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Acetylcholinesterase activity and biogenic amines in phenylketonuria.

Phenylketonuria phenylketonuria (fĕn'əlkēt'ənr`ēə) (PKU), inherited metabolic disorder caused by the absence of a specific enzyme (phenylalanine hydroxylase).  (PKU PKU: see phenylketonuria. ) is a disorder in which the aromatic amino acid Phe cannot be converted to Tyr (1, 2). Unfortunately, many PKU patients do not adhere to their low-Phe diet (off diet), which leads to high concentrations of the amino acid in their blood (1, 2). High Phe concentrations interfere with the production of adrenaline (A), noradrenaline noradrenaline /nor·adren·a·line/ (nor?ah-dren´ah-lin) norepinephrine.
noradrenaline (nōrˈ·
 (NA), and dopamine (DA) (1, 3). Furthermore, Krause et al. (4) reported an inverse relationship between NA and DA plasma concentrations and Phe because high Phe concentrations decrease the availability of the amino acids Tyr and Trp, the precursors of catecholamines Catecholamines
Family of neurotransmitters containing dopamine, norepinephrine and epinephrine, produced and secreted by cells of the adrenal medulla in the brain.
 and serotonin [5-hydroxytryptamine (5HT)], respectively (5-7).

Acetylcholinesterase acetylcholinesterase /ac·e·tyl·cho·lin·es·ter·ase/ (AChE) (-ko?li-nes´ter-as) an enzyme present in the central nervous system, particularly in nervous tissue, muscle, and red cells, that catalyzes the hydrolysis of acetylcholine to  (AChE) is a membrane-bound enzyme with its active side exposed at the external leaflet of the bilayer bilayer /bi·lay·er/ (bi´la-er) a membrane consisting of two molecular layers.

bi·lay·er
n.
A structure, such as a film or membrane, consisting of two molecular layers.
 (ectoenzyme). When the enzyme is inhibited, it can no longer participate in the hydrolysis of acetylcholine (ACh) (8), involving parasympathetic parasympathetic /para·sym·pa·thet·ic/ (-sim?pah-thet´ik) see under system.

par·a·sym·pa·thet·ic
adj.
Of, relating to, or affecting the parasympathetic nervous system.
, sympathetic, peripheral, and central nervous system function (8-10). Alterations of the above substances in the cerebrospinal fluid are correlated with AChE activity in the cerebrospinal fluid of patients with mental impairment (11).

In our previous study (12), incubation of high Phe concentrations with human AChE type XIII led to inhibition of the enzyme (40-60%). The effect of Phe on AChE of rat diaphragm and rat brain showed a concentration-dependent enzyme inhibition (13, 14). We therefore aimed to evaluate AChE activities in the erythrocyte membranes from patients with PKU and to correlate the enzyme activities with blood concentrations of the biogenic biogenic /bi·o·gen·ic/ (-jen´ik) having origins in biological processes.

biogenic

having the property of originating in a biological process.
 amines A, NA, DA, and 5HT as well as with the precursors Tyr and Trp.

The study was approved by the Greek ethics committee and was conducted according to the principles expressed in the Helsinki Declaration.

The study population consisted of 23 PKU patients who were divided into two groups: group A (n = 12; mean age, 6.8 [+ or -] 1.2 years), who adhered strictly to their special therapeutic diet as evidenced by their almost normal plasma Phe concentrations (Phe, 180.4 [+ or -] 30.7 [micro]mol/L); and group B (n = 11; mean age, 7.2 [+ or -] 2.0 years), who were off diet and had increased Phe concentrations (Phe, 1722 [+ or -] 286 [micro]mol/L). Twenty-three healthy children of comparable age were the controls. All PKU patients were admitted to the day clinic of the Inborn Errors of Metabolism Department of the Institute of Child Health in Athens.

All blood samples were collected from an antecubital vein at the same time of day while both patients and controls were at rest. Blood samples (7.0 mL) were collected 3 h after participants arrived at our hospital, during which time the children fasted and were acclimatized to the hospital environment and staff.

