Next Article in Journal
Assessment of the Nutritional Composition and Environmental Impact of Menus Served in a University Cafeteria During an Academic Year
Previous Article in Journal
The Quantitation of EPA and DHA in Fish Oil Dietary Supplements Sold in the United States
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Dietetics
Volume 4
Issue 1
10.3390/dietetics4010001
dietetics-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Influence of Chronic Dietary Nitrate on Downstream Atherogenic Metabolites and the Enteral Microbiome—A Double-Blind Randomized Controlled Trial

by
Daniel Messiha
1,
Miriam Rinke
1,
Adriana Schultz Moreira Amos
1,
Annika Tratnik
1,
Ulrike Barbara Hendgen-Cotta
1,
Julia Lortz
1,
Kristina Hogrebe
1,
Jan Kehrmann
2,
Jan Buer
2,
Tienush Rassaf
1 and
Christos Rammos
1,*
1
Department of Cardiology and Vascular Medicine, West German Heart and Vascular Center Essen, University of Duisburg-Essen, Hufelandstr. 55, 45147 Essen, Germany
2
Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Hufelandstr. 55, 45147 Essen, Germany
*
Author to whom correspondence should be addressed.
Dietetics 2025, 4(1), 1; https://doi.org/10.3390/dietetics4010001
Submission received: 20 October 2024 / Revised: 4 December 2024 / Accepted: 27 December 2024 / Published: 10 January 2025
Browse Figures
Versions Notes

Abstract

:
Background: Inorganic nitrate is abundant in leafy green vegetables and has been shown to exert positive cardiovascular effects through nitric oxide-related pathways. The enteral microbiome is an emerging key player in cardiovascular diseases and depends on dietary habits. Whether dietary inorganic nitrate impacts on the microbiome and atherosclerosis-associated microbiome-dependent metabolites like short chain fatty acids (SCFA) and trimethylamine N-oxide (TMAO) is unknown. Methods: In a double-blind randomized controlled trial, 30 healthy volunteers were included who either received dietary nitrate (0.12 mmol/kg bodyweight) or placebo (equimolar amounts of sodium chloride) for 30 days. The microbiome metabolites TMAO and SCFA were analyzed. The enteral microbiome was analyzed by 16S-rRNA sequencing at baseline and follow-up. Results: Systolic blood pressure decreased after nitrate supplementation (baseline 124.73 mmHg vs. follow up 120 mmHg, p < 0.05) with no change in controls. Dietary nitrate supplementation increased TMAO levels (nitrate baseline 349.28 μ/L vs. nitrate follow-up 481.15 μ/L, p < 0.05), while SCFA levels remained unchanged. The relative abundance of Akkermansia and taxa of Clostridiales were higher in individuals with high compared to normal TMAO levels after nitrate supplementation, while Shannon diversity, richness and evenness did not differ between both groups. Conclusions: Our results indicate that dietary nitrate supplementation is associated with alterations to the enteral microbiome with an impact on proatherogenic metabolites. Further work is warranted to investigate the causal relationship between dietary nutrients, the microbiome and downstream metabolites.

1. Introduction

Atherosclerotic cardiovascular diseases (ACVDs) are the leading cause of morbidity and mortality in the industrialized world. In the past decades, a set of risk factors has been identified that promotes the development and progression of ACVD, such as diabetes, arterial hypertension, hyperlipidemia and tobacco use [1]. The enteral microbiome has been introduced as a new player in the pathogenesis of ACVD, and it acts like a double-edged sword. Mediated by microbiome-dependent metabolites, such as short chain fatty acids (SCFAs) or Trimethylamine N-Oxide (TMAO), the enteral microbiome has the capacity to promote or alleviate the progression of ACVD [2,3,4,5]. SCFAs are a result of bacterial enzymatic fermentation of nutritional elements, like dietary fiber, that are indigestible by the human host [2]. SCFAs are fatty acids with less than six carbons that are a product of bacterial fermentation of dietary fiber in the colon [3]. One of the main effects of SCFAs are local and systemic anti-inflammatory effects [4].
TMAO is a proatherogenic agent that is generated by the enteral microbiome from cholin-rich animal products (for example egg, beef, dairy products and fish) and has been described as being associated with higher risks in major adverse cardiac events (MACEs) [5,6,7,8].
In addition to those metabolites, cellular components of enteral bacteria contribute to an inflammatory enteral milieu, which has been described as “leaky gut”, that in the case of enteral barrier disorders can induce a systematic inflammatory state in the host [9]. Subclinical inflammation has been described as a contributory factor in the pathogenesis of ACVD [10]. Mechanistically, it has been shown in in vivo animal experiments that alterations of the enteral microbiome through antibiotic or probiotic treatment influenced the severity of myocardial, renal infarction and stroke in terms of infarct size and functional recovery [11,12,13,14,15,16].
A vegetarian diet, which is rich in vegetables and green leaflets, was associated with a beneficial enteral microbiome composition and improved cardiovascular risk factors [17]. One of the main abundant micronutrients in a vegetarian diet is inorganic nitrate.
Dietary nitrate has cardioprotective capacities after being metabolized to nitrite by the microbiome of the oral cavity and consequently being further reduced to nitric oxide [18,19,20,21,22,23]. Specifically, dietary nitrate is being reabsorbed in the enterosalivary circulation and thus returns after swallowing from the gut to the salivary glands in the oral cavity. Here, the oral microbiome continues to reduce nitrate to nitrite and nitric oxide [24]. This contributes to a circulating nitric oxide plasma pool [25]. Eradication of the microbiome in the oral cavity with an antiseptic mouthwash abolished those effects of dietary nitrate [26]. Nitric oxide has been extensively studied and proven to be cytoprotective in myocardial ischemia/reperfusion injury [27,28,29,30], to reverse vascular dysfunction in the elderly [31,32,33,34] and to have beneficial effects on blood pressure levels and physical fitness [32,35,36,37,38]. In the setting of microbiome dysregulation, however, others have shown that nitrate had a detrimental effect and was associated with exacerbated outcomes after ischemic stroke in an in vivo mouse experiment [39].
The influence of chronic nitrate supplementation on microbiome-dependent metabolites and the composition of the enteral microbiome has not been elucidated in depth so far.
The aim of this study was to investigate the influence of dietary inorganic nitrate intake on atherosclerosis-associated microbiome dependent metabolites and the upstream enteral microbiome in a cohort, with no pre-existing medical conditions and no regular medication intake.

