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Identification of fatty aldehyde dehydrogenase in the breakdown of phytol to phytanic acid
Molecular Genetics and Metabolism 82 (2004) 33–37 www.elsevier.com/locate/ymgme
IdentiWcation of fatty aldehyde dehydrogenase in the breakdown of phytol to phytanic acid Daan M. van den Brink,a,b Joram N.I. van Miert,a,b Georges Dacremont,c Jean-François Rontani,d Gerbert A. Jansen,a,b and Ronald J.A. Wandersa,b,¤ a
Department of Clinical Chemistry, University of Amsterdam, Academic Medical Center, Emma Children’s Hospital, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands b Department of Pediatrics, University of Amsterdam, Academic Medical Center, Emma Children’s Hospital, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands c University of Ghent, Ghent, Belgium d Laboratoire d’Océanographie et de Biogéochimie (UMR 6535), Centre d’Océanologie de Marseille (OSU), Campus de Luminy, 13288 Marseille, France Received 18 November 2003; received in revised form 19 January 2004; accepted 20 January 2004
Abstract Phytol is a branched chain fatty alcohol, which is abundantly present in nature as part of the chlorophyll molecule. In its free form, phytol is metabolized to phytanic acid, which accumulates in patients suVering from a variety of peroxisomal disorders, including Refsum disease. The breakdown of phytol to phytanic acid takes place in three steps, in which Wrst, the alcohol is converted to the aldehyde, second the aldehyde is converted to phytenic acid, and Wnally the double bond is reduced to yield phytanic acid. By culturing Wbroblasts in the presence of phytol, increases in the levels of phytenic and phytanic acid were detected. Interestingly, Wbroblasts derived from patients aVected by Sjögren Larsson syndrome (SLS), known to be deWcient in microsomal fatty aldehyde dehydrogenase (FALDH) were found to be deWcient in this. In addition, Wbroblast homogenates of these patients, incubated with phytol in the presence of NAD+ did not produce any phytenic acid. This indicates that FALDH is involved in the breakdown of phytol. 2004 Elsevier Inc. All rights reserved. Keywords: Phytol; Phytanic acid; Phytenic acid; Branched chain fatty acids; Fatty acid oxidation; Fatty aldehyde dehydrogenase; Sjögren Larsson syndrome; Refsum disease
Introduction Phytol (3,7,11,15-tetramethylhexadec-2-en-1-ol, Fig. 1) is a branched chain fatty alcohol abundantly found in nature as part of the chlorophyll molecule. Studies in the 1960s have shown that phytol is metabolized to phytanic acid, a fatty acid that accumulates in a variety of peroxisomal diseases. Phytanic acid is degraded by a process called -oxidation, because a methyl group on the three position makes -oxidation impossible [1,2]. The enzyme responsible for the Wrst step of -oxidation is phytanoylCoA hydroxylase (PAHX), which has a peroxisomal
¤
Corresponding author. Fax: +31-20-696-2596. E-mail address: [email protected] (R.J.A. Wanders).
1096-7192/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2004.01.019
localization. In patients suVering from Refsum disease, the large majority of whom are deWcient in PAHX, phytanic acid accumulates to very high levels, which leads to a variety of severe, progressive clinical symptoms, including retinitis pigmentosa, peripheral neuropathy, anosmia, and cerebellar ataxia [3,4]. To avoid the progression of symptoms, Refsum disease patients are prescribed a diet low in phytanic acid. However, despite the fact that relatively large amounts of phytol are taken in via the diet, little attention has been paid to this precursor of phytanic acid. This is mainly because phytol is part of the chlorophyll molecule, which as a whole cannot be digested. Studies with animals fed radio-labeled chlorophyll have shown that only a small percentage of phytol is released and absorbed in the digestive tract [5]. Most likely, this is due
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D.M. van den Brink et al. / Molecular Genetics and Metabolism 82 (2004) 33–37
Phytol
OH
NADPH NADP+
+
NAD
NADH
O Phytenal
NAD+
NAD
NADH
NADH
+
OH Dihydrophytol
O OH Phytenic acid
O Phytanal
NADPH NADP+ NAD+ O
NADH OH
Phytanic acid
Fig. 1. Pathway for the degradation of phytol to phytanic acid. On the left-hand side the degradation pathway proposed for mammals is shown, whereas on the right that for ruminant animals is depicted.
