Assay of 3-hydroxybutyrate in the picomolar range

A radioisotopic procedure for the assay of 3-hydroxybutyrate is presented. It is based on the measurement of NADH, generated in the 3-hydroxybutyrate dehydrogenase reaction, through the conversion of 2-[U-14C]ketoglutarate to 14C-labeled L-glutamate in the presence of beef liver glutamate dehydrogenase. The assay is linear in the range of 2.5 to 20.0 pmole/sample and about 100-times more sensitive than previous methods. The procedure proved useful for the measurement of 3-hydroxybutyrate in liver samples not exceeding 25 micrograms wet weight.     

Effect of glycerol on gluconeogenesis in isolated rabbit kidney cortex tubules

In renal tubules isolated from fed rabbits glycerol is not utilized as a glucose precursor, probably due to the rate-limiting transfer of reducing equivalents from cytosol to mitochondria. Pyruvate and glutamate stimulated an incorporation of [14C]glycerol to glucose by 50- and 10-fold, respectively, indicating that glycerol is utilized as a gluconeogenic substrate under these conditions. Glycerol at concentration of 1.5 mM resulted in an acceleration of both glucose formation and incorporation of [14C]pyruvate and [14C]glutamate into glucose by 2- and 9-fold, respectively, while it decreased the rates of these processes from lactate as a substrate. In the presence of fructose, glycerol decreased the ATP level, limiting the rate of fructose phosphorylation and glucose synthesis. As concluded from the 'cross-over' plots, the ratios of both 3-hydroxybutyrate/acetoacetate and glycerol 3-phosphate/dihydroxyacetone phosphate, as well as from experiments performed with methylene blue and acetoacetate, the stimulatory effect of glycerol on glucose formation from pyruvate and glutamate may result from an acceleration of fluxes through the first steps of gluconeogenesis as well as glyceraldehyde-3-phosphate dehydrogenase. As inhibition by glycerol of gluconeogenesis from lactate is probably due to a marked elevation of the cytosolic NADH/NAD+ ratio resulting in a decline of flux through lactate dehydrogenase.     

Effects of ischaemia on metabolite concentrations in rat liver

1. Changes in the concentrations of ammonia, glutamine, glutamate, 2-oxoglutarate, 3-hydroxybutyrate, acetoacetate, alanine, aspartate, malate, lactate, pyruvate, NAD(+), NADH and adenine nucleotides were measured in freeze-clamped rat liver during ischaemia. 2. Although the concentrations of most of the metabolites changed rapidly during ischaemia the ratios [glutamate]/[2-oxoglutarate][NH(4) (+)] and [3-hydroxybutyrate]/[acetoacetate] changed equally and the value of the expression [3-hydroxybutyrate][2-oxoglutarate][NH(4) (+)]/[acetoacetate][glutamate] remained approximately constant, indicating that the 3-hydroxybutyrate dehydrogenase and glutamate dehydrogenase systems were at near-equilibrium with the mitochondrial NAD(+) couple. 3. The value of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] was about 0.7 in vivo and remained fairly constant during the ischaemic period of 5min, although the concentrations of alanine and oxoglutarate changed substantially. No explanation can be offered why the value of the ratio differed from that of the equilibrium constant of the alanine aminotransferase reaction, which is 1.48. 4. Injection of l-cycloserine 60min before the rats were killed increased the concentration of alanine in the liver fourfold and decreased the concentration of the other metabolites measured, except that of pyruvate. During ischaemia the concentration of alanine did not change but that of aspartate almost doubled. 5. After treatment with l-cycloserine the value in vivo of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] rose from 0.7 to 2.4. During ischaemia the value returned to 0.8. 6. The effects of l-cycloserine are consistent with the assumption that it specifically inhibits alanine aminotransferase. 7. Most of the alanine formed during ischaemia is probably derived from pyruvate and from ammonia released by the deamination of adenine nucleotides and glutamine. The alanine is presumably formed by the combined action of glutamate dehydrogenase and alanine aminotransferase. 8. The rate of anaerobic glycolysis, calculated from the increase in the lactate concentration, was 1.3mumol/min per g fresh wt. 9. Although the concentrations of the adenine nucleotides changed rapidly during ischaemia, the ratio [ATP][AMP]/[ADP](2) remained constant at 0.54, indicating that adenylate kinase established near-equilibrium under these conditions.     

