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PubMed2003

A novel pyrroline-5-carboxylic acid and acetoacetic acid adduct in hyperprolinaemia type II.

BACKGROUND: From investigations of a child with hyperprolinaemia type II, we demonstrated in vitro that pyridoxal phosphate forms a novel adduct with a proline metabolite, pyrroline-5-carboxylic acid, through Claisen condensation. Studies indicated that this was a previously unsuspected generic reaction of aldehydes and some ketones. We have subsequently found the acetoacetic acid adduct in both plasma and urine from the affected child. METHODS: Mixtures of acetoacetic acid and pyrroline-5-carboxylic acid were co-incubated at pH 7.4 and 37 degrees C, dried, or extracted and dried, derivatised and analysed by gas chromatography/mass spectrometry (GC/MS). Urine and plasma from the child were analysed. RESULTS: Fourteen new peaks were found in derivatised pyrroline-5-carboxylic acid/acetoacetic acid co-incubates. From accurate molecular mass data, the four largest peaks were probably diastereoisomers of tri-trimethylsilyl (tri-TMS) derivatives of alcohol adducts formed by Claisen condensation. Eight other peaks were mono- and di-trimethylsilyl derivatives of the adduct and a decarboxylated product. The adduct was demonstrated unequivocally in the child's acute urine and traces in plasma. CONCLUSIONS: Pyrroline-5-carboxylic acid forms an adduct with acetoacetic acid, which was present in urine of a sick child with hyperprolinaemia type II. Evidence suggests it formed in vivo. The biological significance of this novel reaction of aldehydes and ketones merits investigation.

Acetoacetates↗
PubMedDate not supplied

Acetoacetate utilization by human placental mitochondria.

It has been shown that mitochondria from human placenta incubated in the presence of 2-oxoglutarate or its precursors utilize acetoacetate at the rate about I nmol/min/mg protein. Utilization of acetoacetate is completely inhibited by arsenite. Mitochondria from human placental tissue show activity of 3-oxoacid-CoA transferase and acetoacetyl-CoA thiolase. It is proposed that acetoacetate utilization by placental mitochondria proceeds via the conversion to acetoacetyl-CoA catalysed by 3-oxoacid-CoA transferase, and then to acetyl-CoA, catalysed by acetoacetyl-CoA thiolase.

Acetoacetates↗
PubMed2003

Acetoacetate protects hippocampal neurons against glutamate-mediated neuronal damage during glycolysis inhibition.

Glucose is the main substrate that fulfills energy brain demands. However, in some circumstances, such as diabetes, starvation, during the suckling period and the ketogenic diet, brain uses the ketone bodies, acetoacetate and beta-hydroxybutyrate, as energy sources. Ketone body utilization in brain depends directly on its blood concentration, which is normally very low, but increases substantially during the conditions mentioned above. Glutamate neurotoxicity has been implicated in neurodegeneration associated with brain ischemia, hypoglycemia and cerebral trauma, conditions related to energy failure, and to elevation of glutamate extracellular levels in brain. In recent years substantial evidence favoring a close relation between glutamate neurotoxic potentiality and cellular energy levels, has been compiled. We have previously demonstrated that accumulation of extracellular glutamate after inhibition of its transporters, induces neuronal death in vivo during energy impairment induced by glycolysis inhibition. In the present study we have assessed the protective potentiality of the ketone body, acetoacetate, against glutamate-mediated neuronal damage in the hippocampus of rats chronically treated with the glycolysis inhibitor, iodoacetate, and in hippocampal cultured neurons exposed to a toxic concentration of iodoacetate. Results show that acetoacetate efficiently protects against glutamate neurotoxicity both in vivo and in vitro probably by a mechanism involving its role as an energy substrate.

Acetoacetates↗
PubMed1974

Formation of acetoacetate from 3-hydroxy-3-methylglutarate by rat liver and isolation of a mitochondrial coenzyme A-transferase activity involved.

1. Formation of acetoacetate from 3-hydroxy-3-methylglutarate was observed in the perfused rat liver. Production of 3.5mumol of acetoacetate/h per g of tissue was obtained. 2. Formation of acetoacetate was catalysed mainly by the mitochondrial fraction of the homogenized liver, at a rate of 62nmol/h per mg of protein. 3. Experiments with hydroxy-[3-(14)C]methylglutarate demonstrated that the acetoacetate formed was derived mainly from this compound. 4. A mitochondrial transferase activity catalysing the transfer of a CoA molecule from succinyl-CoA (3-carboxypropionyl-CoA) to hydroxymethylglutarate was shown. The K(m) value for hydroxymethylglutarate was 5x10(-3)m.

