Effect of albumin binding and amino acid competition on tryptophan uptake into brain.

Abstract— In the present study we examine the influence of pH, palmitate, and neutral amino acids on the passage of tryptophan from blood into brain during a single capillary pass, and on the partitioning of tryptophan between free and albumin-bound forms. The results show that a considerable fraction of albumin-bound tryptophan is stripped from albumin sites during passage, that uptake is concentration-dependent, and that amino acid competition for carrier sites is quantitatively the most important factor in regulating tryptophan uptake into brain. The interaction between tryptophan concentration, tryptophan binding, and competing amino acids is of considerable influence on brain serotonin biosynthesis.     

The metabolism of L-tryptophan by isolated rat liver cells. Effect of albumin binding and amino acid competition on oxidatin of tryptophan by tryptophan 2,3-dioxygenase.

1. Novel methods, using L-[ring-2-14C]tryptophan, are described for the measurement of tryptophan 2,3-dioxygenase activity and tryptophan accumulation in isolated rat liver cells. 2. The effects of bovine serum albumin, non-esterified fatty acids and neutral amino acids on tryptophan oxidation by hepatocytes and on the partition of tryptophan between free and albumin-bound forms were investigated. 3. Oxidation of physiological concentrations (0.1 mM) of tryptophan was inhibited by approx. 50% in the presence of 2% (w/v) bovine serum albumin; no effects were found at tryptophan concentrations of 0.5 mM and above. 4. Increases in free tryptophan concentrations produced by displacement of 0.1 mM-tryptophan from albumin-binding sites by palmitate resulted in increased flux through tryptophan dioxygenase. 5. Addition of a mixture of neutral amino acids, at plasma concentrations, to hepatocyte incubations had no effect on the rate of tryptophan oxidation. 6. It is concluded that alterations in free tryptophan concentrations consequent to changes in albumin binding may be an important factor in regulating tryptophan uptake and catabolism by the liver. The results are briefly discussed with reference to possible consequences on brain tryptophan metabolism.     

Involvement of the liver in the regulation of tryptophan availability. Possible role in the responses of liver and brain to starvation

The concentrations of free and total (free plus albumin bound) tryptophan were measured in plasma of blood taken from the portal vein, hepatic vein and abdominal aorta of male rats, fed, and starved for one and three days. Liver and brain tryptophan concentrations were measured in similar groups of rats.On starvation, there was an increase in arterial plasma free tryptophan concentration which took place peripherally and was paralleled by an increase in brain tryptophan. In both the fed and starved rats, the portal vein concentrations of free tryptophan were high and as the blood flowed through the liver they were reduced to relatively low levels not directly related to the arterial values. All these changes were due to alterations in degree of binding of tryptophan to plasma albumin.The measurements of plasma total tryptophan concentrations showed that postabsorptively and during starvation there was a net uptake of tryptophan by the peripheral tissues (which included brain), but no overall fall in plasma concentration. At the same time, there was a net release from the liver, and to a lesser extent from the portal-drained tissues. The released tryptophan largely entered the albumin bound plasma pool. Accompanying the hepatic output was a fall in tryptophan concentration in the liver which was apparently caused by altered cell membrane transport.The results suggest (1) that the liver protects the brain from the high free tryptophan level in portal blood, (2) that the availability of tryptophan to the brain is maintained postabsorptively and during starvation by hepatic output into the albumin bound pool and (3) that this release of tryptophan from the liver and the fall in intracellular tryptophan concentration are initiated by altered membrane transport. The pattern of changes is consistent with a role for tryptophan in the mediation of changes in liver protein synthesis and gluconeogenesis and cerebral serotonin turnover on starvation.     

