Effects of vitamins on hepatic nuclear binding of L-tryptophan.

This study investigated the in vitro effects of selected vitamins on nuclear L-tryptophan receptor binding of rat liver. Our results revealed that some fat-soluble vitamins, beta-carotene, retinyl acetate, calciferol, alpha-tocopherol, and Trolox, as well as some water-soluble vitamins, thiamine and riboflavin, acted to inhibit in vitro 3H-tryptophan binding to hepatic nuclei. On the other hand, pyridoxine had little or no effect. The addition of dithiothreitol, a protective agent for sulfhydryl groups, along with each vitamin decreased the vitamin's inhibitory effect on in vitro 3H-tryptophan binding to nuclei, with the exception of riboflavin and calciferol. The addition of L-leucine, which alone had no inhibitory effect on in vitro 3H-tryptophan binding to hepatic nuclei but when added with unlabeled L-tryptophan negated the effect of unlabeled L-tryptophan, caused a markedly diminished inhibitory binding effect due to each of the following vitamins, thiamine, beta-carotene, retinyl acetate, and alpha-tocopherol and Trolox, but no effect on riboflavin and calciferol.

Effects of altered tonicity by sodium chloride on L-tryptophan binding to hepatic nuclei.

This study was concerned with the effects of NaCl administered in vivo or added in vitro to isolated nuclei on [(3)H]tryptophan binding to rat hepatic nuclei assayed in vitro. Hypertonic (10.7%) NaCl administered in vivo to rats caused at 10 min a marked decrease in in vitro binding (total and specific) of [(3)H]tryptophan to hepatic nuclei. In vitro incubation of isolated hepatic nuclei, but not of isolated nuclear envelopes, with added NaCl (particularly at 0.125 x 10(-4) M and 0.25 x 10(-4) M) revealed significant inhibition of [(3)H]tryptophan binding. However, isolated hepatic nuclear envelopes prepared after in vitro incubation of isolated nuclei with added NaCl did show inhibition of [(3)H]tryptophan binding (total and specific) compared with controls. Other salts (KCl, MgCl(2), NaHCO(3), NaC(2)H(3)O(2), NaF, or Na(2)SO(4)), at similar concentrations to that of NaCl except for MgCl(2), when added to isolated nuclei did not appreciably inhibit nuclear tryptophan binding. Kinetic studies of in vitro nuclear [(3)H]tryptophan binding in the presence of 0.125 x 10(-4) M NaCl revealed that binding decreased at 0.5 h and continued to 2 h compared with nuclear [(3)H]tryptophan binding with controls (without NaCl addition). The results obtained in vivo in rats and those obtained in vitro with isolated hepatic nuclei revealed NaCl-induced inhibitory effects on [(3)H]tryptophan binding to hepatic nuclei. Although the inhibitory effects were similar under the two different experimental conditions, the mechanism for each may be different in that the NaCl concentration in hepatic cells after administration of NaCl in vivo was appreciably higher than the low levels added in vitro to the isolated hepatic nuclei.

Effect of cycloheximide on tryptophan binding to rat hepatic nuclei.

This study evaluated whether cycloheximide, an inhibitor of protein synthesis, would affect the binding of L-tryptophan to rat hepatic nuclei or nuclear envelopes. Previous reports have indicated that the binding of L-tryptophan to hepatic nuclear envelope protein was saturable, stereospecific, and of high affinity. Also, the administration of L-tryptophan rapidly stimulated hepatic protein synthesis. In this study, we determined that the addition of cycloheximide in vitro inhibited 3H-tryptophan binding to hepatic nuclei or nuclear envelopes. Heat-treated cycloheximide failed to have this inhibitory binding effect. In vivo treatment of rats with cycloheximide diminished in vitro 3H-tryptophan binding to hepatic nuclei of treated rats compared to controls. Puromycin, another inhibitor of hepatic protein synthesis, when added in vitro did not affect 3H-tryptophan binding to hepatic nuclei but did diminish in vitro binding after in vivo treatment. Thus, cycloheximide added in vitro diminished 3H-tryptophan binding to hepatic nuclei probably by its structural effect on the receptor while cycloheximide administered in vivo may also act in part by inhibiting protein synthesis.

Influence of lead acetate and selected metal salts on tryptophan binding to rat hepatic nuclei.

