Dodecanedioic Acid Research Articles

Uptake of dodecanedioic acid by isolated rat liver

The uptake of dodecanedioic acid (C12), a dicarboxylic acid with 12 carbon atoms, was studied in the isolated perfused rat liver. Fifty μ mol of C12 were injected as a bolus into the perfusing liver solution. The concentration of C12 in perfusate samples taken over 2 h from the beginning of the experiments were analyzed by high performance liquid chromatography. An in vitro experimental session was performed to determine the binding curve of C12 to defatted bovine serum albumin. These data were then used to compute the perfusate C12 free fraction. The number of binding sites on the albumin molecule was equal to 4.29 ± 0.21 (S.E.), while the affinity constant was 6.33 0.87 × 103 M −. Experimental values of perfusate C12 concentration versus time were individually plotted and fitted to a monoexponential decay for each liver perfused. The predicted C12 concentration at time zero averaged 0.354 ± 0.0375 μmol/ml. Prom this value the apparent volume of distribution of C12 was obtained and corresponded to 153.02 ± 14.56 ml. The disappearance rate constant from the perfusate was 0.0278 ± 0.0030 min −1. The C12 half life was 26.6 ± 2.3 min. The mean hepatic clearance from the perfusate was 4.08 0.38 ml/min. In conclusion, C12 is quickly taken up by the liver so that in about 100 rein it was completely cleared from the perfusate.

The Omega-Hydroxylation of Lauric Acid: Oxidation of 12-Hydroxylauric Acid to Dodecanedioic Acid by a Purified Recombinant Fusion Protein Containing P450 4A1 and NADPH–P450 Reductase

The recombinant fusion protein rF450[mRat4A1/mRatOR]L1, containing the heme domain of P450 4A1 and the flavin domains of NADPH–P450 reductase, when incubated with dilaurylphosphatidylcholine (DLPC), Chaps, cytochromeb5, and a 20-fold excess of purified NADPH–P450 reductase, catalyzes the omega-oxidation of lauric acid at a rate of about 300 nmol/min/nmol P450. This is the first report of a mammalian P450 enzyme with such a high turnover number. The resultant 12-hydroxydodecanoic acid [12-hydroxylauric acid (12-OH LA)] is further oxidized by the P450 oxygenase reaction to dodecanedioic acid (decane-1,10-dicarboxylic acid) via 12,12-dihydroxydodecanoic acid. Spectral binding studies show that 12-OH LA inhibits the binding of lauric acid to the active site of P450 with aKiof about 1.9 μM. The construction and expression of recombinant P450 4A1 containing a six-member polyhistidine domain at the carboxy-terminus of the protein is described. Reconstitution experiments with this purified recombinant P450 4A1, DLPC, Chaps,b5, and purified NADPH–P450 reductase show results similar to those obtained with the purified fusion protein, albeit at lower turnover rates. The requirement for normal-phase HPLC in resolving the metabolites formed during lauric acid metabolism is demonstrated.

Plasma clearance and oxidation of dodecanedioic acid in humans.

