Neuroradiology Roland Kreis, Zvi Ackerman,

PhD #{149} Brian MD

Metabolic in Chronic Detected Proton

D. Ross,

Disorders Hepatic with H-i

magnetic

resonance (MR) of the brain was performed in 11 patients with chronic hepatic encephalopathy (CHE), and the results were compared with those of patients with liver disease but without CHE; clinical control subspectroscopy

jects

with

diabetes,

atrophy; technique lated-echo troscopy

uremia,

inositol changes included cerebral a 23%

choline

metaboand myo-

is described. Specific in the brain of CHE patients the anticipated elevation in glutamine levels (P .0001), reduction in choline metabolite (P .0001), and a more than

levels 50%

or cortical

and healthy subjects. The of water-suppressed stimuhydrogen-i MR specfor detection of cerebral glutamine, glucose,

glutamate, N-acetylaspartate, lites, (phospho)creatine,

reduction

in cerebral

myo-inosi-

tol levels (P .0001). In four of the 15 patients with liver disease but without clinical CHE, a significant reduction in the myo-inositol level was detected, and in two of these patients an elevation in the glutamine concentration was also observed. These findings indicate a role for image-guided H-i MR spectroscopy in the diagnosis and monitoring of both overt and preclinical CHE. Index terms: MR. 10.1214 netic resonance Radiology

I

From

Brain,

1992;

the

diseases,

#{149} Liver, cirrhosis, (MR),

10.599 761.794

#{149} Brain, #{149} Mag-

spectroscopy

182:19-27

Magnetic

FRCS,

Resonance

Spectroscopy

Laboratory, Huntington Medical Research Institutes, 660 5 Fair Oaks Aye, Pasadena, CA 91105 (R.K., B.D.R., N.A.F.); the California Institute of Technology, Pasadena, Calif(R.K., B.D.R.); and the Liver Unit, University of Southern California, Los Angeles (Z.A.). Received June 14, 1991; revision requested July 26; revision received August 1; accepted August 7. Supported by the L.K. Whittier Foundation and the Norris Foundation; R.K. supported by the Boswell Foundation. Address reprint requests to B.D.R. RSNA, 1992 See also the editorial by Bottomley (pp 6-7) in this issue.

DPhil

(Oxon),

FRCPath

#{149} Neil

A. Farrow,

BSc

ofthe Brain Encephalopathy MR Spectroscopy’

H

encephalopathy (HE), also known as portal-systemic encephalopathy, is a well-recognized complication of cirrhosis and other severe liver diseases (i,2). Chronic HE (CHE) is a relapsing condition in which changing mood or behavior, tremor (asterixis) and dysarthria, cortical atrophy, myelopathy, and dementia may occur. Individual patients display a variety of neuropsychiatnc abnormalities, culminating in motor disturbances and loss of conscious-

ness.

EPATIC

The

syndrome

is therefore

com-

monly

accorded a grade (grade 0 = imperceptible neurologic changes, grade 4 = coma). The prevalence of CHE in cirrhotic patients is uncertain, but it occurs in over 80% of such patients after portacaval shunt surgery (3). Despite the frequency with which CHE may occur in confirmed cirrhotic patients, there is often difficulty in establishing a definite diagnosis on the basis of clinical cnteria alone. Additional tests are often employed. These include psychomoton tests (number-connection test), standard electroencephalographic methods, and specialized techniques

such as the evoked (4). The effectiveness

potential of these

and P300 tests is

still in some doubt (5). In particular, there is controversy concerning the existence of a subclinical form of CHE and its prevalence (6-8). In one study, 85% of cirrhotic patients had sufficient neuropsychiatric disturbance to impair their ability to drive a car (9). The pathobiochemistry of CHE is probably multifactorial, with a variety of circulating neurotoxins combining to affect the previously “sensitized” brain (2). Mechanisms of sensitization, however, have not been determined. Almost certainly the syndrome involves the accumulation of neurotoxic concentrations of ammonia (10), even though levels of ammonia in the circulating blood are not always elevated (ii). Patients with moderate to severe HE have increased cerebrospi-

nal fluid glutamine (Gln) concentrations (i2) arising from ammonia and glutamate (Glu) by the action of the enzyme Gln synthetase (Enzyme Commission 6.3.i.2) located in the

astrocytes

(i3).

Treatment

of CHE

is

directed at removing excess ammonia from the systemic circulation by means of a low-protein diet, stenlization of the gut flora with neomycin

( 14), or prevention

of ammonia

ab-

sorption by using lactulose (i5,i6). As it is possible that treatment of CHE at an early stage may prevent the late sequelae, there is a need for new techniques of cerebral metabolic analysis to further elucidate this complex organic brain syndrome. Proton magnetic resonance (MR) spectroscopy is a noninvasive examination performed in a clinical MR imager at a minimum field strength of 1.5 T, without the need for additional special equipment. A proton image of the brain is obtained in the usual way. Then, a volume of interest is identifled within the cerebral cortex. With suppression of the water signal, which is normally exploited in MR imaging, a spectrum of this localized region is recorded. In experimental animals, hydrogen-i MR spectroscopy has been used to detect the expected increase in cerebral Gln concentration in models of acute HE (1719). At the lower magnetic field strengths used clinically, the detection of Gin, and in particular its separation from Glu, presents some diffi-

culty

(20). However,

we recently

reported that this distinction was achieved at i.5 T by using short echo times and difference spectra (21). Metabolites now routinely identified in the H-i spectrum of the human brain

=

Abbreviations: CHE chronic hepatic encephalopathy, Cho choline, Cr creatine, Gln glutamine, Glu = glutamate, Clx sum of Gln and Glu, HE hepatic encephalopathy, MI rnyo-inositol, NAA N-acetylaspartate.