to the strong ester bond linking the phytol moiety to chlorophyllin. Bacteria present in the gut of ruminant animals are able to break this bond, which explains the high levels of phytanic acid found in tissues of these animals. In contrast, when free phytol is administered to the diet, it is absorbed eYciently [5]. However, there is little insight in the amounts of free phytol present in meat and dairy products derived from ruminant animals. Furthermore, there are some reports that free phytol is also present in vegetable oils and nuts ([6] and references therein). A high level of free phytol in the diet leads to an increase of phytanic acid and its -oxidation product pristanic acid. The mechanism of the conversion of phytol to phytanic acid is not well known. Animal studies have shown that feeding of a diet supplemented with phytol is associated with an increase of phytenic acid in addition to phytanic acid [7,8]. From this Wnding it was proposed that the degradation pathway of phytol Wrst involves the conversion of the alcohol into the acid, after which the double bond is reduced to form phytanic acid (Fig. 1). An alternative pathway was thought to exist in ruminant animals, in which the double bond is removed Wrst to yield phytanal, which in turn is converted to the acid. Support for the former model came from studies by Muralidharan and Muralidharan in the 1980s [9,10], in which dependence on NAD+ as cofactor was found and some investigation of the substrate kinetics was carried out. However, there is no information in literature with respect to the nature of the individual enzymes catalyzing the diVerent reactions. It is suggested that for the production of phytenic acid, phytol is Wrst converted to the aldehyde phytenal
by an alcohol dehydrogenase, followed by an aldehyde dehydrogenase step, with both steps being NAD+ dependent [10]. The conversion of phytenic acid to phytanic acid, which involves the reduction of the double bond, is thought to be NADPH dependent, although no clear evidence has ever been produced. To investigate the degradation of phytol in more detail, we have set up a gas chromatography–mass spectrometry (GC–MS) method to measure the intermediate products in cultured human Wbroblasts as well as in Wbroblast homogenates. Using this experimental set-up, we found that the conversion of phytol to phytenic acid was deWcient in Wbroblasts derived from patients suVering from Sjögren Larsson syndrome (SLS), characterized by a deWciency of a microsomal fatty aldehyde dehydrogenase (FALDH, ALDH10) due to mutations in the encoding gene ALDH10 [11]. This demonstrates that FALDH is required for the conversion of phytenal to phytenic acid and provides evidence supporting the degradation mechanism of phytol via phytenal to phytenic acid and ultimately to phytanic acid.
Materials and methods Cultured skin Wbroblasts Fibroblasts were cultured in Nutrient mixture Ham’s F-10 with L-glutamine and 25 mM Hepes (Gibco, Invitrogen, Merelbeke, Belgium) supplemented with 10% fetal calf serum (Gibco), penicillin (100 U/ml), and streptomycin (100 g/ml, Gibco) at 37 °C and with 5% CO2. The FALDH-deWcient Wbroblasts were from established
D.M. van den Brink et al. / Molecular Genetics and Metabolism 82 (2004) 33–37
SLS patients as concluded from the clinical history, deWcient FALDH activity and distinct mutations in the ALDH10 gene [12]. Incubations of cultured Wbroblasts with phytol Phytol (mixture of Z- and E-isomers, Merck, Darmstadt, Germany) was dissolved in ethanol, one hundred times diluted in medium to the desired concentration and subsequently added to cells for the indicated time periods. The Wbroblasts were harvested and branched chain fatty acid composition was quantiWed by GC– MS using deuterated phytanic acid as an internal standard, as described [13]. BrieXy, samples were subjected to acidic and alkaline hydrolysis, after which fatty acids were extracted with hexane. The organic layer was evaporated to dryness under nitrogen at 40 °C. The samples were derivatized with N-tert-butyldimethylsilyl-N-methyl-triXuoroacetamide (MTBSTFA, Aldrich, Steinheim, Germany) and pyridine (50 L each) at 80 °C for 30 min on an Agilent Technologies Model 5890/5973 GC–MS system equipped with a CPsil 19CB capillary column (25 m £ 0.25 mm I.D., Wlm thickness 0.25 mm, Varian, Palo Alto, CA), with electron impact ionization applied at 70 eV. MS acquisition was performed in the single ion monitoring mode, monitoring the [M-57]+ ions of the various compounds. Phytenic acid was synthesized as described, and isomers were
35
separated using high performance liquid chromatography (HPLC) [14]. Incubations of Wbroblast homogenates with phytol Fibroblasts were harvested, taken up in phosphatebuVered saline (PBS), and homogenized by sonication (2 cycles of 10 s at 9 W) on ice. The incubation mixture consisted of 40 g/mL protein, 50 mM glycine buVer (pH 9.2), 1 mM NAD+, 0.1% sodium cholate, and 1 mg/ml methyl--cyclodextrin (Fluka, Buchs, Switzerland) in a total volume of 500 L. Reactions were performed at 37 °C and initiated by the addition of 200 M phytol dissolved in dimethyl sulfoxide (DMSO). After 60 min, the incubation was terminated by the addition of 100 L of 1 N HCl. Then 2 mL of hexane was added, after which the organic layer was evaporated to dryness under nitrogen at 40 °C. The sample was then derivatized with MTBSTFA and analyzed as described in the previous section.
