Báo cáo khoa học: Fatty acid omega-oxidation as a rescue pathway for fatty acid oxidation disorders in humans pot

Keywords adrenoleukodystrophy; cytochrome P450; fatty acids; mitochondria; peroxisomes; Refsum disease; Zellweger syndrome; a-oxidation; b-oxidation; x-oxidation Correspondence R.. The s

Fatty acid omega-oxidation as a rescue pathway for fatty acid oxidation disorders in humans

Ronald J A Wanders, Jasper Komen and Stephan Kemp

Academic Medical Center, University of Amsterdam, The Netherlands

Introduction

In general, fatty acids (FAs) can be degraded via

dif-ferent mechanisms, including a-, b- and x-oxidation

(Fig 1) In humans a-oxidation takes place in

peroxi-somes only, whereas both peroxiperoxi-somes and

mitochon-dria are able to b-oxidize FAs Importantly, in recent

years a great number of genetically determined

disor-ders in humans has been described in which either FA

a-oxidation or FA b-oxidation in mitochondria or

per-oxisomes is deficient

As discussed in more detail below, treatment options

for each of the different groups of FA oxidation

disor-ders is limited, which prompted us to investigate

x-oxi-dation as a rescue pathway for these disorders This is

based on the notion that if it was possible to upregulate the x-oxidation of specific FAs known to accumulate in the different disorders, one could reduce the accumula-tion of these FAs under in vivo condiaccumula-tions and thereby counteract the detrimental effects associated with the accumulation of these FAs and their derivatives, which are the basis of the clinical signs and symptoms observed in the different (groups of) disorders

We first briefly describe FA a- and b-oxidation path-ways and the disorders involved and then describe the current state of knowledge regarding x-oxidation as

a rescue pathway for Refsum disease and X-linked adrenoleukodystrophy (X-ALD)

Keywords

adrenoleukodystrophy; cytochrome P450;

fatty acids; mitochondria; peroxisomes;

Refsum disease; Zellweger syndrome;

a-oxidation; b-oxidation; x-oxidation

Correspondence

R J A Wanders, Genetic Metabolic

Diseases, Room F0-226, Academic Medical

Center, University of Amsterdam,

Meibergdreef 9, 1105 AZ Amsterdam,

The Netherlands

Fax: +31 (0)20 6962596

Tel: +31 (0)20 5665958 ⁄ 5664197

E-mail: r.j.wanders@amc.uva.nl

(Received 22 June 2010, revised 28

September 2010, accepted 3 November

2010)

doi:10.1111/j.1742-4658.2010.07947.x

Fatty acids (FAs) can be degraded via different mechanisms including a-, b- and x-oxidation In humans, a range of different genetic diseases has been identified in which either mitochondrial FA b-oxidation, peroxisomal

FA b-oxidation or FA a-oxidation is impaired Treatment options for most

of these disorders are limited This has prompted us to study FA x-oxida-tion as a rescue pathway for these disorders, based on the nox-oxida-tion that if the x-oxidation of specific FAs could be upregulated one could reduce the accumulation of these FAs and the subsequent detrimental effects in the different groups of disorders In this minireview, we describe our current state of knowledge in this area with special emphasis on Refsum disease and X-linked adrenoleukodystrophy

Abbreviations

ATRA, all-trans-retinoic acid; CCALD, childhood cerebral adrenoleukodystrophy; FA, fatty acid; LTB4, leukotriene B4; PPAR, peroxisome proliferator-activated receptor; VLCFA, very long-chain fatty acid; X-ALD, X-linked adrenoleukodystrophy.

General aspects of fatty acid oxidation

Beta-oxidation is the preferred way of oxidizing FAs

In principle, each FA can be b-oxidized, including

straight- and branched-chain FAs, as well as

mono-and polyunsaturated FAs There is one exception,

however, and that is if the carbon-3 has a methyl- or

any other functional group attached In such cases,

degradation can only occur by a- or x-oxidation

Until a few years ago, the enzymology of the

a-oxi-dation system remained unresolved and its subcellular

localization heavily disputed This has now changed;

the basic chemistry of the pathway has been delineated

and the enzymes involved in a-oxidation and their

sub-cellular localization have been identified and

character-ized, although some questions remain [1,2]

