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At least 19 recordsLinked to original sources
PubMed1990

Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli.

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an approximately 2.1 kb EcoRI/Bg/II fragment. A polypeptide with a molecular weight of approximately 28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although the expression of the gene is tightly regulated in C. acetobutylicum, it was well expressed in E. coli, although from a promoter sequence of clostridial origin.

Amino Acid Sequence↗
PubMed1975

Metabolic interactions of glucose, acetoacetate and adrenaline in rat submaxillary gland in vitro.

1. The metabolic interactions between glucose, acetoacetate and adrenaline were studied in submaxillary-gland slices. 2. Acetoacetate (2.5 mM) inhibited glucose removal by 22% and entry of glucose carbon into the tricarboxylic acid cycle by 54%. 3. Acetoacetate caused an increase in (glucose 6-phosphate) together with an increase in (citrate), a finding that suggests that the phosphofructokinase step might be inhibited by the elevated (citrate). Support for this suggestion was obtained in experiments in which fluoracetate was used to elevate (citrate). 4. A further site of action of acetoacetate at the pyruvate dehydrogenase step was suggested by an increase in the lactate+pyruvate pool, and the finding that pyruvate removal and (3-14C)pyruvate oxidation were inhibited by acetoacetate. 5. Adrenaline, a stimulator of secretion by this tissue, increased glucose removal by 25%. Adrenaline increased glucose removal to the same extent when acetoacetate was also present in the incubation medium. In both cases the increase was accompanied by a fall in (glucose 6-phosphate). 6. Adrenaline also overcame the inhibition of pyruvate removal caused by acetoacetate. 7. The tissue (ATP) decreased by about 50% on addition of adrenaline, and a similar fall was observed in vivo after adrenergic stimulation by isoproterenol. 8. Omission of Ca-2+ from the medium prevented the fall in (glucose 6-phosphate) and (ATP) caused by adrenaline, although adrenaline was still able to stimulate glucose removal. The inhibitory effect of acetoacetate on gluocse removal was reversed by adrenaline, but there was no stimulation above the control rates. Inhibition of pyruvate removal by acetoacetate was not overcome by adrenaline in the absence of Ca-2+. 9. Dibutyryl cyclic AMP had no effect on glucose removal or on (ATP). 10. Possible mechanisms by which adrenaline can bring about its metabolic effects are discussed.

Acetoacetates↗
PubMed1991

Changes in citric acid cycle flux and anaplerosis antedate the functional decline in isolated rat hearts utilizing acetoacetate.

To determine the temporal relationship between changes in contractile performance and flux through the citric acid cycle in hearts oxidizing acetoacetate, we perfused isolated working rat hearts with either glucose or acetoacetate (both 5 mM) and freeze-clamped the tissue at defined times. After 60 min of perfusion, hearts utilizing acetoacetate exhibited lower systolic and diastolic pressures and lower cardiac outputs. The oxidation of acetoacetate increased the tissue content of 2-oxoglutarate and glutamate and decreased the content of succinyl-CoA suggesting inhibition of citric acid cycle flux through 2-oxoglutarate dehydrogenase. Whereas hearts perfused with either acetoacetate or glucose were similar with respect to their function for the first 20 min, changes in tissue metabolites were already observed within 5 min of perfusion at near-physiological workloads. The addition of lactate or propionate, but not acetate, to hearts oxidizing acetoacetate improved contractile performance, although inhibition of 2-oxoglutarate dehydrogenase was probably not diminished. If lactate or propionate were added, malate and citrate accumulated indicating utilization of anaplerotic pathways for the citric acid cycle. We conclude that a decreased rate of flux through 2-oxoglutarate dehydrogenase in hearts oxidizing acetoacetate precedes, and may be responsible for, contractile failure and is not the result of decreased cardiac work. Further, anaplerosis play an important role in the maintenance of contractile function in hearts utilizing acetoacetate.

Acetoacetates↗
PubMed1975

Interactions of glucose, acetoacetate and insulin in mammary-gland slices of lactating rats.

1. Utilization of 5mM-glucose by slices of lactating mammary gland was decreased 33% on addition of acetoacetate (2mM) to the incubation medium. This inhibition was accompanied by increases in the intracellular concentrations of citrate and glucose 6-phosphate. 2. In the presence of acetoacetates the accumulation of pyruvate in the medium approximately doubled. 3. Insulin completely reversed the inhibitory effect of acetoacetates on glucose utilization, without altering the amount of acetoacetate removed or pyruvate formed. 4. Similar results were obtained with mammary-gland slices from diabetic rats, except that insulin did not completely reverse the effects of acetoacetates. 5. Acetoacetate inhibited the formation of 14CO2 from [1-14C]pyruvate; this effect was not overcome by insulin. 6. Insulin increased the proportion of [3-14C]acetoacetate that was converted into lipid and decreased that oxidized to CO2.7. The physiological significance of these findings is discussed.

