Lecture 8(1) - nitrate Flashcards
Nitrate (NO3 -) intro:
NO3 - is found throughout the body in most tissues: saliva, skeletal muscle, heart muscle, blood, kidneys, urine etc
Can improve and augment BP regulation
The people who consume the most NO3 - (Japanese and Mediterranean’s) have the longest life expectancy. Correlation not causation
Sources of dietary nitrate:
- 60-80% of NO3 - consumption through the diet is in the form of green leafy vegetables
- 2 beetroot juice shots or 200-300 g of green leafy vegetables will provide > 500-750 mg NO3 –
- 400mg per shot of beetroot 92 x shots is recommended in the literature for CV health)
- High in - Spinach, rocket, cress, beetroot
- Low nitrate content in - asparagus, garlic, onion etc
- Plants store nitrate in their vacuole - Boiling vegetables causes the release of stored NO3 into the surrounding fluid – pour the water down the sink and therefore lose the nitrate content. This can decrease the NO3 - content of vegetables by up to 50%. Must consider how you prepare the veg (preparing a fresh salad of green leafy veg is best)
2 main pathways for nitrate metabolism:
1) Endogenous synthesis citrulline diet- Oxygen dependant – l-arginine synthesised endogenously or from citrulline (an AA), or can be released from the diet.
* L-arginine is reduced into citrulline via Nitric oxide synthase. As a by-product of this we get nitric oxide formation
- This pathway cannot occur in hypoxic environments.
2) Ingested nitrate (Potassium nitrate, sodium nitrate or beetroot juice/ green leafy veg) into the oral cavity - nitrate passes down in the stomach – some of it then passes into systemic circulation – but some is actively transported back into the oral cavity where the oral bacteria reduce nitrate to nitrite. Nitrite in the mouth is then swallowed and its further reduced to NO in the stomach. This reaction is potentiated when there is low oxygen and pH. This is why it happens well in the stomach as it’s an acidic environment
- Oral bacteria have different properties, and everyone has different oral nitrate reducing capabilities. Oral bacteria are affected by oral hygiene habits
- Mouth wash obliterates the oral microbiome – this pathway depends on the biome to reduce nitrate to nitrite.
Inorganic nitrate ad Beetroot juice supplementation reduces BP in adults- A systematic review and meta-analysis
Overall, when you consume a sufficient dose of dietary nitrate you get a 4.4mmHG decrease in systolic BP – this is clinically relevant
How nitrate works:
- Nitric oxide in the stomach activates the enzyme Guanylate Cyclase, which causes cGMP upregulation – this causes vasodilation. A more relaxed blood vessel is under less tension and therefore BP is reduced.
Ergogenic effect of nitrate supplementation: A systematic review and meta-analysis:
- Overall found a 3% increase in exercise performance from nitrate supplementation
- Improve exercise economy and endurance exercise performance – shown more so in moderately trained individual than elite athletes – elite athletes may have mitochondrial adaptations (cross bridge formations already optimised).
- Reduction in oxygen cost of exercise with nitrate
- Nitrate causes a decrease in intramuscular perturbation: slower depletion of energy sources: ATP/PCr, slower accumulation fatiguing metabolites: ADP/Pi (carry on for longer before you reach the critical threshold)
- Nitrate reduces the ATP cost of cross bridge formation/ the cross bridges that are needed for muscle contraction & the ATP cost of ATP resynthesis
- Nitrate causes performance improvements (10-12%) - Time to exhaustion tests improve and a time trial perspective (1-2% improvement in 5k time)
- Nitrate causes vasodilation: preferential blood flow distribution to type 2 fibres (working muscles)
- Nitrate can Increase expression of calcium handling proteins: needed for cross bridge formation and muscle contraction.
Factors influencing nitrate metabolism:
- Dietary sources e.g., Beetroot drinks better that increasing plasma nitrate levels than flapjack etc containing beetroot
- Affinity for transport: vegetable type,– nitrate moves up through the oral cavity via sialin
- Sialin has different affinities for different molecules– nitrate is at the bottom of the pecking order- thiocyanate and iodide are transported before nitrate . if you consume something that is rich in thiocyanate or iodide, they will be preferentially taken into the oral cavity before nitrate. This will affect nitrate uptake into the oral cavity and ultimately NO formation
- Oral: hydration, mouthwash kills bacteria, tongue brushing is good for the bacteria (allows the proliferation of nitrite producing bacteria), antibiotics can disrupt the bacterial community
- Environmental temperature, circadian rhythms, biological sex: lab temperature, biological fluctuations in salivary flow rate, pH and sex hormones, ethnicity, age, medication use, genetics, training status (less trained individuals see a more positive response)
- Analytical considerations: tourniquet use (creates a hypoxic environment – causing a preferential reduction of nitrite to NO (artificially induces the process)– may not reflect what is going on with their physiology), posture, vacutainers, centrifugation, temperature, freezer duration, assay
Rate-limiting steps in nitrate metabolism- salivary nitrate uptake:
- Affinity for salivary uptake: perchlorate > thiocyanate > iodide > nitrate
- By-product of smoking is thiocyanate production. Thiocyanate content is therefore elevated in smokers compared to non-smokers (due to detoxification of cyanide).
