Membrane phospholipids and high-energy ...

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The authors thank Dr. Hugo Moser of Kennedy Krieger. Institute, Baltimore, MD, and members of the United Leukodys- trophy Foundation for helpful discussions, ...
Membrane phospholipids and high-energy metabolites in childhood ataxia with CNS hypomyelination S. Blüml, PhD; M. Philippart, MD; R. Schiffmann, MD; K. Seymour, PhD; and B.D. Ross, MD

Abstract—Background: Childhood ataxia with CNS hypomyelination (CACH) is a leukodystrophy with extreme rarefaction of white matter caused by mutations in one of the five subunits of the translation initiation factor 2B (eIF2B). Methods: Seven children with this disease and nine age-matched control subjects were studied with proton-decoupled phosphorus magnetic resonance (MR) spectroscopy. Results: In patients with CACH, cerebral concentrations of high-energy phosphate metabolites were abnormal. Of the metabolites involved in biosynthesis and catabolism of membrane phospholipids, glycerophosphorylethanolamine was reduced (0.24 ⫾ 0.18 mmol/kg brain vs 0.44 ⫾ 0.14; p ⬍ 0.02), and phosphorylethanolamine was increased (2.32 ⫾ 0.53 vs 1.53 ⫾ 0.22; p ⬍ 0.01), whereas the choline-containing phosphorylated metabolites were unchanged. Nucleoside triphosphate (NTP) was reduced (2.44 ⫾ 0.34 mmol/kg brain tissue vs 3.09 ⫾ 0.58; p ⬍ 0.01), phosphocreatine was elevated (4.11 ⫾ 0.63 vs 3.27 ⫾ 0.33; p ⬍ 0.01), and inorganic phosphate was reduced (0.77 ⫾ 0.32 vs 1.06 ⫾ 0.26; p ⬍ 0.05). Intracellular pH was elevated in patients (7.03 ⫾ 0.04 vs 6.99 ⫾ 0.02; p ⬍ 0.02). Conclusions: The authors found an altered energy state of the residual cell population investigated. Together with previously identified replacement of white matter by CSF, the present findings raise the possibility that the genetic defect in eIF2B may result in impairment of myelin membrane synthesis or myelin membrane transport in the in vivo CACH brain. Ethanolamine metabolites constitute the plasmalogens, and the present findings may include a defect in plasmalogen metabolism. NEUROLOGY 2003;61:648–654

Childhood ataxia with CNS hypomyelination (CACH), also known as vanishing white matter disease (VWMD)1-5 is a leukodystrophy caused by mutations in one of the five subunits of the translation initiation factor 2B (eIF2B).6,7 This study was undertaken to determine if metabolic alterations are associated with the mutant genes. An extensive rarefaction of cerebral white matter and a commensurate loss of axons and myelin sheaths were reported.5 The authors of that study concluded that in vivo magnetic resonance spectroscopy (MRS) and histopathology are compatible with a primary axonopathy rather than primary demyelination. Conversely, oligodendrocytes with typical signs of apoptosis were detected in an active demyelinating lesion in the brainstem.8 This group suggested that death of mature oligodendrocytes is the critical event in the disease and that damage to the axons appears only later. Unusual cells with “foamy” cytoplasm of oligodendroglial phenotype and myelinopathy with abnormal lipid inclusions in oligodendrocytes were found in tissue samples from the brains of patients with CACH.9 In vivo MRI shows characteristic changes in central white matter, whereas in the proton (1H) MR

spectrum of affected white matter, the usual brain metabolites are replaced by the CSF markers glucose and lactate. Although 1H MRS can detect alterations of the myelin metabolites choline and myo-inositol with high sensitivity, a limitation of 1H MRS is the lack of specificity. For example, the choline resonance is a complex peak. Proton-decoupled 31P MRS ([1H]-31P MRS)10-12 separates and quantifies the phosphomonoesters phosphorylcholine (PC) and phosphorylethanolamine (PE) and the phosphodiesters glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC). These metabolites are involved in myelin biosynthesis by methyl group metabolism and lipid transport and are components of a number of important biologic compounds, including the membrane phospholipids lecithin, sphingomyelin, and plasmalogen.13 Similarly, the energy state of brain tissue is only incompletely defined by the total creatine (Cr) peak in 1H MRS, whereas 31P MRS allows the quantitation of ATP, among other nucleoside triphosphates (GTP, ITP, and UTP), inorganic phosphate (Pi), phosphocreatine (PCr), and, from the chemical shift of Pi, the determination of tissue pH. In this study, proton-decoupled 31P MRS ([1H]31 P) was applied with three principal goals:

