Interrelations between C4 Ketogenesis, C5 Ketogenesis, and Anaplerosis in the Perfused Rat Liver

We investigated the interrelations between C₄ ketogenesis (production of β-hydroxybutyrate + acetoacetate), C₅ ketogenesis (production of β-hydroxypentanoate + β-ketopentanoate), and anaplerosis in isolated rat livers perfused with ¹³C-labeled octanoate, heptanoate, or propionate. Mass isotopomer analysis of C₄ and C₅ ketone bodies and of related acyl-CoA esters reveal that C₄ and C₅ ketogenesis share the same pool of acetyl-CoA. Although the uptake of octanoate and heptanoate by the liver are similar, the rate of C₅ ketogenesis from heptanoate is much lower than the rate of C₄ ketogenesis from octanoate. This results from the channeling of the propionyl moiety of heptanoate into anaplerosis of the citric acid cycle. C₅ ketogenesis from propionate is virtually nil because acetoacyl-CoA thiolase does not favor the formation of β-ketopentanoyl-CoA from propionyl-CoA and acetyl-CoA. Anaplerosis and gluconeogenesis from heptanoate are inhibited by octanoate. The data have implications for the design of diets for the treatment of long chain fatty acid oxidation disorders, such as the triheptanoin-based diet.The regulation of the metabolism of C₄ ketone bodies, i.e. β-hydroxybutyrate (BHB)² and acetoacetate (AcAc) has been extensively investigated in vivo in isolated livers, hepatocytes, and subcellular preparations (for reviews, see Refs. 1–4). In contrast, very little information is available on the metabolism of C₅ ketone bodies, i.e. β-hydroxypentanoate (BHP) and β-ketopentanoate (BKP), which are known in the clinical literature as 3-hydroxyvalerate and 3-ketovalerate (5, 6). The C₅ ketone bodies are formed in liver from the partial oxidation of odd-chain fatty acids (see Fig. 1, center column). C₅ ketogenesis uses the same enzymes of the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) cycle as C₄ ketogenesis. The counterpart of HMG-CoA in C₅ ketogenesis is 3-hydroxy-3-ethylglutaryl-CoA (HEG-CoA). We only found one report on the formation of [¹⁴C]HEG-CoA in liver extract incubated with propionyl-CoA and [1-¹⁴C]acetyl-CoA (7).Open in a separate windowFIGURE 1.Scheme of C₄ ketogenesis and C₅ ketogenesis in the liver. Numbers refer to the following enzymes: 3-ketoacyl-CoA thiolase (1), HMG-CoA synthase (2), HMG-CoA lyase (3), and β-hydroxybutyrate dehydrogenase (4). The figure also shows the link between propionyl-CoA and the CAC via anaplerosis.Because odd-chain fatty acids are absent from the diet of non-ruminant mammals, body fluids contain only traces of C₅ ketone bodies. However, C₅ ketone bodies and hydroxyethylglutarate are found in body fluids of patients with disorders of the anaplerotic pathway, propionyl-CoA → methylmalonyl- CoA → succinyl-CoA, such as deficiency in propionyl-CoA carboxylase and methylmalonyl-CoA mutase as well as biotin or vitamin B12 deficiency (5, 6, 8). The formation of C₅ ketone bodies in these pathological states involves either the conversion of propionyl-CoA to BKP-CoA via 3-ketoacyl-CoA thiolase (Fig. 1, reaction 1) or the β-oxidation of odd-chain fatty acids synthesized in these patients (9) using propionyl-CoA as a primer (10).Like their C₄ counterparts, the C₅ ketone bodies are interconverted by mitochondrial BHB dehydrogenase (11). In peripheral tissues, C₅ ketone bodies are converted to propionyl-CoA (which is anaplerotic) + acetyl-CoA via 3-oxoacid-CoA transferase (12) and 3-ketoacyl-CoA thiolase. Peripheral tissues have a high capacity to utilize exogenous C₅ ketone bodies (13), especially heart, kidney, and brain, which have high activities of 3-oxoacid-CoA transferase (14, 15).Our interest in C₅ ketone body metabolism arose from an ongoing clinical trial where patients with long chain fatty acid oxidation disorders are treated with a diet containing triheptanoin (16, 17) instead of the classical treatment with the even-chain triglyceride trioctanoin. These patients suffer from muscle weakness and rhabdomyolysis, manifested by the release of creatine kinase in plasma. It was hypothesized that the accumulation of long chain acyl-CoAs and long chain acylcarnitines results in membrane damage with release of large and small molecules from cells. The leakage of small molecules would deplete intermediates of the citric acid cycle (CAC) which carry acetyl groups as they are oxidized. It was further hypothesized that boosting anaplerosis with a suitable substrate would compensate for the chronic cataplerosis and improve heart and muscle function. The catabolism of heptanoate yields propionyl-CoA, which can be used for anaplerosis in most tissues, and C₅ ketone bodies in liver. C₅ ketone bodies are converted to propionyl-CoA, which can be used for anaplerosis in peripheral tissues. The marked improvement of the patients'' conditions after switching from a trioctanoin- to a triheptanoin-based diet supported the hypothesis.After ingestion of meals containing triheptanoin as the only lipid component, both C₅ ketone bodies and C₄ ketone bodies accumulated in the plasma of patients that have been diagnosed with disorders of long chain fatty acid oxidation (16). This suggested that acetyl groups derived from heptanoate can be used for the synthesis of C₄ and C₅ ketone bodies. Alternatively, the accumulation of C₄ ketone bodies after triheptanoin ingestion might result from the inhibition of the utilization of C₄ ketone bodies in peripheral tissues by C₅ ketone bodies.The aim of the present study was to investigate the interaction between C₄ and C₅ ketogenesis in rat livers perfused with octanoate and/or heptanoate. To gain insight on the fates of the acetyl groups of both fatty acids and on the fate of the propionyl-CoA moiety of heptanoate, we conducted the experiments with a series of labeled substrates: [1-¹³C]octanoate, [8-¹³C]octanoate, [5,6,7-¹³C₃]heptanoate, [1-¹³C]heptanoate, and [¹³C₃]propionate. The outcome of the propionyl-CoA moiety of [5,6,7-¹³C₃]heptanoate and [¹³C₃]propionate was traced by measurements of anaplerosis and glucose labeling by mass isotopomer³ analysis (18). In previous studies on the metabolism of odd-chain fatty acids in liver or hepatocytes (19, 20), ketone bodies were assayed with BHB dehydrogenase. This assay does not differentiate C₄ from C₅ ketone bodies. In the present study we used gas chromatography-mass spectrometry to specifically assay C₄ and C₅ ketone bodies (13).     

