Visual comet scoring revisited: a guide to scoring comet assay slides and obtaining reliable results.

Measurement of DNA migration in the comet assay can be done by image analysis or visual scoring. The latter accounts for 20%-25% of the published comet assay results. Here we assess the intra- and inter-investigator variability in visual scoring of comets. We include three training sets of comet images, which can be used as reference for researchers who wish to use visual scoring of comets. Investigators in 11 different laboratories scored the comet images using a five-class scoring system. There is inter-investigator variation in the three training sets of comets (i.e. coefficient of variation (CV) = 9.7%, 19.8%, and 15.2% in training sets I-III, respectively). However, there is also a positive correlation of inter-investigator scoring in the three training sets (r = 0.60). Overall, 36% of the variation is attributed to inter-investigator variation and 64% stems from intra-investigator variation in scoring between comets (i.e. the comets in training sets I-III look slightly different and this gives rise to heterogeneity in scoring). Intra-investigator variation in scoring was also assessed by repeated analysis of the training sets by the same investigator. There was larger variation when the training sets were scored over a period of six months (CV = 5.9%-9.6%) as compared to 1 week (CV = 1.3%-6.1%). A subsequent study revealed a high inter-investigator variation when premade slides, prepared in a central laboratory, were stained and scored by investigators in different laboratories (CV = 105% and 18%-20% in premade slides with comets from unexposed and hydrogen peroxide-exposed cells, respectively). The results indicate that further standardization of visual scoring is desirable. Nevertheless, the analysis demonstrates that visual scoring is a reliable way of analysing DNA migration in comets.

Inter-laboratory variation in measurement of DNA damage by the alkaline comet assay in the hCOMET ring trial.

The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) in % Tail DNA units indicates the importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern.

DNA strand break levels in cryopreserved mononuclear blood cell lines measured by the alkaline comet assay: results from the hCOMET ring trial.

The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%-2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern.

Long-term cryopreservation of potassium bromate positive assay controls for measurement of oxidatively damaged DNA by the Fpg-modified comet assay: results from the hCOMET ring trial.

The formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay is widely used for the measurement of oxidatively generated damage to DNA. However, there has not been a recommended long-term positive control for this version of the comet assay. We have investigated potassium bromate as a positive control for the Fpg-modified comet assay because it generates many Fpg-sensitive sites with a little concurrent generation of DNA strand breaks. Eight laboratories used the same procedure for the treatment of monocytic THP-1 cells with potassium bromate (0, 0.5, 1.5, and 4.5 mM) and subsequent cryopreservation in a freezing medium consisting of 50% foetal bovine serum, 40% RPMI-1640 medium, and 10% dimethyl sulphoxide. The samples were analysed by the Fpg-modified comet assay three times over a 3-year period. All laboratories obtained a positive concentration-response relationship in cryopreserved samples (linear regression coefficients ranging from 0.79 to 0.99). However, there was a wide difference in the levels of Fpg-sensitive sites between the laboratory with the lowest (4.2% Tail DNA) and highest (74% Tail DNA) values in THP-1 cells after exposure to 4.5 mM KBrO3. In an attempt to assess sources of inter-laboratory variation in Fpg-sensitive sites, comet images from one experiment in each laboratory were forwarded to a central laboratory for visual scoring. There was high consistency between measurements of %Tail DNA values in each laboratory and the visual score of the same comets done in the central laboratory (r = 0.98, P < 0.001, linear regression). In conclusion, the results show that potassium bromate is a suitable positive comet assay control.

A mix of chlorogenic and caffeic acid reduces C/EBPß and PPAR-γ1 levels and counteracts lipid accumulation in macrophages.

