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Proton spectroscopy of brain glutamine in acute liver failure
Proton Spectroscopy of Brain Glutamine in Acute Liver Failure JAMES R.
MCCONNELL,DEAN L. ANTONSON,CHIN
SIONG ONG,WEI-KOM CHU, IRA J. FOX, THOMAS G. HEFFRON, ALAN N. LANGNAS, AND BYERS W. SHAW, JR
MRS) in an animal model of ALF showed a significant increase in brain GLN and a concomitant decrease in GLU using a high-field-strength m a g n e t (8.5 Tesla). ~° More recently, 1H MRS investigations of patients with liver failure have identified increased brain GLN using routine clinical imaging magnets, and a report of a child with Reye's syndrome also dem on stra te d elevation of brain GLN in ALF. 2125 The evidence presented in these various studies indirates the importance of GLN in the pathogenesis of the encephalopathy and suggests t h a t reliable determination of brain GLN could be helpful in the neurologic evaluation of patients with encephalopathy and ALF. The current study was u n d e r t a k e n to assess the feasibility and clinical utility of noninvasive ~H MRS to m easure brain GLN in patients with ALF and to test the premise t h a t GLN quantitation can predict the progression of encephalopathy and the onset of brain edema.
E v i d e n c e indicates that t h e a c c u m u l a t i o n o f glutam i n e in t h e brain plays an i m p o r t a n t role in t h e pathog e n e s i s a n d s e v e r i t y o f the e n c e p h a l o p a t h y o f a c u t e liver failure (ALF). This s t u d y u s e s in vivo p r o t o n m a g n e t i c r e s o n a n c e s p e c t r o s c o p y (1H MRS) to assess brain glutam i n e (GLN) in five c a s e s o f a c u t e liver failure. The findings are c o n s i s t e n t w i t h prior i n v e s t i g a t i o n s a n d suggest that the a l p h a 1H o f t h e GLN m o l e c u l e c a n be u s e d for n o n i n v a s i v e s p e c t r o s c o p i c q u a n t i t a t i o n of brain GLN in p a t i e n t s w i t h ALF. (HEPATOLOGY 1995;22:69-74.)
The neurologic manifestations of encephalopathy in patients with acute liver failure (ALF) can progress from mild confusion to stupor and coma. Death m ay result from brain edema in ALF. ~'2 The pathogenesis of the progressive encephalopathy and brain edema is unknown, b u t evidence suggests t h a t increased brain glutamine (GLN) plays an i m p o r t a n t role. A n in vivo microdialysis study of the brain found significantly increased GLN in the cerebral cortex and in the extracellular fluid of the brain in experimental ALF. ~ Research models of ALF have d e m o n s t r a t e d t h a t increased brain GLN is also associated with the development of brain edema. 4-s Inhibition of glutamine synthetase, an enzyme in astrocytes t h a t converts ammonia and g l u t a m a t e (GLU) to GLN, can prevent the development of brain e de m a in experimental ALF. 9-1G Investigators in 1955 d e m o n s t r a t e d t h a t GLN concentration was elevated in the cerebrospinal fluid of patients in hepatic coma, and later clinical studies have suggested t h a t increased concentration of cerebrospinal fluid GLN correlates with the severity of hepatic encephalopathy (HE). 17-19 In vivo proton magnetic resonance spectroscopy (~H
PATIENTS AND METHODS
Noninvasive brain 1H MRS was performed on four patients with ALF before and after successful liver transplantation (LT) and one patient with halothane-induced ALF, who recovered on supportive treatment. Five normal young adult volunteers served as controls. The patients also served as their own prospective controls before and after LT. The study patients were recruited from the Gastrointestinal and Liver Transplant Surgery Services of the University of Nebraska Medical Center. This study was approved by the Institutional Review Board. All 1H MRS examinations were done in the same manner using a clinical MR imager (Signa, GE Medical Systems, Milwaukee, WI) operating at a magnetic field strength of 1.5 Tesla. A preliminary Tl-weighted axial brain magnetic resonance image (MRI) was obtained for the selection of a normalappearing volume of tissue (voxel size, 3 × 3 × 3 cm) in the left parietal lobe (gray and white matter). The cerebral ventricle was not included in the voxel, and the 1H MRS signal contribution from cerebrospinal fluid was negligible. All 1H MRS examinations, pulse sequences, and data processing were done in the same manner for the study group of patients, the controls, and the in vitro GLN sample. The point-resolved spectroscopy (PRESS) sequence was used with a repetition time of 2,000 ms and echo times (TEs) of 40 ms and 270 ms to obtain the spectroscopy signal. The spectral width was 2,000 Hz. Phase modulation on long echo time (270 ms TE) spectroscopy demonstrates the alpha 1H singlet of GLN at 3.75 ppm. 26'27 The prominent N-acetyl-
Abbreviations: ALF, acute liver failure; GLN, glutamine; GLU, glutamate; HE, hepatic encephalopathy; 1H MRS, proton magnetic resonance spectroscopy; LT, liver transplantation; MRI, magnetic resonance imaging; TE, echo time; NAA, N-acetylaspartate; CHO, choline-containing compounds; INS, myoinositel; CR, creatine/phosphocreatine. From the University of Nebraska Medical Center, Omaha, NE. Received May 18, 1994; accepted January 26, 1995. Address reprint requests to: James R. McConnell, MD, Associate Professor, Department of Radiology, University of Nebraska Medical Center, 600 South 42nd St, Omaha, NE 68198-1045. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2201-001053.00/0
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70 McCONNELL ET AL
HEPATOLOGYJuly 1995 The spectral data were processed on a SUN Sparc II computer using SA/GE software (GE Medical Systems). Processing used exponential multiplication line broadening of 1 Hz, zero filling to 8 K, and one-dimensional Fourier transform. Student's t-test was used for statistical analysis of the data.
