Plasma Free Tryptophan Research Articles

Amino acid analysis demonstrates that increased plasma free tryptophan causes the increase of brain tryptophan during exercise in the rat.

Rats were trained to run on a horizontal treadmill for 2 h at 20 m/min. This activity considerably increased plasma free tryptophan (TRP) (+70%) but did not alter plasma total TRP levels and had little or no effect on plasma concentrations of the other large neutral amino acids (LNAAs) that compete with TRP for entry into the brain. Brain TRP levels increased by 80%. The only other brain LNAA to be affected by exercise was threonine, which rose moderately. The results indicate that increased plasma free TRP was specifically responsible for the increase of brain TRP after 2 h of exercise. Brain lysine was also increased whereas glycine, alanine, and gamma-aminobutyric acid were decreased. The differences between the present findings and those previously obtained following 2 h immobilization stress are discussed.

Ventricular Size and CSF Transmitter Metabolite Concentrations in Severe Endogenous Depression

The relationship between neurotransmitter metabolite concentrations and measurements of ventricular size on CAT scans and pneumoencephalographs was investigated in 15 patients with severe affective disorder. An association was identified between reduced levels of plasma free tryptophan and ventricular enlargement, and also between raised ventricular CSF levels of 5 HIAA and ventricular enlargement.

Plasma free tryptophan concentration in autistic Children

The plasma free and total tryptophan (TRP) and blood serotonin levels in autistic children were examined simultaneously in order to establish a relationship between these parameters and the pathophysiology of infantile autism. The subjects were 37 autistic children, 28 normal adults and 12 normal children. Quantitative evaluation of the global severity, hyperkinetic behavior and intellectual function was performed by means of the Children's Psychiatric Rating Scale (CPRS-1), the Werry-Weiss-Peters Activity Scale (WWPAS) and the Developmental Quotient (DQ), respectively. The mean total TRP level in autistic children was not significantly different from those in normal children and adults. The mean plasma free TRP level in autistic children was significantly higher than those in normal children and adults. A significant positive correlation was found between the plasma free TRP level and CPRS-1 or WWPAS score in autistic children. Moreover, there tended to be a negative correlation between the plasma free TRP level and DQ. These findings suggest the presence of some defect in the metabolic system for TRP-serotonin in the brain of autistic children.

Immobilization decreases amino acid concentrations in plasma but maintains or increases them in brain.

Immobilization for 2 h significantly decreased plasma concentrations of 13 of 16 amino acids assayed, including the transmitter amine precursors tyrosine and total tryptophan. The level of plasma free tryptophan, however, was increased. Despite the reduced plasma levels, corresponding brain concentrations of many large neutral amino acids (LNAAs) were increased (tryptophan, phenylalanine, valine, leucine, and isoleucine). Brain concentrations of tyrosine and the other amino acids measured were unaltered. The results for the LNAAs were not explained by calculated brain influx rates. Therefore, altered influx kinetics or perhaps altered brain protein metabolism or efflux may be responsible. Comparison of calculated brain influxes and brain concentrations of LNAAs suggests that the rise in level of plasma free tryptophan during immobilization is not responsible for the increase in level of brain tryptophan and that the mechanism responsible for the maintenance of or increase in brain concentrations of the other LNAAs is probably involved. Maintenance of brain concentrations of basic amino acids is explicable by reduced competition for brain uptake.

Plasma free tryptophan concentration in children with attention deficit disorder.

In order to examine the serotonin metabolism in children with attention deficit disorder (ADD), plasma tryptophan, which is the precursor of serotonin, was measured in 10 children with ADD and 12 normal children. The mean plasma total tryptophan level in the children with ADD was not significantly different from that of the normal children. The mean plasma free tryptophan level in the children with ADD was significantly higher than that in the normal children. There tended to be a positive correlation between the plasma free tryptophan level and the Werry-Weiss-Peters Activity Scale in children with ADD. In other words, the more severe the hyperactivity of ADD, the higher the plasma free tryptophan level. The mean ratio of plasma free to total tryptophan levels in the children with ADD was significantly higher than that in the normal children, which means that the children with ADD showed a high amount of free tryptophan in the total tryptophan level. These results suggest that there might be some disturbance in the tryptophan-serotonin metabolism in the brain of a child with ADD.

