Peroxisomal Fatty Acid Oxidation

Peroxisomal β-oxidation enzymes are distinct from their mitochondrial counterparts. The biochemical steps are similar, involving several distinct enzymes including an oxidase, multifunctional enzyme (displaying enoyl-CoA hydratase and 3-hydroxy acyl-CoA dehydrogenase activity), and 3-oxo acyl-CoA thiolase. It recently has become clear that several distinct oxidases, multifunctional proteins, and enzymes catalyzing the thiolytic cleavage exist. The two pathways, l– and d-specific, are depicted in Figure 38-1.

Oxidation of Unsaturated Fatty Acids

Degradation of monounsaturated and polyunsaturated fatty acids has been demonstrated to occur in peroxisomes, in addition to chain shortening of long-chain dicarboxylic acids [Ferdinandusse et al., 2001; Hiltunen et al., 1986; Kolvraa and Gregersen, 1983; Schulz and Kunau, 1987]. An example of a polyunsaturated fatty acid that requires peroxisomal oxidation is docosahexaenoic acid, which has an important role in retina and brain development, and is known to be deficient in certain peroxisomal disorders [Moser et al., 1999; Watkins et al., 2001].

Branched-Chain Fatty Acid Oxidation and Phytanic Acid α-Oxidation

Branched-chain fatty acids are channeled into the d-specific pathway. The relevant enzymes include branched-chain acyl-CoA oxidase, MFE2, and sterol carrier protein, which functions as thiolase (see Figure 38-1). Phytanic acid has a β-methyl group that blocks β-oxidation and therefore must be α-oxidized first. After activation to its CoA derivative, phytanic acid is α-hydroxylated by phytanoyl CoA α-hydroxylase, the enzyme defective in classic Refsum’s disease [Jansen et al., 1997; Mihalik et al., 1997]. The α-hydroxy phytanoyl CoA is then degraded by a lyase to pristanic acid and formate [Croes et al., 1997]. The pristanic acid then can enter β-oxidation through the branched-chain pathway.

Bile Acid Synthesis

Cholesterol is converted in the liver to cholic acid and chenodeoxycholic acid by a series of enzymatic steps localized to several subcellular compartments. Pedersen and Gustafsson [1980] first demonstrated that the peroxisomal fraction of rat liver catalyzes the conversion of an intermediate product of this pathway, tri- or tetrahydroxy-5-β-cholestanoic acid (trihydroxycholestanoic acid), to cholic acid. Subsequently, Kase et al. [1986] reported that the conversion of trihydroxycholestanoic acid to cholic acid, and of dihydrocholestanoic acid to chenodeoxycholic acid also took place in peroxisomes and was deficient in patients with Zellweger’s syndrome. Shortening of the cholesterol side chain by β-oxidation resulting in the formation of bile acids is analogous to fatty acid oxidation and uses the enzymes of the d-specific peroxisomal β-oxidation pathway. The oxidase for trihydroxycholestanoic acid–CoA is separate from that of fatty acids and in humans also is believed to function as a branched-chain fatty acid oxidase (see Figure 38-1). A cytochrome P-450 catalyzing the hydroxylation of the C26/27 carbon of cholesterol in bile acid synthesis and formation of taurine conjugates of bile acid also has been localized to peroxisomes [Gutierrez et al., 1988; Kase and Bjorkheim, 1989]. In peroxisomal disorders, an increase in plasma and urine bile acid intermediates can be used as a diagnostic criterion.

Plasmalogen Synthesis

Plasmalogens are ether phospholipids and constitute 5–20 percent of phospholipids in mammalian cell membranes [Snyder, 1972]. They are abundant in myelin, in which they constitute one-third of the myelin phospholipids [Norton and Autilio, 1966]. Although the role of ether phospholipids is not fully elucidated, they appear to have important roles as antioxidants in membrane dynamics, storage of polyunsaturated fatty acids, and signal transduction [Brites et al., 2004].

Plasmalogen synthesis is initiated by the acylation of dihydroxyacetone phosphate by dihydroxyacetone phosphate acyltransferase. Experimental studies in cultured skin fibroblasts demonstrate that ATP is required to overcome the latency of dihydroxyacetone phosphate acyltransferase, a finding that is compatible with an ATP-linked translocation of the substrate across the peroxisomal membrane [Wolvetang et al., 1990]. The second enzyme, alkyl-dihydroxyacetone phosphate synthase, replaces the acyl group with a long-chain alcohol [Hajra and Bishop, 1982]. Both enzymes involved in these initial steps of introducing the ether bond into ether phospholipids are located on the inner surface of peroxisome membranes. The third step is the reduction of the alkyl-dihydroxyacetone phosphate to 1-alkyl glycerol-3-phosphate by the enzyme acyl/alkyl-dihydroxyacetone phosphate reductase, which is present in both peroxisomes and microsomes [Datta et al., 1990]. All subsequent steps of synthesis occur in the microsomes. A method developed by Roscher et al. [1985] determines the ratio of the activities in peroxisomal and microsomal components, and has proved to be of value in the study of peroxisomal disorders, many of which feature a marked reduction in plasmalogen synthesis.

