A specific method for determination of free tryptophan and endogenous tryptophan in Escherichia coli.

A sensitive, simple spectrofluorometric technique for determination of tryptophan inamounts as small as 10 pmol is described. It is based on tryptophanase hydrolysis of tryptophan and spectrofluorometric analysis of the resulting indole. The relationship between released indole and fluorescence is linear over three orders of magnitude. The method is free from interference by other amino acids, polar indole derivatives, and a number of other compounds found in cell extracts or used in bacterial growth media. The method is rapid, reproducible, and accurate. A simple method for extraction and measurement of endogenous free tryptophan from bacterial cells is also described.     

Daily changes of free serum tryptophan in humans

In normal subjects the concentration of free tryptophan in serum was about 45% higher at midnight than at noon. However, total concentration of tryptophan in serum showed no significant change. Since previous experiments in rats indicated that free serum tryptophan reflects the rate of brain serotonin synthesis, the present results suggest that in humans, brain serotonin synthesis is greater at midnight that at noon.     

Automated fluorometric determination of free and total tryptophan in plasma

We have developed a sensitive automated fluorometric method based upon the manual procedure of Denckla and Dewey [(1967) J. Lab. Clin. Med.69, 160–169] for determining both free and total tryptophan in plasma. Free tryptophan is measured in a series of increasingly diluted aliquots of a sample of plasma after tryptophan bound to albumin is removed by continuous-flow dialysis. Free and total tryptophan are then derived from Scatchard plots of the data. The method can be used for nutritional assessments, clinical investigation of behavioral disorders in which serotonin is implicated in the pathogenesis, and studies on tryptophan transport and metabolism.     

Accumulation of free tryptophan and tryptairiine in zinc deficient maize seedlings

Maize seedlings were grown in culture solutions containing variouslevels of zinc. The changes in free tryptophan content due to the differentzinc nutritions were estimated by using an amino acid analyzer.Free tryptophan was detected in normal leaves, but the amountswere very small relative to those in zinc deficient plants.The maximum accumulation coincided with the development of severesymptoms associated with advanced zinc deficiency. Changes in tryptamine content due to different zinc nutritionswere estimated by paper chromatography and thin layer chromatography.The content of tryptamine was highest in maize leaves, whichdeveloped symptoms of zinc deficiency. Under conditions of adequatezinc nutrition, tryptamine was also found in maize leaves, butthe amounts were very small in contrast to those in zinc deficientplants. (Received March 25, 1970; )     

Interferon-gamma-induced degradation of tryptophan by human cells in vitro

Several human cells were investigated for their ability to degrade tryptophan and to synthesize neopterin upon induction by interferon-gamma (500 units/ml for 48 h). Concentrations of tryptophan, kynurenine, 3-hydroxykynurenine, anthranilic acid, 3-hydroxyanthranilic acid, 7,8-dihydroneopterin and neopterin were assessed in the culture supernatants by HPLC. Fibroblasts, A-22 arachnoidea, HK-2351 scalp, T-2346 meningeom and HeLa cervical carcinoma cells but not HL-60 promyelocytic leukaemia cells were found to degrade tryptophan upon induction by interferon-gamma. Tryptophan is converted to kynurenine by fibroblasts, A-22 arachnoidea and HK-2351 scalp cells and to kynurenine and anthranilic acid by HeLa cervical carcinoma and T-2346 meningeom cells. Kynurenine and anthranilic acid always make up more than 82% of the tryptophan degraded. None of these cells synthesizes 3-hydroxyanthranilic acid, 3-hydroxykynurenine, 7,8-dihydroneopterin or neopterin. Human macrophages form 3-hydroxyanthranilic acid and neopterin, but not 3-hydroxykynurenine, beside kynurenine and anthranilic acid upon activation by interferon-gamma. These data indicate that several human cells can be induced by interferon-gamma to degrade tryptophan. The interferon-gamma induced synthesis of 3-hydroxyanthranilic acid and neopterin, however, appears to be restricted to human macrophages. A hypothesis explaining these findings is presented.     

