Levels of amino acids in 52 discrete areas of postmortem brain of adult and aged humans

Summary In a series of studies we have analyzed the regional distribution of the free amino acid pool in 52 discrete areas of postmortem brain of adult and aged humans. Here we show the distribution of eleven amino acids: alanine, methionine, valine, leucine, isoleucine, glutamine, asparagine, lysine, arginine, ornithine, and histidine. As found previously for other amino acids, the distribution of these amino acids was seen to be heterogeneous, the level of the area of highest level being 3.4 to 10.7 times that of the area of the lowest level. On average we found a five- or six-fold difference in concentration between the highest and lowest level areas in the brain samples from adult and old respectively. The distribution patterns were found to be different for each amino acid; they were not similar even in the same class (amides, branched chain, basic amino acids), and they were different from those recently found in rat brain. Only a few changes, mostly increases, were found in the aged brain, such as increases in alanine and valine levels in cortical areas. In studies of changes in cerebral amino acid levels, the great regional heterogeneity of distribution has to be taken into account since changes in whole brain values may not reflect regional changes. The functional significance and the control of this regional heterogeneity are under investigation.     

Tissue cultures from adult human postmortem subcortical brain areas

Animal models used to study human aging and neurodegeneration do not display all symptoms of these processes as they are found in humans. Recently, we have shown that many cells in neocortical slices from adult human postmortem brain may survive for extensive periods in vitro. Such cultures may enable us to study age and disease related processes directly in human brain tissue. Here, we present observations on subcortical brain tissue.     

Changes with aging in the levels of amino acids in rat CNS structural elements II. Taurine and small neutral amino acids

Taurine (Tau) and the small neutral amino acids glycine (Gly), serine (Ser), threonine (Thr), and alanine (Ala) were measured in 53 brain areas of 3- and 29-month-old male Fisher 344 rats. The ratio of highest to lowest level was 34 for Tau, 9.1 for Thr, 7.6 for Gly and Ser, and 6.5 for Ala. The heterogeneity was found in numerous areas; for example, Tau levels were more than 90 nmol/mg protein in 6 areas, and less than 20 nmol/mg protein in 10 areas. Similar heterogeneity was found with the other amino acids. The relative distribution of the small neutral amino acids showed several similarities; Tau distribution was different. With age, four amino acids decreased in 10–18 areas, and increased in only 1–3, while Thr increased in more areas than it decreased. The five amino acids of this paper, and the four of the previous paper, are among the amino acids at highest level in the brain; the sequence in their levels shows considerable regional heterogeneity.     

Changes with aging in the levels of amino acids in rat CNS structural elements I. Glutamate and related amino acids

Glutamate and related amino acids were determined in 53 discrete brain areas of 3-and 29-month-old male Fischer 344 rats microdissected with the punch technique. The levels of amino acids showed high regional variation-the ratio of the highest to lowest level was 9 for aspartate, 5 for glutamate, 6 for glutamine, and 21 for GABA. Several areas were found to have all four amino acids at very high or at very low level, but also some areas had some amino acids at high, others at low level. With age, in more than half of the areas, significant changes could be observed, decrease occurred 5 times more frequently than increase. Changes occurred more often in levels of aspartate and GABA than in those of glutamate or glutamine. The regional levels of glutamate and its related amino acids show severalfold variations, with the levels tending to decrease in the aged brain.     

Changes in brain temperature and free amino acids in normal and protein deprived suckling rats exposed to room temperature

Previous reports on early-induced protein-calorie malnutrition (PCM) in rats have indicated alterations in the concentration of free amino acids and of protein synthesis in the brain. Recently it was shown that early-induced protein deprivation (PD) retards the development of thermoregulation. This resulted in a failure to maintain a normal rectal temperature after short exposure to room temperature (+22°C) still at the age of 20–25 days corresponding to changes seen in normal rats at an age of 10–15 days. In the present study, 20-day old PD and normal rats where examined with regard to the effect of exposure to room temperature on brain temperature and on brain free amino acids. The results show a similar reduction in brain and rectal temperature of the PD rats occuring within 30 minutes after exposure to room temperature. The reduction was in the range of 5°C. PD rats kept in room temperature for 5 hours and then allowed to recover at 32.5°C showed a slow increase in brain and rectal temperature but normal temperatures were not reached even after 1 hour. The concentration of free amino acids in the brain was examined in rats kept for 1 hour at room temperature or at 32.5°C. In the PD rats kept at 32.5°C, free aspartate and glutamate were reduced whereas taurine, GABA and glycine were increased as compared to their corresponding control rats. As a result of the reduced brain temperature in PD rats exposed to room temperature there was a reduction in free asparagine. The lability of the pool of asparagine may be related to the low levels of aspartate and glutamate in PD rats. On the basis of the present findings it is recommended that temperature-sensitive parameters are examined in PCM rats at a normal body temperature.Special Issue dedicated to Prof. Holger Hydén     