Multiple Mass Isotopomer Tracing of Acetyl-CoA Metabolism in Langendorff-perfused Rat Hearts: CHANNELING OF ACETYL-CoA FROM PYRUVATE DEHYDROGENASE TO CARNITINE ACETYLTRANSFERASE*

We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [¹³C₂,²H₃]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [¹³C₆]glucose or [1,2-¹³C₂]palmitate) or/and M1 acetyl-CoA (e.g. [1-¹³C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA.     

Metabolic Modeling of Dynamic Brain 13C NMR Multiplet Data: Concepts and Simulations with a Two-Compartment Neuronal-Glial Model

Metabolic modeling of dynamic ¹³C labeling curves during infusion of ¹³C-labeled substrates allows quantitative measurements of metabolic rates in vivo. However metabolic modeling studies performed in the brain to date have only modeled time courses of total isotopic enrichment at individual carbon positions (positional enrichments), not taking advantage of the additional dynamic ¹³C isotopomer information available from fine-structure multiplets in ¹³C spectra. Here we introduce a new ¹³C metabolic modeling approach using the concept of bonded cumulative isotopomers, or bonded cumomers. The direct relationship between bonded cumomers and ¹³C multiplets enables fitting of the dynamic multiplet data. The potential of this new approach is demonstrated using Monte-Carlo simulations with a brain two-compartment neuronal-glial model. The precision of positional and cumomer approaches are compared for two different metabolic models (with and without glutamine dilution) and for different infusion protocols ([1,6-¹³C₂]glucose, [1,2-¹³C₂]acetate, and double infusion [1,6-¹³C₂]glucose?+?[1,2-¹³C₂]acetate). In all cases, the bonded cumomer approach gives better precision than the positional approach. In addition, of the three different infusion protocols considered here, the double infusion protocol combined with dynamic bonded cumomer modeling appears the most robust for precise determination of all fluxes in the model. The concepts and simulations introduced in the present study set the foundation for taking full advantage of the available dynamic ¹³C multiplet data in metabolic modeling.     

