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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

CYP4 Isoform Specificity in the -Hydroxylation of Phytanic Acid, a Potential Route to Elimination of the Causative Agent of Refsum's Disease

Departments of Pharmaceutical Chemistry (F.X., P.R.O.d.M.) and Biopharmaceutical Sciences (V.Y.N., D.L.K.), University of California, San Francisco, California

Received March 24, 2006; accepted May 16, 2006.

	Abstract

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The saturated C20 isoprenoid phytanic acid is physiologically derived from phytol released in the degradation of chlorophyll. The presence of a C-3 methyl group in this substrate blocks normal -oxidation, so phytanic acid degradation primarily occurs by initial peroxisomal -oxidation to shift the register of the methyl group. However, individuals with Refsum's disease are genetically deficient in the required phytanoyl-CoA -hydroxylase and suffer from neurological pathologies caused by the accumulation of phytanic acid. Recent work has shown that phytanic acid can also be catabolized by a pathway initiated by -hydroxylation of the hydrocarbon chain, followed by oxidation of the alcohol to the acid and conventional -oxidation. However, the enzymes responsible for the -hydroxylation of phytanic acid have not been identified. In this study, we have determined the activities of all of the rat and human CYP4A enzymes and two of the rat CYP4F enzymes, with respect to the -hydroxylation of phytanic acid. Furthermore, we have shown that the ability to -hydroxylate phytanic acid is elevated in microsomes from rats pretreated with clofibrate. The results support a possible role for CYP4 enzyme elevation in the elimination of phytanic acid in Refsum's disease patients.

View larger version (9K): [in this window] [in a new window]   Fig. 1. Alternative modes of catabolism of phytanic acid: -oxidation (after conversion to phytanoyl-CoA) followed by -oxidation, or -hydroxylation followed by conversion to the acid and -oxidation.

Linear fatty acids are oxidized by cytochrome P450 enzymes of the CYP4 family at the -carbon of the chain, producing the -hydroxylated derivatives (Okita and Okita, 2001). Further oxidation of the terminal hydroxyl group to the acid, a reaction catalyzed by both the CYP4 enzymes and alcohol/aldehyde dehydrogenases (Sanders et al., 2005), produces dicarboxylic acids that can undergo -oxidation from either end. It has been reported that phytanic acid can also be -hydroxylated, giving rise to an -hydroxy alcohol that, after oxidation to the acid, can be degraded normally by -oxidation (Komen et al., 2004, 2005). Studies of phytanic acid -hydroxylation have so far only been carried out with microsomal systems that do not allow identification of the actual enzymes involved in the reaction (Komen et al., 2004, 2005).

Hydroxylation of the terminal carbon of a hydrocarbon chain is a difficult reaction relative to hydroxylation at the next to the last (-1) carbon and, in most instances, is only effectively catalyzed by P450 enzymes of the CYP4 family (He et al., 2005). Here, we report studies of the oxidation of phytanic acid by the four known CYP4A enzymes in rats, the single active CYP4A enzyme in humans, and two of the rat CYP4F enzymes, all of which are known to catalyze linear fatty acid -hydroxylation. We also demonstrate that, in rats, administration of clofibrate, which specifically elevates levels of the CYP4 enzymes, results in enhanced -hydroxylation of phytanic acid. Induction of CYP4 enzymes may therefore provide a mechanism for the attenuation of Refsum's disease.

	Materials and Methods

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Materials. Phytanic acid, 16-hydroxyhexadecanoic acid, and methyl--cyclodextrin were purchased from Sigma-Aldrich (St. Louis, MO). Rat cytochrome P450 reductase was expressed and purified as previously reported (Dierks et al., 1998). Purified rat liver cytochrome b₅ was a gift from Lester Bornheim (University of California, San Francisco). Ampicillin, -aminolevulinic acid, glycerol, lysozyme, dilauroylphosphatidylcholine, catalase, and NADPH were obtained from Sigma-Aldrich. Emulgen 913 was from Karlan Research Products (Santa Rosa, CA).

