The effect of nitrate supplementation: source of supplementation

Oral ingestion of nitrate (NO3-) has been shown to increase plasma nitrate and
nitrite levels. Significant lowering of pulmonary oxygen uptake during
exercise, as well as significantly lower blood pressure values at rest have
been observed after supplementation with nitrate. These effects are attributed
to increased endogenous NO production, which is related to the increase in
plasma nitrite levels. So far, studies have primarily used sodium nitrate
(NaNO3) and red beetroot juice to induce these metabolic effects. However, it
is unknown whether similar effects on plasma nitrite levels can be expected
when ingesting the juice that is derived from other nitrate-rich vegetables.
Indeed, no study has yet described a direct comparison between different
nitrate-rich beverages with respect to the bioavailability of nitrate/nitrite
and the possible hemodynamic effects. Based on the gaps in current literature,
our main goal will therefore be to gain further insight into the effects of
nitrate supplementation with different sources on plasma nitrite levels. This
will be investigated in healthy recreationally active men and women by
exploring the ability of NaNO3- and natural nitrate-rich food products to
increase plasma nitrite levels, and to decrease blood pressure as secondary
parameter.

The main aim of the current study will be to compare the ability of different
nitrate sources to increase plasma nitrite levels. The effects of oral
ingestion of NaNO3 will be compared to that of three natural nitrate-rich food
sources matched for total nitrate content (i.e. concentrated red beetroot
juice, a spinach based beverage, and rocket salad based beverage). In addition,
we will investigate whether possible effects on plasma nitrite levels are
accompanied by changes in blood pressure.

Single centre, cross-over study with 4 arms

Acute bolus ingestion containing 800 mg (12.9 mmol) nitrate in the form of
NaNO3- in a water solution, red beetroot juice, a spinach based beverage, or a
rocket salad based beverage. All 4 trials will be performed in a cross-over
manner separated by a 1 week washout period.

Subjects will be required to report to the laboratory for a total of 5 visits,
including screening. Prior to the first experimental visit (visit 2), subjects
will be asked to record their diet and activity for 48 h and refrain from
strenuous physical exercise or labour for the 48 h preceding the visit.
Subjects will be asked to avoid caffeine and alcohol for 12 h and 24 h
preceding each visit, respectively. They will be asked to replicate their food
intake and refrain from exercise for the 48 h prior to subsequent visits. Also,
subjects will be asked not to eat or drink after 22pm the evening before the
test day. They will be asked to fill in a medical questionnaire on the day of
the screening.

The subjects will be asked to give 11 blood samples of 7.5 mL every visit (44
blood samples or 330 mL in total over 4 visits). For blood collection, a small
teflon catheter will be inserted into a forearm vein with the assistance of a
small needle, which is subsequently removed. The discomfort of this procedure
is transient and is very similar to having an injection by a needle, or when
donating blood. Once the needle is removed, there should be no sensation from
the catheter. During the course of the experiment, blood will be drawn
periodically from the catheter. Some minor discomfort may occur and a small
hematoma may result. The researchers will also be aware of the possibility of
the subject fainting when placing the catheter.

The administered dose of nitrate in the natural food products (red beetroot
juice, rocket salad, spinach) has been administered in multiple clinical and
exercise studies and all the products are commercially available
supplements/food products. The most commonly reported side effect of red
beetroot juice is beeturia (red-colored urine/feces, 15-20% of the population).
In general, a common reported side effect of ingestion of nitrate-rich food
products such as red beetroot juice is mild gastrointestinal distress
(bloating, belching). The dose of NaNO3- used in the current study has been
administered in multiple clinical and exercise studies. No adverse events were
reported, except for reports of mild headache (which may or may not be
associated with ingestion of the sodium nitrate). The long term effects of
nitrate ingestion still need to be fully investigated.