= =

=

=

=

=

=

are all in the millimole per liter concentration range and include Gln and Glu, N-acetylaspartate (NAA), creatine (Cr) (as the sum of creatine plus phosphocreatine), choline (Cho) (a composite peak consisting of free choline, choline phosphates, and choline phosphatides), and inositol. The latter peak is assigned to myo-inositol (MI), a hexol present at a concentration of over 5 mmol/L in human and animal brain (22-25). Lactate (26), alanine (27), ethanol (28), glucose, and several other metabolites can also be identifled, and to some extent quantified, in the human brain spectrum when present in pathologically increased concentrations. The present report summarizes the results of H-i MR spectroscopy in 58 persons, including patients with liver disease (with and without CHE), patients with related metabolic disorders, and healthy control subjects.

MATERIALS

AND

METHODS

Table

1

and Laboratory

Clinical

Findings

in 26 Patients

Parameter No.

Group

of patients

Male/female Age (y) Patients

Patients

with with

glucose

abuse

28 51 46

308

level (mmol/L)

6.0 2.1

Urea level (mmol/L) Bicarbonate level (mol/L) Estimated osmolality (mosm/kg) Child class A/B/C

Lactulose/neomycin/furosemide

26.4 296

treatment

I

Group

11 7/4 (30-75) 5 5 (17-34) (20-90) (20-83) (S8S27)t (4.1_15.8)t (1.5-3.7) (22.0-35.8) (2764340k) 0/6/5

56 ascites history

of alcohol Albumin level (gIL) Prothrombin level (%) Bilirubin level (mol/L) Ammonia level (i.mol/L)

Blood

Liver Disease

with

Reference Range

2

15 10/5 51 (25-86) 6 10 34 (21-43) 81 (75-100) 29 (2-128) 105 (fi#{216}..163)t 6.2 (4.4-8.0) 3.7 (0.8-5.5) 24.6 (8.2-38.2) 295 (267-35tY) 9/3/3

7/5**/6

NA* NA NA NA NA 35-50 100

3-17 #{216} 4-6 2.5-6.7 24-30 278-305 NA

1/0/2

NA

Note-Patients were assigned to group 1 if HE was clinically evident at the time of the H-i MR spectroscopic examinations or had been noted at any time in the past. The remaining patients (group 2) had shown no clinical evidence of HE at any time. Values are means (numbers in parentheses are ranges). * NA = not applicable. t n 5. *n= 10. n = 13. Includes glucose in diabetic patients. I Lactulose: Cephulac, Marion Merrell Dow Pharmaceuticals, Cincinnati. Neomycin sulfate: Mycifradin, Upjohn, Kalamazoo, Mich. Furosemide: Lasix, Hoechst-Roussel Pharmaceuticals, Somerville, NJ. ** One patient received both lactulose and neomycin.

=

Patients A total of 58 subjects were examined for this study. Twenty-six patients with liver disease-i 1 with and 15 without clinical evidence of CHE-were selected. A further 17 patients had related clinical conditions including diabetes mellitus, chronic renal failure, and cortical atrophy, all with normal liver function. Fifteen apparently healthy control subjects were also included. Informed consent was obtained, and the study was approved by the Internal Review Board of Huntington Memonial Hospital. The usual exclusions for MR examination,

such

as metallic

implants

or

cardiac pacemaker, were observed. Clinical and laboratory findings in the patients described in the present study are summarized in Table 1. Patients with biopsy-proved cirrhosis or other liver diseases confirmed by means of laboratory tests were clinically evaluated by one or more hepatologists (Z.A.) to define the presence or absence of CHE. Clinical critena of CHE used were (a) changes in behavior, (b) disturbances of consciousness, and (c) existence of astenixis. Blood ammonia

level

determinations

and

number-con-

nection tests were performed in some but not all of the patients and control subjects. Because patients with a history of one on more episodes of HE and patients with CHE at the time of the examination had similar results at H-I MR spectroscopy, they were pooled to yield group I. At the time these patients were examined, they were all receiving medication for their CHE in the form of neomycin or lactulose, with a low-protein diet and diuretics for ascites.

Their

general

hepatic

state,

as de-

termined by liver-function tests and clinical examination, was stable, and they did not have evident gastrointestinal hemorrhage, azotemia, or intercurrent infection. 20 #{149} Radiology

Patients with acute HE induced by diuretics or other causes were not included. All of the patients were ambulant at the time of the MR examination. All were fully conscious and alert, with grade 0-i HE, and none showed lethargy or disorientation, the minimum criterion for grade 2 HE (1). Treatment was not modified for the purpose of this study. Two patients in group 1 had myelopathy solely attributable to their chronic HE. Two, including one of those with myelopathy, had received a surgical portacaval shunt. One patient in this group had insulin-dependent diabetes. Group 2 was composed of patients with mild to severe liver disease but without clinical evidence of CHE at present or at any

time

in the

patients with drug-induced rhosis

induced

past.

The

group

included

chronic inflammatory liver disease, as well by

alcohol

on as cir-

or hepatitis.

Two also had insulin-dependent diabetes. The severity of liver disease was graded on the basis of the Child classification (modified by Pugh) (29). A further modification of this classification (omitting the score for HE) did not change the grouping of patients. To determine the specificity of changes observed in CHE, patients with diseases that might involve altered MI metabolism (23,24,30) were examined. These included subjects with type 1 or type 2 diabetes mellitus (mean blood glucose level, 13.2 mmol/L), patients with uremia (mean blood urea level, 11.0 mmol/L), and others receiving furosemide. Because CHE is reported to be commonly associated with cerebral atrophy (31-33), we included five subjects without diabetes or liver disease but with evidence of cerebral atrophy on

MR images.Ten control subjects five

younger

normal

MR

age- and sex-matched (group 0) and a group control

subjects

of

provided

the

data.