Acetoacetates↗
PubMed1982

Oxaloacetate- and acetoacetate-induced calcium efflux from mitochondria occurs by reversal of the uptake pathway.

1. Addition of oxaloacetate or acetoacetate to isolated rat liver mitochondria results in an efflux of Ca2+. Concomitant with this efflux is an immediate oxidation of endogenous nicotinamide nucleotides, a fall in the mitochondrial membrane potential and an increase in the rate of respiration. The primary effect in this sequence may be either (a) physiologically important stimulation of a Ca2+-efflux carrier, followed by Ca2+ re-uptake, a fall in membrane potential and increased respiration, or (b) physiologically unimportant damage to mitochondrial integrity, followed by a fall in membrane potential, increased respiration and Ca2+ efflux. 2. Ruthenium Red and EGTA will restore the increased respiratory rate to one approximating to the control rate of respiration. However, addition of lanthanide, at a concentration which inhibits the uptake but not the normal efflux of Ca2+, inhibits the rate of Ca2+ efflux induced by oxaloacetate or acetoacetate. Therefore the observed efflux is occurring by a reversal of the uptake pathway (uniporter) and thus follows the fall in membrane potential. 3. From these results we conclude that the decrease in membrane potential and increase in the rate of respiration seen during oxaloacetate- or acetoacetate-induced Ca2+ efflux cannot be accounted for by rapid Ca2+ cycling, but are due to damage to mitochondrial integrity.

Acetoacetates↗
PubMed1983

On the relationship between calcium and phosphate transport, transmembrane potential and acetoacetate-induced oxidation of pyridine nucleotides in rat-liver mitochondria.

Acetoacetate addition to rat liver mitochondria induces a complete oxidation of pyridine nucleotides, a collapse of membrane potential, a release of mitochondrial Ca2+ and a loss of respiratory control only in the presence of external phosphate. Acetoacetate also enhances the efflux of mitochondrial Mg2+ promoted by phosphate. All these effects are not only prevented but also reversed, except the oxidation of pyridine nucleotides, by the combined addition of Mg2+, ADP and dithioerythritol to damaged mitochondria. It is concluded that acetoacetate, through the oxidation of mitochondrial pyridine nucleotides, potentiates the action of phosphate in altering the mitochondrial permeability barrier, which is closely dependent on the maintenance of membrane thiol groups in a reduced form.

Acetoacetates↗
PubMed1995

14CO2 exchange with acetoacetate catalyzed by dialyzed cell-free extracts of the bacterial strain BunN grown with acetone and nitrate.

The nitrate-reducing bacterial strain BunN is able to grow with acetone and nitrate under anoxic conditions. Dialyzed crude cell-free extracts of acetone-plus-nitrate-grown cells of strain BunN catalyzed the exchange of 14CO2 into acetoacetate in an ADP-dependent reaction. The rates of exchange catalyzed by extracts of acetate-grown or 3-hydroxybutyrate-grown cells were only 13% of that catalyzed by extracts of acetone-grown cells. The activity was enzymic since it was destroyed by boiling and was proportional to the amount of added extract. The optimal acetoacetate concentration was 100 mM and the apparent Km was 11.1 mM. The pH optimum was 6.5, the exchange was not dependent on the addition of biotin, and the activity was not inhibited by avidin. The exchange activity was not stimulated (less than two fold) by a variety of metal ions or by a range of possible cofactors. Under optimal conditions (100 mM acetoacetate, 5 mM ADP, 10 mM NaHCO3, pH 6.5, under N2), the exchange activity was 2.7 nmol.min-1.mg protein-1; 2% of the in vivo carboxylation activity of acetone-plus-nitrate-grown cultures. It is suggested that the exchange reaction is a partial reaction catalyzed by the enzyme (or enzyme complex) that carboxylates acetone, and that the methods developed in this study provide a means with which to investigate this reaction further.

Acetoacetates↗
PubMed1995

Metabolism of (R,S)-1,3-butanediol acetoacetate esters, potential parenteral and enteral nutrients in conscious pigs.

