glutamate to maintain Al(3+) ions in solution under normal dietary conditions is predicted to be intermediate between glycine-like amino acids and succinate on the one hand, and tartrate and malate on the other hand, its Al(3+) neutralization effect being similar to that of succinate, tartrate and malate. These results, which point to a potential aggravating role of glutamate on aluminum gastrointestinal absorption, substantiate recent observations made on rats. In spite of the moderate effect expected from glutamate on aluminum bioavailability under most aluminum-based therapies investigated, attention is therefore called to the risk of glutamic acid ingestion simultaneously to any aluminum therapeutic form. Incidentally, the former implication of 'the' aluminum glutamate complex in the transfer of aluminum through the blood-brain barrier of aluminum loaded rats may effectively be attributed to one of the species characterized here, but is of no significance at all to aluminum contamination in humans, even at most extreme levels.     

Aluminum speciation studies in biological fluids. Part 7. A quantitative investigation of aluminum(III)-malate complex equilibria and their potential implications for aluminum metabolism and toxicity.

As a nonessential element, aluminum may be toxic at both environmental and therapeutic levels, depending on ligand interactions. Dietary acids that normally occur in fruits and vegetables and commonly serve as taste enhancers are good ligands of the Al(3+) ion. Malic acid is one of these and also one of the most predominant in food and beverages. The present paper reports an examination of its potential influence on aluminum bioavailability through speciation calculations based on Al(III)-malate complex formation constants especially determined for physiological conditions. According to the results obtained, malate appears to be extremely effective in maintaining Al(OH)(3) soluble over the whole pH range of the small intestine under normal dietary conditions. In addition, two neutral Al(III)--malate complexes are formed whose percentages are maximum from very low malate levels. When aluminum is administered therapeutically as its trihydroxide, the amount of metal neutralized by malate peaks as its solubility pH range regresses to its original limits in the absence of malate. The enhancing effect of malate towards aluminum absorption is therefore virtually independent of the aluminum level in the gastrointestinal tract. The presence of phosphate in the gastrointestinal juice is expected to limit the potential influence of malate on aluminum absorption. Under normal dietary conditions, phosphate effectively reduces the fraction of aluminum neutralized by malate but without nullifying it. Aluminum phosphate is predicted to precipitate when aluminum levels are raised as with the administration of aluminum hydroxide, but a significant amount of neutral aluminum malate still remains in solution. Even therapeutic aluminum phosphate is not totally safe in the presence of malate, even at low malate concentrations. As plasma simulations predict that no compensatory effect in favor of aluminum excretion may be expected from malate, simultaneous ingestion of malic acid with any therapeutic aluminum salt should preferably be avoided.     

Aluminum speciation studies in biological fluids. Part 3. Quantitative investigation of aluminum-phosphate complexes and assessment of their potential significance in vivo

Following the discovery that specific health disorders affecting patients with renal disease were due to their excessive body accumulation of aluminum, it was established that aluminum toxicity was mainly due to the ingestion of aluminum-containing phosphate binders. Suspicion of toxicity was thus cast on aluminum-containing antacids, and subsequent tests held on healthy subjects did reveal that aluminum hydroxide gels were also potential oral sources of aluminum, especially in the presence of citric acid. Nevertheless, authors of these tests concluded that there was only marginal absorption of aluminum phosphate. In contrast with these clinical conclusions, it has recently been contended on theoretical grounds that aluminum phosphate represents a serious health hazard. To help elucidate this issue, this paper first deals with a quantitative investigation of aluminum-phosphate equilibria under physiological conditions. Then appropriate computer simulations based on corresponding results are used to assess the actual extent to which phosphate can influence aluminum bioavailability. These simulations confirm that aluminum phosphate is not expected to induce absorption of high amounts of aluminum when administered by itself. Nevertheless, this result may no longer apply in the presence of food, whose various acidic components are likely to modify the involved chemical equilibria. Moreover, it is shown that rising blood plasma phosphate levels should tend to increase aluminum tissue penetration and hence favor its potential toxicity.     

