Effect of infusing the branched-chain amino acids on concentrations of amino acids in plasma and brain and on brain catecholamines after total hepatectomy in the rat

Life Sciences, Vol. 30, pp. 1361-1368 Printed in the U.S.A.

Pergamon Press

EFFECT OF INFUSING THE BRANCHED-CHAIN AMINO ACIDS ON CONCENTRATIONS OF AMINO ACIDS IN PLASMA AND BRAIN AND ON BRAIN CATECHOLAMINES AFTER TOTAL HEPATECTOMY IN THE RAT J. Howard James, Per M. Herlin, Laura Edwards, Craig A. Nachbauer and Josef E. Fischer Department of Surgery University of Cincinnati Medical Center 231 Bethesda Avenue Cincinnati, Ohio 45267 (Received in final form February I, 1982) Sun~nary In totally hepatectomized rats supported by infusion of glucose, the concentrations of many amino acids in plasma and brain rose progressively over time, while the brain levels of norepinephrine decreased. Infusion of a solution containing glucose, leucine, isoleucine, and valine after hepatectomy greatly reduced the accumulation of other essential amino acids in plasma and brain. However, the decrease in brain norepinephrine content was not significantly affected by this infusion, suggesting that high brain concentrations of monoamine precursor amino acids are not the primary cause of norepinephrine depletion after hepateetomy. In acute hepatic failure in man or after total hepatectomy in animals, the plasma concentrations of most amino acids rise progressively (1,2) while the concentrations of the branched-chain amino acids (BCAA), leucine, isoleucine and valine, usually remain normal (i), presumably because the metabolism of the BCAA is primarily extrahepatic (3). In patients with acute hepatic failure, hyperaminoacidemia probably reflects lysis of necrotic liver tissue, protein breakdown in peripheral tissues and the absence of normal liver metabolic functions. After total hepatectomy, only the latter two factors are present, so that protein breakdown in peripheral tissues contributes to hyperaminoacidemia. Hepatic failure or total hepatectomy also results in extremely high concentrations of amino acids in brain (4,5). Many of these amino acids belong to the group of large neutral amino acids (LNAA) which includes the BCAA, threonine, methionine, tyrosine, phenylalanine, tryptophan and histidine. The LNAA compete for transport across the blood-brain barrier via a common transport system (6). High brain concentrations of some of these amino acids may disturb neurotransmitter metabolism in brain, thereby contributing to hepatic coma and death (7). Thus, serotonin synthesis is greatly accelerated by high brain tryptophan levels after hepatectomy (8). The brain content of norepinephrine (NE) is low in hepatic failure, perhaps due to inhibition of dopamine ~-hydroxylase or to release of NE from nerves by amines such as tyramine and octopamine (9,10). These observations suggest that reducing the accumulation in brain of potentially toxic monoamine precursor amino acids might lengthen survival or reduce morbidity after total hepatectomy. In the 0024-3205/82/161361-08503.00/0 Copyright (c) 1982 Pergamon Press Ltd.