2. Methods

We investigated the effects of inorganic nitrate on microbiome-dependent downstream pathways and conducted this explorative randomized controlled trial with 30 healthy participants to exclude confounding medications that might interfere with microbiome-derived metabolites. Cohort size was based on previous similar dietary research [31]. To minimize confounding factors on microbiome-dependent metabolites and upstream microbiome composition, we thus focused on a healthy cohort that did not have any significant past medical history and did not take any medications including antibiotics during the time of the study and 60 days prior to randomization. Participants older than 18 years were included in this double-blind randomized controlled trial. Participants were recruited via flyers and via electronic newsletters. They were block-randomized 1:1 into two cohorts. One cohort received dietary inorganic nitrate (0, 12 mmol/kg bodyweight), while the other cohort received equimolar dietary sodium chloride as a placebo each day over a time frame of 30 days, using established protocols [31] (Figure 1). Double-blind randomization was performed, and samples and data were collected in the West German Heart and Vascular Centre of the University Clinic Essen, Germany. Participants and investigators were blinded, since the weighing and bottling of dietary inorganic nitrate and placebo were performed by our Cardio Science Labs, which were not involved in further assessment of those specific participants. The samples were stored at room temperature for the duration of the follow-up period and were prepared right before the study entry of the participant.
The participants’ past medical histories were taken, and dietary habits were noted and recorded with an established standardized Mediterranean Diet Adherence Screener (MEDAS) questionnaire [40]. We chose that questionnaire to evaluate possible confounding effects, as a Mediterranean diet is particularly rich in green, leafy nitrate-rich food. Patients were asked to not change their dietary patterns during the course of the trial. Examination at baseline and follow-up including measurement of basic cardiovascular parameters like ankle brachial index, pulse wave velocity, blood pressure, heart rate, collection of stool samples and blood work, including routine-of-care analysis and analysis of TMAO and SCFA panels.
Blood samples were directly analyzed by our laboratory for routine-of-care analysis. Samples for downstream metabolite analysis were centrifuged according to our protocol, and then snap frozen.
SCFA levels were measured by gas chromatograph–mass spectrometry (GC-MS) (MS-Omics, Vedbæk, Denmark). TMAO serum levels were measured by gas chromatography–mass spectrometry (GC-MS) (Ganzimmun, Mainz, Germany). Circulating nitric oxide levels were measured by chemiluminescence detector (CLD) analysis (Eco Physics, Huerth, Germany), as previously explained [38,41].
Stool samples were collected in “DNA/RNA Shield Collection & Lysis Tubes” (ZymoResearch, Irvine, CA, USA) and DNA was isolated according to the manufacturers protocol (ZymoBiomics DNA Miniprep Kit, Irvine, CA, USA). Sequencing of the V3/V4 region of the 16S rRNA gene was performed in collaboration with the biochip core facility of the Institute for Cell Biology (University Duisburg-Essen, Duisburg, Germany) using a 300 bp paired-end approach.
The paired-end fastq files and a mapping file including sample and patient metadata were used as input for data analyses performed with the Quantitative Insights into Microbial Ecology (QIIME2) pipeline [21]. Cutadapt was used for the removal of adapters and primers using [22]. The Dada2 (V 1.26) software package [23], wrapped in QIIME2, was used for modeling and correcting fastq files, including the elimination of chimeras with the consensus method. The q2-diversity plugin was used for computing Shannon’s diversity, observed features and evenness with a sampling depth of 15064. We used a Naïve Bayes classifier, trained on the Greengenes 13_8_99% OTUs 16S rRNA gene full-length sequences, for assigning taxonomy. Differences in bacterial taxa between groups were assessed using LEfSe (Linear discriminant effect size analysis) [42] with a p < 0.05 for bacterial class comparison using Kruskal–Wallis test and a linear discriminant analysis (LDA) score >3.
Statistical analysis was performed with GraphPad Prism utilizing the Student’s t-test and ANOVA.
The trial was conducted in accordance with the Declaration of Helsinki, was approved by the local ethics committee of the medical faculty of the university of Duisburg-Essen (21-9913-BO) and registered at Clinical Trials with the registration No: NCT05122689. Written consent of all participants was obtained prior to randomization.

3. Results

The aim of this study was to investigate the influence of dietary inorganic nitrate intake on atherosclerosis-associated microbiome-dependent metabolites and the upstream enteral microbiome in a cohort with no pre-existing medical conditions and no regular medication (Table 1). Dietary habits were not statistically different between the cohorts as assessed by the MEDAS questionnaire (5.7 ± 2.2 vs. 6.4 ± 2.6, p > 0.05).

3.1. Impact of Dietary Nitrate Supplementation on Microbiome-Related Metabolites

Baseline TMAO levels between the control and nitrate groups did not differ at baseline (p > 0.05). After 30 days of dietary nitrate supplementation, serum levels of the microbiome-dependent metabolite TMAO were elevated significantly compared to the control group (nitrate 270 ± 226.1 μL/L to 355 ± 274 μL/L (p < 0.05) vs. control 331.4 ± 243.8 μL/L to 310 ± 143.9 μL/L; p > 0.05; Figure 2).
Serum levels of the SCFAs acetic acid, propanoic acid, butanoic acid and hexanoic acid did not change during the observed time frame after dietary inorganic nitrate supplementation. In the control group, however, sodium chloride supplementation was associated with decreased levels of propionic acid at follow-up (control baseline 2.2 μM ± 0.99 μM vs. control follow-up 1.7 μM ± 1.13 μM; p < 0.05; Figure 3).

3.2. Dietary Nitrate Supplementation Is Associated with a Difference in Enteral Microbiome Composition

To evaluate the effect of dietary nitrate supplementation on the enteral microbiome, we analyzed the microbiome composition at baseline and follow-up within the nitrate supplementation cohort. While the genus Parabacteroides and the family Porphyromonaceae were overrepresented taxa after nitrate supplementation, a genus and family of Bacteroidales were overrepresented before nitrate supplementation (Figure 4).
In the next step, we analyzed differences in the microbiome composition at follow-up between individuals of the control group treated with placebo and the nitrate supplementation group. The LEfSe analysis identified differences in the enteral microbiome composition, with five taxa that were more abundant at follow-up after nitrate supplementation (Figure 5).

3.3. High TMAO Levels After Dietary Nitrate Supplementation Are Associated with a Distinct Enteral Microbiome Composition

To investigate changes in the microbiome as potential biomarkers for high TMAO levels at follow-up, we further analyzed differences in the microbiome composition between individuals’ high compared to normal TMAO levels after nitrate supplementation. We identified a distinct enteral microbiome composition in participants with high TMAO levels at follow-up after nitrate supplementation compared to those with normal TMAO levels. A total of 27 taxa discriminated individuals with high TMAO levels from those with normal TMAO levels after treatment with nitrate supplements (Figure 6).