PURPOSE: Chlorogenic acid (CGA) and caffeic acid (CA) are bioactive compounds in whole grains, berries, apples, some citrus fruits and coffee, which are hypothesized to promote health-beneficial effects on the cardiovascular system. This study aimed to evaluate the capacity of CGA and CA to reduce lipid accumulation in macrophages, recognized as a critical stage in the progression of atherosclerosis. Furtherly, the modulation of CCAAT/enhancer-binding protein ß (C/EBPß) and peroxisome proliferator-activated receptor- γ1 (PPAR-γ1), as transcription factors involved in lipid metabolism, was evaluated. METHODS: THP-1-derived macrophages were treated for 24 h with 0.03, 0.3, 3 and 30 µM of CGA and CA, tested alone or in combination, and a solution of oleic/palmitic acid (500 µM, 2:1 ratio). Lipid storage was assessed spectrophotometrically through fluorescent staining of cells with Nile red. C/EBPß and PPAR-γ1 mRNA and protein levels were evaluated by RT-PCR and enzyme-linked immunosorbent assay, respectively. RESULTS: The mix of CGA + CA (1:1 ratio) reduced lipid accumulation at all concentrations tested, except for the highest one. The greatest effect ( - 65%; p < 0.01) was observed at the concentration of 0.3 µM for each compound. The same concentration significantly (p < 0.01) downregulated C/EBPß and PPAR-γ1 gene expression and reduced their protein levels at 2 h and 24 h, respectively. CONCLUSION: The results indicate that the capacity of CGA + CA mix to reduce lipid storage in macrophages is mediated by a reduction in the expression of transcription factors C/EBPß and PPAR-γ1.

Potassium bromate as positive assay control for the Fpg-modified comet assay.

The comet assay is a popular assay in biomonitoring studies. DNA strand breaks (or unspecific DNA lesions) are measured using the standard comet assay. Oxidative stress-generated DNA lesions can be measured by employing DNA repair enzymes to recognise oxidatively damaged DNA. Unfortunately, there has been a tendency to fail to report results from assay controls (or maybe even not to employ assay controls). We believe this might have been due to uncertainty as to what really constitutes a positive control. It should go without saying that a biomonitoring study cannot have a positive control group as it is unethical to expose healthy humans to DNA damaging (and thus potentially carcinogenic) agents. However, it is possible to include assay controls in the analysis (here meant as a cryopreserved sample of cells i.e. included in each experiment as a reference sample). In the present report we tested potassium bromate (KBrO3) as a positive comet assay control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. Ten laboratories used the same procedure for treatment of monocytic THP-1 cells with KBrO3 (0.5, 1.5 and 4.5 mM for 1 h at 37°C) and subsequent cryopreservation. Results from one laboratory were excluded in the statistical analysis because of technical issues in the Fpg-modified comet assay. All other laboratories found a concentration-response relationship in cryopreserved samples (regression coefficients from 0.80 to 0.98), although with different slopes ranging from 1.25 to 11.9 Fpg-sensitive sites (%DNA in tail) per 1 mM KBrO3. Our results demonstrate that KBrO3 is a suitable positive comet assay control.

Nutritional and Phytochemical Characterization of Freeze-Dried Raspberry (Rubus idaeus): A Comprehensive Analysis.

Several studies have highlighted the beneficial effects of consuming red raspberries on human health thanks to their high content of phytochemicals. However, the products used in these studies, both in the raw or freeze-dried form, were not fully characterized for nutrient and phytochemical composition. In this study, we aimed to determine the nutrient and non-nutrient compounds present in a freeze-dried red raspberry powder widely used by the food industry and consumers. The main sugars identified were fructose (12%), glucose (11%), and sucrose (11%). Twelve fatty acids were detected, with linoleic acid (46%), α-linolenic acid (20%), and oleic acid (15%) being the most abundant. Regarding micronutrients, vitamin C was the main hydro-soluble vitamin, while minerals, potassium, phosphorous, copper and magnesium were the most abundant, with concentrations ranging from 9 up to 96 mg/100 g, followed by manganese, iron and zinc, detected in the range 0.1-0.9 mg/100 g. Phytochemical analysis using UHPLC-DAD-HR-MS detection revealed the presence of Sanguiin H6 (0.4%), Lambertianin C (0.05%), and Sanguiin H-10 isomers (0.9%) as the main compounds. Among anthocyanins, the most representative compounds were cyanidin-3-sophoroside, cyanidin-3-glucoside and cyanidin-3-sambubioside. Our findings can serve as a reliable resource for the food industry, nutraceutical applications and for future investigations in the context of human health.

