Low Citrate Concentrations Research Articles

The citrate ion increases the conformational stability of α 1-antitrypsin

Sodium citrate has previously been shown to convert native α 1-antitrypsin into the inactive latent state and cause α 1-antitrypsin to polymerize via the C-sheet pathway instead of the more common A-sheet pathway. In order to begin to understand these dramatic effects, we have examined the influence of low concentrations of sodium citrate upon the structure, stability and function of α 1-antitrypsin. In 0.5 M citrate, the midpoint of guanidine hydrochloride-induced unfolding was increased by 1.8 M and the rate of heat inactivation was decreased approximately 30-fold compared with Tris or phosphate buffer. α 1-Antitrypsin was fully active in the presence of a range of citrate concentrations (0.1–0.5 M), forming a stable 1:1 complex with chymotrypsin. The association rate constant between α 1-antitrypsin and chymotrypsin was decreased with increasing citrate concentration. Fluorescence and circular dichroism spectroscopy demonstrated no significant changes in the tertiary structure due to the presence of citrate. However, the insertion rate of exogenous reactive-center loop peptide increased with increasing citrate concentration, indicating some structural changes in the A β-sheet region. Taken together, these data suggest that in the presence of 0.5 M citrate α 1-antitrypsin adopts a highly stable but active conformation.

A proposed biochemical mechanism for citric acid accumulation by Aspergillus niger Yang No. 2 growing in solid state fermentation

Aspergillus niger Yang No. 2 and its mutant strain SL1 were grown in solid state fermentation. Samples were taken after 2, 4 and 6 days of incubation and the mycelia were analysed for their intracellular concentrations of some organic acids and adenylates and the activities of selected enzymes. Strain Yang No. 2 contained high concentrations of citrate with very little oxalate, while strain SL1 contained lower concentrations of citrate but considerably higher concentrations of oxalate. As the fermentation proceeded, strain Yang No. 2 showed a much higher ratio of ATP:AMP than did strain SL1. In addition, the enzyme ATP:citrate lyase became undetectable during citrate accumulation in strain Yang No. 2, while its activity remained high during oxalate accumulation in strain SL1. It is proposed that citrate accumulation by strain Yang No. 2 during solid state fermentation is due to blockage of its metabolism in the mitochondrion via inhibition of isocitrate dehydrogenase by the high ATP:AMP ratio, and in the cytosol by repression of ATP:citrate lyase activity.

Analysis of low concentration sufentanil citrate/bupivacaine hydrochloride admixtures, using solid phase extraction followed by high-performance liquid chromatography

A method has been developed for the quantitative determination of low concentrations of sufentanil citrate (1.0 μg/ml), in the presence of bupivacaine hydrochloride (0.125%), in the quality control of pharmaceutical preparations. The main problem in analysis of this combination is the low concentration of sufentanil citrate in the presence of relatively high concentrations of bupivacaine hydrochloride. This paper describes the validation of a HPLC method of sufentanil citrate in an admixture with bupivacaine hydrochloride using solid phase extraction (SPE). The optimized method shows good linearity, precision and accuracy. The limits of detection (0.09 μg/l) and quantification (0.29 μg/l) for sufentanil citrate are lower than the maximal accepted limits. This method is currently used in stability studies.

The influence of citrate concentration on the quality of plasma obtained by automated plasmapheresis: a prospective study

