Metabolism of isovanillin by aldehyde oxidase, xanthine oxidase, aldehyde dehydrogenase and liver slices

Aromatic aldehydes are good substrates of aldehyde dehydrogenase activity but are relatively poor substrates of aldehyde oxidase and xanthine oxidase. However, the oxidation of xenobiotic-derived aromatic aldehydes by the latter enzymes has not been studied to any great extent. The present investigation compares the relative contribution of aldehyde dehydrogenase, aldehyde oxidase and xanthine oxidase activities in the oxidation of isovanillin in separate preparations and also in freshly prepared and cryopreserved liver slices. The oxidation of isovanillin was also examined in the presence of specific inhibitors of each oxidizing enzyme. Minimal transformation of isovanillin to isovanillic acid was observed in partially purified aldehyde oxidase, which is thought to be due to residual xanthine oxidase activity. Isovanillin was rapidly metabolized to isovanillic acid by high amounts of purified xanthine oxidase, but only low amounts are present in guinea pig liver fraction. Thus the contribution of xanthine oxidase to isovanillin oxidation in guinea pig is very low. In contrast, isovanillin was rapidly catalyzed to isovanillic acid by guinea pig liver aldehyde dehydrogenase activity. The inhibitor studies revealed that isovanillin was predominantly metabolized by aldehyde dehydrogenase activity. The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared or cryopreserved liver slices has not been previously reported. In freshly prepared liver slices, isovanillin was rapidly converted to isovanillic acid, whereas the conversion was very slow in cryopreserved liver slices due to low aldehyde dehydrogenase activity. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. It is therefore concluded that isovanillin is predominantly metabolized by aldehyde dehydrogenase activity, with minimal contribution from either aldehyde oxidase or xanthine oxidase.     

Phenylacetaldehyde oxidation by freshly prepared and cryopreserved guinea pig liver slices: the role of aldehyde oxidase

Phenylacetaldehyde is formed when the xenobiotic and biogenic amine 2-phenylethylamine is inactivated by a monoamine oxidase-catalyzed oxidative deamination. Exogenous phenylacetaldehyde is found in certain foodstuffs such as honey, cheese, tomatoes, and wines. 2-Phenylethylamine can trigger migraine attacks in susceptible individuals and can become fairly toxic at high intakes from foods. It may also function as a potentiator that enhances the toxicity of histamine and tyramine. The present investigation examines the metabolism of phenylacetaldehyde to phenylacetic acid in freshly prepared and in cryopreserved guinea pig liver slices. In addition, it compares the relative contribution of aldehyde oxidase, xanthine oxidase, and aldehyde dehydrogenase in the oxidation of phenylacetaldehyde using specific inhibitors for each oxidizing enzyme. The inhibitors used were isovanillin for aldehyde oxidase, allopurinol for xanthine oxidase, and disulfiram for aldehyde dehydrogenase. In freshly prepared liver slices, phenylacetaldehyde was converted mainly to phenylacetic acid, with traces of 2-phenylethanol being present. Disulfiram inhibited phenylacetic acid formation by 80% to 85%, whereas isovanillin inhibited acid formation to a lesser extent (50% to 55%) and allopurinol had little or no effect. In cryopreserved liver slices, phenylacetic acid was also the main metabolite, whereas the 2-phenylethanol production was more pronounced than that in freshly prepared liver slices. Isovanillin inhibited phenylacetic acid formation by 85%, whereas disulfiram inhibited acid formation to a lesser extent (55% to 60%) and allopurinol had no effect. The results in this study have shown that, in freshly prepared and cryopreserved liver slices, phenylacetaldehyde is converted to phenylacetic acid by both aldehyde dehydrogenase and aldehyde oxidase, with no contribution from xanthine oxidase. Therefore, aldehyde dehydrogenase is not the only enzyme responsible in the metabolism of phenylacetaldehyde, but aldehyde oxidase may also be important and thus its role should not be ignored.     

Substrate specificity of guinea pig liver aldehyde oxidase and bovine milk xanthine oxidase for methyl- and nitrobenzaldehydes.

