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The distribution of cholesterol plasma is regulated by complex interactions between genes and environmental factors. Mutations of PCSK9 gene seem to modulate the levels of LDLC. Mutations of PCSK9 gene with gain of function are associated to hypercholesterolemia and mutations with loss of function determine hypocholesterolemia.

Retinoic acid is regarded as the retinol metabolite that controls proliferation and differentiation of epithelial cells. In the present study, we investigated the potential role of xanthine dehydrogenase (XDH) in retinoic acid biosynthesis in human thyroid glandular cells (HTGC). In particular, we observed that cellular retinoids binding proteins (CRBPs) are also implicated in the biosynthetic pathway leading to retinoic acid formation in primary cultures of HTGC, as we have already reported for human mammary epithelial cells (HMEC). After partial protein purification, the enzyme responsible for retinoic acid biosynthesis was identified and quantified as XDH by immunoassay, by its ability to oxidize xanthine to uric acid and its sensitivity to the inhibitory effect of oxypurinol. The evidence of XDH-driven formation of retinoic acid in HTGC cultures further corroborates the potential role of XDH in retinoic acid biosynthesis in the epithelia.

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 2014

P422 Purine metabolism and multiple sclerosis: pattern varies according to disease stage and clinical form G. Salemi, M. Gueli, V. Cusimano, M. Lo Re, V. Lo Re, M.A. Mazzola, S. Realmuto, P. Ragonese, G. Savettieri University (Palermo, IT) Background: Serum concentration of uric acid (sUA) was variably associated with Multiple Sclerosis (MS). Many papers reported lower sUA in MS patients respect to healthy controls, expecially in course of relapse. This lower sUA was considered as a marker of oxidative stress. However, this association was not confirmed by many other papers. Recently, an increase in the concentration of sUA, hypoxanthine, xanthine, and sum of oxypurine (sPU) was reported in a population of MS patients by HPLC detection. The authors suggested that this could be an index of a sustained purine catabolism, possibly due to imbalance in ATP homeostasis. Objective: To compare sUA and sPU between MS during stable disease and healthy controls. To confront sUA and sPU among different MS subtypes. To search for modification of sUA and sPU during and after a clinical relapse. Materials and Methods: We included MS patients with relapsing- remitting MS (RRMS), secondary-progressive MS (SPMS), and primary-progressive MS (PPMS) during stable disease and a group of healthy controls comparable for gender and age. Venous blood samples were obtained and the concentration of purine compounds was determined by HPLC separation. Moreover, subjects with MS were identified in course of a clinical relapse. Blood samples were obtained within two days from the onset, after 30 and 60 days and processed by HPLC. Non parametric tests were used to compare results between the groups. Results: We included 94 MS patients (74 RRMS, 14 SPMS, 6 PPMS) and 94 healthy controls. Median serum xanthine (p = 0.002), sUA (p = 0.0004) and sPU (p = 0.0002) concentrations were higher in MS sample. In RRMS median hypoxanthine concentration was lower respect to SPMS (0.002) and PPMS (0.01), while median sUA (p = 0.004) and sPU (p = 0.005) concentration was higher than in SPMS. Ten patients were included in course of a clinical relapse. No significant changes in the concentration of sUA aor sPU were observed comparing the first days after a relapse and the period post-relapse. Conclusion: Our data support the thesis that the changes at sUA and sPU observed in course of MS could be an index of a sustained purine catabolism, possibly due to imbalance in ATP homeostasis.