This study evaluated whether lead acetate or other selected metal salts would influence the binding of L-tryptophan to rat hepatic nuclei. Lead salts and other salts of cadmium, zinc, mercury, and molybdenum, when added alone, had only small effects on 3H-tryptophan binding to hepatic nuclei in vitro. However, each of these salts, when added along with unlabeled L-tryptophan (excess, 10(-4) M), caused significantly less inhibition of 3H-tryptophan binding to hepatic nuclei than did unlabeled L-tryptophan alone. Lead acetate (10(-10) to 10(-4) M), when added along with unlabeled L-tryptophan, abrogated the inhibition of binding related to unlabeled L-tryptophan alone. Sodium arsenite (but not potassium arsenate) as well as sodium selenite (at 10(-4) M concentrations) inhibited to a moderate degree the in vitro 3H-tryptophan binding to hepatic nuclei, but addition of 10(-4) dithiothreitol, a protective agent for sulfhydryl groups, diminished this inhibition. Rats receiving a high dose of lead acetate before being tube-fed L-tryptophan displayed a decrease in hepatic protein synthesis compared with the stimulatory response connected with L-tryptophan alone. Thus, the addition of lead acetate and of other metal salts appears to have an inhibitory effect on L-tryptophan binding to hepatic nuclei. Lead acetate was investigated in in vivo experiments and was found to negate the stimulation of hepatic protein synthesis related to L-tryptophan alone.

Hormonal influences on tryptophan binding to rat hepatic nuclei.

This study evaluated whether selected hormones, 3,5,3'-triiodothyronine (T3), hydrocortisone (HC), or insulin, would influence the binding of L-tryptophan to rat hepatic nuclei or nuclear envelopes. The first two hormones have nuclear receptors that belong to the same superfamily, while insulin belongs to a different unrelated superfamily of receptors. Previous reports have indicated that the binding of L-tryptophan to hepatic nuclear proteins was saturable, stereospecific, and of high affinity. Also, previous studies showed that administration of L-tryptophan rapidly stimulated hepatic protein synthesis. In this study, we investigated whether each hormone alone or together with unlabeled tryptophan would influence tryptophan binding to rat hepatic nuclei or nuclear envelopes as assayed by in vitro L-5-(3)H-tryptophan binding. Our results indicate that T3 10(-14) to 1(-10) mol/L appreciably inhibited in vitro 3H-tryptophan binding to hepatic nuclei and T3 10(-16) to 10(-4) mol/L appreciably ameliorated the inhibitory effect of unlabeled tryptophan (10(-4) mol/L) on in vitro 3H-tryptophan binding. In vivo administration (1 hour) of tryptophan alone stimulated hepatic protein synthesis, but addition of T3 negated such stimulation. Addition of HC 10(-12) to 10(-4) mol/L had no effect and addition of insulin 10(-16) to 10(-4) mol/L had only a small inhibitory effect on in vitro 3H-tryptophan binding to rat hepatic nuclei, but each (10(-12) to 10(-4) mol/L), when added to unlabeled tryptophan (10(-4) mol/L), diminished the inhibitory binding effect of unlabeled tryptophan alone. Our study indicates that T3 competes with tryptophan for hepatic nuclear tryptophan binding, and it also appears to negate tryptophan's stimulatory effect on hepatic protein synthesis.

Hormonal influences on tryptophan binding to rat hepatic nuclei.

We evaluated whether selected hormones, 3,5,3'-triiodothyronine (T3), hydrocortisone (HC) or insulin, would influence the binding (saturable, stereospecific, and of high affinity) of L-tryptophan to rat hepatic nuclei or nuclear envelopes. T3 (10(-14) to 10(-10) M) appreciably inhibited in vitro L-(5-3H) tryptophan binding to hepatic nuclei and T3 (10(-16) to 10(-4) M) appreciably ameliorated the inhibitory effect of unlabeled tryptophan (10(-4) M) on such binding. In vivo tryptophan administration (1 h) stimulated hepatic protein synthesis but the addition of T3 negated such stimulation. HC (10(-12) to 10(-4) M) did not affect and insulin (10(-16) to 10(-4) M) had only a small inhibitory effect on 3H-tryptophan binding to hepatic nuclei, but each (10(-12) to 10(-4) M) when added to unlabeled tryptophan (10(-4) M) diminished the inhibitory binding effect of unlabeled tryptophan alone. Thus, T3 competes with tryptophan for hepatic nuclear tryptophan binding and also negates tryptophan's stimulatory effect on hepatic protein synthesis.