There is a need for more comprehensive work dealing with the quality of plasma collected by automated plasmapheresis using different final concentrations of citrate anticoagulant. A prospective study was performed to examine the influence of three concentrations of sodium citrate on the levels of clotting factors and markers of activated hemostasis and fibrinolysis. Fifty-one experienced plasma donors were recruited for subsequent 750-mL plasmapheresis procedures using 4-percent (wt/vol) sodium citrate. Anticoagulant-to-blood ratios of 1:16.6, 1:14.2, and 1:12.5 were used, corresponding to sodium citrate concentrations of 6 percent, 7 percent, and 8 percent (vol/vol), respectively. Between two plasmapheresis procedures, there was a washout period of 7 days. Determinations were made of the plasma levels of fibrinogen and factors V, VII, VIII, and IX, as well as antithrombin, tissue-type plasminogen activator, and several markers of activated hemostasis and fibrinolysis: activated factor VII, prothrombin splits products, D-dimers, and beta-thromboglobulin. The plasma samples anticoagulated with 6-percent citrate contained significantly higher levels of factors V, VIII, and IX than the samples anticoagulated with 8-percent citrate (p<0.0001, p< or =0.0001 and p = 0.009, respectively). The citrate concentration had no influence on the levels of fibrinogen, factor VII, antithrombin, or tissue-type plasminogen activator. There was no evidence that the plasma samples containing lower citrate concentrations were more prone to activation of hemostasis or fibrinolysis. A reduction in the final citrate concentration of plasma collected by automated plasmapheresis results in higher yields of factors V, VIII, and IX without activation of hemostasis. More comprehensive studies should confirm previous work dealing with the establishment of the lowest citrate concentration acceptable in plasma used as therapeutic fresh-frozen plasma or as starting material for the manufacture of plasma derivatives.

Formation of Colloidal Silver Nanoparticles: Capping Action of Citrate

Colloidal silver sols of long-time stability are formed in the γ-irradiation of 1.0 × 10-4 M AgClO4 solutions, which also contain 0.3 M 2-propanol, 2.5 × 10-2 M N2O, and sodium citrate in various concentrations. The reduction of Ag+ in these solutions is brought about by the 1-hydroxyalkyl radical generated in the radiolysis of 2-propanol; citrate does not act as a reductant but solely as a stabilizer of the colloidal particles formed. Its concentration is varied in the range from 5.0 × 10-5 to 1.5 × 10-3 M, and the size and size distribution of the silver particles are studied by electron microscopy. At low citrate concentration, partly agglomerated large particles are formed that have many imperfections. In an intermediate range (a few 10-4 M), well-separated particles with a rather narrow size distribution and little imperfections are formed, the size slightly decreasing with increasing citrate concentration. At high citrate concentrations, large lumps of coalesced silver particles are present, due to destabilization by the high ionic strength of the solution. These findings are explained by two growth mechanisms: condensation of small silver clusters (type-I growth), and reduction of Ag+ on silver particles via radical-to-particle electron transfer (type-II growth). The particles formed in the intermediate range of citrate concentration were studied by high-resolution electron microscopy and computer simulations. They constitute icosahedra and cuboctahedra.

The effects of divalent cations in the presence of phosphate, citrate and chloride on the aggregation of soy protein isolate

The protein aggregation–dissociation process induced by divalent cations was studied using diluted aqueous dispersions of a native soy isolate. Turbidity, as a protein aggregation indicator, was quantified at a fixed time after addition of increasing amounts of CaCl 2, MgCl 2 or an equimolar mixture of them. In all cases turbidity values attained with Ca 2+ were higher than these obtained with Mg 2+. The effects of phosphate, citrate and chloride presence were also determined. Phosphate, becomes incorporated into Ca 2+-induced aggregates, but not in Mg 2+ ones. Both citrate and chloride ions inhibit protein aggregation and contribute to the dissociation of previously formed aggregates. Low citrate concentrations allowed aggregate formation, even at high CaCl 2 concentrations; this behavior was not observed in the presence of MgCl 2. A sedimentation based test was developed as a stability parameter of dispersions of soy protein aggregates. Results showed that even though aggregation was induced by divalent cations, competition between phosphate, citrate and soy protein could modify the aggregates' properties. Optimum aggregation conditions were established from combinations of these variables.

The mitochondrial dicarboxylate carrier is essential for the growth of Saccharomyces cerevisiae on ethanol or acetate as the sole carbon source.