Free amino acids in some sponges

Most invertebrates, particularly those of marine origin, have relatively high concentrations of free amino acids which are considered an important constituent of their osmoregulatory mechanisms [1]. Very little information is available on the free amino acid distribution in Porifera [2,3]. Common amino acids in some sponges were recognised by paper chromatography by Inskip and Cassidy [4] and Ackermann et al. [5,6] included a few sponges in their survey of the occurence of nitrogen compounds in marine invertebrates. More recently Bergquist and Hartman [7] surveyed semiquantitatively the distribution of free amino acids in several sponges. In the present paper we report on the amino acid composition of 12 species of sponges belonging to the class Demospongiae as a part of a study on the metabolites of Porifera [8]. Fresh sponges were extracted with aqueous ethanol. The organic solvent was removed and the aqueous solution, after removal of the ether soluble compounds, was separated into cationic, anionic and neutral fractions by ion-exchange chromatography. The cation fraction was analysed for amino acids using an automatic amino acid analyser. The results, which are presented in Table 1, show that all species of sponges examined have a similar composition in common amino acids. Glycine almost always appears as the dominant protein amino acid, followed by high concentrations of alanine and glutamic acid, whereas relatively lower concentrations of basic amino acids are present. In Axinella cannabina, Chondrosia reniformis, Chondrilla nucula, Cliona viridis and Hymeniacidon sanguinea, glycine represents more than 77% of the total amino acids. The high percentage of free glycine (90.4%) in Chondrosia reniformis is noteworthy. The anionic and the neutral fractions were examined for sulfur-containing amino acids using PC. Taurine (Table 2) was detected in all the Porifera examined; this is in agreement with previous observations [5–7]. N-Methyltaurine was identified in some of the species examined, whereas neither N,N-dimethyltaurine nor N,N,N-trimethyltaurine were found.     

Lipophilicity of amino acids

Summary The lipophilicity (or hydrophobicity) of amino acids is an important property relevant for protein folding and therefore of great interest in protein engineering. For peptides or peptidomimetics of potential therapeutic interest, lipophilicity is related to absorption and distribution, and thus indirectly relates to their bioactivity. A rationalization of peptide lipophilicity requires basic knowledge of the lipophilicity of the constituting amino acids. In the present contribution we will review methods to measure or calculate the lipophilicities of amino acids, including unusual amino acids, and we will make a comparison between various lipophilicity scales.     

Plasma acylcarnitines and gut-derived aromatic amino acids as sex-specific hub metabolites of the human aging metabolome

Aging biology entails a cell/tissue deregulated metabolism that affects all levels of biological organization. Therefore, the application of “omic” techniques that are closer to phenotype, such as metabolomics, to the study of the aging process should be a turning point in the definition of cellular processes involved. The main objective of the present study was to describe the changes in plasma metabolome associated with biological aging and the role of sex in the metabolic regulation during aging. A high-throughput untargeted metabolomic analysis was applied in plasma samples to detect hub metabolites and biomarkers of aging incorporating a sex/gender perspective. A cohort of 1030 healthy human adults (45.9% females, and 54.1% males) from 50 to 98 years of age was used. Results were validated using two independent cohorts (1: n = 146, 53% females, 30–100 years old; 2: n = 68, 70% females, 19–107 years old). Metabolites related to lipid and aromatic amino acid (AAA) metabolisms arose as the main metabolic pathways affected by age, with a high influence of sex. Globally, we describe changes in bioenergetic pathways that point to a decrease in mitochondrial β-oxidation and an accumulation of unsaturated fatty acids and acylcarnitines that could be responsible for the increment of oxidative damage and inflammation characteristic of this physiological process. Furthermore, we describe for the first time the importance of gut-derived AAA catabolites in the aging process describing novel biomarkers that could contribute to better understand this physiological process but also age-related diseases.     

