Evidence for substrate channeling in the early steps of cholesterogenesis

We have directly tested the ability of acetoacetate, upon activation to the CoA thioester, to channel into the cholesterogenic pathway prior to scrambling of its carbon skeleton with the acetate pool. The approach relies upon trapping [3-13C]acetoacetate-derived hydroxymethylglutaryl-CoA, hydrolyzing this metabolite, and esterifying the resulting hydroxymethylglutaric acid to allow gas chromatography/mass spectrometry analysis of the dimethyl esters for the 13C enrichment and labeling pattern. 99% enriched [3-13C] and [1,3,5-13C]hydroxymethylglutaric acid samples were synthesized, providing standards against which physiological samples could be compared. Cytosolic extracts from brain and liver of cholestyramine-fed rats were incubated with [3-13C]acetoacetate (2 mM) or with [1-13C]acetate (5 mM). In contrast to [13C]acetate-derived hydroxymethylglutarate, which shows the expected triple labeling pattern, [13C]acetoacetate-derived hydroxymethylglutarate from both liver and brain extracts is predominantly monolabeled. These data suggest that, after acetoacetate is activated to the CoA thioester, cytosolic hydroxymethylglutaryl-CoA synthase effectively commits much of this acetoacetyl-CoA to cholesterogenesis before thiolase can scramble the carbon skeleton of the acetoacetyl moiety into the acetate pool. This chemical approach represents an alternative method for testing the channeling of metabolites through sequential steps in a metabolic pathway. Such a method may be useful when physical or kinetic techniques prove to be unsuitable.     

Intracellular localization of the 3-hydroxy-3-methylglutaryl coenzme A cycle enzymes in liver. Separate cytoplasmic and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A generating systems for cholesterogenesis and ketogenesis.

Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl coenzyme synthase which comprise the 3-hydroxy-3-methylglutaryl-CoA-generating system(s) for hepatic cholesterogenesis and ketogenesis exhibit dual mitochondrial and cytoplasmic localization. Twenty to forty per cent of the thiolase and synthase of avian and rat liver are localized in the cytoplasmic compartment, the remainder residing in the mitochondria. In contrast, 3-hydroxy-3 methylglutaryl-CoA lyase, an enzyme unique to the "3-hydroxy-3-methylglutaryl-CoA cycle" of ketogenesis, appears to be localized in the mitochondrion. The small proportion, 4 to 8 percent, of this enzyme found in the cytoplasmic fraction appears to arise via leakage from the mitochondria during cell fractionation in that its properties, pI and stability, are identical to those of the mitochondrial lyase. These results are consistent with the view that ketogenesis which involves all three enzymes, acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA lyase, occurs exclusively in the mitochondrion, whereas cholesterogenesis, a pathway which involves only the 3-hydroxy-3-methylglutaryl-CoA synthesizing enzymes, is restricted to the cytoplasm. Further fractionation of isolated mitochondria from chicken and rat liver showed that all three of the 3-hydroxy-3-methylglutaryl-CoA cycle enzymes are soluble and are localized within the matrix compartment of the mitochondrion. Likewise, cytoplasmic acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase are soluble cytosolic enzymes, no thiolase or synthase activity being detectable in the microsomal fraction. Chicken liver mitochondrial 3-hydroxy-3methylglutaryl-CoA synthase activity consists of a single enzymic species with a pI of 7.2, whereas the cytoplasmic activity is composed of at least two species with pI values of 4.8 and 6.7. Thus it is evident that the mitochondrial and cytoplasmic species are molecularly distinct as has been shown to be the case for the mitochondrial and cytoplasmic acetoacetyl-CoA thiolases from avian liver (Clinkenbeard, K. D., Sugiyama, T., Moss, J., Reed, W. D., and Lane, M. D. (1973) J. Biol. Chem. 248, 2275). Substantial mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase activity is present in all tissues surveyed, while only liver and kidney possess significant mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Therefore, it is proposed that tissues other than liver and kidney are unable to generate acetoacetate because they lack the mitochondrial synthase.     