Mitochondrial fatty acid b-oxidation

and its disorders

Mitochondria catalyze the b-oxidation of the majority

of FAs and contain the full enzymatic machinery to

oxidize straight-chain, 2-methyl-branched-chain, and

mono- and polyunsaturated FAs After uptake of FAs

into the cells via a mechanism which remains

incom-pletely understood, but probably involves CD36 [3],

FAs are rapidly converted into coenzyme A

(CoA)-esters by one of the many acyl-CoA synthetases either

of the long-chain or very long-chain acyl-CoA

synthe-tase family [4] Subsequently, the acyl-CoA esters are

transferred across the mitochondrial membrane by

means of the carnitine cycle, which involves carnitine

palmitoyltransferase I, mitochondrial carnitine

acylcar-nitine translocase and caracylcar-nitine palmitoyltransferase II

[5–7] In case of the straight-chain and 2-methyl-branched

chain FAs, b-oxidation can start right away via the well-established cascade of four steps involving dehy-drogenation, hydratation, dehydrogenation again and thiolytic cleavage of the acyl-CoA esters Each step of the b-oxidation spiral is not catalyzed by one single enzyme but by multiple chain-length-specific enzymes For example, at least three different acyl-CoA dehy-drogenases are involved in the oxidation of saturated long-chain FAs These include very long-chain acyl-CoA dehydrogenase, medium-chain acyl-acyl-CoA dehy-drogenase and short-chain acyl-CoA dehydehy-drogenase The same is true for the third step in mitochondrial fatty acid b-oxidation, with at least two different enzymes involved including short-chain CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase The latter enzyme is part of a larger enzyme complex called mitochondrial trifunc-tional protein with additrifunc-tional enoyl-CoA hydratase and 3-ketothiolase activities Defects in each of these enzymes have been identified (see Table 1) Although the clinical signs and symptoms of patients vary depending on the type of enzyme defect and the extent

of the deficiency, a general characteristic of all disor-ders of mitochondrial FA oxidation is hypoketotic hypoglycemia which may be life threatening, and car-diomyopathy, especially in the case of the long-chain fatty oxidation defects such as mitochondrial carnitine acylcarnitine translocase deficiency, carnitine palmito-yltransferase II deficiency, very long-chain acyl-CoA dehydrogenase deficiency and long-chain 3-hydroxya-cyl-CoA dehydrogenase⁄ mitochondrial trifunctional protein deficiency [8,9] With the exception of dietary measures consisting of a diet rich in carbohydrates and low in fat taken at frequent intervals, there are virtu-ally no realistic treatment options

Fig 1 Schematic diagram depicting the

different mechanisms by which fatty acids

can be oxidized (see text).

Peroxisomal a-oxidation and its

disorders

FA a-oxidation allows the chain-shortening of FAs by

one carbon atom and takes place in peroxisomes only

A typical 3-methyl-branched-chain FA like phytanic

acid (3,7,11,15-tetramethylhexadecanoic acid), is

com-pletely dependent on a normal functioning a-oxidation

system in order to be oxidized A defect in the

a-oxida-tion system is reflected in the accumulaa-oxida-tion of phytanic

acid in the tissues and body fluids of patients [1,2,10]

Alpha-oxidation of phytanic acids starts with the

formation of the CoA-ester, i.e phytanoyl-CoA,

fol-lowed by hydroxylation to generate

2-hydroxyphy-tanoyl-CoA, a reaction catalyzed by the enzyme

phytanoyl-CoA 2-hydroxylase Subsequently,

oxyphytanoyl-CoA is cleaved by the enzyme

2-hydr-oxyacyl-CoA lyase to pristanal and formyl-CoA, which

is then hydrolyzed to formic acid and coenzyme A

(CoASH) Pristanal is then oxidized to pristanic acid

(2,6,10,14-tetramethylpentadecanoic acid), as catalyzed

by a yet undefined peroxisomal aldehyde

dehydroge-nase After activation to its CoA-ester, pristanoyl-CoA

undergoes three cycles of b-oxidation in peroxisomes,

after which the end-products are transported to

mito-chondria for full oxidation [11,12]