Both aldehyde oxidase and xanthine oxidase catalyze the oxidation of a wide range of N-heterocycles and aldehydes. These enzymes are important in the oxidation of N-heterocyclic xenobiotics, whereas their role in the oxidation of xenobiotic aldehydes is usually ignored. The present investigation describes the interaction of methyl- and nitrosubstituted benzaldehydes, in the ortho-, meta- and parapositions, with guinea pig liver aldehyde oxidase and bovine milk xanthine oxidase. The kinetic constants showed that most substituted benzaldehydes are excellent substrates of aldehyde oxidase with lower affinities for xanthine oxidase. Low Km values for aldehyde oxidase were observed with most benzaldehydes tested, with 3-nitrobenzaldehyde having the lowest Km value and 3-methylbenzaldehyde being the best substrate in terms of substrate efficiency (Ks). Additionally, low Km values for xanthine oxidase were found with most benzaldehydes tested. However, all benzaldehydes also had low Vmax values, which made them poor substrates of xanthine oxidase. It is therefore possible that aldehyde oxidase may be critical in the oxidation of xenobiotic and endobiotic derived aldehydes and its role in such reactions should not be ignored.     

Metabolism of 2-phenylethylamine and phenylacetaldehyde by precision-cut guinea pig fresh liver slices.

2-Phenylethylamine is an endogenous constituent of human brain and is implicated in cerebral transmission. It is also found in certain foodstuffs and may cause toxic side-effects in susceptible individuals. Metabolism of 2-phenylethylamine to phenylacetaldehyde is catalyzed by monoamine oxidase and the oxidation of the reactive aldehyde to its acid derivative is catalyzed mainly by aldehyde dehydrogenase and perhaps aldehyde oxidase, with xanthine oxidase having minimal transformation. The present investigation examines the metabolism of 2-phenylethylamine to phenylacetaldehyde in liver slices and compares the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activity in the oxidation of phenylacetaldehyde with precision-cut fresh liver slices in the presence/absence of specific inhibitors of each enzyme. In liver slices, phenylacetaldehyde was rapidly converted to phenylacetic acid. Phenylacetic acid was the main metabolite of 2-phenylethylamine, via the intermediate phenylacetaldehyde. Phenylacetic acid formation was completely inhibited by disulfiram (specific inhibitor of aldehyde dehydrogenase), whereas isovanillin (specific inhibitor of aldehyde oxidase) inhibited acid formation to a lesser extent and allopurinol (specific inhibitor of xanthine oxidase) had little or no effect. Therefore, in liver slices, phenylacetaldehyde is rapidly oxidized by aldehyde dehydrogenase and aldehyde oxidase with little or no contribution from xanthine oxidase.     

Oxidation of selected pteridine derivatives by mamalian liver xanthine oxidase and aldehyde oxidase.

Considerable information is available concerning the oxidation of pteridine derivatives by bovine milk xanthine oxidase, but few investigations have been carried out on the oxidation of such compounds by mammalian liver xanthine oxidase and the related aldehyde oxidase. Xanthine oxidase, obtained from rat liver, oxidizes a variety of substituted amino- and hydroxypteridines in a manner identical to that previously observed for milk xanthine oxidase. For example, 2-aminopteridine and its 4- and 7-hydroxy derivatives were oxidized efficiently to 2-amino-4,7-dihydroxypteridine (isoxanthopterin) by the rat liver enzyme, and 4-aminopteridine and its 2- and 7-hydroxy derivatives were oxidized to 4-amino-2,7-dihydroxypteridine.4-Hydroxypteridine and the isomeric 2- and 7-hydroxypteridines were oxidized by rat liver xanthine oxidase to 2,4,7-trihydroxypteridine. Rabbit liver aldehyde oxidase, but not rat liver xanthine oxidase, was able to catalyze the oxidation in position 7 of 2,4-diaminopteridine and its 6-methyl and 6-hydroxymethyl derivatives. 2-Aminopteridine and 4-aminopteridine were both oxidized to the corresponding 7-hydroxy derivatives in the aldehyde oxidase system; 2-amino-4-hydroxypteridine appeared to be a minor product in the oxidation of 2-aminopteridine by rabbit liver aldehyde oxidase. Both aldehyde oxidase and xanthine oxidase were able to catalyze the oxidation of 2-amino-6,7-disubstituted pteridines to the corresponding 4-hydroxy derivatives; 4-hydroxy-6,7-disubstituted pteridines were oxidized in position 2 by both enzymes. 4-Amino-6,7-disubstituted pteridines were not oxidized by either enzyme. 2-Amino-4-methylpteridine was oxidized in position 7 by aldehyde oxidase but was not an effective substrate for xanthine oxidase; 2-hydroxypteridine and 7-hydroxypteridine were not oxidized to a detectably extent by aldehyde oxidase. All oxidations mediated by xanthine oxidase were strongly inhibited by allopurinol (4-hydroxypyrazolo[3,4-d]pyrimidine), and all oxidations mediated by aldehyde oxidase were inhibited by menadione (2-methyl-1,4-naphthoquinone). Rat liver xanthine oxidase and, to a lesser extent, rabbit liver aldehyde oxidase were inhibited by 4-chloro-6,7-dimethylpteridine; 2-amino-3-pyrazinecarboxylic acid inhibited xanthine oxidase but not aldehyde oxidase. The oxidations of 2- and 4-aminopteridines by aldehyde oxidase resulted in concomitant reduction of cytochrome c.     