A major unanswered question is whether direct cellular toxicity of homocysteine (Hcy) is causally involved in cardiovascular disease (CVD) in humans or whether homocysteinemia is simply a passive and indirect indicator of a more complex mechanism. Alternatively or additionally, the association between Hcy and CVD may result from its metabolic precursor S-adenosyl-Hcy (SAH), or from the altered ratio of S-adenosylmethionine SAM/SAH. This competition is based on the known effects of these adenosylated metabolites on cellular methylation, disturbances of which may lead to endothelial dysfunction and/ or CVD. Thus, it is of great significance to know how these adenosylated metabolites (SAM, SAH) and Hcy are interrelated, and how they relate to CVD. We measured SAM, SAH, their molar ratio, and tHcy in plasma and in red blood cells (RBCs) of 30 patients with proven CVD (age 64.2  8.4) and 29 healthy controls (age 61  7.5). Plasma and RBCs SAM, SAH and tHcy levels were measured by HPLC method. We soon confirmed a significant increased of plasma levels of tHcy, SAM, and SAH in patients compared to the controls (13.1 vs. 10.9 micromol/l; 134 vs. 62.0 nmol/l; 79.2 vs. 24.0 nmol/l, respectively), while the molar ratio of SAM/SAH was decreased in patients (1.69 vs. 2.58). In RBCs patients exhibited increased levels of SAH compared with controls (325 vs. 201 nmol/l) whereas SAM (3327 vs. 3635 nmol/l) and the molar ratio of SAM/SAH (10.2 vs. 18.0) were significantly decreased. Folic acid and vitamin B12 in plasma were similar in patients and controls. We suppose that what we noticed might be due to the lack of remethylation in RBCs additionally to the inhibition of transmethylation reactions by elevation of SAH. Since the ratio of SAM/SAH is strongly linked with the activity of numerous enzymatic methylation reactions, these results suggest that methylation may be impaired in these patients. This increased intracellular SAH, when occurs in endothelial cells, would lead to excessive endothelial apoptosis, which is both pro-atherogenic and pro-thrombotic. Together, these results point out the importance of remethylation pathway in CVD.

Curcumin (CUR) can be considered as a good lead compound for the design of new anticancer drugs. Further, structure-activity relationship studies may clarify the importance of the redox activities in the antitumor effects of the drug. We have elaborated the alpha,beta-unsaturated 1,3-diketone moiety of CUR into the isoxazole (ISO) and pyrazole (PYR) derivatives. These derivatives should be much less prone to nucleophilic addition than CUR and benzyl mercaptan addition analyses showed that indeed they do not form isolable conjugated products. When compared with CUR, ISO and PYR exhibited increased cell growth inhibitory and pro-apoptotic effects in liver cancer HA22T/VGH cells as well as in other tumor cell types; in contrast to CUR, the antitumor effects of ISO or PYR were not influenced by concomitant administration of N-acetylcysteine, as a source of -SH groups, or buthionine sulfoximine, as an inhibitor of glutathione synthesis. Further, treatment with CUR, but not with ISO or PYR, significantly decreased the content of reduced glutathione in the HA22T/VGH cells. Finally, ISO and PYR lacked the ability of the parent compound to sensitize the HA22T/VGH cells to cisplatin (CIS), an effect which appeared to occur through an interaction of CUR and CIS at the level of the -SH groups. Thus, the ability of interacting with cell thiols might not be requested for the more potent antitumor activities of new diketone modified CUR derivatives, which might rely on other mechanisms, though possibly devoid of chemosensitization capabilities.

Blood levels of total homocysteine (tHcy), cysteine (Cys), total and reduced glutathione (tGSH and rGSH), folic acid (FA), and vitamin B(12) (B(12) change during ischemic stroke as accompaniment of the tissue damage. The relationship between these changes remains scantly investigated. We evaluated the variation of these molecules in the 48 h after acute large artery atherothrombotic stroke (LAAS) and searched for the presence of matched variation of them. The study involved 50 subjects affected by acute LAAS and 49 healthy controls. Plasma levels of tHcy and Cys were significantly higher and serum levels of FA and B(12) and plasma levels of rGSH were significantly lower in the patients than in the control group. Acute LAAS was associated with increased Hcy-decreased tGSH and decreased FA/tGSH. Pathways involved in cellular stress and in tissue repair are activated during acute LAAS.

