Are the total antioxidant capacity and superoxide dismutase (SOD) activity levels different between adrenoleukodystrophy (ALD) phenotypes?
In a prospective sample of 30 prospective samples and 30 independent retrospective samples, plasma SOD and monocyte total antioxidant capacity levels were progressively reduced between healthy controls, heterozygote female carriers, and patients with adrenomyeloneuropathy or cerebral ALD. Plasma SOD activity levels correlated inversely with brain magnetic resonance imaging severity score and, in addition, showed a reduction over time prior to and at cerebral onset.
Decreased SOD activity in plasma may be a potential biomarker preceding cerebral ALD onset.
X-linked adrenoleukodystrophy (ALD) may switch phenotype to the fatal cerebral form (ie, cerebral ALD [cALD]), the cause of which is unknown. Determining differences in antioxidant capacity and superoxide dismutase (SOD) levels between phenotypes may allow for the generation of a clinical biomarker for predicting the onset of cALD, as well as initiating a more timely lifesaving therapy.
To identify variations in the levels of antioxidant capacity and SOD activity between ALD phenotypes in patients with cALD or adrenomyeloneuropathy (AMN), heterozygote female carriers, and healthy controls and, in addition, correlate antioxidant levels with clinical outcome scores to determine a possible predictive value.
Design, Setting, and Participants
Samples of monocytes and blood plasma were prospectively collected from healthy controls, heterozygote female carriers, and patients with AMN or cALD. We are counting each patient as 1 sample in our study. Because adrenoleukodystrophy is an X-linked disease, the affected group populations of cALD and AMN are all male. The heterozygote carriers are all female. The samples were assayed for total antioxidant capacity and SOD activity. The data were collected in an academic hospital setting. Eligibility criteria included patients who received a diagnosis of ALD and heterozygote female carriers, both of which groups were compared with age-matched controls. The prospective samples (n = 30) were collected between January 2015 to January 2016, and existing samples were collected from tissue storage banks at the Kennedy Krieger Institute (n = 30). The analyses were performed during the first 3 months of 2016.
Main Outcome and Measures
Commercially available total antioxidant capacity and SOD assays were performed on samples of monocytes and blood plasma and correlated with magnetic resonance imaging severity score.
A reduction in antioxidant capacity was shown between the healthy controls (0.225 mmol trolox equivalent) and heterozygote carriers (0.181 mmol trolox equivalent), and significant reductions were seen between healthy controls and patients with AMN (0.102 mmol trolox equivalent; P < .01), as well as healthy controls and patients with cALD (0.042 mmol trolox equivalent; P < .01). Superoxide dismutase activity in human blood plasma mirrored these reductions between prospectively collected samples from healthy controls (2.66 units/mg protein) and samples from heterozygote female carriers (1.91 units/mg protein), patients with AMN (1.39 units/mg protein; P = .01), and patients with cALD (0.8 units/mg protein; P < .01). Further analysis of SOD activity in biobank samples showed significant reductions between patients with AMN (0.89 units/mg protein) and patients with cALD (0.18 units/mg protein) (P = .03). Plasma SOD levels from patients with cALD demonstrated an inverse correlation to brain magnetic resonance imaging severity score (R2 = 0.75, P < .002). Longitudinal plasma SOD samples from the same patients (n = 4) showed decreased activity prior to and at the time of cerebral diagnosis over a period of 13 to 42 months (mean period, 24 months).
Conclusions and Relevance
Plasma SOD may serve as a potential biomarker for cerebral disease in ALD following future prospective studies.
X-linked adrenoleukodystrophy (ALD) is a rare progressive neurometabolic disorder with an incidence of 1 in 17 000.1 The underlying mutations are located in the ABCD1 gene, which encodes a peroxisomal membrane protein associated with very-long-chain fatty acid (VLCFA) metabolism. The ALD protein putatively transports the coenzyme A derivatives of VLCFA, allowing for VLCFA degradation.2,3 Dysfunction of the ALD protein results in VLCFA accumulation, which is seen in the nervous system of all ALD phenotypes, including female carriers. In affected males, accumulation is additionally seen in adrenal glands, testes, and blood irrespective of mutation.
