A comprehensive review of healthy effects of vegetarian diets.

AIMS: A comprehensive review comparing the effect of vegetarian (V) and non-vegetarian (NV) diets on the major cardiometabolic diseases' outcomes was performed. DATA SYNTHESIS: We performed literature research (up to December 31, 2022) of the evidence separately for vascular disease (VD), obesity (OB), dyslipidemia (Dysl), hypertension (HPT), type 2 diabetes (T2D), metabolic syndrome (MetS), analyzing only cohort studies and randomized controlled studies (RCTs) and comparing the effect of V and NV diets. Cohort studies showed advantages of V diets compared to NV diets on incidence and/or mortality risk for ischemic heart disease, overweight and OB risk. Most cohort studies showed V had lower risk of HPT and lower blood pressure (BP) than NV and V diets had positive effects on T2D risk or plasma parameters. The few cohort studies on the risk of MetS reported mixed results. In RCTs, V diets, mainly low-fat-vegan ones, led to greater weight loss and improved glycemic control than NV diets and in the only one RCT a partial regression of coronary atherosclerosis. In most RCTs, V diets significantly reduced LDL-C levels (but also decreased HDL-C levels) and BP. CONCLUSIONS: In this comprehensive review of the association between V diets and cardiometabolic outcomes, we found that following this type of diet may help to prevent most of these diseases. However, the non-uniformity of the studies, due to ethnic, cultural, and methodological differences, does not allow for generalizing the present results and drawing definitive conclusions. Further, well-designed studies are warranted to confirm the consistency of our conclusions.

Position paper on vegetarian diets from the working group of the Italian Society of Human Nutrition.

BACKGROUND: Interest in vegetarian diets is growing in Italy and elsewhere, as government agencies and health/nutrition organizations are emphasizing that regular consumption of plant foods may provide health benefits and help prevent certain diseases. METHODS AND RESULTS: We conducted a Pubmed search, up to September, 2015, for studies on key nutrients (proteins, vitamin B12, iron, zinc, calcium, vitamin D, and n-3 fatty acids) in vegetarian diets. From 295 eligible publications the following emerged: Vegetarians should be encouraged to supplement their diets with a reliable source of vitamin B12 (vitamin-fortified foods or supplements). Since the plant protein digestibility is lower than that of animal proteins it may be appropriate for vegetarians to consume more proteins than recommended for the general population. Vegetarians should also be encouraged to habitually consume good sources of calcium, iron and zinc - particularly vegetables that are low in oxalate and phytate (e.g. Brassicaceae), nuts and seeds, and calcium-rich mineral water. Calcium, iron, and zinc bioavailability can be improved by soaking, germination, and sour-dough leavening that lower the phytate content of pulses and cereals. Vegetarians can ensure good n-3 fatty acid status by habitually consuming good sources of a-linolenic acid (walnuts, flaxseeds, chia seeds, and their oils) and limiting linoleic acid intake (corn and sunflower oils). CONCLUSIONS: Well-planned vegetarian diets that include a wide variety of plant foods, and a reliable source of vitamin B12, provide adequate nutrient intake. Government agencies and health/nutrition organizations should provide more educational resources to help Italians consume nutritionally adequate vegetarian diets.

Iron and copper alter tight junction permeability in human intestinal Caco-2 cells by distinct mechanisms.

Human intestinal Caco-2 cells differentiated for 15-17 days on transparent filter inserts were treated for up to 3 h with 50 and 100 microM CuCl(2) or FeSO(4) in the AP compartment at pH 6.0. Trans-epithelial electrical resistance (TEER) showed a progressive decrease during the course of the experiment that was slower in cells treated with 50 microM CuCl(2) than in those treated with 100 microM CuCl(2). Both 50 and 100 microM FeSO(4) produced a similar decrease in TEER over time, tailing off after 120 min. F-actin localization by fluorescent phalloidin binding in control cells and in cells treated for 3 h with 50 microM CuCl(2) or FeSO(4) highlighted striking differences in the two treatments. Cu(II) led to an overall reduction in F-actin staining with extensive depolymerization in areas of the monolayer, in the absence of cellular loss. Conversely, Fe(II) treatment produced disorganization of F-actin and decreased staining of the perijunctional actin filaments. No changes in the localization and intensity of staining of the junctional proteins ZO1, occludin and E-cadherin were observed after treatment with 100 microM FeSO(4) in analogy with previous observations in Cu(II)-treated cells. The data presented suggest that different mechanisms are responsible for the changes to tight junction permeability produced by the two metals.

