Postnatal diagnosis of peroxisomal disorders: A biochemical approach

Biochimie (1993) 75, 269-279 © Soci6t6 franeaise de biochimie et biologie mol6culaire / Elsevier, Paris

269

Postnatal diagnosis of peroxisomal disorders: A biochemical approach R J A W a n d e r s a, b, R B H S c h u t g e n s

a,b, P G B a r t h b, c J M Tagerd, H v a n d e n B o s c h e

Departments of aClinical ~liochemistry, bpediatrics and CNeurology, University Hospital Amsterdam, AMC, Meibergdreef 9, 1105 AZ Amsterdam; aDepartment of Biochemistry, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam; eCentre for Biomembranes and Lipid Enzymology, Padualaan 8, 3584 CH Utrecht, Netherlands (Received 22 December 1992; accepted 14 January 1993)

Summary m In recent years an increasing number of inherited diseases in man has been identified in which there is an impairment of one or more peroxisomal functions. Sofar 15 different peroxisomal disorders have been identified which can be subdivided into three distinct groups depending upon whether there is a generalized (group A), multiple (group B) or single (group C) loss of peroxisomal functions. In this paper we will briefly describe the functions of peroxisomes in man which are of direct relevance for the peroxisomal disorders known up to now. Based upon the biochemical characteristics of the different peroxisomal disorders, we will describe a straightforward approach for the postnatal identification of patients suspected to suffer from a peroxisomal disorder. Furthermore, a detailed analysis of the biochemical procedures which should be used preferably, is given. peroxisomes / peroxisomal disorders / ether lipid synthesis / fatty acid oxidation Introduction The organelle that we now know as 'peroxisome' was discovered in the early fifties as 'spheric or oval bodies' in the cytoplasm of mouse proximal kidney tubules. Thanks to the pioneering work of De Duve and Baudhuin [1] it became clear that this organelle contained a number of H202-generating enzymes as well as catalase which decomposes H202 to H20 and 02. The association of H202-generating enzymes and catalase in one single organelle led De Duve to introduce the name 'peroxisome'. These studies gave little indication that peroxisomes would prove of relevance to human disease. Even the discovery by Goldfischer et al [2] that morphologically distinguishable peroxisomes were absent in liver and kidney from Zellweger patients did not immediately lead to the appreciation of peroxisomes as indispensable subcellular organelles. It is now clear, however, that peroxisomes catalyze a number of essential metabolic functions. The importance of peroxisomes in man is stressed by the existence of a group of inherited diseases in man

in which there is an impairment in one or more peroxisomal functions. At present we recognize 15 different peroxisomal disorders with neurological involvement in 13 of them (table I). The peroxisomal disorders identified sofar are usually subdivided into three groups depending upon whether there is a generalized, multiple or single loss of peroxisomal functions (table I). In this paper we will describe the current state-of-the-art with regard to peroxisomal disorders paying particular attention to the most recent developments including a critical discussion of methods for postnatal diagnosis. In order to provide the necessary background knowledge we will first describe the functional properties of peroxisomes relevant to human disease.

Functional properties of peroxisomes in man Rather than giving a full account of all the reactions taking place in peroxisomes, we will only describe those functions which are of direct relevance to human disease.

Abbreviations: ZS, Zellweger syndrome; NALD, neonatal adrenoleukodystrophy; IRD, infantile Refsum disease; HPA, hyperpipecolic acidaemia; RCDP, rhizomelic chondrodysplasia punctata; X-ALD, X-linked adrenoleukodystrophy; DHCA, dihydroxycholestanoic acid; THCA, trihydroxycholestanoic acid; VLCFA, very-long-chain fatty acids; DHAPAT, dihydroxyacetonephosphate acyltransferase; alkyl DHAP synthase, alkyldihydroxyacetonephosphate synthase.

270 Table I. Classification of peroxisomal disorders Group A: peroxisomes deficient, generalized loss of peroxisomal fimctions Cerebro-hepato-renal (Zellweger)syndrome(ZS) Neonatal adrenoleukodystrophy(NALD) Infantile Refsum disease (IRD) Hyperpipecolic acidaemia (HPA) Group B: peroxisomes present, multiple loss of peroxisomal fimctions Rhizomelic chondrodysplasia punctata (RCDP) Zellweger-like syndrome Group C: peroxisomes present, single loss of peroxisomal functions X-linked adrenoleukodystrophy or one of its phenotypic variants (X-ALD) Acyl-CoA oxidase deficiency (pseudo-NALD) Bifunctional protein deficiency Peroxisomal thiolase deficiency (pseudo-ZS) Dihydroxyacetone phosphate acyltransferase deficiency (DHAPAT-deficiency)(pseudo-RCDP) Glutaryl-CoA oxidase deficiency Di- and trihydroxycholestanoicacidaemia Hyperoxaluria type I Acatalasaemia

