Acta Neurol. Scandinav. 55, 213-225, 1977

The Departments of Neurology and Pathology, University Hospital, Lund, Sweden.

BRAIN PROTEINS IN HEPATIC ENCEPHALOPATHY

ARNE BRUN, SIGMUND DAWISKIBA, BENGT HINDFELT and JAN-EDVIN OLSON

ABSTRACT

Brain proteins were analyzed in supra- and infratentorial structures of 6 patients dying from liver failure. An equal number of patients, Iack- ing any evidence of liver disease or neurological disorder, served as controls. The results were related to regional light microscopic findings. Hepatic coma was associated with a marked reduction of soluble brain proteins, particularly in areas of grey matter. The protein loss is prob- ably neuronal and may be secondary to abnormalities in glial function. Implications for the pathogenesis of hepatic encephalopathy are dis- cussed .

The early syndrome of hepatic encephalopathy consists of defects in cognition and mentation. The condition is potentially reversible and often runs a protracted, undulating course. With deterioration, motor abnormalities ( asterixis, hyperactive stretch reflexes, decerebrate rigid- ity etc.) ensue and consciousness is lost. Despite these omnious symp- toms and signs the brain does not exhibit any gross morphologic changes. Microscopically, astrocytosis including abnormal Alzheimer type I1 astrocytes (u . Hosslin & Alzheimer 1912) constitutes the neuro- pathological paradigm of hepatic encephalopathy (Adams & Foley 1953).

Hyperammonemia, secondary to liver failure and portal-systemic shunting of intestinal blood, is generally accepted as one of the main pathogenetic factors in hepatic encephalopathy (Breen & Schenker 1972, Plum & Hindfelt 1976). Experimentally, sustained hyperammon- emia reproduces the neuropathology of hepatic coma (Cauanagh & K y u 1971, Cole e f al. 1972) and there is some evidence that ammonia affects brain function by interfering in particular with glial metabolism (Cauanagh 1974). The metabolic mechanism has not been convincingly clarified since ammonia induces a multitude of changes and affects pH regulation (Hindfelt & Siesjo 1971 a ) , carbohydrate and amino acid metabolism (Hindfelf 1972, 1975), electrolyte and energy homeostasis

214

(Hindfelt & Siesjo 1971 b, Hindfelt 1972, Cavanagh 1974). Any of these disturbances, singly or in combination, may tentatively explain dys- function of the brain.

The cerebral detoxification of ammonia occurs by amination of a-ketoglutarate to glutamate which is amidated to glutamine, the non- toxic end-product ( Weil-Malherbe 1962). The prompt development of AIzheimer type I1 astrocytes in hyperammonemia (Norenberg et al. 1974) or with impaired detoxification of ammonia (Gutierrez & Noren- berg 1975) emphasizes the close coupling between the astrocyte and the intermediary amino acid metabolism. Under pathological conditions, accompanied by abnormalities of the astrocytic glia, disturbances of amino acid metabolism occur which have immediate effects on cerebral energy metabolism and transmitter release (Hindfelt 1972, Hindfelt e f al. 1976). An altered amino acid metabolism, especially when sustained, may also affect the cerebral metabolism of proteins, which tentatively may contribute to the cerebral dysfunction in metabolic brain disorder.

In order to test such a possibility, soluble brain proteins were analyzed regionally in patients who had died from liver insufficiency. The total protein content, the concentrations of albumin, IgG and beta- trace protein were measured and correlated with regional neuro- pathological alterations. Beta-trace protein was included since it is predominantly a glial protein (Olsson et al. 1974) and consequently of interest in conditions with an abnormal astroglia.

MATERIAL AND METHODS

Maferial. The patient material for this study was derived from autopsy cases where section was performed within 24 h of death. The patients included were only those without any previous disorder of the central nervous system, and the brain had to be grossly normal. Of the 6 cases of liver cirrhosis chosen, 3 had had recurrent episodes of hepatic coma. To match the cases with liver pathology an equal number of controls were selected. Besides being neurologically normal prior to death, no case with suspectcd liver disease was accepted among the controls. The post-mortem periods did not differ between the two groups.

