Acid Succinate Research Articles

Lipoic acid and diabetes—Part III: Metabolic role of acetyl dihydrolipoic acid

Rat liver lipoyl transacetylase catalyzes the formation of acetyl dihydrolipoic acid from acetyl coenzyme A and dihydrolipoic acid. In an earlier paper the formation of acetyl dihydrolipoic from pyruvate and dihydrolipoic acid catalyzed by pyruvate dehydrogenase has been reported. Acetyl dihydrolipoic acid is a substrate for citrate synthase, acetyl coenzyme A carboxylase and fatty acid synthetase. The Vmax. for citrate synthase with acetyl dihydrolipoic acid was identical to acetyl coenzyme A (approximately 1 μmol citrate formed/min/mg protein) while the apparent Km was approximately 4 times higher with acetyl dihydrolipoic acid as the substrate. This may be due to the fact that synthetic acetyl dihydrolipoic acid is a mixture of 4 possible isomers and only one of them may be the substrate for the enzymatic reaction. While dihydrolipoic acid can replace coenzyme A in the activation of succinate catalyzed by succinyl coenzyme A synthetase, the transfer of coenzyme A between succinate and acetoacetyl dihydrolipoic acid catalyzed by succinyl coenzyme A: 3 oxo-acid coenzyme A transferase does not occur.

Production of L-malic acid by immobilized bacterial cells of the genus Brevibacterium

A HPLC procedure was developed permitting an exact and rapid determination of L-malate, fumarate, succinate, and other dicarboxylis acids. The method was used to assay the products of fumarate metabolism in two Brevibacterium strains, obtained by screening, and showing a high fumarate conversion both in the cell suspension and in the immobilized system. Polyacrylamide and Ca-alginate were employed as polymeric matrices. The conditions were determined (pH, temperature, ion concentration, effect of inhibitors and detergents) under which L-malate is predominantly formed from fumarate and the conversion degree is the highest (8.60 mmol of L-malate h/g cells). The dependence of the conversion of various types of the polymeric matrix and the stability of the system in a continuous experimental arrangement were also examined. The results of these experiments will be utilized in L-malate technology.

Chemotaxis to aromatic and hydroaromatic acids: comparison of Bradyrhizobium japonicum and Rhizobium trifolii

Rhizobia are bacteria well known for their ability to fix nitrogen in symbiosis with leguminous plants. Members of diverse rhizobial species grow at the expense of hydroaromatic and aromatic compounds commonly found in plant cells and plant litter. Using a quantitative capillary assay to measure chemotaxis, we tested the ability of hydroaromatic acids, selected aromatic acids, and their metabolites to serve as chemoattractants for two distantly related rhizobial species, Bradyrhizobium japonicum and Rhizobium trifolii. Slow-growing B. japonicum I-110 demonstrated positive chemotaxis to shikimate, quinate, protocatechuate, and vanillate; threshold concentrations for the compounds were as low as 10(-6) M. The dicarboxylic acids succinate and beta-ketoadipate, metabolites in the catabolism of many aromatic compounds, were positive chemoattractants with low threshold concentrations as well. Taxis to beta-ketoadipate occurred constitutively and, of the tested compounds, beta-ketoadipate gave the strongest peak response. Taxis to shikimate or quinate was induced by growth on either substrate but not by growth on protocatechuate or succinate. In contrast, fast-growing R. trifolii 2066 was only weakly attracted to quinate and other aromatic and dicarboxylic acids that were strong attractants for B. japonicum. The R. trifolii strain exhibited positive chemotaxis to shikimate, but the threshold concentration of shikimate required to elicit a response (10(-4) M) was 2 orders of magnitude higher than that for the B. japonicum strain.

Stimulation of phosphate transport in the proximal tubule by metabolic substrates.