Metabolism of 2-phenylethylamine to phenylacetic acid, via the intermediate phenylacetaldehyde, by freshly prepared and cryopreserved guinea pig liver slices

BACKGROUND: 2-Phenylethylamine is an endogenous amine, which acts as a neuromodulator of dopaminergic responses. Exogenous 2-phenylethylamine is found in certain foodstuffs and may cause toxic side-effects in susceptible individuals. MATERIALS AND METHODS: The present investigation examined the metabolism of 2-phenylethylamine to phenylacetic acid, via phenylacetaldehyde, in freshly prepared and cryopreserved liver slices. Additionally, it compared the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase by using specific inhibitors for each oxidizing enzyme. RESULTS: In freshly prepared and cryopreserved liver slices, phenylacetic acid was the main metabolite of 2-phenylethalamine. In freshly prepared liver slices, phenylacetic acid was completely inhibited by disulfiram (inhibitor of aldehyde dehydrogenase), whereas isovanillin (inhibitor of aldehyde oxidase) inhibited acid formation to a lesser extent and allopurinol (inhibitor of xanthine oxidase) had no effect. In cryopreserved liver slices, isovanillin inhibited phenylacetic acid by 85%, whereas disulfiram inhibited acid formation to a lesser extent and allopurinol had no effect. CONCLUSION: In liver slices, 2-phenylethylamine is rapidly oxidized to phenylacetic acid, via phenylacetaldehyde, by aldehyde dehydrogenase and aldehyde oxidase with no contribution from xanthine oxidase.     

Tissue distribution of the molybdenum hydroxylases, aldehyde oxidase and xanthine oxidase, in male and female guinea pigs

The activity of the molybdenum hydroxylase, aldehyde oxidase, was determined in crude homogenates and (NH4)2SO4 fractions prepared from guinea pig liver, lung, kidney, intestine, spleen and heart. Xanthine oxidase was also measured in (NH4)2SO4 fractions. In each case, xanthine oxidase levels were lower than those of aldehyde oxidase; activity of the latter enzyme was highest in the liver, whereas xanthine oxidase was predominant in the small intestine. There was no significant difference in the activity of either molybdenum hydroxylase between tissues taken from male and female guinea pigs.     

Species variation in hepatic aldehyde oxidase activity

The activity of hepatic aldehyde oxidase from rabbit, guinea pig, rat, marmoset, dog, baboon and man was investigated in vitro with charged and uncharged N-heterocyclic substrates: Km and Vmax values were determined for phthalazine, 6,7-dimethoxy-1-[-4-(ethylcarbamoyloxy)piperidino]phthalazine (carbazeran), quinine and quinidine. The oxidation of N-phenylquinolinium chloride to N-phenyl-2-quinolone and N-phenyl-4-quinolone was followed spectrophotometrically. Rat or dog liver showed low and negligible enzyme activity respectively, whereas baboon liver contained a highly active aldehyde oxidase. Enzyme from marmoset and guinea pig liver had the closest spectrum of activity to human liver aldehyde oxidase. Unlike that from man, rabbit hepatic aldehyde oxidase was refractory towards carbazeran and converted N-phenylquinolinium chloride predominantly to the 2-quinolone. N-Phenyl-4-quinolone was the major oxidation product with enzyme from guinea pig, marmoset, baboon and man.     