Spectroscopic studies of aqueous gallium(III) and aluminum(III) citrate complexes

Aqueous gallium(III) citrate complexes have been studied in the 10(-2) M concentration range with extended X-ray absorption fine structure (EXAFS) and FTIR techniques. From EXAFS data, one mononuclear and one oligomeric species were identified at different Ga(III) to citrate ratios. The first shell of the mononuclear complex was found to be distorted, with average Ga-O bond lengths of 1.95 and 2.06 A, in agreement with the solid-state structure of Ga(Cit)2(3-) (Cit=citrate). Also the oligomeric species was found to have a distorted first shell, with average Ga-O bond lengths of 1.95 and 2.04 A. This complex was found to contain two Ga-Ga distances at 3.03 and 3.56 A, typical for edge and corner sharing GaO6 octahedra, respectively. The gallium(III) and aluminum(III) citrate systems were compared by means of FTIR, and were found to be analogous. The IR results suggest that the bond lengths derived from EXAFS for the 1:2 gallium(III) citrate complex also provide a good estimate of the corresponding distances in the mononuclear 1:1 complex. Direct coordination of citrate to the metal ions in the oligomeric gallium(III) citrate complex was indicated from both EXAFS and IR results, and this complex is stoichiometrically analogous to the Al3(H-1Cit)3(OH)(H2O)4- complex, which has been structurally determined. However, while the formation of the aluminum trimer has been shown to be slow, the gallium trimer was significantly more labile with a rate of formation indicated to be in the order of seconds or faster.     

Characterization of citrate transport through the plasma membrane in a carrot mutant cell line with enhanced citrate excretion

The superior ability of citrate excretion in a carrot (Daucus carota L.) mutant cell line, namely IPG (insoluble phosphate grower) [Takita et al. (1999a) Plant Cell Physiol. 40: 489] cells has been characterized in terms of citrate transport at the plasma membrane. IPG cells released about a 20-fold increase in citrate in comparison with malate, while the concentration of malate was only 35% lower than that of citrate in the cell sap. Citrate excretion was sensitive to anion channel blockers, such as niflumic acid and anthracene-9-carboxylic acid. These results indicate that IPG cells release citrate through the plasma membrane using citrate specific anion channels. The rate of citrate release from IPG cells was not affected by the concentration of aluminum (0 and 50 micro M), soluble P(i) (0 or 2 mM) and the pH (4.5-5.6) of the medium, suggesting that anion channels would not be regulated by such external conditions. Citrate excretion correlated with the H(+) efflux, possibly from the action of H(+)-ATPase on the plasma membrane. The activity of plasma membrane H(+)-ATPase was about three times higher in IPG cells than in wild-type cells, and might be involved in the high citrate excretion ability.     

The effect of acid pH and citrate on the release and exchange of iron on rat transferrin.

The effect of acid pH and citrate on the exchange of iron between binding sites of rat transferrin has been studied. In the absence of citrate, diferric transferrin shows stepwise loss of iron atoms with the first atom of iron released at approximately pH 5.2. Citrate at physiologic concentrations (1.10(-3) M) or greater allows random iron removal at pH 6.5 or less. Iron dissociation from monoferric transferrin at acid pH, with or without citrate, is a random process. At pH 7.4, randomization of iron on transferrin takes from 3 to 6 h in the presence of millimolar concentrations of citrate. We conclude that at acid pH and in the presence of citrate concentrations likely to occur in vivo in the rat there is little scrambling of iron bound to transferrin.     

The effect of acid pH and citrate on the release and exchange of iron on rat transferrin

The effect of acid pH and citrate on the exchange of iron between binding sites of rat transferrin has been studied. In the absence of citrate, diferric transferrin shows stepwise loss of iron atoms with the first atom of iron released at approximately pH 5.2. Citrate at physiologic concentrations (1 · 10^?3 M) or greater allows random iron removal at pH 6.5 or less. Iron dissociation from monoferric transferrin at acid pH, with or without citrate, is a random process. At pH 7.4, randomization of iron on transferrin takes from 3 to 6 h in the presence of millimolar concentrations of citrate. We conclude that at acid pH and in the presence of citrate concentrations likely to occur in vivo in the rat there is little scrambling of iron bound to transferrin.     

Mechanism of Na(+)-dependent citrate transport in Klebsiella pneumoniae.