3.4. Dietary Nitrate Supplementation Does Not Change Microbiome Alpha Diversity

Dietary nitrate supplementation over a period of 30 days did not change alpha diversity metrics of the enteral microbiome, either in the control or in the nitrate group. Shannon diversity (before placebo 7.3 ± 1.9 vs. after placebo 7.2 ± 2 vs. before verum 7.2 ± 2.1 vs. after verum 7.9 ± 0.6, p > 0.05), richness (before placebo 354.4 ± 133 vs. after placebo 327.1 ± 111.4 vs. before verum 325 ± 134.6 vs. after verum 394.3 ± 104; p > 0.05) and evenness (before placebo 0.9 ± 0.2 vs. after placebo 0.9 ± 0.2 vs. before verum 0.9 ± 0.2 vs. after verum 0.9 ± 0.2; p > 0.05; Figure 7) were not significantly different between the different groups.

3.5. Dietary Supplementation of Inorganic Nitrate Increases Circulating Nitric Oxide Serum Levels and Reduces Systolic Blood Pressure

Circulating serum nitric oxide levels were low and did not differ significantly between the control and nitrate groups at baseline (5.06 ppb ± 24.56 ppb vs. 5.0 ppb ± 2.45 ppb, p > 0.05). To verify the supplementation adherence of the participants, we measured nitric oxide levels from peripheral blood samples at follow-up. As expected, the nitric oxide serum levels increased significantly, 20-fold at follow-up in the nitrate group, while they did not change in the control group after placebo supplementation (37.2 ppb ± 45.86 ppb vs. 101.5 ppb ± 28.26 ppb, *** depicts p < 0.005; Figure 8).
Correspondingly, increased nitric oxide serum levels led to significantly reduced systolic blood pressures (control group 124 ± 14.22 mmHg to 127 ± 17.54 mmHg, p > 0.05, and nitrate group 125 ± 10.59 mmHg to 121 ± 10.23 mmHg, p < 0.05). In the control group, blood pressure levels remained unchanged between baseline and follow-up (Figure 9).

4. Discussion

This is the first double-blind randomized controlled trial that shows the direct effect of a chronic dietary intervention with inorganic nitrate focusing on enteral microbiome composition and downstream metabolites. Confounding factors such as regular medications, pre-existing health conditions and relevant differences in nutritional habits were a priori excluded by only including healthy participants without any medications or known preexisting medical conditions. Adherence to the Mediterranean diet was not statistically different between the cohorts, as assessed by the established Mediterranean Diet Adherence Screener (MEDAS) questionnaire.
We detected a distinct regulation in the enteral microbiome composition after dietary nitrate supplementation over 30 days. We identified a high abundance of the Gram-negative bacteria Akkermansia at follow-up as a potential biomarker for high TMAO levels after nitrate supplementation and increased levels of Clostridiales upon comparing the nitrate supplementation and the control cohort at follow-up. In the longitudinal comparison within the nitrate supplementation cohort between baseline and follow-up, we identified the taxa Bacteroidales to be decreased in abundance.
Akkermansia muciniphila has been described as a protective probiotic with beneficial effects on metabolism, immune function and atherosclerosis [43,44]. It has been shown to reduce TMAO levels, and thus is seen as a cardioprotective bacteria [45]. Supplementation of synthetic Akkermansia muciniphila to mice who were set on a high-fat diet could reduce TMAO levels significantly, presumably via downregulation of Flavin-containing monooxidase 3 (FMO3), the enzyme that converts TMA to TMAO [46,47]. In line with those findings, we found increased Akkermansia muciniphila levels in patients with elevated TMAO serum levels at follow-up after nitrate supplementation, which could be a compensatory effect mediated by dietary nitrate. One possible explanation could be a compensatory upregulation of Akkermansia muciniphila, due to elevated nitric oxide levels after dietary nitrate supplementation. It has been shown that Akkermansia muciniphila moderates its antihypertensive and cardioprotective effects in part through increasing nitric oxide concentrations via the nitrate–nitrite–nitric oxide axis [48]. Mediated through its outer membrane protein Amuc_110, Akkermansia muciniphila actively increases nitric oxide levels by metabolizing L-Arginin. Thus, it is possible that the exogeneous administration of nitric oxide through dietary nitrate supplementation in our healthy cohort could in turn further increase Akkermansia muciniphila levels [48,49]. However, due to the exploratory design of our study, we can only detect associations and further work is warranted to examine the underlying mechanisms and causalities, especially in a collective of patients with cardiovascular disease.
Our findings of increased TMAO levels after nitrate supplementation are intriguing and suggest a double-edged function. Others have demonstrated that gut dysbiosis-derived nitrate exerts negative outcomes after stroke through microbiome-dependent metabolites [38,39,41]. It has been hypothesized that a high-fat diet influences TMAO levels through nitrate metabolism. A fatty diet reduces mitochondrial activity, which in turn induces the inducible nitric oxide synthase (iNOS) and leads to elevated nitric oxide concentrations in the colonic mucus layer. Elevated colonic nitric oxide concentration could promote Escherichia coli choline catabolism, which is a central step in TMAO production [44,50]. Dietary exogenous nitrate supplementation could interact with Escherichia coli choline metabolism directly through this mechanism.
We found a high abundance of Clostridiacea in individuals with high TMAO levels after nitrate supplementation. Clostridiacea has been described before in the literature to be a TMAO producer through its cutC gene. This is in line with the elevated TMAO levels we found at follow-up after nitrate supplementation [51,52,53].
Our study has several limitations. While sodium chloride serves as a placebo, an equimolar quantity of sodium may not fully control for potential effects of nitrate-specific metabolic pathways. Secondly, although participants were asked to maintain their diets, subtle changes could still affect outcomes, particularly microbiome composition. Incorporating a more detailed dietary log than MEDAS could further enhance data accuracy. Previous research on the effects of dietary nitrate supplementation on enteral microbiome dynamics are scarce.
Taken together, dietary nitrate supplementation does not yield a one-sided storyline; it seems to have many facets. While it is cytoprotective in myocardial ischemia/reperfusion injury [27], reverses vascular dysfunction in the elderly [31] and has beneficial effects on blood pressure levels [35,36], our randomized controlled trial now highlights that in the gut, dietary nitrate promotes a microbiome dysregulation that is associated with an increased abundance of Akkermansia muciniphila, a known antiatherogenic bacteria, which could be a compensatory mechanism for elevated proatherogenic metabolites such as TMAO after nitrate supplementation. Whether this finding is clinically relevant in the context of an overall cardioprotective nitrate–nitrite–nitric oxide metabolism needs to be further examined. Also, further work with longer follow-up periods is warranted to investigate the exact causal relationships between the nitrate–nitrite–nitric oxide metabolism and gut microbiome composition.