Enzymatic oxidation of 2-phenylethylamine to phenylacetic acid and 2-phenylethanol with special reference to the metabolism of its intermediate phenylacetaldehyde

2-phenylethylamine is an endogenous constituent of the human brain and is implicated in cerebral transmission. This bioactive amine is also present in certain foodstuffs such as chocolate, cheese and wine and may cause undesirable side effects in susceptible individuals. Metabolism of 2-phenylethylamine to phenylacetaldehyde is catalysed by monoamine oxidase B but the oxidation to its acid is usually ascribed to aldehyde dehydrogenase and the contribution of aldehyde oxidase and xanthine oxidase, if any, is ignored. The objective of this study was to elucidate the role of the molybdenum hydroxylases, aldehyde oxidase and xanthine oxidase, in the metabolism of phenylacetaldehyde derived from its parent biogenic amine. Treatments of 2-phenylethylamine with monoamine oxidase were carried out for the production of phenylacetaldehyde, as well as treatments of synthetic or enzymatic-generated phenylacetaldehyde with aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase. The results indicated that phenylacetaldehyde is metabolised mainly to phenylacetic acid with lower concentrations of 2-phenylethanol by all three oxidising enzymes. Aldehyde dehydrogenase was the predominant enzyme involved in phenylacetaldehyde oxidation and thus it has a major role in 2-phenylethylamine metabolism with aldehyde oxidase playing a less prominent role. Xanthine oxidase does not contribute to the oxidation of phenylacetaldehyde due to low amounts being present in guinea pig. Thus aldehyde dehydrogenase is not the only enzyme oxidising xenobiotic and endobiotic aldehydes and the role of aldehyde oxidase in such reactions should not be ignored.     

Identification of mouse liver aldehyde dehydrogenases that catalyze the oxidation of retinaldehyde to retinoic acid

NAD(P)-linked aldehyde dehydrogenases catalyze the oxidation of a wide variety of aldehydes. Thirteen of these enzymes have been identified in mouse tissues; eleven are found in the liver. Some are substrate-nonspecific; others are relatively substrate-specific. The present investigation sought to determine which of these enzymes are operative in catalyzing the oxidation of retinaldehyde to retinoic acid, a metabolite of vitamin A that promotes the differentiation of epithelial and other cells. Spectrophotometric and HPLC assays were used for this purpose. Enzyme-catalyzed oxidation of retinaldehyde (25 microM) was restricted to the cytosol (105,000 g supernatant fraction) and occurred at a rate of 211 nmol/min/g liver; oxidation of acetaldehyde (4 mM) by this fraction proceeds about ten times faster. At least 90% of this activity was NAD dependent. Of the approximately 10% that was apparently NAD independent, two-thirds was inhibited by 1 mM pyridoxal, a known inhibitor of aldehyde oxidase. Of the six cytosolic aldehyde dehydrogenases, only two, viz. AHD-2 and AHD-7, catalyzed the oxidation of retinaldehyde to retinoic acid. An additional NAD-dependent enzyme, viz. xanthine oxidase (dehydrogenase form), also catalyzed the reaction. Catalysis by AHD-2 accounted for more than 90% of the total NAD-dependent activity. Km values were 0.7, 0.6 and 0.9 microM, respectively, for the AHD-2-, AHD-7- and xanthine oxidase (dehydrogenase form)-catalyzed reaction. AHD-4, an aldehyde dehydrogenase found in the cytosol of mouse stomach epithelium and cornea, did not catalyze the reaction.     

Hydralazine: a potent inhibitor of aldehyde oxidase activity in vitro and in vivo

The interaction of the vasodilator, hydralazine, with the molybdenum hydroxylases, aldehyde oxidase and xanthine oxidase has been investigated. A potent progressive inhibition of rabbit liver aldehyde oxidase, in the presence of substrate, by low concentrations of hydralazine (0.1-1 microM) was observed in vitro but no effect was seen with bovine milk xanthine oxidase. This activity was mirrored in vivo when levels of aldehyde oxidase were significantly decreased in rabbits administered hydralazine (10 mg/kg/day for seven days) whereas hepatic xanthine oxidase activity was unaltered by hydralazine treatment. Various metabolites of hydralazine were synthesized but found to be devoid of in vitro inhibitory activity. Aldehyde oxidase prepared from either guinea pig or baboon liver was inhibited in a similar way to that of rabbit liver.     