a'H
A,
CASE R E P O R T S
¢ I
I
3
2 ppm alH
B
H H
HTN
H
FIG. 1. (A) In vitro 1H MRS (270 ms TE) of 20 mmol/L GLN sample shows the refocused alphalH peak (arrow) at 3.75 ppm. The peaks between 2 and 3 ppm represent the complex phase modulations of the beta and gamma protons of GLN. These signal peaks are not used for quantitation in this study. (B) GLN model shows the position of the alpha 1H in the molecule. aspartate (NAA) peak at 2.0 ppm was used as the internal chemical shift reference for the in vivo 1H MRS. The i n vivo spectra exhibit the choline-containing compounds (CHO) at 3.2 ppm, myoinositol (INS) at 3.6 ppm, the GLN and GLU alphalH at 3.75 ppm, and the creatine/phosphocreatine (CR) peak at 3.0 ppm. 2s Using NAA as the internal reference signal, water resonates at approximately 4.7 ppm. The measurements are given as peak intensities ratios of NAA, CHO, and INS with respect to the CR peak. The NAA:CR, CHO:CR, and INS:CR ratios were measured on IH MRS at 40 ms TE, and the alphalH:CR ratios were measured at 270 ms TE for cases and controls. I n v i t r o 1H MRS of a liter sample of 20 mmol/L GLN (buffered at pH 7.1) demonstrates the phase modulation of the GLN alpha 1H on the long echo time (270 ms TE) (Fig. 1).
C a s e N o . 1. Case I is a 9-year-old boy with a 10-day history of acute hepatitis characterized by diarrhea, jaundice, increasing lethargy, and anorexia. Acute serology (immunoglobulin M) was positive for hepatitis A. Liver function tests demonstrated a marked increase in transaminase levels with an aspartate transaminase of 3,850 and an alanine transaminase of 2,100, an increase in bilirubin levels with a total of 15.9 and a direct fraction of 14.5, and a mild increase in protime to 16 seconds. The patient developed ALF with prolongation of the protime to greater than 16 seconds, hepatic fetor, ascites, and stage 1 HE. The patient underwent heterotopic whole-liver auxiliary LT. After LT the liver function tests improved and the HE resolved. Case No. 2. Case 2 is a 17-year-old boy with a 2- to 3week history of lethargy, jaundice, pruritus, and profound thrombocytopenia. Liver function tests demonstrated hyperbilirubinemia with a total bilirubin of 20.8 and a direct fraction of 15.8, hypoalbuminemia of 3.5, and an elevated protime of 13.8 seconds. Platelet count was 23,000. Transaminase levels decreased, and bilirubin increased to 24.1/26.9, the protime increased to 19.4, thrombocytopenia continued, neutropenia developed, and he developed progressive stage 2 HE. He had presumed postviral ALF with bone marrow suppression and underwent orthotopic auxiliary left lobe LT. After LT the liver function tests improved and the HE resolved. Case No. 3. Case 3 is an ll-month-old boy with ALF after a 10-day history of fever, jaundice, and increasing lethargy, presumed to be of viral origin. Liver function tests demonstrated a marked increase in transaminases with an aspartate transaminase of 996 and an alanine transaminase of 1,652, an elevated bilirubin with a total of 20.8 over a direct fraction of 15.7, and an elevated protime of 21 seconds. Transaminases decreased while bilirubin levels remained elevated. The protime was greater than 25 seconds. The patient developed hypoalbuminemia, bone marrow suppression, and stage 2 HE. He underwent orthotopic LT. After LT the liver function tests improved and the HE resolved. Follow-up brain MRI showed ventriculomegaly and prominent sulcal spaces of the cerebral cortex consistent with mild central and cortical atrophy. The cause of the atrophy is unknown. The postoperative 1H MRS showed a reduced alpha 1H (Fig. 2). Case No. 4. Case 4 is a 13-month-old boy with ALF after a preceding 2-week viral prodrome characterized by lethargy, poor feeding, and low-grade fever. Liver tests showed a marked increase in transaminases, with an aspartate transaminase 1,600 and an alanine transaminase of 850, and hypoalbuminemia with a level of 3.2. Transaminases decreased but bilirubin levels increased to 21.4 total with 15.7 direct. The protime was greater than 20 seconds. He developed stage 1 HE, neutropenia, thrombocytopenia, and suspected viral bone marrow aplasia. At the time of the preoperative ~H MRS the patient had developed increasing lethargy, which suggested progressive HE (Fig. 3). The patient underwent orthotopic LT but died before a follow-up 1H MRS was obtained. Case No. 5. Case 5 is a 4-year-old boy who had a mole removed under general anesthesia with halothane. One week
HEPATOLOGYVol. 22, No. 1, 1995
McCONNELL ET AL
NAA
71
NAA
A
B CR alH
j
CR
l
I
4
I
I
I
I
I
|
I
3
2
1
4
3
2
1
ppm
ppm
FIG. 2. (A) In vivo brain 1H MRS (270 ms TE) of case 3 in ALF before LT. Note the alpha 1H peak (arrow) at 3.75 ppm is markedly increased (i.e., over 4 SD above control). (B) In vivo brain 1H MRS (270 ms TE) of case 3 after LT and recovery from ALF. Note the alpha 1H peak (arrow) at 3.75 ppm has decreased (i.e., 1 SD above control).
l a t e r he developed fever, vomiting, d i a r r h e a , a n d i n c r e a s i n g fatigue, a n d a p p e a r e d pale. A n e m e r g e n c y room visit noted a m a r k e d i n c r e a s e in t r a n s a m i n a s e s , w i t h an a s p a r t a t e t r a n s a m i n a s e of 10,388 a n d an a l a n i n e t r a n s a m i n a s e of 5,233. P l a t e l e t s were 58,000. His p r o t i m e was e l e v a t e d at 23.2, as was the p a r t i a l t h r o m b o p l a s t i n t i m e at 38.6. Total bilirubin level was 2.6, with a direct fraction of 1.6. He was a d m i t t e d w i t h ALF, a n d liver biopsy was consistent w i t h halo t h a n e toxicity. His liver functions recovered with supportive t r e a t m e n t , a n d he did not become clinically e n c e p h a l o p a t h i c
I
I
II
4
3
w I
2
In
lUllWU
I l l l i l l
1
li
|
0
(i.e., stage 0 HE). The 1H MRS showed a m i l d l y i n c r e a s e d a l p h a 1H p e a k in this p a t i e n t (Fig. 4). He did not r e q u i r e LT. RESULTS T h e p a t i e n t d a t a a r e s h o w n i n T a b l e 1. T h e c o n t r o l d a t a a r e s h o w n i n T a b l e 2. The brain MRI examinations in the cases were normal and showed no morphologic or signal changes,
' ' ' I .........