Studies of phosphate transport in the proximal tubule have recently focused on interactions with cellular metabolism. The present studies demonstrate that two fatty acids, valerate and butyrate, and two tricarboxylic acid cycle intermediates, succinate and malate, stimulate net phosphate transport in the rabbit proximal tubule by 34-117%. Valerate had no effect on the total uptake of inorganic [32P]phosphate into suspensions of proximal tubules but did enhance the initial rate of influx. Net fluid transport was unaffected by these substrates although glucose absorption increased by 10-15% following the addition of either valerate or succinate. Since valerate, butyrate, and succinate are known to stimulate gluconeogenesis and respiration, we evaluated the role of gluconeogenesis in the stimulation of phosphate transport. The addition of 3-mercaptopicolinate (1 mM), an inhibitor of gluconeogenesis, did not alter phosphate transport, nor did it prevent the valerate-induced stimulation of phosphate transport. We conclude that valerate, butyrate, succinate, and malate enhance phosphate transport by the proximal convoluted tubule. This action appears to be unrelated to effects on gluconeogenesis and may be related to close links between phosphate transport and oxidative metabolism.

Organic acids and penicillin production

Fed-batch fermentations with Penicillium chrysogenum, strain S 3723, were fed with glucose as carbon source or with a mixture of glucose and an organic acid (citrate, lactate, propionate, succinate, formate, or caproate). Increased yields of penicillin-V were observed with, propionate, and formate, 50% higher than in fermentations without acid in the feed. The yield with succinate was improved to the level previously observed with acetate (25%).

Mutagenic investigation of flavourings: Dimethyl succinate, ethyl pyruvate and aconitic acid are negative in theSalmonella/mammalian‐microsome test

Three flavourings: dimethyl succinate, ethyl pyruvate and aconitic acid, commonly used in candy, beverages, and baked goods, were tested in the Salmonella/mammalian-microsome test. Tester strains were TA 1535, TA 100, TA 1537 and TA 98 and doses were 32, 160, 800, 4000 and 20 000 micrograms per plate. All tests were performed with and without the S9 fraction from Aroclor induced rat liver. None of the flavourings showed mutagenic potential. These results support the classification made by the Council of Europe, List I (1981).

Acute effect of mercury toxicity on some enzymes in liver of teleost Sarotherodon mossambicus

The effects of 1.5 ppm of mercuric chloride ( LC 50 48 hr ) on some enzymes and organic substances of liver tissue of Sarotherodon mossambicus were studied. Significant decreases in the activities of succinate dehydrogenases, lactate dehydrogenases, glucose-6-phosphate dehydrogenase, alkaline phosphatase, and acid phosphatase and in the levels of organic substances like total anthrone-positive substances, glycogen, and total ninhydrin-positive substances were observed. The results indicate impaired oxidative and transphosphorylative activities and utilization of carbohydrates during acute mercury toxicosis in fish.

Histochemical investigation of the folliculogenesis in the ovary of the Mongolian gerbil (Meriones unguiculatus).

The ovaries of 70 mature Mongolian gerbils (Meriones unguiculatus) were investigated morphologically and enzyme histochemistry for the appearance of acid phosphatase, non-specific esterases, succinate dehydrogenase and thiamine pyrophosphatase. In the oocyte two particular enzyme active zones exist depending on the state of development. In young oocytes acid phosphatase, succinate dehydrogenase and thiamine pyrophosphatase can be found only in the perinuclear zone. From the late secondary follicle on, activity of acid phosphatase, succinate dehydrogenase and non-specific esterases can be detected only in a peripheral area of cytoplasm, whereas thiamine pyrophosphatase is present in the entire ooplasm. In the follicular epithelium a different pattern of enzyme distribution suggests a functional differentiation of the epithelial cells during folliculogenesis.

Anaerobic metabolism in the lugworm Arenicola Marina L.: The transition from aerobic to anaerobic metabolism

After onset of anaerobic conditions Arenicola Marina does not switch immediately to typical anaerobiosis. The adaptation occurs in three steps. 1. 1. In the first 3 hr the phosphagen stores are mobilized, glycogen is degraded to alanine, aspartate is metabolized to succinate and volatile fatty acids ( transition period). 2. 2. Starting approximately after 3 hr, the route of glycogen degradation at the phosphoenolpyruvate-branchpoint is changed gradually from metabolizing via pyruvate kinase to phosphoenolpyruvate carboxykinase. At the same time the metabolic rate is reduced significantly ( switching period). 3. 3. After more than 12 hr the energy supply occurs exclusively via the known pathways typical for long-term anaerobiosis.