Examination

One

or more

volume

brain

of interest

spectra located

from in the

a single panietal

cortex were acquired in each subject. A second volume of interest, placed symmetnically and contralatenally, was also exammed in eight patients and seven control subjects. MR spectroscopic examinations were repeated on two or more occasions in eight patients with liver disease and four control subjects. Five examinations over the course of 6 months were performed in one patient with CHE. Thus, the total number of MR spectroscopic examinations

performed

approximately

Data

was

165 individual

79,

involving

spectra.

Acquisition

Proton

MR

spectra

were

obtained

from

nominal volumes of i2-27 cm3 localized in the left or right parietal cortex. The localization was guided by Ti-weighted, axial MR images, obtained as the first step in the MR spectroscopic examination. A stimuiated-echo sequence (34-36) combined with chemical shift selective pulses was used for localization and water suppression,

yielding

a signal

only

from

the

re-

gion of interest at the spatial intersection of three section-selective pulses. Expenimental parameters were as follows: echo time, 30 msec; mixing time, 13.7 msec; repetition time, 1.5 seconds (0.75-12 seconds for

relaxation

measurements);

number

January

of

1992

peaks (1.0-1.7 x linewidth) was used to fit a gaussian line. Because this choice may render the fitted linewidths inaccurate, peak amplitudes were used for further analysis (36). At a field strength of 1.5 T, most other components contributing substantially to the cerebral spectrum yield complex spectral patterns, caused by strong coupling. Some of these spectra are included in Figure 1. To estimate the contribution of Gin, an integration over a small spectral range dominated by Gln and Glu was performed (value Ai [2.31-2.41 ppm]). Accompanying changes in the alpha-proton (H0) region of Glu and Gln were measured by an integration A2 (3.72-3.80 ppm). Two further integrations over the spectral ranges dommated by glucose were performed (A3 [3.40-3.49 ppm] and A4 [3.74-3.86 ppm]). The integration ranges for Al, A2, A3, and A4 are indicated in Figure 1. 4. Difference spectra were constructed for each patient. A normal control spectrum, obtained by averaging the spectra from all of the age-matched control sub-

Cr

Cr

NAA+GLU+Cr control

subject

+Cho+MI

Al

A2

Li

U

A2

Al

A4

A3

f-i

fl

-

A2

Al

glucose

*

Jtaunnerine

A2

A4

Al

A3

ethanol

jedts

myo-inositol I 3

2

1

0

4

‘

‘

3

2

,

I...’’,,,,

I

1

0

ppm

ppm

Figure 1. Localized in vivo and in vitro H-i MR spectra, obtained with a stimulated-echo sequence at 1 .5 T, illustrate the identification of the cerebral metabolites discussed in this anticle. At the top left is a normal brain spectrum (the sum of results from 10 age-matched control subjects). At the top right is a reference spectrum from an aqueous solution composed of 36.7 mmol/L of NAA, 25.0 mmol/L of Cr, 6.3 mmol/L of choline chloride, 30.0 mmol/L of Glu, and 22.5

mmol/L

of MI (adjusted

to a pH

of 7.15

were recorded from solutions of individual all spectra were subjected to a line-shape mately

4-Hz

linewidth.

The

integration

in a phosphate

biochemicals. transformation ranges

used

buffer).

The

remaining

To simulate the in vivo yielding gaussian peaks

to detect

changes

in the

cerebral

conditions, of approxiof

Clx (Glu or Gin) (Ai + A2) and glucose (A3 + A4) are indicated. The peaks labeled * nated from giycine, NAA, or acetate, which were added to the various solutions as chemical shift references (the methyl peak of NAA was set to 2.02 ppm). All spectra were scaled individually

and

cannot

be used

for

direct

quantitation.

experiments, 128-512. The water-suppression pulses (nominal bandwidth, 75 Hz) did not distort the measured ratios appreciably (suppression < 2% for all metabolites considered). The total examination time per patient and session was 30-60 minutes. All spectra were acquired with a i.5-T

Signa

imager

(GE

Medical

Systems,

Milwaukee) with aging quadrature

use of the standard imhead coil. The data analysis was performed on a Sun 3/470 cornputer (Sun Microsystems, Mountain View, Calif) equipped with a TAAC1 application acceleration board (Sun Microsystems) and the SAGE data analysis package (GE Medical Systems).

Data

Processing

A visual comparison of all images to assess the degree of atrophy and any differences in the appearance of the basal ganglia was made by a neuroradiologist who was unaware of the clinical diagnosis and the results of the MR spectroscopic exarnination (37). A relatively simple and largely automatic and operator-unbiased processing

Volume

182

#{149} Number

1

subjects

with

a total

of 23 spectra),

Reproducibility

and

Possible

Artifacts

spectra

levels origi-

(10

was aligned to the patient spectrum and then subtracted. Features observed as positive (increased) or negative (reduced) were then identified by comparison with the spectra of the pure compounds in Figure i.

scheme, explained elsewhere (36), was employed to analyze the spectra recorded for this study. It consisted of four steps: 1. Digital low-frequency filtering of the free induction decay for additional water suppression. 2. Lorentz-Gauss transformation to obtam similar linewidths in all spectra (4 Hz) and to enhance spectral resolution, and zero filling of the free induction decay to 8,i92 points (0.004 ppm per point) to make an accurate frequency alignment between different spectra possible. 3. After Fourier transformation and manual zero-order phase correction, different regions in the spectrum were separately analyzed. In Figure 1, the summed spectra of 10 healthy volunteers are cornpared with the spectra obtained from model solutions of pure compounds, line broadened in the above manner to mimic the in vivo conditions. The main resonances in the cerebral spectrum are each attributable to a single major component and appear singletlike (2.02 ppm = NAA, 3.03 ppm = Cr, 3.22 ppm = Cho, and 3.56 ppm = MI). To minimize the effects of overlap, the central part of each of these