Acetoacetates↗
PubMed1977

Effect of acetoacetate on glucose metabolism in the soleus and extensor digitorum longus muscles of the rat.

1. The effect of acetoacetate on glucose metabolism was compared in the soleus, a slow-twitch red muscle, and the extensor digitorum longus, a muscle composed of 50% fast-twitch red and 50% white fibres. 2. When incubated for 2h in a medium containing 5 mM-glucose and 0.1 unit of insulin/ml, rates of glucose uptake, lactate release and glucose oxidation in the soleus were 19.6, 18.6 and 1.47 micronmol/h per g respectively. Acetoacetate (1.7 mM) diminished all three rates by 25-50%; however, it increased glucose conversion into glycogen. In addition, it caused increases in tissue glucose, glucose 6-phosphate and fructose 6-phosphate, suggesting inhibition of phosphofructokinase. The concentrations of citrate, an inhibitor of phosphofructokinase, and of malate were also increased. 3. Rates of glucose uptake and lactate release in the extensor digitorum longus were 50-80% of those in the soleus. Acetoacetate caused moderate increases in tissue glucose 6-phosphate and possibly citrate, but it did not decrease glucose uptake or lactate release. 4. The rate of glycolysis in the soleus was approximately five times that previously observed in the perfused rat hindquarter, a muscle preparation in which acetoacetate inhibits glucose oxidation, but does not alter glucose uptake or glycolysis. A similar rate of glycolysis was observed when the soleus was incubated with a glucose-free medium. Under these conditions, tissue malate and the lactate/pyruvate ratio in the medium were decreased, and acetoacetate did not decrease lactate release or increase tissue citrate or glucose 6-phosphate. An intermediate rate of glycolysis, which was not decreased by acetoacetate, was observed when the soleus was incubated with glucose, but not insulin. 5. The data suggest that acetoacetate glucose inhibits uptake and glycolysis in red muscle under conditions that resemble mild to moderate exercise. They also suggest that the accumulation of citrate in these circumstances is linked to the rate of glycolysis, possibly through the generation of cytosolic NADH and malate formation.

Acetoacetates↗
PubMed1991

Acetoacetate and malate effects on succinate and energy production by O2-deprived liver mitochondria supplied with 2-oxoglutarate.

Acetoacetate provision to Ca(2+)-loaded liver mitochondria (less than 40 micrograms-ion Ca2+ x g protein-1), supplied with 2 mM Pi and 2-oxoglutarate as substrate, was found to prevent the mitochondrial deenergization and Ca2+ release induced by either rotenone during aerobic incubations or by O2 deprivation. Under the latter condition, the acetoacetate-promoted Ca2+ retention was entirely supported by ATP produced anaerobically at the succinylthiokinase step of the tricarboxylic acid cycle and was therefore abolished by addition of oligomycin. Surprisingly, oligomycin was also found to trigger Ca2+ release in rotenone-inhibited mitochondria in the presence of acetoacetate under aerobic conditions, unless a Pi acceptor was supplied. ADP deprivation at the succinylthiokinase step is likely to be involved. As estimated from rates of succinate production in O2-deprived mitochondria or from respiration rates in rotenone-inhibited mitochondria at supramaximal acetoacetate concentrations (above 1.2 mM) in the presence of a Pi acceptor, ATP production by substrate-level phosphorylation was close to 10 mumol.g protein-1.min-1 and appeared to be limited by rates of ketone body transport across the inner membrane. The rates of anaerobic energy production obtained by coupling 2-oxoglutarate oxidation to acetoacetate reduction were markedly higher than those obtained by reactions involved in the anaerobic metabolism of amino acids, simulated by providing 2-oxoglutarate and malate to mitochondria. Energy production was limited by rates of oxidant equivalent generation under the latter condition. Our data suggest that acetoacetate could effectively contribute to sustaining anaerobic energy production from endogenous substrates in liver tissue.

Acetoacetates↗
PubMed1975

Corticosteroids in nocturnal blood plasma of cows in the field related to stage of lactation and plasma acetoacetate.

Two materials of nocturnal blood plasma samples, each representing more than 100 cows unselected with respect to health condition, showed acetoacetate variations within a wide range, overlapping with the range for animals with clinical ketosis. Within each of the two materials a statistically significant (P smaller than 0.001) negative correlation (r equal to minus 0.30) was obtained between plasma corticosteroids and log acetoacetate. The lowest corticosteroid and the highest acetoacetate averages were obtained for animals which were within the first month of the lactation cycle. With increasing time post-partum the two components varied inversely, a maximum in corticosteroids coinciding with a minimum in acetoacetate in cows which had calved 2 1/2 to 3 months before the date of sampling. At this stage the mean plasma sugar was about 10 per cent higher than in early lactation. The corticosteroid ranges for different levels of acetoacetate overlapped extensively. Still cows with acetoacetate smaller than or equal to 0.6 mg/100 ml gave significantly higher corticosteroid averages than the rest of the material. But in animals exceeding the 0.6 mg limit no relationship was found between the severity of the ketonaemia and the steroid level. Thus, no difference was obtained between cows with clinical ketosis and animals which were moderately ketonaemic. Some change in adrenal function may be associated with the development of ketonaemic conditions in cows. But no proof has been provided of a direct adrenal involvement in the transformation of a case of subclinical ketosis to the clinical stage.