vation of VLCFAs [5], the two cholestanoic acids [6] and pristanic acid [7] is brought about by three distinct synthetases located in different subcellular organelles. Furthermore, at least three different acyl-CoA oxidases are involved in the subsequent ~-oxidation of the three CoA-esters. Indeed, the cholestanoyl-CoA esters are handled by a distinct cholestanoyl-CoA oxidase expressed in liver only [8]. Furthermore, pristanoyl-CoA is not a substrate for the clofibrate-inducible acyl-CoA oxidase but reacts almost exclusively with the non-inducible acyl-CoA oxidase [9-11]. Very-long-chain fatty acyl-CoA esters such as lignoceroyl-CoA (C24:0-COA) are substrates for both the clofibrate-inducible as well as the clofibrate-non-inducible acyl-CoA oxidase [ 11]. With regard to the subsequent steps involved in the 13-oxidation of these compounds, available evidence suggests that these reactions are carried out by the peroxisomal multifunctional 13-oxidation enzyme protein (formerly called bifunctional protein) in conjunction with peroxisomal thiolase. This concept is largely derived from studies on patients with defects at the level of bifunctional protein and peroxisomal thiolase, respectively (see [12] for discussion). Ether-phospholipid biosynthesis

Fatty acid ~-oxidation One of the major functions of peroxisomes is the 13-oxidative chain-shortening of fatty acids and fatty acid derivatives. Rather than being a functional duplicate of the mitochondrial 13-oxidation system, peroxisomes are involved in the 13-oxidation of a distinct set of substrates. Indeed, although some fatty acids are handled by mitochondria as well as peroxisomes, a number of fatty acids and fatty acid derivatives depend upon the peroxisomal 13-oxidation system for their ~-oxidation. These includes the following compounds: 1) very-long-chain fatty acids (VLCFA); 2) di- and trihydroxycholestanoic acid (DHCA and THCA); 3) pristanic acid; 4) long chain dicarboxylic acids; 5) certain prostaglandins (eg prostaglandin F2~ [3]); 6) certain leukotrienes (eg c0-carboxy-N-acetylLTE4 [41); 7) 12-and 15-hydroxyeicosatetraenoic acid; 8) certain mono- and polyunsaturated fatty acids. For the subject of peroxisomal disorders only the first three compounds are of direct diagnostic relevance since no patients with an isolated defect in the 13-oxidation of compounds 4--8 have been described sofar, In recent years much has been learned concerning the individual enzymes involved in the activation and subsequent ~-oxidation of compounds 1-3, ie verylong-chain fatty acids, di- and trihydroxycholestanoic acid and pristanic acid. Available evidence, mostly obtained from studies with rat liver, suggests that acti-

A second major function of peroxisomes concerns their role in the synthesis of ether-phospholipids. Indeed, the two enzyme activities responsible for the introduction of the characteristic ether-linkage in ether-phospholipids, ie dihydroxyacetonephosphate acyltransferase (DHAPAT) and alkyldihydroxyacetone-phosphate synthase (aikyI-DHAP synthase) are localized in peroxisomes (see [12] for more information). In mammals the main end products of ether-phospholipids biosynthesis are the plasmalogens (l-0-alkl-enyl-2-acylphosphoglycerides), which possess an o~,13-unsaturated ether bond at the sn-! position of the glycerol backbone. Although ether-phospholipids have long been known to be widely distributed in mammalian cell membranes, their physiological function has remained obscure except for platelet-activating-factor (PAF). Interestingly, Raetz et al [13] recently suggested that plasmalogens may protect cells against reactive oxygen species. Glyoxylate metabolism A third major function of peroxisomes is the detoxification of glyoxylate via alanine glyoxylate aminotransferase (AGT), a deficiency of which causes hyperoxaluria type I [14]. The subcellular distribution of AGT varies markedly from species to species, being exclusively peroxisomal in human liver (see [12] for more information).

