Alzheimer's disease: amino acid levels and brain metabolic status

Abstract To study brain free amino acids and their relation with dementia we measured, by high-performance liquid chromatography (HPLC), the concentration of eight free amino acids, amines and related compounds. We used temporal cortex (TC) samples obtained from 13 Alzheimer’s disease (AD) patients and an equal number of agematched controls (AC). The patterns of free amino acids, amines and related compounds showed significant quantitative changes in AD conditions with respect to healthy ones. In Alzheimer patients, lower levels of GABA were found in the TC (-57 %). Amino acids glutamate (Glu), and aspartate (Asp) concentrations, also appeared significantly reduced in the TC of AD patients (Glu: -30 %; Asp: -40 %) when compared with controls. The significant gap between methionine (Met: -30 %) and cystathionine (Cysta: ?60 %) levels in TC of AD people to controls, might suggest an under/over activity of the transmethylation and transsulphuration pathways, respectively. Glutamine (Gln) and Urea were an exception to this trend because their content was higher in AD patients than in controls. Albeit these compounds may have particular physiological roles, including the possible mediation of synaptic transmission, changes in amino acid levels and related compounds (detected in steady state) suggest a modified metabolic status in brains of AD patients that reveals a reduced function of synaptic transmission. Because several evidences show that patients might display quite different concentrations of neurotransmitters in brain areas, assessing metabolites in different and well-characterized AD stages should be investigated further

Impairment of Methylation cycle in treated patients with Parkinson's disease

L-3,4-dihydroxyphenylalanine (L-DOPA) alone or in combination with a peripheral dopa decarboxylase inhibitor (DDI) is the most effective therapeutic agent to improve motor function in most of patients with Parkinson's disease (PPD). However, chronic L-DOPA therapy is associated with of side-effects arising particularly during long-term therapy. Only a small percentage of an exogenous dose of L-DOPA is converted into dopamine (DA) in the brain. The majority is either decarboxylated in peripheral tissues by aromatic amino acid decarboxylase (AAD) to DA, which does not cross the blood-brain barrier, or is O-methylated by catechol-O-methyltransferase (COMT) in both peripheral and brain tissue to yield 3-O-methyldopa (3-OMD). To effectively raise brain DA levels, a large amount of L-DOPA must be administered, often in an oral dose. It has been reported that such large doses of L-DOPA can significantly affect sulphur amino acid metabolite levels. During long-term clinical practice, the decarboxylation of L-DOPA is inhibited, the role of COMT is accentuated and circulating L-DOPA is largely converted into 3-OMD. Therefore, O-methylation of L-DOPA to 3-OMD is linked with conversion of SAM to S-adenosylhomocysteine (SAH). SAH is split into adenosine and Hcy. We evaluated the impact of long-term application of L-DOPA/DDI formulations on plasma methionine (MET), SAM, SAH and tHcy levels in PPD. Patients were from the Institute of Neuropsychiatry, Palermo University. All patients entering the study were examined by neurologists to confirm or exclude the diagnosis of Parkinson’s disease. There were 10 PPD treated with L-DOPA/DDI formulation and 10 healthy controls. Peripheral blood samples were taken in the morning after the subjects had fasted and were off medication for at least 12 hrs. Thus we avoided impact of acute L-DOPA/DDI intake. Plasma tHcy and sulphur metabolite levels were determined by high-performance liquid chromatography (HPLC) as reported. The levels of MET and SAM (approximately 1.21 and 1.32 fold, respectively) in the treated PPD were significantly lower than in the controls while the levels of tHcy (mean 16.6 mmol/L; SD 4.4) were higher compared with controls (mean 9.8 mmol/L; SD 3.4). No significant differences in SAH levels appeared. Based on these findings, we hypothesized that another consequence of high-dose e/or long-term L-DOPA administration might be hyperhomocysteinaemia and may also represent a risk factor for both ischaemic heart and cerebrovascular disease in treated PPD. Besides, the resulting hyperhomocysteinaemia might be increased if L-DOPA therapy is superimposed on a condition known to impair Hcy metabolism, such an enzyme defect or B/acid folic vitamin deficiency