Dicarboxylic acids are water-soluble, contrary to monocarboxylic acids, and have a metabolic pathway intermediate between those of lipids and carbohydrates. Our goal was to investigate the plasma turnover and oxidation rate of dodecanedioic acid (C12) in eight healthy male volunteers. A simultaneous infusion of both cold (0.24 mmol/min corresponding to 0.396 kcal/min) and radiolabeled (1.62 microCi/min) C12 free acid was performed. Blood specimens were sampled over a period of 360 minutes, and 24-hour urine samples were collected to measure the levels of C12 by high-performance liquid chromatography and liquid scintillation. Indirect calorimetry was continuously performed, and expired 14CO2 was collected. Binding of C12 in human plasma was determined in separate experiments using equilibrium dialysis. A linear one-compartment model was used to describe the kinetics of labeled C12. Its volume of distribution was 139.02 +/- 10.84 mL/kgbw (mean +/- SE), and its plasma elimination constant was 0.01 +/- 0.004 min-1. The 24-hour urinary excretion of C12 was 3.14 +/- 0.96 mmol, corresponding to about 7% of the administered dose. The amount of C12 oxidized, expressed as percent oxidation, was equal to 35.44 +/- 1.64%. The mean basal value of npRQ (0.80 +/- 0.006) significantly (p < .02) decreased during the infusion to 0.78 +/- 0.01, which is a value close to that theoretically calculated (0.77). The oxidation of free fatty acids was significantly increased at the end of the C12 infusion, whereas the glucose oxidation was reduced to about 50%. The experimental data suggest that C12 might represent a fuel substrate immediately available for tissue energy requirements, because a relevant amount of C12 is promptly oxidized. Its prompt oxidation and its conversion to succinic acid support the use of dodecanedioic acid in parenteral nutrition, especially in insulin-resistance conditions in which glucose uptake and oxidation is impaired.

Pharmacokinetic analysis of dodecanedioic acid in humans from bolus data.

Excretion and tissue uptake of dodecanedioic acid (C12), a proposed alternative fuel substrate, was investigated in humans by bolus experiments. Seven overnight-fasting healthy male volunteers received i.v. a bolus (1 g) of C12. Blood samples were collected after C12 administration at intervals of 15 minutes, and C12 serum concentration was measured by high-performance liquid chromatography. C12 excretion in 24-hour urine was measured. Binding of C12 in human serum was determined in separate equilibrium dialysis experiments by means of an isotopic compound (disodic salt of (1,12)14C-dodecanedioic acid). A two-compartment model was used for describing C12 kinetics. The excreted amount of C12 in 24-hour urine was found to be, on the average, 1.62% of administered dose. The apparent number of binding sites per albumin molecule was 3.1 +/- 0.2 (estimate +/- SE) with an affinity constant of 6.4 +/- 1.8 mM-1. The distribution volume of central compartment was 5.56 +/- 3.13 L and that of peripheral compartment was 87.4 +/- 30.4 L. The rate constant of exchange between compartments was 4.60 +/- 3.50 L/min, that or urinary excretion 25.6 +2- 15.5 mL/min, and that of tissue uptake 2.17 +/- 0.86 L/min. These results are promising for C12 utilization in parenteral nutrition, because C12 elimination in urine is low whereas tissue uptake appears to be rather efficient.

Extraction of mono- and dicarboxylic acids from a curative water.

A method for the analysis of mono- and dicarboxylic acids from water is presented. For this purpose two techniques, a C(18) solid phase extraction (SPE) and a combination method of liquid-liquid extraction (LLE) and aminopropyl SPE, were tested. With the combination method all analytes, short-chain mono- and long-chain dicarboxylic acids, could be analysed in one approach. The C(18) SPE was not suitable for short-chain mono- but for dicarboxylic acids. Concentrations in the investigated water ranged from 315 mg/l (butanoic acid) to 2.9 mg/l (octanoic acid). Dicarboxylic acids were found from 5 mg/l (octanedioic acid) to 0.5 mg/l (dodecanedioic acid).

In VivoReconstitution of Highly ActiveCandida maltosaCytochrome P450 Monooxygenase Systems in Inducible Membranes ofSaccharomyces cerevisiae