Acetoacetates↗
PubMed1976

Lipogenesis from ketone bodies in rat brain. Evidence for conversion of acetoacetate into acetyl-coenzyme A in the cytosol.

The metabolism of acetoacetate via a proposed cytosolic pathway in brain of 1-week-old rats was investigated. (-)-Hydroxycitrate, an inhibitor of ATP citrate lyase, markedly inhibited the incorporation of carbon from labelled glucose and 3-hydroxybutyrate into cerebral lipids, but had no effect on the incorporation of labelled acetate and acetoacetate into brain lipids. Similarly, n-butylmalonate and benzene-1,2,3-tricarboxylate inhibited the incorporation of labelled 3-hydroxybutyrate but not of acetoacetate into cerebral lipids. These inhibitors had no effect on the oxidation to 14CO2 of the labelled substrates used. (-)-Hydroxycitrate decreased the incorporation of 3H from 3H2O into cerebral lipids by slices metabolizing either glucose or 3-hydroxybutyrate, but not in the presence of acetoacetate. (-)-Hydroxycitrate also differentially inhibited the incorporation of [2-14C]-leucine and [U-14C]leucine into cerebral lipids. The data show that, although the acetyl moiety of acetyl-CoA generated in brain mitochondria is largely translocated as citrate from these organelles to the cytosol, a cytosolic pathway exists by which acetoacetate is converted directly into acetyl-COA in this cellular compartment.

Acetates↗
PubMed1991

Pyruvate carboxylation prevents the decline in contractile function of rat hearts oxidizing acetoacetate.

Acetoacetate, when present as the only fuel for respiration in rat hearts, causes an impairment in contractile function that is reversible with the addition of substrates that can contribute to anaplerosis. To determine the importance of pyruvate carboxylation via NADP(+)-dependent malic enzyme on metabolism and function in hearts oxidizing acetoacetate, isolated working rat hearts were perfused with [1-14C]pyruvate and acetoacetate. While the cardiac power output after 60 min of perfusion in hearts utilizing acetoacetate alone had fallen to 44% of the initial value, the addition of pyruvate resulted in a stable performance with no fall in the work output. When hydroxymalonate, an inhibitor of NADP(+)-dependent malic enzyme and malate dehydrogenase, was added to the two substrates, function at 60 min was similar to the value for hearts oxidizing acetoacetate alone. Measurements of the specific activities of malate, aspartate, and citrate confirm inhibition of both pyruvate carboxylation and malate oxidation. The findings are consistent with a mechanism in which the enrichment of malate by pyruvate improves function by increasing the production of reducing equivalents by the malate dehydrogenase and the isocitrate dehydrogenase reactions increase flux through the span of the tricarboxylic acid cycle from malate to 2-oxoglutarate. The present study demonstrates the physiological importance of anaplerotic pathways in maintaining contractile function in the heart.

Acetoacetates↗
PubMed1990

An enzymatic assay method for D(-)-3-hydroxybutyrate and acetoacetate involving acetoacetyl coenzyme A synthetase from Zoogloea ramigera.

An enzyme assay method for D(-)-3-hydroxybutyrate and acetoacetate involving acetoacetyl coenzyme A (CoA) synthetase was developed. To determine the concentration of D-3-hydroxybutyrate, it was oxidized with D-3-hydroxybutyrate dehydrogenase in the presence of nicotinamide adenine dinucleotide (NAD+) to acetoacetate, which was then converted to acetyl CoA via acetoacetyl CoA through the combined actions of acetoacetyl CoA synthetase and 3-ketothiolase in the presence of adenosine triphosphate (ATP) and CoA. To determine the concentration of acetoacetate, acetoacetyl CoA generated from acetoacetate with acetoacetyl CoA synthetase was reduced to 3-hydroxybutyryl CoA with 3-hydroxyacyl CoA dehydrogenase in the presence of NADH. The amount of D-3-hydroxybutyrate or acetoacetate was estimated from the increase or decrease in the absorbance at 340 nm, respectively. The present assay method seemed to be accurate and quick. Furthermore, as to the assaying of D-3-hydroxybutyrate, the omission of hydrazine, which is included for the standard method, may be preferable for routine assaying.