The interconversion and disposal of ketone bodies in untreated and injured post-absorptive rats

[3-(14)C]Acetoacetate and beta-hydroxy[3-(14)C]butyrate were used to investigate the kinetics of ketone body metabolism in rats 3h after bilateral hind-limb ischaemia and in controls, both groups being in the post-absorptive state and in a 20 degrees C environment. Calculations were carried out as described by Heath & Barton (1973) and the following conclusions were reached. 1. In both injured and control rats, the rates of irreversible disposal (extrahepatic utilization) of beta-hydroxybutyrate and acetoacetate were proportional within experimental error to their blood concentrations up to at least 0.4mm (the maximum found in these rats), implying that they were determined, via these concentrations, by the rates of production by the liver. 2. Conversion of blood beta-hydroxybutyrate into blood acetoacetate took place mainly in the liver, but the reverse process occurred mainly in extrahepatic tissues. 3. The ;metabolic clearance rate' (the volume of blood which, if completely cleared of substrate in unit time, would give a disposal rate equal to that in the whole animal) was calculated for beta-hydroxybutyrate and acetoacetate. Comparison with the cardiac output showed that in control rats the proportion of circulating beta-hydroxybutyrate extracted was lower than that of acetoacetate, clearance of which appeared almost complete. After injury both metabolic clearance rates decreased, probably because of the lower cardiac output. 4. After injury, because the average blood concentrations of ketone bodies, especially acetoacetate, were higher, the mean total rate of disposal also increased. Assuming complete oxidation, the mean contribution of ketone bodies to the whole body O(2) consumption rose from 7 to 15%.     

Carbon-13 and phosphorus-31 NMR study of hepatic metabolism in the perfused rat liver

Phosphorus-31 nuclear magnetic resonance (NMR) has been used to determine non-invasively absolute concentrations of phosphorylated metabolites in the perfused rat liver. It has been shown that the NMR method does detect cytoplasmic ATP and ADP (ATP:ADP ratio of 15 +/- 3) with no contribution from mitochondrial adenine nucleotides. The concentration of ATP was 7.2 +/- 0.3 mM in the cytosol of well-oxygenated liver, after two hours of perfusion with a Krebs-Ringer buffer. Other phosphorylated metabolites were detected, mainly inorganic phosphate (1.1 mumol/g liver wet weight), phosphorylcholine (1.0 mumol/g wet weight), glycerophosphorylethanolamine (0.34 mumol/g wet weight) and glycerophosphorylcholine (0.30 mumol/g wet weight). The intracellular pH measured from the position of the Pi resonance has a value of 7.2 +/- 0.1. It is likely that the detectable Pi originates from the cytosolic compartment since a pH value of 7.4-7.6 would be expected for the mitochondrial matrix. Natural abundance carbon-13 NMR has also been used to follow the glycogen breakdown in situ by measuring the intensity of the glycogen C-1 resonance in the perfused liver spectrum as a function of the perfusion time. The glycogenolytic process has been studied as a function of the glucose content of the perfusate. Rate of glycogenolysis from 2.7 to 0.16 muEq glycosyl units g wet weight-1 min-1 were found when glucose concentration in the perfusate was varied from 0 to 50 mM. The fate of 90% enriched [2-13C] acetate has been studied in the perfused rat liver by 13C-NMR in order to investigate the mitochondrial metabolism and the interrelations between cytosolic and mitochondrial pools of metabolites. Some compounds of the intermediary metabolism where found to be extensively labelled, e.g. glutamate, glutamine, acetoacetate and beta-hydroxybutyrate. Under our experimental conditions, labelling of glutamate reached a steady-state within 30 min after the onset of perfusion of 20 mM acetate. In addition, the observed incorporation of carbon-13 isotope into glutamine can be linked to the operation of the glutamate-glutamine antiporter and to the high activity of the cytosolic glutamate synthetase. The finding of both active glutaminase and glutamine synthetase activity in the same liver cells is an evidence of the existence of an active glutamine-glutamate futile cycle.     

Proton-nuclear-magnetic-resonance studies of serum, plasma and urine from fasting normal and diabetic subjects.

