Rates of exchange of free amino acids between plasma and brain in mice

The rate of appearance of label in the brain in mice following the intraperitoneal or intravenous injection of tracer doses of amino acids was measured in short time periods (1–8 min). Amino acid flux varied between 2 and 10 nmol/min per g brain for the amino acids used. Defining half-life as the uptake of labeled amino acid amounting to 50% of endogenous levels, a short half-life (between 3 and 30 min) was found for the essential amino acids. The half-life of the nonessential amino acids varied between 2 and 24 h, depending on their level in brain. Flux (exchange) of an amino acid was increased when the level of amino acids belonging to the same transport class was increased by intracerebral injection. Protein-free diet resulted in decrease in some amino acids, increase in others; flux was altered parallel to changes in brain levels in animals on this diet. The stercospecificity of exchange and the substrate specificity of effects of altered brain amino acids indicate that exchange occurs via mediated transport. Mediated exchange was present in immature brain. Heteroexchange (flow of one amino acid causing the counterflow of a related amino acid) may play an important part in cerebral homeostasis.     

Kinetic analysis of blood-brain barrier transport of amino acids.

The Michaelis-Menten kinetics of blood-brain barrier transport of fourteen amino acids was investigated with a tissue-sampling, single-injection technique in the anesthetized rat. Tracer quantities of 14C-labelled amino acids and 3H2O, used as a freely diffusible internal reference, were mixed in 0.2 ml of buffered Ringer's solution and injected rapidly into a common carotid artery. Circulation was terminated by decapitation at 15s following injection. A brain uptake index (Ib) was determined from the ratio of 14C dpm in the brain tissue and the injection mixture divided by the same ratio for the 3H2O reference. Brain clearance of tracer concentration of amino acid was saturable when various concentrations of unlabeled amino acid were added to the injection solution. Double reciprocal plots of the saturation data yielded Km (mM) values that ranged from a low of 0.09 mM for arginine to a high of 0.75 mM for cycloleucine. Transport V values were determined from the relationship P = V/Km where P is the blood-brain barrier permeability constant (ml/g per min): P was calculated from the Ib for each amino acid based on a cerebral blood flow of 0.56 ml/g per min and a fractional extraction of 0.75 for the 3H2O reference 15s following carotid injection. The V values ranged from a low of 6.2 nmol/g per min for lysine to a high of 64 nmol/g per min for l-DOPA. Efflux of the tracer amino acid during the 15-s period after injection was assumed to be slow, since the rate constant of cycloleucine from brain to blood was low, 0.11 min-1.     

Effect of γ-glutamyl cycle inhibitors on brain amino acid transport and utilization

Two inhibitors of the -glutamyl cycle, methionine sulfoximine (MSO) and 2-imidazolidone-4-carboxylic acid (ICA) were administered to C57BL/6J mice. Both agents resulted in a reduced rate of transport of tyrosine from blood to brain and a decreased rate of incorporation of tyrosine from plasma into brain protein. MSO administration also diminished the concentrations of brain tyrosine, dopamine, and norepinephrine. MSO decreased the transport rate of valine by brain as well as the rate of its incorporation into protein when expressed in relation to the plasma specific activity. The results demonstrate a significant role for the -glutamyl cycle in the transport of large neutral amino acids from blood to brain.Presented in part in the April 1977 meeting of the American Academy of Neurology, Atlanta, Georgia.     