Catabolism of Branched Chain Amino Acids Contributes Significantly to Synthesis of Odd-Chain and Even-Chain Fatty Acids in 3T3-L1 Adipocytes

The branched chain amino acids (BCAA) valine, leucine and isoleucine have been implicated in a number of diseases including obesity, insulin resistance, and type 2 diabetes mellitus, although the mechanisms are still poorly understood. Adipose tissue plays an important role in BCAA homeostasis by actively metabolizing circulating BCAA. In this work, we have investigated the link between BCAA catabolism and fatty acid synthesis in 3T3-L1 adipocytes using parallel ¹³C-labeling experiments, mass spectrometry and model-based isotopomer data analysis. Specifically, we performed parallel labeling experiments with four fully ¹³C-labeled tracers, [U-¹³C]valine, [U-¹³C]leucine, [U-¹³C]isoleucine and [U-¹³C]glutamine. We measured mass isotopomer distributions of fatty acids and intracellular metabolites by GC-MS and analyzed the data using the isotopomer spectral analysis (ISA) framework. We demonstrate that 3T3-L1 adipocytes accumulate significant amounts of even chain length (C14:0, C16:0 and C18:0) and odd chain length (C15:0 and C17:0) fatty acids under standard cell culture conditions. Using a novel GC-MS method, we demonstrate that propionyl-CoA acts as the primer on fatty acid synthase for the production of odd chain fatty acids. BCAA contributed significantly to the production of all fatty acids. Leucine and isoleucine contributed at least 25% to lipogenic acetyl-CoA pool, and valine and isoleucine contributed 100% to lipogenic propionyl-CoA pool. Our results further suggest that low activity of methylmalonyl-CoA mutase and mass action kinetics of propionyl-CoA on fatty acid synthase result in high rates of odd chain fatty acid synthesis in 3T3-L1 cells. Overall, this work provides important new insights into the connection between BCAA catabolism and fatty acid synthesis in adipocytes and underscores the high capacity of adipocytes for metabolizing BCAA.     

Fatty Acid Chain Elongation in Palmitate-perfused Working Rat Heart: MITOCHONDRIAL ACETYL-CoA IS THE SOURCE OF TWO-CARBON UNITS FOR CHAIN ELONGATION*

Rat hearts were perfused with [1,2,3,4-¹³C₄]palmitic acid (M+4), and the isotopic patterns of myocardial acylcarnitines and acyl-CoAs were analyzed using ultra-HPLC-MS/MS. The 91.2% ¹³C enrichment in palmitoylcarnitine shows that little endogenous (M+0) palmitate contributed to its formation. The presence of M+2 myristoylcarnitine (95.7%) and M+2 acetylcarnitine (19.4%) is evidence for β-oxidation of perfused M+4 palmitic acid. Identical enrichment data were obtained in the respective acyl-CoAs. The relative ¹³C enrichment in M+4 (84.7%, 69.9%) and M+6 (16.2%, 17.8%) stearoyl- and arachidylcarnitine, respectively, clearly shows that the perfused palmitate is chain-elongated. The observed enrichment of ¹³C in acetylcarnitine (19%), M+6 stearoylcarnitine (16.2%), and M+6 arachidylcarnitine (17.8%) suggests that the majority of two-carbon units for chain elongation are derived from β-oxidation of [1,2,3,4-¹³C₄]palmitic acid. These data are explained by conversion of the M+2 acetyl-CoA to M+2 malonyl-CoA, which serves as the acceptor for M+4 palmitoyl-CoA in chain elongation. Indeed, the ¹³C enrichment in mitochondrial acetyl-CoA (18.9%) and malonyl-CoA (19.9%) are identical. No ¹³C enrichment was found in acylcarnitine species with carbon chain lengths between 4 and 12, arguing against the simple reversal of fatty acid β-oxidation. Furthermore, isolated, intact rat heart mitochondria 1) synthesize malonyl-CoA with simultaneous inhibition of carnitine palmitoyltransferase 1b and 2) catalyze the palmitoyl-CoA-dependent incorporation of ¹⁴C from [2-¹⁴C]malonyl-CoA into lipid-soluble products. In conclusion, rat heart has the capability to chain-elongate fatty acids using mitochondria-derived two-carbon chain extenders. The data suggest that the chain elongation process is localized on the outer surface of the mitochondrial outer membrane.     