Venous blood samples were collected into heparin-containing blood collection tubes from PKU patients and controls. The washed erythrocytes were lysed, as described by Galbraith and Watts (15) and Kamber et al. (16), after being frozen (-80 [degrees]C) and thawed (50 [degrees]C) five times. Membranes were suspended in 0.1 mol/L Tris-HCl, pH 7.4, to a final protein concentration of 2 g/L (17). The minor hemoglobin that remained attached to the membrane surface was measured by reagent set 527-A (Sigma Chemical Co.), and the value was subtracted from the total protein concentration.

AChE activity was determined according to the method of Ellman et al. (18) as modified by Tsakiris (19).

Quantitative analysis of amino acids, including Phe and Tyr, was carried out with an automatic amino acid analyzer (Biotronic LC 5001). Results were calculated with norleucine as internal calibrator. The CVs for Phe and Tyr were 3.1% and 2.9%, respectively. Trp plasma concentrations were measured as described by Faggiano et al. (20), and the CV was 1.9%. Plasma catecholamine (A, NA, and DA) concentrations were evaluated by HPLC HPLC high-performance liquid chromatography.

HPLC

high performance liquid chromatography.

HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed
 with electrochemical detection (21). The CVs for DA, A, and NA were 3.4%, 2.9%, and 3.2%, respectively. 5HT concentrations were measured in a platelet-rich plasma with a new HPLC method (22). The CV for 5HT was 2.8%.

Data were analyzed by t-test and multiple regression analysis for the correlation coefficients. All analyses were performed with the SPSS 10.0 statistical package on an IBM personal computer
''This article discusses to the original IBM PC. For IBM-like PCs in general ("clones"), see IBM PC compatible.


? IBM 5120 IBM PC Series IBM Personal Computer XT • IBM Portable Personal Computer • IBM PCjr ?

The
.

As shown in Table 1, blood Tyr, Trp, DA, NA, A, 5HT, and AChE concentrations in group A of the PKU patients were not statistically different from controls, whereas in group B, plasma amino acids (except Phe), their biogenic amines, and AChE activities were significantly decreased. Additionally, Tyr, Trp, DA, NA, 5HT, and AChE showed strong inverse correlations with Phe concentrations (P <0.001).

Alterations in synaptic transmission are implicated in brain dysfunction in PKU, and several experimental data suggest that the principal cause for this dysfunction is the impairment in biogenic amine synthesis (5). Increased plasma Phe concentrations, such as we found in PKU patients (group B), by decreasing the availability of the precursors Tyr and Trp, which are also decreased in the same group of patients, might be the primary cause of their catecholamine and 5HT depletion (6, 7, 23). It is highly likely that the decreased Tyr and Trp seen in the plasma of PKU patients (group B) might cause decreased uptake by the adrenal medulla and platelets, leading to low production of catecholamine and 5HT. Additionally, a large excess of large neutral amino acids, such as Phe, in the same group of patients will saturate sat·u·rate
v. Abbr. sat.
1. To imbue or impregnate thoroughly.

2. To soak, fill, or load to capacity.

3. To cause a substance to unite with the greatest possible amount of another substance.
 the carrier system across the blood-brain barrier, excluding Tyr and Trp from entry into the brain (4, 23). Thus, conversions of the above amino acids to the biogenic amines are possibly lowered in the central nervous system (4, 5, 23).