The (R,S)-1,3-butanediol-acetoacetate monoesters and diester are nonionized sodium-free precursors of ketone bodies (beta-hydroxybutyrate and acetoacetate). They represent a convenient form of ketone body administration for parenteral and enteral nutrition. We have studied the metabolism of the esters in the conscious pig, an animal in which ketogenesis is congenitally impaired. Some pigs were infused for 3 h, intravenously or intragastrically, with the esters or with (R,S)-1,3-butanediol at 30% of the hourly caloric requirement. Other pigs were given intragastric boluses of esters or of (R,S)-1,3-butanediol at 15% of the daily caloric requirement. Our data show that continuous infusion of the esters at 30% of the caloric requirement leads to low concentrations of (R,S)-1,3-butanediol (0.1 mM) and total ketone bodies (0.5 mM). In pigs given intragastric boluses of esters at 15% of the daily caloric requirement, concentrations of (R,S)-1,3-butanediol and total ketone bodies peaked briefly at 2-3 and 5 mM, respectively. No deleterious side effects were observed in any group, including no hypoglycemia and no acidosis. Thus the (R,S)-1,3-butanediol acetoacetate esters appears to be well utilized as a nutrient by the pig despite its impaired ketogenesis.

3-Hydroxybutyric Acid↗
PubMed2004

Hypochlorous acid inhibition by acetoacetate: implications on neutrophil functions.

Type-1 diabetic patients experience hyperketonemia caused by an increase in fatty acid metabolism. Thus, the aim of this study was to measure the effect of ketone bodies as suppressors of oxidizing species produced by stimulated neutrophils. Both acetoacetate and 3-hydroxybutyrate have suppressive effect on the respiratory burst measured by luminol-enhanced chemiluminescence. Through measurements of hypochlorous acid production, using neutrophils or the myeloperoxidase/H2O2/Cl- system, it was found that acetoacetate but not 3-hydroxybutyrate is able to inhibit the generation of this antimicrobial oxidant. The superoxide anion scavenging properties were confirmed by ferricytochrome C reduction and lucigenin-enhanced chemiluminescence assays. However, ketone bodies did not alter the rate of oxygen uptake by stimulated neutrophils, measured with an oxygen electrode. A strong inhibition of the expression of the cytokine IL-8 by cultured neutrophils was also observed; this is discussed with reference to the antioxidant-like property of acetoacetate.

Acetoacetates↗
PubMed1987

Metabolic control of kidney hemodynamics in normal and insulin-dependent diabetic subjects. Effects of acetoacetic, lactic, and acetic acids.

Diabetes mellitus is associated with important changes in renal hemodynamics. The purpose of this study was to determine whether an increase in blood concentration patterns of ketone bodies and lactic acid, organic acids often elevated in poorly controlled insulin-dependent diabetes mellitus (IDDM), could contribute to increase glomerular filtration rate (GFR) and renal plasma flow (RPF) regardless of changes in circulating levels of glucose and insulin. Six IDDM patients and six normal subjects were given a saline infusion (15 mumol.min-1.kg-1) for 2 h, an acetoacetic acid infusion (15 mumol.min-1.kg-1) for another 2 h, and then a saline infusion after an overnight fast during euglycemic insulin-glucose clamp. Acetoacetic acid infusion resulted in an increase of blood ketone bodies in the range of 0.7-1.5 mM from a basal value of 0.1-0.3 mM. GFR was 125 +/- 16 and 136 +/- 17 ml.min-1.1.73 m-2 in normal and IDDM subjects, respectively, during baseline saline infusion and 138 +/- 21 (P less than .01 vs. basal level) and 158 +/- 15 ml.min-1.1.73 m-2 (P less than .001 vs. basal level) during acetoacetic acid infusion. During the last saline infusion, renal hemodynamic patterns decreased again to baseline levels. Another six IDDM patients and six normal subjects were given saline, lactic acid, and saline infusions at the same rates of infusion after an overnight fast during euglycemic insulin-glucose clamp. Lactic acid concentration increased from approximately 0.5-0.8 to 1.0-1.5 mM in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1992

Comparative effect of fasting on acetoacetate and D-3-hydroxybutyrate metabolism in the newborn chick.

The effect of 24 h fasting on ketone body utilization by three extranervous tissues, liver, duodenum and kidney, was studied in two critical ages of neonatal chick: 4 and 9 days. In 4-day-old chick, plasma concentration of 3-hydroxybutyrate increased about 9-fold after 24 h starvation, while in 9-day-old chick this parameter increased about 23-fold in the same conditions. Hepatic lipogenesis from both precursors sharply decreased by fasting. Changes in the lipogenic activity of duodenum were less patent. However, we have found a clear increase in lipogenesis in chick kidney after 24 h starvation. CO2 production from acetoacetate was higher than that found from hydroxybutyrate. No significant differences in the acetoacetate oxidation to CO2 was observed in any tissue assayed after 24 h fasting. 14C incorporation from ketone bodies into amino acids was clearly decreased in kidney from 9-day-old chick by fasting. In liver and duodenum, acetoacetate incorporation into amino acids was higher than that from hydroxybutyrate.

