Gut bacteria can transform dietary nitrate and non-heme iron into protective molecules known as dinitrosyl iron complexes (DNICs), potentially lowering risks for cardiovascular and metabolic diseases, according to a new study published in The Cell. Researchers from Karolinska Institutet in Sweden combined findings from mouse experiments and human samples to show that these complexes are absorbed by key organs like the liver and kidneys, and that supplementing with nitrate and iron improved health markers in animal models.
How the gut microbiota works
Nitrate is naturally found in vegetables, especially beetroot and leafy greens, while non-heme iron appears in plant-based foods like beans, whole grains, and green vegetables. The researchers found that iron and nitrate work together rather than having equal effects. Iron compounds can be harmful because they have the potential for a destructive redox reaction, and nitrate may also bring undesirable effects due to oxidation of hemoglobin and other hemoproteins.
However, gut bacteria uniquely convert these two potentially dangerous compounds into new protective molecules. The study’s first author and co-corresponding author Andrei Kleschyov, senior researcher at the Department of Physiology and Pharmacology at Karolinska Institutet, explains that nitrate is the starting substrate that nitrate reductase (NR)-expressing bacteria reduce and use in the pathway leading to DNIC formation. Non-heme iron provides the iron component needed to form the DNIC. Iron alone did not increase DNIC levels or produce the same benefits.
The strongest effects occurred when nitrate and iron were combined, and the resulting DNIC is the protective signaling molecule. Previous studies on dietary nitrate have found it to boost the body’s production of nitric oxide (NO), which widens blood vessels and is linked to lower blood pressure and better exercise performance. In many previous studies, dietary nitrate supplementation has been used to support the nitrate–nitrite–NO pathway, while dietary iron levels have received comparatively little attention.
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It is possible that some of the beneficial effects observed following nitrate supplementation may actually be attributable to DNIC formation rather than to the generation of free NO, posits co-corresponding author Mattias Carlström, Pharm.D., Ph.D., professor of Cardiorenal Physiology at Karolinska Institutet. Moreover, the variable outcomes reported across studies using similar or identical doses of nitrate could potentially be explained, at least in part, by differences in dietary iron availability.
Testing the findings
The team used electron paramagnetic resonance (EPR), which enabled them to directly detect and quantify DNICs in tissues and cells. Because they found that DNIC was absent in germ-free mice, they concluded that the gut microbiota is essential for their formation. The study deliberately combined in vivo, in vitro, biochemical, cellular, and human-sample approaches so that the same mechanism could be tested from several angles.
Human fecal samples and Escherichia coli showed that the mechanism is not restricted to mice and identified NR as essential. Vascular, biochemical, and cellular experiments established how DNICs act. HepG2 [human liver] cells and primary human liver spheroids provided evidence that the protective effect can occur in human liver cells.
Employing several complementary methods strengthens their conclusions. Kleschyov adds that EPR also enabled spotting molecules in tissues without needing to break them down. No other methods are specific and sensitive enough to do so. So far, researchers have concentrated on the larger signals, while the small peak, which might belong to DNIC, was generally ignored.
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The team demonstrated that this small EPR feature is due to DNIC, which is constitutively present in the livers and kidneys of healthy animals but was absent in germ-free mice. Furthermore, they found that the DNIC signal is sharply raised in control animals consuming nitrate in combination with nonheme iron in a gut microbiota-dependent process. The role of the EPR was absolutely indispensable in this work.
The researchers revealed that key gut bacteria express NR, making dietary nitrate and non-heme iron essential to boost DNIC formation. The paper shows that nitrate alone was ineffective in mice on a near-zero-iron diet. The study does not, however, establish a particular named human diet, such as Mediterranean versus vegetarian, as the optimal diet.
Practically, the findings suggest that a diet providing nitrate-rich foods together with adequate non-heme iron would provide the substrates needed for this pathway. Importantly, the paper shows that nitrate alone was ineffective in mice on a near-zero-iron diet, whereas nitrate plus iron produced metabolic benefits and increased DNIC formation.
For people with low iron levels, this discovery suggests a mechanistic link between nutrient availability and how the body handles nitrate. If the body lacks the iron component needed to pair with nitrate, the protective pathway essentially shuts down, meaning that improving iron status could be as important for heart health as simply eating more vegetables.
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The most important bacterial species, as well as specific diet supplements busting endogenous DNIC formation, are going to be determined in the future, Kleschyov adds. According to Carlström, the study suggests that an iron deficiency might hinder gut bacteria’s ability to convert dietary nitrate into DNICs, thus potentially reducing the microbiota-dependent signaling pathway.
In the mouse experiments, nitrate supplementation provided no metabolic benefit when dietary iron was nearly absent, whereas nitrate plus iron restored the beneficial effects. However, these findings do not yet justify recommending nitrate or iron supplementation to deficient individuals specifically to increase DNIC production, he warns.
Our data suggest that DNIC is efficiently absorbed in the gut and transported with the blood as an intact entity, independently of the common Fe transport systems. Thus, it is not excluded that nitrate supplementation may help people with iron deficiency. According to Carlström, if human studies could prove the cardiometabolic benefits of nitrate or iron supplementation, it could potentially lead to specific dietary recommendations aimed at supporting DNIC generation.
An alternative would be finding specific bacteria carrying nitrate reductase that can effectively generate DNICs from nitrate and iron. This could lead to new probiotic development. In short, adequate iron and dietary nitrate may be important substrates for this newly identified microbiota pathway, but whether correcting iron or nitrate deficiency in humans increases DNICs and improves health remains to be tested clinically, concludes Carlström.
