Metabolism of phytol to phytanic acid in the mouse, and the role of PPARα in its regulation

Phytol, a branched-chain fatty alcohol, is the naturally occurring precursor of phytanic and pristanic acid, branched-chain fatty acids that are both ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα). To investigate the metabolism of phytol and the role of PPARα in its regulation, wild-type and PPARα knockout (PPARα−/−) mice were fed a phytol-enriched diet or, for comparison, a diet enriched with Wy-14,643, a synthetic PPARα agonist. After the phytol-enriched diet, phytol could only be detected in small intestine, the site of uptake, and liver. Upon longer duration of the diet, the level of the (E)-isomer of phytol increased significantly in the liver of PPARα−/− mice compared with wild-type mice. Activity measurements of the enzymes involved in phytol metabolism showed that treatment with a PPARα agonist resulted in a PPARα-dependent induction of at least two steps of the phytol degradation pathway in liver. Furthermore, the enzymes involved showed a higher activity toward the (E)-isomer than the (Z)-isomer of their respective substrates, indicating a stereospecificity toward the metabolism of (E)-phytol. In conclusion, the results described here show that the conversion of phytol to phytanic acid is regulated via PPARα and is specific for the breakdown of (E)-phytol. Phytol, a branched-chain fatty alcohol, is the naturally occurring precursor of phytanic and pristanic acid, branched-chain fatty acids that are both ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα). To investigate the metabolism of phytol and the role of PPARα in its regulation, wild-type and PPARα knockout (PPARα−/−) mice were fed a phytol-enriched diet or, for comparison, a diet enriched with Wy-14,643, a synthetic PPARα agonist. After the phytol-enriched diet, phytol could only be detected in small intestine, the site of uptake, and liver. Upon longer duration of the diet, the level of the (E)-isomer of phytol increased significantly in the liver of PPARα−/− mice compared with wild-type mice. Activity measurements of the enzymes involved in phytol metabolism showed that treatment with a PPARα agonist resulted in a PPARα-dependent induction of at least two steps of the phytol degradation pathway in liver. Furthermore, the enzymes involved showed a higher activity toward the (E)-isomer than the (Z)-isomer of their respective substrates, indicating a stereospecificity toward the metabolism of (E)-phytol. In conclusion, the results described here show that the conversion of phytol to phytanic acid is regulated via PPARα and is specific for the breakdown of (E)-phytol. Phytol is a branched-chain fatty alcohol (3,7,11,15-tetramethylhexadec-2-en-1-ol) that is abundantly present in nature as part of the chlorophyll molecule. The release of phytol from chlorophyll occurs effectively in the digestive system of ruminant animals only, presumably by bacteria present in the gut (1Mackey R.H. Kuller L.H. Sutton-Tyrrell K. Evans R.W. Holubkov R. Matthews K.A. Lipoprotein subclasses and coronary artery calcium in postmenopausal women from the Healthy Women Study..Am. J. Cardiol. 2002; 90: 71i-76iAbstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). As a result, a relatively high amount of free phytol is present in dairy products (2Koba S. Hirano T. Kondo T. Shibata M. Suzuki H. Murakami M. Geshi E. Katagiri T. Significance of small dense low-density lipoproteins and other risk factors in patients with various types of coronary heart disease..Am. Heart J. 2002; 144: 1026-1035Crossref PubMed Scopus (62) Google Scholar). In mammals, free phytol is readily absorbed in the small intestine and is metabolized to phytanic acid, a fatty acid that accumulates in a number of metabolic disorders. Increased levels of phytanic acid in the body are toxic, so this fatty acid needs to be broken down (3Bittner V. Lipoprotein abnormalities related to women's health..Am. J. Cardiol. 2002; 90: 77i-84iAbstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 4de Graaf J. Hak-Lemmers H.L. Hectors M.P. Demacker P.N. Hendriks J.C. Stalenhoef A.F. Enhanced susceptibility to in vitro oxidation of the dense low density lipoprotein subfraction in healthy subjects..Arterioscler. Thromb. 1991; 11: 298-306Crossref PubMed Google Scholar, 5Dejager S. Bruckert E. Chapman M.J. Dense low density lipoprotein subspecies with diminished oxidative resistance predominate in combined hyperlipidemia..J. Lipid Res. 1993; 34: 295-308Abstract Full Text PDF PubMed Google Scholar, 6Bjornheden T. Babyi A. Bondjers G. Wiklund O. 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The Strong Heart Study..Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2713-2720Crossref PubMed Google Scholar). Because the methyl-group at the 3 position prevents β-oxidation, phytanic acid first has to undergo a round of α-oxidation. This results in the formation of pristanic acid, which is one carbon atom shorter than phytanic acid and can be normally β-oxidized (10Mykkanen L. Kuusisto J. Haffner S.M. Laakso M. Austin M.A. LDL size and risk of coronary heart disease in elderly men and women..Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2742-2748Crossref PubMed Scopus (94) Google Scholar). A deficiency in α-oxidation, such as in Refsum disease, leads to increased levels of phytanic acid in plasma and tissues of patients, and this is thought to cause the main clinical symptoms of this disorder: retinitis pigmentosa, peripheral neuropathy, and cerebellar ataxia (3Bittner V. Lipoprotein abnormalities related to women's health..Am. J. Cardiol. 2002; 90: 77i-84iAbstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 4de Graaf J. Hak-Lemmers H.L. Hectors M.P. Demacker P.N. Hendriks J.C. Stalenhoef A.F. Enhanced susceptibility to in vitro oxidation of the dense low density lipoprotein subfraction in healthy subjects..Arterioscler. Thromb. 1991; 11: 298-306Crossref PubMed Google Scholar). Because the breakdown of phytol will contribute to the phytanic and pristanic acid levels in these patients, it is important to study its metabolism and regulation. In many animal studies, phytol is used as a precursor of phytanic acid. Addition of phytol to the diet results in an increase of phytol metabolites in tissues and plasma (6Bjornheden T. Babyi A. Bondjers G. Wiklund O. Accumulation of lipoprotein fractions and subfractions in the arterial wall, determined in an in vitro perfusion system..Atherosclerosis. 1996; 123: 43-56Abstract Full Text PDF PubMed Scopus (204) Google Scholar, 11Otvos J.D. Jeyarajah E.J. Cromwell W.C. Measurement issues related to lipoprotein heterogeneity..Am. J. Cardiol. 2002; 90: 22i-29iAbstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 12Blake G.J. Otvos J.D. Rifai N. Ridker P.M. Low-density lipoprotein particle concentration and size as determined by nuclear magnetic resonance spectroscopy as predictors of cardiovascular disease in women..Circulation. 2002; 106: 1930-1937Crossref PubMed Scopus (324) Google Scholar, 13Avogaro P. Bon G.B. Cazzolato G. Presence of a modified low density lipoprotein in humans..Arteriosclerosis. 1988; 8: 79-87Crossref PubMed Google Scholar). This has been used as a model to study the effects of the accumulation of phytol metabolites on fatty acid metabolism, in particular via the activation of the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα), which is an important transcription factor in the regulation of fatty acid metabolism. Both phytanic and pristanic acid have been shown to activate PPARα in vitro (14Cazzolato G. Avogaro P. Bittolo-Bon G. Characterization of a more electronegatively charged LDL subfraction by ion exchange HPLC..Free Radic. Biol. Med. 1991; 11: 247-253Crossref PubMed Scopus (101) Google Scholar, 15Chen C.H. Jiang T. Yang J.H. Jiang W. Lu J. Marathe G.K. Pownall H.J. Ballantyne C.M. McIntyre T.M. Henry P.D. et al.Low-density lipoprotein in hypercholesterolemic human plasma induces vascular endothelial cell apoptosis by inhibiting fibroblast growth factor 2 transcription..Circulation. 2003; 107: 2102-2108Crossref PubMed Scopus (135) Google Scholar), and more recently, PPARα was also shown to be activated in vivo in mice fed a phytol-enriched diet (13Avogaro P. Bon G.B. Cazzolato G. Presence of a modified low density lipoprotein in humans..Arteriosclerosis. 1988; 8: 79-87Crossref PubMed Google Scholar). Figure 1 shows a schematic representation of the metabolism of phytol to phytanic acid and the enzymes involved. First, phytol is converted into phytenal via a yet unknown alcohol dehydrogenase, and subsequently, phytenal is converted into phytenic acid by the microsomal enzyme fatty aldehyde dehydrogenase (FALDH), which is encoded by the Aldehyde Dehydrogenase 3A2 gene (16Sanchez-Quesada J.L. Camacho M. Anton R. Benitez S. Vila L. Ordonez-Llanos J. Electronegative LDL of FH subjects: chemical characterization and induction of chemokine release from human endothelial cells..Atherosclerosis. 