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Twin brothers were born with clinical symptoms indicating that they were suffering from Zellweger syndrome. However, instead of a generalized peroxisomal dysfunction, only very long-chain fatty acids and the pristanic acid/phytanic acid ratio were elevated in plasma and decreased oxidation of very long-chain fatty acids and pristanic acid was the only impairment found in fibroblasts. The other peroxisomal parameters tested were normal, including normal oxidation of phytanic acid and normal activity of dihydroxyacetonephosphate acyltransferase in fibroblasts as well as normal plasma bile acids. Although the biochemical results point to a defect in peroxisomal beta-oxidation, the isolated finding of impaired oxidation of very long-chain fatty acids and pristanic acid has to our knowledge not been reported previously and is difficult to explain by a deficiency of a known peroxisomal beta-oxidation enzyme.

In recent years an increasing number of inherited diseases in man have been identified in which there is an impairment in one or more peroxisomal functions. This paper discusses the current state of knowledge on these disorders with particular emphasis on the metabolic abnormalities in these diseases.

A case is reported of a male fetus at risk of X-linked adrenoleucodystrophy who showed a normal cultured chorionic villus cell very long chain fatty acid (VLCFA) profile but at birth exhibited grossly abnormal plasma and cultured fibroblast VLCFAs. Maternal contamination or a sample mix-up was excluded by chromosome analysis and analysis of polymorphic markers. This is the second report of a fetus affected with this disorder who showed normal cultured chorionic villus cell VLCFAs. It highlights the importance of a proper audit of all prenatal diagnoses to evaluate method reliability.

One of the main functions of mammalian peroxisomes is the beta-oxidation of a variety of fatty acids and fatty acid derivatives, including very long-chain fatty acids. Oxidation of these fatty acids is deficient in a number of different peroxisomal disorders, including the disorders of peroxisome biogenesis (Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease), X-linked adrenoleukodystrophy and a number of other disorders of peroxisomal beta-oxidation of known and unknown aetiology. Accurate measurement of peroxisomal fatty acid oxidation is of utmost importance for correct postnatal and prenatal diagnosis of these disorders. In this paper we describe a straightforward and accurate assay method to measure the beta-oxidation of palmitic acid (C16:0), hexacosanoic acid (C26:0) and pristanic acid in intact fibroblasts.

Profiles of saturated very-long-chain (> C22) fatty acids were studied in plasma, fibroblasts, erythrocytes, platelets, and leukocytes of patients affected by peroxisomal disorders such as Zellweger syndrome, X-linked adrenoleukodystrophy (X-ALD), and classic rhizomelic chondrodysplasia punctata (RCDP) and in controls. In Zellweger patients, the concentration of hexacosanoic acid (C26:0) and the C26:0/C22:0 ratio are greatly increased in plasma and fibroblasts. However, the plasma concentration of docosanoic acid (C22:0) is greatly decreased. Also in platelets, leukocytes, and to a lesser extent erythrocytes, the C26:0 concentrations and both the C26:0/C22:0 and C24:0/C22:0 ratios are greatly increased. The C24:0/C22:0 ratio is significantly increased in plasma, platelets, and leukocytes, but not in erythrocytes. In X-ALD, the C26:0 concentration and the C26:0/C22:0 and C24:0/C22:0 ratios are significantly increased in plasma, fibroblasts, platelets, and leukocytes, but the erythrocytes show substantial overlap in the 5-90% ranges between controls and patients. In RCDP, slightly increased C26:0 and C26:0/C22:0 ratios are found in erythrocytes, platelets, and leukocytes, but not in plasma and fibroblasts. We conclude that plasma and fibroblasts are the specimens of choice for biochemical diagnosis of Zellweger syndrome and X-ALD, respectively. The slight increase in C26:0 in blood cells of RCDP patients suggests a decreased flux of very-long-chain fatty acids through the peroxisomal beta-oxidation pathway in liver in this genetic disorder.

Since very long-chain fatty acids with a chain length of 24 carbons or more are known to accumulate in tissues and body fluids from patients with the cerebrohepato-renal (Zellweger) syndrome, infantile Refsum disease, neonatal adrenoleukodystrophy and X-linked adrenoleukodystrophy, we studied very long-chain fatty acid oxidation in cultured skin fibroblasts from these patients. In this paper, we report that in accordance with earlier results the first step in the beta-oxidation of the very long-chain fatty acid lignoceric acid (C24:0) primarily occurs in peroxisomes in control human skin fibroblasts. Furthermore, it was found that peroxisomal lignoceric acid beta-oxidation was strongly deficient in fibroblasts from patients with Zellweger syndrome, infantile Refsum disease, neonatal and X-linked adrenoleukodystrophy, which explains for the accumulation of very long-chain fatty acids in all four disease entities. In Zellweger syndrome, infantile Refsum disease and neonatal adrenoleukodystrophy the impairment in peroxisomal very long-chain fatty acid beta-oxidation is probably caused by a strong deficiency of all peroxisomal beta-oxidation enzyme proteins due to a deficiency of peroxisomes.

We have investigated the pathways involved in the peroxisomal oxidation of palmitate and lignocerate, measured as the cyanide-insensitive formation of acetyl units, in rat-liver homogenates. The peroxisomal beta-oxidation of both fatty acids is dependent on the presence of ATP, coenzyme A, NAD+ and Mg2+. However, there is a striking difference in the dependence of the rate of oxidation of the two substrates on the concentration of the individual cofactors, especially ATP. The peroxisomal beta-oxidation of lignocerate was inhibited to a progressively greater extent by increasing concentrations of palmitate and vice versa. Activation of lignoceric acid to lignoceroyl-CoA, however, was not inhibited by increasing concentrations of palmitate, and vice versa. It can be concluded that the peroxisomal palmitate and lignocerate beta-oxidation pathways differ in at least one enzymic reaction (the synthetase), but that the two pathways share at least one common step.

We investigated the peroxisomal fatty acid beta-oxidation system in liver and cultured skin fibroblasts from patients with X-linked adrenoleukodystrophy known to accumulate very long chain fatty acids. In order to examine whether the deficient peroxisomal oxidation of very long chain fatty acids in these patients results from a deficiency in one of the peroxisomal beta-oxidation enzyme proteins (acyl-CoA oxidase, bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase) we carried out immunoblotting experiments using antibodies directed against the peroxisomal beta-oxidation enzyme proteins from rat liver. Furthermore, we studied the oxidation of palmitoyl-CoA and lignoceroyl-CoA in homogenates of fibroblasts from the patients. The results indicate that the peroxisomal beta-oxidation enzyme proteins are not only present immunologically but also functionally active which suggests that the defect in X-linked adrenoleukodystrophy is, indeed, as recently suggested by Hashmi and coworkers (FEBS Lett 1986;196:247-250) at the level of a deficient peroxisomal activation of very long chain fatty acids.

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 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.

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 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).