Red raspberry (Rubus idaeus) preserves intestinal barrier integrity and reduces oxidative stress in Caco-2 cells exposed to a proinflammatory stimulus.

Growing evidence showed the capacity of (poly)phenols to exert a protective role on intestinal health. Nevertheless, the existing findings are still heterogeneous and the underlying mechanisms remain unclear. This study investigated the potential benefits of a red raspberry (Rubus idaeus) powder on the integrity of the intestinal barrier, focusing on its ability to mitigate the effects of tumor necrosis factor-α (TNF-α)-induced intestinal permeability. Human colorectal adenocarcinoma cells (i.e., Caco-2 cells) were used as a model to assess the impact of red raspberry on intestinal permeability, tight junction expression, and oxidative stress. The Caco-2 cells were differentiated into polarized monolayers and treated with interferon-γ (IFN-γ) (10 ng mL-1) for 24 hours, followed by exposure to TNF-α (10 ng mL-1) in the presence or absence of red raspberry extract (1-5 mg mL-1). The integrity of the intestinal monolayer was evaluated using transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FITC-D) efflux assay. Markers of intestinal permeability (claudin-1, occludin, and zonula occludens-1 (ZO-1)) and oxidative stress (8-hydroxy-2-deoxyguanosine (8-OHdG) and protein carbonyl) were assessed using ELISA kits. Treatment with red raspberry resulted in a significant counteraction of TEER value loss (41%; p < 0.01) and a notable reduction in the efflux of FITC-D (-2.5 times; p < 0.01). Additionally, red raspberry attenuated the levels of 8-OHdG (-48.8%; p < 0.01), mitigating the detrimental effects induced by TNF-α. Moreover, red raspberry positively influenced the expression of the integral membrane protein claudin-1 (+18%; p < 0.01), an essential component of tight junctions. These findings contribute to the growing understanding of the beneficial effects of red raspberry in the context of the intestinal barrier. The effect of red raspberry against TNF-α-induced intestinal permeability observed in our in vitro model suggests, for the first time, its potential as a dietary strategy to promote gastrointestinal health.

Anthocyanins and their metabolites promote white adipose tissue beiging by regulating mitochondria thermogenesis and dynamics.

High-fat diet (HFD) consumption and excess nutrient availability can cause alterations in mitochondrial function and dynamics. We previously showed that anthocyanins (AC) decreased HFD-induced body weight gain and fat deposition. This study investigated: i) the capacity of AC to mitigate HFD-induced alterations in mitochondrial dynamics, biogenesis, and thermogenesis in mouse subcutaneous white adipose tissue (sWAT), and ii) the underlying mechanisms of action of cyanidin-3-O-glucoside (C3G), delphinidin-3-O-glucoside (D3G), and their gut metabolites on mitochondria function/dynamics in 3T3-L1 adipocytes treated with palmitate. Mice were fed control or HFD diets, added or not with 40 mg AC/kg body weight (BW). Compared to control and AC-supplemented mice, HFD-fed mice had fewer sWAT mitochondria that presented alterations of their architecture. AC supplementation prevented HFD-induced decrease of proteins involved in mitochondria biogenesis (PPARγ, PRDM16 and PGC-1α), and thermogenesis (UCP-1), and decreased AMPK phosphorylation. AC supplementation also restored the alterations in sWAT mitochondrial dynamics (Drp-1, OPA1, MNF-2, and Fis-1) and mitophagy (BNIP3L/NIX) caused by HFD consumption. In mature 3T3-L1, C3G, D3G, and their metabolites protocatechuic acid (PCA), 4-hydroxybenzaldehyde (HB), and gallic acid (GA) differentially affected palmitate-mediated decreased cAMP, PKA, AMPK, and SIRT-1 signaling pathways. C3G, D3G, and metabolites also prevented palmitate-mediated decreased expression of PPARγ, PRDM16, PGC-1α, and UCP1. Results suggest that consumption of select AC, i.e. cyanidin and delphinidin, could promote sWAT mitochondriogenesis and improve mitochondria dynamics in the context of HFD/obesity-induced dysmetabolism in part by regulating PKA, AMPK, and SIRT-1 signaling pathways.

