Mouse hepatic microsomal oxidation of aliphatic aldehydes (C8 to C11) to carboxylic acids.

Addition of saturated and alpha, beta-unsaturated aliphatic aldehydes (C8 to C11) significantly increased NADPH oxidation with mouse hepatic microsomes, and the aldehydes themselves were oxidized to the corresponding carboxylic acids. When these aldehyde substrates were incubated similarly under oxygen-18 gas and the carboxylic acids formed were analyzed by GC-MS after methylation, it was indicated that oxygen-18 was significantly incorporated into the carboxylic acids formed from alpha, beta-unsaturated aldehydes, but not significantly into the carboxylic acids formed from saturated aldehydes. These results indicate that enzyme and/or mechanism responsible for the oxidation of these two types of aldehydes is different from each other.     

Oxygenation mechanism in conversion of aldehyde to carboxylic acid catalyzed by a cytochrome P-450 isozyme.

The oxygenation of an aldehyde, 11-oxo-delta 8-tetrahydrocannabinol to a carboxylic acid, delta 8-tetrahydrocannabinol-11-oic acid was catalyzed by cytochrome P-450 MUT-2 purified from hepatic microsomes of male ddN mice. The oxygenation mechanism was confirmed by the incorporation of oxygen-18 from molecular oxygen into the carboxylic acid formed. An aldehyde form but not a hydrated form of 11-oxo-delta 8-tetrahydrocannabinol may be a substrate for the cytochrome P-450. The oxygenation of aldehyde catalyzed by cytochrome P-450 might be a common metabolic reaction in biological systems, and should be considered as an additional role of cytochrome P-450 in biotransformation of endogenous compounds and xenobiotics.     

An efficient conversion of carboxylic acids to one-carbon degraded aldehydes via 2-hydroperoxy acids

After the formation of dianions of a carboxylic acid with lithium diisopropylamide, oxygen was bubbled into the solution to produce 2-hydroperoxy acid. Then the reaction mixture was acidified with a 2 N HCl solution and subsequently elevated to 50 degrees C to afford the aldehyde with the loss of one carbon atom. Even saturated (C(10)-C(20)) and unsaturated (C(18:1)) carboxylic acids were converted into the odd aldehydes (C(9)-C(19), C(17:1)) in high yields. This conversion was found to be an efficient method for the preparation of carboxylic acids (Cn) to one-carbon degraded aldehydes (Cn-1) via 2-hydroperoxy acids.     

Oxygenation mechanism in the oxidation of xenobiotic aldehyde to carboxylic acid by mouse hepatic microsomes

11-Oxo-delta 8-tetrahydrocannabinol was oxidized to delta 8-tetrahydrocannabinol-11-oic acid by mouse hepatic microsomes. The oxygenation mechanism in the reaction was confirmed by the incorporation of oxygen-18 from molecular oxygen into delta 8-tetrahydrocannabinol-11-oic acid. The oxygenation of aldehyde to carboxylic acid represents a novel mechanism in biological oxidation of aldehyde to carboxylic acid.     

An Efficient Conversion of Carboxylic Acids to One-Carbon Degraded Aldehydes via 2-Hydroperoxy Acids

After the formation of dianions of a carboxylic acid with lithium diisopropylamide, oxygen was bubbled into the solution to produce 2-hydroperoxy acid. Then the reaction mixture was acidified with a 2 N HCl solution and subsequently elevated to 50 °C to afford the aldehyde with the loss of one carbon atom. Even saturated (C₁₀–C₂₀) and unsaturated (C_18:1) carboxylic acids were converted into the odd aldehydes (C₉–C₁₉, C_17:1) in high yields. This conversion was found to be an efficient method for the preparation of carboxylic acids (Cn) to one-carbon degraded aldehydes (Cn-1) via 2-hydroperoxy acids.     

