Bruno Berra

A sensitive enzymatic assay for determination of cholesterol in lipid extracts

A procedure for the determination of free and total cholesterol in lipid extracts is described. The method for free cholesterol employs cholesterol oxidase to generate H2O2 and peroxidase to catalyze the reaction of H2O2 with o-dianisidine to yield a colored product. For the determination of total cholesterol, cholesterol ester hydrolase is included.

Effects of n-3 PUFAs on breast cancer cells through their incorporation in plasma membrane.

BackgroundPUFAs are important molecules for membrane order and function; they can modify inflammation-inducible cytokines production, eicosanoid production, plasma triacylglycerol synthesis and gene expression. Recent studies suggest that n-3 PUFAs can be cancer chemopreventive, chemosuppressive and auxiliary agents for cancer therapy. N-3 PUFAs could alter cancer growth influencing cell replication, cell cycle, and cell death. The question that remains to be answered is how n-3 PUFAs can affect so many physiological processes. We hypothesize that n-3 PUFAs alter membrane stability, modifying cellular signalling in breast cancer cells.MethodsTwo lines of human breast cancer cells characterized by different expression of ER and EGFR receptors were treated with AA, EPA or DHA. We have used the MTT viability test and expression of apoptotic markers to evaluate the effect of PUFAs on cancer growth. Phospholipids were analysed by HPLC/GC, to assess n-3 incorporation into the cell membrane.ResultsWe have observed that EPA and DHA induce cell apoptosis, a reduction of cell viability and the expression of Bcl2 and procaspase-8. Moreover, DHA slightly reduces the concentration of EGFR but EPA has no effect. Both EPA and DHA reduce the activation of EGFR.N-3 fatty acids are partially metabolized in both cell lines; AA is integrated without being further metabolized. We have analysed the fatty acid pattern in membrane phospholipids where they are incorporated with different degrees of specificity. N-3 PUFAs influence the n-6 content and vice versa.ConclusionsOur results indicate that n-3 PUFA feeding might induce modifications of breast cancer membrane structure that increases the degree of fatty acid unsaturation. This paper underlines the importance of nutritional factors on health maintenance and on disease prevention.

Effect of omega-3 fatty acids supplementation on depressive symptoms and on health-related quality of life in the treatment of elderly women with depression: a double-blind, placebo-controlled, randomized clinical trial.

Objective: In elderly individuals, depression is one of the most frequently missed diagnoses with negative effects on quality of life. The authors investigated whether a supplement containing long-chain omega-3 polyunsaturated fatty acids (n-3 LCPUFA) improves depressive symptoms and health-related quality of life (HRQoL) in depressed elderly patients. Design: Eight-week, randomized, double-blind, placebo-controlled trial. Setting: Nursing home in Pavia, Italy. Participants: Forty-six depressed women, aged 66–95 years. Intervention: Twenty-two depressed women were included in the intervention group (n-3 group, which received 2.5 g/d of n-3 LCPUFA, with 1.67 g of eicosapentaenoic acid [EPA] and 0.83 g of docosahesaenoic acid [DHA]), and 24 patients were included in the placebo group. The primary endpoint was the improvement of depressive symptoms, as evaluated by the Geriatric Depression Scale (GDS). Secondary endpoints were the evaluation of HRQoL, by using the Short-Form 36-Item Health Survey (SF-36), and modifications of erythrocyte membrane phospholipids fatty acid profile. All variables were assessed before and after the treatment period of 8 weeks. Results: The mean GDS at 8 weeks was significantly lower compared with the n-3 group. The SF-36 physical and mental components were significantly increased in the intervention group. Compliance was good, as confirmed by erythrocyte membrane phospholipid FA concentrations, with a significant increase of EPA and DHA in the intervention group. Conclusion: Supplementation with n-3 LCPUFA is efficacious in the amelioration of depressive symptoms and quality of life in the treatment of depressed elderly female patients.