Resonances for the ketone bodies 3-D-hydroxybutyrate, acetone and acetoacetate are readily detected in serum, plasma and urine samples from fasting and diabetic subjects by 1H n.m.r. spectroscopy at 400 MHz. Besides the simultaneous observation of metabolites, the major advantage of n.m.r. is that little or no pretreatment of samples is required. N.m.r. determinations of 3-D-hydroxybutyrate, acetoacetate, lactate, valine and alanine were compared with determinations made with conventional assays at six 2-hourly intervals after insulin withdrawal from a diabetic subject. The n.m.r. results closely paralleled those of other assays although, by n.m.r., acetoacetate levels continued to rise rather than reaching a plateau 4h after insulin withdrawal. The 3-D-hydroxybutyrate/acetoacetate ratio in urine during withdrawal gradually increased to the value observed in plasma (3.0 +/- 0.2) as determined by n.m.r. The acetoacetate/acetone ratio in urine (17 +/- 6) was much higher than in plasma (2.5 +/- 0.7). Depletion of a mobile pool of fatty acids in plasma during fasting, as seen by n.m.r., paralleled that seen during insulin withdrawal. These fatty acids were thought to be largely in chylomicrons, acylglycerols and lipoproteins, and were grossly elevated in plasma samples from a non-insulin-dependent diabetic and in cases of known hyperlipidaemia.     

Inhibition of d(-)-3-hydroxybutyrate dehydrogenase by malonate analoges

(1) d(-)-3-Hydroxybutyrate dehydrogenase activity from guinea pig, rat, and bovine heart and from guinea pig liver is inhibited by malonate and tartronate, and more potently by the analogs methylmalonate, bromomalonate, chloromalonate, and mesoxalate. Little or no inhibitory effect was found for aminomalonate, ethylmalonate, dimethylmalonate, succinate, glutarate, oxaloacetate, malate, propionate, pyruvate, d- and l-lactate, n-butyrate, isobutyrate, and cyclopropanecarboxylate. (2) In initial velocity kinetics at pH 8.1 with a soluble enzyme preparation from bovine heart, the inhibition by the active malonate derivatives is competitive with respect to 3-hydroxybutyrate and uncompetitive with respect to acetoacetate, NAD⁺ or NADH. With d-3-hydroxybutyrate as the variable reactant (K_{m app} = 0.26 mM) the inhibition constant of methylmalonate (K_is) was 0.09 mm. (3) The rate of utilization of d-3-hydroxybutyrate (78 μm) by coupled rat heart mitochondria in the presence of ADP was inhibited 50% by 150 μm methylmalonate. (4) With coupled guinea pig liver mitochondria oxidizing n-octanoate in the absence of added ADP, methylmalonate (1–3 mm) depressed 3-hydroxybutyrate formation substantially more than total ketone production. However, the intramitochondrial NADH (or NADPH) levels were unchanged by the addition of methylmalonate, indicating that the changes in ratios of accumulated 3-hydroxybutyrate and acetoacetate were caused by direct inhibition of 3-hydroxybutyrate dehydrogenase. Methylmalonate had the same effect on 3-hydroxybutyrate/acetoacetate ratios and ketone body formation with pyruvate or acetate as the source of acetyl groups. Similar results were obtained with malonate (10 mm) although the inhibition of total ketone formation from octanoate was more severe.     

Enhanced ketone body uptake by perfused skeletal muscle in trained rats

Training effect on exercise-induced hyperketonemia was investigated in normal post-absorptive rats subjected to running exercise on a treadmill. Furthermore, rat hindlimb-muscle perfusion was performed to elucidate the mechanism of the training effect. A medium intensity prolonged exercise (running at 15 m/min for 90 min) caused a greater increase in plasma 3-hydroxybutyrate than in acetoacetate both during and after the exercise. Training with medium-intensity exercise (15 m/min) for 90 min 3 times per week for 14 wks or 28 wks caused 1) a reduction of the increase in plasma ketone body (mainly 3-hydroxybutyrate), free fatty acids and glucagon induced by the exercise, and 2) an increase in ketone body (mainly acetoacetate) uptake by perfused skeletal muscle. The present study demonstrates that the reduction of exercise-induced hyperketonemia by prolonged training is caused by increased ketone body utilization in skeletal muscle, and suggested that inhibition of hepatic ketogenesis might also participate in this reduction.     

Purification and Properties of Succinyl-CoA:3-Oxo-Acid CoA-Transferase from Rat Brain

Abstract: Rat brain succinyl-CoA:3-oxo-acid CoA-transferase (3-Oxo-acid CoA-transferase, EC 2.8.3.5), the first committed enzyme in the oxidation of ketone bodies in mitochondria, was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme has an apparent molecular weight of 90,000 as determined by (3-150 Sephadex chromatography, and an apparent subunit molecular weight of 53,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was approximately 161 μmol/min/mg of protein. Initial velocity studies of the forward reaction (acetoacetate → acetoacetyl-CoA) are consistent with a "ping pong" mechanism. Substrate inhibition appears above approximately 1 m M acetoacetate. Apparent K_m, values were 70 μM for acetoacetate and 156 μ M for succinyl-CoA (the forward reaction), and 59 μ M for acetoacetyl-CoA and 25 m M for succinate (the reverse reaction). These values are markedly different from those reported for this enzyme from pig heart.     