From the Magnetic Resonance Spectroscopy Unit (Drs. Blüml, Seymour, and Ross), Huntington Medical Research Institutes, Pasadena, CA; Rudi Schulte Research Institute (Drs. Blüml, Seymour, and Ross), Santa Barbara, CA; Brain Research Institute (Dr. Philippart), University of California, Los Angeles, CA; and Developmental and Metabolic Neurology Branch (Dr. Schiffmann), National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD. S.B.’s current affiliation is Department of Radiology, Children’s Hospital, Los Angeles, CA. Received September 16, 2002. Accepted in final form June 5, 2003. Address correspondence and reprint requests to Dr. Brian D. Ross, Huntington Medical Research Institutes, 660 South Fair Oaks Avenue, Pasadena, CA 91105; e-mail: [email protected] 648

Copyright © 2003 by AAN Enterprises, Inc.

Table 1 Demographics and clinical diagnosis of CACH patients Patient no.

Sex

Age, y

Presenting symptoms and neurology

MRI finding

1

Female

2

Seizures; Severe developmental delay; Cannot speak; Cannot feed herself; Metachromatic leukodystrophy excluded

Severely abnormal MRI; Cavitation on FLAIR MRI

2

Female

4

Slowly deteriorating leukodystrophy after mild head trauma at age three; Hypophonia and slowed speech; Normal cranial nerves; Moderately severe spastic ataxia with dysmetria in upper extremities; Weakness and hyperreflexia of lower extremities

MRI consistent with CACH

3

Male

4

Seizures; Severe developmental delay; Blind

Severely abnormal MRI; Cavitation on FLAIR MRI

4

Male

9

Modest developmental delay; Some motor spasticity and speech impediment; Mild dysarthria and tongue weakness; Proximal muscle weakness; Mild hyperreflexia and mild dysmetria in the upper extremities; Mild gait ataxia

MRI consistent with CACH; no cortical thinning

5

Male

12

Ataxia after head trauma at age two; Mild dysarthria; Spastic paraparesis with dysmetria in upper extremities; Weakness and hyperreflexia of lower extremities; Normal cognition

Cavitation on FLAIR MRI, no cortical thinning

6

Male

13

Asymptomatic sibling of patient no. 4; Mild hyperreflexia; Low IQ (75), Possible Babinski signs; Normal gait and coordination

Diffuse MRI changes mainly at posterior and frontal angles of lateral ventricles

7

Male

17

Hemiparesis post mild head trauma at 4.5 years; Mild dysarthria; Moderate spastic paraparesis with dysmetria in upper extremities; Normal cognition

Ventricular enlargement; Cavitation on FLAIR MRI

CACH ⫽ CNS hypomyelination; FLAIR ⫽ fluid-attenuated inversion recovery.

1) To quantify 31P metabolites in mmol/kg brain tissue. This is essential to determine whether systematically altered cerebral concentration of metabolites in CACH detected by 1H MRS are primarily caused by altered intracellular concentrations or are the result of reduced volume of cells (and an increased of CSF) within the volume of interest. To achieve this goal, we have combined 31P MRS with an MR assay of the fractions of tissue and CSF within a region of interest (ROI) based on the different T2 relaxation characteristics of tissue water and CSF.14 2) To identify abnormalities in the cerebral concentrations of PC, PE, GPC, and GPE, precursors and catabolites of membrane phospholipids involved in membrane and myelin sheath metabolism in patients with CACH and in control subjects. 3) To determine any abnormality in phosphorylated energy metabolites, including ATP or GTP, which may result from the translation initiation factor mutation now identified as the cause of this disease. A preliminary account of these findings was presented at the International Society of Magnetic Resonance in Medicine (ISMRM) meeting in April 2000.15 Methods. Patients and control subjects. Seven children aged 2 to 17 years (mean age, 8.7 ⫾ 5.6 years), with clinical and radiologic diagnosis of CACH, were examined by MRI, quantitative 1H, 31 P MRS, and [1H]-31P MRS. Details of the subjects examined are

