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Taurine in the interphotoreceptor matrix
No full textTAURINE 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
GSH: A MARKER FOR OXIDATIVE STRESS IN HUMAN CELL CULTURES
No full textGSH: 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
Correlation between the effects of retinoic acid and dexamethasone on liver tyrosine aminotransferase
No full textA single dose of 50 μg of trans-retinoic acid administered to rats significantly raised the level of hepatic tyrosine after a few hours. This effect was compared with that of dexamethasone and a possible correlation between these effecters was also investigated. An equal increase in enzyme activity level caused by retinoic acid was observed in adrenalectomized rats, leading to the suggestion that the effect of retinoic acid on liver tyrosine aminotransferase is independent of glucocorticoids. However, the study of the binding activity of the liver nuclear glucocorticoid receptors vs dexamethasone showed that this activity is favoured by retinoic acid, whereas no variation was evidenced for retinoic acid receptors caused by dexamethasone. In the adrenalectomized rat, the synergistic effect produced by the association of retinoic acid and dexamethasone at the lowest doses used led us to conclude that retinoic acid is an efficient effector of liver tyrosine aminotransferase. It probably affects tyrosine aminotransferase activity in a dependent and an independent way, regulated respectively by the glucorticoid status and by the provision of retinoic acid
SIMULTANEOUS DETERMINATION OF ATP, ITS METABOLITES AND NAD+ IN BLOOD BY HPLC WITH PHOTODIODE ARRAY DETECTOR
No full textSIMULTANEOUS DETERMINATION OF ATP, ITS METABOLITES AND NAD+ IN
BLOOD BY HPLC WITH PHOTODIODE ARRAY DETECTOR
Gueli Maria Concetta, Cusimano Vincenza, Lo Re Marianna, Giuseppe Salemi
Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche (BioNEC), Università
degli Studi di Palermo.
Nucleotides are major high-energy phosphate carriers, subunits of nucleic acids and
precursors for the synthesis of nucleotide cofactors such as NAD+ and SAM. The study of
purine nucleotides metabolism is very important topic for a right understanding for the
cellular life. Living cells rely on ATP for growth, differentiation, and response to
physiological stimuli and environmental stress.
We propose a fast isocratic HPLC system with photodiode array detector (PDA) for the
simultaneous separation and quantification of major components of high-energy metabolism:
purine nucleotides (ATP, ADP, AMP), degradation products (nucleosides, bases)
simultaneously with NAD+ in human plasma in a single run.
Blood samples were obtained from the healthy adult volunteers (University workers/30). For
stability study, 200 mL of plasma was deproteinized through a Millipore-Amicon Ultracel.
Waters-HPLC system consisted of a 600E Pump; 2998 PDA; Empower TM2 DS; Atlantis T3
analitycal column (10μL loop). The m.f. was a 40 mmol/L potassium phosphate buffer, pH
2.2 with methanol 20% at a flow rate of 1.0 mL/min. The spectral range of the PDA was 200-
400 nm and the optimal wavelength was 254 nm.
We have obtained an execellent base-line separation of high-energy phospates, including
NAD+ as well as of their catabolic products. All the peaks were identified in order of elution:
ADP, ATP, adenine, AMP, hypoxantine, uric acid, xanthine, NAD+ , adenosine and inosine
(RT = 3.5; 3.6; 4.3; 5.4; 5.6; 6.3; 8.2; 10.2; 12.1; 19.1 min), respectively. Peaks were
identified by spiking the samples with authentic standards. This method makes use of a fastsingle-
step sample pre-treatment procedure and provides the assay of the key metabolites in
small amounts of plasma extracts. Therefore, this HPLC-PDA system is suitable to evaluate
the energetic state in a variety of cell types, both under normal and pathological events and is
very useful tool both in research and in clinical laboratories.
57° SIB FERRARA 18-19 Settembre 201
Similar turnover in the metabolic appenings of central histaminergic system in Down syndrome and Alzheimer disease
No full text
Impairment of Methylation cycle in treated patients with Parkinson's disease
No full textL-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
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