Citrate transport via CitS of Klebsiella pneumoniae has been shown to depend on the presence of Na+. This transport system has been expressed in Escherichia coli, and uptake of citrate in E. coli membrane vesicles via this uptake system was found to be an electrogenic process, although the pH gradient is the main driving force for citrate uptake (M. E. van der Rest, R. M. Siewe, T. Abee, E. Schwartz, D. Oesterhelt, and W. N. Konings, J. Biol. Chem. 267:8971-8976, 1992). Analysis of the affinity constants for the different citrate species at different pH values of the medium indicates that H-citrate2- is the transported species. Since the electrical potential across the membrane is a driving force for citrate transport, this indicates that transport occurs in symport with at least three monovalent cations. Citrate efflux is stimulated by Na+ concentrations of up to 5 mM but inhibited by higher Na+ concentrations. Citrate exchange, however, is stimulated by all Na+ concentrations, indicating sequential events in which Na+ binds before citrate for translocation followed by a release of Na+ after release of citrate. CitS has, at pH 6.0 and in the presence of 5 mM citrate on both sides of the membrane, an apparent affinity (K(app)) for Na+ of 200 microM. The Na+/citrate stoichiometry was found to be 1. It is postulated that H-citrate2- is transported via CitS in symport with one Na+ and at least two H+ ions.     

Interactions of aluminium(III) with glycerolphosphates and glycerophosphorylcholine

The complexation of aluminium(III) with glycerol-1-phosphate (G1P) and glycerol-2-phosphate (G2P) in aqueous solutions has been studied as a function of pH, by pH-potentiometry, 31P NMR spectroscopy and ESI mass spectrometry. Various mononuclear complexes (MLH(2)(3+), MLH(2+), ML(+), ML(2)H, ML(2)(-)) and polynuclear species (M(3)L(3)H(-1)(2+), M(3)L(2)H(-n)((n-5)-) with n=5, 6, 7, M(2)L(2)H(-1)(+) ) are formed in the system where the full protonated ligands are noted LH(2). NMR experiments clearly show that G1P and G2P already interact with Al(III) at pH 1. The potentiometric results are confirmed by ESI measurements and 31P NMR studies. No metal ion-induced deprotonation and coordination of the alcoholic-OH functions seem to occur during the complexation. The situation is very different for the glycerophosphorylcholine ligand (GPC identical with LH). Only the complex ML(3+) is formed in aqueous solution with a relatively low formation constant (K=5 at 37 degrees C). This species is clearly identified in 31P and 27Al NMR spectra. The complexation study as a function of the temperature allowed us to determine the thermodynamic parameters of the complex formation. The complexation is not governed by the reaction enthalpy that is found to be positive but by the entropy that is largely positive.     

Ultrastructural alterations of Erwinia carotovora subsp. atroseptica caused by treatment with aluminum chloride and sodium metabisulfite

Aluminum and bisulfite salts inhibit the growth of several fungi and bacteria, and their application effectively controls potato soft rot caused by Erwinia carotovora. In an effort to understand their inhibitory action, ultrastructural changes in Erwinia carotovora subsp. atroseptica after exposure (0 to 20 min) to different concentrations (0.05, 0.1, and 0.2 M) of these salts were examined by using transmission electron microscopy. Plasma membrane integrity was evaluated by using the SYTOX Green fluorochrome that penetrates only cells with altered membranes. Bacteria exposed to all aluminum chloride concentrations, especially 0.2 M, exhibited loosening of the cell walls, cell wall rupture, cytoplasmic aggregation, and an absence of extracellular vesicles. Sodium metabisulfite caused mainly a retraction of plasma membrane and cellular voids which were more pronounced with increasing concentration. Bacterial mortality was closely associated with SYTOX stain absorption when bacteria were exposed to either a high concentration (0.2 M) of aluminum chloride or prolonged exposure (20 min) to 0.05 M aluminum chloride or to a pH of 2.5. Bacteria exposed to lower concentrations of aluminum chloride (0.05 and 0.1 M) for 10 min or less, or to metabisulfite at all concentrations, did not exhibit significant stain absorption, suggesting that no membrane damage occurred or it was too weak to allow the penetration of the stain into the cell. While mortality caused by aluminum chloride involves membrane damage and subsequent cytoplasmic aggregation, sulfite exerts its effect intracellularly; it is transported across the membrane by free diffusion of molecular SO2 with little damage to the cellular membrane.     

