263

Proton NMR Spectroscopy ofCanavan's Disease By P. B. Barkerl, R. N. Bryan1, A.]. Kumar1 and S. Naidu2 Departments of lRadiology and 2Neurology, lohns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, MD 21205, USA

Proton Nuclear Magnetic Resonance (NMR) spectroscopy was used to quantitatively determine cerebral N-Acetyl Aspartate (NAA) concentrations in four patients with Canavan's disease and in four age-matched control subjects. Macroscopic NAA concentrations ()lmol! gm wet weight) were not found to be significantly different from controls. Reduced levels of choline and creatine were observed in all patients, and increased levels of lactate and inositol in the eldest three patients.

Keywords N-acetyl-aspartate - NMR spectroscopy Canavan's disease

Introduction

Canavan's disease (spongy degeneration of the brain) is a rare form of leukodystrophy inherited as an autosomal recessive disorder, which was previously thought to mainly effect the children of J ewish families, but has since been reported in other ethnic groups. Described by Canavan (8) in 1931 as megaencephaly due to degeneration, it was further docurnented by van Bogaert and Bertrand (27) in 1949 who brought attention to the pathological changes, especially the spongy degeneration, and established it as a nosological entity. Symptoms, which include mental retardation, blindness, and spasticity, become apparent within the first few months of life, and become progressively worse as age increases. Both CT and MRI scans exhibit severe white matter disease (7, 21, 25), which autopsy has shown to be due to demyelination. Within the last few years, reports have appeared linking Canavan' s disease to increased levels of N-Acetyl-Aspartate (NAA) in plasma and urine (11,12,16, 17, 19, 20), and a deficiency of aspartoacylase (N-acyl-L-aspartate amidohydrolase, EC 3.5.1.15) in cultured skin fibroblasts (11, 12, 16, 19,20). Aspartoacylase is the enzyme which mediates the breakdown of NAA to aspartic acid and acetate. In initial re-

Received September 6, 1991, accepted September 27,1991 Neuropediatrics 23 (1992) 263-267 © Hippokrates Verlag Stuttgart

Although the function of NAA is not weIl understood (6), it has been suggested that it may act as a source of acetyl groups for the process of lipid synthesis (9). There appears to be a correspondence between the time course of increasing NAA concentrations in the developing brain and the onset of the myelination process (4). A lack of cerebral aspartoacylase probably leads to an increase in cerebral NAA levels. It is possible, therefore, that a deficiency of· cerebral aspartoacylase could be the cause of the abnormal myelination in Canavan's disease. There have been few direct measurements of cerebral NAA levels in Canavan's disease. A figure of 20.2 )lmol!gm protein has been reported (20) (compared to 3.9 )lmol/gm protein for normal brain), although no details were provided about statistical variations or the age range of patients studied. Qualitative measurements of cerebra! NAA levels have also been made using Proton (lH) Nuclear Magnetic Resonance (NMR) spectroscopy (1, 14, 18). Two of the three studies were on single patients (14, 18), and the other study reported findings in 2 patients (1). All three studies demonstrated increased ratios of NAA to creatine and choline, but no absolute quantitation of NAA (in molar concentration units) was performed. This paper describes quantitative NAA measurements in four patients with Canavan's disease using IH NMR spectroscopy, and a comparison with four age matched control subjects. Materials and methods All patients were diagnosed on the basis of clinical symptoms, urine NAA, and aspartoacylase activity. All subjects (with the exception of the 18-year-old control) were sedated with chloral hydrate prior to the magnetic resonance studies. Patient ages ranged from 9 months to 16 years. A brief summary of each patient's medical history is given in Table 1. Control subjects were recruited from patients referred for routine MRI studies, who had no clinical, biochemical or radiographic evidence for any white matter disease or cerebral metabolic abnormalities.Urine NAA levels were determined by gas chromatography and mass spectroscopy. Aspartoacylase activity from cultured skin fibroblasts was assayed by spectrophotometry (20).

Downloaded by: National University of Singapore. Copyrighted material.

