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Citrate content of liver and kidney of rat in various metabolic states and in fluoroacetate poisoning.

The citrate content of rat liver changes little when normal rats are starved, when starved rats are re-fed with various diets and when normal animals are made diabetic with alloxan. The citrate content of rat kidney changes little on starvation, but it doubles on induction of diabetes. Fluoroacetate poisoning has relatively little effect on the citrate content of liver under a variety of conditions except that normal female rats show a 2.4-fold increase. Fluoroacetate poisoning leads to increases in the citrate content of kidney under all conditions. The relevance of these observations to the regulation of fatty acid synthesis is discussed. The acetic anhydride-pyridine method and the pentabromoacetone method for the estimation of citrate are compared.

Animals↗

The kinetic properties of citrate synthase from rat liver mitochondria.

1. Citrate synthase (EC 4.1.3.7) was purified 750-fold from rat liver. 2. Measurements of the Michaelis constants for the substrates of citrate synthase gave values of 16mum for acetyl-CoA and 2mum for oxaloacetate. Each value is independent of the concentration of the other substrate. 3. The inhibition of citrate synthase by ATP, ADP and AMP is competitive with respect to acetyl-CoA. With respect to oxaloacetate the inhibition by AMP is competitive, but the inhibition by ADP and ATP is mixed, being partially competitive. 4. At low concentrations of both substrates the inhibition by ATP is sigmoidal and a Hill plot exhibits a slope of 2.5. 5. The pH optimum of the enzyme is 8.7, and is not significantly affected by ATP. 6. Mg(2+) inhibits citrate synthase slightly, but relieves the inhibition caused by ATP in a complex manner. 7. At constant total adenine nucleotide concentration made up of various proportions of ATP, ADP and AMP, the activity of citrate synthase is governed by the concentration of the sum of the energy-rich phosphate bonds of ADP and ATP. 8. The sedimentation coefficient of the enzyme, as measured by activity sedimentation, is 6.3s, equivalent to molecular weight 95000.

Adenine Nucleotides↗

Lipid metabolism by rat lung in vitro. Utilization of citrate by normal and starved rats.

1. The utilization of [1,5-(14)C(2)]citrate by lung slices and cell cytosol preparations, and the activities of liver and lung cytosol citrate-cleavage enzyme (EC 4.1.3.8), l-malate-NAD oxidoreductase (malate dehydrogenase, EC 1.1.1.37) and phosphoenolpyruvate carboxylase (EC 4.1.1.32) were examined in normal and starved rats. 2. Lipogenesis from citrate was decreased by approx. 70% in both the phospholipid and neutral lipid fractions of lung slices from starved rats as compared with fed controls. 3. Incorporation of citrate by lung cytosol preparations into fatty acids was decreased by approx. 35% in the starved rats. The apparent inhibition by avidin of fatty acid synthesis was overcome partially by preincubation of lung cytosol preparations with biotin. These results are consistent with the presence in lung tissue of the malonyl-CoA pathway for fatty acid synthesis. 4. Lung citrate-cleavage enzyme activity decreased in rats that had been starved for 72h whereas malate dehydrogenase and phosphoenolpyruvate carboxylase activities remained unchanged. The results suggest that the pattern of utilization of lipid precursors by rat lung may be altered during various nutritional states.

Animals↗

Activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates.

1. The activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were measured in muscles from a large number of animals, in order to provide some indication of the importance of the citric acid cycle in these muscles. According to the differences in enzyme activities, the muscles can be divided into three classes. First, in a number of both vertebrate and invertebrate muscles, the activities of all three enzymes are very low. It is suggested that either the muscles use energy at a very low rate or they rely largely on anaerobic glycolysis for higher rates of energy formation. Second, most insect flight muscles contain high activities of citrate synthase and NAD+-linked isocitrate dehydrogenase, but the activities of the NADP+-linked enzyme are very low. The high activities indicate the dependence of insect flight on energy generated via the citric acid cycle. The flight muscles of the beetles investigated contain high activities of both isocitrate dehydrogenases. Third, other muscles of both vertebrates and invertebrates contain high activities of citrate synthase and NADP+-liniked isocitrate dehydrogenase. Many, if not all, of these muscles are capable of sustained periods of mechanical activity (e.g. heart muscle, pectoral muscles of some birds). Consequently, to support this activity fuel must be supplied continually to the muscle via the circulatory system which, in most animals, also transports oxygen so that energy can be generated by complete oxidation of the fuel. It is suggested that the low activities of NAD+-linked isocitrate dehydrogenase in these muscles may be involved in oxidation of isocitrate in the cycle when the muscles are at rest. 2. A comparison of the maximal activities of the enzymes with the maximal flux through the cycle suggests that, in insect flight muscle, NAD+-linked isocitrate dehydrogenase catalyses a non-equilibrium reaction and citrate synthease catalyses a near-equilibrium reaction. In other muscles, the enzyme-activity data suggest that both citrate synthase and the isocitrate dehydrogenase reactions are near-equilibrium.