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present study, we attempted to reduce the influx into brain and, hence, the brain levels of the LNAA after total hepatectomy by raising the concentrations of the BCAA in blood. We also studied the effects of this treatment on brain levels of NE and dopamine (DA). Materials and Methods Male Sprague-Dawley rats (.initial weight 212-263 g) underwent hepatectomy in three stages (ii) under ether anesthesia as follows: (a) division of inferior vena cava above the renal veins, (b) four weeks later, end-to-side portacaval anastomosis without ligation of the pancreatico-duodenal vein and (c) one week later, removal of all liver parenchyma after ligation of the hepatic artery, biliary duct and the inferior vena cava. Control groups underwent either steps a and b and a sham operation at step c (Group C) or step ~ and sham operations at steps b and ! (Group D). After the third operation, a silastic catheter was placed in the jugular vein for infusion of solutions. Infusion was begun no later than i0 min afterwards. Food was not available during the infusions. Hepatectomized animals were infused either with a solution containing 20% glucose, 145 mM NaCI, 13 mM KoHPO L, 2.3 mM calcium gluconate, 2 m M MgSOA, and 1.5 mM ZnCI2, pH 7.3(Group A)~ or~the same solution plus 0.24 M BCAA (0508 M each of vallne, leucine and isoleucine, Group B). The control groups C and D received the glucose solution. All solutions were infused at a rate of 0.60 ml/hr/100 g body weight while the rats were housed in individual cages and warmed by proximity to a light bulb. Rectal temperature was maintained at 35-37°C. The rats were decapitated 6 and 18 hrs after hepatectomy or sham operation. Blood from the neck was collected into heparinized beakers and plasma was separated by centrifugation and kept frozen at -72°C until analysis. The brain was removed, divided sagittally into two hemispheres and immediately frozen on dry ice. Amino acids were determined using a Beckman 121-MB amino acid analyzer after deproteinization in 5% sulfosalicylic acid. NE and DA in the other brain hemisphere were assayed by reverse phase liquid chromatography using an Altex Ultrasphere C-18 column, 4.6 mm x 25 cm, and a Model LC-4 electrochemical detector (Bioanalytical Systems) with a glassy carbon electrode. Brain tissue was homogenized in 5 volumes of 0.4 M HCIO~ containing sodium metabisulfite, EDTA and i00 ~g/ml of ~-methyl norepinepNrine as internal standard. After centrifugation catecholamines in the supernatant were adsorbed onto alumina. The alumina was washed twice with water and catecholamines were eluted with 1 ml of 0.i N HCI. The mobile phase buffer (10% methanol, 90% 0.i M potassium phosphate buffer, pH 3.0, 0.15 m M sodium octanesulfonate, 0.i mM EDTA) was delivered at a flow rate of i.i ml/min. Plasma glucose was determined by standard laboratory techniques. A multivariate analysis of variance (MANOVA) for plasma or for brain was performed for these factors: treatment, time, treatment x time. The following amino acids were studied: aspartic acid (ASP), threonine (THR), serine (SER), glutamic acid (GLU), glutamine (GLN), glycine (GLY), alanine (ALA), valine (VAL), methionine (MET), isoleucine ~ILE), leucine (LEU), tyrosine (TYR), phenylalanine (PHE), lysine (LYS), histidine (HIS) and arginine (ARG). Overall significance in this MANOVA was precedent for doing univariate 4 x 2 factorial analysis of variance (ANOVA) for each amino acid and catecholamine in plasma or brain. Comparisons between groups utilized the Newman-Keuls multiple-range test. Differences between groups with p
Vol. 30, No. 16, 1982

Hepatectomy and Amino Acids

1363

Results Rats sacrificed at six hrs after hepatectomy were all alert. In Group A, 9 of 14 rats survived 18 hrs and 8 of these 9 rats were in coma (unresponsive to tail-pinch) at sacrifice. In Group B, 9 of 15 rats survived 18 hrs and 4 of these 9 rats were in coma at sacrifice. The difference in incidence of coma was not statistically significant (X 2 test). Rats in coma (n = 12) had significantly less NE in brain than rats not in coma (n = 6) (244+20 vs 373+30 ng/g, respectively, x + SEM; p<0.01 by Student's t-test). Hypoglycemia was prevented by glucose infusion (overall mean 196+12 mg/dl, range: 81-341) with no significant differences between the groups. The MANOVA results indicated overall significant differences (p<0.001) among amino acids and catecholamines in both plasma and brain. All differences noted below were significant, p<0.05 at least. No comparisons were made between different groups at different times. Tables I and II show results for plasma and brain, respectively. Control groups ~C and D) - Effect of time of infusion and of treatment In Groups C and D, no significant differences were observed in plasma between 6 and i8 hrs. In both Groups C and D, THR, GLY, and MET in brain were higher at 18 hrs than at 6 hrs. In Group C only, TYR in brain was also higher at 18 hrs. In Group C, brain GLU was lower at 18 hrs. Comparing Groups C and D, no significant differences were observed in plasma at any time. Brain GLN, PHE and HIS were higher in Group C than in Group D both at 6 and 18 hrs. After 18 hrs, MET and TYR were also higher in Group C. Hepatectomized grpups (A and B) - Effect of time In Group A, ASP, THR~ SER, GLN, ALA, MET, TYR, PHE, LYS, HIS, and ARG were higher in plasma at 18 hrs as compared with 6 hrs. In brain, THR, GLN, GLY, ALA, MET, TYR, PHE, LYS, HIS, and ARG were higher at 18 hrs. In plasma of Group B, ASP, SER, GLN, GLY, ALA, TYR, LYS, HIS, ARG, and the BCAA were higher at 18 hrs than at 6 hrs. In brain, GLN, GLY, ALA, TYR, LYS, HIS, and ARG were higher at 18 hrs, while THR was lower at 18 hrs. Hepatectomy and $1ucose infusion (Group A) vs. Controls Effect of time