The possible role of some enzymes in sclerotia formation in Aspergillus ochraceus

The role of some enzymes in sclerotia production by Aspergillus ochraceus was studied using a sclerotia-producing strain grown under conditions in which sclerotia production was either favoured or inhibited. In addition, a mutant strain incapable of producing sclerotia was used. No significant differences in patterns of soluble proteins, polyphenol oxidase, and esterases could be detected electrophoretically by gel electrophoresis, while the peroxidase pattern of both the sclerotia-producing strain and the mutant showed three bands as compared with two bands that appeared when sclerotia formation was inhibited. The activities of the tricarboxylic acid cycle enzymes, malate dehydrogenase and succinate dehydrogenase, and those of the pentose-phosphate pathway, glucose-6 phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were almost identical in sclerotia- and nonsclerotia-producing mycelia. The activities of isocitrate lyase and malate synthetase, key enzymes of the glyoxylate cycle, and that of glyoxylate dehydrogenase which is related to this cycle were significantly reduced when sclerotia formation was inhibited either by methionine or by high levels of CO2. It is suggested that the glyoxylate cycle plays an important role in sclerotia formation in the fungus.

Succinate transport by free-living forms of Rhizobium japonicum.

We have demonstrated that the transport of succinate into the cells of Rhizobium japonicum strains USDA 110 and USDA 217 is severely inhibited by cyanide, azide, and 2,4-dinitrophenol, but not by arsenate. These results suggest an active mechanism of transport that is dependent on an energized membrane, but does not directly utilize ATP. The apparent Km for succinate was 3.8 microM for strain USDA 110 and 1.8 microM for strain USDA 217; maximal transport velocities were 1.5 and 3.3 nmol of succinate per min per mg of protein, respectively. The expression of the succinate uptake activity was inducible rather than constitutive, with succinate and structurally related compounds being the most effective inducers. The mechanism showed some specificity for succinate and similar organic acids; fumarate and L-malate were classical competitive inhibitors of the system. In general, the best competing compounds were also the best carbon substrates for induction of succinate uptake activity. EDTA inhibited the transport of succinate, implying a role for divalent cations in the system. When various divalent cations were used to reconstitute EDTA-inhibited activity, Ca2+ was most effective, followed by Mg2+, which restored activity at about half the efficiency of Ca2+. Growth media that were supplemented with increased Ca2+ concentration supported more rapid growth with succinate as the carbon substrate, and cells from such media showed higher specific activities of succinate transport.

Comparative study of the formation and excretion of anaerobic fermentation products in bivalves and gastropods

1. 1. Marine bivalves and gastropods, freshwater bivalves and gastropods and land gastropods were studied for accumulation of lactate, alanine, aspartate, succinate and volatile fatty acids during 24 hr of anaerobiosis at 14°C. 2. 2. Lactate was the major fermentation product in land gastropods and was formed in significant amounts in the freshwater gastropod Lymnaea stagnalis and the marine bivalve Cardium edule. 3. 3. Accumulation of alanine occurred concomitantly with the conversion of aspartate to oxaloacetate in marine bivalves and gastropods. Land gastropods use other sources of NH 3 for pyruvate→alanine conversion in addition to aspartate. 4. 4. Alanine and acetate accumulation are interrelated. Freshwater animals with low free aspartate levels convert more pyruvate into acetate, than into alanine. 5. 5. All species accumulate succinate. 6. 6. Conversion of succinate to propionate within 24 hr of anaerobiosis was observed in all bivalves, but was absent in the gastropods, except Buccinum undatum. 7. 7. Many species excrete volatile fatty acids, but some species, e.g. Lymnaea stagnalis, also excrete lactate and succinate.

Cytochrome P-450 dependent deethylase activity in rat and hairless mouse skin microsomes

