Oxygenation Mechanism in Conversion of Aldehyde to Carboxylic Acid Catalyzed by a Cytochrome P-450 Isozyme”

The oxygenation of an aldehyde, 1 l-oxo-A’-tetra- hydrocannabinol to a carboxylic acid, A”-tetrahydro-cannabinol- 1 1 -oic acid was catalyzed by cytochrome P-450 MUT-2 purified from hepatic microsomes of male ddN mice. The oxygenation mechanism was con- firmed by the incorporation of oxygen-18 from molecular oxygen into the carboxylic acid formed. An alde- hyde form but not a hydrated form of 11-oxo-A”-te-trahydrocannabinol may be a substrate for the cytochrome P-450. The oxygenation of aldehyde catalyzed by cytochrome P-450 might be a common met- abolic reaction in biological systems, and should be considered as an additional role of cytochrome P-450 in biotransformation of endogenous compounds and xenobiotics. (alde-hyde:NAD+

The biological oxidation of aldehydes to carboxylic acids, in general, is catalyzed by aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3 and aldehyde:NADP+ oxidoreductase, EC 1.2.1.5). The enzyme has been studied extensively and reviewed (1). The mechanism of this reaction has been postulated as involving the initial formation of the binary complex of the enzyme with an oxidized pyridine nucleotide, followed by formation of the ternary complex of the enzyme, cofactor, and an aldehyde substrate, and finally by hydrolysis of this complex to form a carboxylic acid (2,3). In the course of metabolic study with tetrahydrocannabinol (THC),' a constituent of marijuana, we found for the first time that the microsomal aldehyde oxygenase could catalyze the oxygenation of aldehydes to carboxylic acids (4,5). Cytochrome P-450s (P-450s) are involved in a variety of biological oxidations (6), although their participation in the biological oxidation of aldehyde has not been elucidated. This ' The abbreviations used are: THC, tetrahydrocannabinol; P-450, cytochrome P-450; GC-MS, gas chromatography-mass spectrometry; TMS, trimethylsilyl; HPLC, high performance liquid chromatography. communication describes a rather novel role and a part of mechanism of P-450 isozyme in the oxidation of a xenobiotic aldehyde to a carboxylic acid.

MATERIALS AND METHODS
Enzymes-An enzyme designated P-450 MUT-2 which has a catalytic activity for the oxidation of 1l-oxo-A8-THC to As-THC-11-oic acid was purified from hepatic microsomes of male ddN mice using the methods described by Funae and Imaoka (7) with a slight modification. Briefly, the microsomes (1050 mg of protein) were solubilized with sodium cholate and applied to an w-aminooctyl Sepharose 4B column. A fraction containing P-450 (338 nmol, 125 mg of protein) was eluted with 0.1 M potassium phosphate buffer (pH 7.25) containing 1 mM EDTA, 0.1 mM dithiothreitol, 20% glycerol, 0.4% (w/v) sodium cholate, and 0.08% (w/v) Emulgen 913. The P-450 fraction was subjected to HPLC using a DEAE-5PW column (7). The microsomal aldehyde oxygenase activity monitored ll-oxo-A*-THC as a substrate was eluted in a pass-through fraction. When reconstituted with NADPH, ~-oc-dilauroylglycero-3-phosphocholine and NADPH P-450 reductase purified from hepatic microsomes of male ddN mice by the method of Shephard et al. (8), the pass-through fraction after hydroxylapatite column chromatography showed the specific activity of 12.2 nmol of As-THC-11-oic acid formed per min/nmol P-450. The pass-through fraction was further purified by HPLC using an SP-5PW column (7). P-450 MUT-2 was finally purified using CM-Sephadex (2-50 and hydroxylapatite columns by stepwise elution with potassium phosphate buffer. The purified enzyme (2.76 nmol, 0.19 mg of protein) showed a single protein-staining band with a minimum molecular weight of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (9), and spectral peaks at 417 and 451 nm, respectively, on the oxidized and reduced CO-complex forms. The NH2terminal amino acid sequence of P-450 MUT-2 was determined to be following order:

