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Originally published In Press as doi:10.1074/jbc.M513481200 on March 17, 2006

J. Biol. Chem., Vol. 281, Issue 19, 13180-13187, May 12, 2006
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{omega}-Oxidation of Very Long-chain Fatty Acids in Human Liver Microsomes

IMPLICATIONS FOR X-LINKED ADRENOLEUKODYSTROPHY*

Robert-Jan Sanders, Rob Ofman, Marinus Duran, Stephan Kemp, and Ronald J. A. Wanders1

From the Laboratory of Genetic Metabolic Diseases, University of Amsterdam, Academic Medical Center, 1105 AZ, Amsterdam, The Netherlands

Received for publication, December 19, 2005 , and in revised form, March 16, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder biochemically characterized by elevated levels of very long-chain fatty acids (VLCFA). Excess levels of VLCFAs are thought to play an important role in the pathogenesis of X-ALD. Therefore, therapeutic approaches for X-ALD are focused on the reduction or normalization of VLCFAs. In this study, we investigated an alternative oxidation route for VLCFAs, namely {omega}-oxidation. The results described in this study show that VLCFAs are substrates for the {omega}-oxidation system in human liver microsomes. Moreover, VLCFAs were not only converted into {omega}-hydroxy fatty acids, but they were also further oxidized to dicarboxylic acids via cytochrome P450-mediated reactions. High sensitivity toward the specific P450 inhibitor 17-octadecynoic acid suggested that {omega}-hydroxylation of VLCFAs is catalyzed by P450 enzymes belonging to the CYP4A/F subfamilies. Studies with individually expressed human recombinant P450 enzymes revealed that two P450 enzymes, i.e. CYP4F2 and CYP4F3B, participate in the {omega}-hydroxylation of VLCFAs. Both enzymes belong to the cytochrome P450 4F subfamily and have a high affinity for VLCFAs. In summary, this study demonstrates that VLCFAs are substrates for the human {omega}-oxidation system, and for this reason, stimulation of the in vivo VLCFA {omega}-oxidation pathway may provide an alternative mode of treatment to reduce the levels of VLCFAs in patients with X-ALD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammalian cells, fatty acid oxidation plays a major role in the production of energy, particularly in the heart and skeletal muscle, and is the main energy source during periods of fasting. Both mitochondria and peroxisomes are capable of degrading saturated fatty acids via beta-oxidation. Short-, medium-, and long-chain saturated fatty acids are degraded predominantly by mitochondria, whereas very long-chain fatty acids (VLCFA,2 >22 carbons) are beta-oxidized exclusively in peroxisomes (1, 2). Moreover, peroxisomes also metabolize certain branched chain fatty acids, bile acid precursors, eicosanoids, and dicarboxylic acids (3).

X-linked adrenoleukodystrophy (X-ALD: MIM 300100 [OMIM] ), the most common peroxisomal disorder, is a progressive neurodegenerative disease that affects the cerebral white matter, spinal cord, peripheral nerves, adrenal cortex, and testis (4). X-ALD is caused by mutations in the ABCD1 gene that encodes ALDP, an ATP-binding cassette transporter located in the peroxisomal membrane with an unknown function (5). Biochemically, X-ALD is characterized by elevated levels of saturated and monounsaturated VLCFAs in plasma and tissues due to the impaired beta-oxidation of VLCFAs in peroxisomes (6–8). Since the pathogenesis of X-ALD is probably due to the increased levels of VLCFAs, correction of VLCFA levels is one of the primary objectives in therapeutic approaches. These include gene replacement therapy, bone marrow transplantation, lovastatin treatment, inhibition of VLCFA biosynthesis by mono-unsaturated fatty acids, notably oleic acid (C18:1{omega}9) and erucic acid (C22:1{omega}9) (Lorenzo's oil), and induction of the expression of ALDP-related protein (see Ref. 4 for an overview). Despite the increasing knowledge about X-ALD, there is currently no effective therapy for this disease.