Possible interrelationship between gluconeogenesis and ketogenesis in the liver

Effeects of various ketogenic substrates on gluconeogenesis from lactate were examined. D,L-3-Hydroxybutyrate (5 mM) stimulated gluconeogenesis by 41%, the effect being the same as that of 5 mM acetate (49%). No stimulating effect of acetoacetate was observed; conversely, acetoacetate (up to 40 mM) partially or completely abolished the observed stimulating effects of acetate, oleate, and 3-hydroxybutyrate. The results suggest that, in intact liver cells, pyruvate is transported into mitochondria in exchange for acetoacetate and that an interrelationship between gluconeogenesis and ketogenesis at the level of mitochondrial pyruvate carrier may exist in the liver.     

Ketone body and fatty acid metabolism in sheep tissues. 3-Hydroxybutyrate dehydrogenase, a cytoplasmic enzyme in sheep liver and kidney

1. 3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) activities in sheep kidney cortex, rumen epithelium, skeletal muscle, brain, heart and liver were 177, 41, 38, 33, 27 and 17μmol/h per g of tissue respectively, and in rat liver and kidney cortex the values were 1150 and 170 respectively. 2. In sheep liver and kidney cortex the 3-hydroxybutyrate dehydrogenase was located predominantly in the cytosol fractions. In contrast, the enzyme was found in the mitochondria in rat liver and kidney cortex. 3. Laurate, myristate, palmitate and stearate were not oxidized by sheep liver mitochondria, whereas the l-carnitine esters were oxidized at appreciable rates. The free acids were readily oxidized by rat liver mitochondria. 4. During oxidation of palmitoyl-l-carnitine by sheep liver mitochondria, acetoacetate production accounted for 63% of the oxygen uptake. No 3-hydroxybutyrate was formed, even after 10min anaerobic incubation, except when sheep liver cytosol was added. With rat liver mitochondria, half of the preformed acetoacetate was converted into 3-hydroxybutyrate after anaerobic incubation. 5. Measurement of ketone bodies by using specific enzymic methods (Williamson, Mellanby & Krebs, 1962) showed that blood of normal sheep and cattle has a high [3-hydroxybutyrate]/[acetoacetate] ratio, in contrast with that of non-ruminants (rats and pigeons). This ratio in the blood of lambs was similar to that of non-ruminants. The ratio in sheep blood decreased on starvation and rose again on re-feeding. 6. The physiological implications of the low activity of 3-hydroxybutyrate dehydrogenase in sheep liver and the fact that it is found in the cytoplasm in sheep liver and kidney cortex are discussed.     

Glucagon regulation of gluconeogenesis and ketogenesis in periportal and perivenous rat hepatocytes. Heterogeneity of hormone action and of the mitochondrial redox state.

Hepatocytes isolated from the periportal or perivenous zones of livers of fed rats were used to study the long-term (14 h) and short-term (2 h) effects of glucagon on gluconeogenesis and ketogenesis. Long-term culture with glucagon (100 nM) resulted in a greater increase (P less than 0.01) in gluconeogenesis in periportal than in perivenous cells (93 +/- 16 versus 30 +/- 14 nmol/h per mg of protein; 72% versus 30% increase), but short-term incubation (2 h) with glucagon resulted in similar stimulation in the two cell populations. Rates of ketogenesis (acetoacetate and D-3-hydroxybutyrate production) were not significantly higher in periportal cells cultured without glucagon, compared with perivenous cells. However, after long-term culture with glucagon, the periportal cells had a significantly higher rate of ketogenesis (from either palmitate or octanoate as substrate), but a lower 3-hydroxybutyrate/acetoacetate production ratio, suggesting a more oxidized mitochondrial NADH/NAD+ redox state despite the higher rate of beta-oxidation. Periportal hepatocytes had a higher activity of carnitine palmitoyltransferase but a lower activity of citrate synthase than did perivenous cells. These findings suggest that: (i) glucagon elicits greater long-term stimulation of gluconeogenesis in periportal than in perivenous hepatocytes maintained in culture; (ii) after culture with glucagon, the rates of ketogenesis and the mitochondrial redox state differ in periportal and perivenous hepatocytes.     

