- Email: [email protected]
INSULIN, PLASMA AMINOACID IMBALANCE, AND HEPATIC COMA
722
tinues, our findings clearly indicate the value mithramycin therapy in osteolytic myelomatosis.
of
We are grateful
to Dr J. R. Nassim for his early care of Sister M. C. Dunsdon and the nurses of the metabolic ward, Miss Ann Hilb, and Miss Elizabeth Stainthorpe for their help throughout. Patients were referred after diagnosis by Mr A. W. F. Lettin and Mr E. L. Trickey. case
2; and
to
REFERENCES 1. Medical Research Council. Br. med. J. 1971, i, 641. 2. Cline, M. J., Berlin, N. I. Am. J. Med. 1962, 33, 510. 3. Bergsagel, D. E. Cancer, 1972, 30, 1588. 4. Hoogstraten, G. Med. Clin. N. Am. 1973, 57, 1321. 5. Malpas, J. S. Br. med. J. 1974, iv, 520. 6. Cuttner, J. Proc. Am. Soc. clin. Oncology, 1969. 7. Watson, L. Br. med. J. 1962, ii, 150. 8. Parsons, V., Barim, M., Self, M. ibid. 1967, i, 474. 9. Singer, F. R., Neer, R. M., Murray, T. M., Keutmann, H. T., Deftos, L. J., Potts, J. T. New Engl. J. Med. 1970, 283, 634. 10. Perlia, C. P., Jubish, J., Wolter, J., Edelberg, D., Dederick, M. M., Taylor, S. G. Cancer, 1970, 25, 389. 11. Godfrey, T. D. Calif. Med. 1971, 155, 1. 12. Condon, J. R., Reith, S. B. M., Nassim, J. R., Millard, F. J. C., Hilb, A., Stainthorpe, E. M. Br. med. J. 1971, i, 421. 13. Russell, A. S., Lentle, B. C. Can. med. Ass. J. 1974, 110, 397. 14. Minkin, C. Calcif. Tissue Res. 1973, 13, 249. 15. Munday, G. R., Raisz, L. G., Cooper, R. A., Schechter, G. P., Salmon, S. E. New Engl. J. Med. 1974, 291, 1041. 16. Slayton, R. E., Shnider, B. I., Elias, E., Horton, J., Perlia, C. P. Clin. Pharmac. Ther. 1971, 12, 833. 17. Kennedy, B. J. Am. J. Med. 1970, 49, 494. 18. Monto, R. S., Talley, T. W., Caldwell, M. J., Levin, W. C., Guest, M. M. Cancer Res. 1969, 29, 697.
Hypothesis INSULIN, PLASMA AMINOACID IMBALANCE, AND HEPATIC COMA
JOHN D. FERNSTROM J. WURTMAN of Physiological Chemistry and
HAMISH N. MUNRO RICHARD
Laboratories Neuroendocrine Regulation, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U.S.A.
produce consistent changes in the electroencephalogram. This view has been replaced by suggestions that hepatic coma is due to other nitrogenous compounds formed by intestinal organisms and normally removed by the liver, particularly amines. Fischer and Baldessarini2 have suggested that amines (e.g., tyramine) produced by bacterial action in the gut, and entering the general circulation in cases of hepatic failure, may become " false neurotransmitters " in the brain, displacing the normal adrenergic transmitters dopamine and norepinephrine (noradrenaline). Using rats with ligated hepatic blood-supply, Fischer and his colleagues3 have demonstrated depletion of brain norepinephrine accompanied by an increase in the brain level of octopamine, a hydroxylated derivative of tyramine. Hepatic failure is associated with considerable distortion in the plasma concentrations of free aminoacids. Tyrosine, phenylalanine, and methionine levels disease failed
to
elevated and those of the branched-chain aminoacids are lowered. This pattern has considerable significance for cerebral function. Wehave found that entry into the brain of tryptophan, the precursor of serotonin, and probably the uptake of other aminoacid precursors of neurotransmitters, is regulated by the concentrations of other neutral aminoacids competing for cerebral uptake, notably the branchedchain aminoacids. Without any change in plasmatryptophan level, a decrease in branched-chain aminoacid levels increases entries of tryptophan into brain. The, distorted plasma aminoacid pattern observed in cirrhosis thus favours increased cerebral uptake of tryptophan with consequent elevation in serotonin level. Since increased serotonin is associated with sleepy it may therefore contribute to the coma of hepatic failure. This thesis is supported by the reversal of experimental hepatic coma following administration of branched-chain aminoacids, as Fischer has demonstrated.6 The genesis of the imbalance of plasma aminoacid concentrations in cirrhosis due to hyperinsulinxmia and its effects on neurotransmitter metabolism are described in the following are
hypothesis. It is proposed that severe impairment Summary of liver function in cirrhosis or portacaval shunt results in unrestricted entry of insulin into the peripheral circulation. The ensuing high level of insulin promotes excessive removal of the branched-chain aminoacids by muscle, thereby lowering the plasma levels of these aminoacids. In consequence, the competitive action of the branchedchain aminoacids on the entry of tryptophan into the brain is reduced, more tryptophan enters the brain, and serotonin is synthesised in excess, thus facilitating
hepatic
coma. INTRODUCTION