2003; 166: 261-270Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Recently, it was shown that phytenic acid is activated to its corresponding CoA ester, either at the endoplasmic reticulum or at the peroxisome, which is then reduced by a reductase converting phytenoyl-CoA to phytanoyl-CoA either in the peroxisome or at the mitochondrion (17Yang C.Y. Raya J.L. Chen H.H. Chen C.H. Abe Y. Pownall H.J. Taylor A.A. Smith C.V. Isolation, characterization, and of modified subfractions of low-density Thromb. Vasc. Biol. 2003; PubMed Scopus Google Scholar). In this the metabolism of phytol to phytanic acid in the and the role of PPARα in its regulation. To this phytol were determined and the of the enzymes involved in phytol metabolism were in tissues of wild-type and (PPARα−/−) mice fed a phytol-enriched To investigate the role of the of the enzymes involved in phytol metabolism were in tissues of wild-type and PPARα−/− mice fed a diet enriched with Wy-14,643, a synthetic PPARα agonist. or enriched with phytol of and Wy-14,643, or was from The Phytol was from was from and was from acid was from H. J. the was A of and acid was as described E. C. S. Cazzolato G. M. Bon G. Electronegative low density lipoprotein is increased in 2 patients and is with LDL susceptibility to PubMed Scopus Google Scholar). were from a of and acid as described S. P.M. Chapman M.J. and particle in human LDL for LDL receptor PubMed Scopus Google Scholar). was from the for and the were from was from were from J. T. and were from The was from The and were from and were from with were from a of were from was from and was from The other were of wild-type and PPARα−/− mice on a were used to study the effects of phytol and treatment A. J. H. Cazzolato G. Avogaro P. Bittolo-Bon G. of an in vivo LDL to LDL oxidation and its with dense LDL Thromb. Vasc. Biol. 1996; PubMed Scopus Google Scholar), and wild-type mice on a were used to study the effects of wild-type and PPARα−/− mice were fed or phytol for 1 or of wild-type and PPARα−/− mice were fed or for 2 of wild-type mice were fed or for of the of the mice were and tissues were The animals were at the of and free to and that were in and at animal were by the of was from its precursor phytol as described E. C. S. Cazzolato G. M. Bon G. Electronegative low density lipoprotein is increased in 2 patients and is with LDL susceptibility to PubMed Scopus Google Scholar). phytol of and was to the corresponding with in a of to and The was in of by of of by of in small magnetic After of the 2 of phytol in of was The was for 1 the was into a and with for of the for the of for and After the was and a of The was then a and with which was a of was in at of the showed two corresponding to the and of phytenal with a ion at and a specific for phytol levels were determined as described (13Avogaro P. Bon G.B. Cazzolato G. Presence of a modified low density lipoprotein in humans..Arteriosclerosis. 1988; 8: 79-87Crossref PubMed Google Scholar). tissues were in of a in was to of and the was to by 2 of 1 in for at After to the was by of phytol was with 2 of and the was to at were in of and on a as The was to at and with and at for were to at in of and to as described J.L. Benitez S. C. M. Ordonez-Llanos J. of LDL in and Lipid Res. 2002; Full Text Full Text PDF PubMed Google Scholar). The metabolites were a of enzyme activity measurements were in activity measurements in liver were as described J.L. Benitez S. C. M. Ordonez-Llanos J. of LDL in and Lipid Res. 2002; Full Text Full Text PDF PubMed Google Scholar). activity was with both phytol and phytenal as The of liver and for phytol and phytenal 1 and 1 in a of were at and by the of in of and for phytol and After the were by the of of 2 As of acid, in was 2 of was and the were which the was to a of at After with and at for the were to at in of and to as described J.L. Benitez S. C. M. Ordonez-Llanos J. of LDL in and Lipid Res. 2002; Full Text Full Text PDF PubMed Google Scholar). activation of phytenic acid to its CoA was as described (17Yang C.Y. Raya J.L. Chen H.H. Chen C.H. Abe Y. Pownall H.J. Taylor A.A. Smith C.V. Isolation, characterization, and of modified subfractions of low-density Thromb. Vasc. Biol. 2003; PubMed Scopus Google Scholar). of for liver of for and heart and of for small intestine and was with a 1 and phytenic acid of and in a of The was to for the activity was in liver and for in other were by the of of and on were for at at and were by of phytenoyl-CoA to phytanoyl-CoA was determined as described (17Yang C.Y. Raya J.L. Chen H.H. Chen C.H. Abe Y. Pownall H.J. Taylor A.A. Smith C.V. Isolation, characterization, and of modified subfractions of low-density Thromb. Vasc. Biol. 2003; PubMed Scopus Google Scholar). the 1 phytenoyl-CoA of and and for for liver and for small intestine, and in a of were by the of and at for for liver and for for other were by the of of and on were for at at and were by was from liver and which was a for of and in liver and was the The were used for for were used as described ion of a system for density of PubMed Scopus Google Scholar). was to the of a of a of the size was also by were for were as described by et C.Y. Yang Yang M. Smith C.V. of by of low density lipoproteins to in Radic. Biol. Med. 1997; PubMed Scopus Google Scholar). To for in the amount of the for were the for the gene A was an corresponding to acids of human as a with The for this was from human liver by the a and a are The was into