Oxygen Consumption - Science topic

If the ROS originate during your Sea Horse Measurement either from the mitochondrial source or extra-mitochondrial sources of the cells, definitely there will be an increase in O2 consumption. This is mainly because of the generation of ROS (superoxide) form molecular Oxygen (O2). In my earlier days, we used to differentiate from the CN-sensitive and CN-insensitive O2 consumption. You should try to block each step of the mitochondrial ET (Complex-I to IV using Cyanide, Oligomycin, FCCP, Rotenone, and Antimycin) and measure O2 consumption and simultaneously the specific ROS (Superoxide and Hydrogen Peroxide) formation. Simply, use SOD-inhibitable Epinephrine (adrenaline) oxidation for superoxide generation in cells under the treatment of different complex-specific inhibitors with cells and under identical conditions, measure oxygen consumption by cells with Sea Horse. Similarly, measure the formation of H2O2 under identical conditions. While measuring the H2O2 production by cells, you may also include a GSH-Peroxidase inhibitor or Catalase inhibitor (azide).

Definitely, ROS production leads to increase in oxygen consumption due to uncoupling of the respiratory ET chain.

Note:

Cyanide is a potent poison that inhibits cellular respiration by binding to cytochrome c oxidase (CCO) in the mitochondria. This inhibition leads to an accumulation of electrons in the electron transport chain, resulting in the production of reactive oxygen species (ROS).

Physiol Rev

. 2014 Jul;94(3):909–950. doi: 10.1152/physrev.00026.2013

Plant Physiol

. 2002 Apr;128(4):1271–1281. doi: 10.1104/pp.010999

There is ample work done in this area.

I just suggested a couple of papers. Please read.

Please read earlier papers published by Dr. Britton Chance and Dr. Hagihara.

I spent decades on this topic. This is my 2 cents worth suggestion.

Good area to investigate, especially in the area of Ferroptosis. Since I am a lipidologist, I am interested in the lipid peroxides and mitochondrial lipids.

Good question (for me).

Parinandi

The Ohio State University Wexner Medical Center

Columbus, OH

I was wondering about the thickness of your tissue sample. Considering the limited depth of the sensor to the plate bottom in the XF96, it would be much better to try it in the XF24.

Best of luck.

Felix Sempf Unfortunately, i do not have some of the equipment needed for the study. Gas analyser is a major equipment and its not available here

Amadeo M Parissenti Thank you for your reply. I'm going to do research on that.

We’re talking about children in a paediatric ICU. Is that a confounding variable?

By fixing the algorithm

It depends on the specific design of the Loligo System respirometer and the Autoresp program you are using. The fractional error you mention likely refers to the difference between the actual flow velocity and the desired flow velocity. If the Autoresp program is already accounting for this error and adjusting the flow velocity accordingly, then you do not need to make any additional corrections. However, if the program is not accounting for the error, then you may need to make corrections to the input BL to achieve the desired Uswim. It is best to consult the documentation for the respirometer and program or reach out to the manufacturer for clarification on how the fractional error is handled.

but VO2max makes you an elite athlete, but as an elite athlete, VO2max doesnt make the difference. Especially with running it is more about running economy and such

This will happen with hearty cells/isolated mito when OCR exceeds around 500 pmol O2/min or so (in the XF96). You can usually notice this happening when you see your O2 level data starts to look like a backwards 'J'. This is nicely described with 'Fig 7' from the following protocol article about respirometry in frozen mitochondria samples.

To get your pO2 in your hydrogels over time, you may want to see the couple papers below that use fast responding bare-fibre oxygen sensors. The papers below measure pO2 in Hydrogels using the OxyLite over time.