Effect of Age on Variables Influencing the Supply of Tryptophan to the Brain

Abstract: The relations of plasma concentrations of substances claimed to influence brain tryptophan concentration (total tryptophan, free tryptophan, large neutral amino acids) with the concentrations of tryptophan, 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in the forebrain were investigated in rats of different ages (from 8 days to 16 months after birth). In brain, tryptophan fell by 46%, whereas 5-HT rose by 20% between 8 and 40/42 days after birth. Thereafter, the levels of both tryptophan and 5-HT remained essentially constant. Brain 5-HIAA showed a more complex pattern, rising by 63% between 8 and 19 days, falling between 19 and 40/42 days, and then gradually rising until values at 16 months were significantly higher than those at 40/42 days. In plasma, the concentrations of free fatty acids, free and total tryptophan, and large neutral amino acids all decreased between 8 and 19 days and thereafter either remained constant or increased slowly, the exception being total tryptophan values, which showed large increases between 28/30 and 60/70 days. Also, the unidirectional uptake of tryptophan from blood to brain was determined using a carotid artery injection technique. Uptake values obtained using a tracer concentration of tryptophan in the injection solution decreased progressively with age. Kinetic analysis of the data in terms of the Michaelis-Menten equation for carrier-mediated transport indicated significantly lower values for V_max and K_D (a component for nonsaturable transport) in 6-month-old rats as compared to 19-day-old suckling rats, whereas K_m values were the same at both ages. Detailed analysis of these results indicated that the age-related changes in brain tryptophan were largely explicable in terms of plasma free tryptophan in association with blood-brain transport characteristics; moderate differences in concentration of amino acids competing for transport were without apparent effect between 19 days and 16 months. The larger differences between 8 and 19 days after birth could be important.     

Increased uptake of fatty acids by the isolated rat liver after raising the fatty acid binding protein concentration with clofibrate

Following the administration of clofibrate to rats, the concentration of Z protein or fatty acid binding protein in liver cytosol increases by 98 %. Ligandin concentration remains unchanged. Isolated perfused livers of clofibrate-treated rats take up free fatty acids from the perfusate at a significantly higher rate (+ 76 %) than controls. Lipid synthesis from radioactive fatty acids is not modified by clofibrate administration. The yield of plasma membranes obtained from liver homogenates as well as their lipid composition are similar in control and clofibrate treated livers. These results seem to exclude the possibility that the enhancement of FFA uptake could result from an indirect effect of the drug on FFA metabolism and/or plasma membrane surface and thus support the view that Z protein plays a role in intracellular fatty acid transport in the liver.     

Characteristics of Tryptophan Accumulation by Isolated Rat Forebrain Synaptosomes

Uptake of 10 microM L-tryptophan into isolated rat brain synaptosomes was studied to assess its effect on the rate of serotonin synthesis from tryptophan. The initial rate of uptake was rapid, being two orders of magnitude above the rate of tryptophan hydroxylation. Uptake was highly concentrative, the concentration ratio across the plasma membrane at equilibrium being approximately 9. This concentration ratio was decreased to about 1 in the presence of high concentrations of amino acids transported by the L-type neutral amino acid uptake system. A mixture of the large neutral amino acids at physiological concentrations decreased the internal tryptophan concentration to 58% of that in their absence. Large tryptophan concentration ratios were observed in experiments in which Na+ in the medium was replaced with choline+. The concentrative uptake of tryptophan was energy-dependent, being decreased by inclusion of cyanide and omission of glucose. The concentration gradient was abolished by veratridine or rotenone. Time courses of the changes in ATP content and tryptophan concentration ratio on addition of these and other agents established that tryptophan uptake is probably not driven by ATP hydrolysis or efflux of other amino acids, but by the plasma membrane potential.     

Correlation between brain tryptophan and plasma neutral amino acid levels following food consumption in rats

Brain tryptophan increases significantly within two hr of the time that rats begin to consume a diet containing carbohydrate and fat, but fails to rise if the diet also contains 18–24% protein. The effects of particular diets on brain tryptophan are not well correlated with plasma tryptophan concentrations alone, but do correlate well with the ratio of plasma tryptophan to individual neutral amino acids (leucine, isoleucine, valine, tyrosine, phenylalanine) or to their sums. (These amino acids compete with tryptophan for uptake into the brain.) Carbohydrate ingestion raises brain tryptophan by elevating plasma tryptophan and depressing the plasma levels of the competing neutral amino acids; protein consumption prevents an increase in brain tryptophan by raising the plasma concentrations of the competing amino acids more than of tryptophan.     