Except for the material necessary for the routine work-up of the case, the following tissues were always included in the study: multiple pieces of liver, brain tissue from the frontal lobe (cortex and white matter), thalamus and the cerebellum (the dentate nucleus and white matter). Great care was taken to obtain tissue from identical sites in different cases. The frontal lobe section was taken from the frontal pole, cut in the frontal plane. Cerebellar tissue was obtained from a slice cut a t a 45" angle to the sagittal plane. Corresponding pieces of tissue were simultaneously removed from the contralateral hemispheres (cerebral and rerebel- lar) for biochemical analysis.

Neuropathological methods. The tissues for microscopical study were fixed in 10 per cent formalin and, after a few days, were embedded in paraffin and

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Figure 1. Hilus of the dentate nucleus in a case of liver disease. Among the larger, darkl!l staining neurons there are mang enlarged astrocgtes of the Alzheimer tgpe 11. Only their nuclei are uisible, being pale w i th irregular but distinct outlines (arrows).

Cresgl-uiolet staining. X 500.

sectioned at 5 ,$, stained with hematoxylin, eosine and cresyl violet. The slides stained with cresyl violet were used for counting of glial cells. This was done in a Zeiss microscope at a magnification of x 500, with a grid inserted into one ocular to allow counting within fields of identical size and shape. Cells were counted in 5 randomly choosen fields of respective regions. Each field was counted twice, the first time noting the number of Alzheimer type I1 astrocytes, the second time the total number of glial cells. Alzheimer type 11 cells were identified as slightly or considerably enlarged astrocytes with a pale nucleus and a distinct nuclear membrane, the shape of which was often lobulated (Figure 1 ) . The average was calculated of the numbers obtained.

Biochemical niethods. The hrain specimens were stored at - 60" C until prepared for analysis. The water soluble protein fraction was obtained by homogenization at 4" C with 0.25 M sucrose. 1 ml of the sucrose solution was used for 3 g (wet weight) of brain tissue. The proteins were separated by centrifugation at 38,000 G for 30 min at 0" C. The supernatant was pipetted off and the procedure was repeated twice with resuspension of the pellet with the initial volume of 0.25 M sucrose. The three supernatants of each sample were pooled and stored at -60" C until analyzed.

The protein concentrations of the pooled supernatants were determined by a modification (Legget-Bail!] 1962) of the method of Lowrg et al. (1951) . Quantifica- tions of albumin, IgG and beta-trace protein (BTP) were performed with single radial immunodiffusion on agar gelmedium (Mancini e f al. 1965). Each protein was analyzed simultaneously in all studied hrain regions. Serial dilutions of pooled

216

Table f. Clinical and autopsg data o f the cases w i th l iver disease and controls. ( M , F : male and female) .

Recurrent Portal

coma tension Sex Age Clinical diagnosis hepatic hyper- Autopsy findings

Liver disease 1. M 36 Liver failure

Bronchopneumonia X X

2. M 45 Liver failure - - Haemorrhagic diathesis

3. M 49 Hepato-renal syndrome -_ Alcoholism

4. M 56 Liverfailure Alcoholism

5. M 61 Liverfailure Diabetes mellitus

X

X 6. F 65 Liverfailure Chronic hepatitis Bronchopneumonia

Controls 7. M 62 Ruptured aortic aneurysm -

8. M 62 Acute myocardial -

infarction

9. M 67 Malignant lymphoma __

X

X

Cirrhosis, ascites splenomegaly, eosophageal varices.

Extensive haemorrhages within the lungs and gastrointestinal tract. Hepatic steatosis, centroacinar necrosis and periportal fibrosis.

Hepatic steatosis, necrosis and early cirrhosis. Mild splenomegaly, cholemic nephrosis. Erosive and membranous gastroenteritis.

Hepatic steatosis and cirrhosis. Chronic pancheatitis.

Cirrhosis. splenomegaly, esophageal varices. Tubular nephritis. Peripheral neuropathy.

Cirrhosis. Splenorenal shunt,

Cystic medical necrosis. Ruptured aortic aneurysm.

MI with heart rupture.

Histiocytary lymphoma. Liver involvement restricted to minor infiltrations in portal zones and sinusoids.

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Table 1 (continued).