A mean variation of 0.03 units for all peak ratios was recorded for spectra obtamed consecutively. This value was similar to that of the fluctuations caused by the noise in the observed spectra. Spectral variations, approximately 0.07 units for all tabulated values, were found for contralateral voxels in the same individual. Larger differences between individuals represent the effects of biologic variation and are seen in the figures and tables. Caution has to be used in interpreting changes in relative peak intensities and areas as changes in metabolite concentrations. When we refer to concentration changes, it is with the factors listed below in mind; they can alter the tabulated measurements, but none of them is expected to modify the main conclusions of this artide: 1. Relaxation effects: No obvious linewidth variations attributable to T2 effects were observed. Steady-state relaxation measurements were obtained in two patients and two control subjects. No significant variations in Ti were found. 2. A spillover of the intense lipid resonances originating from bone marrow and subcutaneous fat and of signals from waten protons can occur. In one case, noted in Table 2, the values for NAA and peak integral Al were rejected for this reason. 3. The spectra of gray and white matter are different (38,39). However, the composition of the voxels selected in this study was consistent, as shown by the small variations between spectra acquired from

Radiolotrv

#{149} 21

Table 2 Peak Intensities

Relative

t o Those

of C r and Pho sphocreatine

for All Groups

of Patients

Healthy

and

Peak Intensities Group

Age

(y)

M/F

NAA

Cho

Group Group P

0 (n I (n

= 10) = 11)

53 ± 12 57 ± 15

6/4

7/4

0.83 ± 0.05 0.64 ± 0.07

Group

2 (n

= 15)

± 0.07 1.39 ± 0.14 .8

51 ± 18

10/5

1.39

0.74

54 ± 10

3/1

50 ± 18

7/4

I Preclinical P Group

CHE

2M

= 4)

(n

ii)

(71

P Furosemide (n = 3) P Uremia (H = 4) P Aged (atrophy) (ii = 5) P Young (n = 5) P Diabetic (n = 5) P

1.40

± 0.13 .8 1.43 ± 0.07 .6 1.38 ± 0.14

.0001 ± 0.10 .02 0.66 ± 0.03 .000l 0.74 ± 0.07*

.6 57 ± 3

0/3

1.47

.004

± 0.04

0.85

.2 49 ± 21

2/2

1.42

4/1

± 0.13

1.45

25 ± 2

2/3

48 ± 22

2/3

± 0.10 .4 1.56 ± 0.07 .002 1.48 ± 0.17*

48±18

4/3

1.40±0.07’

P

.99

=9

(excluding

4. Including 0 6. 0

two

diabetic

two patients

with

Statistical

liver

.18 0.77 ± 0.10 .17 0.92 ± 0.03

.5 0.64 ± 0.09 .6 0.68 ± 0.04

.003 0.88±0.08 .16

.06 0.70±0.06 .01

t test comparison

with

group

0.26 0.43

± 0.05 ± 0.09 .0001 0.28 ± 0.09 .43 0.31 ± 0.09 .19 0.27 ± 0.10 .64 0.31 ± 0.07 .14 0.29 ± 0.04 .2 0.37 ± 0.05 .002 0.29 ± 0.03 .3 0.28 ± 0.06 .5 0.27±0.05’ .6 0, the age-matched

0.46 0.62

± 0.07 ± 0.06

0.54

.0001 ± 0.09

0.59 0.52 0.50 0.45

0.44

.02 ± .01 ± .09 ± .4 ± .9 ±

0.07

0.09 0.02 0.02

0.08

.7 ± 0.10 .4 0.59 ± 0.05 .004 0.56±0.06 0.50

.008

control

subjects.

Sub-

disease.

Methods

Quantitative of pained on linear

assays

were

on unpaired regression

compared

by

Student analysis.

RESULTS Main

.02

A2

patients).

slightly different locations within the panetal cortex. 4. The (phospho)cneatine peak at 3.03 ppm was used as an internal standard. Earlier studies with phosphorus-3i MR spectroscopy (20,40) had shown no changes in phosphocreatine concentrations in patients with mild CHE. 5. Foreign compounds (eg, ethanol) or normal metabolites (eg, glucose) can contribute to several regions of the spectrum and have generally been accounted for.

tests

.0001 ± 0.16 .35 0.34 ± 0.05 .000l 0.62 ± 0.06t .9 0.71 ± 0.03 0.56

Al

=

=

means

± 0.06

0.28 ± 0.06

0.68 ± 0.04 .06 0.59 ± 0.09

Note-Mean values ± one standard deviation are given. P values are for a two-tailed tracting the four patients with preclinical CHE from group 2 yields group 2M#{149} t n

0.61

0.92 ± 0.13 .07 0.79 ± 0.06

.23

Diabetic(n=7)

MI

.6

.8 79 ± 14

± 0.07

Subjects

(ppm)

Findings

in CHE

In Figure 2, a representative brain spectrum from a patient with CHE (group 1) is presented along with that from a healthy, age-matched control subject. The spectra are presented before line-shape transformation. Three outstanding differences of diagnostic value are readily observed: 1. The spectral regions around 3.75 ppm and from 2.1 to 2.5 ppm are elevated, which is consistent with an increased Gln or Glu concentration. The corresponding difference spec22 #{149} Radiology

trum indicated that the change was due to an elevation in the Gln concentration (see below). 2. The peak at 3.56 ppm attributed predominantly to MI is drastically reduced. 3. The signal at 3.22 ppm, ascribed to the methyl protons of compounds containing Cho, is substantially reduced. The additional spectral analysis is illustrated in Figure 3, which consists of three parts. rebral spectra