(Groups C and D) -

In Group A compared either to Group C or D, plasma levels of THR, GLN, GLY, MET, TYR, PHE, LYS, and HIS were higher both at 6 and 18 hrs; at 18 hrs, SER, ALA and ARG were also higher in plasma of Group A compared either to Group C or D. In brain of Group A compared either to Group C or D, THR, GLN, GLY, ALA, MET, TYR, PHE, LYS, and HIS were higher at both 6 and 18 hrs; at 18 hrs, ARG was also higher in brain of Group A compared either to Group C or D. At both 6 and 18 hrs, ASP in brain of Group A was lower than in either Group C or D. Hepatectomy and BCAA (Group B) vs. Controls (Groups C and D) - Effect of treatment In plasma of Group B compared either to Group C or D, GLN, GLY, ALA, the BCAA, and LYS were higher both at 6 and 18 hrs; at 18 hrs, ASP, SER, TYR, HIS, and ARG were also higher in plasma of Group B compared to Groups C and D. In Group B compared only to Group D, TYR was higher at 6 hrs. In brain of Group B Compared either to Groups C or D, GLN, the BCAA and LYS were higher both at 6 and 18 hrs. In Group B compared only to Group D, GLY was higher at 6 hrs. At 18 hrs, GLY, ALA and ARG were also higher in Group B compared either to Group C or D. At 18 hrs only, SER and TYR were also higher in Group B compared to Group D. In brain of Group B compared either to Group C or D,

0.18+0 0.0250 0.0550 0.7650 0.i050 0.2450 0.2250 0.0550 0.0750

Concentrations

ALA~ ARG GLU GLN GLY SER TYR

0.86+0 0 4850 0 1250 3 4050 0 9850 0 4650 0 4650

0.31+0.04 0.1850.02 0.0650.01 0.8350.04 0.3350.02 0.2050.02 0.i0+0.01

0,07+0 01 0.0350 01 0,0650 01 0.3850 04 0.0550 01 0.0950 01 0.1350 01 l--a 0.08+0.01

18hrs(6)

difficulties.

0.22+0.02 0.i050.01 0.0650.01 0.6950.02 0.2650.01 0.1550.01 0.0650.01

0.08+0.01 0.0450.01 0.0750.01 0.2650,02 0.0350.01 0.0950.01 0.0850.01 0.0350.01 0.ii$0.02

6hrs(7)

0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

and

32+0.01 1050.01 0650.01 5250.02 2450.01 1650.01 0450.01

06+0.01 0550.01 I050.01 2450.01 04¥0.01 0750.01 1350.02 4050.01 1350.01

6hrs(6)

SHAM Glucose

Hepatectomy

PORTACAVAL SHUNT Glucose (C)

to technical

2.60+0.15 0.7150.05 0.1250.01 7.5450.28 1.0950.09 0.4850.03 0.2350.01

0.24+0.02 0,3550.03 0.4150.04 1.1050.06 0.0450.01 0.1550.03 0.i0+0.01 0.0150.01 0.6750.05

lost due

1.34+0.13 0.1850.03 0.0850.01 2.3550.09 0.5350.03 0.2550.03 0.1250.01

I

6 and 18 Hours After

(B)

18hrs(9)

+ BCAA

(~mol/ml)

0.13+0.01 0.2150.02 0.2750.02 0.4950.04 0.0350.01 0.0850.01 0.0750.01 0.0150.01 0.4550.03

6hrs(7)

a = data

08 06 03 32 08 07 03

0.42+0.04 0.0350.01 0.0750.01 1.3950.06 0.2050.02 0.5050.05 0.3650.04 0.0650.01 0.0750.01

are x + SEM,

0.54+0.06 0.2350.03 0.0650.01 1.4950.09 0.5550.04 0.2450.04 0.2450.02

02 01 01

02

01 05 01

01

01

in Plasma

HEPATECTOMY CA) Glucose

Acids

18hrs(9)

Glucose

of Amino

6hrs(7)

Nonessential

HIS ILE LEU LYS MET PHE THR TRP VAL

Essential

TIME(n)

Treatment

Concentrations

Table

0.42+0.06 0.1650.01 0.0650.01 0.5550.04 0.3150.02 0.2050.01 0.0850.01

0.06+0.01 0.'0550.01 0.0950.01 0.4050.04 0.0650.01 0.0850.01 0.1650.02 Z--a 0.i0+0.01

18hrs(6)

