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Urea, citrate and orotate excretion in growing rats fed amino acid-deficient diets.

Male, weanling rats were fed a control purified amino acid diet or the same diet with lysine, phenylalanine, tryptophan, valine, or arginine omitted singly, or both arginine and lysine omitted. Blood urea reached three to four times that of control levels with all deficient diets. Urea excretion increased almost linearly with time during the first 3 days of amino acid deficiency. Rates of urea excretion on day 3 in decreasing order for various deficiencies were as follows: lysine and arginine combined are more than lysine is more than tryptophan equals valine is more that phenylalanine equals arginine. Urinary citrate was 26 and 21.8 times that of control values without arginine and 11.4 and 6.2 times that of control values without lysine on days 2 and 3, respectively. By day 8 citrate excretion had returned to control levels without lysine but not without arginine. Citrate excretion was unchanged with other deficiencies. Orotic acid excretion increased markedly only without arginine and slightly without tryptopahn. A deficiency of arginine and lysine increased urea and citrate excretions to a greater extent than either deficiency alone. Two injections of arginine or homoarginine (0.50 mmole/injection) given at 12-hour intervals to rats fed no lysine and arginine for 3 days decreased citrate excretion immediatedly and on the following day. Urea excretion decreased with injected homoarginine, but not with arginine. Orotic acid excretion increased more than four times on the day of homoarginine injection compared with that of the preceding day. Arginine injection returned orotic acid excretion to nearly control levels within 24 hours. Urea degradation in the gastrointestinal tract was increased in animals fed amino acid-deficient diets.

Amino Acids, Essential↗

Decreased citrate improves iron availability from infant formula: application of an in vitro digestion/Caco-2 cell culture model.

We have applied an in vitro digestion/Caco-2 cell culture model to the assessment of iron availability from human milk and a generic cow's milk-based infant formula. Experiments were designed to determine the availability of iron from human milk relative to infant formula and whether known promoters of iron absorption would increase Caco-2 cell iron uptake and availability from the infant formula. In addition, we sought to determine if decreasing the citrate concentration in the infant formula would increase the iron uptake. Although approximately twice as much iron was in solution from digests of the infant formula relative to that of human milk, smaller or equal amounts of iron were taken up from the infant formula relative to the human milk digest. These results are qualitatively similar to in vivo studies. Addition of known iron uptake promoters to infant formula did not enhance Caco-2 cell iron uptake from the infant formula digest, indicating that the iron in the infant formula existed predominantly in a tightly bound unavailable form(s). Enzymatic pretreatment of the infant formula with citrate lyase and oxalacetate decarboxylase decreased the citrate concentration by 67% and resulted in a 64% increase of iron in solution, which corresponded to a 46% increase in the cell iron uptake. Iron uptake from the "low citrate" formula plus cysteine was 102% greater relative to the nontreated formula. The results indicate that too much citrate can reduce iron uptake, particularly if it is present at concentrations greater than promoters such as ascorbic acid and cysteine.

Animals↗

The dual functions of Tetrahymena citrate synthase are due to the polymorphism of its isoforms.

Previous studies have shown that Tetrahymena citrate synthase and the Tetrahymena 14-nm filament protein are encoded by a single gene and translated from one species of mRNA, and that they are identical in terms of molecular weight, antigenicity, and some enzymatic properties. In this study, using two-dimensional gel electrophoresis, we demonstrated that the citrate synthase comprised pI 7.7 and 8.0 isoforms, while the 14-nm filament protein comprised three isoforms with isoelectric points of 7.7, 8.0, and 8.4. The amino acid sequences of the NH2-terminal portions of all isoforms were identical and the peptide maps with V8 protease were almost the same. In addition, when the citrate synthase activity of each isoform was measured after separation by non-urea isoelectric focusing without denaturing treatment, the pI 7.7 and/or pI 8.0 isoforms exhibited the citrate synthase activity, but the pI 8.4 isoform only found for the 14-nm filament protein did not possess this activity. These results suggest that the polymorphism of these isoforms is caused by some posttranslational modifications, and that it may have resulted in the different compartmentalization and functions of Tetrahymena citrate synthase and the 14-nm filament protein.

Amino Acid Sequence↗

Cloning and nucleotide sequence of the gene (citA) encoding a citrate carrier from Salmonella typhimurium.