To establish a system for functional characterization of individual Candida maltosa cytochrome P450 monooxygenases, the NADPH-cytochrome P450 reductase from this yeast species was co-expressed in Saccharomyces cerevisiae with each of the following cytochrome P450 forms; P450Cm1 (CYP52 A3), P450Cm2 (CYP52 A4), and P450AlK2A (CYP52 A5). For this purpose, a multicopy plasmid was constructed that contained two independent expression units controlled by the galactose-inducible GAL10 promoter. As shown by spectral and immunological methods, large amounts of the desired monooxygenase components could be simultaneously produced in the respective S. cerevisiae transformants. It was important, however, to adjust semi-anaerobic cultivation conditions during induction by galactose to minimize a mutual impairment of cytochrome P450 and NADPH-cytochrome P450 reductase formation. Compared to the specific cellular content of the host-own enzyme, a 75- to 100-fold overproduction of the reductase component was obtained resulting in P450/reductase molar ratios of about 1:3 in the microsomal fractions prepared from the co-expression strains. At the same time, the rates of cytochrome P450-dependent lauric acid hydroxylation increased more than 10-fold, showing a proper reconstitution of the C. maltosa monooxygenase systems in S. cerevisiae. Using intact cells, an efficient biotransformation of lauric acid to omega-hydroxylauric acid and dodecanedioic acid was found. S. cerevisiae cells coexpressing cytochrome P450 and NADPH-cytochrome P450 reductase were characterized by a marked proliferation of the endoplasmic reticulum. Immunoelectron microscopy revealed a colocalization of the monooxygenase components produced to these newly formed membrane structures.

Partial Suppression of SOS-Inducing Activity of Furylfuramide by Dibasic Acids from Ipomoea nil in the Salmonella typhimurium TA1535/pSK1002 umu Test

An acidic fraction from Ipomoea nil showed suppression of SOS-inducing activity of the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) in the Salmonella typhimurium TA1535/pSK1002 umu test. The suppressive compounds in I. nil were fractionated by SiO 2 column chromatography. The active compounds in the suppressing fraction were identified as butanedioic acid, pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, 3,4-dimethoxycinnamic acid, and linoleic acid by GC and GC-MS. These dibasic acids partially suppressed the SOS-inducing activity of AF-2. The dose-response values of all dibasic acids were in the range 0.3-1.5 μmol/mL. Tetradecanedioic acid suppressed 70% of the SOS-inducing activity of AF-2 under 1.5 μmol/mL. In addition tetradecanedioic acid was assayed with other mutagens (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]indole (Glu-P-1)), and it also showed a suppressive effect on each mutagen. The ID 50 (50% inhibitory dose) values of tetradecanedioic acid for AF-2, Trp-P-1, and Glu-P-1 were estimated as 0.44, 0.80, and 0.65 μmol/mL, respectively

ChemInform Abstract: Tetrahydrofurans and γ‐Lactones. Part 7. Synthesis and Reactions of γ,γ‐Disubstituted γ‐Lactones ‐ An Approach to 4,5‐ Functionalized Dodecanedioic Acid Derivatives.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Dicarboxylic acids affect the growth of dermatophytes in vitro.

Azelaic acid is a dicarboxylic acid with known antimycotic activity. In this study we have used an agar dilution technique to test the effect of six other dicarboxylic acids (sebacic, undecanedioic, dodecanedioic, tridecanedioic, tetradecanedioic and hexadecanedioic acid, 10(-4)-10(-2) mol/l, pH 5.5) on in vitro growth of Trichophyton (T.) rubrum, T. mentagrophytes and Microsporum (M.) canis. Furthermore, the fungicidal activity of 10(-2) mol/l undecanedioic and sebacic acid was tested using a T. rubrum growth assay. Undecanedioic acid proved fungistatic at 10(-2) mol/l for all species and fungicidal for T. rubrum. A minor fungistatic effect on T. rubrum and T. mentagrophytes was also seen with the other acids at this concentration. M. canis was inhibited only by high concentrations of four acids, whereas low concentrations of all six agents resulted in enlarged thallus diameters. We conclude that among dicarboxylic acids fungistatic activity is not limited to azelaic acid. Undecanedioic acid appears promising for further investigations.