(D)

in Controls

CO ~o

o

z

LO O

O

m o

m

==

O~

0

0 0 0 0 0 0 0 0

Concentrations

735o 355o

0450

6350

07+0 1950 9250 2051

0.0850.01 0.3650.03 o.o15o.ol 0.22~0.01

0.0450.01

0.12+0.01 0.1150.01 0.1750.01 0.3850.01

6hrs(7)

0.41+0.01 0.8250.03

0.51+O.03 0.1250.01 11.0850.24 12.0850.62 1.1050.04 0.8050.04 0.1450.01 3.3150.14

0.1650.01 0.4650.05 o.o25o.ol 0.0750.01

0.0650.01

0.17+0.03 0.0250.01 0.0750.01 0.2850.02

6hrs(7)

SHUNT (C)

0.50+0.01 1.2750.05

0.56+0.01 0.1450.01 8.5450.67 10.2651.05 1.3550.02 0.9350.01 0.2550.02 3.5450.12

0.1750.01 0.6550.03 ---a 0.06+0.01

0.1050.01

0.18+0.01 0.0250.01 0.0650.01 0.3350.02

18hrs(6)

PORTACAVAL Glucose

difficulties.

0.32+0.03 1.1050.05

2.27+0.36 0.2450.02 9.5950.51 33.4151.92 1.7950.06 1.0750.04 0.2150.02 2.4050.18

0.1250.02 0.2250.02 o.o15o.ol 0.3750.04

0.0350.01

0.24+0.01 0.2150.03 0.2950.04 0.4550.02

18hrs(9)

(B)

are x ~ = data lost due to technical

0.48+0.01 1.2350.08

47 1.06+0.08 Ol 0.1450.01 55 10.5450.40 82 19.3451.79 03 1.2450.05 05 0.9750.04 04 0.1250.01 ii 3.0750.07

0.26+0.03 1.1550.08

3 0 8 31 1 1 0 2

0 0 o 0

1.44+0.31 0.1550.01 10.2450.44 23.4051.49 1.3050.04 0.9950.06 0.5350.02 2.8750.17

0 2650.01

2o5o Ol

4850 06 7850 03 o65o Ol o85o 01

7050.03 9250.06 o55ool 09~0.01

0.76+0.04 0.025-.01 0.0950.02 0 5850.02

18hrs(9)

HEPATECTOMY (A) Glucose + BCAA

42+0.02 0350 Ol 0850 01 4450 02

6hrs(7)

Glucose

Catecholamines NE 0.44+0.03 DA 1.2450.04

ALA ARG GLU GLN GLY SER TYR ASP

Nonessential

HIS ILE LEU LYS MET PHE THR TRP VAL

Essential

TIME(n)

Treatment

SHAM

0.50+0.01 0.9650.06

0.56+0.01 0.1450.01 10.7150.30 5.1350.17 0.9950.04 1.0350.03 0.0650.01 3.0150.05

0.0950.01 0.5350.05 o OlSOOl 0.0850.01

0.0550.01

0.09+0.01 0.01+0.01 0.0650.01 0.3350.02

6hrs(6)

Glucose

Concentrations of Amino Acids (~mol/g) and Catecholamines (P8/8) in Brain 6 and 18 Hours After Hepateetom X

Table II

0.50+0.02 1.1650.03

03 01 13 38 05 0.8450 04 0.1150 01 3.4950 14

0.65+0 0.1350 9.9550 4.9850 1.2250

0.07~0.01

---a

0.0950.01 0.6150.02

0.0750.01

0.08+0.01 0.0250.01 0.0650.01 0.2950.02

18hrs (6)

CD)

~o

o~

>

o

m.

=n

O

z

O

o

1366

ASP,

Hepatectomy

THR and MET were

and Amino Acids

Vol.

30, No.

16, 1982

lower at 18 hrs.