Tryptophan transport through the blood-brain barrier: in vivo measurement of free and albumin-bound amino acid

The principles of the competitive ligand binding assay have been extended to the in vivo state to study the competition for tryptophan binding between albumin and the tryptophan transport system localized in the brain capillary wall, i.e., the blood-brain barrier (BBB). Based on the concentration of albumin (1.4 mM) which yields 50% inhibition of BBB tryptophan transport, the dissociation constant of tryptophan binding to albumin (K_D = 0.13 mM), and the affinity constant of the BBB tryptophan transport system (K_M = 0.19 mM), the apparent binding capacity of the BBB (1.9 mM), may be calculated. The high apparent binding capacity of the BBB enables the capillary transport system to compete with albumin for tryptophan binding; the inhibition of albumin binding by the carrier increases the fraction of tryptophan that is free in vivo to values greater than the fraction that is free in vitro. Depending on physiological changes in K_M (e.g., due to plasma amino acid competition) or K_D (due to plasma free fatty acid), the apparent free fraction of tryptophan in vivo may approximate either one of two extremes, i.e., the in vitro free fraction or the total tryptophan.     

Transport of Tryptophan into Brain from the Circulating, Albumin-Bound Pool in Rats and in Rabbits

Tryptophan is the only amino acid in the circulation that is bound by albumin, and previous studies have suggested that the brain tryptophan supply is a function of either the free or the albumin-bound pool of tryptophan in blood. Since the albumin molecule per se does not cross the brain capillary wall, i.e., the blood-brain barrier (BBB), the transport of tryptophan from the circulating albumin-bound pool may involve enhanced dissociation of tryptophan from the albumin binding sites within the cerebral microcirculation. This hypothesis was confirmed in the present studies wherein the dissociation constant (KaD) of albumin binding of tryptophan in the rat or rabbit brain microcirculation was measured in vivo. Brain extraction data for [14C]tryptophan determined with the carotid artery injection technique were fit to the Kety-Renkin-Crone equation modified for protein-bound solute. The KaD of albumin binding in the rat or rabbit brain microcirculation under pentobarbital anesthesia was 1.7 +/- 0.1 and 3.9 +/- 1.0 mM, respectively, as compared to the KD value measured in vitro with equilibrium dialysis, 0.13 +/- 0.03 mM. In contrast, the KaD value of albumin binding of tryptophan in vivo in the rabbit brain microcirculation was reduced by ether anesthesia to a value of 2.1 +/- 0.4 mM. This reduction in the KaD under ether anesthesia was associated with a 2.5-fold increase in cerebral blood flow. In addition, dialyzed rabbit serum caused a statistically significant inhibition in [14C]tryptophan influx during ether, but not pentobarbital, anesthesia.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 STUDY OF PROPOSED DETERMINANTS OF BRAIN TRYPTOPHAN CONCENTRATION IN RATS AFTER PORTOCAVAL ANASTOMOSIS OR SHAM OPERATION

Liver dysfunction was produced in rats by surgical portocaval anastomosis (PCA), and the time-course of changes in brain tryptophan and 5-HT metabolism studied in relation to plasma changes possibly influencing brain tryptophan concentration. Brain tryptophan and 5-hydroxyindolylacetic acid (5-HIAA) levels were increased greatly and maximally on the day after PCA and remained high. 5-HT changes were less marked but had a similar time-course. Plasma total tryptophan was little changed but plasma free tryptophan was raised. The latter change showed a similar time-course to that of brain tryptophan but was not large enough to account completely for it. Sham operation was followed by significant but transient increases in plasma free tryptophan, brain tryptophan and 5-HIAA but these were much smaller than after PCA. Brain tryptophan did not correlate with plasma total tryptophan either in control or PCA rats but it correlated significantly with plasma free tryptophan in both groups. However brain levels were much higher in PCA rats than in controls with similar plasma free tryptophan levels at all times from the first day after operation. The increase of brain tryptophan in anastomosed rats not accounted for by plasma free tryptophan was explained neither by insulin changes nor by an increase of the insulin/glucagon ratio nor by changes in plasma concentrations of those amino acids which compete with tryptophan for entry into brain. The results therefore indicate an unknown influence on brain tryptophan concentration in PCA rats. As tyrosine changes in brain and plasma after PCA were very similar to those of tryptophan this influence may not be specific to tryptophan. Results suggest that under the conditions used brain tryptophan concentrations of both PCA and control rats are more influenced by changes of plasma free tryptophan concentration than by changes of plasma concentrations of competing amino acids.     

The effects of diet, lipolysis and limb ischaemia on the distribution of plasma tryptophan in the rat.

