In a study led by Gladwin in 2003, it was shown that slightly supraphysiological amounts of infused nitrite (going from 180 nM at baseline to 2,600 nM after infusion) led to increased forearm blood flow [2
]. More recently, the Gladwin lab showed that infusion of nitrite to achieve a level of only 350 nM results in increased forearm blood flow [3
]. Others have since confirmed that nitrite acts as a vasodilator in various tissues and that its activity is heightened in hypoxia [36
]. Rifkind and coworkers showed that nitrite infusions led to increases in cerebral blood flow in rats as measured by laser Doppler flowmetry [38
]. Thus, there is growing evidence that nitrite, rather than being relatively biologically inert, can act as a potent vasodilator harnessed primarily under hypoxic conditions. In this paper, we demonstrated the ability of oral nitrate to increase plasma nitrite and to increase cerebral blood flow within white matter in older adult humans using perfusion MRI.
Others have previously shown that substantial elevations in plasma nitrite occur through increasing dietary nitrate intake [4
]. Nitrate from the diet, once absorbed from the intestine, is taken up from the plasma by salivary glands and concentrated in saliva; nitrate is subsequently reduced to nitrite by symbiotic, oral bacteria and ultimately absorbed into the circulatory system [4
]. Documented physiological effects from increasing dietary nitrate include reduction in blood pressure [6
], improvement in intestinal health [10
], and increases in exercise performance [9
]. These effects are eliminated when volunteers either spit or use mouthwash, thereby implicating the importance of nitrate reduction to nitrite by oral bacteria. The level of nitrate utilized in the current study (12.4 mmol) from the dietary manipulations falls within the range shown by others to reduce blood pressure and improve exercise performance (22.5 mmol and 5.5 mmol, respectively).
To our knowledge, we are the first to demonstrate that these pathways also function in older adults. One might think that due to xerostomia, altered oral bacterial colonization, and increased gastric pH, the effect of dietary nitrate on plasma nitrite levels of older adults would not be as great as for younger adults. However, the increases in plasma nitrite that we observed in these older adults (an average of 630 nM increase one hour after the high nitrate breakfast compared to before the breakfast, and an average of 830 nM increase in post high nitrate breakfast compared to post low nitrate breakfast, ) are comparable to what has been observed in younger adults [6
]. Moreover, the plasma levels of nitrite achieved in our study using a dietary nitrate intervention (950 ± 470 nM, with a range of 280 to 1890 nM measured 1 hour after the high nitrate breakfast) are similar in magnitude to those obtained in nitrite infusions which led to increased forearm blood flow [2
]. Thus, as dietary nitrate is a natural variant, our data support the idea that nitrite is a physiological (rather than simply therapeutic) modulator of blood flow.
Results from the initial kinetics study presented here demonstrate the time course of changes in nitrate and nitrite after consuming the high nitrate breakfast containing 500 ml of beetroot juice. There was an increase in plasma nitrite levels by over 5 fold above fasting values in one hour, and this level was maintained for at least an additional two hours. These findings suggest that the plasma nitrite concentrations are maintained at a high level for the time participants in the perfusion study underwent imaging for perfusion measures.
In fact, the effects of dietary nitrate on plasma nitrite and nitrate levels were observed to be sustained after the overnight fast (compare levels of different diets pre-breakfast in ), suggesting that the effects of dietary nitrate are prolonged. Consumption of the low nitrate breakfast led to increases in plasma nitrite, but nitrate levels decreased. This could possibly be due to increased swallowing and gastric activity leading to more nitrate conversion to nitrite without consumption of substantial amounts of additional nitrate.
Although the effects of the nitrate in the diet on plasma nitrite and nitrate levels was highly significant, there was a substantial amount of variability, particularly in the levels of nitrite. The increase in individual subjects in plasma nitrite after the high nitrate breakfast compared to the low nitrate breakfast ranged from none in one case and a few fold in a couple of cases to up to 20 fold. This variance is not grossly dissimilar to that previously reported in a study using beet juice in younger adults [8
]. The cause for this large variance could be related to variability in the nitrate reductase activity of oral bacteria in different individuals, rates of nitrate uptake, or other causes. Additionally, gastric pH may lead to alterations in nitrate and nitrite metabolism. The presence of achlorhydria and use of pharmacological agents that can affect gastric pH, such as proton pump inhibitors were not controlled in the present study and this may have contributed to the variability observed. Clearly, this individual difference in response deserves further study.