given in table 1. The diagnoses of CACH were confirmed by the finding of mutations in eIF2B subunits in all patients. The control group, from which MRS changes that accompany normal development of myelin and brain12,16 were determined, comprised agematched children (n ⫽ 9; mean age, 8.6 ⫾ 3.6 years) with negative previous MRI and MRS examinations performed for diagnosis. These subjects were referred to our facility for clinically indicated MRI studies, and [1H]-31P MRS was offered to the parents as an “add-on.” Control subjects showed no clinical neurologic or developmental abnormalities after a follow-up period of more than 3 years. Informed parental consent was obtained for each child. The Institutional Review Board of Huntington Memorial Hospital approved all studies. Children aged less than 8 years received conscious sedation, using oral chloral hydrate, 50 mg/kg body weight. All MRI and MRS examinations were performed on a 1.5-T clinical MR scanner (GE Medical Systems, Waukesha, WI) equipped with a broadband radiofrequency (RF) receiver and a second RF channel for proton decoupling. A double-tuned linear 1 H quadrature 31P birdcage head coil was used. A complete examination was done, including MRI, quantitative 1H MRS and 31P MRS, and [1H]-31P MRS, and lasted approximately 65 minutes. Quantification of brain water and CSF compartments. The discrimination between brain water (BWMRS) and CSF (CSFMRS) is based on the difference in transverse (T2) relaxation times of brain water (T2bw) and CSF (T2csf). Extracellular and intracellular compartments are not separated with this assay. The proportions of CSF and of intracellular plus extracellular brain water (referred to in this article as brain water [BW]) in the 1H and 31P MRS ROIs were determined with the 1H MRS “T2-7 points” assay described previously.14 Fractions of normal- and abnormal-appearing tissue and CSF were also determined visually by segmentation analysis of T2weighted fast spin-echo (FSE), and fluid-attenuated inversion recovery (FLAIR) MRIs using standard software provided by the manufacturer (GE Medical Systems). In the ROIs selected for 1H MRS areas of CSF (CSFMRI), abnormal- and normal-appearing brains were manually traced. MRS and visual tissue fractions were compared. Quantitation of phosphorylated brain metabolites. 31P and [1H]-31P MRS measurements were carried out using the PRESS sequence, with echo time (TE) of 12 ms and repetition time (TR) of September (1 of 2) 2003

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Figure 2. 1H MRS OF patient with childhood ataxia with CNS hypomyelination (CACH). 1H magnetic resonance spectroscopy (MRS) in severely affected white matter in a patient with CACH is characterized by a depletion of the resonances usually observed in normal brain and an excess of glucose and lactate. 1H spectra acquired from lessseverely affected white matter or gray matter showed a normal or close to normal pattern. Figure 1. MRI of patient with childhood ataxia with CNS hypomyelination (CACH). (A) T2-weighted MRI of Patient 2 with superimposed magnetic resonance spectroscopy (MRS) voxel used for 31P MRS. Positioning of the 31P voxel was standardized and thus contained gray matter and varying amounts of white matter with diffuse changes on MRI. (B and C) T2-weighted and fluid-attenuated inversion recovery (FLAIR) MRI of Patient 6 with diffuse white matter changes predominantly at the posterior and frontal angles of the lateral ventricles. (D) T2-weighted MRI of Patient 1 shows disappearance of large portions of white matter.