An alternative method to degrade VLCFAs would be the {omega}-oxidation followed by beta-oxidation of the dicarboxylic acids produced. Different fatty acids are known to undergo {omega}-oxidation, but no data in literature are available with respect to the {omega}-oxidation of VLCFAs (9). The first step in {omega}-oxidation of fatty acids involves the conversion of the {omega}-methyl group of the fatty acid into an {omega}-hydroxyl group. This reaction is carried out by one or more cytochrome P450 enzymes mainly belonging to the CYP4 subfamily and requires NADPH and molecular oxygen (9, 10). Subsequently, the {omega}-hydroxy fatty acid may be oxidized further into a {omega}-carboxylic acid either via an NAD+-dependent alcohol and aldehyde dehydrogenase system or via a cytochrome P450-mediated route (11–14). Finally, dicarboxylic acids can be beta-oxidized in peroxisomes and/or mitochondria to shorter-chain dicarboxylic acids followed by excretion into the urine (15, 16). Accumulation of dicarboxylic acids has not been detected in X-ALD patients, whereas in patients with a peroxisomal biogenesis disorder, elevated levels of medium- and long-chain dicarboxylic acids were found in urine (17). Furthermore, beta-oxidation of long-chain dicarboxylic acids was normal in fibroblasts from X-ALD patients and deficient in fibroblasts from peroxisomal biogenesis disorder patients (15). These studies indicate that peroxisomes play an essential role in dicarboxylic acid degradation and that this metabolic route does not require ALDP. Under normal physiological conditions, fatty acid {omega}-oxidation is a minor oxidation pathway that accounts for 5–10% of total fatty acid oxidation in the liver, but the expression levels of many cytochrome P450s can be induced by a variety of different agents (18, 19). Therefore, if VLCFAs can indeed undergo {omega}-oxidation, then stimulation of this activity could be a means to reduce the levels of these fatty acids in patients with X-ALD.

In this study, we investigated the VLCFA {omega}-oxidation capacity of human liver microsomes. Until now, {omega}-oxidation of VLCFAs has not been studied in humans, and none of the enzymes potentially involved in this system have been characterized. We have studied the {omega}-oxidation pathway for several saturated fatty acids known to be of relevance to X-ALD, which includes docosanoic acid (C22:0), tetracosanoic acid (C24:0), and hexacosanoic acid (C26:0).


Figure 1
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  		FIGURE 1.
Formation of {omega}-hydroxy-C26:0 and the dicarboxylic acid of C26:0 in human liver microsomes as a function of time (A), pH (B), and protein (C). Human liver microsomes (50 µg/ml) were incubated in a buffered medium containing 100 mM glycine, 100 mM HEPES, pH 8.4, 1 mM NADPH, and {alpha}-cyclodextrin. Reactions were carried out at 37 °C. After termination, the reaction products were analyzed as described under "Experimental Procedures." Symbols used are as follows: {blacksquare}, total product formation; •, {omega}-hydroxy-C26:0; {diamondsuit}, C26:0-DCA.

 


Figure 2
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  		FIGURE 2.
Hydroxylation of (A) C22:0, (B) C24:0, and (C) C26:0 by human liver microsomes with the standard VLCFA hydroxylation assay as described under "Experimental Procedures." Reactions were carried out at 37 °C and terminated after 10 min. All data shown represent the means of two independent experiments. Symbols used are as follows: •, {omega}-hydroxy fatty acid; {diamondsuit}, dicarboxylic acid.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Pooled human liver microsomes and the recombinant human P450-containing insect cell microsomes and control microsomes (SupersomesTM) used in this study were purchased from BD Biosciences/Gentest. The cytochrome P450 content of SupersomesTM as provided by the manufacturer was as follows: CYP2E1 (588 pmol/mg), CYP2J2 (185 pmol/mg), CYP3A4 (606 pmol/mg), CYP4A11 (120 pmol/mg), CYP4F2 (556 pmol/mg), CYP4F3A (33 pmol/mg), CYP4F3B (435 pmol/mg), CYP4 F12 (213 pmol/mg), and control (not detectable). 22-Hydroxy-docosanoic acid ({omega}-hydroxy-C22:0) and hexacosanedioic acid (C26:0-DCA) were purchased from Larodan Fine Chemicals (Malmö, Sweden). Sulfaphenazole, quinidine, ketoconazole, furafylline, trimethoprim, diethyldithiocarbamate, and 17-octadecynoic acid were purchased from Sigma. N, O-bis-(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane was from Pierce. All other chemicals used were of analytical grade.