Because high Phe concentrations in the plasma of PKU patients (group B) could lead to brain dysfunction (2) and AChE inhibition can influence cholinergic cholinergic /cho·lin·er·gic/ (ko?lin-er´jik)
1. parasympathomimetic; stimulated, activated, or transmitted by choline (acetylcholine); said of the sympathetic and parasympathetic nerve fibers that liberate acetylcholine at a
 transmission, a more detailed study of Phe action on AChE seemed worthwhile. In our in vitro previous studies (12-14), various concentrations of Phe on human AChE, rat homogenized diaphragm, pure eel (Electrophorus electrophorus, device used to generate static electric charges. It has two parts: a nonconducting plate (e.g., of hard rubber) that is negatively charged and a metal plate with an insulated handle.  electricus) AChE, and rat homogenized brain AChE showed that Phe induced a similar concentration-dependent inhibition of AChE activities. We therefore assumed that Phe directly inhibited AChE, possibly interacting with its positively charged sites, and/or indirectly by changing the membrane lipid-bilayer microenvironment microenvironment /mi·cro·en·vi·ron·ment/ (-en-vi´ron-ment) the environment at the microscopic or cellular level. , causing functional modulation of the enzyme (8, 13). It could be also that the high degree of AChE inhibition in erythrocyte membranes from PKU patients off diet may be caused by the long-term indirect influence of high Phe concentrations on the enzyme membrane bilayer through lipid-protein interactions (24). Experiments on the effects of incubation of red cells with various Phe concentrations and evaluation of AChE protein concentration, such as by Western blot measurements or direct antigen assays, would be useful for understanding the mechanism of this effect.

High Phe concentrations could also induce changes in brain electrical function, which may be mediated in part through inhibition of biogenic amine production (4). Regarding cholinergic brain systems, experimental results showed their possible involvement during Phe action. Additionally, an increase in Phe concentration can cause an increase in the GTP-cyclohydrolase-stimulating protein. The latter increases de novo the synthesis of tetrahydrobiopterin, leading to its high uptake into the red cells. 6R-L-Erythro-5,6,7,8-tetrahydrobiopterin, a natural cofactor cofactor

An atom, organic molecule, or molecular group that is necessary for the catalytic activity (see catalysis) of many enzymes. A cofactor may be tightly bound to the protein portion of an enzyme and thus be an integral part of its functional structure, or it may
 for Phe hydroxylases, has direct ACh-releasing action in vivo in the rat hippocampus (25).

In conclusion, (a) high plasma Phe concentrations caused marked in vivo inhibition of erythrocyte-membrane AChE activity in PKU (the latter is reinforced by our studies on the in vitro effect of Phe on AChE), (b) AChE inhibition could affect ACh hydrolysis and its consequences in nervous system functions, (c) high Phe concentrations may explain the decreased concentrations of biogenic amines in PKU, and (d) our data showed for the first time that the evaluation of erythrocyte-membrane AChE activity in relation to biogenic amine blood concentrations could be a useful peripheral marker for evaluation of the effects of high Phe concentrations in the brains of PKU patients.

We thank Anna Stamatis for assistance with this manuscript.

References

(1.) Scriver C, Kaufman S, Eisensmith R, Woo S. The hyperphenylalaninemias. In: Scriver C, Beaudet A, Sly W, Valle D, eds. The metabolic and molecular bases of inherited disease, 8th ed. New York: McGraw-Hill, 2000:1775-875.

(2.) Missiou-Tsagaraki S, Soulpi K, Loumakou M. Phenylketonuria in Greece: 12 years experience. J Ment Defic Res 1988;32:271-87.

(3.) Curtius H, Wiederwieser C, Viscontini G, Leimbacher M, Wegman H, Schmidt H. Serotonin and dopamine synthesis in phenylketonuria. Adv Exp Med Biol 1981;133:277-91.

(4.) Krause W, Halminski M, McDonald L. Biochemical and neuropsychological effects of elevated plasma phenylalanine phenylalanine (fĕn'əlăl`ənēn'), organic compound, one of the 22 α-amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein.  in patients with treated phenylketonuria. J Clin Invest 1985;75:40-8.

(5.) Blau K. Phenylalanine hydroxylase deficiency: biochemical, psychological and clinical aspects of phenylketonuria and related hyperphenylalaninemias. In: Youdim MBH, ed. Aromatic amino acids Aromatic amino acids are amino acids which include an aromatic ring.

Examples include:
  • phenylalanine, tryptophan, and tyrosine
  • thyroxine
See also
  • Aromatic L-amino acid decarboxylase
External links
  • MeSH
, hydroxylases, and mental diseases. New York: Wiley, 1979:77-139.