Author Contributions

Conceptualization, D.M., U.B.H.-C., T.R. and C.R.; Data curation, M.R. and A.S.M.A.; Formal analysis, D.M., M.R., A.T., U.B.H.-C., J.L., K.H., J.K., J.B. and C.R.; Investigation, D.M., M.R., A.S.M.A., A.T., J.K., J.B. and C.R.; Methodology, D.M., U.B.H.-C. and C.R.; Project administration, T.R. and C.R.; Resources, T.R. and C.R.; Supervision, D.M., T.R. and C.R.; Validation, D.M. and C.R.; Visualization, M.R. and J.K.; Writing—original draft, D.M. and C.R.; Writing—review and editing, A.S.M.A., U.B.H.-C., J.L., K.H., J.K., J.B. and T.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The trial was conducted in accordance with the Declaration of Helsinki, was approved by the local ethics committee of the medical faculty of the university of Duisburg-Essen (21-9913-BO) and was registered at Clinical Trials with the registration no. NCT05122689. Written consent of all participants was obtained prior to randomization.

Informed Consent Statement

Written consent of all participants was obtained prior to randomization.

Data Availability Statement

All data generated or analyzed during this study are included in this published article or available upon request from the corresponding author.

Conflicts of Interest

The authors declare that they have no competing interests.