The analysis of a patient

(group i) and 2 is presented

of a patient on the left

of the cewith CHE from group and on the

right, respectively, whereas the middie part contains spectra of model solutions with and without Gln. The similarity between the positive contnibutions in the difference spectrum of the CHE patient on the left and that of the Gin solution in the regions of the 3 and y protons (2.i-2.5 ppm) and the area of the a proton (3.6-3.9 ppm) is striking. The negative features of this difference spectrum are clearly identified with MI and Cho. The conresponding stick spectrum in Figure 3 further emphasizes these spectra of the appropriate tions in the middle panel the effect of an increased on the original spectrum degree of reliability that

points. The model soludemonstrate Gin content and also the can at best

be achieved for the difference spectra. In contrast to the features of the spectrum from the CHE patient, the featunes of the spectrum from a patient with liver disease but without CHE are almost indistinguishable from normal. The difference spectrum shows a slight reduction in the Cho peak and atypically also in the NAA peak. Results illustrated by these representative spectra were substantiated by analysis of the spectra obtained from all of the patients and control subjects. The quantitative analyses discussed below are summarized in Table 2 and presented individually in Figure 4, in which the distribution of values for each metabolite can be compared. The NAA/Cr ratio was the same in the control subjects and the two groups of patients, a fact that supports the assumption that the cerebral Cr content, which was used as an internal standard, remains constant in CHE. The MI and Cho peaks were significantly decreased in spectra from CHE patients-by 54% and 23%, respectively, compared with normal values (Table 2)-and these two changes were present in all patients with CHE (Fig 4). The integral value Al, used to detect changes in the Gln and Glu concentrations, was increased by 65% January

1992

NAA

Cr

changes associated with CHE. Patients with severe liver dysfunction (Child class C) showed all three of the MR spectroscopic changes (with one exception). No statistically significant differences between MI, Cho, or Glx levels in those with liver disease classified as grade B (n = 9) or C (n = 6) could be demonstrated in the 15 patients with CHE (either overt or preclinical as defined above); these patients were henceforth called group

Cho

Gix

Cr

I

2. Excessive alcohol consumption (Table 3): A slightly lower cerebral concentration and a relatively relipids

2

0

4

Figure

control

2.

Cerebral

subject

2

3

ppm

1

0

ppm MR

spectra

(left spectrum)

H-l

and

demonstrate

a patient

the

with

major

CHE

differences

(right

spectrum):

between

a healthy

The spectral

contnbu-

tions from Gln (2.1-2.5 ppm and 3.75 ppm) are increased in the patient with CHE, whereas the levels of MI (3.56 ppm) and Cho (3.23 ppm) are drastically reduced. Acquisition and processing parameters used for both spectra were as follows: nominal voxel size, 2.5 cm3; echo

time, 30 msec; mixing time, 14 msec; repetition time, 1.5 seconds; number of experiments, 256; spectral width, 2,000 Hz; number of points, 2,048 (zero-filled to 8,092); postacquisition water suppression as described; and exponential line broadening, 0.4 Hz.

in patients with CHE (Table 2). Difference spectra revealed that the increase in this value was mostly due to an increase in Gin contribution, while the Glu level remained approximately constant. From spectra of model solutions of Glu and Gin mixtures (the sum of which is described here as “Glx”), it was estimated that the average cerebral Gln concentration in patients with CHE increased to about 10-12 mmol/L, so that the sum of GLx was doubled. The effect of CHE on the cerebral Gln level varied considerably between individuals. The maximum increase was about double the average, and no increase in Gln was observed in one patient in whom the reduction in MI and choline levels was nevertheless fully expressed. Patients with liver disease but without CHE (group 2) showed a significant 11% decrease in the Cho peak and an increase in the A2 value of 17% (Table 2). However, the results in this group were heterogeneous (Fig 4). Four patients showed changes that were very similar to those observed in the patients in group i. The changes in MI and Cho levels in this subset of group 2 (summarized in row 4 of Table 2) were within the range of the Vnliinic’

#{149} NIiinihr

1

CHE group (decreases of 44% and 20%, respectively), while a definite elevation of the Gln level was found in only two of the four patients. We suggest that all four of these patients might be described as having preciinical CHE. When these four patients were excluded from Group 2 (yielding group 2M5 in Table 2), MI and Clx integrals remained constant.

Correlations

and

Further

Control

Groups The

elevation

of the

cerebral

Gin

concentration in patients with CHE was anticipated (12). But because of individual variations, the direct, noninvasive determination of Gln concentrations in patients may be of diagnostic value. The reduction in the MI level was a novel and entirely unexpected finding. To explore the cause of the MI depletion in CHE, several factors including clinical history, treatment, and related diseases were examined. The results are summarized in Table 2 (lower half) and Table 3: 1 . Severity of hepatic disease: Patients with mild liver disease (Child class A) did not show the spectral

MI

duced value for the integral A2 were observed in alcoholics with CHE. As the value for the integral Al did not follow the same pattern, the effect was probably attributable to a higher cerebral glucose concentration in the nonalcoholic patients with CHE. 3. Lactulose versus neomycin treatment (Table 3): The Al level was lower in patients treated with neomycm. However, this decrease was not accompanied by any change in A2, so it was unlikely to have been due to an effect of the Gln concentration (41). MI and Cho concentrations were comparable in patients receiving diffening CHE treatments and also in four subjects in group 2 receiving no CHE therapy. 4. Furosemide treatment: By altering intracellular osmolarity, diuretics might affect MI concentrations in the brain (42). Furosemide treatment resuited in a 16% higher cerebral MI content (P < .02) (Table 2), and patients with CHE receiving furosemide tended to have a higher MI level (P < .07) than their appropriate control subjects (Table 3). 5. Uremia (Table 2): Patients with elevated blood urea nitrogen levels due to renal failure showed a tendency toward higher Cho and MI concentrations, but the results did not achieve statistical significance. 6. Cortical atrophy and age (Table 2): Mild to moderate cortical atrophy was detected with MR imaging in the majority (seven of 10) of our patients with CHE, in nine of 13 patients in group 2, and in two of the 10 agematched control subjects. Gln and MI levels were unaltered by the presence of cortical atrophy. Compared with the age-matched control subjects, patients with cortical atrophy showed an increase in the Al value but not in the A2 value; the cause of this increase is unknown. The volunteers in the younger age group displayed a significantly increased NAA/Cr ratio. This may be an indication of inR2,linlnw

