Search

The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities including the accumulation of the bile acid intermediates di- and trihydroxycoprostanoic acid. In this paper we show that there is a marked deficiency in the oxidative side chain cleavage of cholesterol in liver from Zellweger patients. These findings not only provide an explanation for the low levels of the major naturally occurring bile acids, cholic acid and chenodeoxycholic acid and the accumulation of di- and trihydroxycoprostanoic acid in Zellweger patients, but also suggest that peroxisomes are essential in bile acid synthesis in man.

Neonatal adrenoleukodystrophy belongs to the newly recognized group of inherited diseases, the peroxisomal disorders. Based on the reported similarities between neonatal adrenoleukodystrophy and the cerebro-hepato-renal (Zellweger) syndrome, we have studied peroxisomal functions in cultured skin fibroblasts from 5 neonatal adrenoleukodystrophy patients. The results indicate that multiple peroxisomal enzyme activities are deficient in fibroblasts from neonatal adrenoleukodystrophy patients. Digitonin titration experiments revealed that peroxisomes are strongly deficient in these fibroblasts as found earlier in fibroblasts from Zellweger patients. These findings not only explain the generalized loss of peroxisomal functions in neonatal adrenoleukodystrophy, but also provide an explanation for the observed resemblance in clinical and biochemical abnormalities between neonatal adrenoleukodystrophy and Zellweger syndrome. The implications for the pre- and postnatal detection of this disease will be discussed.

The Zellweger syndrome is a lethal hereditary disease characterized by the absence of peroxisomes (microbodies) in liver and kidney, and variable abnormalities in mitochondria. We show here that tissues from five infants that had died of this syndrome contain less than 10% of the normal levels of phosphatidylethanolamine plasmalogen (pPE), a major phospholipid component of cellular membranes. Heart and muscle, but not other tissues, also contain a substantial fraction of phosphatidylcholine plasmalogen (pPC), and this fraction is also strongly reduced in the Zellweger patients. No other abnormalities in cellular phospholipids were detected. Key enzymes of the biosynthesis of plasmalogens have previously been shown to be exclusively located in the peroxisomes of rodent liver and the microperoxisomes of rodent brain. We infer that the corresponding enzymes are also located in peroxisomes in man and that the absence of peroxisomes in Zellweger patients leads to their inability to synthesize plasmalogens. Our results support the notion that the biosynthetic role of peroxisomes in mammals has thus far been underestimated. We suggest that the defect in plasmalogen synthesis and possibly as yet unknown peroxisomal reactions are responsible for the diverse abnormalities observed in Zellweger patients.

A 17-year-old girl with progressive lipid-storage myopathy for 2 years had low muscle carnitine levels. There was no therapeutic response to prednisone and DL-carnitine-HCl. Chemical findings indicated glutaric aciduria type II. Riboflavin therapy and a fat-restricted, carbohydrate-enriched diet resulted in dramatic improvement. Low carnitine concentrations in plasma and muscle were observed in three asymptomatic sisters who had normal urinary excretion patterns. There was impaired mitochondrial beta-oxidation in cultured skin fibroblasts from the index patient and all three siblings.

Activity of acyl-CoA: dihydroxyacetone phosphate acyltransferase, a membrane-bound peroxisomal enzyme in mammalian liver cells catalyzing the first step in the biosynthesis of etherphospholipids, is detectable in thrombocytes isolated from blood of human controls. However, in thrombocytes from patients affected by the cerebro-hepato-renal (Zellweger) syndrome, the activity of this enzyme is severely reduced, permitting rapid postnatal biochemical detection of this severe inborn disease, by measuring the enzyme activity in patients' thrombocytes.

Infantile Refsum's disease was diagnosed in three male patients, presenting with facial dysmorphia, retinitis pigmentosa, neurosensory hearing loss, hepatomegaly, osteopenia and delayed growth and psychomotor development. An elevated plasma phytanic acid concentration and a deficient phytanic acid oxidase activity in fibroblasts were found with an accumulation of very long chain fatty acids in plasma and fibroblasts. There were elevated pipecolic acid levels in plasma, urine and CSF, and abnormal bile acid metabolites in plasma. Deficient activity of acylCoA: dihydroxyacetone phosphate acyl transferase was found in thrombocytes and fibroblasts of these patients as well as an impaired de novo plasmalogen biosynthesis in fibroblasts. These biochemical abnormalities, previously described in the Zellweger syndrome, suggest multiple peroxisomal dysfunction in our patients.