A cryptic citrate transport gene (citA) from Salmonella typhimurium chromosome was cloned and its nucleotide sequence was determined. The cloned plasmid conferred citrate-utilizing ability on wild-type Escherichia coli, which cannot grow on citrate as the sole source of carbon. The resultant E. coli transformant was able to transport citrate. A 1,302-base-pair open reading frame with a preceding ribosomal binding site was found in the cloned DNA fragment. The 434-amino-acid protein that could be translated from this open reading frame is highly hydrophobic (69% nonpolar amino acid residues), consistent with the fact that the transport protein is an intrinsic membrane protein. The molecular weight of this protein was calculated to be 47,188. The gene sequence determined is highly homologous to those of Cit+ plasmid-mediated citrate transport gene, citA, from E. coli, the chromosomal citA gene from Citrobacter amalonaticus and the chromosomal cit+ gene from Klebsiella pneumoniae. The hydropathy profile of the deduced amino acid sequence suggests that this carrier has 12 hydrophobic segments, which may span the membrane lipid bilayer.

Amino Acid Sequence↗

Fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase from Thermus aquaticus YT-1, an extreme thermophile: activation by citrate and modification reagents and comparison with Thermus caldophilus GK24 L-lactate dehydrogenase.

Heat-stable fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase [EC 1.1.1.27] was purified from an extremely thermophilic bacterium, Thermus aquaticus YT-1. The amino acid composition and NH2-terminal 34 amino acid sequence of the enzyme were determined. Its NH2-terminal sequence shows high homology with those of Thermus caldophilus GK24 (82% identity) and some other bacterial L-lactate dehydrogenases (44-53% identity), indicating the close phylogenic relationship of the two Thermus species. At the same time, the two Thermus L-lactate dehydrogenases were found not to be identical not only chemically but also kinetically and immunologically. Citrate activated the T. aquaticus enzyme in the weak acidic pH region, while fructose 1,6-bisphosphate did in both acidic and neutral pH regions. The maximum activity obtained with citrate at pH 5.0 was about 2.5 times higher than that in the presence of fructose 1,6-bisphosphate at pH 6.7. The enzymes modified with 2,3-butanedione, acetic anhydride and diethyl pyrocarbonate in the presence of both NADH and oxamate were desensitized to fructose 1,6-bisphosphate, and the modified enzymes were active even in the absence of fructose 1,6-bisphosphate. All of the modified enzymes examined were still activated by citrate similarly to the native enzyme. These results suggest that the mechanism of activation by citrate is different from that by fructose 1,6-bisphosphate, and that the citrate-binding site is different from the fructose 1,6-bisphosphate-binding site.

Amino Acids↗

Magnesium enhances aluminum-induced citrate secretion in rice bean roots (Vigna umbellata) by restoring plasma membrane H+-ATPase activity.

We demonstrated that magnesium (Mg) can alleviate aluminum (Al) toxicity in rice bean [Vigna umbellata (Thunb.) Ohwi & Ohashi] more effectively than is expected from a non-specific cation response. Micromolar concentrations of Mg alleviated the inhibition of root growth by Al but not by lanthanum, and neither strontium nor barium at the micromolar level alleviates Al toxicity. Aluminum also induced citrate efflux from rice bean roots, and this response was stimulated by inclusion of 10 microM Mg in the treatment solution. The increase in the Al-induced citrate efflux by Mg paralleled the improvement in root growth, suggesting that the ameliorative effect of Mg might be related to greater citrate efflux. Vanadate (an effective H+-ATPase inhibitor) decreased the Al-induced citrate efflux, while addition of Mg partly restored the efflux. Mg addition also increased the activity of Al-reduced plasma membrane H+-ATPase, as well as helping to maintain the Mg and calcium contents in root apices. We propose that the addition of Mg to the toxic Al treatment helps maintain the tissue Mg content and the activity of the plasma membrane H+-ATPase. These changes enhanced the Al-dependent efflux of citrate which provided extra protection from Al stress.

Aluminum↗

Sequence alignment of citrate synthase proteins using a multiple sequence alignment algorithm and multiple scoring matrices.

The alignment of Escherichia coli citrate synthase to pig heart citrate synthase and the multiple alignment of the known sequences of the citrate synthase family of enzymes have been performed using six different amino acid similarity scoring matrices and a large range of gap penalty ratios for insertions and deletions of amino acids. The alignment studies have been performed as the first step in a project aimed at homology modelling E. coli citrate synthase (a hexamer) from pig heart citrate synthase (a dimer) in a molecular modelling approach to the study of multi-subunit enzymes. The effects of several important variables in producing realistic alignments have been investigated. The difference between multiple alignment of the family of enzymes versus simple pairwise alignment of the pig heart and E. coli proteins was explored. The effects of initial separate multiple alignments of the most highly related or most homologous species of the family of enzymes upon a subsequent pairwise alignment between species was evaluated. The value of 'fingerprinting' certain residues to bias the alignment in favour of matching those residues, as well as the worth of the computerized approach compared to an intuitive alignment technique, were assessed.

