Putative intestinal permeability markers do not correlate with cardiometabolic health and gut microbiota in humans, except for peptides recognized by a widely used zonulin ELISA kit.

BACKGROUND AND AIMS: Cardiometabolic diseases refer to a group of interrelated conditions, sharing metabolic dysfunctions like insulin resistance, obesity, dyslipidemia, and hypertension. The gut microbiota has been associated with CMD and related conditions. Alterations in the intestinal epithelium permeability triggered by chronic stress and diet could bridge gut microbiota with inflammation and CMD development. Here, we assessed the relationship between intestinal permeability and circulating SCFAs with cardiometabolic health status (CMHS) and gut microbiota in a sample of 116 Colombian adults. METHODS AND RESULTS: Plasma levels of lipopolysaccharide-binding protein (LBP), intestinal fatty acid-binding protein (I-FABP), claudin-3, and purported zonulin peptides (PZP) were measured by ELISA, whereas plasmatic levels of acetate, propionate, butyrate, isobutyrate, and valerate were measured by gas chromatography/mass spectrometry. In addition, for further statistical analysis, we took data previously published by us on this cohort, including gut microbiota and multiple CMD risk factors that served to categorize subjects as cardiometabolically healthy or cardiometabolically abnormal. From univariate and multivariate statistical analyses, we found the levels of I-FABP, LBP, and PZP increased in the plasma of cardiometabolically abnormal individuals, although only PZP reached statistical significance. CONCLUSIONS: Our results did not confirm the applicability of I-FABP, LBP, claudin-3, or SCFAs as biomarkers for associating intestinal permeability with the cardiometabolic health status in these subjects. On the other hand, the poorly characterized peptides detected with the ELISA kit branded as "zonulin" were inversely associated with cardiometabolic dysfunctions and gut microbiota. Further studies to confirm the true identity of these peptides are warranted.

Consumption of golden berries (Physalis peruviana L.) might reduce biomarkers of oxidative stress and alter gut permeability in men without changing inflammation status or the gut microbiota.

Golden berry (Physalis peruviana) is a tropical fruit rich in antioxidants that has been proposed to be able to control the lipid profile in hypercholesterolemic patients. Dyslipidemia is an independent risk factor for cardiometabolic diseases. The gut microbiota is strongly associated with cardiometabolic risk and is involved in redox balance, intestinal permeability, and inflammation. However, the impacts of golden berry on some of these factors, including the human gut microbiota, have never been tested, and there are no tools for compliance monitoring or dietary intake assessment regarding nutritional interventions with this fruit. In the pre-post quasi-experimental nutritional intervention presented here, 18 adult men (27-49 years old) consumed golden berries (Dorada variety) for three weeks. We evaluated putative biomarkers of exposure through an untargeted metabolomics approach (liquid chromatography-mass spectrometry LC-MS), quantified the biomarkers of oxidative stress, gut permeability, and inflammation in plasma, and assessed the effects of fruit intake on the gut microbiota through 16S rRNA gene sequencing of feces (Illumina MiSeq V2). First, syringic acid and kaempferol were identified as putative biomarkers of golden berry consumption. Intervention with this fruit promoted physiological changes in the participants after three weeks, reducing the level of the oxidative stress marker 8-isoprostane (-148 pg/ml; 36.1 %; p = 0.057) and slightly altering gut permeability by increasing the plasma levels of LBP (2.91 µg/ml; 54.6 %; p = 0.0005) and I-FABP (0.15, 14.7 %, p = 0.04) without inducing significant inflammation; i.e., the levels of IL-1ß, TNF-α and IL-8 changed by 0.7 (2.0 %), -4.0 (-9.6 %) and -0.4 (-1.8 %) pg/ml, respectively. Notably, the consumption of golden berries did not affect the gut microbiota of the individuals consistently but instead shifted it in a personalized manner. The compositions of the gut microbiota of a given individual at the end of intervention and one month after the end of intervention were statistically more similar to their own baseline than to a corresponding sample from a different individual. This intervention identified putative biomarkers of golden berry intake along with potential benefits of its consumption relevant to cardiometabolic disease risk reduction. Golden berries are likely to positively modulate redox balance, although this effect must be proven in a future controlled clinical trial.

