Plasma polyenoic very-long-chain fatty acids in peroxisomal disease: biochemical discrimination of Zellweger's syndrome from other phenotypes

The plasma of patients with inherited defects in peroxisomal biogenesis (ie, Zellweger's syndrome, infantile Refsum's disease, and neonatal adrenoleukodystrophy) shows evidence of a disturbance in the metabolism of saturated and monoenoic fatty acids with carbon chain lengths greater than 22 (VLCFA). Zellweger's syndrome plasma alone contains, in addition, increased amounts of a number of n-6 polyenoic VLCFA including 24:5, 26:5, 28:5, 30:5, and 30:6 fatty acids. These fatty acids facilitate the biochemical discrimination of Zellweger's syndrome from other related phenotypes.     

Plasmalogen deficiency in cultured skin fibroblasts from neonatal adrenoleukodystrophy

The plasmalogen ratio (defined as area ratio of lysophosphatidylethanolamine to the diacyl form of phosphatidylethanolamine) was investigated in cultured skin fibroblasts from neonatal adrenoleukodystrophy (N = 4) and X-linked recessive (N = 3) in addition to Zellweger syndrome (N = 3) because plasmalogen was reported to be reduced in Zellweger syndrome. The ratio was markedly decreased in all cases of Zellweger syndrome studied and in three of the four cases of neonatal adrenoleukodystrophy, whereas it was normal in the X-linked cases. This is the first documentation of a plasmalogen deficiency in neonatal adrenoleukodystrophy.     

Analysis of very long-chain fatty acids and plasmalogen in the erythrocyte membrane: a simple method for the detection of peroxisomal disorders and discrimination between adrenoleukodystrophy and Zellweger syndrome

We analyzed the sphingomyelin very long-chain fatty acids (VLCFAs) and phosphatidylethanolamine (PE) plasmalogen contents of the erythrocyte membrane in patients suffering from peroxisomal disorders. In a patient with Zellweger syndrome, both a decrease in the PE plasmalogen content and an increase in the sphingomyelin VLCFAs content of the erythrocyte membrane were noted. In patients with adrenoleukodystrophy, however, there was no decrease in PE plasmalogen, although the sphingomyelin VLCFAs content of the membrane was significantly increased in comparison with control values. Analyses of both sphingomyelin VLCFAs and PE plasmalogen were carried out simultaneously, using both the same process and the same sample.     

Plasma and skin fibroblast C26 fatty acids in infantile Refsum's disease

In infantile and adult Refsum's disease, the activity of phytanic acid oxidase is low in skin fibroblasts, but plasma phytanic acid levels are high. Cultured skin fibroblasts and plasma from patients with the infantile, but not the adult, disorder show marked increases in the concentration of the long-chain fatty acid, hexacosanoic acid (C26), a feature once thought pathognomonic of adrenoleukodystrophy or Zellweger's syndrome.     

Abnormal profiles of polyunsaturated fatty acids in the brain, liver, kidney and retina of patients with peroxisomal disorders.