Enzymatic Assay for VLCFA {omega}-Hydroxylation—The experimental conditions used in this study to study the hydroxylation of different VLCFAs were adapted from previous experiments with minor modifications (14). Briefly, incubations were carried out for 30 min at 37 °C in a reaction mixture that contained Tris buffer, pH 8.4 (100 mM), protein (50 µg), {alpha}-cyclodextrin (1 mg/ml), and NADPH (1 mM) in a total volume of 200 µl, unless otherwise stated. The reaction was initiated by the addition of the fatty acid at a final concentration of 200 µM and terminated by the addition of 1 ml of hydrochloric acid to a final concentration of 1.7 M. The reaction products were extracted as described previously and analyzed by electrospray ionization mass spectrometry (8, 14).

Characterization of {omega}-Hydroxy Fatty Acids by GC-MS—Incubations were carried out under the same conditions as described above in a total volume of 2 ml. After acidification of the mixture, fatty acids were extracted four times with 5 ml of hexane and subsequently dried under a stream of nitrogen. Prior to gas chromatography-mass spectrometry (GC-MS), the residue was incubated with 40 µlof N, O-bis-(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane and 10 µl of pyridine at 80 °C for 1 h. After derivatization, the mixture was used directly for GC-MS analysis on a Hewlett-Packard 6890 gas chromatograph coupled to a Hewlett-Packard 5973 mass-selective detector (Palo Alto, CA). The samples (1 µl) were injected in the splitless mode (Hewlett Packard 7683 injector) and separated on a CP-Sil 5 CB-MS low bleed column (25 m x 0.25 mm x 0.30 µm: Chrompack, Middelburg, The Netherlands). The oven temperature was programmed as follows: 2 min at 85 °C followed by a linear increase of 20 °C per min to 320 °C and held at 320 °C for 10 min. The identities of the reaction products were verified by taking mass spectra in the scanning electron impact mode. The single ion monitoring mode was applied for the detection of the respective (M-15)+ ions, with m/z 485, 513, and 541 of the trimethylsilyl derivatives of {omega} and ({omega}-1)-hydroxy C22:0, C24:0, and C26:0, respectively. The (M-15)+ ions, with m/z 499, 427, and 555, were monitored for the (M-15)+ ions of the trimethylsilyl derivatives of the dicarboxylic acid of C22:0, C24:0, and C26:0, respectively. The ratio of{omega} and ({omega}-1) hydroxy fatty acids was calculated by comparing the peak areas of the single ion current of the{omega}-hydroxy fatty acid and dicarboxylic acid, with the peak area of the ({omega}-1)-hydroxy fatty acid.


Figure 3
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  		FIGURE 3.
GC-MS chromatogram (single-ion monitoring mode) of the products from C26:0 hydroxylation in human liver microsomes. Spectrum analysis was performed as described under "Experimental Procedures." The two peaks labeled I and II correspond to the (M-15)+ with m/z 541 of the hydroxylated C26:0 metabolites. Peak III corresponds to the (M-15)+ of the dicarboxylic acid of C26:0 (m/z 555).

 
Curve-fitting Procedure—The best-fit curves to the product formation data in human liver microsomes were obtained from the kinetic equation for the dual-enzyme Michaelis-Menten model,

Formula(Eq. 1)
where Km1 and Km2 are the high and low affinity Michaelis-Menten constants, respectively, and Vmax1 and Vmax2 are the corresponding maximal catalytic activities. The best-fit curves to the product formation with individually expressed human recombinant P450 enzymes were calculated using a cooperative single-enzyme model with two binding sites (Equation 2) in which product can be formed either from the single-substrate-bound form or from the two-substrate-bound form of the enzyme (20).