4
I'
' ' '''
' ' ' I .........
3
2
I
'
"'
'
'"
'
'
1
ppm
ppm
FIG. 3. In vivo brain 1H MRS (270 ms TE) of case 4 in ALF before LT. Note the alpha 1H peak (arrow) at 3.75 ppm is markedly increased (i.e., over 6 SD above control). This patient died before follow-up ~H MRS.
FIG. 4. In vivo brain 1H MRS (270 ms TE) of case 5 in halothaneinduced ALF. Note the alpha 1H peak (arrow) at 3.75 ppm is only modestly increased above control. This patient had stage 0 HE and recovered with supportive therapy. He did not require LT.
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McCONNELL ET AL
HEPATOLOGY July 1995 TABLE 1. P a t i e n t D a t a Pre-LT
Post-LT
Case
NAA:CR
CHO:CR
INS:CR
NAA:CR
CHO:CR
INS:CR
Pre-LT ~IH:CR
Post-LT ~IH:CR
1 2 3 4 5* Mean SD
1.64 1.58 1.60 1.62 1.52 1.59 0.05
0.90 0.75 0.76 0.75 0.86 0.80 0.07
0.70 0.72 0.68 0.53 0.79 0.72 0.05
1.65 1.60 1.38 Died 1.54 1.54 0.12
1.03 0.80 1.00
0.88 0.63 0.86
0.60 0.86 0.20
0.74 0.78 0.12
0.54 0.73 0.66 0.77 0.62 0.66 0.09
0.43 0.37 0.44 expired 0.55 0.45 0.08
NOTE. The peak intensities of the a l H were measured at 3.75 ppm. The means of the samples are different at P < .01. * Case 5 recovered without LT.
except in case 3. Compared with the preoperative examination, the postoperative MRI showed ventriculomegaly and prominent sulcal spaces of the cerebral cortex, suggesting that mild cerebral atrophy had developed in case 3. There was a concomitant decrease in the postoperative brain NAA on spectroscopy in case 3. This is consistent with neuronal loss and atrophy. 2s The cause of the atrophy was not determined. Pallidal T1 signal hyperintensities reported on brain MRIs in patients with chronic hepatic encephalopathy were not found in these cases of ALF. Preoperative 1H MRS showed decreased brain CHO:CR ratios in all cases compared with control. The postoperative ~H MRS results showed recovery of the choline-containing metabolites to control levels. The brain CHO:CR ratios had increased in all cases after LT. However, no significant differences were found in the INS:CR ratios in our ALF patients before and after LT or compared with controls. The changes in the CHO metabolites are consistent with prior 1H MRS studies of patients with HE before and after L T 9 '3° More case studies are needed to assess possible differences in brain INS in ALF and chronic HE. Comparison of the NAA:CR ratio measurements before and after LT showed no significant variance in cases 1, 2, and 5. Case 4 died before the follow-up ~H MRS. The mean alpha~H:CR ratio was markedly increased preoperatively (i.e., 4.5 SD above control). The postoperative alpha~H:CR mean substantially decreased to about one SD above control. These results are consistent with decreasing brain GLN after successful LT and recovery from ALF.
TABLE 2. C o n t r o l D a t a Controls 1 2 3 4 5 Mean SD
NAA:CR
CHO:CR
INS:CR
~IH:CR
1.53 1.68 1.64 1.56 1.65 1.61 0.06
1.01 1.09 1.01 0.95 0.80 0.97 0.10
0.81 0.80 0.76 0.71 0.73 0.76 0.04
0.44 0.42 0.41 0.29 0.41 0.39 0.06
The prominent resonance peak at 3.75 ppm on in vivo spectra directly matches the in vitro GLN alphalH. The in vitro 1H MRS of the GLN sample showed the chemical shift of the GLN alphalH at 3.75 ppm as measured from the water peak (4.7 ppm). This is consistent with published data. 31 DISCUSSION
The neurologic evaluation of patients with ALF is a formidable task. Signs and symptoms of progressive encephalopathy and impending brain edema are difficult to assess in the obtunded or comatose patient. Electroencephalography is helpful in the clinical management of patients, but the results are operator dependent and nonspecific for HE. Liver function tests, including blood ammonia levels, do not always correlate with the presence or the severity of HE. Spinal puncture for determination of cerebrospinal fluid metabolites and other tests is invasive and dangerous in patients with possible brain edema and increased intracranial pressure. Computed tomography and MR neuroimaging studies are useful to exclude other abnormalities, but may appear normal in the early stages of brain edema. The MRI pallidal T1 hyperintensities, which can be found in chronic HE, do not correlate with the clinical severity of encephalopathy and were not present in this group of patients with acute HE. Transcranial Doppler is helpful in assessing cerebral blood flow in ALF, but irreversible brain damage m a y be present before Doppler results are positive. Dural pressure monitors can accurately measure intracranial pressure but require neurosurgical intervention with its possible complications of hemorrhage and infection. Considering these difficulties in neurologic evaluation, there is a definite need for an accurate and noninvasive test to monitor the encephalopathy in ALF. Brain GLN and GLU (similar molecular structure and spectral overlap) are difficult to resolve at field strengths currently available on clinical imaging magnets. However, these metabolites have been successfully separated and quantitated in experimental ALF using a high-field-strength magnet (8.5 Tesla).20 Recent ~H MRS technological developments in automated water suppression, magnetic field shims, and computer