HEPATIC coma continues to be a metabolic phenowithout a satisfying explanation. The early theory of intoxication, which held that ammonia was formed in the gut by bacterial action and accumulated in the peripheral blood because of the lack of hepatic removal, now seems inadequate because of studies such as those of Cohn and Castell in which acute hyperammonsemia induced in patients with liver menon
THE HYPOTHESIS
Because of its strategic position astride the portal vein, the liver exercises an important regulatory function
over the passage of many substances into the circulation. general First, it is the principal or exclusive site for the catabolism of the aminoacids
methionine, lysine, threonine, phenylalanine, tyrosine, and tryptophan, whereas the branched-chain aminoacids leucine, isoleucine, and valine mostly pass through the liver and are largely degraded in muscle. Second, amines formed in the gut from aminoacids by bacterial action are removed in the liver by monoamine-oxidase action. Third, some hormones such as insulin are partly inactivated by the liver. As suggested by others, hepatic coma is partly due the failure of the liver to remove amines from the portal blood; these amines may become converted in the brain to derivatives that interfere with the function of monoaminergic neurons. In addition, however, the plasma level of insulin is high in fasting cirrhotics and rises considerably after a meal. This to
723
hyperinsulinaemia
can
be
expected
to cause
excessive
formation of serotonin for the following reasons. As pointed out above, catabolism of branched-chain aminoacids occurs mainly in muscle, into which aminoacid entry is controlled by insulin. A sustained high level of insulin in the plasma would therefore result in excessive removal of branched-chain aminoacids by muscle, thus accounting for the low plasma concentrations of leucine, isoleucine, and valine observed in cirrhotics. The entry of neutral aminoacids into the brain is competitive, branched-chain aminoacids exerting an especially large effect on entry of other neutral aminoacids such as tryptophan. Consequently, the reduction in the plasma levels of the branched-chain aminoacids found in cirrhotic subjects will result in excessive uptake of tryptophan by the brain. Since the level of non-protein tryptophan in the brain determines the rate of serotonin formation, more of this neurotransmitter will be formed in the brain of the cirrhotic patient. High levels of brain serotonin are associated with sleep, and in the cirrhotic will precipitate coma. Administration of branched-chain aminoacids to patients in hepatic coma would therefore be expected to alleviate the coma by reducing brain serotonin levels. DISCUSSION
The above observations.
hypothesis is supported by
the
following
(1) The main or exclusive site of catabolism for most of the essential aminoacids (lysine, methionine, phenylalanine, threonine, and tryptophan) is the liver, whereas the branched-chain aminoacids are chiefly catabolised in muscle.7.8 The liver thus monitors entry of the first group into the general circulation, whereas the plasma levels of branched-chain aminoacids are controlled by metabolism in the peripheral tissues. This explains why plasma concentrations of branched-chain aminoacids are especially elevated after a protein-rich meal.8 (2) Secretion of insulin controls removal by muscle of plasma aminoacids, notably the branched-chain aminoacids. The levels of the latter are markedly reduced following ingestion of carbohydrate to stimulate insulin secretion 9-12 or after insulin injection.13,14 and are elevated under conditions associated with lack of insulin secretion or insulin resistance.12,lS.I6 The metabolism of the branched-chain aminoacids is thus considerably affected by insulin, and prolonged excessive levels of insulin might be expected to cause their concentrations in plasma to remain unusually low. (3) About 40-50% of the insulin secreted into the portal circulation is normally extracted during a single transit through the liver, even when insulin secretion has been stimulated several-fold by glucose administration.17.I8 Thus it is not surprising that cirrhosis is commonly associated with elevated concentrations of insulin in the peripheral circulation, both in the fasting state and in response to carbohydrate administration,l9,20 reflecting the impaired extraction by the damaged liver. (4) In cirrhotic subjects, a distinctive pattern of plasma aminoacid concentrations has been observed.21-23 Usually, there are considerable elevations in methionine, phenylalanine, and tyrosine levels, while tryptophan concentration remains within normal limits and the levels of the branchedchain aminoacids are markedly reduced. After a protein meal the branched-chain aminoacids are unusually rapidly cleared from the blood and their metabolism in muscle appears to be accelerated.24 These observations on cirrhotic subjects are consistent with loss of hepatic control over