References

  1. Stone, N.J.; Smith, S.C.; Orringer, C.E.; Rigotti, N.A.; Navar, A.M.; Khan, S.S.; Jones, D.W.; Goldberg, R.; Mora, S.; Blaha, M.; et al. Managing Atherosclerotic Cardiovascular Risk in Young Adults. J. Am. Coll. Cardiol. 2022, 79, 819–836. [Google Scholar] [CrossRef] [PubMed]
  2. Flint, H.J.; Scott, K.P.; Duncan, S.H.; Louis, P.; Forano, E. Microbial degradation of complex carbohydrates in the gut. Gut Microbes 2012, 3, 289–306. [Google Scholar] [CrossRef] [PubMed]
  3. Hu, T.; Wu, Q.; Yao, Q.; Jiang, K.; Yu, J.; Tang, Q. Short-chain fatty acid metabolism and multiple effects on cardiovascular diseases. Ageing Res. Rev. 2022, 81, 101706. [Google Scholar] [CrossRef]
  4. Arpaia, N.; Campbell, C.; Fan, X.; Dikiy, S.; Van Der Veeken, J.; DeRoos, P.; Liu, H.; Cross, J.R.; Pfeffer, K.; Coffer, P.J.; et al. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature 2013, 504, 451–455. [Google Scholar] [CrossRef] [PubMed]
  5. Cho, C.E.; Taesuwan, S.; Malysheva, O.V.; Bender, E.; Tulchinsky, N.F.; Yan, J.; Sutter, J.L.; Caudill, M.A. Trimethylamine-N-Oxide (Tmao) Response to Animal Source Foods Varies among Healthy Young Men and Is Influenced by Their Gut Microbiota Composition: A Randomized Controlled Trial. Mol. Nutr. Food Res. 2017, 61, 1600324. [Google Scholar] [CrossRef]
  6. Ilyas, A.; Wijayasinghe, Y.S.; Khan, I.; El Samaloty, N.M.; Adnan, M.; Dar, T.A.; Poddar, N.K.; Singh, L.R.; Sharma, H.; Khan, S. Implications of trimethylamine N-oxide (TMAO) and Betaine in Human Health: Beyond Being Osmoprotective Compounds. Front. Mol. Biosci. 2022, 9, 964624. [Google Scholar] [CrossRef] [PubMed]
  7. Koeth, R.A.; Wang, Z.; Levison, B.S.; Buffa, J.A.; Org, E.; Sheehy, B.T.; Britt, E.B.; Fu, X.; Wu, Y.; Li, L.; et al. Intestinal Microbiota Metabolism of L-Carnitine, a Nutrient in Red Meat, Promotes Atherosclerosis. Nat. Med. 2013, 19, 576–585. [Google Scholar] [CrossRef] [PubMed]
  8. Li, N.; Zhou, J.; Wang, Y.; Chen, R.; Li, J.; Zhao, X.; Zhou, P.; Liu, C.; Song, L.; Liao, Z.; et al. Association between trimethylamine N-oxide and prognosis of patients with acute myocardial infarction and heart failure. ESC Hear. Fail. 2022, 9, 3846–3857. [Google Scholar] [CrossRef] [PubMed]
  9. Brandsma, E.; Kloosterhuis, N.J.; Koster, M.; Dekker, D.C.; Gijbels, M.J.; van der Velden, S.; Ríos-Morales, M.; van Faassen, M.J.; Loreti, M.G.; de Bruin, A.; et al. A Proinflammatory Gut Microbiota Increases Systemic Inflammation and Accelerates Atherosclerosis. Circ. Res. 2019, 124, 94–100. [Google Scholar] [CrossRef]
  10. Sánchez-Cabo, F.; Fuster, V.; Silla-Castro, J.C.; González, G.; Lorenzo-Vivas, E.; Alvarez, R.; Callejas, S.; Benguría, A.; Gil, E.; Núñez, E.; et al. Subclinical atherosclerosis and accelerated epigenetic age mediated by inflammation: A multi-omics study. Eur. Hear. J. 2023, 44, 2698–2709. [Google Scholar] [CrossRef]
  11. Lam, V.; Su, J.; Koprowski, S.; Hsu, A.; Tweddell, J.S.; Rafiee, P.; Gross, G.J.; Salzman, N.H.; Baker, J.E. Intestinal microbiota determine severity of myocardial infarction in rats. FASEB J. 2011, 26, 1727–1735. [Google Scholar] [CrossRef] [PubMed]
  12. Andrade-Oliveira, V.; Amano, M.T.; Correa-Costa, M.; Castoldi, A.; Felizardo, R.J.; de Almeida, D.C.; Bassi, E.J.; Moraes-Vieira, P.M.; Hiyane, M.I.; Rodas, A.C.; et al. Gut Bacteria Products Prevent AKI Induced by Ischemia-Reperfusion. J. Am. Soc. Nephrol. 2015, 26, 1877–1888. [Google Scholar] [CrossRef]
  13. Organ, C.L.; Li, Z.; Sharp, T.E., 3rd; Polhemus, D.J.; Gupta, N.; Goodchild, T.T.; Tang, W.H.W.; Hazen, S.L.; Lefer, D.J. Nonlethal Inhibition of Gut Microbial Trimethylamine N-Oxide Production Improves Cardiac Function and Remodeling in a Murine Model of Heart Failure. J. Am. Heart Assoc. 2020, 9, e016223. [Google Scholar] [CrossRef]
  14. Singh, V.; Roth, S.; Llovera, G.; Sadler, R.; Garzetti, D.; Stecher, B.; Dichgans, M.; Liesz, A. Microbiota Dysbiosis Controls the Neuroinflammatory Response after Stroke. J. Neurosci. 2016, 36, 7428–7440. [Google Scholar] [CrossRef]
  15. Tang, T.W.; Chen, H.-C.; Chen, C.-Y.; Yen, C.Y.; Lin, C.-J.; Prajnamitra, R.P.; Chen, L.-L.; Ruan, S.-C.; Lin, J.-H.; Lin, P.-J.; et al. Loss of Gut Microbiota Alters Immune System Composition and Cripples Postinfarction Cardiac Repair. Circulation 2019, 139, 647–659. [Google Scholar] [CrossRef]
  16. Wang, Z.; Klipfell, E.; Bennett, B.J.; Koeth, R.; Levison, B.S.; DuGar, B.; Feldstein, A.E.; Britt, E.B.; Fu, X.; Chung, Y.-M.; et al. Gut Flora Metabolism of Phosphatidylcholine Promotes Cardiovascular Disease. Nature 2011, 472, 57–63. [Google Scholar] [CrossRef] [PubMed]
  17. Djekic, D.; Shi, L.; Brolin, H.; Carlsson, F.; Särnqvist, C.; Savolainen, O.; Cao, Y.; Bäckhed, F.; Tremaroli, V.; Landberg, R.; et al. Effects of a Vegetarian Diet on Cardiometabolic Risk Factors, Gut Microbiota, and Plasma Metabolome in Subjects With Ischemic Heart Disease: A Randomized, Crossover Study. J. Am. Hear. Assoc. 2020, 9, e016518. [Google Scholar] [CrossRef]
  18. Lundberg, J.O.; Weitzberg, E.; Lundberg, J.M.; Alving, K. Intragastric nitric oxide production in humans: Measurements in expelled air. Gut 1994, 35, 1543–1546. [Google Scholar] [CrossRef]
  19. Hyde, E.R.; Andrade, F.; Vaksman, Z.; Parthasarathy, K.; Jiang, H.; Parthasarathy, D.K.; Torregrossa, A.C.; Tribble, G.; Kaplan, H.B.; Petrosino, J.F.; et al. Metagenomic Analysis of Nitrate-Reducing Bacteria in the Oral Cavity: Implications for Nitric Oxide Homeostasis. PLoS ONE 2014, 9, e88645. [Google Scholar] [CrossRef] [PubMed]
  20. Lundberg, J.O.; Weitzberg, E.; Gladwin, M.T. The Nitrate-Nitrite-Nitric Oxide Pathway in Physiology and Therapeutics. Nat. Rev. Drug Discov. 2008, 7, 156–167. [Google Scholar] [CrossRef]
  21. Gladwin, M.T.; Schechter, A.N.; Kim-Shapiro, D.B.; Patel, R.P.; Hogg, N.; Shiva, S.; Cannon, R.O., 3rd; Kelm, M.; Wink, D.A.; Espey, M.G.; et al. The Emerging Biology of the Nitrite Anion. Nat. Chem. Biol. 2005, 1, 308–314. [Google Scholar] [CrossRef] [PubMed]
  22. Cosby, K.; Partovi, K.S.; Crawford, J.H.; Patel, R.P.; Reiter, C.D.; Martyr, S.; Yang, B.K.; Waclawiw, M.A.; Zalos, G.; Xu, X.; et al. Nitrite Reduction to Nitric Oxide by Deoxyhemoglobin Vasodilates the Human Circulation. Nat. Med. 2003, 9, 1498–1505. [Google Scholar] [CrossRef]
  23. Koch, C.D.; Gladwin, M.T.; Freeman, B.A.; Lundberg, J.O.; Weitzberg, E.; Morris, A. Enterosalivary Nitrate Metabolism and the Microbiome: Intersection of Microbial Metabolism, Nitric Oxide and Diet in Cardiac and Pulmonary Vascular Health. Free Radic. Biol. Med. 2017, 105, 48–67. [Google Scholar] [CrossRef]
  24. Lundberg, J.O.; Govoni, M. Inorganic nitrate is a possible source for systemic generation of nitric oxide. Free. Radic. Biol. Med. 2004, 37, 395–400. [Google Scholar] [CrossRef]
  25. Rassaf, T.; Preik, M.; Kleinbongard, P.; Lauer, T.; Heiss, C.; Strauer, B.E.; Feelisch, M.; Kelm, M. Evidence for in Vivo Transport of Bioactive Nitric Oxide in Human Plasma. J. Clin. Invest. 2002, 109, 1241–1248. [Google Scholar] [CrossRef]
  26. Hendgen-Cotta, U.B.; Luedike, P.; Totzeck, M.; Kropp, M.; Schicho, A.; Stock, P.; Rammos, C.; Niessen, M.; Heiss, C.; Lundberg, J.O.; et al. Dietary Nitrate Supplementation Improves Revascularization in Chronic Ischemia. Circulation 2012, 126, 1983–1992. [Google Scholar] [CrossRef] [PubMed]