Synaptosomal uptake

Using purified synaptosomal preparations from rat brain, the uptake ofl-tryptophan and norepinephrine was studied. We were unable to replicate some of the results of the experiments obtained with crude mitochondrial, fractions (P₂). Thus we examined the validity of the results of uptake studies obtained with the crude synaptosomes and established conditions which would simulate the biochemical milieu in which the nerve terminals functionin vivo, such as active substrate-dependent respiration, respiratory coupling on addition of ADP, low impurity of noncharacteristic markers, exogenous added proteins (e.g. bovine serum albumin), and verification by electron microscopy. All uptake studies withl-TRP and NE were completed in a system designed for simultaneous recording of respiration and the effect of added ADP. This system was also employed in comparative studies with mitochondria purified by multiple density gradients derived either from the perikaryon or from synaptosomes. Synaptosomal or mitochondrial preparations which did not conform to the above criteria invariably showed significantly lowered ability of uptake ofl-TRP or NE. This was found to be related to impairment in their respiratory and coupling ability. When the experimental conditions of others were employed, the time course of uptake of TRP for crude synaptosomes (P₂) was 100 nmol/g/min and was linear for 2.5 min, while for the purified synaptosomes it was 20 nmol/g/min with a l-min linearity. The mitochondria purified from P₂ displayed 30 nmol/g/min uptake withl-TRP with a linearity of 2.5 min. Reconstituted system of purified synaptosomes and mitochondria gave 60 nmol/g/min ofl-TRP transport with 2.5 min linearity. Also examined was the effect of eight different media. It was found that Krebs-Ringer solution containing glucose (40 mM), pyruvate and malate (10 mM), and ADP (250 nmol) gave optimal uptake of TRP both for synaptosomes and for mitochondria, increasing it to 60 and 86 nmol/g/min. The above conditions also enhanced the uptake of NE by synaptosomes and mitochondria. Uptake of NE was not proportional to protein concentration when the protein content exceeded 0.4 mg. Purified synaptosomal mitochondria accumulated NE more actively than the purified nonsynaptic free mitochondria, albeit at the same rate. Synaptic and free mitochondria had an impaired uptake of NE in presence of DNP, antimycin A, and rotenone, and unlike withl-TRP, pyruvate and malate also reduced uptake of NE. Significant differences were noted for the cytochrome oxidase activity between the synaptosomal and free michondria when compared to that of the homogenate.     

Age related changes in blood-to-brain amino acid transport and incorporation into brain protein

Blood-to-brain amino acid transport consists of at least two components: 1. a fast rate or early process, commonly measured by the intra-carotid bolus injection method and attributed to transport across the capillary endothelium and entry into the astrocytes, and, 2. a slow rate or later component measured over 2 to 15 minutes probably associated with exit from the astrocytes and entry into the neurons. Incorporation into brain protein is temporally related to the second process. In the present study the slow and fast rate transport components and the incorporation into brain protein of tyrosine (Tyr) and Valine (Val) was measured in young adult and aged male C57BL/6 mice. The results indicate that the fast rate transport component is unaffected by age while the rates of the slow process and protein turnover show an exponential decline most marked between 3 and 8 months of age. Changes in the relative incorporation of Tyr and Val suggest that brain protein metabolism is altered qualitatively as well as quantitatively in aging, in these animals.     

Pathways for alanine transport in intestinal basal lateral membrane vesicles

Summary Membrane vesicles obtained from the basal lateral membranes of the rat intestinal epithelium were used to study the pathways for neutral amino acid transport.In the absence of sodium there was a stereospecific uptake ofl-alanine which exhibited saturation kinetics (K _m 0.73mm andV _max 5.3 nmol/mg min at 22°C). The activation energy for this process was 8.1 kcal/mole between 5 and 25°C. Preloading the vesicles with alanine increased the unidirectional influx of alanine into the vesicle. Competition experiments indicated that the affinity of the sodium-independent transport system was glutamine > threonine > alanine > phenylalanine > valine > methionine > glycine > histidine > proline, N-MeAIB. These are the characteristics of the classical L transport system.External sodium increased the rate of the stereospecificl-alanine uptake. The Na-dependent flux had aK _m of 0.04mm and aV _max of 0.26 nmol/mg min at 22°, and an activation energy of 9.1 kcal/mole between 5 and 25°C. Competition experiments suggest the existence of three separate pathways for alanine transport in the presence of sodium. A major pathway is shared by all other amino acids tested (i.e., threonine, glutamine, methionine, phenylalanine, valine, proline and N-MeAIB). This resembles the classical A system. A second pathway is unavailable to either phenylalanine or N-MeAIB; this is reminiscent of the classical ASC system; and the third is a novel pathway which is shared by N-MeAIB but not phenylalanine.The sodium-independent and the sodium-dependent transport ofl-alanine was blocked by PCMBS and significantly inhibited by DTP and NEM. It is concluded that the sodium-independent system (the L-like system) accounts for the efflux of neutral amino acids from the epithelium to the blood during the absorption of amino acids from the gut, and that the sodium-dependent transport processes may play an important role in the supply of amino acids to the epithelium in the absence of amino acids from the gut lumen.     