Hydroperoxy-arachidonic acid mediated n-hexanal and (Z)-3- and (E)-2-nonenal formation in Laminaria angustata

In higher plants, C6 and C9 aldehydes are formed from C18 fatty acids, such as linoleic or linolenic acid, through formation of 13- and 9-hydroperoxides, followed by their stereospecific cleavage by fatty acid hydroperoxide lyases (HPL). Some marine algae can also form C6 and C9 aldehydes, but their precise biosynthetic pathway has not been elucidated fully. In this study, we show that Laminaria angustata, a brown alga, formed C6 and C9 aldehydes enzymatically. The alga forms C9 aldehydes exclusively from the C20 fatty acid, arachidonic acid, while C6 aldehydes are derived either from C18 or from C20 fatty acid. The intermediates in the biosynthetic pathway were trapped by using a glutathione/glutathione peroxidase system, and subjected to structural analyses. Formation of (S)-12-, and (S)-15-hydroperoxy arachidonic acids [12(S)HPETE and 15(S)HPETE] from arachidonic acid was confirmed by chiral HPLC analyses. These account respectively for C9 aldehyde and C6 aldehyde formation, respectively. The HPL that catalyzes formation of C9 aldehydes from 12(S)HPETE seems highly specific for hydroperoxides of C20 fatty acids.     

Fluorometric assay of microsomal aldehyde oxygenase activity: 9-anthraldehyde as a substrate.

With regard to hepatic microsomal oxidation of 9-anthraldehyde (9-AA), a fluorometric method for determination of 9-anthracene carboxylic acid (9-ACA) is described. 9-AA was incubated with hepatic microsomes prepared from male ddN mice. 9-ACA formed was fluorometrically (excitation and emission wavelengths of 255 and 458 nm, respectively) quantitated after the separation from 9-AA by an alkali extraction and ethyl acetate reextraction. Hepatic microsomes less than 0.1 mg protein were enough to assay the microsomal aldehyde oxidation. The enzyme in the microsomes that catalyzes the oxidation of 9-AA to 9-ACA has been characterized by this method.     

Enzymatic Improvement of Food Flavor I. Purification and Characterization of Bovine Liver Mitochondrial Aldehyde Dehydrogenase

Bovine liver mitochondrial aldehyde dehydrogenase (aldehyde: NAD⁺ oxidoreductase, EC 1.2.1.3) has been purified to homogeneity by conventional purification procedures. The enzyme was found to have a molecular weight of 215,000 based on gel filtration. The protein is composed of polypeptides having the same molecular weight, 54,000 and thus it appears to consist of four subunits of equal size. The enzyme exhibited a broad aldehyde specificity, oxidizing irreversibly a wide variety of aliphatic and aromatic aldehydes to corresponding carboxylic acids. Km values for straight-chain saturated aldehydes were below 0.1 µm, and relatively constant independent of the carbon chain lengths of the aldehydes. The maximum velocities for saturated aldehydes also did not vary appreciably with their carbon chain lengths. Maximum activity was observed at pH 9.3 and 50°C. The enzyme activity was affected by some divalent cations. Ca²⁺ enhanced the activity, while Mg²⁺ inhibited it. The enzyme was quite stable at neutral pH, but was unstable above pH 9 or below pH 6. Bovine liver has three isozymes of aldehyde dehydrogenase which are located in the mitochondrial, cytosolic, and microsomal fractions. Comparison of enzymic properties among these isozymes and yeast enzyme indicates that the mitochondrial enzyme is very suitable for improving the objectionable flavor due to aldehydes in foods.     

Oxidation of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids by Acetobacter and Serratia strains

Conversion of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids was carried out using Acetobacter rancens IFO3297, A. pasteurianus IFO13753 and Serratia liquefaciens LF14. IFO3297 produced 110 g 2-furoic acid l(-1) from furfural with a 95% molar yield. 5-Hydroxymethyl-2-furancarboxylic acid was produced from the corresponding aldehyde by using whole cells LF14. IFO13753 and LF14 both converted isophthalaldehyde, 2,5-furandicarbaldehyde, 2,5-thiophenedicarbaldehyde and 2,2' biphenyldicarbaldehyde to the corresponding formylcarboxylic acid with 86-91% molar yields.     