Adrenoleukodystrophy presents as multiple phenotypes. Symptoms do not appear during the first 4 years of life, but adrenomyeloneuropathy (AMN), a slow progressive dying-back axonopathy of the spinal cord, will eventually develop in all patients, with symptoms typically appearing in the third and fourth decade of life. Patients with ALD are at risk of developing deadly rapid demyelinating cerebral disease (cerebral ALD [cALD]), both in childhood and later in life. Symptoms of AMN vary between patients and commonly include spastic paraparesis, sensory ataxia, sphincter dysfunction, and impotence, which are sometimes accompanied by pain.
While cALD manifests in about 60% of patients with the diagnosis, a genotype-phenotype correlation has not been shown, nor is the cALD onset trigger known.4 Phenotype switching to cALD typically peaks in either between 4 and 8 years (30%-40% of cases) or in adult years (20% of cases). Cerebral ALD typically results in complete disability or death within 4 years of diagnosis.2,5 Only traumatic brain injury has been shown to induce brain disease in a few cases.6
A hematopoietic stem cell transplant provides the only established lifesaving therapy option for cALD, halting disease progression some months after intervention. A timely diagnosis of cerebral onset is therefore of critical value in avoiding neurological deficits and lowering mortality. A diagnosis of cALD is confirmed by magnetic resonance imaging (MRI) in conjunction with clinical suspicion of progression.7
Magnetic resonance imaging findings have been shown to either predate brain disease or appear simultaneously with symptomatic onset. T2-weighted hyperintensity of the posterior white matter is most common, whereas other presentations include frontal periventricular or projection fiber patterns.8 A 34-point ALD MRI severity score was proposed by Loes et al9 and is now the gold standard in determining cALD disease severity. Any cerebral involvement constitutes a positive score (≥1). Very early disease may be a score below 4, early stage scores between 4 and 8, whereas late stage scores between 9 and 13, and advanced cALD scores above 13. Disease severity has been shown to determine viability of therapeutic options because therapy outcome is dependent on timely intervention, and a score below 10 is desirable.10
Oxidative stress has been portrayed as taking multiple central roles in the cellular pathology of ALD. Excess C26:0 has been shown to generate reactive oxygen species (ROS) and decreases mitochondrial membrane potential in human cALD fibroblasts.11 Furthermore, dysfunction of the ubiquitin-proteasome “misfolded protein clearance system” has been described as a result of the accumulation of oxidized proteins.12 Oxidative lesions may transform lipid properties and induce mitochondrial and nuclear DNA mutations, leading to further mitochondrial dysfunction, and thereby enhance oxidative stress. A proposed mechanism has been hypothesized positing that mitochondrial membrane permeability or microviscosity is altered by C26:0 accumulation, leading to electron leakage and ROS.12
Polymorphisms of superoxide dismutase (SOD), an enzymatic antioxidant, have been associated with the cALD phenotype.13 We hypothesize that SOD levels vary between phenotypes and within cALD.
A diagnosis of ALD is confirmed via measurement of elevated VLCFA in blood samples.14 Recent implementation of newborn screening in some North American states will allow those with an ABCD1 mutation to receive regular consultation and MRI, ameliorating current initial detection paradigms.15 A more timely, even predictive detection of cALD onset may reduce the posttransplant neurological deficit and mortality.
Our objective was to determine antioxidant levels, specifically SOD activity and whether these SOD levels affected the total antioxidant capacity, in blood samples from patients with ALD. In addition, we aim to assess their suitability as potential biomarkers.
Prospective Blood Sample Collection
The studies conducted herein were approved by the Johns Hopkins Medical Institution’s institutional review board. Thirty milliliters of fresh blood were obtained via venipuncture into heparinized and nonheparinized collection tubes from patients at the Kennedy Krieger Institute after obtaining written informed consent. The blood samples were transported immediately, uncooled, to the laboratory for processing. Samples were collected from January 2015 to January 2016.
Plasma was obtained by centrifugation of whole blood at 400 g for 15 minutes at room temperature. Aliquots were stored at −80°C.