A decrease of antioxidants, of neuroprotective and immunoregulatory vitamins and an increase of total-Homocysteine, Cholesterol, HDL-cholesterol, and of cellular stress markers [1] was reported in patients affected by Multiple Sclerosis. Recently, considering their unreliability, mainly due to the variability of the samples investigated, the attention focused on clinical relapse that results associated to a decrease of Uric acid and an increase of Cholesterol and stress markers. Aim. To identify the biochemical status during Multiple Sclerosis (MS) in a phase of clinical stability (PCS), we compared the blood levels of Urico acid (UA), Folic acid (FA), vitamins B12, A, and E, total-Homocysteine (t-Hcy), total Cholesterol (CHL) , HDL-CHL, and Triglycerides (TG) in 20 MS stable patients with those of 40 healthy controls. Methods. Consecutive MS patients, with relapsing-remitting or secondary-progressive courses, in a (PCS), were included. Plasma t-Hcy levels were determined by Ubbink method [1]. Technicon Immuno autoanalyser was used for serum FA and vitamin B12 assays. HPLC and fluorometry were used for UA, vitamin A and E, CHL, HDL-CHL, and TG assays. The ratio of E/CHL was valued. Results and Discussions. We found that MS patients in a PCS have higher blood levels of vitamin B12, t-Hcy, CHL, and HDL-CHL and lower blood levels of vitamin E and of the ratio E/CHL. The blood level of UA, FA, vitamin A, and TG do not differ from controls during this phase of MS. The increased level of CHL could be expression of both an its increased synthesis by neural cells and a chronic damage of myelin and axons. HDL-CHL concentration might arise to counteract the increase of CHL and assure its transport to the liver. Plasma levels of vitamin E, the major hydrophobic chain-breaking antioxidant, are decreased and our data confirm and support the view that it is consumed to counterbalance MS chronic neurodegeneration. Also, the increased levels of t-Hcy and vitamin B12 match previous studies performed in MS patients outside relapse. No significant differences in UA, FA, vitamin A, and TG levels appeared, making unprobable their involvement in the degenerative process of the stable phase of MS.

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.

S-thiolation of plasma proteins have been detected in healthy humans, in patients with cardiovascular diseases and it is a recurrent phenomenon in oxidative stress elicited by reactive oxygen species. The low-molecular-weight aminothiols homocysteine (Hcy) and cysteine (Cys) can exist in the plasma either free or bound to thiol-combining groups, many of which are present in proteins (Pb), particularly albumin. Recent studies have demonstrated that also plasma lipoproteins (LP) are susceptible to form disulfide-linked products with Hcy and Cys and that the N-homocysteinylation is accompanied by structural and functional alteration and could increase the atherogenicity of LDL. However, the mechanism through which Hcy contributes to these abnormalities remains however still undefined. In order to ascertain the link between physiologic thiols and lipoproteins (LP), we determined simultaneously the distribution of Hcy and Cys, in vivo, bound to different plasma protein fractions by disulfide linkage. Human VLDL, LDL, HDL and LPDS (lipoprotein-free protein fraction) were obtained from plasma of normolipemic subjects volunteers (University workers) prepared by differential ultracentrifugation. The purity of the isolated subfractions were controlled by electrophoresis on agarose gel and no significant albumin content was found in the isolated lipoprotein fractions. The Hcy and Cys bound to single plasma protein fraction was calculated as difference between total and free aminothiols. Hcy and Cys in plasma and in lipoprotein fractions were analyzed by the HPLC method (1). Protein-bound Hcy and Cys (Pb-Hcy and Pb-Cys) (9.10 and 196.79 µmol/L, respectively) represents about 76% and 79% of total plasma Hcy and Cys (11.98 and 249.33 µmol/L, respectively). The absolute Pb-Hcy and Pb-Cys distribution between the single LP fractions was as follows (µmol/L): VLDL=0.59 (6.4%) and 36.78 (18.7%) respectively; LDL=0.77 (8.5%) and 9.13 (4.6%) respectively; HDL=1.44 (15.8%) and 6.63 (3.4%) respectively; LPDS=6.30 (69.3%) and 144.24 (73.3) respectively. The Hcy and Cys/protein ratios in each single LP fraction (nmol/mg of protein) suggest a higher binding capacity for Hcy and Cys by VLDL and LDL probably due to their content in Apo B. The detection of all aminothiols (Hcy, Cys, Cys-Gly, GSH) linked to Apo B may be important to understand the mechanisms of thiol-disulfide exchange reactions and to evaluate if this binding may have a complex role in promoting lipoprotein oxidation or structural and/or functional modifications in lipoprotein thiolation both in vivo and in vitro.