We have recently reported on plasmalogen deficiency in tissues and fibroblasts from patients with Zellweger syndrome. In this paper we have analyzed the activity of the first enzyme in the pathway leading to plasmalogen biosynthesis, i.e. acyl-CoA:dihydroxyacetone phosphate acyltransferase in liver, brain and cultured skin fibroblasts from Zellweger patients and controls. The results indicate a severe deficiency of this enzyme in Zellweger patients. Thus, the Zellweger syndrome constitutes the first inborn error of metabolism with a deficiency in an enzyme involved in phospholipid biosynthesis. Cultured amniotic fluid cells contained an enzymatic activity comparable to that of control fibroblasts. These findings suggest a method for prenatal diagnosis of this disease.

The presence of a beta-oxidation system in peroxisomes has been well documented. Rather than a duplicate of the mitochondrial beta-oxidation system, peroxisomes seem specially equipped to initiate the oxidation of very-long-chain fatty acids. Thus, the accumulation of very-long-chain fatty acids in tissues and body fluids from patients with a limited (X-linked adrenoleukodystrophy) or generalized (cerebro-hepato-renal (Zellweger) syndrome, infantile Refsum disease, neonatal adrenoleukodystrophy) peroxisomal dysfunction probably results from an impairment in the peroxisomal beta-oxidation system. In order to study this, we have developed an original assay which allows measurement of the overall peroxisomal beta-oxidation activity in human liver homogenates. Compared to controls, a strong deficiency of this activity was detected in liver from Zellweger patients using palmitoyl-CoA as a substrate.

The cerebro-hepato-renal (Zellweger) syndrome is an autosomal recessive disorder biochemically characterized by the absence of morphologically distinguishable peroxisomes. Key enzymes involved in the biosynthesis of ether phospholipids, i.e., dihydroxyacetone phosphate acyltransferase and alkyl dihydroxyacetone phosphate synthase, are located in mammalian (micro)peroxisomes. We have previously shown a strikingly reduced activity of dihydroxyacetone phosphate acyltransferase in liver, brain, and cultured skin fibroblasts from Zellweger patients (Schutgens et al. 1984. Biochim. Biophys. Res. Commun. 120: 179-184). We have now extended these investigations by studying alkyl dihydroxyacetone phosphate synthase in cultured human skin fibroblasts. Enzymatic activity was determined by measuring the formation of radioactive alkyl dihydroxyacetone phosphate from palmitoyl dihydroxyacetone phosphate and [1-14C]hexadecanol as substrates. The enzyme was optimally active at pH 8.5 and was stimulated (about 2-3-fold) by the presence of 0.05% (v/v) Triton X-100. The apparent KM values for the enzyme in control fibroblasts amounted to 35 microM for palmitoyl dihydroxyacetone phosphate and 90 microM for hexadecanol. The reaction became inhibited at higher concentrations of both Triton X-100 and palmitoyl dihydroxyacetone phosphate. Control skin fibroblasts showed alkyl dihydroxyacetone phosphate synthase activity of 69 +/- 28 pmol X min-1 X mg-1 (n = 7), while fibroblasts from patients had an activity of only 6.3 +/- 1.7 pmol X min-1 X mg-1 (n = 7). Alkyl dihydroxyacetone phosphate synthase was also found to be deficient in tissue homogenates of Zellweger patients. The specific activity of this enzyme in liver, kidney, and brain homogenates from Zellweger patients was less than 15% of that in the corresponding tissues from controls.

An improved quantitative method for measuring galactose-1-phosphate uridyltransferase (EC 2.7.7.12, Gal-PUT) activity in erythrocytes was developed based on the detection of glucose 1-phosphate generated under the catalytic influence of the enzyme. This is achieved by incubating the enzyme with galactose 1-phosphate and uridyldiphosphoglucose during 15 min, followed by deproteinisation. The glucose 1-phosphate generated is quantitated subsequently by measuring NADPH formation from added NADP+ in a second incubation step with added phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Gal-PUT activity is calculated from the increment in absorption at 340 nm. Because it is technically a simple assay that is sensitive, specific and not affected by UDPgalactose-4-epimerase (EC 5.1.3.2) activity in the erythrocytal lysates it is suggested to be the method of choice for measuring Gal-PUT activity. Activities in erythrocytes of controls varied from 264 to 556 U/kg hemoglobin; in obligate heterozygotes from 53 to 190 U/kg Hb and in homozygous deficient patients less than 5 U/kg Hb was measured at 37 degrees C.