Animals↗

Models of proteolysis of oligomeric enzymes and their applications to the trypsinolysis of citrate synthases.

A simple statistical approach was used to generate predictive models of the proteolysis of multisubunit enzymes in order to correlate the loss of enzyme activity with the loss of native subunit. The models were applied to the trypsinolysis of the citrate synthases of pig heart, Bacillus megaterium and Escherichia coli. With the dimeric citrate synthases (pig heart and B. megaterium) trypsinolysis of one of the subunits appears to destroy the activity of the whole enzymic molecule. The hexameric E. coli citrate synthase behaves like a trimer of dimeric units, each of the dimers behaving similarly to the B. megaterium and pig heart enzymes. Palmitoyl-CoA is required for the trypsinolysis of pig heart citrate synthase, and at relatively high concentrations of this compound trypsinolysis of one subunit leaves the other subunit fully active. Palmitoyl-CoA is not required for the trypsinolysis of the other citrate synthases, and high concentrations of this metabolite do not affect the correlation of proteolysis with inactivation of these enzymes.

Animals↗

The CIT3 gene of Saccharomyces cerevisiae encodes a second mitochondrial isoform of citrate synthase.

We have identified a third citrate synthase gene in Saccharomyces cerevisiae which we have called CIT3. Complementation of a citrate synthase-deficient strain of Escherichia coli by lacZ::CIT3 gene fusions demonstrated that the CIT3 gene encodes an active citrate synthase. The CIT3 gene seems to be regulated in the same way as CIT1, which encodes the mitochondrial isoform of citrate synthase. Deletion of the CIT3 gene in a delta cit1 background severely reduced growth on the respiratory substrate glycerol, whilst multiple copies of the CIT3 gene in a delta cit1 background significantly improved growth on acetate. In vitro import experiments showed that cit3p is transported into the mitochondria. Taken together, these data show that the CIT3 gene encodes a second mitochondrial isoform of citrate synthase.

Amino Acid Sequence↗

Lactoferrin and citrate concentrations at drying-off and during early mammary involution of dairy cows.

Quarter milk samples were taken from 48 clinically healthy, pregnant Finnish Ayrshire and Friesian dairy cows at the last milking before drying-off, and 2 and 6 days later to determine the lactoferrin and citrate concentrations in the udder secretion. The mean lactoferrin concentration in the milk increased from 5.29 mg ml(-1) on the last day of drying-off process to 8.09 and 11.26 mg ml(-1), 2 and 6 days later, respectively. Citrate concentration decreased from 1.85 mg ml(-1) to 1.54 and 1.09 mg ml(-1), respectively. Median molar ratio (citrate to native-Lf) decreased from 153 to 86 and further to 44. Lf and citrate concentrations in milk varied greatly among cows. Sampling time had a statistically significant effect on lactoferrin and citrate concentrations in milk while the breed and the parity of the cows had no effect on either of these variables.

Animals↗

Inhibition of an anti-Pr1d cold agglutinin by citrate present in commercial red cell preservative solutions.

A patient with known cold autoimmune hemolyticanemia was admitted for surgery. Routine cold agglutinin evaluations, using commercial red cells (RBCs) in modified Alsever's preservative solution, revealed a cold agglutinin titer of 4 to 16. However, using RBCs washed four times with saline, a high-titer (greater than 2000 at 4 degrees C) cold autoagglutinin was demonstrated. The cold agglutinin was shown to be an IgM kappa paraprotein with anti-Pr1d specificity. The addition of Alsever's solution to washed RBCs inhibited the cold agglutinin. Each major component of Alsever's solution (neomycin, chloramphenicol, inosine, dextrose, and citrate) was tested individually; only citrate inhibited the patient's cold agglutinin. Various compounds structurally related to citrate were tested and found to cause various degrees of inhibition. The strongest inhibition correlated with the presence of either three carboxyl groups on molecules devoid of double-bonded carbon atoms or two carboxyl groups in cis configuration. A panel of 54 cold agglutinins, including 7 with anti-Pr specificity, was analyzed. None was significantly inhibited by Alsever's solution, although one with anti-Pr2 specificity was weakly inhibited. In summary, these studies describe an anti-Pr1d cold autoagglutinin that was inhibited by citrate in RBC preservative solutions. The failure to detect such a cold agglutinin can result from not washing RBCs free of citrate before testing.