Tetrahydrofurane und γ‐Lactone, VII. Synthese und Reaktionen von γ,γ‐disubstituierten γ‐Lactonen – ein Weg zu funktionalisierten Dodecandisäure‐Derivaten

Tetrahydrofurans and γ‐Lactones, VII. – Synthesis and Reactions of γ,γ‐Disubstituted γ‐Lactones – an Approach to 4,5‐Functionalized Dodecanedioic Acid DerivativesThe title compounds 5 are obtained by Diels‐Alder reaction of cyclooctyne and 2‐substituted furans, subsequent hydrogenation, ozonolysis and oxidation with air. The lactone ring of 6c is opened by treatment with sodium iodide and zinc to give the methylene carbonyl derivative 9a. Compound 9a is converted into the cyclopropane 13 via the pyrazoline 12. magnified image

Pharmacokinetic profile of dodecanedioic acid, a proposed alternative fuel substrate.

Dodecanedioic acid (C12), a saturated, aliphatic dicarboxylic acid with 12 carbon atoms, was given as an intravenous bolus (800 mumol/kg of body weight [kgBW]) in male Wistar rats to study its pharmacokinetic profile. Because total plasma C12, which results from the sum of both free and albumin binding fractions, was measured by high-performance liquid chromatography, an in vitro experimental session was carried out to determine the binding curve of C12 in rat plasma. These data were then used to calculate the plasma C12 free fraction in in vivo experiments. The best fit obtained for the experimental data of albumin binding was obtained with the equation of reversible, saturable binding to one, two, or three classes of noninteracting equivalent sites. Only a single binding site was clearly identified with a dissociation constant of 147 mumol/L and a maximal predicted binding of 1.57 mol/mol albumin. The urinary excretion of C12 was 3.90 +/- 1.62% of the administered dose. The pharmacokinetic analysis was performed by one-compartment model with linear transfer to the tissues, taking into account simultaneously both plasma concentration and urine excretion data. The apparent volume of distribution of C12 was 0.248 +/- 0.035 L/kgBW, the apparent first order rate constant to the tissues was 0.0535 +/- 0.0123 min-1 and that from plasma to urine was 0.00206 +/- 0.00051 min-1. The C12 plasma half-life was 12.47 minutes. Renal clearance was 0.00051 L/kgBW per minute, whereas the systemic clearance was 0.0138 L/kgBW per minute. Because the renal clearance was much less than the rat inulin clearance reported in literature, the presence of C12 passive back-diffusion was hypothesized.

Cytochrome P450BM-3 (CYP102): regiospecificity of oxidation of omega-unsaturated fatty acids and mechanism-based inactivation.

Cytochrome P450BM-3 preferentially oxidized fatty acids with terminal double or triple bonds to the omega-2 hydroxylated fatty acids rather than, respectively, to the epoxide or diacid metabolites. The enzyme is inactivated during catalytic turnover of long, terminally unsaturated fatty acids but not by the analogous medium-length fatty acids. Enzyme inactivation by 17-octadecynoic acid and 16-hydroxy-17-octadecynoic acid is due to alkylation of the prosthetic heme group to given an adduct tentatively identified as N-(2-oxo-3-hydroxy-17-carboxyheptadecyl)protoporphyrin IX by its chromatographic and spectroscopic properties. Catalytic turnover of 17-octadecenoic acid also results in heme modification. Fatty diacid monoethyl thioesters are introduced as a new class of irreversible inhibitors that exploit the omega-2 oxidation specificity of cytochrome P450BM-3. Catalytic oxidation of the monoethyl thioesters of dodecanedioic and hexadecanedioic acids results in enzyme inactivation and formation of the parent diacids as metabolites. Limited tryptic digestion of the enzyme after incubation with the monoethyl thioester of [14C]hexadecanedioic acid shows that the inactivating agent binds covalently to both the heme and flavin domains. This finding, and the observation that glutathione prevents inactivation of the enzyme by the monoethyl thioesters, indicate that a diffusible metabolite, probably the sulfoxide, is responsible for enzyme inactivation. The strong preference for omega-2 allylic or propargylic hydroxylation over terminal pi-bond oxidation is opposite to the usual cytochrome P450 pattern and requires that the enzyme actively suppress terminal pi-bond oxidation. The inference that the enzyme binds and sequesters the terminal carbon in a lipophilic pocket is consistent with the crystal structure of the hemoprotein domain of cytochrome P450BM-3.