A non-linear relationship between the plasma non-esterified fatty acid concentration and the percentage of free plasma tryptophan was found in rats in different nutritional states, although non-esterified fatty acids are not the only factors determining the percentage of free tryptophan. This relationship was not seen in rats injured by limb ischaemia. The effect of drugs causing rapid increases in the plasma non-esterified fatty acid concentration was also studied. Isoprenaline decreased the total plasma tryptophan concentration. Dichloroisoprenaline caused a sustained increase in the plasma non-esterified fatty acid concentration which was accompanied by an increase in the concentration of free plasma tryptophan and followed by a fall in the concentration of total tryptophan. The loss of tryptophan from the plasma was attributed to an altered distribution of tryptophan in the extracellular space rather than to increased metabolism. This interpretation was supported by determinations of the irreversible disposal rate of plasma tryptophan which in uninjured rats was unaffected by the concentration of free plasma tryptophan. In the injured rats this rate was unaltered during limb ischaemia but was decreased after removal of the tourniquets; increased competition for tissue entry by other neutral amino acids and the fall in body temperature could be factors in this fall.     

Studies on the uptake of radioactive copper by sheep erythrocytes in vitro

From previous studies different mechanisms for uptake of Cu by mammalian tissues from buffer and media containing proteins have been proposed. The interpretation of some of these investigations may have been complicated by the binding of Cu to proteins in the media.The uptake of ⁶⁴Cu from both buffer and plasma has therefore been studied using sheep erythrocytes in order to determine the mechanism and the effects of protein. Cu uptake was proportional to concentration of added Cu and the kinetics were those of a first order reaction for both media. There was no evidence for the participation of a membrane carrier and studies with inhibitors indicated that active transport was not involveed. For a given concentration of added Cu the rate of uptake was much slower in plasma and the effective concentration was calculated to be about 13% of that added. Efflux from labelled cells was much faster into plasma than into buffer. The addition of histidine to the medium increased uptake from dialysed plasma but not from buffer.It is concluded that Cu is taken up by the erythrocyte by simple diffusion, and the reduced rate in plasma is due to the binding of Cu to plasma protein thus reducing the effective concentration. The effect of histidine is attributed to the formation of a Cu-histidine complex which exists in equilibrium with Cu bound to albumin and facilitates removal of Cu from the latter.     

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.     

CORRELATION OF PLASMA AND BRAIN AMINO ACID AND PUTATIVE NEUROTRANSMITTER ALTERATIONS DURING ACUTE HEPATIC COMA IN THE RAT

During acute hepatic coma following two-stage hepatic devascularization in the rat, profound changes occurred in plasma and whole-brain amino acids and putative neurotransmitters. Brain ammonia, glutamine and GABA were increased, aspartate was decreased, while glutamate was unchanged. An increase in brain tryptophan was accompanied by a similar increase in plasma unbound tryptophan but decreased plasma total tryptophan. These changes occurred in the presence of high plasma levels of the other neutral amino acids, including the branched chain amino acids. Plasma insulin was unchanged while glucagon levels rose, resulting in a decreased insulin to glucagon ratio. These results suggest that while plasma unbound tryptophan may influence brain tryptophan levels, altered plasma concentrations of neutral amino acids which compete with tryptophan for transport into the brain do not contribute to the increase in brain tryptophan observed during acute hepatic coma.     

EFFECTS OF CHRONIC PORTO-CAVAL ANASTOMOSIS ON BRAIN TRYPTOPHAN, TYROSINE AND 5-HYDROXYTRYPTAMINE

—Three weeks after porto-caval anastomosis, tryptophan and 5-hydroxyindolylacetic acid concentrations were-greatly increased in rat brain regions. 5-Hydroxytryptamine showed smaller increases. Midbrain tyrosine and muscle tyrosine and tryptophan concentrations were also increased. Striatal dopa-mine concentration was not significantly changed. Unlike previous results from acute liver failure, brain tryptophan changes in this chronic study did not simply reflect plasma-free tryptophan changes. Midbrain tryptophan/plasma-free tryptophan ratio and midbrain tyrosine/plasma tyrosine ratio both rose, suggesting increased effectiveness of uptake of these amino acids from plasma by brain. Corresponding muscle/plasma ratios were unaltered by the porto-caval anastomosis. Uptake of tryptophan from buffer by cerebral cortex slices was unaffected. Results on control animals illustrate the importance of plasma-free tryptophan in the normal physiological control of brain tryptophan.     