Alpha-oxidation is deficient in different

peroxi-somal disorders including the peroxisome biogenesis

disorders, in which the primary genetic defect is in one

of the many genes involved in peroxisome biogenesis [13–15] To date, however, only one single enzyme defi-ciency in the a-oxidation pathway per se has been described This is phytanoyl-CoA hydroxylase defi-ciency with Refsum disease as its disease counterpart [10] Patients suffering from Refsum disease show a late-onset phenotype, dominated by retinitis pigmen-tosa, culminating in blindness with anosmia, cerebellar ataxia and a range of other more variable abnormali-ties The only therapy available to date is a life-long diet low in phytanic acid, which may stop further pro-gression of some, but not all, of the symptoms provided the diet is meticulously maintained

Peroxisomal fatty acid b-oxidation and its disorders

Peroxisomes contain a FA b-oxidation system just like mitochondria, but the individual reactions of the b-oxi-dation spiral are catalyzed by different enzymes encoded by distinct genes compared with the mito-chondrial b-oxidation system [11] Importantly, peroxi-somes oxidize a unique set of FAs which cannot be b-oxidized in mitochondria Most important from a clinical point of view are: (a) very long-chain fatty acids (VLCFAs), notably C24:0 and C26:0; (b) pris-tanic acid (2,6,10,14-tetramethylpentadecanoic acid), as

Table 1 The mitochondrial and peroxisomal beta-oxidation deficiencies

Mitochondrial fatty acid oxidation disorders Mutant gene Deficient enzyme Locus OMIM

Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency HADHA LCHAD 2p23 600890

Peroxiomal fatty acid oxidation disorders

derived directly from dietary sources and indirectly

from phytanic acid upon a-oxidation; and (c) di- and

trihydroxycholestanoic acid (see [11] for review)

Per-oxisomes are unable to b-oxidize FAs to completion

Instead FAs are only chain-shortened to shorter chain

FAs followed by the transfer of these chain-shortened

FAs to mitochondria for full oxidation This has been

established most firmly for pristanic acid which

under-goes three cycles of b-oxidation in peroxisomes to

produce propionyl-CoA, acetyl-CoA and

4.8-dimethyl-nonanoyl-CoA followed by the transfer of these

CoA-esters either as carnitine-ester or as free fatty acid

to mitochondria for full oxidation to CO2 and H2O

[16,17]

At present, five different genetically determined

sin-gle-enzyme deficiencies have been described in humans

These include: (a) X-ALD, (b) acyl-CoA oxidase

defi-ciency, (c) D-bifunctional protein defidefi-ciency, (d) sterol

carrier protein x deficiency and (e) 2-methylacyl-CoA

racemase deficiency [18] All five disorders are

rela-tively rare with sterol carrier protein x deficiency

described in a single patient only to date [19],

2-meth-ylacyl-CoA racemase-deficiency described in six

patients [20] and acyl-CoA oxidase deficiency described

in 30 patients [21] Most frequent is X-ALD with an

incidence of 1 : 15 000, followed by D-bifunctional

protein deficiency [22] X-ALD is a devastating

neuro-logical disease which comes in two main phenotypes

including childhood cerebral ALD (CCALD) and

adrenomyeloneuropathy, together constituting > 80%

of all X-ALD patients The most devastating

pheno-type is CCALD which is characterized by a rapidly

progressive cerebral demyelination causing severe

dis-ability and death, usually within 2 years after the onset

of symptoms Adrenomyeloneuropathy has a much milder course characterized by a gradually progressive myelopathy and peripheral neuropathy, causing severe disability

X-ALD is caused by mutations in the ABCD1 gene which codes for a peroxisomal half-ABC transporter adrenoleukodystrophy protein (ALDP), localized in the peroxisomal membrane as a homodimer ALDP catalyzes the transport of very long-chain FAs across the peroxisomal membrane in the CoA-ester form [23,24] If ALDP is absent or dysfunctional, oxidation

of VLCFA is impaired and this leads to the accumula-tion of VLCFAs in plasma and tissues including the brain of X-ALD patients The VLCFAs that accumu-late are not so much derived from the diet, but are synthesized endogenously via chain elongation [24], which explains why a diet low in VLCFAs is of no benefit for X-ALD patients (Fig 2) The only thera-peutic options for X-ALD are bone-marrow transplan-tation and gene therapy, as recently reported by Cartier and Aubourg [25] in three X-ALD boys