HEPATOLOGYVol. 22, No. 1, 1995 p r o c e s s i n g p r o v i d e r e l a t i v e l y f a s t d a t a acquisition a n d h a n d l i n g . ~2'33 T h e t e c h n o l o g y is c u r r e n t l y a v a i l a b l e for u s e on clinical i m a g i n g m a g n e t s a n d h a s significantly e n h a n c e d t h e u t i l i t y of in vivo 1H MRS. T h e s o f t w a r e a p p l i c a t i o n s a r e u s e r f r i e n d l y a n d c a n give a c c u r a t e b i o c h e m i c a l i n f o r m a t i o n a b o u t t h e b r a i n in a p p r o x i m a t e l y 15 to 20 m i n u t e s s c a n time. P r e v i o u s 1H M R S i n v e s t i g a t i o n s of p a t i e n t s h a v e found, in a d d i t i o n to i n c r e a s e s in b r a i n G L N , c h a n g e s in o t h e r i m p o r t a n t m e t a b o l i t e s . T h e s e s t u d i e s identified d e c r e a s e d b r a i n I N S a n d C H O a n d i n d i c a t e d a c h a r a c t e r i s t i c p a t h o b i o c h e m i c a l p a t t e r n of m e t a b o l i t e s in H E . 2125 T h e c u r r e n t s t u d y confirms t h e s e i m p o r t a n t o b s e r v a t i o n s except t h a t no significant differences w e r e f o u n d in I N S : C R r a t i o s in o u r c a s e s of A L F before a n d a f t e r L T or c o m p a r e d w i t h controls. T h i s finding sugg e s t s t h a t m o r e s t u d y is n e e d e d to a s s e s s possible differences in t h e b r a i n m y o i n o s i t o l c o n c e n t r a t i o n s in a c u t e v e r s u s chronic h e p a t i c e n c e p h a l o p a t h y . T h e m a j o r a s s e r t i o n of t h e c u r r e n t s t u d y is t h a t long T E 1H M R S m e a s u r e m e n t of b r a i n G L N c a n be u s e d clinically to m o n i t o r t h e p r o g r e s s i v e e n c e p h a l o p a t h y of ALF. T h e signal p e a k of t h e a l p h a ~H (3.75 p p m ) of G L N is r e a d i l y d e t e c t e d a n d q u a n t i t a t e d on long TE spectroscopy, a n d its a m p l i t u d e is directly r e l a t e d to t h e c o n c e n t r a t i o n of G L N in t h e b r a i n . I n addition, t h e p h a s e m o d u l a t i o n a n d c h e m i c a l shift of t h e a l p h a ~ H o b s e r v e d on in vitro G L N s p e c t r o s c o p y directly m a t c h t h e r e s u l t s in p a t i e n t s . N o r m a l l y , t h e m a j o r signal c o n t r i b u t i o n to t h e a l p h a ~H (3.75 p p m ) p e a k c o m e s f r o m G L U , w h i c h is m o r e c o n c e n t r a t e d in n o r m a l b r a i n t h a n G L N . H o w e v e r , t h e a b n o r m a l a c c u m u l a t i o n of b r a i n G L N in H E a l t e r s t h e r e l a t i v e c o n c e n t r a t i o n s of G L N a n d G L U . I n H E , t h e i n c r e a s e d signal i n t e n s i t y of t h e a l p h a ~H (3.75 p p m ) a r i s e s f r o m t h e p a t h o l o g i c a l l y i n c r e a s e d G L N . T h i s is p e r h a p s b e s t d e m o n s t r a t e d in t h i s s t u d y b y t h e longitudinal c h a n g e of t h e a l p h a 1H p e a k before a n d a f t e r LT. T h e p e a k i n t e n s i t y of t h e a l p h a 1H w a s m a r k e d l y i n c r e a s e d in t h e cases of A L F b u t c o n v e r t e d to control v a l u e s a f t e r LT a n d r e c o v e r y f r o m H E . T h e s e r e s u l t s a r e h i g h l y c o n s i s t e n t w i t h t h e c h a n g e s of b r a i n G L N f o u n d in e x p e r i m e n t a l ALF. C a n 1H M R S q u a n t i t a t i o n of G L N p r e d i c t b r a i n e d e m a in A L F ? M o r e p r o s p e c t i v e s t u d i e s a r e n e e d e d to a n s w e r t h i s i m p o r t a n t clinical question. T h e c u r r e n t s t u d y does s u g g e s t t h a t 1H M R S q u a n t i t a t i o n of b r a i n G L N is clinically feasible a n d useful for m o n i t o r i n g e n c e p h a l o p a t h y in ALF. REFERENCES
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troscopy of major brain metabolites. Magn Reson Med 1991;21:82-96. 27. Ernst T, McConnell JR, Michaelis T, Ross BD. Detection of glutamate and glutamine in long echo time: 1H MRS in vivo. Proceedings of the Twelfth Annual Scientific Meeting of the Society of Magnetic Resonance in Medicine, Book of Abstracts 3, 1993; 1532. 28. Urenjak J, Williams SR, Gadian DG, Noble M. Proton nuclear magnetic resonance spectroscopy unambiguously identifies different neural cell types. J Neurosci 1993; 13:981-989. 29. Geissler A, Ernst T, Ross BD, Kreis R, Moats R, Shonk T, Villamil F. Reversal of hepatic encephalopathy after liver transplantation: a proton MRS study. Hepatogastroenterology Current Medical and Surgical Trend. 1993;40:37.