Mouse strain and source of L-tryptophan affects hepatic nuclear tryptophan binding.

This study describes that the affinity for specific L-tryptophan binding to hepatic nuclei in vitro is markedly decreased in NZBWF1 mice in comparison to that in Swiss mice. Also, the hepatic nuclei of NZBWF1 mice have a significantly decreased binding response in vitro to Showa Denko L-tryptophan (implicated in the eosinophilia-myalgia syndrome) or to its contaminants, 1,1'-ethylidenebis(tryptophan) or 3-phenylamino-L-alanine, when each is added to control, non-implicated L-tryptophan compared with hepatic nuclei of Swiss mice. Enhanced hepatic protein synthesis induced by tube-feeding control L-tryptophan is much less in NZBWF1 mice than in Swiss mice. Tube-feeding of Showa Denko L-tryptophan induced less stimulation of hepatic protein synthesis than did control L-tryptophan in Swiss mice but essentially none in NZBWF1 mice. NZBWF1 mice have a genetically altered response to L-tryptophan which may prove to be useful is studying the role of L-tryptophan in health and in disease.

Differences in tryptophan binding to hepatic nuclei of NZBWF1 and Swiss mice: insight into mechanism of tryptophan's effects.

We have observed that in NZBWF1 mice the affinity for L-tryptophan binding to hepatic nuclei in vitro is markedly less than that of Swiss mice. In vitro binding of [3H]tryptophan to hepatic nuclei from both strains was determined without and with unlabeled L-tryptophan (10(-4) mol/L). The relative specific binding of L-tryptophan to hepatic nuclei in vitro was 60.9 +/- 4.4% for Swiss mice and 35.8 +/- 5.4% (P < 0.01) in NZBWF1 mice. The total specific binding (bound radioactivity/mg nuclear protein) of L-tryptophan to hepatic nuclei in vitro was 74.9% (P < 0.05) lower in NZBWF1 mice than in Swiss mice. Other strains (DBA, SJL and BALB/c) had specific binding affinities similar to that of Swiss mice. Serum and hepatic free tryptophan concentrations and hepatic tryptophan dioxygenase activity in mice that were food-deprived overnight or 1 h after tube-feeding L-tryptophan (20 mg/100 g body weight) were similar in the strains of mice. In vitro [14C] leucine incorporation into protein using hepatic microsomes of mice 1 h after tube-feeding L-tryptophan (20 mg/100 g body weight) revealed a significantly greater (P < 0.05) increase relative to food-deprived controls in Swiss mice (76.8 +/- 19.2%) than the increase in NZBWF1 mice (26.5 +/- 2.6%). Nuclear [14C]-labeled RNA release in vitro was increased 77.2 +/- 18.0% by tube-feeding of L-tryptophan in Swiss but only 7.6 +/- 5.8% (P < 0.02) in NZBWF1 mice. Liver nuclear poly(A)-polymerase and nucleoside triphosphatase activities were variably increased by the administration of L-tryptophan in both strains. In summary, compared with Swiss mice, NZBWF1 mice have a lower specific binding affinity for L-tryptophan by hepatic nuclei, and this alteration may account for the other differences in responses to L-tryptophan by the two strains.

Tryptophan and carcinogenesis: review and update on how tryptophan may act.

This review covers the historical developments of the consideration that tryptophan may influence the induction of cancer in experimental studies. Studies relating to stimulatory effects, as well as to inhibitory effects, of tryptophan or tryptophan-related compounds are described. Also the effects of pyrolysis products of tryptophan on carcinogenesis are covered. In consideration that new L-tryptophan-related contaminants may be involved in a recently described human disease, a description is given of the eosinophilia-myalgia syndrome, which has been attributed to the ingestion of L-tryptophan-containing related contaminants. Whether these new L-tryptophan-related contaminants alone or together with L-tryptophan may prove to be carcinogenic remains to be determined. Lastly, recent developments relating to regulatory effects of L-tryptophan on liver metabolism are reviewed and then considered as possibly playing a role in carcinogenesis.

Toxic effect of valproic acid on tryptophan binding to rat hepatic nuclei.