Results To study the degradation mechanism of phytol, human Wbroblasts were cultured for 4 days in the presence of 5, 25, and 50 M phytol. GC–MS analysis of the fatty acid composition of the harvested cells showed an
Fig. 2. Production of (A, D) pristanic acid, (B, E) phytanic acid, and (C, F) phytenic acid in Wbroblasts cultured in the presence of phytol. (A–C) Cells were cultured for 4 days in the presence of 0, 5, 25 or 50 M phytol. (D–F) Control cells and cells derived from SLS patients were cultured for 4 days in the presence of 25 M phytol. Branched chain fatty acids were measured as described under Materials and methods. Values represent means § standard deviation (SD) of duplicates expressed in nmol/mg protein. *P 0 0.05; **P 0 0.01 as calculated by Student’s t test, compared with control Wbroblasts incubated with 25 M phytol.
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acid was formed, even though cells had been incubated with a racemic mixture of Z- and E-phytol (Fig. 3). Since phytenal is an intermediate in the conversion of phytol to phytenic acid, an aldehyde dehydrogenase is required in the second step to form phytenic acid. A candidate enzyme for this reaction would be FALDH, encoded by the ALDH10 gene, which has been shown to be reactive with a closely related compound, dihydrophytal as a substrate [15]. Therefore, phytol incubations were performed using Wbroblast cell lines derived from SLS patients, deWcient in FALDH. As shown in Figs. 2F and 3B, these cell lines were deWcient in the production of phytenic acid from phytol. To further substantiate this observation, enzyme assays in Wbroblast homogenates were performed. Incubation of homogenates with phytol in the presence of NAD+ resulted in a protein and time dependent production of phytenic acid (Figs. 4A and B). Homogenates of FALDH-deWcient Wbroblasts were found to be deWcient in the production of phytenic acid (Fig. 4C).
Fig. 3. Separation of isomers of phytenic acid by GC–MS. (A) Analysis of standards for (Z)- and (E)-phytenic acid separately and in an racemic mixture and (B) phytenic acid formed by control Wbroblasts or Wbroblasts derived from a SLS-patient, cultured for 0 or 4 days in the presence of 25 M phytol.
increase in the levels of pristanic and phytanic acid, which was not observed in cells cultured without phytol (Figs. 2A and B). The amount of these metabolites directly correlated with the concentration of phytol present in the medium (Figs. 2A and B). In addition, a third peak was observed with a similar response to phytol as seen for pristanic and phytanic acid. Using an authentic standard as a reference, this peak was identiWed as phytenic acid (Fig. 2C). The Z- and E-isomers of phytenic acid have distinct retention times on the GC-column, which led to the observation that only E- and no Z-phytenic
Discussion Although a model for the degradation of phytol was proposed as early as the 1960s, very little research has been done to elucidate the precise reaction mechanism and to characterize the enzymes involved. In the present study, using Wbroblast incubations with phytol, it was shown conclusively that phytenic acid is a speciWc metabolite of phytol degradation. Furthermore, the Wnding that FALDH-deWcient Wbroblast lines are deWcient in phytenic acid production implies that this enzyme is part of the degradation pathway. Using a GC–MS method to analyze branched chain fatty acid composition in Wbroblast homogenates incubated with phytol, a deWciency in phytenic acid formation was found in FALDH-deWcient cells from SLS patients, which strongly suggests that phytenal is a speciWc substrate for
Fig. 4. Degradation of phytol to phytenic acid in Wbroblast homogenates. (A) Dependence of protein concentration and (B) time course of the phytol degradation, with reaction conditions as described in Materials and methods. (C) Fibroblast homogenates derived from SLS-patients are deWcient in phytenic acid production. Values in the bars represent means § SD of the activity expressed in nmol/min/mg of protein (n D 5 and 9 for control and SLS-patient derived cell lines, respectively). **P 0 0.01 as calculated by Student’s t test, compared with control Wbroblasts.