A lipid-hydrolysing activity involved in hexenal formation

Short-chain aldehydes such as (3Z)-hexenal and n-hexanal are formed from lipids through sequential actions of lipid-hydrolysing, lipoxygenase and fatty acid hydroperoxide lyase activities. The aldehydes are formed upon wounding of plant tissues, and are reported to have bactericidal and fungicidal activities. Furthermore, it has been reported that the aldehydes can induce expression of a subset of genes involved in disease resistance and that they are involved in a defence response against insect herbivores. Although several genes encoding lipoxygenases and the lyases have been isolated, and characterized to some extent, only little is known about the enzyme accountable for the lipid-hydrolysing step. In this study, we tried to characterize the lipid-hydrolysing activity involved in the short-chain aldehyde formation in Arabidopsis. When Arabidopsis leaves were homogenized, (3Z)-hexenal was formed rapidly within a few minutes. During this time period, the amount of alpha-linolenic acid and C(16:3) rapidly decreased. Such a rapid increase of the aldehyde was repressed almost completely when the leaves were homogenized under a nitrogen stream, and instead free trienoic acids accumulated. A lipase inhibitor, quinacrine, successfully repressed the hydrolysis. It was revealed that trienoic acids in monogalactosyldiacylglycerol were predominantly hydrolysed during the formation of short-chain aldehydes. Collectively, it is suggested that the lipolytic enzyme involved in the short-chain aldehyde formation is a galactolipid-specific lipase.     

Roles of rat and human aldo-keto reductases in metabolism of farnesol and geranylgeraniol

Farnesol (FOH) and geranylgeraniol (GGOH) with multiple biological actions are produced from the mevalonate pathway, and catabolized into farnesoic acid and geranylgeranoic acid, respectively, via the aldehyde intermediates (farnesal and geranylgeranial). We investigated the intracellular distribution, sequences and properties of the oxidoreductases responsible for the metabolic steps in rat tissues. The oxidation of FOH and GGOH into their aldehyde intermediates were mainly mediated by alcohol dehydrogenases 1 (in the liver and colon) and 7 (in the stomach and lung), and the subsequent step into the carboxylic acids was catalyzed by a microsomal aldehyde dehydrogenase. In addition, high reductase activity catalyzing the aldehyde intermediates into FOH (or GGOH) was detected in the cytosols of the extra-hepatic tissues, where the major reductase was identified as aldo-keto reductase (AKR) 1C15. Human reductases with similar specificity were identified as AKR1B10 and AKR1C3, which most efficiently reduced farnesal and geranylgeranial among seven enzymes in the AKR1A-1C subfamilies. The overall metabolism from FOH to farnesoic acid in cultured cells was significantly decreased by overexpression of AKR1C15, and increased by addition of AKR1C3 inhibitors, tolfenamic acid and R-flurbiprofen. Thus, AKRs (1C15 in rats, and 1B10 and 1C3 in humans) may play an important role in controlling the bioavailability of FOH and GGOH.     

Carboxylic acid reductase: a new tungsten enzyme catalyses the reduction of non-activated carboxylic acids to aldehydes

An enzyme which we call carboxylic acid reductase (aldehyde dehydrogenase) seems to be the first which is able to reduce non-activated carboxylic acids to aldehydes at the expense of reduced viologens. There is no further reduction of the aldehydes to the corresponding alcohols. In the presence of oxidized viologens aldehydes can be dehydrogenated to carboxylic acids roughly 20 times faster than the latter are reduced. The specific enzyme activity in crude extracts is about 100 times increased if 10 microM tungstate and a sulphur source in addition to sulphate is given to the growth medium of Clostridium thermoaceticum. Carboxylic acid reductase seems to be present in two forms. One has an apparent molecular mass of about 240 kDa and is bound to red-Sepharose, whereas, the other, a form of an apparent molecular mass of about 60 kDa, is not bound. SDS gel electrophoresis shows a higher complexity. The very labile enzyme has been enriched by a factor of about 145 by binding to octyl-Sepharose and further chromatographic separation by red-Sepharose and FPLC using Mono-Q and phenyl-Superose columns. After cell growth in the presence of [185W]tungstate, radioactivity coincides with the two forms of enzyme activity during all purification steps. This is also the case when the enzyme is electrophoretically separated on polyacrylamide slab gels.     

Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646.

An aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646 was purified 196-fold by a combination of Mono-Q, Reactive Green 19 agarose affinity, and hydroxyapatite chromatographies. The purified enzyme runs as a single band of 140 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass was estimated to be 163 +/- 3.8 kDa by gel filtration, indicating that this enzyme is a monomeric protein. The binding of the enzyme to Reactive Green 19 agarose was Mg2+ dependent. The binding capacity was estimated to be about 0.2 mg of Reactive Green agarose per ml in the presence of 10 mM MgCl2. This enzyme can catalyze the reduction of a wide range of aryl carboxylic acids, including substituted benzoic acids, phenyl-substituted aliphatic acids, heterocyclic carboxylic acids, and polyaromatic ring carboxylic acids, to produce the corresponding aldehydes. The Km values for benzoate, ATP, and NADPH were determined to be 645 +/- 75, 29.3 +/- 3.1, and 57.3 +/- 12.5 microM, respectively. The Vmax was determined to be 0.902 +/- 0.04 micromol/min/mg of protein. Km values for (S)-(+)-alpha-methyl-4-(2-methylpropyl)-benzeneacetic acid (ibuprofen) and its (R)-(-) isomer were determined to be 155 +/- 18 and 34.5 +/- 2.5 microM, respectively. The Vmax for the (S)-(+) and (R)-(-) isomers were 1.33 and 0.15 micromol/min/mg of protein, respectively. Anthranilic acid is a competitive inhibitor with benzoic acid as a substrate, with a Ki of 261 +/- 30 microM. The N-terminal and internal amino acid sequences of a 76-kDa peptide from limited alpha-chymotrypsin digestion were determined.     

Oxidation of aldehydic products of lipid peroxidation by rat liver microsomal aldehyde dehydrogenase

Lipid peroxidation in microsomal membranes produces a large number of aldehydes, alcohols, and ketones, some of which have been shown to be cytotoxic. This study has determined the kinetic parameters for the oxidation of aldehyde lipid peroxidation products by purified rat hepatic microsomal aldehyde dehydrogenase (ALDH). Livers were obtained from male Sprague-Dawley rats for preparation of microsomal ALDH which was purified 400-fold. Kinetic parameters, Vmax and V/K, were determined for saturated and unsaturated aldehydes of three to nine carbons in length in the presence of NAD+. Of the aldehydes examined, only acrolein and 4-hydroxynonenal were not oxidized by ALDH. The Vmax values (mumol NADH produced/min/mg protein) increased linearly with carbon chain length and ranged from 6.5 to 23 for the saturated series and 4.0 to 9.0 for the unsaturated aldehydes. The affinity constant V/K (nmol NADH produced/min/mg protein/nmol aldehyde/liter) also increased with carbon chain length and ranged from 12 to 9000 for the saturated aldehydes and 13 to 5300 for the unsaturated aldehydes. These results suggest that microsomal ALDH may serve a biological role for detoxification of reactive aldehydes produced by lipid peroxidation of microsomal membranes.     

Characterization of degradation products from alkaline wet oxidation of wheat straw