Fatty acid x-oxidation by CYP450 proteins in humans

Early work on FA x-oxidation dates back to the 1930s when Verkade et al [26,27] performed a series of systematic studies that revealed the formation of dicar-boxylic acids after administration of medium-chain tri-glycerides to healthy individuals It was the 1960s before enzymatic studies could be performed using subcellular fractions prepared from guinea-pig, rat and human livers This allowed identification of the path-way intermediates and the subsequent discovery that

Fig 2 Schematic diagram illustrating the

homeostatic mechanisms involved in C26:0

metabolism Very long-chain fatty acids are

predominantly derived from long-chain fatty

acids via chain-elongation and degraded via

b-oxidation in the peroxisome Several

diseases are known in which b-oxidation

is deficient including X-linked

adrenoleukodystrophy Omega-oxidation of

C26:0 involves the participation of different

enzymes including CYP4F2 and CYP3FB

plus ALDH3A2 The latter converts the

x-keto form of C26:0 into the dicarboxylic

acid.

the enzyme catalyzing the first step of the pathway was

in fact a hemoprotein belonging to the ubiquitous

dis-covered family of CYP450s, with members in

eukary-otic and prokaryeukary-otic species [28–30]

The CYP4A subfamily

After the successful cloning of CYP4A1 coding for

clofibrate-inducible arachidonic acid⁄ lauric acid

x-hydroxylase from rat liver [31], the human homolog

of this enzyme was identified and named CYP4A11

[32,33] CYP4A11 turned out to have a broad

sub-strate spectrum and is able to x-hydroxylate the

satu-rated FAs lauric acid, myristic acid (tetradecanoic

acid), palmitic acid (hexadecanoic acid) and the

unsat-urated FAs oleic acid [(Z)-octadec-9-enoic acid] and

arachidonic acid (all-cis-5,8,11,14-eicosatetraenoic acid)

[34] Recently, another CYP4A subfamily member in

humans was identified and designated CYP4A22 [35]

This protein is highly homologous with CYP4A11

and not abundantly present in tissues Kawashima

et al have expressed the CYP4A22 protein in

Escheri-chia coli, and showed that this protein has lauric acid

x-hydroxylase activity [36], but erroneously reported it

to be CYP4A11

The CYP4F subfamily

CYP4A is not the only subfamily of CYP4 proteins in

humans that are capable of x-hydroxylation of fatty

acids During the early 1980s, Hansson et al reported

on the x-oxidation of leukotriene B4 (LTB4) in human

leukocytes [37] The x-oxidation pathway is necessary

for the degradation (and thereby inactivation) of this

compound, which plays an important role in the

inflam-mation process The CYP450 involved in this pathway

belonged to a, at that moment, unidentified subfamily,

the CYP4F subfamily (reviewed in [38,39]), and was

designated as CYP4F3 Later it was found that the

CYP4F3gene could give rise to two different transcripts

by alternative promoter usage and tissue-specific gene

splicing, which results in two different proteins [40,41]

The isoform originally detected in leukocytes was

desig-nated CYP4F3A and the other, which was detected in

liver, was designated CYP4F3B These proteins differ

from each other due to the alternative use of only one

exon However, this leads to a substantial difference in

substrate specificity, with CYP4F3A being specific for

LTB4, whereas CYP4F3B has a higher specificity

towards arachidonic acid [40,42]

Shortly after the cloning of CYP4F3A from human

leukocytes in 1993, Kikuta et al identified a novel

LTB4-hydroxylating CYP450 in human liver [43] This

isoform was named CYP4F2 and has a high homology with the CYP4F3B protein CYP4F2 was shown to be the major arachidonic acid x-hydroxylase in human liver and kidney with a higher substrate specificity for arachidonic acid than the already established arachi-donic acid x-hydroxylase CYP4A11 [44,45] The formation of x-hydroxylated arachidonic acid (20-hy-droxyeicosatetraenoic acid) plays an important role in the regulation of the cardiovascular system because it

is a known vasoconstrictor (reviewed in [46]) CYP4F2 was also shown to x-hydroxylate LTB4 in liver, which suggests that this protein might play a role in the inflammatory system [47] Furthermore, CYP4F2 is responsible for x-hydroxylation of the phytyl tail of the tocopherols and tocotrienols that are collectively called vitamin E [48] Omega-hydroxylation is the initial step for the degradation of vitamin E via x-oxi-dation and subsequent b-oxix-oxi-dation [48,49]