the and was to by The was into the to the and of the The E. was with the were at in of with and to an density at of and was to a concentration of 1 to of the were by on for at an of with an of 1 The was on to the of the was to from the and used to To this a was with of the with an of After 1 the was by of in a was of were in a of of was on a and to a L. P. S. M. Chapman M.J. is a of the oxidative susceptibility of dense Thromb. Vasc. Biol. 1999; 19: PubMed Scopus Google Scholar). After of with and in 1 the was for 2 with in in to were used for are as was an The results were at Phytol levels were in tissues to investigate the of phytol in of the a phytol-enriched As phytol could be detected in of the tissues of mice a In the liver and small intestine of mice fed a phytol-enriched diet, was an accumulation of phytol could be detected in and In 2 the of and detected in liver and small intestine of wild-type and PPARα−/− mice are The phytol levels in small intestine to the that the animals were fed this high the amount of was higher in PPARα−/− mice than in wild-type animals In a determined the phytol levels in liver and showed that of diet the accumulation of phytol in PPARα−/− mice was more than in wild-type animals (13Avogaro P. Bon G.B. Cazzolato G. Presence of a modified low density lipoprotein in humans..Arteriosclerosis. 1988; 8: 79-87Crossref PubMed Google Scholar). In this determined the levels of both and in liver. After 1 on a phytol-enriched diet, were in phytol levels wild-type and PPARα−/− mice After on the phytol-enriched diet, the amount of in to the was increased significantly in PPARα−/− mice compared with wild-type In wild-type the phytol level increase with it increase in PPARα−/− mice. Because only into acid, the accumulation of in liver of PPARα−/− mice in with levels of phytanic acid in PPARα−/− mice compared with wild-type animals (13Avogaro P. Bon G.B. Cazzolato G. Presence of a modified low density lipoprotein in humans..Arteriosclerosis. 1988; 8: 79-87Crossref PubMed Google a of the phytol degradation pathway in PPARα−/− mice. To investigate the of the enzymes of this pathway in liver 3 First, the conversion of phytol and phytenal into acid. phytol is used as a the first and steps of the pathway are by alcohol dehydrogenase and phytenal as a only the by is The specific activity of acid formation with both was higher in wild-type mice than in PPARα−/− mice. the of the phytol-enriched diet, both significantly increased acid formation in wild-type mice Because phytol metabolites are ligands for this that PPARα a role in the regulation of and also be involved in the regulation of the of the alcohol Because the induction of the activity with phytol and phytenal as was the it is that the by is the a small induction of acid formation also was with both in PPARα−/− this was compared with wild-type indicating that are both PPARα-dependent and of showed a induction of in wild-type mice fed a phytol diet compared with the diet, induction was in PPARα−/− mice To that is the of was in wild-type and PPARα−/− mice with the PPARα Figure shows that induction of in wild-type in PPARα−/− fed a diet enriched with of liver levels showed a induction of treatment in wild-type mice. The induction of is in with the of of Wy-14,643, which PPARα to the transcription of its the activation of acid to its CoA was significantly increased in liver from and wild-type animals compared with animals on a diet The specific activity for the formation of was than for 1 and it was higher in and wild-type this was The increase in formation was in PPARα−/− mice on a diet enriched with phytol or Wy-14,643, indicating that also the induction of phytenoyl-CoA is for the activation of and acid to their corresponding CoA and for the of phytenoyl-CoA in tissues of wild-type mice on a in a the specific activity of phytenoyl-CoA the of the was significantly increased in wild-type mice on a phytol-enriched diet, induction was in PPARα−/− mice. This in to phytenoyl-CoA phytenoyl-CoA reductase is regulated in a PPARα-dependent induction of phytenoyl-CoA reductase activity was a The in phytenoyl-CoA reductase regulation the phytol diet and treatment could be to activation of PPARα or to the that phytenoyl-CoA reductase is via PPARα activation is in animals as a of metabolites from the phytol breakdown To investigate the of synthetic PPARα on phytenoyl-CoA reductase activity was treatment in an increase of phytenoyl-CoA reductase activity in wild-type mice. The specific activity in liver from wild-type mice was and the specific activity treatment was results show that phytenoyl-CoA reductase is via PPARα activation that metabolites of the phytol breakdown which is PPARα cause an increase in phytenoyl-CoA reductase the of the reductase the specific activity for the of was to be higher than