Alkaline wet oxidation pre-treatment (water, sodium carbonate, oxygen, high temperature and pressure) of wheat straw was performed as a 2(4-1) fractional factorial design with the process parameters: temperature, reaction time, sodium carbonate and oxygen. Alkaline wet oxidation was an efficient pre-treatment of wheat straw that resulted in solid fractions with high cellulose recovery (96%) and high enzymatic convertibility to glucose (67%). Carbonate and temperature were the most important factors for fractionation of wheat straw by wet oxidation. Optimal conditions were 10 min at 195 degrees C with addition of 12 bar oxygen and 6.5 g l(-1) Na2CO3. At these conditions the hemicellulose fraction from 100 g straw consisted of soluble hemicellulose (16 g), low molecular weight carboxylic acids (11 g), monomeric phenols (0.48 g) and 2-furoic acid (0.01 g). Formic acid and acetic acid constituted the majority of degradation products (8.5 g). The main phenol monomers were 4-hydroxybenzaldehyde, vanillin, syringaldehyde. acetosyringone (4-hydroxy-3,5-dimethoxy-acetophenone), vanillic acid and syringic acid, occurring in 0.04-0.12 g per 100 g straw concentrations. High lignin removal from the solid fraction (62%) did not provide a corresponding increase in the phenol monomer content but was correlated to high carboxylic acid concentrations. The degradation products in the hemicellulose fractions co-varied with the pre-treatment conditions in the principal component analysis according to their chemical structure, e.g. diacids (oxalic and succinic acids), furan aldehydes, phenol aldehydes, phenol ketones and phenol acids. Aromatic aldehyde formation was correlated to severe conditions with high temperatures and low pH. Apart from CO2 and water, carboxylic acids were the main degradation products from hemicellulose and lignin.     

On the role of microsomal aldehyde dehydrogenase in metabolism of aldehydic products of lipid peroxidation

To elucidate a possible role of membrane-bound aldehyde dehydrogenase in the detoxication of aldehydic products of lipid peroxidation, the substrate specificity of the highly purified microsomal enzyme was investigated. The aldehyde dehydrogenase was active with different aliphatic aldehydes including 4-hydroxyalkenals, but did not react with malonic dialdehyde. When Fe/ADP-ascorbate-induced lipid peroxidation of arachidonic acid was carried out in an in vitro system, the formation of products which react with microsomal aldehyde dehydrogenase was observed parallel with malonic dialdehyde accumulation.     

Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution.

1. The properties and distribution of the NAD-linked unspecific aldehyde dehydrogenase activity (aldehyde: NAD+ oxidoreductase EC 1.2.1.3) has been studied in isolated cytoplasmic, mitochondrial and microsomal fractions of rat liver. The various types of aldehyde dehydrogenase were separated by ion exchange chromatography and isoelectric focusing. 2. The cytoplasmic fraction contained 10-15, the mitochondrial fraction 45-50 and the microsomal fraction 35-40% of the total aldehyde dehydrogenase activity, when assayed with 6.0 mM propionaldehyde as substrate. 3. The cytoplasmic fraction contained two separable unspecific aldehyde dehydrogenases, one with high Km for aldehydes (in the millimolar range) and the other with low Km for aldehydes (in the micromolar range). The latter can, however, be due to leakage from mitochondria. The high-Km enzyme fraction contained also all D-glucuronolactone dehydrogenase activity of the cytoplasmic fraction. The specific formaldehyde and betaine aldehyde dehydrogenases present in the cytoplasmic fraction could be separated from the unspecific activities. 4. In the mitochondrial fraction there was one enzyme with a low Km for aldehydes and another with high Km for aldehydes, which was different from the cytoplasmic enzyme. 5. The microsomal aldehyde dehydrogenase had a high Km for aldehydes and had similar properties as the mitochondrial high-Km enzyme. Both enzymes have very little activity with formaldehyde and glycolaldehyde in contrast to the other aldehyde dehydrogenases. They are apparently membranebound.     

Cytotoxic aldehydes originating from the peroxidation of liver microsomal lipids. Identification of 4,5-dihydroxydecenal

During the NADPH-Fe-induced peroxidation of liver microsomal lipids products are formed which are provided with cytopathological activities. In a previous study one of the major products was identified as an aldehyde of the 4-hydroxyalkenal class, namely 4-hydroxynonenal. In the present study another cytotoxic product has been isolated and identified as 4,5-dihydroxy-2,3-decenal. The isolation was performed by means of thin-layer chromatography and high-pressure liquid chromatography and the structure was ascertained mainly by means of mass spectroscopy of the free aldehyde and of its derivatives. In the absence of NADPH-Fe liver microsomes produced no 4,5-dihydroxydecenal. The inhibitory activity of 4,5-dihydroxydecenal on microsomal glucose-6-phosphatase is somewhat lower than that exhibited by 4-hydroxynonenal. This lower inhibitory activity correlates with the lower capacity to bind to the microsomal protein of 4,5-dihydroxydecenal as compared to 4-hydroxynonenal. The reactivities of the two aldehydes with cysteine were comparable. The production of toxic aldehydes may represent a mechanism by which lipid peroxidation causes deleterious effects on cellular functions.     