Formula 2(Eq. 2)
The values of the kinetic parameters were calculated by fitting the experimental data in the appropriate enzyme model using the IGOR Pro 5 software program (Wavemetrics).


Figure 4
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  		FIGURE 4.
Characterization of the C26:0 {omega}-oxidation intermediates from the peaks in Fig. 3. Based on the fragmentation spectra, the trimethylsilyl derivative was identified as peak II, {omega}-hydroxy-C26:0 (A); peak I, ({omega}-1)-hydroxy-C26:0 (B); and peak III, C26:0-DCA (C).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Optimization of the VLCFA Hydroxylation Assay—Previous studies have demonstrated that C22:0 is a substrate for the {omega}-oxidation system in rat liver microsomes (14). To study the {omega}-hydroxylation capacity of human liver microsomes for VLCFAs, the hydroxylation assay was optimized for the {omega}-oxidation of C26:0. To this end, human microsomal protein was incubated in a buffered medium containing NADPH and {alpha}-cyclodextrin to solubilize C26:0. The production of {omega}-hydroxy-C26:0 was linear with time up to 30 min (Fig. 1A). The effect of pH on the hydroxylation activity was determined using a combined buffer system that contained 100 mM HEPES and 100 mM glycine to cover the pH range 6.6–10.5. Fig. 1B shows that the formation of {omega}-hydroxy-C26:0 was maximal at pH 8.4.

Interestingly, the oxidation of C26:0 was not limited to the production of {omega}-hydroxy-C26:0, but also, the corresponding dicarboxylic acid (C26:0-DCA) was detected. The identity of C26:0-DCA was confirmed by two signals that appeared in the electrospray ionization mass spectra, which corresponded to its single and double negative charged state (data not shown), as well as by GC-MS studies (see below). The formation of C26:0-DCA from C26:0 was linear with time and maximal at the same pH as for the formation of {omega}-hydroxy-C26:0 (Fig. 1, A and B). Total product formation was linear with protein up to 60 µg/ml (Fig. 1C). At higher protein concentrations, the production of {omega}-hydroxy-C26:0 decreased, whereas C26:0-DCA formation increased slightly. Several buffer systems at pH 8.4 were tested: 100 mM Tris, 100 mM Tricine, and 100 mM Hepes. Hydroxylation of C26:0 was maximal with Tris as buffer (results not shown), which has therefore been used in subsequent experiments.

Kinetic Analysis of VLCFA Hydroxylation—The enzyme kinetics for the hydroxylation of C22:0, C24:0, and C26:0 were analyzed. To determine apparent Km and Vmax values for the different fatty acids, rates of product formation were determined at different substrate concentrations (Fig. 2). Interestingly, at the highest substrate concentrations used, the ratio of {omega}-hydroxy/dicarboxylic acid produced decreased with increasing chain length of the substrate. Furthermore, the rate of total product formation versus substrate concentration of all fatty acids used did not follow simple Michaelis-Menten kinetics. Different models were tested for data fitting. However, the model for dual-enzyme Michaelis-Menten kinetics as described under "Experimental Procedures" produced the best fit of the data points of Fig. 2. The kinetic parameters calculated from the experimental data of each of the different substrates using this model are listed in Table 1. At low substrate concentrations, the highest activity was observed with C22:0 with an apparent Vmax of 0.8 nmol · min–1 · mg–1, whereas the highest affinity was observed for C26:0 with an apparent Km of <1 µM. The results described in Table 1 show that the highest catalytic efficiency, Vmax/Km ratio, is observed for C26:0. The hydroxylation efficiency for C24:0 and C22:0 is 10-fold lower as compared with C26:0. Overall, these results demonstrate that VLCFAs are substrates for the human microsomal {omega}-oxidation system.