271 Phytanic acid catabolism

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is an isoprenoid-derived 3-methyl fatty acid which is solely derived from external sources. In Zellweger patients there is an age- and diet-dependent accumulation of phytanic acid due to a deficient itoxidation activity (see [15,16] for discussion). These results were taken as evidence that phytanic acid txoxidation proceeds in peroxisomes. Direct tx-oxidation measurements, however, showed that peroxisomes from rat liver as well from human liver lack ix-oxidation activity. Instead, o~-oxidation was found to occur in mitochondria only. Recently, Huang et al [17] presented evidence suggesting that phytanic acid it-oxidation proceeds in the endoplasmic reticulum. We have recently reinvestigated the question of the subcellular site of phytanic acid a-oxidation in rat liver and human cultured skin fibroblasts and discovered that both peroxisomes and mitochondria are indispensable for phytanic acid tx-oxidation [ 18]. If o~oxidation, indeed, proceeds via hydrophytanic acid and ketophytanic acid which would imply the functional participation of three enzyme activities, it is our current hypothesis that one (or two) of these enzyme activities occur in peroxisomes, the other enzyme activity (or activities) being localized in mitochondria. Pipecolic acid metabolism

Peroxisomes play a n indispensable role in the degradation of L-pipecolic acid, a metabolite derived from L-lysine. Indeed, L-pipecolate oxidase which catalyzes the dehydrogenation of L-pipecolic acid, is a peroxisomal enzyme in man [19, 20]. Although mammalian peroxisomes are known to be involved in a great number of additional metabolic pathways, we will not discuss these functions here since no specific defects in any of these pathways have so far been described.

The peroxisomal disorders The cerebro-hepato-renal (Zellweger) syndrome (ZS) is a multi-system disorder involving both embryofetopathy and regressive changes which continue into postnatal life. The clinical presentation of ZS is dominated by the typical craniofacial dysmorphia (high forehead, large anterior fontanel, hypoplastic supraorbital ridges, epicanthal folds and deformed ear lobes in > 90% of cases) and profound neurological abnormalities. Impaired hearing, and retinopathy are probably present in all cases whereas cataracts are frequently found. Furthermore, there is usually liver disease, calcific stippling of the epiphyses and small renal cysts. Brain abnormalities in ZS include cortical

dysplasia and neuronal heterotopia but also regressive changes. There is hypomyelination rather than demyelination. In ZS peroxisomes are strongly deficient. A similar deficiency of peroxisomes is also found in the other disorders of group A which include neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD) and the four cases of hyperpipecolic acidaemia (HPA) described in literature (see [ 15,16]). In neonatal adrenoleukodystrophy (NALD) degenerative changes take predominance over errors in morphogenesis. Patients may show some psychomotor development and develop certain milestones before regression sets in. Kelley et al [21] have suggested criteria to discriminate between ZS and NALD. Indeed, NALD patients demonstrate adrenal atrophy, cerebral demyelination, systemic infiltration of lipidladen macrophages and elevated levels of saturated VLCFAs whereas ZS patients have chondrodysplasia, glomerulocystic kidney disease, CNS dysmyelination rather than demyelination and accumulation of saturated and unsaturated VLCFAs. The clinical course of infantile Refsum disease (IRD) is even milder with the absence of distinct abnormalities in the neonatal period, minor facial dysmorphy and often survival into the second decade of life (see [22]). Accordingly, it can be concluded that the deficiency of peroxisomes is associated with a wide spectrum of clinical abnormalities, ranging from severe in ZS to much milder in IRD. The severity of clinical abnormalities may reflect the extent of peroxisomal malfunction (see [15]). Group B comprises rhizomelic chondrodysplasia punctata and Zellweger-like syndrome. RCDP is clinically characterized by a disproportionally short stature primarily affecting the proximal parts of the extremities, typical facial appearance, congenital contractures, characteristic ocular involvement, severe growth deficiency and mental retardation. Most patients survive beyond the first year of life, sometimes well into their second decade. Four distinct abnormalities have been found in RCDP including a deficient activity of DHAPAT, alkyl DHAP synthase and phytanic acid oxidase (table II). Furthermore, peroxisomal thiolase occurs in an abnormal molecular form [15]. Recently we identified a patient with all the clinical signs and symptoms of RCDP but with a single deficiency of DHAPAT only [23]. The identification of this new peroxisomal disorder (pseudoRCDP) exemplifies the functional importance of ether-phospholipids although no unique function has yet been identified for these lipids. Group C includes X-linked adrenoleukodystrophy (X-ALD) as most frequent disorder. Extensive studies by Moser and Moser [24] have shown that the clinical presentation of X-ALD is also very diverse ranging from the lethal childhood form to an 'Addison-only'