Prenatal diagnosis of the cerebro-hepato-renal (Zellweger) syndrome has been performed in 10 pregnancies at risk by measuring both the activity of acyl CoA: dihydroxyacetone-phosphate acyltransferase (DHAP-AT) and the de novo plasmalogen biosynthesis, either in cultured amniotic fluid cells or in fibroblasts cultured from a chorionic villus biopsy. In 7 of the pregnancies both tests indicated no abnormality. All 7 continued to term and normal infants were delivered. However, in amniotic fluid cells from 2 fetuses affected by Zellweger syndrome unequivocal differences from control values were found. The activity of DHAP-AT was clearly deficient and the de novo plasmalogen biosynthesis was impaired. In one pregnancy at risk prenatal diagnosis was performed during the first trimester by measuring both the DHAP-AT activity and the de novo plasmalogen biosynthesis in fibroblasts cultured from a chorionic villi biopsy. From the deficient DHAP-AT activity and the impaired de novo plasmalogen biosynthesis it was concluded that the fetus was affected. This was confirmed biochemically after induced abortion. It can be concluded that measurement of the DHAP-AT activity and the de novo plasmalogen biosynthesis provides convenient methods for the early prenatal detection of Zellweger syndrome.

Peroxisomes play an essential role in human cellular metabolism. Peroxisomal disorders, a group of genetic diseases caused by peroxisomal dysfunction, can be classified in three groups namely a group of disorders with a general peroxisomal dysfunction (Zellweger syndrome; infantile type of Refsum's disease; neonatal adrenoleukodystrophy, hyperpipecolic acidemia), a group with an impairment of some, but not all peroxisomal functions (rhizomelic chondrodysplasia punctata) and a group with impairment of only a single peroxisomal function (acatalasemia, X-linked adrenoleukodystrophy/adrenomyeloneuropathy; adult type of Refsum's disease; peroxisomal thiolase deficiency; peroxisomal acyl-CoA oxidase deficiency; hyperoxaluria type I). In this paper we report the typical findings in ophthalmological examinations of patients suspected of Zellweger syndrome contributing to the clinical diagnosis of this disorder. In biochemical studies using a rapid gaschromatographic detection method for plasmalogens we confirmed that plasmalogens are severely deficient in all tissues of Zellweger patients studied. Moreover, using a recently developed radiochemical method, de novo plasmalogen biosynthesis was found to be impaired in fibroblasts from patients with Zellweger syndrome, infantile Refsum's disease, neonatal adrenoleukodystrophy or rhizomelic chondrodysplasia punctata, this in contrast to X-linked chondrodysplasia in which a normal plasmalogen biosynthesis was found. From the literature it is known that peroxisomal beta-oxidation with both long-chain (C16:0) and very long-chain (C24:0; C26:0) fatty acids is deficient in Zellweger syndrome, infantile Refsum's disease and neonatal adrenoleukodystrophy. In contrast, in X-linked adrenoleukodystrophy only the peroxisomal beta-oxidation of the very long chain fatty acids is impaired. As a result very long-chain fatty acids accumulate in tissues, plasma, fibroblasts and amniotic fluid cells from patients with Zellweger syndrome, infantile Refsum's disease, neonatal and X-linked adrenoleukodystrophy, but not in rhizomelic chondrodysplasia punctata or X-linked chondrodysplasia. Finally we confirmed that the peroxisomal enzyme alanine glyoxylate aminotransferase is severely deficient in liver from a patient that died because of the neonatal type of hyperoxaluria type I, but not in liver from Zellweger patients.

In patients with cerebro-hepato-renal (Zellweger) syndrome, the absence of peroxisomes results in an impairment of metabolic processes in which peroxisomes are normally involved. These include the catabolism of very long chain (greater than C22) fatty acids, the biosynthesis of ether-phospholipids and of bile acids, the catabolism of phytanic acid and the catabolism of pipecolic acid. Many diagnostic tests for Zellweger syndrome have become available in recent years. In classic Zellweger syndrome abnormal C27-bile acids, very long chain fatty acids, dicarboxylic acids and pipecolic acid accumulate in the plasma of the patients. Moreover, depending upon the diet, plasma phytanic acid concentrations may be elevated. In platelets the activity of acyl-CoA: dihydroxyacetone phosphate acyltransferase is deficient; in erythrocytes from young (less than 4 months) patients the plasmalogen content of the phospholipids is decreased. In cultured fibroblasts from skin and from chorionic villus and cultured amniotic fluid cells from Zellweger patients the plasmalogen level is lowered; there is a decreased activity of acyl-CoA: dihydroxyacetone phosphate acyltransferase, alkyl dihydroxyacetonephosphate synthase and phytanic acid oxidase; the de novo biosynthesis of plasmalogens and the peroxisomal beta-oxidation of fatty acids are impaired and the intracellular localization of catalase is abnormal. Dietary treatment of patients with Zellweger syndrome has not so far resulted in an objective clinical improvement. As Zellweger syndrome is usually fatal in early life, prenatal diagnosis of the disease is important.