Nonhomogeneous labeling of liver mitochondrial acetyl-CoA

The specific activity of carbons 1 and 2 of plasma acetoacetate has been used as a measure of the specific activity of liver mitochondrial acetyl-CoA in tracer studies. To test whether or not acetoacetate actually reflects acetyl-CoA, livers were perfused with a mixture of substrates that are converted to mitochondrial acetyl-CoA: 1 mM lactate, 0.2 mM pyruvate, 0.2 mM acetate, and, where indicated, 0.2 mM octanoate or 0.2 mM alpha-ketoisocaproate. In each experiment, one of these substrates was 13C-labeled. Labeling of mitochondrial acetyl-CoA was assessed by three methods: (i) molar percent enrichment of total tissue acetyl-CoA; (ii) molar percent enrichment of carbons 4 and 5 of tissue citrate, the precursor of which is acetyl-CoA; and (iii) molar percent enrichment of carbons 1 and 2 of perfusate ketone bodies. Nonhomogeneous labeling of liver mitochondrial acetyl-CoA occurred under most conditions, i.e. the enrichments of carbons 4 and 5 of citrate were different from enrichments of carbons 1 and 2 of ketone bodies. Thus, based upon our results obtained in perfused livers, we question the validity of measuring the labeling of carbons 1 and 2 of acetoacetate as a noninvasive probe of liver mitochondrial acetyl-CoA.     

Enzymes involved in anaerobic degradation of acetone by a denitrifying bacterium

The pathway of anaerobic acetone degradation by the denitrifying bacterial strain BunN was studied by enzyme measurements in extracts of anaerobic acetone-grown cells. An ADP- and MgCl₂-dependent decarboxylation of acetoacetate was detected which could not be found in cell-free extracts of acetate-grown cells. It is concluded that free acetoacetate is formed by ATP-dependent carboxylation of acetone. Acetoacetate was converted into its coenzyme A ester by succinyl-CoA: acetoacetate CoA transferase, and cleaved by a thiolase into acetyl-CoA. The acetyl residue was completely oxidized in the citric acid cycle. The ADP-dependent decarboxylation of acetoacetate was inhibited by EDTA, but not by avidin. High myokinase activities led to equilibrium amounts of ATP, ADP, and AMP in the reaction mixtures, and prevented determination of the decarboxylase reaction stoichiometry, therefore.Abbreviations ADP adenosine diphosphate - AMP adenosine monophosphate - ATP adenosine triphosphate - BSA bovine serum albumine - MOPS 3-(N-morpholino)propanesulfonic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PHB poly--hydroxybutyrate - Tris Tris-(hydroxymethyl-) aminomethane     

Quantitation of ketogenesis in periportal and pericentral regions of the liver lobule

A method has been devised to quantitate rates of ketogenesis (acetoacetate + beta-hydroxybutyrate production) in discrete regions of the liver lobule based on changes in NADH fluorescence. In perfused livers from fasted rats, ketogenesis was inhibited nearly completely with either 2-bromoctanoate (600 microM) or 2-tetradecylglycidic acid (25 microM). During inhibition of ketogenesis, a linear relationship (r = 0.90) was observed between decreases in NADH fluorescence detected from the liver surface and decreases in ketone body production. NADH fluorescence was monitored subsequently from individual regions of the liver lobule by placing microlight guides on periportal and pericentral regions of the liver lobule visible on the liver surface. Rates of ketogenesis in sublobular regions were calculated from regional decreases in NADH fluorescence and changes in the rate of ketone body formation by the whole liver during infusion of inhibitors. In the presence of bromoctanoate, ketogenesis was reduced 80% and local rates of ketogenesis were decreased 31 +/- 4 mumol/g/h in periportal areas and 28 +/- 3 mumol/g/h in pericentral regions. Similar results were observed with tetradecylglycidic acid. Therefore, it was concluded that submaximal rates of ketogenesis from endogenous, mainly long-chain fatty acids are nearly equal in periportal and pericentral regions of the liver lobule in liver from fasted rats. Rates of ketogenesis and NADH fluorescence were strongly correlated during fatty acid infusion. Infusion of 250 microM oleate increased NADH fluorescence maximally by 8 +/- 1% over basal values in periportal regions and 17 +/- 4% in pericentral areas. Local rates of ketogenesis, calculated from these changes in fluorescence, increased 35 +/- 6 mumol/g/h in periportal areas and 55 +/- 5 mumol/g/h in pericentral regions. Thus, oleate stimulated ketogenesis nearly 60% more in pericentral than in periportal regions of the liver lobule.     

