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PubMed1989

Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC).

The phbC gene encoding the third enzyme of the poly-beta-hydroxybutyrate biosynthetic pathway, poly-beta-hydroxybutyrate polymerase, in Alcaligenes eutrophus H16 has been identified by the complementation of poly-beta-hydroxybutyrate negative mutants of A. eutrophus H16. These results demonstrate that the three enzymes of the poly-beta-hydroxybutyrate biosynthetic pathway are organized phbC-phbA-phbB. Expression of all three genes in Escherichia coli results in a significant level (50% dry cell weight) of poly-beta-hydroxybutyrate production. phbC encodes a polypeptide of Mr = 63,900 which has a hydropathy profile distinct from typical membrane proteins indicating that poly-beta-hydroxybutyrate biosynthesis probably does not involve a membrane complex.

Alcaligenes↗

PubMed1986

Is there Ca2+(Sr2+)-3-hydroxybutyrate symport in rat-liver mitochondria? A reappraisal.

The observation in this laboratory that respiration and Sr2+ import were stimulated by the addition of 3-hydroxybutyrate to suspensions of N-ethylmaleimide-treated mitochondria respiring in state 6, after the addition of Sr2+, in a sucrose medium containing choline as substrate, led to the proposal by Moyle and Mitchell [(1977) FEBS Lett. 84, 135-140] that there is a Ca2+(Sr2+)-3-hydroxybutyrate symporter in rat liver mitochondria. However, experiments described in the present paper support a different interpretation. Under the conditions of the experiments by Moyle and Mitchell, the rate of respiration and the poise of Sr2+ accumulation are mainly limited, not by delta mu H+, but by lack of respiratory substrate. Even though N-ethylmaleimide is a potent inhibitor of 3-hydroxybutyrate dehydrogenase, we have found that, somewhat surprisingly, under the special conditions of these experiments, sufficient 3-hydroxybutyrate dehydrogenase activity remains available to account for the 3-hydroxybutyrate-dependent respiratory stimulation and Sr2+ import.

3-Hydroxybutyric Acid↗

PubMed1988

Kinetic measurement of the combined concentrations of acetoacetate and beta-hydroxybutyrate in serum.

This is an automated method for the kinetic measurement of the combined concentrations of acetoacetate and beta-hydroxybutyrate in a single channel of the "Multistat III" centrifugal analyzer. Acetoacetate is first reduced with high concentrations of NADH by catalysis with 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30). This reaction mixture is diluted with excess NAD+. The endogenous beta-hydroxybutyrate and that resulting from acetoacetate are then measured kinetically. Comparing the combined concentration of acetoacetate and beta-hydroxybutyrate (y) with the sum of acetoacetate and beta-hydroxybutyrate measured as described by Hansen and Freier (Clin Chem 1978;24:475) (x) yielded the relationship: y = 0.99x - 0.57 (r = 0.93, n = 25). The run-to-run CVs for low (5 mmol/L) and high (15 mmol/L) acetoacetate controls were 12% and 6%, respectively. The method is useful for determining the concentration of ketone bodies in 2-microL samples of serum of patients with diabetic ketoacidosis. The sensitivity can be increased to determine ketone body concentration in nonketotic individuals by increasing sample volume to 10 microL.

3-Hydroxybutyric Acid↗

PubMed1984

Automated kinetic method for D-3-hydroxybutyrate in plasma or serum.