3,000 ms. To compensate for the low signal-to-noise ratio (S/N) of 31 P MRS relative to 1H MRS, a large voxel of 97.5 cm3 (6.5 ⫻ 5.0 ⫻ 3.0 cm3) was selected in a standard region in frontoparietal mixed white and gray matter, superior to the lateral ventricles (figure 1). The 128 –256 scans were averaged for the nondecoupled (conventional), and the 512–768 scans were averaged for the [1H]31 P MR spectra. For automated fitting and metabolite quantitation, a model function was subtracted from the spectra to produce a flat baseline.12 From the baseline function, the amount of membrane phospholipids underlying the resonances of PE and PC (PME-PL) and GPE and GPC (PDE-PL) were determined, and their concentrations were expressed as arbitrary institutional units. 31P MRS signal intensities were expressed as concentrations by attributing intracellular metabolites to the volume of the BW fraction. Nondecoupled 31P MRS was used to quantify PCr, combined phosphomonoesters (PME), and phosphodiesters (PDE) in mmol/kg brain tissue. PCr was then applied as the internal reference from which the concentrations of NTP, Pi, PE, PC, GPE, and GPC were determined in proton-decoupled spectra. Intracellular pH (pHICF) and extracellular/CSF pH (pHCSF) were determined from the chemical shift difference between Pi resonances and PCr. 1 H MRS. 1H MR spectra were acquired from standardized ROIs in parietal white matter and occipital gray matter containing varying amounts of affected tissue. These spectra were processed and quantified as explained in detail in previous publications.14,16,17 A STEAM sequence with a TR of 1.5 s, TE of 30 650

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ms, and a mixing time (TM) ⫽ 13.7 ms was used, and 256 averages were collected. Statistics. Means and SDs were calculated, and significance tests were performed using Student’s t-tests for unpaired samples.

Results. MRI and 1H MRS in patients with CACH. MRI in patients with CACH (see figure 1) represented a range of severity, with diffuse white matter signal predominantly at the angles of the lateral ventricles in two patients and severe diffuse changes with disappearance of a large proportion of the white matter in the remainder, confirming the findings of previous investigators.1-3,18 FLAIR images demonstrated further heterogeneity within the high-signal regions of white matter, with lower signal possibly caused by cavitation at the center. In keeping with earlier reports,1-3 1H MRS spectra acquired from severely affected white matter in children with CACH were abnormal, showing excess glucose and lactate (figure 2). 1H spectra acquired from less-severely affected white matter or gray matter showed a normal or close to normal pattern. Brain water and CSF compartments in CACH patients and control subjects. Excess CSF was apparent in MRIs of all CACH patients. When identical ROIs were analyzed in patients using the T2-7 points assay and image segmentation, a larger compartment of CSF was determined by the T2-7 points assay (53% ⫾ 17%) than by segmentation (22% ⫾ 19%; p ⬍ 0.002, paired t-test). T2bw was larger in patients with CACH than in control subjects (113 ⫾ 16 vs 82 ⫾ 6; p ⬍ 0.001, unpaired t-test), whereas T2csf in patients with CACH was within normal limits. Concentrations of phosphorylated brain metabolites. Conventional 31P MR spectra and proton-decoupled 31P spectra obtained from control subjects and patients with CACH are shown in figure 3, and the concentrations of

Figure 3. 31P and proton-decoupled 31 P magnetic resonance spectroscopy (MRS) of patients with childhood ataxia with CNS hypomyelination (CACH) and age-matched control subjects. (A) In the standard 31P MR spectra, phosphocreatine (PCr), inorganic phosphate (Pi), the phosphomonoester (PME), and the phosphodiester (PDE) peaks were quantified. (B) Protondecoupled 31P MRS in patients with CACH and in control subjects resolves the metabolite peaks of phosphorylethanolamine (PE), phosphorylcholine (PC), glycerophosphorylethanolamine (GPE), and glycerophosphorylcholine (GPC). Shown are the baseline corrected spectra after the subtraction of the baseline function (see Methods section and reference 13). Note the apparent increase in PME, the reduction in PDE, and the increase of PCr in the nondecoupled spectrum. Proton-decoupled 31P MRS with improved spectral resolution reveals elevated PE, two Pi peaks, reduced GPE, and slightly but significantly reduced ATP in patients with CACH. All spectra are scaled to absolute concentrations to allow direct comparison.