S

normal

jJJ\normai

mature

Gln

Cho

Gin

(\

J\kJJLAj;;jHE Gin

ddM

Gin

.l.._

I

I

MI

I

I

I

j

\ /\

Gln

Gln

i

Group2

I

i

1

I

I

-

Cho

NAA

difference

pVffence

+

difference

Gly*

Gin +

25

ppm

GIy*

:i

35

+

Gly*

25

ppm

ppm

Original and difference spectra that help identify metabolites with altered levels in patients with CHE are shown. On the left, the normal spectrum (cf Fig 1) at the top is to be compared with the spectrum of a patient with CHE below. The corresponding difference is plotted with a scaling factor of two. Interpretation is facilitated by the “stick” spectrum above it and the spectrum of a Gln-glycine solution at the bottom. (Glycine has the same chemical shift as the main MI peak.) The positive features in the difference spectrum are interpreted as a surplus in cerebral GIn, whereas the negative peaks indicate a drastic reduction in the cerebral inositol level and a lower concentralion of Cho compounds. The spectral differences obtained from model solutions are illustrated in the central column of the figure. The solution for the top spectrum contained NAA, Cr, Cho, MI, and Glu (cf Fig 1), and the spectrum below was obtained from the same solution after the Figure

3.

averaged spectrum

addition

of 30.0

mmol/L

of Gln.

In the

difference

metabolic changes in a patient with liver disease and NAA peaks, indicating lower cerebral levels the NAA resonance was generally unchanged.

creased neuronal number or NAA content (43,44). 7. Diabetes mellitus (Table 2): Inositol phosphate metabolism has been implicated in peripheral neuropathy in both humans and animals with diabetes mellitus (45,46). In the diabetic patients studied, levels of choline (11%), MI (15%) and A2 (28%) were all significantly elevated. The increase in the A2 value in diabetic patients is most readily explained by an increased cerebral glucose content. This was confirmed when two separate partial spectral integrations (A3 and A4, as described above), covering the most prominent peaks in the glucose spectrum, were carried out for 17 healthy and six diabetic subjects. Both of these values were increased in diabetic patients by 83% and 32%, respectively (P < .0005). After correcting for the increased cerebral glucose contributions, the MI peak in diabetic patients remained higher (by 11%) than that in the control subjects (P < .03).

24

#{149} Radiology

spectrum,

the

changes

but without CHE. of these metabolites.

assigned

The difference A reduced

to GIn

are

identified.

DISCUSSION Proton spectroscopy of the human brain has previously yielded several interesting new findings in localized lesions, including tumors (47), stroke lesions (48,49), and multiple sclerosis plaque (50). Use of short echo times, however, considerably increases the range of metabolites that can be assayed (36,51,52), and it has now proved useful in examining the metabolic basis of CHEF a global, organic brain syndrome, and in making initial comparisons with other generalized cerebral metabolic disorders. A number of interesting points arise from this study 1. A diagnostic

of patients elevation

On

the

right

are

spectrum is almost featureless, except cerebral Cho peak is typical of patients

with CHE: in the Gln

level was observed in the great majority of patients with liver disease in whom CHE had been established dinically. 2. A new metabolic defect, the depletion of cerebral MI, was found. The peak identified as MI probably includes a small proportion as inositol-

spectra

illustrating

for small negative with liver disease,

the

Cho but

1-phosphate and an even smaller contribution from membrane-bound phosphatidyl-inositol. The important intracellular messengers 1P3 and 1P4 contribute to a negligible extent due to their very low concentration (i0iO_8 mol/L). MI itself is reported to have a concentration of 5-7 mmol/L in the human brain and thus would account for up to 70% of our present estimate for the observed MI peak (circa 10 mmol/L, based on the assumption that Cr plus phosphocreatme contribution is 10 mmoi/L). A furthen 10% at most may be due to glycine (36). The evidence of cerebral MI depletion in CHE appears to be well established by the present results, but neither treatment nor clinical history factors point to the cause of this abnormality. Previous interest in this hexol has focused on two possible roles. MI, by virtue of being metabolically inert, could be an osmolyte and one of the “ idiogenic” osmoles in hyperosmolar states (42). Also, as mositol monophosphate, an intermediate

January

1992

been

Al

NAA

#{149}#{149}

.5

‘I

noted

related

Os

S

develops %

#{149}.#{149}“.! #{149} .

.%

S

0.4

1.2

I

S..’.

0

:

1

2

3

4

5

S

7

#{149}

I

.

#{149} #{149} #{149}

.5

I

o.@

-h

-

.

I

-

I

-

#{149} #{149}I #{149} #{149}. #{149} #{149} #{149} . . #{149} #{149} .1

in CHE.

However,

cortical

atrophy,

Cho

peak

intensities

Cho

depletion

tias

0.a

#{149}

:.

.:

#{149}::

:

:#{149}::

0.4

0

2

1

I

0.0

-

4.

Figure 1

.1

i

lI.....

#{149}

. #{149}

1qlP

I

::

o

0.0

#{149}

.

#{149}

. 0.4

evaluated

I

1PLu:.