The dicarboxylate carrier (DIC) is an integral membrane protein that catalyses a dicarboxylate-phosphate exchange across the inner mitochondrial membrane. We generated a yeast mutant lacking the gene for the DIC. The deletion mutant failed to grow on acetate or ethanol as sole carbon source but was viable on glucose, galactose, pyruvate, lactate and glycerol. The growth on ethanol or acetate was largely restored by the addition of low concentrations of aspartate, glutamate, fumarate, citrate, oxoglutarate, oxaloacetate and glucose, but not of succinate, leucine and lysine. The expression of the DIC gene in wild-type yeast was repressed in media containing ethanol or acetate with or without glycerol. These results indicate that the primary function of DIC is to transport cytoplasmic dicarboxylates into the mitochondrial matrix rather than to direct carbon flux to gluconeogenesis by exporting malate from the mitochondria. The delta DIC mutant may serve as a convenient host for overexpression of DIC and for the demonstration of its correct targeting and assembly.

Citrate: a Component of Bile and Calcium Chelator in Gallbladder Disease

Citrate, a calcium chelator, was found to be a regular component of bile in the majority of patients with cholelithiasis. The elevation of citric acid levels was investigated in patients with indwelling T-tubes that were indicative of bile duct exploration for retained bile duct stones following cholecystectomy. The concentration of citrate in gallbladder bile in patients at cholecystectomy was variable. The mean (SEM) 95% confidence interval (CI) concentration in gallbladder bile was 57.8 (4.9) 48.2-67.3 with a range of 0-406 μmoles l-1, and in bile duct bile was 39.6 (11.2) 17.5-61.6 with a range of 0-200 μmoles l-1. Gallbladder or common duct bile infected with bacteria with at least one species with a CFU of ≥ 105 recorded significantly lower citrate concentrations. It was not possible in this study significantly to infer that gallbladder bile associated with a particular stone type had an increased or decreased citric acid content. In 7 of 12 patients with indwelling T-tubes owing to choledocholithia...

The Role of pH in the Adsorption of Citrate Ions on Hydroxyapatite

The adsorption of citrate on hydroxyapatite (HA) has been studied at 25°C, pH 6 and 8, and at 37°C, pH 8. The experimental results agree with the Langmuir adsorption model at low citrate concentrations, with similar values of the affinity coefficientK. The amount adsorbedQdecreases with the increase of pH and the rise in temperature. However, the fraction of sites occupied on the HA surface is the same at both values of pH. On the other hand, the desorption is higher at pH 8 and zeta potential values of suspensions prepared from HA preadsorbed with citrate are negative and decrease with the rise of pH. The adsorption takes place by ionic exchange of phosphate by citrate ions at the solid-solution interface, caused by a higher affinity of citrate than phosphate species for the Ca-sites on the HA surface. To explain experimental data we propose a model where citrate species interact in different ways: cit3−interacts weakly in a bidentate manner (1 citrate per 2 Ca sites), whereas the Hcit2−interaction is stronger (1 citrate per 1 Ca site) than the previous one, possibly due to certain resonance between a monodentate (using 1 -COO−group) and a surface–chelate interaction (using 2 -COO−groups).

Characterization of adhesive and aggregating functions of pig platelets: comparative studies with human platelets and differential effects of calcium chelation

We have characterized functional responses of pig platelets in citrate-anticoagulated blood and compared results with those of human platelets. Platelet aggregation was induced with known activating agents using citrated platelet-rich plasma. Adhesive and cohesive functions of platelets were evaluated using a perfusion flow system with whole anticoagulated blood (10 min, 800/s). To test the differential sensitivity to free calcium levels, two citrate concentrations (19 mM {standard} vs 13 mM) were used as anticoagulants. The ability of platelets to produce thromboxane A2 was also quantified, using radioimmunoassay. In the presence of 19 mM citrate concentration, pig platelets responded poorly to ADP and collagen, and did not respond to arachidonic acid and U46619. Pig platelets adhered well onto subendothelium, but failed to form aggregates on previously spreaded platelets. At 13 mM citrate concentration, the aggregating responses of pig platelets improved, but some of the aggregation patterns were still reversible. The lower citrate concentration had a dramatic impact on perfusion studies, where the formation of platelet aggregates was restored up to levels found in humans. Thromboxane levels in pig serum were only 20 to 30% of that found in humans. So, pig platelets possess functional properties that clearly differ from human platelets: they are more sensitive to the calcium chelating effects of citrate. Arachidonic acid metabolism and thromboxane amplification of platelet responses seem less preponderant in the pig species.

