In vitro oxidation of aldehyde oxidase from rabbit liver: Specificity toward endogenous substrates

1 Introduction

Xenobiotics are compounds that are foreign to the body, which include drugs, pollutants and other substances that are not normally present in the body and are potentially toxic. Xenobiotic metabolism is the series of metabolic reactions that change the chemical structure of xenobiotics; generally acting to detoxify the toxic chemical compounds. Sometimes, however, the product of xenobiotic metabolism can be the cause of toxic effects (Hodgson and Smart, 2001). Of the biotransformations that occur in animals oxidation plays a major role in the metabolism of foreign compounds. Although the microsomal cytochrome P-450 mono-oxygenase system is of major importance in this respect, enzymes present in the cytosol also contribute to this process. This study is concerned with the enzyme aldehyde oxidase (AO; EC 1.2.3.1) which is a molybdo-flavoenzyme found in nearly every organism from bacteria to humans (Beedham, 2001; Garattini et al., 2003, 2008, 2009; Garattini and Terao, 2011). AO catalyzes the oxidation of many different N-heterocyclic compounds as well as aliphatic and aromatic aldehydes to their corresponding lactam and carboxylic acids respectively (Beedham, 2001; Garattini et al., 2003, 2008; Garattini and Terao, 2011, 2012). Although AO catalyzes the biotransformation of several endogenous compounds, the absolute primary physiological function of AO is yet to be determined. The physiological importance of aldehyde oxidase’s role in aldehyde oxidation is in question due to the fact that the Michaelis constant (K_m) for AO is higher for aliphatic aldehydes than is that of another mammalian enzyme, aldehyde dehydrogenase (ALDH) [EC; 1.2.1.3] (Jakoby and Ziegler, 1990; Panoutsopoulos et al., 2004). Two notable endogenous substrates for AO include retinaldehyde and pyridoxal (Beedham, 2001; Garattini et al., 2003, 2008; Garattini and Terao, 2011, 2012; Huang et al., 1999; Kitamura et al., 2006). Retinaldehyde is the principle component of visual pigments and for this reason it has been suggested that aldehyde oxidase may play an important part in the overall visual process since it catalyzes the biotransformation of this aldehyde to its corresponding carboxylic acid, and retinoic acid, which is the active form of vitamin A (Calzei et al., 1995; Garattini et al., 2008; Garattini and Terao, 2011, 2012; Huang et al., 1999; Stanulovic and Chaykin, 1971). The involvement of AO in all-trans retinaldehyde oxidation to all-trans retinoic acid was first seen in rabbit liver cytosol, where it was observed that a fraction of the oxidizing activity did not require an addition of NAD⁺ and was due to a molybdo-flavoenzyme (Garattini et al., 2008; Tomita et al., 1993; Tsujita et al., 1994). As well as its ability to catalyze the biotransformation of vitamin A metabolite (Fig. 1) AO also converts vitamin B6 (pyridoxal) to 4-pyridoxic acid (Fig. 1) (Tomita et al., 1993). Vitamin B6 is a water-soluble compound that contains a pyridine ring. Vitamin B6 is present in nature in several different forms such as pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM) and their active form pyridoxal 5′-phosphate (PLP) (Fitzpatrick et al., 2007). PLP is the coenzymatically active form of vitamin B6 and plays an important role in maintaining the biochemical homeostasis of the body (Meister, 1990). There are more than 100 PLP-dependent enzymes in a cell that perform essential roles in various metabolic pathways including amino acid metabolism (such as amino acid synthesis and degradation), fatty acid metabolism (such as synthesis of polyunsaturated fatty acids) and carbohydrate metabolism (such as breakdown of glycogen) (Mooney et al., 2009). The preferred degradation route from PLP to 4-pyridoxic acid involves the dephosphorylation of PLP by phosphatase (Jang et al., 2003) followed separately by the actions of aldehyde oxidase and β-nicotinamide adenosine dinucleotide-dependent dehydrogenase (Schwartz and Kjeldgaard, 1951; Stanulovic et al., 1976). In mice Garattini et al. (2008) reported that pyridoxal can be oxidized by purified mouse aldehyde oxidase AOX₁ and AOH1, although it is not an efficient substrate in the case of AOH2. Although a wealth of data is available on endogenous substrates of AO they are still being sought (Garattini et al., 2009).