The polyunsaturated fatty acid (PUFA) composition of the brain was studied in 8 patients with Zellweger's syndrome (ZS), 3 with neonatal adrenoleukodystrophy (NALD), one with bifunctional enzyme deficiency (BED), one with X-linked adrenoleukodystrophy (X-ALD), and one with adrenomyeloneuropathy (AMN). The PUFA composition of the liver, kidney and retina was studied in 8, 6 and 1 patients with ZS, respectively. An infant with NALD and a child with rhizomelic chondrodysplasia punctata (RCDP) were also studied for the PUFA composition of the liver. The liver and kidney of the patient with X-ALD and the liver of the patient with AMN were included in the study. The fatty acid values in the peroxisomal patients were compared with control data obtained in the normal developing brain (38 cases), liver (9 cases), kidney (7 cases) and retina (16 cases). The brain of a patient with metachromatic leukodystrophy (MLD) and the liver of a child with Krabbe's disease (KD) were also studied for comparison. The most constant and severe abnormality in all the peroxisomal patients was a drastic decrease in the total amount of docosahexaenoic acid (22:6 omega 3), especially in the brain. The other product of delta 4-desaturation, 22:5 omega 6, was generally decreased in the brain, liver and kidney of the ZS patients, but very much increased in the brain of two patients with NALD. The 22:6 omega 3/22:4 omega 6 ratio, which remains quite constant throughout normal brain development, was consistently decreased in the peroxisomal brain, in ZS as well as in NALD. This study confirms that, in classical Zellweger's syndrome, the two products of delta 4-desaturation are affected. In contrast, in neonatal adrenoleukodystrophy the deficiency is probably restricted to the omega 3 product of delta 4-desaturation, docosahexaenoic acid, especially in the brain, while the other product, 22:5 omega 6, is either normal or increased, perhaps in an attempt to compensate for the 22:6 omega 3 deficiency in brain membranes.     

Gas chromatography/mass spectrometry analysis of very long chain fatty acids, docosahexaenoic acid, phytanic acid and plasmalogen for the screening of peroxisomal disorders

Very long chain fatty acids (VLCFAs) and docosahexaenoic acid (DHA), phytanic acid, and plasmalogens are usually measured individually. A novel method for the screening of peroxisomal disorders, using gas chromatography/mass spectrometry (GC/MS), was developed. Saturated and unsaturated fatty acids, including VLCFAs and DHA, phytanic acid, and plasmalogen were detected by a selected ion monitoring-electron impact method, using 100 microl of serum or plasma. Methyl-esterification and extraction could be done in one tube, and data were obtained within 4 h. All patients with Zellweger syndrome (ZS), X-linked adrenoleukodystrophy (ALD), isolated deficiency of peroxisomal beta-oxidation enzyme, and most ALD carriers showed increased VLCFA ratios, including C24:0/C22:0, C25:0/C22:0 and C26:0/C22:0. The ratio of DHA to palmitic acid (C16:0) and plasmalogen (measured as hexadecanal dimethyl acetal) to C16:0 in ZS patients was significantly lower than for the controls (P<0.001 for healthy high school students, P<0.05 for infants with other disorders). Plasmalogen was also decreased in patients with isolated deficiency of plasmalogen biosynthesis. Two of eight patients with ZS, two of four with RCDP, and all of three classical Refsum patients showed increased levels of phytanic acid. This method will simplify the screening for peroxisomal disorders.     

Clinical and biochemical heterogeneity in conditions with phytanic acid accumulation

Phytanic acid accumulation has for more than 20 years been used as a diagnostic criterion of Refsum's disease. Recently, however, phytanic acid has also been found in peroxisomal disorders (Zellweger's syndrome, neonatal adrenoleukodystrophy, infantile Refsum's syndrome, rhizomelic chondrodysplasia punctata). The 17 patients with Refsum's disease in the present study had serum phytanic acid values differing from 73 to less than 0.5 mg/dl (normal). alpha-Oxidation of phytanic acid in skin fibroblast cultures showed a defective capacity in all, with only small differences in residual activity. Phytanic acid determinations in serum from 3 of the 7 patients with peroxisomal disorders showed slightly elevated levels in 2. The alpha-oxidation capacity in the fibroblasts was defective in all, with a residual activity similar to that of Refsum's disease. An assay of the alpha-oxidation capacity may be useful in the diagnosis of both Refsum's disease and the peroxisomal disorders. The distinction between Refsum's disease and the peroxisomal disorders can easily be done on a clinical basis.     

Neonatal adrenoleukodystrophy. Impaired plasmalogen biosynthesis and peroxisomal beta-oxidation due to a deficiency of catalase-containing particles (peroxisomes) in cultured skin fibroblasts

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.     