The chemical state of the calcium reacting in the coagulation of blood

1. The widely accepted theory that calcium participates in the coagulation mechanism in the form of Ca(++) and acts as a catalyst is not in accord with several important experimental findings: (a) The anticoagulant action of sodium oxalate is much slower than the precipitation of ionized calcium as the oxalate salt. (b) Sodium citrate begins to depress prothrombin activity at a concentration at which ionized calcium is still present. The inability of tricalcium phosphate to adsorb prothrombin from citrated plasma indicates that citrate forms a complex with prothrombin and it is postulated that prothrombin is thereby inactivated. (c) In plasma which is decalcified, i.e. in which the Ca(++) is markedly reduced, the labile factor of prothrombin rapidly decreases. A concentration of 0.01 M sodium citrate sufficient to inhibit coagulation does not depress Ca(++) enough to cause diminution of the labile factor, whereas when the concentration is increased to 0.02 M the labile factor decreases as rapidly as in oxalated plasma. 2. It is postulated that calcium functions in coagulation not as Ca(++) but as combined with a component which is part of the prothrombin complex that is not adsorbed by tricalcium phosphate. A concentration of sodium citrate just sufficient to inhibit coagulation is not enough to remove calcium from the essential prothrombin component. The primary anticoagulant action of sodium citrate is therefore not decalcification but antiprothrombic. 3. It has been shown that citrated plasma is basically different from oxalated plasma in several important aspects. Unless cognizance is taken of these differences, serious errors and misinterpretations of experimental findings may be made.     

Complexes of aluminium(III) with glucose-6-phosphate in aqueous solutions

The interaction of aluminium(III) with glucose-6-phosphate (GP: LH2) in aqueous solutions has been studied from pH 1 to pH 8, by pH-potentiometry and multinuclear (31P, 27Al, 13C) NMR spectroscopy. Various mononuclear species (MLH2, MLH, ML, ML2H, ML2 and MLH(-3)) and dinuclear complexes M2L2H-n (n=1-4) are formed in the system. NMR clearly indicates that GP is already bound to Al(III) at pH 1. The potentiometric speciation results are confirmed and completed by spectroscopic experiments. Many peaks are observed in the 31P NMR spectra suggesting the formation of isomeric species. An attempt to assign the signals to the corresponding complexes is made, allowing a discussion about their structure. Interestingly enough no metal ion-induced deprotonation and coordination of the alcoholic-OH functions have been observed.     

The 35 kDa acid metallophosphatase of the frog Rana esculenta liver: studies on its cellular localization and protein phosphatase activity

The cellular localization of the 35 kDa, low molecular mass acid metallophosphatase (LMW AcPase) from the frog (Rana esculenta) liver and its activity towards P-Ser and P-Tyr phosphorylated peptides were studied. This enzyme was localized to the cytoplasm of hepatocytes but did not appear in other cells of liver tissue (endothelium, macrophages, blood cells). This LMW AcPase does not display activity towards (32)P-phosphorylase a under conditions standard for the enzymes of PPP family. Proteins containing P-Ser: rabbit (32)P-phosphorylasea and phosvitin are hydrolysed only at acidic pH and are poor substrates for this enzyme. The frog AcPase is not inhibited by okadaic acid and F(-) ions, the Ser/Thr protein phosphatase inhibitors. Moreover, the frog enzyme does not cross-react with specific antisera directed against N-terminal fragment of human PP2A and C-terminal conserved fragment of the eukaryotic PP2A catalytic subunits. These results exclude LMW AcPase from belonging to Ser/Thr protein phosphatases: PP1c or PP2Ac. In addition to P-Tyr, this enzyme hydrolyses efficiently at acidic pH P-Tyr phosphorylated peptides (hirudin and gastrin fragments). K(m) value for the hirudin fragment (7.55 +/- 1.59 x 10(-6) M) is 2-3 orders of magnitude lower in comparison with other substrates tested. The enzyme is inhibited competitively by typical inhibitors of protein tyrosine phosphatases (PTPases): sodium orthovanadate, molybdate and tungstate. These results may suggest that the LMW AcPase of frog liver can act as PTPase in vivo. A different cellular localization and different response to inhibition by tetrahedral oxyanions (molybdate, vanadate and tungstate) provide further evidence that LMW AcPase of frog liver is distinct from the mammalian tartrate-resistant acid phosphatases.     

Ganglioside GM3 sialidase activity in fibroblasts of normal individuals and of patients with sialidosis and mucolipidosis IV. Subcellular distribution and and some properties.