Tryptophan Determination in Tissue Homogenates and Biological Fluids Using HPLC-EC

Abstract A method for quantitating tryptophan in tissue homogenates and biological fluids using 6-hydroxy-tryptamine as internal standard is described. Tryptophan in CSF and free tryptophan in serum or plasma can be quantitated by directly injecting CSF or ultrafiltrate into the HPLC system with the internal standard. Serum, plasma, amniotic fluid, and saliva are deproteinized by addition of equal volume of acetonitrile. Urine samples are processed through Biorex-70 columns. Cross validation was done by comparing the values of column procedures with direct injection method.

Lowering of plasma free tryptophan in normal and portacaval shunt dogs

The purpose of this study was to develop a method which would produce immediate and sustained lowering of plasma free tryptophan. Glucose, insulin, and mixtures of the two in different concentrations were infused into dogs over a period of 6 hr. Blood was sampled during the course of the infusion for assay of plasma free and total tryptophan, free fatty acids, glucose, and insulin. The infusate found to produce the most significant and sustained reduction in plasma free tryptophan in normal dogs was a mixture of 25% glucose and insulin. The lowering ranged from 26 to 47% during the course of the infusion. When the same infusion mixture was administered to dogs with established portacaval shunts, and elevated plasma free tryptophan concentrations (3.3 ± 0.3 μg/ml compared to normal dogs which had 1.9 ± 0.1 μg/ml), there was a 35% reduction to normal levels (2.0–2.2 μg/ml). A glucose-insulin mixture has, therefore, been found which, upon infusion into dogs, significantly lowers plasma free tryptophan levels. The effect of such an infusion on the plasma levels of free tryptophan, and the clinical course of patients with hepatic encephalopathy remains to be determined.

Variables influencing the effect of a meal on brain tryptophan.

Previous work from our laboratory points to plasma free tryptophan being a useful predictor of brain tryptophan concentration in many circumstances. Other work, in particular various studies on the acute effects of food intake, has emphasized the roles of plasma total tryptophan and of plasma large neutral amino acids that compete with tryptophan for transport to the brain. We have now studied associations between the above variables under different dietary conditions. Rats were allowed to feed for restricted periods during a 12-h light-12-h dark cycle. In the first study, rats were given access to a carbohydrate diet for 2 h midway through the light cycle and following an 18-h fast. The resultant rise of brain tryptophan was explicable largely by the associated fall in large neutral amino acids. In a second study, rats were adapted to a regimen whereby they were allowed access to the standard laboratory diet for 4 h during the dark cycle for 3 weeks. A postprandial decrease in brain tryptophan was associated with a fall in free tryptophan and of its ratio to competing amino acids. The brain change could be attributed neither to changes in plasma total tryptophan (which increased) nor to changes of its ratio to the competers (which remained unchanged). Results as a whole are thus consistent with changes of plasma free tryptophan and large neutral amino acid concentrations affecting brain tryptophan concentration under different dietary circumstances. It is suggested that these influences serve to maintain brain tryptophan when dietary supplies are defective.

A controlled clinical trial of l-tryptophan in acute mania

In a 2-week study, 24 newly admitted manic patients were treated for 1 week with l- tryptophan (12 g/day); during the second week, half the patients, chosen at random, continued to receive tryptophan, while placebo was substituted in the other half under double-blind conditions. In the open phase of the study, there was a clinically and statistically ( p < 0.001) significant reduction in manic symptom scores, with little need for haloperidol prn. Patients who continued to be treated with tryptophan showed no significant change in mean scores during the second week, but those who were switched to placebo tended ( p < 0.10) to show an increase in the mean scores for manic symptoms. There was a significant ( p < 0.05) increase in the geometric mean of morning fasting total and free plasma tryptophan concentrations in men, but not in women. These results suggest that increasing the synthesis of 5-hydroxytryptamine has some therapeutic effect in mania.