Study of the resolution of amino acids and aminoalcohols in organic solvents

Summary The enzymatic resolution of racemic phenylglycine, phenylglycinol and phenylalaninol has been studied in organic solvents under a variety of experimental conditions. Subtilisin in 3-methyl-3-pentanol was effective for the resolution of phenylglycine esters, via N-acylation with trifluoroethyl butyrate. Porcine pancreatic lipase in ethyl acetate gave satisfactory results in the resolution of phenylglycinol and phenylalaninol; the or position of the phenyl group was found to influence both the rate and the chemioselectivity of the reaction.     

Effect of hyperammonemia on brain amino acids in young and adult ferrets

Summary Effects of arginine deficiency and hyperammonemia on the brain concentrations of amino acids and urea cycle enzyme activities in young and adult ferrets were investigated. Only young ferrets developed hyperammonemia and encephalopathy immediately after consuming the arginine-free diet. Brain ornithine and citrulline concentrations in young ferrets fed arginine containing diet were significantly lower than those in adult ferrets. Compared to rats and other animals, young and adult ferrets had lower concentrations of brain glutamic acid and glutamine. Unlike in other species, brain glutamine was not elevated in young, hyperammonemic ferrets. Brain arginase and glutamate dehydrogenase activities were significantly increased in young ferrets fed arginine-free diet. Young ferrets provide a useful animal model for investigating the neurotoxicity of acute hyperammonemia.Abbreviations ACD Arginine-containing diet - AFD Arginine-free diet This work was presented, in part, at the annual meeting of the Midwest Society for Pediatric Research, Chicago, IL, 1991.     

Comparative study of extracellular amino acids in culture of human liver and fibroblastic cells

Summary Amino acid concentrations are studied in the extracellular media of ten series of human fibroblast and liver cell monolayer cultures. These two cell types consume and produce ostensively the same amino acids. Among the nonessential amino acids, the most significant variations involve serine and aspartate which are decreased; α-alanine, glutamate, ornithine and proline are, on the contrary, increased. Among the essential amino acids, leucine, isoleucine and glutamine are preferentially decreased. The variations of some amino acids are correlated with the cell density. The interrelations which may exist between the variations of these different amino acids are discussed. Furthermore, the glycolytic activity of the cells studied is very high: 85% of glucose consumed is found in the form of lactate. Unité d’Hépatologie Infantile I.N.S.E.R.M. (U56) Laboratoire Central de Biochimie This work was supported by grant: INSERM CRL 73-5-057-04 and 74-5-075-04.     

Sugar amino acids in designing new molecules

Emulating the basic principles followed by Nature to build its vast repertoire of biomolecules, organic chemists are developing many novel multifunctional building blocks and using them to create ‘nature-like’ and yet unnatural organic molecules. Sugar amino acids constitute an important class of such polyfunctional scaffolds where the carboxyl, amino and hydroxyl termini provide an excellent opportunity to organic chemists to create structural diversities akin to Nature’s molecular arsenal. This article describes some of our works on various sugar amino acids and many other related building blocks, like furan amino acids, pyrrole amino acids etc. used in wide-ranging peptidomimetic studies. Published in 2005.Based on the invited lecture presented at the XVII International Symposium on Glycoconjugates held in January 12–16, 2003 at Bangalore, India.     

Calpain activity in adult and aged human brain regions

We assayed calpain activity in 27 human brain regions from adult (43–65 years of age) and aged (66–83 years of age) postmortem tissue samples. Calpain I (M Ca-requiring) activity was 10% or less of the total activity; it was below detectable levels in a number of areas, and so data are are expressed as total (M+mM Ca-dependent) calpain activity. The distribution of the enzyme was regionally heterogeneous. Highest activity was found in the spinal cord, followed by the amygdala, and levels in mesencephalic areas and in cerebellar grey matter were also high. Levels in cerebellar white matter, tegmentum, pons, and putamen were low, and activity in cortical areas was also relatively low. Although in some areas activity seemed higher with aging, the differences were not statistically significant. We previously found that the regional distribution of cathepsin D in human and in rat brain is similar, this seems to be true for calpain activity as well. The increase of protease activity with age found in rat brain is not found in human areas, as was shown previously with cathepsin D, and in the present study with calpain.Special issue dedicated to Dr. Bernard W. Agranoff.     