certain aminoacids (methionine, tyrosine, phenylalanine), combined with excessive removal of the branched-chain aminoacids by muscle due to the hyperinsulinaemia. (5) The distorted pattern of essential aminoacids in cirrhosis will result in changes in aminoacid availability to the brain. Entry of aminoacids into the brain is dependent on several carriers, each shared by groups of aminoacids.25 Tryptophan, tyrosine, phenylalanine, and the branched-chain aminoacids utilise a common transport Fernstrom and Wurtman 2s have shown that system. of entry tryptophan into the brain varies directly with total plasma tryptophan content and inversely with the concentrations of the other competing aminoacids, particularly the branched-chain ones. This view conflicts with the theory 27,28 that tryptophan uptake by the brain depends solely on the plasma concentrations of " free " tryptophan (i.e., not bound to albumin). Tryptophan is unique among plasma aminoacids in being transported largely bound to albumin. While the unbound form is almost certainly the immediate scource of tryptophan available to cells, extensive changes in the proportion of tryptophan bound to albumin have been induced by us by manipulation of the diet of the rat without causing corresponding changes in the brain levels of free tryptophan.29 Instead, the ratio of tryptophan to other neutral aminoacids in the plasma is the primary determinant of tryptophan entry into brain.26 (6) Since brain serotonin formation is regulated by brain tryptophan level,3O changes in the plasma concentrations of aminoacids competing with tryptophan for brain uptake will affect serotonin synthesis rate. In cirrhosis, the reduction in branched-chain aminoacid levels should thus increase passage of tryptophan into the brain and elevate serotonin levels. This conclusion is supported by the findingthat brain serotonin levels are increased in animals in coma due to experimental porta-caval shunt, and fall in animals recovering from coma following the injections of branched-chain aminoacids, without a change in plasma tryptophan level. It is significant that serotonincontaining neurons have been associated with sleep,31 and the central catecholamine-containing neurons with arousal.32 (7) Finally, the high plasma phenylalanine levels observed in cirrhotics may raise brain phenylalanine levels sufficiently to inhibit the activity of neuronal tyrosine hydroxylase,33 and thus suppress the synthesis of the brain neurotransmitters dopamine and norepinephrine which would contribute to the low levels of brain catecholamines observed in hepatic insufficiency.s CONCLUSION
The above evidence suggests that the inability of the diseased liver to metabolise the aromatic aminoacids and insulin, together with the resulting excessive uptake of branched-chain aminoacids into skeletal muscle, is the cause of the abnormal plasma aminoacid patterns observed in cirrhotics. These patterns, by changing the uptake of tryptophan and phenylalanine into the brain, could be predicted to accelerate brain serotonin synthesis and depress the formation of catecholamines; Other neurochemical consequences of hepatic disease (e.g., impaired metabolism of tyramine formed in the gut, leading to storage in brain of its beta-hydroxylated derivative, octopamine) might be expected to enhance the vulnerability of the brain to the neurotransmitter imbalances resulting from this mechanism. The administration of insulin antagonists, branched-chain aminoacids, or catecholamine precursors might thus be expected to make some contribution towards improving the clinical state of cirrhotics with hepatic encephalopathy. We wish to
acknowledge the stimulus provided by discus-
724 sions with Dr Josef E. Fischer, whose clinical and studies provide some of the evidence cited here. expressed in our article are not necessarily his.
experimental The views
REFERENCES
Cohn, R., Castell, D. O. J. Lab. clin. Med. 1966, 68, 195. Fischer, J. E., Baldessarini, R. Lancet, 1971, ii, 75. Dodsworth, J. M., James, J. H., Cummings, M. C., Fischer, J. E. Surgery, St. Louis, 1974, 75, 811. 4. Fernstrom, J. D., Wurtman, R. J. Science, 1972, 178, 414. 5. Jouvet, M., Pujol, J. F. Adv. Biochem. Psychopharm. 1974, 11, 1. 2. 3.
disintegration of mural cells is due to the same hæmodynamic stresses which induce degenerative changes in the vascular connective tissues, and that the lipid accumulation within the vesicular debris is a non-specific change common to necrotic tissue and cellular debris which have not undergone resolution or phagocytosis.
199. 6. 7. 8. 9.