A method was developed to prepare peroxisome-enriched fractions depleted of microsomes and mitochondria from cultured skin fibroblasts. The method consists of differential centrifugation of a postnuclear supernatant followed by density gradient centrifugation on a discontinuous Metrizamide gradient. The activity of hexacosanoyl-CoA synthetase was subsequently measured in postnuclear supernatants and peroxisome-enriched fractions prepared from cultured skin fibroblasts from control subjects and patients with X-linked adrenoleukodystrophy. Whereas the hexacosanoyl-CoA synthetase activity in postnuclear supernatants of X-linked adrenoleukodystrophy fibroblasts was only slightly decreased (77.8 +/- 4.4% of control (n = 15], enzyme activity was found to be much more markedly reduced in peroxisomal fractions isolated from the mutant fibroblasts (19.6 +/- 6.7% of control (n = 5]. This is a direct demonstration that the defect in X-linked adrenoleukodystrophy is at the level of a deficient ability of peroxisomes to activate very long chain fatty acids, as first suggested by Hashmi et al. [Hashmi, M., Stanley, W. and Singh, I. (1986) FEBS Lett. 86, 247-250].

The nonspecific lipid transfer protein (i.e., sterol carrier protein 2) from human liver was purified to homogeneity using ammonium sulfate precipitation, CM-cellulose chromatography, molecular sieve chromatography and fast protein liquid chromatography. Its amino acid composition was determined and found to be very similar to that of the nonspecific lipid transfer protein from bovine and rat liver with, as main feature, the absence of arginine, histidine and tyrosine. By way of a specific enzyme immunoassay using affinity-purified antibodies, the levels of nonspecific lipid transfer protein were determined in human livers. Levels varied from approximately 150 ng nonspecific lipid transfer protein per mg 105,000 X g supernatant protein for juvenile and adult humans to 40 ng per mg supernatant protein for a young infant. Levels of nonspecific lipid transfer protein in livers of infants with cerebro-hepato-renal (Zellweger) syndrome were extremely low (i.e., 2 ng per mg supernatant protein). Immunoblotting revealed the presence of crossreactive proteins of molecular masses of 40,000 and 58,000. The 40 kDa and 58 kDa proteins occurred in control livers, whereas only the 40 kDa protein was present in Zellweger livers. As in rat the 58 kDa protein could be demonstrated in a peroxisomal preparation isolated from an adult liver. A possible link between the occurrence of nonspecific lipid transfer protein and the presence of peroxisomes is discussed.

The peroxisomal oxidation of the long chain fatty acid palmitate (C16:0) and the very long chain fatty acids lignocerate (C24:0) and cerotate (C26:0) was studied in freshly prepared homogenates of cultured skin fibroblasts from control individuals and patients with peroxisomal disorders. The peroxisomal oxidation of the fatty acids is almost completely dependent on the addition of ATP, coenzyme A (CoA), Mg2+ and NAD+. However, the dependency of the oxidation of palmitate on the concentration of the cofactors differs markedly from that of the oxidation of lignocerate and cerotate. The peroxisomal oxidation of all three fatty acid substrates is markedly deficient in fibroblasts from patients with the Zellweger syndrome, the neonatal form of adrenoleukodystrophy and the infantile form of Refsum disease, in accordance with the deficiency of peroxisomes in these patients. In fibroblasts from patients with X-linked adrenoleukodystrophy the peroxisomal oxidation of lignocerate and cerotate is impaired, but not that of palmitate. Competition experiments indicate that in fibroblasts, as in rat liver, distinct enzyme systems are responsible for the oxidation of palmitate on the one hand and lignocerate and cerotate on the other hand. Fractionation studies indicate that in rat liver activation of cerotate and lignocerate to cerotoyl-CoA and lignoceroyl-CoA, respectively, occurs in two subcellular fractions, the endoplasmic reticulum and the peroxisomes but not in the mitochondria. In homogenates of fibroblasts from patients lacking peroxisomes there is a small (25%) but significant deficiency of the ability to activate very long chain fatty acids. This deficient activity of very long chain fatty acyl-CoA synthetase is also observed in fibroblast homogenates from patients with X-linked adrenoleukodystrophy. We conclude that X-linked adrenoleukodystrophy is caused by a deficiency of peroxisomal very long chain fatty acyl-CoA synthetase.