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  		TABLE 1
Kinetic parameters derived from the experimental data in Fig. 2 of VLCFA hydroxylation by human liver microsomes

Values were calculated using a dual-enzyme Michaelis-Menten model as described under "Experimental Procedures." The {omega} versus ({omega}-1) hydroxylation ratio was determined by GC-MS analysis as described.

 
Characterization of VLCFA {omega}-Oxidation Products—Hydroxylation of VLCFAs by cytochrome P450 enzymes may occur at the {omega}-position as well as at the adjacent positions (9, 21). To determine the position of the hydroxyl group, reaction products of the incubations with C22:0, C24:0, and C26:0 were analyzed by GC-MS. Three products appeared in the gas chromatogram after incubating human liver microsomes with C26:0 (Fig. 3). The major peak (Fig. 3, peak II) with a retention time of 14.5 min was analyzed by mass spectral analysis and identified as {omega}-hydroxy-C26:0 (Fig. 4A). The fragment at m/z 103 is characteristic of {omega}-hydroxy fatty acids, representing the terminal CH2OSi(CH3)3 moiety (22). The fragments observed in this mass spectrum were in analogy with those of the commercially available 22-hydroxy-C22:0 (data not shown). The minor peak in the GC chromatogram with a retention time of 14.25 min (Fig. 3, peak I) was identified by mass spectral analysis as ({omega}-1)-hydroxy-C26:0 (Fig. 4B). It was deduced from the mass spectrum that the peak at m/z 117 represents the CH3CHOSi(CH3)3 due to cleavage of the molecule adjacent to the hydroxyl group. Hydroxylated products of C26:0 with the hydroxyl group at other positions than {omega} and ({omega}-1) were not detected. In addition, the third peak (Fig. 3, peak III) with retention time of 14.85 min was identified as C26:0-DCA (Fig. 4C). The fragmentation pattern of this compound was identical to that of the commercial available C26:0-DCA (not shown). Product analysis of incubations with C22:0 and C24:0 as substrates resulted in similar patterns of product formation and analogous mass spectra as observed for C26:0 (data not shown). The ratio {omega}/({omega}-1)-hydroxylation for each substrate is listed in Table 1. Apparently, cytochrome P450 enzymes preferably hydroxylate VLCFAs at the methyl group.


Figure 5
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  		FIGURE 5.
Effect of several P450 isoform specific inhibitors on the hydroxylation of C26:0 by human liver microsomes. Microsomal protein was preincubated in the standard reaction mixture in the presence of inhibitor for 10 min. Subsequently, reactions were initiated by the addition of the substrate and were allowed to proceed for 30 min. The data represent the relative inhibition of C26:0 hydroxylation as compared with the activity observed in the absence of inhibitors. The results are the mean of two independent experiments, which did not vary by more than 10%. The colors of the bars represents the final inhibitor concentration: white, 1 µM; gray, 10 µM; black, 100 µM. The key is as follows: SP, sulfaphenazole; QD, quinidine; KET, ketoconazole; TA, troleandomycin, FF, furafylline; DDC, diethyldithiocarbamate; TMP, trimethoprim; 17-ODYA, 17-octadecynoic acid.

 


Figure 6
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  		FIGURE 6.
Hydroxylation activity of several human recombinant P450 isoforms toward C26:0. Recombinant P450 protein (5 pmol) was incubated for 30 min in the standard VLCFA hydroxylation reaction mixture with C26:0 followed by determination of both {omega}-hydroxy-C26:0 and C26:0-DCA.