This assay for 3-hydroxybutyrate in plasma or serum, based on kinetic initial-rate techniques, has been optimized with respect to initial rate and linearity as follows: pH 8.5, hydroxybutyrate dehydrogenase 62.5 U/L, and NAD+ concentration 20 mmol/L. We have used the assay satisfactorily with both the Gilford 103 and Hitachi 705 discrete analyzers, obtaining results that compare well with those by a segmented-flow method. Within-assay precision (CV) varied from 7.8 to 0.6%, depending on both the analyzer used and on the concentration of 3-hydroxybutyrate. Analytical recovery was also dependent on 3-hydroxybutyrate concentration, varying from 99% at 2 mmol/L to 91% at 9.5 mmol/L. Lactate dehydrogenase/lactate interference in this direct assay is eliminated by incorporating oxalate in the assay reagents. Hydrazine, commonly used in equilibrium methods of analysis for 3-hydroxybutyrate, produced no significant advantage in this assay and was omitted. The mixed reagents for the assay are stable at 4 degrees C for at least a week. The advantages of analysis for this metabolite may now be realized, so that clinical and subclinical ketosis can be identified in humans and animals.

3-Hydroxybutyric Acid↗

PubMed1983

An extracellular D(-)-3-hydroxybutyrate oligomer hydrolase from Alcaligenes faecalis.

A strain of Alcaligenes faecalis secretes an extracellular D(-)-3-hydroxybutyrate oligomer hydrolase, in addition to poly(3-hydroxybutyrate) depolymerase, when it is grown in a medium containing poly(3-hydroxybutyrate) as the sole carbon source. The oligomer hydrolase (EC 3.1.1.22), which has been purified to electrophoretic homogeneity, has a molecular weight of 68 000, as estimated by Sephadex G-100 gel filtration, and of 74 000, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric point of the enzyme is approx. 6.0 and the pH optimum for the enzyme reaction is 8.5. The purified oligomer hydrolase has high affinity for oligomeric esters (apparent Km for the D(-)-3-hydroxybutyrate dimer = 32.8 microM; for the dodecamer = 1.3 microM), but does not attack poly(3-hydroxybutyrate) (average molecular weight, 32 500) at all. Analysis of hydrolysates of the oligomeric esters suggests that the enzyme hydrolyzes these substrates from the carboxyl terminus, releasing D(-)-3-hydroxybutyrate units one by one.

Alcaligenes↗

PubMed2004

Protection by the GABAB receptor antagonist, SCH 50911, of gamma-hydroxybutyric acid-induced mortality in mice.

Different effects of moderate to high doses of gamma-hydroxybutyric acid, including sedation/hypnosis, have been found to be blocked by gamma-aminobutyric acidB (GABAB) receptor antagonists. The present study investigated whether the protective effect of GABAB receptor antagonists extends also to gamma-hydroxybutyric acid-induced mortality. To this aim, the present study investigated the effect of the GABAB receptor antagonist, (2S)(+)-5,5-dimethyl-2-morpholineacetic acid (SCH 50911; 100 mg/kg, ip), on mortality induced by gamma-hydroxybutyric acid (1-6 g/kg, ip) in DBA mice. Pretreatment with SCH 50911 resulted in a significant shift to the right of the dose-response curve of gamma-hydroxybutyric acid-induced mortality. Accordingly, the LD50 in SCH 50911-pretreated mice was significantly higher than that obtained in water-pretreated mice. The results of the present study support the hypothesis that (a) the GABAB receptor is a relevant site of action of gamma-hydroxybutyric acid, and (b) GABAB receptor antagonists may constitute potentially effective therapeutic interventions for gamma-hydroxybutyric acid intoxication.

Anesthetics, Intravenous↗

PubMed2000

Neuroprotective effect of gamma-hydroxybutyrate in transient global cerebral ischemia in the rat.