INTRODUCTION
Fischer, J. E., Aguirre, A., Funovicks, J. M. Surgery, St. Louis (in the press). Miller, L. L. in Amino Acid Pools (edited by J. T. Holden); p. 708. Amsterdam, 1962. Elwyn, D. H. Mammalian Protein Metabolism, 1970, 4, 523. Munro, H. N., Thomson, W. S. T. Metab. clin. Exp. 1953, 2, 354.
Crofford, O. B., Felts, P. W., Lacy, W. W. Proc. Soc. exp. Biol. Med. 1964, 117, 11. 11. Zinneman, H. H., Nuttal, F. Q., Goetz, F. C. Diabetes, 1966, 15, 5. 12. Felig, P., Marliss, E., Cahill, G. F., New Engl. J. Med. 1969, 281, 10.
811. 13. Lotspeich, W. D., J. biol. Chem. 1949, 179, 175. 14. Pozefsky, T., Felig, P., Tobin, J. D., Soeldner, J. S., Cahill, G. F., Jr. J. clin. Invest. 1969, 48, 2278. 15. Swendseid, M. E., Umezawa, C. Y., Drenick, E. J. Am. J. clin. Nutr. 1969, 22, 740. 16. Carlsten, A., Hallgren, B., Jagenburg, R., Svanborg, A., Werkö, L. Acta med. scand. 1967, 181, 195, 199. 17. Kaden, M., Harding, P., Field, J. B. J. clin. Invest. 1973, 52, 2016. 18. Krass, E., Bittner, R., Meves, M., Beger, H. G. Klin. Wschr. 1974, 52, 404. 19. Creutzfeldt, W., Frerichs, H., Sickinger, K. Prog. Liver Dis. 1970, 20.
21. 22.
23. 24.
3, 371. Marco, J., Diego, J., Villanueva, M. L., Diaz-Fierros, M., Valverde, I., Segovia, J. M. New Engl. J. Med. 1973, 289, 1107. Ning, M., Lowenstein, L. M., Davidson, C. S. J. Lab. clin. Med. 1967, 70, 554. Fischer, J. E., Yoshimura, N., Aguirre, A., James, J. H., Cummings, M. G., Abel, R. M., Deindorfer, F. Am. J. Surg. 1974, 127, 40. Sherwin, R., Joshi, P., Hendler, R., Felig, P., Conn, H. O. New Engl. J. Med. 1974, 290, 239. Iob, V., Coon, W. W., Sloan, M. J. surg. Res. 1966, 6, 233; 1967, 7, 41.
25. Blasberg, R., Lajtha, A. Archs Biochem. Biophys. 1965, 112, 365. 26. Fernstrom, J. D., Wurtman, R. J. Science, 1972, 178, 414. 27. Knott, P. J., Curzon, G. Nature, 1972, 239, 452. 28. Tagliamonte, A., Biggio, G., Vargiu, L., Gessa, G. L. Life Sci. 1973, 12 (part II), 277. 29. Madras, B. K., Cohen, E. L., Messing, R., Munro, H. N., Wurtman, R. J. Metabolism, 1974, 23, 1107. 30. Fernstrom, J. D., Wurtman, R. J. Sci. Am. 1974, 230, 84. 31. Wyatt, R. J., Neff, N. H., Vaughn, T., Franz, T., Omaya, A. 32. 33.
Adv. Biochem. Psychopharm. 1974, 11, 193. Reis, D. J. Ass. Res. nerv. ment. Dis. 1972, 50, 266. Wurtman, R. J., Larin, F., Mostafapour, S., Fernstrom, J. D. Science, 1974, 185, 183.