Changes in n-6 and n-3 polyunsaturated fatty acids during lipid-peroxidation of mitochondria obtained from rat liver and several brain regions: effect of alpha-tocopherol

The effect of intraperitoneal administration of alpha-tocopherol (100 mg/kg weight/24 h) on ascorbate (0-0.4 mM) induced lipid peroxidation of mitochondria isolated from rat liver, cerebral hemispheres, brain stem and cerebellum was examined. The ascorbate induced light emission in hepatic mitochondria was nearly completely inhibited by alpha-tocopherol (control-group: 114.32+/-14.4; vitamin E-group: 17.45+/-2.84, c.p.m.x10(-4)). In brain mitochondria, 0.2 mM ascorbate produced the maximal chemiluminescence and significant differences among both groups were not observed. No significant differences in the chemiluminescence values between control and vitamin E treated groups were observed when the three brain regions were compared. The light emission produced by mitochondrial preparations was much higher in cerebral hemispheres than in brain stem and cerebellum. In liver and brain mitochondria from control group, the level of arachidonic acid (C20:4n6) and docosahexaenoic acid (C22:6n3) was profoundly affected. Docosahexaenoic in liver mitochondria from vitamin E group decreased by 30% upon treatment with ascorbic acid when compared with mitochondria lacking ascorbic acid. As a consequence of vitamin E treatment, a significant increase of C22:6n3 was detected in rat liver mitochondria (control-group: 6.42 +/-0.12; vitamin E-group: 10.52 +/-0.46). Ratios of the alpha-tocopherol concentrations in mitochondria from rats receiving vitamin E to those of control rats were as follows: liver, 7.79; cerebral hemispheres, 0.81; brain stem, 0.95; cerebellum, 1.05. In liver mitochondria, vitamin E shows a protector effect on oxidative damage. In addition, vitamin E concentration can be increased in hepatic but not in brain mitochondria. Lipid peroxidation mainly affected, arachidonic (C20:4n6) and docosahexaenoic (C22:6n3) acids.     

Formation and utilization of 3-hydroxy-3-methylglutarate in liver mitochondria of starved and streptozotocin-diabetic rats.

Kidney and liver mitochondria of rat, rabbit and guinea pig are able to transform 3-hydroxy-3-methylglutarate into acetoacetate, whereas ox liver mitochondria and rat mitochondria of heart, diaphragm and brain do not exhibit such an activity. Starvation and streptozotocin treatment decreases the formation of acetoacetate from 3-hydroxy-3-methylglutarate. Addition of acetoacetate and succinate to the incubation media of mitochondria results in a decrease in the transformation of 3-hydroxy-3-methylglutarate into acetoacetate. A 3-hydroxy-3-methylglutaryl-CoA hydrolase is present in rat liver mitochondria; the activity does not show appreciable changes after starvation or streptozotocin treatment.     

The Stability and Automatic Determination of Ketone Bodies in Blood Samples Taken in Field Conditions

An automated, spectro-photometric determination of blood acetoacetate and β-hydroxybutyrate was developed with a Gilford 3500 autoanalyzer. The stability of ketone bodies was studied in different conditions. An immediate precipitation with 0.6 M perchloric acid and cooling the sample effectively prevent the loss of acetoacetate from samples during transport to the laboratory (at 4°C a 6 % loss of acetoacetate was noted during 24 h). Freezing the sample makes it practically stable (less than 2 % loss of acetoacetate per week during a study lasting 2 months). At room temperature (20°C) the sample’s acetoacetate was instable and disappeared with a rate of 6 % per h. β-hydroxybutyrate was stable in precipitated samples. Because the precipitation also retains the sample’s glucose, 3 main parameters for the indication of ketosis could be analyzed automatically from the same sample with a total capacity of 40 samples in 2½ h.     