Recurrent Portal

coma tension Sex Age Clinical diagnosis hepatic hyper- Autopsy findings

10. F 68 Acutemyocardial - __ Coronary sclerosis. infarction MI with heart rupture.

11. M 79 Acute myocardial infarction

12. iLI 42 Aortic stenosis (op.) ~-

Cardiac failure

Coronary sclerosis. MI. Multiple pulmonary emboli.

Coronary and general arteriosclerosis Cor bovinum. Op. aortic valve.

Table 2. The total number of glial cells and Alzheimer type I1 astrocytes in each microscopic field within different regions (see Methods). Means k S.E.M.

Cerebellar Frontal Thalamus white matter Dentate nucleus

Alz. I1 glial cells

white matter Frontal cortex

A h . 11 glial ~ 1 ~ . 11 glial A h . 11 glial II glial cells cells cells cells

Controls 3 45 11 128 5 62 3 76 8 94

f l f 3 + 2 + 8 k 2 + 6 + 1 & 6 2 3 f 6

Liuer disease 11 54 20 140 16 68 1 0 70 25 92

f 2 k 8 f 3 + 8 " . 1 f 3 f 2 & 7 f 3 + 8

blood donor serum were used as standards for quantification of albumin and IgG, whereas the standard for beta-trace protein consisted of known dilutions of the purified protein, isolated from human cerebrospinal fluid (Link 1967) .

Statistical methods. The Wilcoxon's rank sum test was used for the statistical evaluation and the levels of significance are given in the test.

RESULTS

The clinical data on the patients with liver disease and the controls are presented in Table 1, along with autopsy findings. Among the patients in the first category, 3 males were chronic alcoholics; 1 female suffered from chronic hepatitis and in the remaining 2 patients the pathogenesis of the liver disease was obscure. 3 patients had had previous symptoms

14 ACTA NEUROL. SCAND. 55, 3

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Table 3. The concentration of total soluble proteins and beta-trace protein in various structures of ihe central nervous s{]stem in cases w i th liuer failure ( L ) and controls

( C ) . Means f S.E.M.

Total protein (TP) Beta-trace protein

g/l mg/l mg/g TP

Frontal cortex C 12.0 * 1.0 10.0 f 0.7 0.8 t 0.1 1, 8.6 f 0.3 10.9 t 1.2 1.3 * 0.1

Frontal white matter C 9.7 t 1.2 21.5 f 2.1 2.3 f 0.3 L 8.7 t 0.5 21.6 f 2.5 2.5 t 0.3

Thalamus C L

13.1 k 1.6 12.4 t 1.4 0.9 f 0.1 10.1 t 1.0 10.7 f 1.3 1.1 * 0.1

Cerebellar white matter C 10.7 t 0.7 10.4 t 1.4 1.0 * 0.2

1.3 f 0.2 L 9.8 * 0.7 12.8 f 1.7

Dentate nucleus C 11.2 2 1.4 12.2 t 1.8 1.1 t 0.1 L 10.5 * 1.1 14.6 f 2.9 1.5 f 0.2

of hepatic encephalopathy with brief episodes of stupor and/or coma of a couple of years. Portal-systemic shunting was the common de- nominator in these cases.

None of the patients had any evidence, morphologically o r histo- logically, of cerebrovascular disease or other relevant brain disorder. Microscopically, there were differences between the two groups and the patients dying from liver disease exhibited in all regions a three- fold increase in the number of Alzheimer type I1 astrocytes (Table 2 ) . In single controls high numbers of Alzheimer type I1 cells were en- countered regionally but never uniformly. The total number of glial cells did not differ significantly between the groups (Table 2 ) . The abundance of Alzheimer I1 glia in cases with liver disease allowed a clear separation between the two categories.

The concentrations of soluble brain proteins within various regions are summarized in Tables 3 and 4. The total protein concentration (Table 3) was consistently lower among the liver cases and the reduc- tions varied from 7 to 28 per cent, being most pronounced in areas of grey matter, i.e. the frontal cortex and the thalamus. As exemplified for the frontal cortex (Figure 2) there seemed to be a relation between

219

Total protein 15- concentration dl

10.

5-

0-

0

0 0

0

0

0 .X .X

b b 0

.X

0

I I I 1 0 10 20 30 4'0

% Alzheimer type P glia.