Ricinoleic Acid Catabolism in Peroxisomes

Peroxisomes from castor bean endosperm and mung bean hypocotyl completely degrade ricinoleic acid (12-D-hydroxy-9-cis-octadecenoic acid) to acetyl-CoA. Concomitant NADH formation occurred with a stoichiometry of 9 nmol NADH formed per 1 nmol ricinoleate degraded. At the C₈-intermediate level, where the hydroxy group of ricinoleic acid forms a barrier to β-oxidation, 2-hydroxyoctanoate and 2-oxooctanoate were detected as intermediates. 2-Hydroxyoctanoate was oxidized to 2-oxooctanoate with H₂O₂ producing a reaction exhibiting 1:1 stoichiometry of the products. The peroxisomes appeared to oxidize both isomers of racemic 2-hydroxyoctanoate. 2-Oxooctanoate was metabolized to heptanoyl-CoA (propionyl-CoA and acetyl-CoA) in a NAD-dependent, but ATP-independent, reaction. Heptanoate was not detected as an intermediate. Imidazole, an inhibitor of α-oxidation, did not effect the degradation of ricinoleate or 2-oxooctanoate. Arsenite, an inhibitor of oxidative decarboxylation, inhibited the metabolism of ricinoleate at the C₈-intermediate level, according to the accumulation of 2-oxooctanoate and the stoichiometry of concomitant NADH formation. Arsenite completely inhibited the metabolism of 2-oxooctanoate. It is concluded that the barrier caused by the hydroxy group of ricinoleic acid and prevention of β-oxidation at the C₈-intermediate level, is circumvented by an α-hydroxy acid oxidase reaction followed by an oxidative decarboxylation allowing return to the β-oxidation track.     

Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation

The pathway of autotrophic CO₂ fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO₂-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either ¹⁴CO₂ or [¹⁴C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for C. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus and Acidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula, S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO₂ fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.     

Effect of Organic Acids on Shrimp Pathogen, <Emphasis Type="Italic">Vibrio harveyi</Emphasis>

Shrimp farming accounts for more than 40% of the world shrimp production. Luminous vibriosis is a shrimp disease that causes major economic losses in the shrimp industry as a result of massive shrimp kills due to infection. Some farms in the South Asia use antibiotics to control Vibrio harveyi, a responsible pathogen for luminous vibriosis. However, the antibiotic-resistant strain was found recently in many shrimp farms, which makes it necessary to develop alternative pathogen control methods. Short-chain fatty acids are metabolic products of organisms, and they have been used as food preservatives for a long time. Organic acids are also commonly added in feeds in animal husbandry, but not in aquaculture. In this study, growth inhibitory effects of short-chain fatty acids, namely formic acid, acetic acid, propionic acid, and butyric acid, on V. harveyi were investigated. Among four acids, formic acid showed the strongest inhibitory effect followed by acetic acid, propionic acid, and butyric acid. The minimum inhibitory concentration (MIC) of 0.035% formic acid suppressed growth of V. harveyi. The major inhibitory mechanism seems to be the pH effect of organic acids. The effective concentration 50 (EC₅₀) values at 96 h inoculation for all organic acids were determined to be 0.023, 0.041, 0.03, and 0.066% for formic, acetic, propionic, and butyric acid, respectively. The laboratory study results are encouraging to formulate shrimp feeds with organic acids to control vibrio infection in shrimp aquaculture farms.     