Peripheral blood monocyte cultures were derived using double gradient Ficoll (Sigma-Aldrich) and Percoll (Sigma-Aldrich) separation as described by Menck et al.16 Monocytes were cultured for 7 days in Minimum Essential Medium (Sigma-Aldrich) to guarantee adherence and thereby increase purity, with 5% fetal bovine serum (Sigma-Aldrich), 1% penicillin-streptomycin solution (Sigma-Aldrich), 1% oral passive immunization (Sigma-Aldrich), and 1% nonessential amino acid solution (Sigma-Aldrich). Cell homogenization was performed via probe sonication at room temperature for 10 seconds (Branson Model W185D Sonifier Cell Disruptor) followed by immediate assay.
Total SOD activity was detected using a commercially available kit (Cayman Chemical) and performed at least 3 times in duplicate per sample. Total antioxidant capacity was assessed. Spectrophotometry was performed using a Spectramax M5 (Molecular Devices). For each patient, 2 independent assays were performed in triplicate, and mean (SEM) values were calculated.
Initially, prospectively collected fresh tissue samples were assayed. To further validate our results with these samples, tissue storage banks at the Kennedy Krieger Institute were searched for samples from patients with AMN or cALD (patients ranged in age from 18 months to 15 years). Eligible patients with cALD required a positive MRI severity score. Blood plasma was stored at −80°C, having undergone multiple freeze/thaw cycles for numerous other experiments.
Freeze/Thaw Cycle of Blood Plasma
To address the degradation of enzyme activity due to multiple thawing and refreezing, 3 plasma samples obtained from healthy controls were frozen 6 times. To thaw, the sample was placed on ice for 2 hours, then left at room temperature for a maximum of 6 hours, and then aliquoted. For freezing, the sample was stored at −80°C for at least 16 hours.
One-way analysis of variance was performed to determine statistical significance between multiple phenotypes. The Pearson correlation was used for modeling the relationship between clinical MRI severity score and antioxidant sample values. An unpaired t test was used to discern significance between biobank AMN and cALD plasma SOD samples. Findings were considered statistically significant at P < .05.
Monocyte Total Antioxidant Capacity
We prospectively sought to determine whether total antioxidant capacity would differ between healthy controls and patients with AMN or cALD. An investigation of ALD phenotypes measured the total antioxidant capacity of monocytes; a tissue’s ability to reduce radical species provided an initial perspective in discriminating between the ALD phenotypes. As shown in Figure 1, the 9 samples obtained from healthy controls showed the highest antioxidant capacity at 0.225 mmol trolox equivalent, with progressively lower capacity levels in the 3 samples obtained from heterozygous female carriers (0.181 mmol trolox equivalent), the 10 samples obtained from patients with AMN (0.102 mmol trolox equivalent), and the 8 samples obtained from patients with cALD (0.042 mmol trolox equivalent). The decreases in capacity in patients with AMN or cALD vs healthy controls were statistically significant (P = .01 and P < .001, respectively). The difference in capacity between patients with cALD and heterozygous female carriers was also significant (P = .02) (Figure 1).
SOD Activity in Blood Plasma
To test our initial hypothesis, SOD activity was determined in prospectively collected blood plasma samples. Superoxide dismutase plays a crucial role, catalyzing the dismutation of superoxide radicals to hydrogen peroxide or oxygen. This prevents the dangerous Haber-Weiss reaction, which generates hydroxyl radicals.17
The levels of SOD activity were determined in the same samples previously used in isolating monocytes (Figure 2A). Reductions are shown between the prospectively collected samples from healthy controls (2.66 units/mg protein) and the samples from heterozygote female carriers (1.91 units/mg protein). Significant reductions were also shown between healthy controls and patients with AMN (1.39 units/mg protein; P = .01), as well as patients with cALD (0.8 units/mg protein; P < .01).
Next, to independently validate these data, 10 biobank plasma samples from patients with AMN and 20 biobank plasma samples from patients with cALD were obtained from the Peroxisomal Diseases Laboratory and the Kennedy Krieger Institute’s Intellectual and Developmental Diseases Research Center, and SOD activity levels were determined. These samples showed a significant reduction in the biobank AMN cohort (0.89 units/mg protein) compared with the cALD cohort (0.18 units/mg protein) (P = .03) (Figure 2B).