This is from my thesis for the best working protocol I managed to get: Jesse Gerard Meyer

A XFe96 Cell Culture Microplate (Agilent) was coated with 60 μl 0.1% (w/v) Poly-L- Lyine (Sigma) for one hour, washed twice with 100 μl of PBS and stored at room temperature overnight. For starvation experiments, 0.25 OD600 of yeast were added to the precoated plate with starvation medium; 0.1 OD600 for growing cells in complete medium, span down at 500 rpm for 5 minutes and then stored for 1 hour at 30’c. The sensor cartridge was loaded with 20 μl of 250 μM of FCCP (Tebu-bio) in port A, to a final working concentration of 25 μM, and 250 μM of Antimycin A (Sigma) in Port B, to a final working concentration of 25 μM. The vector for the drugs was the corresponding growth/starvation media. The measuring protocol included 3 initial mixing steps, and 3 measurements for the basal levels. Port A and B injection protocols were 3 mixing steps, waiting 5 minutes and three measurements. After the assay was completed, the yeast optical density was measured an additional time by transferring the cultures to a Nunclon Delta Surface 96 well plate and measuring OD600 on a Varioskan Flash plate reader (ThermoFischer). The data was analysed and normalised using the Agilent WAVE software version 2.6.1.

Using a circle breathing circuit in critically ill patients must be carefully considered. Most operations under general anaesthesia are relatively short, lasting a few hours at most. Critically ill patients may be ventilated for days to weeks. In this context there are potential benefits of rebreathing exhaled gases. However, rebreathing of exhaled toxins such as carbon monoxide may be problematic with prolonged use of a circle circuit.

It is a question of supply and demand. If the initial DO level is small, only sufficient for endogenous respiration, then the consumption rate would be low (cell economy principle). But if the initial DO is sufficient for normal aerobic respiration, then the consumption rates are equal for both cases (R is not dependent of oxygen availability) but the DO level in the first case (aerated) will start to drop more slowly until the supply rate is balanced by the microbial demand, whereas the DO level in the second case (non-aerated) would drop in a linear fashion until the DO is completely depleted whereupon the system will turn septic i.e. anaerobic respiration will take over.

Please see the following attached article.

Here are some things to consider in response to your questions:

- Is there a generally accepted protocol for investigating running economy with measuring oxygen consumption during sub-maximal running (with controlling speed, i.e. on a treadmill)?

Answer: Not really. What you exactly want to investigate will determine the exact protocol (e.g. duration, speed, condition).

- Specifically, how long would you have runners run in each condition (assuming you want to compare 3 to 4 different conditions) in order to prevent possible fatigue effects?

A steady-state oxygen consumption is usually reached after 2-3 minutes, so if you collect data during 1 minute you could use 4-minute conditions. 3-4 minutes is therefore also the minimum period required per condition. The number and order of conditions and rest between conditions can be manipulated to minimize fatigue.

- Given they know their 10k race performance (e.g. 40 minutes), how fast would you ask them to run?

Also this depends on what you want to know. Runners are most economical at the speed where they run most at. But if you want to have a valid comparison of their technique then you should match the speed. Usually, studies use a speed of up to 12 km/h because this is a speed where most recreational runners also have a RER <1.0

The easiest one, that does not require practically anything, is to observe the color of culture medium. If it turns yellow after 2-4 days of culture, there is an intense glycolytic metabolism, if cell metabolism is affected by your treatment, you will see the color change. If you want more quantitative data, harvest the medium and measure the pH.

It depends if you are fixing the cells for cellular markers or keeping them viable for live cell imaging markers. Also, whether you want to measure expression induction markers (mRNA expression), or on-going cellular processes (proteins, subcellular organelle activity like mitochondria and lysosomes).

Thank you Moayyad Shawaqfah

In G*Power, the following should be the appropriate menu:

F-tests -> ANOVA: Repeated measures, within factors

I don't know if this will help, but when arterial blood gases are drawn during a cardiopulmonary exercise test, the results of the blood in the syringe are 15 - 20 seconds behind what the heart and lungs are pumping out.

I think you can do the same experiments on sulfite solution and determine K_La. In this case dissolved oxygen is zero.