Fermentation of 4-aminobutyrate by Clostridium aminobutyricum: cloning of two genes involved in the formation and dehydration of 4-hydroxybutyryl-CoA

Clostridium aminobutyricum ferments 4-aminobutyrate via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA and crotonyl-CoA to acetate and butyrate. The genes coding for the enzymes that catalyse the interconversion of these intermediates are arranged in the order abfD (4-hydroxybutyryl-CoA dehydratase), abfT (4-hydroxybutyrate CoA-transferase), and abfH (NAD-dependent 4-hydroxybutyrate dehydrogenase). The genes abfD and abfT were cloned, sequenced and expressed as active enzymes in Escherichia coli. Hence the insertion of the [4Fe-4S]clusters and FAD into the dehydratase required no additional specific protein from C. aminobutyricum. The amino acid sequences of the dehydratase and the CoA-transferase revealed close relationships to proteins deduced from the genomes of Clostridium difficile, Porphyromonas gingivalis and Archaeoglobus fulgidus. In addition the N-terminal part of the dehydratase is related to those of a family of FAD-containing mono-oxygenases from bacteria. The putative assignment in the databank of Cat2 (OrfZ) from Clostridium kluyveri as 4-hydroxybutyrate CoA-transferase, which is thought to be involved in the reductive pathway from succinate to butyrate, was confirmed by sequence comparison with AbfT (57% identity). Furthermore, an acetyl-CoA:4-hydroxybutyrate CoA-transferase activity could be detected in cell-free extracts of C. kluyveri. In contrast to glutaconate CoA-transferase from Acidaminococcus fermentans, mutation studies suggested that the glutamate residue of the motive EXG, which is conserved in many homologues of AbfT, does not form a CoA-ester during catalysis.     

Utilization of 3-hydroxybutyrate by chick cerebral hemispheres during postnatal maturation.

1. The utilization of 3-hydroxybutyrate has been studied in the chick telencephalon during its post-hatching maturation. 2. In the 1-day-old chick the blood concentration of 3-hydroxybutyrate appears to be relatively high and its value is 5 times that estimated in the 4- and 30-day-old chicks. 3. The determination of the cerebral arteriovenous differences of 3-hydroxybutyrate shows that the brain of the newly-hatched chick takes up 3 times more actively this ketone body than the brain of the 4-day-old bird does. 4. During incubation in a non-oxygenated and an oxygenated physiological medium, in the presence of 3-hydroxy [3-14C]butyrate, the specific radioactivity of the dicarboxylic amino acids in the 1-day-old chick brain slices is higher than in those of the 30-day-old chick, particularly in the oxygenated medium. 5. Thirty minutes after a subcutaneous injection of 3-hydroxy [3-14C]butyrate, the specific radioactivity of the dicarboxylic amino acids in the 1-day-old chick telencephalon is 3-4 times higher than that in the 4- and 30-day-old chick. 6. In conclusion, in the brain of the newly hatched chick, 3-hydroxybutyrate is an efficient precursor in the biosynthesis of dicarboxylic amino acids, particularly glutamate, and, as glucose, it is metabolically related to the "large compartment" of glutamate. 7. These results have been discussed comparatively to those previously obtained in the developing rodent brain.     

Effects of riboflavin deficiency on oxidative phosphorylation, flavin enzymes and coenzymes in rat liver.

Riboflavin deficiency in rats caused a decrease in the activities of hepatic succinate dehydrogenase (50 %), L-α-glycerophosphate dehydrogenase (50 %) and xanthine oxidase (70 %). It also reduced to 50 % the rate of mitochondrial oxidation of succinate, β-hydroxybutyrate, α-ketoglutarate, glutamate, pyruvate and malate without changing ADP : O ratios, thus showing that riboflavin deficiency interferes with electron transport along the respiratory chain without noticably affecting phosphorylation.     