D.M. van den Brink et al. / Molecular Genetics and Metabolism 82 (2004) 33–37
FALDH. Due to the presence of some phytenic acid in the phytol used as substrate (Fig. 3B), it is diYcult to say whether this is a complete deWciency or whether some residual activity exists. Interestingly, only the production of E-phytenic acid was observed, even though incubations were carried out with a racemic mixture of Z- and E-isomers of phytol as a substrate. It remains to be established how this apparent stereospeciWcity of the reaction is achieved and whether FALDH or the preceding alcohol dehydrogenase is responsible. A deWciency of FALDH has been shown to be the cause of SLS, a metabolic disorder characterized by ichthyosis, mental retardation, and spastic diplegia or tetraplegia [11,16]. In tissues and plasma of SLS patients an accumulation of long chain fatty aldehydes and alcohols has been shown, which is thought to play a role in the onset of the symptoms [17]. Levels of phytol have never been investigated in these patients, but it is interesting to speculate on possible contribution of a phytol accumulation to the disease, since phytol has been shown to be toxic to cells [7]. Earlier reports have suggested that FALDH is part of the microsomal fatty alcohol:NAD+-oxidoreductase complex, also consisting of an alcohol dehydrogenase part [18]. This complex would therefore be a good candidate for the catalysis of the entire phytol to phytenic acid conversion. Although FALDH has been characterized in great detail [15], relatively little research has focused on the alcohol dehydrogenase domain. PuriWcation of this domain will make it clear which alcohol dehydrogenase is involved in the phytol degradation pathway.
Acknowledgments This work was supported by a grant from the Meelmeijer Fund and a grant from the European Commission (QLG3-2002-00696).
References [1] R.J. Wanders, G.A. Jansen, M.D. Lloyd, Phytanic acid alphaoxidation, new insights into an old problem: a review, Biochim. Biophys. Acta 1631 (2003) 119–135. [2] M. Mukherji, C.J. SchoWeld, A.S. Wierzbicki, G.A. Jansen, R.J. Wanders, M.D. Lloyd, The chemical biology of branched-chain lipid metabolism, Prog. Lipid Res. 42 (2003) 359–376.
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[3] A.S. Wierzbicki, M.D. Lloyd, C.J. SchoWeld, M.D. Feher, F.B. Gibberd, Refsum’s disease: a peroxisomal disorder aVecting phytanic acid alpha-oxidation, J. Neurochem. 80 (2002) 727– 735. [4] R.J. Wanders, C. Jakobs, O.H. Skjeldal, Refsum disease, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill, New York, 2001, pp. 3303–3321. [5] J.H. Baxter, Absorption of chlorophyll phytol in normal man and in patients with Refsum’s disease, J. Lipid Res. 9 (1968) 636–641. [6] R.P. Hansen, Phytol: its metabolic products and their distribution. A review, N. Z. J. Sci. 23 (1980) 259–275. [7] D. Steinberg, J. Avigan, C.E. Mize, J.H. Baxter, J. Cammermeyer, H.M. Fales, P.F. Highet, EVects of dietary phytol and phytanic acid in animals, J. Lipid Res. 7 (1966) 684–691. [8] C.E. Mize, J. Avigan, J.H. Baxter, H.M. Fales, D. Steinberg, Metabolism of phytol-U-14C and phytanic acid-U-14C in the rat, J. Lipid Res. 7 (1966) 692–697. [9] F.N. Muralidharan, V.B. Muralidharan, In vitro conversion of phytol to phytanic acid in rat liver: subcellular distribution of activity and chemical characterization of intermediates using a new bromination technique, Biochim. Biophys. Acta 835 (1985) 36–40. [10] F.N. Muralidharan, V.B. Muralidharan, Characterization of phytol–phytanate conversion activity in rat liver, Biochim. Biophys. Acta 883 (1986) 54–62. [11] W.B. Rizzo, G. Carney, Z. Lin, The molecular basis of SjogrenLarsson syndrome: mutation analysis of the fatty aldehyde dehydrogenase gene, Am. J. Hum. Genet. 