Aged↗

Storage of platelets in additive solutions: the effect of citrate and acetate in in vitro studies.

The in vitro effects of storage of platelets prepared from 6 pooled buffy coat units and stored in a platelet storage medium consisting of CPD and plasma and different platelet additive solutions were evaluated. The total count of platelets per pooled unit included in the present investigation (n = 20) was 335 +/- 35 x 10(9) (mean +/- SD). Measurements of pH, pO2, pCO2, glucose, lactate, ATP, total adenine nucleotide content, and extracellular adenylate kinase activity were performed in a three-part study. The observations were 1) During storage in saline and citrate (10 mmol/L of citrate), the consumption of glucose and the production of lactate were significantly increased over the values with storage in saline, which were used as a reference. The values for pH at Day 6 were significantly lower. 2) The effects of different concentrations (10, 20, and 30 mmol/L) of acetate in saline were studied. With the exception of significantly higher pH values in saline and acetate, no significant differences were seen in the effects with saline and those with saline and acetate. 3) The combined effect of citrate and acetate was evaluated. The consumption of glucose and the production of lactate, the values for pO2, and extracellular adenylate kinase activity were significantly lower with saline and citrate and acetate than with saline and citrate. Significantly higher values for pH were found at Day 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗

Unexpected citrate toxicity and severe hypocalcemia during apheresis.

BACKGROUND: The citrate anticoagulant used during apheresis procedures is considered a safe medication because it is rapidly metabolized by the donor. However, acute, life-threatening hypocalcemia is possible if the infusion rate of citrate is increased. CASE REPORT: A 54-year-old woman with metastatic breast cancer, but otherwise in good health, had just begun a fifth collection of hematopoietic peripheral blood progenitor cells by leukapheresis. The instrument's self-loading apheresis kit was primed uneventfully. Seven minutes into the procedure, the patient developed signs and symptoms suggesting severe hypocalcemia, including muscle spasms, chest pain, and hypotension. The citrate bag was discovered to have emptied, and a section of the anticoagulant tubing was protruding outside of the rotary pump. The patient's ionized calcium level was 0.64 mmol per L (normal range, 1.18-1.38 mmol/L). In subsequent experiments where the anticoagulant tubing was either improperly loaded at the outset or partially pulled out of the rotary pump, no instrument alarms sounded. CONCLUSION: Citrate toxicity and life-threatening hypocalcemia can occur if the anticoagulant line of an apheresis instrument is not properly housed in its rotary pump or becomes disengaged during the procedure. Instrument manufacturers are encouraged to consider designs that allow the direct measurement of the volume of citrate delivered. In the interim, periodic visual and tactile confirmation of tubing placement during apheresis procedures is prudent.

Blood Component Removal↗

Regional citrate anticoagulation for hemodialysis using a conventional calcium-containing dialysate.

Regional citrate anticoagulation is currently a frequently applied technique for hemodialysis patients at increased risk of bleeding. Most experience exists with isotonic citrate in combination with a calcium-free dialysate and separate substitution with calcium chloride. This method is effective, but rather cumbersome and laborious. In search for a less demanding, but equally safe and effective technique, we performed 203 double-needle hemodialysis sessions in 45 patients at high risk of bleeding using regional anticoagulation with hypertonic trisodium citrate (TSC) and a conventional calcium-containing dialysate. At the start of dialysis, citrate was infused at a rate of 75 mL/h; adjustments were made during dialysis according to the degree of anticoagulation and level of ionized calcium within the systemic circuit. The efficacy and short-term safety of regional anticoagulation with TSC as compared with heparin anticoagulation was ascertained in a cohort of 19 stable hemodialysis patients. Systemic anticoagulation did not occur, and plasma-ionized calcium remained on a stable level. Manifestations of citrate toxicity or hypocalcemia were not observed. Clotting within the dialyzer was noted in 18 of the 203 sessions (8.87%) and resulted in early termination of dialysis in only 3 cases (1.48%). In conclusion, the use of hypertonic TSC and a conventional calcium-containing dialysate was shown to be safe and effective. The risk of clotting of the extracorporeal circuit is limited and outweighed by the advantage of reduced procedural complexity. Compared with the use of a calcium-free dialysate, the number of analyses can be reduced substantially, making this method financially attractive.