3-Hydroxybutyric Acid↗
PubMed1992

A 28-day feeding study with ethyl acetoacetate in rats.

Ethyl acetoacetate encapsulated in gum arabic was administered in rodent diet for a minimum of 28 consecutive days to groups of 16 male and 16 female rats (Sprague-Dawley strain) at levels of approximately 100, 300 and 1000 mg/kg body weight/day. A further group of 16 male and 16 female rats was given rodent diet containing gum arabic as a control. The administration of ethyl acetoacetate in the diet did not adversely affect the growth or general health of the animals or their food intakes. None of the minor variations observed in the haematology, serum chemical analyses or urine analyses are considered to be indicative of a treatment-related toxic effect. Caecal enlargement was seen in male rats treated with the top dose of ethyl acetoacetate, but this was accompanied by a normal histopathology. Few histopathological abnormalities were observed. Proteinaceous casts were found in the bladder of approximately half the male rats given 1000 mg ethyl acetoacetate/kg, and nephrocalcinosis was a common occurrence in female rats in this dose group. Renal function was unimpaired in treated male and female rats, and the histopathological findings are common in the strain of rats chosen for this study. Although the caecal enlargement and the changes in kidney and bladder of rats given 1000 mg ethyl acetoacetate/kg are noted, it is considered that ethyl acetoacetate did not produce treatment-related adverse effects in rats during this study.

Acetoacetates↗
PubMed1977

A kinetic spectrophotometric assay for rapid determination of acetoacetate in blood.

We describe an automated kinetic assay for acetoacetate in blood. Acetoacetate is enzymatically reduced to D-beta-hydroxybutyrate and the reaction is monitored for 60 s with a reaction-rate analyzer. This technique allows low concentrations of acetoacetate to be measured with good precision and overcomes many of the problems associated with other automated techniques. Our studies on the stability of acetoacetate emphasize the need for care in handling specimens. The use of a reaction-rate analyzer, an item of equipment common to most laboratories, allows for rapid handling of samples in small or large batches, depending on the needs of the laboratory.

Acetoacetates↗
PubMed1976

Mammalian acetoacetate decarboxylase activity. Its distribution in subfractions of human albumin and occurrence in various tissues of the rat.

In this article further information is presented about the characteristics of the mammalian enzyme acetoacetate decarboxylase (acetoacetate carboxylase, EC 4.1.1.4). The Michaelis-Menten plot shows a sigmoidal relationship between the enzyme activity (v) and the substrate concentration (s) indicating an allosteric hindrance. Because of this, the KM value can only be predicted to be equal to or less than 1 X 10(-1) M. Cysteine and glutathione, although activating the spontaneous decarboxylation, have no effect upon the enzyme activity. From experiments with human albumin by means of gel filtration with Sephadex G-200, it can be concluded that the acetoacetate decarboxylase activity does not depend upon the degree of polymerisation of albumin. From experiments performed by means of ion exchange chromatography the enzyme activity may be localized in the non-mercaptalbumin fraction. Investigation of enzyme activity in homogenates of various rat tissues, as well as in their respective subfractions, reveals that: (1) the specific activity of brain tissue exceeds those of liver and kidney and (2) most of the activity in liver tissue is localized in the 20 000 X g supernatant, containing the endoplasmatic reticulum, the ribosomes and the soluble part of the cytoplasm, while in brain tissue a high activity is found in the nuclei fraction.

Acetoacetates↗
PubMed1989

Determination of acetoacetate in urine by solid-phase spectrophotometry.

A method for the determination of acetoacetate has been developed based on solid-phase spectrophotometry (SPS). The acetoacetate reacts with nitroprusside and glycine and the reaction product is sorbed on Dowex 1-X8 resin. The absorbance of the resin phase at 590 and 720 nm is measured directly. The calibration graph is linear up to 3.3 mg l-1 and the RSD is 1.9%. The detection limit is 7.6 micrograms l-1. The method has been applied to the determination of acetoacetate in normal and diabetic subjects' urine without pretreatment of the samples, and the results compared with those of 1H-NMR and homogeneous nitroprusside methods.

Acetoacetates↗
PubMed1990

An improved synthesis of carbon-11 labeled acetoacetic acid and an evaluation of its potential for the investigation of cerebral pathology by positron emission tomography.

1-11C-acetoacetic acid was synthesized by carboxylation of the acetone carbanion. Purification was carried out using HPLC. The product was obtained with a radiochemical yield of up to 58%, corrected for decay, in a total preparation time of 30 min. The distribution of 1-11C-acetoacetic acid after injection into adult Wistar rats and cats was investigated by PET. When the tracer was injected into cats, 3 weeks after inflicting a unilateral freezing lesion upon the brain, accumulation of 1-11C-acetoacetic acid in the ipsilateral brain hemisphere was observed.

Acetoacetates↗
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↗