The effects of the Green-Mediterranean diet on cardiometabolic health are linked to gut microbiome modifications: a randomized controlled trial.

BACKGROUND: Previous studies have linked the Mediterranean diet (MED) with improved cardiometabolic health, showing preliminary evidence for a mediating role of the gut microbiome. We recently suggested the Green-Mediterranean (Green-MED) diet as an improved version of the healthy MED diet, with increased consumption of plant-based foods and reduced meat intake. Here, we investigated the effects of MED interventions on the gut microbiota and cardiometabolic markers, and the interplay between the two, during the initial weight loss phase of the DIRECT-PLUS trial. METHODS: In the DIRECT-PLUS study, 294 participants with abdominal obesity/dyslipidemia were prospectively randomized to one of three intervention groups: healthy dietary guidelines (standard science-based nutritional counseling), MED, and Green-MED. Both isocaloric MED and Green-MED groups were supplemented with 28g/day walnuts. The Green-MED group was further provided with daily polyphenol-rich green tea and Mankai aquatic plant (new plant introduced to a western population). Gut microbiota was profiled by 16S rRNA for all stool samples and shotgun sequencing for a select subset of samples. RESULTS: Both MED diets induced substantial changes in the community structure of the gut microbiome, with the Green-MED diet leading to more prominent compositional changes, largely driven by the low abundant, "non-core," microorganisms. The Green-MED diet was associated with specific microbial changes, including enrichments in the genus Prevotella and enzymatic functions involved in branched-chain amino acid degradation, and reductions in the genus Bifidobacterium and enzymatic functions responsible for branched-chain amino acid biosynthesis. The MED and Green-MED diets were also associated with stepwise beneficial changes in body weight and cardiometabolic biomarkers, concomitantly with the increased plant intake and reduced meat intake. Furthermore, while the level of adherence to the Green-MED diet and its specific green dietary components was associated with the magnitude of changes in microbiome composition, changes in gut microbial features appeared to mediate the association between adherence to the Green-MED and body weight and cardiometabolic risk reduction. CONCLUSIONS: Our findings support a mediating role of the gut microbiome in the beneficial effects of the Green-MED diet enriched with Mankai and green tea on cardiometabolic risk factors. TRIAL REGISTRATION: The study was registered on ClinicalTrial.gov ( NCT03020186 ) on January 13, 2017.

Effect of green-Mediterranean diet on intrahepatic fat: the DIRECT PLUS randomised controlled trial.

OBJECTIVE: To examine the effectiveness of green-Mediterranean (MED) diet, further restricted in red/processed meat, and enriched with green plants and polyphenols on non-alcoholic fatty liver disease (NAFLD), reflected by intrahepatic fat (IHF) loss. DESIGN: For the DIRECT-PLUS 18-month randomized clinical trial, we assigned 294 participants with abdominal obesity/dyslipidaemia into healthy dietary guidelines (HDG), MED and green-MED weight-loss diet groups, all accompanied by physical activity. Both isocaloric MED groups consumed 28 g/day walnuts (+440 mg/day polyphenols provided). The green-MED group further consumed green tea (3-4 cups/day) and Mankai (a Wolffia globosa aquatic plant strain; 100 g/day frozen cubes) green shake (+1240 mg/day total polyphenols provided). IHF% 18-month changes were quantified continuously by proton magnetic resonance spectroscopy (MRS). RESULTS: Participants (age=51 years; 88% men; body mass index=31.3 kg/m2; median IHF%=6.6%; mean=10.2%; 62% with NAFLD) had 89.8% 18-month retention-rate, and 78% had eligible follow-up MRS. Overall, NAFLD prevalence declined to: 54.8% (HDG), 47.9% (MED) and 31.5% (green-MED), p=0.012 between groups. Despite similar moderate weight-loss in both MED groups, green-MED group achieved almost double IHF% loss (-38.9% proportionally), as compared with MED (-19.6% proportionally; p=0.035 weight loss adjusted) and HDG (-12.2% proportionally; p<0.001). After 18 months, both MED groups had significantly higher total plasma polyphenol levels versus HDG, with higher detection of Naringenin and 2-5-dihydroxybenzoic-acid in green-MED. Greater IHF% loss was independently associated with increased Mankai and walnuts intake, decreased red/processed meat consumption, improved serum folate and adipokines/lipids biomarkers, changes in microbiome composition (beta-diversity) and specific bacteria (p<0.05 for all). CONCLUSION: The new suggested strategy of green-Mediterranean diet, amplified with green plant-based proteins/polyphenols as Mankai, green tea, and walnuts, and restricted in red/processed meat can double IHF loss than other healthy nutritional strategies and reduce NAFLD in half. TRIAL REGISTRATION NUMBER: NCT03020186.