0.5

:!:2NINus

-

in sonbitol importance

-

-

-

-

-

-

metabolism, MI may in the development

be of of

cataracts and peripheral neuropathy in diabetes mellitus (45,46). We have not formally investigated the effects of hyperor hypoosmolar states on human MI metabolism, as in our patients estimated serum osmolality did not differ from that of the control subjects. Modest alterations in serum glucose, sodium, or urea values associated with diabetes, furosemide therapy, or uremia had the effect of increasing the cerebral MI level. Gin, too, is an osmolyte; Takahashi et al (53) postulated that Gin accumulation could cause cell swelling in HE. We might surmise that depletion of MI occurred as an osmotic response to Gln accumulation. As shown in Figure 5, there is an apparent linear relationship between the cerebral Gix and MI concentrations (R = 0.6), as would be anticipated from such an interdependence. However, within each group of patients no such relationship

or control between

subjects MI and

Glx could be demonstrated. istence of MI depletion and cess points to a link between

The coexGln exthe met-

abolic pathways of their biosynthesis. Observations in two children with hyperammonemia and proved onnithine-transcarbamoylase deficiency lend support to this view. In both, a

Volume

182

3

4

5

6

7

I

MI

#{149} Number

1

be

that in pernormal

were

results

observed.

in an Alzheimer

syndrome acetylcholmne

with reduced in rats (57). The

between

point

‘I.

cerebral

Cho

Spectral data for all 58 subjects in this study are shown. Each

corresponds

to an intensity

subdivided

on

the

basis

of their

(NAA,

MI and

Cho,

Cho

peaks. Excluding the four patients with tentative diagnoses of preclinical CHE from group 2 yields group 2MINUS; adding them to group I results in group A systematic reduction in MI and Cho levels and a more variable elevation in the cerebral GIn level characterize the patients with CHE. Statistical data and details of the changes noted in the patients with CHE and the other patients are given in Tables 2 and 3.

marked decrease in the cerebral MI level compared with that in agematched control subjects was found (B.D.R., R.K., and J.C. Williams, unpublished data, 1990-1991). An alternative role for MI may lie in its function as a possible precursor of glucuronic acid (via MI oxygenase [Enzyme Commission 1.13.99.1]) (54). In the brain, as in the liver and kidney, conjugation of xenobiotics with glucuronic acid provides an important pathway of detoxification (55). Depletion of MI might be the result of the excessive detoxification that probably occurs in patients with long-term portosystemic shunts. This hypothesis also implies that MI depletion contnibutes to the observed sensitization of the brain to neurotoxins in HE (2). 3. The small but significant depleof Cho

in the

brain

in CHE

to be established.

clinical

early

diagnosis

encephalopathy?

of preIf the

occur-

of MI depletion in patients 2 is taken as the metabolic

in

counterpart

of preclinical

CHE,

then

MR spectroscopy showed a remarkable prevalence of this disorder (four of 15 [27%]). In the absence of detailed neuropsychiatric evaluation

-.1.-

MI) or area (Al and A2 for Glu plus GIn) in one subject and is the average of all spectra recorded from that individual. The patients were grouped according to their diagnosis: 0= age-matched control subjects; I = patients with CHE; 2 = patients with liver disease but without CHE; 3 = patients receiving furosemide; 4 = patients with uremia; 5 = aged healthy subjects with mild atrophy; 6 = young control subjects; and 7 = diabetic patients. The position along the abscissa in each of the graphs is the same for each individual patient. The patients of group 2 are further

tion

remains 4. Possible

rence group

06

.

might

concentration and neuropsychiatnic changes in CHE and in other demen-

A2 I

:

and atrophy

with

relationship On

(56)

cortical

sons

disease-like cerebral

! 1

1.

before

to the

has

and with this small patient series, we cannot draw a firm conclusion, but we suggest that the increasing availability of clinical H-i MR spectroscopy

makes

such

sible. 5. A trivial application technique ethanol

but

early

diagnosis

potentially

fea-

useful

of the MR spectroscopic is the detection of cerebral in patients drinking alcohol

some time before the examination. We observed the methyl-proton let at 1.2 ppm (see Fig 1) in two patients in group 2 and confirmed

tripof the the

assignment in two of the control subjects. 6. Finally, in diabetes mellitus, elevated cerebral glucose levels can be readily determined, although the contnbution lar glucose

from vascular cannot yet

or extracellube defined. The

observed increase in MI levels in the diabetic brain, although small, is a challenging finding. Although it is consistent with the results of animal studies reported 24 years previously (24), this finding appears to contradict current ropathy, tol

thinking in which

(phosphate)

about diabetic neua decrease in inosiin nerve

for the use of aldose tons in the treatment peripheral obvious

is the

reductase of cataracts

neuropathy reasons, there

basis inhibiand

(45,46). For have not been

any parallel studies in the brain in man. H-i MR spectroscopy offers an ideal noninvasive means of addressing this problem. In conclusion, we have described a noninvasive olites

assay

that

demonstrates

for

cerebral complex

metabbio-

chemical abnormalities in the brain in patients with liver disease and sheds new light on the mechanism of CHE.

U

Radiolor-

‘

“

Table

3

Peak Intensities within Groups

Relative

to Those

of Cr and Phosphocreatine:

Differentiation

According

to Treatment

Peak Intensities Criterion

Child

Age

M/F

59 ± 14 51 ± 15 NA

6/3 4/2 NA

1.37 1.45

51 ± 12 62 ± 15 NA

8/0 2/5 NA

1.45 1.35

48 ± 18

4/3

55 ± 25

3/1

NA

NA

57 ± 17 62 ± 6

4/2 2/2

NA

NA

52 ± 20 62 ± 5 NA

4/2 3/2 NA

Cho

NAA

History

(ppm)

Ml

Al

A2

class, GI’

Class

=

B (n

Class C P Alcohol

(,i

=

abuse,

Yes (n No (n P

9) 6)

= 8) = 7)

abuse, Yes (n = 7) No (n = 4)

(n = 6) (n = 4)