Acyl, pseudotetra‐, tri‐ and dipeptide active‐core analogs of insect neuropeptides

Pseudopeptides of the achetakinin insect neuropeptide family were synthesized by replacing the amino acid blocks Phe-, Phe-Tyr-, and Phe-Tyr-Pro- of the active-core pentapeptide Phe-Tyr-Pro-Trp-Gly-NH2 with hydrocinnamic acid, 6-phenylhexanoic acid, and both 9-phenylnonanoic and 6-phenylhexanoic acid, respectively. All four of these analogs retained myotropic activity, demonstrating that the active core could be reduced from a pentapeptide to a modified dipeptide. Most notable of these was the pseudotetrapeptide hydrocinnamyl-Tyr-Pro-Trp-Gly-NH2, which retained 70% of the potency and over 85% of the maximal activity of the parent pentapeptide. The N-terminal amino group, the phenol ring of the Tyr residue, the sulfate moiety and the Gly residue of the insect sulfakinin active core Tyr(SO3H)-Gly-His-Met-Arg-Phe-NH2 were all replaced by dodecanedioic acid. The resulting pseudotetrapeptide, dodecandioyl-His-Nle-Arg-Phe-NH2, elicited myostimulatory activity. Conversely, the related acyl pseudopentapeptide azelayl-Gly-His-Nle-Arg-Phe-NH2 proved myoinhibitory. A possible explanation for these disparate biological responses is discussed. These acyl pseudopeptides are important advances towards the eventual development of stable, potent mimetic agonists and antagonists of insect neuropeptides.

The enzymology of dicarboxylic acid formation by Corynebacterium sp. strain 7E1C grown on n-alkanes

Cultures of the Gram-positive bacterium Corynebacterium sp. strain 7E1C contained up to 300 mg dodecanedioic acid l−1 after growth on dodecane. Small amounts of tetradecanedioic acid (17 to 45 mg l−1) were produced during growth on tetradecane or methyl tetradecanoate. No dicarboxylic acids were detected after growth on hexadecane, hexadecanoic acid or 16-hydroxyhexadecanoic acid. Studies on the rates of degradation of exogenous dicarboxylic acids showed that this is not a significant factor influencing the accumulation of dodecanedioic and tetradecanedioic acids. The activities and substrate specificities of a number of enzyme activities involved in dicarboxylic acid metabolism were investigated. The specificities of the long-chain acyl-CoA synthetase and thioesterase, alcohol dehydrogenases and β-oxidation are consistent with the accumulation of dodecanedioic acid from dodecane and the lack of production of hexadecanedioic acid from hexadecane. The ω-hydroxy fatty acid may occupy a pivotal position in determining whether significant production of dicarboxylic acid occurs with this organism.

Solid‐state 13C and 1H NMR study of anomalous acid salts of dibasic carboxylic acids

AbstractHigh‐resolution solid‐state 13C and 1H NMR techniques were used to study the structure of a number of anomalous acid salts of adipic, azelaic, dodecanedioic, maleic and fumaric acids. To assist in the understanding of these results, solid‐state NMR data were also obtained for the corresponding acids and a number of their ‘normal’ salts. The resulting information provides insights into the solid structure of these complexes, including approximations of the length of the hydrogen bonds present in these systems. Strong evidence is given for the presence of a symmetrical hydrogen bond for tetramethylammonium didodecanedioate. Carbon‐13 NMR measurements carried out over a range of temperatures have revealed differences in the behavior of several of these compounds which may provide clues regarding the nature of the hydrogen bond in these systems.

Induction of omega-oxidation of monocarboxylic acids in rats by acetylsalicylic acid.