Sensitive assays for the determination of the ganglioside sialidase activity of fibroblast homogenates were established using ganglioside GM3, 3H-labelled in the sphingosine moiety, as a substrate. Ganglioside GM3 sialidase activity was greatly stimulated by the presence of the non-ionic detergent Triton X-100 and was further enhanced by salts such as NaCl; the optimal pH was 4.5. The subcellular localization of this activity was determined by fractionation using free-flow electrophoresis and found to be exclusively associated with the marker for the plasma membrane, but not with that for lysosomes. This Triton-stimulated ganglioside sialidase activity was selectively inhibited by preincubating intact cells in the presence of millimolar concentrations of Cu2+, suggesting that the activity resides on the external surface of the plasma membrane. In normal fibroblasts homogenates, ganglioside GM3 sialidase was also greatly stimulated by sodium cholate. In contrast to the Triton X-100-activated reaction, however, it was not diminished by prior incubation of intact cells in the presence of Cu2+. Only after cell lysis was Cu2+ inhibitory. the cholate-stimulated ganglioside sialidase activity thus paralleled the behaviour of the lysosomal 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (4-MU-NeuAc) sialidase. In fibroblasts from sialidosis patients, the cholate-stimulated ganglioside GM3 sialidase activity, but not that of the Triton-activated enzyme, was profoundly diminished. In fibroblasts from patients with mucolipidosis IV (ML IV), both the Triton X-100- and the cholate-stimulated ganglioside GM3 sialidase activities were in the range of normal controls. The Triton-activated enzyme was associated with the plasma membrane in the same manner as in normal cells. Our findings suggest that, in human fibroblasts, there exist two sialidases that degrade ganglioside GM3: one on the external surface of the plasma membrane, and another that is localized in lysosomes and seems identical with the activity that acts on sialyloligosaccharides and 4-MU-NeuAc. As neither activity was found to be deficient in ML IV fibroblasts, our results argue against the hypothesis of a primary involvement of a ganglioside GM3 sialidase in the pathogenesis of ML IV.     

Identification of quercetin 3-O-beta-D-glucuronide as an antioxidative metabolite in rat plasma after oral administration of quercetin

The potential beneficial effect of dietary quercetin (3,3',4',5,7-pentahydroxyflavone) has attracted much attention in relation to the prevention of cardiovascular disease. It is generally recognized that dietary quercetin is subject to metabolic conversion resulting in conjugated forms during absorption and circulation. However, no quercetin conjugates have yet been identified from biological fluids or tissues. In the present study, we isolated and characterized two quercetin conjugates from the plasma of quercetin-administered rats. The blood plasma was collected from 26 rats 30 min after oral administration of quercetin (250 mg/kg body weight), concentrated, dissolved in 2% acetic acid aqueous solution (pH 2.65), and extracted with ethyl acetate. Two compounds (P2, P3) were obtained from the extract by repeated reversed-phase HPLC. On the other hand, two quercetin glucuronides were synthesized chemically and identified as quercetin 3-O-beta-D-glucuronide (Q3GA) and quercetin 4'-O-beta-D-glucuronide (Q4'GA), as determined from FABMS, 1H- and 13C-NMR, and HMBC data. The retention times of P2 and P3 in the HPLC chromatogram corresponded to those of Q3GA and Q4'GA, respectively. FABMS data demonstrated that P2 and P3 are quercetin monoglucuronides. 1H-NMR data for P2 were completely in agreement with those for Q3GA. P2 was therefore identified as Q3GA. This is, to our knowledge, the first evidence that Q3GA accumulates in vivo after oral administration of quercetin. Q3GA is likely to act as an effective antioxidant in blood plasma low-density lipoprotein, because this conjugated metabolite was found to possess a substantial antioxidant effect on copper ion-induced oxidation of human plasma low-density lipoprotein as well as 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity.     

Short-term aluminium uptake by tobacco cells: Growth dependence and evidence for internalization in a discrete peripheral region