Concrement formation and urease-induced crystallization in urine from patients with continent ileal reservoirs.

To study the relationship between urinary tract infection, urine composition and concrement formation in patients with continent ileal reservoirs for urinary diversion. The study comprised 27 patients (seven men and 20 women, mean age 47 years, range 23-76) with continent ileal reservoirs who were followed for a mean of 67 months (range 13-146) by annual reservoiroscopy, intravenous urography and urine culture; at the final follow-up, a sample of their morning urine was analysed for a range of compounds and the number and size of any particles present or produced in response to incubation with urease. The presence of urease-producing bacteria was associated with the formation of concrement. However, a few patients in whom an infection with urease-producing organisms was not detected also formed concrement. Urine from those patients forming stones tended to have a high calcium and a low citrate concentration. After incubation with urease, significantly more and larger particles were formed in the urine from stone formers. There was a strong correlation (r = 0.8) between urinary calcium content and urinary pH when the urease-induced precipitation commenced, and between urinary calcium and the size and volume of the crystals developed (r = 0.9) after 4 h of incubation. There are many factors which might influence the formation of concrement, e.g. outflow conditions, the presence of staples or infection in the reservoir, and the composition of the urine is also important. It thus appears appropriate to determine if measures to reduce urinary calcium and increase urinary citrate can decrease the episodes of stone formation in those patients with continent ileal reservoirs for urinary diversion who frequently form stones.

Assessing the Effect of Dissolved Organic Ligands on Mineral Dissolution Rates: an Example from Calcite Dissolution

ABSTRACTExperiments suggest that dissolved organic ligands may primarily modify mineral dissolution rates by three mechanisms: (1) metal-ligand (M-L) complex formation in solution, which increases the degree of undersaturation, (2) formation of surface M-L complexes that attack the surface, and (3) formation of surface complexes which passivate or “protect” the surface. Mechanisms (1) and (2) increase the dissolution rate and the third decreases it compared with organic-free solutions. The types and importance of these mechanisms may be assessed from plots of dissolution rate versus degree of undersaturation.To illustrate this technique, calcite, a common repository cementing and vein-filling mineral, was dissolved at pH 7.8 and 22°C in Na-Ca-HCO3-Cl solutions with low concentrations of three organic ligands. Low citrate concentrations (50 μm) increased the dissolution rate consistent with mechanism (1). Oxalate decreased the rate, consistent with mechanism (3). Low phthalate concentration (&lt; 50 μM) decreased calcite dissolution rates; however, higher concentrations increased the dissolution rates, which became faster than in inorganic solutions. Thus, phthalate exhibits both mechanisms (2) and (3) at different concentrations. In such cases linear extrapolations of dissolution rates from high organic ligand concentrations may not be valid.

Prevalence of Endemic Distal Renal Tubular Acidosis and Renal Stone in the Northeast of Thailand

We have previously reported a large group of patients with endemic distal renal tubular acidosis (EdRTA) admitted to the hospitals in the northeast of Thailand. Since large number of patients were identified in a relatively short period of time, and in an area whose population is homogeneous, we were led to investigate the prevalence of the condition in the area. A survey was conducted in five villages (total population of 3,606) within the northeast of Thailand. 3,013 villagers were examined for urinary citrate concentration and short acid loading test was performed in those with low urinary citrate. 2.8% of the population (2.2-3.4%, 95% confidence interval) failed to lower their urine pH after acid loading; within this group, 0.8% of the population had serum potassium less than or equal to 3.5 mEq/l. In addition a large number of villagers were found to have low urinary citrate concentration and there was concurrent high prevalence of renal stone. The prevalence of EdRTA and renal stone was higher in villagers with poorer socioeconomic status, suggesting that environmental factors play a major role in their pathogenesis. Villagers with acidification defect have 2.4 times the chance of having renal stone and/or nephrocalcinosis. EdRTA is therefore one of the important factors responsible for the high prevalence of renal stone in the area. In conclusion we have confirmed the high prevalence of EdRTA in the northeast of Thailand and provided data showing high prevalence of renal stone and hypocitraturia in the same population.