3-Hydroxybutyric Acid↗
PubMed1975

Acetoacetate coenzyme A transferase activity in rat hepatomas.

The presence of succinyl-coenzyme A:acetoacetate CoA transferase (CoA transferase) (EC 2.8.3.5), an initiator of ketone body utilization in nonhepatic tissue, was examined in liver from normal, partly hepatectomized, neonatal, and tumor-bearing rats, as well as in a series of transplantable rat hepatomas ranging widely in growth rate. While levels of CoA transferase are extremely low in normal, host, and regenerating liver, considerable amounts of activity are detectable in neonatal liver and in the hepatomas. In fact, the content of CoA transferase in the series of Morris hepatomas increases progressively with increase in tumor-growth rate. The fastest-growing tumor studied (7288Ctc) contains about the same amount of CoA transferase activity as rat skeletal muscle (i.e., an activity of about 0.1 mumole of acetoacetate used per min per g tissue). These results clearly indicate that the faster-growing hepatomas have adequate capacity to utilize ketone bodies in bioenergetic or biosynthetic activities. Furthermore, the enzymes from normal and hepatoma 7288Ctc tissues are quite similar with respect to (a) size of about 10(5) daltons, (b) reaction mechanism requiring formation of an enzyme:CoA intermediate (from ping-pong kinetic data), and (c) various kinetic parameters (such as Michaelis constants, product competitive inhibition constants, and acetoacetate substrate inhibition). The enzymes from rat skeletal muscle and Morris hepatoma 7288Ctc have the same isoelectric point (7.6), which differs from that for the rat heart enzyme (6.8).

Acetoacetates↗
PubMed1989

Ketone body utilization in duodenum. Differential effect of fasting on lipogenesis from acetoacetate and 3-hydroxybutyrate.

The effect of fasting and refeeding on oxidation, lipogenesis and amino acid synthesis from ketone bodies has been studied in neonatal chick duodenal mucosa. Oxidation and amino acid synthesis were higher from acetoacetate and were stimulated by fasting from both 3-hydroxybutyrate and acetoacetate. On the contrary, lipogenesis was always higher from 3-hydroxybutyrate and fasting reduced lipogenesis rate from acetoacetate (by 66%) but not from 3-hydroxybutyrate. Results suggests the existence of a cytosolic fast-dependent acetoacetyl-CoA synthetase in chick duodenal mucosa which is involved in phospholipid synthesis.

3-Hydroxybutyric Acid↗
PubMedDate not supplied

[The effect of acetoacetate on 3-hydroxybutyrate oxidation by rat liver mitochondria].

Effect of acetoacetate on 3-hydroxybutyrate oxidation by rat liver mitochondria is described. State 3 respiration is inhibited by acetoacetate, while state 4 respiration is not inhibited, though cytochrome c reduction was decreased. Acetoacetate is also non-competitive inhibitor of 3-hydroxybutyrateoxidase and 3-hydroxybutyrate dehydrogenase activity in frozen-thawed mitochondria. The results are discussed in terms of the thermodynamic hypothesis and control strength method.

3-Hydroxybutyric Acid↗
PubMed1982

Pathways of acetoacetate's formation in liver and kidney.

Specifically 14C-labeled palmitic acids were perfused through livers and incubated with slices of kidneys from rats in diabetic ketosis. The distribution of 14C in the hydroxybutyric acid formed was determined. In liver, the ratio of incorporation of 14C from [13-14C]palmitic acid into carbon 1 to carbon 3 of the hydroxybutyric acid was the same as the ratio in carbon 2 to carbon 4 from [6-14C]palmitic acid. In kidney, the carbon 1-to-carbon 3 ratio was more than twice the carbon 2-to-carbon 4 ratio. In both tissues, 14C from [16-14C] palmitic acid was preferentially incorporated into carbon 4 compared to carbon 2 of the hydroxybutyric acid, but more so in liver than kidney. These results mean that in liver, the sole pathway of acetoacetate formation is via hydroxymethylglutaryl-CoA, while in kidney it is not. Rather in kidney, acetoacetyl-CoA is converted to acetoacetate to a large extent by direct deacylation, presumably via a transferase- and/or deacylase-catalyzed reaction. In liver, most of the palmitic acid utilized is converted to acetoacetate while in kidney it is not. We previously estimated that, as a minimum, 11% of the hydroxybutyric acid excreted by the rat in diabetic ketosis is formed without hydroxymethylglutaryl-CoA as an intermediate. The kidney appears to be the source of this hydroxybutyric acid if the pathways operative in these tissues in vitro are those that also operate in vivo.