This study evaluated whether valproic acid, a branched-chain fatty acid which has been used in the treatment of seizures, would influence the binding Of L-tryptophan to rat hepatic nuclei. Previous studies have indicated that binding of L-tryptophan to hepatic nuclear envelope protein was saturable, stereospecific, and of high affinity. In this study, we investigated whether valproic acid, which under certain conditions is heptatoxic, would influence L-tryptophan binding to rat hepatic nuclei as assayed by in vitro L-(5-3H)tryptophan binding. Our results indicate that the addition of valproic acid to hepatic nuclei or nuclear envelopes in vitro has little influence on their L-(5-3H)tryptophan binding. On the other hand, when valproic acid (80 mg/100 g body weight) is tube-fed 2 h before killing, the isolated nuclei show decreased specific L-tryptophan binding (total binding minus non-specific binding using unlabeled L-tryptophan (10(-4)M), at 2000-fold excess) compared with controls. Other fatty acids (oleic, palmitic or linoleic acid at 10(-4)M) when added with excess, unlabeled L-tryptophan (10(-4)M) in vitro to hepatic nuclei revealed some (but less than with valproic acid) decreased specific binding compared with controls. At high doses, valproic acid (80 mg/100 g body weight) appears to decrease tryptophan-induced stimulation of hepatic protein synthesis, probably in a hepatotoxic manner.

Eosinophilia-myalgia syndrome: a recent syndrome serving as an alert to new diseases ahead.

A recently recognized disease, the eosinophilia-myalgia syndrome, is described and presented as a new condition attributable to nutritional toxicology. Its etiology is related to the ingestion of L-tryptophan, manufactured by a single Japanese supplier who had modified its production system, which, though of high purity, contained in minute concentrations a number of contaminants or impurities. Patients with eosinophilia-myalgia syndrome develop an eosinophilia with pathologic changes mainly involving skin, muscle, and connective tissue. The findings suggest an autoimmune response. Experimental studies with the implicated L-tryptophan as well as with some contaminants have as yet failed to develop a suitable animal model of eosinophilia-myalgia syndrome. Further studies are needed to unravel the pathogenesis of this complex syndrome. At present, physicians need to be cognizant of this recent syndrome and be aware that other new diseases, induced by nutritional toxicological alterations and possibly related to technicological developments, lie ahead.

Comparative studies on tryptophan binding to hepatic nuclear envelopes in Sprague-Dawley and Lewis rats.

Since Lewis rats are susceptible to many inflammatory diseases and have been used in an experimental model of the eosinophilia-myalgia syndrome, we investigated whether Lewis rats would respond to L-tryptophan as have Sprague-Dawley rats reported earlier. In this comparative study using females of both strains, we observed a decrease in the affinity of in vitro L-tryptophan binding to hepatic nuclei and nuclear envelopes of Lewis rats compared with Sprague-Dawley rats. However, in vivo stimulatory effects of administering L-tryptophan on hepatic polyribosomal aggregation, protein synthesis, and nuclear RNA release were similar in both strains. In vitro [3H]tryptophan binding to hepatic nuclear envelopes, using L-tryptophan implicated in cases of the eosinophilia-myalgia syndrome, revealed less specific binding than when using nonimplicated L-tryptophan in both strains. The possible significance of the quantitative difference in the binding affinity of L-tryptophan to hepatic nuclei of Lewis rats compared with those of Sprague-Dawley rats is as yet undetermined.

Indolic compounds affect tryptophan binding to rat hepatic nuclei.

This study evaluates the effects of indolic or indole-related compounds on binding of L-tryptophan (saturable, stereospecific and of high affinity) to rat hepatic nuclei or nuclear envelopes. Addition of any one of many indolic or indole-related compounds, and particularly of 3-methylindole, does not inhibit in vitro binding of [3H]tryptophan to hepatic nuclear envelopes. However, when 3-methylindole (10(-10) to 10(-4) mol/L) is added in combination with unlabeled L-tryptophan (10(-4) mol/L), it diminishes the inhibitory effect of unlabeled L-tryptophan alone. Scatchard analysis of the binding affinities of in vitro [3H]tryptophan binding to hepatic nuclear envelopes using L-tryptophan in the absence or presence of 3-methylindole reveals similar dissociation constants (KD) under the two conditions, but the binding concentrations (Bmax) were greater in the combined group compared with that in the L-tryptophan alone group. Addition of 3-methylindole to liver before homogenization decreases specific [3H]tryptophan binding to nuclei compared with controls (without addition). L-Tryptophan tube-fed to rats with or without 3-methylindole administration increases in vitro hepatic protein synthesis compared with that of saline tube-fed controls. 3-Methylindole itself does not affect protein synthesis. Our report describes the effects of 3-methylindole on specific tryptophan binding to hepatic nuclear envelope receptor and discusses the possible implications thereof.