I'm not sure about telemetry but there are chronic implantable O₂ sensors for the OxyLite system that might work for you.

Please see the manuscripts in Attachment. In the articles there is scant information on your topic. Good luck.

I want to follow this discussion.

DEAR GABER

The attached content has some relevent information which might be useful to your good self.

Ann Bot. 2005 Sep; 96(4): 625–638.Published online 2005 Aug 10. doi:  10.1093/aob/mci215PMCID: PMC4247030

Rice: Sulfide-induced Barriers to Root Radial Oxygen Loss, Fe2+ and Water Uptake, and Lateral Root Emergence

Hi,

many years ago, Prof. Thomas Miller at the University of California at Riverside created a very nice and complete website devoted to insect physiology. You could find there all kind of useful stuff, information, illustrations, didactical material, laboratory guides, practicals, etc. I cannot find it anymore in the web, but it could have evolved (repository?). In any case, it would be worth contacting Tom (thomas.miller@ucr.edu).

Best regards,

Claudio

I think that physical activity should be an inevitable and irreplaceable part of everyday routine in maintaining good health, both physically and psychologically, in all age groups. But unfortunately, in Serbia, I believe that this attitude is widespread in urban areas among young people, but not among elderly people, only in those who are engaged in recreational sports. While in rural areas, physical activity is considered optional for young people, and even unnecessary and inappropriate for elderly people, which is highly harmful attitude. 

I think that raising awareness, education, organizing public trainings and similar activities should be at a higher level in rural areas, because there are large differences in understanding that physical activity is necessary, among the elderly population in urban and rural areas.

Well. We (laboratory of structure and functions of mitochondria) use oxygen electrode and confocal microscopy mostly.

So if you have good confocal (or very good fluorescent) microscope, which can resolve mitochondria - you can work with different fluoresent probes. For potential (tmre), ros (dcf), different mitotracers and so on. If you can resolve individual mitochondria you can do quite a lot of things - measure quantity, do 3d segmentation (if you have z-stacks) and have their volume, check their movement and shape (fragmentation process). And you can check dynamics of this processes and reaction to some changes be it chemical, or physical (like oxygen depletion). You can try to fixate and use immunohistochemistry.

Plus you can isolate individual mitochondria and use flow cytometry on functioning mito or check something via western blotting or similar methods.

Much depends on your microscope and object.

Hope it helps, I can try to think more if you specify task a little.

Thanks for the input! 

Dear Prof,

go through the link they are having instruments to measure ozygen.

We use different oxygen sensors from Ocean Optics. I would advise you to look at their products. They custom made for us the probe for strongly alkaline solutions. I know the person from Ocean Optic who can help you. If you are interested, send me an e-mail.

I have no experiance with that

Since you put whole piece of tissue (contains layers of cells) in the well, do you know how efficiently those drugs can get into cells and function on mitochondria? 

I think based on what I have read about SMO2 analysis, it will be too expensive.

Hopefully there's still someone following this question.. Do you know somehow recent article(s) supporting double product as an index of myocardial oxygen consumption?

The results from longitudinal studies are equivocal, with some showing increased HRV after training but an equal number of studies showing no differences. Further, Supra exercise was shown to worsen the reliability of HRV-spectral indices........"For example, in elite athletes, studies have revealed both increases and decreases in HRV to be associated with negative adaptation. Additionally, signs of positive adaptation, such as increases in cardiorespiratory fitness, have been observed with atypical concomitant decreases in HRV. As such, practical ways by which HRV can be used to monitor training status in elites are yet to be established"

"In these competitive athletes, short-term interventions resulted in a moderate increase in both resting HR and low frequency/high frequency ratio , and a moderate decrease in maximal HR. Long-term interventions resulted in a small decrease in HR during submaximal and maximal exercise ), without alteration of resting values. The small to moderate amplitude of these alterations limits their clinical usefulness, as expected differences may fall within the day-to-day variability of these markers. Consequently, correct interpretation of HR or HRV fluctuations during the training process requires the comparison with other signs and symptoms of over-reaching to be meaningful"

"Increases in post-exercise HRV and HRR also occur in response to overreaching, demonstrating that additional measures of training tolerance may be required to determine whether training-induced changes in these parameters are related to positive or negative adaptations"

"The results from longitudinal studies are equivocal, with some showing increased HRV after training but an equal number of studies showing no differences"

Thank you very much sir

I wish this guideline will show me the proper way.