Acetoacetates↗
PubMed1982

Lipogenesis from ketone bodies in the isolated perfused rat liver. Evidence for the cytosolic activation of acetoacetate.

The production of ketone bodies by the isolated perfused rat liver has been measured by the dilution of the specific activity of tracer amounts of beta-hydroxy[3-14C]butyrate and by accumulation in the perfusate. The latter method has been found to underestimate ketogenesis by 12 to 44% because it does not take into account acetoacetate utilization by the liver. Incorporation of ketone bodies into fatty acids and 3-beta-hydroxysterols was compared to total lipid synthesis measured by incorporation of tritium from tritiated water. A preferential labeling of 3-beta-hydroxysterols over fatty acids was observed, which is consistent with the activation of acetoacetate in the cytosol by acetoacetyl-CoA synthetase. Ketone bodies contribute 19 to 80% of the carbon incorporated into sterols and up to 22% of the carbon incorporated into fatty acids, depending upon the metabolic status of the liver. The activity of acetoacetyl-CoA synthetase is more than sufficient to account for the rate of ketone body utilization. Conditions that decrease the citrate cleavage pathway of acetyl group translocation through the mitochondrial membrane are associated with an increase in carbon flux through acetoacetyl-CoA synthetase. Formation of acetoacetate in the mitochondria and its utilization in the cytosol thus appear to be a secondary pathway of acetyl group translocation operating concurrently with the predominant citrate cleavage pathway.

Acetoacetates↗
PubMedDate not supplied

Regulation of coenzyme A transferase and acetoacetate decarboxylase activities in Clostridium acetobutylicum.

The activity of two enzymes involved in acetone production in Clostridium acetobutylicum, acetoacetate decarboxylase and coenzyme A transferase, was studied under acidogenic or solventogenic conditions. Acetoacetate decarboxylase activity was low under acidogenic conditions and after pyruvate addition. Under the same conditions, coenzyme A transferase activity was high. A mutant which lacked acetoacetate decarboxylase activity but was positive for coenzyme A transferase activity was isolated.

Acetone↗
PubMed1990

Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum.

Acetoacetate decarboxylase (ADC) (EC4.1.1.4) of Clostridium acetobutylicum DSM 792 was purified to homogeneity, and its first 25 N-terminal amino acids were determined. Oligonucleotide probes deduced from this sequence were used to detect positive clones in partial gene banks derived from Sau3A and HaeIII digests with following ligation into the vector pUC9. In Escherichia coli, the 2.1-kbp HaeIII clones expressed high levels of ADC activity. The expression was independent of the orientation of the insert with respect to the lac promoter of the vector and also of the addition of isopropyl-beta-D-thiogalactopyranoside, thus indicating that sequences located on the clostridial DNA controlled transcription and translation. From the E. coli clone with the recombinant plasmid pUG93 containing the 2.1-kbp HaeIII fragment, the ADC protein was purified and compared with the native enzyme. Both were indistinguishable with respect to the molecular mass of subunits and native protein as well as to activity stain. The 2.9-kbp Sau3A fragment could be shown to contain the amino terminus of the acetoacetate decarboxylase (adc) gene but did not express enzyme activity. It partially overlapped with the HaeIII fragment, spanning together 4,053 bp of the clostridial genome that were completely sequenced. Four open reading frames (ORFs) could be detected, one of which was unambiguously assigned to the acetoacetate decarboxylase (adc) gene. Amino acid sequences of the N terminus and the catalytic center as deduced from the nucleotide sequence were identical to sequences obtained from direct analysis of the protein. Typical procaryotic transcriptional and translational start and stop signals could be found in the DNA sequence. Together with these regulatory sequences, the adc gene formed a single operon. The carboxyl terminus of the enzyme proved to be rather hydrophobic. In vitro transcription-translation assays resulted in formation of ADC and ORF3 gene product; the other two ORFs were not expressed. Whereas no homology of the adc gene and ORF2 could be detected with sequences available in the EMBL or GenBank data bases, the obviously truncated ORF1 showed significant similarity to alpha-amylase of Bacillus subtilis. The restriction pattern and N-terminal amino acid sequence (as deduced from the nucleotide sequence) of ORF3 proved to be identical to those of the large subunit of acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase.

Amino Acid Sequence↗