Dietary antioxidant intake is inversely associated with 2,3-dinor oxylipin metabolites, the major excreted oxylipins in overweight and obese subjects.

Cardiometabolic disease risk factors, including obesity, insulin resistance, high blood pressure, and dyslipidemia, are associated with elevated oxidative stress biomarkers like oxylipins. Increased adiposity by itself induces various isomers of this oxidized lipid family, while dietary polyphenols show benefits in its regulation. Previously, we showed that specific co-abundant microorganisms characterized the gut microbiota of Colombians and associated differentially with diet, lifestyle, obesity, and cardiometabolic health status, which led us to hypothesize that urinary oxylipins would reflect the intensity of oxidative metabolism linked to gut microbiota dysbiosis. Thus, we selected a convenience sample of 105 participants (age: 40.2 ± 11.9 years, 47.6% women), grouped according to microbiota, cardiometabolic health status, and body mass index (BMI); and evaluated 33 urinary oxylipins by HPLC-QqQ-MS/MS (e.g., isoprostanes, prostaglandins, and metabolites), paired with anthropometry and blood chemistry information and dietary antioxidants estimated from a 24-h food recall. In general, oxylipins did not show differences among individuals who differed in gut microbiota. While the unmetabolized oxylipin levels were not associated with BMI, the total content of oxylipin metabolites was highest in obese and cardiometabolically abnormal subjects (e.g., insulin resistant), mainly by prostaglandin-D (2,3-dinor-11ß-PGF2α) and 15-F2t-IsoPs (2,3-dinor-15-F2t-IsoP and 2,3-dinor-15-epi-15-F2t-IsoP) metabolites. The total polyphenol intake in this cohort was 1070 ± 627 mg/day. After adjusting for body weight, the polyphenol intake was significantly higher in lean than overweight and showed an inverse association with dinor-oxylipin levels in principal component analysis. These results suggest that the 2,3-dinor-oxylipins could be more specific biomarkers associated with BMI than their parent oxylipins and that are sensitive to be regulated by dietary antioxidants.

Changes in the plasma lipidome of healthy subjects after coffee consumption reveal potential cardiovascular benefits: A randomized controlled trial.

Lipid metabolism dysregulation is associated with cardiovascular disease (CVD) risk. Specific oxidized lipids are recognized CVD biomarkers involved in all stages of atherosclerosis, including foam cell formation. Moderate coffee intake is positively associated with cardiovascular health. A randomized, controlled (n = 25) clinical trial was conducted in healthy subjects to assess the changes in lipid species relevant to CVD (main inclusion criteria: coffee drinkers, nonsmokers, with no history and/or diagnosis of chronic disease and not consuming any medications). Volunteers consumed a coffee beverage (400 mL/day) containing either 787 mg (coffee A; n = 24) or 407 mg (coffee B; n = 25) of chlorogenic acids for eight weeks. We measured the total plasma levels of 46 lipids, including fatty acids, sterols, and oxysterols, at baseline and after eight weeks and assessed the effects of chlorogenic and phenolic acids, the major coffee antioxidants, in an in vitro foam cell model via targeted lipidomics. At baseline (n = 74), all participants presented oxysterols and free fatty acids (FFAs) (CVD risk markers), which are closely correlated to among them, but not with the classical clinical variables (lipid profile, waist circumference, and BMI). After eight weeks, the control group lipidome showed an increase in oxysterols (+7 ± 10%) and was strongly correlated with FFAs (e.g., arachidonic acid) and cholesteryl ester reduction (-13 ± 7%). Notably, the coffee group subjects (n = 49) had increased cholesteryl esters (+9 ± 11%), while oxysterols (-71 ± 30%) and FFAs (-29 ± 26%) decreased. No differences were found between the consumption of coffees A and B. Additionally, coffee antioxidants decreased oxysterols and regulated arachidonic acid in foam cells. Our results suggest that coffee consumption modulates the generation of oxidized and inflammatory lipids in healthy subjects, which are fundamental during CVD development. The clinical trial was registered on the International Clinical Trials Registry Platform, WHO primary registry (RPCEC00000168).

