Acid Succinate Research Articles

Treatment of mammalian spinal cord injury with antioxidants

After spinal cord injury, two groups of cats were treated with a combination of methylprednisolone sodium succinate (MP, 35 mg/kg) and epsilon-aminocaproic acid (EACA, 350 mg/kg), and guanabenz acetate (0.65 mg/kg). Guanabenz acetate was administered twice daily for 8 weeks. In the first group, the treatment significantly increased blood flow in the abdominal aorta. All cats treated with guanabenz acetate 3 hr after spinal cord contusion had return of micturition and none suffered complete paraplegia. Four animals had partial and the other four had complete motor recovery. A superoxide (O2−) generating system, horseradish peroxidase, decreased [14C]gamma amino butyric acid uptake by mouse cortical slices by 33% but when superoxide dismutase was added to the medium, the uptake was reduced by only 9%. The nerve endings were also protected by superoxide dismutase from morphologic damage by (O2−) as observed by electron microscopy. The agents used in these studies produce their ameliorating effects by virtue of their anti-inflammatory, antioxidant. and membrane stabilizing properties, and enhancing the regional microcirculation. In addition to having these properties, guanabenz acetate is also an alpha2 adrenoceptor agonist.

Bacterial siderophores : structure and NMR assignment of pyoverdins Pa, siderophores ofPseudomonas aeruginosa ATCC 15692

In iron-deficient conditions,Pseudomonas aeruginosa ATCC 15692 synthesizes two major siderophores, pyoverdins Pa and pyoverdin Pa B. Two other compounds, pyoverdin Pa A (occurring from hydrolysis of pyoverdin Pa during the culture) and pyoverdin Pa C (occurring artifactually during the purification procedure) were also isolated. All these compounds possess the same partly cyclic peptide chain wherel-Orn(δOH · HCO) isN δ-formyl,N δ-hydroxy-l-ornithine. The chain is bound to a chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and having the (S) configuration. The four pyoverdins differ only in the acyl substituent bound to the nitrogen atom bound to carbon C3 of the chromophore. This is succinamide (pyoverdin Pa), succinic acid (pyoverdin Pa A), methyl succinate (pyoverdin Pa C) and 2-oxoglutaric acid (pyoverdin Pa B). The complete1H- and13CNMR assignments, using two-dimensional total correlation NMR spectroscopy (TOCSY) and rotating-frame Overhauser enhancement spectroscopy (ROESY) procedures, as well as1H-13C correlations, are reported. The complete sequence of the peptide using CHα-NH correlations was achieved by NMR and confirmed the partly cyclic structure earlier reported using fast-atom-bombardment mass spectrometry (FAB-MS) on the siderophores and their dansylated fragments [Briskot G, Taraz K, Budzikiewicz H (1989)Liebigs Ann Chem: 375–384]. The use of these NMR procedures appears to be a tool of choice and a complementary approach to FAB-MS in the structure determination of some complex pyoverdins.

Acid succinates of diterpene alcohols — A new type of components of conifer oleoresins

Acid succinates of diterpene alcohols — A new type of components of conifer oleoresins

EFFECTS OF MONOSODIUM SUCCINATE, DISODIUM SUCCINATE, AND SUCCINIC ACID ON URINARY BLADDER CARCINOGENESIS IN <i>N</i>-BUTYL-<i>N</i>-(4-HYDROXYBUTYL)-NITROSAMINE-INITIATED, UNILATERALLY URETER-LIGATED RATS

The effects of monosodium succinate, disodium succinate, and succinic acid on the development of hyperplastic or preneoplastic lesions in the urinary bladder of rats were investigated. These test chemicals were fed to rats after pretreatment for 2 weeks with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) as 0.05% of the drinking water. Rats were subjected to unilateral ureter ligation at week 3 and killed at week 20 of the experiment. In the groups given disodium succinate or sodium citrate (positive control), the incidence of simple hyperplasia was significantly higher than that of the control group given the basal diet alone. The incidence and number per 10cm basal membrane of papillary or nodular hyperplasia of the urinary bladder were higher than the control in the groups given disodium succinate or sodium citrate, although the changes were not statistically significant, probably due to the short administration period of the test chemicals. The results support the significance of the presence of sodium ion and increased urinary pH as promoting factors for urinary bladder carcinogenesis.

