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R e f e r e n c e s

Allen I. V. and Kirk I. (1992) Demyelinating diseases. In Adams JH, Duchen LW. Greenfield's Neuropathology, ed 5. New York, Oxford University Press, p 447.

Raine C. S. (1991) Demyelinating diseases, in Textbook ofNeuropathology, 2nd ed. (Davis R. L. and Robertson D. M., eds.), Williams and Wilkins, Baltimore, p. 535.

Becker L. E. and Yates A. J. (1991) Inherited metabolic disease, in Textbook of Neuropathology, (Davis R.L. and Robertson D.M., eds.), Williams and Wilkins,Baltimore, p. 331.

SESSION IV: MAGNETIC RESONANCE SPECTROSCOPY

Chairman's Introduction Bryan Ross

Magnetic resonance spectroscopy (MRS) has the following possible roles in dealing with inborn errors of metabolism:

1. Detection of carriers; 2. Monitoring of subclinical cases; 3. Help in judgment of clinical cases; [and] 4. Grading of these diseases.

MRS is an orphan branch of MRI and it is unusual to get a focussed session on spectroscopy at [an] MR meeting like this. In my talk, I shall give some background to MRS by discussing the technique, the metabolites iden- tified and their role, the normal evolution of infant MRS, and a case of adrenoleukodystrophy.

There are now some 20 different techniques that can be used in MRS including:

Localized 1H MRS Localized .~lp MRS Localized 13C MRS (Localized) 15N MRS

23Na MRS Localized 19F MRS

MRS techniques enable one to detect metabolites present in tissue at a concentration 1 / 10,000 that of water. We use MRS much less often than MRI so that we have much less experience with the former technique. You can use long echo times or short echo times (equivalent to T2 or T1 weighted images in MRI). Radiologists have been asking for many years, don't pro- duce peaks and valleys and troughs and numbers; give us pictures (and I suspect that the neuropathologists would rather have that too). You will see from two of our speakers this morning that we have come a long way. We now produce images. These images may not satisfy Dr. Valk because the spa- tial resolution is very limited. But since metabolites at a concentration 1 / 10,000 that of water are used to produce the images, we are not doing too badly.

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Developments in automation in MRS mean that these techniques, which have been very esoteric so far, are going to be available on every street corner as it were. What I mean by that is that in a 7-minute sequence in a standard MRS scanner, you can produce spectra of the kind I am going to show you. I'm even going to show spectra done by my secretary, not to belittle my secretary, but to show that anyone can do it.

What are the results? The normal proton spectrum measures peaks. Each peak is proportional to the concentration plotted against the frequency and the frequency identifies the chemical. N-acetylaspartate (NAA) is the major peak in brain. The area with glutamate and glutamine is well-defined. Other peaks include creatine + creative phosphate (indicator of the energy status); choline (which is an aggregate of a number of metabolites); and myoinositol.

Dr. Valk has demonstrated the power of pattern recognition of MRI; I think we can expect the same in MRS. Here are some patterns:

1. Hypoxic encephalopathy: Lipid peaks appear; NAA goes down; glutamine goes up.

2. Hepatic encephalopathy: Although there are no changes in MRI, the spectra change dramatically. Glutamine goes up 2-3 fold; cholines go down; and myoinositol drops to almost zero, which is characteristic for this condition.

3. Alzheimer disease: Myoinositol goes up, which is surprising; NAA, the neuronal marker, decreases, which is not a surprise--we see that constantly in the childhood dementias as well. Preliminary results indicate that this pattern (decrease in NAA and increase in myoinosi- tol) is also seen in elderly Down syndrome patients with dementia.

The techniques of quantitation are still rather controversial, but every- one is agreed on the need for quantitation. You need numbers in order to be able to talk to biochemists. The question Dr. van der Knaap raised yesterday is an important one: if you are looking at ratios you need to know what is going up and what is going down. There are many methods for quantitation available now. The net result is that, in rats, the biochemical concentrations measured by these noninvasive techniques are very close to those obtained by real biochemists grinding up the tissue (see Table 5).

These results stress that, when you acquire spectra from children of different ages, you must look at age-related controls.

