Fatty acids, as structural components of membranes and inflammation/anti-inflammatory mediators, have well-known protective and regulatory effects. They are studied as biomarkers of pathological conditions, as well as saturated and unsaturated hydrophobic moieties in membrane phospholipids that contribute to homeostasis and physiological functions. Lifestyle, nutrition, metabolism and stress—with an excess of radical and oxidative processes—cause fatty acid changes that are examined in the human body using blood lipids. Fatty acid-based membrane lipidomics represents a powerful diagnostic tool for assessing the quantity and quality of fatty acid constituents and also for the follow-up of the membrane fatty acid remodeling that is associated with different physiological and pathological conditions. This review focuses on fatty acid biomarkers with two examples of recent lipidomic research and health applications: (i) monounsaturated fatty acids and the analytical challenge offered by hexadecenoic fatty acids (C16:1); and (ii) the cohort of 10 fatty acids in phospholipids of red blood cell membranes and its connections to metabolic and nutritional status in healthy and diseased subjects.

Palmitic acid metabolism involves delta-9 and delta-6 desaturase enzymes forming palmitoleic acid (9cis-16:1; n-7 series) and sapienic acid (6cis-16:1; n-10 series), respectively. The corresponding biological consequences and lipidomic research on these positional monounsaturated fatty acid (MUFA) isomers are under development. Furthermore, sapienic acid can bring to the de novo synthesis of the n-10 polyunsaturated fatty acid (PUFA) sebaleic acid (5cis,8cis-18:2), but such transformations in cancer cells are not known. The model of Caco-2 cell line was used to monitor sapienic acid supplementation (150 and 300 μM) and provide evidence of the formation of n-10 fatty acids as well as their incorporation at levels of membrane phospholipids and triglycerides. Comparison with palmitoleic and palmitic acids evidenced that lipid remodelling was influenced by the type of fatty acid and positional isomer, with an increase of 8cis-18:1, n-10 PUFA and a decrease of saturated fats in case of sapienic acid. Cholesteryl esters were formed only in cases with sapienic acid. Sapienic acid was the less toxic among the tested fatty acids, showing the highest EC50s and inducing death only in 75% of cells at the highest concentration tested. Two-photon fluorescent microscopy with Laurdan as a fluorescent dye provided information on membrane fluidity, highlighting that sapienic acid increases the distribution of fluid regions, probably connected with the formation of 8cis-18:1 and the n-10 PUFA in cell lipidome. Our results bring evidence for MUFA positional isomers and de novo PUFA synthesis for developing lipidomic analysis and cancer research.

Abstract. BACKGROUND:Red blood cells (RBC) are obtained by non-invasive methods and widely used for diagnostic tests of health status. Hyperspectral Dark Field Microscopy (HDFM) is a promising technique for nanoscale bio imaging and spectral analysis without additional sample preparation. OBJECTIVE: Develop a protocol for human RBC characterization by HDFM, checking the feasibility of a reference spectral library that can image and afford a new comprehensive descriptor of RBC status. METHOD: A step-by-step protocol for HDFM measurement of human RBC was for the first time established using 5 µl of EDTA-treated whole blood from healthy adults (n = 30). Hyperspectral characteristics of solutions/suspensions at biological concentrations of phospholipids, hemoglobin, spectrin, cholesterol and protoporphyrin IX, as the most relevant RBC components, were also determined. RESULTS: A library made of 8 end-member spectra and classification of their spectral distribution carried out by Single Angle Mapper (SAM) were determined, furnishing a comprehensive mapping and descriptor of healthy human RBC. The spectra of single components allowed some of the RBC spectral bands to be attributed. CONCLUSIONS: This work reports for the first time the hyperspectral optical imaging of the human RBC by a library made of 8 scattering spectra, whose spectral signatures are compared with those of the main RBC molecular components. The percent distribution of the spectral end-members was also achieved, thus giving for the first time the HDFM mapping of human healthy RBCs. The protocol developed herein allows the clinical potential of hyperspectral imaging to be developed for the use of RBC mapping in health and disease.

Docosahexaenoic acid (DHA) is a semiessential polyunsaturated fatty acid (PUFA) for eukaryotic cells that is found in natural sources such as fish and algal oils and widely used as an ingredient for omega-3 containing foods or supplements. DHA effects are connected to its natural structure with six cis double bonds, but geometrical monotrans isomers can be formed during distillation or deodorization processes, as an unwanted event that alters molecular characteristics and annihilates health benefits. The characterization of the six monotrans DHA regioisomers is an open issue to address for analytical, biological, and nutraceutical applications. Here we report the preparation, separation, and first identification of each isomer by a dual approach consisting of the following: (i) the direct thiyl radical-catalyzed isomerization of cis-DHA methyl ester and (ii) the two-step synthesis from cis-DHA methyl ester via monoepoxides as intermediates, which are separated and identified by nuclear magnetic resonance spectroscopy, followed by elimination for the unequivocal assignment of the double bond position. This monotrans DHA isomer library with NMR and GC analytical characterization was also used to examine the products of thiyl-radical-catalyzed isomerization of a fish oil sample and to evaluate the trans isomer content in omega-3 containing supplements commercially available in Italy and Spain.