Blood-brain barrier transport ofL-pipecolic acid in various rat brain regions

Blood-brain barrier transport ofL-[l-¹⁴C]pipecolic acid was studied in the rat by single intracarotid injection using³H₂O as a diffusible internal standard. Brain uptake index (BUI) forL-[¹⁴C]pipecolic acid (0.036 mM) was found to be 18.1, 10.5, and 12.6 for the cerebral cortex, brain stem, and cerebellum, respectively which was substantially higher than that reported for its analogL-proline in the whole brain. Influx ofL-pipecolic acid into the brain was concentration dependent and differed significantly between the cerebral cortex and the brain stem, and between the cerebral cortex and the cerebellum, but not between the brain stem and the cerebellum. Kinetic study ofL-pipecolic acid influx revealed a low- and a high-capacity uptake mechanisms. The low-capacity saturable component hasK _m values ranging from 38 to 73 μM, andV _max values ranging from 10 to 13 nmol/g/min for the three brain regions. The nonsaturable component has aK _m of 4 mM, aV _max of 200 nmol/g/min and similar diffusion constant (K _d) (0.03 to 0.06 mlg^?1 min^?1) for all three brain regions. A possible role of the two-component brain uptake mechanism in the regulation of the neuronal function ofL-pipecolic acid was suggested.     

Kinetic analysis of blood-brain barrier transport of amino acids

The Michaelis-Menten kinetics of blood-brain barrier transport of fourteen amino acids was investigated with a tissue-sampling, single-injection technique in the anesthetized rat. Tracer quantities of ¹⁴C-labelled amino acids and ³H₂O, used as a freely diffusible internal reference, were mixed in 0.2 ml of buffered Ringer's solution and injected rapidly into a common carotid artery. Circulation was terminated by decapitation at 15 s following injection. A brain uptake index (I_b) was determined from the ratio of ¹⁴C dpm in the brain tissue and the injection mixture divided by the same ratio for the ³H₂O reference. Brain clearance of tracer concentration of amino acid was saturable when various concentrations of unlabeled amino acid were added to the injection solution. Double reciprocal plots of the saturation data yielded K_m (mM) values that ranged from a low of 0.09 mM for arginine to a high of 0.75 mM for cycloleucine. Transport V values were determined from the relationship $\text{P =}\text{V}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ where P is the blood-brain barrier permeability constant (ml/g per min): P was calculated from the I_b for each amino acid based on a cerebral blood flow of 0.56 ml/g per min and a fractional extraction of 0.75 for the ³H₂O reference 15 s following carotid injection. The V values ranged from a low of 6.2 nmol/g per min for lysine to a high of 64 nmol/g per min for l-DOPA. Efflux of the tracer amino acid during the 15-s period after injection was assumed to be slow, since the rate constant of cycloleucine from brain to blood was low, 0.11 min^?1.     

Differential effects of 1-deoxynojirimycin on the intracellular transport of secretory glycoproteins of human hepatoma cells in culture

To investigate our earlier hypothesis that carbohydrates play a regulatory role in the intracellular transport of secretory glycoproteins, we used 1-deoxynojirimycin (DNJ), and inhibitor of glucosidase I and II of the rough endoplasmic reticulum (RER), to modify the structure of N-linked glycan moieties of secretory glycoproteins of human hepatoma (Hep G2) cells in culture. Using a pulse-chase protocol, we found that treatment of Hep G2 cultures with 1.25 mM DNJ markedly reduced the rate of secretion of ₁-protease inhibitor, ceruloplasmin, and ₂-macroglobulin, but had no effect on the export of fibronectin, -fetoprotein and transferrin, nor on albumin which lacks carbohydrate. For example, 50% of newly synthesized ₁-protease inhibitor, the glycoprotein most dramatically affected, was secreted by 27 min in control cultures versus 110 min in DNJ-treated cultures. Percoll gradient cell fractionation analyses revealed that DNJ inhibited transport of the affected secretory glycoproteins in the RER segment of the ER/Golgi pathway. For example, 50% of newly synthesized ₁-protease inhibitor was lost from the RER fraction by 10 min in untreated cells, but 70 min was required for the transport of a similar amount of protein in DNJ-treated cells. DNJ treatment also inhibited the rate at which the N-linked glycan moieties of the affected glycoproteins became resistant to endo H in the Golgi. Since the glycan moiety of secreted forms of the affected glycoproteins were fully processed to the complex structure, suggesting escape from DNJ inhibition, we concluded that removal of terminal glucose residues from the glycan chain of secretory glycoproteins is required for their transport from the RER to the Golgi. We suggest that the oligosaccharide moieties on ₁-protease inhibitor, ceruloplasmin and ₂-macroglobulin form part of the binding site for a receptor which regulates transport of these glycoproteins.     

