Metabolic origin of urinary 3-hydroxy dicarboxylic acids

3-Hydroxy dicarboxylic acids with chain lengths ranging from 6 to 14 carbons are excreted in human urine. The urinary excretion of these acids is increased in conditions of increased mobilization of fatty acids or inhibited fatty acid oxidation. Similar urinary profiles of 3-hydroxy dicarboxylic acids were also observed in fasting rats. The metabolic genesis of these urinary 3-hydroxy dicarboxylic acids was investigated in vitro with rat liver postmitochondrial and mitochondrial fractions. 3-Hydroxy monocarboxylic acids ranging from 3-hydroxyhexanoic acid to 3-hydroxyhexadecanoic acid were synthesized. In the rat liver postmitochondrial fraction fortified with NADPH, these 3-hydroxy fatty acids with carbon chains equal to or longer than 10 were oxidized to (omega - 1)- and omega-hydroxy metabolites as well as to the corresponding 3-hydroxy dicarboxylic acids. 3-Hydroxyhexanoic (3OHMC6) and 3-hydroxyoctanoic (3OHMC8) acids were not metabolized. Upon the addition of mitochondria together with ATP, CoA, carnitine, and MgCl2, the 3-hydroxy dicarboxylic acids were converted to 3-hydroxyoctanedioic, trans-2-hexenedioic, suberic, and adipic acids. In the urine of children with elevated 3-hydroxy dicarboxylic acid levels, 3OHMC6, 3OHMC8, 3-hydroxydecanoic, 3,10-dihydroxydecanoic, 3,9-dihydroxydecanoic, and 3,11-dihydroxydodecanoic acids were identified. On the basis of these data, we propose that the urinary 3-hydroxy dicarboxylic acids are derived from the omega-oxidation of 3-hydroxy fatty acids and the subsequent beta-oxidation of longer chain 3-hydroxy dicarboxylic acids. These urinary 3-hydroxy dicarboxylic acids are not derived from the beta-oxidation of unsubstituted dicarboxylic acids.     

Abnormal urinary excretion of unsaturated dicarboxylic acids in patients with medium-chain acyl-CoA dehydrogenase deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently described metabolic disorder of fatty acid oxidation in humans. Acute episodes are usually characterized biochemically by the appearance of nonketotic dicarboxylic aciduria. In addition, other abnormal metabolites, such as suberylglycine, n-hexanoylglycine, 3-phenylpropionylglycine, and octanoylcarnitine, are excreted in the urine. Urinary organic acids were determined using dual capillary column gas-liquid chromatography and gas-liquid chromatography/mass spectrometry. In three cases of MCAD deficiency we observed a disproportionate increase in the excretion of unsaturated dicarboxylic acids compared to either fasting control children with expected ketotic dicarboxylic aciduria or patients with nonketotic dicarboxylic aciduria not associated with MCAD deficiency. The most significant increase was in the urinary excretion of cis-4-decendioic acid. Additionally, the urinary excretions of cis-3-octenedioic and cis-5-decenedioic acids were slightly decreased whereas the excretion of cis-5-dodecenedioic acid was increased. These data are consistent with the notion that as a result of MCAD deficiency the metabolic oxidation of unsaturated fatty acids such as linoleate and oleate is inhibited more than saturated fatty acids.     

Production of dicarboxylic acids from novel unsaturated fatty acids by laccase-catalyzed oxidative cleavage

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids in Lactobacillus plantarum as substrates. Hydroxy or oxo fatty acids with a functional group near the carbon–carbon double bond were cleaved at the carbon–carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids.     

Metabolic engineering for the production of dicarboxylic acids and diamines

Microbial production of chemicals and materials from renewable carbon sources is becoming increasingly important to help establish sustainable chemical industry. In this paper, we review current status of metabolic engineering for the bio-based production of linear and saturated dicarboxylic acids and diamines, important platform chemicals used in various industrial applications, especially as monomers for polymer synthesis. Strategies for the bio-based production of various dicarboxylic acids having different carbon numbers including malonic acid (C3), succinic acid (C4), glutaric acid (C5), adipic acid (C6), pimelic acid (C7), suberic acid (C8), azelaic acid (C9), sebacic acid (C10), undecanedioic acid (C11), dodecanedioic acid (C12), brassylic acid (C13), tetradecanedioic acid (C14), and pentadecanedioic acid (C15) are reviewed. Also, strategies for the bio-based production of diamines of different carbon numbers including 1,3-diaminopropane (C3), putrescine (1,4-diaminobutane; C4), cadaverine (1,5-diaminopentane; C5), 1,6-diaminohexane (C6), 1,8-diaminoctane (C8), 1,10-diaminodecane (C10), 1,12-diaminododecane (C12), and 1,14-diaminotetradecane (C14) are revisited. Finally, future challenges are discussed towards more efficient production and commercialization of bio-based dicarboxylic acids and diamines.     

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.     

Characterization of dicarboxylic acids for cellulose hydrolysis

In this paper, we show that dilute maleic acid, a dicarboxylic acid, hydrolyzes cellobiose, the repeat unit of cellulose, and the microcrystalline cellulose Avicel as effectively as dilute sulfuric acid but with minimal glucose degradation. Maleic acid, superior to other carboxylic acids reported in this paper, gives higher yields of glucose that is more easily fermented as a result of lower concentrations of degradation products. These results are especially significant because maleic acid, in the form of maleic anhydride, is widely available and produced in large quantities annually.     