Short-term uptake and initial localization of aluminium (Al) were investigated in cultured cells of Nicotiana tabacum L. cv. BY-2. Graphite furnace atomic absorption spectrometry and an in vivo Al-sensitive fluorometric assay, employing morin, yielded similar results in all experiments. Aluminium uptake was critically dependent on cell growth. As opposed to negligible uptake in stationary-phase cells, Al uptake (20 μ M AlCl₃, pH 4.5, 23°C) by actively growing cells was detectable within 5 min, with an initial rate of 16 nmol Al (10⁶ cells)⁻¹ h⁻¹. Increased CaCl₂ levels (up to 20 m M ), low temperature (4°C), and pre-chelation of Al to citrate greatly reduced Al uptake (by 75–90%). A pH-associated permeabilization of cells at pH 4.5, as monitored by trypan blue, was observed in some growing cells. Although permeability to trypan blue was not a requirement for Al uptake, enhanced membrane permeability at pH 4.5, relative to pH 5.6, may contribute to Al uptake. Aluminium was observed to localize mainly in a pronounced and discrete fluorescent zone at the cell periphery (2–30 μm wide), presumably in the cortical cytosol and/or the adjoining plasma membrane section, although the possibility cannot be excluded that some Al resided in the cell wall apposing this discrete region. However, as judged by the Al-morin assay, there were no detectable Al levels in the remaining, larger portion of the cell wall. The potential of the Al-morin method in Al toxicity studies is illustrated.     

Metal ion-tetracycline interactions in biological fluids. Part 6. Formation of copper(II) complexes with tetracycline and some of its derivatives and appraisal of their biological significance

It was previously established by computer simulation that the interactions of tetracyclines with calcium and magnesium play a paramount role in the bioavailability of these antibiotics in blood plasma. More recently, the influence of zinc was found to be insignificant in this biofluid at drug therapeutic levels but clinical data, relative to the gastrointestinal absorption of tetracyclines as well as zinc in the presence of each other, could be interpreted on the basis of the appropriate simulations.The present paper deals first with the experimental determination of the formation constants for the complexes of copper(II) with tetracycline, oxytetracycline, doxycyline, minocycline, chlortetracycline and demethylchlortetracyclin in aqueous NaCl 0.15 mol dm⁻³ at 37 °C.The data was then used for simulating the distribution of copper and each of these drugs in blood plasma as well as in gastrointestinal fluid. No influence can be expected from copper on the bio- availability of tetracyclines in blood plasma, the reverse also being true. For the therapeutic doses under consideration, copper cannot interfere with gastrointestinal absorption of these antibiotics but the presence of the latter tends to favour copper absorption to a determining extent.     

Isomerization of lycopene in the gastric milieu

There is considerable interest in the bioavailability of carotenoids from the diet and their bioactivity in vivo. Little is known, however, of the preabsorption events in the gastric lumen on the breakdown or isomerisation of dietary carotenoids. In this study the effects of the acidic environment found in the gastric milieu on lycopene have been investigated. The results show that under these conditions all-trans-lycopene is isomerised to cis-isomers, which may be implicated in enhanced absorption from the small intestine. Furthermore the pH, as well as the food matrix, seems to have an influence on the level of isomerisation of this carotenoid.     

Ultrastructural Alterations of Erwinia carotovora subsp. atroseptica Caused by Treatment with Aluminum Chloride and Sodium Metabisulfite

Aluminum and bisulfite salts inhibit the growth of several fungi and bacteria, and their application effectively controls potato soft rot caused by Erwinia carotovora. In an effort to understand their inhibitory action, ultrastructural changes in Erwinia carotovora subsp. atroseptica after exposure (0 to 20 min) to different concentrations (0.05, 0.1, and 0.2 M) of these salts were examined by using transmission electron microscopy. Plasma membrane integrity was evaluated by using the SYTOX Green fluorochrome that penetrates only cells with altered membranes. Bacteria exposed to all aluminum chloride concentrations, especially 0.2 M, exhibited loosening of the cell walls, cell wall rupture, cytoplasmic aggregation, and an absence of extracellular vesicles. Sodium metabisulfite caused mainly a retraction of plasma membrane and cellular voids which were more pronounced with increasing concentration. Bacterial mortality was closely associated with SYTOX stain absorption when bacteria were exposed to either a high concentration (0.2 M) of aluminum chloride or prolonged exposure (20 min) to 0.05 M aluminum chloride or to a pH of 2.5. Bacteria exposed to lower concentrations of aluminum chloride (0.05 and 0.1 M) for 10 min or less, or to metabisulfite at all concentrations, did not exhibit significant stain absorption, suggesting that no membrane damage occurred or it was too weak to allow the penetration of the stain into the cell. While mortality caused by aluminum chloride involves membrane damage and subsequent cytoplasmic aggregation, sulfite exerts its effect intracellularly; it is transported across the membrane by free diffusion of molecular SO₂ with little damage to the cellular membrane.