SOD Activity and ALD MRI Score Correlation
To determine whether SOD activity differed within 1 phenotype and whether these correlated clinically, SOD activity levels were measured in both biobank and fresh blood plasma samples obtained from patients with cALD who underwent MRI within the 6-month period of the blood sample collection. An inverse correlation (Pearson R2 = 0.75, P = .002) is shown in decreasing SOD activity vs an increasing ALD MRI severity score (Figure 3).
We examined longitudinal data to determine whether differences in SOD activity were consistent over time. The Peroxisomal Diseases Laboratory database was searched for patients who had simultaneous MRI and blood samples obtained on multiple visits, prior to developing a positive MRI severity score. Four patients fulfilled these criteria, and SOD activity levels were measured in these biobank plasma samples and plotted as a function of time. All patients showed a decrease in SOD activity over time. Three patients had 2 or more time points: one showed no SOD activity 172 days prior to cALD diagnosis (defined by an MRI severity score of at least 1), another showed no SOD activity at cerebral onset, and another showed SOD activity (0.66 units/mg protein) at cALD diagnosis (MRI severity score of 3.5) (Figure 4).
Degradation of SOD Activity
To determine whether a reduction in SOD activity was the result of denaturation processes expedited by thawing and refreezing and by time, freshly prepared plasma samples obtained from 3 healthy controls were subjected to multiple freeze/thaw cycles prior to measurement of SOD activity. The levels SOD activity show a steeper reduction in the first 2 freeze/thaw cycles, asymptotically stabilizing 25% below initial levels (eFigure in the Supplement).
Our initial investigation indicated significant differences in antioxidant capacity between ALD phenotypes in prospectively collected plasma samples in which we measured monocyte cell lysate and SOD levels. Then, to independently validate these data, stored AMN and cALD plasma samples were assayed for SOD activity, replicating previous prospective results. Both fresh and biobank cALD plasma SOD levels were correlated with their MRI severity score and showed a significant inverse correlation to increasing disease severity. Finally, patients with samples from multiple time points before and after cALD onset were plotted.
Monocyte antioxidant capacity appears to be in lockstep with plasma SOD activity. While SOD activity is encompassed in the antioxidant capacity assay, an assortment of other potentially interesting redox molecules may additionally play a role in the reduced capacity. The total antioxidant assay relies on the ability of antioxidants to inhibit the oxidation of ABTS (2,2’-azino-di-[3-ethylbenzthiazoline-6-sulfonic acid]) to ABTS+ by metmyoglobin. The degree to which intracellular SOD contributes to the assay in monocyte cell lysate is not known.
Physiologically, ROS and their derivative products have been shown to induce compensatory proinflammatory pathway mediators, including NF-κB.18 In migrating and binding to intranuclear gene promoter region elements, NF-κB induces the expression of genes encoding proinflammatory cytokines and enzymes, one of which is SOD2.19 In addition, increases in ROS-induced SOD expression are mediated by the proinflammatory cytokines tumor necrosis factor and IL-1.20 Superoxide dismutase levels have been shown to be reduced in the erythrocytes of patients with Down syndrome, Alzheimer disease, or Parkinson disease, with levels continuing to decrease as the disease advances.21 Interestingly, the level of SOD has been shown to decrease in erythrocytes and plasma in other inflammatory states and in smokers vs nonsmokers,22 A downregulation may be the case in chronic oxidative stress or as a result of ALD-specific pathology.
Vargas et al23 showed increased SOD levels in cALD erythrocytes, attributing the higher levels to a compensatory mechanism in the presence of sustained oxidative stress. Although these data seem contrary to our monocyte and plasma findings, we suggest that it is possible that while erythrocytes show higher levels of intracellular SOD activity, mature anucleated erythrocytes, unlike phagocytic cells, are not responsive to epigenetic modulation during the phenotype shift in responding to oxidative stress. As ALD protein expression is shown to differ between immune cells,24 differing levels of VLCFA accumulation and resultant oxidative stress may too affect SOD levels in these species. Future investigations should compare longitudinal erythrocyte, monocyte, and plasma antioxidant levels.