Enzyme Activities in Regions of the Hypothalamus

Abstract: The activities of certain key enzymes have been measured in the ventral medial and ventral lateral areas of the hypothalamus, which are implicated in feeding behaviour, and compared with enzyme activities in the cortex and brainstem. The enzymes measured are concerned with glucose metabolism [hexokinase (EC 2.7.1.1) and glucosesphosphate dehydrogenase (EC 1.1.1.49)], ketone body metabolism [3-hydroxybutyrate dehydrogenase (EC 1.1.1.30)], fatty acid utilisation [carnitine palmitoyl transferase (EC 2.3.1.7)], citric acid cycle activity [pyruvate dehydrogenase (EC 1.2.4.2) and citrate synthase (EC 4.1.3.7)] and neurotransmitter synthesis [glutamate dehydrogenase (EC 1.4.1.3)].     

Effect of methylmalonate on ketone body metabolism in developing rat brain

The oxidation of 3-hydroxy[3-¹⁴C]butyrate to CO₂ and its incorporation into cerebral lipids by cortex slices from one-week old rats were markedly inhibited by methylmalonate. However, methylmalonate had no effect on the metabolism of labelled aceto- acetate, glucose and acetate by brain slices. Addition of propionate in the incubation medium reduced cerebral lipogenesis from labelled 3-hydroxybutyrate and acetate. Acute methylmalonic acidemia induced in one-week old pups by injecting 3% methylmalonate solution caused a reduction in the incorporation of labelled 3-hydroxybutyrate into cerebral lipids. However, acute methylmalonic acidemia had no effect on cerebral lipogensis $\text{in vivo}$ from labelled acetate. These findings show (i) the conversion of 3-hydroxybutyrate to acetoacetate by 3-hydroxybutyrate dehydrogenase in the brain is inhibited by methylmalonate, and (ii) an inhibition of cerebral lipid synthesis by propionate, which also accumulates in patients with methylmalonic aciduria.     

Hepatic uptake of amino acids at mid-lactation in the rat.

The enzymic determination of D-3-hydroxybutyrate and acetoacetate normally involves the use of 3-hydroxybutyrate dehydrogenase (HBDH, EC 1.1.1.30) of bacterial origin. We show that HBDH from Rhodopseudomonas spheroides (BCL, grade II) contains a 3-hydroxyisobutyrate dehydrogenase (HIBDH) activity: activity with 3-hydroxyisobutyrate as substrate was greater than 10% of that with 3-hydroxybutyrate. However, HBDH could be prepared essentially free of HIBDH activity by incubation at 37 degrees C in the presence of 1 mM-CaCl2, to produce an enzyme preparation that may be used for the specific determination of 3-hydroxybutyrate. Use of the purified enzyme preparations indicated that a major product of valine metabolism in hemidiaphragms from 40 h-starved rats was 3-hydroxyisobutyrate rather than 3-hydroxybutyrate.     

Effects of 2-mercaptoacetate in isolated liver mitochondria in vitro. Competitive inhibition of 3-hydroxybutyrate dehydrogenase and depression of the beta-oxidation pathway.

The effects of 2-mercaptoacetate on the respiration rates induced by different substrates were studied in vitro in isolated liver mitochondria. With palmitoyl-L-carnitine or 2-oxoglutarate as the substrate, the ADP-stimulated respiration (State 3) was dose-dependently inhibited by 2-mercaptoacetate. with glutamate or succinate as the substrate. State-3 respiration was only slightly inhibited by 2-mercaptoacetate. In contrast, the oxidation rate of 3-hydroxybutyrate was competitively inhibited by 2-mercaptoacetate in both isolated mitochondria and submitochondrial particles. In uncoupled mitochondria and in mitochondria in which ATP- and GTP-dependent acyl-CoA biosynthesis was inhibited, the inhibitory effect of 2-mercaptoacetate on palmitoyl-L-carnitine oxidation was abolished; under the same conditions, however, inhibition of 3-hydroxybutyrate oxidation by 2-mercaptoacetate still persisted. These results led to the following conclusions: 2-mercaptoacetate itself enters the mitochondrial matrix, inhibits fatty acid oxidation through a mechanism requiring an energy-dependent activation of 2-mercaptoacetate and itself inhibits 3-hydroxybutyrate oxidation through a competitive inhibition of the membrane-bound 3-hydroxybutyrate dehydrogenase. This study also strongly suggests that the compound responsible for the inhibition of fatty acid oxidation is 2-mercaptoacetyl-CoA.     