A 7 and one half-year-old boy with a massive excretion of argininosuccinic acid is described. He exhibited only moderate mental retardation, cerebellar ataxia and both abnormal hair and skin. Argininosuccinate lyase activity in the erythrocytes of his parents and his sister was in the range expected for heterozygotes. The patient was put on a low protein diet with arginine supplementation and improved clinically and biochemically on this regime. The variability of the phenotypic expression of argininosuccinate lyase deficiency is stressed.

We have recently identified four complementation groups in fibroblasts from patients deficient in peroxisomes. Here we describe a kinetic analysis of the complementation process. The kinetics of peroxisome assembly was assessed in heterokaryons of complementary cell lines by measuring the rate of incorporation of catalase, initially present in the cytosol, into particles. In two combinations of cell lines assembly was rapid and insensitive to cycloheximide. Thus the components required for peroxisome assembly must have been present in the parental cell lines, at least one of which presumably contained peroxisomal ghosts. In three other combinations of cell lines assembly of peroxisomes was slow and sensitive to cycloheximide.

The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities, including an accumulation of very long-chain fatty acids. We show by immunoblotting that there is a marked deficiency in livers from patients with the Zellweger syndrome of the peroxisomal beta-oxidation enzyme proteins acyl-CoA oxidase, the bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase. Using anti-(acyl-CoA oxidase), increased amounts of cross-reactive material of low Mr were seen in the patients. With anti-(oxoacyl-CoA thiolase), high Mr cross-reactive material, presumably representing precursor forms of 3-oxoacyl-CoA thiolase, was detected in the patients. Catalase protein was not deficient, in accordance with the finding that catalase activity is not diminished in the patients. Thus in contrast to the situation with catalase functional peroxisomes are required for the stability and normal activity of peroxisomal beta-oxidation enzymes.

In recent years a growing number of inherited diseases have been recognized to originate from an impairment in one or more peroxisomal functions. Since it is well established that the first two steps in the biosynthesis of plasmalogens proceed in peroxisomes, we studied the biosynthesis of plasmalogens in cultured skin fibroblasts from patients with different peroxisomal and related disorders. When de novo plasmalogen biosynthesis was studied by growing the cells in the presence of [14C]hexadecanol, impaired plasmalogen biosynthesis was found in rhizomelic chondrodysplasia punctata, cerebrohepatorenal (Zellweger) syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease. In all these cases, alkyl-acyl phospholipids, the precursors of plasmalogens, did not accumulate and 1-O-[9,10-3H2]octadecylglycerol was converted into plasmalogens with equal efficiency as in controls. This indicated that impaired de novo plasmalogen biosynthesis as measured by [14C]hexadecanol incorporation was due to a deficient formation of the glycero-ether bond. Using this procedure, normal de novo plasmalogen biosynthesis was found in X-linked adrenoleukodystrophy, adrenomyeloneuropathy, X-linked chondrodysplasia punctata, adult Refsum disease, as well as in heterozygotes for Zellweger syndrome and infantile Refsum disease. The data have indicated that the average extent of the deficiency in glycero-ether bond formation is different in Zellweger syndrome, chondrodysplasia punctata, neonatal adrenoleukodystrophy, and infantile Refsum disease.

In this paper we show that cultured chorionic villous fibroblasts efficiently catalyse the peroxisomal beta-oxidation of hexacosanoic acid (cerotic acid), a saturated very long chain fatty acid containing 26 carbon atoms. Hexacosanoic beta-oxidation was found to be strongly impaired in cultured chorionic villous fibroblasts from a Zellweger foetus. This finding indicates that measurement of peroxisomal beta-oxidation can be used (in addition to measurement of acyl-CoA:dihydroxyacetone phosphate acyltransferase, de novo plasmalogen biosynthesis, the amount of particle-bound catalase and phytanic acid oxidase) for prenatal diagnosis in the first trimester of Zellweger syndrome, infantile Refsum disease and neonatal adrenoleukodystrophy. The method should be equally applicable to the early prenatal diagnosis of disorders in which there is a deficiency of a single peroxisomal beta-oxidation enzyme. Such diseases include X-linked adrenoleukodystrophy (peroxisomal very long chain fatty acyl CoA ligase deficiency), 'pseudo-Zellweger syndrome' (peroxisomal 3-oxoacyl-CoA thiolase deficiency) and 'pseudo-neonatal adrenoleukodystrophy' (acyl-CoA oxidase deficiency).