Prostaglandin Degradation

Peroxisomes are involved in the chain shortening of prostaglandins F_2a [Diczfalusy and Alexson, 1988] and E₂ [Schepers et al., 1986]. Tiffany et al. [1991] demonstrated that basal and interleukin-1-stimulated synthesis of prostaglandin E₂ increased in a group of patients with X-linked adrenoleukodystrophy.

Amino Acid Metabolism

Molecular Etiology of Disorders of Peroxisome Assembly

Cell complementation studies were instrumental in the early understanding of the genetics of peroxisomal assembly disorders. Identification of complementation groups provided cell lines that are homogeneous with respect to their gene defect. In conjunction with the use of other molecular biology tools, including the study of yeast and other cell types and the emergence of computerized genomic databases, a new understanding of the molecular underpinnings of the peroxisome assembly disorders has been attained [Lazarow, 1995]. These disorders result from defects in the PEX genes. A complex interaction of peroxins is necessary for the biogenesis of peroxisomes, and a defect in any of these proteins impairs the process. The final common pathway is peroxisomal dysfunction, with the respective clinical syndromes.

From the study of complementation groups, it was determined that peroxisomal disorders comprise up to 16 complementation groups [Moser et al., 1995]. A majority of the identified groups included persons with Zellweger’s syndrome and milder variations of that condition. One complementation group, however, contained all of the patients with rhizomelic chondrodysplasia punctata, and no overlap between this clinical group and the others was identified. It has subsequently been determined that Zellweger spectrum disorders are secondary to PTS1-mediated pathways including PEX5, and that rhizomelic chondrodysplasia punctata is secondary to mutations in PEX7, the receptor for PTS2 proteins.

The most common causes of Zellweger spectrum disorders are mutations in either PEX1 or PEX6, although to date, 13 PEX genes have been identified as potential causes of these disorders (Table 38-1). PEX1 and PEX6 encode AAA ATPases. Their exact role in matrix protein importation is not certain, but it is evident that they interact. Overexpression of PEX6 or PEX1 can correct mild deficiencies of the other [Germain-Lee et al., 1997; Reuber et al., 1997]. PEX1 defects account for more than 65 percent of cases of such defects [Steinberg et al., 2003; Weller et al., 2003]. A degree of phenotype correlation with the causative mutation has been noted. A single base-pair deletion results in a severe phenotype, and G843D allows a milder phenotype [Collins and Gould, 1999]. Because it is known that PEX1 and PEX6 interact, the mutation G843D affects this interaction [Weller et al., 2003].

Table 38-1 Disorders of Peroxisome Biogenesis

Clinical and Pathologic Features

Zellweger’s syndrome

Zellweger’s syndrome is a multiple congenital anomaly syndrome characterized by craniofacial abnormalities, eye abnormalities, neuronal migration defects, hepatomegaly, chondrodysplasia punctata, and near-complete absence of peroxisomes. The craniofacial features include a high forehead, hypoplastic supraorbital ridges, epicanthal folds, midface hypoplasia, and a large fontanel (Figure 38-3). The head circumference usually is normal. Reported ocular abnormalities include cataracts, glaucoma, corneal clouding, Brushfield spots, optic nerve hypoplasia, and pigmentary retinal abnormalities. Severe weakness and hypotonia manifest in the newborn period, often accompanied by seizures and apnea [Heymans, 1984; Wilson et al., 1986]. Most affected infants have oromotor dysfunction and require tube feeding. Little psychomotor development ensues, and the average life span is limited, with most affected children surviving for 12–24 months. The facial appearance, Brushfield spots, and profound hypotonia may lead to a consideration of Down syndrome, although the chromosomal determination will eliminate that as a consideration.

Fig. 38-3 Cerebrohepatorenal syndrome of Zellweger.

A, Note the prominent forehead, large fontanel, hypertelorism, epicanthal folds, hypotonia, and hepatomegaly. B, Milder form of generalized peroxisomal disorder: 10-year-old boy with ataxia, significant mental retardation, visual loss secondary to retinitis pigmentosa, absence of speech, and sensorineural hearing loss. Liver biopsy showed micronodular cirrhosis and very few, abnormally small peroxisomes.