HEPATOLOGYJuly 1995 30. Geissler A, Farrow NA, Villamil F, Makowka L, Ernst T, Kreis R, Ross BD. Is hepatic encephalopathy reversed by liver transplantation? Proceeding of the Eleventh Annual Scientific Meeting of the Society of Magnetic Resonance in Medicine, Book of Abstracts 1, 1992:647. 31. Gordon AJ, Ford RA. The chemist's companion: a handbook of practical data, techniques and references. New York: Wiley & Sons, 1972. 32. Webb PG, Sai]asuta N, Kohler SJ, Raidy T, Moats RA, Hurd RE. Automated sing]e-voxel proton MRS: technical development and multisite verification. Magn Reson Med 1994;31:365-373. 33. Kohler S. PROBE/SV Single-voxel proton brain exam. PROBE/ SV Application Guide Vol V. 199 GE Medical Systems, 1993.
MCCONNELL,DEAN L. ANTONSON,CHIN
SIONG ONG,WEI-KOM CHU, IRA J. FOX, THOMAS G. HEFFRON, ALAN N. LANGNAS, AND BYERS W. SHAW, JR
MRS) in an animal model of ALF showed a significant increase in brain GLN and a concomitant decrease in GLU using a high-field-strength m a g n e t (8.5 Tesla). ~° More recently, 1H MRS investigations of patients with liver failure have identified increased brain GLN using routine clinical imaging magnets, and a report of a child with Reye's syndrome also dem on stra te d elevation of brain GLN in ALF. 2125 The evidence presented in these various studies indirates the importance of GLN in the pathogenesis of the encephalopathy and suggests t h a t reliable determination of brain GLN could be helpful in the neurologic evaluation of patients with encephalopathy and ALF. The current study was u n d e r t a k e n to assess the feasibility and clinical utility of noninvasive ~H MRS to m easure brain GLN in patients with ALF and to test the premise t h a t GLN quantitation can predict the progression of encephalopathy and the onset of brain edema.
E v i d e n c e indicates that t h e a c c u m u l a t i o n o f glutam i n e in t h e brain plays an i m p o r t a n t role in t h e pathog e n e s i s a n d s e v e r i t y o f the e n c e p h a l o p a t h y o f a c u t e liver failure (ALF). This s t u d y u s e s in vivo p r o t o n m a g n e t i c r e s o n a n c e s p e c t r o s c o p y (1H MRS) to assess brain glutam i n e (GLN) in five c a s e s o f a c u t e liver failure. The findings are c o n s i s t e n t w i t h prior i n v e s t i g a t i o n s a n d suggest that the a l p h a 1H o f t h e GLN m o l e c u l e c a n be u s e d for n o n i n v a s i v e s p e c t r o s c o p i c q u a n t i t a t i o n of brain GLN in p a t i e n t s w i t h ALF. (HEPATOLOGY 1995;22:69-74.)
The neurologic manifestations of encephalopathy in patients with acute liver failure (ALF) can progress from mild confusion to stupor and coma. Death m ay result from brain edema in ALF. ~'2 The pathogenesis of the progressive encephalopathy and brain edema is unknown, b u t evidence suggests t h a t increased brain glutamine (GLN) plays an i m p o r t a n t role. A n in vivo microdialysis study of the brain found significantly increased GLN in the cerebral cortex and in the extracellular fluid of the brain in experimental ALF. ~ Research models of ALF have d e m o n s t r a t e d t h a t increased brain GLN is also associated with the development of brain edema. 4-s Inhibition of glutamine synthetase, an enzyme in astrocytes t h a t converts ammonia and g l u t a m a t e (GLU) to GLN, can prevent the development of brain e de m a in experimental ALF. 9-1G Investigators in 1955 d e m o n s t r a t e d t h a t GLN concentration was elevated in the cerebrospinal fluid of patients in hepatic coma, and later clinical studies have suggested t h a t increased concentration of cerebrospinal fluid GLN correlates with the severity of hepatic encephalopathy (HE). 17-19 In vivo proton magnetic resonance spectroscopy (~H
PATIENTS AND METHODS
Noninvasive brain 1H MRS was performed on four patients with ALF before and after successful liver transplantation (LT) and one patient with halothane-induced ALF, who recovered on supportive treatment. Five normal young adult volunteers served as controls. The patients also served as their own prospective controls before and after LT. The study patients were recruited from the Gastrointestinal and Liver Transplant Surgery Services of the University of Nebraska Medical Center. This study was approved by the Institutional Review Board. All 1H MRS examinations were done in the same manner using a clinical MR imager (Signa, GE Medical Systems, Milwaukee, WI) operating at a magnetic field strength of 1.5 Tesla. A preliminary Tl-weighted axial brain magnetic resonance image (MRI) was obtained for the selection of a normalappearing volume of tissue (voxel size, 3 × 3 × 3 cm) in the left parietal lobe (gray and white matter). The cerebral ventricle was not included in the voxel, and the 1H MRS signal contribution from cerebrospinal fluid was negligible. All 1H MRS examinations, pulse sequences, and data processing were done in the same manner for the study group of patients, the controls, and the in vitro GLN sample. The point-resolved spectroscopy (PRESS) sequence was used with a repetition time of 2,000 ms and echo times (TEs) of 40 ms and 270 ms to obtain the spectroscopy signal. The spectral width was 2,000 Hz. Phase modulation on long echo time (270 ms TE) spectroscopy demonstrates the alpha 1H singlet of GLN at 3.75 ppm. 26'27 The prominent N-acetyl-
Abbreviations: ALF, acute liver failure; GLN, glutamine; GLU, glutamate; HE, hepatic encephalopathy; 1H MRS, proton magnetic resonance spectroscopy; LT, liver transplantation; MRI, magnetic resonance imaging; TE, echo time; NAA, N-acetylaspartate; CHO, choline-containing compounds; INS, myoinositel; CR, creatine/phosphocreatine. From the University of Nebraska Medical Center, Omaha, NE. Received May 18, 1994; accepted January 26, 1995. Address reprint requests to: James R. McConnell, MD, Associate Professor, Department of Radiology, University of Nebraska Medical Center, 600 South 42nd St, Omaha, NE 68198-1045. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2201-001053.00/0
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70 McCONNELL ET AL
HEPATOLOGYJuly 1995 The spectral data were processed on a SUN Sparc II computer using SA/GE software (GE Medical Systems). Processing used exponential multiplication line broadening of 1 Hz, zero filling to 8 K, and one-dimensional Fourier transform. Student's t-test was used for statistical analysis of the data.