What about the pathological things you can measure? Take hypoxia as an example. We have already heard that repeated, sequential MRI is far preferable to single-shot MRI (like in morbid anatomy and histology) and I think it is clearly going to be the same for MRS too. If you look at a con- tinuum of spectra during hypoxia after drowning you see that the changes are slow. These results show on the one hand that you can learn something about the pathology of the events in hypoxia and, on the other hand, that you should not rely on a single spectrum unless it is diagnostic.

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Table 5 Concentrations of NAA and Myoinositol in Rat Brain

Concentration (mM) in brain of

Metabolite Newborn Adult

NAA 5 9 Myoinositol 10 5

I a m showing you some spectra done by Dr. Thomas Ernst in our labo- ratory of the brain of a pat ient wi th that rare disorder , Reye syndrome. (Actually, in this case a single spec t rum can be diagnostic.) What you see is the following: lactate appears; NAA goes down; Gln increases to 15-20 rnM; an u n k n o w n peak appears, which later disappears . The child recovered bu t wi th only 50% of normal NAA. Perhaps this fits wi th wha t is k n o w n about Reye; the chi ldren fall into two categories: those w h o have no neurona l deficit and those w h o have a serious deficit.

What have we learned? Let us first look at NAA. We take the publ i shed histochemical data which says that NAA, and in part icular NAAG, is con- f ined to neurons and cell bodies, and we say NAA is a marker for neurons . But, as we now know, NAA is also found in cultures of ol igodendrocytes . This biosynthesis of NAA occurs in o l igodendrog l i a /neuron . The biosyn- thesis, which is slow, precedes myelination. It is located in neurons and axons. NAAG is found in spinal cord and cerebellum. A loss of NAA is equa ted wi th a loss of neurons; NAA is low in gliomas, MS, stroke, and hypoxia. NAA is certainly present in neurons , but we find it in whi te mat ter so that it mus t be in axons as well. We do not know how it gets there. It seems to be t ranspor ted d o w n the axon so that this is w h y we see it in whi te mat ter and w h y NAA is lost in whi te-mat ter disease and even in MS. The results wi th hypoxia indicate that NAA is first lost from gray matter and then at a later stage from whi te matter.

Does NAA recover? In pos thypoxia and in MS there is both neuronal and axonal recovery.

N A A responds to other metabolic events. It goes d o w n in diabetes. It goes up, or dow n , in hyperna t remia , and it goes d o w n in hypona t remia .

We k n o w a lot about creatine. The creatine peak is the s u m of creatine plus phosphocreat ine , which are in rapid exchange, since the creatine kinase reaction is in equilibrium as we can show with other MRS techniques. There is unl ikely to be an invisible pool of creatine + phosphocrea t ine , as some people have suggested. I do not share the feeling some MR spectroscopists have that one set of cells have creatine whereas a second set does not. We find the same concentrat ion of creatine in gray and whi te matter. What controls creatine + phosphocrea t ine is not energy metabol i sm per se, bu t e n z y m e equil ibria. So in early hypox ia you lose creat ine and in mi to-

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chondrial diseases you lose phosphocreatine; but total creatine + phos- phocreatine may not change. Metabolites like phosphocreatine may also be important osmolytes.

What have we learned about myoinositol? Fluctuations over a 10-fold range are seen in this metabolite. There is some evidence that myoinositol can be a marker for astrocytes. Mysinositol is at the center of a very complex and important biochemical network of reactions involved in control mecha- nisms in metabolism.

What have we learned about choline? The choline peak comprises not only choline but also derivatives like phosphoglycerylcholine and glycerylcholine. The concentration of cholines in white matter (1.6 mM) is about equal to that in gray matter (1.4 mM), in contrast to what is generally believed. The headgroup of phosphatidylcholine is not visible in MRS. An additional technique, decoupled 31p MRS, can be used to measure choline and choline derivatives separately. Dissection of the cho- lines is a logical step to apply if one observes that the choline peak changes in a diseased state.

In conclusion, we have on our hands a powerful tool which has reached the stage now where it is readily available for those who need it. We should see a lot more of it being performed in the future.

Discuss ion

E. Boltshauser: Does your choline peak include sphingomyelin? B. Ross: No. Compounds like sphingomyelin are too big to "spin" in the

NMR experiment; they are NMR invisible. There is in every 31p spectrum a hump which includes the macromolecules of choline. The new techniques may get us there. Macromolecules are not always invisible. Glycogen, which is a big molecule, is visible in the carbon spectrum.