Availability of neurotransmitter glutamate is diminished when β-hydroxybutyrate replaces glucose in cultured neurons

Ketone bodies serve as alternative energy substrates for the brain in cases of low glucose availability such as during starvation or in patients treated with a ketogenic diet. The ketone bodies are metabolized via a distinct pathway confined to the mitochondria. We have compared metabolism of [2,4-¹³C]β-hydroxybutyrate to that of [1,6-¹³C]glucose in cultured glutamatergic neurons and investigated the effect of neuronal activity focusing on the aspartate–glutamate homeostasis, an essential component of the excitatory activity in the brain. The amount of ¹³C incorporation and cellular content was lower for glutamate and higher for aspartate in the presence of [2,4-¹³C]β-hydroxybutyrate as opposed to [1,6-¹³C]glucose. Our results suggest that the change in aspartate–glutamate homeostasis is due to a decreased availability of NADH for cytosolic malate dehydrogenase and thus reduced malate–aspartate shuttle activity in neurons using β-hydroxybutyrate. In the presence of glucose, the glutamate content decreased significantly upon activation of neurotransmitter release, whereas in the presence of only β-hydroxybutyrate, no decrease in the glutamate content was observed. Thus, the fraction of the glutamate pool available for transmitter release was diminished when metabolizing β-hydroxybutyrate, which is in line with the hypothesis of formation of transmitter glutamate via an obligatory involvement of the malate–aspartate shuttle.     

Metabolism of S-3-hydroxybutyrate in the perfused rat liver

The metabolism of millimolar concentrations of S-3-hydroxybutyrate (the unnatural enantiomer) has been studied in perfused livers from fed and starved rats. Protocols were designed to test whether S-3-hydroxybutyrate is metabolized in the cytosol or in the mitochondria via a racemase, a dehydrogenase, or a ligase. Our data show that only a minor fraction of S-3-hydroxybutyrate metabolism could occur via L-3-hydroxyacid dehydrogenase. Most of the metabolism of S-3-hydroxybutyrate proceeds via mitochondrial activation. In rat liver, S-3-hydroxybutyrate is converted to physiological ketone bodies (i.e., R-3-hydroxybutyrate, acetoacetate, acetone), lipids, and CO2. Carbons from S-3-hydroxybutyrate are transferred from the mitochondria to the cytosol mostly via citrate and the citrate cleavage pathway.     

Activities of NAD-specific and NADP-specific isocitrate dehydrogenases in rat-liver mitochondria. Studies with D-threo-alpha-methylisocitrate.