65 (1999) 1547–1560. [12] M.A. Willemsen, L. Ilst, P.M. Steijlen, J.J. Rotteveel, J.G. de Jong, P.H. van Domburg, E. Mayatepek, F.J. Gabreels, R.J. Wanders, Clinical, biochemical and molecular genetic characteristics of 19 patients with the Sjogren-Larsson syndrome, Brain 124 (2001) 1426–1437. [13] P. Vreken, A.E. van Lint, A.H. Bootsma, H. Overmars, R.J. Wanders, A.H. van Gennip, Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry, J. Chromatogr. B. Biomed. Sci. Appl. 713 (1998) 281–287. [14] J.F. Rontani, P.C. Bonin, J.K. Volkman, Biodegradation of free phytol by bacterial communities isolated from marine sediments under aerobic and denitrifying conditions, Appl. Environ. Microbiol. 65 (1999) 5484–5492. [15] T.L. Kelson, M. Secor Jr., W.B. Rizzo, Human liver fatty aldehyde dehydrogenase: microsomal localization, puriWcation, and biochemical characterization, Biochim. Biophys. Acta 1335 (1997) 99–110. [16] W.B. Rizzo, D.A. Craft, Sjogren-Larsson syndrome. DeWcient activity of the fatty aldehyde dehydrogenase component of fatty alcohol:NAD+ oxidoreductase in cultured Wbroblasts, J. Clin. Invest. 88 (1991) 1643–1648. [17] W.B. Rizzo, D.A. Craft, Sjogren-Larsson syndrome: accumulation of free fatty alcohols in cultured Wbroblasts and plasma, J. Lipid Res. 41 (2000) 1077–1081. [18] T. Lee, Characterization of fatty alcohol:NAD+ oxidoreductase from rat liver, J. Biol. Chem. 254 (1979) 2892–2896.
IdentiWcation of fatty aldehyde dehydrogenase in the breakdown of phytol to phytanic acid Daan M. van den Brink,a,b Joram N.I. van Miert,a,b Georges Dacremont,c Jean-François Rontani,d Gerbert A. Jansen,a,b and Ronald J.A. Wandersa,b,¤ a
Department of Clinical Chemistry, University of Amsterdam, Academic Medical Center, Emma Children’s Hospital, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands b Department of Pediatrics, University of Amsterdam, Academic Medical Center, Emma Children’s Hospital, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands c University of Ghent, Ghent, Belgium d Laboratoire d’Océanographie et de Biogéochimie (UMR 6535), Centre d’Océanologie de Marseille (OSU), Campus de Luminy, 13288 Marseille, France Received 18 November 2003; received in revised form 19 January 2004; accepted 20 January 2004
Abstract Phytol is a branched chain fatty alcohol, which is abundantly present in nature as part of the chlorophyll molecule. In its free form, phytol is metabolized to phytanic acid, which accumulates in patients suVering from a variety of peroxisomal disorders, including Refsum disease. The breakdown of phytol to phytanic acid takes place in three steps, in which Wrst, the alcohol is converted to the aldehyde, second the aldehyde is converted to phytenic acid, and Wnally the double bond is reduced to yield phytanic acid. By culturing Wbroblasts in the presence of phytol, increases in the levels of phytenic and phytanic acid were detected. Interestingly, Wbroblasts derived from patients aVected by Sjögren Larsson syndrome (SLS), known to be deWcient in microsomal fatty aldehyde dehydrogenase (FALDH) were found to be deWcient in this. In addition, Wbroblast homogenates of these patients, incubated with phytol in the presence of NAD+ did not produce any phytenic acid. This indicates that FALDH is involved in the breakdown of phytol. 2004 Elsevier Inc. All rights reserved. Keywords: Phytol; Phytanic acid; Phytenic acid; Branched chain fatty acids; Fatty acid oxidation; Fatty aldehyde dehydrogenase; Sjögren Larsson syndrome; Refsum disease
Introduction Phytol (3,7,11,15-tetramethylhexadec-2-en-1-ol, Fig. 1) is a branched chain fatty alcohol abundantly found in nature as part of the chlorophyll molecule. Studies in the 1960s have shown that phytol is metabolized to phytanic acid, a fatty acid that accumulates in a variety of peroxisomal diseases. Phytanic acid is degraded by a process called -oxidation, because a methyl group on the three position makes -oxidation impossible [1,2]. The enzyme responsible for the Wrst step of -oxidation is phytanoylCoA hydroxylase (PAHX), which has a peroxisomal
¤
Corresponding author. Fax: +31-20-696-2596. E-mail address: [email protected] (R.J.A. Wanders).