THE correlation of premature severe atherosclerosis with hypercholesterolaemic states (e.g., diabetes mellitus, hypothyroidism and familial hyperlipidxmia) led some 60 years ago to the observation that chronic administration of egg-yolk or cholesterol induced lipid-containing lesions in rabbit arteries and veins. Since then the lipid hypothesis has dominated nearly all research into the aetiology and pathogenesis of atherosclerosis. The theory has met with such general acceptance that the accumulation of histologically demonstrable lipid in the blood-vessel wall, although only one of the manifestations of atherosclerosis, has come to be regarded as the sine qua non of atherosclerosis, and investigations have been focused on the source, nature, and fate of this lipid. The accumulation of lipid in the blood-vessel wall has been attributed variously to excessive quantities in the serum, to an augmented permeability of the vessel wall especially of the endothelium, to the impenetrability of the internal elastic lamina, and also to a local metabolic deficiency of the cellular constituents of the wall in dealing with lipid infiltration from the lumen. However, some maintain that atherosclerosis is a systemic metabolic disorder.1 It is disturbing that most workers have assumed that the lipid hypothesis is valid and that only the mechanism underlying the lipid deposition and the therapy of atherosclerosis require clarification. Indeed, Dock2 declared that infiltration of the blood-vessel wall with lipid from hyperlipidaemic serum had been unequivocally confirmed as the cause of atherosclerosis. However, although there is some evidence to support the lipid hypothesis, the following serious and fundamental inconsistencies detract from its
validity: THE ROLE OF LIPID IN THE PATHOGENESIS OF ATHEROSCLEROSIS W. E. STEHBENS
Department of Pathology, Wellington Clinical School, and the Wellington Cancer and Medical Research Institute, Wellington 2, New Zealand
experimental model of atherosclerosis in sheep veins identical to the human disease indicates (i) that ingestion of an atherogenic diet is not a prerequisite in atherosclerosis and (ii) that hæmodynamic stress must be the dominant ætiological factor in atherosclerosis. Ultrastructural studies reveal that the early lipid deposition in spontaneous human atherosclerosis and in hæmodynamically induced atherosclerosis is related to the transformation of extracellular vesicular debris into closely packed membranous profiles with electrontranslucent centres. It is postulated that the vesicular Summary
An
In diet-induced lesions there are distinct, unreconcilable, histological differences from human atherosclerosis. Moreover, even in the same species, diet-induced lesions differ histologically from spontaneous lipid-containing 1.
lesions.
,
2. In diet-induced disease there are attendant extravascular lesions which are not found in the spontaneous disease but which have the hallmarks of a lipid-storage disease. 3. In diet-induced disease there are no complications such as intimal tears, ulceration, and thrombosis, all of which are integral features of the human disease.
Epidemiologically, total serum-cholesterol is regarded as the most useful criterion in determining the risk of coronary heart-disease,3 and in national or community studies levels of fat intake, blood-cholesterol levels, and deaths from ischaemic heart-disease are said to be associated. However, the correlation between fat consumption and cholesterol levels in individualsand between the level of the serum-
tinues, our findings clearly indicate the value mithramycin therapy in osteolytic myelomatosis.
of
We are grateful
to Dr J. R. Nassim for his early care of Sister M. C. Dunsdon and the nurses of the metabolic ward, Miss Ann Hilb, and Miss Elizabeth Stainthorpe for their help throughout. Patients were referred after diagnosis by Mr A. W. F. Lettin and Mr E. L. Trickey. case
2; and
to
REFERENCES 1. Medical Research Council. Br. med. J. 1971, i, 641. 2. Cline, M. J., Berlin, N. I. Am. J. Med. 1962, 33, 510. 3. Bergsagel, D. E. Cancer, 1972, 30, 1588. 4. Hoogstraten, G. Med. Clin. N. Am. 1973, 57, 1321. 5. Malpas, J. S. Br. med. J. 1974, iv, 520. 6. Cuttner, J. Proc. Am. Soc. clin. Oncology, 1969. 7. Watson, L. Br. med. J. 1962, ii, 150. 8. Parsons, V., Barim, M., Self, M. ibid. 1967, i, 474. 9. Singer, F. R., Neer, R. M., Murray, T. M., Keutmann, H. T., Deftos, L. J., Potts, J. T. New Engl. J. Med. 1970, 283, 634. 10. Perlia, C. P., Jubish, J., Wolter, J., Edelberg, D., Dederick, M. M., Taylor, S. G. Cancer, 1970, 25, 389. 11. Godfrey, T. D. Calif. Med. 1971, 155, 1. 12. Condon, J. R., Reith, S. B. M., Nassim, J. R., Millard, F. J. C., Hilb, A., Stainthorpe, E. M. Br. med. J. 1971, i, 421. 13. Russell, A. S., Lentle, B. C. Can. med. Ass. J. 1974, 110, 397. 14. Minkin, C. Calcif. Tissue Res. 1973, 13, 249. 15. Munday, G. R., Raisz, L. G., Cooper, R. A., Schechter, G. P., Salmon, S. E. New Engl. J. Med. 1974, 291, 1041. 16. Slayton, R. E., Shnider, B. I., Elias, E., Horton, J., Perlia, C. P. Clin. Pharmac. Ther. 1971, 12, 833. 17. Kennedy, B. J. Am. J. Med. 1970, 49, 494. 18. Monto, R. S., Talley, T. W., Caldwell, M. J., Levin, W. C., Guest, M. M. Cancer Res. 1969, 29, 697.