Three additional members of the CYP4F subfamily have been identified in humans, as reviewed by Kalso-tra and Strobel [38] These were recently discovered and have been only partially characterized CYP4F8

is present in epithelial linings and catalyzes the (x-1)-hydroxylation of prostaglandin H2 CYP4F11 is mainly expressed in liver, followed by kidney, heart, brain and skeletal muscle No endogenous substrates have been found to date for CYP4F11, but it has been shown that recombinant CYP4F11 is quite active in hydroxylating some xenobiotics Finally, the CYP4F12 protein detected in human liver, heart, gastrointestinal and urogenital epithelia is active towards both eicosa-noids and xenobiotics

Other CYP4 homologs

The CYP4B1 protein, which is predominantly expressed in lung, forms another subfamily of x-hy-droxylases However, this protein has no clear sub-strate spectrum, but it is capable of x-hydroxylating medium-chain FAs and xenobiotics (see [50] for review) Other CYP450s belonging to family 4 have been identified in humans Their homology with the known CYP4 subfamilies suggests that these orphans (i.e CYPs with unknown substrate specificity) might

be able to x-hydroxylate FAs and⁄ or FA-like com-pounds [51]

The most important and well-characterized x-hy-droxylases, the CYP450s belonging to the CYP4A and 4F subfamilies, are present not only in humans; CYP4A⁄ F homologs are also well represented in other animals, such as the mouse, rat and rabbit [38,52,53] Moreover, these animals contain more CYP4A and CYP4F subfamily members than humans, which

makes interpretation of results found in studies using

these animals problematic

Induction of fatty acid x-hydroxylases

CYP4A gene regulation and the role of

peroxisome proliferator-activated receptor a in

CYP4A induction

The induction of drug-metabolizing enzymes by

for-eign compounds has been the topic of many research

studies during the last several decades Halfway the

20th century it was already known that the

x-hydroxy-lase activity in microsomal fractions prepared from rat

livers was much higher when laboratory animals were

fed with certain kinds of xenobiotics, including: (a)

polycyclic aromatic hydrocarbons and (b) barbiturates,

as described in Conney [54] A third type of metabolic

enzyme inducers are the hypolipidemic drugs of the

fibrate class, which have been in use since the early

1960s, and were found to upregulate the

x-hydroxyl-ation of lauric acid [55,56] All of these compounds are

able to induce one or more CYP450s However, the

precise mechanism which is the basis of this induction

has remained unclear for several decades The

discov-ery that a receptor (the aryl or aromatic hydrocarbon

receptor) was involved in the induction by polycyclic

aromatic hydrocarbons of the CYP450s responsible for

the hydroxylation of polycyclic aromatic hydrocarbons

(CYP1 family) was the first step in unraveling the

com-plex mechanism of CYP450 regulation (reviewed in

[57]) Another breakthrough in CYP450 regulation was

the finding that peroxisome proliferator-activated

receptor alpha (PPARa) was involved in the induction

of CYP4A enzymes (reviewed by Johnson et al [58])

PPARa is a member of the large receptor superfamily

of ligand-activated transcription factors (also referred

to as the nuclear receptor family) [59] Moreover,

many members of this superfamily have been found to

be involved in the regulation of multiple CYP450s

[60,61]

PPARa is a member of the larger family of PPARs

which also consists of a b- (d) and c-isoform All

iso-forms play important roles in physiological processes

as lipid sensors and regulators of lipid and glucose

homeostasis However, the different PPARs have

spe-cific substrate spespe-cificities and tissue distributions, and

control specific subsets of transcriptional profiles (see

[62,63] for review) Activation of the PPARs by the

so-called peroxisome proliferators (a structurally

unre-lated class of compounds among which are FAs,

plast-icizers, herbicides and the fibrate class of

hypolipidemic drugs) enables the receptor to dimerize

with another nuclear receptor, the retinoid X receptor [64] The ligand-activated heterodimer can bind to spe-cific sequences of DNA known as peroxisome prolifer-ator responsive elements in the promoter regions of target genes, thereby inducing gene expression of the target gene Most of these target genes are involved in lipid metabolism Particularly pronounced is the induc-tion of proteins involved in peroxisomal fatty acid metabolism, which leads to an increase in peroxisomal number (i.e peroxisome proliferation) and size [65] Induction of hepatic peroxisome proliferation by PPARa activation in rodents ultimately leads to hepa-tomegaly and hepatocarcinogenesis (see Gonzalez [66] for review) Fortunately, these toxic effects of PPARa ligands are not observed in humans [67] Therefore, fibrates are still in use as important drugs for the treat-ment of patients with dyslipidemia and⁄ or metabolic syndrome (reviewed in [68,69])