GSH: A MARKER FOR OXIDATIVE STRESS IN HUMAN CELL CULTURES

GSH: A MARKER FOR OXIDATIVE STRESS IN HUMAN CELL CULTURES Gueli Maria Concetta Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche (BioNEC), Università degli Studi di Palermo. Reactive oxygen species (ROS) play an important role in physiological processes, but when being in excess ROS react readily with proteins, lipids, carbohydrates, and nucleic acids, often inducing irreversible functional alterations or even complete destruction. In cells, under physiological conditions, the production and detoxification of ROS are more or less balanced. GSH functions as antioxidant and the oxidative conversion of GSH to GSSG is widely recognized as a reliable index of oxidative stress. Therefore great interest exists in the determination of GSH and GSSG in various biological tissues, organs, and cells. We propose a rapid, user-friendly, HPLC with fluorescence detection (HPLC-FD) method to quantify GSH and GSSG using human epatoma HA22T/VGH cells. Dubecco’s Eagle’s modified medium, TNBP, SBDF, GSH, Bradford reagent (Sigma). HA22T/VGH cells were grown in appropriate medium. The number of cells per flask was determined by Reverse Microscope to assess cell growth rate. After 7 days of incubations HA22T/VGH cells at 80-90% of confluency were washed with cold PBS, scraped in NaCl 0.9% and centrifuged. The pellet cells were resuspended in 1 ml Lysis buffer, sonicate and centrifuged at 13000 rpm for 15 min. Afterwards the supernatants were collected and used for HPLC assay. Briefly:100mL of HA22T/VGH cell estracts and TNBP reagent was incubated for 30 min at 4°C after which TCA was added. The clear supernatant was added to an eppendorf containing 1.55mol/L NaOH; 0.125mol/L borate buffer, pH 9.5, SBDF solution, then incubated at 60°C. Waters-HPLC system consisted of a 600E Pump, 474 fluorescence detector (FD) and Empower TM2 Software. Separation of the SBDF derivatized thiols was performed on a Spherisorb ODS2, 0.1mol/L acetic acid-acetate buffer, pH 4.0 as mf. The R.T. for GSH was 11.57 ± 0.01 min (means ± SD). Calibration curve for GSH was linear up to 100 mmol/L. Levels of tGSH, GSH, and GSSG levels in HA22T/VGH cells were (means ± SEM) 26.87 ± 0.02; 23.18 ± 0.01 and 3.69 ± 0.01 μmol/L, respectively and 33.37 ± 0.01; 28.79 ± 0.01 and 4.58 ± 0.01 nmol/mg prot., respectively. This HPLC-FD method developed in our laboratory is very useful for research purposes and for routine clinical use. Gueli MC (2000) IBTS 15, 167. 4° SIBIOC Interr. SORRENTO 9-11 Ottobre 201

Taurine in the interphotoreceptor matrix

TAURINE IN THE INTERPHOTORECEPTOR MATRIX Gueli Maria Concetta Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche (BioNEC), Università degli Studi di Palermo Taurine (Tau) is the most abundant amino compound free in the retina. It is concentrated in the photoreceptor inner segment, in the outer nuclear layer and in the synapses. The retina synthesizes and receives Tau from choroidal blood via the pigment epithelium (PE). The high content in the retina suggest the possibily of verifying whether it was present in the interphotoreceptor matrix (IPM), which occupies the subretinal space. In this study we have determined the Tau level in the IPM, separating it from other soluble amino compounds. Bovine eyes were obtained from local slaughterhouses and were bisected in darkness. After removal of the vitreos body, the eye cup was washed with 0.14 M NaCl-5mM sodium phosphates, pH 7.4. Preparation of the IPM was carried out by detaching the retina from eye cup using the method of Pfeffer,1983. PE were collected using the method of Feeney-Burns, 1982. Retinas deprived of IPM were homogenized using 0.32 M sucrose-50 mM phosphates, pH 7.2. Free amino compounds in the various preparations were separated using the procedure described by Borum, 1985. Levels of Tau in bovine IPM; PE (homogenate and sonicated); retina (homogenate and sonicated) were (804.10 ± 79.22; 83.91 ± 7.90 and 85.30 ± 8.20; 5,170.50 ± 314.82 and 5,209.00 ± 498.00 nmoles/eye), respectively. The chromatographic profile of a.a. in the IPM was qualitatively more similar to that of retina than to that of PE. As expected, GABA was absent in the PE preparations. It was not surprising to find Tau and amino compounds in the IP space because of the transit role of this retinal area. We believe that three sites could be considered for the origin of Tau in the IPM. One is the PE, which takes up Tau from the blood and accumulates it avidly, to send it via the membrane apical process to photoreceptor cells. The other possible sources are Mùller cells and photoreceptor cells, which have the largest Tau pool. In conclusion, the great similarity between the amino acid profile in the IPM and in the retina suggests that a pool of amino compounds and Tau might be present in the subretinal space. Among the roles suggested for the IPM is that of a route by which nutrients and other small molecules reach the retinal photoreceptor from the apical process of the PE cells. 46° SIBIOC ROMA 13-15 Ottobre 201