Rise in salivary [NO3 -] and plasma [NO2 -] following dietary nitrate supplementation attenuated in smokers. Resting SBP reduced in non smokers but not smokers. - Uptake with brassica vegetables (cabbage, cauliflower, sprouts and broccoli): these are rich in glucosinolates and these are hydrolysed to form thiocyanate, which is preferentially taken up over and above nitrates.
- Saw a reduction in systolic blood pressure after ingestion of high nitrate, low thiocyanate smoothie, but a reduction in BP did not occur when it was consumed alongside brassica veg.
- To increase dietary nitrate to benefit CV profile – ingest it in isolation
Nitrate ingestion blunts the increase in BP during cool air exposure.
A double-blind placebo, controlled, randomised, crossover trial:
- Nitrate-rich beetroot juice is more effective at increasing salivary and plasma (NO2-) in cool compared to normothermic conditions and blunts the rise in systolic blood pressure following acute cool air exposure. This might have implications for attenuating the increased cardiovascular strain in the cold.
- Nitrate levels higher in the warm compared to cool condition. The reverse occurred for nitrite levels
- Environmental temperate affects salivary flow rates. In cooler environments you have a higher salivary flow rate than you do in warm environments.
- Inverse relationship between salivary flow rate and environmental temperature
- In cold environments you have an elevated cardiovascular risk profile because there is an elevation in BP (vasoconstriction).
- After 30mm consumption of nitrate (via beetroot juice), more of the nitrate was reduced to nitrite in a cool condition. The mechanism behind this = the upregulation of salivary flow rate
Does exposure to chlorine-sterilised pool water influence nitrate metabolism:
- Swimmers given nitrate-rich / nitrate-depleted mouth rinse solutions
- Oral nitrite production assessed before and after morning and afternoon pool-based training.
- Thought that chlorine is sterilising so will kill microbiome bacteria
- No difference was seen in oral nitrite production
Is there diurnal variability in response to dietary NO3 supplementation
- Salivary flow rate peaks in the afternoon
- Better metabolism when salivary flow rate is increased
- Salivary pH is upregulated in the afternoon – alkaline environment is more preferential for the nitrite producing bacteria
- CV risk is heightened in the morning due to sudden postural changes, psychological stress, arousal from sleep etc
Research suggests exercise performance is augmented in the afternoon: lactate concentrations, calcium handling, muscle damage, core temperature
Influence of acute dietary nitrate supplementation timing on nitrate metabolism, central and peripheral blood pressure and exercise tolerance in young men:
- Found no detectable circadian variability in salivary flow rate or pH across the conditions (morning, afternoon, evening)
- Similar plasma nitrate and nitrite concentrations across the morning, afternoon and evening – all saw an elevation after beetroot
- Acute dietary nitrate supplementation was ineffective at improving exercise time to exhaustion in the morning, afternoon and evening
- No difference in BP between the times of day
- We cannot conclusively give a time when it is best to consume nitrate
Dietary nitrate supplementation enhances performance and speeds muscle deoxyhaemoglobin kinetics during an end-sprint after prolonged moderate intensity exercise:
- 2 h of moderate-intensity cycling, which immediately transitioned into a 60 s maximal-intensity end-sprint. Supplements ingested 2 h before and 1 h into the moderate-intensity exercise bout. Tried to simulate a maximal sprint that occurs at the end of a race
- Takes 1hr for nitrate to be taken back into the oral cavity to be reduced into nitrite and further into NO.
- We see peak plasma nitrite several hours after ingestion (~3hrs)
- Found that Higher power output during final 30s of the sprint with beetroot juice consumed before and during exercise compared to placebo condition.
Nitrate & biological sex:
- Females have higher nitrite levels at baseline and after nitrate supplementation
- Women have better bioconversion at any given dose of nitrate
- No sex differences in the oral microbiome composition
- Women have higher NO2- in all biological matrices at baseline and after NO3- supplementation
- Women have lower renal excretion of nitrate compared to men
- Women have higher oral bacterial NO3- reducing capacity
- Women have higher NO3- reabsorption by the kidneys
- However, women have Lower salivary flow rates
Oestrogen circulates in higher levels in females – this is a key regulator in endothelial NO production – therefore a regulator of NO production
BP regulation in males and females is different – women have lower resting BP compared to aged, matched men
Variability within females and between females:
- Variations in hormone cycles
- Endogenous nitric oxide production varies day to day in a female
Estradiol = regulator of eNOS (alternative pathway for NO production