Heterologous Expression of CYP4A and CYP4F Isoforms. CYP4A1, CYP4A2, CYP4A3, CYP4A8, CYP4A11, CYP4F1, and CYP4F4 hexahistidine-tagged proteins were expressed in XL1-blue cells, purified on a Ni²⁺-nitrilotriacetic acid agarose column from Qiagen (Valencia, CA), and desalted on a PD-10 column (Amersham Biosciences AB, Uppsala, Sweden) as described previously (Hoch et al., 2000; Xu et al., 2004). The P450 content was determined using the method of Omura and Sato (1964).

Animals and Liver Microsome Preparation. All procedures relating to the care and treatment of animals were approved by the University of California San Francisco Committee on Animal Research and followed the National Institutes of Health guidelines. Nine-week-old Fischer 344 male rats were purchased from Charles River Laboratories (Wilmington, MA). The animals were injected i.p. once a day for 3 days with corn oil or 200 mg/kg clofibrate dissolved in 2.5 ml of corn oil. On the 4th day, the animals were sacrificed using an i.m. injection of ketamine/xylazine/acepromazine. The abdominal cavities were opened, and the vascular system was perfused with ice-cold 0.9% NaCl. The livers were removed, snap-frozen in liquid nitrogen, and stored at -80°C until used for the preparation of microsomes. Microsomes were prepared from the liver of a single animal as described previously (Su et al., 1998). Microsomal protein concentrations were measured with the Pierce bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL) with bovine serum albumin as the standard.

Phytanic Acid -Hydroxylation. Phytanic acid -hydroxylation was measured in a reaction mixture of 20 µg/ml dilauroylphosphatidylcholine, 0.2 mg/ml sodium cholate, 50 pmol/ml CYP4A or CYP4F, 500 pmol/ml cytochrome P450 reductase, 50 pmol/ml cytochrome b₅, and 10 µg/ml catalase. This mixture was incubated for 10 min at room temperature before 100 mM potassium phosphate buffer, pH 7.4, 5 mM MgCl₂, and 1 mg/ml cyclodextrin were added. Phytanic acid was dissolved in dimethyl sulfoxide and was then added to the incubation to a final concentration of 10 to 300 µM. Phytanic acid -hydroxylation in liver microsomes was measured in incubations containing 200 µM phytanic acid, 1 mg/ml microsomal protein, 100 mM potassium phosphate buffer, pH 7.4, 5 mM MgCl₂, 8 mM sodium isocitrate, 0.5 IU of isocitrate dehydrogenase, and 1 mg/ml methyl--cyclodextrin. After a 3-min preincubation at 37°C, the reaction was initiated by addition of NADPH to a final concentration of 1 mM. The reaction was stopped after 60 min by adding 1 N HCl and immediately mixing the contents of the vial before placing it on ice. The internal standard (16-hydroxyhexadecanoic acid) was then added, and the samples were mixed again before they were transferred to solid-phase extraction.

Solid-Phase Extraction. In a modification of a published procedure (Holmes et al., 2004), Bond-Elut C18 cartridges (100 mg, 3 ml; Varian, Inc., Palo Alto, CA) were sequentially washed with 2 ml of ethyl acetate, methanol, and water. The incubation samples were transferred to the cartridges, and the solvent was slowly pushed through under nitrogen pressure. The cartridges were then washed with 2 ml of water and were thoroughly dried for 1 h by nitrogen at 20 p.s.i. The analytes were eluted into 5-ml glass screw-top tubes with Teflon-lined caps with two 1-ml aliquots of ethyl acetate. The ethyl acetate was evaporated to dryness under a stream of nitrogen. The samples were then converted to the trimethylsilyl derivatives by incubation with 50 µl of 15% N,O-bis(trimethylsilyl)-trifluoroacetamide in acetonitrile at 80°C for 10 to 15 min. The derivatized samples were allowed to cool, vortexed, and transferred to sealed Teflon-capped glass vials for GC-MS.