The effect of gamma-hydroxybutyrate on the histological and behavioral consequences of transient brain ischemia was studied in the four vessel occlusion rat model. In saline-treated animals, 30 min ischemia caused a massive loss of neurons in the hippocampal CA1 subfield (normal neurons: 14%, 5%, 23% and 30% on the 3rd, 10th, 15th and 65th day after ischemia, respectively). gamma-Hydroxybutyrate - 300 mg/kg intraperitoneally (i.p.) 30 min before or 10 min after arteries occlusion, followed by 100 mg/kg i.p. twice daily for the following 10 days - afforded a highly significant protection (normal neurons on the 3rd, 10th, 15th and 65th day after ischemia: 88% and 91%, 80% and 80%, 91% and 90%, 72% and 71% in rats receiving the first dose before or after arteries occlusion, respectively). The ischemia-induced sensory-motor impairment was significantly attenuated in rats receiving the first dose of gamma-hydroxybutyrate before arteries occlusion. Finally, the ischemia-induced impairment in spatial learning and memory, evaluated starting 27 days after the ischemic episode, was significantly attenuated by gamma-hydroxybutyrate, either injected first at 30 min before or 10 min after arteries occlusion. Lower doses of gamma-hydroxybutyrate had no significant effect. In conclusion, these results indicate that gamma-hydroxybutyrate provides significant protection against both histological and behavioral consequences of transient global cerebral ischemia in rats.

Animals↗

PubMed1996

What is the role of the gamma-hydroxybutyrate receptor?

A possible functional role for endogenous gamma-hydroxybutyrate has been disputed. However, there are receptor sites for this molecule, which are highly enriched in the synaptosomal membrane fraction in the rat brain and are functionally linked to a guanosine triphosphate-binding protein. These data suggest that they play a neurological role. The binding sites recognize some drug molecules that bear no structural similarity to gamma-hydroxybutyrate. Recent experimental evidence indicates the existence of endogenous hydrophobic ligands. As a minor brain metabolite directly or indirectly involved in scavenging oxygen-derived free radicals, gamma-hydroxybutyrate demonstrates similarities with melatonin. The gamma-hydroxybutyrate receptor is compared to the cannabis receptor, for which an endogenous hydrophobic ligand has been identified. Structurally similar molecules to this ligand are believed to be implicated in the sleep process. As gamma-hydroxybutyrate itself can induce sleep, a search amongst these molecules as possible ligands for the gamma-hydroxybutyrate receptor might be enlightening.

Animals↗

PubMed1988

Floc Formation by Azospirillum lipoferum Grown on Poly-beta-Hydroxybutyrate.

Azospirillum lipoferum RG6xx was grown under conditions similar to those resulting in encystment of Azotobacter spp. A. lipoferum produced cells of uniform shape when grown on nitrogen-free beta-hydroxybutyrate agar. Cells accumulated poly-beta-hydroxybutyrate and often grew as chains or filaments that eventually lost motility and formed capsules. Within 1 week, vegetative A. lipoferum inocula were converted into microflocs arising from filaments or chains. Cells within microflocs were pleomorphic, contained much poly-beta-hydroxybutyrate, and were encapsulated. Some cells had a cystlike morphology. Up to 57% of the dry weight of encapsulated flocs was poly-beta-hydroxybutyrate, whereas vegetative cells grown in broth with combined nitrogen had only 3% of their dry weight as poly-beta-hydroxybutyrate. Neither encapsulated cells in flocs nor nonencapsulated vegetative cells were significantly desiccation resistant. Under starvation conditions (9 days) only 25% of encapsulated cells remained viable, whereas vegetative cells multiplied severalfold. In short-term germination experiments with encapsulated flocs, nitrate, ammonium, and soil extract promoted formation of motile vegetative cells. Most cells in treatments lacking combined nitrogen eventually depleted their visible poly-beta-hydroxybutyrate reserves without germinating. The remaining cells retained the reserve polymer and underwent size reduction.

Journal Article↗

PubMed1983

Studies of cerebral beta-hydroxybutyrate transport by carotid injection; effects of age, diet and injectant composition.