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Figure 1

Conversion of retinal (vitamin A) to its corresponding carboxylic acid (retinoic acid) and oxidation of vitamin B6 (pyridoxal) to its corresponding carboxylic acid (4-pyridoxic acid) catalyzed by aldehyde oxidase. Based on Macrae et al., 1984; Tomita et al., 1993.

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The activity of AO between animal species varies depending on the substrate considered. Sugihara et al. found the activity of AO in monkeys is higher than in humans when using N¹-methylnicotinamide and benzaldehyde as substrates (Sugihara et al., 2006). Klecker et al. (2006) found the activity of AO is highest in mouse toward zebularine substrate than in monkeys and humans (Klecker et al., 2006). Species differences have also been found when using cinchonidine as a substrate where rabbits have higher AO activity than monkeys (Fukiya et al., 2010).

Therefore, the present study investigates the role of AO in the metabolism of pyridoxal and all-trans retinaldehyde in partially purified rabbit liver fractions. The specificity of liver rabbit AO for these substrates was quantitatively explored by determining kinetic constants for a variety of endogenous compounds.

2 Materials and methods

3 Results and discussion

Activities of the molybdenum hydroxylase fraction from rabbit liver were measured spectrophotometrically using 0.1 mM final substrate concentrations of all-trans retinal and pyridoxal specific substrates of aldehyde oxidase (Tomita et al., 1993; Macrae et al., 1984). The ratio of oxidation rates of all-trans retinal and pyridoxal was calculated to reflect the relative aldehyde oxidase activity in the rabbit liver investigated (Table 2). The results demonstrate the high aldehyde oxidase activity of rabbit liver toward pyridoxal was much higher than all-trans retinal substrate (Table 2). In order to determine that there was definitely activity present in the rabbit liver for these endogenous substrates a HPLC assay was performed with these substrates. This is shown in Fig. 2, where the product (4-pyridoxic acid and retinoic acid) was detected even after extended incubation periods. In agreement with the present result Schwartz and Kjeldgaard, 1951 found that pyridoxal, the vitamin B6 precursor, is oxidized to 4-pyridoxic acid by the human enzyme (Schwartz and Kjeldgaard, 1951), pyridoxal was rapidly oxidized to 4-pyridoxic acid by the liver (28.1 ± 19.3 nmol/min/mg protein) (Merrill et al., 1994) and by the two mouse liver aldehyde oxidases (Garattini et al., 2009). Pyridoxal is currently the sole example of a substrate that shows a certain degree of selectivity for a specific aldehyde oxidase protein as it is not recognized by purified mouse (Terao et al., 2009). These findings are consistent with Huang and Ichikawa (1994) and Tsujita et al. (1994) who first observed the role of AO in the oxidation of all-trans-retinaldehyde to retinoic acid without NAD⁺ in rabbit by liver AO cytosol (Huang and Ichikawa, 1994; Tsujita et al., 1994).

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Figure 2

HPLC-UV analysis of the in vitro biotransformation of endogenous compounds (pyridoxal and all-trans retinal) by partially purified rabbit liver fraction. HPLC chromatograms are offset on the vertical axis to allow comparison between different incubation times. (a) Incubation rabbit liver fraction with pyridoxal at 37 °C for 0 (blue line) and 15 min (red line) (b) Incubation rabbit liver fraction with all-trans retinal. Blue line is a zero minute incubation and red line for 15 min incubation.

