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PubMed1989

Metabolic studies on citrate synthase mutants of yeast. A change in phenotype following transformation with an inactive enzyme.

We have studied the growth on acetate, the metabolism of acetate enzymes, and respiration of a series of citrate synthase mutants of Saccharomyces cerevisiae. The results confirmed and extended our previous observation that cytosolic citrate synthase is not necessary for growth on acetate. Deletion of mitochondrial citrate synthase (CS1) protein resulted in changes in metabolites, decrease in the amounts of pyruvate and alpha-ketoglutarate dehydrogenase complexes, reduced mitochondrial respiration of citrate and isocitrate, and an inability to grow on acetate. Using site-directed mutagensis, we constructed two separate CS1 proteins with mutations in the enzyme's active site. The mitochondria of cells carrying either site-directed mutagenized CS1 contained the inactive citrate synthase protein. With one mutant in which His313 was replaced with a glycine (CS1/H313G), growth on acetate was restored, and mitochondrial respiration of citrate and isocitrate increased toward parental levels as did the levels of several enzymes. With the other mutant CS1 in which Asp414 was replaced with a glycine (CS1/D414G), no growth on acetate or changes in other parameters was observed. We propose that the characteristics of the strain carrying the CS1 with a H313G mutation result from the formation of an intact Krebs cycle complex by the inactive but structurally unchanged H313G protein.

Acetates↗
PubMed1989

The efficacy of topical citrate after alkali injury is dependent on the period of time it is administered.

The neutrophilic response in the alkali-injured rabbit cornea can be separated into two peaks: the first from 12-24 hr and the second at 21 days. In the experiments detailed here, alkali-injured rabbit eyes were treated for 0-7 days (early peak), 6-35 days (late peak), or 0-35 days (both peaks) with 6.5% or 10% topical citrate. Clinical evaluation of these eyes showed that treatment with 10% citrate (0-35 days) significantly reduced the incidence and severity of corneal ulceration from the control level. When 10% citrate was administered to block the early neutrophilic peak, the incidence of corneal ulceration was also significantly reduced (33.3% from 72.2%). The average ulcer depth increased gradually, reaching an intermediate level well below the control group by day 35. In contrast, 10% citrate treatment of the late peak had no significant effect on the incidence of ulceration; however, these ulcers were less severe than those in the control. When 6.5% topical citrate (0-35 days) was used, there was a significant reduction in the severity but not the incidence of ulceration. In addition, the beneficial effects obtained from separate treatment of the early and late PMN peaks were mostly lost. These experiments suggest that both the early and late peak of the PMN response after alkali injury are important to the development of corneal ulcers. We hypothesize that the early use of 10% citrate diminishes the initial peak of PMNs, interfering with the release of self-perpetuating inflammatory mediators, thereby decreasing the later accumulation of PMNs that cause ulceration.

Administration, Topical↗
PubMed1988

In vitro mutagenesis of Escherichia coli citrate synthase to clarify the locations of ligand binding sites.

In vitro mutagenesis techniques have been used to investigate two structure-function questions relating to the allosteric citrate synthase of Escherichia coli. The first question concerns the binding site of alpha-keto-glutarate, which is a structural analogue of the substrate oxaloacetate and yet has been suggested to be an allosteric inhibitor of the enzyme. Using oligonucleotide-directed mutagenesis of the cloned E. coli citrate synthase gene, we prepared missense mutants, designated CS226H----Q and CS229H----Q, in which histidine residues at positions 226 and 229, respectively, were replaced by glutamine. In the homologous pig heart citrate synthase it is known (Wiegand, G., and Remington, S. J. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 97-117) that the equivalent of His-229 helps to bind oxaloacetate, while the equivalent of His-226 is nearby. Kinetic and ligand binding measurements showed that CS226H----Q had a reduced affinity for oxaloacetate and alpha-ketoglutarate, while CS229H----Q bound oxaloacetate even less effectively, and was not inhibited by alpha-ketoglutarate at all under our conditions. This parallel loss of binding affinities for oxaloacetate and alpha-ketoglutarate, in two mutants altered in residues at the active site of E. coli citrate synthase, strongly suggests that inhibition of this enzyme by alpha-ketoglutarate is not allosteric but occurs by competitive inhibition at the active site. The second question investigated was whether the known inhibition by acetyl-CoA of binding of NADH, an allosteric inhibitor of E. coli citrate synthase, occurs heterotropically, as an indirect result of acetyl-CoA binding at the active site, or directly, by competition at the allosteric NADH binding site. Using existing restriction sites in the cloned E. coli citrate synthase gene, we prepared a deletion mutant which lacked 24 amino acids near what is predicted to the acetyl-CoA-binding portion of the active site. The mutant protein was inactive, and acetyl-CoA did not bind to the active site but still inhibited NADH binding. Thus acetyl-CoA can interact with both the allosteric and the active sites of this enzyme.