Berries and Human Health: Mechanisms and Evidence.

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Human Milk Fatty Acid Composition and Its Effect on Preterm Infants' Growth Velocity.

This study aimed to analyze the fatty acid content in human milk and to find its relationship with the growth velocity of preterm infants. Mature milk samples from 15 mothers of preterm infants were collected from three different hospitals, followed by lipid extraction, fatty acid methylation, and finally gas chromatography analysis to determine the fatty acids composition. The average total lipid content was 3.61 ± 1.57 g/100 mL with the following classes of fatty acids: saturated fatty acids 43.54 ± 11.16%, unsaturated fatty acids 52.22 ± 10.89%, in which monounsaturated fatty acids were 36.52 ± 13.90%, and polyunsaturated fatty acids were 15.70 ± 7.10%. Polyunsaturated fatty acid sub-class n-6 was 15.23 ± 8.23% and n-3 was 0.46 ± 0.18%. Oleic acid, palmitic acid, and linoleic acid were the most abundant fatty acids. The n-6/n-3 ratio was 32.83:1. EPA and DHA fatty acids were not detected. As gestational age and birth weight increase, C20:2n6 content increases. The growth velocity increases with the decrement in C16 and increment in C20:2n6. The lipid profile of preterm human milk was found to be low in some essential fatty acids, which may affect the quality of preterm infants' nutrition.

Effect of caper fruit (Capparis spinosa L.) consumption on liver enzymes, lipid profile, fasting plasma glucose, and weight loss. A systematic review and a preliminary meta-analysis of randomized controlled trials.

This systematic review and meta-analysis aimed to evaluate the overall effect of caper fruit on the modulation of glycemic, lipid profile, liver enzymes, and body mass. Google Scholar, PubMed, and Scopus were explored to collect relevant studies in the last 10 years. RCTs with caper fruit supplementation or consumption in different cohorts of subjects with non-alcoholic fatty liver disease (NAFLD), Type-2-Diabetes (T2D), metabolic syndrome, and hyperlipidemia were included in this systematic review with a mean intervention duration from 2 to 12 weeks. The outcomes measured in this meta-analysis were liver enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT), the lipid profile represented by triglycerides, total cholesterol (TC) with LDL and HDL and also, weight, and fasting blood glucose. Five randomized controlled trials, which involved a total of 178 adults, were included. According to the results, caper fruit seems to decrease liver enzymes ALT -12.29 U/L [-24.47, -0.11], AST -2.20 U/L [-4.70, 0.31]. Furthermore, the lipid profile seems to improve with a decrease in triglycerides. -11.89 mg/dL [-33.73, 9.95], LDL -4.80 mg/dL [-16.34, 6.74], HDL 0.72 mg/dL [0.10, 1.34], total cholesterol -7.83 mg/dL [-20.04, 4.38], FPG -17.93 [-42.66, 6.79], weight -1.00 kg [-1.44, -0.56]. Significant modulations were found only for ALT, HDL, and weight. In conclusion, this systematic review and meta-analysis showed the paucity of data available on the topic while showing the potential role of caper fruit as a promising food for improving the liver-lipid profile axis in patients with metabolic syndrome and diabetes. Further studies are required to confirm these results.

Wild blueberry (V. angustifolium) improves TNFα-induced cell barrier permeability through claudin-1 and oxidative stress modulation in Caco-2 cells.