that for the (Z)-isomer To more into the site of phytol metabolism in the the of the enzymes involved in this pathway in The of was described M. S. H. K. H. C. Pownall H.J. Chen H. M. Yang C. of in endothelial by low density lipoproteins subfractions from patients with Full Text Full Text PDF PubMed Scopus Google Scholar). and activity were in by small intestine, and and activity were also present in and of in and is in In heart and levels are induction of was the In was significantly in wild-type mice the diet, indicating regulation via of levels in showed an induction of in wild-type mice treatment in PPARα−/− which is in with the In both and are In tissues the specific activity for the formation of was higher than that for the The activity of was in liver. In and the specific activity was of the for liver. In small intestine and low specific of could be activity was In in and heart of mice in the are After treatment of wild-type mice with Wy-14,643, a increase of activity was in A increase of activity also was in heart of wild-type mice treatment with Wy-14,643, this was The increase in of wild-type animals fed a phytol diet was also In other in activity was As for phytenoyl-CoA the specific activity of phytenoyl-CoA reductase was in liver. In the specific activity was of the for liver. In other tissues of phytenoyl-CoA reductase were In the phytenoyl-CoA reductase activity was by phytol or by results show that PPARα an important role in the regulation of the enzymes involved in phytol In from PPARα−/− phytol was to to higher levels compared with from wild-type animals longer duration of the phytol-enriched diet, a for phytol metabolism in PPARα−/− mice compared with wild-type showed that the regulation of at least two of the enzymes of this breakdown pathway and phytenoyl-CoA is their increase phytol or in from wild-type mice from PPARα−/− for was it that can be by the of peroxisome has been yet W. Pownall H.J. of human low-density lipoproteins to 2003; PubMed Scopus (28) Google Scholar). Both and are absorbed in the small intestine of the as shown by the of both Upon longer duration of the phytol-enriched diet, increase in the accumulation of phytol was in small In the accumulation of phytol in the liver increased with which was to an increase in The accumulation of the (E)-isomer of phytol that the phytol degradation pathway is which the in the breakdown of acid. The and steps of the phytol degradation by phytenoyl-CoA and phytenoyl-CoA also show a for as The reductase by and of low density lipoprotein particle with 15: PubMed Scopus Google was as a for the of phytenoyl-CoA to phytanoyl-CoA (17Yang C.Y. Raya J.L. Chen H.H. Chen C.H. Abe Y. Pownall H.J. Taylor A.A. Smith C.V. Isolation, characterization, and of modified subfractions of low-density Thromb. Vasc. Biol. 2003; PubMed Scopus Google Scholar). This reductase has been shown to be toward the of (17Yang C.Y. Raya J.L. Chen H.H. Chen C.H. Abe Y. Pownall H.J. Taylor A.A. Smith C.V. Isolation, characterization, and of modified subfractions of low-density Thromb. Vasc. Biol. 2003; PubMed Scopus Google Scholar, of low density lipoprotein particle with 15: PubMed Scopus Google and has been shown to to G. Y. G. J. M.J. of small and dense LDL concentration in and men and 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). to be this enzyme is the reductase the of which the reductase by and of low density lipoprotein particle with 15: PubMed Scopus Google Scholar), are also to In conclusion, results show that the phytol degradation pathway is for the breakdown of to phytanic acid. of the enzymes involved in the breakdown of phytol to phytanic acid high activity in liver. The first by the alcohol dehydrogenase, be the of this is least in this enzyme be phytol was converted into acid in liver The enzyme activity measurements of the other enzymes involved in the breakdown of with the of phytol levels in tissues of the that the main site of phytol degradation is the liver. Phytol could be detected in liver and small intestine only the phytol-enriched the phytol levels in small intestine are a of the phytol that has been absorbed from the diet As a increase in small phytol levels was with in to the phytol which increase with In the results described here show that the degradation of phytol to phytanic acid is regulated via Furthermore, phytol accumulates in and the enzymes involved in phytol degradation their in indicating that the liver is the main site of phytol metabolism in the the pathway shows for the breakdown of this accumulates in liver of PPARα−/− mice on a phytol-enriched diet and at least of the enzymes of the pathway have been shown to be toward this The R. for and J. for This was by from the for The and by from the fatty aldehyde dehydrogenase peroxisome proliferator-activated receptor α