Regional localization of [14C]mescaline in rabbit brain after intraventricular administration

Albino rabbits of either sex were anesthetized, and a cannula was implanted permanently into the lateral ventricle. About 1 week later, the distribution of [¹⁴C]mescaline and its deaminated metabolite, [¹⁴C]trimethoxyphenylacetic acid ([¹⁴C]TMPA) in 12 brain regions was examined at 15, 60, and 180 min after the intraventricular injection of [¹⁴C]mescaline (0.5 mol in 0.05 ml saline).¹⁴C-radioactivity was rapidly distributed in all regions, reaching peak levels within 15 min. The spinal cord, superior colliculus, pons, hypothalamus, caudate, medulla oblongata, and inferior colliculus contained 23–57 nmol/g of mescaline; the thalamus, tegmentum, and cerebellum, 12–15 nmol/g; and the cerebrum and hippocampus, less than 10 nmol/g; the levels of [¹⁴C]TMPA ranged from 0.5 to 5 nmol/g. The levels of [¹⁴C]mescaline and of [¹⁴]TMPA in all brain areas were considerably decreased 180 min after its injection. Pretreatment with chlorpromazine (15 mg/kg, i.p., 30 min) lowered [¹⁴C]mescaline concentrations in the hippocampus, caudate, thalamus, and cerebrum and elevated them in the spinal cord, medulla oblongata, pons, and tegmentum; [¹⁴C]TMPA levels as the percentage of total radioactivity were not affected. Pretreatment with iproniazid (150 mg/kg, i.p., 18 h), on the other hand, uniformly reduced the TMPA levels in all brain areas, with the resultant increases in mescaline levels. The CPZ-effect in lowering the mescaline concentrations in the areas belonging to the limbic system may have significance in explaining its antihallucinogenic effect in humans and its ability to block the altered behavior induced by the latter drug in laboratory animals.     

The effect of inhibition of hexosemonophosphate shunt on the metabolism of glucose and function in rat brain in vivo

Rats treated 4 hr previously with 6-aminonicotinamide showed a twenty-four fold increase of [¹⁴C]phosphogluconate in the adult brain at 30 min after injection of [U-¹⁴C]glucose indicating a blockade of the hexosemonophosphate shunt. There was a significant increase in the¹⁴C-content of glucose and glucose 6-phosphate, and a decrease in that of amino acids. [¹⁴C]Phosphoglycerate content showed no consistent change after 6-aminonicotinamide treatment. The concentration of glucose and glucose 6-phosphate increased significantly without a significant change in the lactate pool in the brain of 6-aminonicotinamide treated rats. The rate of utilization of glucose in the brain of control rats was 0.73 mol/min per g of brain. It decreased by 16% in rats treated with 6-aminonicotinamide; the results suggested that both glycolysis and pyruvate oxidation were affected. The amount of glucose utilized in the brain by the hexosemonophosphate shunt was approximately 0.0093 mol/min per g of brain, i.e. 1.3% of the total rate of utilization of glucose. The observed changes were not due to hypothermia. The rate of glucose utilization was higher in animals exposed to higher ambient temperature and to stress caused by handling. The results were explained by postulating a role for the hexosemonophosphate shunt in providing neurotransmitter amino acids glutamate and -aminobutyrate, and interdependence of brain function and glucose utilization.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

LYSINE METABOLISM IN THE RAT BRAIN: BLOOD—BRAIN BARRIER TRANSPORT, FORMATION OF PIPECOLIC ACID AND HUMAN HYPERPIPECOLATEMIA