Binding Sites↗
PubMed1987

Compensatory regulation in metabolic pathways--responses to increases and decreases in citrate synthase levels.

The level of citrate synthase was varied in Escherichia coli by recombinant DNA methods to elucidate regulatory interactions between the individual steps of the citric acid cycle. The effects of overproduction and underproduction of citrate synthase were assessed by measuring metabolite levels, rates of carbon flow, the phosphorylation state of isocitrate dehydrogenase, and the growth rate of the culture. This analysis revealed that the levels of citrate synthase and isocitrate dehydrogenase activity are co-ordinated for efficient growth on acetate. When citrate synthase was overproduced the isocitrate dehydrogenase reaction became rate limiting and prevented large increases in the flux through the citric acid cycle. Furthermore, changes in the level of citrate synthase were found to modulate the phosphorylation state of isocitrate dehydrogenase which regulates the distribution of carbon flow between the citric acid cycle and the glycoxylate shunt. These adjustments allowed the organism to maintain a relatively constant metabolic state despite changes in the level of a central metabolic enzyme. The interplay between citrate synthase and isocitrate dehydrogenase illustrates how living systems can compensate for variations in their internal environment.

Acetates↗
PubMed1987

Structural basis for regulation in gram-negative bacterial citrate synthases.

The citrate synthases of Gram-negative bacteria, unlike those of eukaryotes, are inhibited allosterically by NADH, but the two kinds of citrate synthase are about 30% homologous in amino acid sequence--the two Gram-negative citrate synthase sequences so far determined, from Escherichia coli and Acinetobacter anitratum, are about 70% identical. A model for the NADH-sensitive E. coli citrate synthase has been constructed using sequence homology and the known structure of the pig heart enzyme. The most reactive cysteine in the E. coli enzyme, which probably marks the NADH binding site, has now been identified as Cys-206. The model places this residue far from the active site. An E. coli citrate synthase mutant, from which a stretch of 24 amino acids has been deleted near the active site, still binds NADH normally. Two active site missense mutants of this enzyme, generated by oligonucleotide-directed mutagenesis, have lower affinities for one substrate, oxaloacetate, but also are much less sensitive to 2-oxoglutarate, an oxaloacetate analogue hitherto believed to be an allosteric inhibitor. These results confirm that NADH binds to a truly allosteric site in E. coli citrate synthase, the features of which are still to be defined; while 2-oxoglutarate is really an active-site directed inhibitor, although it may still play a regulatory role in vivo.

Binding Sites↗
PubMed1988

Citrate or ascorbate/citrate treatment of established corneal ulcers in the alkali-injured rabbit eye.

