Molec. Aspects Med. Vol. 6, pp. 101-197, 1982 0098-2997/82/020101-97548.50/0 Printed in Great Britain. All rights reserved. Copyright © Pergamon Press Ltd. BIOCHEMISTRY OF TRYPTOPHAN IN HEALTH AND DISEASE David A. Bender Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School, London WIP 7PN, U.K. Contents Chapter 1 THE DISCOVERY OF TRYPTOPHAN, ITS PHYSIOLOGICAL SIGNIFICANCE AND METABOLIC FATES 103 Tryptophan and glucose metabolism 105 Xanthurenic acid and insulin 105 The glucose tolerance factor 106 Inhibition of gluconeogenesis by tryptophan metabolites i07 Metabolic fates of tryptophan 108 Protein synthesis 108 Oxidative metabolism Ii0 5-Hydroxyindole synthesis 111 Intestinal bacterial metabolism iii Chapter 2 THE 5-HYDROXYINDOLE PATHWAY OF TRYPTOPHAN METABOLISM; SEROTONIN AND OTHER CENTRALLY ACTIVE TRYPTOPHAN METABOLITES 112 Tryptophan 5-hydroxylase 112 Inhibition of tryptophan hydroxylase and the carcinoid syndrome 116 Aromatic amino acid decarboxylase 118 The specificity of aromatic amino acid decarboxylase 120 Tryptophan metabolism in the pineal gland 121 Monoamine oxidase 124 The uptake of tryptophan into the brain 124 The binding of tryptophan to serum albumin 127 Competition for uptake by other neutral amino acids 129 Changes in tryptophan metabolism in response to food intake 129 Tryptophan uptake into the brain in liver failure 131 Sleep and tryptophan metabolism 134 101 102 D.A. Bender Tryptophan and serotonin in psychiatric disorders 135 Affective disorders 136 Evidence for a deficit of serotonin or tryptophan in depression 138 The use of tryptophan as an anti-depressant drug 140 Schizophrenia 141 Chapter 3 THE OXIDATIVE PATHWAY OF TRYPTOPHAN METABOLISM 145 Tryptophan oxygenase 145 Induction by hormones 145 Stabilisation and activation by tryptophan 149 Product inhibition 150 The availability of haem 151 Indoleamine dioxygenase (D-tryptophan oxygenase) 152 The metabolism of D-tryptophan 152 Formylkynurenine formamidase 153 The metabolism of kynurenine 154 The tryptophan load test for vitamin B 6 status 156 The tryptophan load test in women receiving oestrogens 158 The synthesis of nicotinamide nucleotides 160 The control of tissue concentrations of nicotinamide nucleotides 163 Pellagra 165 Non-nutritional pellagra 168 Hartnup disease 169 Carcinoid syndrome 169 Isoniazid therapy for tuberculosis 170 The pellagragenic effect of excess dietary leucine 171 Picolinic acid and the absorption of zinc 173 REFERENCES 177 Chapter I The Discovery of Tryptophan, Its Physiological Significance and Metabolic Fates The discovery of the amino acid tryptophan by Hopkins and Cole at the beginning of this century was an example of scientific serendipity - the facility for making happy and unexpected discoveries by accident and sagacity. One of the class practical exercises that medical students were expected to perform was the identification of proteins by means of the Adamkiewicz reaction - the formation of a coloured derivative when proteins were reacted with glacial acetic acid. The reaction worked well for some students, and failed for others. Perhaps today we would dismiss this as reflecting the inability of medical students to perform simple biochemical exercises. However, Hopkins was more persistent, and set out to investigate the reasons for the inconsistent performance of the reaction. He and Cole [I] showed that the active reagent was not acetic acid, but rather glyoxylic acid that was present as an impurity in some samples of acetic acid, but not in others. Today the chromogenic reaction of proteins with glyoxylic acid is generally known as the Hopkins-Cole reaction. They went on to identify the component of proteins that was responsible for the reaction with glyoxylic acid, and found it was the same compound as was responsible for another colour reaction of proteins, the so-called 'tryptophane' reaction. 'The substance also gives the tryptophane reaction. If bromine water be cautiously added to aqueous solutions of the compound, excess being avoided, a fine rose-red colour is produced; and if the mixture be shaken with amyl alcohol the coloured product of the reaction is character- istically taken up by the latter solvent, showing in this the exact spectro- scopic absorption which is seen where the 'tryptophane' (proteinchrome) reaction has been obtained from the original mixture of tryptic digestion products. The tryptophane reaction, no less than the glyoxylic reaction, is seen to be associated with the compound that we are describing at each stage of the separation of the latter from the original digestion mixture .... there can be no doubt that our substance is the hitherto unknown precursor of the red colour of this familiar reaction ..... there is no doubt that our substance is the much