Biochemical markers predicting survival in peroxisome biogenesis disorders

OBJECTIVE: To identify prognostic markers reflecting the extent of peroxisome dysfunction in primary skin fibroblasts from patients with peroxisome biogenesis disorders (PBD). BACKGROUND: PBD are a genetically heterogeneous group of disorders due to defects in at least 11 distinct genes. Zellweger syndrome is the prototype of this group of disorders, with neonatal adrenoleukodystrophy and infantile Refsum disease as milder variants. Common to these three disorders are liver disease, variable neurodevelopmental delay, retinopathy, and perceptive deafness. Because genotype-phenotype studies are complicated by the genetic heterogeneity among patients with PBD, the authors evaluated a series of biochemical markers as a measure of peroxisome dysfunction in skin fibroblasts. METHODS: Multiple peroxisomal functions including de novo plasmalogen synthesis, dihydroxyacetonephosphate acyltransferase (DHAPAT) activity, C26:0/C22:0 ratio, C26:0 and pristanic acid beta-oxidation, and phytanic acid alpha-oxidation were analyzed in fibroblasts from a series of patients with defined clinical phenotypes. RESULTS: A poor correlation with age at death was found for de novo plasmalogen synthesis, C26:0/C22:0 ratio, and phytanic acid alpha-oxidation. A fairly good correlation was found for pristanic acid beta-oxidation, but the best correlation was found for DHAPAT activity and C26:0 beta-oxidation. A mathematic combination of DHAPAT activity and C26:0 beta-oxidation showed an even better correlation. CONCLUSIONS: DHAPAT activity and C26:0 beta-oxidation are the best markers in predicting life expectancy of patients with PBD. Combination of both markers gives an even better prediction. These results contribute to the management of patients with PBD.     

Severe deficiency of docosahexaenoic acid in peroxisomal disorders: a defect of delta 4 desaturation?

In confirmation of previous findings, patients with Zellweger's syndrome had extremely low levels of docosahexaenoic acid (22:6 omega 3) in the brain, liver, and kidneys. The other product of delta 4 desaturation, 22:5 omega 6, was also very significantly decreased, as were the ratios 22:6 omega 3/22:5 omega 3 and 22:5 omega 6/22:4 omega 6, especially in the brain and liver of the Zellweger patients. The infant with pseudo-Zellweger's syndrome also had very low levels of 22:6 omega 3 and of the ratio 22:6 omega 3/22:5 omega 3 in all tissues, especially in the brain, where the index 22:5 omega 6/22:4 omega 6 was also very significantly reduced. The ratio 22:6 omega 3/22:4 omega 6 was markedly decreased in all tissues, in Zellweger's as well as in pseudo-Zellweger's syndrome. The findings reported here strongly reinforce the hypothesis of a new enzymatic defect in peroxisomal disorders involving the desaturation of long polyunsaturated fatty acids, especially of the omega 3 family.     

A comparison of erythrocytes, lymphocytes and blood plasma as samples in fatty acid analysis for the diagnosis of adrenoleukodystrophy

We studied the very-long-chain fatty acids of blood plasma, erythrocyte membranes and lymphocytes in 4 adrenoleukodystrophy patients, 5 adrenoleukodystrophy obligate carriers, 12 normal controls and 81 patients with various neurological disorders by high-performance liquid chromatography and compared the reliabilities in the diagnosis of adrenoleukodystrophy of these 3 components of peripheral blood. Of 81 patients with various neurological disorders, 2 myotonic dystrophy and 2 spinocerebellar degeneration patients showed increased ratios of C26:0 to C22:0 in erythrocyte membranes, but not in blood plasma and lymphocytes. None of the 12 normal controls showed increased ratios of C26:0 to C22:0 in erythrocyte membranes, blood plasma and lymphocytes. These results suggest that fatty acid analysis for the diagnosis of adrenoleukodystrophy is more reliable when blood plasma and lymphocytes are used than when erythrocyte membranes are used.     