Deficient Deficient Deficient Deficient

Deficient Deficient Deficient

Present NA

Deficient NA Deficient

Normal Normal Normal

Deficient

Present Present

Normal Normal Normal

Normal

NA

NA

Elevated c Normal Normal Normal

Elevated Elevated NA

Deficiem

Deficient Deficient

Deficient Deficient Impaired

Elevated Elevated Eievateda E!evatedb Elevatedb

Peroxisome Zeliweger- X-linked deficiency like ALl) disorders syndrome

Type of peroxisomal disorder

Deficient Normal Normal

Deficient

Present Present

Normal Normal Normal

Elevated Normal Normal Normal Normal

Acyl-CoA oxidase deficiency

Normal Deficient Normal

Deficient

Present Present

Normal Normal Normal

Elevated Elevated Normal Elevateda Elevatedd

Bifunctional protein deficiency

Normal Normal Deficient

Deficient

Present Present

Normal Normal Normal

Elevated Elevated Normal Elevated d Elevatedd

Thiolase deficiency

Normal Normal Normal

Normal

Present Present

Normal Normal Normal

Normal Elevated Normal Elevatedd Elevatedd

Normal Normal Normal Normal Normal

Pseudo RCDP

Normal

Present Present

Normal Normal Normal Normal Abnormale Normal

Normal

Present Present

Deficient Deficient Deficient Normal Deficient Deficient

Normal Normal Normal Elevated b Normal

Di/trihydroxy RCDP cholestanoic acidaemia

a Age dependent; b age- and diet-dependent; c elevated except in some cases; d may be normal depending on age and diet; e abnormal mo~:.:ular form, 44 kDa rather than 41 kDa. NA = not analyzed.

Activity with C26:0 Enzyme proteins: - acyl-CoA oxidase - bifunctional protein - peroxisomal thiolase

Peroxisomal ~-oxidation

Hepatic peroxisomes Particle-bound catalase

Peroxisomes

DHAPAT Alkyl DHAP synthase De novo synthesis

Plasmalogen synthesis

Very-long-chain fatty acids Bile acid intermediates Pipecolic acid Phytanic acid Pristanic acid

Metabolites in body fluids

Parameter measured

Table ii. Biochemical characteristics of the peroxisomal disorders

I,,2 -..d tO

273 form with no neurological involvement. This phenotypic heterogeneity may occur within the same pedigree. Other disorders belonging to group C are acylCoA oxidase deficiency, bifunctional protein deficiency and peroxisomal thiolase deficiency, all reported in single cases only (see [ 15,16] for review). Other diseases belonging to group C are hyperoxaluria type I (alanine: glyoxylate aminotransferase deficiency [14]) and glutaryl-CoA oxidase deficiency in a variant case of glutaric aciduria type I [25].

Additional (presumed) peroxisomai disorders of unknown etiology In recent years an increasing number of patients has been identified with a defect in peroxisomal ~-oxidation of unknown etiology (see [26] for review). These patients all demonstrate accumulation of VLCFAs in plasma with or without di- and trihydroxycholestanoic acidemia. Importantly, the clinical presentation of these patients is comparable to that of patients affected by ZS, NALD or IRD. Finally, several patients have recently been recognized who were suspected to suffer from a peroxisomal disorder on clinical grounds but with normal plasma VLCFA levels. Instead, there was di- and trihydroxycholestanoic acidemia in all four patients [27-29]. This latter finding has important consequences for the biochemical work-up of suspected patients (see below).

Biochemical id~ntiflcation of peroxisomal disorders First, it must be stressed that there is no such thing as a single, unequivocal biochemical test allowing the identification of all peroxisomal disorders. Accordingly, the clinical symptomatology of each presenting patient must be considered first. It will be clear that the clinical presentation of patients affected by hyperoxaluria E'pe I or acatalasaemia lacking neurological involvement is quite different from any of the other disorders. In patients suspected to suffer from hyperoxaluria type I, oxalate, glycolate and glyoxylate must be determined in body fluids followed by measurement of alanine glyoxylate aminotransferase in a liver biopsy specimen [14]. In case of acatalasaemia catalase activity must be measured in erythrocytes. Inspection of table I and II reveals that in nine out of the 13 peroxisomal disorders which remain after discarding hyperoxaluria type I and acatalasaemia, there is accumulation of very-long-chain fatty acids. Accordingly, analysis of VLCFA levels in plasma or serum has generally been regarded as a good screening method for peroxisomal disorders. Most laboratories use the original procedure deve-