Figure 2. T h e correlation between the toial protein content and the occurrence o f Alzheimer type I I cells i n the f ronta l cortex. Open circles denote controls, f i l led dots the cases with liver failure. T h e latter connotations prouided with a cross indicate the patients who had su f fered f r o m recurrent episodes o f hepatic coma. The number of Alzheimer cells are expressed as percentages in relation to the total number o f

glial cells.

the extent of neuropathological changes (i.e. the number of Alzheimer type I1 astrocytes) and the reduction in total protein content. In cases with more than 8 per cent Alzheimer type I1 cells the total protein concentration fell below 10 g/l. The single control patient who had a high number of Alzheimer type I1 astrocytes in his frontal cortex also had a marked reduction in total protein content. The patients who had had episodic stupor and coma did not differ in total protein content from those who had had previous symptoms of hepatic encephalopathy (Figure 2 ) .

The data on beta-trace protein in the cases with liver pathology and the controls are shown in Table 3. The absolute concentration of this protein was slightly higher in the liver group, with the exception of thalamus. When the amount of BTP was considered in relation to the total protein content of the structure (Table 3 expressed in mg per g of total protein) the ratio was higher among the liver cases, particularly in the frontal cortex and the dentate nucleus ( p < 0.01 and p < 0.05, respectively).

14*

220

BTP 25 mg/l

20

15

10

5

I rn I I 0 50 100 150

Total number of glial cells

per visual field

Figure 3. The correlation between f h e total number o f glial cells and the concentra- tion of beta trace protein in various regions. Means fk S.E.M. Connotations are as follows: Frontal cortex (0) frontal white maifer (A), the thalamus (a), the dentate nucleus (0) and the cerebellar white matter ( 6 1. Open symbols indicate controls; filled ones cases wi th liver disease. The line is drawn bll best visual f i t .

In Table 4 the concentrations of albumin and IgG are presented. In the ‘liver group’ the albumin content amounted to two thirds that of the controls. The proportion of albumin to total protein content did not differ statistically for any region though the mean calues for the liver cases fell below those of the controls. The concentrations of IgG were not significantly different in the two groups although there may have been a tendency towards higher mean concentrations among the cases with liver failure. With the amount of IgG expressed in relation to the total protein content, higher values were consistently obtained

22 1

for the liver group. The albumin/IgG ratios of the controls were higher than those of the liver cases (range: 2.94-4.20 and 1.90-2.09, respec- tively).

Table 4 . The regional concentrations of albumin and ZgG. Means f S.E.M. Connotations as in Table 3.

Albumin JgG mg/l mg/g TP mg/l mg/g TP

Frontal cortex c 153 f 26 12.6 f 2.0 52 f 12 4.4 f 0.9 L 106 f 25 12.6 3~ 3.1 54 f 12 6.1 f 1.3

Frontal white matter c 183 f 49 18.0 f 2.9 57 f 19 5.4 f 1.1 L 122 f 23 14.9 f 3.8 62 f 13 7.0 f 1.3

Thalamus C 287 f 84 20.2 f 4.1 8 3 + 9 6.5 f 1.0 L 190 f 35 18.9 f 4.3 91 f 22 9.6 f 2.8

Cerebellar white matter c 194 f 48 17.6 f 3.4 4 6 f 2 4.3 f 0.3 L 145 f 22 15.2 f 2.5 73 f 16 8.1 f 2.1

Dentate nucleus C 181 f 48 15.8 f 2.8 4 6 + 9 4.0 f 0.6 L 120 f 24 12.2 f 2.7 63 f 18 6.1 f 1.6

DISCUSSION

Although the Alzheimer type I1 glia is not pathognomonic of liver insufficiency, its abundance in the patients with liver pathology pro- vides neuropathological confirmation of hepatic encephalopathy. The brain histology allowed a clear separation between the two groups. However, it was not possible by neuropathological means to decide which cases had had a chronic course, interpunctuated by hepatic coma, and which had not. In this respect the limitations were similar to those of liver pathology by which i t is impossible confidently to predict the existence or future development of hepatic encephalopathy (Popper 1951).