Characteristics of interferon induced tryptophan metabolism in human cells in vitro

Interferon-gamma-induced tryptophan metabolism of human macrophages was compared to ten human neoplastic cell lines of various tissue origin and to normal dermal human fibroblasts. Tryptophan and metabolites were determined in supernatants of cultures, after incubation for 48 h, by high-performance liquid chromatography with ultraviolet and fluorescence detection. With the exception of two cell lines (Hep G 2, hepatoma and CaCo 2, colon adenocarcinoma) in all of the ten other cells and cell lines tryptophan degradation was induced by interferon-gamma. Five of these ten formed only kynurenine (SK-N-SH, neuroblastoma; T 24, J 82, bladder carcinoma; A 431, epidermoid carcinoma; normal dermal fibroblasts), three formed kynurenine and anthranilic acid (U 138 MG, glioblastoma; SK-HEP-1, hepatoma; A 549, lung carcinoma). Only one line, A 498 (kidney carcinoma) showed the same pattern of metabolites as macrophages (kynurenine, anthranilic acid and 3-hydroxyanthranilic acid). Interferon-gamma regulated only the activity of indoleamine 2,3-dioxygenase. All other enzyme activities detected were independent of interferon-gamma, as shown by the capacity of the cells to metabolize L-kynurenine or N-formyl-L-kynurenine. Increasing the extracellular L-tryptophan concentration resulted in a marked induction of tryptophan degradation by macrophages. Contrarily, a significant decrease of the tryptophan degrading activity was observed when the extracellular L-tryptophan concentration was increased 2-fold with SK-N-SH, T 24 and J 82, 4-fold with A 431 and A 549 and 10-fold with U 138 MG and SK-HEP-1. The activity was unaffected by extracellular L-tryptophan with dermal fibroblasts and A 498. Though interferon-gamma was the most potent inducer of tryptophan metabolism, interferon-alpha and/or -beta showed small but distinct action on some of the cells. In all cells which reacted to interferon-gamma by enhanced expression of class I and/or class II major histocompatibility complex antigens tryptophan degradation was also inducible. These results demonstrate that induction of indoleamine 2,3-dioxygenase is a common feature of interferon-gamma action, that the extent of this induction is influenced by extracellular L-tryptophan concentrations and that indoleamine 2,3-dioxygenase is the only enzyme in the formation of 3-hydroxyanthranilic acid from tryptophan which is regulated by interferon-gamma.     

Changes in total and free tryptophan levels in serum following acute morphine administration in arthritic rats

In ‘arthritic’ rats a decrease in total tryptophan and an increase in free tryptophan levels was observed in serum after morphine administration (10 mg kg, s.c.). These changes were maximum within 15 and 30 min after injection.A decrease in total and an increase in free tryptophan levels in serum were observed 30 min after naloxone administration (1 mg/kg, i.m.).An increase in tryptophan and 5-hydroxyindoleacetic acid levels was also observed in the brain after morphine and naloxone.These observations suggest that the rise in 5-hydroxytryptamine synthesis provoked by morphine may be partly related to an increase in the availability of tryptophan from blood. However, the analgesia induced by the opiate appears unlikely to be directly related to this effect.     

Effect of long-term Intralipid administration in mice

Intralipid was administered intravenously to mice at a level of 2 g kg-1 day-1 for 23 days. No alterations in phagocytic index, liver or spleen size were observed in the chronically injected mice as compared with control mice that received saline injections. Tissue distribution of 0.45 micron multilamellar liposomes of egg phosphatidylcholine:cholesterol (2:1) was similar in mice that had been chronically injected with Intralipid to that in control mice. Mice chronically given the same total amount of phospholipid in the form of 0.2 micron liposomes of phosphatidylcholine:cholesterol (2:1) rather than as a lipid-triglyceride emulsion showed altered tissue distribution of entrapped label with decreased liver uptake and increased splenic uptake, which is indicative of reticuloendothelial blockade. Tissue distribution of [14C]dipalmitoylphosphatidylcholine Intralipid was compared with that of [14C]dipalmitoylphosphatidylcholine 0.2 micron MLV of phosphatidylcholine:cholesterol (2:1). Intralipid was taken up 2- to 3-fold less by liver and 5- to 10-fold less by spleen than liposomes. Blood levels of Intralipid were higher than those of liposomes. [14C]dipalmitoylphosphatidylcholine Intralipid was eliminated from the body at a faster rate than [14C]dipalmitoylphosphatidylcholine liposomes. The lack of reticuloendothelial blockade caused by Intralipid as compared with liposomes appears to be related to its diminished uptake into reticuloendothelial tissues. This diminished uptake may be related to differences in apolipoprotein uptake of Intralipid, which is primarily in the form of a phospholipid monolayer, and liposomes, which have their phospholipid organized into a bilayer.