Hypobetalipoproteinemia (HBL) is characterized by cholesterol levels below the 5th percentile of the population distribution. Familial HBL (FHBL) is characterized by an inherited HBL trait within a family. In the last years gene defects underlying some cases of FHBL, but not all, have been identified. The molecular basis of some gene defect and the clinical onset of FHBL are presented in this paper.

The distribution of cholesterol plasma is regulated by complex interactions between genes and environmental factors. Mutations of PCSK9 gene seem to modulate the levels of LDL-C. Mutations of PCSK9 gene with gain of function are associated to hypercholesterolemia and mutations with loss of function determine hypocholesterolemia.

Milk xanthine oxidase (xanthine: oxygen oxidoreductase; XO; EC 1.1.3.22) was found to catalyze the conversion of retinaldehyde to retinoic acid. The ability of XO to synthesize all trans-retinoic acid efficiently was assessed by its turnover number of 31.56 min-1, determined at pH 7.0 with 1 nM XO and all trans-retinaldehyde varying between 0.05 to 2 microM. The determination of both retinoid and purine content in milk was also considered in order to correlate their concentrations with kinetic parameters of retinaldehyde oxidase activity. The velocity of the reaction was dependent on the isomeric form of the substrate, the all trans- and 9-cis-forms being the preferred substrates rather than 13-cis-retinaldehyde. The enzyme was able to oxidize retinaldehyde in the presence of oxygen with NAD or without NAD addition. In this latter condition the catalytic efficiency of the enzyme was higher. The synthesis of retinoic acid was inhibited 87% and 54% by 4 microM and 2 microM allopurinol respectively and inhibited 48% by 10 microM xanthine in enzyme assays performed at 2 microM all trans-retinaldehyde. The Ki value determined for xanthine as an inhibitor of retinaldehyde oxidase activity was 4 microM.

Multiple Sclerosis (MS) patients present a decrease of antioxidants and neuroprotective and immunoregulatory vitamins and an increase of total homocysteine (tHcy), cholesterol (CHL), HDL-cholesterol, and of cellular stress markers, variably associated with the different phases of the disease. We compared the blood levels of uric acid, folic acid, vitamins B12, A, and E, tHcy, CHL, HDL-cholesterol, and triglycerides in forty MS patients during a phase of clinical inactivity with those of eighty healthy controls, matched for age and sex. We found higher levels of tHcy (p = 0.032) and of HDL-cholesterol (p = 0.001) and lower levels of vitamin E (p = 0.001) and the ratio vitamin E/CHL (p = 0.001) in MS patients. In conclusion, modifications of some biochemical markers of cell damage were detected in MS patients during a phase of clinical inactivity.

1. A nonspecific nucleoside phosphotransferase (nucleotide : 3'-deoxynucleotide 5'-phosphotransferase, EC 2.7.1.77), purified from chick embryos, catalyzes the transfer of phosphate ester from a nucleotide donor to a nucleoside acceptor. 2. The enzyme exhibits sigmoidal kinetics with respect to nucleoside monophosphate donors, but with respect to nucleoside di- or triphosphate donors and nucleoside acceptors hyperbolic kinetics were obtained. 3. The nucleoside phosphotransferase of chick embryo is unstable to heat and is protected from inactivation by a large number of nucleosides. 4. Nucleoside di- and triphosphates lower both the concentration of nucleoside monophosphates required for half-maximal velocity and the kinetic order of reaction measured with these phosphate donors. On the contrary, nucleoside di- or triphosphate do not modify the kinetic parameters evaluated for nucleoside acceptors. 5. We suggest that the nucleoside phosphotransferase contains both substrate and regulatory sites. It seems that the free apoenzyme is converted, by means of cooperative interactions between regulatory sites, into an enzyme-nucleotide complex, which is particularly stable at 37 degrees C.