Although peroxisomes were initially believed to play only a minor role in mammalian metabolism, it is now clear that they catalyse essential reactions in a number of different metabolic pathways and thus play an indispensable role in intermediary metabolism. The metabolic pathways in which peroxisomes are involved include the biosynthesis of ether phospholipids and bile acids, the oxidation of very long chain fatty acids, prostaglandins and unsaturated long chain fatty acids and the catabolism of phytanate and (in man) pipecolate and glyoxylate. The importance of peroxisomes in cellular metabolism is stressed by the existence of a group of inherited diseases, the peroxisomal disorders, caused by an impairment in one or more peroxisomal functions. In the last decade our knowledge about peroxisomes and peroxisomal disorders has progressed enormously and has been the subject of several reviews. New developments include the identification of several additional peroxisomal disorders, the discovery of the primary defect in several of these peroxisomal disorders, the recognition of novel peroxisomal functions and the application of complementation analysis to obtain information on the genetic relationship between the different peroxisomal disorders. The peroxisomal disorders recognized at present comprise 12 different diseases, with neurological involvement in 10 of them. These diseases include: (1) those in which peroxisomes are virtually absent leading to a generalized impairment of peroxisomal functions (the cerebro-hepato-renal syndrome of Zellweger, neonatal adrenoleukodystrophy, infantile Refsum disease and hyperpipecolic acidaemia); (2) those in which peroxisomes are present and several peroxisomal functions are impaired (the rhizomelic form of chondrodysplasia punctata, combined peroxisomal beta-oxidation enzyme protein deficiency); and (3) those in which peroxisomes are present and only a single peroxisomal function is impaired (X-linked adrenoleukodystrophy, peroxisomal thiolase deficiency (pseudo-Zellweger syndrome), acyl-CoA oxidase deficiency (pseudo-neonatal adrenoleukodystrophy) and probably, the classic form of Refsum disease.

In the present study we investigated peroxisomal functions in cultured human muscle cells from control subjects and from a patient with the Zellweger syndrome, a genetic disease characterized by the absence of morphologically distinguishable peroxisomes in liver and kidney. In homogenates of cultured muscle cells from control subjects, catalase is contained within subcellular particles, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity is present and palmitoyl-CoA can be oxidized by a peroxisomal beta-oxidative pathway; these findings are indicative of the presence of peroxisomes in the cells. In homogenates of cultured muscle cells from the patient with the Zellweger syndrome, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity was deficient, peroxisomal beta-oxidation of palmitoyl-CoA was impaired and catalase was not particle-bound. These findings indicate that functional peroxisomes are absent in muscle from patients with the Zellweger syndrome. We conclude that cultured human muscle cells can be used as a model system to study peroxisomal functions in muscle and the consequences for this tissue of a generalized dysfunction of peroxisomes.

Deficient mitochondrial acetoacetyl-CoA thiolase in fibroblasts from four patients with 3-ketothiolase deficiency was studied using immunochemical methods. We also examined fibroblasts from two heterozygotes, the mother and the brother of the case 1 patient, identified on the basis of the results of the enzyme activity measurements, using 2-methylacetoacetyl-CoA as substrate. The results were as follows: 1) in fibroblasts from all four patients, the thiolase activity using acetoacetyl-CoA was not activated by K+, although that of the controls and the heterozygotes was activated about 2-fold. 2) by immunoblot analyses, mitochondrial acetoacetyl-CoA thiolase was not detectable in fibroblasts from cases 2 and 3, although a very faint band was seen in tissues from cases 1 and 4. However, the band of mitochondrial 3-ketoacyl-CoA thiolase was clearly detected in all patients to the same extent as in the controls. 3) mitochondrial acetoacetyl-CoA thiolase was observed after pulse labeling for 1-h and a 72-h chase period in three cell lines (cases 1, 2, and 4), but was fainter compared to the controls. In another cell line (case 3), a fluorographic band at the same position was detected following a 1-h pulse, but disappeared following a 6-h chase. These results demonstrate heterogeneity in the enzyme defect resulting in a deficiency of mitochondrial acetoacetyl-CoA thiolase in fibroblasts from patients with 3-ketothiolase deficiency.