Effects of Acidic Monomers, Proposed Components of Self-etching Dentin Primers, on Dentin Surface, Surface Tension and Rat Erythrocytes

An experimental self-etching dentin primer, prepared by dissolving an acidic monomer, methacryloxyethyl succinate (MES), dimethacryloxyethyl phosphate (DMEP) or tertiarybutyl acrylamide sulfonic acid (TBAS), in an aqueous solution of 2.9M 2-hydroxyethyl methacrylate (HEMA), has been shown to promote adhesion by a bonding agent between a composite resin and dentin. To clarify the mechanism of this promotion we examined the effects of acidic monomers on dentin cleansing, surface tension of water and hypotonic hemolysis of rat erythrocytes. In scanning electron microscope observation of the dentin surface, the smear layer remained slightly evident after MES-HEMA treatment. DMEP-HEMA or TBAS-HEMA caused complete removal of the smear layer, and openings of dentinal tubules were clearly evident. MES and DMEP, 1×10-6 M and 1×10-4 M, respectively, decreased the surface tension of distilled water (72 dyne/cm) . The surface tension depression by 0.3M HEMA was apparently not further affected by MES, DMEP or TBAS at 1×10-7 to 1×10-2M. The osmotic pressure of hypotonic buffer was increased by MES, DMEP, TBAS and HEMA at concentrations greater than 1×10-2M. The hypotonic hemolysis of erythrocytes was inhibited by MES and TBAS at 4×10-3 to 1×10-2M and by HEMA at 4×10-1M. DMEP promoted hypotonic hemolysis at 4×10-5 to 1×10-4M, but caused inhibition in the concentration range of 4×10-4 to 1×10-3M. The protective effect of acidic monomers on the erythrocytes was promoted by the presence of 0.1 M HEMA, while DMEP at 1×10-2 M showed only a lytic effect. These results suggest that the mechanism of promotion of the adhesive ability of a bonding agent by self-etching dentin primers containing these acidic monomers is related to the increased surface activity of water and membrane stability of erythrocytes, since these effects of acidic monomers might be caused by the reduction in surface tension of secretion from dentin and inhibition of secretion from dentinal tubules.

General mechanism for the bacterial toxicity of hypochlorous acid: abolition of ATP production

The adenylate energy charges (EC) of Escherichia coli 25922, Pseudomonas aeruginosa 27853, and Streptococcus lactis 7962 rapidly fell in nutrient-rich media from values in excess of 0.9 to below 0.1 when the organisms were exposed to lethal levels of HOCl. The same cells maintained in energy-depleted states were incapable of attaining normal EC values necessary for biosynthesis and growth when challenged with nutrient energy sources after HOCl exposure. These changes correlated quantitatively with loss of replicative capabilities. Initial rates of transport of glucose, succinate, and various amino acids that act as respiratory substrates and the ATP hydrolase activity of the F1 complex from the ATP synthase of E. coli 25922 also declined in parallel with or preceded loss of viability. These results establish that cellular death is accompanied by complete disruption of bacterial ATP production by both oxidative and fermentative pathways as a consequence of inhibition of inner membrane bound systems responsible for these processes.

Increased therapeutic efficacy of Zidovudine in combination with vitamin E

Antiviral activity and bone marrow toxicity of 3′-azido-3′ deoxythymidine (Zidovudine; AZT) was evaluated in the presence of α-D-tocopherol acid succinate (ATS) in the MT4 cell line and in murine hematopoietic progenitor cells, respectively. At varying concentrations (.016 to .125 μM) of AZT, addition of ATS (5 to 15 μg/ml) showed a dose-dependent increase in anti-HIV activity. The ED 90 of AZT in this test system was 0.37 μM, whereas in the presence of ATS (15 μg/ml) it was 0.06 μM, thus producing an approximately 6-fold increase in anti-HIV activity. In contrast, in murine bone marrow cells, ATS (4 μg/ml) showed significant protection (p<0.05) against AZT-induced toxicity as measured by CFU-E and CFU-GM assays. The IC 50 values in the presence and absence of ATS for CFU-E were 3.7 and 1.5 μM, whereas for CFU-GM were 6.0 and 2.7 μM, respectively. Overall, these data suggest that AZT in combination with ATS has greater therapeutic efficacy against HIV-1.

