Clinical characteristics are listed in . The mean EDSS for ACALD patients was 3.3 ± 1.9 (range = 1.5 to 6). The mean EDSS for AMN patients was 4.5 ± 3.1 (range = 1 to 8). The mean EDSS for female heterozygotes was 2.8 ± 0.9 (range = 2 to 4).
All 4 ACALD patients had characteristic T1 hypointense lesions in the splenium of the corpus callosum on MPRAGE images (see ). Two of the 4 AMN patients had T1 hypointense lesions confined to the bilateral corticospinal tracts. The remaining 2 AMN patients, all 4 female heterozygotes and all controls had no brain abnormalities on conventional sequences. In contrast to routine 1.5 Tesla images, MPRAGE at 7 Tesla showed high resolution of individual fiber tracts (resolution 0.56 × 0.56 × 1mm3).
1.5 Tesla versus 7 Tesla in adult X-ALD
All MRSI voxels were assigned to either predominantly white matter, or predominantly gray matter voxels based on visual inspection by a neuroradiologist. On average, 12 ± 5 (mean ± SD) voxels were assigned to predominantly white matter and 5 ± 2 voxels were assigned to predominantly gray matter. In addition, on three ACALD patients, 5 ± 1 voxels were assigned to lesions. Peaks of NAA, Cho, Cr, MI and Glu were detected in all patients and controls, and average values for metabolite ratios within normal appearing white matter and gray matter were determined and are shown in the bar graphs in and . No significant age difference between the 13 ALD patients (37±13) and 9 controls (32±8) was observed (p = 0.3). Neither in our control population nor in our X-ALD population did we find any significant correlations between age and metabolite ratios.
Metabolite Ratios in White Matter
Metabolite Ratios in Gray Matter
Metabolite Ratios in White Matter
Initially, MANOVA was performed using a model including four metabolite ratios, NAA/Cr, Cho/Cr, Glu/Cr and MI/Cr, in two tissue types, gray matter and NAWM. There was a significant difference between ACALD, AMN, female heterozygotes and controls (p = 0.01). Based on these results, we analyzed the individual four metabolite ratios in the two regions using ANOVA and used Holm's t-tests to isolate differences between the phenotypes (see ).
Compared to controls, we found significantly decreased NAA/Cr ratios in the normal appearing white matter of ACALD and AMN patients (p=0.003 and p = 0.028, respectively), but not in female heterozygotes. By contrast, Cho/Cr and MI/Cr metabolite ratios were increased in ACALD compared to controls (p = 0.014 and p = 0.002, respectively), but not in AMN patients and female heterozygotes. In all X-ALD patients, including female heterozygotes, white matter Glu/Cr showed a trend to be lower compared to normal controls (p = 0.09).
Among adult X-ALD phenotypes, MI/Cr was 46% higher in ACALD compared to AMN and female heterozygotes (p = 0.015 and p = 0.028, respectively). The Cho/Cr ratio was 21% higher in ACALD compared to AMN (p = 0.006). There was a trend for NAA/Cr to be lower in ACALD compared to AMN and female heterozygotes, although this was not significant. There were no significant differences between AMN and female heterozygotes.
Metabolite Ratios in Gray Matter
Compared to controls we found a decreased NAA/Cr ratio in the cortical gray matter (p = 0.022) () in male X-ALD patients. There was no significant elevation of the Cho/Cr, Glu/Cr and MI/Cr ratios in cortex compared with healthy volunteers.
Among adult X-ALD patients, ACALD patients had lower values of NAA/Cr compared to female heterozygotes (p = 0.022). There were no significant differences between AMN and ACALD or AMN and female heterozygotes.
A significant correlation between NAA/Cr and Glu/Cr (Spearman ρ = 0.66, p = 0. 002) was found. NAA/Cr and MI/Cr (Spearman ρ = - 0.49, p = 0. 03) as well as Glu/Cr and MI/Cr (Spearman ρ = -0.55, p = 0. 015) showed a significant negative correlation.
Relationship to Clinical Disability and PLASMA VLCFA
The scatter-plot matrix in demonstrates the interrelationship between the global metabolic estimates and clinical disability index (EDSS). Global metabolic ratios were defined as the metabolite ratio of all gray and white matter voxels combined per patient. EDSS demonstrated a significant association with the global MI/Cr ratio (Spearman ρ = 0.66, p = 0.039). Assessment of individual scale components revealed significant correlations between motor function and global metabolite ratios of Glu/Cr (Spearman ρ = -0.64, p = 0.003), MI/Cr (Spearman ρ = 0.67, p = 0.002), and NAA/Cr (Spearman ρ = -0.61, p = 0.002). This correlation was also present for bladder function (with Glu/Cr: Spearman ρ = -0.67, p = 0.002; with MI/Cr: Spearman ρ = 0.67, p = 0.002; with NAA/Cr: Spearman ρ = -0.59, p = 0.002) but not for sensory or mental function.
Metabolite Ratios, EDSS and plasma VLCFA
We found an inverse correlation between the patients' plasma VLCFA (absolute C26:0 levels) and their global NAA/Cr (Spearman ρ = -0.67, p = 0.02) (see ). On the other hand, we do not find any correlations between C26:0 and any of the other metabolic markers (MI/Cr – C26: Spearman ρ = 0.36, p = 0.38; Cho/Cr – C26: Spearman ρ = 0.11, p = 0.7; Glu/Cr – C26: Spearman ρ = -0.52, p = 0.18).