Abstract

ports, patients with NAA aciduria and aspartoacylase deficiency were not diagnosed with Canavan's disease (16, 17), but with generalleukodystrophy. Subsequently, it has since become generally accepted that aspartoacylase deficiency and elevated urine and plasma NAA is specifically associated with Canavan's disease (11, 12, 19, 20). However, there has also been one report of a patient with radiologically and biopsy-proven Canavan's disease, where urine, plasma and CSF NAA was undetectable at 6 months of age, but was elevated at 6 years (10).

P. B. Barker et al

Neuropediatrics 23 (1992)

labte 1

Patient histories.

1. AP

AP is a 9-month-old male of Jewish descent. By 2 months of age developmental delay was apparent and he was noted to be very hypotonie. Head circumference was in the 95th percentile by 7 months, at which time MRI showed abnormal and delayed myelination.

2. EG

EG is a 13-month-old male of Jewish descent. Development was normal until 4 months, at which time he had an onset of apathy and passivity. At 13 months head circumference was in 98th percentile. Despite severe hypotonia, he had 3+ DTR's with bilateral Babinski's.

3. RS

RS is a 3-year-old female of Jewish descent. Developmental delay was first noted at 2 months of age when patient had visual tracking difficulties. At 3 months head circumference was in the 98th percentile. MRI at age 16 months showed abnormal white matter and ventricular enlargement. At age 3 years patient had prominent hypotonicity and increased DTR's and persistent Babinski responses.

4. AT

AT is a 15 and one half-year-old male who is developmentally delayed with spastic quadriplegia and cerebral atrophy. His mother is of Ashkenazi Jewish ancestry. Developmental delay was apparent at 6 months, and at 1 year head circumference was in 75th percentile. Examination at 5 years showed macrocephaly (98th percentile head circumference), mild cerebral atrophy and enlarged ventricels. At age 14 years head circumference was considerably greater than 98th percentile, and cranial MRI showed marked corticalloss throughout the cerebral hemispheres and cerebellum, with diffuse loss of white matter.

All magnetic resonance studies were performed on a General Electric 1.5 Tesla Signa scanner (General Electric Medical Systems,. Milwaukee, WI) with the standard quadrature head coil and actively shielded field gradient coils. Magnetic Resonance Imaging (MRI) in most subjects consisted of sagittal T1-weighted images (TR 500, TE 20 msec), and axial double-echo spin density/T2-weighted images (TR 3000, TE 30/100 msec). 1H NMR spectra were recorded from regions identified as predominantly frontal lobe white matter using the STEAM pulse sequence (13). Frequency-selective single-lobe sinc pulses of 15 msec duration (15) were applied prior to and between the 2nd and 3rd slice-selection pulses of the STEAM sequence for water-suppression. Acquisition parameters were TR 3000, TE 270, TM 80 msec, and 64, 128 or 256 scans de-

pending on voxel size, which ranged from 8 to 27 cm3 . Spectra were processed with a 3 Hz line-broadening, and analyzed using a time domain non-linear least squares fitting procedure (2). Quantitation was performed using a fully relaxed water signal (TR 10000, TE 50, TM 80 msec) from the localized volume as an internal intensity reference (3). The water signal was corrected for the receiver attenuation value which was used to record it, Tl and T2 relaxation times measured from the double-echo MR images. NAA signal intensities were corrected for Tl and T2 losses, partial saturation effects due to the wQ.ter suppression pulses, and the number of protons per functional group. Concentrations were calculated assuming a cerebral water content of 80 %, and a tissue density of 1049 g/cm3 (23).

Results Figure 1 shows T2-weighted MR images from one control subject and one patient of 36 months age. All patients showed hyperintensity in the white matter indicating lack of myelination. The boxes drawn on each image show the voxel locations used to record the NMR spectra. The spectra from all patients are shown in Figure 2 and control subjects Figure 3. The spectra are plotted on a vertical scale normalized relative to the water signal from the localized volume. Concentrations of cerebral NAA, urine NAA and aspartoacylase activity are given in Table 2. In all subjects, signals were detected from NAA (2.01 ppm, used as a chemical shift reference), creatine (3.03 ppm), and choline-containing compounds (3.20 ppm). In the three oldest patients, additional signals were observed from lactate (1.34 ppm) and inositol (3.54 ppm). In the oldest patient, peaks were also observed at 3.44 and 3.70 ppm, which were tentatively assigned to taurine and glutamate/glutamine respectively. In both controls and patients, NAA conceIitration increased with age except in the oldest patient. Contrary to expectations, NAA concentrations (measured in units of J..lmoles/ gm wet weight) were not significantly different in Canavan patients compared to controls. However, reductions in both creatine and choline were observed. In general, inositol, taurine, glutamate/glutamine and lactate peaks were undetectable at