1096-7192/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2004.01.019
localization. In patients suVering from Refsum disease, the large majority of whom are deWcient in PAHX, phytanic acid accumulates to very high levels, which leads to a variety of severe, progressive clinical symptoms, including retinitis pigmentosa, peripheral neuropathy, anosmia, and cerebellar ataxia [3,4]. To avoid the progression of symptoms, Refsum disease patients are prescribed a diet low in phytanic acid. However, despite the fact that relatively large amounts of phytol are taken in via the diet, little attention has been paid to this precursor of phytanic acid. This is mainly because phytol is part of the chlorophyll molecule, which as a whole cannot be digested. Studies with animals fed radio-labeled chlorophyll have shown that only a small percentage of phytol is released and absorbed in the digestive tract [5]. Most likely, this is due
34
D.M. van den Brink et al. / Molecular Genetics and Metabolism 82 (2004) 33–37
Phytol
OH
NADPH NADP+
+
NAD
NADH
O Phytenal
NAD+
NAD
NADH
NADH
+
OH Dihydrophytol
O OH Phytenic acid
O Phytanal
NADPH NADP+ NAD+ O
NADH OH
Phytanic acid
Fig. 1. Pathway for the degradation of phytol to phytanic acid. On the left-hand side the degradation pathway proposed for mammals is shown, whereas on the right that for ruminant animals is depicted.
to the strong ester bond linking the phytol moiety to chlorophyllin. Bacteria present in the gut of ruminant animals are able to break this bond, which explains the high levels of phytanic acid found in tissues of these animals. In contrast, when free phytol is administered to the diet, it is absorbed eYciently [5]. However, there is little insight in the amounts of free phytol present in meat and dairy products derived from ruminant animals. Furthermore, there are some reports that free phytol is also present in vegetable oils and nuts ([6] and references therein). A high level of free phytol in the diet leads to an increase of phytanic acid and its -oxidation product pristanic acid. The mechanism of the conversion of phytol to phytanic acid is not well known. Animal studies have shown that feeding of a diet supplemented with phytol is associated with an increase of phytenic acid in addition to phytanic acid [7,8]. From this Wnding it was proposed that the degradation pathway of phytol Wrst involves the conversion of the alcohol into the acid, after which the double bond is reduced to form phytanic acid (Fig. 1). An alternative pathway was thought to exist in ruminant animals, in which the double bond is removed Wrst to yield phytanal, which in turn is converted to the acid. Support for the former model came from studies by Muralidharan and Muralidharan in the 1980s [9,10], in which dependence on NAD+ as cofactor was found and some investigation of the substrate kinetics was carried out. However, there is no information in literature with respect to the nature of the individual enzymes catalyzing the diVerent reactions. It is suggested that for the production of phytenic acid, phytol is Wrst converted to the aldehyde phytenal
by an alcohol dehydrogenase, followed by an aldehyde dehydrogenase step, with both steps being NAD+ dependent [10]. The conversion of phytenic acid to phytanic acid, which involves the reduction of the double bond, is thought to be NADPH dependent, although no clear evidence has ever been produced. To investigate the degradation of phytol in more detail, we have set up a gas chromatography–mass spectrometry (GC–MS) method to measure the intermediate products in cultured human Wbroblasts as well as in Wbroblast homogenates. Using this experimental set-up, we found that the conversion of phytol to phytenic acid was deWcient in Wbroblasts derived from patients suVering from Sjögren Larsson syndrome (SLS), characterized by a deWciency of a microsomal fatty aldehyde dehydrogenase (FALDH, ALDH10) due to mutations in the encoding gene ALDH10 [11]. This demonstrates that FALDH is required for the conversion of phytenal to phytenic acid and provides evidence supporting the degradation mechanism of phytol via phytenal to phytenic acid and ultimately to phytanic acid.
