Biomarker Identification, Safety, and Efficacy of High-Dose Antioxidants for Adrenomyeloneuropathy: a Phase II Pilot Study.

X-Adrenoleukodystrophy (X-ALD) and its adult-onset, most prevalent variant adrenomyeloneuropathy (AMN) are caused by mutations in the peroxisomal transporter of the very long-chain fatty acid ABCD1. AMN patients classically present spastic paraparesis that can progress over decades, and a satisfactory treatment is currently lacking. Oxidative stress is an early culprit in X-ALD pathogenesis. A combination of antioxidants halts the clinical progression and axonal damage in a murine model of AMN, providing a strong rationale for clinical translation. In this phase II pilot, open-label study, 13 subjects with AMN were administered a high dose of α-tocopherol, N-acetylcysteine, and α-lipoic acid in combination. The primary outcome was the validation of a set of biomarkers for monitoring the biological effects of this and future treatments. Functional clinical scales, the 6-minute walk test (6MWT), electrophysiological studies, and cerebral MRI served as secondary outcomes. Most biomarkers of oxidative damage and inflammation were normalized upon treatment, indicating an interlinked redox and inflammatory homeostasis. Two of the inflammatory markers, MCP1 and 15-HETE, were predictive of the response to treatment. We also observed a significant decrease in central motor conduction time, together with an improvement or stabilization of the 6MWT in 8/10 subjects. This study provides a series of biomarkers that are useful to monitor redox and pro-inflammatory target engagement in future trials, together with candidate biomarkers that may serve for patient stratification and disease progression, which merit replication in future clinical trials. Moreover, the clinical results suggest a positive signal for extending these studies to phase III randomized, placebo-controlled, longer-term trials with the actual identified dose. ClinicalTrials.gov Identifier: NCT01495260.

Protective effect of antioxidants on DNA damage in leukocytes from X-linked adrenoleukodystrophy patients.

Toxic metabolites accumulation and oxidative stress have been associated to the pathophysiology of X-linked adrenoleukodystrophy (X-ALD), an inborn error of peroxisome metabolism. Parameters of oxidative damage to proteins and lipids in X-ALD patients were already described in literature; however, DNA injuries were not studied yet. Considering that, the aims were to investigate DNA damage by comet assay in heterozygotes and symptomatic X-ALD patients, to look for associations between DNA damage and lipid peroxidation as measured by urinary 15-F2t-isoprostane; and to evaluate the in vitro effect of N-acetyl-l-cysteine (NAC), trolox (TRO) and rosuvastatin (RSV) on DNA damage in leukocytes from symptomatic patients. Symptomatic patients presented higher DNA damage levels than those found in heterozygotes and controls; heterozygotes and controls showed similar results. In order to investigate the in vitro antioxidant effect on DNA damage, whole blood cells from symptomatic patients were incubated with NAC (1 and 2.5mM), TRO (25 and 75 µM) and RSV (0.5, 2 and 5 µM) before DNA damage analysis. NAC, TRO and RSV, at all tested concentrations, were all capable to reduce DNA damage in symptomatic X-ALD patients until control levels. Finally, DNA damage correlated with urinary isoprostanes and plasmatic levels of TBA-RS and DCFH-DA, allowing to hypothesize that DNA damage might be induced by lipid peroxidation in symptomatic patients. The present work yields experimental evidence that NAC, TRO and RSV reduce the in vitro DNA injury in symptomatic X-ALD patients, what may suggest that the administration of these antioxidants might be considered as an adjuvant therapy for X-ALD.