Studies with 1,1'-ethylidenebis(tryptophan), a contaminant associated with L-tryptophan implicated in the eosinophilia-myalgia syndrome.

L-Tryptophan binds to a rat liver nuclear envelope protein, and this binding is saturable, stereospecific, and of high affinity. Utilizing an in vitro assay of [3H]tryptophan binding to rat hepatic nuclear envelopes, we have previously determined that the L-tryptophan obtained from Showa Denko and which was implicated in cases of the eosinophilia-myalgia syndrome (EMS) inhibited [3H]tryptophan binding differently than did control L-tryptophan (not implicated in EMS). Therefore, in this study we investigated whether the addition of 1,1'-ethylidenebis(tryptophan) (EBT), a contaminant or impurity in L-tryptophan implicated in EMS, would have an effect. Our results indicate that EBT alone has little inhibitory binding effect compared with that of control L-tryptophan and that when EBT was added to control L-tryptophan the inhibitory binding effort was similar to that of control L-tryptophan alone. On the other hand, in vitro addition of EBT plus L-tryptophan to nuclei of cultured murine macrophages (WLG5) results in less inhibition of [3H]-tryptophan binding than does addition of L-tryptophan alone. Similar in vitro additions to nuclei of rat brain reveal little effect on binding, as was also the case for hepatic nuclear envelopes. Adding EBT to an in vitro hepatic protein synthesis system and measuring [3H]tryptophan incorporation into acid-precipitable proteins reveal that it competes similarly to that found with equimolar concentrations of unlabeled L-tryptophan. It does not affect [14C]leucine incorporation into proteins. [14C]EBT becomes incorporated in vitro into proteins (acid-precipitable), and this incorporation is diminished in the presence of equimolar concentrations of unlabeled EBT or L-tryptophan. This suggests that EBT or possibly a breakdown product becomes incorporated into proteins. Speculation as to how EBT may affect tissues in experimental animals is presented.

Effect of metyrapone on tryptophan binding to rat hepatic nuclei.

This study evaluated whether metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone), an inhibitor of endogenous adrenal corticosteroid synthesis via inhibition of cytochrome P-450-mediated steroid hydroxylation, would influence the binding of L-tryptophan to rat hepatic nuclei or nuclear envelopes. Previous publications have indicated that binding of L-tryptophan to hepatic nuclear envelope proteins was saturable, stereospecific, and of high affinity. In this study, we investigated whether metyrapone would influence L-tryptophan binding to rat hepatic nuclei or nuclear envelopes as assayed by in vitro L-(5-3H)tryptophan binding. Our results indicate that the addition of metyrapone in vitro has little influence on L-(5-3H)tryptophan binding to hepatic nuclei or nuclear envelopes. On the other hand, when metyrapone (1 mg/100 g body weight) is tube-fed 30 minutes before killing, the isolated hepatic nuclei show decreased specific L-tryptophan binding (total binding minus nonspecific binding [using 2,000-fold excess of unlabeled L-tryptophan]) compared with controls. Also, addition of metyrapone in vitro to rat liver before homogenization and preparation of nuclei caused the nuclei to show decreased specific tryptophan binding compared with controls. Under these in vitro conditions, SKF 525A, another inhibitor of hydroxylation, showed inhibitory effects similar to those of metyrapone. Thus, metyrapone can interfere with rat liver nuclear envelope receptor binding to L-tryptophan, and possibly acts via its effects on hydroxylation. At high doses, metyrapone (20 mg/100 g body weight) appears to inhibit tryptophan-induced stimulation of hepatic protein synthesis.

Effect of 3-phenylamino-L-alanine on tryptophan binding to rat hepatic nuclear envelopes.