(6.) Aragon MC, Gimenez G, Valdinieso F. Inhibition by phenylalanine of tyrosine transport by synaptosomal plasma membrane vesicles: implication in the pathogenesis of phenylketonuria. J Neurochem 1982;39:1185-7.

(7.) Herrero E, Aragon MC, Gimenez C, Valdivieso F. Inhibition by L-phenylalanine of tryptophan tryptophan (trĭp`təfăn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein.  transport by synaptosomal plasma membrane vesicles: implication in the pathogenesis of phenylketonuria. J Inherit Metab Dis 1983;6:32-5.

(8.) Lotti M. Cholinesterase cholinesterase /cho·lin·es·ter·ase/ (-es´ter-as) serum cholinesterase, pseudocholinesterase; an enzyme that catalyzes the hydrolytic cleavage of the acyl group from various esters of choline and some related compounds; determination of  inhibition: complexities in interpretation. Clin Chem 1995;41:1814-8.

(9.) Sussman JL, Harrel M, Frolow F, Goldman A. Atomic structure of acetylcholinesterase from Torpedo california: a prototypic acetylcholine binding protein. Science 1991;253:872-80.

(10.) Deliconstantinos G, Tsakiris S. Differential effect of anionic and cationic drugs on the synaptosome associated acetylcholinesterase activity of dog brain. Biochem J 1985;229:81-9.

(11.) Egashira T, Goto H, Takda H, Takada K, Matsumiya T. Alterations in neurotransmitter, amino acid and free radical related substances in cerebrospinal fluid in patients with cerebrovascular diseases. Nippon Ronen Igakkai Asshi 1999;36:256-61.

(12.) Schulpis KH, Karikas GA, Tjamouranis J. In vitro influence of phenylalanine on acetylcholinesterase activity and DNA DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
. Z Naturforsch 1998;53c:291-3.

(13.) Tsakiris S, Krontiri P, Schulpis KH. L-Phenylalanine effect on rat diaphragm acetylcholinesterase and [Na.sup.+],[K.sup.+]-ATPase. Z Naturforsch 1998;53c:1055-60.

(14.) Tsakiris S, Krontiri P, Schulpis KH, Stavridis JC. L-Phenylalanine effect on rat brain acetylcholinesterase and [Na.sup.+],[K.sup.+]-ATPase. Z Naturforsch 1998;58c: 163-8.

(15.) Galbraith DA, Watts DC. Human erythrocyte acetylcholinesterase in relation to cell age. Biochem J 1981;195:221-8.

(16.) Kamber E, Poyigi A, Deliconstantinos G. Modifications in the activities of membrane-bound enzymes during in vivo ageing of human and rabbit erythrocytes. Comp Biochem Physiol 1984;77B:95-9.

(17.) Lowry OH, Rosebrough NJ, Farr AL, Randal RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.

(18.) Ellman GL, Gourtney D, Andres V, Featherstone RM. A new rapid colorimetric col·or·im·e·ter  
n.
1. Any of various instruments used to determine or specify colors, as by comparison with spectroscopic or visual standards.

2.
 determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7: 88-95.

(19.) Tsakiris S. Effects of L-phenylalanine on acetylcholinesterase and [Na.sup.+],[K.sup.+]-ATPase activities in adult and aged rat brain. Mech Ageing Dev 2001;122: 491-501.

(20.) Faggiano A, Pivonello R, Melis D, Alfiori R, Filippela M, Spagnuolo G, et al. Evaluation of circulating levels and renal clearance of natural amino acids in patients with Cushing disease. J Endocrinol Invest 2002;25:142-151.

(21.) Candito M, Albertini M, Politanos S, Deville A, Mariani S. Plasma catecholamine levels in children. J Chromatogr 1993;617:304-7.

(22.) Kema J, Meijer WG, Meiborg G, corns B, Willense PA, De Vries E. Profiling of tryptophan-related plasma indoles in patients with carcinoid carcinoid /car·ci·noid/ (kahr´si-noid) a yellow circumscribed tumor arising from enterochromaffin cells, usually in the gastrointestinal tract; the term is sometimes used to refer specifically to the gastrointestinal tumor  tumors by automated on line solid extraction and HPLC with fluorescence detection. Clin Chem 2001;47:1811-20.