Transport of beta-hydroxybutyrate at the blood--brain barrier was studied by the carotid-injection technique of Oldendorf. beta-hydroxybutyrate permeability declined sharply with age (80-300 g rats) and, in adult rats, increased 5-fold during one week on a high fat diet. Acetoacetate and lactate permeabilities showed age and diet dependences which were similar in direction whereas DMO, urea and mannitol did not show age and diet dependent permeabilities. There was no apparent increase of beta-hydroxybutyrate Km with age, so the decline of permeability was attributed to a decline of Vmax. beta-Hydroxybutyrate permeability was inversely related to pH of the injectant in the alkaline range but not in the acid range, suggesting that the pH dependence reflected titration of a carrier rather than titration of the permeant. Permeability was independent of [Na+], [K+], [Cl-] and [SO2-(4)]. Replacing a portion of the Na+ with ammonium enhanced beta-hydroxybutyrate uptake. This effect appeared to be due to trans alkalization, and was as expected of an A-/H+-symport or A-/OH- -antiport mechanism. Pyruvate, 4-hydroxy-alpha-cyanocinnamate and tetracaine inhibited, but SITS, DIDS, phloretin and methyl-isobutylxanthine did not. The data are consistent with transport by an A-/H+ -symporter or A-/OH- -antiporter with properties similar to those found in erythrocytes and other cells. Induction of its activity during ketosis would spare carbohydrate both by favoring ketoacid uptake and by favoring lactate output.

3-Hydroxybutyric Acid↗

PubMed1995

Peripheral 3-hydroxybutyrate and food intake in a model of dietary-fat induced obesity: effect of vagotomy.

We have examined the effect of peripheral 3-hydroxybutyrate injections on food intake and the contribution of the vagus nerve in the resistance to dietary fat-induced obesity in a rodent model. S 5B/Pl rats, which are resistant to dietary-fat induced obesity, and Osborne-Mendel rats, which are sensitive, were adapted to reverse light cycle. Food intake was measured for 24 h following the injection of 3-hydroxybutyrate, lactate, or glycerol (all 5 mMol/kg0.75, SC) at the onset of dark. Three-hydroxybutyrate reduced food intake (p < 0.0001) in S 5B/Pl rats only. Lactate reduced food intake slightly (p < 0.009) in both strains and glycerol had no effect on food intake. In a second experiment, S 5B/Pl and Osborne-Mendel rats were adapted to a high-fat diet and were then subjected to either selective hepatic vagotomy or sham operation. Vagotomy had no effect on weight gain of Osborne-Mendel rats but allowed weight gain in S 5B/Pl rats (p < 0.0001). Even in vagotomized S 5B/Pl rats, however, blood 3-hydroxybutyrate levels were inversely associated (r = -0.50) with food intake. These data suggest that the hepatic vagus nerve may contribute to the resistance of S 5B/Pl rats to dietary-fat induced obesity, but the data do not rule out a strictly central role for the regulation of food intake by 3-hydroxybutyrate in this strain.

3-Hydroxybutyric Acid↗

PubMed1981

beta-Hydroxybutyrate as a precursor to the acetyl moiety of acetylcholine.

Rat brain cortex slices were incubated with 10 mM-glucose and trace amounts of [6-3H]glucose and [3-14C]beta-hydroxybutyrate. The effects of (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase; methylmalonate, an inhibitor of beta-hydroxybutyrate dehydrogenase; and increasing concentrations of unlabeled acetoacetate were examined. The incorporation of label into lactate, citrate, malate, and acetylcholine (ACh) was measured and 3H:14C ratios calculated. Incorporation of [14C]beta-hydroxybutyrate into lactate was limited because of the low activity of gluconeogenic enzymes in brain, whereas incorporation of 14C label into Krebs cycle intermediates and ACh was higher than in previous experiments with [3H-,14C]-glucose. (-)-Hydroxycitrate (5.0 mM) reduced incorporation of [3H]glucose and [14C]beta-hydroxybutyrate into ACh. In contrast, slices incubated with methylmalonate (1 mM) showed a decrease in 14C incorporation without appreciably affecting glucose metabolism. The effects of high concentrations of methylmalonate were nonselective and yielded a generalized decrease in metabolism. Acetoacetate (1 mM) also produced a decreased 14C incorporation into ACh and its precursors. At 10 mM, acetoacetate reduced 3H and 14C incorporation into ACh without substantially affecting total ACh content. From the results, it is suggested that in adult rats beta-hydroxybutyrate can contribute to the acetyl moiety of ACh, possibly via the citrate cleavage pathway, though it is quantitatively less important than glucose and pyruvate. This contribution of ketone bodies could become significant should their concentration become abnormally high or glucose metabolism be reduced.