Fig. 1

T2-weighted MR images (TR = 3000, TE

= 100 msec) from (A) a 36-month-old contral

a

b

subject, and (B) a 36-month-old Canavan patient demonstrating diffuse abnormal signal intensity of white matter. Voxels drawn on image represent volumes used to record NMR spectra.

Downloaded by: National University of Singapore. Copyrighted material.

264

Proton NMR Spectroscopy ojCanavan's Disease

A

B.

c.

D.

A

B.

Fig. 2 Proton NM R spectra from patients with Canavan's disease. (A) 9 months, voxel size = 27 cm 3 , 66 scans, (8) 13 months, voxel size = 27 cm 3 , 64 scans, (C) 41 months, voxel size = 15.6 cm 3 , 128 scans, (D) 186 months, voxel size = 27 cm 3 , 256 scans. Peaks are assigned to residual water (4.70 ppm), inositol (3.54 ppm), choline-containing compounds (3.20 ppm), creatine (3.03 ppm). NAA (2.01 ppm), and lactate (1.34 ppm).

4

o

2

-2

-4

c.

4

o

-2

-4

o

-2

-4

D.

* Fig. 3

Proton NM R spectra from control subjects. (A) 6 months, voxel size = 8 cm 3 , 224 scans, (8) 15 months, voxel size = 15.6 cm 3 , 128 scans, (C) 36 months, voxel size = 27 cm 3 , 128 scans, (D) 228 months, voxel size = 27 cm 3 , 64 scans.

4

2

-2

-4

265

Downloaded by: National University of Singapore. Copyrighted material.

Neuropediatrics 23 (1992)

4

2

P. B. Barker et al

266 Neuropediatrics 23 (1992) Control Subject

Age (months)

Cerebral NAA

1. 2. 3. 4.

LM OS NJ DA

6 15 36 228

10.3 12.6 13.4 20.8

Canavan 1. AP 2. EG 3. RS 4. AT

9 13 41 186

M M

F M

10.9 13.4 16.1 11.0

Urine NAA

Aspartoacylase activity

775 606 3073 1031

0.008 0.016 0.038 0.000

Table 2 Cerebral and urine NAA concentrations and aspartoacylase activity.

Notes (1) Cerebral NAA concentrations in units of J.1molejgm wet weight. (2) Urine NAA in units of mgjg creatinine (normal range for control subjects = 4-40) (3) Aspartoacylase activity from cultured skin fibroblasts in units of mUjmg (normal range for control subjects = 0.4 0.1)

long echo times in control subjects (the broad peak in the upfield region of the 36 month control subject arises from lipid signals in sub-cutaneous fat which slightly overlapped one corner ofthe STEAM voxel).

Discussion

The Canavan spectra are significantly different from controls, and are similar to those reported previously (1, 14, 18). The most prominent features are an elevated ratio of NAA to creatine and choline, and elevated lactate and inositol compared to controls, indicating severely perturbed cerebral metabolism. Although the ratio of NAA to creatine (or choline) is higher than normal, absolute quantitation indicates that this is due to decreased creatine and choline, as opposed to increased NAA. Cerebral NAA, measured in terms of J,lmoles/ gm wet weight, is not significantly different from normal despite the large increase in urine NAA in the patients. As noted previously (1), cerebral NAA is believed to be located primarily within neurons. The reduced cellularity of Canavan's disease may lead to a reduction of macroscopic (J,lmoles/gm wet weight) NAA concentrations even if intracellular NAA concentrations are normal or elevated. A reduction in wet weight concentrations compared to dry weight measurements may occur if tissue water content is increased or cell density reduced. A reduction in the number of cells per unit volurne would be consistent with the decreased creatine and choline levels observed by NMR spectroscopy, although it has also been suggested that reduced choline signal could be associated with demyelination, since choline is a major component of lecithin and sphingomyelin (18). However, there is some uncertainty regarding the visibility of choline signals from these compounds in proton NMR spectra because of their limited mobility (22). Although the lactate peak was generally too small to provide reliable quantitation, it did appear to increase with age in agreement with the suggestion that mitochondrial oxidative activity decreases with increasing age in Canavan's disease (18). The inositol peak in 1H NMR spectra is believed to constitute primarily myo-inositol (26), which is a "growth" requirement for mammalian cells (26), and mayaIso be a storage form for the inositol phosphates which play important roles in