Immediate treatment of alkali-injured eyes with citrate or ascorbate has previously been shown to prevent corneal ulceration and perforation in the rabbit. Other experiments showed that while ascorbate treatment of established ulcers did not appear to lead to significant healing it did reduce perforations by prolonging the presence of descemetoceles. In the present experiment with established ulcers, alkali injuries were created with 1 N NaOH in a 12 mm corneal well for 35 seconds. Eyes were entered into the study with anterior, middle or posterior ulcers. When compared to controls, 10% citrate (q1/2 hr) significantly reduced the deepening of anterior stromal ulcers while 10% ascorbate/10% citrate (q1 hr/q1 hr-30 min apart) showed only a trend toward reduction of these ulcers (14 hr of dropping). The demonstration of healing (total vascularization or no ulcer) is significant when comparing the control group (8.3%) to the citrate treated group (58.3%, 0.01 less than P less than 0.009), but not the ascorbate/citrate group (18.2%). Sixty-seven percent of anterior stromal ulcers in the control group progressed to descemetocele or perforation, compared to 8.3% in the citrate treated (0.003 less than P less than 0.004), and 45.5% in the ascorbate/citrate treated group (not significant). While the numbers of ulcers entered as middle stromal were too few to analyze statistically, the reduced numbers of perforations and increased stability prior to perforation in both treatment groups suggest a positive effect by both citrate and ascorbate/citrate. Treatment of posterior stromal ulcers did not prevent the development of descemetoceles and perforations in either treatment group; however, the numbers in this category were too few to analyze.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

[Indication of the urinary citrate levels in idiopathic renal calculosis].

The reported incidence of hypocitraturia among stone formers (SF) varies between 15 and 50%. It is not known whether this incidence depends on the subtype of SF. The recent simplification of the method of measurement of U-citrate (Boehringer-Mannheim kit) led us to address this issue in 118 male idiopathic (I) SF, with either idiopathic hypercalciuria of the renal (n = 6), absorptive (n = 19) or dietary (n = 41) type, or with normocalciuria (n = 52). Results were compared with those of 42 normal male volunteers. 9 patients had hypocitraturia (less than 215 mg/24 h), all of whom belonged to the group of normocalciuric ISF; only this group of patients had mean U-citrate X V significantly lower than controls (p = 0.025). The cause of hypocitraturia in these patients remains unknown: there were no significant correlations between U-citrate X V and U-pH or blood HCO3 or Cl. Moreover, the patients with hypocitraturia had neither urinary infection nor signs of distal tubular acidification dysfunction as assessed by fasting morning urinary pH. Extended to our whole population of ISF (250 patients) 75% of whom have idiopathic stone disease, it appears that measurement of U-citrate decreased the number of ISF without detectable disorder from 34 to 26, i.e. to 10% of the whole population of stone formers. Since hypocitraturia can easily be corrected by oral administration of citrate, routine measurement of U-citrate in normocalciuric ISF appears a promising method.

Calcium↗
PubMed1983

Kinetics of activation of acetyl-CoA carboxylase by citrate. Relationship to the rate of polymerization of the enzyme.

The kinetics of citrate-induced activation and polymerization (into filaments) of the 450,000-dalton protomeric form of acetyl-CoA carboxylase were compared to assess the concertedness of the two processes. Rapid-quench techniques were employed to measure the time course of activation by citrate of the carboxylase-catalyzed reaction. When enzyme was preincubated with citrate prior to initiating the steady state turnover reaction with acetyl-CoA in the rapid-quench device, the observed rate of carboxylation of acetyl-CoA was apparently linear from the moment of mixing. However, when enzyme was mixed with citrate to initiate the reaction, a lag (t1/2 = 0.7 s) occurred in the approach to steady state carboxylation rate. This lag was independent of enzyme concentration over a 230-fold range and was marginally dependent upon citrate concentration. Over the same range of enzyme concentration, polymerization of carboxylase protomers, as determined by right angle light scattering, was enzyme concentration-dependent in a manner predicted by a single protomer activation step, followed by a rate-limiting dimerization of active protomer and subsequent polymerization. Based on these results, it is concluded that activation of catalysis and the polymerization of carboxylase protomers are not concerted. Furthermore, activation of carboxylation leading to the formation of an active protomer was faster than polymerization under all conditions, and therefore precedes polymerization. It was also shown that the activation constant (Kact) for citrate is altered in a predictable manner by the accumulation of the reaction product, malonyl-CoA, the Kact increasing with increasing malonyl-CoA concentration. Additional evidence is presented indicating that this change in Kact was not caused by autophosphorylation of the enzyme under these conditions and that phosphorylation does not affect the mechanism of activation elucidated above.