We investigated the oxidation of L-pipecolate in human liver. The results obtained with L-pipecolate from which traces of D-pipecolate had been removed by a preincubation with D-aminoacid oxidase indicate that a distinct L-pipecolate oxidase rather than D-aminoacid oxidase is responsible for the L-pipecolate dependent H2O2-production in human liver. Importantly, L-pipecolate oxidase was found to be localized in peroxisomes which adds to the growing number of enzymes and metabolic functions which can be ascribed to peroxisomes.

The distribution of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency in ethnic minorities in the Netherlands was studied in a random sample of 668 healthy pregnant women and 754 healthy full term neonates. The overall prevalence of G-6-PD deficiency was 6.6% in males and 5.2% in females. Highest frequencies were found in sub-Saharan blacks. The hematological data in severe deficient women of African descent suggest slight hemolysis in the first trimester of pregnancy. Mean hemoglobin concentrations in pregnant women and neonates of African descent were lower as compared to hemoglobin concentrations in persons of Asian or Mediterranean origin. G-6-PD deficiency was found to be the only cause of neonatal jaundice in 6% of the non-Caucasian neonates who underwent exchange transfusion for severe neonatal hyperbilirubinaemia. Mean bilirubin in cordblood, however, was not found to be significantly higher in severe deficient neonates of African, Asian and Mediterranean descent.

A mild variant of Zellweger (cerebro-hepato-renal) syndrome was diagnosed in male and female siblings aged 7 and 2 years. They had mild facial dysmorphia, moderate psychomotor retardation, tapetoretinal degeneration, sensorineural deafness and hepatomegaly. Ultrastructural examination of a liver biopsy in the younger patient revealed the absence of recognizable peroxisomes. In both patients plasma levels of pipecolic acid, phytanic acid, trihydroxycoprostanoic acid and dihydroxycoprostanoic acid were elevated. The very long chain fatty acid C26:0 and the C26:0/C22:0 fatty acid ratio were elevated in plasma, but less than in classical Zellweger syndrome. In cultured fibroblasts, deficient acyl-CoA:dihydroxyacetone phosphate acyltransferase and increased concentrations of C26:0 as well as C26:1 very long chain fatty acids were found within the ranges previously established for patients with classical Zellweger syndrome. Particle-bound catalase was absent in fibroblasts. Despite the relatively mild clinical expression the biochemical abnormalities found in these patients are the result of a general peroxisomal dysfunction similar to the changes in classical Zellweger syndrome.

The administration of phenylalanine (Phe) combined with UVA exposure was found to be effective in vitiligo. Phe is an amino acid which constitutes part of the daily dietary protein, and when orally administered in a dose of 50 mg/kg body weight, it results in an elevated plasma level. Since peak concentrations of Phe in the blood are reached between 30 and 45 min after ingestion, UVA exposure was administered at this time. After 4 months (32 treatments) reasonable repigmentation preferentially occurred in the skin area of subcutaneous fat (adipose tissue). Apart from the repigmentation of hypo-pigmented macules, vitiligo patients can tolerate more sun than usual, especially at the vitiliginous lesion, and they experience no sunburn as a result of Phe-UVA therapy. Normal skin also tans very well.

Biochemical studies have been performed in muscle, liver, leukocytes, and fibroblasts from patients suffering from the Zellweger syndrome. Oxidation rates of [1-14C]pyruvate, [U-14C]malate, and [1-14C]2-oxoglutarate were strongly reduced in skeletal muscle homogenate. Oxygen consumption in isolated skeletal muscle mitochondria could only be stimulated by ADP in the presence of ascorbate and N,N,N1,N1-tetramethyl-p-phenylenediamine. Cytochrome contents in heart muscle and liver mitochondria were normal. A very low activity of succinate-ubiquinone oxidoreductase was found in liver homogenate of two patients. From the effect of 2-thenoyltrifluoroacetone on the succinate-phenazine methosulphate oxidoreductase activity, a nearly competitive inhibition with respect to phenazine methosulphate was demonstrated in contrast with a non-competitive inhibition in controls. Normal oxidation rate of [1-14C]pyruvate and [2-14C]pyruvate was found in leucocytes and fibroblasts. Lactate and pyruvate levels were normal in serum and cerebrospinal fluid and beta-hydroxybutyrate and acetoacetate levels were normal in blood. The ratios lactate/pyruvate and beta-hydroxybutyrate/acetoacetate were normal as well. These findings point to a defect in the electron transport chain at the succinate-ubiquinone oxidoreductase level. This defect might be related to the absence of peroxisomes in the cells of Zellweger patients.