Ethanol increases rat liver tryptophan oxygenase: Evidence for corticosterone mediation

Acute administration of ethanol (4.0 g/kg) intragastrically or intraperitoneally induced rat liver tryptophan oxygenase (TO) activity 3–4 fold 4–5 hr later. Ethanol administration increased the concentration of plasma free tryptophan and free fatty acids. Pretreatment with a beta-receptor blocker, propranolol, modified the latter responses without affecting the TO induction due to ethanol. The rise of the level of plasma free tryptophan due to ethanol was too small to influence TO activity. Liver tryptophan concentration and TO half-life was unchanged after ethanol administration. Ethanol administration increased the concentration of plasma corticosterone sufficiently to increase TO activity. Pretreatment with a glucocorticoid antagonist blocked this TO response to ethanol. The increased TO activities found after ethanol or corticosterone treatment were influenced in the same manner and to the same extent by cycloheximide. Taken together it is concluded that ethanol induces TO through a rise of glucocorticoid hormones and not by a tryptophan-linked mechanism.

血漿遊離トリプトファン微量定量法に関する研究

Summary: Free tryptophan in plasma was separated by centrifugation through an Amicon ultrafiltration membrane cone. The value obtained without control of pH was found to be lower than that obtained with control of pH by an improved method (Hijikata et al. (1981) Anal. Biochem.118,10-16).For determina tion of the total tryptophan concentration in the plasma, high performance liquid chromatography (HPLC) was better than the method of Denckla and Dewey as modified by Bloxam and Warren ( (1974) Anal. Biochem.60,621-625), as judged on the basis of sensitivity, recovery rate and coefficient of variance.The total tryptophan concentration in the plasma determined by HPLC was lower than that determined by the Bloxam and Warren method. The total tryptophan concentration (t-Trp), free tryptophan concentration (f-Trp) and f-Trp/t-Trp ratio were 55.8±10.2μmol/1,11.6 ± 1.5μmol/1 and 0.211 ± 0.03 (mean ± 1 SD) respectively, in healthy subjects (controls). No significant difference was observed between the values of controls and those of patients with liver cirrhosis, hepatocellular carcinoma, liver cirrhosis with hepatocellular carcinoma and hepatic encephalopathy of liver cirrhosis without bleeding. But in liver cirrhosis with bleeding, free tryptophan concentration (48.0 ± 23.3 prno1/1, p<0.001) and f-Trp/t-Trp ratio (0.645 ± 0.289, p<0.001) were significantly highe r than those of controls. In fulminant hepatitis free tryptophan concentration (106.6 ± 79.9 pmo1/1, p<0.02), and f-Trp/t-Trp ratio (0.973 ± 0.017, p<0.001) were significantly higher than those of controls, and free tryptophan concentration (p<0.005) and f-Trp/t-Trp ratio (p<0.005)were significantly higher than in liver cirrhosis with bleeding. In other words the increase of free tryptophan concentration in plasma may be related to the bleeding, and in fulminant hepatitis the binding ability of free tryptophan to albumin may decrease.In the examined cases of liver cirrhosis, the correlatio n of the concentration of plasma tryptophan with those of other amino acids and indicators of liver function was changed when hepatocellular carcinoma was also present.