  27. Hendgen-Cotta, U.B.; Merx, M.W.; Shiva, S.; Schmitz, J.; Becher, S.; Klare, J.P.; Steinhoff, H.-J.; Goedecke, A.; Schrader, J.; Gladwin, M.T.; et al. Nitrite reductase activity of myoglobin regulates respiration and cellular viability in myocardial ischemia-reperfusion injury. Proc. Natl. Acad. Sci. USA 2008, 105, 10256–10261. [Google Scholar] [CrossRef]
  28. Rassaf, T.; Heiss, C.; Mangold, S.; Leyendecker, T.; Kehmeier, E.S.; Kelm, M.; Lauer, T. Vascular Formation of Nitrite After Exercise Is Abolished in Patients With Cardiovascular Risk Factors and Coronary Artery Disease. Circ. 2010, 55, 1502–1503. [Google Scholar] [CrossRef]
  29. Rassaf, T.; Poll, L.W.; Brouzos, P.; Lauer, T.; Totzeck, M.; Kleinbongard, P.; Gharini, P.; Andersen, K.; Schulz, R.; Heusch, G.; et al. Positive effects of nitric oxide on left ventricular function in humans. Eur. Hear. J. 2006, 27, 1699–1705. [Google Scholar] [CrossRef]
  30. Rassaf, T.; Totzeck, M.; Hendgen-Cotta, U.B.; Shiva, S.; Heusch, G.; Kelm, M. Circulating Nitrite Contributes to Cardioprotection by Remote Ischemic Preconditioning. Circ. Res. 2014, 114, 1601–1610. [Google Scholar] [CrossRef] [PubMed]
  31. Rammos, C.; Hendgen-Cotta, U.B.; Sobierajski, J.; Bernard, A.; Kelm, M.; Rassaf, T. Dietary Nitrate Reverses Vascular Dysfunction in Older Adults With Moderately Increased Cardiovascular Risk. Circ. 2014, 63, 1584–1585. [Google Scholar] [CrossRef] [PubMed]
  32. Rammos, C.; Hendgen-Cotta, U.B.; Totzeck, M.; Pohl, J.; Lüdike, P.; Flögel, U.; Deenen, R.; Köhrer, K.; French, B.A.; Gödecke, A.; et al. Impact of dietary nitrate on age-related diastolic dysfunction. Eur. J. Hear. Fail. 2016, 18, 599–610. [Google Scholar] [CrossRef]
  33. Rammos, C.; Luedike, P.; Hendgen-Cotta, U.; Rassaf, T. Potential of dietary nitrate in angiogenesis. World J. Cardiol. 2015, 7, 652–657. [Google Scholar] [CrossRef] [PubMed]
  34. Rammos, C.; Totzeck, M.; Deenen, R.; Köhrer, K.; Kelm, M.; Rassaf, T.; Hendgen-Cotta, U.B. Dietary Nitrate Is a Modifier of Vascular Gene Expression in Old Male Mice. Oxidative Med. Cell. Longev. 2015, 2015, 1–12. [Google Scholar] [CrossRef] [PubMed]
  35. Larsen, F.J.; Ekblom, B.; Sahlin, K.; Lundberg, J.O.; Weitzberg, E. Effects of Dietary Nitrate on Blood Pressure in Healthy Volunteers. New Engl. J. Med. 2006, 355, 2792–2793. [Google Scholar] [CrossRef] [PubMed]
  36. Rassaf, T.; Ferdinandy, P.; Schulz, R. Nitrite in organ protection. Br. J. Pharmacol. 2013, 171, 1–11. [Google Scholar] [CrossRef]
  37. Totzeck, M.; Hendgen-Cotta, U.B.; Rammos, C.; Frommke, L.-M.; Knackstedt, C.; Predel, H.-G.; Kelm, M.; Rassaf, T. Higher endogenous nitrite levels are associated with superior exercise capacity in highly trained athletes. Nitric Oxide 2012, 27, 75–81. [Google Scholar] [CrossRef] [PubMed]
  38. Rassaf, T.; Lauer, T.; Heiss, C.; Balzer, J.; Mangold, S.; Leyendecker, T.; Rottler, J.; Drexhage, C.; Meyer, C.; Kelm, M. Nitric oxide synthase-derived plasma nitrite predicts exercise capacity. Br. J. Sports Med. 2007, 41, 669–673. [Google Scholar] [CrossRef] [PubMed]
  39. Xu, K.; Gao, X.; Xia, G.; Chen, M.; Zeng, N.; Wang, S.; You, C.; Tian, X.; Di, H.; Tang, W.; et al. Rapid gut dysbiosis induced by stroke exacerbates brain infarction in turn. Gut 2021, 70, 1486–1494. [Google Scholar] [CrossRef] [PubMed]
  40. Hebestreit, K.; Yahiaoui-Doktor, M.; Engel, C.; Vetter, W.; Siniatchkin, M.; Erickson, N.; Halle, M.; Kiechle, M.; Bischoff, S.C. Validation of the German version of the Mediterranean Diet Adherence Screener (MEDAS) questionnaire. BMC Cancer 2017, 17, 341. [Google Scholar] [CrossRef] [PubMed]
  41. Hendgen-Cotta, U.; Grau, M.; Rassaf, T.; Gharini, P.; Kelm, M.; Kleinbongard, P. Reductive Gas-Phase Chemiluminescence and Flow Injection Analysis for Measurement of the Nitric Oxide Pool in Biological Matrices. Methods Enzymol. 2008, 441, 295–315. [Google Scholar]
  42. Segata, N.; Izard, J.; Waldron, L.; Gevers, D.; Miropolsky, L.; Garrett, W.S.; Huttenhower, C. Metagenomic biomarker discovery and explanation. Genome Biol. 2011, 12, R60. [Google Scholar] [CrossRef] [PubMed]
  43. Zhang, T.; Li, Q.; Cheng, L.; Buch, H.; Zhang, F. Akkermansia muciniphila is a promising probiotic. Microb. Biotechnol. 2019, 12, 1109–1125. [Google Scholar] [CrossRef]
  44. Li, J.; Lin, S.; Vanhoutte, P.M.; Woo, C.W.; Xu, A. Akkermansia Muciniphila Protects against Atherosclerosis by Preventing Metabolic Endotoxemia-Induced Inflammation in Apoe-/- Mice. Circulation 2016, 133, 2434–2446. [Google Scholar] [CrossRef]
  45. Luo, Y.; Zhang, Y.; Han, X.; Yuan, Y.; Zhou, Y.; Gao, Y.; Yu, H.; Zhang, J.; Shi, Y.; Duan, Y.; et al. Akkermansia muciniphila prevents cold-related atrial fibrillation in rats by modulation of TMAO induced cardiac pyroptosis. EBioMedicine 2022, 82, 104087. [Google Scholar] [CrossRef]
  46. Plovier, H.; Everard, A.; Druart, C.; Depommier, C.; Van Hul, M.; Geurts, L.; Chilloux, J.; Ottman, N.; Duparc, T.; Lichtenstein, L.; et al. A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice. Nat. Med. 2016, 23, 107–113. [Google Scholar] [CrossRef] [PubMed]
  47. Gao, X.; Du, L.; Randell, E.; Zhang, H.; Li, K.; Li, D. Effect of different phosphatidylcholines on high fat diet-induced insulin resistance in mice. Food Funct. 2021, 12, 1516–1528. [Google Scholar] [CrossRef]
  48. Lakshmanan, A.P.; Murugesan, S.; Al Khodor, S.; Terranegra, A. The potential impact of a probiotic: Akkermansia muciniphila in the regulation of blood pressure—the current facts and evidence. J. Transl. Med. 2022, 20, 1–15. [Google Scholar] [CrossRef]
  49. Catry, E.; Bindels, L.B.; Tailleux, A.; Lestavel, S.; Neyrinck, A.M.; Goossens, J.-F.; Lobysheva, I.; Plovier, H.; Essaghir, A.; Demoulin, J.-B.; et al. Targeting the Gut Microbiota with Inulin-Type Fructans: Preclinical Demonstration of a Novel Approach in the Management of Endothelial Dysfunction. Gut 2018, 67, 271–283. [Google Scholar] [CrossRef]
  50. Yoo, W.; Zieba, J.K.; Foegeding, N.J.; Torres, T.P.; Shelton, C.D.; Shealy, N.G.; Byndloss, A.J.; Cevallos, S.A.; Gertz, E.; Tiffany, C.R.; et al. High-fat diet–induced colonocyte dysfunction escalates microbiota-derived trimethylamine N -oxide. Science 2021, 373, 813–818. [Google Scholar] [CrossRef] [PubMed]
  51. Skye, S.M.; Zhu, W.; Romano, K.A.; Guo, C.-J.; Wang, Z.; Jia, X.; Kirsop, J.; Haag, B.; Lang, J.M.; DiDonato, J.A.; et al. Microbial Transplantation With Human Gut Commensals Containing CutC Is Sufficient to Transmit Enhanced Platelet Reactivity and Thrombosis Potential. Circ. Res. 2018, 123, 1164–1176. [Google Scholar] [CrossRef] [PubMed]
  52. Cao, Y.; Leng, C.; Lin, K.; Li, Y.; Zhou, M.; Zhou, M.; Shu, X.; Liu, W. Tangeretin Mitigates Trimethylamine Oxide Induced Arterial Inflammation by Disrupting Choline–Trimethylamine Conversion through Specific Manipulation of Intestinal Microflora. Molecules 2024, 29, 1323. [Google Scholar] [CrossRef] [PubMed]
  53. Koeth, R.A.; Lam-Galvez, B.R.; Kirsop, J.; Wang, Z.; Levison, B.S.; Gu, X.; Copeland, M.F.; Bartlett, D.; Cody, D.B.; Dai, H.J.; et al. l-Carnitine in omnivorous diets induces an atherogenic gut microbial pathway in humans. J. Clin. Investig. 2018, 129, 373–387. [Google Scholar] [CrossRef] [PubMed]
Dietetics 04 00001 g001
Figure 1. Study design.
Figure 1. Study design.
Dietetics 04 00001 g001
Dietetics 04 00001 g002
Figure 2. TMAO levels before and after supplementation between control group and intervention group. Chronic dietary nitrate supplementation increases TMAO levels, with no effect in controls; * depicts p < 0.05.