There have been several mechanisms proposed for nitrite’s vasodilatory action, all involving conversion of nitrite to nitric oxide, although sometimes through the intermediacy of other nitrogen oxides [13
]. Most (but not all [41
]) of these involve the action of a particular protein and those proposed include hemoglobin [2
], myoglobin [43
], xanthine oxidoreductase [45
], nitric oxide synthase [47
], cytochrome c oxidase [48
], aldehyde oxidase [50
] and cytochrome c [52
]. It is likely that several of these act to reduce nitrite in different tissues and different conditions. Importantly, all proposed mechanisms of nitrite reduction to nitric oxide include a potentiation under hypoxic conditions, consistent with observed physiological nitrite action.
Earlier work has shown that aging is associated with progressive impairment of endothelial function [54
]. This may lead to a reduction in NO produced from arginine via the nitric oxide synthase (NOS) reaction in the endothelium and contribute to the reduced perfusion in tissues with aging. Supplementing L-arginine in older adults has produced equivocal results with some groups indicating improvements in flow-mediated dilation and other showing no effect with L-arginine supplementation [55
]. The current work indicates improvement in tissue perfusion via an endothelium-independent treatment. It would be informative to determine if L-arginine supplementation would increase cerebral blood flow independent of the effect from dietary nitrate, or if there is a potentiating effect from these two agents.
Historically, very high concentrations of nitrate in drinking water were thought to cause methemoglobinemia in infants, but more recent research has questioned whether nitrate or nitrite alone is the cause or if bacterial infection is also required [57
]. Furthermore, in the 1970s, concern over the possibility of nitrite causing cancer arose from the theoretical association between the ability of nitrite to form nitrosamines in the gut and studies showing that nitrosoamines are carcinogenic [58
]. However, the evidence that high levels of nitrate in diet causes any type of cancer is weak and most studies have found no link between dietary nitrate and cancer at all [58
]. The highest sources of nitrate in our diet are found in certain vegetables like spinach, celery, and beetroot, and epidemiologic evidence does not support these foods as causing diseases from nitrate. It may be that in addition to nitrate, vegetables also have antioxidants which may protect against nitrosamine formation. This being said, it is possible that some human subpopulations may be especially sensitive to cancer causing effects of nitrate and continued monitoring of this potential untoward health effect is advisable.
Chronic ischemia in the white matter is associated with aging. Chronic ischemia appears to be the fundamental process that leads to so-called white matter hyperintensities (WMHs). Several underlying mechanisms have been shown to contribute to this chronic ischemic state, including abnormal arterioles, capillaries and venules, and increased tortuosity of arterioles as we age [18
]. However, whatever the cause and regardless of the clinical diagnosis, chronic ischemia in the white matter appears to be the complex endgame leading to cognitive decline [21
]. As poor cerebral perfusion and ischemia have been associated with cognitive decline and dementia [14
], our results support the proposal that oral nitrate therapy may be beneficial in treating cognitive decline that is often observed with aging. Towards that end, we show a direct effect of dietary nitrate on cerebral blood flow within the subcortical and deep white matter of the frontal lobes. This finding is intriguing as there is evidence for an anterior-posterior gradient in age-related degeneration of white matter [62
] suggesting frontal regions are particularly compromised by aging. Moreover, there is a strong relationship between losses in white matter integrity and declines in aspects of executive function, including working memory, task-switching and episodic memory retrieval [25
], which are important for older adults’ performance of instrumental activities of daily living, such as writing checks, using appliances, and shopping [64
As already discussed, nitrite has been shown to not only increase blood flow to certain areas of the body, but also acts preferentially in hypoxic conditions, allowing nitrite to increase blood flow precisely in the areas where it is needed
]. Based on this notion, our data suggest that a diet high in nitrate might allow increased perfusion to those areas of the brain known to be at risk in the elderly and important for cognitive function —the deep white matter in the frontal lobe.
The diets used in our studies were designed to be isocaloric with similar protein levels, but differing levels of nitrate. The measured levels of nitrate were more than 1,000 fold different but the nitrite levels were similar between the low and high nitrate treatments. Based on the different food groups that compose each diet, it is expected that the level of antioxidants and other phytochemicals in the diets differ. We recognize that other dietary components besides nitrate may be partially responsible for the differential response in cerebral perfusion we observed. However, previous studies using a treatment of beetroot juice only or nitrate by itself have found that minimizing the conversion of nitrate to nitrite, by using mouthwash to kill bacteria or spitting to lower the absorption of nitrite, eliminated the hypotensive effect of the nitrate or juice [6
]. Thus, we suggest that the differences in nitrate in the diet and nitrite in the plasma are primarily responsible for the physiological effect we observed. Nevertheless, using a bactericidal mouthwash or spitting would need to be performed to substantiate the effect observed with the current diet is from the nitrate content in the diet.