Interference of aldehyde metabolizing enzymes with diamine oxidase/histaminase/activity as determined by 14C putrescine method.

The Δ¹ pyrroline formation, as an indicator of diamine oxidase activity according to Okuyama and Kobayashi ¹⁴C putrescine test [1], has been investigated in several tissue homogenates. When guinea pig liver homogenate was used as a source of enzyme in the presence of aldehyde dehydrogenase inhibitors chloral hydrate and acetaldehyde the level of formation Δ¹ pyrroline was strongly increased in a dose-dependent manner. Also inhibition of aldehyde reductase by phenobarbital enhanced Δ¹ pyrroline formation, but to a lesser degree. In other tissues, with very high initial diamine oxidase activity (rat intestine, dog kidney) or with very low diamine oxidase activity (guinea pig skin, dog liver) the influence of these inhibitors was only slight. Pyrazole, an inhibitor of alcohol dehydrogenase exerted only a small effect on Δ¹ pyrroline formation. All aldehyde-metabolizing enzymes inhibitors, except pyrazole, were without effect on purified pea seedling and hog kidney diamine oxidases. The use of aldehyde-metabolizing enzymes inhibitors may help to reveal the real values of diamine oxidase activity, when tissues homogenates are used as a source of enzyme.     

In vitro study of 6-mercaptopurine oxidation catalysed by aldehyde oxidase and xanthine oxidase

In spite of over 40 years of clinical use of 6-mercaptopurine, many aspects of complex pharmacology and metabolism of this drug remain unclear. It is thought that 6-mercaptopurine is oxidized to 6-thiouric acid through 6-thioxanthine or 8-oxo-6-mercaptopurine by one of two molybdenum hydroxylases, xanthine oxidase (XO), however, the role of other molybdenum hydroxylase, aldehyde oxidase (AO), in the oxidation of 6-mercaptopurine and possible interactions of AO substrates and inhibitors has not been investigated in more details. In the present study, the role of AO and XO in the oxidation of 6- mercaptopurine has been investigated. 6-mercaptopurine was incubated with bovine milk xanthine oxidase or partially purified guinea pig liver molybdenum hydroxylase fractions in the absence and presence of XO and AO inhibitor/substrates, and the reactions were monitored by spectrophotometric and HPLC methods. According to the results obtained from the inhibition studies, it is more likely that 6- mercaptopurine is oxidized to 6-thiouric acid via 6-thioxanthine rather than 8-oxo-6-mercaptopurine. The first step which is the rate limiting step is catalyzed solely by XO, whereas both XO and AO are involved in the oxidation of 6-thioxanthine to 6-thiouric acid.     

Role of guinea pig and rabbit hepatic aldehyde oxidase in oxidative in vitro metabolism of cinchona antimalarials.

Cinchona alkaloids (quinine, quinidine, cinchonine, and cinchonidine) were incubated with partially purified aldehyde oxidase from rabbit or guinea pig liver. Reversed-phase HPLC methods were developed to separate the oxidation products from the parent drugs, and the metabolites were identified on the basis of their infrared and mass spectral characteristics. All four alkaloids were oxidized at carbon 2 of the quinoline ring to give the corresponding lactams. In addition, the dihydro contaminants of the cinchona alkaloids were also metabolized by aldehyde oxidase to the 2-quinolone derivatives. Kinetic constants for the oxidation reactions were determined spectrophotometrically and showed that these substrates have a low affinity (KM values of around 10(-5) M) for hepatic aldehyde oxidase, coupled with a relatively low oxidation rate. However, the overall efficiency of the enzyme (Vmax/KM) toward this group of compounds indicates that in vivo biotransformation by aldehyde oxidase will be a significant pathway. Microsomal metabolites were also isolated from quinine and quinidine incubations with rabbit or guinea pig liver fractions. 3-Hydroxyquinine (quinidine) and O-desmethylquinine (quinidine) were identified in microsomal and 10,000g supernatant extracts from quinine and quinidine, respectively. Oxidation of quinine via aldehyde oxidase appeared to be the predominant pathway in rabbit 10,000g fractions, because 2'-quininone was the major metabolite under these conditions with lower concentrations of the microsomal metabolites produced along with a dioxygenated derivative thought to be 3-hydroxy-2'-quininone.     