Acetyl-CoA Carboxylase↗
PubMed1982

Insulin-stimulated phosphorylation of ATP-citrate lyase in isolated hepatocytes. Stoichiometry and relation to the phosphoenzyme intermediate.

We have estimated the insulin-stimulated phosphorylation of ATP-citrate lyase by two methods. Isolated hepatocytes incorporate extracellular 32P into [gamma-35P] ATP and immunoprecipitated ATP-citrate lyase to steady state levels by 1 h. The content of acid-stable 32P in hepatocyte ATP-citrate lyase at steady state is 0.33 +/- 0.038 mol of P/mol (tetrameric) holoenzyme. Insulin (1 milliunit/ml) increases the 32P content of immunoprecipitated lyase 2- to 3-fold in 10 min. Over 90% of acid-stable 32P on lyase is 32P-serine in enzyme isolated from both control and insulin-treated cells. ATP-citrate lyase isolated from hepatocytes contains 0.95 +/- 0.1 mol of alkali-labile phosphate/mol of holoenzyme. Insulin treatment of hepatocytes (1 milliunit/ml for 10 min) increases the alkali-labile P content by 45%. Evidence is presented which indicates that the insulin-stimulated phosphorylation does not arise by intramolecular migration from the catalytic phosphoenzyme intermediate. These observations support the conclusion that insulin-stimulated phosphorylation of ATP-citrate lyase is mediated either by an insulin-induced increase in the activity of lyase kinase and/or decrease in a lyase phosphatase. The functional role of the substoichiometric phosphorylation of ATP-citrate lyase remains unknown.

ATP Citrate (pro-S)-Lyase↗
PubMedDate not supplied

Evaluation of activated charcoal and magnesium citrate in the prevention of aspirin absorption in humans.

Inhibition of aspirin absorption by activated charcoal and magnesium citrate solution was compared with the inhibition produced by activated charcoal alone. Following an overnight fast, eight healthy male volunteers were given three 325-mg aspirin tablets under four study regimens: (1) water 360 ml; (2) water 300 ml and activated charcoal 10 g in water 60 ml; (3) water 105 ml, activated charcoal 10 g in water 60 ml, and magnesium citrate solution 200 ml; and (4) same as (3) except that administration of magnesium citrate was delayed 30 minutes. At least one week separated each regimen. Urine samples were collected at 0, 2, 4, 8, 12, 24, 36, and 48 hours, and percent of the aspirin dose excreted in the urine was determined. The data were analyzed using analysis of variance for Latin-square design and Newman-Keuls test. The salicylate excreted with regimens 2, 3, and 4 was each significantly less compared with that excreted following regimen 1 (p less than 0.001). Salicylate excretion percentages when magnesium citrate was given with activated charcoal in regimens 3 and 4 were not significantly different from each other or the salicylate excretion with activated charcoal alone (p greater than 0.05). These findings apply only to the dose of aspirin tested, and it is possible that magnesium citrate and activated charcoal may decrease aspirin absorption to a greater extent when aspirin is taken in overdose. Hence, it is recommended that this study not be used to justify discontinuing the combined use of magnesium citrate and activated charcoal to treat aspirin overdose.

Adult↗
PubMed1995

Measurement of urinary oxalate and citrate by capillary electrophoresis and indirect ultraviolet absorbance.

We describe a method for measuring urinary oxalate and citrate with capillary electrophoresis (CE) and indirect ultraviolet absorbance detection. Sample preparation is minimal, requiring an acidification, brief centrifugation, and dilution. The method is rapid, with oxalate and citrate having mean migration times of 4.02 and 4.50 min, respectively. The minimal detectable concentration (signal-to-noise ratio of 7) of both oxalate and citrate in urine was 7 mg/L. Total imprecisions (CV) were 1.2-5.6% for three urine samples with oxalate and citrate concentrations of 8-60 mg/L and 80-860 mg/L, respectively. The recovery of added oxalate ranged from 94% to 101%. Results of CE analyses agreed well with enzymatic determinations of oxalate and citrate. Rapid analysis time, accuracy, and reproducibility make this procedure well suited for routine urinary oxalate and citrate determinations.