Lactulose p

No (n P Note-Mean = group * 0.25 versus

± 0.06 ± 0.07 .99

0.32 0.27

± 0.16 ± 0.05 .14

0.64 0.65

± 0.08 ± 0.05 .7

0.29

± 0.16 ± 0.06 .4

0.74 ± 0.08 0.82 ± 0.14

0.65 0.63

± 0.16 ± 0.07

0.65 0.64

± 0.07 ± 0.04 .09

0.38 ± 0.09 0.42

± 0.08 ± 0.04

0.62 0.59

± 0.12 .5

.4

± 0.06

0.37

± 0.11

0.57

± 0.03

0.32 ± 0.06

0.43 ± 0.10

0.66 ± 0.06

4*

.3

.003

1.41 1.33

± 0.10 ± 0.06 .8

0.26 0.29

0.29 0.26

± 0.08 ± 0.04 .5

0.47 0.38

0.32 0.24

± 0.06 ± 0.04 .07

0.43 0.43

.3

± 0.08 ± 0.13

0.50 0.55

± 0.10 ± 0.07 .4

± 0.05 ± 0.04 .01

0.62 0.63

± 0.07 ± 0.07 .8

± 0.10 ± 0.08 .9

0.63 0.60

± 0.06 ± 0.07 .4

.7

Cl

Neomycin Furosemide, Yes (n

0.65 0.65

G2

P Treatment,

± 0.10 ± 0.16 .3

Cl’

Alcohol

C2

(y)

and Clinical

1.44 1.32

.2

± 0.06 ± 0.09

.8

Cl

= 6) = 5)

1.45 1.32

± 0.16 ± 0.08 .2

0.64 0.64

values ± one standard deviation are given. P values are for a two-tailed NA = not applicable. 0.34 ppm with P < .01 for 10 patients in the original group I.

± 0.07 ± 0.08 .9 t test comparison

within

each

group.

Cl

group

=

1, Cl

‘

=

group

l,

2M,

Acknowledgments:

We are grateful to the physicians and nurses of Huntington Memorial Hospital, in particular the staff of the Liver Unit (Myron Tong, MD, director), and to the staff of the Liver Unit at the University of Southern California (Telfer Reynolds, MD, director) for permission to examine patients under their care. Analysis of MR images was performed by Robert Crouch, MD. We thank Jennifer Bellinger for excellent secretarial support.

Al

2. 3. 4.

5.

6.

7.

8.

9.

10. 11.

12.

Sherlock S. Diseases of the liver and biliary system. Oxford, England: Blackwell Scientific, 1985; 91-107. Zieve L. Hepatic encephalopathy. In: Schiff L, Schiff ER, eds. Diseases of the liver. Philadeiphia: Lippincott, 1987; 925-948. Malt RA. Portal systemic shunts. N Engl Med 1976; 295:24-29. Zeneroli ML, Pinelli C, Gollini C, et al. Visual evoked potential: a diagnostic tool for the assessment of hepatic encephalopathy. Gut 1984; 25:291-299. Casellas F, Sagales T, Calzada MD, Accarino A, Vargas V. Guarner L. Visual evoked potentials in hepatic encephalopathy. Lancet 1985; I :394-395. Rekkers L, Jenko P. Rudman D, Freides D. Subclinical hepatic encephalopathy: detection, prevalence, and relationship to nitrogen metabolism. Gastroenterology 1978; 75:462-469. Tarter RE, Hegedus AM, Van Thiel DH, Schade RR, GavalerJS, Starzl TE. Nonalcoholic cirrhosis associated with neuropsychiatnc dysfunction in the absence of overt cvidence of hepatic encephalopathy. Gastroenterologv 1984; 86:1421-1427. Gitlin N, Lewis DC, Hinkley L. The diagnosis and prevalence of subclinical hepatic encephalopathy in apparently healthy, ambulant, nonshunted patients with cirrhosis. J Hepatol 1986; 3:7-82. Schomerus H, Hamster W, Blunck H, Reinhard U, Mayer K, DOlle W. Latent portasystemic encephalopathy. I. Nature of cerebral functional defects and their effect on fitness to drive. Dig Dis Sci 1981; 26:622. Butterworth R. Ammonia in hepatic encephalopathy. Neurochem Pathol 1987; 6(12):l-1 57. Jones EA. Hepatic encephalopathy: an update. Viewpoint Dig Dis 1986; 18:1-4. Hourani B, Hamlin E, Reynolds T. Cerebrospinal fluid glutamine as a measure of hepatic

26 #{149} Radiology

U

.5

U

0

.4

0 D

U

.3

Dr

o

o

o

Do

.2 References 1.

U

.6

0

0 .1

0

A

0

.1

.2

.3

.4

.5

.6

.7

.8

.9

1

MI Figure 5. Levels of MI are plotted against the integral value Al, corresponding to the sum Gin and Glu, for all subjects in groups 0 (control subjects) (0), 1 (patients with liver disease and CHE) (U), and 2 (patients with liver disease but without CHE) (LI). On the basis of the levels,

there

is a clear

separation

with liver disease but without of those in the CHE patients ues

of Al

showed

some

overlap

between

CHE. The (+) are thought between

between MI and Al values exists (Al points shown), no significant correlation other groups considered singly.

13.

patients with CHE and control subjects four patients in group 2 with MI values

=

the 0.51

to suffer -

was observed

encephalopathy. Arch Intern Med 1971; 127: 1033-1036. Yamamoto H, Konno H, Yamamoto T, Ito K, Mizugaki M, Iwasaki Y. Glutamine synthetase of the human brain: purification and characterization. J Neurochem 1987; 49:603-

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Dawson AM, McLaren J, Sherlock S. Neomycosis in the treatment of hepatic coma. Lancet 1957; 2:1262-1268. Ingelfinger F. Editorial comments. In: Beeson PB, ed. Year Book of Medicine, 1964-65. Chicago: Year Book Medical, 1965; 591-592. Bircher J, MUller J, Guggenheim P. et al. Treatment of chronic portal-systemic encephalopathy with lactulose. Lancet 1966; 1:890-893.

from

pre- or subclinical

groups. Although 0.38 . MI and R

19.

a significant = 0.6, on the

in patients

with

CHE

of MI

or patients in the range

CHE.

The

val-

linear correlation basis of all the

or in either

of the

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