3-Hydroxybutyric Acid↗

PubMed2006

Isolation of poly-3-hydroxybutyrate metabolism genes from complex microbial communities by phenotypic complementation of bacterial mutants.

The goal of this study was to initiate investigation of the genetics of bacterial poly-3-hydroxybutyrate (PHB) metabolism at the community level. We constructed metagenome libraries from activated sludge and soil microbial communities in the broad-host-range IncP cosmid pRK7813. Several unique clones were isolated from these libraries by functional heterologous complementation of a Sinorhizobium meliloti bdhA mutant, which is unable to grow on the PHB cycle intermediate D-3-hydroxybutyrate due to absence of the enzyme D-3-hydroxybutyrate dehydrogenase activity. Clones that conferred D-3-hydroxybutyrate utilization on Escherichia coli were also isolated. Although many of the S. meliloti bdhA mutant complementing clones restored D-3-hydroxybutyrate dehydrogenase activity to the mutant host, for some of the clones this activity was not detectable. This was also the case for almost all of the clones isolated in the E. coli selection. Further analysis was carried out on clones isolated in the S. meliloti complementation. Transposon mutagenesis to locate the complementing genes, followed by DNA sequence analysis of three of the genes, revealed coding sequences that were broadly divergent but lay within the diversity of known short-chain dehydrogenase/reductase encoding genes. In some cases, the amino acid sequence identity between pairs of deduced BdhA proteins was <35%, a level at which detection by nucleic acid hybridization based methods would probably not be successful.

Cloning, Molecular↗

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↗

PubMedDate not supplied

Effect of hyperthermia on the lactic acid and beta-hydroxybutyric acid content in tumour.

The effects of hyperthermia on the content of lactic acid and beta-hydroxybutyric acid in the SCK mammary carcinoma and the leg muscle of A/J mice were studied. The contents of lactic acid in the SCK tumour before heating was 9.32 mumol/g, and the content of beta-hydroxybutyric acid was only 0.013 mumol/g. The lactic acid content in the tumour increased to 17.5 mumol/g at 0 h after heating at 41.5 degrees C for 30 min and then decreased to the control level 3 h later. When heated at 43.5 degrees C for 30 min, the lactic acid content in the tumour increased to 24 mumol/g at the end of heating and remained elevated for 24 h. The content of beta-hydroxybutyric acid increased continuously reaching 0.45 mumol/g at 5 h after heating at 43.5 degrees C for 30 min, and then declined thereafter. The contents of lactic acid and beta-hydroxybutyric acid in the muscle also increased after heating, but these increases were far less than those observed in the tumours. The absolute amount of lactic acid in the heated tumours was far greater than that of beta-hydroxybutyric acid, and thus appeared to play the major role for the increased acidity in the heated tumours.

3-Hydroxybutyric Acid↗

PubMed1983

Influence of the beta-hydroxybutyrate/acetoacetate ratio on the redox states of mitochondrial NAD(P) and cytochrome c systems, extramitochondrial ATP/ADP ratio and the respiration of isolated liver mitochondria in the resting state.