Capillary Action↗
PubMed1993

The routine measurement of platelet size using sodium citrate alone as the anticoagulant.

Mean platelet volume (MPV), a measure of platelet size, is becoming recognised as an important marker of platelet function. However, platelets swell in edetic acid (EDTA), the standard haematology anticoagulant, in a time-dependent manner making such measurements potentially unreliable. The effect of incubation time on MPV, and platelet distribution width (PDW), as measured in EDTA, low (1:9 volume/volume with blood) or high (1:4 v/v with blood) concentration sodium citrate was studied. MPV measured in high concentration sodium citrate did not change with time in contrast to MPV measured in either low concentration sodium citrate or EDTA which both increased in an inverse exponential fashion. MPV and PDW, measured in high concentration sodium citrate, had similar within-assay and between-assay coefficients of variation as other platelet, red cell and white cell haematology variables measured in EDTA: MPV 1.4%, 2.1%; PDW 1.4%, 1.5%; MCV 0.4%, 0.7%; PC 3.1%, 6.1%; WCC 1.5%, 7.3%; Hb 2.1%, 2.4% respectively. MPV measured in EDTA and corrected for incubation time approximated to, but was higher than, the MPV measured in high concentration sodium citrate. PDW correlated inversely with platelet count (r = -0.415, 2p < 0.001). MPV may be measured in sodium citrate (at 1:4 v/v with blood) alone with a better accuracy and reproducibility than similar measurements made in EDTA. Furthermore, such measurements are not influenced by incubation time, unlike for EDTA.

Anticoagulants↗
PubMed1976

Studies of the mechanism by which hepatic citrate synthase activity increases in vitamin B12 deprivation.

Hepatic citrate synthase activity has been shown to be increased 2- to 3-fold in vitamin B12 deficiency. Immunochemical titrations of the affinity chromatography-purified enzyme obtained from liver of animals with B12 deprivation demonstrated that this increase in activity was the result of a true increase in enzyme protein content. When fixed ratios of aliquots of normal and B12-deprived rat liver homogenates were mixed, the activity measured showed no change from the expected total citrate synthase activity based on the admixture ratios. Partial purification of the enzyme resulted in the expected recovery of the enzyme at each of the purification steps. Thus, it is unlikely that the change in enzyme activity in B12 deprivation was due to the presence of a soluble or easily dissociable normally occurring activator or inhibitor. Ouchterlony double diffusion studies, immunochemical titration, and determination of Km vlaues for exalacetate and acetyl-CoA (substrates for citrate synthase) and Ki values for ATP (inhibitor of citrate synthase) all indicated that the enzyme from the B12-deprived livers was structurally the same as that from normal liver. Hepatic citrate synthase degradation rate constants were shown to be essentially unchanged in B12deficiency. The rate of hepatic citrate synthase synthesis, under steady state conditions, was shown to be 2.8-fold greater in the B12-deficient animal than in the normal animal. The increased rate of synthesis appeared to explian the increased enzyme content. Finally, no change in specific activity of the enzyme was seen in brain, heart, or kidney in the B12-deprived animal.

Animals↗
PubMed1993

13C nuclear magnetic resonance studies of malate and citrate synthesis and compartmentation in higher plant cells.

The synthesis of malate and citrate by sycamore cells (Acer pseudoplatanus L.) perfused with KH13CO3 was analyzed using 13C NMR. To perform in vivo experiments, cells were compressed in a 25-mm tube and perfused with an arrangement enabling tight control of the circulating nutrient medium. An original method using paramagnetic Mn2+ that induced a complete loss of the vacuolar malate and citrate signals was developed to discriminate between cytoplasmic and vacuolar pools of malate and citrate. Our results indicated the following. (a) The accumulation of appreciable amounts of malate in sycamore cells required rather high (1 mM) concentrations of bicarbonate at all the pH values tested. (b) Malate was equally labeled at C-1 and C-4, suggesting that malate labeled at C-1 was produced by randomization of C-1 and C-4 by mitochondrial fumarase. Indeed, the separation of the intact organelles from the lysed protoplasts indicated that fumarase activity was essentially limited to the mitochondria. Similarly, citrate was equally enriched at C-1 and C-5 + C-6 carboxyls. (c) Malate appeared first in the cytoplasmic compartment; and when a threshold of cytoplasmic malate concentration was attained, malate molecules were expelled into the vacuole, where they accumulated. On the other hand, citrate accumulated steadily in the vacuole. Pulse-chase experiments demonstrated the central role played by the tonoplast in governing the vacuolar influx of citrate and the permanent exchange of malate between the cytoplasm and the vacuole.