Algorithms↗

The value of Tc-99m citrate scintigraphy in chronic osteomyelitis. An indicator of the involved bone.

The authors present a prospective analysis of Tc-99m MDP and Tc-99m citrate scintigraphy in 29 patients who were clinically suspected of having chronic osteomyelitis. All of the patients showed increased Tc-99m MDP uptake on bone scintigraphy involving the area of concern. However, Tc-99m citrate scintigraphy clearly identified the patients with osteomyelitis (N = 18) confirmed either by biopsy or clinical follow-up. Eleven patients who were free of osteomyelitis showed only minimal radiotracer uptake on Tc-99m citrate scintigraphy. These patients had other benign musculoskeletal diseases that were diagnosed by other imaging modalities. In addition to visually interpreting the scintigraphic images, a semi-quantitative analysis was performed by drawing regions of interest over the areas involved and the normal contralateral side, and patients with osteomyelitis were found to have lesion to nonlesion ratios of greater than 1.70. Because Tc-99m MDP is a nonspecific radiopharmaceutical for the evaluation of bone infection, other imaging agents have been investigated. Tc-99m citrate is a small molecule that makes it suitable to penetrate injured capillaries to the edema fluid known to be present in areas of infection. The authors conclude that Tc-99m citrate is a promising agent for localizing and showing the extent of bone infection that will help the surgeon to determine areas of debridement before surgery.

Adult↗

Milk lactose, citrate, and glucose as markers of lactogenesis in normal and diabetic women.

A study was undertaken to define an appropriate marker of lactogenesis II (the onset of copious milk secretion) in mothers, and to determine the effect of diabetes on this marker. Changes in the concentrations of three milk components--lactose, citrate, and glucose--were measured in 38 normal mothers and 6 type I diabetic mothers up to 10 days after birth. Milk yield was measured in 12 of the normal mothers, and all mothers were asked to note the time of milk "coming in" (the feeling of overfullness of the breasts). The average concentrations of lactose, citrate, and glucose in milk were low for the first 24 h after birth, then between 24 and 48 h after birth there was a rapid increase in the concentrations of lactose and citrate, and this transitional period was followed by a plateau period that began between 60 and 84 h after birth. For individual mothers the transitional period for citrate began 32 +/- 9 h (n = 13) and finished 77 +/- 10 h (n = 17) after birth, and for lactose the transitional period finished at 53 +/- 12 h (n = 29) after birth. For diabetic mothers these times were significantly later. The average 24-h milk intake by infants increased from 82 to 556 ml/24 h between 24 and 144 h after birth. Milk intakes were correlated with the concentration of lactose (r = 0.52, n = 51, p less than 0.001), citrate (r = 0.47, n = 47, p less than 0.001), and glucose (r = 0.69, n = 50, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Studies in renal preservation using a rat kidney transplant model: II. The effect of reflushing with citrate.

This study investigated the possible beneficial effects of reflushing renal grafts with isotonic citrate solution. Rat kidneys were initially flushed with isotonic citrate or with Hartmann's solutions at O C. After 2 hr, half the kidneys of each group were reflushed with isotonic citrate; 22 hr later, all kidneys were transplanted into rats of the same inbred strain. All animals receiving kidneys flushed with Hartmann's solution died, whereas reflushing such kidneys with isotonic citrate significantly ameliorated the deleterious effects of Hartmann's solution. All animals receiving citrate-flushed kidneys survived with relatively good renal function and morphology. However, reflushing itself is not a beneficial procedure and is only of value where an ineffective preserving solution has been used to flush the kidneys initially. There is evidence that some of the adverse effects of flushing develop in the renal medulla.

Animals↗

Meta-analysis of calcium bioavailability: a comparison of calcium citrate with calcium carbonate.