血漿遊離トリプトファン微量定量法に関する研究

Previous methods for measuring plasma free tryptophan (f-Trp) are not completely satisfactory, and therefore we developed an improved method for this purpose. Free tryptophan was separated from bound tryptophan b y centrifugation through Amicon ultrafiltration membrane cones in a quite short period (within 2 minutes). The first fraction of filtrate obtained in 30 seconds by centrifugation at 3,000g at controlled pH was assayed with a high performance liquid chromatography system.The first filtrate showed a rath er higher tryptophan concentration than that obtained by prolonged centrifugation. We propose that tryptophan concentration in the first filtrate is nearest value to f-Trp concentration in plasma, since a change of equilibrium between bound and free tryptophan during separation procedure is quite small.Furthermore we compared our improved met h od with previous methods and got following results.1. The fresh human plasma after replacing the air in the test tube with CO2-N2 gas mixture kept pH and pCO2 almost constant but without replacing the air, pCO2 decreased a n d pH increased.2. The linear correlation between Trp concentration and the intensity of fluorescence was observed upto 1000 μmol/1 of Trp, but with the Denckla and Dewey method modified by Bloxam and warren it was observed upto 20 μmol/l.3. In case of ultrafiltration under pressure to obtain free tryptophan fraction, the pCO2gradually increased and pH decreased from 7.4 to 7.0 with the progress of ultrafiltratio n. The f-Trp concentration determined by this method showed lower value t han that by our method.4. Our improved method proved to keep pH and pCO2 of plasma almost constant for more than 30 minutes and the necessary sample to determine the f-Trp concentration was obtained in a short time.5. The f-Trp concentration increased as the temperature of plasma rose, and it changed after freezing without showing definite tendency.The dialysis method to determi n e f-Trp concentration was not proper, because as the volume of outer solution increased, the f-Trp in plasma increased until all bound tryptopha n got free.

Ethanol-induced increase in liver tryptophan oxygenase activity in the starved rat: Evidence against tryptophan mediation

A single oral dose of ethanol (4.0 g/kg) increased the activity of liver tryptophan oxygenase in starved male rats. The peak increase of 340% for the total activity and 400% for the holoenzyme activity occurred 6 hr after ethanol administration. At or after these peaks, the levels of tryptophan in plasma and brain but not in liver, decreased significantly. Plasma total tryptophan and brain tryptophan started to decrease significantly as early as 0.5–1.0 hr after the ethanol treatment, while the activity of liver tryptophan oxygenase was still at the control level. These findings suggest that not all the changes in tissue tryptophan concentrations seen after acute ethanol treatment are caused by increased liver tryptophan oxygenase activity. Prior to the increase in liver tryptophan oxygenase activity, an increase of 104 and 50% in plasma corticosterone and free tryptophan, respectively, were seen 15 min after ethanol treatment. However, the increase in liver tryptophan at this time appeared to be small (13%) and statistically insignificant. With tryptophan treatment, the initial peak levels of liver tryptophan and plasma free tryptophan required to stimulate an increase in tryptophan oxygenase activity were 170 times higher than those caused by ethanol. It was therefore concluded that increases in plasma and liver tryptophan after acute ethanol ingestion, probably mediated by the lipolytic action of ethanol, are too small to cause the increase in liver tryptophan oxygenase activity seen after ethanol administration. However, experiments with different corticosterone doses showed that ethanol-induced increases in plasma corticosterone concentrations are high enough to cause an increase in liver tryptophan oxygenase activity.

Blood serotonin and free tryptophan concentration in autistic children.

In 37 autistic children and 67 normal control subjects, determinations of plasma free and total tryptophan and blood serotonin levels were made simultaneously in order to establish a relationship between these parameters and the clinical rating scales: Children's Psychiatric Rating Scale (CPRS-1), Werry-Weiss-Peters Activity Scale (WWPAS), and Developmental Quotient (DQ). The plasma free tryptophan level was significantly higher in autistic children than in normal control subjects. There tended to be a significant positive correlation between the plasma free tryptophan level and CPRS-1 or WWPAS score and a negative correlation between the plasma free tryptophan level and DQ. The blood serotonin level was significantly higher in autistic children than in normal control subjects. No correlation was established, however, between the blood serotonin level and CPRS-1, WWPAS score or DQ, and hence the clinical symptoms. Nor was there a correlation between blood serotonin and free tryptophan levels in these children. These results suggest that autistic children have some defect in tryptophan-serotonin metabolism in the brain, which is responsible for the clinical manifestations and behavioral abnormalities of infantile autism.