Transformations of Halogenated Aromatic Aldehydes by Metabolically Stable Anaerobic Enrichment Cultures

Metabolically stable enrichment cultures of anaerobic bacteria obtained by elective enrichment of sediment samples from the Baltic Sea and Gulf of Bothnia have been used to study the oxidation and reduction of the aldehyde group of various halogenated aromatic aldehydes. During the transformation of 5- and 6-chlorovanillin, 6-bromovanillin, 3-chloro-4-hydroxybenzaldehyde, 3,5-dichloro-4-hydroxybenzaldehyde, and 3,5-dibromo-4-hydroxybenzaldehyde, it was shown that synthesis of the corresponding carboxylic acids, which were the principal metabolites, was invariably accompanied by partial reduction of the aldehyde to a hydroxymethyl group in yields of between 3 and 30%. Complete reduction to a methyl group was observed with some of the halogenated vanillins, but to an extremely limited extent with the halogenated 4-hydroxybenzaldehydes. One consortium produced both the hydroxymethyl and methyl compounds from both 5- and 6-chlorovanillin: it was therefore assumed that the methyl compound was the ultimate reduction product. On the basis of the kinetics of formation of the metabolites, it was concluded that the oxidation and reduction reactions were mechanistically related. In addition to these oxidations and reductions, dehalogenation was observed with one of the consortia. In contrast to the transformations of 5- and 6-chlorovanillin, which produced chlorinated methylcatechols, the corresponding compounds were not observed with 5- and 6-bromovanillin: the former was debrominated, forming 4-methylcatechol, whereas the latter produced 6-bromovanillyl alcohol without demethylation. Similarly, although 3-chloro-4-hydroxybenzaldehyde formed the chlorinated carboxylic acid and the benzyl alcohol, the 3-bromo compound was debrominated with formation of 4-hydroxybenzoic acid and, ultimately, phenol. On prolonged incubation, the halogenated carboxylic acids were generally decarboxylated, so that the final products from these substrates were halogenated catechols or phenols. Reductive processes of the type revealed in this study might therefore plausibly occur in the environment during anaerobic transformation of halogenated aromatic aldehydes containing hydroxyl and/or methoxyl groups.     

Biosynthesis of alkanes by particulate and solubilized enzyme preparations from pea leaves (Pisum sativum)

Enzymatic activity responsible for the conversion of fatty acids to alkanes catalyzed by pea leaf homogenate was found to be mainly in the microsomal fraction. This particulate preparation catalyzed alkane formation from n-C18, n-C22, and n-C24 acids at rates comparable to that observed with n-C32 acid with O2 and ascorbate as required cofactors. In each case the major alkane contained two carbon atoms less than the precursor acid. Since the preparation also catalyzed alpha-oxidation, it was suspected that some alpha-oxidation intermediate, with one less carbon atom than the substrate acid, might lose another carbon to generate the alkane. Thin-layer and radio-gas-liquid chromatographic analysis of the products generated from [U-14C]stearic acid by the particulate preparation after different periods of incubation showed that, at all time periods, alpha-hydroxy C18 acid, C17 aldehyde, and C17 acid were the major products. Since C16 alkane was the major product even after short periods of reaction, the C17 aldehyde might have been the immediate precursor of the alkane. Exogenous labeled C18 and C24 aldehyde were converted to alkanes. The alkane-synthesizing activity was solubilized from the microsomal preparation using Triton X-100. The solubilized preparation was retarded in a Sepharose 6-B column, but the hydrocarbon-forming activity was not resolved from alpha-oxidation. The solubilized preparation produced alkane with two carbon atoms less than the parent acid in a time- and protein-dependent manner. The soluble preparation also required O2 and ascorbate and, like the microsomal preparation, was inhibited by dithioerythritol and metal ion chelating agents.