Bicarbonates↗
PubMed2004

Changes in urinary parameters after oral administration of potassium-sodium citrate and magnesium oxide to prevent urolithiasis.

OBJECTIVES: To examine urinary parameters among normal individuals and patients with calcium oxalate (CaOx) stones after oral administration of potassium-sodium citrate (KNa-Cit) and magnesium oxide (MgO). Urinary citrate and magnesium have been known as the inhibitors of CaOx stone formation. Supplementation with potassium-magnesium citrate prevents the recurrence of CaOx stones. METHODS: Twenty-five male volunteers aged 21 to 42 years without a history of urinary stones were given either KNa-Cit or MgO, or both. Fourteen patients with recurrent CaOx stones were also given both supplements, and 24-hour urine samples were collected to determine the urinary parameters. RESULTS: The administration of both KNa-Cit and MgO to the normal individuals increased the excretion of citrate, magnesium, and potassium by 70.0%, 44.2%, and 50.0%, respectively. These parameters increased less when KNa-Cit or MgO was administered individually. After administration of both supplements to the patients with stones, the citrate, magnesium, and potassium levels increased by 62.1%, 63.3%, and 25.3%, respectively, and oxalate decreased by 66.5%. In both normal individuals and patients, the ion activity product index of CaOx decreased significantly more after administration of the combination than with either compound alone or before administration. CONCLUSION: The combination of KNa-Cit and MgO is more effective than either supplement alone in inhibiting the crystallization of CaOx stones. The combination may improve the urinary parameters of patients with stones accompanied by hypocitraturia and/or hypomagnesuria.

Administration, Oral↗
PubMed2000

Clomiphene citrate for ovulation induction in women with oligo-amenorrhoea.

BACKGROUND: The administration of clomiphene citrate is followed by an enhanced release of pituitary gonadotropins resulting in follicular recruitment. After the drug is stopped, there is continuing secretion of estradiol, selection of the dominant follicle and, in successful cases, ovulation. Clomiphene is indicated as the initial treatment in the majority of women with amenorrhoea and oligomennorhoea. In women with irregular ovulation it seems to re-establish typical frequency of ovulation. Its effectiveness in oligo-amenorrhoeic women was tested in a number of randomized controlled trials at that time. These trials form the basis for the following review. OBJECTIVES: Clomiphene citrate enhances the release of pituitary hormones, often resulting in ovulation. The objective of this review was to assess the effects of clomiphene citrate on ovulation and pregnancy in women with oligo-ovulatory subfertility. SEARCH STRATEGY: The Cochrane Subfertility Review Group specialised register of controlled trials was searched. SELECTION CRITERIA: Randomised trials of clomiphene compared with placebo or no treatment in women with oligo-ovulatory subfertility of at least 12 months duration. DATA COLLECTION AND ANALYSIS: Trial quality was assessed and data were extracted independently by two reviewers. MAIN RESULTS: Four studies were included. They were all of crossover design. Since it was not possible to separate data from the first and second phases of these trials, the effect of clomiphene may be overestimated. Compared with placebo, clomiphene citrate was associated with increased ovulation. The odds ratio for high doses (50-250 milligrams per day) was 6.82 (95% confidence interval 3.92 to 11.85). This dropped to a non-significant odds ratio of 1.29 (95% confidence interval 0.48 to 3.49) with low doses (10 milligrams per day). Clomiphene citrate (all doses) was associated with an increased pregnancy rate per treatment cycle (odds ratio 3.41, 95% confidence interval 4.23 to 9.48). REVIEWER'S CONCLUSIONS: Clomiphene citrate (at doses between 50 to 250 milligrams per day) appears to be an effective method of inducing ovulation and improving fertility in oligo-ovulatory women. However adverse effects include possible ovarian cancer risk and risk of multiple pregnancy.

