Cinzia Mascia

An inter-laboratory validation of methods of lipid peroxidation measurement in UVA-treated human plasma samples

Abstract Lipid peroxidation products like malondialdehyde, 4-hydroxynonenal and F2-isoprostanes are widely used as markers of oxidative stress in vitro and in vivo. This study reports the results of a multi-laboratory validation study by COST Action B35 to assess inter-laboratory and intra-laboratory variation in the measurement of lipid peroxidation. Human plasma samples were exposed to UVA irradiation at different doses (0, 15 J, 20 J), encoded and shipped to 15 laboratories, where analyses of malondialdehyde, 4-hydroxynonenal and isoprostanes were conducted. The results demonstrate a low within-day-variation and a good correlation of results observed on two different days. However, high coefficients of variation were observed between the laboratories. Malondialdehyde determined by HPLC was found to be the most sensitive and reproducible lipid peroxidation product in plasma upon UVA treatment. It is concluded that measurement of malondialdehyde by HPLC has good analytical validity for inter-laboratory studies on lipid peroxidation in human EDTA-plasma samples, although it is acknowledged that this may not translate to biological validity.

Pro-oxidant and proapoptotic effects of cholesterol oxidation products on human colonic epithelial cells: A potential mechanism of inflammatory bowel disease progression

With the aim of investigating whether cholesterol oxidation products could contribute to the pathogenesis of the intestinal epithelial barrier dysfunction that occurs in human inflammatory bowel disease (IBD), differentiated versus undifferentiated CaCo-2 cells, an accepted model for human intestinal epithelial cells, were challenged with a dietary-representative mixture of oxysterols. Only differentiated colonic cells were susceptible to the proapoptotic action of the oxysterol mixture, checked both by enzymatic and by morphological methods, mainly because of a very low AKT phosphorylation pathway compared to the undifferentiated counterparts. Enhanced production of reactive oxygen species by a colonic NADPH oxidase hyperactivation seemed to represent the key event in oxysterol-induced up-regulation of the mitochondrial pathway of programmed death of differentiated CaCo-2 cells. These in vitro findings point to the pro-oxidant and cytotoxic potential of cholesterol oxidation products, of both dietary and endogenous origin, as an important mechanism of induction and/or worsening of the functional impairment of enteric mucosa that characterizes IBD.

The contribution of animal fat oxidation products to colon carcinogenesis, through modulation of TGF-β1 signaling

It is now unanimously accepted that neoplastic cells tend to become less susceptible to the growth regulatory effects of transforming growth factor-beta1 (TGF-beta1), mainly because of reduced expression and/or activity of TGF-beta1-specific receptors, as reported for many human cancers including colon cancer. Consequently, a sustained increase of TGF-beta1 in the intestinal mucosa, like that caused by inflammatory processes and/or high dietary intake of animal fat, might become crucial for the progression of a neoplastic clone. In fact, this proapoptotic and prodifferentiating cytokine could eliminate neoplastic cells still susceptible to TGF-beta1s antiproliferative action (TGF-beta1 receptor-positive cells), indirectly favoring the expansion of TGF-beta1 resistant ones (TGF-beta1 receptors deficient or negative cells). The actual concentration of TGF-beta1 in the colonic mucosa undergoing neoplastic transformation is still debated, and the phase of the relevant carcinogenetic process in which a reduced susceptibility to this antiproliferative molecule first occurs has not been precisely established yet. However, no doubt that TGF-beta1 level and activity may be upregulated in cells of the macrophage lineage by animal fat oxidation products, such as oxysterols and aldehydes, as reviewed here. But phagocytes as well as fibroblasts constitutively express TGF-beta1 and are accumulating in tumor-associated stroma. Thus, upregulation of this cytokine system within colonic tumor-associated stroma by excess dietary intake of cholesterol and n-6 polyunsaturated fatty acids appears as a primary mechanism of cancer progression at least in neoplastic lesions of the digestive tract.

N-acetylcysteine is able to reduce the oxidation status and the endothelial activation after a high-glucose content meal in patients with Type 2 diabetes mellitus

Post-prandial hyperglycemia seems to play a pivotal role in the pathogenesis of the cardiovascular complications of diabetes mellitus, as it leads to an oxidative stress which in turn causes a reduced NO bioavailability. These conditions produce an endothelial activation. Aim of the study: The aim of this study was to assure that the administration of N-acetylcysteine (NAC), thiolic antioxidant, is able to decrease the oxidation status and endothelial activation after a high-glucose content meal. Subjects and methods: Ten patients with Type 2 diabetes mellitus (DMT2) (Group 1) and 10 normal subjects (Group 2) were studied. They assumed a high-glucose content meal without (phase A) or after (phase B) the administration of NAC. Glycemia, insulinemia, intercellular adhesion molecule 1, vascular adhesion molecule 1 (VCAM-1), E-selectin, malonaldehyde (MDA), and 4-hydroxynonenal (HNE) were assessed at −30, 0, +30, +60, +90, +120, and +180 min with respect to the meal consumption. Results: During the phase A in Group 1, only HNE and MDA levels increased after the meal assumption; all parameters remained unchanged in Group 2. During the phase B, in Group 1, HNE, MDA, VCAM-1, and E-selectin levels after the meal were lower than those in phase A, while no change for all variables were observed in Group 2. Conclusions: A high-glucose meal produces an increase in oxidation parameters in patients with DMT2. The administration of NAC reduces the oxidative stress and, by doing so, reduces the endothelial activation. In conclusion, NAC could be efficacious in the slackening of the progression of vascular damage in DMT2.

TGFβ1 expression in colonic mucosa: modulation by dietary lipids

Transforming growth factor beta1 (TGFβ1) is fundamental to maintain the intestinal epithelial cell homeostasis through its control action on cell proliferation, differentiation and apoptosis. TGFβ1 dysregulation has been observed in several chronic human diseases, including ulcerative colitis, Crohn’s disease and colon carcinoma. In the first two conditions, a marked oxidative stress is consistently present, while in the third one, levels of reactive oxygen species tend to be significantly lower than in the surrounding normal tissue. Lipid-derived compounds such as the aldehyde 4-hydroxynonenal (HNE) or cholesterol oxidation products (oxysterols) were shown able to induce expression and synthesis of TGFβ1, an event which can be detrimental or beneficial, essentially depending on its actual intensity. Understanding how specific dietary lipids may influence the complex molecular signaling underlying this cytokine expression, may provide new indications for therapeutic and preventive strategies in inflammatory bowel diseases and colon carcinoma.