The interpretation of the biomechanical data necessitates a brief discussion of some factors which may have critically influenced the results. The main factors to be considered are 1. autolysis and 2. con- tamination from extracellular (plasma) proteins. Admittedly, it is always difficult to evaluate metabolic changes in postmortem speci-

222

mens, because of autolytic phenomena. Nothing is known about how such processes affect the total brain protein and its composition with time, nor do we know if the temporal profile of autolysis is different in cases with metabolic brain disorders prior to death. These objections cannot be met. However, one important parameter, i.e. the post-mortem periods, did not differ between the controls and the group with liver failure.

The amount of plasma proteins by far exceeds the contents of soluble brain proteins (Table 3 ) . The total blood volume of the brain is prob- ably about 3 per cent of the brain weight (Flock et al. 1966) and major contributions of extracellular proteins are possible when analyzing brain tissue proteins. Such errors may invalidate the results. In this study significant contamination from plasma proteins, albumin and immunoglobulins, seems untenable for the following reasons. The con- centrations of albumin and IgG in the brain were low (Table 4 ) . Fur- thermore, the regional distribution of these proteins contradict extra- cellular contamination since in that case the contents in grey matter areas (the frontal cortex and the dentate nucleus) should exceed those of the white matter (the frontal and cerebellar white matter) because of the differences in vascularity. As is evident from Table 4 the con- centrations were similar in grey and white matter structures and this pertains to both controls and liver cases. Consequently, the protein data should not be markedly influenced by extracellular contributions.

The reduction in brain protein content in cases of liver disease (Table 3) is remarkable but may be compatible with earlier clinical and experimental results. Thus, advanced liver failure or hepatectomy are accompanied by raised concentrations of amino acids in both brain and extracellular fluids (plasma and cerebrospinal fluid-Mattson ef al. 1970, Breen & Schenker 1972), indicating either an accelerated catabol- ism of proteins or a defect in protein synthesis. Any of these alterna- tives may relate to the present findings.

The total protein contents were particularly reduced in supraten- torial areas of grey matter (the frontal cortex and the thalamus), which could mean that the protein loss is predominantly neuronal. Anyway, the topographic distribution of the fall in protein content is interesting from a clinical point of view, since the early symptomato- logy of hepatic encephalopathy consists of a “frontal lobe syndrome” with personality changes, defects in mentation etc. The electroence- phalographic abnormalities of impending encephalopathy also occur initially over the frontal regions and consist of progressive slowing of the EEG activity, which subsequently spreads all over the cortical surface (Bickford & Butt 1955).

The contents of beta-trace protein were not reduced along with the total soluble proteins (Table 3 ) . Two alternative explanations are pos- sible: the cerebral metabolism of BTP could be different from that of the proteins which were reduced, or beta-trace protein could be con- fined to a compartment, unaffected by liver failure. Nothing is known about the metabolism of BTP in the brain, except that i t is synthetized within the central nervous system (Olsson & Sandberg 1975). The molecule is a single polypeptide chain of low molecular weight (Link 1967) and there is a priori nothing indicating a peculiar metabolism of this protein. This leaves the second possibility as the more plausible explanation. Available evidence suggest that BTP in the central nervous system is a glial protein. High amounts occur in the white matter, in enriched glial fractions and in gliomas (Olsson & Nord 1973, Olsson et al. 1974). Our data confirm such a distribution of BTP since the amount of the protein in the brain tissue rises with increasing number of glial cells (Figure 3 ) . As the concentration of beta-trace protein did not reflect the occurrence of Alzheimer type I1 cells (Tables 2 and 3 ) , the protein is probably confined to normal, unaffected glia.

Although definite conclusions are premature the results are com- patible with a disturbed cerebral metaboIism of proteins in liver failure, complicated by severe cerebral dysfunction. Whether the metabolic abnormality should be considered a cause or a consequence of the encephalopathy remains to be clarified. However, the findings add another aspect to the enigmatic pathogenesis of hepatic coma.

ACKNOWLEDGEMENTS

The study was supported hy grants from the Medical Faculty, University of Lund, and from the Swedish Medical Research Council (12X-2037).

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Received September 23, accepted October 19, 1976

Bengt Hindfe l t , M.D. Department of Neurology University Hospital of Lund S-221 85 Lund Sweden