Materials and methods Cultured skin Wbroblasts Fibroblasts were cultured in Nutrient mixture Ham’s F-10 with L-glutamine and 25 mM Hepes (Gibco, Invitrogen, Merelbeke, Belgium) supplemented with 10% fetal calf serum (Gibco), penicillin (100 U/ml), and streptomycin (100 g/ml, Gibco) at 37 °C and with 5% CO2. The FALDH-deWcient Wbroblasts were from established
D.M. van den Brink et al. / Molecular Genetics and Metabolism 82 (2004) 33–37
SLS patients as concluded from the clinical history, deWcient FALDH activity and distinct mutations in the ALDH10 gene [12]. Incubations of cultured Wbroblasts with phytol Phytol (mixture of Z- and E-isomers, Merck, Darmstadt, Germany) was dissolved in ethanol, one hundred times diluted in medium to the desired concentration and subsequently added to cells for the indicated time periods. The Wbroblasts were harvested and branched chain fatty acid composition was quantiWed by GC– MS using deuterated phytanic acid as an internal standard, as described [13]. BrieXy, samples were subjected to acidic and alkaline hydrolysis, after which fatty acids were extracted with hexane. The organic layer was evaporated to dryness under nitrogen at 40 °C. The samples were derivatized with N-tert-butyldimethylsilyl-N-methyl-triXuoroacetamide (MTBSTFA, Aldrich, Steinheim, Germany) and pyridine (50 L each) at 80 °C for 30 min on an Agilent Technologies Model 5890/5973 GC–MS system equipped with a CPsil 19CB capillary column (25 m £ 0.25 mm I.D., Wlm thickness 0.25 mm, Varian, Palo Alto, CA), with electron impact ionization applied at 70 eV. MS acquisition was performed in the single ion monitoring mode, monitoring the [M-57]+ ions of the various compounds. Phytenic acid was synthesized as described, and isomers were
35
separated using high performance liquid chromatography (HPLC) [14]. Incubations of Wbroblast homogenates with phytol Fibroblasts were harvested, taken up in phosphatebuVered saline (PBS), and homogenized by sonication (2 cycles of 10 s at 9 W) on ice. The incubation mixture consisted of 40 g/mL protein, 50 mM glycine buVer (pH 9.2), 1 mM NAD+, 0.1% sodium cholate, and 1 mg/ml methyl--cyclodextrin (Fluka, Buchs, Switzerland) in a total volume of 500 L. Reactions were performed at 37 °C and initiated by the addition of 200 M phytol dissolved in dimethyl sulfoxide (DMSO). After 60 min, the incubation was terminated by the addition of 100 L of 1 N HCl. Then 2 mL of hexane was added, after which the organic layer was evaporated to dryness under nitrogen at 40 °C. The sample was then derivatized with MTBSTFA and analyzed as described in the previous section.
Results To study the degradation mechanism of phytol, human Wbroblasts were cultured for 4 days in the presence of 5, 25, and 50 M phytol. GC–MS analysis of the fatty acid composition of the harvested cells showed an
Fig. 2. Production of (A, D) pristanic acid, (B, E) phytanic acid, and (C, F) phytenic acid in Wbroblasts cultured in the presence of phytol. (A–C) Cells were cultured for 4 days in the presence of 0, 5, 25 or 50 M phytol. (D–F) Control cells and cells derived from SLS patients were cultured for 4 days in the presence of 25 M phytol. Branched chain fatty acids were measured as described under Materials and methods. Values represent means § standard deviation (SD) of duplicates expressed in nmol/mg protein. *P 0 0.05; **P 0 0.01 as calculated by Student’s t test, compared with control Wbroblasts incubated with 25 M phytol.