References Cady E. B., Dawson M. F., Hope P. L., et al. (1983) Non-invasive investigation of cerebral

metabolism in newborn infants by phosphorus nuclear magnetic resonance spectros- copy. Lancet 1, 1059-1062.

Hope P. L., Cady E. B., Tofts P. S., et al. (1984) Cerebral energy metabolism studied with phosphorus NMR spectroscopy in normal and birth-asphyxiated infants. Lancet 1, 366-370.

Michaelis T., Merboldt K-D., Hanicke W., Gyngell M. L., Bruhn H., and Frahm J. (1991) On the identification of cerebral metabolites in localized ~H NMR spectra of human brain in vivo. NMR Biomed. 4, 90--98.

Ross B. D., Kreis R., and Ernst T. (1992) Clinical tools for the 90's: Magnetic resonance spectroscopy and metabolite imaging. Eur. J. Radiol. 14, 128-140.

Kries R., Ernst T., and Ross B. D. (1993) Development of the human brain: In vivo quanti- fication of metabolite and water content with proton magnetic resonance spectros- copy. Magn. Reson. Med. 30, 1-14.

van der Knaap M. S., Ross B. D., and Valk J. (1993) Inborn errors of metabolism, in Magnetic Resonance Neuroimaging (Kucharcyzk I., Barkovich A. J., and Moseley M., eds.), CRC, Boca Raton, FL.

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Grodd W., Kragelah-Mann |., Klose U., and Sauter R. (199l) Metabolic and destructive brain disorders in children: Findings with localized proton MR spectroscopy. Radiol- ogy 181, 173.

Zimmerman R. A. and Wang Z. (1992) Proton spectroscopy of the pediatric brain. Riv. Neuroradiol. 5, 5.

Ernst T., Ross B. D., and Flores R. (1992) Cerebral MRS in infant with suspected Reye's syndrome [Letter]. Lancet 340, 486.

Boesch C., Gruetter R., Martin E., Duc G., and Wuthrich K. (1989) Variations in the in vivo 31P MR spectra of the developing human brain during postnatal life. Radiology 172, 197-199.

Heerschap A. and van den Berg P. (1993) Proton MR spectroscopy of the human fetus in utero, in Proceedings, 12th Society of Magnetic Resonance in Medicine, Society of Magnetic Resonance in Medicine, 318.

Magnetic Resonance Spectroscopy of the Human Brain Peter Barker

At Johns Hopkins University, in the period 1990-1993, we examined about 300 patients by proton magnetic resonance spectroscopy. Some of these patients have also been examined by MR spectroscopic imaging. Among the reasons for choosing proton spectroscopy is its sensitivity, the spatial breakdown it affords, and the fact that no significant hardware is required. And in spectroscopic imaging you map out the metabolite levels over much of the brain.

When spectroscopic imaging is done on a control brain and you use long echo times, you see signals for NAA, creatines, and cholines. There is varia- tion in the levels with age so that in studying a disease like Canavan disease, which I will talk about during the first part of my talk, it is important to use age-matched controls. The studies with Canavan disease illustrate nicely the importance of doing quantitative MRS studies. At Johns Hopkins we have developed a scheme for calculating concentrations from the spectra based on the cerebral water content.

As discussed at various times during this meeting, Canavan disease is a white-matter disease caused by a deficiency of N-acetylaspartate acylase. Elevation of NAA in the urine can be used as a diagnostic feature. There have been several reports of nonquantitative studies indicating that NAA is also elevated in brain. Together with Dr. Naidu we have studied 6 patients using both long echo times and short echo times. The NAA peak does not look very different from that of the controls. What is different, though, is that practically all of the other peaks have decreased. If you calculate the ratio NAA/creatines, as some people have done, you would get the errone- ous impression that NAA had increased. Of importance is that there is a large increase in myoinositol in the patients. There is also a little lactate. There is an age-related increase in NAA in the brain and this is almost exactly the same in the patients and in the controls. How do you reconcile this apparent lack of an increase of NAA in the brains of Canavan patients

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