Besides increasing peroxisomal FA oxidation, PPARa is also involved in the upregulation of mito-chondrial b-oxidation, FA transport and the already mentioned FA x-hydroxylation via the CYP4A sub-family Initial studies, which focused on the induction

of the CYP4A subfamily in rats and mice, showed that levels of certain subtypes did indeed increase in these rodent animal models after PPARa activation [70,71]

In humans, uncertainties remain with respect to the induction of the CYP4A subtype Overexpression of PPARa in the hepatoma cell line HepG2 led to an increase in CYP4A11⁄ A22 under specific growth con-ditions, suggesting the involvement of PPARa in the regulation of human CYP4A expression [72] Another study showed that fibrates are able to induce CYP4A11 mRNA expression in primary cultures of human hepatocytes [73] By contrast, the peroxisome proliferators responsive elements present in the promo-tor regions of the genes coding for members of the CYP4A subfamily in rodents have not been identified

in human CYP4A genes [36]

Recently, another regulatory pathway for CYP4A11 gene expression was discovered Activation of a differ-ent member of the nuclear hormone receptor family, retinoic acid receptor, by all-trans retinoic acid (ATRA) in the hepatoma cell line HepaRG was shown

to decrease CYP4A11 gene and protein expression, ultimately leading to a decrease in catalytic activity (lauric acid hydroxylation) in this cell line [74]

In mice, three different CYP4A genes have been identified Cyp4a10 is highly expressed in both sexes, whereas Cyp4a12 (consisting of two gene products, Cyp4a12a and Cyp4a12b) is predominantly male spe-cific and Cyp4a14 is a female-spespe-cific isoform Further-more, the protein levels of these Cyp4a isoforms vary

in different mouse strains and tissues PPARa also

plays an important role in the regulation of the

expres-sion of the different Cyp4a isoforms in mice Fibrates

are able to induce gene expression of Cyp4a10 and

Cyp4a14 [71,75] in both liver and kidney Cyp4a12 is

constitutively expressed in kidney and liver of male

mice, whereas in kidney and liver of female mice

Cyp4a12is expressed at low levels Moreover, Cyp4a12

gene expression cannot be induced by fibrates in

kid-ney and liver of male mice, whereas in female mice,

kidney and liver Cyp4a12 RNA levels were increased

to male levels after treatment with fibrates In

addi-tion, Cyp4a12 gene expression is also upregulated in

female mice by treatment with androgens via an as yet

unknown mechanism [75,76]

CYP4F induction

By contrast to the CYP4A subfamily, relatively few

studies have appeared on the regulation of genes

comprising the CYP4F subfamily (reviewed in

[37,38,77]) Regulation of the CYP4F2 gene has been

studied most intensively of all human isoforms

Zhang et al [78] found that CYP4F2 gene expression

was regulated by retinoic acid and fibrates

Peroxi-some proliferators suppressed CYP4F2 promotor

activity, whereas both 9-cis-retinoic acid and ATRA

induced promoter activity through activation of

reti-noic acid receptor and retinoid X receptor However,

further research revealed that protein expression of

CYP4F2 was increased by 9-cis-retinoic acid in the

hepatoma cell line HepG2, in marked contrast to

ATRA, which only gave rise to an induction of

CYP4F2 promotor activity [79] From these results,

Zhang and Hardwick concluded that CYP4F2 gene

expression is regulated by 9-cis-retinoic acid and

ATRA Activation of retinoid X receptor induces

gene expression (and protein content) and retinoic

acid receptor activation results in repression of gene

expression Recently, Hsu et al showed that in

HepG2 cells and primary hepatocytes, CYP4F2 gene

expression and protein content could be induced by

statins, which are well-known drugs used for the

treatment of hypercholesterolemia [80] Furthermore,

Hsu et al showed that the CYP4F2 transcriptional

activation is mediated by sterol regulatory element

binding proteins (SREBP; reviewed in [81]) and that

activation of the sterol regulatory element binding

protein-2 isoform is involved in the induction

CYP4F2 by statins [80]