Striking abnormalities of neuronal migration unique to Zellweger’s syndrome are evident in the cerebral hemispheres as areas of pachygyria or polymicrogyria localized to the opercular region. In the cerebellum, the Purkinje cells form scattered heterotopias throughout the cortex and in the granule cell layer. Laminar discontinuities involving the olivary nucleus are noted, which also are unique to Zellweger’s syndrome [Evrard et al., 1978; Volpe and Adams, 1972]. Studies of PEX gene knockout mice have demonstrated similar neuropathologic changes [Baes et al., 1997; Faust, 2003; Faust et al., 2001; Gressens et al., 2000; Janssen et al., 2000, 2003]. N-methyl-d-aspartate (NMDA)-mediated abnormalities may contribute to the clinical findings seen in the most severely affected persons [Gressens et al., 2000].

Multiple other abnormalities have been reported. The eyes demonstrate loss of retinal ganglion cells and gliosis of the optic nerve [Cohen et al., 1983]. Retinal pigmentary degenerative changes are associated with absent electroretinograms [Hittner et al., 1981]. Hepatomegaly (present in 78 percent of patients) with periportal fibrosis may result in significant cholestasis (in 59 percent) and jaundice, micronodular cirrhosis (in 37 percent), and hypoprothrombinemia [Heymans, 1984]. Excessive iron deposition has been noted, which diminishes with age [Gilchrist et al., 1976]. Renal cortical cysts of varied sizes are present in 97 percent of patients studied pathologically but may be missed by ultrasound analyses [Bernstein et al., 1974; Heymans, 1984]. The adrenal gland demonstrates changes similar to those in X-linked adrenoleukodystrophy, with cytoplasmic lamellar inclusions consisting of cholesterol esterified with very long chain fatty acids [Goldfischer et al., 1983]. Skeletal abnormalities include clubfoot, thumb rotation, and stippled chondral calcification of the patella and acetabulum in 50 percent of patients [Heymans, 1984].

Laboratory Diagnosis

The diagnostic abnormalities present in patients with Zellweger spectrum disorders are listed in Table 38-2. A major diagnostic biochemical abnormality is the increased amount of very long chain fatty acids, which are fatty acids with carbon chains of more than 22. They also are elevated in single-enzyme defects of peroxisomal β-oxidation, so their presence is nonspecific, and other studies are required for this diagnosis. Very long chain fatty acid accumulation here is due to the reduction of peroxisomal β-oxidation, which is greater in Zellweger’s syndrome than in milder forms. A virtual absence of peroxisomal acyl-CoA oxidase and bifunctional enzyme has been demonstrated in the liver of patients with Zellweger’s syndrome [Suzuki et al., 1986; Tager et al., 1985]. It should be noted, however, that very long chain fatty acids are not elevated in rhizomelic chondrodysplasia punctata and other peroxisomal disorders (e.g., acatalasemia) that do not involve lipid oxidation, and normal levels do not exclude all peroxisomal disorders.

Table 38-2 Diagnostic Biochemical Plasma Profile of Peroxisomal Disorders

Because the first two steps of plasmalogen synthesis are peroxisomal functions, a reduction of plasmalogen levels is noted in patients with peroxisome biogenesis defects. Deficient levels of dihydroxyacetone phosphate acyltransferase have been used for diagnostic purposes in skin fibroblasts, amniocytes, chorionic villus samples, red blood cells, leukocytes, and platelets [Besley and Broadhead, 1987; Datta et al., 1984; Schutgens et al., 1985; Wanders et al., 1985, 1986a]. Webber et al. [1987] have demonstrated that the K_m values and other properties of the residual enzyme are normal, suggesting that the enzyme is not defective but is abnormally labile in the cytosol because of failure of transport into the peroxisome. Plasmalogens are reduced to 5 percent of control values in Zellweger’s syndrome [Heymans et al., 1984; Wanders et al., 1986a].

An age-related increase in the levels of phytanic acid occurs in all of the peroxisome biogenesis disorders, including rhizomelic chondrodysplasia punctata, although not to the extent seen in classic adult Refsum’s disease. The accumulation of phytanic acid was demonstrated first in infantile Refsum’s disease [Scotto et al., 1982] and later in Zellweger’s syndrome [Poulos et al., 1984]. Poulos et al. [1988] demonstrated that pristanic acid is elevated in disorders of peroxisome biogenesis. This elevation was later confirmed to be due to an impaired capacity to oxidize pristanic acid [Singh et al., 1990]. These findings are unlike those in classic Refsum’s disease, in which the defect involves the conversion of phytanic acid to pristanic acid. Thus, oxidation of pristanic acid is a peroxisomal function that is disrupted in disorders of peroxisome biogenesis [Watkins et al., 1990].