We have determined that the addition of 3-phenylamino-L-alanine (PAA), a recently reported contaminant in L-tryptophan implicated in the eosinophilia-myalgia syndrome, affects tryptophan binding by utilizing an in vitro measurement of 3H-tryptophan binding to hepatic nuclei or nuclear envelopes. PAA (10(-10) to 10(-4) M) diminishes the inhibitory effect of binding due to excess unlabeled L-tryptophan (10(-4) M). PAA alone has no inhibitory effect on binding. The effect of PAA on in vitro tryptophan binding is in contrast to that of another contaminant, 1,1'-ethylidenebis(tryptophan), which together with excess unlabeled L-tryptophan does not appreciably affect the binding. In vitro addition of PAA and L-tryptophan to nuclei of rat brain or of cultured murine macrophages does not affect [3H]tryptophan binding in comparison to L-tryptophan alone as is the case with hepatic nuclear envelopes. Adding PAA to an in vitro protein synthesis system and measuring [3H]tryptophan or [3H]alanine incorporation into acid-precipitable proteins reveals that it competes similarly, but somewhat less, than does equimolar concentrations of unlabeled L-tryptophan or L-alanine, respectively. This suggests that PAA or a breakdown compound becomes incorporated into proteins. Speculation as to how PAA may affect tissues in experimental animals is presented.

Effect of tryptophan on rat hepatic nuclear poly(A)polymerase activity.

Addition of poly(A) to hnRNA in the cell nucleus is a post-transcriptional event and is presumed to be brought about by a specific poly(A)polymerase. Since it is known that tryptophan rapidly increases the cytoplasmic levels of polyadenylated mRNA, it was of interest to investigate whether the essential amino acid, tryptophan, affects the enzyme responsible for polyadenylation. Tryptophan (300 mg/kg body wt.) tube-fed for 10 min elevated the hepatic nuclear enzymatic activities of both the chromatin-bound nuclear poly(A)polymerase (44%, n = 7) as well as that of the free solubilized form (48%, n = 7). Hepatic nuclear proteins separated under denaturing conditions, transferred to nitrocellulose sheets, and then probed with antibody raised against hepatic nuclear poly(A)polymerase showed no differences between the hepatic nuclei of control and tryptophan-treated rats.

Tryptophan binding to nuclei of rat brain.

Nuclei purified from whole rat brain specifically bind [3H]tryptophan ([3H]Trp) under in vitro conditions. Excess unlabeled Trp (10(-4) M) is an effective inhibitor of in vitro [3H]Trp binding to brain nuclei. Rats tube-fed L-tryptophan (Trp) (30 mg/100 g body wt) 30 min to 4 hr before killing revealed decreased specific binding of [3H]Trp to purified brain nuclei in vitro. By Scatchard analysis, the nuclei from whole brain appear to contain one binding site for [3H]Trp, and the KD is 263 nM. A number of Trp-related compounds, Trp metabolites, or other amino acids and their analogues were observed to compete for in vitro [3H]Trp binding to brain nuclei. The ability of Trp analogues, metabolites, and other cognate compounds to inhibit in vitro [3H]Trp binding to brain nuclei was evaluated and utilized to map the active site of Trp binding.

Competitive studies relating to tryptophan binding to rat hepatic nuclear envelopes as a sensitive assay for unknown compounds.

Our laboratory has reported that L-tryptophan binds to a rat liver nuclear envelope protein and this binding is saturable, stereospecific and of high affinity. Utilizing an in vitro [3H]tryptophan binding assay to hepatic nuclear envelopes, we have determined the effects of using excess unlabeled L-tryptophan from a number of different suppliers. This study reports that, based on our in vitro binding assay, some significant differences were observed when implicated L-tryptophan in cases of the eosinophilia-myalgia syndrome obtained from a Japanese manufacturer, Showa Denko, was assayed, in contrast to non-implicated L-tryptophan from other suppliers. An isolated impurity of Showa Denko L-tryptophan, 1,1'-ethylidenebis(tryptophan) alone or together with non-implicated L-tryptophan or its breakdown product, 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, did not appreciably affect the in vitro [3H]tryptophan binding to hepatic nuclear envelopes as did the Showa Denko L-tryptophan. Our data, derived with our in vitro binding assay system, suggests that implicated L-tryptophan from Showa Denko contains a compound/s (unknown at present) other than 1,1'-ethylidenebis(tryptophan), which alters in vitro [3H]tryptophan binding. The significance of the impurity/ies involved remains to be determined.