In tissues of patients with the cerebro-hepato-renal (Zellweger) syndrome the plasmalogen content is very low. In order to study the biosynthesis of plasmalogens, skin fibroblasts of Zellweger patients, controls and heterozygotes, and amniotic fluid cells of controls were cultured in a medium supplemented with [1-14 C]hexadecanol or 1-O-[9,10-3H2]octadecylglycerol. The incorporation of 14C-label into the alkenyl moiety of plasmalogens was strongly reduced in Zellweger patients as compared to controls. The low concentration of 14C-labeled plasmalogens was not compensated for by an elevated levels of 14C-labeled alkyl phospholipids. Hexadecanol was partly oxidized to fatty acid in all cell lines and the incorporation of 14C-labeled fatty acid into phospholipids was comparable for patients and controls. [3H]Alkylglycerol was incorporated into plasmalogens with the same efficiency in Zellweger patients as in controls. These results indicate that only the reaction(s) involved in the introduction of the ether bond in the process of plasmalogen synthesis are deficient in Zellweger patients. The results also suggest that the hexadecanol incorporation patterns can be used for the (prenatal) diagnosis of the Zellweger syndrome.

A 13-year-old girl with severe mental retardation, tapetoretinal degeneration, an extinguished electroretinogram and sensoneurinal hearing loss is described. In early life the diagnosis of Zellweger (cerebro-hepato-renal) syndrome was considered because of hypotonia, craniofacial dysmorphia, abnormal liver functions and pipecolic aciduria. Biochemical studies in fibroblasts from the patient revealed a general peroxisomal dysfunction comparable to the findings in Zellweger Syndrome. As the clinical presentation of this patient is essentially different from that in classical Zellweger patients, who usually die early in life, we recommend the study of peroxisomal functions in all patients with severe mental retardation, tapetoretinal degeneration and sensoneurinal hearing loss.

Peroxisomes play an essential role in human cellular metabolism. Peroxisomal disorders, a group of genetic diseases caused by peroxisomal dysfunction, can be classified into three groups: (1) disorders of peroxisome biogenesis with a generalized loss of peroxisomal functions (Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease, hyperpipecolic acidaemia); (2) disorders with a loss of multiple peroxisomal functions (rhizomelic chondrodysplasia punctata and Zellweger-like syndrome; (3) disorders with loss of a single peroxisomal function (X-linked adrenoleukodystrophy, peroxisomal thiolase deficiency, bifunctional protein deficiency, acyl-CoA oxidase deficiency, classic Refsum disease, hyperoxaluria type I and acatalasaemia). Prenatal diagnosis is indicated in all these genetic disorders with the exception of classic Refsum disease, most types of hyperoxaluria type I and acatalasaemia. A variety of techniques is available now for the prenatal diagnosis of peroxisomal disorders in the first or second trimester of gestation. Prenatal diagnosis was performed by us in 70 pregnancies at risk for a disorder of peroxisome biogenesis, three for rhizomelic chondrodysplasia punctata, four for X-linked adrenoleukodystrophy and two for a defect in peroxisomal beta-oxidation. Fourteen affected fetuses were identified; no false negative cases were obtained.

Phosphatidylethanolamine plasmalogen levels were determined in erythrocytes from controls and 13 patients with the cerebro-hepato-renal (Zellweger) syndrome. It was found that in Zellweger patients 20 weeks of age or younger, erythrocyte phosphatidylethanolamine plasmalogen levels were lowered whereas in older patients (except in one) normal levels were found. The results obtained suggest a close relationship between the age of the patients at sampling and the phosphatidylethanolamine plasmalogen levels in their erythrocytes. A possible explanation for these findings and the implications for the postnatal detection of Zellweger syndrome are discussed.

A micromethod for the quantitative determination of L-alanine in blood and urine is described. No interference of D-alanine, D-cysteine, L-proline, L-glutamine, L-valine, L-histidine or glycine in concentrations up to 5 mmol/l was found. Using this method fasting blood levels of L-alanine in children were determined. Defects in gluconeogenesis can be easily established by measuring with this method the in vivo L-alanine metabolism in an oral, or intravenous L-alanine loading test.