(23.) Schulpis KH, Papakonstantinou E, Michelakakis H, Theodoridis T, Papadreou U, Constantopoulos A. Elevated serum prolactin prolactin /pro·lac·tin/ (-lak´tin) a hormone of the anterior pituitary that stimulates and sustains lactation in postpartum mammals, and shows luteotropic activity in certain mammals.

pro·lac·tin
n.
 concentrations in phenylketonuric patients on a "loose diet". Clin Endocrinol 1998;48:99-101.

(24.) Shafferman A, Ordentlich A, Barak D, Stein D, Ariel N, Velan B. Aging of phosphorylated human acetylcholinesterase: catalytic processes mediated by aromatic and polar residues of the active centre. Biochem J 1998;318: 833-40.

(25.) Ohue T, Koshimura K, Lee K, Watanabe Y, Miwa S. A novel action of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin, a cofactor of hydroxylases of phenylalanine, tyrosine and tryptophan: enhancement of acetylcholine release in vivo in the rat hippocampus. Neurosci Lett 1991;128:93-6.

Kleopatra H. Schulpis, [1] * George A. Karikas, [2] Joanna Tjamouranis, [1] Helen Michelakakis, [1] and Stylianos Tsakiris [3]

[1] Institute of Child Health and

[2] Pharmacokinetics and Parenteral Nutrition Unit, Aghia Sophia Children's Hospital, 11527 Athens, Greece;

[3] Department of Experimental Physiology, Medical School, University of Athens, 15401 Athens, Greece;

* address correspondence to this author at: Institute of Child Health, Aghia Sophia Children's Hospital, PO Box 65257, 11527 Athens, Greece; fax 3010-7700111, e-mail inchildh@otenet.gr)
Table 1. Phe, Tyr, Trp, and biogenic amine concentrations and AChE
activities in PKU patients vs controls. (a)

                        Group A (n = 12)      Group B (n = 11)

Phe, (b) [micro]mol/L   180.4 [+ or -] 30.7   1722 [+ or -] 286
Tyr, (c) [micro]mol/L   115.3 [+ or -] 26.5   45.8 [+ or -] 12.5
Trp, (c) [micro]mol/L   62.7 [+ or -] 13      36.2 [+ or -] 10
DA, (c) pmol/L          130 [+ or -] 25       46 [+ or -] 23
NA, (c) nmol/L          290 [+ or -] 1.30     1.46 [+ or -] 0.80
A, (c) pmol/L           860 [+ or -] 148      148 [+ or -] 68
5HT (c,d)               4.26 [+ or -] 0.90    2.10 [+ or -] 1.00
AChE activity (c,e)     3.01 [+ or -] 0.18    1.19 [+ or -] 0.05

                        Controls (n = 23)

Phe, (b) [micro]mol/L   66.5 [+ or -] 33.5
Tyr, (c) [micro]mol/L   139.6 [+ or -] 32.1
Trp, (c) [micro]mol/L   68.5 [+ or -] 12
DA, (c) pmol/L          138 [+ or -] 26
NA, (c) nmol/L          3.10 [+ or -] 1.20
A, (c) pmol/L           890 [+ or -] 168
5HT (c,d)               4.98 [+ or -] 0.90
AChE activity (c,e)     3.13 [+ or -] 0.16

(a) Values are expressed as mean [+ or -] SD.

(b) P <0.0001 for group A vs group B, group A vs controls, and group
B vs controls.

(c) P <0.001 for group A vs group B and group B vs controls;
differences between group A and controls were not significant.

(d) Platelet 5HT content is expressed as nmol of 5HT/[10.sup.9]
platelets.

(e) AChE activity is expressed as [Delta]absorbance/min * mg protein.
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Title Annotation:Technical Briefs
Author:Schulpis, Kleopatra H.; Karikas, George A.; Tjamouranis, Joanna; Michelakakis, Helen; Tsakiris, Styl
Publication:Clinical Chemistry
Date:Oct 1, 2002
Words:2378
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