 
VLCFA Hydroxylation Inhibition Studies—The first step of the {omega}-oxidation of fatty acids is catalyzed by cytochrome P450 enzymes. The human cytochrome P450 family consists of at least 57 CYP isoforms that are able to oxidize a broad spectrum of chemical compounds (23). To identify the P450 subfamily and/or the individual CYP isoform that is involved in VLCFA hydroxylation, various inhibitors were tested, which include: sulfaphenazole (selective for CYP2C) (24), quinidine (for CYP2D6) (24), ketoconazole (for CYP3A) (25), troleandomycin (for CYP3A4) (24), furafylline (for CYP1A2) (26), diethyldithiocarbamate (for CYP2E1) (26), trimethoprim (for CYP2C8) (27), and 17-octadecynoic acid (for CYP4A/F) (10, 28). In Fig. 5, the effect of these inhibitors on the {omega}-oxidation of C26:0 is shown. Hydroxylation of C26:0 was inhibited markedly by 17-octadecynoic acid already at the lowest concentration used (1 µM), and no {omega}-oxidation products were detected at the highest concentration of inhibitor used (100 µM). In the presence of 1 µM troleandomycin, product formation was decreased by 20%. Diethyldithiocarbamate and ketoconazole, inhibitors of CYP2E1 and CYP3A, respectively, had some inhibitory effect at the highest concentration inhibitor used (100 µM). All other inhibitors tested did not affect the hydroxylation of C26:0 to any significant extent.

Identification of P450 VLCFA Hydroxylases—The results from the VLCFA inhibition studies indicated that cytochrome P450 enzymes belonging to the CYP2E1, CYP3A, and CYP4A/F subfamily are likely to be involved in the hydroxylation of these fatty acids. To confirm this, several human recombinant P450 enzymes (SupersomesTM) were tested for hydroxylation activity toward C26:0. SupersomesTM are microsomes from baculovirus-infected insect cells expressing a single human CYP isoform. These experiments revealed that CYP4F2 as well as CYP4F3B catalyzed the hydroxylation of C26:0 (Fig. 6). All other recombinant CYP isoforms tested, as well as non-CYP-containing SupersomesTM, did not hydroxylate C26:0 to any appreciable extent.


Figure 7
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  		FIGURE 7.
Hydroxylation of VLCFAs by human recombinant CYP4F2 at different substrate concentrations. Reactions were initiated by the addition of the fatty acid C22:0 (A), C24:0 (B), or C26:0 (C) for 10 min at 37 °C. The results are the mean of two independent experiments. Symbols used are as follows: •, {omega}-hydroxy fatty acid; {diamondsuit}, dicarboxylic acid.

 
Kinetic Analysis of VLCFA Hydroxylation by CYP4F2 and CYP4F3B—The enzyme kinetics of CYP4F2 and CYP4F3B were analyzed for the hydroxylation of different VLCFAs. Figs. 7 and 8 show that hydroxylation of VLCFAs was not limited to the production of the {omega}-hydroxylated product, but dicarboxylic acids were produced as well. The rate of total product formation versus substrate concentration again did not follow Michaelis-Menten kinetics. The plots were hyperbolic, and product formation was not saturable, even at the highest substrate concentration used (200 µM). The corresponding Eadie-Hofstee plots were biphasic (data not shown), indicating apparent multiple binding site kinetics (20). To determine the kinetic parameters of CYP4F2 and CYP4F3B for the fatty acids tested, different enzyme kinetic models were analyzed. A cooperative single-enzyme model with two binding sites as described under "Experimental Procedures" resulted in the best fit of the data points of Figs. 7 and 8. The kinetic parameters calculated for each of the different substrates are listed in Table 2. Apparent Km values of both enzymes for VLCFAs were in the micromolar range. The highest VLCFA hydroxylation activity was observed for CYP4F3B. Although CYP4F2 was found to be less active toward VLCFAs, Vmax/Km ratios were similar to those of CYP4F3B.


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  		TABLE 2
Kinetic parameters for VLCFA hydroxylation by CYP4F2 and CYP 4F3B

The values were obtained from the experimental data in Figs. 7 and 8 using a two-substrate binding site model as described under "Experimental Procedures." The {omega}:({omega}-1) hydroxylation ratio was determined by GC-MS analysis as described.