Effect of esters of succinic acid and other citric acid cycle intermediates on insulin release and inositol phosphate formation by pancreatic islets

Esters of carboxylic acids are permeable to cells and once inside the cell are hydrolyzed to carboxylic acids. Methyl and ethyl esters of succinate and other citric acid cycle intermediates were tested to find out whether they are insulin secretagogues. Monomethyl succinate stimulated insulin release from pancreatic islets in a concentration-dependent manner with maximal release attained at a concentration of 10 m m. Dimethyl succinate (10 m m) was as effective as monomethyl succinate, but pyruvate methyl ester, monoethyl succinate, and dimethyl fumarate were ineffective as primary secretagogues. However, dimethyl fumarate potentiated both leucine- and leucine-plus-glutamine-induced insulin release. Glucose, leucine, leucine plus glutamine, and monomethyl succinate increased inositol tris-, bis- and monophosphate formation in pancreatic islets and antimycin A inhibited this formation. Since mitochondrial metabolism is probably essential for glucose-induced insulin release and the metabolism of succinate and leucine (without or with glutamine) involves mitochondrial respiration exclusively, these results might indicate that mitochondrial metabolism generates conditions or factors that are transmitted to the cytosol to increase inositol trisphosphate formation and thus calcium mobilization and insulin release. Since succinate is believed to enter metabolism at site II of the mitochondrial respiratory chain, it is interesting that rotenone, an inhibitor of NADH dehydrogenase and site I of the respiratory chain, was a potent inhibitor of monomethyl succinate-induced insulin released. Rotenone also inhibited leucine (plus or minus glutamine)-induced insulin release. These results indicate that β cell metabolism of monomethyl succinate and leucine, like glucose, influences dehydrogenases that produce NADH.

Regression of experimental cancer by oral administration of combined α‐tocopherol and β‐carotene

alpha-Tocopherol (vitamin E) and beta-carotene have been shown to be capable of regressing established epidermoid carcinomas of hamster buccal pouch when injected locally into the tumor site. Neither has yet been shown to be effective in regressing cancer when administered by oral route. However, a combination of both alpha-tocopherol and beta-carotene was shown to be effective in regressing epidermoid carcinomas of hamster buccal pouch when the mixture was administered orally in vegetable oil. The epidermoid carcinomas were induced in the right buccal pouch of 100 Syrian hamsters by painting three times weekly for 14 weeks with a 0.5% solution of 7,12-dimethylbenz[a]anthracene in mineral oil. The animals were then divided into five equal groups of 20 animals. Group 1 animals received no further treatment and represented tumor controls. Group 2 animals received 200 micrograms beta-carotene and 200 micrograms dl-alpha-tocopherol acid succinate combined in 0.2 ml vegetable oil. Animals received the mixture daily by mouth using a 1-ml syringe. Groups 3 and 4 received beta-carotene and alpha-tocopherol individually in double amounts (400 micrograms in 0.2 ml vegetable oil). Group 5 animals received only the vegetable oil (0.2 ml daily) and were controls for vehicle. The animals in Groups 1, 3, 4, and 5 were killed after 22 weeks because the tumors were extensive, large, and necrotic and the animals were weak and cachectic. After 22 weeks, the tumors in Group 2 animals were small in 15 out of 20 animals. The tumors were reduced in size compared with tumor burden at 14 weeks, the point at which the beta-carotene/alpha-tocopherol was started.

Different effects of vitamin E on the growth and morphological differentiation of 4 murine neuroblastoma cell lines in vitro.

The effects of vitamin E (vit.E, d-alpha-tocopherol acid succinate) on the growth and the morphological differentiation of 4 representative murine neuroblastoma cell lines: C1300 (wild type), NS-20 (cholinergic type), N-18 (inactive type), N1E-115 (adrenergic type), and the combined effects of vit.E and cis-diamminedichloroplatinum(II)(cisplatin, CDDP) were studied in culture. Vitamin E inhibited the growth of all clonal cells in a dose-dependent manner, especially that of NS-20 cells, and it caused significant morphological differentiation only in NS-20 cells. In addition, vit.E enhanced the growth inhibition of C1300 cells by cisplatin in an additive manner, but not that of NS-20 cells. These suggested that both phenomena induced by vit.E may not always occur in parallel in each clonal neuroblastoma, and both mechanisms might be different.