Increasing evidence links the impairment of intestinal permeability (IP), a feature of the intestinal barrier, to numerous dysmetabolic and dysfunctional conditions. Several host and environmental factors, including dietary factors, can negatively and/or positively affect IP. In this regard, polyphenol-rich foods including berries have been proposed as potential IP modulators. However, the exact mechanisms involved are not yet fully elucidated. The aim of the present study was to evaluate the effect of a wild blueberry (WB; V. angustifolium) powder, naturally rich in polyphenols, to affect Caco-2 cell monolayer permeability and to identify the potential mechanisms in modulating the IP process. Caco-2 cells were incubated with TNF-α (10 ng mL-1), as a pro-inflammatory stimulus, and supplemented for 24 hours with different concentrations (1 and 5 mg mL-1) of WB powder. The integrity of the intestinal cell monolayer was evaluated by measuring the transepithelial electrical resistance (TEER) and the paracellular transport of FITC-dextran. In addition, the production of the tight junction proteins, such as claudin-1 and occludin, as well as protein carbonyl and 8-hydroxy 2 deoxyguanosine, as oxidative stress markers, were quantified in the supernatant by ELISA kits. Overall, the treatment with WB powder (5 mg mL-1) mitigated the loss of Caco-2 cell barrier integrity, as documented by an increase in TEER and a reduction in FITC values. This modulation was accompanied by an upregulation of claudin-1 and a reduction of 8-OHdG. Conversely, no effect was documented for the lower concentration (1 mg mL-1) and the other IP markers, as well as oxidative stress markers analysed. In conclusion, our findings suggest a potential role of WB in the modulation of cell barrier integrity. This modulation process could be attributed to an increase in claudin-1 expression and a reduction in 8-OHdG. Further studies should be performed to corroborate the results obtained. In addition, since the effects were observed at doses of WB achievable with the diet, these findings should be substantiated also through in vivo approaches.

Blueberries and their bioactives in the modulation of oxidative stress, inflammation and cardio/vascular function markers: a systematic review of human intervention studies.

Blueberries represent a rich source of (poly)phenols and other bioactive compounds. Numerous in vitro and animal model studies documented the potential health-promoting properties of blueberries and blueberry-bioactives, while little is still known about their effects in humans. The objective of the present systematic review is to provide main evidence and the potential mechanisms of action of blueberry and its (poly)phenols in the regulation of markers related to oxidative stress, inflammation, vascular and cardiometabolic function in health and disease states. A total of 45 human intervention studies were included in this review. Overall, the evidence suggests that blueberries may play a role in the improvement of markers of vascular function. Their effects were observed following both post-prandial and long-term consumption, particularly in subjects with risk factors and/or disease conditions. Conversely, the conflicting results on inflammation, oxidative stress and cardiometabolic risk markers were most likely due to differences among studies in terms of study design, subject characteristics, duration of intervention, dosage, and type of biomarkers analyzed. For these reasons, high-quality, well-designed, human intervention studies are warranted to strengthen the current findings on vascular function and provide more evidence about the impact of blueberries on the different markers considered. In addition, studies focusing on the relationship between the structure and the function of (poly)phenols will be fundamental for a better comprehension of the mechanisms behind the health effects observed.

Berry Dietary Interventions in Metabolic Syndrome: New Insights.

Metabolic Syndrome (MetS) is characterized by a group of dysmetabolic conditions, including abdominal obesity, dyslipidemia, glucose intolerance and/or insulin resistance, and hypertension. Generally, MetS is accompanied by an exacerbation of oxidative stress, inflammation, and vascular dysfunction. Increasing evidence suggests that berries and berry bioactives could play a potential role in the prevention and mitigation of the risk factors associated with MetS. The present systematic review summarizes the more recently available evidence deriving from human intervention studies investigating the effect of berries in subjects with at least three out of five MetS parameters. The PubMed, Scopus, and Embase databases were systematically searched from January 2010 until December 2022. A total of 17 human intervention trials met the inclusion criteria. Most of them were focused on blueberry (n = 6), cranberry (n = 3), and chokeberry (n = 3), while very few or none were available for the other berries. If considering MetS features, the main positive effects were related to lipid profile (low and high-density lipoproteins, cholesterol, and triglycerides) following blueberries and chokeberries, while conflicting results were documented for anthropometric parameters, blood pressure, and fasting blood glucose levels. Other markers analyzed within the studies included vascular function, oxidative stress, and inflammation. Here, the main positive effects were related to inflammation with a reduction in interleukin 6 and tumor necrosis factor-alpha following the intake of different berries. In conclusion, although limited, the evidence seems to support a potential role for berries in the modulation of lipid profile and inflammation in subjects with MetS. Furthermore, high-quality intervention trials are mandatory to demonstrate the role of berries in reducing risk factors for MetS and related conditions. In the future, such a demonstration could bring the adoption of berries as a potential dietary strategy to prevent/counteract MetS and related risk factors.