Metabolism of dicarboxylic acids in rat hepatocytes

[carboxyl-14C]Dodecanedioic acid (DC12) is metabolized in hepatocytes at a rate about two thirds that of [1-14C]palmitate. Shorter dicarboxylates (sebacic (DC10), suberic (DC8), and adipic (DC6) acid) are formed, mainly DC6, less DC8 and only a little DC10. In hepatocytes from clofibrate-treated rats, more polar products account for most of the breakdown products, presumably because the beta-oxidation proceeds all the way to succinate and acetyl-CoA. [carboxyl-14C]Suberic acid (DC8) is oxidized at a rate only one fifth that of dodecanedioic acid. (+)-Decanoylcarnitine inhibits palmitate oxidation but not the oxidation of dodecanedioic acid. At low concentrations of [carboxyl-14C]dodecanedioic acid or of [1-14C]palmitate, acetylsulfanilamide is more efficiently labeled by the former. High concentrations of dodecanedioic acid inhibit palmitate oxidation and the acetylation of sulfanilamide, presumably because their CoA-esters accumulate in the cytosol. These results indicate that medium-chain dicarboxylic acids are beta-oxidized mainly in the peroxisomes.     

Synthesis of unsaturated fatty acids

This article reviews synthetic routes leading to polyunsaturated fatty acids having “skipped” double bonds. Emphasis is placed on the “acetylenic approach”.The suitability of building blocks, their condensation reactions as well as the controlled reduction of triple bonds to cis double bonds are discussed. In addition, the application of the various methods to the preparation of polyunsaturated fatty acids labelled with ³H and/or ¹⁴C at distinct positions of the molecules is reviewed.     

Urinary excretion of dicarboxylic acids from patients with the Zellweger syndrome. Importance of peroxisomes in beta-oxidation of dicarboxylic acids

The urinary excretion of adipic acid, suberic acid and sebacic acid from two patients with the cerebrohepato-renal syndrome of Zellweger was studied. The patients had a complete lack of peroxisomes in the liver as judged by electron microscopy. In the non-ketotic state, the total excretion of free and conjugated adipic acid, suberic acid and sebacic acid was increased by about 100%, 200% and 350%, respectively, as compared to the corresponding excretion from six healthy infants of the same age. The excretion of free dicarboxylic acid was increased to a considerably lesser extent than the free + conjugated dicarboxylic acid. In view of the presence of adipic acid in urine of the Zellweger patients, it is concluded that peroxisomes are not obligatory for beta-oxidation of medium-chain dicarboxylic acids in vivo. The relative accumulation of suberic acid and sebacic acid as compared to adipic acid is, however, consistent with a relative block in the conversion of suberic acid and sebacic acid into adipic acid in patients with the Zellweger syndrome.     

Transport of dicarboxylic acids in castor bean mitochondria

Mitochondria from castor bean (Ricinus communis cv Hale) endosperm, purified on sucrose gradients, were used to investigate transport of dicarboxylic acids. The isolated mitochondria oxidized malate and succinate with respiratory control ratios greater than 2 and ADP/O ratios of 2.6 and 1.7, respectively. Net accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate into the mitochondrial matrix during substrate oxidation was examined by the silicone oil centrifugation technique. In the presence of ATP, there was an appreciable increase in the accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate accompanied by an increased oxidation rate of the respective dicarboxylate. The net accumulation of dicarboxylate in the presence of ATP was saturable with apparent K_m values of 2 to 2.5 millimolar. The ATP-stimulated accumulation of dicarboxylate was unaffected by oligomycin but inhibited by uncouplers (2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) and inhibitors of the electron transport chain (antimycin A, KCN). Dicarboxylate accumulation was also inhibited by butylmalonate, benzylmalonate, phenylsuccinate, mersalyl and N-ethylmaleimide. The optimal ATP concentration for stimulation of dicarboxylate accumulation was 1 millimolar. CTP was as effective as ATP in stimulating dicarboxylate accumulation, and other nucleotide triphosphates showed intermediate or no effect on dicarboxylate accumulation. Dicarboxylate accumulation was phosphate dependent but, inasmuch as ATP did not increase phosphate uptake, the ATP stimulation of dicarboxylate accumulation was apparently not due to increased availability of exchangeable phosphate.

The maximum rate of succinate accumulation (14.5 nanomoles per minute per milligram protein) was only a fraction of the measured rate of oxidation (100-200 nanomoles per minute per milligram protein). Efflux of malate from the mitochondria was shown to occur at high rates (150 nanomoles per minute per milligram protein) when succinate was provided, suggesting dicarboxylate exchange. The uptake of [¹⁴C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [¹⁴C]succinate into mitochondria preloaded with malate was rapid (27 nanomoles per 15 seconds per milligram protein at 4°C) and inhibited by butylmalonate, benzylmalonate, and phenylsuccinate. Uptake of [¹⁴C]succinate into mitochondria preloaded with malonate showed saturation kinetics with an apparent K_m of 2.5 millimolar and V_max of 250 nanomoles per minute per milligram protein at 4°C. The measured rates of dicarboxylate-dicarboxylate exchange in castor bean mitochondria are sufficient to account for the observed rates of substrate oxidation.