84 THE DISTRIBUTION OF ENZYME ACTIVITIES IN CIRRHOTIC LIVERS OF DEVELOPING RATS SUGGESTS A SIMILAR METABOLIC ROLE FOR THE PERIPHERY OF THE RECENERATING NODULES &amp; ZONE 3 OF THE NORMAL LIVER ACINUS

To assess whether the development of experimental cirrhosis was associated with changes in the intralobular distribution of enzyme activities, we have used microspectrophotometry to quantitate in periportal (PPA) and centrilobular areas (CLA) of the liver succinate dehydrogenase (SDH), glutamate dehydrogenase (GDH), acid phosphatase (AP), glucose-6 phosphatase (GP) and NADPH diaphorase (ND). Rats were treated with phenobarbitone (PB) 30mg/kg/day from day 4 and CCl4 0.5mg/kg twice weekly from day 8 to 62 and developed centrilobular necrosis followed by fibrous tissue deposition from day 25 and cirrhosis by 45 days. During drug exposure, all enzymes showed decreased activity, particularly in the CLA, with SDH and GP falling by 85-90%, AP and GDH by 30-50% and ND by 10%. in the PPA, AP and ND showed little change, SDH and GDH decreased by 10-30% and GP by 40%. Although cirrhosis persisted after both drugs were stopped and enzymatic activity returned to normal, the distribution had changed, with the cells adjacent to the vascular fibrous septa at the periphery of the regenerating nodules expressing activities similar to the cells in the CLA of the controls, while enzyme activity in the centre of the nodules were similar to the PPA of the controls. Further application of this technique may elucidate metabolic and pharmacological complications of both experimental and clinical cirrhosis.

Wood anatomy and heartwood formation in Neem (Azadirachta indica A. Juss.)

The wood of Azadirachta indica is diffuse porous. Axial parenchyma is paratracheal banded or vasicentric. Rays are uniseriate to multiseriate and heterocellular with procumbent and upright cells. There is a strong negative correlation between vessel member length and diameter. The vulnerability and mesomorphic values are different in two trees of almost the same age growing in the same locality. The vessel member wall has spiral thickenings on its inner surface. Axial and ray parenchyma cells and sometimes vessels and fibres of the heartwood show the presence of extractives. The necrobiosis of the parenchyma cells occurs at the heartwood boundary. Senescence and death of parenchyma cells are associated with depletion of starch grains and accumulation of extractives. There is a climacteric rise in succinate dehydrogenase and acid phosphatase activities at the sapwood-heartwood interface which may be associated with heartwood formation. Peroxidase activity is greater near the cambial zone, indicating its probable role in lignification.

Alpha tocopheryl succinate inhibits melanocyte-stimulating hormone (MSH)-sensitive adenylate cyclase activity in melanoma cells.

D-alpha tocopheryl succinate (vitamin E succinate), which is known to induce differentiation and growth inhibition in murine B-16 melanoma cells, reduced basal and melanocyte-stimulating hormone (MSH)-stimulated adenylate cyclase (AC) activity in vitro. Vitamin E succinate treatment also reduced sodium fluoride- and forskoline-stimulated AC activity of melanoma cells in vitro. Treatment of cells with vitamin E succinate (6 micrograms/ml] for a period of 24 hours was sufficient to reduce MSH-stimulated AC activity. Other forms of vitamin E, such as d1-alpha tocopheryl nicotinate, d1-alpha tocopheryl acetate, and d1-alpha tocopherol, which did not affect growth or morphology of melanoma cells, were relatively less effective in altering basal and MSH-stimulated AC activity. Retinoic acid, which inhibited the growth of B-16 melanoma cells, also reduced basal and MSH-, NaF-, and forskolin-stimulated AC activity in vitro. Prostaglandin A2, which inhibited growth and altered morphology, did not change basal or MSH-stimulated AC activity. These results show that one of the mechanisms of action of vitamin E succinate and retinoic acid on melanoma cells may involve reduction of basal and MSH-sensitive AC activity, and this vitamin effect is not necessarily related to growth inhibition.

Filomicrobium fusiforme gen. nov., sp. nov., a Slender Budding, Hyphal Bacterium from Brackish Water

Summary A strain of a fusiform, aerobic bacterium was isolated from brackish water of the Baltic Sea. The cells carried a hypha on each pole and formed non-motile buds at the tips of these hyphae. During logarithmic growth the hyphae increased to a length of 40 μm. Cells of enrichment cultures carried hyphae of more than 300 μm. the stenohaline organisms utilized only a few organic acids as a carbon source: acetate, propionate, fumarate, malate, succinate, and glutamic acid. The cells were sensitive to penicillin G, tetracyclin, streptomycin, and chloramphenicol. While the G+C content of 61.9 mol% was in the range of other hyphal, budding bacteria, morphological and physiological properties clearly showed that strain IFAM 1315 (IFAM = Institut fur Allgemeine Mikrobiologie) represents a new genus and species: Filomicrobium fusiforme gen. nov., sp. nov.