a'H
A,
CASE R E P O R T S
¢ I
I
3
2 ppm alH
B
H H
HTN
H
FIG. 1. (A) In vitro 1H MRS (270 ms TE) of 20 mmol/L GLN sample shows the refocused alphalH peak (arrow) at 3.75 ppm. The peaks between 2 and 3 ppm represent the complex phase modulations of the beta and gamma protons of GLN. These signal peaks are not used for quantitation in this study. (B) GLN model shows the position of the alpha 1H in the molecule. aspartate (NAA) peak at 2.0 ppm was used as the internal chemical shift reference for the in vivo 1H MRS. The i n vivo spectra exhibit the choline-containing compounds (CHO) at 3.2 ppm, myoinositol (INS) at 3.6 ppm, the GLN and GLU alphalH at 3.75 ppm, and the creatine/phosphocreatine (CR) peak at 3.0 ppm. 2s Using NAA as the internal reference signal, water resonates at approximately 4.7 ppm. The measurements are given as peak intensities ratios of NAA, CHO, and INS with respect to the CR peak. The NAA:CR, CHO:CR, and INS:CR ratios were measured on IH MRS at 40 ms TE, and the alphalH:CR ratios were measured at 270 ms TE for cases and controls. I n v i t r o 1H MRS of a liter sample of 20 mmol/L GLN (buffered at pH 7.1) demonstrates the phase modulation of the GLN alpha 1H on the long echo time (270 ms TE) (Fig. 1).
C a s e N o . 1. Case I is a 9-year-old boy with a 10-day history of acute hepatitis characterized by diarrhea, jaundice, increasing lethargy, and anorexia. Acute serology (immunoglobulin M) was positive for hepatitis A. Liver function tests demonstrated a marked increase in transaminase levels with an aspartate transaminase of 3,850 and an alanine transaminase of 2,100, an increase in bilirubin levels with a total of 15.9 and a direct fraction of 14.5, and a mild increase in protime to 16 seconds. The patient developed ALF with prolongation of the protime to greater than 16 seconds, hepatic fetor, ascites, and stage 1 HE. The patient underwent heterotopic whole-liver auxiliary LT. After LT the liver function tests improved and the HE resolved. Case No. 2. Case 2 is a 17-year-old boy with a 2- to 3week history of lethargy, jaundice, pruritus, and profound thrombocytopenia. Liver function tests demonstrated hyperbilirubinemia with a total bilirubin of 20.8 and a direct fraction of 15.8, hypoalbuminemia of 3.5, and an elevated protime of 13.8 seconds. Platelet count was 23,000. Transaminase levels decreased, and bilirubin increased to 24.1/26.9, the protime increased to 19.4, thrombocytopenia continued, neutropenia developed, and he developed progressive stage 2 HE. He had presumed postviral ALF with bone marrow suppression and underwent orthotopic auxiliary left lobe LT. After LT the liver function tests improved and the HE resolved. Case No. 3. Case 3 is an ll-month-old boy with ALF after a 10-day history of fever, jaundice, and increasing lethargy, presumed to be of viral origin. Liver function tests demonstrated a marked increase in transaminases with an aspartate transaminase of 996 and an alanine transaminase of 1,652, an elevated bilirubin with a total of 20.8 over a direct fraction of 15.7, and an elevated protime of 21 seconds. Transaminases decreased while bilirubin levels remained elevated. The protime was greater than 25 seconds. The patient developed hypoalbuminemia, bone marrow suppression, and stage 2 HE. He underwent orthotopic LT. After LT the liver function tests improved and the HE resolved. Follow-up brain MRI showed ventriculomegaly and prominent sulcal spaces of the cerebral cortex consistent with mild central and cortical atrophy. The cause of the atrophy is unknown. The postoperative 1H MRS showed a reduced alpha 1H (Fig. 2). Case No. 4. Case 4 is a 13-month-old boy with ALF after a preceding 2-week viral prodrome characterized by lethargy, poor feeding, and low-grade fever. Liver tests showed a marked increase in transaminases, with an aspartate transaminase 1,600 and an alanine transaminase of 850, and hypoalbuminemia with a level of 3.2. Transaminases decreased but bilirubin levels increased to 21.4 total with 15.7 direct. The protime was greater than 20 seconds. He developed stage 1 HE, neutropenia, thrombocytopenia, and suspected viral bone marrow aplasia. At the time of the preoperative ~H MRS the patient had developed increasing lethargy, which suggested progressive HE (Fig. 3). The patient underwent orthotopic LT but died before a follow-up 1H MRS was obtained. Case No. 5. Case 5 is a 4-year-old boy who had a mole removed under general anesthesia with halothane. One week
HEPATOLOGYVol. 22, No. 1, 1995
McCONNELL ET AL
NAA
71
NAA
A
B CR alH
j
CR
l
I
4
I
I
I
I
I
|
I
3
2
1
4
3
2
1
ppm
ppm
FIG. 2. (A) In vivo brain 1H MRS (270 ms TE) of case 3 in ALF before LT. Note the alpha 1H peak (arrow) at 3.75 ppm is markedly increased (i.e., over 4 SD above control). (B) In vivo brain 1H MRS (270 ms TE) of case 3 after LT and recovery from ALF. Note the alpha 1H peak (arrow) at 3.75 ppm has decreased (i.e., 1 SD above control).