 
The reaction products after incubation of CYP4F2 or CYP4F3B with the different fatty acids were analyzed by GC-MS. Both enzymes predominantly hydroxylate the methyl group of the VLCFAs and to a lesser extent the ({omega}-1) group (data not shown). The {omega}: ({omega}-1)-hydroxylation ratio for each substrate is listed in Table 2.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we investigated whether the {omega}-oxidation pathway may provide an alternative oxidation route for VLCFAs. Our results show that these fatty acids are substrates for the {omega}-oxidation system in human liver microsomes. Moreover, C26:0 was not only converted into {omega}-hydroxy-C26:0 but also further to its dicarboxylic acid by cytochrome P450 enzymes. Our previous studies on C22:0 {omega}-oxidation demonstrated the existence of a cytochrome P450-mediated hydroxylation system for the production of dicarboxylic acids in rat liver microsomes (14). The results described in this study clearly show that in humans, a similar pathway is present for the {omega}-oxidation of VLCFAs. Based on the inhibition studies (Fig. 5) and the experiments with SupersomesTM containing individual human cytochrome P450 enzymes (Fig. 6), we conclude that CYP4F2 and CYP4F3B are able to hydroxylate C26:0. Moreover, both enzymes are able to hydroxylate C26:0 all the way to its dicarboxylic acid (Figs. 7 and 8). CYP4F2 and CYP4F3B have a high affinity for saturated VLCFAs with Km values in the micromolar range and are therefore interesting from a physiological point of view.

CYP4F2 and CYP4F3 are distinct genes, and both are located on the short arm of chromosome 19. The CYP4F2 and CYP4F3 proteins contain 520 amino acids, and they share a 94% identity. Both CYP4F2 and CYP4F3B catalyze the {omega}-hydroxylation of various eicosanoids including arachidonic acid, prostaglandins, and leukotriene B4 (29). These enzymes are expressed predominantly in human liver and kidney, and to lesser extent, in brain, testis, skin, and various other tissues (29–32). Alternative splicing of the CYP4F3 gene generates two distinct isoforms, CYP4F3A and CYP4F3B, and these proteins differ in tissue distribution and biological function (32). CYP4F3A does not contain exon 3 and inactivates leukotriene B4 by {omega}-hydroxylation. In contrast, CYP4F3B does not contain exon 4 and has high {omega}-hydroxylation activity toward arachidonic acid (32–34). The substrate specificity of CYP4F3 is apparently determined by the protein domains encoded by exon 3 (CYP4F3B) and exon 4 (CYP4F3A). Our data are in line with this; CYP4F3B has high {omega}-hydroxylation activity toward VLCFAs, whereas no product formation was observed with CYP4F3A.


Figure 8
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  		FIGURE 8.
{omega}-Oxidation of VLCFAs by human recombinant CYP4F3B at different substrate concentrations. Hydroxylation of C22:0 (A), C24:0 (B), and C26:0 (C) by human recombinant CYP4F3B at different substrate concentrations, and reactions were allowed to proceed for 10 min. All data shown represent the means of two independent experiments. Symbols used are as follows: •, {omega}-hydroxy fatty acid; {diamondsuit}, dicarboxylic acid.

 
Biochemically, X-ALD is characterized by elevated levels of VLCFAs in plasma and tissues due to the impaired beta-oxidation of these fatty acids in peroxisomes. At present, there is no effective therapy for this disease. Based on the hypothesis that the accumulation of VLCFAs in X-ALD patients may be reduced by stimulating VLCFA {omega}-oxidation, we have now studied the {omega}-oxidation of VLCFAs. Long-chain dicarboxylic acids can undergo beta-oxidation in X-ALD fibroblasts, whereas fibroblasts from patients with a peroxisomal biogenesis disorder were unable to oxidize these compounds (15). Furthermore, accumulation of dicarboxylic acids has not been detected in X-ALD patients, whereas elevated levels of medium- and long-chain dicarboxylic acids were detected in the urine of peroxisomal biogenesis disorder patients (17, 35). These studies indicate that peroxisomes are essential for the degradation of long-chain dicarboxylic acids and that ALDP is not involved in the beta-oxidation of these fatty acids.