Proton MR spectroscopic imaging at 7 Tesla was used to study adult patients with X-ALD. Metabolite ratios of NAA/Cr, Cho/Cr, Glu/Cr and MI/Cr were analyzed in both white and gray matter. We hypothesized that adult phenotypes with X-ALD would differ in their neurochemistry, and we have found that among adult X-ALD phenotypes, MI/Cr was 46% higher and Cho/Cr 21% higher in normal appearing white matter of ACALD compared to AMN (p < 0.05). There were no significant differences between AMN and female heterozygotes.
Traditional methods to distinguish adult phenotypes of X-ALD include clinical and biochemical assessment as well as imaging at 1.5 Tesla. However, these methods remain inadequate to explain the dramatic differences in pathology and symptoms severity among ALD patients. We demonstrate that MR imaging at 7 Tesla allows for better visualization of X-ALD lesion architecture, white matter tracts and gray-white matter distinction compared to 1.5 Tesla (). Improved SNR and chemical-shift dispersion result in better spectral resolution and more reliable detection of metabolites, such as Glu and MI.
The best correlation with the EDSS, a validated clinical rating scale, was with the MI/Cr ratio (). Since microglia produce myo-inositol [14
] and ALD protein is highly expressed in glial cells [16
], the increased myoinositol may reflect microglial activation and gliosis. Our findings of elevated myoinositol and choline, the latter an established marker of cell membrane turnover and demyelination, further support this notion [9
Consistent with prior reports [5
], NAA was decreased in normal appearing white matter of all adult X-ALD phenotypes, suggesting a tight link between the mutated peroxisome and axonal dysfunction. Interestingly, higher plasma VLCFA levels were associated with lower NAA/Cr. Recent findings in a conditional PEX5 knockout mouse suggest the degree of peroxisomal dysfunction in oligodendrocytes alone may regulate the onset of axonal degeneration [18
The normal choline levels in patients with axonopathy indicate structural integrity of myelin despite the mutated organelle in the oligodendrocyte. In many neurodegenerative conditions there is a decrease in glutamate associated with neuroaxonal loss [20
], and our finding of concurrent decreases in NAA and Glu within white matter of X-ALD patients supports this observation.
A novel finding in our study is that of decreased NAA in cortex of X-ALD patients, which appears greater in male hemizygotes than female heterozygotes, and most pronounced with the occurrence of white matter lesions in males. Although the cytoarchitecture of cerebral cortex generally appears normal in X-ALD, scattered neuronal loss can be seen in gray matter during pathological examination [22
]. Dementia, depression and emotional disturbances are common in male hemizygote X-ALD patients [23
], and may also be a manifestation of altered cortical metabolism. Unfortunately, EDSS is a crude measure of cognition and careful correlation of neuropsychological function and cerebral metabolism needs to be undertaken.
Neurological abnormalities in female heterozygotes range from severe disability to hyperreflexia and impaired vibration sense. In our study the neurochemistry in female heterozygotes was similar to that of AMN patients, suggesting that the normal X-chromosome may protect against the inflammatory brain disease but not the noninflammatory axonopathy. Prior reports have shown that NAA levels are reduced within the corticospinal tract fibers, suggesting axonal dysfunction [5
]. We found a similar trend, although this did not reach significance. Of note, we did not find decreases in NAA/Cr in gray matter, as we found in males with X-ALD.
Despite the advantages at 7 Tesla, the interpretation of 7T MRSI data sets requires care. Voxels closest to the scalp were necessarily excluded due to poor water and lipid suppression as determined by visual inspection of spectra along the edge of the PRESS volume. Further, the quantification of spectral data in the presence of substantial RF excitation field (B1) variations is a challenging task, and in this study we relied on ratios of metabolites to Cr, which, while improving robustness for conditions where the Cr signal is stable, limits the interpretation of the findings in the presence of unknown Cr variations. In childhood ALD, high concentrations of creatine have been reported in brain lesions but not in normal appearing white matter, the subject of our study [10
This study demonstrates the feasibility of proton MRSI at 7 Tesla and takes advantage of improved signal dispersion and higher signal-to-noise ratio of 7 Tesla compared to lower field. Limitations of our study are the small sample size as well as the limited spatial coverage of regions of interest, which we are addressing with full brain spiral spectroscopic imaging [25
]. The expanded coverage of such methods, in combination with segmented high-resolution structural images, will allow us to analyze the tract-specific nature of metabolic abnormalities in X-ALD [26
] and obtain improved-quality cortical data.
The utility of MI/Cr as a biomarker in adult X-ALD will require further investigation. Specifically, to determine its disease specificity and predictive ability with early diagnosis of ALD, a study is required that follows male hemizygotes with normal MRI prospectively. How the degree of VLCFA accumulation in plasma contributes to the neurochemical changes in various phenotypes is currently unknown. A better understanding of the regional metabolic impact of VLCFA in brain in various phenotypes will help elucidate this question. Hence, a longitudinal study with sequential 7 Tesla proton MRSI examinations will be necessary to establish the sensitivity of the MI/Cr ratio in predicting disease progression.