OBJECTIVE: To perform a meta-analysis of data from available published trials comparing the bioavailability of calcium carbonate with that of calcium citrate. DATA SOURCES: The whole set was comprised of 15 studies involving 184 subjects who underwent measurement of calcium absorption from calcium carbonate and calcium citrate. Category A excluded four studies for lack of physiological relevance, use of a mixed preparation with low content of calcium carbonate, or wide variability in results. Category B was comprised of five studies (from Category A) involving 71 subjects who took calcium supplements on an empty stomach. Category C was comprised of six studies (from Category A) involving 65 subjects who took calcium preparations with meals. METHOD: The meta-analysis of calcium absorption data from calcium carbonate and calcium citrate, with calculation of effect size and 95% confidence intervals. RESULTS: Calcium absorption from calcium citrate was consistently significantly higher than that from calcium carbonate by 20.0% in the whole set, by 24.0% in Category A, by 27.2% on an empty stomach, and by 21.6% with meals. CONCLUSION: Calcium citrate is better absorbed than calcium carbonate by approximately 22% to 27%, either on an empty stomach or co-administered with meals.

Biological Availability↗

Citrate synthase and 2-methylcitrate synthase: structural, functional and evolutionary relationships.

Following the complete sequencing of the Escherichia coli genome, it has been shown that the proposed second citrate synthase of this organism, recently described by the authors, is in fact a 2-methylcitrate synthase that possesses citrate synthase activity as a minor component. Whereas the hexameric citrate synthase is constitutively produced, the 2-methylcitrate synthase is induced during growth on propionate, and the catabolism of propionate to succinate and pyruvate via 2-methylcitrate is proposed. The citrate synthases of the psychrotolerant eubacterium DS2-3R, and of the thermophilic archaea Thermoplasma acidophilum and Pyrococcus furiosus, are approximately 40% identical in sequence to the Escherichia coli 2-methylcitrate synthase and also possess 2-methylcitrate synthase activity. The data are discussed with respect to the structure, function and evolution of citrate synthase and 2-methylcitrate synthase.

Bacterial Proteins↗

Early copper-induced leakage of K(+) from Arabidopsis seedlings is mediated by ion channels and coupled to citrate efflux.

Copper tolerance among Arabidopsis ecotypes is inversely correlated with long-term K(+) leakage and positively correlated with short-term K(+) leakage (A. Murphy, L. Taiz [1997] New Phytol 136: 211-222). To probe the mechanism of the early phase of K(+) efflux, we tested various channel blockers on copper and peroxide-induced K(+) efflux from seedling roots. The K(+) channel blockers tetraethyl ammonium chloride and 4-aminopyridine (4-AP) both inhibited short-term copper-induced K(+) efflux. In contrast, peroxide-induced K(+) efflux was insensitive to both tetraethyl ammonium chloride and 4-AP. Copper-induced lipid peroxidation exhibited a lag time of 4 h, while peroxide-induced lipid peroxidation began immediately. These results suggest that short-term copper-induced K(+) efflux is mediated by channels, while peroxide-induced K(+) efflux represents leakage through nonspecific lesions in the lipid bilayer. Tracer studies with (86)Rb(+) confirmed that copper promotes K(+) efflux rather than inhibiting K(+) uptake. Short-term K(+) release is electroneutral, since electrophysiological measurements indicated that copper does not cause membrane depolarization. Short-term K(+) efflux was accompanied by citrate release, and copper increased total citrate levels. Since citrate efflux was blocked by 4-AP, K(+) appears to serve as a counterion during copper-induced citrate efflux. As copper but not aluminum selectively induces citrate production and release, it is proposed that copper may inhibit a cytosolic form of aconitase.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

Ranitidine bismuth citrate and aspirin-induced gastric mucosal injury.

The aim of this study was to investigate the protective action of a new compound, ranitidine bismuth citrate, in the prevention of aspirin-induced acute mucosal injury to the upper gastrointestinal tract of healthy human volunteers. In a double-blind randomized three-way cross-over study 24 male volunteers received placebo, 900 mg aspirin or 900 mg aspirin and 800 mg ranitidine bismuth citrate at 12-h intervals for nine doses with a 2-week wash-out period between each treatment. The median (interquartile range) number of erosions seen at endoscopy when ranitidine bismuth citrate was given with aspirin (1 [0-4]) was significantly lower than aspirin alone (24 [16-32]) (P < 0.001) and not significantly different from either baseline or placebo (0 [0-2]). These findings were similarly reflected in the effects on microbleeding following the ninth dose: 12.1 (7.1-21.0) microL/10 min following aspirin alone compared to levels with placebo of 1.2 (0.4-2.9), and with aspirin and ranitidine bismuth citrate of 1.6 (0.8-2.6) (P < 0.005). Ranitidine bismuth citrate conferred substantial protection from aspirin-induced injury to the gastric and duodenal mucosa as determined by both endoscopic assessment and microbleeding rates, reducing injury to placebo levels.