Does the Mediterranean Diet Have Any Effect on Lipid Profile, Central Obesity and Liver Enzymes in Non-Alcoholic Fatty Liver Disease (NAFLD) Subjects? A Systematic Review and Meta-Analysis of Randomized Control Trials.

The effectiveness of the Mediterranean diet (MD) in non-alcoholic fatty liver disease (NAFLD) subjects has been evaluated in several randomized controlled trials (RCTs). This systematic review and meta-analysis aimed to evaluate the overall effects of MD intervention in a cohort of NAFLD patients targeting specific markers such as central obesity, lipid profile, liver enzymes and fibrosis, and intrahepatic fat (IHF). Google Scholar, PubMed, and Scopus were explored to collect relevant studies from the last 10 years. RCTs with NAFLD subjects were included in this systematic review with a mean intervention duration from 6 weeks to 1 year, and different intervention strategies, mainly including energy restriction MD (normal or low glycaemic index), low-fat MD with increased monounsaturated and polyunsaturated fatty acids, and increased exercise expenditure. The outcomes measured in this meta-analysis were gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), total cholesterol (TC), waist circumference (WC), and liver fibrosis. Ten randomized controlled trials, which involved a total of 737 adults with NAFLD, were included. According to the results, the MD seems to decrease the liver stiffness (kPa) by -0.42 (CI95% -0.92, 0.09) (p = 0.10) and significantly reduce the TC by -0.46 mg/dl (CI95% -0.55, -0.38) (p = 0.001), while no significant findings were documented for liver enzymes and WC among patients with NAFLD. In conclusion, the MD might reduce indirect and direct outcomes linked with NAFLD severity, such as TC, liver fibrosis, and WC, although it is important to consider the variations across trials. Further RCTs are necessary to corroborate the findings obtained and provide further evidence on the role of the MD in the modulation of other disorders related to NAFLD.

Vitamin D Counteracts Lipid Accumulation, Augments Free Fatty Acid-Induced ABCA1 and CPT-1A Expression While Reducing CD36 and C/EBPß Protein Levels in Monocyte-Derived Macrophages.

The biologically active form of vitamin D, calcitriol (VD3), has received great attention for its extraskeletal effects, such as a protective role on the cardiovascular system. The aim of the present work is to test the capacity of VD3 to affect lipid metabolism and fatty acid accumulation in an in vitro model of monocyte (THP-1)-derived macrophages. Cells were treated for 24 h with oleic/palmitic acid (500 µM, 2:1 ratio) and different VD3 concentrations (0.1, 1, 10, 50 and 100 nM). Lipid accumulation was quantified spectrophotometrically (excitation: 544 nm, emission: 590 nm). C/EBPß, PPAR-γ1, CD36, CPT-1A, and ABCA1 protein levels were assessed by ELISA kits at different time-points (1, 2, 4, 8, and 24 h). VD3 at 50 and 100 nM significantly reduced fatty acids accumulation in macrophages by 27% and 32%, respectively. In addition, tested at 50 nM, VD3 decreased CD36, PPAR-γ1, and C/EBPß, while it increased ABCA1 and CPT-1A protein levels in free fatty acid-exposed cells. In conclusion, VD3 reduced fatty acid accumulation in THP-1-derived macrophages exposed to lipid excess. The anti-atherogenic effect of VD3 could be ascribable to the regulation of proteins involved in lipid transport and clearance.

Plant-Based Foods and Vascular Function: A Systematic Review of Dietary Intervention Trials in Older Subjects and Hypothesized Mechanisms of Action.