While these SOD and antioxidant capacity data supported our a priori hypothesis, a post hoc hypothesis was generated proposing that SOD levels within cALD may correspond to disease severity. To view SOD activity as a function of disease severity, MRI severity scores were used, and an inverse correlation between SOD activity and MRI severity score was observed in the cALD population. Lower SOD levels may be a response to chronic oxidative stress processes or may result from upstream ALD-specific pathogenic processes, even the ABCD1 mutation itself.
No conclusive stance regarding the predictive value of SOD for cerebral onset may be ascertained from the plotted patient samples from multiple time points. However, these data do indicate that there is a reduction in SOD levels precipitating, preceding, or during cerebral onset and not an initially lower SOD activity level in those patients with AMN who ultimately develop cerebral symptoms.
Concerns of confounding data or bias due to the degradation of SOD activity as a result of long-term storage of samples may be cautiously dismissed because the older stored samples from the individuals with longitudinal data did not show decreased activity levels compared with the more recent biobank samples prior to cerebral disease. When stored at −80°C, SOD activity in samples of erythrocyte lysate has been reported to be comparable at 21 months to that of fresh samples.25 The intraindividual biological variability of SOD in blood was shown to maximally 15% over 6 months, supporting the use of the enzyme as a biomarker.26 Although SOD activity has been shown to differ between the sexes27 and to decrease significantly with advanced age (≥60 years of age),28 cerebral onset is seen only in male patients with ALD, typically peaking in early childhood or young adulthood.
Future plasma samples should be obtained systematically at each visit from all patients with ALD who provided informed consent, and these samples should be aliquoted prior to initial freezing at −80°C. In addition, if MRI is performed, blood samples should be obtained and stored in a similar fashion.
Our data highlight a possible predictive value of SOD. The reduction in SOD level may temporally precipitate a phenotype shift and allow a prognostic-diagnostic value for cerebral onset, or it may reveal early differences in SOD levels, thus enabling the determination of a “low SOD level/high-risk cALD” subgroup. Future prospective studies investigating the reduction in longitudinal SOD activity may aid us in understanding the natural history of the AMN phenotype and may aid us in determining whether the rate of reduction of SOD activity varies within the AMN phenotype, accompanying symptom onset and progression, and whether a sharp decrease in SOD activity is detectable prior to cALD onset.
Superoxide dismutase shows promise as a potential biomarker and as a tool for expanding our understanding of ALD pathology. Our data establish phenotype-specific SOD activity levels, reducing over time in both AMN and cALD and correlating with cerebral disease severity quantified by the MRI severity score. Future studies may consider investigating the clinical use of plasma SOD activity as a predictive measure of cALD.
Corresponding Author: Ali Fatemi, MD, Moser Center for Leukodystrophies, Kennedy Krieger Institute, 707 N Broadway, Baltimore, MD 21218 (firstname.lastname@example.org).
Accepted for Publication: November 23, 2016.
Published Online: March 13, 2017. doi:10.1001/jamaneurol.2016.5715
Author Contributions: Dr Fatemi had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Turk, Theisen, Tiffany, Fatemi.
Acquisition, analysis, or interpretation of data: All authors.
Drafting of the manuscript: Turk.
Critical revision of the manuscript for important intellectual content: All authors.
Statistical analysis: Turk, Nemeth, Shi, Fatemi.
Obtained funding: Fatemi.
Administrative, technical, or material support: Turk, Nemeth, Marx, Shi, Jones, Moser, Watkins, Tiffany, Fatemi.
Study supervision: Turk, Raymond, Fatemi.
Conflict of Interest Disclosures: Dr Fatemi is a paid member of the drug and safety monitoring committees of Bluebird Bio Inc and Teva Pharmaceutical Industries Ltd and a paid consultant for Vertex Pharmaceuticals. No other disclosures are reported.
Funding/Support: This study was funded by the Brian’s Hope Foundation and the National Institutes of Health (grant P30HD024061 to Drs Fatima, Nemeth, and Jones).
Role of the Funder/Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, or interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Additional Contributions: We would like to thank the patients and their families for their participation in this study and Kim Hollandsworth, RN, at Kennedy Krieger Institute, John Hopkins Medical Institutions, for collecting blood samples. No compensation was received for her contributions.
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