Metabolism of cryptolepine and 2-fluorocryptolepine by aldehyde oxidase

Objectives To investigate the metabolism of cryptolepine and some cryptolepine analogues by aldehyde oxidase, and to assess the implications of the results on the potential of cryptolepine analogues as antimalarial agents. Methods The products resulting from the oxidation of cryptolepine and 2‐fluorocryptolepine by a rabbit liver preparation of aldehyde oxidase were isolated and identified using chromatographic and spectroscopic techniques. The antiplasmodial activity of cryptolepine‐11‐one was assessed against Plasmodium falciparum using the parasite lactate dehydrogenase assay. Key findings Cryptolepine was oxidized by aldehyde oxidase give cryptolepine‐11‐one. Although 2‐fluorocryptolepine was found to have less affinity for the enzyme than cryptolepine, it was a better substrate for aldehyde oxidase than the parent compound. In contrast, quindoline, the 11‐chloro‐ , 2,7‐dibromo‐ and 2‐methoxy analogues of cryptolepine were not readily oxidized. Cryptolepine‐11‐one was found to be inactive against P. falciparum in vitro raising the possibility that the effectiveness of cryptolepine as an antimalarial, may be compromised by metabolism to an inactive metabolite by liver aldehyde oxidase. Conclusions Cryptolepine and 2‐fluorocryptolepine are substrates for aldehyde oxidase. This may have implications for the design and development of cryptolepine analogues as antimalarial agents.     

Potent inhibition of human liver aldehyde oxidase by raloxifene.

The selective estrogen receptor modulator, raloxifene, has been demonstrated as a potent uncompetitive inhibitor of human liver aldehyde oxidase-catalyzed oxidation of phthalazine, vanillin, and nicotine-Delta1'(5')-iminium ion, with K(i) values of 0.87 to 1.4 nM. Inhibition was not time-dependent. Raloxifene has also been shown to be a noncompetitive inhibitor of an aldehyde oxidase-catalyzed reduction reaction of a hydroxamic acid-containing compound, with a K(i) of 51 nM. However, raloxifene had only small effects on xanthine oxidase, an enzyme related to aldehyde oxidase. In addition, several other compounds of the same therapeutic class as raloxifene were examined for their potential to inhibit aldehyde oxidase. However, none were as potent as raloxifene, since IC(50) values were orders of magnitude higher and ranged from 0.29 to 57 micro M. In an examination of analogs of raloxifene, it was shown that the bisphenol structure with a hydrophobic group on the 3-position of the benzthiophene ring system was the most important element that imparts inhibitory potency. The relevance of these data to the mechanistic understanding of aldehyde oxidase catalysis, as well as to the potential for raloxifene to cause drug interactions with agents for which aldehyde oxidase-mediated metabolism is important, such as zaleplon or famciclovir, is discussed.     

Substrate preference of a cytosolic aldehyde dehydrogenase inducible in rat liver by treatment with 3-methylcholanthrene

The substrate preference of an aldehyde dehydrogenase induced in rat liver cytosol by 3-methylcholanthrene was examined. This enzyme, T-ALDH, is identical to the aldehyde dehydrogenase inducible in rat liver by 2,3,7,8-tetrachloro-dibenzo-p-dioxin and the tumor-associated aldehyde dehydrogenase found in rat hepatocellular neoplasms. With either NAD or NADP as coenzyme, the preferred substrates were the aliphatic aldehydes n-hexanal, n-nonanal, and isobutyraldehyde and the aromatic aldehydes 2,5-dihydroxybenzaldehyde, benzaldehyde, and 3-hydroxybenzaldehyde. The results indicate that T-ALDH may play a role in oxidizing a variety of aldehydes produced in physiological lipid metabolism. On the contrary, this isozyme does not seem to participate in the oxidation of small aliphatic aldehydes generated during lipid peroxidation. Similarly, no significant activity could be detected when the enzyme was tested with aldehydes produced in carbohydrate, amino acid, polyamine, steroid, and vitamin metabolism.     

Tissue distribution and characteristics of xanthine oxidase and allopurinol oxidizing enzyme.