Hypothesis INSULIN, PLASMA AMINOACID IMBALANCE, AND HEPATIC COMA
JOHN D. FERNSTROM J. WURTMAN of Physiological Chemistry and
HAMISH N. MUNRO RICHARD
Laboratories Neuroendocrine Regulation, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U.S.A.
produce consistent changes in the electroencephalogram. This view has been replaced by suggestions that hepatic coma is due to other nitrogenous compounds formed by intestinal organisms and normally removed by the liver, particularly amines. Fischer and Baldessarini2 have suggested that amines (e.g., tyramine) produced by bacterial action in the gut, and entering the general circulation in cases of hepatic failure, may become " false neurotransmitters " in the brain, displacing the normal adrenergic transmitters dopamine and norepinephrine (noradrenaline). Using rats with ligated hepatic blood-supply, Fischer and his colleagues3 have demonstrated depletion of brain norepinephrine accompanied by an increase in the brain level of octopamine, a hydroxylated derivative of tyramine. Hepatic failure is associated with considerable distortion in the plasma concentrations of free aminoacids. Tyrosine, phenylalanine, and methionine levels disease failed
to
elevated and those of the branched-chain aminoacids are lowered. This pattern has considerable significance for cerebral function. Wehave found that entry into the brain of tryptophan, the precursor of serotonin, and probably the uptake of other aminoacid precursors of neurotransmitters, is regulated by the concentrations of other neutral aminoacids competing for cerebral uptake, notably the branchedchain aminoacids. Without any change in plasmatryptophan level, a decrease in branched-chain aminoacid levels increases entries of tryptophan into brain. The, distorted plasma aminoacid pattern observed in cirrhosis thus favours increased cerebral uptake of tryptophan with consequent elevation in serotonin level. Since increased serotonin is associated with sleepy it may therefore contribute to the coma of hepatic failure. This thesis is supported by the reversal of experimental hepatic coma following administration of branched-chain aminoacids, as Fischer has demonstrated.6 The genesis of the imbalance of plasma aminoacid concentrations in cirrhosis due to hyperinsulinxmia and its effects on neurotransmitter metabolism are described in the following are
hypothesis. It is proposed that severe impairment Summary of liver function in cirrhosis or portacaval shunt results in unrestricted entry of insulin into the peripheral circulation. The ensuing high level of insulin promotes excessive removal of the branched-chain aminoacids by muscle, thereby lowering the plasma levels of these aminoacids. In consequence, the competitive action of the branchedchain aminoacids on the entry of tryptophan into the brain is reduced, more tryptophan enters the brain, and serotonin is synthesised in excess, thus facilitating
hepatic
coma. INTRODUCTION
HEPATIC coma continues to be a metabolic phenowithout a satisfying explanation. The early theory of intoxication, which held that ammonia was formed in the gut by bacterial action and accumulated in the peripheral blood because of the lack of hepatic removal, now seems inadequate because of studies such as those of Cohn and Castell in which acute hyperammonsemia induced in patients with liver menon
THE HYPOTHESIS
Because of its strategic position astride the portal vein, the liver exercises an important regulatory function
over the passage of many substances into the circulation. general First, it is the principal or exclusive site for the catabolism of the aminoacids
methionine, lysine, threonine, phenylalanine, tyrosine, and tryptophan, whereas the branched-chain aminoacids leucine, isoleucine, and valine mostly pass through the liver and are largely degraded in muscle. Second, amines formed in the gut from aminoacids by bacterial action are removed in the liver by monoamine-oxidase action. Third, some hormones such as insulin are partly inactivated by the liver. As suggested by others, hepatic coma is partly due the failure of the liver to remove amines from the portal blood; these amines may become converted in the brain to derivatives that interfere with the function of monoaminergic neurons. In addition, however, the plasma level of insulin is high in fasting cirrhotics and rises considerably after a meal. This to
723
hyperinsulinaemia
can
be
expected
to cause
excessive
formation of serotonin for the following reasons. As pointed out above, catabolism of branched-chain aminoacids occurs mainly in muscle, into which aminoacid entry is controlled by insulin. A sustained high level of insulin in the plasma would therefore result in excessive removal of branched-chain aminoacids by muscle, thus accounting for the low plasma concentrations of leucine, isoleucine, and valine observed in cirrhotics. The entry of neutral aminoacids into the brain is competitive, branched-chain aminoacids exerting an especially large effect on entry of other neutral aminoacids such as tryptophan. Consequently, the reduction in the plasma levels of the branched-chain aminoacids found in cirrhotic subjects will result in excessive uptake of tryptophan by the brain. Since the level of non-protein tryptophan in the brain determines the rate of serotonin formation, more of this neurotransmitter will be formed in the brain of the cirrhotic patient. High levels of brain serotonin are associated with sleep, and in the cirrhotic will precipitate coma. Administration of branched-chain aminoacids to patients in hepatic coma would therefore be expected to alleviate the coma by reducing brain serotonin levels. DISCUSSION
The above observations.