phosphorylated brain metabolites in patients with CACH and in age-matched control subjects are summarized in table 2. Energy metabolites and tissue pH. The most significant abnormality in patients with CACH was the reduced NTP/PCr ratio caused by an increase in PCr and a reduction of cerebral NTP (principally ATP) concentration. [1H]31 P MRS resolved two Pi peaks; the first (Pii) indicated a close to normal intracellular pH of 7.03; the second (PiCSF), not seen in the brain of healthy control subjects, was at pH of 7.35, equal to that of CSF.19 This pH distribution in patients with CACH was consistent with the expanded CSF compartment demonstrated previously. Duplication of Pi peaks was observed in all patients, and the extent of the alkaline CSF compartment increased with the severity of disease (figure 4A). The fraction of Pi at alkaline pH (PiCSF/[Pii ⫹ PiCSF]), measured with [1H]-31P MRS, showed a close to linear correlation with the proportion of CSF within the voxel assayed (figure 4B; R2 ⫽ 0.90). The mean ratio PCr/Pii was significantly increased in patients with CACH. Tissue pH determined from the chemical shift difference between the Pii and the PCr resonances was slightly but significantly more alkaline in patients with CACH. When individual data were examined, PCr/Pi increased and NTP/PCr decreased with the severity of tissue abnormalities in the ROI, quantified by the extent of the replacement of tissue by CSF (figure 4, C and D). Phosphomonoesters and phosphodiesters. The PMEs PE and PC and the PDEs GPE and GPC are believed to be involved in a cycle of phospholipid membrane synthesis and breakdown, with the monoesters on the anabolic limb and the diesters as their catabolites. Significantly reduced concentrations of PDE were found in patients with CACH. In the nondecoupled spectrum, this peak comprises the less mobile membrane phospholipids and the lower molecular weight metabolites GPE and GPC. Decoupled 31P MRS shows that the overall reduction in PDE is the result of reduction of the metabolite GPE and of the underlying

membrane lipid resonance (PDE-PL), but not GPC (see table 2). In patients with CACH, PE was higher and GPE was lower than in control subjects. GPC and PC were normal within the current accuracy of the method, consistent with downregulation of a biosynthetic step between PE and GPE.

Discussion. Translation initiation is a tightly regulated process central to cell function. With the recent discovery of a mutation in the five genes encoding the subunits of eIF2B in patients with CACH and VWMD, a disorder of white matter, it has become necessary to define the metabolic abnormalities observed in the brain of these children more closely. In this study, we applied noninvasive MRS methods to explore the biochemical basis of the leukodystrophy in children with CACH. On MRI, CACH is characterized by diffuse white matter abnormalities on T1- and T2-weighted images, and normal cortex. The study of tissue samples revealed unusual cells with abundant cytoplasm, lacking lysosomal structures. Further analysis with antibodies demonstrated that these cells are of oligodendroglial type.2 These foamy oligodendrocytes were recently found in patients with a severe form of CACH.20 Previous in vivo 1H MRS studies detected a uniform decrease of all metabolites in the affected brain regions. 1H MRS carried out in each subject in this study confirmed previously reported findings and is not discussed in detail. In this report, we focus on 31 P MRS and proton-decoupled 31P MRS. 31P MRS was used to define cerebral energy status from PCr, NTP (including ATP and GTP), Pi, intracellular pH, and free ADP concentrations. The proton-decoupled 31 P MRS technique was essential to assay separately the PMEs PC and PE and PDEs GPE and GPC, September (1 of 2) 2003

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Table 2 Cerebral concentrations of phosphorylated metabolites CACH (n ⫽ 7)

Control (n ⫽ 9)

2.32† ⫾ 0.53

1.53 ⫾ 0.22

PC

0.56 ⫾ 0.20

0.61 ⫾ 0.17

Pi

0.77* ⫾ 0.32

1.06 ⫾ 0.26

GPE

0.24* ⫾ 0.18

0.44 ⫾ 0.14

GPC

0.81 ⫾ 0.26

0.85 ⫾ 0.26

PE

PDE-PL (a.u.)