Through the use of intravenous pulse injection of L-[U-¹⁴C] lysine, the blood-brain barrier transport of L-lysine was studied. The uptake of L-lysine plus metabolites in the brain remained essentially unchanged at approx 0.002–0.005 nmol/g in the low dose (3μg per kg body weight) injection, and 20–40 nmol/g in the high dose (30 mg/kg) injection throughout the time intervals of up to 60 min. The uptake of L-lysine plus metabolites in the heart, however, decreased substantially from 0.03 to 0.003 nmol/g in the low dose injection and from 320 to 62 nmol/g in the high dose injection. The plasma to heart uptake ratio only decreased slightly through the 60 min period: from 6 to 2 in either the low or high dose L-lysine injection. The plasma to brain uptake ratio, however, decreased rapidly from a high of 62 to a low of about 4 in either the low or high dose injection throughout the 60-min time course. Study of labeled L-pipecolate formation in the plasma and individual organs indicates that this compound was formed only in the brain to a significant level within 0.5 min of ¹⁴C-L-lysine intravenous pulse injection. Labeled pipecolate was recovered from heart, liver, kidney and plasma in significant quantities only at 2 min or later after pulse-injection. It is concluded that the blood-brain barrier of L-lysine in the rat is not particularly strong and that the rat brain may be primarily responsible for L-pipecolate synthesis from L-lysine. The possible etiology of human hyperpipecolatemia is also discussed in light of the current findings.     

Procedure for measurement of amino acid transport from blood to brain in small animals

The exponential plasma specific activity curve 2.5 to 12.5 min after injection (sc) of [¹⁴C]tyrosine was integrated and divided by time to obtain the mathematical relationship between the average equivalent specific activity $\text{S}$ and the measured specific activity S in any individual animal. $\text{S}$ is the constant, average value of S that is equivalent to the curvllinearly varying quantity that the body tissues are actually exposed to. Dividing the total brain radioactivity by $\text{S}$ gave the tissue Tyr uptake U. The function $\text{dU}\text{dt}$ is linear from 2.5 to 12.5 min and represents the rate of uptake of the amino acid. Incorporation into protein was similarly measured. Brain uptake of Tyr averaged 7.06, and the apparent protein incorporation was 1.99 nmol/g of brain per min. The γ-glutamyl cycle inhibitor l-methionine-RS-sulfoximine reduced total brain uptake of tyrosine by 42.8% and the apparent rate of protein incorporation by 39.0%.     

Choline Transport and Metabolism in Soman-or Sarin-Intoxicated Brain

The metabolism and blood-brain transport of choline (Ch) were investigated in perfused canine brain under control conditions and for 60 min after inhibition of brain cholinesterases by the organophosphorus (OP) compounds soman (pinacolylmethylphosphonofluoridate). Ch and acetylcholine (ACh) in blood and brain samples were analyzed using gas chromatography-mass spectrometry methods. Net transport of Ch was determined by Ch analysis in arterial and venous samples. Unidirectional transport of [3H]Ch was determined using the indicator dilution method. During control perfusion periods of 90 min, net efflux of brain Ch occurred at a rate of 1.6 +/- 0.4 nmol/g/min, and the Ch content of the recirculated perfusate increased 10-fold to approximately 8 microM. Brain Ch content increased in proportion to the increase in perfusate Ch level, but brain ACh was unaltered. Rapid administration of soman (100 micrograms) or sarin (400 micrograms) into the arterial perfusate after a 40-min control period resulted in a greater than 10-fold increase in ACh content in cerebral cortex, brainstem, and hippocampus. The ACh content of cerebellum increased only slightly. The Ch level in all four brain regions studied also increased two- to fourfold above control levels. Ch efflux from brain, however, decreased to 0.2 +/- 0.1 nmol/g/min during the 60 min after OP exposure. Unidirectional influx of [3H]Ch was 0.49 +/- 0.07 nmol/g/min before and did not change significantly 10 or 40 min after OP exposure, thus indicating that the Ch transporter of the brain endothelial cell is not directly inhibited.2+ Based on these results, it is proposed that (a) efflux of brain Ch occurs from the extracellular compartment, which becomes depleted when ACh breakdown is inhibited;(ABSTRACT TRUNCATED AT 250 WORDS)     