GC-MS. Product analysis was performed on an Agilent GC6850 Series II GC coupled to an Agilent 5973 Network Mass Spectrometry Detector. This GC-MS system was equipped with an Agilent 7683 Series autosampler interfaced with a personal computer running Agilent MSD Productivity ChemStation Revisions D.01.02 software. A 30-m DB-5 capillary column coupled with a 5-m guard column (Agilent Technologies, Palo Alto, CA) (0.25-mm inner diameter, 0.1-µm film) was used with helium as the carrier gas at a head pressure of 50 p.s.i. Injection (3 µl) was in the split mode (10:1) with the all-glass injector temperature at 250°C. The GC oven temperature was programmed for 2 min at 70°C followed by a rise to 170°C at 20°C/min, to 260°C at 5°C/min, and to 280°C at 20°C/min. The oven was finally held at 280°C for 2 min. The MS source and interface were maintained at 250 and 280°C, respectively, and a solvent delay of 4 min was used. The MS was set for a full scan from 30 to 500 mass units. The analytes were identified from a total ion chromatogram. The following are the retention times and molecular masses of the trimethylsilyl derivatives of phytanic acid, 16-hydroxyhexadecanoic acid (internal standard), and - and -1 hydroxylated phytanic acid minus one methyl group, respectively: phytanic acid, 17.8 min, MR = 369; 16-hydroxyhexadecanoic acid, 20.8 min, MR = 416; (-1)-hydroxyphytanic acid (-1-OH-PA), 21.6 min, MR = 457; and -hydroxyphytanic acid (-OH-PA), 22.4 min, MR = 457, respectively.

Western Immunoblotting. Liver microsomes (10 µg) were separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose in 25 mM Tris-192 mM glycine-20% methanol using a semidry transfer system (Bio-Rad, Hercules, CA). Goat anti-rat CYP4A1 antisera were obtained from Gentest (Woburn, MA). A goat actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Western blots were incubated with a 1:1000 dilution of the primary antibody followed by a 1:10,000-fold dilution of Alexa Fluor 680 donkey anti-goat IgG. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection kit (Amersham Life Science).

View larger version (28K): [in this window] [in a new window]   Fig. 2. GC-MS chromatograms of phytanic acid metabolism with CYP4A or CYP4F isoforms (A) and rat liver microsomes (B). Peaks corresponding to phytanic acid (PA), internal standard (IS), -OH-PA, and -1-OH-PA are labeled. The chromatographic analysis was done as described under Materials and Methods. Mass spectra corresponding to -1-OH-PA (C) and -OH-PA (D) are shown.

	Results

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Phytanic Acid Hydroxylation by CYP4A Enzymes. All of the CYP4A and CYP4F isoforms catalyze the formation of -OH-PA, but -1-hydroxyphytanic acid (-1-OH-PA) was not detected (Fig. 2A). Rat microsomes were used as a control to confirm the formation of -1-OH-PA, which is clearly separated from -OH-PA in the analytical system. As shown in Fig. 2B, -1-OH-PA was formed in the incubations with rat liver microsomes (retention time = 21.6 min, m/z = 457, with a characteristic fragment of m/z = 131, Fig. 2C), although the peak is small in comparison with that of the -OH-PA (retention time = 22.4 min, m/z = 457, with a characteristic fragment of m/z = 103, Fig. 2D) (Komen et al., 2004).

Optimization of Phytanic Acid Metabolism. Incubation conditions were optimized to increase the turnover of phytanic acid. An aliquot of 0 to 2 mg/ml methyl--cyclodextrin was added to the incubation to increase the solubility of phytanic acid, and the concentration of methyl--cyclodextrin was optimal at 1.0 mg/ml (Fig. 3). At 1.0 mg/ml methyl--cyclodextrin, the formation of -HO-PA was increased by 10-fold.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Optimization of the methyl--cyclodextrin concentration in phytanic acid metabolism. The effect of different concentrations (0-2 mg/ml) of methyl--cyclodextrin on the formation of -OH-PA by CYP4A11 was determined as described under Materials and Methods.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Concentration-dependent activity of CYP4A1 () and CYP4A11 () in -hydroxylation of phytanic acid. The formation of -OH-PA was measured as described under Materials and Methods.