Heavy metal toxicity following apical and basolateral exposure in the human intestinal cell line Caco-2.

Caco-2 is a cell line, derived from a human colon carcinoma, that retains the ability to differentiate in culture into absorptive intestinal cells. Caco-2 cells were used to evaluate the toxicity of three heavy metals-the essential trace elements zinc and copper, and the xenobiotic cadmium. The cells were cultivated on permeable filters until differentiated and were then exposed to the metals either from the apical (luminal) or from the basolateral (serosal) side. Toxicity was measured in dose-effect experiments with reference to cell survival and integrity of the cell monolayer. The metals were more toxic when presented to the basolateral than to the apical cell side. The toxicity ranking was cadmium > > copper > zinc. The cell's ability to transport each metal across the monolayer and the resulting intracellular accumulation could account for the cytotoxic effects. A specific toxic effect observed on a specialized function of these cells was the interference of cadmium in tight-junction integrity as shown by changes in the transepithelial electrical resistance, in the rate of transport of a specific marker across the cell monolayer, and by morphological alterations of the tight junctions.

Optimization of chemically defined cell culture media--replacing fetal bovine serum in mammalian in vitro methods.

Quality assurance is becoming increasingly important. Good laboratory practice (GLP) and good manufacturing practice (GMP) are now established standards. The biomedical field aims at an increasing reliance on the use of in vitro methods. Cell and tissue culture methods are generally fast, cheap, reproducible and reduce the use of experimental animals. Good cell culture practice (GCCP) is an attempt to develop a common standard for in vitro methods. The implementation of the use of chemically defined media is part of the GCCP. This will decrease the dependence on animal serum, a supplement with an undefined and variable composition. Defined media supplements are commercially available for some cell types. However, information on the formulation by the companies is often limited and such supplements can therefore not be regarded as completely defined. The development of defined media is difficult and often takes place in isolation. A workshop was organised in 2009 in Copenhagen to discuss strategies to improve the development and use of serum-free defined media. In this report, the results from the meeting are discussed and the formulation of a basic serum-free medium is suggested. Furthermore, recommendations are provided to improve information exchange on newly developed serum-free media.

The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics.

The human intestinal Caco-2 cell line has been extensively used over the last twenty years as a model of the intestinal barrier. The parental cell line, originally obtained from a human colon adenocarcinoma, undergoes in culture a process of spontaneous differentiation that leads to the formation of a monolayer of cells, expressing several morphological and functional characteristics of the mature enterocyte. Culture-related conditions were shown to influence the expression of these characteristics, in part due to the intrinsic heterogeneity of the parental cell line, leading to selection of sub-populations of cells becoming prominent in the culture. In addition, several clonal cell lines have been isolated from the parental line, exhibiting in general a more homogeneous expression of differentiation traits, while not always expressing all characteristics of the parental line. Culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often make it extremely difficult to compare results in the literature. This review is aimed at summarizing recent, or previously unreviewed, data from the literature on the effects of culture-related factors and the influence of line sub-types (parental vs. different clonal lines) on the expression of differentiation traits important for the use of Caco-2 cells as a model of the absorptive and defensive properties of the intestinal mucosa. Since the use of Caco-2 cells has grown exponentially in recent years, it is particularly important to highlight these methodological aspects in order to promote the standardization and optimisation of this intestinal model.

The Fao cell. A tissue culture model for lipoprotein synthesis and secretion. I. Characterization of the system.