Oxylipin regulation by phenolic compounds from coffee beverage: Positive outcomes from a randomized controlled trial in healthy adults and macrophage derived foam cells.

Oxylipins are considered biomarkers related to cardiovascular diseases (CVDs). They are generated in vivo via the oxygenation of polyunsaturated fatty acids as a result of oxidative stress and inflammation. Oxylipins are involved in vascular functions and are produced during foam cell formation in atherogenesis. Additionally, the consumption coffee is associated with the regulation on a particular oxylipin group, the F2t-isoprostanes (F2t-IsoPs). This function has been attributed to the chlorogenic acids (CGAs) from the coffee beverage. Considering the anti-inflammatory and antioxidant properties of CGAs, we evaluated the effects of two types of coffee that provided 787 mg CGAs/day (Coffee A) and 407 mg CGAs/day (Coffee B) by reducing 35 selected oxylipins in healthy subjects. Furthermore, we assessed the effect of CGAs on the cellular proatherogenic response in foam cells by using an oxidized LDL (oxLDL)-macrophage interaction model. After eight weeks of coffee consumption, the contents of 12 urine oxylipins were reduced. However, the effect of Coffee A showed a stronger decrease in IsoPs, dihomo-IsoPs, prostaglandins (PGs) and PG metabolites, probably due to its higher content of CGAs. Neither of the two coffees reduced the levels of oxLDL. Moreover, the in vitro oxylipin induction by oxLDL on foam cells was ameliorated by phenolic acids and CGAs, including the inhibition of IsoPs and PGs by caffeoylquinic and dicaffeoylquinic acids, respectively, while the phenolic acids maintained both antioxidant and anti-inflammatory activities. These findings suggest that coffee antioxidants are strong regulators of oxylipins related to CVDs. The clinical trial was registered on the International Clinical Trials Registry Platform, WHO primary registry (RPCEC00000168).

The Traditional Medicinal Plants Cuphea calophylla, Tibouchina kingii, and Pseudelephantopus spiralis Attenuate Inflammatory and Oxidative Mediators.

Aerial parts of Cuphea calophylla, Tibouchina kingii, and Pseudelephantopus spiralis have been used in Colombian traditional medicine for inflammation. However, the underlying mechanisms that could explain the anti-inflammatory actions remain unknown. This study aimed to elucidate the anti-inflammatory and cytoprotective effects of hydroalcoholic extracts from C. calophylla (HECC), T. kingii (HETK), and P. spiralis (HEPS) in LPS-stimulated THP-1 macrophages. Reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA) were monitored as inflammatory and oxidative markers. The inhibition of lipoxygenase (LOX) and cyclooxygenase (COX) activities in a cell-free system were also investigated. Antioxidant activities were determined using standard in vitro methods. All extracts inhibited the NO, ROS, and MDA levels. HETK showed the highest ROS production inhibition and the highest antioxidant values, whereas HETK and HEPS significantly decreased the cytotoxicity mediated by LPS. The release of MDA was reduced significantly by all extracts. Moreover, the catalytic activity of LOX was inhibited by HECC and HETK. HECC was a more potent reducer of COX-2 activity. All extracts effectively suppressed COX-1 activity. In summary, these results suggest that HECC, HEPS, and HETK possess anti-inflammatory properties. Therefore, these plants could provide a valuable source of natural bioactive compounds for the treatment of inflammatory-related diseases.

Oxidized LDL triggers changes in oxidative stress and inflammatory biomarkers in human macrophages.