loped by Moser and Moser (see [30] for a detailed description of the method) which involves preparation of a total lipid extract, followed by acid methanolysis, chromatography on TLC plates and finally, analysis by gas-liquid-chromatography with or without mass spectrometry. Recently, a simple, one-step procedure was developed [30, 31 ] based on direct transesterification of total lipid fatty acids with acetyl chloride in the presence of methanol and methylene chloride. The fatty acid methyl esters are extracted, purified by silicic acid column chromatography and quantitated by capillary GLC. According to Moser and Moser [30] the results with the new assay p,ocedure are identical to those obtained with the earlier procedure. In our own laboratories we use the original procedure described by Moser and Moser (see [30]) to analyse very-long-chain fatty acid levels in plasma (or serum) and cultured skin fibroblasts. If the patient is suspected to suffer from X-linked adrenoleukodystrophy or one of its phenotypic variants, it is usually sufficient to analyze plasma VLCFA-Ievels only, although we prefer to do additional studies in cultured skin fibroblasts especially if prenatal diagnosis is wanted in future. The results of table III show that in virtually all cases of X-linked adrenoleukodystrophy or adrenomyelo-neuropathy all three parameters, ie C26:0, C24:0/C22:0 and C26"0/C22:0 are abnormal in plasma, although great variability is seen among individual patients (table III). However, we recently came across two patients showing all the clinical signs and symptoms of X-ALD, but with nomaal plasma VLCFA-Ievels (patients V and VI of the series of ALD-patients listed in table III). Subsequent studies in fibroblast from the two patients revealed clearly abnormal values indicating that C26:0 13-oxidation was, indeed, impaired in these patients as confirmed by direct C26:0 [3-oxidation activity measurements (see [32]). Table III further depicts results of VLCFAanalyses in plasma and fibroblasts from obligate heterozygotes. The results show that plasma and fibroblast VLCFAs are usually abnormal although not in all cases. Indeed, according to Moser and Moser [24] 80% of obligate heterozygotes show abnormal plasma VLCFAs. Interestingly, this percentage is higher (95%) if fibroblast VLCFAs are considered which suggests that heterozygote detection can best be done in cultured skin fibroblasts. If plasma VLCFAs are found to be abnormal and the patient is suspected to suffer from a peroxisomai disorder different from XALD or one of its phenotypic variants, we proceed by carrying out additional studies in plasma, erythrocytes, platelets and fibroblasts from patients. This allows one to ascertain whether the accumulation of plasma VLCFAs is due to a generalized loss of peroxisomal functions as in the disorders of peroxisome

274 Table IlL Biochemical findings in patients with X-linked adrenoleukodystrophy or one of its variants. Patient studied

ALD-patients

I II 11I IV V VI

AMN -patients

I I! III IV

(~tg/ml )

C24:0 C22:0 Plasma

C26:0 C22:0

C26:0 (~tg/mg )

C24:0 C22:0 F ibrob lasts

C26:0 C22:0

0.83 0.74 2.16 1.27 0.51 0.45

1.23 0.66 1.63 1.32 0.96 0.73

0.05 0.03 0.09 0.05 0.02 0.02

NA 0.29 NA NA 0.27 0.90

NA 2.21 NA 2.68 2.27 3.18

NA 0.20 NA 0.65 0.25 0.38

0.78 0.73 2.27 0.68

1.56 1.08 1.21 1.41

0.05 0.05 0.04 0.03

0.31 0.39 0.70 0.24

2.56 2.64 3.19 1.98

0.34 0.37 0.28 0.17

0.60 1.26 1.24 0.81

0.99 1.27 1.34 1.13

0.04 0.03 0.04 0.04

0.33 0.39 0.23 0.12

2.29 2.03 2.16 1.94

0.18 0.26 0.16 0.08

0.31 0.11-0.62 (109)

0.73 0.48-0.89 (109)

0.01 0.004).02 (109)

0.06 0.02-0.10 (57)

1.49 1.17-1.83 (57.)

0.03 0.02-0.05 (57)

C:26:0

Obligate heterozygotes

I II III IV

Control values

Mean Range (5-90 %) (n) NA, not analyzed.