Comparison of gas chromatography–mass spectrometry and gas chromatography–combustion–isotope ratio mass spectrometry analysis for in vivo estimates of metabolic fluxes

Gas chromatography–mass spectrometry (GC–MS) was compared with gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for measurements of cholesterol ¹³C enrichment after infusion of labeled precursor ([¹³C_1,2]acetate). Paired results were significantly correlated, although GC–MS was less accurate than GC–C–IRMS for higher enrichments. Nevertheless, only GC–MS was able to provide information on isotopologue distribution, bringing new insights to lipid metabolism. Therefore, we assessed the isotopologue distribution of cholesterol in humans and dogs known to present contrasted cholesterol metabolic pathways. The labeled tracer incorporation was different in both species, highlighting the subsidiarity of GC–MS and GC–C–IRMS to analyze in vivo stable isotope studies.     

Effect in the cow of intraruminal infusions of volatile fatty acids and of lactic acid on the secretion of the component fatty acids of the milk fat and on the composition of blood

1. The effects in the cow of intraruminal infusions of acetic acid, propionic acid or butyric acid on the secretion of the component fatty acids of the milk fat, and of these acids and of lactic acid on the composition of the blood plasma of the jugular vein, have been studied. 2. The infusion of acetic acid or butyric acid increased the yield of the C₄–C₁₆ acids of milk fat but decreased the yield of C₁₈ acids. The infusion of propionic acid decreased the yields of all major component acids except palmitic acid and possibly lauric acid. 3. The changes in the concentrations in blood plasma of glucose and of ketone bodies were consistent with the glucogenic effect of propionic acid and the ketogenic effects of butyric acid and acetic acid. The effects of lactic acid were not consistent from cow to cow. Only with the infusion of acetic acid was a significant increase in the concentration of total volatile fatty acids in blood plasma found. Infusions of butyric acid and of propionic acid tended to depress the concentration of citric acid in the blood plasma and infusion of acetic acid increased it. No consistent effects of the infused acids on the concentration in blood plasma of esterified cholesterol, free cholesterol, triglyceride or phospholipid were observed. 4. The possibility is discussed that the effects of the infused acids on milk-fat secretion are caused through an alteration of the concentrations of precursors of milk fat in mammary arterial blood.     

Elongation of acyl-CoAs by microsomes from etiolated leek seedlings

Long chain fatty acid synthesis was studied using etiolated leek seedling microsomes. In the presence of ATP, [2-¹⁴C]malonyl-CoA was incorporated into fatty acids of C₁₆C₂₆. The omission of ATP, even in the presence of acetyl-CoA, led to a complete loss of activity, which was restored by addition of exogeneous acyl-CoAs. Comparison of acyl-CoA (C₁₂C₂₄) elongation showed that stearoyl-CoA, in the presence of [2-¹⁴C]malonyl-CoA, was the more efficient precursor leading to the formation of fatty acids having a chain length of C₂₀C₂₆. [1-¹⁴C]C₁₆CoA and [1-¹⁴C]C₁₈CoA were elongated in the presence of malonyl-CoA, without degradation of the acyl chain. The time-course and the malonyl-CoA concentration curves showed that [1-¹⁴C]C₁₈CoA was a better primer than [1-¹⁴C]C₁₆CoA. Acyl-CoA elongation was also studied over the concentration range 4.5–45 μM [1-¹⁴C]C₁₈CoA. Comparison of the radioactivity incorporated into the fatty acids formed using [2-¹⁴C]malonyl-CoA in the presence of C₁₈CoA, on the one hand, and [1-¹⁴C]C₁₈CoA in the presence of malonyl-CoA, on the other, demonstrated clearly that the acyl chain of the acyl-CoA was elongated by malonyl-CoA.     