Parallel studies on the induction of CYP4F3 showed

that this enzyme was induced in HL60 cells and

human leukocytes after treatment of these cells with

ATRA [82,83] However, the mechanism behind this induction remains to be determined since the receptor for ATRA, i.e retinoic acid receptor, seems only indi-rectly involved in this process

Studies in rats and mice have shown that the expres-sion of some isoforms of the CYP4 subfamily changes during inflammation During an inflammatory response, induction of CYP4F isoforms occurs in rodents needed for the breakdown of inflammatory mediators such as the eicosanoid LTB4 (reviewed in [38,77]) Recent studies by Kalsotra et al [84] provided evidence that specific cytokines are involved in regula-tion of the CYP4F enzymes levels during inflamma-tion The pro-inflammatory cytokines interleukin-1b, interleukin-6 and tumor necrosis factor-a are able to induce CYP4Fs, whereas the anti-inflammatory cyto-kine interleukin-10 suppresses CYP4F expression [84]

Peroxisomal fatty acid b-oxidation disorders including X-ALD and x-oxidation

Despite the profound increase in our knowledge about X-ALD, treatment options are very limited and are mostly symptomatic Lorenzo’s oil reduces plasma C26:0 but does not halt progression of the disease [85,86] Lovastatin also lowered plasma VLCFA [87], but a recent placebo-controlled trial revealed that lova-statin has no effect on C26:0 levels in peripheral blood lymphocytes and erythrocytes, or on the VLCFA con-tent of the low-density lipoprotein fraction [88] Hematopoietic stem cell transplantation can halt or reverse clinical deterioration [89] However, it is only effective in patients at the earliest stage of CCALD Recent breakthroughs in gene therapy have to date been applied to CCALD only [25] Therefore new therapeutic options aimed at the reduction of VLCFA are warranted

We have previously demonstrated that VLCFA can undergo x-oxidation [90] This would provide an alter-native mode of degradation We demonstrated that CYP4F2 and CYP4F3B are key enzymes in this pathway [91] In the first step of the metabolism of VLCFA via x-oxidation, VLCFAs are converted into x-hydroxy-VLCFA by CYP4F2 or CYP4F3B (Fig 1) Subsequently, this product is readily oxidized to a dicarboxylic-VLCFA by an alcohol and aldehyde dehydrogenase or via subsequent hydroxylation reactions by CYP4F2 and CYP4F3B [92] The dicarboxylyl-VLCFAs that are generated can be metabolized further in peroxisomes via b-oxidation Beta-oxidation of dicarboxylyl VLCFA takes place in peroxisomes and this process is not deficient in

X-ALD This is concluded from the finding that

b-oxidation of long-chain dicarboxylic acid is not

affected in fibroblasts from X-ALD patients, whereas

oxidation was deficient in fibroblasts from patients

with a peroxisomal biogenesis disorder [93] These

findings indicate that peroxisomes are essential for the

degradation of dicarboxylic acids, but that ALDP is

not required for the transport of dicarboxylic acids

across the peroxisomal membrane Because the

trans-port of dicarboxylic acids may involve other ABC

half-transporters, i.e ALDRP or PMP70, the

x-oxida-tion of VLCFA may funcx-oxida-tion as an escape route

Under normal physiological conditions, the

x-tion pathway accounts for 5–10% of total FA

oxida-tion Because expression levels of cytochrome P450

enzymes can be induced by a variety of drugs and

chemicals [94], stimulation of VLCFA x-oxidation

may reduce or normalize VLCFA levels and might

therefore be beneficial for X-ALD patients (Fig 1)

The possibility of upregulating VLCFA x-oxidation

and its consequences for VLCFA homeostasis are now

being studied in a mouse model of X-ALD

Mitochondrial b-oxidation and

x-oxidation

In the case of mitochondrial FA b-oxidation disorders,

there is accumulation of certain FAs either as free

FAs, or in an esterified form as in CoA and carnitine esters The types of FAs and FA derivates that accu-mulate are determined by the site of the enzyme defect The different acylcarnitine profiles observed in the var-ious mitochondrial b-oxidation deficiencies emphasize this notion [95] Specific induction of the capacity to x-oxidize these FAs would reduce the FA burden and may ameliorate the signs and symptoms in these patients No studies on this point have been published