Oxidized low-density lipoprotein (oxLDL) is a well-recognized proatherogenic particle that functions in atherosclerosis. In this study, we established conditions to generate human oxLDL, characterized according to the grade of lipid and protein oxidation, particle size and oxylipin content. The induction effect of the cellular proatherogenic response was assessed in foam cells by using an oxLDL-macrophage interaction model. Uptake of oxLDL, reactive oxygen species production and expression of oxLDL receptors (CD36, SR-A and LOX-1) were significantly increased in THP-1 macrophages. Analyses of 35 oxylipins revealed that isoprostanes (IsoP) and prostaglandins (PGs) derived from the oxidation of arachidonic, dihomo gamma-linolenic and eicosapentaenoic acids were strongly and significantly induced in macrophages stimulated with oxLDL. Importantly, the main metabolites responsible for the THP1-macrophage response to oxLDL exposure were the oxidative stress markers 5-epi-5-F2t-IsoP, 15-E1t-IsoP, 8-F3t-IsoP and 15-keto-15-F2t-IsoP as well as inflammatory markers PGDM, 17-trans-PGF3α, and 11ß-PGF2α, all of which are reported here, for the first time, to function in the interaction of oxLDL with THP-1 macrophages. By contrast, a salvage pathway mediated by anti-inflammatory PGs (PGE1 and 17-trans-PGF3α) was also identified, suggesting a response to oxLDL-induced injury. In conclusion, when THP-1 macrophages were treated with oxLDL, a specific induction of biomarkers related to oxidative stress and inflammation was triggered. This work contributes to our understanding of initial atherogenic events mediated by oxLDL-macrophage interactions and helps to generate new approaches for their modulation.

Natural Biflavonoids Modulate Macrophage-Oxidized LDL Interaction In Vitro and Promote Atheroprotection In Vivo.

The accumulation of oxidized ApoB-100-containing lipoproteins in the vascular intima and its subsequent recognition by macrophages results in foam cell formation and inflammation, key events during atherosclerosis development. Agents targeting this process are considered potentially atheroprotective. Since natural biflavonoids exert antioxidant and anti-inflammatory effects, we evaluated the atheroprotective effect of biflavonoids obtained from the tropical fruit tree Garcinia madruno. To this end, the pure biflavonoid aglycones morelloflavone (Mo) and volkensiflavone (Vo), as well as the morelloflavone's glycoside fukugiside (Fu) were tested in vitro in primary macrophages, whereas a biflavonoid fraction with defined composition (85% Mo, 10% Vo, and 5% Amentoflavone) was tested in vitro and in vivo. All biflavonoid preparations were potent reactive oxygen species (ROS) scavengers in the oxygen radical absorbance capacity assay, and most importantly, protected low-density lipoprotein particle from both lipid and protein oxidation. In biflavonoid-treated macrophages, the surface expression of the oxidized LDL (oxLDL) receptor CD36 was significantly lower than in vehicle-treated macrophages. Uptake of fluorescently labeled oxLDL and cholesterol accumulation were also attenuated in biflavonoid-treated macrophages and followed a pattern that paralleled that of CD36 surface expression. Fu and Vo inhibited oxLDL-induced ROS production and interleukin (IL)-6 secretion, respectively, whereas all aglycones, but not the glucoside Fu, inhibited the secretion of one or more of the cytokines IL-1ß, IL-12p70, and monocyte chemotactic protein-1 (MCP-1) in lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, in macrophages primed with low-dose LPS and stimulated with cholesterol crystals, IL-1ß secretion was significantly and comparably inhibited by all biflavonoid preparations. Intraperitoneal administration of the defined biflavonoid fraction into ApoE-/- mice was atheroprotective, as evidenced by the reduction of the atheromatous lesion size and the density of T cells and macrophages infiltrating the aortic root; moreover, this treatment also lowered the circulating levels of cholesterol and the lipid peroxidation product malondialdehyde. These results reveal the potent atheroprotective effects exerted by biflavonoids on key events of the oxLDL-macrophage interphase: (i) atheroligand formation, (ii) atheroreceptor expression, (iii) foam cell transformation, and (iv) prooxidant/proinflammatory macrophage response. Furthermore, our results also evidence the antioxidant, anti-inflammatory, hypolipemiant, and atheroprotective effects of Garcinia madruno's biflavonoids in vivo.