intracellular signal transduction (5). Inositollevels also qualitatively increased with age, except in the oldest patient. It should be noted that the NMR quantitation methodology used here relies upon assummed values for the tissue water content and density (3). In pediatric brain, and especially in Canavan's disease (because of the spongy degeneration), the actual water content is likely to be higher than the 80 % used here, and tissue density slightly lower. Although this would lead to an underestimation of the NAA concentrations, it would appear unlikely that the maximum error would be larger than 10-200/0. The NAA concentrations reported here for control subjects are somewhat higher than those found in the literature from conventional biochemical analysis of tissue extracts (6), although they are in reasonable agreement with those oI Frahm (13). The difference between in vivo NMR measurement of NAA concentrations and values determined from tissue extracts is not readily explained, but presumably originates from either biochemical changes occurring during the extraction procedure, or from the overlap of the NAA signal with other unidentified species in the in vivo NMR spectra. Summary

Despite the fact that there could be some small (10-20%) underestimation of NAA concentrations with the quantitation protocol used in this study, there is certainly not a large increase in macroscopic cerebral NAA concentrations in Canavan's disease compared to age matched controls. However, because of the reduced cellularity in Canavan's disease, wet weight concentration measurements may not accurately reflect intracellular NAA levels. 1H NMR spectroscopy will probably not be a useful diagnostic test for Canavan' s disease (1) since macroscopic cerebral NAA levels are not as markedly elevated as in urine. Urine testing is also relatively simple and inexpensive compared to NMR spectroscopy, and also shows promise for pre-natal detection (24). However, IH NMR spectroscopy has provided information about cerebral metabolite concentrations in Canavan's disease which would only otherwise be obtained from brain biopsy. Lactate (and inositol) levels increase with age indicating progressively decreasing mitochondrial oxidative activity. It appears that NMR spectroscopy may therefore be used to evaluate changes occurring over periods of time which would otherwise be impossible for ethical or practical reasons.

Downloaded by: National University of Singapore. Copyrighted material.

±

Acknowledgments

The authors would like to thank Dr. Chrit Moonen and Dr. Peter van Zijl (NIH in vivo NMR Center, Bethesda, MD) for the STEAM pulse sequence which was used to record some of the spectra in this study, Dr. Richard Kelley (Kennedy Institute, Johns Hopkins Hospital) for performing the urine NAA assays, and Dr. Reuben Matalon (Department of Pediatrics, University of Illinois College of Medicine, Chicago, Illinois) for performing the aspartoacylase assays.