The accumulation of dicarboxylic acids, particularly long chain, is a prominent feature of Reye's syndrome and diseases of peroxisomal metabolism. We assessed the omega-oxidation of a spectrum of fatty acids in rats and asked whether pretreatment of rats with aspirin, which is known to predispose children to Reye's syndrome, would affect omega-oxidation of long chain fatty acids. We found that aspirin increased liver free fatty acids and increased the capacity for omega-oxidation three- to sevenfold. Omega-oxidation of long chain substrate was stimulated to a greater degree than medium chain substrate and was apparent within one day of treatment, at serum aspirin concentrations below the therapeutic range in humans. The apparent Km for lauric acid was 0.9 microM and 12 microM for palmitate. We also found a difference in the storage stability of activity toward medium and long chain substrate. Saturating concentrations of palmitate had no effect on the formation of dodecanedioic acid, whereas laurate decreased but never eliminated the omega-oxidation of palmitate. 97% of the total laurate omega-oxidative activity recovered was found in the microsomes, but 32% of palmitate omega-oxidative activity was present in the cytosol. These results demonstrate that aspirin is a potent stimulator of omega-oxidation and suggest that there may be multiple enzymes for omega-oxidation with overlapping substrate specificity.

Characterization of the binding sites for dicarboxylic acids on bovine serum albumin

Dicarboxylic acids are prominent features of several diseases, including Reye's syndrome and inborn errors of mitochondrial and peroxisomal fatty acid oxidation. Moreover, dicarboxylic acids are potentially toxic to cellular processes. Previous studies [Tonsgard, Mendelson & Meredith (1988) J. Clin. Invest. 82, 1567-1573] demonstrated that long-chain dicarboxylic acids have a single high-affinity binding site and between one and three lower-affinity sites on albumin. Medium-chain-length dicarboxylic acids have a single low-affinity site. We further characterized dicarboxylic acid binding to albumin in order to understand the potential effects of drugs and other ligands on dicarboxylic acid binding and toxicity. Progesterone and oleate competitively inhibit octadecanedioic acid binding to the single high-affinity site. Octanoate inhibits binding to the low-affinity sites. Dansylated probes for subdomain 2AB inhibit dodecanedioic acid binding whereas probes for subdomain 3AB do not. In contrast, low concentrations of octadecanedioic acid inhibit the binding of dansylated probes to subdomain 3AB and 2AB. L-Tryptophan, which binds in subdomain 3AB, inhibits hexadecanedioic acid binding but has no effect on dodecanedioic acid. Bilirubin and acetylsalicylic acid, which bind in subdomain 2AB, inhibit the binding of medium-chain and long-chain dicarboxylic acids. Our results suggest that long-chain dicarboxylic acids bind in subdomains 2C, 3AB and 2AB. The single low-affinity binding site for medium-chain dicarboxylic acids is in subdomain 2AB. These studies suggest that dicarboxylic acids are likely to be unbound in disease states and may be potentially toxic.

CDNA cloning and expression of the mRNA for cytochrome P‐450kd which shows a fatty acid ω‐hydroxylating activity

We have recently purified three distinct forms of fatty acid omega-hydroxylase cytochrome P-450 (P-450), designated P-450ka-1, P-450ka-2 and P-450kd, from rabbit kidney cortex microsomes, and isolated and sequenced cDNA clones corresponding to P-450ka-1 and P-450ka-2 [Yokotani, N., Bernhardt, R., Sogawa, K., Kusunose, E., Gotoh, M., Kusunose, M. & Fujii-Kuriyama, Y. (1989) J. Biol. Chem. 264, 21,665-21,669]. The present paper describes cloning, sequencing and expression of a cDNA for the third fatty acid, omega-hydroxylase, P-450kd, from a rabbit kidney cDNA library. The cDNA for P-450kd encodes a polypeptide of 511 amino acids with sequence similarity of 87% to P-450ka-1. Its deduced NH2-terminal sequence of amino acids 5-24 is in complete agreement with the NH2-terminal sequence of P-450kd. The identity of the cDNA was further confirmed by its expression in COS-7 cells. When 14C-labeled lauric acid was added to the culture medium of COS-7 cells transfected with the cDNA, significant amounts of radioactive dodecanedioic acid, together with omega- and (omega-1)-hydroxylauric acids, were produced. Microsomes prepared from the transfected cells also efficiently catalyzed the omega- and (omega-1)-hydroxylation of lauric acid without formation of dodecanedioic acid. RNA blot analysis demonstrated that the mRNA for P-450kd gave a single band at the approximately 2.6-kb position. The mRNA for P-450kd was expressed in the liver and kidney, but not in many other tissues examined. Treatment of rabbits with clofibrate resulted in a elevated level of mRNA for P-450kd in both liver and kidney. Furthermore, the mRNA was remarkably increased in the kidney by the administration of cyclosporin A.