Resting oxygen uptake is about 3.5 mL/kg/minute, STPD. A 70 kg person would be estimated to have an oxygen uptake of about 245 mL per minute. The conditions inside the chamber would need to be corrected to STPD For calculations and if people were moving around inside the chamber, the oxygen use and CO2 volume exhaled will increase.  

As Philip mentioned above, without a system to scrub CO2 from the chamber, you will probably run into problems with re-breathing CO2 before hypoxia becomes a problem. At rest, humans exhale around 4.5% CO2, while the normal inspired CO2 level is about .03%

Dear Dr Marisa,

see articles that might be worth reading. 

Kind regards,

Olaf 

Dear Thibaut, 

Two questions in one. Check these papers out: 

I agree with Sergei. All the states of respiration should be ascertained. Please include specific inhibitors for respiratory complexes. If you see the increase in oxygen consumption with claritho and penicillin even after blocking the state 3 respiration, must be an uncoupling phenomenon.

Thank you, Ariadne. I had this review literature in this field. But seems it did not point out the effective experimental method to directly measure the [O2] in the porous media domain.   

Most tumors are associated with tumor promoting immune cells where blood vessels bring these cells right into tumors. Mainly for cytokine signals.

Optical oxygen sensors do not consume oxygen.

Thank you so much for the answers! I am trying the Clark electrode :)

Godfrey protocol, work rate increments depending on the child's height.

Thank you so much, dear colleague.  Searching the internet, I found a simple recent equipment for measurement of bacterial respiration and other applications.  I think you might be interested in. 

 the following link may be useful to you.

Hi Guys.

 I would like to know if you have experience with arm-crank exercise tests in patients with vascular disease, such as Peripheral Artery Disease patients.

Thanks

Mark, do not trust any method that can assess VO2max without the person work out. The evaluation of resting HR does not allow that the VO2max can be calculated indirectly because many factors affect the values of HR at rest, therefore invalidating your data or your value of VO2max. You can use submaximal tests that allow, by inference, that VO2max can be established. They are more reliable, but even so, the real value of VO2max can only get in maximum dynamic effort.

y

Hi,

I do not know much about oxygen consumption experiments but I do ATPase assays on sub mitochondrial particles which requires NADH. The buffer that we use in our lab for the assay is 50mM Tris Acetate at pH7.5. Maybe the pH of the buffer is too high which causes NADH degradation. Is this the pH that is required for your O2 consumption experiments? If using a UV spec, check that the wavelength is at 340nm because that is the region of NADH absorption. Also, try using NADH as quickly as possible as it degrades really soon. I hope this helps!!!

Hydrogen peroxide -- as mentioned by others -- if present in solution will contribute to COD.  A mg/L of H2O2 increases COD by about 0.393 mg/L ; this value was obtained employing Milli-Q water spiked with varying concentrations of H2O2; you may do your own measurements employing distilled and deionized water or Milli-Q water, etc.

Addition of catalase also introduces error in COD measurement that has to be accounted for; this can be done by the following relation.

COD_COR = COD_S+C - COD_C

Where COD_S+C is COD of water sample after catalase addition and COD_C is COD of Milli-Q water with the same amount of catalase.

 I could probably help but you did not provide details. Check my publications in my profile. Ask specific questions. 

Grettings.

The acid production it's because this enzimatic systems use co-factors (NAD+ and NADP+) that the producto of that reaction is an acid equivalent.