Aged↗

The role of citrate in the regulation of hepatic fatty acid synthesis by insulin and glucagon.

Incubation of intact rat hepatocytes with insulin and glucagon resulted in increased and decreased rates of de novo fatty acid biosynthesis, respectively. These changes were paralleled by corresponding alterations in the activity of acetyl-CoA carboxylase, an important regulatory enzyme of this pathway. The hormonal conditions imposed on the hepatocytes did not change the cellular or the cytosolic level of citrate. Incubation of hepatocytes with octanoate showed a parallel increase in the rate of fatty acid synthesis and acetyl-CoA carboxylase with a concomitant elevation of the cellular citrate level. The increase in whole-cell citrate was mainly due to a marked increase in the level of cytosolic citrate. Collectively, our data indicate that insulin and glucagon-determined changes in acetyl-CoA carboxylase are not mediated by changes in cytosolic citrate levels.

Acetyl-CoA Carboxylase↗

Inhibition of citrate synthase by oleoyl-CoA: a regulatory phenomenon.

Fatty acyl-CoAs are good detergents (dritical micelle concentrations = 3-4 muM) and can inhibit a number of enzymes, including some involved in fatty acid biosynthesis. The regulatory significance of fatty acyl-CoAs as negative effectors has been questioned largely because of the difficulties in distinguishing possible nonspecific detergent effects from more specific regulatory interactions with these enzymes. A new analogue of oleoyl-CoA, oleoyl-(1, N6-etheno)-CoA, is a better detergent (critical micelle concentration = 3.2 muM) than oleoyl-CoA (critical micelle concentration = 4.7 muM). This new analogue is not as good (by an order of magnitude) an inhibitor of citrate synthase [citrate oxaloacetatelyase (pro-3S-CH2-COO-vectoracetyl-CoA); EC 4.1.3.7] nor is it bound as well oleoyl-CoA. Since the only difference between these two compounds is substitution of 1,N6-ethenoadenine for the adenine of CoA, the difference in inhibition and binding implies a specific interaction between the adenine moiety of oleoyl-CoA and citrate synthase. Moreover, since oleoyl-(1,N6-etheno)CoA is a better detergent than oleoyl-CoA, the detergency of oleoyl-CoA is not the sole cause of the fatty acyl-CoA inhibition of citrate synthase. These results support a physiological role for oleoyl-CoA as a negative effector for citrate synthase. An analogous physiological role for fatty acyl-CoA as negative effectors for other enzymes seems reasonable.

Animals↗

The identification of ATP-citrate lyase as a protein kinase B (Akt) substrate in primary adipocytes.

Protein kinase B (Akt) plays a central role in cellular regulation, although many of the physiologically relevant substrates for the kinase remain to be identified. In this study, we have isolated a protein from primary epididymal adipocytes with an apparent molecular weight of 125,000. This protein exhibited immunoreactivity, in an insulin-dependent manner, with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) as well as a phosphospecific antibody that recognizes serine 21/9 of GSK-3alpha/beta. MALDI-TOF mass spectrometry revealed the protein to be ATP-citrate lyase, suggesting that the two phosphospecific antibodies recognize phosphoserine 454, a previously reported insulin- and isoproterenol-stimulated ATP-citrate lyase phosphorylation site. Indeed, both insulin and isoproterenol stimulated the phosphorylation of this protein on the site recognized by the phosphospecific antibodies in a wortmannin-sensitive and -insensitive manner, respectively. In addition, transient expression of a constitutively active protein kinase B in primary adipocytes mimicked the effect of insulin on ATP-citrate lyase phosphorylation. Furthermore, ATP-citrate lyase was phosphorylated in vitro by recombinant protein kinase B on the same site. Taken together, these results demonstrate that serine 454 of ATP-citrate lyase is a novel and major in vivo substrate for protein kinase B.

ATP Citrate (pro-S)-Lyase↗

The physiological and ventilatory responses to repeated 60 s sprints following sodium citrate ingestion.