Effects of branched chain amino acids,pyruvate, or ketone bodies on the free amino acid pool and release from brain cortex slices of normal and streptozotocindiabetic rats

Brain cortex slices from diabetic rats incubated in Krebs-Ringer-bicarbonate (KRB)-glucose medium show, compared to the normals, a 75% higher glutamine content. Branched chain amino acids (BCAA) added, at 0.5mM each, to this medium increase (53%) the glutamine content in the normal slices but have no effect on the glutamine content in the slices from diabetic rats. When the incubation medium is KRB-pyruvate, glutamine and glutamate contents are lower than in the KRB-glucose medium. The addition of BCAA in the KRB-pyruvate medium partially restores the contents of glutamine in the normal and of glutamine plus glutamate in the diabetic. Keto acids or BCAA added to the incubation medium of normal slices decrease the pool of most of the neutral and acidic amino acids but they do not affect this pool in slices from the diabetic rats. In addition keto acids increase the ratio glutamate in the tissue: glutamate in the medium.Abbreviations used BCAA branched chain amino acids - 3-OHB d,l-3-hydroxybutyrate - AcAc acetoacetate - KRB Krebs-Ringer-bicarbonate     

Feeding pigs amino acids as protein-bound or in free form influences postprandial concentrations of amino acids,metabolites, and insulin

Dietary proteins need to be digested first while free amino acids (AAs) and small peptides are readily available for absorption and rapidly appear in the blood. The rapid postprandial appearance of dietary AA in the systemic circulation may result in inefficient AA utilisation for protein synthesis of peripheral tissues if other nutrients implicated in AA and protein metabolism are not available at the same time. The objective of this experiment was to compare the postprandial concentrations of plasma AA and other metabolites after the ingestion of a diet that provided AA either as proteins or as free AA and small peptides. Twenty-four male growing pigs (38.8 ± 2.67 kg) fitted with a jugular catheter were assigned to one of three diets that provided AA either in protein form (INT), free AA and small peptides (HYD), or as free AA (FAA). After an overnight fast and initial blood sampling, a small meal was given to each pig followed by serial blood collection for 360 min. Postprandial concentrations of plasma AA, glucose, insulin, and urea were then measured from the collected blood. Non-linear regression was used to summarise the postprandial plasma AA kinetics. Fasting concentrations of urea and some AA were higher (P < 0.05) while postprandial plasma insulin and glucose were lower (P < 0.01) for INT than for HYD and FAA. The area under the curve of plasma concentration after meal distribution was lower for INT for most AAs (P < 0.05), resulting in a flatter curve compared to HYD and FAA. This was the result of the slower appearance of dietary AA in the plasma when proteins are fed instead of free AA and small peptides. The flatter curve may also result from more AAs being metabolised by the intestine and liver when INT was fed. The metabolism of AA of the intestine and liver was higher for HYD than FAA. Providing AA as proteins or as free AA and small peptides affected the postprandial plasma kinetics of AA, urea, insulin, and glucose. Whether the flat kinetics when feeding proteins has a positive or negative effect on AA metabolism still needs to be explored.     

Application of PIXE analysis to the study of the regional distribution of trace elements in normal human brain

A particle-induced X-ray emission (PIXE) analysis method is presented, which allows measurement of eight elements (i.e., K, Ca, Mn, Fe, Cu, Zn, Se, and Rb) in human brain samples of only a few mg dry weight. The precision and accuracy of the method were investigated by analyzing animal brain matter with both PIXE and instrumental neutron activation analysis (INAA). The method was applied to measure the 8 elements in 46 different regions of 3 human brains. The sections analyzed originated from either the left or the right cerebral hemisphere, brain stem, and cerebellum. For one of the brains, sections were also analyzed from 26 corresponding regions of both hemispheres. For all elements, similar concentrations were found in the corresponding areas of the left and right sides of the brain. The concentrations (in μg/g dry weight) of the elements K, Fe, Cu, Zn, Se, and Rb were consistently higher in cortical structures than in white matter. Deep nuclei and brain stem, which have a mixed composition, showed intermediate values for K, Zn, Se, and Rb. A hierarchical cluster analysis indicated that the various brain regions clustered into two large groups, one comprising gray and mixed matter regions and the other, white and mixed matter brain areas.     