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Palmitic acid metabolism involves delta-9 and delta-6 desaturase enzymes forming palmitoleic acid (9cis-16:1; n-7 series) and sapienic acid (6cis-16:1; n-10 series), respectively. The corresponding biological consequences and lipidomic research on these positional MUFA isomers are under development. Furthermore, sapienic acid can bring to the de novo synthesis of the n-10 polyunsaturated fatty acid (PUFA) sebaleic acid (5cis,8cis-18:2), but such transformations in cancer cells are not known. The model of Caco-2 cell line was used to monitor sapienic acid supplementation (150 and 300 μM) and evidence the formation of n-10 fatty acids as well as their incorporation at levels of membrane phospholipids and triglycerides. Comparison with palmitoleic and palmitic acids evidenced that lipid remodeling was influenced by the type of fatty acid and positional isomer, with increase of 8cis-18:1, n-10 PUFA and decrease of saturated fats in case of sapienic acid. Cholesteryl esters were formed only in case of sapienic acid. EC50 of sapienic acid (232.3 μM at 96 hrs) was the highest found among the tested fatty acids, thus influencing cell viability that was only reduced at 25% at 300 μM, whereas palmitoleic acid induced cell death. Two-photon fluorescent microscopy with Laurdan as a fluorescent dye provided information on membrane fluidity, highlighting that sapienic acid increases the distribution of fluid regions, probably connected with the formation of 8cis-18:1 and the n-10 PUFA in cell lipidome. Our results bring evidence for MUFA positional isomers and de novo PUFA synthesis for developing lipidomic analysis and cancer research.

Background: Breast cancer is still the leading cause of death in women worldwide. Study question:Our purpose is to improve survival, objective response and quality of life with a non-toxic biological therapy. Study design:The DBM (Di Bella Method) with MLT (Melatonin), Retinoids, solubilized in vitamin E, D3, and C, Folates, proteoglycans and calcium, has a differentiating, cytostatic, anti-angiogenic, immune-modulating, factorially synergic effect, simultaneously reinforcing the functions which physiology considers to be essential for life. With Somatostatin/Octreotide, prolactin and estrogen inhibitors, DBM has an antiproliferative, antiangiogenic and antimetastatic effect. These molecules govern negatively pituitary secretions like GH-PRL, whose mitogenic properties are enhanced by the combination of ovarian hormones such as estrogen. The negative regulation of GH extends to IGF-1, EGF (Epidermal Growth Factor), VEGF (Vascular Endothelial Growth Factor), and GHdependent growth factors. The DBM (Di Bella Method) uses minimal metronomic apoptotic, non-cytotoxic and non-mutagenic doses of cyclophosphamide. The tolerability of which is enhanced by MLT and by the vitamins included in the DBM. We report a retrospective observational study with 5-year follow-up, carried out on 297 patients affected by breast cancer and treated with the Di Bella Method biological therapy.Results and conclusion: complete and stable objective responses without cytolytic chemotherapy, in some cases even without surgery or radiotherapy, with a generalized improvement in quality of life and no significant and/or prolonged toxicity. The most important 5-year survival rate was 69.4% at stage IV cancer versus 26.3% reported by the National Cancer Institute.

Background:The interaction between pituitary hormones (Growth Hormone-Prolactin), ovarian hormones (Estradiol) and growth factors forms the basis of the mechanisms underlying the growth of tumors of the breast. The literature contains reports of the increased expression of the mitogenic GH (Growth Hormone)/IGF1 (Insulin-Like Growth Factor) axis in tumor tissues compared to healthy tissues, with a directly proportional dose-dependent relationship between GH/IGF1, proliferative index and invasive ability in numerous types of tumors. Methods and Findings:We carried out this experimental research on the mitogenic role of GH and consequently on the rationale of the anticancer use of its inhibition. The levels of expression of several genes, GH and GHR, were evaluated in 39 cases of breast cancer, divided according to different risk levels on the basis of immunohistochemical and histological tests with nuclear grade. Conclusion:Research showed that breast cancers with a high and intermediate risk of recurrence are characterized by over-expression of GH and of its receptor (GHR). The expression was limited in cases with a low risk. The overexpression of GH-GHR in breast cancer with a ratio proportional to the level of aggressiveness is a rationale that can encourage a therapeutic intervention with inhibition of the mitogenic GH-IGF1-PRL axis and estrogen.