l a t e r he developed fever, vomiting, d i a r r h e a , a n d i n c r e a s i n g fatigue, a n d a p p e a r e d pale. A n e m e r g e n c y room visit noted a m a r k e d i n c r e a s e in t r a n s a m i n a s e s , w i t h an a s p a r t a t e t r a n s a m i n a s e of 10,388 a n d an a l a n i n e t r a n s a m i n a s e of 5,233. P l a t e l e t s were 58,000. His p r o t i m e was e l e v a t e d at 23.2, as was the p a r t i a l t h r o m b o p l a s t i n t i m e at 38.6. Total bilirubin level was 2.6, with a direct fraction of 1.6. He was a d m i t t e d w i t h ALF, a n d liver biopsy was consistent w i t h halo t h a n e toxicity. His liver functions recovered with supportive t r e a t m e n t , a n d he did not become clinically e n c e p h a l o p a t h i c
I
I
II
4
3
w I
2
In
lUllWU
I l l l i l l
1
li
|
0
(i.e., stage 0 HE). The 1H MRS showed a m i l d l y i n c r e a s e d a l p h a 1H p e a k in this p a t i e n t (Fig. 4). He did not r e q u i r e LT. RESULTS T h e p a t i e n t d a t a a r e s h o w n i n T a b l e 1. T h e c o n t r o l d a t a a r e s h o w n i n T a b l e 2. The brain MRI examinations in the cases were normal and showed no morphologic or signal changes,
' ' ' I .........
4
I'
' ' '''
' ' ' I .........
3
2
I
'
"'
'
'"
'
'
1
ppm
ppm
FIG. 3. In vivo brain 1H MRS (270 ms TE) of case 4 in ALF before LT. Note the alpha 1H peak (arrow) at 3.75 ppm is markedly increased (i.e., over 6 SD above control). This patient died before follow-up ~H MRS.
FIG. 4. In vivo brain 1H MRS (270 ms TE) of case 5 in halothaneinduced ALF. Note the alpha 1H peak (arrow) at 3.75 ppm is only modestly increased above control. This patient had stage 0 HE and recovered with supportive therapy. He did not require LT.
72
McCONNELL ET AL
HEPATOLOGY July 1995 TABLE 1. P a t i e n t D a t a Pre-LT
Post-LT
Case
NAA:CR
CHO:CR
INS:CR
NAA:CR
CHO:CR
INS:CR
Pre-LT ~IH:CR
Post-LT ~IH:CR
1 2 3 4 5* Mean SD
1.64 1.58 1.60 1.62 1.52 1.59 0.05
0.90 0.75 0.76 0.75 0.86 0.80 0.07
0.70 0.72 0.68 0.53 0.79 0.72 0.05
1.65 1.60 1.38 Died 1.54 1.54 0.12
1.03 0.80 1.00
0.88 0.63 0.86
0.60 0.86 0.20
0.74 0.78 0.12
0.54 0.73 0.66 0.77 0.62 0.66 0.09
0.43 0.37 0.44 expired 0.55 0.45 0.08
NOTE. The peak intensities of the a l H were measured at 3.75 ppm. The means of the samples are different at P < .01. * Case 5 recovered without LT.
except in case 3. Compared with the preoperative examination, the postoperative MRI showed ventriculomegaly and prominent sulcal spaces of the cerebral cortex, suggesting that mild cerebral atrophy had developed in case 3. There was a concomitant decrease in the postoperative brain NAA on spectroscopy in case 3. This is consistent with neuronal loss and atrophy. 2s The cause of the atrophy was not determined. Pallidal T1 signal hyperintensities reported on brain MRIs in patients with chronic hepatic encephalopathy were not found in these cases of ALF. Preoperative 1H MRS showed decreased brain CHO:CR ratios in all cases compared with control. The postoperative ~H MRS results showed recovery of the choline-containing metabolites to control levels. The brain CHO:CR ratios had increased in all cases after LT. However, no significant differences were found in the INS:CR ratios in our ALF patients before and after LT or compared with controls. The changes in the CHO metabolites are consistent with prior 1H MRS studies of patients with HE before and after L T 9 '3° More case studies are needed to assess possible differences in brain INS in ALF and chronic HE. Comparison of the NAA:CR ratio measurements before and after LT showed no significant variance in cases 1, 2, and 5. Case 4 died before the follow-up ~H MRS. The mean alpha~H:CR ratio was markedly increased preoperatively (i.e., 4.5 SD above control). The postoperative alpha~H:CR mean substantially decreased to about one SD above control. These results are consistent with decreasing brain GLN after successful LT and recovery from ALF.