TRYPTOPHAN TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER DURING ACUTE HEPATIC FAILURE

Abstract— Tryptophan transport across the blood-brain barrier was studied using a single injection dual isotope label technique, in the following three conditions: normal rats, rats with portacaval shunts, and rats with portacaval shunts followed 65 h later by hepatic artery ligation. In both normal rats and those with acute hepatic failure the tryptophan transport system was found to be comprised of two kinetically distinct components. One component was saturable and obeyed Michaelis-Menten kinetics (normal: V_max= 19.5 nmol.min^?1.g^?1. K_m= 113 μM; hepatic failure: V_max, = 33.8 nmol.min^?1.g^?1, K_m= 108 μM), and the second was a high capacity system which transported tryptophan in direct proportion to concentration over the range tested (normal: K= 0.026 ml.min^?1.g^?1; hepatic failure: K= 0.067 ml.min^?1.g^?1). Since the saturable low capacity component transports several neutral amino acids, and their collective plasma concentration is high in relation to the individual K_ms, tryptophan transport by this component is reduced by competitive inhibition under physiological conditions. Thus it was calculated that in normal rats approx 40% of tryptophan influx occurs via the high capacity system. During acute hepatic failure transport via both components was increased substantially, approximately doubling the rate of tryptophan penetration of the blood-brain barrier at all concentrations tested. The contribution by the high capacity component became even more significant than in normal rats, accounting for about 75% of all tryptophan passage from plasma to brain. Brain tryptophan content was 29.9 nmol/g in normal rats and rose to 45.2 nmol/g in rats with portacaval shunts and 50.5 nmol/g in those with acute hepatic failure, correlating with the increased rate of tryptophan transport. In a previous study we found that plasma competing amino acids were greatly increased during acute hepatic failure. Calculations predict that these increased concentrations would cause a reduction in tryptophan transport by the low capacity system. However, because of the increase in the rate of transport by the high capacity component, net tryptophan entry across the blood-brain barrier was actually increased. This increased rate of transport clearly contributes to the increased content of brain tryptophan found during hepatic failure.     

Amino acid and monoamine transport in primary astroglial cultures from defined brain regions

The uptake ofl-[³H]glutamate,l-[³H]aspartate, -[³H]aminobutric acid (GABA), [³H]dopamine,dl-[³H]norepinephrine and [³H]5-hydroxytryptamine (5-HT) was studied in astrocytes cultured from the cerebral cortex, striatum and brain stem of newborn rat and grown for 2 weeks in primary cultures. The astrocytes exhibited a high-affinityl-glutamate uptake withK _m values ranging from 11 to 110 M.V _max values were 4.5 in cerebral cortex, 39.1 in striatum, and 0.4 in brain stem, nmol per mg cell protein per min. There was a less prominent high-affinity uptake ofl-aspartate withK _m values from 88 to 187 M.V _max values were 7.4 in cerebral cortex, 37.1 in striatum, and 3.1 in brain stem, nmol per mg cell protein per min. The high-affinity GABA uptake exhibitedK _m values ranging from 5 to 17 M andV _max values were 0.01 for cerebral cortex, 0.04 for striatum, and 0.1 for brain stem, nmol per mg cell protein per min. No high-affinity, high-capacity uptake was found for the monoamines. The results demonstrate a heterogeneity among the astroglial cells cultivated from the different brain regions concerning the uptake capacity of amino acid neurotransmitters. Furthermore, amino acid transmitters and monoamines are taken up by the cells in different ways.     

Accumulation and metabolism of pipecolic acid in the brain and other organs of the mouse

The long-term accumulation of pipecolic acid, as well as its disappearance following exogenous administration was studied in brain and other organs of the mouse. Mice were pulse-injected intraperitoneally or intravenously with 1Ci[³H]D,l-pipecolic acid (6.9 nmol/mouse=2.9 g/kg). The total radioactivity retained in tissues was measured in brain, liver, and kidney, as well as in plasma during the period 1 min to 24 hr. TLC separation of DNP-derivatives was performed. Three features of the pattern of retention of pipecolic acid are most salient; first the rapid accumulation in brain, second the rapid secretion of this compound in the urine, and third the long-lasting steady levels of radioactivity maintained in brain.Sixty minutes after i.v. injection, the brain/plasma ratio is approximately 0.2 and approaches unity only at 5 hr. Following intraperitoneal injection the percent recovered as pipecolic acid in brain is 78% at 30 min and 71% at 120 min, suggesting a slow metabolic activity. Liver shows a different trend than brain with a slower accumulation and a faster disappearance. Kidney shows a pattern similar to plasma with a rapid secretion of radioactivity into urine which correlates well with the exponential decrease in plasma and urine. The administration of probenecid significantly increases radioactivity due to pipecolic acid in brain, liver, and urine. Formation of -aminoadipic acid, a known metabolite of pipecolic acid, can be demonstrated in kidney 30 min after intraperitoneal injection. The present data together with results obtained previously with intracarotid injections suggest that pipecolic acid is taken up in the mouse brain from the circulation. Most of the pipecolic acid taken up is rapidly removed through the circulation and secreted in the urine; however, a small part is retained and probably metabolized by brain and kidney.     