hypothesis is supported by
the
following
(1) The main or exclusive site of catabolism for most of the essential aminoacids (lysine, methionine, phenylalanine, threonine, and tryptophan) is the liver, whereas the branched-chain aminoacids are chiefly catabolised in muscle.7.8 The liver thus monitors entry of the first group into the general circulation, whereas the plasma levels of branched-chain aminoacids are controlled by metabolism in the peripheral tissues. This explains why plasma concentrations of branched-chain aminoacids are especially elevated after a protein-rich meal.8 (2) Secretion of insulin controls removal by muscle of plasma aminoacids, notably the branched-chain aminoacids. The levels of the latter are markedly reduced following ingestion of carbohydrate to stimulate insulin secretion 9-12 or after insulin injection.13,14 and are elevated under conditions associated with lack of insulin secretion or insulin resistance.12,lS.I6 The metabolism of the branched-chain aminoacids is thus considerably affected by insulin, and prolonged excessive levels of insulin might be expected to cause their concentrations in plasma to remain unusually low. (3) About 40-50% of the insulin secreted into the portal circulation is normally extracted during a single transit through the liver, even when insulin secretion has been stimulated several-fold by glucose administration.17.I8 Thus it is not surprising that cirrhosis is commonly associated with elevated concentrations of insulin in the peripheral circulation, both in the fasting state and in response to carbohydrate administration,l9,20 reflecting the impaired extraction by the damaged liver. (4) In cirrhotic subjects, a distinctive pattern of plasma aminoacid concentrations has been observed.21-23 Usually, there are considerable elevations in methionine, phenylalanine, and tyrosine levels, while tryptophan concentration remains within normal limits and the levels of the branchedchain aminoacids are markedly reduced. After a protein meal the branched-chain aminoacids are unusually rapidly cleared from the blood and their metabolism in muscle appears to be accelerated.24 These observations on cirrhotic subjects are consistent with loss of hepatic control over
certain aminoacids (methionine, tyrosine, phenylalanine), combined with excessive removal of the branched-chain aminoacids by muscle due to the hyperinsulinaemia. (5) The distorted pattern of essential aminoacids in cirrhosis will result in changes in aminoacid availability to the brain. Entry of aminoacids into the brain is dependent on several carriers, each shared by groups of aminoacids.25 Tryptophan, tyrosine, phenylalanine, and the branched-chain aminoacids utilise a common transport Fernstrom and Wurtman 2s have shown that system. of entry tryptophan into the brain varies directly with total plasma tryptophan content and inversely with the concentrations of the other competing aminoacids, particularly the branched-chain ones. This view conflicts with the theory 27,28 that tryptophan uptake by the brain depends solely on the plasma concentrations of " free " tryptophan (i.e., not bound to albumin). Tryptophan is unique among plasma aminoacids in being transported largely bound to albumin. While the unbound form is almost certainly the immediate scource of tryptophan available to cells, extensive changes in the proportion of tryptophan bound to albumin have been induced by us by manipulation of the diet of the rat without causing corresponding changes in the brain levels of free tryptophan.29 Instead, the ratio of tryptophan to other neutral aminoacids in the plasma is the primary determinant of tryptophan entry into brain.26 (6) Since brain serotonin formation is regulated by brain tryptophan level,3O changes in the plasma concentrations of aminoacids competing with tryptophan for brain uptake will affect serotonin synthesis rate. In cirrhosis, the reduction in branched-chain aminoacid levels should thus increase passage of tryptophan into the brain and elevate serotonin levels. This conclusion is supported by the findingthat brain serotonin levels are increased in animals in coma due to experimental porta-caval shunt, and fall in animals recovering from coma following the injections of branched-chain aminoacids, without a change in plasma tryptophan level. It is significant that serotonincontaining neurons have been associated with sleep,31 and the central catecholamine-containing neurons with arousal.32 (7) Finally, the high plasma phenylalanine levels observed in cirrhotics may raise brain phenylalanine levels sufficiently to inhibit the activity of neuronal tyrosine hydroxylase,33 and thus suppress the synthesis of the brain neurotransmitters dopamine and norepinephrine which would contribute to the low levels of brain catecholamines observed in hepatic insufficiency.s CONCLUSION
The above evidence suggests that the inability of the diseased liver to metabolise the aromatic aminoacids and insulin, together with the resulting excessive uptake of branched-chain aminoacids into skeletal muscle, is the cause of the abnormal plasma aminoacid patterns observed in cirrhotics. These patterns, by changing the uptake of tryptophan and phenylalanine into the brain, could be predicted to accelerate brain serotonin synthesis and depress the formation of catecholamines; Other neurochemical consequences of hepatic disease (e.g., impaired metabolism of tyramine formed in the gut, leading to storage in brain of its beta-hydroxylated derivative, octopamine) might be expected to enhance the vulnerability of the brain to the neurotransmitter imbalances resulting from this mechanism. The administration of insulin antagonists, branched-chain aminoacids, or catecholamine precursors might thus be expected to make some contribution towards improving the clinical state of cirrhotics with hepatic encephalopathy. We wish to
acknowledge the stimulus provided by discus-
724 sions with Dr Josef E. Fischer, whose clinical and studies provide some of the evidence cited here. expressed in our article are not necessarily his.