sought tryptophane' [2]. The name tryptophan (the final '-e' was gradually dropped, for obscure reasons, during the decades following its discovery) seems to be derived from the fact that it is that which is 'revealed by tryptic digestion of a protein' The Greek ~cpoo means visible or evident. Acid hydrolysis of proteins destroys tryptophan, so that it can only be detected in enzymic or alkaline hydrolysates of proteins. Tryptophan was the first of the amino acids to be shown to be a dietary essential, despite the fact that six of the other amino acids that we now know to be essential or indispensible had already been identified and isolated by the turn of the century. This reflects both the interest of Hopkins in essential nutrients, and also the fortunate chance that there were two specific reactions available for the detection and quantification of tryptophan - the glyoxylic (Hopkins-Cole) reaction and the 'tryptophane' 103 104 D. A, Bender reaction. Furthermore, the amino acid could be destroyed by heating in strong acid, so that diets could be prepared for experimental animals that were essentially tryptophan-free. In fact, the early work establishing the indispensibility of tryptophan used diets based on zein, the principal protein of maize, as the sole protein source, since it had been established that zein was a poor source of tryptophan. Willcock and Hopkins [3] showed that adding tryptophan to such a diet led to an increase in the rate of growth of their animals. In view of this early work showing that zein, and hence maize, was deficient in tryptophan, it is perhaps somewhat surprising that the connection between tryptophan and pellagra arising in areas where maize was the dietary staple was not realised sooner than it was (see Chapter 3). The discovery and early investigation of tryptophan also marked another milestone in the development of the newly emerging science of biochemistry - the first use of bacterial degradation of a naturally-occurring compound as a means of determining its chemical structure [4]. Tryptophan probably has more entries in Index Medicus than any other single amino acid, and interest in this amino acid covers a wide range of scientific and medical specialties. A meeting on tryptophan may well attract biochemists, chemists and nutritionists on the one hand, and such diverse clinical special- ists as dermatologists, ophthalmologists, neurologists, psychiatrists, oncolo- gists and endocrinologists on the other. The metabolism of tryptophan is affected by a number of medical conditions, and conversely, tryptophan and its metabolites have a number of profound effects on other metabolic and endocrine systems. The naturally occurring form of tryptophan is the L-enantiomer, as with other amino acids. L-Tryptophan has a strong bitter flavour, which has caused some problems in the administration of doses of the amino acid to volunteers in laboratory studies, and, more seriously, to patients in various clinical trials. There are those who prefer to take the amino acid mixed with a strongly fruit-flavoured yoghourt, or even raspberry jam, to mask the flavour, and one preparation made available in Britain was mixed with cocoa powder (Optimax, Cambrian). By contrast, the synthetic D-tryptophan has a pleasant, if somewhat lingering, sweet flavour. It is about 40-times as sweet as sucrose on a weight basis, and therefore might be of interest as a 'non-metabolisable' sweetener for use in energy or carbohydrate restricted diets. Of greater potential importance for such a use are two derivatives of D-tryptophan, 7-chloro- and 7-methyl-D-tryptophan. Both of these are several hundred times sweeter than sucrose. However, 7-chloro-D-tryptophan gives rise to metabolites that are potent inhibitors of the normal pathway of tryptophan oxidative metabolism [5], so that it is unlikely that this compound will be developed for use as a food additive. The interest of oncologists in tryptophan is principally that when the amino acid is fed to animals at relatively high levels, over a prolonged period, it has some slight co-carcinogenic or promoter action in association with known chemical carcinogens [6]. It is difficult, if not impossible, to interpret these data in terms of human cancer and tryptophan metabolism. However, there is also evidence that one or more of the tryptophan metabolites that are excreted in increased amounts in vitamin B 6 deficiency (kynurenine, hydroxykynurenine, xanthurenic and kynurenic acids) may be associated with the development of bladder tumours. It has been suggested that vitamin B 6 supplementation may be appropriate for patients from whom such tumours have been removed surgically, in view of the normal high rate of recurrence [7]. Excretion of abnormally large amounts