Figure 2. TMAO levels before and after supplementation between control group and intervention group. Chronic dietary nitrate supplementation increases TMAO levels, with no effect in controls; * depicts p < 0.05.
Dietetics 04 00001 g002
Dietetics 04 00001 g003
Figure 3. Serum levels of the SCFAs before and after a chronic dietary supplementation with inorganic nitrate or placebo. Acetic acid (A), propanoic acid (B), butanoic acid (C) and hexanoic acid (D) at baseline and follow-up in the control and nitrate group (** depicts p < 0.005).
Figure 3. Serum levels of the SCFAs before and after a chronic dietary supplementation with inorganic nitrate or placebo. Acetic acid (A), propanoic acid (B), butanoic acid (C) and hexanoic acid (D) at baseline and follow-up in the control and nitrate group (** depicts p < 0.005).
Dietetics 04 00001 g003
Dietetics 04 00001 g004
Figure 4. Linear discriminant analysis effect size (LEfSe) analysis used for the identification of microbial taxa that are overrepresented and biomarkers for the enteral microbiome of individuals before (green) and after (red) nitrate supplementation (A), and a cladogram (B) visualizing taxa that are biomarkers for individuals before (green) and after (red) nitrate supplementation.
Figure 4. Linear discriminant analysis effect size (LEfSe) analysis used for the identification of microbial taxa that are overrepresented and biomarkers for the enteral microbiome of individuals before (green) and after (red) nitrate supplementation (A), and a cladogram (B) visualizing taxa that are biomarkers for individuals before (green) and after (red) nitrate supplementation.
Dietetics 04 00001 g004
Dietetics 04 00001 g005
Figure 5. Linear discriminant analysis effect size (LEfSe) analysis (A) and cladogram (B) of microbial taxa that differentiate individuals of the control from the nitrate supplementation cohort after treatment with placebo or nitrate. Biomarkers for the placebo group are visualized in red and biomarkers for the nitrate supplementation group are visualized in green.
Figure 5. Linear discriminant analysis effect size (LEfSe) analysis (A) and cladogram (B) of microbial taxa that differentiate individuals of the control from the nitrate supplementation cohort after treatment with placebo or nitrate. Biomarkers for the placebo group are visualized in red and biomarkers for the nitrate supplementation group are visualized in green.
Dietetics 04 00001 g005
Dietetics 04 00001 g006
Figure 6. Analysis of microbial taxa that differentiate patients with high and normal TMAO serum levels after supplementation of nitrate. Linear discriminant analysis effect size (LEfSe) analysis shows taxa enriched in individuals after receiving nitrate supplementation with high (red) and normal (green) TMAO serum levels with LDA > 3 (A), and a cladogram visualizes the phylogenetic relatedness of discriminant taxa identified by the LEfSe method (B).
Figure 6. Analysis of microbial taxa that differentiate patients with high and normal TMAO serum levels after supplementation of nitrate. Linear discriminant analysis effect size (LEfSe) analysis shows taxa enriched in individuals after receiving nitrate supplementation with high (red) and normal (green) TMAO serum levels with LDA > 3 (A), and a cladogram visualizes the phylogenetic relatedness of discriminant taxa identified by the LEfSe method (B).
Dietetics 04 00001 g006
Dietetics 04 00001 g007
Figure 7. Alpha diversity does not differ between control and nitrate groups and between baseline and follow-up. Alpha diversity is depicted for (A) Shannon diversity, (B) richness and (C) evenness (p > 0.05).
Figure 7. Alpha diversity does not differ between control and nitrate groups and between baseline and follow-up. Alpha diversity is depicted for (A) Shannon diversity, (B) richness and (C) evenness (p > 0.05).
Dietetics 04 00001 g007
Dietetics 04 00001 g008
Figure 8. Nitric oxide serum levels before and after supplementation between intervention and control groups at baseline and follow-up. There is no effect in the control group after placebo supplementation but a significant increase in nitrate oxide serum levels in the intervention group. Baseline control 5.06 ± 24.56 vs. baseline nitrate 5.0 ppb ± 2.45 ppb, p > 0.05, follow-up placebo 37.2 ppb ± 45.86 ppb vs. follow-up nitrate 101.5 ppb ± 28.26 ppb, *** depicts p < 0.005.
Figure 8. Nitric oxide serum levels before and after supplementation between intervention and control groups at baseline and follow-up. There is no effect in the control group after placebo supplementation but a significant increase in nitrate oxide serum levels in the intervention group. Baseline control 5.06 ± 24.56 vs. baseline nitrate 5.0 ppb ± 2.45 ppb, p > 0.05, follow-up placebo 37.2 ppb ± 45.86 ppb vs. follow-up nitrate 101.5 ppb ± 28.26 ppb, *** depicts p < 0.005.
Dietetics 04 00001 g008
Dietetics 04 00001 g009
Figure 9. Systolic blood pressure before and after placebo and nitrate supplementation between control group and intervention group. There is no effect in the control group after placebo supplementation, yet the blood pressure is significantly lowered after nitrate intake. Control group 124 ± 14.22 mmHg to 127 ± 17.54 mmHg, p > 0.05, and nitrate group 125 ± 10.59 mmHg to 121 ± 10.23 mmHg, * depicts p < 0.05.
Figure 9. Systolic blood pressure before and after placebo and nitrate supplementation between control group and intervention group. There is no effect in the control group after placebo supplementation, yet the blood pressure is significantly lowered after nitrate intake. Control group 124 ± 14.22 mmHg to 127 ± 17.54 mmHg, p > 0.05, and nitrate group 125 ± 10.59 mmHg to 121 ± 10.23 mmHg, * depicts p < 0.05.
Dietetics 04 00001 g009
Table 1. Baseline characteristics of the study cohort.
Table 1. Baseline characteristics of the study cohort.
Characteristic (Mean ± SD)Both GroupsNitratePlacebop-Value
(Nitrate vs. Placebo)
Sex, n (%)
Male8 (26.7)4 (26.7)4 (26.7)>0.9999
Female22 (73.3)11 (73.3)11 (73.3)>0.9999
Age (years)50 ± 7.551 ± 6.748 ± 8.20.2611
Height (cm)165.9 ± 32.7169.1 ± 11.3162.7 ± 45.40.6034
Weight (kg)77.5 ± 18.471.8 ± 13.883.1 ± 20.90.0925
BMI (kg/m2)26 ± 4.724.9 ± 2.927.2 ± 5.90.1872
Omnivore, n (%)29 (96.7)14 (93.3)15 (100)>0.9999
Hemodynamics baseline (mean ± SD)Both groupsNitratePlacebop-value (Nitrate vs. Placebo)
Systolic blood pressure (mmHg)124.8 ± 13.9125.2 ± 14.8124.3 ± 13.60.6666
Heart rate (bpm)61.9 ± 9.362.8 ± 8.260.9 ±10.50.5923
ABI right1.09 ± 0.11.11 ± 0.091.06 ± 0.090.0877
ABI left1.06 ± 0.11.07 ± 0.091.02 ±0.110.6101
PWV (m/s)6.9 ± 1.8 7.3 ± 1.76.5 ± 1.80.1891
TMAO (µg/L)399.6 ± 253.3393.6 ± 270.3406 ± 243.80.8986
High-sensitive CRP (mg/L)0.1 ± 0.10.1 ± 0.08 0.2 ± 0.1 0.1183
LDL (mg/dL)142.6 ± 40.7142.3 ± 43.5142.8 ± 39.20.9756
Cholesterin (mg/dL)205.7 ± 38204.7 ± 39.4206.6 ± 37.80.8956
Abbreviations: BMI: body mass index, ABI: ankle-brachial index, PWV: pulse wave velocity, CRP: C-reactive protein, LDL: low-density lipoprotein.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Messiha, D.; Rinke, M.; Schultz Moreira Amos, A.; Tratnik, A.; Hendgen-Cotta, U.B.; Lortz, J.; Hogrebe, K.; Kehrmann, J.; Buer, J.; Rassaf, T.; et al. Influence of Chronic Dietary Nitrate on Downstream Atherogenic Metabolites and the Enteral Microbiome—A Double-Blind Randomized Controlled Trial. Dietetics 2025, 4, 1. https://doi.org/10.3390/dietetics4010001