View this table: [in this window] [in a new window]   TABLE 1 Kinetic characterization of phytanic acid -hydroxylation by CYP4 enzymes Purified recombinant CYP4 enzymes were incubated with varying concentrations of phytanic acid, and the -hydroxylated product was detected by GC-MS as described under Materials and Methods. The Km and Vmax values were estimated by fitting the data to a single-substrate Michaelis-Menten equation. The values are the averages of two independent measurements that differed by no more than 10%.

Induction of -Hydroxylation of Phytanic Acid by Clofibrate in Rat Liver Microsomes. The hepatic and renal expression of the CYP4A genes is highly inducible in rats by a diverse group of compounds referred to as peroxisome proliferators. These agents include widely prescribed lipid-lowering drugs of the fibrate class such as clofibrate, fenofibrate, and nafenopin (Reddy et al., 1980). Induction of phytanic acid -hydroxylation may be an alternative approach to controlling the tissue levels of phytanic acid in Refsum's disease. The rats were pretreated with clofibrate, and the resulting changes in the -hydroxylation of phytanic acid by liver microsomes were measured. As shown in Fig. 5, the -hydroxylation of phytanic acid was significantly (P < 0.001) elevated more than 3-fold in liver microsomes from rats treated with clofibrate. Western blotting studies indicated that the increase in CYP4 proteins due to clofibrate treatment was greater than 10-fold (Fig. 6).

View larger version (9K): [in this window] [in a new window]   Fig. 5. Induction of phytanic acid -hydroxylation by clofibrate. Rats were pretreated with 200 mg/kg clofibrate, liver microsomes were prepared, and the formation of -OH-PA was measured as described under Materials and Methods. Values are expressed as means ± S.D. from four to six animals. *, significant difference between control and clofibrate treatment (P < 0.0001).

View larger version (33K): [in this window] [in a new window]   Fig. 6. Induction of CYP4A immunoreactive proteins by clofibrate in the rat liver. Liver microsomal proteins (10 µg) were separated on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and blotted with antisera against rat CYP4A1. Immunoreactive proteins were detected by chemiluminescence. The blots shown are representative of results from three individual animals. Ctl, control; CF, clofibrate.

	Discussion

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The CYP4 P450 enzymes have a wide tolerance for the chain length of their fatty acid substrates and are unique among P450 enzymes in that they preferentially oxidize the terminal () carbon rather than internal carbons of the chain. Although the mechanism for this preference remains unclear, recent evidence showing that the enzyme oxidizes terminal chloro and bromo but not iodo atoms suggests that the specificity is enforced by steric control of access to the catalytic iron atom (He et al., 2005). In view of this steric control, it is not surprising that introduction of steric bulk near the terminal carbon can interfere with -hydroxylation. Thus, CYP4 enzymes cannot oxidize a terminal cyclopropyl or phenyl group even though one or even two methyl substituents are acceptable on the -1 carbon (Alterman et al., 1995; Bambal and Hanzlik, 1996). Because the substitution on phytanic acid is a single methyl group on the -1 carbon, the structure of this branched fatty acid is compatible with the -hydroxylation regiospecificity of the CYP4 enzymes. It is notable in this regard that the only product detected in our assay was the result of -hydroxylation, with no detectable amount of the -1 hydroxylated fatty acid (Fig. 2A). If the -1 hydroxylation occurs, it accounts for only a very low fraction of the hydroxylation reaction. Thus, the CYP4 enzymes specifically favor formation of the terminal hydroxylated phytanic acid that can undergo oxidation to the terminal carboxylic acid required for entry into the -oxidation pathway.