Hepatectomy and G l u c o s e of treatment

(Group A) vs. Hepatectomy

and BCAA

(Group B) - Effect

In plasma of Group B compared to Group A, THR, MET, TYR, PHE, LYS, and HIS were lower both at 6 and 18 hrs. Both at 6 and 18 hrs, GLN, ALA, and the BCAA were higher in Group B; at 18 hrs, ASP and ARG were also higher. In brain of Group B, THR, MET, TYR, PHE, LYS, and HIS were lower than in Group A both at 6 and 18 hrs; at 18 hrs in Group B, ALA was also lower. At both 6 and 18 hrs, the BCAA were higher in Group B; at 18 hrs in Group B, GLY and ARG were also higher. Both in plasma and brain, tryptophan was higher in Group A compared to Groups C and D at 6 hrs. Concentrations of TRP in plasma and brain were lower in Group B compared to Group A at both 6 and 18 hrs and lower than the Control Groups C and D at 6 hrs. Catecholamines

in Brain

- Effect

In Group C, DA was higher B, NE was lower at 18 hrs than B, NE was lower than in either differences in brain NE or DA Correlation

of time and treatment

at 18 hrs than at 6 hrs. Both in Groups A and at 6 hrs. At 18 hrs only, both in Groups A and Group C or D. There were no significant levels between Groups A and B.

of brain amino acid and NE concentrations

Attempts were made to find correlations between brain NE levels and brain concentrations of amino acids in the h e p a t e c t o m i z e d rats. Significant inverse correlations were observed between NE and brain GLN (r=-0.744, n=32, p<0.O01) and between NE and lOglO A L A [r=-0.872, n=32, p
600

0 6hr GLUC • 6hr BCAA O IBhr GLUC

0 •

• IBhr BCAA

FIG.

400

:'olio2. 0

200

•

Relationship between alanine and NE in brain of hepatectomized rats only.

00

r = O 87

O

i

O

O

~oo

i

,ooo

I

2ooo

BRAIN ALA

~o'oo 80'o0

(nmol/g)

Discussion Treatments of acute hepatic failure often involve w h o l e s a l e clearing of solutes from the blood in an attempt to remove an unknown toxin or toxins w h i c h

Vol. 30, No. 16, 1982

Hepatectomy and Amino Acids

1367

may contribute to coma. Thus, hemoperfusion through columns of treated charcoal (12) or hemodialysis utilizing special membranes (13,14) have been proposed as techniques which may offer support to the failing liver. Although such techniques delay or reduce the accumulation of amino acids and other substances in blood, they also deplete the body of many nutrients, such as essential amino acids, which are presumably necessary for hepatic regeneration. The results of the present study suggest that raising the blood levels of the BCAA after hepatectomy reduces the rate of accumulation of many amino acids in blood, either by stimulating protein synthesis, by inhibiting protein ~breakdown or both. In the present study, by 18 hrs after hepatectom~, rats receiving only glucose infusion had significantly higher plasma concentrations of most amino acids as compared with either control group; diffe=ences in concentrations of 3 to 5 times the control levels were common. In b~a~ns of glucose-treated, hepatectomized rats, the accumulation of GLN, ALA and the LNAA (except the BCAA) was particularly notable; the concentrations Of MET, PHE, HIS, and TYR were higher in brain than in plasma both at 6 and 18 hrs. These effects of hepatectomy confirm previous reports (8). In hepatectomized rats receiving glucose and BCAA, the plasma concentrations of BCAA were 5- to 10-fold higher than in hepatectomized rats receiving only glucose. Infusion of BCAA apparently greatly stimulated the release of ALA and GLN from peripheral tissues. Leueine has been reported to stimulate the release of ALA and GLN from isolated perfused rat hind-quarters and from the human forearm (15,16). The release of GLN and ALA from rat muscle has been reported to be stimulated by the BCAA (17). Normally, the rat intestine takes up GLN from the blood and releases ALA (18), a substrate for gluconeogenesis in the liver. The concentrations of LYS, MET, PHE, THR, HIS, TYR, and TRP were much lower in plasma of hepatectomized rats receiving BCAA and glucose than in rats receiving glucose alone, suggesting an effect of BCAA on protein metabolism. The BCAA, but not other amino acids, stimulate protein synthesis and inhibit breakdown of protein in incubated or perfused skeletal muscle (19). The present, results do not identify which tissue(s) or which aspect of protein metabolism (synthesis or breakdown) was specifically affected by infusion of the BCAA; however, it is likely that the BCAA both stimulated protein synthesis and inhibited protein breakdown in skeletal muscle. Thus BCAA infusion may be a useful adjunct to other therapy aimed at clearing the blood of potential toxins. BCAA infusion greatly reduced the accumulation of THR, MET, TYR, PHE, and HIS in brain after hepatectomy (Table II). However, the difference between the concentrations of those five amino acids in the two hepatectomized groups was much greater in brain than in plasma. This difference suggests that high concentrations of BCAA in blood reduced the uptake of other LNAA by brain by competing for transport across the blood-brain barrier. High concentrations of PHE and TYR in brain have been suggested to alter catecholamine synthesis (7). At 18 hrs after hepatectomy, regardless of treatment, NE levels were reduced while DA levels were unchanged. Infusion of BCAA to hepatectomized rats resulted in levels of PHE and TYR similar to those obtained in the controls with portacaval shunt, in which brain NE was near normal levels. Thus~ high concentrations o~ PHE and TYR do not appear to be the primary cause of NE depletion after hepatectomy. The present results are consistent with previous studies in hepatectomized rats and suggest an inhibition of the enzymatic conversion of DA to NE (9). Our results suggest that such inhibition, if it occurs, is not mediated