Metabolic Pathway for Propionate Utilization by Phosphorus-Accumulating Organisms in Activated Sludge: 13C Labeling and In Vivo Nuclear Magnetic Resonance

In vivo ¹³C and ³¹P nuclear magnetic resonance techniques were used to study propionate metabolism by activated sludge in enhanced biological phosphorus removal systems. The fate of label supplied in [3-¹³C]propionate was monitored in living cells subjected to anaerobic/aerobic cycles. During the anaerobic phase, propionate was converted to polyhydroxyalkanoates (PHA) with the following monomer composition: hydroxyvalerate, 74.2%; hydroxymethylvalerate, 16.9%; hydroxymethylbutyrate, 8.6%; and hydroxybutyrate, 0.3%. The isotopic enrichment in the different carbon atoms of hydroxyvalerate (HV) produced during the first anaerobic stage was determined: HV₅, 59%; HV₄, 5.0%; HV₃, 1.1%; HV₂, 3.5%; and HV₁, 2.8%. A large proportion of the supplied label ended up on carbon C-5 of HV, directly derived from the pool of propionyl-coenzyme A (CoA), which is primarily labeled on C-3; useful information on the nature of operating metabolic pathways was provided by the extent of labeling on C-1, C-2, and C-4. The labeling pattern on C-1 and C-2 was explained by the conversion of propionyl-CoA to acetyl-CoA via succinyl-CoA and the left branch of the tricarboxylic acid cycle, which involves scrambling of label between the inner carbons of succinate. This constitutes solid evidence for the operation of succinate dehydrogenase under anaerobic conditions. The labeling in HV₄ is explained by backflux from succinate to propionyl-CoA. The involvement of glycogen in the metabolism of propionate was also demonstrated; moreover, it was shown that the acetyl moiety to the synthesis of PHA was derived preferentially from glycogen. According to the proposed metabolic scheme, the decarboxylation of pyruvate is coupled to the production of hydrogen, and the missing reducing equivalents should be derived from a source other than glycogen metabolism.     

The loss of carbon-20 in C19-gibberellin biosynthesis in a cell-free system from Pisum sativum L.

The fate of the carbon-20 atom in gibberellin (GA) biosynthesis was studied in a cell-free system from Pisum sativum. This carbon atom is lost at the aldehyde stage of oxidation when C₂₀-GAs are converted to C₁₉-GAs. Gibberellin A₁₂ labeled with ¹⁴C at C-20 was prepared from [3-¹⁴C]mevalonic acid with a cell-free system from Cucurbita maxima and incubated with the pea system. Analysis of the gas and aqueous phases showed that ¹⁴CO₂ was formed at the same rate and in nearly equivalent amounts as ¹⁴C-labeled C₁₉-GAs whereas [¹⁴C]formic acid and [¹⁴C]formaldehyde were not detectable. The possibility that C-20 had been lost as formic acid which had then been converted to CO₂ was investigated by control incubations with [¹⁴C]formic acid. The rate of release of ¹⁴CO₂ from [¹⁴C]formic acid was only one fiftieth of the rate of ¹⁴CO₂ release from [¹⁴C]GA₁₂ as the substrate. We conclude that in the formation of C₁₉-GAs from C₂₀-GAs, the C-20 is removed directly as CO₂.Abbreviations GA_n Gibberellin A_n     

Propionyl-CoA carboxylase of Myxococcus xanthus: catalytic properties and function in developing cells

An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase. The apparent K _m values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0.2, 0.03, and 1.0 mM, respectively. The native enzyme has a molecular mass of 605–615 kDa and is composed of nonidentical subunits (α and β) with molecular masses of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0–7.5 and at 25–30°C, and was affected by variation in concentrations of ATP and Mg²⁺. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage. Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development. Received: 24 February 1998 / Accepted: 25 May 1998     

A microsomal fatty acid synthetase coupled to acyl-CoA reductase in Euglena gracilis