All of the enzymes have significant phytanic acid -hydroxylase activity. Of the rat enzymes examined, rat CYP4A1, 4A2, and 4A3 are the most efficient in the -hydroxylation of phytanic acid (Table 1). Rat CYP4A8, CYP4F1, and CYP4F4 are 4- to 5-fold less efficient, but the efficiency of human CYP4A11 is similar to that of the rat CYP4A isoforms. The activities of all these enzymes for phytanic acid hydroxylation are relatively low but are in line with the activities of the enzymes for other long-chain fatty acids. Thus, the oxidation of lauric acid by CYP4F1 is not detectable and that of arachidonic acid is 9 min^-1, and the corresponding activities for CYP4F4 are 1 and 11 min^-1 (Xu et al., 2004). The CYP4 enzymes are much more active with shorter fatty acids, with CYP4A1 oxidizing lauric acid with a K_cat of 649 min^-1, but only oxidizing oleic acid with K_cat of 1.4 min^-1. The corresponding rates for CYP4A11, the human enzymes, are 42 and 0.4 min^-1, respectively (Hoch et al., 2000). The oxidation of phytanic acid is thus well within the range of rates observed for the oxidation of other long-chain fatty acids by the CYP4 enzymes.

Administration of clofibrate to rats causes the induction of CYP4 enzymes through a peroxisomal receptor-mediated process (Savas et al., 1999). As shown in Figs. 5 and 6, this induction results in a more than 10-fold increase in the CYP4 protein content of the hepatic microsomes and a 3-fold increase in the corresponding phytanic acid hydroxylation activity. Induction of CYP4 enzymes, perhaps even by phytanic acid itself, could therefore increase the contribution of the -hydroxylation pathway to phytanic acid metabolism. The relevance of the increased oxidation of phytanic acid seen in clofibrate treated rats to the human situation depends on both the role of CYP4 proteins in phytanic acid oxidation and the inducibility of these proteins. Exposure of primary human hepatocyte cultures showed that CYP4A11 mRNA was elevated 2.4-fold and CYP4A11 protein 1.5-fold upon exposure to clofibrate (Raucy et al., 2004). The peroxisome proliferator Wy14643 has also been shown to elevate the levels of CYP4A11 in HepG2 cells (Savas et al., 2003). Finally, a very powerful peroxisome proliferator has been reported to elevate CYP4A11 mRNA 4-fold in human hepatocytes (Lawrence et al., 2001). These results clearly show that the CYP4 enzyme can be induced in human liver cells, but the level to which induction occurs in vivo in humans is unclear because cultured cells are rarely truly representative of the in vivo situation. It is informative in this context that the levels of hepatic CYP4A11 vary 10-fold among human individuals because this suggests that the levels may be susceptible to alteration by environmental exposure (Powell et al., 1996; Savas et al., 2003). Overall, the phytanic acid -hydroxylase activities of the CYP4 enzymes are low enough that their contribution to metabolism of this substrate under normal conditions is likely to be limited and in Refsum's disease patients readily overwhelmed by the high levels of phytanic acid. It is worth noting that the CYP4 enzymes are also responsible for the formation of -hydroxyarachidonic acid (Capdevila et al., 2005), a physiological vasoconstrictor, and it is possible that high levels of phytanic acid will out-compete arachidonic acid, giving rise to decreased levels of -hydroxyarachidonic acid and possibly vascular relaxation.

	Footnotes

 
This work was supported by National Institutes of Health Grants GM25515 and HL53994.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.104976.

ABBREVIATIONS: GC, gas chromatography; MS, mass spectrometry; -1-OH-PA, (-1)-hydroxyphytanic acid; -OH-PA, -hydroxyphytanic acid; Wy14643, N-(3-[2-quinolinylmethoxy]phenyl)-trifluoromethanesulfonamide; MR, molecular ratio.

Address correspondence to: Dr. Paul Ortiz de Montellano, University of California, Genentech Hall GH-N572D, 600 16th Street, San Francisco, CA 94143-2280. E-mail: ortiz{at}cgl.ucsf.edu

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This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.104976v1 318/2/835    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xu, F. Articles by de Montellano, P. R. O. Search for Related Content PubMed PubMed Citation Articles by Xu, F. Articles by de Montellano, P. R. O.