References 1 Austin, S.]., et al: Localized 1H NMR spectroscopy in Canavan's disease: Areport of two cases. Magn. Reson. Med. 19 (1991) 439-445 2 Barker, P. B., et al: Non-Linear Least Squares Analysis of in vivo 31p NMR Data. Proceedings of the 9th Annual Meeting Society of Magnetic Resonance in Medicine. New York (1990) 3 Barker, P. B., et al: Quantitation of Proton NMR Spectra of the human brain. 10th Annual Meeting of the Society of Magnetic Resonance in Medicine San Francisco, CA (1991) 4 Bates, T E., et al: 1H NMR study of cerebral development in the rat. NMR Biomed. 2 (1989) 225-229 5 Berridge, M.]., et al: Inositol triphosphate, a novel second messenger in cellular signal transduction. Nature (London) 312 (1984) 315-321 6 Birken, D. L., et al: N-acetyl-L-aspartic acid: a literature review of a compound prominent in 1H -NMR spectroscopic studies of brain. Neurosci. Biobehav. Rev. 13 (1989) 23-31 7 Brismar, ]., et al: Canavan disease: CT and MR imaging of the brain. AJNR 11 (1990) 805-810 8 Canavan, M. M.: Schilder's encephalitis periaxialis diffusa. Arch. Neurol. Psychiatr. 25 (1931) 299-308 9 D'Adamo, A. F., et al: Acetate metabolism in the nervous system. N-acetyl-L-aspartic acid and the biosynthesis of brain lipids. J. Neurochern. 13 (1966) 961-965 10 De Coo, 1. F. M., et al: Canavan disease: Value of N-acetylaspartic aciduria? Neuropediatrics 22 (1991) III 11 Divry, P., et al: Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with Canavan disease [letter]. Am. J. Med. Genet. 32 (1989) 550-551 12 Eekenne, B., et al: Spongy degeneration of the neuraxis (Canavan-van Bogaert disease) and N-acetylaspartic aciduria. Neuropediatrics 20 (1989) 79-81 13 Frakm,]., et al: Localized proton NMR spectroscopy in different regions

Neuropediatrics 23 (1992) of the human brain in vivo. Relaxation times and concentrations of cerebral metabolites. Magn. Reson. Med. 11 (1989) 47-63 14 Grodd, W., et al: In vivo assessment of N-acetylaspartate in brain in spongy degeneration (Canavan's disease) by proton spectroscopy [letter]. Lancet 336 (1990) 437-438 15 Haase, A., et al: 1H NMR chemical shift selective imaging. Phys. Med. Biol. 30 (1985) 341-344 16 Hagenfeldt, L., et al: N-acetylaspartic aciduria due to aspartyoacylase deficiency - a new aetiology of childhood leukodystrophy. J. Inherit. Metab. Di~ 10(1987) 135-141 17 Kvittingen, E. A., et al: N-acetylaspartic aciduria in a child with progressive cerebral atrophy. Clin. Chem. Acta 158 (1986) 217-227 18 Marks, H. G., et al: Use of computed tomography, magnetic resonance imaging, and localized 1H magnetic resonance spectroscopy in Canavan's disease: A case report. Ann. Neurol. 30 (1991) 106-110 19 Matalon, R., et al: Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with Canavan disease. Am. J. Med. Genet. 29 (1988) 463-471 20 Matalon, R., et al: Aspartoacylase deficiency: the enzyme defect in Canavan disease. J. Inherit. Metab. Dis. 2 (1989) 329-331 21 MeAdams, H. P., et al: CT and MR imaging of Canavan disease. AJNR 11 (1990)397-399 22 Miller, B. L.: A review of chemical tissues in 1H NMR spectroscopy: A-acetyl-L-aspartate, creatine and choline. NMR Biomed. 4 (1991) 4752 23 Narayana, P. A., et al: Regional in vivo proton magnetic resonance spectroscopy of brain. J. Magn. Reson. 83 (1989) 44-52 24 Ozand, P. T, et al: Prenatal detection of Canavan disease [letter]. Lancet 337 (1991) 735-736 25 Patel,]. ]., et al: Sonographic and computed tomographic findings in Canavan's disease. Br. J. Radiol. 59 (1986) 1226-1228 26 Ross, B. D.: Biochemical considerations in 1H NMR spectroscopy. Glutamate and glutamine; myoinositol and related metabolites. NMR Biomed. 4 (1991) 59-63 27 Van Bogaert, L., et al: Sur une idiote familiale avec degenerescence spongieuse de neuraxe (note preliminaire). Acta Neurol. Belg. 49 (1949) 572-587

P. B. Barker, D.Pkil. Division of NMR Research, MRI 110 Dept. of Radiology and Radiological Science Johns Hopkins Hospital 600 N. Wolfe Street, Bldg. RM 110 Baltimore, MD 21205 USA

267

Downloaded by: National University of Singapore. Copyrighted material.

Proton NMR Spectroscopy ojCanavan's Disease