Correlation between ganglioside distribution and histological grading of human astrocytomas.

Changes in membrane lipid content and composition have been repeatedly detected in experimentally induced as well as spontaneous tumors. Previous data on ganglioside analysis in human astrocytomas suggested a possible correlation between their content and pattern and the histological grading of these tumors. In our study we could definitely prove this correlation: in fact the level of malignancy, passing from grade 1 to grade IV, is associated with a statistically significant increase of a ganglioside identified as ganglioside GD3. Another important modification in the ganglioside pattern of human astrocytomas is the decrease in polysialylated species with increasing level of malignancy.

Comparison between Extra Virgin Olive Oil and Oleic Acid Rich Sunflower Oil: Effects on Postprandial Lipemia and LDL Susceptibility to Oxidation

The aim of our study was to determine whether the minor polar components of virgin olive oil could have favorable effects (1) on fasting and postprandial lipid profile and (2) on low-density lipoprotein (LDL) composition and susceptibility to oxidation in vitro. Ten normolipidic subjects were included in a crossover study (two diet periods of 3 weeks) and received either virgin olive oil (OO diet) or oleic acid rich sunflower oil. An oral fat load was performed at the end of each period. The plasma lipid levels were not significantly different after both diets in the fasting and postprandial states. A few minor variations of the LDL composition were observed only in the postprandial lipemia, and they were different after both diets. The LDL oxidation susceptibility was evaluated by the formation of conjugated dienes. With LDL isolated in the fasting state, the diene production decreased (p = 0.0573) only after the OO diet. The dienes determined at time 0 and the maximal dienes obtained during the oxidation reaction decreased (p = 0.0145 and p = 0.0184, respectively) only after the OO fat load. Nevertheless, the diene production decrease was not significant (p = 0.0848). Our results suggest a mild effect of minor components of virgin olive oil related to a decrease of LDL susceptibility to oxidation; further analyses are necessary to give clear conclusions about their role.

Endogenous Antioxidants and Radical Scavengers

All living organisms are constantly exposed to oxidant agents deriving from both endogenous and exogenous sources capable to modify biomolecules and induce damages. Free radicals generated by oxidative stress exert an important role in the development of tissue damage and aging. Reactive species (RS) derived from oxygen (ROS) and nitrogen (RNS) pertain to free radicals family and are constituted by various forms of activated oxygen or nitrogen. RS are continuosly produced during normal physiological events but can be removed by antioxidant defence mechanism: the imbalance between RS and antioxidant defence mechanism leads to modifications in cellular membrane or intracellular molecules. In this chapter only endogenous antioxidant molecules will be critically discussed, such as Glutathione, Alpha-lipoic acid, Coenzyme Q, Ferritin, Uric acid, Bilirubin, Metallothioneine, L-carnitine and Melatonin.

Enzyme replacement treatment in type 1 and type 3 Gaucher's disease

The development of intravenous enzyme-replacement treatment for Gauchers disease has changed life expectancy in cases without neurological involvement (type 1). The effects in patients with neurological involvement are unknown. We treated 12 Italian patients, types 1 (9) and 3 (3), with intravenous alglucerase: 70-120 IU/kg per month for type 3 and 30-60 IU/kg per month for type 1. Maintenance infusions were biweekly in patients without neurological symptoms, whereas in one symptomatic type 3 patient, infusion was weekly. All patients improved; a resumption of growth in children with growth retardation was observed and spleen and liver reduced in size. In one type 3 patient, a bone callus formed during treatment and enabled the patient to walk. Laboratory tests showed rapid increase of haemoglobin in anaemic patients, and a slower response in patients with thrombocytopaenia. In 4 patients there was temporary hypocalcaemia immediately after the beginning of treatment. Neurological symptoms were present in 1 of the type 3 patients, and electroencephalogram was abnormal in another. After 2 years of treatment, the patient with symptoms showed an improvement of psychomotor skills and of IQ from 50 to 60. Genotype analysis showed a high frequency of the 1448C mutation (54.5%). The 9 patients carrying this allele came from Italian regions which in the past had been invaded from north Europe and Scandinavia. Enzyme replacement in Gauchers type 1 can also be effective at low doses and even with a 2-week interval between infusions. This makes treatment cheaper, and reduces hospital stay for patients.