The contributions of NAD-specific and NADP-specific isocitrate dehydrogenases to isocitrate oxidation in isolated intact rat liver mitochondria were examined using DL-threo-alpha-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) to specifically inhibit flux through NADP-specific isocitrate dehydrogenase. Under a range of conditions tested with respiring mitochondria, the rate of isocitrate oxidation was decreased by about 20--40% by inhibition of NADP-isocitrate dehydrogenase, and matrix NADP became more oxidized. (a) For mitochondria incubated with externally added DL-isocitrate and citrate, the rate of isocitrate oxidation obtained by extrapolation to infinite alpha-methylisocitrate concentration was approximately 70% of the uninhibited rate in both state 3 and state 4. (b) With pyruvate plus malate added as substrates of citric acid cycle oxidation and isocitrate generated intramitochondrially, a concentration of alpha-methylisocitrate (400 microM) sufficient for 99.99% inhibition of NADP-isocitrate dehydrogenase inhibited isocitrate oxidation in states 4 and 3 by 21 +/- 6% and 19 +/- 11% (mean +/- SEM), respectively. (c) With externally added isocitrate and citrate, the addition of NH4Cl increased isocitrate oxidation by 3--4-fold, decreased NADPH levels by 30--40% and 2-oxoglutarate accumulation by about 40%. The further addition of 600 microM alpha-methylisocitrate decreased the NH4Cl-stimulated isocitrate oxidation by about 40% and decreased NADPH to about 30% of the level prevailing in the absence of NH4Cl; nevertheless, the rate of isocitrate oxidation was still twice as large in the presence of NH4Cl and alpha-methylisocitrate as in their absence. Experiments were also performed with intact mitochondria incubated with respiratory inhibitors to determine additional factors which might affect the flux through the two isocitrate dehydrogenases. (a) In the coupled reduction of acetoacetate by isocitrate, where the rate of reoxidation of reduced pyridine nucleotides is limited by NAD-specific 3-hydroxybutyrate dehydrogenase, 85--100% of the rate of 3-hydroxybutyrate formation was retained in the presence of 400--900 microM alpha-methylisocitrate. (b) In a system where the rate of isocitrate oxidation is limited by the rate of NADPH reoxidation by glutathione reductase, the rate of glutathione reduction extrapolated to infinite alpha-methylisocitrate concentration was from 20--40% of the uninhibited rate. (c) In the coupled synthesis of glutamate from isocitrate and NH4Cl, where the reoxidation of NADPH and NADH can occur via glutamate dehydrogenase, the rate of glutamate production extrapolated to infinite alpha-methylisocitrate concentration was about 60% of the uninhibited rate.     

THE MITOCHONDRIAL REDOX STATE OF RAT BRAIN

The use of the glutamate dehydrogenase (EC 1.4.1.3) and β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) reactions for the calculation of the mitochondrial redox state of brain has been examined. To prevent post-mortem anoxic metabolism, brains were frozen in less than a second by using a new technique. Levels of ketone bodies in brain were so low relative to the contamination by blood and extracellular fluid that calculation of the mitochondrial redox state using the β-hydroxybutyrate dehydrogenase reaction was not practical. The concentrations of the non-nucleotide substrates of the glutamate dehydrogenase reaction could be accurately measured in brain and themitochondrial [NAD⁺]/[NADH] ratio calculated from the ratio [α-oxoglutarate] [NH₄⁺]/[glutamate]. The calculation is valid if the ratio [α-oxoglutarate] [NH₄⁺]/[glutamate] in mitochondria is the same as that measured in whole tissue. The evidence supporting this conclusion is the near-equilibrium of the aspartate aminotransferase (EC 2.6.1.l) reaction in brain and the observation by others that the distribution of label between α-oxoglutarate and glutamate in brain, after administration of labelled precursors, conforms to expectation. The alanine aminotransferase (EC 2.6.1.2) reaction was not near equilibrium in brain, probably because of the low in vivo activity of the enzyme.     

Concerning the decreased D-3-hydroxybutyrate dehydrogenase activity in the liver and heart of hyperthyroid rats

Summary Whereas in rat liver mitochondria the hyperthyroid state causes an increase both in fatty acid unsaturation and in the Ea of D-3-hydroxybutyrate dehydrogenase and a decrease in phase transition temperature, in hyperthyroid rat heart mitochondria these changes are negligible. D-3-hydroxybutyrate dehydrogenase in both the liver and the heart mitochondria of hyperthyroid rats is reduced by about 35% [l2] but this reduction is not due to changes in membrane fluidity in either tissue. Hypothyroidism, on the other hand, affects BDH activity in neither heart nor liver.Abbreviations BDH D-3-hydroxybutyrate dehydrogenase - PTU 6n-propyl-2-thiouracil - T₃,3,3 5-L-triiodothyronine - Tm temperature phase transition - Ea apparent activation energy     

The regulation of pyruvate oxidation during membrane depolarization of rat brain synaptosomes.