Expression of both CYP4F2 and CYP4F3 has been detected in tissues that are affected in patients with X-ALD. The tissue distribution of CYP4F3B is quite similar to that of CYP4F2 (29). At present, little information is known in literature about the regulation of gene expression of CYP4F2 and CYP4F3B. Many cytochrome P450 enzymes are under the control of one or more members of the nuclear hormone receptor family, notably peroxisome proliferator-activated receptor-{alpha}, retinoic acid receptor, retinoid X receptor, pregnane X receptor, and constitutive androstane receptor (18). Unlike P450 enzymes of the CYP4A subfamily, which are induced by peroxisomal proliferators and hypolipidemic drugs such as WY14,643 and clofibrate, the expression of CYP4F enzymes either remains unchanged or is repressed (36, 37). Induction studies on the promoter activity of the CYP4F2 gene demonstrated that it may be mediated via retinoic acid receptor/retinoid X receptor-{alpha} and peroxisome proliferator-activated receptor-{alpha}/retinoid X receptor-{alpha} (38).

CYP4F2 and CYP4F3B predominantly produce {omega}-hydroxy VLCFAs, and to a lesser extent, their corresponding dicarboxylic acids. Further oxidation of {omega}-hydroxy VLCFAs can occur via NAD+-dependent alcohol and aldehyde dehydrogenases localized in the cytosol and endoplasmic reticulum. This pathway has been identified for long-chain {omega}-hydroxy fatty acids, retinoids, and (very) long-chain aldehydes in the endoplasmic reticulum and cytosol (14, 39–42). The enzymes involved in the dehydrogenation of these compounds have a broad substrate specificity (40, 42, 43). Our previous studies have shown that rat liver microsomes readily oxidize {omega}-hydroxy-C22:0 to a dicarboxylic acid in a NAD+-dependent pathway, and we therefore postulate that the dehydrogenases involved in this pathway may utilize {omega}-hydroxy VLCFAs as well (14). At present, there are no data in the literature on the nature of the alcohol and aldehyde dehydrogenases that are active toward {omega}-hydroxy VLCFAs. Future studies are aimed at the characterization of the enzymes involved in the NAD+-dependent {omega}-oxidation of {omega}-hydroxy VLCFAs.

To summarize, VLCFAs are hydroxylated to very long-chain {omega}-hydroxy and dicarboxylic acids in human liver microsomes. We have identified two cytochrome P450 enzymes that catalyze the {omega}-hydroxylation of VLCFAs. Both enzymes are members of the 4F subfamily, namely CYP4F2 and CYP4F3B. Future work is aimed at the identification of the regulatory mechanisms involved in the expression of these enzymes. To generate new therapeutic options for patients with X-ALD, we will investigate whether the VLCFA {omega}-oxidation route can be induced to normalize the levels of VLCFAs in affected tissues.


   FOOTNOTES
 
* This work was supported by Prinses Beatrix Fonds Grant MAR 02-0116, the Netherlands Organization for Scientific Research Grant NWO-MW 903-42-077, and a grant from the European Leukodystrophy Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Laboratory of Genetic Metabolic Diseases (Rm. F0-224), Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands. Tel.: 31-20-5665958; Fax: 31-20-6962596; E-mail: r.j.wanders{at}amc.uva.nl.

2 The abbreviations used are: VLCFA, very long-chain fatty acid; X-ALD, X-linked adrenoleukodystrophy; ALDP, adrenoleukodystrophy protein; {omega}-hydroxy-C26:0, 26-hydroxy-hexacosanoic acid; C26:0-DCA, hexacosanedioic acid; CYP, cytochrome P450; CAR, constitutive androstane receptor; GC-MS, gas chromatography-mass spectrometry; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Back


   ACKNOWLEDGMENTS
 
We thank Henk Overmars, Fredoen Valianpour, and Wim Kulik for technical assistance with the electrospray ionization mass spectrometry and Jasper Komen for stimulating discussions and help at various stages of these studies. We also thank Lia van Lint and Wilma Smit for expert technical assistance with the GC-MS analysis.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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