Combined extraction of acyl carnitines and 26:0 lysophosphatidylcholine from dried blood spots: prospective newborn screening for X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a severe genetic disorder that affects the nervous system, and the adrenal cortex. Newborn screening for X-ALD has been proposed to allow improved diagnosis along with prospective monitoring and treatment for this severe disorder. Newborn dried whole blood spot (DBS) 26:0 lysophosphatidyl choline was validated as a diagnostic marker for X-ALD and other peroxisomal disorders of peroxisomal ß-oxidation. In this study, we developed a new one step extraction procedure that simultaneously extracts acyl carnitines and the lysophosphatidyl cholines from DBS. Further analysis of these metabolites has been performed by two different high throughput LC-MS/MS methods. The 26:0 lysophosphatidyl choline levels in this study were consistent with previously published values and discriminate between healthy and abnormal profiles. There is a very minor modification to the original acyl carnitine extraction procedure and our data indicates that there is no significant effect on acyl carnitine levels in DBS. Our new method potentially can be complementary to the current newborn screening panel. It successfully combines the existing method for acyl carnitine analysis and 26:0 lysophosphatidyl choline that can be applied for prospective X-ALD newborn screening.

Treatment of an adrenomyeloneuropathy patient with Lorenzo's oil and supplementation with docosahexaenoic acid--a case report.

This is a case report of adrenomyeloneuropathy (AMN), the adult variant of adrenoleukodystryphy (ALD). The diagnoses in the patient, aged 34, was confirmed via increased serum very long chain fatty acid concentration (VLCFA). Treatment started with the cholesterol lowering drug, atorvastatin, followed by add-on therapy with Lorenzo's oil (LO) and finally supplementation with docosahexaenoic acid (DHA). The magnetic resonance imaging (MRI) scan of the AMN patient before DHA treatment, already showed abnormal white matter in the brain. Although the MRI showed no neurological improvement after 6 months of DHA treatment, no selective progression of demyelination was detected in the AMN patient. Contrary to what was expected, LO failed to sustain or normalize the VLCFA levels or improve clinical symptoms. It was however, shown that DHA supplementation in addition to LO, increased DHA levels in both plasma and red blood cells (RBC). Additionally, the study showed evidence that the elongase activity in the elongation of eicosapentaenoic acid (EPA) to docosapentaenoic acid (DPA) might have been significantly compromised, due to the increased DHA levels.

Auditory brainstem response findings and peripheral auditory sensitivity in adrenoleukodystrophy.

Measurements of the auditory brainstem response (ABR) were obtained in 96 individuals with X-linked adrenoleukodystrophy (X-ALD). The patients were divided into five diagnostic groups on the basis of neurologic diagnosis. The five groups were cerebral childhood and adolescent, pure adrenomyeloneuropathy (pure AMN), adrenomyeloneuropathy cerebral (AMN cerebral), Addison's only and symptomatic female heterozygotes. Results indicated the presence of marked ABR abnormalities for all groups most frequently involving Wave V, followed by Wave III and Wave I. Abnormalities of all interpeak latency intervals (i.e., I-III, III-V and I-V) were observed for all groups. ABR abnormalities were most frequently seen in the AMN-cerebral and pure AMN groups but were also common in the symptomatic female heterozygote group. The ABRs in the cerebral childhood and adolescent group were the least impaired of the five groups examined. Age was found to be a significant independent predictor of bilateral ABR abnormalities but VLCFA levels, MRI Loes score, and duration of symptoms were not found to be independent predictors of bilateral ABR abnormalities after adjusting for ALD phenotype. Patients with AMN were significantly more likely to have bilateral ABR abnormalities than the cerebral childhood and adolescent group after adjusting for age, duration of symptoms, EDSS score, VLCFA levels and MRI Loes scores. The prevalence of peripheral hearing loss was not found to exceed that present in age and sex matched normal control groups derived from the NHANES (1999-2000), indicating a lack of association between peripheral hearing loss and X-linked adrenoleukodystrophy. It was concluded that: (1) auditory sensitivity in X-ALD is not significantly impaired; (2) ABR abnormalities are a frequent finding and may be caused by abnormalities of fiber tracts in the region of the lateral lemniscus and inferior colliculus; and, (3) the abnormalities progress slowly and appear to be associated mainly with the AMN phenotype.