TABLE 2. C o n t r o l D a t a Controls 1 2 3 4 5 Mean SD
NAA:CR
CHO:CR
INS:CR
~IH:CR
1.53 1.68 1.64 1.56 1.65 1.61 0.06
1.01 1.09 1.01 0.95 0.80 0.97 0.10
0.81 0.80 0.76 0.71 0.73 0.76 0.04
0.44 0.42 0.41 0.29 0.41 0.39 0.06
The prominent resonance peak at 3.75 ppm on in vivo spectra directly matches the in vitro GLN alphalH. The in vitro 1H MRS of the GLN sample showed the chemical shift of the GLN alphalH at 3.75 ppm as measured from the water peak (4.7 ppm). This is consistent with published data. 31 DISCUSSION
The neurologic evaluation of patients with ALF is a formidable task. Signs and symptoms of progressive encephalopathy and impending brain edema are difficult to assess in the obtunded or comatose patient. Electroencephalography is helpful in the clinical management of patients, but the results are operator dependent and nonspecific for HE. Liver function tests, including blood ammonia levels, do not always correlate with the presence or the severity of HE. Spinal puncture for determination of cerebrospinal fluid metabolites and other tests is invasive and dangerous in patients with possible brain edema and increased intracranial pressure. Computed tomography and MR neuroimaging studies are useful to exclude other abnormalities, but may appear normal in the early stages of brain edema. The MRI pallidal T1 hyperintensities, which can be found in chronic HE, do not correlate with the clinical severity of encephalopathy and were not present in this group of patients with acute HE. Transcranial Doppler is helpful in assessing cerebral blood flow in ALF, but irreversible brain damage m a y be present before Doppler results are positive. Dural pressure monitors can accurately measure intracranial pressure but require neurosurgical intervention with its possible complications of hemorrhage and infection. Considering these difficulties in neurologic evaluation, there is a definite need for an accurate and noninvasive test to monitor the encephalopathy in ALF. Brain GLN and GLU (similar molecular structure and spectral overlap) are difficult to resolve at field strengths currently available on clinical imaging magnets. However, these metabolites have been successfully separated and quantitated in experimental ALF using a high-field-strength magnet (8.5 Tesla).20 Recent ~H MRS technological developments in automated water suppression, magnetic field shims, and computer
HEPATOLOGYVol. 22, No. 1, 1995 p r o c e s s i n g p r o v i d e r e l a t i v e l y f a s t d a t a acquisition a n d h a n d l i n g . ~2'33 T h e t e c h n o l o g y is c u r r e n t l y a v a i l a b l e for u s e on clinical i m a g i n g m a g n e t s a n d h a s significantly e n h a n c e d t h e u t i l i t y of in vivo 1H MRS. T h e s o f t w a r e a p p l i c a t i o n s a r e u s e r f r i e n d l y a n d c a n give a c c u r a t e b i o c h e m i c a l i n f o r m a t i o n a b o u t t h e b r a i n in a p p r o x i m a t e l y 15 to 20 m i n u t e s s c a n time. P r e v i o u s 1H M R S i n v e s t i g a t i o n s of p a t i e n t s h a v e found, in a d d i t i o n to i n c r e a s e s in b r a i n G L N , c h a n g e s in o t h e r i m p o r t a n t m e t a b o l i t e s . T h e s e s t u d i e s identified d e c r e a s e d b r a i n I N S a n d C H O a n d i n d i c a t e d a c h a r a c t e r i s t i c p a t h o b i o c h e m i c a l p a t t e r n of m e t a b o l i t e s in H E . 2125 T h e c u r r e n t s t u d y confirms t h e s e i m p o r t a n t o b s e r v a t i o n s except t h a t no significant differences w e r e f o u n d in I N S : C R r a t i o s in o u r c a s e s of A L F before a n d a f t e r L T or c o m p a r e d w i t h controls. T h i s finding sugg e s t s t h a t m o r e s t u d y is n e e d e d to a s s e s s possible differences in t h e b r a i n m y o i n o s i t o l c o n c e n t r a t i o n s in a c u t e v e r s u s chronic h e p a t i c e n c e p h a l o p a t h y . T h e m a j o r a s s e r t i o n of t h e c u r r e n t s t u d y is t h a t long T E 1H M R S m e a s u r e m e n t of b r a i n G L N c a n be u s e d clinically to m o n i t o r t h e p r o g r e s s i v e e n c e p h a l o p a t h y of ALF. T h e signal p e a k of t h e a l p h a ~H (3.75 p p m ) of G L N is r e a d i l y d e t e c t e d a n d q u a n t i t a t e d on long TE spectroscopy, a n d its a m p l i t u d e is directly r e l a t e d to t h e c o n c e n t r a t i o n of G L N in t h e b r a i n . I n addition, t h e p h a s e m o d u l a t i o n a n d c h e m i c a l shift of t h e a l p h a ~ H o b s e r v e d on in vitro G L N s p e c t r o s c o p y directly m a t c h t h e r e s u l t s in p a t i e n t s . N o r m a l l y , t h e m a j o r signal c o n t r i b u t i o n to t h e a l p h a ~H (3.75 p p m ) p e a k c o m e s f r o m G L U , w h i c h is m o r e c o n c e n t r a t e d in n o r m a l b r a i n t h a n G L N . H o w e v e r , t h e a b n o r m a l a c c u m u l a t i o n of b r a i n G L N in H E a l t e r s t h e r e l a t i v e c o n c e n t r a t i o n s of G L N a n d G L U . I n H E , t h e i n c r e a s e d signal i n t e n s i t y of t h e a l p h a ~H (3.75 p p m ) a r i s e s f r o m t h e p a t h o l o g i c a l l y i n c r e a s e d G L N . T h i s is p e r h a p s b e s t d e m o n s t r a t e d in t h i s s t u d y b y t h e longitudinal c h a n g e of t h e a l p h a 1H p e a k before a n d a f t e r LT. T h e p e a k i n t e n s i t y of t h e a l p h a 1H w a s m a r k e d l y i n c r e a s e d in t h e cases of A L F b u t c o n v e r t e d to control v a l u e s a f t e r LT a n d r e c o v e r y f r o m H E . T h e s e r e s u l t s a r e h i g h l y c o n s i s t e n t w i t h t h e c h a n g e s of b r a i n G L N f o u n d in e x p e r i m e n t a l ALF. C a n 1H M R S q u a n t i t a t i o n of G L N p r e d i c t b r a i n e d e m a in A L F ? M o r e p r o s p e c t i v e s t u d i e s a r e n e e d e d to a n s w e r t h i s i m p o r t a n t clinical question. T h e c u r r e n t s t u d y does s u g g e s t t h a t 1H M R S q u a n t i t a t i o n of b r a i n G L N is clinically feasible a n d useful for m o n i t o r i n g e n c e p h a l o p a t h y in ALF. REFERENCES
1. Ede RS, Williams RJ. Hepatic encephalopathy and cerebral edema. Semin Liver Dis 1986;6:352-379. 2. Gazzard BG, Portmann B, Murray-Lyon IM. Causes of death in fulminant hepatic failure and relationship to quantitative histological assessment of parenchymal damage. Q J Med 1975;64: 615-626. 3. Bosman DK, Deutz NEP, Maas MAW, van Eijk HMH, Smit JJH, de Haan JG, Chamuleau RAFM. Amino acid release from cerebral cortex in experimental acute liver failure, studied by in vivo cerebral cortex microdialysis. J Neurochem 1992;59:591-599. 4. Blei AT, Traber PG. Brain edema in experimental fulminant
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