Metabolic conversion of dicarboxylic acids to succinate in rat liver homogenates. A stable isotope tracer study.

The metabolic conversion of dicarboxylic acids into succinate and other gluconeogenic intermediates in rat liver homogenates was investigated using [1,2,4-13C4]dodecanedioic acid as tracer. Isotope enrichments in 3-hydroxybutyrate, succinate, fumarate, and malate, as well as dicarboxylates (dodecanedioic, sebacic, suberic, and adipic acids) were measured with selected ion monitoring capillary column gas chromatograph-mass spectrometry. Significant enrichment in the M + 4 (four labeled carbons) ion of succinate (0.4-2.9%) was detected, unequivocally demonstrating the direct conversion of dicarboxylate into succinate. In addition, significant enrichment of the M + 2 ion of succinate was also observed. This labeled species was generated from labeled acetyl-CoA through the tricarboxylic acid cycle. The partition of acetyl-CoA into the tricarboxylic acid cycle relative to ketone body formation was higher in the beta oxidation of dicarboxylate than monocarboxylate. Therefore, in addition to the production of succinate, the beta oxidation of dodecanedioate resulted in the channeling of the acetyl-CoA produced to the tricarboxylic acid cycle instead of to acetoacetate production. The enrichments in lower chain dicarboxylates are consistent with a partial bidirectional beta oxidation of dodecanedioic acid. In addition to the expected M + 0 and M + 4 labels, significant M + 2 species were detected in suberic and adipic acids. These M + 2-labeled species were produced from the released free dicarboxylate intermediates which were then reactivated and metabolized. In these experiments, the overall succinate production was derived 4% from the direct conversion of dodecanedioic acid and 11% from the indirect route via acetyl-CoA through tricarboxylic acid.

Physical and chemical properties of a biosurfactant synthesized by Rhodococcus species H13-A

The chemical and physical properties of a biosurfactant synthesized by hexadecane-grown Rhodococcus species H13-A are described. The biosurfactant is an anionic glycolipid consisting of 1 major and 10 minor components. The hydrophilic portion of the molecule is trehalose, which is acylated with normal C(10) to C(22) saturated and unsaturated fatty acids, C(35) to C(40) mycolic acids, hexanedioic and dodecanedioic acids, and 10-methyl hexadecanoic and 10-methyl octadecanoic acids. The major glycolipid species was identified as 2,3,4,6,2',3',4',6'-octaacyltrehalose, plus minor glycolipid species of di-, tetra- and hexa-acyltrehalose derivatives. The glycolipid exhibited a critical micelle concentration of 1.5 mg/mL and minimum interfacial tension value of 2 × 10(-2) mN/m against decane, with a further reduction in interfacial tension to 6 × 10(-5) mN/m in the presence of the cosurfactant pentanol. The phase behavior of the glycolipid indicates the formation of a surfactant-rich, "middle-phase" microemulsion containing liquid crystals, both of which are associated with surfactant systems having ultralow interfacial tension values. Key words: trehalose lipids, glycolipids, biosurfactants.