This study examined the influence of sodium citrate on changes in selected blood, ventilatory and performance variables in response to intermittent sprint exercise. Eight moderately active male students completed three tests over a 6 day experimental period. The first test involved incremental exercise to determine VO2 max, while the second and third tests were identical in nature and involved five 60 s sprints cycling against 0.075 kg kg-1 body mass (BM); each of the five sprints was separated by 5 min passive seated recovery. Three days separated the VO2 max test and first interval test, while a further 3 days elapsed between the first and second interval tests. Ninety minutes prior to each interval test, the subjects consumed either a solution of sodium citrate (0.5 g kg-1 BM) or a placebo solution (1 g of calcium carbonate and 4 mg of sodium chloride). These were randomly administered in a double-blind crossover procedure so that every subject consumed each solution prior to the interval test over the 6 day period. Measures of work, VE, VO2, VCO2, post-exercise plasma lactate, and changes in both venous blood pH and venous blood bicarbonate (HCO3-) were measured during each interval test. Although analysis of variance failed to identify differences in performance between the two solutions, both exercise VCO2 and changes in venous blood HCO3- were higher in the citrate condition (P < 0.05). In addition, both peak post-exercise plasma lactate concentrations and post-exercise venous blood pH were significantly higher following citrate ingestion. Although these data are consistent with greater clearance of lactate and H+ from the active muscle cells following citrate ingestion, performance between the two trials was the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

Superior calcium absorption from calcium citrate than calcium carbonate using external forearm counting.

Calcium absorption from calcium citrate was compared with that obtained from calcium carbonate in 20 normal women. It was measured by external forearm counting technique, where the ratio of forearm radioactivity after an oral dose of labeled calcium salt (containing 500 mg calcium) and after an intravenous injection of trace radiocalcium represented fractional calcium absorption. The fractional calcium absorption from calcium citrate was 39.2 +/- 8.6%, which was significantly higher than the 31.2 +/- 9.4% derived from calcium carbonate (p less than 0.001). Seventeen subjects displayed greater calcium absorption from calcium citrate. The remaining three patients, with a lower calcium absorption from the citrate salt, had high levels of calcium absorption from calcium carbonate. It is concluded that calcium is better absorbed from calcium citrate than calcium carbonate when these salts are taken on an empty stomach in most women. The exception might be those with optimum calcium absorption from calcium carbonate.

Absorption↗

Effect of added citrate or malate on calcium absorption from calcium-fortified orange juice.

OBJECTIVE: Calcium absorption was determined from calcium-fortified diluted orange juice, which contained additional citrate or malate, in 16 normal subjects. METHODS: Each load of fortified orange juice with additional citrate (OJ+C) contained 300 mg Ca, 5.7 mEq malate, and 33.6 mEq citrate (10.4 mEq of which were added). Each load of orange juice with additional malate (OJ+M) had 300 mg Ca, 23.2 mEq citrate and 16.1 mEq malate (10.4 mEq of which were added). For each subject, fractional (intestinal) calcium absorption was measured by taking the ratio of fractional forearm radioactivity following an oral administration of OJ+C or OJ+M (labeled with 47Ca) and the fractional forearm radioactivity obtained after intravenous administration of trace 47Ca chloride on a separate occasion. RESULTS: There was no significant difference in fractional calcium absorption from the two calcium-fortified orange juice preparations (40.1 +/- 8.3% for OJ+C and 40.6 +/- 8.6% for OJ+M, p = 0.81). CONCLUSION: Calcium-fortified orange juice with additional citrate provides equivalent bioavailable calcium as the juice with additional malate.

Administration, Oral↗

Large-volume leukapheresis using regional citrate anticoagulation to collect peripheral blood progenitor cells.

The use of peripheral blood progenitor cells (PBPC) for hematopoietic rescue after high-dose chemotherapy is limited by the number of leukaphereses required to collect an adequate number of hematopoietic progenitors. To optimize the collection of PBPC, we evaluated a single large-volume leukapheresis protocol with citrate anticoagulation. A group of 23 patients received cyclophosphamide (4 g/m2) and GM-CSF (5 micrograms/kg/day for 15 days) as PBPC mobilization, with a single outpatient 6 h leukapheresis performed on the COBE Spectra 15 days later. Citrate (0.190 mmol/ml) was infused at 1.2 ml/L of blood/minute with a whole blood to citrate ratio between 17:1 and 25:1. Calcium chloride (50 mM) was administered at a citrate to calcium molar ratio between 10:1 and 5:1 to prevent hypocalcemia. A median 36.6 L (range 24.4-46.4) blood was processed using 338 mM citrate (269-473) and 50 mM calcium (25-75). A median 5 x 10(6) CD34+ cells/kg (< 0.3-24) and 6.2 x 10(5) CFU-GM/kg (< 0.001-29) were collected, representing 5.6 and 5.9 more PBPC, respectively, than were in circulation at the initiation of leukapheresis. We conclude that a 6 h large-volume leukapheresis following cyclophosphamide and GM-CSF mobilization is safe, can recruit hematopoietic progenitors into the circulatory compartment, and allows the collection of high numbers of PBPC in a single procedure.

Adolescent↗