Growth of human tumor cell lines in transferrin-free, low-iron medium.

Iron is essential for tumor cell growth. Previous studies have demonstrated that apart from transferrin-bound iron uptake, mammalian cells also possess a transport system capable of efficiently obtaining iron from small molecular weight iron chelates (Sturrock et al., 1990). In the present study, we have examined the ability of tumor cells to grow in the presence of low molecular weight iron chelates of citrate. In chemically defined serum-free medium, most human tumor cell lines required either transferrin (5 micrograms/ml) or a higher concentration of ferric citrate (500 microM) as an iron source. However, we have also found that from 13 human cell lines tested, 4 were capable of long-term growth in transferrin-free medium with a substantially lower concentration of ferric citrate (5 microM). When grown in medium containing transferrin, both regular and low-iron dependent cell lines use transferrin-bound iron. Growth of both cell types in transferrin medium was inhibited to a certain degree by monoclonal antibody 42/6, which specifically blocks the binding of transferrin to the transferrin receptor. On the contrary, growth of low-iron dependent cell lines in transferrin-free, low-iron medium (5 microM ferric citrate) could not be inhibited by monoclonal antibody 42/6. Furthermore, no autocrine production of transferrin was observed. Low-iron dependent cell lines still remain sensitive to iron depletion as the iron(III) chelator, desferrioxamine, inhibited their growth. We conclude that low-iron dependent tumor cells in transferrin-free, low-iron medium may employ a previously unknown mechanism for uptake of non-transferrin-bound iron that allows them to efficiently use low concentrations of ferric citrate as an iron source. The results are discussed in the context of alternative iron uptake mechanisms to the well-characterized receptor-mediated endocytosis process.

Comparative studies of coupled assays for phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) from higher plants is usually assayed by using malate dehydrogenase (MDH) as a coupling enzyme. To avoid erroneous readings caused by metal ions, which convert oxaloacetate (OAA) to pyruvate, lactic dehydrogenase can be included. Reporting the total NADH used by both coupling enzymes gives the total OAA production. Microbial PEPC has been assayed by employing citrate synthase (CS) as a coupling enzyme which detects the reaction of CoA with Ellman's reagent. Comparable Km values for MgPEP are found with the two assays. When MDH alone is used as the coupling system, the Vmax value is about 60% larger than the one found with the CS assay. However, when MDH is added to the CS assay without the NADH cofactor, Vmax is brought back to the same level as that with the NADH‐coupled enzyme. Malate inhibition of PEPC assayed with the CS coupling system is blocked by low concentrations of citrate in the range produced in the assay. High concentrations of citrate inhibit PEPC. Glucose‐6‐phosphate in concentrations higher than 1 mM blocks the response of PEPC to added MDH in the CS assay.

Clinical significance of hypocitraturia in kidney stone patients.

Although a low concentration of urinary citrate is cited as one of the risk factors promoting stone formation or recurrence among patients with urinary stones, its clinical significance remains obscure. We studied 62 kidney stone patients with a low urinary citrate excretion (hypocitraturia) of less than 320 mg/day, without any apparent cause. The incidence of hypocitraturia in 722 kidney stone patients followed up at our stone clinic was 14.6%. Among the 62 patients, 37 had an uncomplicated hypocitraturia as the sole abnormality, while 25 had other associated stone risk factors, including hypercalciuria in 11% (7/62), hyperuricosuria in 24% (15/62), hyperoxaluria in 5% (3/62) and hypomagnesuria in 24% (15/62). The rate of urinary stone recurrence was 38% (14/37) in uncomplicated hypocitraturia, and 52% (13/25) in complicated hypocitraturia, but no statistical difference was observed. Regarding the outcome of stones, more stones were managed with lithotripsy and more passed spontaneously in the hypocitraturic patients than in the control patients with normal urinary citrate excretion. The diagnosis of hypocitraturia complicated with additional stone risks urged us to treat patients more vigorously with lithotripsy and medication, resulting in a prompt cure.