In this study we found that a differentiated cultured rat hepatocyte cell line, Fao, synthesizes and secretes lipoproteins qualitatively similar to those synthesized by the rat hepatocyte in vivo, but quantitatively differing considerably in apoprotein composition and density distribution. Immunoprecipitation demonstrates that all the major apoproteins are synthesized, including both forms of apoB, apoE and apoA-I. Particles of all density classes are formed, apoB associating with the lighter particles and apoA-I with the heavier. ApoE is a major apoprotein in all but the lightest density classes. The general finding is that most particles formed have a density exceeding 1.10 g/ml, while very few of the lighter, apoB-containing particles form, probably because the normal growth medium of the cultured cells is lipid-poor as compared with rat serum. In ref. [20] we show that the composition of the lipoproteins synthesized can be effectively modulated by lipid depletion and lipid supplementation of the growth medium.

The Fao cell. A tissue culture model for lipoprotein synthesis and secretion. II. Modulation by lipid depletion and supplementation.

We have shown that the Fao cell, a differentiated rat hepatoma line, is an excellent model for the study of the synthesis of lipoproteins (Scarino, M L & Howell, K E, Exp cell res 170 (1987) 1 [1]. Here we demonstrate that variation of the lipid composition of the growth medium significantly modulates the composition and quantity of particles formed. Three growth conditions were compared: normal, lipid-depleted, and lipid-supplemented. The synthesis of both the protein and lipid moieties of the lipoproteins was quantitated using the radioactive metabolic precursors [35S]methionine and [14C]acetate. The total secretion of the cells was collected and fractionated into four density classes equivalent to plasma lipoproteins and a bottom fraction equivalent to plasma proteins. Each density class was evaluated for the apoprotein distribution after separation by SDS-PAGE and for lipid distribution and composition after lipid extraction. ApoE accounts for approx. 15% of the total protein synthesized and is the major apoprotein. The amount synthesized remains relatively constant under all growth conditions. In contrast, the amount of apoB synthesis varies over 600-fold. In lipid-depleted conditions, only 0.01 times the normal amount was synthesized, while in lipid-supplemented conditions 6.2 times the normal amount was synthesized. ApoB was associated with the lighter fraction; therefore the modulation increased the quantity of low-density particles formed. A similar but far less pronounced variation of the heavier particles and the apoA-I concentration was obtained. Under lipid-depleted conditions, 0.75 times the normal amount was synthesized, while under lipid-supplemented conditions 2.6 times the normal quantity was synthesized.

Biphasic effect of iron on human intestinal Caco-2 cells: early effect on tight junction permeability with delayed onset of oxidative cytotoxic damage.

Treatment of differentiated human intestinal Caco-2 cells with Fe(II) ascorbate altered tight junction permeability in a dose and time-dependent way for up to 3 hr of treatment Upon iron removal and transfer to complete culture medium, the effect was reversible up to 10 microM Fe(II), while at higher concentrations a late phase toxic effect was observed. Reduction of intracellular energy abolished the short term effect of iron on tight junction permeability without affecting its cellular uptake, suggesting that active processes, other than transport, were involved. The short term effect of iron the permeability of tight junctions did not appear to result from the generation of reactive oxygen species, as it was not prevented by antioxidant treatment under normal energy conditions. Conversely, the late phase effect leading to both apoptosis and necrosis during the 24 hr following iron removal could be reduced by antioxidant treatment and was exacebated by GSH depletion. Iron induced oxidative stress may therefore be responsible for membrane damage and cellular death occurring in the late phase. The reported effects of iron on intestinal tight junction permeability followed by more widespread cytotoxicity from oxidative events should be considered in light of the extensive use of iron supplementation in different phases of human life.

Evidence that polyunsaturated lecithin induces a reduction in plasma cholesterol level and favorable changes in lipoprotein composition in hypercholesterolemic rats.