Myopathy in an infant with a fatal peroxisomal disorder

An infant with neonatal adrenoleukodystrophy experienced extreme hypotonia and virtually continuous convulsions at four months of age and died. Light and electron microscopic examination revealed evidence of myopathy and the presence of mitochondrial inclusions. Concentrations of very long-chain fatty acids were elevated in blood and fibroblasts and the oxidation of 14C-labeled fatty acids was defective. Urinary pipecolic acid content was increased. Activity of the peroxisomal dihydroxyacetone phosphate acyltransferase, which catalyzes the first step in plasmalogen synthesis, was decreased.     

Adrenoleukodystrophy: survey of 303 cases: biochemistry, diagnosis, and therapy

Adrenoleukodystrophy (ALD) is a genetically determined disorder associated with progressive central demyelination and adrenal cortical insufficiency. All affected persons show increased levels of saturated unbranched very-long-chain fatty acids, particularly hexacosanoate (C26:0), because of impaired capacity to degrade these acids. This degradation normally takes place in a subcellular organelle called the peroxisome, and ALD, together with Zellweger's cerebrohepatorenal syndrome, is now considered to belong to the newly formed category of peroxisomal disorders. Biochemical assays permit prenatal diagnosis, as well as identification of most heterozygotes. We have identified 303 patients with ALD in 217 kindreds. These patients show a wide phenotypic variation. Sixty percent of patients had childhood ALD and 17% adrenomyeloneuropathy, both of which are X-linked, with the gene mapped to Xq28. Neonatal ALD, a distinct entity with autosomal recessive inheritance and points of resemblance to Zellweger's syndrome, accounted for 7% of the cases. Although excess C26:0 in the brain of patients with ALD is partially of dietary origin, dietary C26:0 restriction did not produce clear benefit. Bone marrow transplant lowered the plasma C26:0 level but failed to arrest neurological progression.     

Adrenoleukodystrophy: abnormality of "tightly bound" fatty acids in the erythrocyte membrane proteins

"Tightly bound" and "loosely bound" fatty acids in erythrocyte membranes were analyzed in three patients with adrenoleukodystrophy, three probable carriers, and eight controls. The ratios of C28:0 or C26:0 to C22:0 or C20:0 in the tightly bound fatty acids of three patients were significantly higher than those of controls, and the ratios in two of three probable carriers were higher than those in controls. The ratios of C26:0 to C22:0 or C20:0 in the loosely bound fatty acids of three patients and three probable carriers were also significantly higher than those of controls. Since tightly bound fatty acids in membrane proteins are found not only in erythrocyte membranes but also in myelin proteins, the abnormality of tightly bound fatty acids may be related to the demyelination in adrenoleukodystrophy.     

Immunohistochemistry for a bifunctional protein in patients with peroxisomal disorders

Immunohistochemical studies using antisera against bifunctional protein, a β-oxidation enzyme, were performed on liver, kidney, and brain tissue specimens from patients with peroxisomal disorders and from controls to investigate the distribution and development of peroxisomes. Bifunctional protein-positive granules were not found in patients with Zellweger syndrome or neonatal adrenoleukodystrophy, whereas positive immunoreactivity was observed from 8 and 6 weeks gestation in the liver and kidney, respectively, and in the brain, from 23–25 weeks in the brainstem neurons and from 12–14 weeks in the white matter glia, in controls. Bifunctional protein immunoreactivity then increased with gestation in the brain. These results suggest that bifunctional protein immunohistochemistry is useful for the detection of peroxisomes, which are closely related to neuronal maturation and gliogenesis in premyelination in human brain development.     

Circulating plasmalogen levels and Alzheimer Disease Assessment Scale–Cognitive scores in Alzheimer patients

Background

Plasmalogens, which are key structural phospholipids in brain membranes, are decreased in the brain and serum of patients with Alzheimer disease (AD). We performed this pilot study to evaluate the relation between the levels of circulating plasmalogens and Alzheimer Disease Assessment Scale–Cognitive (ADAS-Cog) scores in patients with AD.