biogenesis or due to an isolated defect in VLCFA I]oxidation. For this purpose we first analyze the bile acid levels by gas-chromatography (see [33] for details on the procedure used) paying particular attention to the C27 bile acid intermediates, ie dihydroxycholestanoic acid (DHCA) and trihydroxycholestanoic acid (THCA). Furthermore, plasma phytanic and pristanic acid levels are measured which is also done by gas-chromatography [341. In the same blood sample we measure the levels of plasmalogens in erythrocytes and the activity of the peroxisomal enzyme dihydroxyacetonephosphate acyl-transferase (DHAPAT) in platelets or leukocytes [35]. The best procedure to measure erythrocyte plasmalogen levels is described by Bj6rkhem et al [36]. After methanolysis of an erythrocyte lipid extract in the presence of hydrochloric acid, the ether-linked chains are released as dimethylacetal derivatives. The relative amount of erythrocyte plasmalogen is thus reflected in the ratio of C16:0 dimethylacetal to methyl palmitate as well as the C18:0 dimethylacetal to methyl stearate ratio. In our hands the method shows a moderate day-to-day variation. Therefore, we express the values relative to the mean value found in 3-5 control erythrocyte preparations carried through the same procedure (see table IV). Finally, detailed studies are done in cultured

skin fibroblasts. The types of analysis done in fibreblasts include the following: VLCFA-analysis [57], de n o v o plasmalogen biosynthesis using a double-label technique based on the use of [l-14C]hexadecanol and [3H]alkylglycerol [38], determination of particlebound catalase [39], enzyme activity measurements (DHAPAT, alkyl DHAP synthase, phytanic acid oxidase, etc) and immunoblot analysis using antibodies directed against several peroxisomal enzyme proteins (acyl-CoA oxidase, bifunctional protein, peroxisomal thiolase, catalase, etc) (see [40]). The strategy described above allows clear-cut identification of the extent of peroxisomal dysfunction. Table IV lists the results of such investigations in patients who turned out to be suffering from a disorder of peroxisome biogenesis, alternatively called peroxisome deficiency disorders [ 16]. The results show that abnormalities are most pronounced in classical Zellweger patients with markedly abnormal VLCFA values, greatly decreased erythrocyte plasmalogen levels, strongly deficient DHAPAT activities and a profound impairment in de nero plasmalogen biosynthesis. Abnormalities are much milder in patients with NALD or IRD. This is exemplified by the findings in patients VII and VIII. Indeed, patient VIII displayed only very mildly abnormal VLCFAs in plasma and fibroblasts with

275 near normal erythrocyte plasmalogen levels, a high residual activity of DHAPAT in platelets and a mild impairment in de novo plasmalogen synthesis. In recent years it has become clear that clinical phenotypes such as Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease do not only result from a generalized loss of peroxisomal functions due to a deficiency of peroxisomes but may also be due to a single defect in peroxisomal [3-oxidation. Indeed, the classical Zellweger phenotype may result from a deficiency of peroxisomes but also from a deficiency of peroxisomal thiolase only [41, 42]. Sofar we have identified more than 30 patients with an isolated defect in peroxisomal ~-oxidation. The

biochemical findings in five of these patients are listed in table V. The results show elevated VLCFA levels in plasma and cultured skin fibroblasts from all patients. Other peroxisomal parameters such as the activity of DHAPAT in platelets and fibroblasts show no abnormalities suggesting that the defect in each of these patients is at the level of the peroxisomal 13-oxidation system. The results of table V further show that there is accumulation of trihydroxycholestanoic acid in all five patients except patient II. This finding immediately suggests that the underlying biochemical defect in patient II is different from that in patients I, III, IV and V. Indeed, accumulation of VLCFAs together with trihydroxycholestanoic acid as in patients I,

Table IV. Biochemical findings in Zellweger patients and other patients suffering from a disorder of peroxisome biogenesis Parameter measured

Patients studied ! <

H I11 Classical ZS

IV >

V VI N-ALD N-ALD

VII IRD

VIII IRD

Controls

VLCFA - C26:0 (Ixg/ml) - C26:0/C22:0 ratio

2.96 0.51

3.21 0.49

2.16 0.57

1.74 0.47

1.78 0.18

2.58 0.41

2.05 0.17

0.79 0.06

0.31 (0.11--0.62) 0.01 (0.00-0.02)

109 109

Bile acids - THCA (l.tg/ml)

4.6

2.4

2.6

0.5

3.1

ND

0.8

0.8

<0.05

ll0

Phytanic acid (~tg/ml) Pristanic acid (~tg/ml)

NA NA

NA NA

NA NA

3.9 0.4

NA NA

16.1 3.0

28.0 13.5

NA NA

<4.0 <1.0

55 55

0.0 0.0

0.03 0.0

0.0 0.0

0.35 0.24

NA NA

NA NA

NA NA

0.83 0.88

1.0 1.0

0.1

0,0

0.0

0.4

1.1

NA

NA

1.4

3.55:0.9

1~

0.19

0.87

0.62

0.46

0.12

0.28

0.21

0.12

0.06 (0.02--0.10)