in the literature

Refsum disease, phytanic acid and x-oxidation

Brenton and Krywawych [96] reported on the excretion

of 3-methyladipic acid in the urine of Refsum patients, which suggested that phytanic acid does undergo x-oxi-dation under in vivo conditions This was soon followed

by another report, which documented the identification

of 2,6-dimethyloctanedioic acid, a metabolite derived from x-oxidation of phytanic acid in Refsum’s patients The finding by Wierzbicki et al [97] that the amounts of 3-methyladipic acid in urine from Refsum’s patients correlated with plasma levels of phytanic acid in these patients, has lent further support to the notion that 3-methyladipic acid is indeed formed upon x-oxidation

of phytanic acid Based on these results, we have begun

to characterize the enzymology of the x-oxidation

Fig 3 Schematic diagram depicting phytanic acid homeostasis Phytanic acid is derived solely from dietary sources and can be oxidized by either a-oxidation or x-oxidation (see text for further details) The product of the peroxisomal a-oxidation of phytanic acid is pristanic acid which first undergoes three cycles of b-oxidation in the peroxisome to produce propionyl-CoA (in the first and third cycle of b-oxidation) and acetyl-CoA (in the second cycle of b-oxidation) plus 4,8-dimethylnonanoyl-CoA These CoA-esters are all transferred to mitochondria for further oxidation.

pathway, first in rat liver microsomes [98] and then in

human liver microsomes [99,100] In rat liver

micro-somes we found that phytanic acid undergoes both (x-)

and (x-1)-hydroxylation [98] In human microsomes,

however, there was a virtually exclusive production of

x-hydroxy phytanic acid [99] In order to identify the

CYP450 involved we first performed studies with

selec-tive inhibitors including 17-octadecynoic acid,

diet-hyldithiocarbamate, ketoconazole, troleandomycin,

omeprazole, trimethoprim, furafylline, quinidine and

sulfaphenozole [100] These studies already pointed to

the CYP4 family of x-hydroxylases as likely candidates

The availability of individually expressed CYP4s

pro-duced in baculovirus-infected insect cells (Supersomes)

allowed this possibility to be tested directly CYP4F3A

turned out to be most reactive towards phytanic acid,

followed by CYP4F3B, CYP4F2 and CYP4A11 with

catalytic efficiencies of 0.87, 0.22, 0.06 and 0.02,

respec-tively [100] The question now is whether upregulation

of one or more of these CYP450s is feasible under in vivo

conditions and if this is associated with an increased rate

of phytanic acid x-oxidation or not [12] (see Fig 3)

With respect to CYP4F3A and CYP4F3B, there is no

information about whether expression can be

upregulat-ed CYP4A11 expression is controlled by PPARa in

conjunction with retinoid X receptor so that fibrates or

other PPAR ligands should be successful in upregulating

CYP4A11 activity Finally, with respect to CYP4F2, it

has been established experimentally that the promoter of

the CYP4F2 gene contains a sterol-regulatory element,

as described above Activation of the classical sterol

regulatory element binding protein (SREBP) route, for

example by means of statins, inhibitors of

3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, would

then lead to the increased expression of CYP4F2 The

availability of a mouse model for Refsum disease allows

for future studies aimed at resolving whether

upregu-lation of CYP4A11 by fibrates and⁄ or CYP4F2 by

statins leads to the enhanced degradation of phytanic

acid and amelioration of the clinical signs and symptoms

[101]

Conclusions

Omega-oxidation as a rescue pathway for different

genetic diseases in humans in which either peroxisomal

or mitochondrial FA oxidation is impaired, is an

attractive possibility to allow breakdown of FAs which

accumulate as a consequence of an enzyme or

trans-porter defect Identification of the specific cytochrome

P450s involved in the x-oxidation of phytanic acid and

VLCFAs, added to the fact that the different CYPs

involved can be induced pharmacologically, now

allows us to study whether our in vitro data can be extrapolated successfully to intact organisms We will first perform such studies in mouse models for Refsum disease and X-ALD

Acknowledgements

This work was supported by grants from the European Leukodystrophy Association [ELA 2008-05111A (RJW)], the Prinses Beatrix Fonds [WAR08-20 (SK)] and the Netherlands Organization for Scientific Research [VIDI-grant No 91786328 (SK)] Mrs Maddy Festen is gratefully acknowledged for preparation of the manu-script and Mr Jos Ruiter for design of the figures

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