Microsomal fractions were prepared from rat and hairless mouse skin. The method of preparation was validated by studying the distribution of succinate dehydrogenase, acid phosphate and UDP-glucuronosyltransferase. Induction of oxidative deethylation activities by 5,6-benzoflavone and 3-methylcholanthrene was investigated. Preparations from hairless mouse skin exhibited higher basal activities but the enzymes were less responsive than those of rat skin to inducers. Species differences were observed in the extent of induction between topical and i.p. administration of 5,6-benzoflavone, the former route being more effective in the hairless mouse and the latter route most effective in the rat. Generally oxidative deethylation activity increased linearly with protein conen up to 2–3 mg protein/ml. The only exception was rat skin microsomes prepared from animals pretreated with 5,6- benzoflavone, where linearly was observed only to 0.75 protein/ml above which oxidative deethylation activity decreased with increasing protein cocn. The inhibition of 7-ethoxycoumarin deethylase by various compounds was investigated; the activity in hairless mouse skin exhibited a greater sensitivity to water-soluble solvents than that in rat skin microsomes. Both hairless mouse and rat skin 7-ethoxycoumarin deethylase were sensitive to inhibition by 5,6-benzoflavone, 7,8-benzoflavone and metyrapone.

Biochemical and toxicological effects of single and repeated exposures to polyacetal thermodegradation products

Male Wistar rats were exposed to the fumes from heated (250°C) polyacetal plastic once or three times (6 hr/night) and killed 4 or 24 hr after the last exposure. The exposure atmosphere contained 196 ppm formaldehyde, 11 ppm formic acid, and 0.07 ppm acrolein. The exposures caused a decrease in the reduced glutathione (GSH) content of the brain, kidney, liver, and lungs. Microsomal mono-oxygenase (7-ethoxycoumarin O-deethylase) activity was significantly suppressed in the lung and increased in the kidney. The fumes decreased the cerebral RNA concentration together with decreases in the succinate dehydrogenase and acid proteinase activities. Acetylcholine esterase, creatine kinase, and glutathione peroxidase activities increased above the respective control ranges with a concomitant restoration of the initially decreased GSH level after three exposure periods. Light and electron microscopic examination revealed slight lipid accumulation and splitting of the rough endoplasmic reticulum in the liver cells and degeneration of tubular cells in the kidney. The biochemical and histopathological findings suggest that hypoxic and peroxidative mechanisms are involved in the fume-induced damage.

Succinate-Induced Morphology of Rhizobium trifolii 0403 Resembles That of Bacteroids in Clover Nodules

Morphological changes which accompany nutrient enrichment of Rhizobium trifolii 0403 were studied. Assays of cell number and size coupled with scanning electron microscopy and immunofluorescence microscopy showed that succinate induces cells to stop dividing in vitro and to swell either in the cell center or at one cell pole. The extent and frequency of in vitro cell swelling were in direct relation to the concentration of succinate added to the enrichment medium. The in vitro swelling of cells in 16.6 mM succinate plus Casamino Acids, glucose, and yeast extract closely resembled that of bacteroids of R. trifolii 0403 in nitrogen-fixing nodules of white clover. We hypothesize that succinate may be involved in the transformation of vegetative bacteria into the bacteroid morphology found in nitrogen-fixing nodules.

Prevention of damage in acute spinal cord injury by peptides and pharmacologic agents

Cats were used as models of traumatic spinal cord injury. Each experimental animal received a 500 g-cm force to the exposed dura at the level of thoracic fourth vertabra. Somatosensory evoked potentials (SEPs), carotid arterial blood pressure (BP), and abdominal aorta blood flow in the treated groups were compared with those of the control group. The three treated groups received naloxone (5 mg/kg), TRH (5 mg/kg), and a combination of methyl-prednisolone sodium succinate (MP, 35 mg/kg) and epsilon-aminocaproic acid (EACA, 350 mg/kg). The SEPs which were done only in the naloxone treated group approached “normalcy” 24–26 hours after trauma as compared with the absence of SEPs in traumatized untreated group. In all three groups, the treatment increased the blood flow in abdominal aorta significantly. Morphine sulfate increased substance P (SP) immunoreactivity in the dorsal and ventral gray matter. Naloxone not only reversed this effect, it depleted SP below the saline control level. In order to establish that lipid free radicals are responsible for damage to biological membranes, their effects were also investigated in vitro: 14C-GABA uptake by mouse cortical slices which had decreased by 33% in the presence of superoxide (·O 2 ) generating system, horseradish peroxidase (HRP), was reduced only by 9% when superoxide dismutase was added to the medium. The latter also protected the nerve endings from damage by (·O 2 ) as examined by electron microscopy. It is concluded that the agents used in this study produce their ameliorating effects by virtue of their anti-inflammatory, anti-oxidant, and membrane stabilizing properties in addition to their effect on enhancing the regional microcirculation. The release of SP by naloxone may be responsible for the increase in blood flow. The consequences of traumatic injury as depicted in Fig. 1 are discussed at length.

