Two opposing theories how metabolic rate affects life span and ROS production
”Rate of living” and ”Uncoupling to survive” are tw o different, completely contrary predictions linking energy metabolism and aging. Both theories highlight the role of reactive oxygen species (ROS). The “rate of living” theory (42) associates high metabolism with a shorter lifespan. The theory is based on the observation that a higher oxygen metabolism results from an increased flux of electrons in the mitochondrial electron transport system (ETS). Of the total oxygen consumption a set amount (0.1-4%) is believed to be used for the formation of ROS. A lower energy metabolism would give a lower ROS formation, meaning less molecular damage and therefore a slower aging rate.
The ”Uncoupling to survive” (43) hypothesis sugges ts a positive association between lifespan and oxygen metabolism. In the ETS, ROS is primarily produced at complex I and complex III (43). The production of ROS at complex III increases if the membrane potential in the mitochondria increases. Thus, a lower membrane potential would effectively decrease ROS production. A lower membrane potential can be achieved by releasing some of the protons through the inner mitochondrial membrane, i.e. uncoupling of the mitochondria. There are several proton leak pathways, two of them are adenine nucleotide translocase (ANT) (44) and the uncoupling proteins (UCP) (45).
First a pilot study was performed to examine the amount of nitrates and nitrite after mouthwash with placebo and antiseptic chlorhexidine mouthwash to determine the efficacy of the antibacterial effect. The end-points of this pilot study were to confirm that the nitrate reducing capacity in the oral cavity were lost using this protocol.
In a randomized, double-blinded, cross-over study we aimed to investigate the effects of antibacterial mouthwash during a three-day period on the basal metabolic rate in 19 healthy non-tobacco using male volunteers.
It is known that the mitochondrial efficiency is one factor that determines BMR (46). With this new data as a background, we intended to investigate if normal levels of nitrate and nitrite were important in the control of BMR. Therefore, we aimed to investigate if the nitrate reducing capacity of oral bacteria, without concurrent nitrate supplementation, is an important determinant of BMR in health male subjects. Our hypothesis was that the antibacterial mouthwash effectively reduced saliva and plasma levels of nitrite and thus increased BMR.
MATERIALS AND METHODS
This work contains a pilot study where the aim was to evaluate the antibacterial effect of chlorhexidine based mouthwash with respect to the levels of nitrate and nitrite. The analysis method was HPLC. The basal metabolic rate was measured using indirect calorimetry.
Four healthy volunteers (three males, one female, range 30-49 years) participated in this study.
A baseline saliva sample was collected, immediately followed by antibacterial mouthwash or placebo for one minute. Saliva was again sampled after 60, 120 and 180 minutes. This procedure was followed with placebo mouthwash and chlorhexidine mouthwash. During these three hours the participants were fasting.
The mouthwash and metabolic rate study 19 healthy, non-tobacco using, male volunteers were recruited with mean age 29 (range 22-43) and mean weight 82.4 kg (range 71.2-107.35). Variations in the metabolic rate because of the menstrual cycle are significant (47), therefore women were excluded. Recruitment was achieved via personal networking. The blinding was performed so the primary researcher and participants were unaware of its content.
The antibacterial mouthwash ingredient is based on the ingredients in Corsodyl® with chlorhexidine as active substance (48). The ingredients used in the antibacterial mouthwash are chlorhexidine (0.2 %), ethanol 95 %, menthol and distilled water. The placebo mouthwash had the same mixture except for chlorhexidine. The amount of menthol was adjusted so it effectively masked the taste of chlorhexidine. The taste and antibacterial effect were tested in the pilot study in a cross-over design.
Mouthwash and BMR
During two separate three-day periods the subjects were instructed to rinse their mouth with the 10 ml mouthwash for two minutes, three times a day after a meal. During these three days they were instructed to follow a nitrate-restricted diet, i.e. they were told to avoid green leafy vegetables, beetroot and cured meats. On day three they were also instructed to avoid protein rich food and to be physically inactive. They were also told to start fasting from 8.00 pm. The next morning (Day 4) they were instructed to wash their mouths with mouthwash one last time immediately when they woke up. Fasting and physically inactive they were told to come to the lab for measuring their basal metabolic rate by indirect calorimetry (see table 1). Each visit lasted two hours.
The basal metabolic rate was measured with an indirect calorimetric system. A ventilated hood is placed over the face of the person and the expired air is led into an instrument which measures ventilation and volume of carbon dioxide and oxygen. The method is based on the difference in inspired and expired air and accounts for the amount of oxygen which is taken up and the amount of carbon dioxide produced in the body.
In the pilot study nitrate and nitrite concentrations in saliva were analyzed with HPLC. Nitrate and nitrite concentrations in plasma and saliva from the BMR study have not yet been analyzed because of time constraints.
Graph Pad Prism 5.0 was used for the statistical analyses. P values <0.05 were considered significant. Students paired t-test was used to analyze group differences for single measurements. A one-way ANOVA was used for repeated measures and time effects.
For this work; results from blood, urine and saliva are not included but the participants in this work did leave such samples and therefore this aspect will be considered.
The mouthwash with the antibacterial agent chlorhexidine can cause mild pain in the mouth and a loss of taste but will go back to normal after the use of mouthwash stops (48).
A blood sample was taken and can be associated with discomfort due to the needle penetrating the skin. It is possible to feel claustrophobic and uneasy during the BMR-measurement because of the ventilated hood.
Each participant received oral and written information about the experimental approach. They gave their written agreement for participating and signed agreements letting us store their blood samples, urine and saliva. The participants could, whenever they wanted to, terminate their participation without any further questions. All participants who finished the experiment received compensation. The ethical approval number from Local Ethics Committee in Stockholm is 2013/520-31/2.
Table of contents :
1.1. Dietary sources of nitrate and nitrite
1.2. Nitrate, nitrite and NO
1.3. Established effects of dietary nitrate administration
1.4. Basal metabolic rate
1.5. Two opposing theories how metabolic rate affects life span and ROS production
3. MATHERIALS AND METHODS
3.1. Study population
The mouthwash and metabolic rate study
3.2. The mouthwash
3.3. Mouthwash and BMR
3.4. Experimental protocol
3.6. Molecular analysis
3.7. Statistical analysis
3.8. Ethical considerations
4.1. Pilot study
4.2. BMR and mouthwash