The effect of changes in hydrogen supply on the rate of energy transformation (measured as respiration rate), on the redox state of the NAD(P) and cytochrome c couples and on the extramitochondrial ATP/ADP was investigated with isolated rat liver mitochondria. Hydrogen supply was changed by variation of the beta-hydroxybutyrate/acetoacetate ratio in the incubation medium. In the active state respiration rate was strongly diminished by lowering the beta-hydroxybutyrate/acetoacetate ratio, whereas in the resting state a minor effect was observed. This resulted in a decrease in the respiratory control index (RCI). Even under a metabolic situation of strongly diminished hydrogen supply corresponding to a beta-hydroxybutyrate/acetoacetate ratio equal to 0.5 added ADP was phosphorylated. However, the ATP/ADP ratio generated extramitochondrially amounted to half that obtained at a beta-hydroxybutyrate/acetoacetate ratio of more than 100. Nevertheless at a beta-hydroxybutyrate/acetoacetate ratio of 0.5 the generated extramitochondrial ATP/ADP ratio is near to 100. A comparison of the changes in the available free redox energy with those in the extramitochondrial phosphorylation potential points to a disequilibrium between them in the resting state, too. The experimental results are discussed in the light of the metabolic conditions in vivo.

3-Hydroxybutyric Acid↗

PubMed1981

Influence of ration composition and energy balance on blood beta-hydroxybutyrate (ketone) and plasma glucose concentrations of dairy cows in early lactation.

The effect of ration composition, with respect to concentrate, crude protein, and methionine hydroxyanalog content, on blood beta-hydroxybutyrate and plasma glucose concentrations was assessed in Holstein cows every 2 weeks over the first 6 weeks of lactation. The correlation of these metabolites with estimated energy balance, and the effects of these ration variables on this correlation were studied. High concentrate diets (60% of dry matter) compared with low concentrate diets (40% of dry matter) increased mean plasma glucose values and reduced mean blood beta-hydroxybutyrate concentration. Variation in crude protein and methionine hydroxyanalog supplementation did not affect metabolite concentration. The correlations between blood beta-hydroxybutyrate and energy balance and between plasma glucose and energy balance were weak and subject to the influence of variation in ration composition. It was concluded that plasma glucose and blood beta-hydroxybutyrate concentrations cannot be used as valid indicators of energy balance. However, it did appear that blood beta-hydroxybutyrate might be used as an indicator of the relative glucogenic potential of dairy rations and that blood concentrations of this metabolite could potentially be used to adjust factors in the ration which influence glucose availability to the cow.

3-Hydroxybutyric Acid↗

PubMed1983

Influence of diabetes on rat liver mitochondria: decreased unsaturation of phospholipid and D-beta-hydroxybutyrate dehydrogenase activity.

Liver mitochondria and submitochondrial vesicles have been prepared from rats made diabetic by treatment with streptozotocin (diabetic membranes). The membranes were characterized in terms of phospholipid and fatty acid composition, electron transport functions, and D-beta-hydroxybutyrate dehydrogenase activity and compared with mitochondria and submitochondrial vesicles prepared from control animals (control membranes). No change in the phospholipid composition (44% lecithin, 35% phosphatidylethanolamine, and 21% diphosphatidylglycerol) was found, but a marked alteration in fatty acid composition of both the total phospholipid and lecithin occurred within 3 weeks after streptozotozin treatment and persisted thereafter. In lecithin, the 18:1/18:0 ratio decreases approximately 33% and the 20:4/18:2 ratio decreases approximately 55%. D-beta-hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which has a specific requirement of lecithin for function. In diabetic membranes, there is a progressive decrease in D-beta-hydroxybutyrate dehydrogenase activity with time after streptozotocin treatment to about 40% of control value at 15 weeks. In contrast, succinate oxidase and succinate- or NADH-cytochrome c reductase activities remain essentially unaltered. Further, the Arrhenius plot characteristics differ for D-beta-hydroxybutyrate dehydrogenase in diabetic membranes as compared with control membranes, in that the break point of the biphasic plot increases from 20 +/- 1 degree C in controls to 29 +/- 1 degree C in samples from diabetic animals. The change occurs about 3 weeks after streptozotocin treatment and is correlatable with the increased saturation of the fatty acid moiety of the phospholipids. The observed changes in D-beta-hydroxybutyrate dehydrogenase function and phospholipid composition were prevented by administration of insulin to the diabetic animals and are therefore referable to insulin insufficiency.

Animals↗