A Green-Mediterranean Diet, Supplemented with Mankai Duckweed, Preserves Iron-Homeostasis in Humans and Is Efficient in Reversal of Anemia in Rats.

BACKGROUND: Decreased dietary meat may deplete iron stores, as plant-derived iron bioavailability is typically limited. OBJECTIVES: We explored the effect of a low-meat Mediterranean (green-MED) diet, supplemented with Wolffia globosa duckweed (Mankai: rich in protein and iron) as a food source for humans, on iron status. We further examined the iron bioavailability of Mankai in rats. METHODS: Two hundred and ninety-four abdominally obese/dyslipidemic [mean age = 51.1 y; body mass index (kg/m2) = 31.3; 88% men] nonanemic participants were randomly assigned to physical activity (PA), PA + MED diet, or PA + green-MED diet. Both isocaloric MED groups consumed 28 g walnuts/d and the low-meat green-MED group further consumed green tea (800 mL/d) and Mankai (100 g green shake/d). In a complementary animal experiment, after 44 d of an iron deficiency anemia-inducing diet, 50 female rats (age = 3 wk; Sprague Dawley strain) were randomly assigned into: iron-deficient diet (vehicle), or vehicle + iso-iron: ferrous gluconate (FG) 14, Mankai 50, and Mankai 80 versions (1.7 mg · kg-1 · d-1 elemental iron), or FG9.5 and Mankai 50-C version (1.15 mg · kg-1 · d-1 elemental iron). The specific primary aim for both studies was changes in iron homeostasis parameters. RESULTS: After 6 mo of intervention, iron status trajectory did not differ between the PA and PA + MED groups. Hemoglobin modestly increased in the PA + green-MED group (0.23 g/dL) compared with PA (-0.1 g/dL; P < 0.001) and PA + MED (-0.1 g/dL; P < 0.001). Serum iron and serum transferrin saturation increased in the PA + green-MED group compared with the PA group (8.21 µg/dL compared with -5.23 µg/dL and 2.39% compared with -1.15%, respectively; P < 0.05 for both comparisons), as did folic acid (P = 0.011). In rats, hemoglobin decreased from 15.7 to 9.4 mg/dL after 44 d of diet-induced anemia. After depletion treatment, the vehicle-treated group had a further decrease of 1.3 mg/dL, whereas hemoglobin concentrations in both FG and Mankai iso-iron treatments similarly rebounded (FG14: +10.8 mg/dL, Mankai 50: +6.4 mg/dL, Mankai 80: +7.3 mg/dL; FG9.5: +5.1 mg/dL, Mankai 50-C: +7.1 mg/dL; P < 0.05 for all vs. the vehicle group). CONCLUSIONS: In humans, a green-MED low-meat diet does not impair iron homeostasis. In rats, iron derived from Mankai (a green-plant protein source) is bioavailable and efficient in reversal of anemia. This trial was registered at clinicaltrials.gov as NCT03020186.

The lack of effectiveness of hyperbaric oxygenation as a treatment for Leishmania major in a mouse model.