Lithoautotrophic growth of sulfate-reducing bacteria, and description of Desulfobacterium autotrophicum gen. nov., sp. nov.

The capacity of mesophilic sulfate-reducing bacteria to grow lithoautotrophically with H2, sulfate and CO2 was investigated with enrichment cultures and isolated species. (a) Enrichments in liquid mineral media with H2, sulfate and CO2 consistently yielded mixed cultures of nonautotrophic, acetate-requiring Desulfovibrio species and autotrophic, acetate-producing Acetobacterium species (cell ratio approx. 20:1). (b) By direct dilution of mud samples in agar, various non-sporing sulfate reducers were isolated in pure cultures that did grow autotrophically. Two oval cell types (strains HRM2, HRM4) and one curved cell type (strain HRM6) from marine sediment were studied in detail. The strains grew in mineral medium supplemented only with vitamins (biotin, p-aminobenzoate, nicotinate). Carbon autotrophy was evident (i) from comparative growth experiments with non-autotrophic, acetate-requiring species, (ii) from high cell densities ruling out a cell synthesis from organic impurities in the mineral media, and (iii) by demonstrating that 96–99% of the cell carbon was derived from 14C-labelled CO2. Autotrophic growth occurred with a doubling time of 16–20 h at 24–28°C. Formate, fatty acids up to palmitate, ethanol, lactate, succinate, fumarate, malate and other organic acids were also used and completely oxidized. The three strains possessed cytochromes of the b-and c-type, but no desulfoviridin. Strain HRM2 is described as a new species of a new genus, Desulfobacterium autotrophicum. (c) The capacity for autotrophic growth was also tested with sulfate-reducing bacteria that originally had been isolated on organic substrates. The incompletely oxidizing, non-sporing types such as Desulfovibrio and Desulfobulbus species and Desulfomonas pigra were confirmed to be obligate heterotrophs that required acetate for growth with H2 and sulfate. In contrast, several of the completely oxidizing sulfate reducers were facultative autotrophs, such as Desulfosarcina variabilis, Desulfonema limicola, Desulfococcus niacini, and the newly isolated Desulfobacterium vacuolatum and Desulfobacter hydrogenophilus. The only incompletely oxidizing sulfate reducer that could grow autotrophically was the sporing Desulfotomaculum orientis, which obtained 96% of its cell carbon from 14C-labelled CO2. Desulfovibrio baarsii and Desulfococcus multivorans may also be regarded as types of facultative autotrophs; they could not oxidize H2, but grew on sulfate with formate as the only organic substrate.

Free radical-mediated membrane depolarization in renal and cardiac cells

Cell membrane potential was measured with a flow cytometer by quantitating the intracellular accumulation of a fluorescent cationic carbocyanine dye. We used this system to demonstrate depolarization upon the addition of hydrogen peroxide (10–1 000 μM) and ferrous chloride (25–100 μM) to cultures of either neonatal rat myocardial or LLC-PK1 renal epithelial cells. Ferrous chloride-induced depolarization was prevented by superoxide dismutase, catalase and dimethyl sulfoxide, suggesting roles for the superoxide anion, hydrogen peroxide and the hydroxyl radical in effecting this depolarization, possibly through a Fenton-type reaction mechanism. Supplementation of either cell type with 2 μM tocopherol acid succinate during growth in tissue culture, prior to exposure to the oxidizing agent, decreased the magnitude of the depolarization in both cell types. The results are consistent with a role for tocopherols in scavenging free radical species responsible for the depolarization of the cell membrane.

Histochemical Studies of the Transition from Sapwood to Heartwood in Tectona Grandis

Histochemistry of the wood of Tectona grandis Linn. f. (Verbenaceae) has been studied during the transition from sapwood to heartwood. Starch, lipids, proteins, nucleic acids, phenolics and the enzymes peroxidase, succinate dehydrogenase, acid phosphatase, adenosine triphosphatase and glucose-6-phosphatase show significant changes during the transition. Peroxidase and adenosine triphosphatase are highly active in the sapwood and moderate in the transition zone while succinate dehydrogenase, glucose-6-phosphatase and acid phosphatase are moderate throughout the sapwood.