0.17† ⫾ 0.02

0.21 ⫾ 0.02

ATP

2.44† ⫾ 0.34

3.09 ⫾ 0.58

NAD

0.69 ⫾ 0.31

0.81 ⫾ 0.18

PCr

4.11† ⫾ 0.63

3.27 ⫾ 0.33

PME

4.30 ⫾ 1.16

4.12 ⫾ 0.77

PDE

6.43‡ ⫾ 1.66

9.74 ⫾ 1.39

ATP/PCr

0.62§ ⫾ 0.12

0.94 ⫾ 0.13

PCr/Pi

5.86* ⫾ 2.38

3.31 ⫾ 1.08

pH

7.03* ⫾ 0.04

6.99 ⫾ 0.02

7.35 ⫾ 2.30

9.29 ⫾ 0.85

NAA*¶

Metabolite concentrations (in mmols/kg brain tissue) measured with 31P MRS and proton-decoupled 31P MRS in CACH patients and in controls. * p ⬍ 0.05. † p ⬍ 0.01. ‡ p ⬍ 0.001. § p ⬍ 0.0001. ¶ Obtained from quantitative 1H MRS in white matter carried out in all patients and controls as explained in detail in previously published work.14,17 MRS ⫽ magnetic resonance spectroscopy; CACH ⫽ CNS hypomyelination; PE ⫽ phosphorylethanolamine; PC ⫽ phosphorylcholine; Pi ⫽ inorganic phosphate; GPE ⫽ glycerophosphorylethanolamine; GPC ⫽ glycerophosphorylcholine; PDE-PL ⫽ GPE and GPC; PCr ⫽ phosphocreatine; PME ⫽ phosphomonoesters; PDE ⫽ phosphodiesters; NAA ⫽ N-acetyl aspartate.

which are involved in myelin and membrane biosynthesis.13 Cholines are also involved in methyl group metabolism and lipid transport and are components of a number of important biologic compounds, including the membrane phospholipids lecithin, sphingomyelin, and plasmalogen. Our first goal was to accurately quantify the concentrations of phosphorylated neurochemicals in mmol/kg of brain tissue. To achieve this objective, we applied an assay (T2-7 points) that allows the discrimination between tissue water and CSF based on the differences in T2 relaxation time between these two compartments. When the 31P MRS signal intensities were corrected for the smaller fraction of tissue remaining within the ROI of the 31P MRS measurement, cerebral concentrations were not generally reduced in patients with CACH. We also found this to be true for 1H MRS15 (not further discussed in this report). We noticed that the fraction of CSF measured by the MRS assay, CSFMRS was higher in each 652

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patient than the amount of CSF determined by segmentation of the MRIs, CSFMRI. This indicates that within individual MRI voxels, small pockets of CSF develop in this disease. There was no correlation between the tissue T2 time and the severity of the disease. The short T2 compartment represents viable tissue, dead cells being replaced by CSF; intracellular metabolites do not disappear in this disease but maintain almost normal concentrations within a reduced cell population. For the determination of metabolite concentrations in this study, we assumed that the NMR properties, such as the T1 and T2 relaxation times, are unchanged in this disease. Differences could alter the actual measured concentrations in patients and control subjects. However, only a small change in tissue water T2 (see above) was observed, and all phosphorylated metabolites, apart from extracellular Pi, are intracellular. Therefore, the impact of hypothetical differences in the relaxation properties would be within the error of the current MRS methods and would not change the principal findings of this study. A reduction of the absolute cerebral concentration of NTP and an increase in PCr were observed in patients with CACH. The ratios NTP/PCr and PCr/Pi were plotted against the fraction of CSF and appeared to correlate with the replacement of tissue by CSF. An increasingly altered metabolic fingerprint, which parallels the advance of disease, could be caused by uniformly altered metabolism of all cells within the ROI, selective loss of a certain cell type changing the partial volume fractions of the different cells within the voxel, or both. It has been found with quantitative 31P spectroscopic imaging21 that NTP/PCr is lower in gray matter than in white matter. This difference was attributed to an approximately 40% higher NTP concentration in white matter while PCr was essentially unchanged. Although a progressive loss of white matter in patients with CACH is consistent with the decrease of NTP/PCr with disease severity, the significant increase in PCr observed in patients with CACH in this study cannot be explained by a selective loss of white matter while gray matter is relatively preserved. Currently, there is an active discussion whether this leukodystrophy is of primary axonopathic5 or primary myelinopathic phenotype.8,9 Mean N-acetyl aspartate (NAA), a neuronal and axonal marker, was reduced because of depletion in the most severe cases. A comparison of 1H MRS (see figure 2) and [1H]-31P MRS (see figure 4A) reveals that NAA in patients with CACH is normal (or close to normal) at a time when profound changes in concentrations of myelin metabolites of the ethanolamine series have occurred. Therefore, the present metabolic study favors primary myelinopathy. GPE and the membrane phospholipids beneath the PDE peaks are decreased, whereas PE is elevated. GPC and PC are normal within the current accuracy of the method. The PMEs PE and PC and the PDEs GPE and GPC are believed to be involved