Effect of dehydroepiandrosterone supplementation on fatty acid and hormone levels in patients with X-linked adrenoleucodystrophy.

OBJECTIVE: In X-linked adrenoleucodystrophy (X-ALD) the peroxisomal beta-oxidation of saturated very long-chain fatty acids (VLCFAs; carbon length > 22 atoms) is impaired. These fatty acids accumulate in blood and tissues, in particular in the nervous system, adrenal cortex and testis. Most patients have a primary adrenocortical insufficiency with low levels of cortisol and dehydroepiandrosterone (DHEA) and its sulphate ester (DHEA-S), collectively called DHEA(S). Surprisingly, very low plasma levels of DHEA(S) may be found when plasma cortisol and ACTH levels are normal. In animal studies DHEA administration had a peroxisome proliferating effect and induced the expression of peroxisomal enzymes involved in the beta-oxidation of fatty acids. PATIENTS AND DESIGN: To study the effect of DHEA on fatty acids in X-ALD patients, we conducted a randomized double-blind study in which 14 men (age range 21-63 years) and one boy (12 years) received 50 mg of DHEA or placebo for 3 months, followed by a 1-month wash-out period, then 3 months of placebo or vice versa. RESULTS: A significant rise was seen in the plasma levels of DHEA-S, Delta4-androstenedione and IGF-I. The elevated saturated VLCFAs in plasma and erythrocytes did not change. However, in erythrocytes significant decreases were found in the total amount of fatty acids, in C16:0, C18:0 and in C20:4omega-6, C22:5omega-6, C18:1omega-9, C20:1omega-9 and C20:3omega-9. In plasma, decreases were found for C18:1omega-9 and increases for C20:1omega-9. CONCLUSIONS: Dehydroepiandrosterone supplementation for 3 months did not lower the elevated plasma levels of saturated very long-chain fatty acids in patients with X-linked adrenoleucodystrophy. Instead, a decrease in saturated and mono- and polyunsaturated fatty acids in erythrocytes and plasma was found. An increase of C20:1omega-9 was found in plasma only.

[Frontal pseudo-tumoral form of adrenoleukodystrophy]. / Adrénoleucodystrophie à forme frontale pseudo-tumorale.

A case of adrenoleukodystrophy in a 9-year old boy revealed by a predominant frontal syndrome is reported. Brain MRI showed an unusual pseudo-tumoral frontal lesion. The diagnosis was confirmed by increased plasma levels of very long chain fatty acids. His young brother had an isolated adrenal insufficiency with normal brain MRI. The frontal predominance of the lesion and the clinical polymorphism of the disease in this family are discussed.

Determination of very-long-chain fatty acids in serum by gas chromatography-nitrogen-phosphorus detection following cyanomethylation.

A sensitive method for very-long-chain fatty acid analysis was developed by gas chromatography-nitrogen-phosphorus detection by using cyanomethyl derivatization. Bromoacetonitrile as alkylating reagent was used to improve nitrogen phosphorus detection detectability of compounds containing non-nitrogen. The carboxyl group of very-long-chain fatty acid was alkylated to cyanomethyl esters. Reaction conditions were 40 min at 60 degrees C under potassium carbonate base. Heptacosanoic acid was used as an internal standard and hexane was used as a solvent of extraction. The extraction yield was 82.8% or more, relative standard deviation of the precision test was 8.3% or more and the result of linearity test showed a good correlation coefficient of r2=0.999 in the range of 0.1-50 microg/ml. The quantification limits were 10 ng/ml when 0.5 ml of serum was used. The present method proved simple, rapid, inexpensive and resistant to contaminants. When it was applied to serum samples taken from patients with X-linked adrenoleukodystrophy which is a hereditary X-linked disorder characterized by progressive demyelination and adrenal insufficiency during childhood, relative increase of the concentration of hexacosanoic acid and the concentration ratios of hexacosanoic, lignoceric to behenic acid was observed in comparison with control samples.