Tissue distribution and levels of allopurinol oxidizing enzyme and xanthine oxidase with hypoxanthine as a substrate were compared with supernatant fractions from various tissues of mice and from liver of mice, rats, guinea pigs and rabbits. The allopurinol oxidizing enzyme activities in liver were quite different among the species and the sex difference of the enzyme activity only in mouse liver. In mice, the highest activity of allopurinol oxidizing enzyme was found in the liver with a trace value in lung, but the enzyme activity was not detected in brain, small intestine and kidney, while the highest activity of xanthine oxidase was detected in small intestine, lung, liver and kidney in that sequence. The allopurinol oxidizing enzyme activity in mouse liver supernatant fraction did not change after storage at -20 degrees C or dialysis against 0.1 M Tris-HCl containing 1.15% KCl, but the activity markedly decreased after dialysis against 0.1 M Tris-HCl. On the contrary, the xanthine oxidase was activated 2 to 3 times the usual activity after storage at -20 degrees C or dialysis of the enzyme preparation. These results indicated that allopurinol was hydroxylated to oxipurinol mainly by the enzyme which is not identical to xanthine oxidase in vivo. A possible role of aldehyde oxidase involved in the allopurinol oxidation in liver supernatant fraction was dicussed.     

The impact of single nucleotide polymorphisms on human aldehyde oxidase

Aldehyde oxidase (AO) is a complex molybdo-flavoprotein that belongs to the xanthine oxidase family. AO is active as a homodimer, and each 150-kDa monomer binds two distinct [2Fe2S] clusters, FAD, and the molybdenum cofactor. AO has an important role in the metabolism of drugs based on its broad substrate specificity oxidizing aromatic aza-heterocycles, for example, N(1)-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. Sequencing the 35 coding exons of the human AOX1 gene in a sample of 180 Italian individuals led to the identification of relatively frequent, synonymous, missense and nonsense single-nucleotide polymorphisms (SNPs). Human aldehyde oxidase (hAOX1) was purified after heterologous expression in Escherichia coli. The recombinant protein was obtained with a purity of 95% and a yield of 50 μg/l E. coli culture. Site-directed mutagenesis of the hAOX1 cDNA allowed the purification of protein variants bearing the amino acid changes R802C, R921H, N1135S, and H1297R, which correspond to some of the identified SNPs. The hAOX1 variants were purified and compared with the wild-type protein relative to activity, oligomerization state, and metal content. Our data show that the mutation of each amino acid residue has a variable impact on the ability of hAOX1 to metabolize selected substrates. Thus, the human population is characterized by the presence of functionally inactive hAOX1 allelic variants as well as variants encoding enzymes with different catalytic activities. Our results indicate that the presence of these allelic variants should be considered for the design of future drugs.     

Metabolism of azoxy derivatives of procarbazine by aldehyde dehydrogenase and xanthine oxidase.

Procarbazine, a 1,2-disubstituted hydrazine, is employed therapeutically in the treatment of Hodgkin's disease and a limited number of other neoplasias. The isomeric azoxy metabolites of procarbazine have recently been identified as the precursors of species responsible for both the anti-cancer efficacy and toxic effects mediated by this drug. This study demonstrates that cytosolic enzymes are involved in the metabolism of the azoxy metabolites of procarbazine. Two azoxy procarbazine oxidase activities were resolved by diethylaminoethyl (DEAE)-cellulose chromatography. The activity which did not bind to this column was purified to homogeneity and was identified as a phenobarbital-inducible form of cytosolic aldehyde dehydrogenase. This protein fraction was shown to metabolize only the azoxy 2 procarbazine isomer to yield N-isopropy-p-formylbenzamide (ALD) in a reaction which did not require NAD+ as cofactor. The ALD product formed was also a substrate for a subsequent NAD(+)-dependent reduction reaction catalyzed by that purified protein. The azoxy 2 procarbazine isomer and ALD were shown to be potent inhibitors of both the dehydrogenase and esterase activities of aldehyde dehydrogenase. The second azoxy procarbazine oxidase activity which was retained by the DEAE-cellulose column co-eluted with xanthine oxidase activity. Both the xanthine dehydrogenase/oxidase and azoxy procarbazine oxidase activities of this protein fraction were inhibited by allopurinol, a specific inhibitor of xanthine dehydrogenase. Xanthine dehydrogenase/oxidase was partially purified by an alternative procedure and was shown to metabolize both the azoxy 2 procarbazine isomer and ALD, ultimately producing N-isopropylterephthalamic acid. The ability of xanthine oxidase to metabolize azoxy 2 procarbazine and ALD was confirmed using commercial, purified milk xanthine oxidase.