Figure 4. Extracellular and intracellular compartments of inorganic phosphate (Pi), phosphocreatine (PCr)/Pi, and ATP/PCr vs fraction of CSF. (A) Phosphomonoester/phosphodiester region of proton-decoupled 31P MR spectra of patients with CACH and a control subject. The inorganic phosphate peak PiCSF at a chemical shift consistent with a pH of 7.35 of extracellular water and CSF increases with the severity of the disease quantified here as the extent by which CSF replaced brain tissue in the region of interest (ROI). (B) When the fraction of PiCSF/total Pi was plotted vs the amount of CSF determined by the T2-7 points assay, a close to linear correlation was found (p ⬍ 0.002). Note that a strict linear correlation would be expected only if intracellular Pi and extracellular/CSF Pi concentrations are equal. (C and D) A significant linear correlation was also found for ATP/PCr (p ⬍ 0.05). Although PCr/Pi appears to correlate with the extension of replacement of brain tissue by CSF, with the small number of subjects studied, it did not reach significance. Patients with CACH ⫽ filled symbols; control subjects ⫽ open symbols.

in membrane synthesis and breakdown with the monoesters on the synthesis pathway and the diesters being catabolites. For a general defect in membrane metabolism, some parallelism of alterations is expected. The fact that only GPE and PE are altered may add some more specific information. Adults with multiple sclerosis (MS), another white matter disease, did not reveal this pattern (unpublished obser-

vation in this laboratory). Choline metabolites principally constitute the lecithins, and ethanolamine metabolites contribute to the plasmalogens of the brain.22 Therefore, the present findings are consistent with a defect in plasmalogen metabolism as an early event in patients with CACH. [1H]-31P MRS may help with the diagnoses of difficult, atypical, or mild cases and may offer a means of monitoring therSeptember (1 of 2) 2003

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apeutic response for this and other demyelinating disorders. The discovery that a rare autosomal recessive neurologic disorder results from mutation in such an ubiquitous gene complex may increase our understanding of more common conditions, including MS.23 It is tempting to hypothesize that alterations in PE and GPE reflect a fundamental neurochemical change, induced by the mutation of translation initiation gene complex, and represent some more generalized cellular processes. Further study of ethanolamine metabolism in the brain and other tissues will be required to confirm such a link. Acknowledgment Dedicated to the late Jean Devlin (Rockefeller University and RSRI), an untiring friend to children with brain disorders. Dr. Jorge Carrera Mardones of Puerto Monnt, Chile, referred two patients. The authors thank Dr. Hugo Moser of Kennedy Krieger Institute, Baltimore, MD, and members of the United Leukodystrophy Foundation for helpful discussions, and the Rudi Schulte Research Institute for travel funds provided to bring two children from Chile for MRS examinations. The authors also thank Alexander P. Lin, BS, for help with the preparation of this manuscript.

References 1. Hanefeld F, Holzbach U, Kruse B, et al. Diffuse white matter disease in three children: an encephalopathy with unique features on magnetic resonance imaging and proton magnetic resonance spectroscopy. Neuropediatrics 1993;24:244 –248. 2. Schiffmann R, Moller JR, Trapp BD, et al. Childhood ataxia with diffuse central nervous system hypomyelination [see comments]. Ann Neurol 1994;35:331–340. 3. van der Knaap MS, Barth PG, Gabreels FJ, et al. A new leukoencephalopathy with vanishing white matter. Neurology 1997;48:845– 855. 4. Rodriguez D, Gelot A, della Gaspera B, et al. Increased density of oligodendrocytes in childhood ataxia with diffuse central hypomyelination (CACH) syndrome: neuropathological and biochemical study of two cases. Acta Neuropathol 1999;97:469 – 480. 5. van der Knaap MS, Kamphorst W, Barth PG, et al. Phenotypic variation in leukoencephalopathy with vanishing white matter [see comments]. Neurology 1998;51:540 –547.

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