Stimulation by 3-hydroxybutyrate of pyruvate carboxylation in mitochondria from rat liver

Isolated rat liver mitochondria incubated in the presence of 3-hydroxybutyrate display a markedly increased rate of pyruvate carboxylation as measured by malate and citrate production from pyruvate. The stimulation was demonstrable both with exogenously added pyruvate, even at saturating concentration, and with pyruvate intramitochondrially generated from alanine. The concentration of DL-3-hydroxybutyrate required for half-maximal stimulation amounted to about 1.5 mM. The intramitochondrial ATP/ADP ratio as well as the matrix acetyl-CoA level was found to remain unchanged by 3-hydroxybutyrate exposure, which, however, lowered the absolute intramitochondrial contents of the respective adenine nucleotides. The effects of 3-hydroxybutyrate were diminished by the concomitant addition of acetoacetate. Moreover, a direct relationship between mitochondrial reduction by proline and the rate of pyruvate carboxylation was observed. The results seem to indicate that the mitochondrial oxidation--reduction state might be involved in the expression of the 3-hydroxybutyrate effect. As to the physiological relevance of the findings, 3-hydroxybutyrate could be shown to activate pyruvate carboxylation in isolated hepatocytes.     

Acetoacetate metabolism in rat brain. Development of acetoacetyl-coenzyme A deacylase and 3-hydroxy-3-methylglutaryl-coenzyme A synthase.

1. Data are provided that indicate that the rat brain acetoacetyl-CoA deacylase is almost exclusively mitochondrial. Developmental studies show that this enzyme more than doubles its activity during suckling (0--21 days) and then maintains this activity in adults (approx. 1.1 units/g wet wt.). 2. Kinetic studies (on the acetoacetyl-CoA deacylase) in a purified brain mitochondrial preparation give a Vmax. of 47 nmol/min per mg of protein, and a Km for acetoacetyl-CoA of 5.2 micron and are compatible with substrate inhibition by acetoacetyl-CoA above concentrations of 47 micron. 3. The total brain 3-hydroxy-3-methyl-glutaryl-CoA synthase remains constant in the developing and adult rat brain (approx. 1.2 units/g wet wt.). This enzyme is located in both the mitochondrial and cytosolic fractions. During suckling (0--21 days) the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase represents approx. one-third of the total, but this increases markedly to about 60% of the total in the adult. The cytosolic enzyme correspondingly falls to approx. 40% of the total. 4. The role of the acetoacetyl-CoA deacylase in providing cytosolic acetoacetate for biosynthetic activities in the developing brain is discussed.     

A possible mechanism for the anti-ketogenic action of alanine in the rat

1. The anti-ketogenic effect of alanine has been studied in normal starved and diabetic rats by infusing l-alanine for 90min in the presence of somatostatin (10μg/kg body wt. per h) to suppress endogenous insulin and glucagon secretion. 2. Infusion of alanine at 3mmol/kg body wt. per h caused a 70±11% decrease in [3-hydroxybutyrate] and a 58±9% decrease in [acetoacetate] in 48h-starved rats. [Glucose] and [lactate] increased, but [non-esterified fatty acid], [glycerol] and [3-hydroxybutyrate]/[acetoacetate] were unchanged. 3. Infusion of alanine at 1mmol/kg body wt. per h caused similar decreases in [ketone body] (3-hydroxybutyrate plus acetoacetate) in 24h-starved normal and diabetic rats, but no change in other blood metabolites. 4. Alanine [3mmol/kg body wt. per h] caused a 72±9% decrease in the rate of production of ketone bodies and a 57±8% decrease in disappearance rate as assessed by [3-¹⁴C]acetoacetate infusion. Metabolic clearance was unchanged, indicating that the primary effect of alanine was inhibition of hepatic ketogenesis. 5. Aspartate infusion at 6mmol/kg body wt. per h had similar effects on blood ketone-body concentrations in 48h-starved rats. 6. Alanine (3mmol/kg body wt. per h) caused marked increases in hepatic glutamate, aspartate, malate, lactate and citrate, phosphoenolpyruvate, 2-phosphoglycerate and glucose concentrations and highly significant decreases in [3-hydroxybutyrate] and [acetoacetate]. Calculated [oxaloacetate] was increased 75%. 7. Similar changes in hepatic [malate], [aspartate] and [ketone bodies] were found after infusion of 6mmol of aspartate/kg body wt. per h. 8. It is suggested that the anti-ketogenic effect of alanine is secondary to an increase in hepatic oxaloacetate and hence citrate formation with decreased availability of acetyl-CoA for ketogenesis. The reciprocal negative-feedback cycle of alanine and ketone bodies forms an important non-hormonal regulatory system.