Pathogenesis of sudden unexplained nocturnal death (lai tai) and endemic distal renal tubular acidosis

Sudden unexplained nocturnal death (SUND), a disorder of unknown cause that occurs in otherwise healthy young adults, mostly male, during their sleep, is prevalent in the north-east region of Thailand, where it has been known for generations as lai tai. It occurs in the same population and area where hypokalaemic periodic paralysis (HPP), endemic distal renal tubular acidosis (EdRTA), and renal stones are also endemic. SUND has occurred in families of patients with EdRTA, and HPP can present as sudden onset of muscle paralysis with potentially lethal cardiac arrhythmias and respiratory failure from severe hypokalaemia occurring in the middle of the night. Surveys in which serum and urinary potassium have been measured indicate a deficiency of the electrolyte in the population. Potassium deficiency is probably the prime factor responsible for SUND and HPP. Low urinary citrate concentrations and the high prevalence of acidification defects in the population indicate that potassium deficiency is also responsible for the prevalence of EdRTA and for renal stones.

Kinetics of iron removal from human serum monoferric transferrins by citrate

The kinetics of release of Fe3+ from human serum diferric transferrin (Fe2Tf) and the Nand C-terminal monoferric transferrins (FeNTf and FkTf) to citrate were studied spectrophotometrically a t pH 7.4,37 OC, and 1.30 M ionic strength. Due to the similarity in rate constants for iron release from the two sites, iron release from Fe2Tf appears to be a monophasic process. Under the present experimental conditions, the C-terminal site is somewhat more labile than the N-terminal site. The observed rate constants for iron release from FeNTf and FecTf show a dependence on citrate concentration (at constant ionic strength) which changes from one apparent linear gradient at low citrate concentration to a second, smaller, apparently linear gradient a t higher citrate concentration. This is described by an equation of the form kob = (k,[Cit][X] + k2K[CitI2]/([X] + K[Cit]}, where k, and k2 are the second-order rate constants for pathways that are dominant a t low and high citrate concentration, respectively, and K is an equilibrium constant reflecting the competition by citrate and an anion, X, from solution for anion-binding sites, distinct from the synergistic-anion-binding sites, which control the kinetics of iron release. The experimental data both for FecTf and FeNTf are adequately fitted by assuming the existence of one such site. The effect of the anions HPO:-, CIOL, Cl-, and SO,*on the kinetics was studied, and it was shown that phosphate, while itself only mobilizing iron very slowly from transferrin, has a marked accelerating effect on metal release to citrate. The present results are compared to the previously reported results for release of AI3+ from AI2Tf to citrate.

The effect of citrate ions on the stress corrosion cracking of Al-Brass in acidic sulphate solutions

The addition of tri-sodium citrate to acidic copper sulphate solutions produces contrasting effects on the occurrence of the stress corrosion cracking (SCC) in Al-brass. Stimulating effects are observed in a wide range of low citrate concentrations (5 × 10−6 to 10−2 M) in which the formation of Cu2O deposits is delayed or suppressed. Inhibitive effects are observed at higher citrate concentrations at which the formation of crystalline copper citrate deposits takes place. The contrasting effects produced by citrates on the corrosive process are discussed in terms of the different weight that the formation of cuprous citrate complexes can have on the activity of the soluble copper species and, consequently, on the chemical and electrochemical equilibria at the metal/solution interface.

Pilot scale (300 kg) fractionation of plasma for factor VIII and factor IX

Pilot-scale processing of plasma to factor VIII and factor IX concentrates has been used to provide advance information for manufacturing at >3000 kg scale, and to confirm or extend data obtained by model (3 kg) fractionation experiments. It has been applied especially to the validation of apheresed plasma procured by various semi-automated procedures and in different anticoagulants. There are inter-Regional differences in the quality of plasma as measured by the recovery of factor VIII in the cryoprecipitate, but stage yields on further processing are constant. The low concentration of citrate in ACD-B plasma leads to insecure anticoagulation during the early stages of processing, and such plasma is not acceptable for the routine production of factor IX concentrate. The sequence in which other clinically useful proteins are removed from plasma influences the quality of factor IX concentrate, as measured by in vitro tests for activated factors.