Amenorrhea↗
PubMed1998

The effect of ascorbic acid and ferric ammonium citrate on iron uptake and storage in lens epithelial cells.

Ferritin is the major intracellular iron storage protein which has been shown to protect cells against oxidative damage. Recent reports that an inherited abnormality in human ferritin synthesis is associated with early bilateral cataracts underscore the importance of understanding ferritin synthesis and iron storage in lens epithelial cells. We previously demonstrated that ascorbic acid greatly increases de novo synthesis of ferritin in lens epithelial cells. The objectives of the present study were to determine: (1) the effects of ascorbic acid and ferric ammonium citrate on iron uptake by canine lens epithelial cells from iron bound to transferrin and from ferric chloride and (2) the incorporation of this element into ferritin. Iron uptake by lens epithelial cells from 59ferric chloride was 20 times higher than from 59iron-transferrin and iron deposition into ferritin was 8-fold higher when 59ferric chloride was the source. Ascorbic acid had a stimulatory effect on iron uptake from transferrin and on incorporation of this element into ferritin. The ascorbic acid-induced increase of iron uptake required de novo protein synthesis but not specifically de novo ferritin biosynthesis. Although ferritin is not directly involved in iron uptake, the level of ferritin protein could control the pool of intracellular iron. The present results indicate that iron homeostasis in lens epithelial cells is affected mainly by changes in apoferritin synthesis, which is greatly stimulated by ascorbic acid, rather than by altering the rate of protein degradation, which is very slow in these cells under all circumstances. Ferric ammonium citrate activates iron uptake from transferrin in a wide range of cell lines by generation of free radicals. Ferric ammonium citrate also increased iron uptake from Tf in lens epithelial cells. Ferric ammonium citrate treated cells incorporated 5 times more iron and deposited 2 times more iron into ferritin than control cells. Increased incorporation of iron into ferritin was due to ferric ammonium citrate-induced stimulation of de novo ferritin synthesis rather than an increased rate of iron deposition into pre-existing ferritin. Ferric ammonium citrate had a different effect on iron uptake from ferric chloride; total iron uptake was not significantly increased while deposition into ferritin was significantly decreased. These results demonstrate that iron homeostasis in lens epithelial cells is regulated by ascorbic acid and by changes in the rate of de novo ferritin synthesis. In addition, the differences in iron uptake from transferrin and ferric chloride and its subsequent incorporation into ferritin suggests that the mechanisms by which iron is incorporated into ferritin are source dependent.

Animals↗
PubMed1992

Stability of sufentanil citrate in a portable pump reservoir, a glass container and a polyethylene container.

The stability of sufentanil citrate (100 ml, 5 micrograms/ml) in an admixture with sodium chloride 0.9% injection was investigated when filled in a portable pump reservoir with PVC wall, a glass container and a polyethylene container, at 32 degrees C, 4 degrees C and -20 degrees C for up to 21 days. No change in colour was visually observed in any of the samples during the 21-day storage period. A slight precipitation was noticed in three out of nine portable pump reservoirs, one at each storage temperature. There was a slight rise in pH at each storage temperature in all samples. There was approximately 13% loss of sufentanil citrate in the portable pump reservoirs stored at 32 degrees C during 2 days and 60% loss after 21 days, due to absorption of sufentanil citrate in the reservoir wall. No loss of sufentanil citrate could be detected in the portable pump reservoirs when stored at -20 degrees C and 4 degrees C. However, a serious inhomogeneity of the sufentanil citrate solution occurred after thawing at room temperature in the portable pump reservoirs which had been kept at -20 degrees C. The homogeneity could be restored by shaking for approximately 10 min. There was no change in the sufentanil citrate concentrations in the glass containers and polyethylene containers stored at the three temperatures. The portable pump reservoirs stored at 32 degrees C also showed a significant loss of vehicle due to evaporation (1.0 +/- 0.1 ml a week). This could not be detected in any of the other samples.

Absorption↗