AMA Style

Messiha D, Rinke M, Schultz Moreira Amos A, Tratnik A, Hendgen-Cotta UB, Lortz J, Hogrebe K, Kehrmann J, Buer J, Rassaf T, et al. Influence of Chronic Dietary Nitrate on Downstream Atherogenic Metabolites and the Enteral Microbiome—A Double-Blind Randomized Controlled Trial. Dietetics. 2025; 4(1):1. https://doi.org/10.3390/dietetics4010001

Chicago/Turabian Style

Messiha, Daniel, Miriam Rinke, Adriana Schultz Moreira Amos, Annika Tratnik, Ulrike Barbara Hendgen-Cotta, Julia Lortz, Kristina Hogrebe, Jan Kehrmann, Jan Buer, Tienush Rassaf, and et al. 2025. "Influence of Chronic Dietary Nitrate on Downstream Atherogenic Metabolites and the Enteral Microbiome—A Double-Blind Randomized Controlled Trial" Dietetics 4, no. 1: 1. https://doi.org/10.3390/dietetics4010001

APA Style

Messiha, D., Rinke, M., Schultz Moreira Amos, A., Tratnik, A., Hendgen-Cotta, U. B., Lortz, J., Hogrebe, K., Kehrmann, J., Buer, J., Rassaf, T., & Rammos, C. (2025). Influence of Chronic Dietary Nitrate on Downstream Atherogenic Metabolites and the Enteral Microbiome—A Double-Blind Randomized Controlled Trial. Dietetics, 4(1), 1. https://doi.org/10.3390/dietetics4010001

Article Metrics

Back to TopTop