We aimed to study the effectiveness of hyperbaric oxygen therapy (HOT) (100% oxygen at 2 ATA for 70 minutes each session for 20 consecutive days) on BALB/c male mice infected with Leishmania major. Fifty-one mice were assigned to six groups. Group 1 was treated with HOT from 1 day after the inoculation. In Groups 2-5, treatment began when the cutaneous lesions appeared. Group 2 received HOT only, Group 3 received topical therapy with Leshcutan only, Groups 4 and 5 received a combination of HOT and Leshcutan for 5 and 10 days respectively, and Group 6 did not receive any treatment (control group). When comparing the control group with Group 1, treatment with HOT in Group 1 did not significantly affect the time of the appearance of the lesions. In contrast, mice treated with Leshcutan demonstrated a significant difference in lesion size and spleen dimensions as compared to the rest of the mice (p<0.001). The results show that HOT treatment has no positive effect on the course of Leishmaniasis in a BALB/c mice model infected with Leishmania major. Further studies are needed with a mouse model closer to humans and with different HOT protocols.

Fecal microbiota transplant for relapsing Clostridium difficile infection using a frozen inoculum from unrelated donors: a randomized, open-label, controlled pilot study.

BACKGROUND: Recurrent Clostridium difficile infection (CDI) with poor response to standard antimicrobial therapy is a growing medical concern. We aimed to investigate the outcomes of fecal microbiota transplant (FMT) for relapsing CDI using a frozen suspension from unrelated donors, comparing colonoscopic and nasogastric tube (NGT) administration. METHODS: Healthy volunteer donors were screened and a frozen fecal suspension was generated. Patients with relapsing/refractory CDI were randomized to receive an infusion of donor stools by colonoscopy or NGT. The primary endpoint was clinical resolution of diarrhea without relapse after 8 weeks. The secondary endpoint was self-reported health score using standardized questionnaires. RESULTS: A total of 20 patients were enrolled, 10 in each treatment arm. Patients had a median of 4 (range, 2-16) relapses prior to study enrollment, with 5 (range, 3-15) antibiotic treatment failures. Resolution of diarrhea was achieved in 14 patients (70%) after a single FMT (8 of 10 in the colonoscopy group and 6 of 10 in the NGT group). Five patients were retreated, with 4 obtaining cure, resulting in an overall cure rate of 90%. Daily number of bowel movements changed from a median of 7 (interquartile range [IQR], 5-10) the day prior to FMT to 2 (IQR, 1-2) after the infusion. Self-ranked health score improved significantly, from a median of 4 (IQR, 2-6) before transplant to 8 (IQR, 5-9) after transplant. No serious or unexpected adverse events occurred. CONCLUSIONS: In our initial feasibility study, FMT using a frozen inoculum from unrelated donors is effective in treating relapsing CDI. NGT administration appears to be as effective as colonoscopic administration. CLINICAL TRIALS REGISTRATION: NCT01704937.

Hyperbaric oxygen treatment reduces mortality in acute iron intoxication in rats.

Acute iron intoxication is one of the leading causes of overdose morbidity and mortality in children. The toxicity of iron has been postulated to be related to free radical formation and subsequent lipid peroxidation. Hyperbaric oxygen treatment can result in a number of beneficial biochemical, cellular and physiological effects, and has recently been shown to induce cellular protection against ischaemia, and in some cases against free radical formation. In the current study, we aimed to investigate the effects of hyperbaric oxygen treatment on mortality in acute iron intoxication in rats. After iron administration, 57 animals were divided into two groups: a treatment group receiving hyperbaric oxygen treatment (n = 30) and a control group (n = 27), and followed for 48 hr for signs of severe intoxication. In the second part of the study, 21 animals were divided into a treatment group receiving hyperbaric oxygen treatment (n = 10) and a control group (n = 11), and markers of oxidative stress were evaluated. We showed a significant reduction in mortality in hyperbaric oxygen-treated animals from 17 of 27 (62.9%) among untreated rats to 6 of 30 (20%). Surprisingly, in the treatment group, levels of oxidative stress markers were higher. We postulate that hyperbaric oxygen has a potentially beneficial effect in acute iron intoxication.