Methods

We evaluated participants’ ADAS-Cog scores and serum plasmalogen levels. For the 40 included AD patients with an ADAS-Cog score between 20 and 46, we retested their ADAS-Cog score 1 year later. The levels of docosahexaenoic acid plasmalogen were measured by use of liquid chromatography–tandem mass spectrometry.

Results

We found that the ADAS-Cog score increased significantly in AD patients with circulating plasmalogen levels that were ≤ 75% of that of age-matched controls at entry into the study. There was no change in score among participants with normal serum plasmalogen levels at baseline (> 75%).

Limitations

This was a pilot study with 40 patients, and the results require validation in a larger population.

Conclusion

Our study demonstrates that decreased levels of plasmalogen precursors in the central nervous system correlate with functional decline (as measured by ADAS-Cog scores) in AD patients. The use of both ADAS-Cog and serum plasmalogen data may be a more accurate way of predicting cognitive decline in AD patients, and may be used to decrease the risk of including patients with no cognitive decline in the placebo arm of a drug trial.     

A pathological study of a peripheral nerve in a case of neonatal adrenoleukodystrophy

Summary The pathological findings for a sural nerve biopsy specimen in a case of neonatal adrenoleukodystrophy are described. The density and total number of myelinated fibers in the patient showed no significant changes compared with controls. On electric microscopy, however, thickness of the myelin was smaller in the patient than in controls. Some linear or trilamellar inclusion bodies were found in Schwann cells and fibroblasts, similar to those found in X-linked adrenoleukodystrophy. Büngner's bands were also seen on electron microscopy, and myelin ovoids and balls were seen in teased fibers. These results show that a sural nerve biopsy is useful for the diagnosis of neonatal adrenoleukodystrophy. We suspect that axonal or neuronal degeneration occurs with changes in myelin in neonatal adrenoleukodystrophy.     

The cerebro-hepato-renal (Zellweger) syndrome: lamellar lipid profiles in adrenocortical, hepatic mesenchymal, astrocyte cells and increased levels of very long chain fatty acids and phytanic acid in the plasma

Clinical, radiological, histological and biochemical aspects of two cases of cerebro-hepato-renal syndrome (CHRS) are reported. CT scan disclosed a demyelinating process and gyral abnormalities reflecting the observed neuropathological findings. Trilamellar and lamellar inclusions were found in brain astrocytes, hepatic mesenchymal and adrenal cells. The morphologic features of these inclusions are similar to those observed in childhood adrenoleukodystrophy, neonatal adrenoleukodystrophy and infantile Refsum's disease. In the two CHRS patients, increased plasma levels of very long chain fatty acids (C26:1, C26:0) and phytanic acid were in the same range as those observed in seven other instances of neonatal adrenoleukodystrophy. The presence of increased plasma levels of phytanic acid in these disorders suggests that phytanate oxidase activity is, at least, partially located in peroxisomes.     

Adrenoleukodystrophy: a correlation between saturated very long-chain fatty acids in mononuclear cells and phenotype

Saturated very long-chain fatty acids in erythrocyte membranes, blood plasma, and mononuclear cells were studied in 4 patients with childhood-adolescent adrenoleukodystrophy and 4 patients with adult adrenoleukodystrophy and 19 normal control subjects by using high-performance liquid chromatography. Ratios of C26:0 to C22:0 in mononuclear cells, erythrocyte membranes, and blood plasma in patients with childhood-adolescent and adult adrenoleukodystrophy were significantly higher than in normal control subjects. Furthermore, ratios of C26:0 to C22:0 in mononuclear cells were significantly higher in patients with childhood-adolescent adrenoleukodystrophy than in patients with adult adrenoleukodystrophy, whereas there was no significant difference in the ratios in erythrocyte membranes and blood plasma between the two groups of patients with adrenoleukodystrophy. These results suggest that there is a correlation between phenotype and ratio of C26:0 to C22:0 within mononuclear cells in patients with adrenoleukodystrophy.