Here i put you the equations:

Catalase:

2H2O2 ---> 2H2O + O2

NAD+ ---> NADH + H+

Glucose oxidase:

D-Glucose (C6H1206) + O2 ---> C6H10O6 + H2O2

NADP+ ----> NADPH + H+

Michal has made a couple of interesting points. He provides a possible reason for nighttime minimal MR and EWL to differ. I am not sure of the mechanism though, because most EWL is achieved through panting or some other active form (e.g., gular flutter), not common at night. Could enough be lost passively through breathing? That itself is an interesting question.

He also gives you a reference for Aschoff. It was Aschoff and Pohl who first recommended that BMR be measured at night in diurnally active birds. This gradually became standard practice to the point that some authors have defined BMR conditions to include measurement during the rho (inactive) phase of the bird's day. However, that is an extension of A & P; I don't think they ever went that far. Today, we recognize that BMR can shift as a function of season, body composition, even diet. That is, as Gabrielsen and I have asserted, BMR should be treated as a parameter (I think we called it a statistic), not a constant. That said, measurement of BMR in the daytime is reasonable, as long as it is identified as such (I make measurements in both the day and night).

Also, Michal suggests you make measurements in the daytime. Daytime measurements may have more ecological relevance, especially when you pair them to simultaneous measurements of EWL. The reason is that EWL, converted to evaporative heat loss, is surely more important to thermoregulation at higher temperatures.

The relatively high MLSS and small reactors associated with MBRs require that more oxygen be transferred per unit reactor volume. However, oxygen-transfer kinetics may decrease with increased MLSS concentration and viscosity. High viscosity can reduce the interfacial area for oxygen transfer by increasing the rate of bubble coalescence. In fact, several MBR studies reported that oxygen demand can exceed the volumetric capacity of the aeration system at high MLSS concentrations (>13000- 15000 mg/L). The influence of mixed liquor constituents on aeration capacity can be quantified by the alpha (α) factor. Because physical features and operating conditions of aeration equipment can vary, the relationship between MLSS and the α value is system-specific. For example increasing activated sludge MLSS concentration from 3 000 to 6000 g/L will reduce the aeration α from 0.77 to 0.59, resulting in 23% reduction in α.

dear kakarala you can calculate the O2 demand via substrate degradation (organic matter : acetate or other compound)

in cease of acetate :

CH3COO- + 2O2---> 2HCO3- + H+

you see the following article about anodic and cathodic reaction for estimation the oxidation reaction and amount of dissolved oxygen

Rozendal, R.A., Hamelers, H.V.M., Rabaey, K. et al. (2008)

Towards practical implementation of bioelectrochemical wastewater

treatment. Trends in Biotechnology 26: 450-459. Doi:10.1016/j.

tibtech.2008.04.008.

I'm sorry - I am not set up for aquatic respirometry.

The use of large muscle mass on a exercise is the goal for obtain a high oxygen consumption. However, like Garstang said the volume and intensity are important. Regarding the intensity the use of high loads are important for increase the VO2. However when we use strength training exercises like Snatch and Jerk the load used for the upper limbs is more high than the used for the lower limbs. Another problem is the used of power movements when performed the Snatch and jerk. The power movement have a low increase of the VO2 when compare with the movements with a cadence of 40 or 60 batmin. My suggestion is use the combination of machine for the lower limbs (e.g. Leg press) with exercises who use dumbbells for the upper limbs (e.g. inclined chest press). In this way you can relativize the load for the lower limbs and upper limbs, used more muscle mass and get a higher VO2 during and after exercise with few time of performing. My research team compared the use of 3 sets of ten repetitions, with 70 1RM and 1 min rest between sets of the incline leg press exercise following the same protocol for the incline chest press with the simultaneous execution of the same exercises, with the same protocol but only 3 sets, in the leg press machine and with dumbbells. We saw a high VO2 in a few time in the simultaneous protocol.

Best Regards for all!