Changes in brain amines associated with cancer anorexia

Analysis of indole amine metabolism within acute (Walker 256 carcinosarcoma) and chronic (methycholanthrene-induced sarcoma) animal models of cancer anorexia demonstrated elevated levels of plasma free tryptophan, whole brain tryptophan, serotonin and 5-hydroxyindoleacetic acid in anorectic tumor-bearing rats. Whole brain levels of catecholamines were not changed within either tumor line. Regional central nervous system determination of tryptophan metabolism in rats bearing Walker 256 tumors revealed elevated tryptophan in the hypothalamus, corpus striatum, mesencephalon, diencephalon, cerebellum and cortex, increased serotonin in the diencephalon and cerebellum and elevated 5-hydroxyindoleacetic acid in the diencephalon, hippocampus, pons-medulla, cerebellum and cortex. Although tryptophan was significantly increased only in the corpus striatum and diencephalon of the more chronic methycholanthrene tumor model, serotonin concentration was elevated in the corpus stiratum, diencephalon, hippocampus, pons-medulla, cerebellum and cortex, while levels of 5-hydroxyindoleacetic acid were significantly increased in all these areas as well as in the mesencephalon. Since similar changes in indole activity were not observed in pair-fed control rats, it is concluded that the elevated serotonin and 5-hydroxyindoleacetic acid levels in tumor-bearing rats did not result from undernutrition alone. Assay of regional catecholamines revealed few food-relevant changes, with norepinephrine being elevated in the corpus striatum and decreased in the pons-medulla of tumor-bearing rats. Therefore, these experiments suggest that the increased serotonin metabolism observed in tumor-bearing rats may be involved in the etiology of the anorexia of cancer.

Tryptophan metabolism in analbuminemic rats.

Studies were made on tryptophan metabolism in Nagase analbuminemic rats (NAR), a mutant strain established from Sprague-Dawley rats that is characterized by lack of plasma albumin and hyperlipidemia. The total tryptophan concentration in NAR plasma is one fifth of normal, but the plasma free tryptophan concentration and liver and brain tryptophan levels are normal. Radioimmunoelectrophoresis showed that the tryptophan binding protein in NAR plasma is alpha 2-macroglobulin. The effect of oral tryptophan loading on plasma tryptophan and urinary excretion of tryptophan and its metabolites were examined. Differences between the values for NAR and normal rats were not significant except that the disappearance rate of tryptophan from NAR plasma was faster than in normal rats. The effects of several drugs that influence the concentration of non-esterified fatty acid (NEFA) or amino acids in the plasma of normal rats on NAR plasma tryptophan and NEFA concentrations and brain tryptophan metabolism were investigated. These drugs did not alter plasma NEFA and free tryptophan in NAR in the same direction as in normal rats. This difference may be caused by the difference of tryptophan binding proteins in the plasma of these two strains.

Studies on the mechanisms by which clenbuterol, a β-adrenoceptor agonist, enhances 5-HT-mediated behaviour and increases metabolism of 5-HT in the brain of the rat

The head twitch response in mice produced by injection of 5-hydroxytryptophan (100 mg/kg i.p.) and carbidopa (25 mg/kg i.p.) was enhanced by administration of clenbuterol (0.5 mg/kg i.p.), a β-adrenoceptor agonist. Clenbuterol also enhanced the hyperactivity syndrome in rats produced by quipazine (25 mg/kg i.p.), a 5-hydroxytryptamine (5-HT) agonist. This enhancement was not prevented by depletion of 5-HT in brain with p-chlorophenylalanine or after pretreatment with prazosin. The behavioural responses of the rats to administration of the α 2-adrenoceptor agonist, clonidine, was unaltered by acute or longer-term administration of clenbuterol. Following chronic administration of clenbuterol (5 mg/kg daily for 14 days), a procedure resulting in down-regulation of central β-adrenoceptors, a larger dose of clenbuterol was necessary to enhance the quipazine-induced hyperactivity, suggesting that the mechanism of enhancement involved central post-synaptic β-adrenoceptors. Further evidence for this conclusion was that a lesion of central noradrenaline pathways produced by 6-hydroxydopamine did not abolish the clenbuterol-induced enhancement of the quipazine-mediated behaviour. The binding characteristics of 5-HT 2-receptors were unchanged by acute or chronic administration of clenbuterol. Clenbuterol (5 mg/kg) increased the percentage of plasma free (non-albumin bound) tryptophan, plasma free fatty acid concentration and the concentration of tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) in the brain. The increase in 5-HT turnover in brain was prevented by pretreatment with the β 1-adrenoceptor antagonist atenolol, which enters the brain poorly. It is therefore suggested that the clenbuterol-induced increase in 5-HT metabolism results from the increase in the concentration of plasma free fatty acid which increases plasma free tryptophan and thus increases the concentration of tryptophan in brain and 5-HT synthesis in brain. The clenbuterol-induced enhancement of 5-HT-mediated behaviour is therefore not associated with its effect on 5-HT metabolism. The data are discussed in relation to that obtained after administration of antidepressant drugs.