Transmembrane transport of fatty acids in the heart

Summary Although fatty acid uptake by the myocardium is rapid and efficient, the mechanism of their transmembrane transport has been unclear. Fatty acids are presented to the plasma membrane of cardiomyocytes as albumin complexes within the plasma. Since albumin is not taken up by the cells, it was postulated that specific high affinity binding sites at the sarcolemma may mediate the dissociation of fatty acids from the albumin molecules, before they are transported into the cells. In studies with a representative long-chain fatty acid, oleate, it was in fact shown that fatty acids bind with high affinity to isolated plasma membranes of rat heart myocytes revealing a K_D of 42 nM. Moreover, a specific membrane fatty acid-binding protein (MFABP) was isolated from these membranes. It had a molecular weight of 40 kD, an isoelectric point of 9.0, and lacked carbohydrate or lipid components. Binding to a specific membrane protein might represent the first step of a carrier mediated uptake process. Therefore, the uptake kinetics of oleate by isolated rat heart myocytes was determined under conditions where only cellular influx and not metabolism occurred. Uptake revealed saturation kinetics and was temperature dependent which were considered as specific criteria for a facilitated transport mechanism. For evaluation whether uptake is mediated by MFABP, the effect of a monospecific antibody to this protein on cellular influx of oleate was examined. Inhibition of uptake of fatty acids but not of glucose by the antibody to MFABP indicated the physiologic significance of this protein as transmembrane carrier in the cellular uptake process of fatty acids. Such a transporter might represent an important site for the metabolic regulation of fatty acid influx into the myocardium.     

PLASMA TRYPTOPHAN AND 5-HT METABOLISM IN THE CNS OF THE NEWBORN RAT

—The relationships between plasma tryptophan and 5-HT metabolism in the CNS were studied in newborn rats and compared with adults. Both the concentration of free tryptophan in plasma and that of the amino-acid in brain were much higher immediately after birth than later on. Drugs such as salicylate and chlordiazepoxide, which increased brain tryptophan concentrations in adults by displacing the plasma amino acid bound to serum albumin, were ineffective in newborn rats: most of the amino acid being already free in their plasma. The study of 5-HT metabolism in brain stem slices revealed that the affinity of the uptake process for tryptophan was higher in newborn than in adult animals, whereas the reverse situation was observed for the enzyme complex involved in 5-HT synthesis (lower apparent K_m in adults). In addition, the catabolism of newly synthesized 5-HT was more rapid in newborn than in adult tissues. Finally, the free state of tryptophan in plasma of newborn animals induced in brain both a high amino acid concentration and, in contrast to the situation observed in adults, a synthesis rate of 5-HT very near its maximal value.     

PLASMA AND BRAIN TRYPTOPHAN CHANGES IN EXPERIMENTAL ACUTE HEPATIC FAILURE

An experimental model for acute hepatic failure in man was obtained in pigs by hepatic devascularization. After operation, liver function was grossly impaired, movements became inco-ordinated and coma ensued. Most animals died 5½–8½ h after operation. Plasma unesteritied fatty acid and free (but not total) tryptophan concentrations rose markedly after operation and correlated significantly with each other. Brain tryptophan concentration increased and correlated significantly with plasma free tryptophan concentration. Increased tryptophan was found in the four brain regions studied (hypothalamus, thalamus, caudate and cortex) and was associated with raised 5-hydroxytryptamine turnover as indicated by raised 5-hydroxyindolylacetic acid concentration. Results are discussed in relation to altered tryptophan metabolism in human hepatic coma and to investigations of the influence of plasma unesterified fatty acid and free tryptophan changes on brain tryptophan metabolism in the rat.     

Ethanol impairs tryptophan transport into the brain and depresses serotonin

Alcoholics were found to have decreased plasma levels of tryptophan, the serotonin precursor, and a decreased ratio of tryptophan over amino acids competing for transport into the brain. Studies conducted in the plasma of rats and baboons with carefully controlled alcohol and dietary intake showed a decreased in the ratio of tryptophan over competing amino acids resulting mostly from increases in valine in the rat and in valine, leucine and isoleucine in the baboon. In the rat concomitant decreases in brain tryptophan and serotonin were noted. Central serotonin dificiency may contribute to the depressive states frequently seen in alcoholics.