For 30 d adult rats were fed a hypercholesterolemic (H) diet (25% saturated fat, 1% cholesterol and 0.5% cholic acid) containing different amounts of saponins (1% or 0.2%) and/or purified polyunsaturated lecithin (2.5% or 0.7%). Lecithin induced a striking reduction in the plasma levels of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) cholesterol as well as an increase in the level of high density lipoprotein (HDL) cholesterol. Saponins had only a very slight effect in lowering the level of VLDL cholesterol. Apoprotein A-I was unexpectedly present in VLDL, IDL and LDL after feeding rats the H diet and disappeared only after lecithin feeding. The activity of plasma lecithin-cholesterol acyltransferase was higher when the two lecithin diets were fed than when the other diets were fed. Fecal excretion of neutral sterols was unmodified by the various diets whereas acid steroid excretion increased after lecithin feeding. Saponins, when added with lecithin to the diet, reduced the beneficial effect of lecithin. The results indicate that polyunsaturated lecithin induced a reduction in plasma cholesterol, possibly through an increased formation of HDL particles.

Modifications in plasma cholesterol and apolipoproteins of hypercholesterolaemic rats induced by ethanol-soluble factors of Vicia faba.

High-fat-high-cholesterol diets containing casein or a Vicia faba bean (faba bean) protein concentrate as the protein source were given to rats for 5 weeks. When the faba bean protein concentrate or its ethanol extract was present in the diet, a marked decrease was found in the level of circulating cholesterol associated with the lower-density lipoproteins (very-low-, intermediate- and low-density lipoproteins) compared with the level found on the diets containing casein or the faba bean protein concentrate deprived of ethanol-soluble factors. Alterations in apoprotein pattern were detected after the different dietary treatments. In particular, apoA-I appeared in an unusual form with electrophoretic mobility faster than normal in all lipoprotein fractions after feeding the diets that did not lower plasma cholesterol. When the diets contained the faba bean protein concentrate or its ethanol extract, the apoA-I disappeared from the lower-density lipoproteins but its normal form and the unusual one were apparent in the high-density lipoproteins. A moderate increase in faecal excretion of acidic steroids was found after feeding the diets containing the ethanol-soluble factors, irrespective of the protein source. The results are discussed in relation to the presence of saponin and polyunsaturated lecithin in the ethanol extract of the faba bean protein concentrate.

Copper treatment alters the permeability of tight junctions in cultured human intestinal Caco-2 cells.

The effects of copper on tight-junction permeability were investigated in human intestinal Caco-2 cells, monitoring transepithelial electrical resistance and transepithelial passage of mannitol. Apical treatment of Caco-2 cells with 10-100 microM CuCl(2) (up to 3 h) produced a time- and concentration-dependent increase in tight-junction permeability, reversible after 24 h in complete medium in the absence of added copper. These effects were not observed in cells treated with copper complexed to L-histidine [Cu(His)(2)]. The copper-induced increase in tight-junction permeability was affected by the pH of the apical medium, as was the apical uptake of (64)CuCl(2), both exhibiting a maximum at pH 6.0. Treatment with CuCl(2) produced a concentration-dependent reduction in the staining of F actin but not of the junctional proteins zonula occludens-1, occludin, and E-cadherin and produced ultrastructural alterations to microvilli and tight junctions that were not observed after treatment with up to 200 microM Cu(His)(2) for 3 h. Overall, these data point to an intracellular effect of copper on tight junctions, mediated by perturbations of the F actin cytoskeleton.

Copper uptake and intracellular distribution in the human intestinal Caco-2 cell line.

The apical uptake of 64CuCl2 was investigated in human differentiated intestinal Caco-2 cells grown on permeable supports. At pH 6.0 in the apical compartment, the uptake of copper was linear over the first 6 min and between 10 and 80 microM CuCl2 exhibited non-saturable transport kinetics. In addition, copper uptake was energy-independent, affected by the valency state of copper, preferring Cu(II) over Cu(I), and not influenced by high (10 mM) extracellular calcium. The intracellular distribution of copper was investigated by FPLC at different times of uptake ('pulse') and of 'chase'. Intracellular copper initially bound predominantly to low molecular weight components (i.e., glutathione). and subsequently shifted to higher molecular weight components such as metallothionein and Cu,Zn superoxide dismutase.