Severity of experimental traumatic brain injury modulates changes in concentrations of cerebral free amino acids

In this study, concentrations of free amino acids (FAA) and amino group containing compounds (AGCC) following graded diffuse traumatic brain injury (mild TBI, mTBI; severe TBI, sTBI) were evaluated. After 6, 12, 24, 48 and 120 hr aspartate (Asp), glutamate (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), histidine (His), glycine (Gly), threonine (Thr), citrulline (Cit), arginine (Arg), alanine (Ala), taurine (Tau), γ‐aminobutyrate (GABA), tyrosine (Tyr), S‐adenosylhomocysteine (SAH), l ‐cystathionine (l ‐Cystat), valine (Val), methionine (Met), tryptophane (Trp), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), ornithine (Orn), lysine (Lys), plus N‐acetylaspartate (NAA) were determined in whole brain extracts (n = 6 rats at each time for both TBI levels). Sham‐operated animals (n = 6) were used as controls. Results demonstrated that mTBI caused modest, transient changes in NAA, Asp, GABA, Gly, Arg. Following sTBI, animals showed profound, long‐lasting modifications of Glu, Gln, NAA, Asp, GABA, Ser, Gly, Ala, Arg, Citr, Tau, Met, SAH, l ‐Cystat, Tyr and Phe. Increase in Glu and Gln, depletion of NAA and Asp increase, suggested a link between NAA hydrolysis and excitotoxicity after sTBI. Additionally, sTBI rats showed net imbalances of the Glu‐Gln/GABA cycle between neurons and astrocytes, and of the methyl‐cycle (demonstrated by decrease in Met, and increase in SAH and l ‐Cystat), throughout the post‐injury period. Besides evidencing new potential targets for novel pharmacological treatments, these results suggest that the force acting on the brain tissue at the time of the impact is the main determinant of the reactions ignited and involving amino acid metabolism.     

Effects of branched-chain amino acids on plasma amino acids in amyotrophic lateral sclerosis

Summary Although the cause of amyotrophic lateral sclerosis (ALS) remains unknown, biological findings suggest that the excitatory amino acid glutamate contributes to the pathogenesis of ALS. In previous studies of ALS, the therapeutic effect of the branched-chain amino acids (BCAAs) leucine, valine and isoleucine has been evaluated. The present study aimed at investigating the acute effect of BCAAs on plasma glutamate levels in ALS patients. Following two oral doses of BCAAs, significantly increased plasma levels were seen for valine (500%), isoleucine (1,377%) and leucine (927%), however the plasma level of glutamate was not affected. The plasma level of several other amino acids (tryptophan, tyrosine, phenylalanine and methionine) were found decreased after oral BCAAs, which may indicate a diminution in the rate of degradation of muscle protein and/or an increase in tissue disposal of amino acids.     

Site-directed mutagenesis of rabbit LAT1 at amino acids 219 and 234

The availability of amino acids in the brain is regulated by the blood-brain barrier (BBB) large neutral amino acid transporter type 1 (LAT1) isoform, which is characterized by a high affinity (low Km) for substrate large neutral amino acids. The hypothesis that brain amino acid transport activity can be altered with single nucleotide polymorphisms was tested in the present studies with site-directed mutagenesis of the BBB LAT1. The rabbit has a high Km LAT1 large neutral amino acid transporter, as compared to the low Km neutral amino acid transporter at the human or rat BBB. The rabbit LAT1 was cloned from a rabbit brain capillary cDNA library. Alignment of the amino acid sequences of rabbit, human, and rat LAT1 revealed two radical amino acid residues that differ in the rabbit relative to the rat or human LAT1. The G219D mutation had a modest effect on the Km and Vmax of tryptophan transport via cloned rabbit LAT1 in frog oocytes, but the W234L variant reduced the Km by 64% and the Vmax by 96%. Conversely, LAT1 transport of either tryptophan or phenylalanine was nearly normalized when the double mutation W234L/G219D variant was produced. These studies show that marked changes in the affinity and capacity of the LAT1 are caused by single nucleotide polymorphisms and that phenotype can be restored with a double mutation.     

Effect of the cationic polypeptide polylysine on neutral amino acid transport in isolated brain microvessels

Summary The functionality of isolated brain microvessels — used as anin vitro model of the blood-brain barrier — can be influenced by interaction with cationic proteins. The various polylysines (M_r ranging from 0.9 to 180 kDa) tested affected the activity of both the Na⁺-dependent (A) and the Na⁺-independent (L) systems for neutral amino acid transport. Exposure to the 180 kDa polylysine caused a conspicuous inhibition of both transport systems, associated to an increased passive permeability. There was a constant, M_r-dependent, inhibition of the the L-system-mediated uptake of hydrophobic neutral amino acids. The activity of the A-system was enhanced, upon exposure to polymers larger than 22 kDa reaching its peak at 68 kDa and and declining at higher M_r values. The effect which was Na⁺-ions dependent and abolished by phloretine, could be essentially ascribed to an increased affinity of the MeAIB for its carrier (K_m value decreasing from 265 to 169µM in presence of 68 kDa polylysine).