57

0.29

0.75

0.48

0.58

0.28

0.35

0.15

0.05

0.03 (0.02-0.05)

57

187 4.7

292 9.9

38.2 8.0

26.7 6.4

10.9 6.4

12.4 5.6

2.1 1.6

5.8 2.9

1.5 + 0.9 1.1 5:0.7

38 38

0. l

0.4

0.2

0.7

0.8

0.7

3. l

0.7

7.8 +_2.0

59

Clinical diagnosis Plasma

Erythrocyws

Plasmalogens -C16 -C18 Platelets

DHAPAT (nmol/30 rain rag) Fibroblasts

VLCFA C26:0 (l.tg/mg protein) C26:0/C22:0 ratio -

-

Plasmalogen synthesis - [3H]1[14C] in PE - [3H]/[t4C] in PC DHAPAT (nmol/2 h • rag)

NA, not analyzed; ND, not detectable; DHAPAT, Dihydroxyacetonephosphate acyltransferase; ZS, Zellweger syndrome; NALD, neonatal adrenoleukodystrophy; IRD, infantile Refsum disease. The control values listed in the table represent mean +_ SD except for C26:0 and C26:0/C22:0 for which the median values plus the 5-90% ranges are given. C 16 and C 18 plasmalogens were measured by gas-chromatography as C16:0 and C18:0 dimethylacetai/methyl ester ratios and the values obtained are expressed relative to those of 3-5 controls.

276 Table V. Biochemical findings in patients with an isolated defect in peroxisoma113-oxidation Parameter measured

Patients studied 1 ZS

H ZS

111 N-ALD

IV IRD

V N-ALD

Controls

n

VLCFA - C26:0 (~tg/ml) - C26:0/C22:0 ratio

1.59 0.29

7.46 0.36

2.75 0.48

1.63 0.15

1.51 0.12

0.31 (0.11-0.62) 0.01 (0.00-0.02)

109 109

Bile acids - THCA (lag/ml)

0.26

ND

0.19

0.51

0.71

< 0.05

110

Phytanic acid (~tg/ml) Pristanic acid (~tg/ml)

NA NA

6.3 NA

NA NA

21 58

11 21

< 4.0 < 1.0

55 55

0.95 1.05

NA NA

1.01 0.91

0.84 0.77

1.05 1.11

1.0 1.0

4. I

NA

NA

3.2

2.4

3.5 + 0.9

136

VLCFA - C26:0 (~tg/mg protein) - C26:0/C22:0 ratio

0.34

0.36

0.58

0.24

0.34

0.06 (0.02--0.10)

57

0.75

0.49

0.39

0.24

0.76

0.03 (0.02-0.05)

57

DHAPAT (nmol/2 h • mg)

7.5

6.4

12.5

4.6

7. !

7.8 :t: 2.0

59

Enzyme proteins - AcyI-CoA oxidase - Bifunctional protein - Peroxisomal thiolase

N N N

N N N

N N N

N N N

N N N

N N N

Clinical phenotype Plasma

Etythrocytes

Plasmalogens -C16 -C18 Platelets

DHAPAT (nmol/30 min ° rag) Fibroblasts

NA, not analyzed; ND, not detectable; N, normal. III, IV and V suggests that the defect in these patients is at the level of bifunctional protein or peroxisomal thiolase whereas patient II is probably suffering from an acyI-CoA oxidase deficiency (compare table II). Direct proof that is, indeed, true must come from additional studies including immunoblot analysis. Table V shows that the three peroxisomal 13-oxidation enzyme proteins were found to be present at their mature size in fibroblasts from all five patients. The apparent discrepancy between this latter finding and the clear impairment in peroxisomal 13-oxidation as reflected in strongly deficient rates of C26:0 13-oxidation is probably explained by the fact that one of the peroxisoma113-oxidation enzyme proteins is enzymatically inactive although immunologically present. The activity of the individual peroxisomal 13-oxidation enzyme proteins is difficult to measure, especially in

fibroblasts, due to the abundant activity of their mitochondrial counterparts. We have therefore developed a technique based upon complementation analysis to identify the true molecular defect in these patients (see eg [43]). Using this strategy we have now found that the molecular defect in patients III and IV from table V is at the level of bifunctional protein. The defect in patients I-III remains to be determined. The results of table V further show that there is accumulation of phytanic acid and pristanic acid in patients IV and V in which these parameters could be measured. The accumulation of pristanic acid in these patients is directly explained from the impairment in pristanic acid ~-oxidation as shown by direct [1-14C] pristanic acid 13-oxidation activity measurements in fibroblasts (Wanders and Denis, unpublished observations). Phytanic acid or-oxidation, however, was found to be