Adult↗

Ranitidine bismuth citrate and ranitidine do not affect gastric emptying of a radio-labelled liquid meal.

Ranitidine bismuth citrate, a new chemical entity which is a salt complex of ranitidine and bismuth citrate, is being developed for the treatment of relapse of benign gastric and duodenal ulcer and eradication of Helicobacter pylori. The aim of the present study was to establish whether ranitidine bismuth citrate (800 mg) or ranitidine hydrochloride (300 mg) have any effect on gastric emptying of a liquid meal using gamma scintigraphy. On three separate occasions, each of twelve subjects received a single oral tablet of 800 mg ranitidine bismuth citrate, 300 mg ranitidine hydrochloride or placebo in random order. Thirty minutes after dosing each subject was given 375 ml of 99mTc-DTPA (diethylene triaminepentaacetic acid) labelled Clinifeed-ISO. The primary endpoint was the time to 50% gastric emptying (t50%). The proportion of the meal remaining was summarised by weighted mean proportion of the meal remaining in the stomach over 0-60 min and 0-180 min, separately. No differences were observed for t50%, weighted mean 0-60 min, and weighted mean 0-180 min between any two treatments. In man, we have detected no significant effect of single oral doses of ranitidine bismuth citrate 800 mg or ranitidine hydrochloride 300 mg on the rate of gastric emptying of a liquid meal when compared with placebo.

Adult↗

Utilization of a new strength citrate anticoagulant during centrifugal plateletpheresis. I. Assessment of donor effects.

A reduction of donor effects during centrifugal plateletpheresis with the Haemonetics Blood Processor was achieved by reducing the concentration of the citrate anticoagulant. Serum citrate and ionized calcium levels, immediately and 1 h post-pheresis, were affected to a lesser extent by using 5.0 g total ionized citrate (TIC) THAN WITH EITHER 8.0 G OR 11.0 G. Total calcium, bicarbonate, prothrombin time, partial thromboplastin time, ECG, and platelet counts were affected to a similar degree by all three TIC formulations. The total number of platelets collected per litre of blood processed was not significantly different among the three TIC formulations. In vitro studies employing the screen filtration pressure (SFP) technique showed no evidence of platelet aggregates in whole blood collected into either 0.01 M or 0.005 M citrate and agitated or left stationary at room temperature for 5 h. The use of different citrate concentrations in plateletpheresis is discussed.

Anticoagulants↗

Studies on a mutant form of Escherichia coli citrate synthase desensitised to allosteric effectors.

Naturally occurring citrate synthases fall into distinct molecular and catalytic types. Gram-negative bacteria produce a 'large' enzyme, allosterically inhibited by NADH and, in the facultative anaerobes such as Escherichia coli, also by 2-oxoglutarate. On the other hand, Gram-positive bacteria and all eukaryotes produce a 'small' citrate synthase which is insensitive to these metabolites. As a complement to structure-function studies we have explored the possibility of genetically altering one type of citrate synthase to the other. By mutagenesis and suitable selection we have succeeded in isolating a mutant of E. coli whose citrate synthase is both 'small' and insensitive to NADH and 2-oxoglutarate. Some characteristics of the enzyme are described. Such mutant enzymes offer a novel approach to the study of citrate synthase, its regulation and its natural diversity.

Allosteric Regulation↗

Citrate synthesis in intact rat-liver mitochondria is irreversible.

Rat-liver mitochondria were incubated with [1,5-14C]citrate in the presence of fluorocitrate to block its oxidation in the Krebs cycle. The reaction products were analysed enzymatically and by anion-exchange chromatography. Incorporation of 14C into acetyl-L-carnitine or ketone bodies via a backward action of citrate synthase was not observed. The optimal rate of citrate synthesis from pyruvate and malate in the presence of fluorocitrate was 15 nmol . mg-1 min-1. In the absence of fluorocitrate, but in the presence of malonate, citrate was oxidized to succinate at a rate of 4 nmol . mg-1 . min-1. We conclude that the synthesis of citrate by intact rat liver mitochondria is an irreversible process. The possible mechanism underlying this phenomenon and the consequence for metabolic regulation are discussed.

Acetyl Coenzyme A↗