GC-MS analyses of methyl ester and TMS derivatives of As-THC-11-oic acid formed under different conditions
The incubation system was same as described in the text except for experiment 4 where the incubation volume was reduced to 0.2 ml.  min; ionizing current 300 PA. The retention time of T M S derivative of A'-THC-11-oic acid methyl ester was 6.9 min under the above conditions. The major fragment ions of the derivative of As-THC-11oic were a t m/z 430 (M+, loo%), 374 (58%), 303 (44%), and 265 (11%). The metabolism of 9-anthraldehyde, cinnamic aldehyde, and veratrum aldehyde with P-450 MUT-2 was also investigated under the same conditions described above. After the extraction with ethyl acetate, the formation of 9-anthracene carboxylic acid was fluorometrically determined (excitation 255 nm, emission 458 nm). The formation of carboxylic acid metabolites of cinnamic aldehyde and veratrum aldehyde was qualitatively analyzed by GC-MS as reported previously (5). Protein concentrations were estimated by the method of Dulley and Grieve (15), and P-450 was measured by the method of Omura and Sato (16).

RESULTS AND DISCUSSION
GC-MS analysis indicated that 11-oxo-As-THC was transformed to As-THC-11-oic acid by the reconstituted system involving P-450 MUT-2. When either P-450 MUT-2 or NADPH P-450 reductase was omitted from the incubation mixture, the carboxylic acid metabolite could not be detected. This indicates an obligatory requirement of the P-450 and NADPH P-450 reductase for the reaction. The specific activity for the oxidation of I~-O X O -A~-T H C was 29.8 nmol/min/ nmol P-450 under the optimal conditions. The addition of cytochrome bs did not significantly affect the specific activity. P-450 MUT-2 could also catalyze the oxidation of other aldehyde substrates such as 9-anthraldehyde, veratrum aldehyde, and cinnamic aldehyde. The catalytic activity of P-450 MUT-2 for 9-anthraldehyde was 6.3 nmol of 9-anthracene carboxylic acid formed per min/nmol P-450. The formation of the carboxylic acid metabolites of cinnamic aldehyde and veratrum aldehyde was qualitatively confirmed by GC-MS analysis.
To clarify the reaction mechanism, several lines of experiments were conducted. The results are summarized in Table  I. In Experiment 2, I~-O X O -A~-T H C was incubated with the reconstituted system under oxygen-18 (97 atom %). The methyl ester and TMS derivative of the carboxylic acid metabolite formed was analyzed by GC-MS, which showed molecular ions a t m/z 430 and 432 with relative abundance of 6: 100, indicating that the reaction is exclusively monooxygenation.
The formation of hydrates (gem-diol) is well known for many aldehydes in aqueous medium (17). It is therefore important for understanding the mechanism to know is a preferable substrate for P-450 MUT-2 whether an aldehyde form or its hydrated form. Thus, 1l-oxo-Aa-THC dimethyl acetal was prepared as a model substrate for the hydrated form and incubated with the reconstituted system. The dimethyl acetal was transformed to a methyl ester of Aa-THC-11-oic acid, in which the specific activity was 8.1 nmol/min/ nmol P-450. Under oxygen-18 gas (97 atom %), one atom of oxygen-18 was exclusively incorporated into the methyl ester However, the hydrated form cannot be the predomi: substrate for this P-450, because the specific activity fol acetal was much lower than that for 1l-oxo-A8-THC.
As shown in experiment 4, oxygen-18 was not significz enriched into As-THC-11-oic acid formed from 11-ox( THC with the reconstituted system in phosphate buffer taining H2"0 (44 atom %). In addition, the aldehyde con1 ing oxygen-18 (79 atom %) in the aldehyde group was prep and incubated with the reconstituted system (Table I, El iment 5). GC-MS analysis revealed that molecular ior methyl ester and TMS derivative of the carboxylic formed were observed at m/z 430 and 432 with relative al dance of 24:lOO. The content of oxygen-18 in the carbo: acid was almost same as that in the substrate. The re! obtained from the above experiments indicate that 11-As-THC is hardly hydrated in the incubation medium an the time scale of these experiments does not exchange solvent water and that the aldehyde form i s preferent oxygenated to the carboxylic acid by the P-450 as show Scheme 1.
In conclusion, it seems very likely that this P-450 isoz could catalyze the oxygenation of lipophilic aldehyde carboxylic acids in common, and might play some rol biotransformation of both xenobiotic and endogenous 1 hydes. The oxygenation of aldehydes catalyzed by P should be considered as an additional function of the en2 because of its physiological and biological importance.