Oxidation of Substrates by Isolated Bacteroids and Free-living Cells of Rhizobium leguminosarum 3841

Free-living cells of Rhizobium leguminosarum 3841 have inducible oxidation systems for the catabolism of histidine, malonate, p-hydroxybenzoate, glycerol, mannitol and sorbitol. In addition to p-hydroxybenzoate, strain 3841 is able to use a variety of other aromatic substrates as sole carbon source. Cultured free-living bacteria of this strain have constitutive systems for the catabolism of acetate, pyruvate, succinate, fumarate, malate, glucose, fructose, sucrose, lactose, ribose and arabinose. However, isolated pea bacteroids of the same strain are unable to oxidize monosaccharides or disaccharides although they can oxidize the organic acids succinate, fumarate, malate and pyruvate.

Practical applications of the microphotometric quantification of histoenzyme reactions.

There are many histochemical reactions which can be used for the accurate topographic detection of enzymes in tissues. There are also many reports of applications of these techniques in which the results have been evaluated subjectively. However, only a few of these methods have been the subject of quantification, using different methodologies, most of them based on microphotometry. A critical analysis of these procedures has generally shown that they are valid either relatively or at an absolute level. Nevertheless, the routine application of these methods is not as widespread as one might expect. In this paper, the methodological developments that have taken place in the histochemical assay of some enzymes by microspectrophotometry are reviewed briefly. The enzymes include acid phosphatase, non-specific esterase, succinate dehydrogenase and cytochrome oxidase. These methods have given consistent and useful results when applied to our experimental models of irradiated skin, and thus seem useful for various other applications. The reasons why there have been so few applications for quantitative histochemistry are also discussed together with possible ways of making its use more general.

Anaphylaxis After Intravenous Methylprednisolone Administration

ALLERGIC type 1 (anaphylactic) reactions to corticosteroid compounds are rare, but a few well-documented case reports of allergic reactions to prednisolone¹and methylprednisolone sodium succinate¹have been reported. We describe a patient who experienced an anaphylactic reaction to systemically administered methylprednisolone. Allergic sensitivity to this compound as well as to hydrocortisone hemisuccinate was documented by results of intradermal and skin prick testing. Furthermore, our patient demonstrated his allergic reaction only to the succinate esters of the parent corticosteroids. This is the first reported case, to our knowledge, of an anaphylactic reaction to only the succinate acid ester of methylprednisolone without reaction to methylprednisolone acetate. <h3>Report of a Case</h3> A 40-year-old man was admitted to the emergency room for an acute asthmatic attack. The patient had a history of childhood asthma that was seasonal in nature, but he had been asymptomatic for the past 30 years. Two days before

The importance of the phosphoenolpyruvate carboxykinase in the anaerobic metabolism of two marine polychaetes. In vivo investigations on Nereis virens and Arenicola marina

1. 1. The importance of the PEPCK reaction in the anaerobic energy metabolism of the marine polychaetes Arenicola marina and Nereis virens was investigated. 2. 2. By the administration of 3-MPA, a specific inhibitor of PEPCK, it could be proved that PEPCK catalyzes the obligate CO 2-fixation in the anaerobic metabolism. 3. 3. Under the influence of the drug the anaerobic formation of succinate, acetate and propionate is clearly reduced in both species. 4. 4. However, in N. virens as well as in A. marina, inhibition of PEPCK promotes—as an alternative possibility to gain energy—anaerobic glycolysis 5. 5. By this pathway, N. virens is able to compensate for the loss of energy, due to the inhibition of PEPCK, at least during an incubation period of 48 hr, thereby increasing glycogen degradation. 6. 6. On the contrary the anaerobic metabolism of A. marina is much more determined to the formation of succinate and volatile fatty acids. 7. 7. The capacity to form lactate is very limited and is insufficient to compensate the loss of energy resulting from the inhibition of PEPCK. 8. 8. It is possible that differences in their way of life are the reasons for the distinct adaptations to anaerobic conditions in Nereis virens and Arenicola marina.