experimental The views
REFERENCES
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disintegration of mural cells is due to the same hæmodynamic stresses which induce degenerative changes in the vascular connective tissues, and that the lipid accumulation within the vesicular debris is a non-specific change common to necrotic tissue and cellular debris which have not undergone resolution or phagocytosis.
199. 6. 7. 8. 9.
INTRODUCTION
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THE correlation of premature severe atherosclerosis with hypercholesterolaemic states (e.g., diabetes mellitus, hypothyroidism and familial hyperlipidxmia) led some 60 years ago to the observation that chronic administration of egg-yolk or cholesterol induced lipid-containing lesions in rabbit arteries and veins. Since then the lipid hypothesis has dominated nearly all research into the aetiology and pathogenesis of atherosclerosis. The theory has met with such general acceptance that the accumulation of histologically demonstrable lipid in the blood-vessel wall, although only one of the manifestations of atherosclerosis, has come to be regarded as the sine qua non of atherosclerosis, and investigations have been focused on the source, nature, and fate of this lipid. The accumulation of lipid in the blood-vessel wall has been attributed variously to excessive quantities in the serum, to an augmented permeability of the vessel wall especially of the endothelium, to the impenetrability of the internal elastic lamina, and also to a local metabolic deficiency of the cellular constituents of the wall in dealing with lipid infiltration from the lumen. However, some maintain that atherosclerosis is a systemic metabolic disorder.1 It is disturbing that most workers have assumed that the lipid hypothesis is valid and that only the mechanism underlying the lipid deposition and the therapy of atherosclerosis require clarification. Indeed, Dock2 declared that infiltration of the blood-vessel wall with lipid from hyperlipidaemic serum had been unequivocally confirmed as the cause of atherosclerosis. However, although there is some evidence to support the lipid hypothesis, the following serious and fundamental inconsistencies detract from its
validity: THE ROLE OF LIPID IN THE PATHOGENESIS OF ATHEROSCLEROSIS W. E. STEHBENS
Department of Pathology, Wellington Clinical School, and the Wellington Cancer and Medical Research Institute, Wellington 2, New Zealand
experimental model of atherosclerosis in sheep veins identical to the human disease indicates (i) that ingestion of an atherogenic diet is not a prerequisite in atherosclerosis and (ii) that hæmodynamic stress must be the dominant ætiological factor in atherosclerosis. Ultrastructural studies reveal that the early lipid deposition in spontaneous human atherosclerosis and in hæmodynamically induced atherosclerosis is related to the transformation of extracellular vesicular debris into closely packed membranous profiles with electrontranslucent centres. It is postulated that the vesicular Summary
An
In diet-induced lesions there are distinct, unreconcilable, histological differences from human atherosclerosis. Moreover, even in the same species, diet-induced lesions differ histologically from spontaneous lipid-containing 1.
lesions.
,
2. In diet-induced disease there are attendant extravascular lesions which are not found in the spontaneous disease but which have the hallmarks of a lipid-storage disease. 3. In diet-induced disease there are no complications such as intimal tears, ulceration, and thrombosis, all of which are integral features of the human disease.
Epidemiologically, total serum-cholesterol is regarded as the most useful criterion in determining the risk of coronary heart-disease,3 and in national or community studies levels of fat intake, blood-cholesterol levels, and deaths from ischaemic heart-disease are said to be associated. However, the correlation between fat consumption and cholesterol levels in individualsand between the level of the serum-