Coffee Consumption Increases the Antioxidant Capacity of Plasma and Has No Effect on the Lipid Profile or Vascular Function in Healthy Adults in a Randomized Controlled Trial.

BACKGROUND: Coffee, a source of antioxidants, has controversial effects on cardiovascular health. OBJECTIVE: We evaluated the bioavailability of chlorogenic acids (CGAs) in 2 coffees and the effects of their consumption on the plasma antioxidant capacity (AC), the serum lipid profile, and the vascular function in healthy adults. METHODS: Thirty-eight men and 37 women with a mean ± SD age of 38.5 ± 9 y and body mass index of 24.1 ± 2.6 kg/m(2) were randomly assigned to 3 groups: a control group that did not consume coffee or a placebo and 2 groups that consumed 400 mL coffee/d for 8 wk containing a medium (MCCGA; 420 mg) or high (HCCGA; 780 mg) CGA content. Both were low in diterpenes (0.83 mg/d) and caffeine (193 mg/d). Plasma caffeic and ferulic acid concentrations were measured by GC, and the plasma AC was evaluated with use of the ferric-reducing antioxidant power method. The serum lipid profile, nitric oxide (NO) plasma metabolites, vascular endothelial function (flow-mediated dilation; FMD), and blood pressure (BP) were evaluated. RESULTS: After coffee consumption (1 h and 8 wk), caffeic and ferulic acid concentrations increased in the coffee-drinking groups, although the values of the 2 groups were significantly different (P < 0.001); caffeic and ferulic acid concentrations were undetectable in the control group. At 1 h after consumption, the plasma AC in the control group was significantly lower than the baseline value (-2%) and significantly increased in the MCCGA (6%) and HCCGA (5%) groups (P < 0.05). After 8 wk, no significant differences in the lipid, FMD, BP, or NO plasma metabolite values were observed between the groups. CONCLUSIONS: Both coffees, which contained CGAs and were low in diterpenes and caffeine, provided bioavailable CGAs and had a positive acute effect on the plasma AC in healthy adults and no effect on blood lipids or vascular function. The group that did not drink coffee showed no improvement in serum lipid profile, FMD, BP, or NO plasma metabolites. This trial was registered at registroclinico.sld.cu as RPCEC00000168.

Proatherogenic macrophage activities are targeted by the flavonoid quercetin.

Many studies have demonstrated that the flavonoid quercetin protects against cardiovascular disease (CVD) and related risk factors. Atherosclerosis, the underlying cause of CVD, is also attenuated by oral quercetin administration in animal models. Although macrophages are key players during fatty streak formation and plaque progression and aggravation, little is known about the effects of quercetin on atherogenic macrophages. Here, we report that primary bone marrow-derived macrophages internalized less oxidized low-density lipoprotein (oxLDL) and accumulated less intracellular cholesterol in the presence of quercetin. This reduction of foam cell formation correlated with reduced surface expression of the oxLDL receptor CD36. Quercetin also targeted the lipopolysaccharide-dependent, oxLDL-independent pathway of lipid droplet formation in macrophages. In oxLDL-stimulated macrophages, quercetin inhibited reactive oxygen species production and interleukin (IL)-6 secretion. In a system that evaluated cholesterol crystal-induced IL-1ß secretion via nucleotide-binding domain and leucine-rich repeat containing protein 3 inflammasome activation, quercetin also exhibited an inhibitory effect. Dyslipidemic apolipoprotein E-deficient mice chronically treated with intraperitoneal quercetin injections had smaller atheromatous lesions, reduced lipid deposition, and less macrophage and T cell inflammatory infiltrate in the aortic roots than vehicle-treated animals. Serum levels of total cholesterol and the lipid peroxidation product malondialdehyde were also reduced in these mice. Our results demonstrate that quercetin interferes with both key proatherogenic activities of macrophages, namely foam cell formation and pro-oxidant/proinflammatory responses, and these effects may explain the atheroprotective properties of this common flavonoid.