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K_m and V_max values for the formation of 4-pyridoxic acid and retinoic acid metabolites, also determined spectrophotometrically as described in Methods, by rabbit enzyme fractions are tabulated in Table 3 and Figs. 3 and 4. The kinetic parameters of pyridoxal and all-trans-retinal were found to be: K_m 21 ± 6.4 and 46 ± 9.1 μM, respectively and V_max 1.84 ± 0.2 and 1.28 ± 0.1 nmol/mg/min, respectively (Mean ± Standard deviation (SD), N = 6) for rabbit liver aldehyde oxidase using molecular oxygen as an electron acceptor although, rabbit liver enzyme gave the lowest k_m value for 4-pyridoxic acid formation (Table 3). The lowest K_m value for an AO substrate was found with pyridoxal and K_m value for pyridoxal was ∼2 lower than that for all-trans-retinal as a substrate. The results of this study indicated that pyridoxal is a better substrate of rabbit AO than all-trans-retinal (Table 3) which has been discovered in rabbit liver cytosol (Tomita et al., 1993) and confirmed using purified preparations of mouse liver AOX1(Huang et al., 1999; Vila et al., 2004). In other hands, retinaldehyde has been one of the best substrates not only of mouse AO, but also of aldehyde dehydrogenases (Duester, 2000; Vasiliou and Nebert, 2005) which have long been known to catalyze the oxidation of retinaldehyde and to play a critical role in the morphogenetic activity of retinoic acid during the development of the vertebrate embryo (Duester, 2008). A similar finding of pyridoxal oxidase has been made in detailed studies of the enzyme purified from rabbit liver, which had higher activity (Choi et al., 1983). This activity agrees well with the pharmacokinetics of vitamin B6 metabolism in humans (Wozenski et al., 1980) and other animals (Colombini and McCoy, 1970; Johansson et al., 1974). Furthermore, this study confirmed that hydrophobicity enhances affinity of pyridoxal and all-trans-retinal (aromatic aldehyde) toward AO as excellent substrates (Beedham, 2001; Garattini and Terao, 2013; Panoutsopoulos et al., 2004).

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Figure 3

Eadie-Hofstee plot for the oxidation of pyridoxal by rabbit liver aldehyde oxidase. The substrate concentrations were between 25–100 μM.

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Figure 4

Eadie-Hofstee plot for the oxidation of all-trans-retinal by rabbit liver aldehyde oxidase. The substrate concentrations were between 25–100 μM.

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However, species variation in the levels of aldehyde oxidase has been reported (Beedham, 1987; Rashidi and Nazemiyeh, 2010). This marked variation in substrate specificity of aldehyde oxidase would make it difficult to extrapolate the results obtained from one animal to another. For instance, carbazeran is a good substrate for guinea-pig, baboon and human liver aldehyde oxidase, whereas it does not serve as a substrate for rabbit enzyme (Beedham, 1987). Conversely, methotrexate is a more efficient substrate for rabbit liver aldehyde oxidase than that of other species (Kitamura et al., 1999). Furthermore, the site of substrate oxidation catalyzed by aldehyde oxidase may be species-dependent. N¹-methylnicotinamide is oxidized to two metabolites, a 2-pyridone (N¹-methyl-2-pyridone-5-carboxamide) and a 4-pyridone (N¹-methyl-4-pyridone-3-carboxamide) by liver aldehyde oxidase from various species; however, the ratio of the 4-pyridone to the 2-pyridone metabolite differs from one species to another (Stanulovic and Chaykin, 1971).

It is concluded that difference in activities of endogenous substrates metabolizing aldehyde oxidase may be controlled by a number o factors such as gene sequence, protein structure and hormonal regulation of AO activity (Beedham, 2001 and Beedham, 2010). Overall, the data presented serve as a guide for predicting the susceptibility of endogenous to oxidation by rabbit liver aldehyde oxidase, because the species variation and substrate specificity of this enzyme indicates that this enzyme has a relatively large active/binding site with a marked flexibility from one animal to another (Beedham, 1987; Garattini and Terao, 2013; Rashidi and Nazemiyeh, 2010).

Conflict of interest

Declared none.