Normally, bile acid intermediates, such as trihydroxycholestanoic acid and dihydroxycholestanoic acid, are absent or present in low concentrations. In Zellweger spectrum disorders, however, they constitute 30–50 percent of total plasma bile acids [Clayton et al., 1987; Eyssen et al., 1985; Gustafsson et al., 1983; Hanson et al., 1979; Mathis et al., 1980; Monnens et al., 1980; Poulos and Whiting, 1985]. Bile acid intermediates also may be abnormal in single-enzyme defects, such as peroxisomal bifunctional enzyme deficiency.

The impaired activity of l-pipecolic acid oxidase results in increased accumulation of pipecolic acid in plasma and increased excretion of pipecolic acid in urine of patients with Zellweger spectrum disorders [Kelley and Moser, 1984; Mihalik et al., 1989; Wanders et al., 1988a]. Pipecolic acid levels in blood and urine are age-dependent, but an excess can be demonstrated in all patients, irrespective of age [Dancis and Hutzler, 1986]. Medium- and long-chain dicarboxylic acids accumulate and are excreted in urine, suggestive of a partial block in the degradation of long-chain dicarboxylic acids [Bjorkheim et al., 1984; Rocchiccioli et al., 1986].

In Zellweger’s syndrome, the number of catalase-containing particles in liver or kidney biopsy specimens is reduced [Goldfischer et al., 1973; Small et al., 1988], although today biopsy rarely is needed for diagnosis. A reduction in the number of peroxisomes also occurs in neonatal adrenoleukodystrophy and infantile Refsum’s disease but, as expected, is less severe than in Zellweger’s syndrome. The catalase is cytosolic rather than particulate in catalase-containing particles. Although, in the past, peroxisomes were considered absent, Santos et al. [1988] demonstrated membranous structures called peroxisomal ghosts that contain peroxisomal membrane proteins. This finding has been substantiated by other investigators, who demonstrated the presence of major membrane proteins in skin fibroblasts from patients with Zellweger’s syndrome, some of which were quite abundant [Gaertner et al., 1991; Suzuki et al., 1989]. These ghosts lack the matrix enzyme catalase and some or all of the matrix proteins. Schram et al. [1986] have reported that peroxisomal β-oxidation enzymes, which normally are located in the peroxisome matrix, are formed at a normal rate but are rapidly degraded in the cytosol. The mitochondrial abnormalities observed are considered secondary [Trijbels et al., 1983].

Prenatal Diagnosis

Cultured amniocytes or chorionic villus cells can be used to diagnose all of the disorders of peroxisome biogenesis. A variety of biochemical strategies have been used and focus on the type and degree of biochemical abnormality. Two independent techniques, the measurement of very long chain fatty acid β-oxidation [Moser et al., 1984a; Solish et al., 1985] and plasmalogen synthesis [Roscher et al., 1985; Schutgens et al., 1985], have demonstrated accurate diagnosis when performed by laboratories experienced in these methodologies. Molecular genetic techniques may be used in families in which the mutation has been identified and pre-implantation genetic diagnosis has been performed [Al-Sayed et al., 2007].

Therapy

Because many of the abnormalities are present in the affected fetus, potential for therapy is limited at present and likely to remain so. Treatment is primarily supportive, targeting, as appropriate, liver dysfunction with vitamin K for prothrombin deficiency, tube feeding, and antiepileptic medication. In the milder phenotypes, rehabilitative approaches, including communication training and physical and occupational therapy, are helpful.

Attempts to normalize some of the biochemical abnormalities include oral ether lipid therapy [Holmes et al., 1987; Poulos et al., 1990; Wilson et al., 1986] and dietary restriction of very long chain fatty acids [Van Duyn et al., 1984] and phytanic acid [Greenberg et al., 1987; Steinberg, 1989]. These approaches may normalize plasma levels of very long chain fatty acids, phytanic acid, and red blood cell plasmalogens. The clinical effectiveness of these interventions, however, has yet to be demonstrated because of the phenotypic variability of these disorders. Ursodeoxycholic acid can reduce levels of bile acid intermediates and has the potential to prevent liver damage [Colombo et al., 1990]. Dietary therapy with docosahexaenoic acid replaces low levels in plasma and red blood cells of patients with Zellweger’s syndrome, neonatal adrenoleukodystrophy, and infantile Refsum’s disease [Martinez et al., 1993, 1994]; however, it is ineffective in improving clinical function [Mahmood et al., 2010]. Peroxisome proliferators, such as clofibrate, do not induce the formation of catalase-containing peroxisomes or improve clinical status [Lazarow et al., 1985].