Vitamin E Enhances the Chemotherapeutic Effects of Adriamycin on Human Prostatic Carcinoma Cells in Vitro

Vitamin E (tocopherol) enhances the growth inhibitory effects of adriamycin (ADR) on a variety of cancer cells in vitro. The role of vitamin E (d-α-tocopheryl) acid succinate in adjuvant chemotherapy with ADR was assessed in DU-145 human prostatic carcinoma cells in culture. Adriamycin produced a dose-dependent growth inhibition of DU-145 cells. The ID50 of DU-145 cells on the criteria: a) of clonal assay was 13ng./ml. and b) of cell count assay was 14ng./ml. Vitamin E succinate also inhibited the growth of DU-145 human prostatic carcinoma cells in a dose-dependent manner. 4.4μg./ml. and 5.4μg./ml. vitamin E succinate in the culture medium produced inhibition of growth to 50 per cent of control (ID50) in the clonal and the cell count assays respectively. When adriamycin and vitamin E succinate were used in combination, both additive and synergistic effects were observed, depending on the concentration of vitamin E succinate used. Doses of vitamin E succinate greater than its ID50 had a synergistic effect while doses smaller than its ID50 had an additive effect. In either case, the presence of vitamin E succinate caused an enhancement of tumor cell cytotoxicity of adriamycin while decreasing its ID50. Equivalent concentrations of sodium succinate and ethanol used to dissolve vitamin E succinate did not have any effect on DU-145 cells. Thus, it is concluded that the effect of vitamin E succinate is due to vitamin E and not due to succinate or ethanol. These results suggest that vitamin E may have a role in the treatment of human prostatic cancer as an adjuvant agent to adriamycin.

Characteristics of the active oxygen in covalent binding of the pesticide methoxychlor to hepatic microsomal proteins

This study examined the characteristics of the active oxygen species involved in generation of the reactive intermediate of methoxychlor which covalently binds to liver microsomal proteins. The possibility that the active oxygen participating in the above reaction is the Superoxide anion (O − 2) or a species generated from O − 2 was examined with the help of Superoxide dismutase (SOD) and with an SOD-mimetic agent, CuDIPS [Cu 2+(3,5-diisopropylsalicyclic acid)2]. It was observed that, whereas CuDIPS inhibited covalent binding of methoxychlor metabolite(s), SOD did not. However, ZnDIPS [Zn 2+(3,5-diisopropylsalicyclic acid);], which exhibits no SOD-mimetic activity, did not inhibit covalent binding. Furthermore, both CuDIPS and ZnDIPS had little or no effect on the formation of demethylated (polar) metabolites of methoxychlor, demonstrating that the inhibition of covalent binding by CuDIPS was not merely due to a general inhibition of the hepatic monooxygenase system. These findings suggested that O 2 − was involved in covalent binding, but was not accessible to SOD. Additional support for O 2 − involvement stems from the observation that a-tocopheryl acid succinate markedly inhibited covalent binding of methoxychlor. The possibility that hydrogen peroxide (H 2O 2) was involved in covalent binding of methoxychlor appears unlikely. Catalase had no effect on covalent binding when NADPH was the cofactor, and the use of H 2O 2 in place of NADPH did not yield covalent binding. Certain scavengers of hydroxyl radical (ethanol, t-butanol and benzoate) inhibited, and other known scavengers (DMSO and mannitol) did not inhibit, covalent binding. EDTA stimulated binding, desferal (desferrioxamine) exhibited no effect on binding, and diethylenetriaminepentaacetic acid (DETAPAC) inhibited binding. A possible explanation for this observation is that the Fe 2− needed for generation of OH is much more easily obtained from Fe 3+-EDTA than from Fe 3+-desferal, which resists reduction. The inhibitory effect by DETAPAC may be due to chelation of another metal which is needed for the reaction. Lastly, certain scavengers of singlet oxygen inhibited covalent binding with little effect on the formation of polar metabolites of methoxychlor. In conclusion, these studies support the involvement of OH and singlet oxygen, possibly derived from O − 2, in the formation of the reactive methoxychlor intermediate. However, the less likely possibility that these compounds, used to probe for active oxygen, merely inhibit the activity of a cytochrome P-450 which specifically catalyzes the formation of the reactive methoxychlor intermediate but not of the demethylation of methoxychlor cannot be ruled out.

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.