Microsomal particles from dark-grown Euglena gracilis incorporated malonyl-CoA into fatty acids and fatty alcohols in the presence of acetyl-CoA, NADH, NADPH, and ATP with an optimum pH of 8.0. Schmidt degradation of the individual fatty acids derived from [l,3-¹⁴C]malonyl-CoA showed that the microsomal fatty acid synthesis was a de novo type. Detailed analysis of the products formed in the absence of various cofactors showed that the role of ATP was specifically in the formation of fatty alcohols and that fatty acid reduction specifically required NADH.The major aliphatic chains synthesized by the microsomes were C₁₆, C₁₈, and C₁₄ in both the acyl portions and alcohols. Although relative concentrations of acetyl-CoA and malonyl-CoA influenced the chain length distribution of products, C₁₆remained the major product in both the alcohol and the acid fractions. Effects of NADPH and NADH concentrations on malonyl-CoA incorporation suggested that the two reductive steps involved in the microsomal fatty acid synthesis have different pyridine nucleotide specificity. The apparent K_m for malonyl-CoA was 4.2 × 10^?4m. Based on the experimental results a mechanism is suggested by which carbon is channeled into wax esters under conditions of nutritional abundance in dark-grown E. gracilis.     

Simplified analysis of acetaminophen glucuronide for quantifying gluconeogenesis and glycogenolysis using deuterated water

Measurement of acetaminophen glucuronide (AG) ²H enrichment from deuterated water (²H₂O) by ²H nuclear magnetic resonance (NMR) analysis of its monoacetone glucose (MAG) derivative provides estimation of gluconeogenic and glycogenolytic contributions to endogenous glucose production (EGP). However, AG derivatization to MAG is laborious and unsuitable for high-throughput studies. An alternative derivative, 5-O-acetyl monoacetone glucuronolactone (MAGLA), was tested. Eleven healthy subjects ingested ²H₂O to 0.5% body water enrichment and 500 mg of acetaminophen. Plasma glucose and urinary glucuronide positional ²H enrichments were measured by ²H NMR spectroscopy of MAG and MAGLA, respectively. A Bland–Altman analysis indicated agreement at the 95% confidence level between glucose and glucuronide estimates.     

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.     

Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets

Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [¹³C₁]propionate and [¹³C₃]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[²H₂]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer studies confirmed that the new acyl-CoA was trans-2-methyl-2-pentenoyl-CoA. Acyl-CoA profiles following treatment of HepG2 cells with mono-unsaturated six-carbon fatty acids also supported this conclusion. Similar results were obtained with human platelets, mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells. Interestingly, trans-2-methyl-2-pentenoyl-CoA corresponds to a previously described acylcarnitine tentatively described in patients with propionic and methylmalonic acidemia. We have proposed a mechanism for this metabolic route consistent with all of the above findings.     

Influence of phytoplankton on the response of bacterioplankton growth to nutrient enrichment

1. Laboratory experiments were conducted to test the effect of nutrient enrichment on bacterioplankton growth in the presence and absence of phytoplankton. 2. In one series of experiments, bacterioplankton growth in terms of specific activity [³H-thymidine incorporation (cell number)^?1] was greater in whole lake water samples than in samples from which phytoplankton had been removed by filtration (1.0 μm), regardless of the nutrient enrichments (control, NH⁺₄ plus PO^3-₄ and mannitol). Organic C enhanced bacterioplankton growth in both whole and filtered lake water. 3. In another series of experiments (with the same nutrient enrichments as in the first experiment except that glucose replaced mannitol), bacterioplankton growth in whole lake water enriched with PO^3-₄ plus NH⁺₄ and incubated in the light was greater than in two treatments designed to inhibit photosynthetic activity (+DCMU and dark). Bacterioplankton response to nutrient addition was greatest in the PO^3-₄ plus NH⁺₄ enrichment under all three conditions (light +DCMU, and dark). 4. These results indicate that bacterioplankton growth could be directly limited by inorganic P and N when these elements are in short supply. Enhancement of bacterioplankton growth by phytoplankton occurs only under PO^3-₄ and NH⁺₄ replete environments.