Plasma, red blood cells phospholipids and clinical evaluation after long chain omega-3 supplementation in children with attention deficit hyperactivity disorder (ADHD)

Abstract Omega-3 and omega-6 long-chain polyunsaturated fatty acids (LCPUFAs), are crucial to brain development and function. Increasing evidence indicates that deficiencies or metabolic imbalances of these fatty acids might be associated with childhood developmental and psychiatric disorders including attention-deficit/hyperactivity disorder (ADHD). Omega-3 are often lacking on modern diets. Moreover preliminary evidences suggest that supplementation with omega-3 LCPUFAs, might help in the management of the ADHD linked behavioural and learning difficulties. However, few studies published to date have involved different populations, study designs, treatments and outcome results. Thus, further researches are required to assess the durability of the treatment effects, to determine optimal composition and dosages of the supplement and to develop reliable ways to identify patients that might have some benefits from this kind of treatment, also because the study of LCPUFAs and their metabolism might offer new approaches to the early identification and management of ADHD. In this paper, we provide new insight on the lipid pattern in plasma and red blood cells (RBC) phospholipids, together with evaluation of the arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio which seems to correlate with the improvement of the patients both from a biochemical and clinical point of view.

The primary structure of UK 114 tumor antigen

UK114 is a tumor antigen expressed by various malignant neoplasms. The complete amino acid sequence of UK114 purified from goat liver has been determined by automated Edman degradation of CNBr and endoproteinase Lys‐C peptides. The protein contains 137 amino acid residues, which corresponds to a molecular mass of 14 229 Da. MALDITOF analysis resulted in a molecular weight of 14 290, suggesting that the N‐terminal Met residue is acetylated. Sequence comparison shows that UK114 from goat liver (1) has 77% identity with a previously described 23 kDa protein from rat liver (Levy‐Favatier et al. (1993) Eur. J. Biochem. 212, 665–673), (2) shares a very high degree of similarity with a family of prokaryotic and eukaryotic hypothetic proteins whose function have not yet been characterized, and (3) exhibits a significant similarity to a group of tumor‐associated antigens which belongs to a superfamily of heat shock proteins, acting as possible targets for the hosts antitumor immunity.

Antioxidant defences in hydrated and desiccated states of the tardigrade Paramacrobiotus richtersi

Reactive oxygen species (ROS) are formed in all aerobic organisms, potentially leading to oxidative damage of all biological molecules. A number of defence mechanisms have developed to protect the organism from attack by ROS. Desiccation tolerance is correlated with an increase in the antioxidant potential in several organisms, but the regulation of the antioxidant defence system is complex and its role in desiccation-tolerant organisms is not yet firmly established. To determine if anhydrobiotic tardigrades have an antioxidant defence system, capable of counteracting ROS, we compared the activity of several antioxidant enzymes, the fatty acid composition and Heat shock protein expression in two physiological states (desiccated vs. hydrated) of the tardigrade Paramacrobiotus richtersi. In hydrated tardigrades, superoxide dismutase and catalase show comparable activities, while in desiccated specimens the activity of superoxide dismutase increases. Both glutathione peroxidase and glutathione were induced by desiccation. The percentage of fatty acid composition of polyunsaturated fatty acids and the amount of thiobarbituric acid reactive substances are higher in desiccated animals than in hydrated ones. Lastly, desiccated tardigrades did not differ significantly from the hydrated ones in the relative levels of Hsp70 and Hsp90. These results indicate that the possession of antioxidant metabolism could represent a crucial strategy to avoid damages during desiccation in anhydrobiotic tardigrades.