1368

Hepatectomy and Amino Acids

Vol. 30, No. 16, 1982

by PHE, TYR or their amine metabolites. An unanticipated inverse correlation was observed between lOgl0 of the brain alanine concentration and brain NE levels. This relationship was also found when BCAA were added to the infusion. Since it is unlikely that the biochemical pathways involving the synthesis of alanine are closely related to the pathway leading to NE synthesis, the observed correlation suggests that hepatectomy results in some unknown disturbance which independently affects the metabolism of alanine and NE in brain. Acknowledgements Supported in part by USPHS Grant AM-25638 and by Lipid-AtherosclerosisNutrition Training Grant USPHS HL-07460. References i.

H.M. ROSEN, N. YOSHIMURA, J.M. HODGMAN and J.E. FISCHER, Gastroenterology 72 483-487 (1977). 2. E.--V. FLOCK, M.A. BLOCK, J.H. GRINDLAY, F.C. MAN and O.L. BOLLMAN, J. Biol. Chem. 198 427-437 (1952). 3. L.L. MILLER, Amino Acid Pools, J.T. HOLDEN, ed., 708-721, Elsevier, A m s t e ~ dam (1961). 4. C.O. RECORD, B. BUXTON, R.A. CHASE, G. CURZON, I.M. MURRAY-LYON and R. WILLIAMS, Eur. J. Clin. Invest. 6 387-394 (1976). 5. E.V. FLOCK, G.M. TYCE and C.A. OWEN, J. Neurochem. 13 1389-1406 (1966). 6. W.H. OLDENDORF, Am. J. Physiol. 221 1629-1639 (1971). 7. J.E. FISCHER and R.J. BALDESSARINI, Progress in Liver Disease, Vol. 5, p. 363-397, Grune and Stratton, New York (1976). 8. G.M. TYCE, E.V. FLOCK and C.A. OWEN, Biochem. Pharmacol. 1 6 979-992 (1967). 9. G.M. TYCE and C.A. OWEN, Life Sci. 2 2 781-785 (1978). i0. B.A. FARAJ, V.M. CAMP, J.D. ANSLEY, J. SCOTT, F.M. ALl and E.J. MALVEAUX, J. Clin. Invest. 67 395-402 (1981). ii. T. HOLMIN, C.O. AGARD, G. ALINDER and P. HERLIN, Amino Acids, Ammonia, and Energy Metabolism in Hepatic Failure and Neoplastic Diseases, p. 91-94, Verlag Gerhard Witzstrock (1981). 12. E. GAZZARD, M.J. WESTON, I.M. MURRAY-LYON, H. FLAX, C.O. RECORD, B. PORTMANN, P.G. LANGBY, E.H. DUNLOP, P.J. MELLON and M.B. WARD, Lancet 1301-1307 (1974). 13. R.A. CHASE, M. DAVIES, P.N. TREWBY, D.B.A. SILK and R. WILLIAMS, Gastroenterology 75 1033-1040 (1978). 14. M.L. DELORME, J. DENIS, B. NORDLINGER, M. BOSCHAF and P. OPOLON. J. Neurochem. 36 1058-1066 (1981). 15. N.B. RUDERMA-N and M. BERGER, J. Biol. Chem. 249(17) 5500-5506 (1974). 16. T.T. AOKI, M.F. BRENNAN, G.F. FITZPATRICK and D.C. KNIGHT, J. Clin. Invest. 68 1522-1528 (1981). 17. A.--J. GARBER, J.E. KARL and D.M. KIPNIS, J. Biol. Chem. 251(3) 836-843 (1976). 18. H.G. WINDMUELLER and A.E. SPAETH. J. Biol. Chem. 249(16) 5070-5079 (1974). 19. R.M. FULKS, J.B. Ll and A.L. GOLDBERG, J. Biol. Chem. 250 290-298 (1975).