Transport ofl-lactate by cultured rat brain astrocytes

Several reports indicate that lactate can serve as an energy substrate for the brain. The rate of oxidation of this substrate by cultured rat brain astrocytes was 3-fold higher than the rate with glucose, suggesting that lactate can serve as an energy source for these cells. Since transport into the astrocytes may play an important role in regulating nutrient use by individuals types of brain cells, we investigated the uptake ofl-[U-¹⁴C]lactate by primary cultures of rat brain astrocytes. Measurement of the net uptake suggested two carrier-mediated mechanisms and an Eadie-Hofstee type plot of the data supported this conclusion revealing 2 Km values of 0.49 and 11.38 mM and Vmax values of 16.55 and 173.84 nmol/min/mg protein, respectively. The rate of uptake was temperature dependent and was 3-fold higher at pH 6.2 than at 7.4, but was 50% less at pH 8.2. Although the lactate uptake carrier systems in astrocytes appeared to be labile when incubated in phosphate buffered saline for 20 minutes, the uptake process exhibited an accelerative exchange mechanism. In addition, lactate uptake was altered by several metabolic inhibitors and effectors. Potassium cyanide and -cyano-4-hydroxycinnamate inhibited lactate uptake, but mersalyl had little or no effect. Phenylpyruvate, -ketoisocaproate, and 3-hydroxybutyrate at 5 and 10 mM greatly attenuated the rate of lactate uptake. These results suggest that the availability of lactate as an energy source is regulated in part by a biphasic transport system in primary astrocytes.This data was presented in part at the meeting of the Federation of American Societies for Experimental Biology in May 1989.     

Effect of experimental hyperphenylalaninemia induced by dietary phenylalanine plus α-methylphenylalanine administration on amino acid concentration in neonatal chick brain,plasma, and liver

Supplementation of 5% phenylalanine plus 0.4% -methylphenylalanine to the standard diet or 1% phenylalanine plus 0.08% -methylphenylalanine to the drinking water produced phenylketonuria-like conditions in 5-day-old chicks. An increase of 10 to 15-fold in the phenylalanine content was observed in plasma or brain of animals after 9 days of both types of treatment. A smaller but significant increase was also observed in liver. However, practically no changes were found in the levels of tyrosine in the same conditions. Thus, the high values of plasma and brain phenylalanine/tyrosine ratio obtained by these treatments were mainly due to an increase in the phenylalanine levels, without increasing those of tyrosine. Chronic hyperphenylalaninemia induced a nonsignificant decrease in the most of amino acid contents in brain, especially after 9 days of treatment, although the levels of glycine and serine were significantly increased. A similar decrease was found in the plasma and liver concentration of various amino acids, although the variations observed in the liver were smaller than those found in plasma and brain.     

Low liver conversion rate of alpha-linolenic to docosahexaenoic acid in awake rats on a high-docosahexaenoate-containing diet

We quantified the rates of incorporation of alpha-linolenic acid (alpha-LNA; 18:3n-3) into "stable" lipids (triacylglycerol, phospholipid, cholesteryl ester) and the rate of conversion of alpha-LNA to docosahexaenoic acid (DHA; 22: 6n-3) in the liver of awake male rats on a high-DHA-containing diet after a 5-min intravenous infusion of [1-(14)C]alpha-LNA. At 5 min, 72.7% of liver radioactivity (excluding unesterified fatty acid radioactivity) was in stable lipids, with the remainder in the aqueous compartment. Using our measured specific activity of liver alpha-LNA-CoA, in the form of the dilution coefficient lambda(alpha-LNA-CoA), we calculated incorporation rates of unesterified alpha-LNA into liver triacylglycerol, phospholipid, and cholesteryl ester as 2,401, 749, and 9.6 nmol/s/g x 10(-4), respectively, corresponding to turnover rates of 3.2, 8.7, and 2.9%/min and half-lives of 8-24 min. A lower limit for the DHA synthesis rate from alpha-LNA equaled 15.8 nmol/s/g x 10(-4) (0.5% of the net in corporation rate). Thus, in rats on a high-DHA-containing diet, rates of beta-oxidation and esterification of alpha-LNA into stable liver lipids are high, whereas its conversion to DHA is comparatively low and insufficient to supply significant DHA to the brain. High incorporation and turnover rates likely reflect a high secretion rate by liver of stable lipids within very low density lipoproteins.