L-Tryptophan: Effects on daytime sleep latency and the waking EEG

The effects of l-tryptophan (4 g) on the waking EEG and daytime sleep were studied in a group of 20 normal adults. Subjects were assigned to a morning or afternoon group, and data were collected on two occasions, after l-tryptophan and after placebo, assigned in a counterbalanced order. l-Tryptophan significantly reduced sleep latency without altering nap sleep stages and elevated plasma total and free tryptophan levels. EEGs were digitized on-line and later analyzed for changes in 5 frequency bands: 16–40 c/sec (beta), 13.0–15.5 c/sec (sigma), 8.0–12.5 c/sec (alpha), 4.0–7.5 c/sec (theta) and 0.5–3.5 c/sec (delta). During waking EEGs, l-tryptophan significantly increased alpha time, theta time, and theta intensity and significantly decreased alpha frequency. No wave bands were altered during sleep. l-Tryptophan is an effective daytime hypnotic which can facilitate sleep onset at clock times which do not coincide with biological sleep times. The hypnotic effects may be mediated by lowering arousal level during the awake state, thus setting the stage for more rapid sleep onset.

Increased central serotonergic activity associated with nocturnal anorexia induced by Walker 256 carcinoma

The role of brain serotonin levels in Walker 256 tumor induced anorexia was investigated. Total and free plasma tryptophan, regional brain serotonin and 5-hydroxyindoleacetic acid were determined at night, and their relationship to nocturnal anorexia assessed by linear regression analysis. No significant difference in tryptophan, serotonin, or 5-hydroxyindoleacetic acid levels was detected between pair fed and tumor bearing rats exhibiting a 20% reduction of nighttime food intake. Tumor bearing rats with a 40% reduction in food intake had higher nighttime plasma free tryptophan and regional 5-hydroxyindoleacetic acid levels than their pair fed malnourished controls. These results indicate that increased plasma free tryptophan and elevated serotonin metabolism may not be the initial dysfunction responsible for nocturnal anorexia. However, it may contribute to the decreasing nocturnal food intake in severely anorexic tumor rats.

Delay of cancer anorexia following intraventricular injection of para-chlorophenylalanine

Serotonergic mediation of cancer anorexia was investigated in immature female rats following the intraventricular injection of parachlorophenylalanine (PCPA) or normal saline. Significant anorexia developed 6 days after the induction (IM) of Walker 256 carcisarcomas in saline-treated rats. Although tumor-bearing rats treated with PCPA ate less than PCPA-injected controls by day 7, their feeding response was significantly greater than that of saline-treated tumor-bearing rats on days 5, 6 and 7. The PCPA treatment had no significant effect on food intake in nontumor-bearing rats. Biochemical analysis revealed significant elevations in plasma free tryptophan, brain tryptophan and brain 5-hydroxyindoleacetic acid in saline-treated tumor-bearing rats. Brain serotonin, 5-hydroxyindoleacetic acid and norepinephrine levels were decreased in PCPA-treated rats. Although these data may provide some support for a serotonergic mediation of cancer anorexia, additional mechanisms are clearly indicated.