Cardiovascular diseases, still the leading cause of mortality in the world, are closely related to vascular function. Older subjects are more susceptible to endothelial dysfunction and therefore it is important to define possible preventive or support strategies, such as consumption of foods with health-promoting effects. This systematic review aims to summarize the currently available evidence on acute or chronic trials testing the effect of selected plant-based foods on vascular function parameters in older subjects, and consider plausible mechanisms that may support the main findings. A total of 15 trials were included and analyzed, testing the effects of beetroot, plum, blueberry, and vegetable oils. We found some interesting results regarding markers of vascular reactivity, in particular for beetroot, while no effects were found for markers of arterial stiffness. The amelioration of vascular function seems to be more related to the restoration of a condition of nitric oxide impairment, exacerbated by diseases or hypoxic condition, rather than the enhancement of a physiological situation, as indicated by the limited effects on healthy older subjects or in control groups with young subjects. However, the overall set of selected studies is, in any case, rather limited and heterogeneous in terms of characteristics of the studies, indicating the need for additional high-quality intervention trials to better clarify the role of vegetable foods in restoring and/or improving vascular function in order to better elucidate the mechanisms through which these foods may exert their vascular health benefits in older subjects.

What Is the Current Direction of the Research on Carotenoids and Human Health? An Overview of Registered Clinical Trials.

Carotenoids have been the object of numerous observational, pre-clinical and interventional studies focused on elucidating their potential impacts on human health. However, the large heterogeneity among the trials, in terms of study duration and characteristics of participants, makes any conclusion difficult to draw. The present study aimed to explore the current carotenoid research trends by analyzing the characteristics of the registered clinical trials. A total of 193 registered trials on ClinicalTrials.gov and ISRCTN were included in the revision. Eighty-three studies were performed with foods, one-hundred-five with food supplements, and five with both. Among the foods tested, tomatoes and tomato-based foods, and eggs were the most studied. Lutein, lycopene, and astaxanthin were the most carotenoids investigated. Regarding the goals, 52 trials were focused on studying carotenoids' bioavailability, and 140 studies investigated the effects of carotenoids on human health. The main topics included eye and cardiovascular health. Recently, the research has focused also on two new topics: cognitive function and carotenoid-gut microbiota interactions. However, the current research on carotenoids is still mostly focused on the bioavailability and metabolism of carotenoids from foods and food supplements. Within this context, the impacts/contributions of food technologies and the development of new carotenoid formulations are discussed. In addition, the research is still corroborating the previous findings on vision and cardiovascular health. Much attention has also been devoted to new research areas, such as the carotenoid-microbiota interactions, which could contribute to explaining the metabolism and the health effects of carotenoids; and the relation between carotenoids and cognitive function. However, for these topics the research is still only beginning, and further studies are need.

Cobalamin status is negatively correlated with vascular endothelial-cadherin in vegetarian and vegan women with vitamin B12 deficiency.

Optimal vitamin B12 status is important for vascular health. Vascular endothelial (VE) cadherin is an adherent junction protein involved in the maintenance of a functional endothelium. We hypothesized that vitamin B12 deficiency can negatively affect markers of vascular function, such as VE-cadherin. Within a human intervention study, we explored the possible association between cobalamin status (i.e., vitamin B12, holotranscobalamin, and homocysteine) and VE-cadherin (as marker of vascular health) in vegetarians/vegans (VEG) with B12 deficiency. The associations were evaluated at baseline and after 90-day supplementation with 2000 µg/wk of vitamin B12. On the whole, an inverse association between VE-cadherin and holotranscobalamin (P = .014) and a positive association between VE-cadherin and homocysteine (P = .041) was documented at baseline. VEG women showed higher levels of VE-cadherin compared with VEG men (P = .044), suggesting an increase in endothelial permeability. The intervention with vitamin B12 restored serum vitamin levels and improved the overall cobalamin status, whereas it did not affect VE-cadherin levels. The inverse association between holotranscobalamin and VE-cadherin was also maintained after intervention in women, corroborating the strong correlation between these 2 parameters. The results obtained seem to suggest a possible association between cobalamin status and VE-cadherin even if the intervention with B12 failed to positively affect VE-cadherin levels. Thus, further studies are needed to corroborate these findings and clarify the contribution of a vitamin B12 intervention on VE-cadherin levels in this target population. This trial was registered at ISRCTN registry (ISRCTN75099618).