Studies were performed to elucidate factors involved in the regulation of pyruvate dehydrogenase activity in rat brain synaptosomes during membrane depolarization. Addition of 24 mM-KCl to synaptosomes resulted in increases in rates of O2 consumption (90%) and [1-(14)C]pyruvate decarboxylation (85%) and in the active/total ratio of extractable pyruvate dehydrogenase (90--100%) within 10 s. Neither pyruvate (10 mM) nor dichloroacetate (10 mM) affected the activation state of the enzyme complex. Also, the activation state of pyruvate dehydrogenase was unaffected by addition of 1 mM-octanoate, L-(--)-carnitine, 3-hydroxybutyrate, glutamate, citrate, lactate, L-malate, acetate, acetaldehyde or ethanol. Removal of Ca2+ by using EGTA lowered the active/total ratio to about 70%, although the rate of O2 consumption and pyruvate decarboxylation was unaffected. Rates of pyruvate decarboxylation in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence and absence of NaF and EGTA demonstrated a linear correlation with changes in the activity of the enzyme complex. This observation indicated that a change in the activation state of pyruvate dehydrogenase from 90 to 100% active could result in a 27% increase in the rate of pyruvate decarboxylation. It is suggested that the pyruvate dehydrogenase complex is an important site for the regulation of substrate utilization in rat brain synaptosomes. Further, the phosphorylation/dephosphorylation system and direct feedback-inhibitory effects on the enzyme complex both play a significant role in rapidly adapting pyruvate decarboxylation to changes in the requirements for mitochondrial energy production.     

A novel radiochemical method for the microdetermination of total and individual ketone bodies in biological materials

A novel radiochemical method has been developed for ultramicrodetermination of acetone based on the principle that ¹²⁵I-labeled iodoform is produced by iodination of acetone with ¹²⁵ICl. [¹²⁵I]Iodoform is readily counted as a measure of acetone after separation from unreacted iodide ions. Quantitative conversion of 3-hydroxybutyrate to acetoacetate takes place when NAD-dependent oxidation of 3-hydroxybutyrate by 3-hydroxybutyrate dehydrogenase is coupled with NADH-dependent reduction of pyruvate (or 2-oxoglutarate) by lactic dehydrogenase (or glutamic dehydrogenase). Acetoacetate thus formed produces acetone spontaneously when the acidified (deproteinized) reaction mixture is maintained at 50°C for 2 hr. Thus, total and individual ketone bodies in plasma are determined conveniently by combining the radiochemical determination of acetone with these conversion procedures.     

Radioisotopic measurement of femtomolar amounts of NAD(P)H in the assay of enzymatic activity at a single cell level

A radioisotopic method for the assay of reduced or oxidized pyridine nucleotides, based on the interconversion of 2-[U-14C]ketoglutarate or 2-keto[3,4-3H]glutarate and labelled L-glutamate in the reaction catalyzed by glutamate dehydrogenase, was applied to the measurement of lactate dehydrogenase activity in rat pancreatic islet homogenates. Using the tritiated tracer, the limit of sensitivity of the procedure for NAD(P)H assay was close to 1.0 fmol/sample, and lactate dehydrogenase activity could be measured in as little as 0.0005 islet/sample i.e., at a single cell level. This radioisotopic procedure, which can be used for the assay of various metabolites and enzymic activities, thus provides a tool for investigating the heterogeneity in metabolic behaviour of individual cells.     

The stimulation of tricarboxylic acid-cycle flux by alpha-adrenergic agonists in perfused rat liver.

Output of 14CO2 from 1-14C-labelled glutamate, 2-oxoglutarate or octanoate and from 4-methyl-2-oxo[2-14C]pentanoate was increased by more than 100% after infusion of phenylephrine into perfused livers of fed rats. Infusion of ethanol or sorbitol raised 3-hydroxybutyrate/acetoacetate ratios and decreased the output of 14CO2. Increases in 14CO2 output induced by phenylephrine were observed in the presence or absence of ethanol or sorbitol and were accompanied by elevated 3-hydroxybutyrate/acetoacetate ratios under all conditions examined. Phenylephrine had no effect on total tissue ATP/ADP ratios in livers from fed or starved rats. The data suggest that phenylephrine-induced increases in tricarboxylic acid-cycle flux do not arise from lowered matrix NADH/NAD+ or ATP/ADP ratios.