277

Table VI. Biochemical findings in patients suffering from rhizomelic chondrodysplasia punctata Parameter measured Phenotype

Patients studied i <,

•

H 111 Classical R C D P

IV >

V VI VII <--Pseudo RCDPm>

Cono'ols

r,:

Plasma

VLCFA - C26:0 (~tg/ml) - C26:0/C22-0 ratio

0.36 0.02

0.31 0.02

0.29 0.02

0.24 0.01

0.61 0.01

0.42 0.02

NA NA

0.31 (0.11-0.62) 0.01 (0.00-0.02)

109 109

Bile acids - THCA (~tg/ml)

ND

ND

ND

ND

ND

ND

ND

< 0.05

110

Phytanic acid (~tg/ml) Pristanic acid (~tg/ml)

99 0.1

38 ND

47 ND

2.1 ND

3.2 ND

ND ND

ND ND

< 4.0 < 1.0

55 55

NA NA

0.0 0.0

0.02 0.01

0.07 0.11

0.10 0.20

0.0 0.0

0.01 0.01

1.0 1.0

0.07

NA

NA

0.3

0.0

0.05

0.0

3.5 + 0.9(136)

136

Erythrocytes

Plasmalogens -C16 -C18 Platelets

DHAPAT (nmol/30 min • mg) Fibroblasts

VLCFA - C26:0 (l.tg/mg protein) C26:0/C22:0 ratio

0.02

0.05

0.04

0.02

NA

NA

0.04

0.06 (0.02--0.10)

57

0.02

0.04

0.04

0.02

NA

NA

0.03

0.03 (0.02-0.05)

57

Plasmalogen synthesis [3H]/[14C] in PE [3H]/[14C] in PC

41.9 2.8

174 7.3

399 3.8

32 8.3

>500 6.0

NA NA

194 13.6

1.5 :!: 0.9 I.I + 0.7

38 38

0.75

1.6

1.8

0.8

0.0

NA

0.0

7.8 + 2.0

59

44

44

44

44

41

NA

41

41

54

DHAPAT (nmol/2 h • mg) Thiolase (M,) (kDa)

For details see legend to table I and text. ND, not detectable; NA, not analyzed. normal in these cells (Wanders and Denis, unpublished observations). These results suggest that the accumulation of phytanic acid in these patients is the secondary consequence of inhibition of the phytanic acid a-oxidation system by pristanic acid itself or one of the intermediary metabolites of the phytanic acid/pristanic acid converting pathway (ie hydroxyand/or ketophytanic acid). As already discussed above, VLCFA levels are normal in rhizomelic chondrodysplasia punctata, its recently discovered variant with an isolated deficiency of dihydroxyacetonephosphate acyltransferase (DHAPAT) and in patients with di- and trihydroxycholestanoic acidaemia (table II). Although it has been advocated that RCDP can be identified by phytanic acid analysis in plasma, we believe that patients are missed if this method is used. Indeed, phytanic

acid levels may be completely normal in classical RCDP patients due to the fact that the accumulation of phytanic acid is age- and diet-dependent (compare case IV in table VI). Furthermore, phytanic acid is normal in patients with pseudo-RCDP due to an isolated DHAPAT-deficiency (cases V, VI and VII in table VI). The method of choice in identifying RCDP, both in its classical as well as its variant form, is analysis of erythrocyte plasmalogen levels. Sofar, in a series of almost 40 cases we found plasmalogens to be deficient in all cases (see also table VI). Finally, in those cases in which there is a strong suspicion for a peroxisomal disorder, but with normal values for plasma VLCFAs and erythrocyte plasmalogens, one should analyze di- and trihydroxycholestanoic acid by gas-chromatography. This allows identification of true cases of di- and trihydroxycho-

278 lestanoic acidaemia (see [27-29]). If such studies also fail to show any abnormalities, we conclude that the patient is not suffering from any of the hitherto known peroxisomal disorders. Detailed biochemical investigations as described above are of great importance since a prenatal diagnosis can be made in case of all peroxisomal disorders known up to now [44, 45].

Acknowledgments The authors gratefully acknowledge The Princess Beatrix Fund (The Hague, the Netherlands) for financial support and A Nijenhuis, W Smit, M Fruman, L van Lint, C Dekker, S Denis, C W T van Roermund, A de Rooy, R Purvis, L Gouw and GJ R o m e y n for expert technical assistance. Mrs let van der Gracht and Mr GJ Romeyn are gratefully thanked for preparation of the manuscript.

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