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acid was formed, even though cells had been incubated with a racemic mixture of Z- and E-phytol (Fig. 3). Since phytenal is an intermediate in the conversion of phytol to phytenic acid, an aldehyde dehydrogenase is required in the second step to form phytenic acid. A candidate enzyme for this reaction would be FALDH, encoded by the ALDH10 gene, which has been shown to be reactive with a closely related compound, dihydrophytal as a substrate [15]. Therefore, phytol incubations were performed using Wbroblast cell lines derived from SLS patients, deWcient in FALDH. As shown in Figs. 2F and 3B, these cell lines were deWcient in the production of phytenic acid from phytol. To further substantiate this observation, enzyme assays in Wbroblast homogenates were performed. Incubation of homogenates with phytol in the presence of NAD+ resulted in a protein and time dependent production of phytenic acid (Figs. 4A and B). Homogenates of FALDH-deWcient Wbroblasts were found to be deWcient in the production of phytenic acid (Fig. 4C).
Fig. 3. Separation of isomers of phytenic acid by GC–MS. (A) Analysis of standards for (Z)- and (E)-phytenic acid separately and in an racemic mixture and (B) phytenic acid formed by control Wbroblasts or Wbroblasts derived from a SLS-patient, cultured for 0 or 4 days in the presence of 25 M phytol.
increase in the levels of pristanic and phytanic acid, which was not observed in cells cultured without phytol (Figs. 2A and B). The amount of these metabolites directly correlated with the concentration of phytol present in the medium (Figs. 2A and B). In addition, a third peak was observed with a similar response to phytol as seen for pristanic and phytanic acid. Using an authentic standard as a reference, this peak was identiWed as phytenic acid (Fig. 2C). The Z- and E-isomers of phytenic acid have distinct retention times on the GC-column, which led to the observation that only E- and no Z-phytenic
Discussion Although a model for the degradation of phytol was proposed as early as the 1960s, very little research has been done to elucidate the precise reaction mechanism and to characterize the enzymes involved. In the present study, using Wbroblast incubations with phytol, it was shown conclusively that phytenic acid is a speciWc metabolite of phytol degradation. Furthermore, the Wnding that FALDH-deWcient Wbroblast lines are deWcient in phytenic acid production implies that this enzyme is part of the degradation pathway. Using a GC–MS method to analyze branched chain fatty acid composition in Wbroblast homogenates incubated with phytol, a deWciency in phytenic acid formation was found in FALDH-deWcient cells from SLS patients, which strongly suggests that phytenal is a speciWc substrate for
Fig. 4. Degradation of phytol to phytenic acid in Wbroblast homogenates. (A) Dependence of protein concentration and (B) time course of the phytol degradation, with reaction conditions as described in Materials and methods. (C) Fibroblast homogenates derived from SLS-patients are deWcient in phytenic acid production. Values in the bars represent means § SD of the activity expressed in nmol/min/mg of protein (n D 5 and 9 for control and SLS-patient derived cell lines, respectively). **P 0 0.01 as calculated by Student’s t test, compared with control Wbroblasts.
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FALDH. Due to the presence of some phytenic acid in the phytol used as substrate (Fig. 3B), it is diYcult to say whether this is a complete deWciency or whether some residual activity exists. Interestingly, only the production of E-phytenic acid was observed, even though incubations were carried out with a racemic mixture of Z- and E-isomers of phytol as a substrate. It remains to be established how this apparent stereospeciWcity of the reaction is achieved and whether FALDH or the preceding alcohol dehydrogenase is responsible. A deWciency of FALDH has been shown to be the cause of SLS, a metabolic disorder characterized by ichthyosis, mental retardation, and spastic diplegia or tetraplegia [11,16]. In tissues and plasma of SLS patients an accumulation of long chain fatty aldehydes and alcohols has been shown, which is thought to play a role in the onset of the symptoms [17]. Levels of phytol have never been investigated in these patients, but it is interesting to speculate on possible contribution of a phytol accumulation to the disease, since phytol has been shown to be toxic to cells [7]. Earlier reports have suggested that FALDH is part of the microsomal fatty alcohol:NAD+-oxidoreductase complex, also consisting of an alcohol dehydrogenase part [18]. This complex would therefore be a good candidate for the catalysis of the entire phytol to phytenic acid conversion. Although FALDH has been characterized in great detail [15], relatively little research has focused on the alcohol dehydrogenase domain. PuriWcation of this domain will make it clear which alcohol dehydrogenase is involved in the phytol degradation pathway.
Acknowledgments This work was supported by a grant from the Meelmeijer Fund and a grant from the European Commission (QLG3-2002-00696).
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